U.S. patent application number 09/050662 was filed with the patent office on 2001-10-25 for water soluble paclitaxel derivatives.
Invention is credited to LI, CHUN, WALLACE, SIDNEY, YANG, DAVID J., YU, DONG-FANG.
Application Number | 20010034363 09/050662 |
Document ID | / |
Family ID | 21966621 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034363 |
Kind Code |
A1 |
LI, CHUN ; et al. |
October 25, 2001 |
WATER SOLUBLE PACLITAXEL DERIVATIVES
Abstract
Disclosed are water soluble compositions of paclitaxel and
docetaxel formed by conjugating the paclitaxel or docetaxel to a
water soluble polymer such as poly-glutamic acid, poly-aspartic
acid or poly-lysine. Also disclosed are methods of using the
compositions for treatment of tumors, auto-immune disorders such as
rheumatoid arthritis. Other embodiments include the coating of
implantable stents for prevention of restenosis.
Inventors: |
LI, CHUN; (MISSOURI CITY,
TX) ; WALLACE, SIDNEY; (HOUSTON, TX) ; YU,
DONG-FANG; (HOUSTON, TX) ; YANG, DAVID J.;
(SUGAR LAND, TX) |
Correspondence
Address: |
RONALD J. KAMIS
FOLEY & LARDNER
3000 K STREET N.W., SUITE 500
WASHINGTON
DC
20007-5109
US
|
Family ID: |
21966621 |
Appl. No.: |
09/050662 |
Filed: |
March 30, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09050662 |
Mar 30, 1998 |
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08815104 |
Mar 11, 1997 |
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5977163 |
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60013184 |
Mar 12, 1996 |
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Current U.S.
Class: |
514/449 ;
424/1.65; 424/9.36; 424/9.364; 527/200; 534/10; 534/11; 549/510;
549/511 |
Current CPC
Class: |
A61K 2123/00 20130101;
A61L 2300/606 20130101; A61K 41/0038 20130101; A61L 31/10 20130101;
A61P 35/00 20180101; A61K 51/065 20130101; A61K 31/337 20130101;
C08G 73/1092 20130101; A61P 35/02 20180101; A61L 31/16 20130101;
A61P 35/04 20180101; A61L 2300/416 20130101; A61K 38/13 20130101;
A61K 51/0497 20130101; A61K 47/645 20170801; A61K 47/59 20170801;
A61K 47/547 20170801; A61K 2121/00 20130101; A61K 31/4375 20130101;
A61K 47/60 20170801 |
Class at
Publication: |
514/449 ;
424/1.65; 424/9.36; 424/9.364; 549/510; 549/511; 534/10; 534/11;
527/200 |
International
Class: |
A61M 036/14; A61K
051/00; A61B 005/055; C07F 001/00; C07F 005/00; A61K 031/335; A01N
043/02; C07D 305/00; C07D 407/00 |
Claims
What is claimed is:
1. A composition comprising an anti-tumor drug selected from
paclitaxel, docetaxel, etopside, teniposide, camptothecin or
epothilone conjugated to polymer of water soluble amino acids.
2. The composition of claim 1 wherein said anti-tumor drug is
paclitaxel.
3. The composition of claim 1, wherein said anti-tumor drug is
docetaxel.
4. The composition of claim 1, wherein said polymer is further
defined as a copolymer of water soluble polyamino acids with
polycaprolactone, polyglycolic acid, and polylactic acid and other
polymers of poly(2-hydroxyethyl 1-glutamine), carboxymethyl
dextran, hyaluronic acid, human serum albumin, polyalginic acid or
a combination thereof.
5. The composition of claim 1, wherein said polymer has a molecular
weight of from about 5,000 to about 100,000.
6. The composition of claim 1, wherein said polymer has a molecular
weight of from about 20,000 to about 80,000.
7. The composition of claim 1, wherein said polymer has a molecular
weight of about 25,000 to about 50,000.
8. The composition of claim 1, wherein said polymer comprises a
water soluble polymer.
9. The composition of claim 8, wherein said water soluble polymer
is conjugated to the 2'-and/or the 7-hydroxyl of paclitaxel or
docetaxel.
10. The composition of claim 8, wherein said water soluble polymer
is poly (l-glutamic acid), poly (d-glutamic acid), or poly
(dl-glutamic acid).
11. The composition of claim 8, wherein said water soluble polymer
is poly (l-aspartic acid), poly (d-aspartic acid), or poly
(dl-aspartic acid).
12. The composition of claim 1, wherein said composition is
dispersed in a pharmaceutically acceptable solution.
13. The composition of claim 1, wherein said composition comprises
from about 2% to about 35% (w/w) anti-tumor drug.
14. The composition of claim 10, wherein said water soluble polymer
contains 1 to 65 molecules of paclitaxel per chain.
15. The composition of claim 14, wherein said water soluble polymer
contains 3 to 15 molecules of paclitaxel per chain.
16. A method of treating cancer in a subject comprising the steps
of: a) obtaining a composition comprising paclitaxel or docetaxel
conjugated to water soluble polyamino acids and dispersed in a
pharmaceutically acceptable solution; b) administering said
solution to said subject in an amount effective to treat said
cancer wherein said treating cancer kills a cancer cell by
mechanisms in addition to apoptosis.
17. The method of claim 16, wherein said composition comprises
paclitaxel conjugated to poly-glutamic acids, poly-aspartic acids
or poly-lysines.
18. The method of claim 17, wherein said composition comprises
paclitaxel conjugated to poly-glutamic acids.
19. The method of claim 16, wherein said cancer is breast cancer,
ovarian cancer, malignant melanoma, lung cancer, gastric cancer,
prostate cancer, colon cancer, head and neck cancer, leukemia, or
Kaposi's Sarcoma.
20. The method of claim 19, wherein said cancer is breast
cancer.
21. The method of claim 19, wherein said cancer is ovarian
cancer.
22. The method of claim 19, wherein said cancer is malignant
melanoma.
23. The method of claim 19, wherein said cancer is lung cancer.
24. The method of claim 19, wherein said cancer is gastric
cancer.
25. The method of claim 19, wherein said cancer is prostate
cancer.
26. The method of claim 19, wherein said cancer is colon
cancer.
27. The method of claim 19, wherein said cancer is head and neck
cancer.
28. The method of claim 19, wherein said cancer is leukemia.
29. The method of claim 19, wherein said cancer is Kaposi's
Sarcoma.
30. A method of decreasing at least one symptom of a systemic
autoimmune disease comprising administering to a subject having a
systemic autoimmune disease an effective amount of a composition
comprising paclitaxel or docetaxel conjugated to water soluble
polyamino acids.
31. The method of claim 30, wherein said composition comprises
paclitaxel conjugated to poly-glutamic acids, poly-aspartic acids
or poly-lysines.
32. The method of claim 31, wherein said composition comprises
paclitaxel conjugated to poly-glutamic acid.
33. The method of claim 30, wherein said systemic autoimmune
disease is rheumatoid arthritis.
34. A method of inhibiting arterial restenosis or arterial
occlusion following vascular trauma comprising administering to a
subject in need thereof, a composition comprising paclitaxel or
docetaxel conjugated to water soluble polyamino acids.
35. The method of claim 34, wherein said composition comprises
paclitaxel conjugated to poly-glutamic acids, poly-aspartic acids
or poly-lysines.
36. The method of claim 35, wherein said composition comprises
paclitaxel conjugated to poly-glutamic acids.
37. The method of claim 34, wherein said subject is a coronary
bypass, vascular surgery, organ transplant, coronary angioplasty,
or arterial angioplasty patient.
38. The method of claim 34, wherein said composition is coated on a
stent and said stent is implanted at the site of vascular
trauma.
39. A pharmaceutical composition comprising paclitaxel conjugated
to water soluble polyamino acids.
40. The pharmaceutical composition of claim 39, wherein said
composition comprises paclitaxel conjugated to poly-glutamic acids,
poly-aspartic acids or poly-lysines.
41. An implantable medical device, wherein said device is coated
with a composition comprising paclitaxel conjugated to water
soluble polyamino acids in an amount effective to inhibit smooth
muscle cell proliferation.
42. The composition of claim 41, wherein said water soluble
polyamino acids comprise poly (l-glutamic acid), poly (d-glutamic
acid), or poly (dl-glutamic acid).
43. The implantable medical device of claim 42, further defined as
a stent coated with said composition.
44. The implantable medical device of claim 43, wherein said stent
is adapted to be used after balloon angioplasty and said
composition is effective to inhibit restenosis.
45. A composition comprising water soluble polyamino acids
conjugated to the 2' or 7 hydroxyl of paclitaxel.
46. The composition of claim 45, wherein said composition comprises
poly-glutamic acids conjugated to the 2' or 7 hydroxyl of
paclitaxel.
47. The composition of claim 45, wherein said composition comprises
poly-aspartic acids conjugated to the 2' or 7 hydroxyl of
paclitaxel.
48. The composition of claim 45, wherein said composition comprises
poly-lysines conjugated to the 2' or 7 hydroxyl of paclitaxel.
49. The composition of claim 46, wherein said poly-glutamic acids
are conjugated to the 2' and the 7 hydroxyl of paclitaxel.
50. The composition of claim 47, wherein said poly-aspartic acids
are conjugated to the 2' and the 7 hydroxyl of paclitaxel.
51. The composition of claim 48, wherein said poly-lysines are
conjugated to the 2' and the 7 hydroxyl of paclitaxel.
Description
[0001] This application is a continuation-in-part of co-pending
application U.S. Ser. No. 08/815,104, which is a continuation of
U.S. Provisional Application No. 60/013,184.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
pharmaceutical compositions to be used in the treatment of cancer,
autoimmune diseases and restenosis. The present invention also
relates to the field of pharmaceutical preparations of anticancer
agents such as paclitaxel (Taxol.TM.) and docetaxel (Taxotere), in
particular making paclitaxel water soluble by conjugating the drug
to water soluble moieties.
BACKGROUND OF THE INVENTION
[0003] Paclitaxel, an anti-microtubule agent extracted from the
needles and bark of the Pacific yew tree, Taxus brevifolia, has
shown a remarkable anti-neoplastic effect in human cancer in Phase
I studies and early Phase II and III trials (Horwitz et al., 1993).
This has been reported primarily in advanced ovarian and breast
cancer. Significant activity has been documented in small-cell and
non-small cell lung cancer, head and neck cancers, and in
metastatic melanoma. However, a major difficulty in the development
of paclitaxel for clinical trial use has been its insolubility in
water.
[0004] Docetaxel is semisynthetically produced from 10-deacetyl
baccatin III, a noncytotoxic precursor extracted from the needles
of Taxus baccata and esterified with a chemically synthesized side
chain (Cortes and Pazdur, 1995). Various cancer cell lines,
including breast, lung, ovarian, and colorectal cancers and
melanomas have been shown to be responsive to docetaxel. In
clinical trials, docetaxel has been used to achieve complete or
partial responses in breast, ovarian, head and neck cancers, and
malignant melanoma.
[0005] Paclitaxel is typically formulated as a concentrated
solution containing paclitaxel, 6 mg per milliliter of Cremophor EL
(polyoxyethylated castor oil) and dehydrated alcohol (50% v/v) and
must be further diluted before administration (Goldspiel, 1994).
Paclitaxel (Taxol.TM.) has shown significant activity in human
cancers, including breast, ovarian, non-small cell lung, and head
and neck cancers (Rowinsky and Donehower, 1995). It has also shown
significant activity in patients with advanced breast cancer who
had been treated with multiple chemotherapeutic agents (Foa et al.,
1994). As with most chemotherapeutic agents, however, the maximum
tolerated dose of paclitaxel is limited by toxicity. In humans,
paclitaxel's major toxic effect at doses of 100-250 mg/m.sup.2 is
granulocytopenia (Holmes et al., 1995); symptomatic peripheral
neuropathy is its principal nonhematologic toxicity (Rowinsky et
al., 1993).
[0006] The amount of Cremophor EL necessary to deliver the required
doses of paclitaxel is significantly higher than that administered
with any other drug that is formulated in Cremophor. Several toxic
effects have been attributed to Cremophor, including
vasodilatation, dyspnea, and hypotension. This vehicle has also
been shown to cause serious hypersensitivity in laboratory animals
and humans (Weiss et al., 1990). In fact, the maximum dose of
paclitaxel that can be administered to mice by i.v. bolus injection
is dictated by the acute lethal toxicity of the Cremophor vehicle
(Eiseman et al., 1994). In addition, Cremophor EL, a surfactant, is
known to leach phthalate plasticizers such as
di(2-ethylhexyl)phthalate (DEHP) from the polyvinylchloride bags
and intravenous administration tubing. DEHP is known to cause
hepatotoxicity in animals and is carcinogenic in rodents. This
preparation of paclitaxel is also shown to form particulate matter
over time and thus filtration is necessary during administration
(Goldspiel, 1994). Therefore, special provisions are necessary for
the preparation and administration of paclitaxel solutions to
ensure safe drug delivery to patients, and these provisions
inevitably lead to higher costs.
[0007] Prior attempts to obtain water soluble paclitaxel have
included the preparation of prodrugs of paclitaxel by placing
solubilizing moieties such as succinate, sulfonic acid, amino
acids, and phosphate derivatives at the 2'-hydroxyl group or at the
7-hydroxyl position (Deutsch et al., 1989; Mathew et al., Zhao and
Kingston, 1991, 1992; Nicolaou et al., 1993; Vyas et al., 1995,
Rose et al., 1997). While some of these prodrugs possess adequate
aqueous solubility, few have antitumor activity comparable to that
of the parent drug (Deutsch et al., 1989; Mathew et al., 1992; Rose
et al., 1997). Several of these derivatives are not suitable for
i.v. injection because of their instability in aqueous solution at
neutral pH. For example, Deutsch et al. (1989) report a
2'-succinate derivative of paclitaxel, but water solubility of the
sodium salt is only about 0.1% and the triethanolamine and
N-methylglucamine salts were soluble at only about 1%. In addition,
amino acid esters were reported to be unstable. Similar results
were reported by Mathew et al. (1992).
[0008] Recently, Nicolaou et al. (1993) reported the synthesis and
in vitro biological evaluation of a novel type of prodrug termed
"protaxols". These compounds possess greater aqueous solubility and
are converted to paclitaxel as the active drug through an
intramolecular hydrolysis mechanism. However, no in vivo data on
the antitumor activity of protaxols are yet available. Greenwald et
al. reported the synthesis of highly water-soluble 2' and
7-polyethylene glycol esters of paclitaxel (Greenwald et al.,
1994). Using the strategy of polymer linkage, others have developed
water-soluble polyethylene glycol (PEG)-conjugated paclitaxel (Li
et al., 1996; Greenwald et al., 1996). Although these conjugates
have excellent water solubility, their therapeutic efficacies are
not better than free paclitaxel. Moreover, PEG has only two
reactive functional groups at each end of its polymer chain, which
effectively limit the amount of paclitaxel that PEG could carry
(U.S. Pat. No. 5,362,831).
[0009] Other attempts to solve these problems have involved
microencapsulation of paclitaxel in both liposomes and nanospheres
(Bartoni and Boitard, 1990). The liposome formulation was reported
to be as effective as free paclitaxel, however only liposome
formulations containing less than 2% paclitaxel were physically
stable (Sharma and Straubinger, 1994). Unfortunately, the
nanosphere formulation proved to be toxic. There is still a need
therefore for a water soluble paclitaxel formulation that can
deliver effective amounts of paclitaxel and docetaxel without the
disadvantages caused by the insolubility of the drug.
[0010] Another obstacle to the widespread use of paclitaxel is the
limited resources from which paclitaxel is produced, causing
paclitaxel therapy to be expensive. A course of treatment may cost
several thousand dollars, for example. There is the added
disadvantage that not all tumors respond to paclitaxel therapy, and
this may be due to the paclitaxel not getting into the tumor. There
is an immediate need, therefore, for effective formulations of
paclitaxel and related drugs that are water soluble with long serum
half lives for treatment of tumors, autoimmune diseases such as
rheumatoid arthritis, as well as for the prevention of restenosis
of vessels subject to traumas such as angioplasty and stenting.
SUMMARY OF THE INVENTION
[0011] The present invention seeks to overcome these and other
drawbacks inherent in the prior art by providing compositions
comprising a chemotherapeutic and/or antiangiogenic drug, such as
paclitaxel, docetaxel, or other taxoid conjugated to a water
soluble polymer such as a water soluble polyamino acid, or to a
water soluble metal chelator. It is a further embodiment of the
present invention that a composition comprising a conjugate of
paclitaxel and poly-glutamic acid has surprising antitumor activity
in animal models, and further that this composition is demonstrated
herein to be a new species of taxane that has pharmaceutical
properties different from that of paclitaxel. These compositions
are shown herein to be surprisingly effective as anti-tumor agents
against exemplary tumor models, and are expected to be at least as
effective as paclitaxel, docetaxel, or other taxoid against any of
the diseases or conditions for which taxanes or taxoids are known
to be effective. The compositions of the invention provide water
soluble taxoids to overcome the drawbacks associated with the
insolubility of the drugs themselves, and also provide the
advantages of improved efficacy and controlled release so that
tumors are shown herein to be eradicated in animal models after a
single intravenous administration, as well as providing a novel
taxane. Poly-(l-glutamic acid) conjugated paclitaxel is shown in
the examples hereinbelow to have a novel drug activity, in addition
to having improved the delivery to the tumor and providing a
controlled release.
