U.S. patent application number 10/119417 was filed with the patent office on 2003-02-20 for drug complex for treatment of metastatic prostate cancer.
This patent application is currently assigned to Beth Israel Deaconess Medical Center. Invention is credited to Bubley, Glenn J., D'Amico, Anthony V., Jebaratnam, David J., Weinberg, James S..
Application Number | 20030035804 10/119417 |
Document ID | / |
Family ID | 22335136 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030035804 |
Kind Code |
A1 |
D'Amico, Anthony V. ; et
al. |
February 20, 2003 |
Drug complex for treatment of metastatic prostate cancer
Abstract
A drug complex for delivery of a drug or other agent to a target
cell, comprising a targeting carrier molecule which is selectively
distributed to a specific cell type or tissue containing the
specific cell type; a linker which is acted upon by a molecule
which is present at an effective concentration in the environs of
the specific cell type; and a drug or an agent to be delivered to
the specific cell type. In particular, a drug complex for
delivering a cytotoxic drug to prostate cancer cells, comprising a
targeting carrier molecule which is selectively delivered to
prostate tissue, bone or both; a peptide which is a substrate for
prostate specific antigen; and a cytotoxic drug which is toxic to
androgen independent prostate cancer cells.
Inventors: |
D'Amico, Anthony V.;
(Weston, MA) ; Bubley, Glenn J.; (Walpole, MA)
; Jebaratnam, David J.; (Lexington, MA) ;
Weinberg, James S.; (Dover, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Beth Israel Deaconess Medical
Center
Boston
MA
02215
|
Family ID: |
22335136 |
Appl. No.: |
10/119417 |
Filed: |
April 9, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10119417 |
Apr 9, 2002 |
|
|
|
09110822 |
Jul 6, 1998 |
|
|
|
6368598 |
|
|
|
|
09110822 |
Jul 6, 1998 |
|
|
|
09003838 |
Jan 7, 1998 |
|
|
|
09003838 |
Jan 7, 1998 |
|
|
|
08713114 |
Sep 16, 1996 |
|
|
|
Current U.S.
Class: |
424/178.1 ;
424/649; 424/650; 514/19.4; 514/19.5; 514/19.8 |
Current CPC
Class: |
A61K 38/06 20130101;
A61K 38/08 20130101; A61P 35/04 20180101; A61K 47/543 20170801;
A61K 47/65 20170801 |
Class at
Publication: |
424/178.1 ;
424/649; 424/650; 514/8; 514/12 |
International
Class: |
A61K 039/395; A61K
038/14; A61K 038/16; A61K 033/24 |
Claims
We claim:
1. A drug complex comprising: a) a targeting carrier molecule
which, when introduced into an individual, is selectively
distributed to a specific cell type or tissue containing the
specific cell type; b) a linker which is acted upon by a molecule
present at an effective concentration in the environs of the
specific cell type; and c) a drug or an agent to be delivered to
the specific cell type.
2. A drug complex comprising: a) a targeting carrier molecule
which, when introduced into an individual, is selectively delivered
to prostate tissue, bone or both; b) a peptide which is a substrate
for prostate specific antigen; and c) a cytotoxic drug which is
toxic to androgen independent prostate cancer cells, wherein the
peptide links the targeting molecule and the cytotoxic drug.
3. The drug complex of claim 2, wherein the targeting carrier
molecule is selected from the group consisting of polyamines; the
peptide linker is selected from the group consisting of SEQ ID NOS.
1-14 and alanine-lysine-phenylalanine-glutamate; and the cytotoxic
drug is selected from the group consisting of: adriamycin,
amonafide, cisplatin, carboplatin (CBDCA), CHIP, cyclophosphamide,
doxorubicin, epirubicin, estramustine, etoposide, 5-fluorouracil,
gallium nitrate, idarubicin, ifosfamide+mesna, ketoconazole,
liarozole (R85,246), methotrexate, mitomycin C, mitoguazone,
mitoxantrone, proscar (finasteride), suramin, taxol, thapsigargin,
trimetrexate, vinblastine (CI), and vinblastine and emcyt.
4. The drug complex of claim 3, wherein the polyamine is selected
from the group consisting of putrescine, spermine, and
spermidine.
5. The drug complex of claim 4, wherein the putrescine is
fluoropropylputrescine.
6. The drug complex of claim 2, wherein the peptide linker
additionally comprises a chemical selected from the group
consisting of para-amino benzoic acid and
para-aminobenzyloxycarbonyl.