[0012] The methods described herein could also be used to make
water soluble polymer conjugates of other therapeutic agents,
contrast agents and drugs, including paclitaxel, tamoxifen,
Taxotere, etopside, teniposide, fludarabine, doxorubicin,
daunomycin, emodin, 5-fluorouracil, FUDR, estradiol, camptothecin,
retinoids, verapamil, epothilones cyclosporin, and other taxoids.
In particular, those agents with a free hydroxyl group would be
conjugated to the polymers by similar chemical reactions as
described herein for paclitaxel. Such conjugation would be well
within the skill of a routine practitioner of the chemical art, and
as such would fall within the scope of the claimed invention. Those
agents would include, but would not be limited to etopside,
teniposide, camptothecin and the epothilones. As used herein,
conjugated to a water soluble polymer means the covalent bonding of
the drug to the polymer or chelator.
[0013] It is also understood that the water soluble conjugates of
the present invention may be administered in conjunction with other
drugs, including other anti-tumor or anti-cancer drugs. Such
combinations are known in the art. The water soluble paclitaxel,
docetaxel, or other taxoid, or in preferred embodiments the
poly-(l-glutamic) acid conjugated paclitaxel (PG-TXL), of the
present invention may, in certain types of treatment, be combined
with a platinum drug, an antitumor agent such as doxorubicin or
daunorubicin, for example, or other drugs that are used in
combination with Taxol.TM. or combined with external or internal
irradiation.
[0014] Conjugation of chemotherapeutic drugs to polymers is an
attractive approach to reduce systemic toxicity and improve the
therapeutic index. Polymers with molecular mass larger than 30 kDa
do not readily diffuse through normal capillaries and glomerular
endothelium, thus sparing normal tissue from irrelevant
drug-mediated toxicity (Maeda and Matsumura, 1989; Reynolds, 1995).
On the other hand, it is well established that malignant tumors
often have disordered capillary endothelium and greater
permeability than normal tissue vasculature (Maeda and Matsumura,
1989; Fidler et al., 1987). Tumors often lack a lymphatic
vasculature to remove large molecules that leak into the tumor
tissue (Maeda and Matsumura, 1989). Thus, a polymer-drug conjugate
that would normally remain in the vasculature may selectively leak
from blood vessels into tumors, resulting in tumor accumulation of
active therapeutic drug. The water soluble polymers, such as, in
preferred embodiments PG-TXL, may have pharmacological properties
different from non-conjugated drugs (i.e. paclitaxel).
Additionally, polymer-drug conjugates may act as drug depots for
sustained release, producing prolonged drug exposure to tumor
cells. Finally, water soluble polymers (e.g., water soluble
polyamino acids) may be used to stabilize drugs, as well as to
solubilize otherwise insoluble compounds. At present, a variety of
synthetic and natural polymers have been examined for their ability
to enhance tumor-specific drug delivery (Kopecek, 1990, Maeda and
Matsumura, 1989). However, only a few are known by the present
inventors to be currently undergoing clinical evaluation, including
SMANCS in Japan and HPMA-Dox in the United Kingdom (Maeda, 1991;
Kopecek and Kopeckova, 1993).
[0015] In the present disclosure, a taxoid is understood to mean
those compounds that include paclitaxels and docetaxel, and other
chemicals that have the taxane skeleton (Cortes and Pazdur, 1995),
and may be isolated from natural sources such as the Yew tree, or
from cell culture, or chemically synthesized molecules, and a
preferred taxane is a chemical of the general chemical formula,
C.sub.47H.sub.5, NO.sub.14, including
[2aR-[2a.alpha.,4.beta.,4.alpha..beta.,6.beta.,9.alpha.(.alpha.R*,.beta.S-
*), 11.alpha., 12.alpha., 12a.alpha.,
12.beta..alpha.,]]-.beta.-(Benzoylam- ino)-.alpha.-hydroxybenzene
propanoic acid 6, 12b,bis(acetyloxy)-12-(benzo-
yloxy)-2a,3,4,4a,5,6,9,10,11,
12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,1-
3,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz-[1,2-b]oxet-9-yl
ester. It is understood that paclitaxel and docetaxel are each more
effective than the other against certain types of tumors, and that
in the practice of the present invention, those tumors that are
more susceptible to a particular taxoid would be treated with that
water soluble taxoid or taxane conjugate.
[0016] In those embodiments in which the paclitaxel is conjugated
to a water soluble metal chelator, the composition may further
comprise a chelated metal ion. The chelated metal ion of the
present invention may be an ionic form of any one of aluminum,
boron, calcium, chromium, cobalt, copper, dysprosium, erbium,
europium, gadolinium, gallium, germanium, holmium, indium, iridium,
iron, magnesium, manganese, nickel, platinum, rhenium, rubidium,
ruthenium, samarium, sodium, technetium, thallium, tin, yttrium or
zinc. In certain preferred embodiments, the chelated metal ion will
be a radionuclide, i.e. a radioactive isotope of one of the listed
metals. Preferred radionuclides include, but are not limited to
.sup.67Ga, .sup.68Ga, .sup.111In, .sup.99mTc, .sup.90Y, .sup.114mSn
and .sup.193mPt.
[0017] Preferred water soluble chelators to be used in the practice
of the present invention include, but are not limited to,
diethylenetriaminepentaacetic acid (DTPA),
ethylenediaminetetraacetic acid (EDTA), 1,4,7,1
0-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA),
tetraazacyclotetradecane-N,N',N"N"'-tetraacetic acid (TETA),
hydroxyethylidene diphosphonate (HEDP), dimercaptosuccinic acid
(DMSA), diethylenetriaminetetramethylenephosphonic acid (DTTP) and
1-(p-aminobenzyl)-DTPA, 1,6-diamino hexane-N,N,N',N'-tetraacetic
acid, DPDP, and ethylenebis (oxyethylenenitrilo)-tetraacetic acid,
with DTPA being the most preferred. A preferred embodiment of the
present invention may also be a composition comprising
.sup.111In-DTPA-paclitaxel, and Na-DTPA-paclitaxel.
[0018] In certain embodiments of the present invention, the
paclitaxel, docetaxel, or other taxoid may be conjugated to a water
soluble polymer, and preferably the polymer is conjugated to the 2'
or the 7- hydroxyl or both of the paclitaxel, docetaxel, or other
taxoid. Poly-glutamic acid (PG) is one polymer that offers several
advantages in the present invention. First, it contains a large
number of side chain carboxyl functional groups for drug
attachment. Second, PG can be readily degraded by lysosomal enzymes
to its nontoxic basic component, 1-glutamic acid, d-glutamic acid
and di-glutamic acid.
[0019] Finally, sodium glutamate has been reported to prevent
manifestations of neuropathy induced by paclitaxel, thus enabling
higher doses of paclitaxel to be tolerated (Boyle et al., 1996).
Preferred polymers include, but are not limited to poly(l-glutamic
acid), poly(d-glutamic acid), poly(dl-glutamic acid),
poly(l-aspartic acid), poly(d-aspartic acid), poly(dl-aspartic
acid), poly(l-lysine), poly(d-lysine), poly(dl-lysine), copolymers
of the above listed polyamino acids with polyethylene glycol,
polycaprolactone, polyglycolic acid and polylactic acid, as well as
poly(2-hydroxyethyl 1-glutamine), chitosan, carboxymethyl dextran,
hyaluronic acid, human serum albumin and alginic acid, with
poly-glutamic acids being particularly preferred. At the lower end
of molecular weight, the polymers of the present invention
preferably have a molecular weight of about 1,000, about 2,000,
about 3,000, about 4,000, about 5,000, about 6,000, about 7,000,
about 8,000, about 9,000, about 10,000, about 11,000, about 12,000,
about 13,000, about 14,000, about 15,000, about 16,000, about
17,000, about 18,000, about 19,000, about 20,000, about 21,000,
about 22,000, about 23,000, about 24,000, about 25,000, about
26,000, about 27,000, about 28,000, about 29,000, about 30,000,
about 31,000, about 32,000, about 33,000, about 34,000, about
35,000, about 36,000, about 37,000, about 38,000, about 39,000,
about 40,000, about 41,000, about 42,000, about 43,000, about
44,000, about 45,000, about 46,000, about 47,000, about 48,000,
about 49,000, to about 50,000 kd. At the higher end of molecular
weight, the polymers of the present invention preferably have a
molecular weight of about 51,000, about 52,000, about 53,000, about
54,000, about 55,000, about 56,000, about 57,000, about 58,000,
about 59,000, about 60,000, about 61,000, about 62,000, about
63,000, about 64,000, about 65,000, about 66,000, about 67,000,
about 68,000, about 69,000, about 70,000, about 71,000, about
72,000, about 73,000, about 74,000, about 75,000, about 76,000,
about 77,000, about 78,000, about 79,000, about 80,000, about
81,000, about 82,000, about 83,000, about 84,000, about 85,000,
about 86,000, about 87,000, about 88,000, about 89,000, about
90,000, about 91,000, about 92,000, about 93,000, about 94,000,
about 95,000, about 96,000, about 97,000, about 98,000, about
99,000, to about 100,000 kd. Within these ranges, the ranges of
molecular weights for the polymers are preferably of about 5,000 to
about 100,000 kd, with about 20,000 to about 80,000 being
preferred, or even about 25,000 to about 50,000 being more
preferred.
[0020] It is a further aspect of the invention that a composition
of the invention such as PG-TXL may also be conjugated to a second
lipophilic or poorly soluble antitumor agent such as camptothecin,
epothilone, cisplatin, melphalan, Taxotere, etoposide, teniposide,
fludarabine, verapamil, or cyclosporin, for example, or even to
water soluble agents such as 5 fluorouracil (5 FU) or
fluorodeoxyuridine (FUDR), doxorubicin or daunomycin.
[0021] It is understood that the compositions of the present
invention may be dispersed in a pharmaceutically acceptable carrier
solution as described below. Such a solution would be sterile or
aseptic and may include water, buffers, isotonic agents or other
ingredients known to those of skill in the art that would cause no
allergic or other harmful reaction when administered to an animal
or human subject. Therefore, the present invention may also be
described as a pharmaceutical composition comprising a
chemotherapeutic or anti-cancer drug such as paclitaxel, docetaxel,
or other taxoid conjugated to a high molecular weight water soluble
polymer or to a chelator. The pharmaceutical composition may
include polyethylene glycol, poly-glutamic acids, poly-aspartic
acids, poly-lysine, or a chelator, preferably DTPA. It is also
understood that a radionuclide may be used as an anti-tumor agent,
or drug, and that the present pharmaceutical composition may
include a therapeutic amount of a chelated radioactive isotope.
[0022] In certain embodiments, the present invention may be
described as a method of determining the uptake of a
chemotherapeutic drug such as paclitaxel, docetaxel, or other
taxoid by tumor tissue. This method may comprise obtaining a
conjugate of the drug and a metal chelator with a chelated metal
ion, contacting tumor tissue with the composition and detecting the
presence of the chelated metal ion in the tumor tissue. The
presence of the chelated metal ion in the tumor tissue is
indicative of uptake by the tumor tissue. The chelated metal ion
may be a radionuclide and the detection may be scintigraphic. The
tumor tissue may also be contained in an animal or a human subject
and the composition would then be administered to the subject.
[0023] The present invention may also be described in certain
embodiments as a method of treating cancer in a subject. This
method includes obtaining a composition comprising a
chemotherapeutic drug such as paclitaxel, docetaxel, or other
taxoid conjugated to a water soluble polymer or chelator and
dispersed in a pharmaceutically acceptable solution and
administering the solution to the subject in an amount effective to
treat the tumor. Preferred compositions comprise paclitaxel,
docetaxel, or other taxoid conjugated to a water soluble polyamino
acids, including but not limited to poly (l-aspartic acid), poly
(d-aspartic acid), or poly (dl-aspartic acid), poly (l-lysine
acid), poly (d-lysine acid), or poly (dl-lysine acid), and more
preferably to poly (l-glutamic acid), poly (d-glutamic acid), or
poly (dl-glutamic acid). The compositions of the invention are
understood to be effective against any type of cancer for which the
unconjugated taxoid is shown to be effective and would include, but
not be limited to breast cancer, ovarian cancer, malignant
melanoma, lung cancer, head and neck cancer. The compositions of
the invention may also be used against gastric cancer, prostate
cancer, colon cancer, leukemia, or Kaposi's Sarcoma. As used herein
the term "treating" cancer is understood as meaning any medical
management of a subject having a tumor. The term would encompass
any inhibition of tumor growth or metastasis, or any attempt to
inhibit, slow or abrogate tumor growth or metastasis. The method
includes killing a cancer cell by non-apoptotic as well as
apoptotic mechanisms of cell death. The method of treating a tumor
may include some prediction of the paclitaxel or docetaxel uptake
in the tumor prior to administering a therapeutic amount of the
drug, by methods that include but are not limited to bolus
injection or infusion, as well as intraarterial, intravenous,
intraperitoneal, or intratumoral administration of the drug.
[0024] This method may include any of the imaging techniques
discussed above in which a paclitaxel-chelator-chelated metal is
administered to a subject and detected in a tumor. This step
provides a cost effective way of determining that a particular
tumor would not be expected to respond to DTPA-paclitaxel therapy
in those cases where the drug does not get into the tumor. It is
contemplated that if an imaging technique can be used to predict
the response to paclitaxel and to identify patients that are not
likely to respond, great expense and crucial time may be saved for
the patient. The assumption is that if there is no reasonable
amount of chemotherapeutic agent deposited in the tumor, the
probability of tumor response to that agent is relatively
small.
[0025] In certain embodiments the present invention may be
described as a method of obtaining a body image of a subject. The
body image is obtained by administering an effective amount of a
radioactive metal ion chelated to a paclitaxel-chelator conjugate
to a subject and measuring the scintigraphic signals of the
radioactive metal to obtain an image.
[0026] The present invention may also be described in certain broad
aspects as a method of decreasing at least one symptom of a
systemic autoimmune disease comprising administering to a subject,
having a systemic autoimmune disease an effective amount of a
composition comprising paclitaxel or docetaxel conjugated to
polymer, with polyamino acids being preferred and poly-glutamic
acid being more preferred. Of particular interest in the context of
the present disclosure is the treatment of rheumatoid arthritis,
which is known to respond in some cases to paclitaxel when
administered in the standard Cremophor formulation (U.S. Pat. No.
5,583,153, incorporated herein by reference). As in the treatment
of tumors, it is contemplated that the effectiveness of the water
soluble taxoids or taxane of the present invention will not be
diminished by the conjugation to a water soluble moiety. Therefore,
the compositions of the present invention are expected to be as
effective as paclitaxel against rheumatoid arthritis. Paclitaxel is
an antiangiogenic agent. Rheumatoid arthritis creates a collection
of newly formed vessels which erode the adjacent joints. It is also
understood that the taxoid or taxane compositions of the present
invention may be used in combination with other drugs, such as an
angiogenesis inhibitor (AGM-1470) (Oliver et al., 1994), or other
anti-cancer drugs, such as methotrexate.
[0027] The finding that paclitaxel also inhibits restenosis after
balloon angioplasty indicates that the water soluble paclitaxels
and docetaxels of the present invention will find a variety of
applications beyond direct parenteral administration (WO 9625176,
incorporated herein by reference). For example, it is contemplated
that water soluble paclitaxel will be useful as a coating for
implanted medical devices, such as tubings, shunts, catheters,
artificial implants, pins, electrical implants such as pacemakers,
and especially for arterial or venous stents, including
balloon-expandable stents. In these embodiments it is contemplated
that water soluble paclitaxel may be bound to an implantable
medical device, or alternatively, the water soluble paclitaxel may
be passively adsorbed to the surface of the implantable device. For
example, stents may be coated with polymer-drug conjugates by
dipping the stent in polymer-drug solution or spraying the stent
with such a solution. Suitable materials for the implantable device
should be biocompatible and nontoxic and may be chosen from the
metals such as nickel-titanium alloys, steel, or biocompatible
polymers, hydrogels, polyurethanes, polyethylenes, ethylenevinyl
acetate copolymers, etc. In a preferred embodiment the water
soluble paclitaxel, especially a PG-TXL conjugate, is coated onto a
stent for insertion into an artery or vein following balloon
angioplasty. The invention may be described therefore, in certain
broad aspects as a method of inhibiting arterial restenosis or
arterial occlusion following vascular trauma comprising
administering to a subject in need thereof, a composition
comprising paclitaxel or docetaxel conjugated to poly-glutamic acid
or other water soluble poly-amino acids. In the practice of the
method, the subject may be a coronary bypass, vascular surgery,
organ transplant or coronary or any other arterial angioplasty
patient, for example, and the composition may be administered
directly, intravenously, or even coated on a stent to be implanted
at the sight of vascular trauma.
[0028] An embodiment of the invention is, therefore, an implantable
medical device, wherein the device is coated with a composition
comprising paclitaxel or docetaxel conjugated to poly-glutamic
acids or water soluble polyamino acids in an amount effective to
inhibit smooth muscle cell proliferation. A preferred device is a
stent coated with the compositions of the present invention as
described herein, and in certain preferred embodiments, the stent
is adapted to be used during or after balloon angioplasty and the
coating is effective to inhibit restenosis.
[0029] In certain preferred embodiments, the invention may be
described as a composition comprising poly-glutamic acids
conjugated to the 2' or 7 hydroxyl or both of paclitaxel,
docetaxel, or other taxoids, or even a composition comprising water
soluble polyamino acids conjugated to the 2' or 7 hydroxyl or both
of paclitaxel, docetaxel, or other taxoids.
[0030] As used herein, the terms "a poly-glutamic acid" or
"poly-glutamic acids" include poly (l-glutamic acid), poly
(d-glutamic acid) and poly (dl-glutamic acid), the terms "a
poly-aspartic acid" or "poly-aspartic acids" include poly
(l-aspartic acid), poly (d-aspartic acid), poly (dl-aspartic acid),
the terms "a poly-lysine" or "poly-lysine" include poly (l-lysine),
poly (d-lysine), poly (dl-lysine), and the terms "a water soluble
polyamino acid", "water soluble polyamino acids", or "water soluble
polymer of amino acids" include, but are not limited to,
poly-glutamic acid, poly-aspartic acid, poly-lysine, and amino acid
chains comprising mixtures of glutamic acid, aspartic acid, and/or
lysine. In certain embodiments, the terms "a water soluble
polyamino acid", "water soluble polyamino acids", or "water soluble
polymer of amino acids" include amino acid chains comprising
combinations of glutamic acid and/or aspartic acid and/or lysine,
of either d and/or l isomer conformation. In certain prefered
embodiments, such a "water soluble polyamino acid" contains one or
more glutamic acid, aspartic acid, and/or lysine residues. Such
"water soluble polyamino acids" may also comprise any natural,
modified, or unusual amino acid described herein, as long as the
majority of residues, i.e. greater than 50%, comprise glutamic acid
and/or aspartic acid and/or lysine. In certain embodiments, a water
soluble polymer of amino acids that contains more than one
different type of amino acid residue is sometimes referred to
herein as a "co-polymer".
[0031] In certain embodiments, various substitutions of naturally
occurring, unussual, or chemically modified amino acids may be made
in the amino acid composition of the "water soluble polyamino
acids", and particularly in "poly-glutamic acids", to produce a
taxoid-polyamino acid conjugate of the present invention and still
obtain molecules having like or otherwise desirable characteristics
of solubility and/or therapeutic efficacy. A polyamino acid such as
poly-glutamic acid, poly-aspartic acid, poly-lysine, or water
soluble amino acids chain or polymer comprising a mixture of
glutamic acid, aspartic acid, and/or lysine, may, at the lower end
of the amino acid substitution range, have about 1, about 2, about
3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,
about 12, about 13, about 14, about 15, about 16, about 17, about
18, about 19, about 20, about 21, about 22, about 23, about 24, or
about 25 or more glutamic acid, aspartic acid, or lysine, residues,
respectively, substituted by any of the naturally occurring,
modified, or unusual amino acids described herein. In other aspects
of the invention, a polyamino acid such as poly-glutamic acid,
poly-aspartic acid, poly-lysine, or a poly-amino acid chain
comprising a mixture of some or all of these three amino acids may,
at the lower end, have about 1%, about 2%, about 3%, about 4%,
about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about
11%, about 12%, about 13%, about 14%, about 15%, about 16%, about
17%, about 18%, about 19%, about 20%, about 21%, about 22%, about
23%, about 24%, to about 25% or more glutamic acid, aspartic acid,
or lysine residues, respectively, substituted by any of the
naturally occurring, modified, or unusual amino acids described
herein.
[0032] In further aspects of the invention, a polyamino acid such
as poly-glutamic acid, poly-aspartic acid, or poly-lysine may, at
the high end of the amino acid substitution range, have about 25%,
about 26%, about 27%, about 28%, about 29%, about 30%, about 31%,
about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,
about 38%, about 39%, about 40%, about 41%, about 42%, about 43%,
about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,
to about 50% or so of the glutamic acid, aspartic acid, or lysine
residues, respectively, substituted by any of the naturally
occurring, modified, or unusual amino acids described herein, as
long as the majority of residues comprise glutamic acid and/or
aspartic acid and/or lysine. In amino acid substitution of the
various water soluble amino acid polymers, residues with a
hydrophilicity index of +1 or more are preferred.
[0033] In certain aspects of the invention, the amount of
anti-tumor drug conjugated per water soluble polymer can vary. At
the lower end, such a composition may comprise from about 1%, about
2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,
about 9%, or about 10%, about 11%, about 12%, about 13%, about 14%,
about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,
about 21% about 22%, about 23%, about 24%, to about 25% (w/w)
antitumor drug relative to the mass of the conjugate. At the high
end, such a composition may comprise from about 26%, about 27%,
about 28%, about 29%, about 30%, about 31% about 32%, about 33%,
about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,
to about 40% or more (w/w) antitumor drug relative to the mass of
the conjugate. Preferred anti-tumor drugs include paclitaxel,
docetaxel, or other taxoids, and preferred water soluble polymers
include water soluble amino acid polymers.
[0034] In certain other aspects of the invention, the number of
molecules of anti-tumor drug conjugated per molecule of water
soluble polymer can vary. At the lower end, such a composition may
comprise from about 1, about 2, about 3, about 4, about 5, about 6,
about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, about 15, about 16, about 17, about 18, about 19, to
about 20 or more molecules of antitumor drug per molecule of water
soluble polymer. At the higher end, such a composition may comprise
from about 21, about 22, about 23, about 24, about 25, about 26,
about 27, about 28, about 29, about 30, about 31, about 32, about
33, about 34, about 35, about 36, about 37, about 38, about 39,
about 40, about 41, about 42, about 43, about 44, about 45, about
46, about 47, about 48, about 49, about 50, about 51, about 52,
about 53, about 54, about 55, about 56, about 57, about 58, about
59, about 60 about 61, about 62, about 63, about 64, about 65,
about 66, about 67, about 68, about 69, about 70, about 71, about
72, about 73, about 74, to about 75 or more molecules or more of
antitumor drug per molecule of water soluble polymer. Preferred
anti-tumor drugs include paclitaxel, docetaxel, or other taxoids,
and preferred water soluble polymers include water soluble amino
acid polymers. The preferred number of anti-tumor drug molecules
conjugated per molecule of water soluble polymer is about 7
molecules of antitumor drug per molecule of water soluble
polymer.
[0035] Water soluble amino acid polymers with various substitutions
of residues conjugated to paclitaxel, docetaxel, or other taxoids
are referred to as "biological functional equivalents". These
"biologically functional equivalents" are part of the definition of
"water soluble polyamino acids" that are conjugated to taxoids, and
may be identified by the assays described herein as well as any
applicable assay that is known to those of skill in the art to
measure improved aqueous solubility relative to the unconjugated
taxoid or taxoids used to produce the particular water soluble
amino acid polymer-taxoid composition. In other aspects of the
invention, "biological functional equivalents" of water soluble
amino acid-taxoid polymers may be further identified by improved
anti-tumor cell activity, relative to the anti-tumor cell activity
of the unconjugated water soluble amino acid polymer used to
produce the particular water soluble amino acid polymer-taxoid
composition by the assays described herein as well as any
applicable assay that is known to those of skill in the art. The
term "biologically functional equivalents" as used herein to
describe this aspect of the invention is further described in the
detailed description of the invention.
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Also as
used herein, the term "a" is understood to include the meaning "one
or more". Although any methods and materials similar or equivalent
to those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are now
described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1A. Chemical structure of paclitaxel, PEG-paclitaxel
and DTPA-paclitaxel.
[0038] FIG. 1B. Chemical structure and reaction scheme for
production of PG-TXL.
[0039] FIG. 2. Effect of paclitaxel, PEG-paclitaxel and
DTPA-paclitaxel on proliferation of B16 melanoma cells.
[0040] FIG. 3. Antitumor effect of DTPA-paclitaxel on MCa-4 mammary
tumors.
[0041] FIG. 4. Median time (days) to reach tumor diameter of 12 mm
after treatment with paclitaxel, DTPA-paclitaxel and
PEG-paclitaxel.
[0042] FIG. 5. Gamma-scintigraphs of mice bearing MCa-4 tumors
following intravenous injection of .sup.111In-DTPA-paclitaxel and
.sup.111In-DTPA. Arrow indicates the tumor.
[0043] FIG. 6. Hydrolytic degradation of PG-TXL as determined in
PBS as a function of time at different pH levels. -.diamond.-
represents percent paclitaxel released, -O- represents metabolite-1
produced.
[0044] FIG. 7A. Anti-tumor effect of PG-TXL against syngeneic OCA-I
ovarian carcinoma tumor in female C3Hf/Kam mice. Drugs were
injected intraveneously in a single dose. Data are presented as
mean .+-. standard deviation of tumor volumes. a, Mice bearing
OCA-l tumor were injected with -z,900 -, PG control (800 mg/kg;
n=9); -.tangle-solidup.-, paclitaxel (80 mg/kg; n=7); -.DELTA.-,
paclitaxel (80 mg/kg) plus PG (800 mg/kg; n=5); -.circle-solid.-,
PG-TXL (80 mg equiv. paclitaxel; n=6); or --, PG-TXL (160 mg equiv.
paclitaxel/kg; n=26).
[0045] FIG. 7B. Anti-tumor effect of PG-TXL against 13762F tumor in
female rats. -- represents PG control (220 mg/kg; n=7),
-.tangle-solidup.- represents paclitaxel (20 mg/kg; n=5), -.DELTA.-
represents paclitaxel (40 mg/kg; n=7), -.circle-solid.- represents
PG-TXL (20 mg equivalent paclitaxel/kg; n=5), -- represents PG-TXL
(40 mg or 60 mg equivalent paclitaxel/kg; n=9).
[0046] FIG. 7C. The antitumor effect of PG-TXL on mice bearing
MCa-4 mammary carcinoma tumors. -.quadrature.- represents the
response to a single i.v. dose of saline, -.DELTA.- represents the
response to a single i.v. dose of PG (0.6 g/kg); -.diamond-solid.-
represents response to PG-TXL (40 mg/kg), -.diamond.- represents
response to PG-TXL (60 mg equiv. paclitaxel/kg), -- represents
response to PG-TXL (120 mg/kg).
[0047] FIG. 7D. The antitumor effect of PG-TXL against soft-tissue
sarcoma tumor (FSa-II) in mice. -.quadrature.- represents the
response to a single i.v. dose of saline, -.diamond.- represents
the response to a single i.v. dose of PG (0.8 g/kg); -- represents
response to paclitaxel (80 mg/kg), -.DELTA.- represents response to
PG-TXL (160 mg equiv. paclitaxel/kg).
[0048] FIG. 7E. The antitumor effect of PG-TXL against syngeneic
hepatocarcinoma tumor (HCa-I) in mice. -.quadrature.- represents
the response to a single i.v. dose of saline, -.DELTA.- represents
the response to a single i.v. dose of PG (0.8 g/kg); -- represents
response to PG-TXL (80 mg/kg), -.DELTA.- represents response to
PG-TXL (160 mg equiv. paclitaxel/kg).
[0049] FIG. 8. Release profile of paclitaxel from PEG-paclitaxel in
phosphate buffer (pH 7.4). Release profiles of paclitaxel
(-.times.-); from PEG-paclitaxel (--) at pH 7.4 is shown.
[0050] FIG. 9. Antitumor effect of PEG-paclitaxel on MCa-4 mammary
tumors. -.quadrature.-represents the response a single i.v.
injection with a saline solution of PEG (60 mg/ml), -.box-solid.-
represents the response to the Cremophor/alcohol vehicle,
--represents a single dose of 40 mg/kg body weight of paclitaxel,
-.circle-solid.- represents PEG-paclitaxel at 40 mg equiv.
paclitaxel/kg body weight.
[0051] FIG. 10. Tubulin polymerization assays performed at
32.degree. C. in the presence of 1.0 mM GTP and 1.0 mg/ml of
tubulin. -- represents paclitaxel (1.0 EM), -.DELTA.- represents
PG-TXL (10 .mu.M equivalent paclitaxel) incubated in PBS (pH 7.4)
at 37.degree. C. for 3 days, -- represents freshly dissolved
PG-TXL.
[0052] FIG. 11. Plasma clearance of radioactivity following an i.v.
injection of PG-[.sup.3H]paclitaxel and [.sup.3H]paclitaxel in
C3Hf/Kam mice. -- represents PG-TXL radioactivity after injection
of 6 .mu.Ci of radiolabeled PG-[.sup.3H]paclitaxel (20 mg
equivalent paclitaxel/kg), -.times.- represents paclitaxel
radioactivity after injection of 6 .mu.Ci of radiolabeled
[.sup.3H]paclitaxel (20 mg equivalent paclitaxel/kg), -- represents
"Paclitaxel" radioactivity released from injected
PG-[.sup.3H]paclitaxel.
[0053] FIG. 12A. Time-dependent OCA-1 tumor content of
radioactivity following injection of either PG-[.sup.3H]paclitaxel
and [.sup.3 H]paclitaxel into mice. Open bars represents PG-TXL
radioactivity after injection of 6 .mu.Ci of radiolabeled
PG-[.sup.3H]paclitaxel (20 mg equivalent paclitaxel/kg), filled
bars represents paclitaxel radioactivity after injection of 6
.mu.Ci of radiolabeled [.sup.3H]paclitaxel (20 mg equivalent
paclitaxel/kg).
[0054] FIG. 12B. Conversion of PG-[.sup.3H]paclitaxel to
[.sup.3H]paclitaxel within OCA-1 tumor. Total radioactivity
measured after injection of 6 .mu.Ci of radiolabeled
PG-[.sup.3H]paclitaxel is shown in open bars, "Paclitaxel" derived
radioactivity released from injected PG-[.sup.3H]paclitaxel is
shown in solid bars.
[0055] FIG. 13. Kinetics of apoptosis in OCA-1 tumors after a
single i.v. dose of 160 mg equiv. paclitaxel/kg of PG-TXL (MTD) and
80 mg/kg paclitaxel (MTD). -.quadrature.- represents the response
to a single i.v. dose of PG-TXL (160 mg equiv. paclitaxel/kg MTD),
-.largecircle.- represents response to paclitaxel (80 mg
paclitaxel/kg MTD).
[0056] FIG. 14. Survival of nude mice with human ovarian cancer
cells (SKOV3ip1) treated with PG-TXL. Five days after tumor
injection, the mice were injected i.v. with the PG-paclitaxel
(PG-TXL), or PG control. Injections of PG-TXL were administered
every seven days (.tangle-soliddn.) in the 120 mg/kg group, but not
the 160 mg/kg group. -.box-solid.- represents untreated mice.
-.tangle-solidup.- represents the response to multiple i.v. doses
of PG. -.tangle-soliddn.- represents the response to an i.v. dose
of PG-TXL (120 mg equiv. paclitaxel/kg), -.diamond-solid.-
represents the response to an i.v. dose of PG-TXL (160 mg equiv.
paclitaxel/kg).
[0057] FIG. 15. Chemical structure and reaction scheme for
production of glutamic acid containing polyamino acids.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention arises from the discovery of novel,
water soluble formulations of paclitaxel and docetaxel, and the
surprising efficacy of these formulations against tumor cells in
vivo. Poly (l-glutamic acid) conjugated paclitaxel (PG-TXL)
administered to mice bearing ovarian carcinoma (OCA-I) caused
significant tumor growth delay as compared to the same dose of
paclitaxel without PG. Mice treated with paclitaxel alone or with a
combination of free paclitaxel and PG showed delayed tumor growth
initially, but tumors regrew to levels comparable to an untreated
control group after ten days. Moreover, at the maximum tolerated
dose (MTD) of the PG-TXL conjugate, (160 mg equiv. paclitaxel/kg),
the growth of tumors was completely suppressed, the tumors shrank,
and mice observed for two months following treatment remained tumor
free (MTD: defined as the maximal dose that produced 15% or less
body weight loss within two wk after a single i.v. injection). In a
parallel study, the antitumor activity of PG-TXL in rats with rat
mammary adenocarcinoma (13762F) was examined. Again, complete tumor
eradication at 40-60 mg equiv. paclitaxel/kg of PG-TXL was
observed. These surprising results demonstrate that the
polymer-drug conjugate, PG-TXL, successfully eradicates well
established solid tumors in both mice and rats after a single
intravenous injection.