7. A method of killing androgen independent prostate cancer cells
in a man with prostate cancer, comprising administering to the man
a therapeutically effective amount of a drug complex which
comprises: a) a targeting carrier molecule which is selectively
delivered to prostate tissue and bone; b) a peptide which is a
substrate for prostate specific antigen; and c) a cytotoxic drug
which is toxic to androgen independent prostate cancer cells,
wherein the peptide links the targeting molecule and the cytotoxic
drug and wherein the drug complex is administered to the man in
such a manner that it is delivered to androgen independent prostate
cancer cells and the cytotoxic drug enters androgen independent
prostate cancer cells, thereby killing the cells.
8. The method of claim 7, wherein the peptide which is a substrate
for prostate specific antigen is selected from the group consisting
of SEQ ID NOS. 1-14 and alanine-lysine-phenylalanine-glutamate.
9. The method of claim 7, wherein the targeting carrier molecule is
selected from the group consisting of polyamines.
10. The method of claim 9, wherein the polyamine is selected from
the group consisting of putrescine, spermine, and spermidine.
11. The method of claim 10, wherein the putrescine is
fluoropropylputrescine.
12. The method of claim 7, wherein the peptide linker additionally
comprises a chemical selected from the group consisting of
para-amino benzoic acid and para-aminobenzyloxycarbonyl.
13. The method of claim 12, wherein the drug complex is
administered intravenously.
14. The method of claim 13, wherein the cytotoxic drug is selected
from the group consisting of: adriamycin, amonafide, cisplatin,
carboplatin (CBDCA), CHIP, cyclophosphamide, doxorubicin,
epirubicin, estramustine, etoposide, 5-fluorouracil, gallium
nitrate, idarubicin, ifosfamide+mesna, ketoconazole, liarozole
(R85,246), methotrexate, mitomycin C, mitoguazone, mitoxantrone,
proscar (finasteride), suramin, taxol, thapsigargin, trimetrexate,
vinblastine (CI), and vinblastine and emcyt.
15. A method of treating metastatic prostate cancer in an man,
comprising administering to the man a therapeutically effective
amount of a drug complex which comprises: a) a targeting carrier
molecule which is selectively delivered to prostate tissue and
bone; b) a peptide which is a substrate for prostate specific
antigen; and c) a cytotoxic drug which is toxic to metastatic
prostate cancer cells, wherein the peptide links the targeting
molecule and the cytotoxic drug and wherein the drug complex is
administered to the man in such a manner that it is delivered to
prostate tissue and bone and the cytotoxic drug enters metastatic
prostate cancer cells, thereby killing the cells.
16. The method of claim 15, wherein the peptide which is a
substrate for prostate specific antigen is selected from the group
consisting of SEQ ID NOS. 1-14 and
alanine-lysine-phenylalanine-glutamate.
17. The method of claim 15, wherein the targeting carrier molecule
is selected from the group consisting of polyamines.
18. The method of claim 17, wherein the polyamine is selected from
the group consisting of putrescine, spermine, and spermidine.
19. The method of claim 18, wherein the putrescine is
fluoropropylputrescine.
20. The method of claim 15, wherein the peptide linker additionally
comprises a chemical selected from the group consisting of
para-amino benzoic acid and para-aminobenzyloxycarbonyl.
21. The method of claim 20, wherein the drug complex is
administered intravenously.
22. The method of claim 21, wherein the cytotoxic drug is selected
from the group consisting of: adriamycin, amonafide, cisplatin,
carboplatin (CBDCA), CHIP, cyclophosphamide, doxorubicin,
epirubicin, estramustine, etoposide, 5-fluorouracil, gallium
nitrate, idarubicin, ifosfamide+mesna, ketoconazole, liarozole
(R85,246), methotrexate, mitomycin C, mitoguazone, mitoxantrone,
proscar (finasteride), suramin, taxol, thapsigargin, trimetrexate,
vinblastine (CI), and vinblastine and emcyt.
Description
RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 09/110,822 filed Jul. 6, 1998, which is a continuation-in-part
of application Ser. No. 09/003,838 filed Jan. 7, 1998, which is a
continuation of application Ser. No. 08/713,114 filed Sep. 16,
1996, now abandoned, the teachings of which are incorporated herein
in their entirety.
BACKGROUND OF THE INVENTION
[0002] Prostate cancer is the second most common malignancy in men
and is the third most common cause of cancer death in men older
than age 55. Harrison's Principles of Internal Medicine, Eds. J. D.
Wilson et al., 11th Edition, pp. 1630-1633, McGraw Hill, New York,
1991. New methods of identifying and treating prostate cancer early
are extremely important in saving the lives of those afflicted.
Presently, only a limited number of treatments are available for
treatment of prostate cancer. These treatments include surgery,
radiation therapy, and androgen deprivation.
[0003] There is a need for additional methods of treatment of
prostate cancer.