[0059] In addition to the remarkable antitumor (breast, ovarian,
etc.) data in syngeneic mice, good activity of PG-TXL against human
breast cancer (MDA-435) and ovarian cancer (SKOV3ipl) in nude mice
has recently been observed. Nude mice are special animals with
incomplete immune systems in which human tumors can grow.
[0060] The data presented herein have led the present inventors to
conclude that PG-TXL is a novel species of taxane that is
pharmacologically distinct from previous paclitaxel or Taxol.TM.
preparations. For example, the distribution of PG-TXL within plasma
is distinct from free paclitaxel. While paclitaxel remains in the
plasma of mice for an extremely short time, PG-TXL appears to
remain for a much longer period. This is contemplated to offer a
distinct advantage in that prolonged exposure of tumors to the drug
may result in an enhanced response. The rate of conversion of
PG-TXL to paclitaxel is slow, with less than 1% of the
radioactivity from radiolabeled PG-TXL being recovered as
radioactive paclitaxel within 48 h after injection of the
paclitaxel-polymer complex. This finding suggests that the novel
drug, PG-TXL, may produce death within tumor cells in a manner
which is not simply due to the gradual release of paclitaxel
itself.
[0061] Further evidence of the novelty of PG-TXL is that relatively
high levels of radioactivity from radiolabeled PG-TXL appear in
tumor tissue shortly after injection. However, only small amounts
of radioactivity within tumor tissue are due to the release of free
paclitaxel. Furthermore, the percent of radioactivity within tumor
tissue due to paclitaxel itself does not appreciably increase with
time suggesting again that PG-TXL is a minimal prodrug for the
gradual release of paclitaxel. Uptake of PG-TXL versus paclitaxel
has also been studied in a specialized human colon adenocarcinoma
cell transport system. While radioactivity associated with
radiolabeled PG-TXL readily gained entry into cells, only 10% of it
was due to free paclitaxel. These data parallel that which was
found in studies of tissue distribution and again suggest that
there are several mechanisms or ways in which PG-TXL may lead to
the death of cancer cells which are different from those for
paclitaxel.
[0062] In another study, it was discovered that freshly prepared
PG-TXL does not support the growth of paclitaxel-dependent cell
lines suggesting that free paclitaxel is only slowly released from
the polymer-paclitaxel complex and that the polymer-paclitaxel
complex itself is not behaving pharmacologically as "Taxol.TM.".
Aging will promote the degradation of PG-TXL and does increase the
relative ability of the resulting material to support the growth of
paclitaxel-dependent cells, but to a lesser extent than compared to
free paclitaxel.
[0063] Recent analyses of tumor tissues from mice treated with
paclitaxel suggests that, as expected, this drug results in the
formation of many apoptotic bodies within the tumor itself.
Apoptosis is a mechanism in which cells commit self-induced death
or programmed cell death, a natural process used by an organism in
wound healing and tissue remodeling. Tumors from mice treated with
PG-TXL had far fewer apoptotic bodies compared to free paclitaxel
but had an increased incidence of tumor necrosis and edema
suggesting that paclitaxel and PG-TXL may result in tumor cell
death by two distinctly different pathways.
[0064] These studies, and those described in the specific examples,
demonstrate that PG-TXL is a new taxane which is not only extremely
active against breast and ovarian cancers, and appears to have
limited side affects. It is now clear that the polymer conjugation
of paclitaxel results in a compound (PG-TXL) that has novel and
greater over-all antitumor activity.
[0065] Another aspect of the present invention is the inclusion of
molecules in the polymeric composition that are effective to target
the therapeutic composition to a disease or tumor site or to a
particular organ or tissue. Many of such targeting molecules are
known in the art and may be conjugated to the water soluble
anti-tumor compositions of the present invention. Examples of such
molecules or agents would include, but not be limited to antibodies
such as anti-tumor antibodies; anti-cell receptor antibodies;
tissue specific antibodies; hormonal agents such as octreotide,
estradiol and tamoxifen; growth factors; cell surface receptor
ligands; enzymes; hypoxic agents such as misonidazole and
erythronitroimidazole; and antiangiogenic agents.
[0066] Another composition of the present invention is
DTPA-paclitaxel, also shown herein to be as effective as paclitaxel
in an in vitro antitumor potency assay using a B16 melanoma cell
line. DTPA-paclitaxel did not show any significant difference in
antitumor effect as compared to paclitaxel against an MCa-4 mammary
tumor at a dose of 40 mg/kg body weight in a single injection.
Furthermore, .sup.111Indium labeled DTPA-paclitaxel was shown to
accumulate in the MCa-4 tumor as demonstrated by
gamma-scintigraphy, demonstrating that the chelator conjugated
anti-tumor drugs of the present invention are useful and effective
for tumor imaging.
[0067] The novel compounds and methods of the present invention
provide significant advances over prior methods and compositions,
as the water-soluble paclitaxels are projected to improve the
efficacy of paclitaxel-based anti-cancer therapy, by providing
water soluble and controlled release paclitaxel derived
compositions that also have different antitumor properties than
unmodified paclitaxel. Such compositions eliminate the need for
solvents that are associated with side effects seen with prior
paclitaxel compositions. In addition, radiolabeled paclitaxel,
which is shown to retain anti-tumor activity, will also be useful
in the imaging of tumors. Further, the present invention allows one
to determine whether a paclitaxel will be taken up by a particular
tumor by scintigraphy, single photon emission computer tomography
(SPECT) or positron emission tomography (PET). This determination
may then be used to predict the efficacy of an anti-cancer
treatment. This information may be helpful in guiding the
practitioner in the selection of patients to undergo
chelator-paclitaxel therapy.
[0068] The paclitaxel may be rendered water-soluble in many ways:
i.e. by conjugating paclitaxel to water-soluble polymers which
serve as drug carriers, and by derivatizing the antitumor drug with
water soluble chelating agents. The latter approach also provides
an opportunity for labeling with radionuclides (e.g., .sup.111In,
.sup.90Y, .sup.166Ho, .sup.68Ga, .sup.99mTc) for nuclear imaging
and/or for radiotherapy studies. The structures of paclitaxel,
polyethylene glycol-paclitaxel (PEG-paclitaxel), poly-glutamic
acid-paclitaxel conjugate (PG-TXL) and
diethylenetriaminepentaacetic acid-paclitaxel (DTPA-paclitaxel) are
shown in FIG. 1.
[0069] In certain embodiments of the present invention,
DTPA-paclitaxel or other paclitaxel-chelating agent conjugates,
such as EDTA-paclitaxel, DTTP-paclitaxel, or DOTA-paclitaxel, for
example, may be prepared in the form of water-soluble salts (sodium
salt, potassium salt, tetrabutylammonium salt, calcium salt, ferric
salt, etc.). These salts will be useful as therapeutic agents for
tumor treatment. Secondly, DTPA-paclitaxel or other
paclitaxel-chelating agents will be useful as diagnostic agents
which, when labeled with radionuclides such as .sup.111In or
.sup.99mTc, may be used as radiotracers to detect certain tumors in
combination with nuclear imaging techniques. It is understood that
in addition to paclitaxel (Taxol.TM.) and docetaxel (Taxotere),
other taxane derivatives may be adapted for use in the compositions
and methods of the present invention and that all such compositions
and methods would be encompassed by the present invention.
[0070] As modifications and changes may be made in the structure of
the water soluble polymer such as a water soluble polyamino acid,
or a water soluble metal chelator, of the present invention and
still obtain molecules having like or otherwise desirable
characteristics, such "biologically functional equivalents" or
"functional equivalents" are also encompassed within the present
invention.
[0071] For example, one of skill in the art will recognize that
certain amino acids may be substituted for other amino acids in a
polyamino acid structure, including water soluble amino acid
polymers such as poly-glutamic acid, poly-aspartic acid, or
poly-lysine, without appreciable loss of interactive binding
capacity with structures such as, for example, a chemotherapeutic
and/or antiangiogenic drug, such as paclitaxel or docetaxel, or
such like. Additionally, amino acid substitutions in a water
soluble polyamino acid conjugated to a chemotherapeutic and/or
antiangiogenic drug, such as paclitaxel or docetaxel, or such like,
as exemplified by but not limited to PG-TXL, may be made and still
maintain part or all of the novel pharmacological properties
disclosed herein. Since it is the interactive capacity and nature
of a protein that defines that protein's biological functional
activity, certain amino acid sequence substitutions can be made in
a polyamino acid sequence and nevertheless obtain a polyamino acid
with like (agonistic) properties. It is thus contemplated by the
inventors that various changes may be made in the sequence of the
water soluble polyamino acids of a drug conjugate, such as, but not
limited to PG-TXL, without appreciable loss of their biological
utility or activity.
[0072] In terms of functional equivalents, it is well understood by
the skilled artisan that, inherent in the definition of a
"biologically functional equivalent of a water soluble polyamino
acid", is the concept that there is a limit to the number of
changes that may be made within a portion of the molecule and still
result in a molecule with an acceptable level of equivalent
biological activity. Biologically functional equivalent of a water
soluble polyamino acids, are thus defined herein as those water
soluble polyamino acids in which certain, not most or all, of the
amino acids may be substituted by non-water soluble amino acids,
whether natural, unusual, or chemically modified.
[0073] In particular, where shorter length water soluble polyamino
acids are concerned, it is contemplated that fewer amino acids
should be made within the given peptide. Longer domains may have an
intermediate number of changes. The longest water soluble polyamino
acid chains, as described herein, will have the most tolerance for
a larger number of changes. Of course, a plurality of distinct
water soluble polyamino acids, such as but not limited to poly
glutamic acid, poly aspartic acid, or poly-lysine, with different
substitutions may easily be made and used in accordance with the
invention.
[0074] It is also well understood that where certain residues are
shown to be particularly important to the biological or structural
properties of a polyamino acid, such residues may not generally be
exchanged. In this manner, functional equivalents are defined
herein as those water soluble polyamino acids which maintain a
substantial amount of their native biological activity.
[0075] Amino acid substitutions are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and the like.
An analysis of the size, shape and type of the amino acid
side-chain substituents reveals that arginine, lysine and histidine
are all positively charged residues; that alanine, glycine and
serine are all a similar size; and that phenylalanine, tryptophan
and tyrosine all have a generally similar shape. Therefore, based
upon these considerations, arginine, lysine and histidine; alanine,
glycine and serine; and phenylalanine, tryptophan and tyrosine; are
defined herein as biologically functional equivalents.
[0076] To effect more quantitative changes, the hydropathic index
of amino acids may be considered. Each amino acid has been assigned
a hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0077] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein, and
correspondingly a polyamino acid, is generally understood in the
art (Kyte & Doolittle, 1982, incorporated herein by reference).
It is known that certain amino acids may be substituted for other
amino acids having a similar hydropathic index or score and still
retain a similar biological activity. In making changes based upon
the hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0078] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. As detailed in U.S. Pat. No. 4,554,101, the
following hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1);
glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). In making changes based
upon similar hydrophilicity values, the substitution of amino acids
whose hydrophilicity values are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. Hence, in reference
to hydrophilicity, arginine, lysine, aspartic acid, and glutamic
acid are defined herein as biologically functional equivalents,
particularly in water soluble amino acid polymers.
[0079] In addition to the water soluble polyamino
acid-chemotherapeutic and/or antiangiogenic drug compounds
described herein, such as paclitaxel or docetaxel conjugated to a
water soluble amino acid, or such like, as exemplified by, but not
limited to PG-TXL compounds described herein, the inventors also
contemplate that other sterically similar compounds may be
formulated to mimic the key portions of the water soluble polyamino
acid structure. Such compounds, which may be termed
peptidomimetics, may be used in the same manner as the peptides of
the invention and hence are also functional equivalents.
[0080] Certain mimetics that mimic elements of protein secondary
structure are described in Johnson et al. (1993). The underlying
rationale behind the use of peptide mimetics is that the peptide
backbone of proteins, including polyamino acids, exists chiefly to
orientate amino acid side chains in such a way as to facilitate
molecular interactions, such as those of antibody and antigen. A
peptide mimetic is thus designed to permit molecular interactions
similar to the natural molecule.
[0081] Some successful applications of the peptide mimetic concept
have focused on mimetics of .beta.-turns within proteins, which are
known to be highly antigenic. Likely .beta.-turn structure within a
polypeptide can be predicted by computer-based algorithms, as
discussed herein. Once the component amino acids of the turn are
determined, mimetics can be constructed to achieve a similar
spatial orientation of the essential elements of the amino acid
side chains.
[0082] The generation of further structural equivalents or mimetics
may be achieved by the techniques of modeling and chemical design
known to those of skill in the art. The art of receptor modeling is
now well known, and by such methods a chemical that binds to water
soluble polyamino acids can be designed and then synthesized. It
will be understood that all such sterically designed constructs
fall within the scope of the present invention.
[0083] In addition to the 20 "standard" amino acids provided
through the genetic code, modified or unusual amino acids are also
contemplated for use in the present invention. A table of
exemplary, but not limiting, modified or unusual amino acids is
provided herein below.
1TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acids Aad
2-Aminoadipic acid bAad 3-Aminoadipic acid bAla beta-alanine,
beta-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu
4-Aminobutyric acid, piperidinic acid Acp 6-Aminocaproic acid Ahe
2-Aminoheptanoic acid Aib 2-Aminoisobutyric acid bAib
3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu
2,4-Diaminobutyric acid Des Desmosine Dpm 2,2'-Diaminopimelic acid
Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn
N-Ethylasparagine Hyl Hydroxylysine aHyl allo-Hydroxylysine 3Hyp
3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine aIle
allo-Isoleucine MeGly N-Methylglycine, sarcosine MeIle
N-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline Nva
Norvaline Nle Norleucine Orn Orinthine
[0084] Toxicity studies, pharmacokinetics and tissue distribution
of DTPA-paclitaxel have shown that in mice the LD.sub.50 (50%
lethal dose) of DPTA-paclitaxel observed with a single dose
intravenous (iv) injection is about 110 mg/kg body weight. Direct
comparison with paclitaxel is difficult to make because of the
dose-volume constraints imposed by limited solubility of paclitaxel
and vehicle toxicity associated with iv administration. However, in
light of the present disclosure, one skilled in the art of
chemotherapy would determine the effective and maximum tolerated
doses (MTD) in a clinical study for use in human subjects.
[0085] In certain embodiments of the invention, a stent coated with
the polymer-paclitaxel conjugates may be used to prevent
restenosis, the closure of arteries following balloon angioplasty.
Recent results in clinical trials using balloon-expandable stents
in coronary angioplasty have shown a significant benefit in patency
and the reduction of restenosis compared to standard balloon
angioplasty (Serruys et al., 1994). According to the
response-to-injury hypothesis, neointima formation is associated
with increased cell proliferation. Currently, popular opinion holds
that the critical process leading to vascular lesions in both
spontaneous and accelerated atherosclerosis is smooth muscle cell
(SMC) proliferation (Phillips-Hughes and Kandarpa, 1996). Since SMC
phenotypic proliferation after arterial injury mimics that of
neoplastic cells, it is possible that anti-cancer drugs may be
useful to prevent neointimal SMC accumulation. Stents coated with
polymer-linked anti-proliferative agents that are capable of
releasing these agents over a prolonged period of time with
sufficient concentration will thus prevent ingrowth of hyperplastic
intima and media into the lumen thereby reducing restenosis.
[0086] Because paclitaxel has been shown to suppress collagen
induced arthritis in a mouse model (Oliver et al. 1994), the
formulations of the present invention are also contemplated to be
useful in the treatment of autoimmune and/or inflammatory diseases
such as rheumatoid arthritis. Paclitaxel binding to tubulin shifts
the equilibrium to stable microtubule polymers and makes this drug
a strong inhibitor of eukaryotic cell replication by blocking cells
in the late G2 mitotic stage. Several mechanisms may be involved in
arthritis suppression by paclitaxel. For example, paclitaxel's
phase specific cytotoxic effects may affect rapidly proliferating
inflammatory cells, and furthermore paclitaxel inhibits cell
mitosis, migration, chemotaxis, intracellular transport and
neutrophil H.sub.2O.sub.2 production. In addition, paclitaxel may
have antiangiogenic activity by blocking coordinated endothelial
cell migration (Oliver et al. 1994). Therefore, the water soluble
polyamino acids conjugated paclitaxel of the present invention are
contemplated to be useful in the treatment of rheumatoid arthritis.
The polymer conjugated formulation disclosed herein would also
offer the advantages of controlled release of the drug and greater
solubility. It is also an aspect of the treatment of arthritis that
the formulations may be injected or implanted directly into the
affected joint areas.
[0087] The pharmaceutical preparations of paclitaxel or docetaxel
suitable for injectable use include sterile aqueous solutions or
dispersions and sterile powders for the preparation of sterile
injectable solutions or dispersions. In all cases the form must be
sterile and must be fluid for injection. It must be stable under
the conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms, such as
bacteria and fungi. The carrier may be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The prevention of the action of microorganisms can be brought
about by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. In many cases, it will be preferable to include isotonic
agents, for example, sugars or sodium chloride.
[0088] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0089] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents and isotonic agents and the
like. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0090] The phrase "pharmaceutically acceptable" also refers to
molecular entities and compositions that do not produce an allergic
or similar untoward reaction when administered to an animal or a
human.
[0091] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous and intraperitoneal administration. In
this connection, sterile aqueous media which can be employed will
be known to those of skill in the art in light of the present
disclosure.
[0092] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE 1
Poly-glutamic Acid-Paclitaxel (PG-TXL)
[0093] The present example concerns a first study involving the
conjugation of paclitaxel to a water-soluble polymer, poly
(l-glutamic acid) (PG) and the efficacy of the preparation against
a variety of tumors in mice and rats. The potential of
water-soluble polymers used as drug carriers is well established
(Kopecek, 1990; Maeda and Matsumura, 1989).
[0094] Synthesis of Poly-Gutamic Acid-Paclitaxel (PG-TXL)
[0095] PG was selected as a carrier for paclitaxel because it can
be readily degraded by lysosomal enzymes, is stable in plasma and
contains sufficient functional groups for drug attachment. Several
antitumor drugs, including Adriamycin (Van Heeswijk et al., 1985;
Hoes et al., 1985), cyclophosphamide (Hirano et al., 1979), Ara-C
(Kato et al., 1984) and melphalan (Morimoto et al., 1984) have been
conjugated to PG. However, poly-aspartic acid may be conjugated to
anti-tumor drugs using the reaction scheme described herein for
PG-TXL.
[0096] The reaction scheme is presented in FIG. 1B. Poly(l-glutamic
acid) (PG) sodium salt was obtained from Sigma (St. Louis, Mo.).
The polymer by viscosity had a molecular weight of 36,200, and
number-average molecular weight (M.sub.n) of 24,000 as determined
by low-angle laser light scattering (LALLS). Lot-specific
polydispersity (M.sub.w/M.sub.n) was 1.15 where M.sub.w is
weight-average molecular weight. PG sodium salt (MW 34 K, Sigma,
0.35 g) was first convened to PG in its proton form. The pH of the
aqueous PG sodium salt solution was adjusted to 2.0 using 0.2 M
HCl. The precipitate was collected, dialyzed against distilled
water, and lyophilized to yield 0.29 g PG.
[0097] To a solution of PG (75 mg, repeating unit FW 170, 0.44
mmol) in dry N,N-dimethylformamide (DMF) (1.5 mL) was added 22 mg
paclitaxel (0.026 mmol, molar ratio PG/paclitaxel=17), 15 mg
dicyclohexylcarbodiimid- e (DCC) (0.073 mmol) and trace amount of
dimethylaminopyridine (DMAP). Paclitaxel was supplied by Hande Tech
(Houston, Tex.), and the purity was 99% and higher as confirmed by
HPLC assay.
[0098] The reaction was allowed to proceed at room temperature for
12-18 h. Thin layer chromatography (TLC, silica) showed complete
conversion of paclitaxel (Rf=0.55) to polymer conjugate (Rf=0,
CHCl.sub.3/MeOH=10:1). To stop the reaction, the mixture was poured
into chloroform. The resulting precipitate was collected and dried
in vacuum to yield 70 mg polymer-drug conjugate. By changing the
weight ratio of paclitaxel to PG in the starting materials,
polymeric conjugates of various paclitaxel concentrations can be
synthesized.
[0099] The sodium salt of PG-TXL conjugate was obtained by
dissolving the product in 1.0 M NaHCO.sub.3. The aqueous solution
of PG-TXL was dialyzed against distilled water (MWCO 10,000) to
remove low molecular weight contaminants and excess NaHCO.sub.3
salt. Lyophilization of the dialysate yielded 98 mg of product as a
white powder. The paclitaxel content in this polymeric conjugate as
determined by UV was 20-22% (w/w). Yield: 98% (conversion to
polymer bound paclitaxel, UV). Solubility in water>20 mg
paclitaxel/ml. A similar method can be used to synthesize PG-TXL
with higher paclitaxel content (up to 35%) by simply increasing the
ratio of paclitaxel to PG used.
[0100] Characterization of Poly-Glutamic Acid-Paclitaxel
(PG-TXL)
[0101] Ultraviolet spectra were obtained on a Beckman DU-70
spectrophotometer, using the same concentration of PG aqueous
solution as reference. PG-TXL showed characteristic paclitaxel
absorption with .lambda..sub.max shifts from 228 to 230 nm. The
concentration of paclitaxel in PG-TXL conjugate was estimated based
on standard curve generated with known concentrations of paclitaxel
in methanol at absorption of 228 nm, assuming that the polymer
conjugate in water at 230 nm and the free drug in methanol at 228
nm have the same molar extinction and both follow Lambert Beer's
law.
[0102] .sup.1H-NMR spectra were recorded with GE model GN 500 (500
MHz) spectrometer in D.sub.2O. Both the PG moieties and the
paclitaxel moieties were discernible. The couplings of polymer
conjugated paclitaxel are too poorly resolved to be measured with
sufficient accuracy. Resonances at 7.75 to 7.36 ppm are
attributable to aromatic components of paclitaxel resonances at
6.38 ppm (C.sub.120-H), 5.97 ppm (C.sub.13-H), 5.63 ppm
(C.sub.2'-H, d), 5.55-5.36 ppm (C.sub.3'-H and C.sub.2-H, m), 5.10
ppm (C.sub.5-H), 4.39 ppm (C.sub.7-H), 4.10 (C.sub.20-H), 1.97 ppm
(OCOCH.sub.3), and 1.18-1.20 ppm (C CH.sub.3) are tentatively
assigned to aliphatic components of paclitaxel. Other resonances
were obscured by the resonances of PG. PG resonances at 4.27 ppm
(H-.alpha.), 2.21 ppm (H-.gamma.), and 2.04 ppm (H-.beta.) are in
accordance with pure PG spectrum. Although a peak at 5.63 ppm could
be tentatively assigned to the C-2' proton of the C-2' ester, the
C-2' proton of unsubstituted paclitaxel at 4.78 ppm was also
present, suggesting that the resulting conjugate may contain
paclitaxel substitutions at both the C-2' and C-7 positions. A 100
mg/ml solution of the conjugate produces a clear, viscous, yet
flowable liquid. This procedure consistently produces PG-TXL
conjugate containing 20% of paclitaxel by weight, i.e.,
approximately 7 paclitaxel molecules are bound to each polymer
chain.
[0103] Gel Permeation Chromatography Studies of Poly-Glutamic
Acid-Paclitaxel (PG-TXL)
[0104] The relative molecular weight of PG-TXL was characterized by
gel permeation chromatography (GPC). The GPC system consisted of
two LDC model III pumps coupled with LDC gradient master, a PL gel
GPC column, and a Waters 990 photodiode array detector. The elutant
(DMF) was run at 1.0 ml/min with ultraviolet (UV) detection set at
270 nm. For PG-TXL sodium salt, a TSK-gel column suitable for
analysis of water-soluble polymer was used, and the system was
eluted with 0.2 mM PBS (pH 6.8) at 1.0 ml/min. Conjugation of
paclitaxel to PG resulted in an increase in the molecular weight of
PG-TXL, as indicated by the shift of retention time from 6.4 min
for PG to 5.0 min for PG-TXL conjugate. The crude product contained
small molecular weight contaminants (retention time 8.0 to 10.0
min, and 11.3 min), which can be effectively removed by convening
PG-TXL to its sodium salt, followed by dialysis.
[0105] Hydrolytic Degradation of a Poly-Glutamic Acid-Paclitaxel
(PG-TXL) Conjugate
[0106] To gain insight on the release kinetics of paclitaxel and
related molecular species from PG-TXL, the hydrolytic stability of
PG-TXL was tested in PBS at various pH. High performance liquid
chromatography (HPLC) revealed that incubation of PG-TXL in PBS
solutions produced paclitaxel and several other species including
one that is more hydrophobic than paclitaxel (metabolite 1). The
fact that these species all were derived from paclitaxel was
confirmed through similar degradation studies using
PG-[.sup.3H]TXL. Based on its retention time on HPLC, metabolite-l
is probably 7-epipaclitaxel, a biologically active isomer of
paclitaxel. In fact, the amount of metabolite 1 recovered in PBS
surpassed that of paclitaxel after 5 days and 1 day of incubation
at pH 7.4 and pH 9.5 respectively (FIG. 6). At pH 5.5 and pH 7.4,
the release profiles of metabolite 1 indicated pseudo-zero order
kinetics and displayed a delay time varying from 3 days (pH 5.5) to
7 h (pH 7.4), suggesting that metabolite-1 is a secondary product.
Apparently, PG-TXL is more stable in acidic solution than in basic
solution.
[0107] In Vivo Antitumor Activity
[0108] All animal work was carried out at the animal facility at
M.D. Anderson Cancer Center in accordance with institutional
guidelines. C3H/Kam mice were bred and maintained in a
pathogen-free facility in the Department of Experimental Radiation
Oncology.
[0109] The tumor growth delay induced by PG-TXL was measured in
mammary ovarian carcinoma (OCA-I) implanted in C3Hf/Kam mice. All
tumors were syngeneic to this strain. Solitary tumors were produced
in the muscle of the right thigh of female C3H/Kam mice (25-30 g)
by injecting 5.times.10.sup.5 murine ovarian carcinoma cells
(OCA-I), mammary carcinoma (MCa-4), hepatocarcinoma (HCa-I) or
fibrous sarcoma (FSa-II). In a parallel study, female Fischer 344
rats (125-150 g) were injected with 1.0.times.10.sup.5 viable
13762F tumor cells in 0.1 ml PBS. Treatments were initiated when
the tumors in mice had grown to 500 mm.sup.3 (10 mm in diameter),
or when the tumors in rats had grown to 2400 mm.sup.3 (mean
diameter 17 mm).
[0110] PG-TXL was disolved in saline (10 mg equivalent
paclitaxel/ml), and paclitaxel was dissolved in Cremophor EL(g
vehicle (6 mg/ml). Data are presented as mean .+-. standard
deviation of tumor volumes. In control studies, saline (0.6 ml),
Cremophor vehicle [50/50 Cremophor/ethanol diluted with saline
(1:4)], PG solution in saline, and paclitaxel plus PG were used.
The maximum tolerated dose (MTD) of PG-TXL and paclitaxel in normal
female C3Hf/Kam mice was estimated to be 160 mg/kg and 80 mg/kg
respectively. A single dose of PG-TXL in saline or paclitaxel in
Cremophor EL vehicle was given in doses varying from 40 to 160 mg
equiv. Paclitaxel/kg body weight. Tumor growth was determined daily
(FIGS. 7A, 7B, 7C, 7D and 7E) by measuring three orthogonal tumor
diameters. Tumor volume was calculated according to formula
(A.times.B.times.C)/2. Absolute growth delay (AGD) in mice is
defined as the time in days for tumors treated with various drugs
to grow from 500 to 2,000 mm.sup.3 in mice minus the time in days
for tumors treated with saline control to grow from 500 to 2,000
mm.sup.3. When the tumor size reached 2000 mm.sup.3, the tumor
growth delay was calculated; the mice were sacrificed when tumors
were approximately 2500 mm.sup.3. The PG-TXL group were (n=6 and
7), other each group were (n=5). Table 2 summarizes acute toxicity
of PG paclitaxel in rats in comparison with paclitaxel/Cremophor.
Table 3 summarizes the data concerning the effect of PG-TXL against
MCa-4, FSa-II and HCa-I tumors in mice. The data are also
summarized in FIG. 7A-FIG. 7E.
2TABLE 2 Acute Toxocity of PG-TXL in Fischer Rats* # of Body Time
of Full Dose Toxic Weight Time at Nadir Recovery Group (mg/kg)
Death Loss in % (days) (days) PG-TXL.sup.a 60 1/4 15.7 7 14
PG-TXL.sup.a 40 0/4 11.1 6 11 Paclitaxel.sup.b 60 1/4 16.7 6 15
Paclitaxel.sup.b 40 0/3 17.9 6 16 Paclitaxel.sup.b 20 0/5 17.0 5
N/A *Drugs were administered intravenously into 13762F
tumor-bearing Fischer rats (female, 130 g) in a single injection.
.sup.aPG-TXL solution was prepared by dissolving the conjugate in
saline (8 mg equiv. paclitaxel/ml). The injected volume at 60 mg/kg
was 0.975 ml per rat. .sup.bPaclitaxel Cremophor solution was
prepared by dissolving paclitaxel in a 1:1 mixture of ethyl alcohol
and Cremophor (30 mg/ml). This stock solution was further diluted
with saline (1:4) before injection. The final concentration of
paclitaxel in the solution was 6 mg/ml. The injected volume at 60
mg/kg was 1.3 ml per rat. .sup.cPG solution was prepared by
dissolving the polymer in saline (22 mg/ml). The injected dose was
0.3 g/kg (1.8 ml per rat), which was equivalent to paclitaxel dose
of 60 mg/kg. .sup.dCremophor vehicle was prepared by diluting a
mixture of ethyl alcohol and Cremophor (1:1) with saline (1:4).
[0111]
3TABLE 3 The Antitumor Effect of PG-TXL Against Different Types of
In vivo Murine Tumors Time to Grow.sup.bb Tumor Drug.sup.a 500-2000
mm.sup.3 AGD.sup.c t-test.sup.d MCa-4 Saline 4.8 .+-. 0.8 (5) -- --
PG (0.6 g/kg) 9.3 .+-. 1.1 (4) 4.5 0.0114 Cremophor Vehicle 6.1
.+-. 0.7 (5) 1.3 0.265 PG-TXL (40 mg/kg) 8.6 .+-. 1.2 (4) 3.8 0.026
PG-TXL (60 mg/kg) 14.2 .+-. 1.1 (5) 9.4 0.0001 PG-TXL (120 mg/kg)
44.4 .+-. 2.9 (5) 39.6 <0.0001 Paclitaxel (40 mg/kg) 9.0 .+-.
0.6 (4) 4.2 0.0044 Paclitaxel (60 mg/kg) 9.3 .+-. 0.3 (5) 4.5
0.0006 FSa-II Saline 1.9 .+-. 0.1 (5) -- -- PG (0.8 g/kg) 2.8 .+-.
0.2 (6) 0.9 0.0043 Cremophor Vehicle 2.2 .+-. 0.2 (6) 0.3 0.122
PG-TXL (80 mg/kg) 3.8 .+-. 0.4 (6) 1.9 0.0016 PG-TXL (160 mg/kg)
5.1 .+-. 0.3 (13) 3.2 <0.0001 Paclitaxel (80 mg/kg) 4.2 .+-. 0.3
(6) 2.3 0.0002 PG + Paclitaxel 3.0 .+-. 0.2 (6) 1.1 0.0008 HCa-I
Saline 7.3 .+-. 0.3 (5) -- -- PG (0.8 g/kg) 7.7 .+-. 0.4 (4) 0.4
0.417 Cremophor Vehicle 6.8 .+-. 0.8 (5) -0.5 0.539 PG-TXL (40
mg/kg) 8.2 .+-. 0.7 (5) 0.9 0.218 PG-TXL (80 mg/kg) 8.6 .+-. 0.2
(5) 1.3 0.0053 PG-TXL (160 mg/kg) 11.0 .+-. 0.8 (4) 3.7 0.0023
Paclitaxel (80 mg/kg) 6.4 .+-. 0.5 (5) -0.9 0.138 PG + Paclitaxel
6.7 .+-. 0.4 (5) -0.6 0.294 .sup.aMice bearing 500 mm.sup.3 tumors
in the right leg were treated with various doses of PG-TXL (40-160
mg equiv. paclitaxel/kg) in saline of paclitaxel in Cremophor
vehicle i.v. in a single injection. Control animals were treated
with saline (0.6 ml), Cremophor vehicle (0.5 ml), PG solution in
saline, or PG g/kg plus paclitaxel (80 mg/kg). .sup.bTumor growth
was determined by daily measurement of three orhtogonal diameters
with calipers and the volume was calculated as (A .times. B .times.
C)/2. Shown in brackets are the number of mice used in each group.
The time in days to grow from 500 mm.sup.3 to 2000 mm.sup.3 are
presented mean .+-. standard deviation. .sup.cAbsolute growth delay
(AGD) defined as the time in days for tumors treated with various
drugs to grow from 500 to 2000 mm.sup.3 minus the time in days for
tumors treated with saline control to grow from 500 to 2000
mm.sup.3. .sup.dThe time in days to grow from 500 to 2000 mm.sup.3
were compared for treatment groups and saline group using Student's
t-Test. P-values are two-sided and were taken to be significant
when less that to equal 0.05.