SUMMARY OF THE INVENTION
[0004] The present invention relates to a method of delivering a
drug to a specific cell type, such as a cancer cell, in an
individual by means of a drug complex which comprises three
components and is also the subject of the present invention. The
three components of the drug complex include the following: a
targeting carrier molecule; a linker which is acted upon by a
molecule present at an effective concentration in the environs of
the specific cell (referred to as a target cell); and a drug (or
agent) to be delivered to the specific cell type. The targeting
carrier molecule is one which is delivered specifically to (has a
biodistribution which favors) a specific cell type, or tissue
containing the specific cell type, to which the drug is to be
delivered. The linker can be any type of molecule such as a
peptide, provided that it is a substrate for a molecule, such as an
enzyme (e.g., a protease), which is found in the environs of the
target cell to which the drug is to be delivered and is present in
sufficient quantities at the site(s) of the target cell to act upon
the linker and release the drug or agent to be delivered, such as
by cleaving the linker. The linker can comprise more than one type
of molecule. For example, the linker can comprise a component which
is a substrate for a molecule which acts upon it and releases the
drug to be delivered and an additional component(s), such as a
molecule which acts as a good leaving group; additional amino acid
residues or other molecule, such as one which acts as a spacer
between the targeting carrier molecule and the drug to be
delivered. The drug or agent to be delivered can be any
therapeutically or diagnostically useful drug or agent, such as a
cytotoxic drug or an imaging agent. It remains essentially
functionally inactive while it is a component of the drug complex;
upon release at the site(s) of the target cells, the drug is
functionally active (exerts or displays its therapeutic or
diagnostic function(s)). In one embodiment, the linker comprises a
substrate for a molecule as described previously, and one or more
other additional components, such as chemical molecules, e.g.
para-amino benzoic acid (PABA) and para-aminobenzyloxycarbonyl
(pABOC), which are good leaving groups. Optionally, there can be
amino acid residues in addition to those acted upon by the enzyme
or other molecule. Such amino acid residues can be at either or
both ends (amino and/or carboxy terminus) of the amino acid
residues acted upon. In one embodiment, additional amino acid
residue (or residues) is/are included between the carboxy terminal
amino acid of the substrate and the drug or agent to be delivered.
Cleavage of the substrate results in a product in which an amino
acid residue(s) is attached to the drug or agent delivered to the
target cell. Such amino acid residues can be, for example, one or
more amino acid residues of the substrate (e.g., if the enzyme
cleaves at an internal amino acid residue in the substrate), one or
more amino acid residues which are not substrate amino acid
residues (e.g., if the enzyme cleaves between the carboxyl terminal
amino acid residue of the substrate and the amino terminal amino
acid residue of additional amino acids) or both. The additional
amino acid residue(s) between the substrate carboxyl terminal amino
acid residue and the drug or agent to be delivered to the target
cell can be linked to the drug or agent in such a manner (e.g.,
through an amide bond, disulfide bond or ester bond) that cellular
enzymes act upon them; resulting in release of the amino acid
residue(s) attached to the drug or agent when it enters the cell.
The drug or agent to be delivered can be any therapeutically or
diagnostically useful drug or agent, such as a cytotoxic drug or an
imaging agent.
[0005] The present invention also relates to a method of delivering
a drug to a specific cell type by means of the drug complex
described herein. In the method, the drug complex is administered
to an individual in need of therapy or in whom a diagnostic
procedure or assessment is to be conducted.
[0006] In one embodiment, the present invention is a method of
killing prostate cancer cells, including metastatic prostate cancer
cells or androgen independent prostate cancer cells, in a man with
prostate cancer. The method is particularly effective because it
makes use of a drug complex whose components provide the basis for
localization to and killing prostate cancer cells, particularly
androgen-independent cancer cells. The method of killing metastatic
prostate cancer cells comprises administering to the man a
therapeutically effective amount of a drug complex which comprises
three components: a targeting carrier molecule which is selectively
delivered to prostate tissue, bone or both; a linker which
comprises a component acted upon by prostate specific antigen (PSA)
which is present in the microenvironment of malignant prostatic
epithelial cells; and a cytotoxic drug which is toxic to prostatic
cancer cells. The present invention also relates to a method of
treating metastatic cancer in an individual, in which the drug
complex described above is administered to a man with prostate
cancer, in a quantity sufficient to deliver the cytotoxic drug to
prostate cancer cells at a concentration sufficient to kill some or
all of the cancer cells or inhibit replication or division of the
cancer cells. Drug complexes useful in the method of treating
prostate cancer and the method of killing prostate cancer cells
(such as androgen independent cells) are also the subject of this
invention.