[0112] Two important findings emerged from these studies. First,
like paclitaxel, there is an intertumor variability of the
antitumor effect of water-soluble PG-TXL. PG-TXL is most effective
against MCa-4 and OCA-1 tumors. Second, PG-TXL is more effective
than paclitaxel on equivalent mg paclitaxel basis in the case of
MCa-4, HCa-I, and on OCA-1 tumors, and is remarkably potent at its
maximum tolerated dose (MTD).
[0113] In a parallel study, the antitumor activity of PG-TXL in
Fischer rats with the well established rat mammary adenocarcinoma
13762F was examined. Female Fischer 344 rats (125-150 g) were
injected with 1.0.times.10.sup.5 viable 13762F tumor cells in 0.1
ml PBS. Once tumors reached a mean volume of 2000 mm.sup.3 (mean
diameter, 1.6 cm), animals were treated using a similar protocol as
described above. Tumor growth was determined daily by measuring
three orthogonal tumor diameters. Tumor volume was calculated
according to the formula (A.times.B.times.C)/2. A single dose of
PG-TXL in saline or paclitaxel in a Cremophor EL.RTM. vehicle was
given in doses varying from 20 to 60 mg equivalent paclitaxel/kg
body weight. In control studies, saline, the Cremophor EL.RTM.
vehicle [50/50 Cremophor/ethanol diluted with saline (1:4)], PG
solution in saline and paclitaxel plus PG were used. Again,
complete tumor eradication at the MTD of PG-TXL (60 mg equivalent
paclitaxel/kg) was observed. PG-TXL given at a lower dose of 40 mg
equivalent paclitaxel/kg also resulted in complete tumor regression
(FIG. 7B). In contrast, the MTD of paclitaxel in Cremophor EL.RTM.
was less than 20 mg/kg. Paclitaxel at this dose caused a tumor
growth delay (Tumor growth delay is defined as the time in days for
tumors treated with the test drugs to grow from 2,000 mm.sup.3 to
10,000 mm.sup.3 minus the time in days for tumors treated with
saline control to grow from 2,000 mm.sup.3 to 10,000 mm.sup.3.) of
only 5 days, whereas the same equivalent paclitaxel dose of PG-TXL
resulted in a tumor growth delay of 23 days (FIG. 7B).
[0114] Studies of Nude Mice Injected with Human Breast Cancer and
Treated with PG-TXL
[0115] Nude mice were injected with 2.times.10.sup.6 MDA-435-Lung2
cells (a variant of the MDA-MB-435 human breast cancer cell line)
into the mammary fatpad. When the tumors reached 5 mm mean
diameter, (27 days after tumor injection), mice were treated with
an i.v. injection of PG-TXL or the various controls (see Table 4).
Tumor measurements were taken weekly. Tumors that reached 1.5 cm
were removed surgically. All mice were killed at 120 days, and
remaining tumors removed and weighed. Mice were examined for
metastases, and lungs processed for histology, with single sections
of the organs scored for the presence of micrometastases.
4TABLE 4 No. Tumor Mean tumor tumors Lung Treatment take.sup.a wt
(g).sup.b regressed.sup.c metastases.sup.d PBS 5/6 1.3 .+-. 0.24 --
4/5 (80%) Cremophor 9/9 1.26 .+-. 0.67 -- 4/8 (50%) PGA 10/10 1.13
.+-. 0.7 -- 4/7 (57%) Taxol .TM./Cremophor 10/10 1.31 .+-. 0.69 --
3/7 (42%) 60 mg/kg PG-TXL 60 mg/kg 10/10 1.23 .+-. 0.38 2/10 5/8
(62.5%) PG-TXL 120 mg/kg 9/10 0.925 .+-. 0.12 4/8 1/4 (25%)
.sup.aNumber of mice with 5 mm tumors at time of therapy/number of
mice injected .sup.bMean weight of tumors removed at time of
autopsy .sup.cNumber of tumors that had regressed at time of
autopsy .sup.dNumber of mice with lung metastases (either
macroscopic or found in histology preparations)/number of mice with
tumors. Some discrepancies between tumor take and number mice with
tumors in this column due to sacrifice or deaths of animals for
non-related reasons, e.g., developing Staphylococcus abscesses. One
mouse in PG-TXL 120 mg group was killed due to extreme weight loss
after treatment; otherwise there were no obvious therapy related
deaths. Nude mice couldn't tolerate 160 mg/kg #equivalent of
PG-TXL.
[0116] From the results of the study in which a single bolus of
PG-conjugated paclitaxel (PG-TXL) was given, at a drug equivalent
of 120 mg/kg paclitaxel, it is apparent that the MDA-435 cancer
cell line responds to the drug and that this formulation of the
drug is much better tolerated than when Cremophor is the
vehicle.
[0117] In the breast cancer study using MDA-MB-435, only the higher
dose of PG-TXL inhibited the growth rate of the mammary fatpad
tumors. From the growth curve it was apparent that tumor growth
resumed approximately 30 days after the single dose of conjugate.
However, the growth curve does not reveal that in the PG-TXL 120
mg/kg group there were a number of tumor regressions. As shown in
Table 3, the incidence of lung metastasis in the mice with residual
tumors was also reduced. While the numbers of mice in the study are
small, they do suggest that the therapy was effective in reducing
both local tumor growth and incidence of metastasis.
[0118] In this study design it is not possible to distinguish
whether a lower incidence of metastasis is due to a reduction of
tumor mass of the primary site, or due to a direct effect on any
micrometastases that may have already been established at the time
of therapy.
[0119] In Vivo Therapy of Human Breast Cancer Using Multiple
Injections of PG-TXL
[0120] To test the effect of multiple injections of PG-TXL, nude
mice were injected with 2.times.10.sup.6; MDA-435-Lung 2 cells (a
variant of the MDA-MB-435 human breast cancer cell line) into the
mammary fatpad. When the tumors reached 5 mm mean diameter, the
treatments were started, and repeated at 14 day intervals (day 24,
38, 52) for a total of three injections. Tumor measurements were
taken weekly. The mice were killed on day 105 after tumor cell
injection, and the tumor weights and incidence of metastasis
recorded. The lungs were processed for histology, and single
sections scored for the presence of micrometastases. The results
are shown in Table 5.
5TABLE 5 Tumors Treatment Tumor take.sup.a Mean weight (g).sup.b
regressed.sup.c Metastasis.sup.d None 4/5 1.83 .+-. 0.15 -- 4/4
(100%) PG-control 6/10 1.7 .+-. 0.11 -- 5/6 (83%) PG-TXL/60 7/10
1.36 .+-. 0.28 -- 6/7 (86%) mg PG-TXL/120 8/10 0.97 .+-. 0.22 p =
2/8 2/6 (33%) mg 0.011e Legend: .sup.aNumber of mice with 5 mm
tumors at the time of therapy/number of mice injected .sup.bMean
weight of tumors (.+-.SEM) .sup.cNumber of tumors that had
regressed at the time of autopsy .sup.dNumber of mice with lung
metastases, either macroscopic or microscopic/number of mice with
tumors .sup.ep value from unpaired t test comparing tumor weight of
treated mice with the control PG group.
[0121] In Vivo Therapy of Human Ovarian Cancer Using PG-TXL
Conjugate
[0122] Nude mice were injected i.p. with the human ovarian cancer
cell line, SKOV3ip1. Five days after tumor injection, the mice were
injected i.v. with the PG-paclitaxel (PG-TXL), at concentrations
equivalent to 120 mg/kg or 160 mg/kg of paclitaxel. Initially the
plan was to repeat these injections at 7-day intervals, but a
single injection of the 160 mg/kg dose killed 5 of the 10 mice.
Only the 120 mg/kg group received three injections. The study was
terminated on day 98, and any surviving mice killed. The results
are shown in FIG. 14, and in Table 6.
[0123] The median survival values for the groups at present are:
untreated=47 days, PC-control=43 days, PG-TXL (120 mg/kg)=83 days,
PG-TXL (160 mg/kg)=83 days [note that this does not include the
mice that died from the initial toxicity of the drug].
6TABLE 6 Median Treatment Tumor take.sup.a survival (range).sup.b
Ascitcs.sup.c Mean vol (ml).sup.d None 10/10 56 (38-98) 8/10 2.2
.+-. 1.6 PG-control 8/9 45 (39-98) 8/8 2.2 .+-. 1.6 PG-120 7/8 82
(59-98) 3/7 2.7 .+-. 1.4 PG-160 .sup. 3/5.sup.c 84 (34.sup.f-98)
0/3 -- Legends: .sup.aIncidence of tumor/number of mice injected
.sup.bmedian survival time in days .sup.cincidence of
ascites/number of mice with tumor .sup.dmean volume (and s.d.) of
ascites .sup.ethese mice only received a single dose of
PG-paclitaxel, 160 mg/kg, and does not include the mice that dies
within 5 days of the treatment .sup.fthe mouse that was killed on
day 34 had minimal tumor burden, but was paraplegic (possible
toxicity?).
[0124] The PG-TXL 120 mg/kg significantly extended the survival of
the mice with intraperitoneal SKOV3ipl, (a human ovarian cancer
cell line which overexpresses HER2/neu), compared with mice
injected with PG alone. Multiple doses and/or increasing the dose
of conjugate may significantly reduce the tumor incidence in
addition to extending survival.
[0125] In the nude mice studies above, the growth curves show that
although breast cancer growth is checked by paclitaxel, especially
with the higher dose conjugated with PG, tumor size continues to
increase about a month after the therapy. A second (or third) round
of therapy may have caused the tumor growth to plateau, or give
more tumor regressions. The growth curves do not include the tumors
that regressed--as shown in Table 4, the tumors shrank/disappeared
in 50% of the mice treated with the highest dose of PG-TXL, and of
the 4 animals with progressively growing tumors at the end of the
study, only one had micrometastases in the lungs. So the treatment
that reduced growth of the primary tumors also reduced the
incidence of metastasis. The incidence of metastasis in all other
therapy groups, including the control groups of Cremophor and PG
were lower than the PBS control, therefore it is probably not valid
to state that the reduction in incidence of metastasis in the
Taxol.TM./Cremophor group is a significant finding.
EXAMPLE 2
DTPA-Paclitaxel
[0126] Synthesis of DTPA-Paclitaxel:
[0127] To a solution of paclitaxel (100 mg, 0.1 17 mmol) in dry DMF
(2.2 ml) was added diethylenetriaminepentaacetic acid anhydride
(DTPA A) (210 mg, 0.585 mmol) at 0.degree. C. The reaction mixture
was stirred at 4.degree. C. overnight. The suspension was filtered
(0.2 .mu.m Millipore filter) to remove unreacted DTPA anhydride.
The filtrate was poured into distilled water, stirred at 4.degree.
C. for 20 min, and the precipitate collected. The crude product was
purified by preparative TLC over C.sub.18 silica gel plates and
developed in acetonitrile/water (1:1). Paclitaxel had an R.sub.f
value of 0.34. The band above the paclitaxel with an R.sub.f value
of 0.65 to 0.75 was removed by scraping and eluted with an
acetonitrile/water (1:1) mixture, and the solvent was removed to
give 15 mg of DTPA-paclitaxel as product (yield 10.4%): mp:
>226.degree. C. dec. The UV spectrum (sodium salt in water)
showed maximal absorption at 228 nm which is also characteristic
for paclitaxel. Mass spectrum: (FAB) m/e 1229 (M+H).sup.+, 1251
(M+Na), 1267 (M+K). In the .sup.1H NMR spectrum (DMSO-d.sub.6) the
resonance of NCH.sub.2CH.sub.2N and CH.sub.2COOH of DTPA appeared
as a complex series of signals at .delta. 2.71-2.96 ppm, and as a
multiplet at .delta. 3.42 ppm, respectively. The resonance of C7-H
at 4.10 ppm in paclitaxel shifted to 5.51 ppm, suggesting
esterification at the 7-position. The rest of the spectrum was
consistent with the structure of paclitaxel.
[0128] The sodium salt of DTPA-paclitaxel was also obtained by
adding a solution of DTPA-paclitaxel in ethanol into an equivalent
amount of 0.05 M NaHCO.sub.3, followed by lyophilizing to yield a
water-soluble solid powder (solubility>20 mg equivalent
paclitaxel/ml).
[0129] Hydrolytic Stability of DTPA-Paclitaxel:
[0130] The hydrolytic stability of DTPA-paclitaxel was studied
under accelerated conditions. Briefly, 1 mg of DTPA-paclitaxel was
dissolved in 1 ml 0.5 M NaHCO.sub.3 aqueous solution (pH 9.3) and
analyzed by HPLC. The HPLC system consisted of a Waters
150.times.3.9 (i.d.) mm Nova-Pak column filled with C18 4 .mu.m
silica gel, a Perkin-Elmer isocratic LC pump, a PE Nelson 900
series interface, a Spectra-Physics UV/Vis detector and a data
station. The eluant (acetonitrile/methanol/0.02M ammonium
acetate=4:1:5) was run at 1.0 ml/min with UV detection at 228 nm.
The retention times of DTPA-paclitaxel and paclitaxel were 1.38 and
8.83 min, respectively. Peak areas were quantitated and compared
with standard curves to determine the DTPA-paclitaxel and
paclitaxel concentrations. The estimated half-life of
DTPA-paclitaxel in 0.5 M NaHCO.sub.3 solution is about 16 days at
room temperature.
[0131] Effects of DTPA-Paclitaxel on the Growth of B16 Mouse
Melanoma Cells In Vitro:
[0132] Cells were seeded in 24-well plates at a concentration of
2.5.times.10.sup.4 cells/ml and grown in a 50:50 Dulbecco's
modified minimal essential medium (DEM) and F12 medium containing
10% bovine calf serum at 37.degree. C. for 24 h in a 97% humidified
atmosphere of 5.5% CO.sub.2. The medium was then replaced with
fresh medium containing paclitaxel or DTPA-paclitaxel in
concentration ranging from 5.times.10.sup.-9 M to
75.times.10.sup.-9 M. After 40 h, the cells were released by
trypsinization and counted in a Coulter counter. The final
concentrations of DMSO (used to dissolve paclitaxel) and 0.05 M
sodium bicarbonate solution (used to dissolve DTPA-paclitaxel) in
the cell medium were less than 0.01%. This amount of solvent did
not have any effect on cell growth as determined by control
studies.
[0133] The effects of DTPA-paclitaxel on the growth of B16 melanoma
cells are presented in FIG. 2. After a 40-h incubation with various
concentrations, DTPA-paclitaxel and paclitaxel were compared as to
cytotoxicity. The IC.sub.50 for paclitaxel and DTPA-paclitaxel are
15 nM and 7.5 nM, respectively.
[0134] Antitumor Effect on Mammary Carcinoma (MCa-4) Tumor
Model:
[0135] Female C3Hf/Kam mice were inoculated with mammary carcinoma
(MCa-4) in the muscles of the right thigh (5.times.10.sup.5
cells/mouse). When the tumors had grown to 8 mm (approx. 2 wks), a
single dose of paclitaxel or DTPA-paclitaxel was given at 10, 20
and 40 mg equivalent paclitaxel/kg body weight. In control studies,
saline and absolute alcohol/Cremophor 50/50 diluted with saline
(1:4) were used. Tumor growth was determined daily, by measuring
three orthogonal tumor diameters. When the tumor size reached 12 mm
in diameter, the tumor growth delay was calculated. The mice were
sacrificed when tumors were approximately 15 mm.
[0136] The tumor growth curve is shown in FIG. 3. Compared to
controls, both paclitaxel and DTPA-paclitaxel showed antitumor
effect at a dose of 40 mg/kg. The data were also analyzed to
determine the mean number of days for the tumor to reach 12 mm in
diameter. Statistical analysis showed that DTPA-paclitaxel delayed
tumor growth significantly compared to the saline treated control
at a dose of 40 mg/kg (p<0.01). The mean time for the tumor to
reach 12 mm in diameter was 12.1 days for DTPA-paclitaxel compared
to 9.4 days for paclitaxel (FIG. 4).
[0137] Radiolabeling of DTPA-Paclitaxel with .sup.111In
[0138] Into a 2-ml V-vial were added successively 40 .mu.l 0.6 M
sodium acetate (pH 5.3) buffer, 40 .mu.l 0.06 M sodium citrate
buffer (pH 5.5), 20 .mu.l DTPA-paclitaxel solution in ethanol (2%
w/v) and 20 .mu.l .sup.111InCl.sub.3 solution (1.0 mCi) in sodium
acetate buffer (pH 5.5). After an incubation period of 30 min at
room temperature, the labeled .sup.111In-DTPA-paclitaxel was
purified by passing the mixture through a C18 Sep-Pac cartridge
using saline and subsequently ethanol as the mobile phase. Free
.sup.111n-DTPA (<3%) was removed by saline, while
.sup.111In-DTPA-paclitaxel was collected in the ethanol wash. The
ethanol was evaporated under nitrogen gas and the labeled product
was reconstituted in saline. Radiochemical yield: 84%.
[0139] Analysis of .sup.111In-DTPA-Paclitaxel:
[0140] HPLC was used to analyze the reaction mixture and purity of
.sup.111In-DTPA-paclitaxel. The system consisted of a LDC binary
pump, a 100.times.8.0 mm (i.d.) Waters column filled with ODS 5
.mu.m silica gel. The column was eluted at a flow rate of 1 ml/min
with a gradient mixture of water and methanol (gradient from 0% to
85% methanol over 15 min). The gradient system was monitored with a
NaI crystal detector and a Spectra-Physics UV/Vis detector. As
evidenced by HPLC analysis, purification by Sep-Pak cartridge
removed most of the .sup.111In-DTPA, which had a retention time of
2.7 min. The .sup.111In-DTPA was probably derived from traces of
DTPA contaminant in the DTPA-paclitaxel. A radio-chromatogram of
.sup.111In-DTPA-paclitaxel correlated with its UV chromatogram,
indicating that the peak at 12.3 min was indeed the target
compound. Under the same chromatographic conditions, paclitaxel had
a retention time of 17.1 min. The radiochemical purity of the final
preparation was 90% as determined by HPLC analysis.
[0141] Whole-Body Scintigraphy:
[0142] Female C3Hf/Kam mice were inoculated with mammary carcinoma
(MCa-4) in the muscles of the right thigh (5.times.10.sup.5 cells).
When the tumors had grown to 12 mm in diameter, the mice were
divided into two groups. In group I, the mice were anesthetized by
intraperitoneal injection of sodium pentobarbital, followed by
.sup.111In-DTPA-paclitaxel (100-200 mCi) via tail vein. A
.gamma.-camera equipped with a medium energy collimator was
positioned over the mice (3 per group). A series of 5 min
acquisitions were collected at 5, 30, 60, 120, 240 min and 24 h
after injection. In group II, the same procedures were followed
except that the mice were injected with .sup.111In-DTPA as a
control. FIG. 5 shows gamma-scintigraphs of animals injected with
.sup.111In-DTPA and .sup.111In-DTPA-paclitaxel. .sup.111In-DTPA was
characterized by rapid clearance from the plasma, rapid and high
excretion in the urine with minimal retention in the kidney and
negligible retention in the tumor, the liver, the intestine and
other organs or body parts. In contrast, .sup.111In-DTPA-paclitaxel
exhibited a pharmacological profile resembling that of paclitaxel
(Eiseman et al., 1994). Radioactivity in the brain was negligible.
Liver and kidney had the greatest tissue:plasma ratios.
Hepatobiliary excretion of radiolabeled DTPA-paclitaxel or its
metabolites was one of the major routes for the clearance of the
drug from the blood. Unlike paclitaxel, a significant amount of
.sup.111In-DTPA-paclitaxel was also excreted through kidney, which
only played a minor role in the clearance of paclitaxel. The tumor
had significant uptake of .sup.111In-DTPA-paclitaxel. These results
demonstrate that .sup.111In-DTPA-paclitaxel is able to detect
certain tumors and to quantify the uptake of
.sup.111In-DTPA-paclitaxel in the tumors, which in turn, may assist
in the selection of patients for the paclitaxel treatment. In
contrast, the smaller PG-TXL conjugate has a different distrubution
than DTPA-paclitaxel, and partly localizes in the liver and tumors
of test animals.
EXAMPLE 3
Polyethylene glycol-Paclitaxel
[0143] Synthesis of Polyethylene Glycol-Paclitaxel
(PEG-Paclitaxel)
[0144] The synthesis was accomplished in two steps. First
2'-succinyl-paclitaxel was prepared according to a reported
procedure (Deutsch et al., 1989). Paclitaxel (200 mg, 0.23 mmol)
and succinic anhydride (288 mg, 2.22 mmol) were allowed to react in
anhydrous pyridine (6 ml) at room temperature for 3 h. The pyridine
was then evaporated, and the residue was treated with water,
stirred for 20 min, and filtered. The precipitate was dissolved in
acetone, water was slowly added, and the fine crystals were
collected to yield 180 mg 2'-succinyl-paclitaxel. PEG-paclitaxel
was synthesized by an N-ethoxycarbonyl-2-ethoxy-1,2-dihydr-
oquinoline (EEDQ) mediated coupling reaction. To a solution of
2'-succinyl-paclitaxel (160 mg, 0.18 mmol) and
methoxypolyoxyethylene amine (PEG-NH.sub.2, MW 5000, 900 mg, 0.18
mmol) in methylene chloride was added EEDQ (180 mg, 0.72 mmol). The
reaction mixture was stirred at room temperature for 4 h. The crude
product was chromatographed on silica gel with ethyl acetate
followed by chloroform-methanol (10:1). This gave 350 mg of
product. .sup.1H NMR (CDCl.sub.3) .delta. 2.76 (m, succinic acid,
COCH.sub.2CH.sub.2CO.sub.2), .delta. 3.63 (PEG,
OCH.sub.2CH.sub.2O), .delta. 4.42 (C7-H) and .delta. 5.51 (C2'-H).
Maximal UV absorption was at 288 nm which is also characteristic
for paclitaxel. Attachment to PEG greatly improved the aqueous
solubility of paclitaxel (>20 mg equivalent paclitaxel/ml
water).
[0145] Hydrolytic Stability of PEG-Paclitaxel
[0146] PEG-Paclitaxel was dissolved in phosphate buffer (0.01M) at
various pHs at a concentration of 0.4 mM and the solutions were
allowed to incubate at 37.degree. C. with gentle shaking. At
selected time intervals, aliquots (200 .mu.l) were removed and
lyophilized. The resulting dry powders were redissolved in
methylene chloride for gel permeation chromatography (GPC
analysis). The GPC system consisted of a Perkin-Elmer PL gel mixed
bed column, a Perkin-Elmer isocratic LC pump, a PE Nelson 900
series interface, a Spectra-Physics UV/Vis detector and a data
station. The elutant (methylene chloride) was run at 1.0 ml/min
with the UV detector set at 228 nm. The retention times of
PEG-paclitaxel and paclitaxel were 6.1 and 8.2 min, respectively.
Peak areas were quantified and the percentage of PEG-paclitaxel
remaining and the percentage of paclitaxel released were
calculated. The half life of PEG-paclitaxel determined by linear
least-squares at pH 7.4 was 54 min. The half-life at pH 9.0 was 7.6
min. Release profiles of paclitaxel from PEG-paclitaxel at pH 7.4
is shown in FIG. 8.
[0147] Cytotoxicity Studies of PEG-Paclitaxel Using B16 Mouse
Melanoma Cells In Vitro
[0148] Following the procedure described in the cytotoxicity
studies with DTPA-paclitaxel, melanoma cells were seeded in 24-well
plates at a concentration of 2.5.times.10.sup.4 cells/ml and grown
in a 50:50 Dulbecco's modified minimal essential medium (DME) and
F12 medium containing 10% bovine calf serum at 37.degree. C. for 24
h in a 97% humidified atmosphere of 5.5% CO,. The medium was then
replaced with fresh medium containing paclitaxel or its derivatives
in concentrations ranging from 5.times.10.sup.-9 M to
75.times.10.sup.-9 M. After 40 h, the cells were released by
trypsinization and counted in a Coulter counter. The final
concentrations of DMSO (used to dissolve paclitaxel) and 0.05 M
sodium bicarbonate solution (used to dissolve PEG-paclitaxel) in
the cell medium were less than 0.01%. This amount of solvent did
not have any effect on cell growth as determined by control
studies. Furthermore, PEG in the concentration range used to
generate an equivalent paclitaxel concentration from
5.times.10.sup.-9 M to 75.times.10.sup.-9 M also did not effect
cell proliferation.
[0149] Antitumor Effect of PEG-Paclitaxel Against MCa-4 Tumor in
Mice
[0150] To evaluate the antitumor efficacy of PEG-paclitaxel against
solid breast tumors, MCa-4 cells (5.times.10.sup.5 cells) were
injected into the right thigh muscle of female C3Hf/Kam mice. As
described in Example 1 with the DTPA-paclitaxel, when the tumors
were grown to 8 mm (Approx. 2 wks), a single dose of paclitaxel or
PEG-paclitaxel was given at 10, 20 and at 40 mg equivalent
paclitaxel/kg body weight. Paclitaxel was initially dissolved in
absolute ethanol with an equal volume of Cremophor. This stock
solution was further diluted (1:4 by volume) with a sterile
physiological solution within 15 min of injection. PEG-paclitaxel
was dissolved in saline (6 mg equiv. paclitaxel/ml) and filtered
through a sterile filter (Millipore, 4.5 elm). Saline, paclitaxel
vehicle, absolute alcohol:Cremophor (1:1) diluted with saline (1:4)
and PEG solution in saline (600 mg/kg body weight) were used in
control studies. Tumor growth was determined daily, by measuring
three orthogonal tumor diameters. When the tumor size reached 12 mm
in diameter, the tumor growth delay was calculated.
[0151] The tumor growth curve is shown in FIG. 9. At a dose of 40
mg/kg, both PEG-paclitaxel and paclitaxel effectively delayed tumor
growth. Paclitaxel was more effective than PEG-paclitaxel, although
the difference was not statistically significant. Paclitaxel
treated tumors required 9.4 days to reach 12 mm in diameter whereas
PEG-paclitaxel-treated tumors required 8.5 days. Statistically,
these values were significant (.rho.>0.05) as compared to their
corresponding controls, which were 6.7 days for the paclitaxel
vehicle and 6.5 days for the saline solution of PEG (FIG. 4).
EXAMPLE 5
Poly(L-glutamic acid)-Paclitaxel (PG-TXL) and Paclitaxel
Pharmacological Properties
[0152] The objective of this study was to compare PG-TXL and
paclitaxel pharmacological properties in their ability to promote
in vitro assembly of tubulin, to inhibit cell growth against rat
mammary tumor cell line 13762F and human breast tumor cell lines,
to induce p53 protein, and to rescue a paclitaxel-dependent mutant
cell line. Paclitaxel's release from PG-TXL in vivo was measured to
determine if PG-TXL's mechanism of action can be attributed to free
pacitaxel.
[0153] Microtubule Polymerization Using Poly-Glutamic
Acid-Paclitaxel (PG-TXL) and Paclitaxel
[0154] To test whether intact PG-TXL has any intrinsic biological
activity in promoting tubulin polymerization, paclitaxel, PG-TXL,
and "aged" PG-TXL were compared for their relative ability to
promote in vitro assembly of purified bovine brain tubulin. The
tubulin assembly reaction was performed at 32.degree. C. in PEM
buffer (80 mM PIPES buffer, 1 mM EGTA, 1 mM MgCl.sub.2, pH 6.9) at
a tubulin (bovine brain, Cytoskeleton Inc., Boulder, Colo.)
concentration of 1 mg/ml (10 .mu.M) in the presence or absence of
drugs (1.0 .mu.M equivalent paclitaxel) and 1.0 mM guanosine
5'-triphosphate (GTP). "Aged" PG-TXL was obtained by placing PG-TXL
in PBS (pH 7.4) at 37.degree. C. for 3 days. Tubulin polymerization
was followed by measuring the absorbance of the solution at 340 nm
over time. Addition of 1 .mu.M paclitaxel to a solution of tubulin
in assembly buffer caused a clear increase in absorbance due to the
increase in light scattering resulting from the polymerization of
tubulin into microtubules. In contrast, a 10 .lambda.M paclitaxel
equivalent of PG-TXL had no effect on polymerization. A solution of
the conjugate that was "aged" for 3 days in PBS (pH 7.4) at
37.degree. C. exhibited enhanced activity although its ability to
promote tubulin polymerization was still markedly less than
paclitaxel (FIG. 10).
[0155] Effects of Poly-Glutamic Acid-Paclitaxel (PG-TXL) on the
Growth of Rat and Human Tumor Cell Lines In Vitro
[0156] To evaluate whether the superior antitumor activity of
PG-TXL observed in animals is due to increased cytotoxicity, PG-TXL
and paclitaxel were compared for their ability to inhibit cell
growth against the established rat mammary tumor cell line 13762F.
The effect of PG-TXL on cell growth was examined by a plating
efficiency assay. Rat 13762F cells were seeded (200 cells) into 60
mm dishes containing drug concentrations varying from 0 to 200 nM
in growth medium (cc modified minimum essential medium
[.alpha.-MEM] containing 5% fetal bovine serum, 50 U/ml of
penicillin, and 50 .mu.g/ml of streptomycin). After 6 days of
growth, the cells were stained with a 0.1% methylene blue solution
and colonies were counted. The drug concentration producing 50%
inhibition of colony formation (IC.sub.50) was then calculated. The
approximate IC.sub.50 values after 6 days of continuous exposure
were: paclitaxel (2 nM), PG-TXL (100 nM), "aged" (see below) PG-TXL
(50 nM). It is clear that PG-TXL is approximately 30-50 fold less
potent than paclitaxel itself. When PG-TXL was incubated in
phosphate buffered saline solution (PBS, pH 74) at 37.degree. C.
for 3 days to obtain an "aged" solution, only about 10% of
paclitaxel was released. Since the "aged" solution is more potent
than freshly dissolved PG-TXL, the in vitro degradation of PG-TXL
or release of active drug appears to be important for PG-TXL to
exert this biological activity. However, even after "aging," PG-TXL
is still 25 times less potent than paclitaxel.
[0157] In a similar study, the effect of PG-TXL on cell growth of
human breast cancer cell lines was examined by MTT assay after 3
days of continuous exposure. While PG-TXL was 8-and 30-fold more
potent than paclitaxel against MDA330 and MDA-MB453 cell lines,
PG-TXL was 2- and 3-fold less potent than paclitaxel against
MCF7/her-2 and MCF7 cell lines. These results suggest that PG-TXL
and paclitaxel have different activity against different cell
lines. PG-TXL may be a product with distinct pharmacological
properties different from that of paclitaxel.
[0158] The Ability of Poly-Glutamic Acid-Paclitaxel (PG-TXL) to
Support a Paclitaxel-Dependent Cell Line In Vitro
[0159] The inventors investigated the ability of PG-TXL to rescue a
paclitaxel-dependent mutant cell line. Tax 18, a CHO cell line
selected for resistance to paclitaxel, is a well characterized
mutant that has been found to require the continuous presence of
paclitaxel for cell division. In the absence of drug, a functional
mitotic spindle apparatus is unable to form (Cabral et al., 1983).
The mitosis phase of the cell cycle is prolonged with subsequent
failure to segregate chromosomes and to divide into daughter cells.
Nonetheless, the cells continue to progress through the cell cycle
and replicate their DNA resulting in the formation of large
polyploid cells that eventually die due to genomic instability
(Cabras and Barlow, 1991). Low concentrations of paclitaxel are
able to rescue the mutant phenotype by permitting microtubule
assembly and the formation of sufficient mitotic spindle fibers.
Thus, these cells provide a convenient bioassay for agents that
promote microtubule assembly. Growth of paclitaxel-dependent CHO
mutant Tax-18 cells was carried out on 24-well tissue culture
dishes. Approximately 100 cells were added to wells containing
growth medium and equivalent concentrations of paclitaxel varying
from 0 to 1.0 .mu.M. After 6 days of incubation at 37.degree. C.,
the medium was removed and the cells were stained with methylene
blue.
[0160] Little or no increase in cell number is seen in the absence
of drug, but concentrations of paclitaxel between 0.05-0.2 .mu.M
clearly support the growth of this cell line. Higher concentrations
of paclitaxel are presumably toxic to the cells because of
overstabilization of the microtubules as is observed for normal
cells. On the other hand, freshly prepared PG-TXL shows little
ability to rescue Tax-18 cell growth even at the highest
paclitaxel-equivalent concentration tested (1 .mu.M). When PG-TXL
was "aged" by incubating in PBS for 6 days at 37.degree. C., its
ability to support Tax-18 cell growth was partially restored. These
data indicate that PG-TXL does not promote microtubule assembly,
and that the in vitro biological activity of "aged" PG-TXL is a
contribution of paclitaxel released from poly-glutamic
acid-paclitaxel (PG-TXL).