[0007] In a further embodiment, the drug complex comprises a
targeting carrier molecule which is an imaging agent for prostate
and prostate derived tumors, such as fluoropropylputrescine (e.g.,
N-3 fluoropropylputrescine); a peptide which serves as a linker and
comprises amino acid residues acted upon by PSA; and a cytotoxic
drug, which is linked to the targeting carrier molecule by the
peptide. The linker can be comprised of more than one type of
molecule, as described herein. The drug complex is administered to
an individual by any route which results in delivery of the
cytotoxic drug to prostate cells. In one embodiment, the drug
complex is administered intravenously.
[0008] Combining a targeting carrier molecule with specificity for
prostate tissue, bone or both with a cytotoxic drug, which are
joined by a linking peptide that is a substrate for PSA, provides a
treatment for cancer having higher efficacy and lower toxicity than
presently available treatments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a graphic representation of growth delay of the
human DU-145 prostate carcinoma xenograft after treatment with
fractionated radiation therapy (RT) on days 7-11 or with
thapsigargin (TG) on days 4-18.
[0010] FIG. 2 is a graphic representation of growth delay of the
human DU-145 prostate carcinoma xenograft after treatment with
fractionated RT on days 7-11 and 14-18 or with TG on days 4-18.
[0011] FIG. 3 is a schematic representation of one embodiment of a
drug complex of the present invention, which comprises a targeting
carrier molecule ("carrier"), a peptide linker ("peptide"), and a
drug ("drug"); the peptide is a substrate for a molecule whose
action at the cleavage site results in cleavage of the drug from
the drug complex.
[0012] FIG. 4 is a schematic representation of one embodiment of a
drug complex of the present invention in which the carrier is a
polyamine, the peptide is a PSA-specific peptide and the drug is a
cytotoxic drug.
[0013] FIG. 5 is a schematic representation of a
tyrosine-containing tripeptide (1) to which a succinyl ester group
(MeO-Suc) is attached and of a para-nitroanilide (pNA) derivative
of the tyrosine-containing peptide (2), with the PSA cleavage site
indicated an arrow.
[0014] FIG. 6 is a schematic representation of a
tyrosine-containing tripeptide with adriamycin attached directly to
the tripeptide via adriamycin's NH.sub.2 group; MeO-Suc represents
a succinyl ester group attached to the tyrosine-containing
tripeptide.
[0015] FIG. 7 is a schematic representation of one embodiment of
the present invention in which the synthesized compound contains
pABA. FIG. 7 shows a model compound (4) that demonstrated that pABA
is a good leaving group and that pABA can activate the tyrosine
carbonyl for hydrolysis. FIG. 7 further shows a compound (5)
comprising a succinyl ester group (MeO-Suc) attached to a
tripeptide (Arg-Pro-Tyr), which is attached to pABA, which is
attached to adriamycin. Finally, FIG. 7 shows a compound (6), which
arises as a result of the cleavage of compound (5) by PSA.
[0016] FIG. 8 is a schematic representation of one embodiment of
the present invention in which the synthesized compound contains
pABOC. FIG. 8 shows a compound (7) comprising a succinyl ester
group (MeO-Suc) attached to a tripeptide (Arg-Pro-Tyr), which is
attached to pABOC, which is attached to adriamycin. FIG. 8 further
shows how the fluorine-containing pABOC, being
electron-withdrawing, activates the tyrosine carbonyl toward
hydrolysis (8). Finally, FIG. 8 shows how the spontaneous breakdown
of 8, results in the release of "native" adriamycin.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention relates to a method of selectively
delivering a drug or other agent to a particular cell type by means
of a drug complex which includes at least three components: a
targeting carrier molecule which is selectively distributed to the
cell type or tissue to which the drug or agent is to be delivered;
a linker which is acted upon (cleaved or otherwise broken or
disrupted) by a molecule present at the site to which the drug or
agent is to be delivered; and a drug or agent to be delivered. The
invention also relates to the drug complex, which, optionally,
includes additional components. In the method, the drug complex is
administered via an appropriate route to an individual in need of
the drug or agent. The drug complex moves to the cells or tissues
to which the targeting carrier molecule is specifically delivered.
The linker is cleaved by the molecule present at the site to which
the drug or agent is to be delivered and the drug or agent is made
available to enter into or otherwise interact with the specific
cell type. The location(s) in the body at which the tissue or cell
type(s) to which a drug or agent is to be delivered are referred to
as site(s) of the target cell(s).
[0018] In a particular embodiment, the invention is a method of
treating prostate cancer by killing prostate cancer cells or
inhibiting replication or division of prostate cancer cells, such
as androgen independent prostate cancer cells. In this embodiment,
the drug complex comprises three components: a targeting carrier
molecule which is any agent or molecule which is selectively
delivered to prostate, bone or both; a linker which is cleaved by
PSA; and a drug which is cytotoxic to cancer cells, particularly
androgen independent prostatic cancer cells. Additionally, the
linker may be comprised of more than one type of molecule. For
example, the linker may comprise a substrate for a molecule as
described previously, and may also include one or more other
molecules, such as one or more desirable chemical molecules, e.g.
para-amino benzoic acid (pABA) and para-aminobenzyloxycarbonyl
(pABOC).