[0161] The Release of [.sup.3H]paclitaxel from PG-[.sup.3H]TXL In
Vivo
[0162] To assess the pharmacokinetic and release characteristics of
paclitaxel in vivo, normal female C3Hf/Kam mice (25-30 g) were
injected with a dose of 20 mg equivalent [.sup.3H]paclitaxel or
PG-[.sup.3H]paclitaxel intravenously into the tail vein. Each mouse
received 6 .mu.Ci of radiolabeled drug. [.sup.3H]paclitaxel was
dissolved in Cremophor EL.RTM. vehicle whereas
PG-[.sup.3H]paclitaxel was dissolved in saline. Volume injected
into each mice was between 0.2 to 0.3 ml. At 0, 5, 15, 30 min, and
1, 2, 4, 8, 16, 24, 48 h postinjection, animals were sacrificed and
blood samples were collected (4-5 mice per time point). Total
radioactivity in plasma was measured by liquid scintillation
counting (Beckman Model LS 6500, Fullerton, Calif.) using 10 .mu.l
aliquots of plasma. Up to 200 .mu.l plasma was extracted with 3
volume of ethyl acetate according to Longnecker et al. (1987). The
extraction efficiency for paclitaxel was 80%. The samples were
centrifuged for 5 min at 2500 rpm, and the supernatant was
separated and brought to dryness. The dried extract was
reconstituted with 195 .mu.l of HPLC mobile phase, mixed with 5
.mu.l of cold paclitaxel (0.2 mg/ml), and 100 .mu.l was injected
onto the HPLC for determination of free paclitaxel radioactivity.
Pharmacokinetic parameters were analyzed by a noncompartmental
model using the WinNonlin software package. Each data point
generated was the mean value of 4-5 mice.
[0163] The clearance of both drugs from plasma is shown in FIG. 11.
While paclitaxel has an extremely short half life in plasma of mice
(t.sub.1/2, 29 min), the apparent half life of PG-TXL is prolonged
(t.sub.1/2, 317 min). Slower clearance of PG-TXL from the blood was
a design feature of the polymer-drug conjugate with the goal of
improving tumor uptake. Surprisingly, the rate of conversion of
PG-TXL to paclitaxel in plasma is slow with less than 0.1% of the
radioactivity from PG-[.sup.3H]TXL being recovered as
[.sup.3H]paclitaxel within 144 h after drug injection (FIG.
11).
[0164] In a separate study, mice bearing OCA-1 tumors were prepared
as described previously. When the tumor reached 500 mm.sup.3,
animals were injected with a dose of 20 mg equivalent paclitaxel/kg
of [.sup.3H]paclitaxel or PG-[.sup.3H]TXL into the tail vein.
Animals were killed at 2, 5, 9, 24, 48, and 144 h postinjection.
Tumors were removed, weighed, and homogenized with 3 volume of PBS
(w/v). Percent of injected dose per gram tissue is calculated based
on total radioactivity associated with the tumor, which was
determined by counting prepared tissue homogenate aliquots. An
aliquot of tissue homogenate was mixed with tissue solubilizer,
followed by addition of scintillation solvent, and counted for
total radioactivity. The counting efficiency was verified by the
method of standard addition. Alternatively, aliquots of tissue
homogenates were extracted with ethyl acetate and analyzed for free
paclitaxel by HPLC. The HPLC system consisted of a 150.times.3.9 mm
Nova-Pak column (Waters, Milford, MA), a liquid chromatography pump
(Waters model 510), a UV/Vis detector set at 228 nm (Waters model
486), a flow scintillation analyzer (Packard model 500TR, Downers
Grove, Ill.), and a Packard radiomatic software for data analysis.
The eluting solvent (methanol:watter=2:1) was run at 1.0 ml/min.
The uptake of total drugs in OCA-1 tumor was expressed as a
percentage of the administered dose per gram of tissue and the
association of radioactivity within OCA-1 tumor as free paclitaxel
was expressed as dpm per gram tissue.
[0165] Quantitative assessment of tumor uptake in C3Hf/Kam mice
showed that relatively high levels of radioactivity from
radiolabeled PG-TXL appear in tumor tissue shortly after injection
(FIG. 12A) as compared to radiolabeled paclitaxel. However, only
small amounts of radioactivity within tumor tissue are due to the
release of free paclitaxel (FIG. 12B). Data are presented in FIG.
12A and FIG. 12B as mean.+-.SD from 3 mice per time point. The
percent of radioactivity within tumor tissue due to paclitaxel does
not appreciably increase with time suggesting that PG-TXL is not
simply a prodrug for the gradual release of paclitaxel.
[0166] In contrast to paclitaxel, in vitro studies with PG-TXL
whether prepared as a fresh solution or even after "aging" in
buffer have clearly shown that the complex is not a potent
cytotoxic species. It neither strongly supports tubulin
polymerization nor the growth and survival of a
paclitaxel-dependent CHO cell line. Furthermore, data obtained from
pharmacokinetic studies indicate that both the extent and rate of
release of paclitaxel in plasma is very low (less than 0.1% in 144
h). While the uptake of PG-TXL material was some 5-fold greater
than that achieved by paclitaxel when using equivalent antitumor
doses, that material which gains entry into tissues exists in the
tissue mainly in form(s) which have been shown not to be free
paclitaxel.
EXAMPLE 6
Effect of Polymer Structure on Activity of Water soluble polyamino
Acid-Paclitaxel Conjugates
[0167] The present study evaluated whether antitumor activities of
polymer-paclitaxel conjugates were affected by the structure of
polyamino acids used for drug conjugation. Paclitaxel was coupled
to poly(l-glutamic acid), poly(d-glutamic acid), and
poly(l-aspartic acid) according to previously described procedures.
These polyamino acid-paclitaxel conjugates had similar paclitaxel
content, aqueous solubility, and molecular weight (30-40K). In
C3Hf/Kam mice bearing murine OCA-1 ovarian cancer (500 mm.sup.3 at
time of treatment), a single i.v. injection of poly(l-glutamic
acid)-paclitaxel at 80 mg equiv. paclitaxel/kg body weight produced
a tumor growth delay of 21 days vs. saline treated controls.
Poly(d-glutamic acid)-paclitaxel was as effective as
poly(l-glutamic acid)-paclitaxel. However, paclitaxel conjugated
with poly(l-aspartic acid) was completely inactive against OCA-1
tumor. In a separate study, the antitumor activity of
polymer-paclitaxel conjugates of different molecular weight (1K,
13K, and 36K) was compared. Conjugates of lower molecular weight
were significantly less effective than conjugate of higher
molecular weight. The higher molecular weights above 50,000 was too
viscous.
EXAMPLE 7
Poly-glutamic Acid-Paclitaxel (PG-TXL) Induces less Apoptosis
Compared to Paclitaxel
[0168] To assess the mechanism of PG-TXL associated antitumor
activity, histological sections of OCA-1 tumors excised from
paclitaxel and PG-TXL treated mice were examined. OCA-1 tumor
bearing mice were prepared as previously described. When tumor
volume reached 500 mm.sup.3, animals were injected with either
paclitaxel (80 mg/kg) or PG-TXL (160 mg equivalent paclitaxel/kg).
At different times ranging from 0 to 144 h after treatment, tumors
were histologically analyzed to quantify mitotic and apoptotic
activity according to Milas et al. (1995). The mice were killed by
cervical dislocation and the tumors were immediately excised and
placed in neutral-buffered formalin. The tissues were then
processed and stained with hematoxylin and eosin. Both mitosis and
apoptosis were scored in coded slides by microscopic examination at
400.times.magnification. Five fields of nonnecrotic areas were
randomly selected in each histological specimen, and in each field
the number of apoptotic nuclei and cells in mitosis were recorded
as numbers per 100 nuclei and were expressed as a percentage. The
values were based on scoring 1500 nuclei obtained from 3 mice per
time point.
[0169] The changes observed in the paclitaxel-treated mice were
qualitatively similar to those previously described (Milas et al.,
1995). The tumor cells showed marked nuclear fragmentation with
formation of apoptotic bodies, which was especially marked on day 1
(FIG. 13). Viable tumor cell clumps with normal mitoses were still
present in these tumors by 144 h, indicating that these tumors
would eventually regrow. Treatment with PG-TXL only resulted in a
mild increase in mitotically arrested cells and apoptotic cells,
presumably due to the small amount of free paclitaxel released from
PG-TXL (FIG. 13). By 96 h, tumors from PG-TXL-treated mice
developed extensive edema and necrosis, and only a small rim of
viable tumor cells remained. By 144 h, the residual tumor clumps as
compared to controls were comprised of cells that were larger, more
pleomorphic, and that displayed less mitotic activity.
[0170] These data suggest that the water-soluble PG-TXL conjugate
of the present disclosure has superior antitumor efficacy with
reduced toxicity as compared to conventional free paclitaxel
preparations. Although originally designed as a water-soluble form
of paclitaxel, it is now clear that the agent used to solubilize
paclitaxel, contributes to the overall anti-tumor activity of this
remarkable new complex. These data indicate that PG-TXL has an
ability to produce cell death in a manner which is separate from
and in addition to the apoptosis produced by released free
paclitaxel.
EXAMPLE 8
Synthesis of Poly-Glutamic Acid-Camptothecin (PG-CPT)
Conjugate.
[0171] The synthesis of PG-CPT followed a similar reaction as
previously described for the synthesis of PG-TXL. Into 80 mg of PG
polymer in 2.5 ml dry DMF was added 20 mg CPT (Hande Tech.), 34 mg
DCC, and trace amount of DMAP as catalyst. After stirred at room
temperature overnight, the reaction mixture was poured into
chloroform, and the precipitate collected. The dried precipitate
was redissoved in sodium carbonate solution, dialyzed against 0.05
M phosphate buffer (pH4.5), filtered, and lyophilized. The content
of CPT in the polymer conjugate was determined by fluorescence
spectrometer (Perkin-Elmer Model MPF-44A) using emission wavelength
of 430 nm and excitation wavelength of 370 nm. Content: 2% to 5%
(w/w), solubility: >200 mg conjugate/ml.
EXAMPLE 9
Synthesis of Poly-Lysine (PL) TXL Conjugate (PL-TXL).
[0172] All accessible amine functional groups of poly-lysine
(MW>30,000, Sigma) will be converted to carboxylic acid
functional groups by reacting poly-lysine with succinic anhydride,
glutaric anhydride, or DTPA. The remaining unreacted NH2 group in
poly-lysine will be blocked by reacting the modified polymer with
acetic anhydride. TXL, docetaxel, other taxiods, etopside,
teniposide, camptothecin, epothilone or other anti-tumor drugs will
be conjugated to the resulting polymer according to previously
described procedures for the synthesis of PG-TXL.
EXAMPLE 10
Synthesis of Other Polyamino Acids to be Used to Conjugate TXL
[0173] Polyamino acid copolymers containing glutamic acid may be
synthesized by the copolymerization of N-carboxyanhydrides (NCAs)
of corresponding amino acid with gamma-benzyl-L-glutamate NCA. The
resulting benzyl glutamate-containing copolymer will be converted
to glutamic acid-containing copolymer by removing the benzyl
protecting group (FIG. 15). TXL, docetaxel, other taxiods,
etopside, teniposide, camptothecin, epothilone or other anti-tumor
drugs will be conjugated to the resulting polymer according to
previously described procedures for the synthesis of PG-TXL and
PG-CPT.
EXAMPLE 11
Use of PG-TXL in Humans
[0174] Introduction
[0175] Poly-L-glutamic acid-Paclitaxel (PG-TXL) is a conjugate of
poly-L-glutamic acid and paclitaxel. This compound is water soluble
and based on early animal studies it appears that it can be
administered as a short, that is several minute, intravenous
injection. Based on the in vitro and early animal work, it appears
that this compound is at least as active against cancer as the
monomeric paclitaxel in Cremophor and may have fewer side effects.
Based on these observations, this drug will be studied in humans.
The study will first require formulation of this compound in a
solvent which is commonly used for intravenous infusion. The
inventors expect that either normal saline, 5% dextrose in water or
sterile water will be used as the solvent. This formulation of
PG-TXL will then be administered to at least two species of test
animals such as rats and dogs to determine the toxicities of the
drug in those animals and to determine a dose of the drug which
then can serve as the lowest starting dose for a Phase I human
study. That Phase I human study will define a dose of PG-TXL which
may be used in subsequent Phase II studies in patients. Phase II
studies will be performed in several tumor types to determine the
activity of PG-TXL in various cancers. One of ordinary skill in the
art will recognize that modifications in administration, selection
of animal models and dose regiments may be made in the methods
disclosed in following example, and such modifications are
encompassed by the invention.
[0176] Animal Studies
[0177] These studies will be performed in rats and Beagle dogs with
approximately 3 animals studied at each dose level of the drug. The
levels will be increased until life threatening toxicity is noted.
The animals will undergo blood testing as well as necropsy to
determine the organ systems which are susceptible to this drug's
toxicity and therefore to expect the side effects in human studies.
Once the dose is determined which causes the death of 10% of
animals then the equivalent of one-tenth of that dose will be
recommended as the starting dose for human studies. This is the
usual recommendation by the Food and Drug Administration (FDA) as
the initial dose for human Phase I studies.
[0178] Phase I Studies
[0179] Phase I study of this drug will be performed using the
starting dose defined in animal studies. The drug will be injected
into the vein by a syringe over several min or alternatively it may
be infused as a short infusion, up to approximately 10 to 15 min.
The volume of the solvent will be from 10 ml to approximately 100
ml depending on which of the two intravenous injection approaches
are used. The drug will be administered every 3 wk. This schedule
is based on the early animal studies and on the schema used with
paclitaxel in Cremophor. Three patients will be started on the
lowest dose level as defined by the animal studies and will be
treated with an injection of PG-TXL. Blood tests will be performed
at baseline and weekly to evaluate blood counts; tests of liver
function and renal function will be performed every 3 wk. It is
expected that the counts and physiological parameters will recover
sufficiently from the PG-TXL to resume the next cycle of treatment
3 wk after the previous one. If this is the case then the treatment
will be repeated every 3 wk. If the first cohort of three patients
tolerates the drug for 3 wk then these patients will be allowed to
have the dose increased by a predetermined schema that is usually
used in the Phase I studies. Once three patients have tolerated the
first cycle, the next cohort of 3 patients will be started on the
next higher dose level. This process of increasing the dose level
will continue until at least 2 out of 3 patients at a dose level
have side effects which are so severe that they prohibit continuing
administration of the drug. In such a circumstance the dose level
just prior to the excessively toxic one will be considered the
level of drug to be administered in subsequent studies. Six to ten
patients shall be treated on the dose level which will be
recommended for Phase II studies to confirm its tolerability. Once
the appropriate dose has been defined and acute toxic side effects
of the drug evaluated, Phase II studies will be initiated.
[0180] Phase II Studies
[0181] Phase II studies of PG-TXL will be performed in several
tumor types. Each study will be designed in a usual standard Phase
II manner following either Gahan's or Simon's design. In brief,
approximately 14 patients of a given tumor type will be treated
initially, if there is no evidence of anti-cancer activity in that
tumor type then further studies of PG-TXL in that tumor type will
be aborted. However, if at least one patient has clinical benefit,
defined as at least 50% decrease in the sum of products of
perpendicular cross-sectional diameters of the tumors, then the
number of patients with that tumor type treated with PG-TXL will be
increased to 30. These studies will allow us to define the activity
of PG-TXL in various cancers and refine the information on the side
effects of the drug. The tumor types of special interest for PG-TXL
will be the ones which have shown good response to paclitaxel and
docetaxel. This will include ovarian cancer, breast cancer, and
lung cancer. Studies comparing poly-glutamic acid-paclitaxel to
paclitaxel in tumors showing response to PG-TXL will be performed.
Such studies are called Phase III studies.
[0182] Phase III Studies of PG Paclitaxel
[0183] Based on the activity of paclitaxel in ovarian cancer,
breast cancer, and lung cancer these will be the tumor types in
which PG-TXL will be compared to paclitaxel. In view of the
necessity to have a large number of patients in such randomized
studies, the inventors expect that a multi-institutional study will
be necessary. The inventors have in their institution access to
Cooperative Community Oncology Program (CDDP) and to many other
multi-institutional study groups. In addition to the potential
clinical benefit of PG-TXL vs. paclitaxel, it would be appropriate
to evaluate the economic impact of the two drugs. It is expected
that a short term infusion of PG-TXL may result in a less costly
treatment. And, therefore, there is an expectation that PG-TXL may
be cost effective relative to paclitaxel monotherapy. Not only is
the infusion going to be shorter, it is expected that in view of
the absence of Cremophor fewer side effects will be experienced by
the patients and therefore the premedication regiment including
steroids and intravenous H2 and H1 blockers may no longer be
necessary. All of these factors will result in a reduction in the
cost of the treatment.
[0184] Summary
[0185] It is expected that the initial animal toxicology evaluation
will require up to 6 months. Subsequent to that, if a drug
formulation is available, human Phase I studies may be completed in
another 6 to 9 months. Once these have been completed, Phase II
studies in various tumor types may take another 6 to 9 months. At
that point, the inventors will have a good idea of the efficacy of
this drug and targeted Phase III studies may be designed and
initiated. It is also possible that the Phase II studies will show
enough clinical activity that abbreviated Phase III studies or no
Phase III studies would be necessary.
[0186] While the compositions and methods of this invention have
been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the compositions, methods and in the steps or in the
sequence of steps of the methods described herein without departing
from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the
appended claims.
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