[0019] A selectively delivered drug is one which is delivered or
distributed to the targeted cell or tissue to an extent sufficient
for delivery of the drug or agent, such as a cytotoxic drug or
agent, in a quantity or concentration sufficient to have the
desired therapeutic or diagnostic effects. In the case of treatment
of cancer, such as the treatment of prostate cancer, the desired
effect is killing of some or all of the cancer cells (e.g.,
prostatic cancer cells) in an individual or at least a reduction in
replication or division of the cancerous cells. In other words, the
targeting carrier molecule generally will be a molecule which is
distributed to a limited number of sites within the body, but it is
not necessary that it be delivered only to those sites. For
example, the targeting carrier molecule can be an imaging agent,
such as N-3 fluoropropylputrescine which is selectively distributed
to prostate and bone, relative to blood and muscle, after
administration to a man. In the embodiment in which prostate cancer
is treated, the targeting carrier molecule is a polyamine (e.g.,
putrescine, spermine or spermidine) because of its affinity to
prostatic cancer cells through the polyamine surface receptor
molecules present on prostatic epithelial cells.
[0020] The linker, which is acted upon by a molecule which is
present at an effective concentration in the environs of the
specific cell, may be any entity which is acted upon by a molecule,
such as an enzyme, at or within a given target cell. Acted upon is
defined as any change in the linker induced by the molecule at or
within the given target cell which results in release of the
drug.
[0021] In the embodiment in which the drug complex is used to treat
prostate cancer, the linker comprises amino acid residues which are
acted upon by PSA. The linker used comprises at least those amino
acid residues necessary for PSA to act, resulting in cleavage of
the linker and release of the cytotoxic drug. The linker may
comprise additional amino acid residues, which may be the same as
those in the PSA peptide substrate or different (e.g., they can be
random or filler amino acid residues, amino acid residues which
enhance uptake of the cytotoxic drug into the prostatic cancer
cell, amino acid residues which stabilize the cytotoxic drug or
enhance its resistance to degradation once inside the cell or amino
acid residues which are substrates for cellular enzymes).
Optionally, the linker can comprise more than one type of molecule.
For example, the linker may comprise amino acid residues acted upon
by PSA as described previously, and may also include one or more
other molecules, such as one or more desirable chemical molecules,
e.g. para-amino benzoic acid (PABA) and para-aminobenzyloxycarbonyl
(pABOC).
[0022] Examples of possible amino acids that can be used in the
subject invention include the following combinations of amino acid
residues, as represented in Table 1 of Denmeade et al., Cancer
Research 57: 4924-4930 (1997): KGISSQY (SEQ ID NO.: 1); SRKSQQY
(SEQ ID NO.: 2); GQKGQHY (SEQ ID NO.: 3); EHSSKLQ (SEQ ID NO.: 4);
QNKISYQ (SEQ ID NO.: 5); ENKISYQ (SEQ ID NO.: 6); ATKSKQH (SEQ ID
NO.: 7); KGLSSQC (SEQ ID NO.: 8); LGGSQQL (SEQ ID NO.: 9); QNKGHYQ
(SEQ ID NO.: 10); TEERQLH (SEQ ID NO.: 11); GSFSIQH (SEQ ID NO.:
12); HSSKLQ (SEQ ID NO.: 13); SKLQ (SEQ ID NO.: 14); KLQ (SEQ ID
NO.: 15) and LQ (SEQ ID NO.: 16).
[0023] Another example of a possible combination of amino acids
that can be used in the subject invention is AKFE (SEQ ID NO.:
17).
[0024] In the bloodstream, the peptide link between the targeting
carrier molecule and the cytotoxic drug will be sustained because
there is insufficient unbound PSA to act to a significant extent on
the linking peptide that is substrate for unbound PSA. In the
microenvironment of the androgen independent prostate cancer cells,
where unbound PSA is found in large concentrations, PSA will cleave
the linking peptide to a great extent and thereby release the
cytotoxic drug into the cell. By having the features of specificity
for prostate and bone and the required presence of unbound PSA for
drug release, the targeting carrier molecule is highly target
specific for the androgen independent prostate cancer cell. Such a
highly target specific targeting carrier molecule will increase the
efficacy of and decrease the toxicity of the cytotoxic drug
component used. Targeting molecules can be, for example,
polyamines, e.g., putrescine, spermine or spermidine, which provide
affinity to prostatic cancer cells through the polyamine surface
receptor molecules present on prostatic epithelial cells. A
specific example of such a targeting carrier molecule is N-3
fluoropropylputrescine. (Hwang, D. et al., J. Nucl. Med.,
30:1205-1210 (1989)).
[0025] The cytotoxic drug which is a component of the drug complex
can be any agent effective in killing prostate cancer cells,
including metastatic prostate cancer cells. For example, any of the
following drugs can be used: adriamycin, amonafide, cisplatin,
carboplatin (CBDCA), CHIP, cyclophosphamide, doxorubicin,
epirubicin, estramustine, etoposide, 5-fluorouracil, gallium
nitrate, idarubicin, ifosfamide+mesna, ketoconazole, liarozole
(R85,246), methotrexate, mitomycin C, mitoguazone, mitoxantrone,
proscar (finasteride), suramin, taxol, thapsigargin, trimetrexate,
vinblastine (CI), and vinblastine and emcyt.
[0026] Adriamycin is particularly desirable for several reasons.
First, it gives a response rate of 15-20% in hormone refractory
metastatic prostate cancer with a median survival of 33 weeks.
These results are among the best noted for single agents in this
disease. (Kreis, W., Cancer Investigation, 13:296-312 (1996)).
Second, the chemistry is well established. For example, adriamycin
can be easily coupled to peptides through its amino (--NH.sub.2)
group. (Nogusa, H. et al., Chem. Pharm. Bull. Jpn., 43:1931-1936
(1995). Moreover, several of its analogs, including N-substituted
ones, are known to display high anticancer activity. (Israel, A. et
al., Cancer Treatment Reviews, 14:163-167 (1987).
[0027] Another cytotoxic drug, Thapsigargin, is a sesquiterpene
lactone extracted from the roots of the umbelliferous plant Thapsia
garganica L. (Thastrup, O. et al., Proc. Nat. Acad. Sci., USA,
87:2466-2470 (1990)). This highly lipophilic agent specifically
inhibits Ca.sup.++-ATPase pumps of the endoplasmic reticulum, but
not the pumps of erythrocytes, plasma, or mitochondrial
membranes.
[0028] In vitro treatment with 500 nm thapsigargin in a series of
both rat and human androgen independent prostate cancer cell lines,
which either express or completely lack p53 protein expression,
results in the elevation of intracellular Ca.sup.++. Within 72 to
96 hours, these cells undergo apoptosis and lose their clonogenic
potential.
[0029] The dose of the drug complex to be used is a sufficient
quantity of complex to result in delivery and availability to
target cells of the cytotoxic drug at a therapeutically effective
level. Dose will be determined empirically and will be determined,
for example, by the stage or condition of the disease for which an
individual is being treated, the individual's general health, size,
age, and sex. In the embodiment of the present invention in which a
man is treated for prostate cancer, the dose will be determined by
taking into consideration the cytotoxic drug being used, the stage
of the cancer, and the man's age, general health and size.
[0030] The timing and number of doses of the drug complex
administered will also be determined empirically. The number of
doses may be at any interval sufficient to promote or to result in
killing of cells and/or inhibition of replication or division. For
example, the drug complex can be administered hourly, daily,
weekly, monthly, or any combination thereof.
[0031] The route of administration can be any route that delivers a
therapeutically effective quantity of the drug complex to the
target cells, such as to prostate cancer cells. The formulation of
the drug complex can be any pharmaceutically effective formulation
or carrier, such as any physiologically acceptable buffer, saline
solution, or water.
[0032] In one embodiment, the prodrug of the invention has the
structure shown below, and is believed to have a prostate specific
antigen (PSA) cleavage site as indicated. 1
[0033] Where R represents a polyarnine attached through the amino
group of the N-terminal amino acid Histidine, and 2
[0034] Adriamycin, indicates attachment of Adriamycin (structure
given below) through its amino group. 3
[0035] One method of forming this prodrug is shown below: 4
[0036] The present invention is illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLE 1
[0037] In Vitro Testing of Drug Complexes
[0038] PC3 and DU-145 androgen-independent human prostate cancer
cell lines obtained from the American Tissue Culture Collection
(ATCC) are used for in vitro studies. Cells are grown as monolayer
cultures in RPMI 1640 medium supplemented with 10% fetal bovine
serum, 2 mM glutamine and antibiotics. Radiation survival curves
and radiation-induced apoptotic DNA fragmentation patterns in these
cell lines are already established in the laboratory. Cells are
treated with this drug, carrier-peptide, or carrier-peptide-drug
complex with or without radiation. After 24 hours, the cells are
trypsinized and plated for clonogenic survival studies. Apoptotic
response to the treatments is determined by: 1) agarose gel
electrophoresis analysis of DNA fragmentation at 48 and 72 hours
after treatment, 2) morphological observations after staining with
DAPI, and 3) terminal deoxytransferase-mediated dUTP Nick End
Labeling (TUNEL) assay.
EXAMPLE 2
[0039] In Vivo Testing of Drug Complexes
[0040] Two human prostate carcinoma tumor lines grown as xenografts
in male SCID mice are used: the human DU-145 prostate carcinoma
which is not androgen dependent and the human LNCaP prostate
carcinoma which produces PSA and has a well characterized androgen
receptor and response to androgens. The drug complexes are
administered up to maximally tolerated doses alone and in
conjunction with fractionated radiation therapy (.sup.137Cs Gamma
Cell 40) delivered to the tumor bearing limb.
[0041] The progress of each tumor is assessed thrice weekly by
caliper measurements until the tumors reach 2000 mm.sup.3. Tumor
growth delay is calculated as the number of days for each tumor to
reach a volume of 500 mm.sup.3 as compared to untreated controls.
The efficacy of combination treatments is assessed using
isobologram analysis for determination of additivity/synergy.
[0042] Phase I Clinical Trial: (Maximal Tolerable Dose
Assessment)
[0043] Based on the results of the in vitro and in vivo studies of
the drug complexes, a drug complex is chosen for testing in ten
patients with hormone refractory metastatic prostate cancer. A
single intravenous dose of the drug complex is given on an
inpatient basis with cardiorespiratory monitoring. Toxicity
evaluation of all major organ systems is evaluated using blood,
urine, stool, and, if necessary, bone marrow studies. Doses are
escalated in 10% increments per patient until the maximal response
with acceptable toxicity is reached. Response is assessed using a
combination of a weekly digital rectal examination (DRE), PSA, LDH,
hemoglobin, and monthly bone scan. The starting dose is selected on
the toxicity data from the preceding animal studies.
EXAMPLE 3
[0044] Effect of Thapsigargin (TG) on Cell Proliferation in
vitro
[0045] Preliminary in vitro data indicate that TG inhibits cell
proliferation (Table 1, DU 145 cells) clonogenic cell survival
(Table 2, PC3 cells) of androgen independent human prostate cancer
cells.
[0046] Table 1 shows the effect of thapsigargin and radiotherapy on
proliferation of DU-145 prostate cancer cells. The results indicate
that thapsigargin inhibits cell proliferation in vitro.
1TABLE 1 Effect of TG and RT on proliferation of DU-145 prostate
cancer cells* Total Number of Cells per Thapsigargin Dish
(10.sup.6) (nM) 0 Gy 2 Gy 4 Gy 8 Gy 0 2.1 1.4 1.2 0.5 20 1.0 0.8
0.3 0.4 100 0.7 0.5 0.5 0.4 400 0.7 0.4 0.3 0.3
[0047] Table 2 shows the effect of thapsigargin and radiotherapy on
clonogenic cell survival of PC3 prostate cancer cells. The results
indicate that thapsigargin inhibits clonogenic survival in
vitro.
2TABLE 2 Effect of TG and RT on clonogenic survival of PC3 cells**
Surviving Fraction (SF) NET survival Thapsigargin (PE treated/PE
control) (SF/TG toxicity) (nM) 0 Gy 2 Gy 4 Gy 2 Gy 4 Gy 0 1 (0.8)
0.57 0.38 0.57 0.38 20 0.69 0.42 0.23 0.61 0.33 50 0.80 0.39 0.23
0.49 0.29 100 0.62 0.25 0.18 0.40 0.30 200 0.58 0.25 0.13 0.43 0.22
400 0.60 0.25 0.13 0.42 0.22 PE (Plating Efficiency) **Cells were
irradiated 2 hours after adding TG or DMSO. At 24 hours cells were
trypsinized and plated for clonogenic assay in a drug free media.
Colonies were stained with 0.4% crystal violet on the 12th day and
viable colonies of >50 cells were counted.
EXAMPLE 4
[0048] Effect of Thapsigargin (TG) on Cell Proliferation in
vivo
[0049] Male SCID mice bearing the human DU-145 prostate carcinoma
Xenograft growing subcutaneously in a hind-limb were treated daily
with TG (0.5 mg/kg) by IP injection from day 4-18 post tumor cell
implantation. Some animal groups also received fractionated RT
locally to the tumor bearing region on days 7-11 or days 7-11 and
14-18. FIG. 1, low dose TG (0.5 mg/kg) administration produced a
measurable tumor growth delay and, when given in conjunction with
RT, the independent effects of TG and RT were additive. Moreover,
when the RT regimen was extended to 2 weeks, the additional benefit
of the TG administration was maintained.
EXAMPLE 5
[0050] PSA Cleaving of pNA Derivative
[0051] A tyrosine tripeptide (compound 1 in FIG. 5) was prepared
using methods known to those in the peptide chemistry art. It is
known that PSA cleaves its para-nitroanilide (pNA) derivative at
the tyrosine site (compound 2 in FIG. 5). (Christensson, A. et al.,
Eur. J. Biochem., 194:755-763 (1990)). It is also known that the
"released" pNA makes it possible to study this reaction
colormetrically. Using this feature, it was demonstrated that the
sonicate from a cell line that is known to produce PSA, i.e. LNCaP,
cleaves compound 2 in FIG. 5. It was also shown that a negative
control, i.e. CV-1 cell line, that does not produce PSA did not
cleave compound 2 in FIG. 5 and that neither newborn calf serum,
nor pooled human serum cleaved compound 2 in FIG. 5. These results
indicate that a "PSA-like" activity exists in the sonicate of LNCaP
(and not in human or fetal calf serum) that can cleave compound 1
in FIG. 5 at the tyrosine site and release pNA.
[0052] Based on the above results, the pNA group was replaced with
adriamycin, i.e. by attaching adriamycin directly to the tyrosine
(FIG. 6) via adriamycin's --NH.sub.2 group. It was hypothesized
that PSA would cleave the molecule of FIG. 6 at the tyrosine site
and release adriamycin. However, there was no cleavage and no
release of adriamycin.
[0053] There are several reasons why cleavage and release did not
occur. First, pNA, because of its electron withdrawing
para-NO.sub.2 group, is a better leaving group than adriamycin.
Second, because pNA is electron-withdrawing, it can greatly
activate the tyrosine carbonyl group toward hydrolysis relative to
adriamycin. Finally, since adriamycin is sterically bulkier than
the pNA group, the drug may have prevented PSA from approaching the
tyrosine site.
EXAMPLE 6
[0054] Construction of Molecules Containing para-Amino Benzoic Acid
and para-Aminobenzyloxycarbonyl
[0055] Attaching adriamycin to a tripeptide molecule (see compound
1 of FIG. 5) through a para-amino benzoic acid can solve the
problems previously discussed for the following reasons: (a) pABA
can function as a good leaving group because it contains an
electron withdrawing carbonyl group in the para-position, (b)
because pABA is electron-withdrawing, it can activate the tyrosine
carbonyl hydrolysis, and (c) PSA activity may not be sterically
hindered as the pABA linker can keep adriamycin away from the
tyrosine cleavage site.
[0056] The results with this molecule indicate that pABA is a good
leaving group and that it can activate the tyrosine carbonyl for
hydrolysis. That is, the sonicate of LNCaP that showed "PSA-like"
activity cleaved compound 4 of FIG. 7 at the tyrosine site and
released pABA.
[0057] Since it is established, based on the above results, that
there is a linker suitable for the attachment of adriamycin,
compound 5 of FIG. 7 can be synthesized. Furthermore, it can be
examined to determine if it can also be cleaved at the tyrosine
site to release an active form of adriamycin, namely compound 6 of
FIG. 7.
[0058] Cleavage of compound 5 can be assessed using the sonicate of
LNCaP that exhibits "PSA-like" activity. It is expected that
cleavage will occur at the tyrosine site of compound 5. However,
what will be "released" is an analog of adriamycin (compound 6 of
FIG. 7) and not adriamycin itself. The ability of this analog to
intercalate DNA can be assessed and compared with that of native
adriamycin using known methods, such as measurement of emissions at
450 nm on ethanol-precipitated DNA. The data is also normalized for
efficiency of precipitation by assaying the DNA at 260 nm.
[0059] An alternative approach takes advantage of a compound that
is similar to pABA. This compound is para-aminobenzyloxycarbonyl
(pABOC), which offers the advantage of breaking down spontaneously
(compound 8 of FIG. 8) to release "native" adriamycin (i.e.
adriamycin lacking N-substituents; compound 9 of FIG. 8). Because
of the similarity between pABA and pABOC, the latter offers many of
the same advantages. First, since the physical size of pABOC is
similar to that of pABA, it can keep the "bulky" adriamycin away
from the tyrosine cleavage site. Second, electron-withdrawing
fluorine substituents to be added at the two ortho-positions (shown
in compounds 7 and 8 of FIG. 8) should make pABOC a good leaving
group without introducing any steric bulk, since fluorine and
hydrogen atoms are essentially the same size. Finally, the
fluorine-containing pABOC is electron-withdrawing and, thus, can
also activate the tyrosine carbonyl toward hydrolysis. Based on
this information, it can be expected that PSA will cleave compound
7 of FIG. 8 at the tyrosine site and release compound 8 of FIG. 8.
Then, as described above, compound 8 of FIG. 8 should break down
spontaneously under physiological conditions to release "native"
adriamycin. This can be assessed using known methods.
[0060] Equivalents
[0061] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *