JP2001288097A - Water-soluble paclitaxel derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a water-soluble composition of paclitaxel and docetaxel. SOLUTION: A method for enhancing the response of tumors to radiation exposure comprises the following steps of (a) administering a radiation- sensitizing dose of a pharmaceutical composition comprising a water-soluble polyamino acid polymer and paclitaxel, docetaxel, etoposide, teniposide, camptothecin or epothilone converted into a conjugate with a pharmaceutically acceptable carrier to a patient requiring the treatment and (b) a step of exposing the tumors to radiation. In the method, the paclitaxel or docetaxel converted into the conjugate is increased in water solubility, efficacies and accumulation in the tumors as compared with those of the corresponding drug unconverted into the conjugate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates generally to the field of pharmaceutical compositions used in the treatment of cancer, autoimmune diseases and restenosis. The invention also relates to the field of pharmaceutical compositions of anti-cancer drugs such as paclitaxel (Taxol ^™ ) and docetaxel (Taxotere), in particular, by conjugating the drug to a water-soluble moiety. To make it water-soluble.

[0002]

2. Description of the Related Art Paclitaxel is a Pacific yew (Pa
civic new, Taxus brevifoli
a) An antimicrotubule agent extracted from the needles and bark of the tree of a), which has shown significant anticancer effects in human cancer phase I studies and early phase II and III studies (Horwitz et al.) , 1993).
It is mainly reported in advanced ovarian and breast cancer. Significant activity has been described in small and non-small cell lung cancer, head and neck cancer, and metastatic melanoma. However, a major difficulty in developing clinical trial use of paclitaxel was that it was insoluble in water.

Docetaxel is semi-synthetically produced from 10-deacetylbaccatin III (a non-cytotoxic precursor extracted from the needles of Taxus baccata) and esterified with chemically synthesized side chains. (Cortes and Pazdur, 1995). Various cancer cell lines, including breast, lung, ovarian and colorectal cancer, and melanoma have been shown to be responsive to docetaxel. In clinical trials, docetaxel has been used to achieve a complete or partial response in breast, ovarian, head and neck cancer, and malignant melanoma.

[0004] Paclitaxel is typically paclitaxel at 6 mg Cremophor EL per ml.
(Polyoxyethylated castor oil) and a concentrated solution containing absolute alcohol (50% v / v) and must be further diluted before administration (Goldsp.
iel, 1994). Paclitaxel (Taxol ^™ )
Has shown significant activity in human (including breast, ovarian, small cell lung, and head and neck cancers) (Rowinsky and Doneh)
power, 1995). Significant activity has also been shown in patients with advanced breast cancer treated with multiple chemotherapeutic agents (Foa et al., 1994). But,
As with most chemotherapeutics, the maximum tolerated dose of paclitaxel is limited by toxicity. In humans, 1
Major toxic effects of paclitaxel at a dose of 00~250mg / m ² is granulocytopenia (Holmes et al., 199
5) is; symptomatic peripheral neurodegeneration is its major non-hematological toxicity (Rowinsky et al., 1993).

[0005] The amount of Cremophor EL required to deliver the required dose of paclitaxel is Cremphor EL.
significantly higher than the amount administered with any other drug prescribed in ophor. Some toxic effects include Cre
Due to mophor, this includes vasodilation, dyspnea, and hypotension. This vehicle has also been shown to produce severe hypersensitivity in laboratory animals and humans (Weiss et al., 1990). In fact, the maximum dose of paclitaxel that can be administered to mice by iv bolus injection is governed by the acute lethal toxicity of Cremophor vehicle (Eiseman et al., 199).
4). Further, Cremophor, a surfactant, is used.
EL is known to leach phthalate plasticizers (eg, di (2-ethylhexyl) phthalate from polyvinyl chloride bags and intravenous tubing) (DEHP). DEHP causes hepatotoxicity in animals,
And it is known to be carcinogenic in rodents.
This preparation of paclitaxel has also been shown to form particles over time, thus indicating that filtration is required during administration (Goldspiel, 199).
4). Therefore, special conditions are required for the preparation and administration of paclitaxel solutions to ensure safe drug delivery to the patient, and these conditions inevitably lead to higher costs.

Previous attempts to obtain water-soluble paclitaxel have included the placement of a prodrug of paclitaxel by placing a solubilizing moiety such as a succinate, sulfonic acid, amino acid, and phosphate derivative at the 2 'or 7 hydroxyl position. Preparation (Deutsch et al., 1989; Mathew et al., Zh)
ao and Kingston, 1991, 1992; N
icolaou et al., 1993; Vyas et al., 1995;
Rose et al., 1997). Some of these prodrugs have adequate water solubility, but most do not have antitumor activity comparable to that of the parent drug (Deuts
ch et al., 1989; Mathew et al., 1992; Ros.
e et al., 1997). Some of these derivatives are not suitable for intravenous injection because of their instability in aqueous solutions at neutral pH. For example, Deutsch et al. (1989)
Reported a 2 'succinate derivative of paclitaxel, but the water solubility of its sodium salt was only about 0.1%, and the triethanolamine and N-methylglutamine salts were only soluble at about 1% . further,
Amino acid esters are reported to be unstable. Similar results were reported by Mathew et al. (1992).

Recently, Nicolaou et al. (1993)
Reported the synthesis and in vitro biological evaluation of a new type of prodrug called "protaxol". These compounds have greater water solubility and are converted to paclitaxel as the active drug by an intramolecular hydrolysis mechanism. However, no in vivo data is available on the antitumor activity of protaxol. Greenwald et al. Reported the synthesis of highly water-soluble 2 ′ and 7 polyethylene glycol esters of paclitaxel (Greenwald).
Et al., 1994). Using a polymer conjugation strategy, other investigators 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 efficacy is not better than free paclitaxel. In addition, PEG has only two reactive functional groups at each end of its polymer chain, which effectively limits the amount of paclitaxel that PEG can have (US Pat. No. 5,362,831). .

[0008]

Another attempt to solve these problems involves the microencapsulation of paclitaxel in both liposomes and nanospheres (Bartoni and Boitard, 1990).
Although liposome formulations were reported to be as effective as free paclitaxel, only liposome formulations containing less than 2% paclitaxel were physically stable (Sharma and Straubinger, 199).
4). Unfortunately, the nanosphere formulation was illuminated to be toxic. Thus, there is still a need for water-soluble paclitaxel formulations that can deliver effective amounts of paclitaxel and docetaxel without the drawbacks caused by the insolubility of this drug.

Another barrier to the widespread use of paclitaxel is the limited source from which it is produced, which makes paclitaxel therapy very expensive. One unit of treatment can cost, for example, thousands of dollars. There is an additional disadvantage that not all tumors respond to paclitaxel treatment, and this may be due to the absence of paclitaxel on the tumor. Thus, it has a long serum half-life for the treatment of tumors, autoimmune diseases (eg rheumatoid arthritis) and for the prevention of restenosis of vessels subjected to trauma such as angioplasty and stenting. There is an immediate need for effective formulations of paclitaxel and related drugs that are soluble and water soluble.

[0010]

SUMMARY OF THE INVENTION The present invention addresses these and other disadvantages inherent in the major arts by providing chemotherapeutics conjugated to water-soluble polymers such as water-soluble polyamino acids or water-soluble metal chelators. It is sought to overcome by providing a composition comprising an agent and / or an anti-angiogenic drug (eg, paclitaxel, docetaxel or other taxoid). A further embodiment of the present invention is that a composition comprising a conjugate of paclitaxel and polyglutamic acid has surprising anti-tumor activity in an animal model, and that the composition herein differs from the properties of paclitaxel by paclitaxel. It is to be demonstrated as a new species of taxane with properties. These compositions have been shown herein to be surprisingly effective as anti-tumor agents against exemplary tumor models, and these compositions are known to be effective for taxanes or taxoids. Is expected to be at least as effective as paclitaxel, docetaxel, or other taxoids for any disease or condition that is The composition of the present invention provides a water-soluble taxoid to overcome disadvantages associated with the insolubility of the drug itself,
And also provide the benefits of improved efficacy and controlled release as shown herein that tumors are eradicated in animal models after a single intravenous administration;
And a new taxane. It has been demonstrated herein that poly (l-glutamic acid) conjugated to paclitaxel has novel drug activity in addition to having improved delivery to tumors and providing controlled release. Shown in the example.

A water-soluble composition of paclitaxel and docetaxel formed by conjugating paclitaxel or docetaxel to a water-soluble polymer such as polyglutamic acid, polyaspartic acid, or polylysine is disclosed. Disclosed are methods of using the compositions for the treatment of tumors, autoimmune diseases (eg, rheumatoid arthritis). Other embodiments include coating an implantable stent for prevention of restenosis.

The present invention relates to a method for enhancing the response of a tumor to irradiation, comprising the steps of: a) providing paclitaxel, docetaxel conjugated to a water-soluble polyamino acid polymer to a patient in need of such treatment; Administering a radiosensitizing amount of a pharmaceutical composition comprising, etoposide, teniposide, camptothecin, or epothilone, and a pharmaceutically acceptable carrier; and b) irradiating the tumor. Include. Here, the conjugated paclitaxel or docetaxel has increased water solubility, potency and intratumoral accumulation as compared to the corresponding unconjugated drug.

[0013] In one embodiment of the method of the present invention, the polymer is polyglutamic acid, polyaspartic acid,
Or selected from polylysine. In further embodiments, the polymer has a molecular weight of about 5,000 to about 100,000 daltons, a molecular weight of about 20,000 to about 80,000 daltons, or a molecular weight of about 25,000 to about 50,000 daltons.

In another embodiment of the method of the present invention, the polymer is polyglutamic acid. In a further embodiment, the conjugate comprises about 2% to about 35% by weight paclitaxel or docetaxel.

In yet another embodiment of the method of the present invention,
Step (b) is performed by administering gamma irradiation to the tumor. In a further embodiment, the composition is administered prior to or after irradiation. In another embodiment, the radiation dose is about 10 Grays per week, and the composition is administered within 1-2 days of radiation.

Preferably, the cancer is breast cancer, ovarian cancer, malignant melanoma, lung cancer, stomach cancer, prostate cancer, colon cancer, head and neck cancer, leukemia or Kaposi's sarcoma.

The invention also relates to a method of treating cancer, comprising: a) providing paclitaxel, docetaxel, etoposide, teniposide, camptothecin, conjugated to a polyglutamic acid polymer to a patient in need of such treatment; Or administering a radiosensitizing amount of a pharmaceutical composition comprising epothilone and a pharmaceutically acceptable carrier; and b) irradiating the tumor.

In one embodiment of the method of the present invention, the amount of the composition is about 0.5 to about 2 times the maximum tolerated amount of paclitaxel at an equivalent paclitaxel dose.

[0019]

DETAILED DESCRIPTION OF THE INVENTION The methods described herein also include other therapeutic agents, contrast agents and drugs (paclitaxel, tamoxifen, taxotere, etoposide (et).
opside), teniposide, fludarabine, doxorubicin, daunomycin, emodin, 5-fluorouracil, FUDR, estradiol, camptothecin,
(Including retinoids, verapamil, epothilone, cyclosporine, and other taxoids) can be used to make water-soluble polymer conjugates. In particular, those drugs with free hydroxyl groups are conjugated to this polymer by a similar chemical reaction as described herein for paclitaxel. Such conjugates are well within the skill of those in the chemical arts, and as such fall within the claimed scope of the present invention. These drugs include etoposide, teniposide, camptothecin,
And epothilones. As used herein, conjugated to a water-soluble polymer refers to the covalent attachment of a drug to the polymer or chelator.

It is also understood that the water-soluble conjugates of the present invention can be administered 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 of the present invention, or poly (l conjugated to paclitaxel in a preferred embodiment)
-Glutamic acid) (PG-TXL) may be combined with a platinum drug, an antitumor drug (eg, doxorubicin or daunorubicin) or other drugs used in combination with Taxol ^™ , in certain types of treatment, or External or internal radiation (ie, administered by an external source, or systematically (eg,
Radioactive materials (eg, formulations containing radioisotopes)
) Administered by injection or ingestion of).

Conjugation of chemotherapeutic drugs to polymers is an attractive approach to reduce systemic toxicity and improve therapeutic indices. Polymers with molecular weights greater than 30 kDa do not readily disperse through normal capillaries and glomerular endothelium, thus protecting normal tissues from irrelevant drug-mediated toxicity (Maeda and Ma)
tsumura, 1989; Reynolds, 199
5). On the other hand, malignant tumors are often well documented to have greater permeability than damaged capillary endothelium and normal tissue vasculature (Maeda and Mats).
umura, 1989; Fidler et al., 1987).
Tumors often lack lymphatic vasculature to remove large molecules that leak into tumor tissue (Maeda and Matsumura, 1989). Thus, the polymer drug conjugate, which normally remains in the vasculature, selectively leaks from the blood vessels to the tumor, resulting in tumor accumulation of the active therapeutic drug. Water-soluble polymers, such as PG-TXL in a preferred embodiment
May have different pharmacological properties than the unconjugated drug (ie, paclitaxel). In addition, the polymer drug conjugate can act as a drug depot for sustained release, resulting in prolonged drug exposure to tumor cells.
Finally, water-soluble polymers (eg, water-soluble polyamino acids) can be used to stabilize drugs, or to solubilize otherwise insoluble compounds. Currently, various synthetic and natural polymers are being tested for their ability to enhance tumor-specific drug delivery (Kopecek, 19
90, Maeda and Matsumura, 198
9). However, it is known to the inventors that only a few are currently undergoing clinical evaluation, including SMANCS in Japan and HPMA-Dox in the United Kingdom (Maeda, 1991; Kopesk and Kopekova). , 1993).

In the present disclosure, taxoid is understood to mean those compounds, including paclitaxel and docetaxel and other chemicals having a taxane skeleton (Cortes and Pazdur, 199).
5) and can be isolated from natural sources such as yew trees or from cell culture, or can be chemically synthesized molecules, and the preferred taxane has the general formula C ₄₇
H ₅₁ NO ₁₄ ([2aR- [2aα, 4β, 4αβ, 6
β, 9α (αR ^* , βS ^* ), 11α, 12α, 12a
α, 12bα]]-β- (benzoylamino) -α-hydroxybenzenepropanoic acid 6,12b, bis (acetyloxy) -12- (benzoyloxy) -2a, 3,
4,4a, 5,6,9,10,11,12,12a, 1
2b-dododecahydro-4,11-dihydroxy-4
a, 8,13,13-Tetramethyl-5-oxo-7,
11-methano-1H-cyclodeca [3,4] benz-
[1,2-b] oxet-9-yl ester. Paclitaxel and docetaxel are each more effective than others against certain types of tumors, and in the practice of the present invention, those tumors that are more sensitive to certain taxoids use water-soluble taxoids or taxane conjugates. It is understood that it is treated.

Paclitaxel is combined with a water-soluble metal chelating agent.
In those embodiments that are conjugated, the composition
The object can further include a chelated metal ion. Book
The chelated metal ions of the invention are aluminum,
U, calcium, chromium, cobalt, copper, dyspros
Um, erbium, europium, gadolinium, gully
, Germanium, holmium, indium, iridium
Um, iron, magnesium, manganese, nickel, platinum,
Rhenium, rubidium, ruthenium, samarium, nato
Lium, technetium, thallium, zinc, yttrium
Or any one of the ionized forms of zinc
You. In certain preferred embodiments, the chelated
Metal ions are radionuclides, that is, of the listed metals.
One radioisotope. Preferred radionuclides are⁶⁷
Ga,⁶⁸Ga,¹¹¹In,^99mTc, ⁹⁰Y,^114mSn and
And^193mIncluding but not limited to Pt.

A preferred water-soluble chelating agent used in the practice of the present invention is diethylenetriaminepentaacetic acid (D
TPA), ethylenediaminetetraacetic acid (EDTA), 1,
4,7,10-tetraazacyclododecane-N, N ',
N ", -N '"-tetraacetic acid (DOTA), tetraazacyclotetradecane-N, N', N ", -N '"-
Tetraacetic acid (TETA), hydroxyethylidene diphosphonate (HEDP), dimercaptosuccinic acid (DMS
A), diethylenetriaminetetramethylenephosphate (D
TTP) and 1- (p-aminobenzyl) -DTP
A, 1,6-diaminohexane-N, N, N ',-N'
-Tetraacetic acid, DPTP, and ethylenebis (oxyethynitrilo) tetraacetic acid, including but not limited to.
DTPA is most preferred. A preferred embodiment of the present invention also comprises ¹¹¹ In-DTPA paclitaxel, and Na
-A composition comprising -DTPA-paclitaxel.

In certain embodiments of the invention, paclitaxel, docetaxel, or other taxoid may be conjugated to a water-soluble polymer, and preferably the polymer is 2 'of paclitaxel, docetaxel, or another taxoid. -Conjugated to hydroxyl or 7'-hydroxyl or both. Polyglutamic acid (PG) is one polymer that offers several advantages in the present invention. First, it contains a large number of side chain carbonyl functions for drug attachment. Second, PG is readily converted to non-toxic base components, L-glutamic acid, d-glutamic acid and dl by lysosomal enzymes.
-Can be degraded to glutamic acid. Finally, it has been reported that sodium glutamate prevents the development of paclitaxel-induced neurodegeneration, so that higher doses of paclitaxel can be tolerated (Boyle et al., 1996). Preferred polymers are poly (l-glutamic acid), poly (d-glutamic acid), poly (dl-glutamic acid), poly (l-aspartic acid), poly (d-aspartic acid), poly (d-aspartic acid), poly (d-
l-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, and poly (2-hydroxyethyl 1-glutamine), chitosan , Carboxymethyl dextran, hyaluronic acid, human serum albumin, and alginic acid. Polyglutamic acid is particularly preferred. At the lower molecular weight limit, the polymers of the present invention preferably have about 1,000 kd, about 2,000
kd, about 3,000 kd, about 4,000 kd, about 5,000
00kd, about 6,000kd, about 7,000kd, about 8,000kd, about 9,000kd, about 10,000k
d, about 11,000 kd, about 12,000 kd, about 1
3,000kd, about 14,000kd, about 15,000
kd, about 16,000 kd, about 17,000 kd, about 1
8,000kd, about 19,000kd, about 20,000
kd, about 21,000 kd, about 22,000 kd, about 2
3,000kd, about 24,000kd, about 25,000
kd, about 26,000 kd, about 27,000 kd, about 2
8,000kd, about 29,000kd, about 30,000
kd, about 31,000 kd, about 32,000 kd, about 3
3,000kd, about 34,000kd, about 35,000
kd, about 36,000 kd, about 37,000 kd, about 3
8,000kd, about 39,000kd, about 40,000
kd, about 41,000kd, about 42,000kd, about 4
3,000kd, about 44,000kd, about 45,000
kd, about 46,000 kd, about 47,000 kd, about 4
8,000kd, about 49,000kd, about 50,000
It has a molecular weight of up to kd. At the upper molecular weight limit, the polymers of the present invention preferably have about 51,000 kd, about 5
2,000kd, about 53,000kd, about 54,000
kd, about 55,000 kd, about 56,000 kd, about 5
7,000kd, about 58,000kd, about 59,000
kd, about 60,000 kd, about 61,000 kd, about 6
2,000kd, about 63,000kd, about 64,000
kd, about 65,000 kd, about 66,000 kd, about 6
7,000kd, about 68,000kd, about 69000
kd, about 70,000 kd, about 71,000 kd, about 7
2,000 kd, about 73,000 kd, about 74,000
kd, about 75,000 kd, about 76,000 kd, about 7
7,000kd, about 78,000kd, about 79,000
kd, about 80,000 kd, about 81,000 kd, about 8
2,000kd, about 83,000kd, about 84,000
kd, about 85,000 kd, about 86,000 kd, about 8
7,000kd, about 88,000kd, about 89,000
kd, about 90,000 kd, about 91,000 kd, about 9
2,000kd, about 93,000kd, about 94,000
kd, about 95,000 kd, about 96,000 kd, about 9
7,000kd, about 98,000kd, about 99000
kd, having a molecular weight of up to about 100,000 kd. Within these ranges, the molecular weight range for the polymer is preferably from about 5,000 to about 100,000 kd.
And preferably about 20,000 to about 80,000,
Alternatively, more preferably about 25,000 to about 50,000.

A further aspect of the invention is that the compositions of the invention (eg, PG-TXL) also include a second lipophilic or poorly soluble antineoplastic agent (eg, camptothecin, epothilone, cisplatin, melamine). Phalan, taxotere, etoposide, teniposide, fludarabine, verapamil, or cyclosporin).
Alternatively, a water-soluble drug (eg, 5-fluorouracil (5FU), or fluorodeoxyuridine (FUD)
R), doxorubicin or daunomycin).

It is understood that the compositions of the present invention can be dispersed in a pharmaceutically acceptable carrier solution as described below. Such solutions may be sterile or sterile, and may contain water, buffers, tonicity agents, or other ingredients known to those of skill in the art, which are useful when administered to animal or human subjects. No allergic or other adverse reactions occur). Thus, the present invention can also be described as a pharmaceutical composition comprising a chemotherapeutic drug or anti-cancer agent (eg, paclitaxel, docetaxel, or other taxoid) conjugated to a high molecular weight water-soluble polymer or chelator. . The pharmaceutical composition comprises polyethylene glycol, polyglutamic acid, polyaspartic acid, polylysine, or a chelating agent, preferably DT
PA may be included. The radionuclide can be used as an anti-tumor agent or drug, and the pharmaceutical composition of the present invention
It is also understood that a therapeutic amount of a chelated radioisotope can be included.

In certain embodiments, the present invention may be described as a method for determining the uptake of chemotherapeutic agents, such as paclitaxel, docetaxel, or other taxoids, by tumor tissue. The method comprises the steps of obtaining a conjugate of the drug and the metal chelator with the metal ion to be chelated, contacting the tumor tissue with the composition, and dissociating the chelated metal ion in the tumor tissue. Detecting the presence may be included. The presence of the chelated metal ion in its tumor tissue is
It is an indicator of uptake by the tumor tissue. The chelated metal ion can be a radionuclide, and its detection can be scintigraphic. The tumor tissue can also be included in an animal or human subject, and the composition is then administered to the subject.

The present invention also provides, in certain embodiments,
It can be described as a method of treating cancer in a subject.
The method comprises obtaining a composition comprising a chemotherapeutic drug (eg, paclitaxel, docetaxel or other taxoid) conjugated to a water-soluble polymer or chelating agent 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 a water-soluble polyamino acid (poly (l-aspartic acid), poly (d-aspartic acid), poly (dl-aspartic acid), poly (l-lysine), poly (d-lysine), or poly (d-lysine). Paclitaxel, docetaxel conjugated with (dl-lysine), and more preferably poly (l-glutamic acid), poly (d-glutamic acid), including but not limited to poly (dl-glutamic acid)). Or contain other taxoids. The composition of the present invention comprises:
It is understood that unconjugated taxoids are effective against any type of cancer that has been shown to be effective, and breast cancer, ovarian cancer, melanoma, lung cancer, head cancer, and Including but not limited to cervical cancer. The compositions of the present invention may also be used against gastrointestinal cancer, prostate cancer, colon cancer, leukemia, or Kaposi's sarcoma. As used herein, the term (cancer)
“Treat” is understood to mean any medical management of a subject having a tumor pair. This term is
Includes any inhibition of tumor growth or metastasis, or any attempt to inhibit, delay or eliminate tumor growth or metastasis. The method involves killing cancer cells by a non-apoptotic mechanism of cell death and an apoptotic mechanism. A method of treating a tumor may include some prediction of paclitaxel or docetaxel uptake in the tumor before administering a therapeutic amount of the drug. Methods of administration include, but are not limited to, bolus injection or infusion of the drug, as well as intraarterial, intravenous, intraperitoneal, or intratumoral administration.

The method may include any of the imaging techniques described above, wherein paclitaxel-chelating agent-
The chelated metal is administered to the subject and is detected in the tumor. This step provides a cost-effective way to determine that a particular tumor is not expected to respond to DTPA-paclitaxel treatment if the drug does not enter the tumor. It is contemplated that if imaging techniques can be used to predict response to paclitaxel and identify patients who do not appear to respond, significant cost and significant time will be saved for the patients. The assumption is that if the amount of chemotherapeutic agent deposited on the tumor is not appropriate, the tumor response to that agent is relatively low.

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 complexed with a paclitaxel chelator conjugate to a subject, measuring a radioactive metal scintigraphic signal, and obtaining an image.

The present invention may also be described in certain broad aspects as a method of reducing at least one symptom of a systemic autoimmune disease. The method comprises providing, to a subject having a systemic autoimmune disease, a composition comprising paclitaxel or docetaxel conjugated to a polymer (preferably having polyamino acids, more preferably having polyglutamic acid). Administering an effective amount of the substance. Of particular interest in the context of the present disclosure is the treatment of rheumatoid arthritis. Rheumatoid arthritis is known to respond to paclitaxel in some cases when administered in standard Cremophor formulations (US Pat. No. 5,5,5).
83,153, incorporated herein by reference). It is contemplated that the effectiveness of the water-soluble taxoids or taxanes of the invention in treating tumors will not be reduced by conjugation with a water-soluble moiety. Thus, the compositions of the present invention are expected to be as effective as paclitaxel on rheumatoid arthritis. Paclitaxel is an anti-angiogenic agent. Rheumatoid arthritis forms a newly formed cluster of vessels that erodes adjacent joints. The taxoid or taxane composition of the present invention may contain other drugs such as angiogenesis inhibitor (AGM-1470) (Olive
It is also understood that they can be used in combination with other anti-cancer drugs such as r. et al., 1994) or methotrexate).

The finding that paclitaxel also inhibits restenosis after balloon angioplasty indicates that the water-soluble paclitaxel and docetaxel of the present invention find various applications beyond direct parenteral administration (WO9625).
176, incorporated herein by reference). For example, medical devices into which water-soluble paclitaxel has been implanted (eg, tubes, shunts, catheters, artificial implants, electrical implants such as pins, pacemakers, and especially arterial or venous stents (such as balloon expandable stents). In these embodiments, the water-soluble paclitaxel can be bound to a implantable medical device, or the water-soluble paclitaxel can be used as a coating for the implantable device. It is contemplated that the surface can be passively absorbed, for example, a stent can be coated with a polymer drug conjugate by dipping the stent into a polymer drug solution or spraying such a solution on the stent. Suitable materials for implantable devices are biocompatible And should be non-toxic and may be selected from metals such as nickel titanium alloys, steel, or biocompatible polymers, hydrogels, polyurethanes, polyethylenes, ethylene vinyl acetate copolymers, etc. In a preferred embodiment, water-soluble paclitaxel In particular, the PG-TXL conjugate is coated on a stent for insertion into an artery or vein after balloon angioplasty.Thus, the present invention is directed to arterial restenosis or arterial closure following vascular trauma. Can be described in certain broad aspects as methods of inhibiting.
The method includes the step of administering to a subject in need of the method a composition containing paclitaxel or docetaxel conjugated to polyglutamic acid or another water-soluble polyamino acid. In the practice of the present invention, the subject may be a patient with coronary artery and vein bypass, a patient with vascular surgery, an organ transplant, or a patient with coronary artery and vein or any other arterial angioplasty, for example, the composition may be Can be administered directly, intravenously, or even coated on a stent that is implanted at the site of vascular trauma.

Accordingly, an embodiment of the present invention is a transplantable medical device, wherein the device is conjugated to an effective amount of polyglutamic acid or a water-soluble polyamino acid that inhibits smooth muscle cell proliferation. Coated with a composition containing paclitaxel or docetaxel. A preferred device is a stent coated with a composition of the invention as described herein,
And in certain preferred embodiments, the stent is adapted for use during and after balloon angioplasty, and the coating is effective to inhibit restenosis.

In certain preferred embodiments, the present invention provides compositions comprising paclitaxel, docetaxel, or other taxoids containing polyglutamic acid conjugated to the 2 'or 7 hydroxyl or both, or paclitaxel, It may even be described as a composition containing a water-soluble polyamino acid conjugated to docetaxel, or other taxoids, at the 2 'or 7 hydroxyl or both.

[0036] As used herein, the term "polyglutamic acid (s)" refers to poly (l
-Glutamic acid), poly (d-glutamic acid) and poly (dl-glutamic acid), and the term "polyaspartic acid" (s) includes poly (1-aspartic acid), poly (d-aspartic acid) and Poly (d
l-aspartic acid) and the term "polylysine"
(Single or multiple) are poly (l-lysine), poly (d
-Lysine) and poly (dl-lysine), as well as the term "water-soluble polyamino acid (s)",
Or "water-soluble polymer of amino acids" includes, but is not limited to, poly-glutamic acid, poly-aspartic acid, poly-lysine, and amino acid chains containing a mixture of glutamic acid, aspartic acid and / or lysine. In certain embodiments, the term "water-soluble polyamino acid (s)" or "water-soluble polymer of amino acids" refers to glutamic acid and / or aspartic acid and / or aspartic acid in either d and / or 1 isoform conformation. Or it contains an amino acid chain containing a combination of lysines. In certain preferred embodiments, such "water-soluble polyamino acids" contain one or more glutamic, aspartic, and / or lysine residues. Such "water-soluble polyamino acids" also include any natural, modified, or unusual amino acid described herein, wherein most of the residues (ie, greater than 50%) are glutamic and / or aspartic acid. And / or as long as it contains lysine. In certain embodiments, the water-soluble polymer of amino acids containing more than one different type of amino acid residue is sometimes
It is referred to herein as a “copolymer”.

In certain embodiments, a naturally occurring,
Various substitutions of unusual or chemically modified amino acids can be made in amino acid compositions of "water-soluble polyamino acids" (particularly "poly-glutamic acid"),
The taxoid-polyamino acid conjugates of the present invention are produced and still obtain molecules having similar or otherwise desirable characteristics of water solubility and / or therapeutic efficacy. Polyamino acids such as poly-glutamic acid, poly-aspartic acid, poly-lysine or a water-soluble amino acid chain, or a polymer containing a mixture of glutamic acid, aspartic acid, and / or lysine have a lower limit of amino acid substitution range of 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,
Each of 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 are described herein. Replaced by any naturally occurring, modified or unusual amino acid described herein. In another aspect of the invention, a polyamino acid such as a poly-glutamic acid, a poly-aspartic acid, a poly-lysine or a polyamino acid chain containing a mixture of some or all of these three amino acids has a lower limit of 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 of each of the glutamic, aspartic, or lysine residues is any of the herein described. Can be substituted with naturally occurring, modified or unusual amino acids of

In a further aspect of the invention, the polyamino acid, such as poly-glutamic acid, poly-aspartic acid, or poly-lysine, has 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% of glutamic acid, aspartic acid, or lysine Each of the residues is replaced by any of the naturally occurring, modified or unusual amino acids described herein, as long as the majority of the residues contain glutamic acid and / or aspartic acid and / or lysine. obtain. In amino acid substitution of various water-soluble amino acid polymers, a residue having a hydrophilicity index of +1 or more is preferable.

In certain aspects of the invention, the amount of the anti-tumor drug conjugated by the water-soluble polymer can vary. At the lower end, such compositions contain 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
%, From about 24% to about 25% (weight / weight) of the conjugate by weight of the antitumor drug. At the upper limit, such compositions comprise 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% (w / w) or more of the conjugate may contain the anti-tumor drug based on the weight of the conjugate. Preferred anti-tumor drugs include paclitaxel, docetaxel, or other taxoids, and preferred water-soluble polymers include water-soluble amino acid polymers.

In certain other aspects of the invention, the number of molecules of the antitumor drug conjugated per molecule of water-soluble polymer can vary. At the lower end, such compositions comprise 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 13, 14,
About 15, about 16, about 17, about 18, about 19 to about 20 or more molecules of the anti-tumor drug per water-soluble polymer molecule may be included. At the upper limit, such a composition comprises about 2
1, about 22, about 23, about 24, about 25, about 26, about 2
7, about 28, about 29, about 30, about 31, about 32, about 3
3, about 34, about 35, about 36, about 37, about 38, about 3
9, about 40, about 41, about 42, about 43, about 44, about 4
5, about 46, about 47, about 48, about 49, about 50, about 5
1, about 52, about 53, about 54, about 55, about 56, about 5
7, about 58, about 59, about 60, about 61, about 62, about 6
3, about 64, about 65, about 66, about 67, about 68, about 6
9, about 70, about 71, about 72, about 73, about 74 to about 7
It may contain 5 or more molecules or more anti-tumor drug per water-soluble polymer molecule. Preferred anti-tumor drugs include paclitaxel, docetaxel, or other taxoids, and preferred water-soluble polymers include water-soluble amino acid polymers. A preferred number of anti-tumor drug molecule conjugates per molecule of water-soluble polymer is about 7 molecules of anti-tumor drug per molecule of water-soluble polymer.

Water-soluble amino acid polymers having various substitutions of residues that are conjugated to paclitaxel, docetaxel, or other taxoids are referred to as "biologically functional equivalents."
That. This "biologically functional equivalent" is part of the definition of a "water-soluble polyamino acid" conjugated to a taxoid, and as described herein, as well as certain water-soluble amino acid polymers- As defined by any applicable assay known to those of skill in the art that measures improved aqueous solubility as compared to the unconjugated taxoid (s) used to produce the taxoid composition. obtain. In other aspects of the invention, the "biologically functional equivalent" of the water-soluble amino acid-taxoid polymer can be prepared by the assays described herein, as well as any applicable assays known to those of skill in the art. It can be further identified by improved anti-tumor cell activity compared to the anti-tumor cell activity of the unconjugated water-soluble amino acid polymer used to produce a particular water-soluble amino acid polymer-taxoid composition. . The term "biologically functional equivalent" used herein to describe this aspect of the invention is further described in detail in the detailed description herein.

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 "one (a)" is understood to include the meaning of "one or more." Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are now described.

DETAILED DESCRIPTION OF THE INVENTION The present invention results 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 with ovarian carcinoma (OCA-I)
Compared to the same dose of paclitaxel without G resulted in significant tumor growth delay. Mice treated with paclitaxel alone or in combination with free paclitaxel and PG initially showed delayed tumor growth, but tumors regrow after 10 days to levels comparable to the untreated control group did. Furthermore, PG-TX
At the maximum tolerated dose (MTD) of the L-conjugate (160 m
g equivalent paclitaxel / kg), tumor growth was completely suppressed, tumors shrank, and mice were observed to remain tumor free for 2 months after treatment (M
TD: single i. v. Within 2 weeks after injection, 1
It is defined as the maximum dose that results in less than 5% weight loss). In a parallel study, PG-T in rats
XL antitumor activity was measured in rat mammary carcinoma (13762F)
Was tested using Again, complete tumor eradication is PG-
TXL was observed at 40-60 mg equivalent paclitaxel / kg. These surprising results demonstrated that the polymeric drug conjugate, PG-TXL, successfully eradicated well-established solid tumors in both mice and rats after a single intravenous infusion.

In addition to significant antitumor (chest, ovary, etc.) data in syngeneic mice, human breast cancer (MDA-435) and ovarian cancer (SKOV) in nude mice
Good activity of PG-TXL on 3ip1) has been observed in recent years. Nude mice are a particular animal with an incomplete immune system in which human tumors can grow.

According to the data shown in this specification, PG
-Concluded in the present invention that TXL is a new species of taxane that is pharmaceutically different from previous paclitaxel or Taxol ^™ preparations. For example, PG-T in plasma
The distribution of XL is different from free paclitaxel. Paclitaxel appears to be retained in mouse plasma for a very short time, whereas PG-TXL appears to be retained for a longer time. This is intended to provide a unique advantage in that prolonged exposure of the tumor to the drug can result in an enhanced response. The rate of conversion of PG-TXL to paclitaxel is low and radiolabeled PG-
Less than 1% of the radioactivity from TXL is recovered as radioactive paclitaxel within 48 hours after injection of the paclitaxel polymer conjugate. This finding indicates that the new drug PG-T
XL can result in death within tumor cells in a manner that is not just due to the slow release of paclitaxel itself.

Further evidence of the novelty of PG-TXL is that
That is, relatively high levels of radioactivity from radiolabeled PG-TXL appear in tumor tissue shortly after injection. However, only a small amount of radioactivity in tumor tissue is due to the release of free paclitaxel. Moreover, the proportion of radioactivity in tumor tissue due to paclitaxel itself does not increase significantly over time. This means
Again, we show that PG-TXL is the least prodrug for the gradual release of paclitaxel. PG-T
XL vs. paclitaxel uptake has also been studied in certain human colon adenocarcinoma cell transport systems. Radioactivity associated with readily available radiolabeled PG-TXL enters the cells, but only 10% is due to free paclitaxel. These data are comparable to those found in tissue distribution studies, and again, PG-TX
There are several mechanisms or methods by which L can lead to cancer cell killing, and this suggests that it is different from that of paclitaxel.

In another study, freshly prepared PG-
It was found that TXL did not support the growth of paclitaxel-dependent cell lines, which indicated that free paclitaxel was released only slowly from the polymer-paclitaxel conjugate, and that the polymer-paclitaxel conjugate itself had Taxol ^™ Does not act pharmaceutically. Aging promotes PG-TXL degradation and increases the relative ability of the resulting substance to support the growth of paclitaxel-dependent cells, but to a lesser extent when compared to free paclitaxel.

Recent analysis of tumor tissue 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 the mechanism by which cells undergo self-induced killing, or programmed cell death, a natural process used by organisms in wound healing and tissue remodeling. Tumors from mice treated with PG-TXL have significantly less apoptotic bodies, but an increased frequency of tumor necrosis and edema, compared to free paclitaxel, indicating that paclitaxel and It suggests that PG-TXL can cause tumor cell death by two distinct pathways.

These studies and the studies described in the specific examples show that PG-TXL is a novel taxane that is not only very active against breast and ovarian cancer but also appears to have limited side effects. Demonstrate that Compounds in which polymer conjugates of paclitaxel are novel and have higher overall anticancer activity (PG
-TXL) is now evident.

Another aspect of the present invention is to include a molecule in a polymeric composition effective to target a therapeutic composition to a disease or tumor site or to a particular organ or tissue. It is. Many such targeting molecules are known in the art and can be conjugated to the water-soluble antitumor compositions of the present invention. Examples of such molecules or drugs include antibodies such as anti-tumor antibodies, anti-cell receptor antibodies, tissue-specific antibodies; octreotide,
Hormonal drugs such as estradiol and tamoxifen; growth factors; cell surface receptor ligands; enzymes; hypoxic agents such as misonidazole and erythronitroimidazole; and anti-angiogenic agents.

Another composition of the present invention is DTPA-paclitaxel, which is also shown herein to be as effective as paclitaxel in an in vitro anti-tumor potency assay using the B16 melanoma cell line. . DTPA-paclitaxel showed no significant difference in antitumor effect when compared to paclitaxel on MCa-4 breast tumors at a dose of 40 mg / kg body weight in a single injection. In addition, it was shown that gamma-ray scintigraphy demonstrated that indium-labeled DTPA-paclitaxel accumulated in MCa-4 tumors. This demonstrates that the chelator-conjugated anti-tumor drugs of the present invention are useful and effective for tumor imaging.

The novel compounds and methods of the present invention offer significant advantages over conventional methods and compositions.
By providing a water-soluble and controlled release paclitaxel derivatized composition, which also has different anti-tumor properties than unmodified paclitaxel, water-soluble paclitaxel is Because it is designed to improve the efficacy of cancer treatment. Such a composition eliminates the need for a solvent associated with the side effects seen in conventional paclitaxel compositions. In addition, radiolabeled paclitaxel, which has been shown to retain antitumor activity, is also useful for tumor imaging. In addition, the present invention provides a method for determining whether paclitaxel is taken up by a particular tumor by scintigraphy, single-photon emission computed tomography (SPECT) or positron emission tomography (PE).
T). This decision can then be used to predict the efficacy of the anti-cancer treatment. This information may be useful in guiding a clinician in the department of a patient receiving chelator-paclitaxel treatment.

Paclitaxel can be rendered water-soluble in a number of ways; that is, by conjugating paclitaxel with a water-soluble polymer (acting as a drug carrier) and using a water-soluble chelating agent to produce an antitumor drug. By derivatization. The latter approach also involves radionuclides (eg, ¹¹¹ In, ⁹⁰ Y, ¹⁶⁶ Ho,
^It offers opportunities for labeling with ⁶⁸ Ga, ^99m Tc), for nuclear imaging, and / or for radiotherapy studies. Paclitaxel, polyethylene glycol-paclitaxel (PEG-paclitaxel), polyglutamic acid-paclitaxel conjugate (PG-TXL)
FIG. 1 shows the structures of diethylenetriaminepentaacetic acid-paclitaxel (DTPA-paclitaxel).

In certain embodiments of the present invention, DTP
A-paclitaxel or other paclitaxel chelator conjugates such as EDTA-paclitaxel, DTTP-paclitaxel, or DOTA-paclitaxel can be prepared, for example, in the form of water-soluble salts (sodium, potassium, tetrabutyl). Ammonium salt, calcium salt, ferric salt, etc.). These salts
Useful as therapeutic agents for the treatment of tumors. Second
In addition, DTPA-paclitaxel or other paclitaxel chelators, when labeled with a radionuclide such as ¹¹¹ In or ^99m Tc, can be used as a radiotracer to detect specific tumors in combination with nuclear imaging techniques. It is useful as a diagnostic agent. Paclitaxel (Taxol ^™ ) and docetaxel (doc)
etaxel) (Taxotere), other taxane derivatives may be adapted for use in the compositions and methods of the present invention, and all such compositions and methods are understood to be encompassed by the present invention. I do.

Modifications and variations can be made in the structure of the water-soluble polyamino acids of the present invention, or water-soluble polymers such as water-soluble metal chelators, to still produce molecules having similar or otherwise desired properties. Such "biologically functional equivalents" or "functional equivalents" that may be obtained are also included in the present invention.

For example, one skilled in the art will recognize that certain amino acids can interact with structures such as chemotherapeutic and / or anti-angiogenic agents, such as, for example, paclitaxel or docetaxel, without appreciable loss of ability to bind. It will be appreciated that other amino acids in the structure of the polyamino acid, including water soluble amino acid polymers such as polyglutamic acid, polyaspartic acid, or polylysine, can be substituted. further,
As exemplified without being limited to PG-TXL, amino acid substitutions in water-soluble polyamino acids conjugated to chemotherapeutic agents and / or anti-angiogenic agents such as paclitaxel or docetaxel are provided. And may still maintain some or all of the novel pharmaceutical properties disclosed herein. Since the interaction capabilities and properties of the protein dictate the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the polyamino acid sequence and nevertheless have similar (agonistic) Polyamino acids having characteristics can be obtained. Thus, various changes can be made in the sequence of a water-soluble polyamino acid of a drug conjugate, such as, but not limited to, PG-TXL, without any apparent loss of their biological utility or activity. That is intended by the present inventors.

In the term functional equivalent, the concept that the number of changes that can be made within a portion of a molecule and still produce a molecule having an acceptable level of equivalent biological activity is limited. To be unique in the definition of "biologically functional equivalent of a water-soluble polyamino acid"
It is well understood by those skilled in the art. Thus, the biologically functional equivalent of a water-soluble polyamino acid is that specific, but few or all, amino acids are replaced by water-insoluble amino acids (either natural, unusual, or chemically modified). Defined herein as those water-soluble polyamino acids that can be substituted.

It is contemplated that fewer amino acids should be made in a given peptide, especially when shorter lengths of water-soluble polyamino acids are considered.
Longer domains may have a moderate number of changes. The longest water-soluble polyamino acid chains described herein are:
Most resistant to a greater number of changes. Of course, another large number of water-soluble polyamino acids with different substitutions, such as, but not limited to, polyglutamic acid, polyaspartic acid, or polylysine, can be readily made and used in accordance with the present invention.

It is also well understood that any particular residue indicates that the biological or structural properties of the polyamino acid are of particular importance, and that such residues generally cannot be interchanged. You. In this manner, the functional equivalent is
Defined herein as those water-soluble polyamino acids that retain a substantial amount of their natural biological activity.

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substitutions (eg, their hydrophobicity, hydrophilicity, charge, size, etc.). Analysis of the size, shape and type of amino acid side chain substitutions revealed that arginine, lysine and histidine were all positively charged residues; alanine, glycine and serine were all of similar size; and phenylalanine , Tryptophan, and tyrosine all show generally similar forms. Thus, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

To effect a further qualitative change, the hydropathic index of amino acids can be considered. Each amino acid was given a hydropathic index based on its hydrophobicity and charge properties. They are isoleucine (+4.5); valine (+4.2);
Leucine (+3.8); Phenylalanine (+2.
8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8);
0.4); threonine (-0.7); serine (-0.
8); tryptophan (-0.9); tyrosine (-1.
3); Proline (-1.6); Histidine (-3.
2); Glutamic acid (-3.5); Glutamine (-3.
5); aspartic acid (-3.5); asparagine (-
3.5); lysine (-3.9); and arginine (-
4.5).

The importance of the hydropathic amino acid index in conferring interactive biological functions on proteins and their corresponding polyamino acids is generally understood in the art (Kyte & Do).
olittle, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score, and still retain a similar biological activity. In making the change based on the hydropathic index, the amino acid substitution preferably has a hydropathic index within ± 2,
It is particularly preferred that they are within ± 1, and even more particularly preferred that they are within ± 0.5.

It is also understood in the art that similar amino acid substitutions can be made effectively on the basis of hydrophilicity. As detailed in U.S. Patent Application No. 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3 0.0 ± 1); glutamic acid (+ 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); (-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 on similar hydrophilicity values, amino acid substitutions are preferred where the hydrophilicity values are within ± 2, they are particularly preferably within ± 1, and even more preferably, they are within ± 0.5. Therefore, with reference to hydrophilicity, arginine, lysine, aspartic acid, and glutamic acid are defined herein as biologically functional equivalents, especially water-soluble amino acid polymers.

The water-soluble polyamino acid chemotherapeutic agents and / or anti-angiogenic drug compounds described herein (eg, but not limited to the PG-TXL compounds described herein) are exemplified by this. (Paclitaxel or docetaxel conjugated to such a water-soluble amino acid, etc.), we also have other sterically similar compounds that mimic key parts of the structure of water-soluble polyamino acids. Intended to be prescribed. Such compounds, which may be referred to as peptidomimetics, may be used in the same manner as the peptides of the invention, and are therefore also functional equivalents.

Certain mimetics, which are mimic elements of protein secondary structure, are described in Johnson et al. (1993). The underlying principle behind the use of peptidomimetics is that amino acid side chains are mainly composed of amino acid side chains in such a way that the peptide backbone of proteins containing polyamino acids facilitates molecular interactions such as antibody and antigen interactions. Is to orientate. Peptidomimetics are therefore designed to allow for similar molecular interactions with the natural molecule.

Some successful applications of the concept of peptidomimetics have focused on mimics of β-turns in proteins known to be highly antigenic. Possibly the β-turn structure within the polypeptide can be predicted by computer-based algorithms, as described herein. Once the amino acid components of the turn have been determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chain.

The generation of additional structural equivalents or mimetics can be achieved by modeling and chemical design techniques known to those skilled in the art. The art of receptor modeling is now well known, and chemicals that bind to water-soluble polyamino acids can be designed by such methods and then synthesized. It is understood that all such sterically designed constructs are included within the scope of the present invention.

In addition to the twenty "standard" amino acids provided via the genetic code, modified or unusual amino acids are also contemplated for use in the present invention. A table of non-limiting examples of modified or unusual amino acids,
Provided herein below.

[0069]

[Table 1]

Toxicity studies (DTPA-paclitaxel pharmacokinetics and tissue distribution) were performed using a single dose of iv (i
v) LD _{50 (50%} lethal dose in mice DPTA- paclitaxel observed in injection), about 110mg
/ Kg body weight. Direct comparisons with paclitaxel indicate the limited solubility of paclitaxel and the dose imposed by vehicle toxicity associated with iv administration-
This is difficult to do because of capacity constraints. However, following the present disclosure, those skilled in the art of chemotherapy will determine the effective and maximum tolerated dose (MTD) in clinical studies for use in human subjects.

In certain embodiments of the present invention, a stent coated with a polymer-paclitaxel conjugate comprises:
It can be used to prevent restenosis (closure of an artery following balloon angioplasty). Recent results in clinical trials using balloon-expandable stents in coronary angioplasty show that standard balloon angioplasty (Serr)
uys et al., 1994) showed a significant advantage in patency and reduced restenosis. According to the hypothesis of response to injury, neointima formation is
Associated with increased cell proliferation. At present, the general view supports that the critical process leading to vascular lesions in both spontaneous and accelerated atherosclerosis is the proliferation of smooth muscle cells (SMC) (Phi
llips-Hughes and Kandapa, 1
996). Since the proliferation of the SMC phenotype after arterial injury is very similar to that of neoplastic cells,
It is possible that it could be useful to prevent MC accumulation. Stents coated with an antiproliferative agent linked to a polymer capable of releasing these agents in sufficient concentration over time, therefore, prevent hyperplasia of the intima and media from entering the lumen, thereby reducing restenosis. Decrease.

Since paclitaxel has been shown to suppress collagen-induced arthritis in a mouse model (Oliver et al., 1994), the formulations of the present invention also provide autoimmune and / or rheumatoid arthritis. It was intended to be useful in the treatment of inflammatory diseases.
Binding of paclitaxel to tubulin makes this agent a potent inhibitor of eukaryotic cell replication by shifting the equilibrium to stable microtubule polymers and blocking cells in the late G2 mitotic phase. Some mechanisms may relate to the suppression of arthritis by paclitaxel. For example, the stage-specific cytotoxic effects of paclitaxel can affect the rapid proliferation of inflammatory cells, and furthermore, paclitaxel has mitosis, migration, chemotaxis, intracellular trafficking and neutrophil H ₂ O ₂ Inhibits production. In addition, paclitaxel may have anti-angiogenic activity by blocking coordinated endothelial cell migration (Oliver et al., 1).
994). Accordingly, it was contemplated that the water-soluble polyamino acids conjugated to paclitaxel of the present invention would be useful in treating rheumatoid arthritis. The polymer conjugated formulations disclosed herein also provide the advantage of controlled release of the drug and greater solubility. It is also an aspect of the treatment of arthritis that the formulation can be injected or implanted directly into the affected joint.

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 dispersion. In all cases,
The form should be sterile and should be liquid 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. Carriers include, for example, water, ethanol, polyol (eg, glycerol, propylene glycol,
And liquid polyethylene glycols and the like), suitable mixtures thereof, and vegetable oils. Prevention of the action of microorganisms includes, for example, parabens,
It can be achieved by various antibacterial and antifungal agents such as 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.

A sterile injectable solution, if necessary, is prepared by incorporating the required amount of the active compound in the appropriate other solvents with the various other ingredients enumerated above, followed by sterile injection. Filtered. 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 method of preparation is
Vacuum drying and lyophilization techniques to produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antibacterial 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 vehicle or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase “pharmaceutically acceptable” also refers to whole molecules and compositions that do not cause an allergic or similar untoward reaction when administered to an animal or human.

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 particularly suitable for intravenous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.

The following examples are included to illustrate preferred embodiments of the present invention. The techniques disclosed in the following examples have been found by the inventors to work satisfactorily in the practice of the present invention, and it should be obvious to those skilled in the art to demonstrate the techniques, and therefore are preferred in their practice. It can be considered to compose the style. However, in light of this disclosure, those skilled in the art will recognize that many modifications may be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention. Understand that.

[0079]

EXAMPLES (Example 1) (Paclitaxel polyglutamate (PG-TXL))
This example relates to the first studies on the conjugation of paclitaxel to a water-soluble polymer (poly (l-glutamic acid) (PG)) and the efficacy of the preparation against various tumors in mice and rats. The potential of water-soluble polymers used as drug carriers is well established (Kopesk, 1990; Maeda).
And Matsumura, 1989).

(Paclitaxel polyglutamate (PG
Synthesis of -TXL) PG was selected as a carrier for paclitaxel. Because it can be easily degraded by lysosomal enzymes, is stable in plasma, and contains sufficient functional groups for drug adsorption. Some anti-neoplastic agents (Adriamycin (Van Heesw)
ijk et al., 1985; Hoes et al., 1985), cyclophosphamide (Hirano et al., 1979), Ara-.
C (Kato et al., 1984) and melphalan (Mo)
rimoto et al., 1984)) were conjugated to PG. However, polyaspartic acid can be conjugated to an anti-tumor agent using the reaction scheme for PG-TXL described herein.

The reaction scheme is shown in FIG. 1B. Poly (1
-Glutamic acid) (PG) sodium salt
(St. Louis, MO). Depending on the viscosity,
This polymer has low angle laser light scattering (LALLS)
Had a molecular weight of 36,200 and a number average molecular weight (Mn) of 24,000, as determined by HPLC. The lot-specific polydispersity (M _w / M _n ) is 1.15, where M
_w is the weight average molecular weight. PG sodium salt (MW
34K, Sigma, 0.35 g) was first converted to its protonated PG. P of aqueous PG sodium salt solution
H was adjusted to 2.0 using 0.2M HCl. The precipitate was collected, dialyzed against distilled water and freeze-dried 0.2%.
9 g PG was obtained.

Anhydrous N, N-dimethylformamide (DM
PG in F) (75 mg, repeating unit FW 17)
0, 0.44 mmol) to a solution (1.5 ml).
mg of paclitaxel (0.026 mmol, molar ratio P
G / paclitaxel = 17), 15 mg of dicyclohexylcarbodiimide (DCC) (0.073 mmol)
l) and traces of dimethylaminopyridine (DMA
P) was added. Paclitaxel for Hande Tec
h (Houston, Tex.) and its purity was 99% and higher than confirmed by HPLC assay.

The reaction was carried out at room temperature for 12 to 18 hours. Thin layer chromatography (TLC, silica) is based on paclitaxel (Rf = 0.55) polymer conjugate (R
f = 0, complete conversion to CHCl ₃ / MeOH = 10: 1). The mixture was poured into chloroform to stop the reaction. Collecting the resulting precipitate,
It was then vacuum dried to produce 70 mg of the polymer-drug conjugate. By varying the weight ratio of paclitaxel to PG starting material, various paclitaxel concentrations of the polymeric conjugate can be synthesized.

The sodium salt of the PG-TXL conjugate was obtained by dissolving the product in 1.0 M NaHCO ₃ . PG
Aqueous solution of TXL with low molecular weight contaminants and excess N
Dialyzed against distilled water to remove aHCO ₃ salts (MWCO 10,000). Lyophilization of the dialysate yielded 98 mg of the product as a white powder. The paclitaxel content of the polymeric conjugate was 20-22% (w / w) as determined by UV. Yield: 9
8% (conversion to polymer-bound paclitaxel, UV).
Solubility in water is greater than 20 mg paclitaxel / ml. A similar method can be used to synthesize PG-TXL with higher paclitaxel content (up to 35%) by simple increase in the ratio of paclitaxel and PG used.

(Paclitaxel polyglutamate (PG
-TXL) Characterization) The UV spectra were taken as a reference using the same concentration of PG aqueous solution and Beckman
Obtained on a DU-70 spectrophotometer. PG-TXL is 22
It showed paclitaxel-specific absorption with a λ _max shift from 8 nm to 230 nm. The concentration of paclitaxel in the PG-TXL conjugate was determined by measuring the concentration of paclitaxel in absorbance at 228 nm with the polymer conjugate in water at 230 nm and the free drug in methanol at 228 nm, based on a standard curve generated with known concentrations of paclitaxel in methanol. Have the same molar absorbance and both are Lamber
Estimation was performed assuming that t Beer's method was followed.

¹ H-NMR spectra were recorded at D ₂ O on a GE model GN500 (500 MHz) spectrophotometer. Both the PG and paclitaxel moieties were distinguishable. The coupling of the polymer conjugated to paclitaxel was too poorly degraded to measure with sufficient accuracy. The resonance from 7.75 to 7.36 ppm may be due to the resonance of the paclitaxel aromatic compound, 6.38 ppm (C ₁₀ -H), 5.97 ppm (C
_{13 -H), 5.63ppm (C 2} '-H, d), 5.5
5~5.36ppm (C ₃ '-H, and C ₂ -H,
_{m), 5.10ppm (C 5 -H} ), 4.39ppm
_{(C 7 -H), 4.10 (} C 20 -H), 1.97ppm
(OCOCH ₃₎ and 1.18~1.20ppm (C
The resonance at CH ₃ ) is experimentally assigned to the aliphatic component of paclitaxel. Other resonances were masked by PG resonances. 4.27 ppm (H-α), 2.21 p
The PG resonance at pm (H-γ) and 2.04 ppm (H-β) is consistent with the spectrum of pure PG.
The peak at 5.63 ppm corresponds to the C-
Can be experimentally assigned to the 2 'proton, but 4.78 pp
The C-2 'proton of unsubstituted paclitaxel at m is also present, suggesting that the resulting conjugate may contain paclitaxel substitution at both C-2' and C-7 positions. A 100 mg / ml solution of the conjugate yields a clear, viscous, and still fluid liquid. This procedure consistently produced a PG-TXL conjugate containing 20% weight paclitaxel (i.e., about 7 molecules of paclitaxel were attached to each polymer chain).

(Polyglutamic acid-paclitaxel (P
G-TXL) Gel Permeation Chromatography Study) Gel Permeation Chromatography (GPC)
Were characterized by relative molecular weight. The GPC system is L
DC gradient master, PL gel GPC column and Wat
It consists of two LDC Model III pumps combined with an ers990 photodiode array detector. The eluate (DMF) was flowed at 1.0 ml / min using ultraviolet (UV) detection at a setting of 270 nm. TSK suitable for analysis of water-soluble polymers due to PG-TXL sodium salt
Using a gel column and using this system at 0.2 mM
Elution was performed at 1.0 ml / min with PBS (pH 6.8). P
Conjugation of paclitaxel to G was demonstrated by a shift in retention time from 6.4 minutes for PG to 5.0 minutes for PG-TXL conjugate, as shown by PG-TX.
L resulted in an increase in molecular weight. The crude product contains low molecular weight contaminants (retention times 8.0-10.0 minutes, and 11.3 minutes), which means that PG-TXL is collected in its own sodium salt, followed by dialysis Can be more effectively removed.

(Polyglutamic acid-paclitaxel (P
G-TXL) Conjugate Hydrolysis) To gain insight into the release kinetics of paclitaxel and related molecular species from PG-TXL, the hydrolysis stability of PG-TXL was measured using PBS.
Tested at different pHs. High performance liquid chromatography (HPLC) showed that incubation of PG-TXL in PBS solution produced paclitaxel and other species, including those more hydrophobic than paclitaxel (metabolite 1). The fact that all of these species were derived from paclitaxel indicates that PG- [ ³ H] TXL
Was confirmed through a similar decomposition experiment using Based on its retention time on HPLC, Metabolite 1 is probably the biologically active isomer of paclitaxel, 7-epipaclitaxel. In fact, metabolite 1 recovered in PBS
At pH 7.4 and pH 9.5 exceeded the amount of paclitaxel after 5 days and 1 day incubation, respectively (FIG. 6). In FIG. 6, the vertical circles indicate the percentage of released paclitaxel, and the horizontal circles indicate the metabolite 1 produced. At pH 5.5 and 7.4, the release profile of Metabolite 1 showed pseudo-zero order kinetics and exhibited a lag time varying from 3 days (pH 5.5) to 7 hours (pH 7.4). , Suggest that metabolite 1 is a secondary product. Clearly, PG-TXL is more stable in acidic solutions than in basic solutions.

(In vivo antitumor activity) All animal studies were D. Anderson Cancer Cen
Performed at ter animal facility according to facility guidelines. C3H / Kam mice were transferred to Depertme
nt of Experimental Radiat
Ion Oncology was bred and maintained in a pathogen-free facility.

The delay in tumor growth induced by PG-TXL was measured in breast ovarian cancer (OCA-I) implanted in C3Hf / Kam mice. All tumors were genetically syngeneic with this system. 5x solitary tumor
By injecting 10 ⁵ mouse ovarian tumor cells (OCA-I), breast tumors (MCa-4), liver cancer (HCa-I) or fibrosarcomas (FSa-II), female C3H
/ Kam mice (25-30 g) in the right thigh muscle. In a parallel study, female Fischer3
0.1 ml PBS for 44 rats (125-150g)
1.0 × 10 ⁵ viable 13762F tumor cells were inoculated. Mouse tumors of 500 mm ³ (10 in diameter)
mm) or when the rat tumor grows to 2400
Treatment began when it grew to mm ³ (17 mm average diameter).

PG-TXL was dissolved in physiological saline (10
(in mg equivalents of paclitaxel / ml), and paclitaxel were dissolved in CremophorEL® vehicle (6 mg / ml). Data are presented as mean ± standard deviation of tumor volume. In control studies,
Saline (0.6 ml), Cremophor vehicle (50/50 Cremophor / ethanol diluted (1: 4) in saline), PG in saline
Solutions and paclitaxel and 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. Single dose of PG-TXL in saline or Cr
A single dose of paclitaxel in the emophorEL vehicle is given as 40-160 mg equivalent paclitaxel / kg
They were given at doses that varied with body weight. Tumor growth was measured daily by measuring three orthogonal diameters (FIGS. 7A, 7B,
7C, 7D and 7E). The volume of the tumor can be calculated by the formula
C) / 2. Absolute growth retardation (AGD) in mice indicates that tumors treated with various drugs in mice began to grow from 500 to 2,000 mm ³ from day 5 on tumors treated with saline control.
It is defined as 00 minus the time to grow to 2,000 mm ³ . When tumor size reached 2000 mm ³ , tumor growth delay was calculated; tumors were approximately 2500 mm ³
At the time, the mice were sacrificed. PG-TXL group (n =
6) and 7), the other respective groups were (n = 5). Table 2 summarizes the acute toxicity of PG paclitaxel in rats compared to paclitaxel / Cremophor. Table 3 shows MCa-4 and FSa-II in mice.
And data on the effect of PG-TXL on HCa-I tumors. This data is also summarized in FIGS. 7A-7E. In FIG. 7A, the drug was injected intravenously in a single dose. Data are shown as mean ± standard deviation of tumor volume. In mice bearing OCA-1 tumors, open squares, PG
Control (800 mg / kg; n = 9);
Paclitaxel (80 mg / kg; n = 7); open triangle,
Paclitaxel (80 mg / kg) and PG (800
mg / kg; n = 5); solid circle, PG-TXL (80 mg
Equivalent paclitaxel; n = 6) or open circle, PG-T
XL (160 mg equivalent paclitaxel / kg; n = 2
6) was injected. In FIG. 7B, open squares indicate PG control (220 mg / kg; n = 7), closed triangles indicate paclitaxel (20 mg / kg; n = 5), and open triangles indicate paclitaxel (40 mg / kg; n = 7). Indicates that
Closed circles indicate PG-TXL (20 mg equivalent paclitaxel / k
g; n = 5), and open circles indicate PG-TXL (40 mg or 60 mg equivalent paclitaxel / kg; n = 9). In FIG. 7C, open squares indicate the response to a single intravenous dose of saline, open triangles indicate the response to a single intravenous dose of PG (0.6 g / kg);
A response to G-TXL (40 mg / kg) is shown, and white diamonds indicate PG-TXL (60 mg equivalent paclitaxel /
kg), the open circles indicate PG-TXL (12
0 mg / kg). In FIG. 7D, open squares indicate the response to a single intravenous dose of saline,
Open diamonds indicate response to a single intravenous dose of PG (0.8 g / kg); open circles indicate paclitaxel (80 mg / kg).
kg), the open triangles indicate PG-TXL (1
3 shows the response to 60 mg equivalent paclitaxel / kg). In FIG. 7E, open squares indicate response to a single intravenous dose of saline, open triangles indicate response to a single intravenous dose of PG (0.8 g / kg); open circles indicate PG-
The response to TXL (80 mg / kg) is shown. The open triangle indicates PG-TXL (160 mg equivalent paclitaxel / k).
g) shows the response to

[0092]

[Table 2]

* The drug is Fis with 13762 tumor
cher rats (130 g female) were administered intravenously in a single injection. a: PG-TXL solution was prepared by dissolving the conjugate in saline (8 mg equivalent of paclitaxel / ml). The injection volume of 60 mg / kg was 0.975 ml per rat. b: Paclitaxel Cremophor solution was mixed with ethyl alcohol and Cremophor (30 mg / m
It was prepared by dissolving paclitaxel in a 1: 1 mixture of 1). The stock solution was further diluted with saline before injection (1: 4). The final concentration of paclitaxel in this solution was 6 mg / ml. The injection volume of 60 mg / kg was 1.3 ml per rat. c: PG solution was prepared by dissolving the polymer in physiological saline (22 mg / ml). The injection dose is 0.3 g / k
g (1.8 ml per rat), which is 60 g
It was equivalent to mg / Kg paclitaxel. d: Cremophor vehicle was prepared by diluting a mixture of ethyl alcohol and Cremophor (1: 1) with physiological saline (1: 4).

[0094]

[Table 3]

A: A mouse having a tumor of 500 mm ^{3 in} the right limb was treated with PG-TXL (40 to 160 m
g of paclitaxel / kg) or Cremoph
Oral single doses of paclitaxel in vehicle were treated with a single injection. Control animals were saline (0.6 ml), Cremophor vehicle (0.5 ml), saline PG solution, or PG
(G / Kg) and paclitaxel (80 mg / kg)
Treated. b: Tumor growth was determined by three orthogonal diameter measurements with calipers daily and the volume was calculated as (A × B × C) / 2. The numbers in parentheses indicate the number of mice used in each group. Time days of growth from 500 mm ³ to 2000 mm ³ are shown as mean ± standard deviation. c: Absolute growth retardation (AGD) indicates that the tumors treated with various drugs grew from 500 hours to 2,000 mm ³ days,
It is defined as 00 minus the time to grow to 2,000 mm ³ . d: Time to grow from 500 mm ³ to 2000 mm ³ Day was compared for treated and saline groups using Student's t-test. The P value was significant when it was 0.05 or less on both sides.

These studies have revealed two important findings. First, like paclitaxel, water-soluble PG-
There is a tumor-to-tumor variability of the antitumor effect of TXL. PG-T
XL is quite effective against MCa-4 and OCA-1 tumors. Second, PG-TXL is MCa-
4. More effective than paclitaxel per mg paclitaxel in its case against HCa-I and OCA-1 tumors, and its maximum tolerated dose (MTD)
Is remarkably powerful.

In a parallel study, the antitumor activity of PG-TXL in Fischer rats with well-established rat mammary tonsillar tumor 13762F was tested. Female Fischer 344 rat (125-150 g)
Was inoculated with 1.0 × 10 ⁵ viable 13762F tumor cells in 0.1 ml of PBS. Once the tumors reached a mean volume of 2000 mm ³ (mean diameter, 1.6 cm), the animals were treated in the same procedure as described above. Tumor growth was determined daily by tri-orthogonal tumor diameter measurements. Tumor volume was calculated according to the formula (A × B × C) / 2. Single dose of PG-TXL in saline or Cre
Paclitaxel in the mophorEL® vehicle was administered at a dose of paclitaxel / kg (body weight) varying from 20 to 60 mg equivalents. In control tests, saline, CremophorEL®
Vehicle [50/50 Cremophor / ethanol diluted with saline (1: 4)] and PG solution in saline and paclitaxel with PG were used. In addition, complete tumor eradication with MTD of PG-TXL (60 mg equivalent paclitaxel / kg) was observed. 4
PG- at a low dose of 0 mg equivalent paclitaxel / kg
Administration of TXL also resulted in complete tumor regression (FIG. 7).
B). Conversely, the MTD of paclitaxel in CremophorEL® was less than 20 mg / kg. This dose of paclitaxel results in tumor growth delay of only 5 days (tumor growth delay is defined as the time at which the tumor treated with the study drug grows from 2,000 to 10,000 mm ³ , treated with saline control). Tumor
The time to grow from 2,000 to 10,000 mm ³ is defined as days minus days. ), While the same paclitaxel equivalent dose of PG-TXL resulted in a 23-day tumor growth delay (FIG. 7B).

(Study of Nude Mice Injected with Human Breast Cancer and Treated with PG-TXL) 2 × 10 ⁶ MDA-435-Lung2 cells (MDA-MB-435 human breast cancer cells) were placed on the mammary fat pad of nude mice. Variant of strain)
Was injected. When this tumor reaches an average diameter of 5 mm,
On day 27 (after tumor injection), mice were treated with PG-TXL or various control intravenous injections (see Table 4). Tumor measurements were taken weekly. Tumors that reached 1.5 cm were surgically removed. All mice are 120
At day killed, residual tumors were removed and weighed. Testing the mice for metastasis, processing the lungs for histology,
The organ-only sections were scored for the presence of micrometastases.

[0099]

[Table 4]

A: number of mice with 5 mm tumor at the time of treatment / number of mice injected b: average weight of tumor removed at necropsy c: number of tumors regressed at necropsy d : Number of mice with lung metastases (either visually or in tissue preparation) / number of mice with tumor. Some discrepancies between tumor removal on this column and the number of mice bearing tumors were for unrelated reasons (eg, Staphylococcus abscess development)
By animal sacrifice or death. PG-TXL 1
One mouse in the 20 mg group died due to extreme weight loss after treatment; otherwise there were no apparent treatment-related deaths. The nude mouse is 160m of PG-TXL
g / kg equivalent could not be tolerated.

120 mg equivalents / kg of paclitaxel drug, PG conjugated paclitaxel (PG-TXL)
The results of a study that administered a single bolus of MDA-4
It is clear that 35 cancer cell lines respond to the drug and that this formulation of the drug is much better tolerated than when Cremophor is the vehicle.

In a breast cancer study using MDA-MB-435, only higher doses of PG-TXL inhibited the growth rate of breast fat pad tumors. From the growth curve,
It was evident that tumor growth had recovered approximately 30 days after a single dose of the conjugate. However, the growth curve is P
There is no indication that there was much tumor regression in the G-TXL 120 mg / kg group. As shown in Table 3, the frequency of lung metastases in mice with residual tumor was also reduced. Although the number of mice in this study is small, they suggest that the treatment was effective in reducing both local tumor growth and metastatic frequency.

In this study design, it was determined whether the lower frequency of metastases was due to a reduction in tumor burden at the primary site or a direct effect on any micrometastases that had already been established at the time of treatment. It is impossible to do.

In Vivo Treatment of Human Breast Cancer with Multiple Injections of PG-TXL To test the effect of multiple injections of PG-TXL, 2 ×
10 ⁶ , MDA-435-Lung 2 cells (a modified human breast cancer cell line) were injected. When the tumors reached an average diameter of 5 mm, treatment was started and a total of three injections were taken for 14 injections.
Repeated at day intervals (24, 38, 52 days). Tumor measurements were taken weekly. The mice were injected 105 days after tumor cell injection.
Days were killed and tumor weight and metastasis frequency were recorded. Lungs were processed for histology and single sections scored for the presence of microtranscripts. Table 5 shows the results.

[0105]

[Table 5]

Legends: a: number of mice with 5 mm tumor at the time of treatment / number of injected mice b: mean weight of tumor (± SEM) c: number of tumors regressed at necropsy d: macroscopic or microscopic Visual, number of mice with lung metastasis / number of mice with tumor e: P value by one-tailed t-test comparing tumor weight of treated mice with control PG group.

(In Vivo Treatment of Human Ovarian Cancer Using PG-TXL Conjugate) A human ovarian cancer cell line, S
KOV3ipl was inoculated intraperitoneally. 5 days after tumor injection
Mice were injected intravenously with PG paclitaxel (PG-TXL) at a concentration of 120 mg eq / kg or 160 mg eq / kg of paclitaxel. Initially, these injections were planned to be repeated every 7 days, but 160 m
A single inoculation of the g / Kg dose killed 5 out of 10 mice.
Only the 120 mg / Kg group was inoculated three times. 98 tests
Ended in days and any surviving mice were killed. The results are shown in FIG. In FIG. 14, mice were injected with PG-paclitaxel (PG-TX) 5 days after tumor injection.
L) or PG control was injected intravenously. PG-
TXL injections were administered every 7 days in the 120 mg / kg group (closed triangles), but not in the 160 mg / kg group. Closed squares indicate untreated mice. The upward triangle indicates the response to multiple intravenous doses of PG. The downward triangle indicates the response to an intravenous dose of PG-TXL (120 mg equivalent paclitaxel / kg). Black diamonds are PG-TXL (160 mg equivalent paclitaxel / k
2 shows the response to the intravenous dose of g).

The median survival value in the group is currently:
No treatment = 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 mice that died from the first toxicity of this drug].

[0109]

[Table 6]

Legend: a: frequency of tumors / number of injected mice b: days of median survival time c: frequency of ascites / number of mice with tumor d: mean volume of ascites fluid (and standard deviation) e: these mice Is PG-paclitaxel, 160m
g only received a single dose of g / kg and did not include mice that died within 5 days of treatment. f: Mice that died at 34 days had minimal tumor burden but were not paraplegic (toxic Is there?).

PG-TXL 120 mg / kg
Intraperitoneal SKOV3 compared to mice inoculated alone
ipl (a human ovarian cancer cell line that overexpresses HER2 / neu) significantly prolonged mouse survival. Multiple doses and / or escalating doses of the conjugate, in addition to prolonging survival, can significantly reduce tumor frequency.

In the above nude mouse study, the growth curves show that breast cancer growth is increased by paclitaxel, especially at higher doses conjugated with PG, and that tumor size continues to increase by about one month after treatment. . The second (or third) treatment may flatten tumor growth,
Or it may result in more tumor regression. The growth curves did not include the regressed tumor (as shown in Table 4),
At 50% of mice treated with the highest dose of PG-TXL had shrunk / disappeared and at the end of the study only 1 of 4 animals with progressively growing tumors had micrometastasis in the lung . Therefore, treatments that reduce primary tumor growth also reduced the frequency of metastases. Cremopho
The frequency of metastases in all other treatment groups, including the r and PG control groups, is lower than in the PBS control group, thus arguing that reducing the frequency of metastases in the Taxol ^™ / Cremophor group is a significant finding. Is probably not valid.

(Example 2: DTPA-paclitaxel) (Synthesis of DTPA paclitaxel)
Solution of paclitaxel in 100 ml (2.2 ml)
g, 0.117 mmol) in diethylenetriaminepentaacetic anhydride (DTPAA) (210 mg, 0.58
5 mmol) was added. The reaction mixture was stirred at 4 ° C. overnight. The suspension is filtered (0.2 μm Millipore
The mixture was filtered to remove unreacted DTPA anhydride. The filtrate was poured into distilled water, stirred at 4 ° C. for 20 minutes, and the precipitate was collected. The crude product was purified by preparative TLC on _C18 silica gel plates and developed in acetonitrile / water (1: 1). Paclitaxel had an Rf value of 0.34. The upper band of paclitaxel with an Rf value of 0.65 to 0.75 was scraped off and eluted with an acetonitrile / water (1: 1) mixture and the solvent was removed.
15 mg of DTPA-paclitaxel was obtained as the product (yield 10.4%): melting point:> 226 ° C. (decomposition). The ultraviolet spectrum (sodium salt in water) showed a maximum absorption at 228 nm, which was also characteristic of paclitaxel. Mass spectrum: (FAB) m / e 1229
(M + H) ⁺ , 1251 (M + Na), 1267 (M +
K). ^{In the 1} H NMR spectrum (DMSO-d ₆ ), the resonances of DTPA NCH ₂ CH ₂ N and CH ₂ COOH were as a complex series of signals at δ 2.71 to 2.96 ppm and at δ 3.42 ppm, respectively. Appeared as multiplets. 4.10 in paclitaxel
The C7-H resonance in ppm shifted to 5.51 ppm, suggesting esterification at position 7. The rest of the spectrum was consistent with the structure of paclitaxel.

The sodium salt of DTPA paclitaxel can also be obtained by adding a solution of DTPA-paclitaxel in ethanol to an equivalent amount of 0.05 M NaHCO ₃ ,
Thereafter, by freeze-drying, a water-soluble solid powder (solubility> 20 mg equivalent paclitaxel / ml) was obtained.

(Hydrolysis stability of DTPA paclitaxel :) The hydrolysis stability of DTPA paclitaxel was studied under accelerated conditions. Briefly, 1 mg of DTPA paclitaxel was dissolved in 1 ml of 0.5 M aqueous NaHCO ₃ (pH 9.3) and analyzed by HPLC. This HPLC system is a Waters 150 x 3.9 (inner diameter) mm Nova-Pak column packed with 4 μm C18 silica gel, Perkin-E
lmer isocratic LC pump, PEN
elson 900 series interface, Sp
It consists of an Etra-Physics UV / Vis detector and a data station. Eluate (acetonitrile / methanol / 0.02M ammonium acetate = 4:
1: 5) was run at 1.0 ml / min with UV detection at 228 nm. Retention times for DTPA-paclitaxel and paclitaxel were 1.38 and 8.83 minutes, respectively. Peak areas were quantified and compared to a standard curve to determine the concentration of DTPA-paclitaxel and paclitaxel. DT in 0.5 M NaHCO ₃ solution
The estimated half-life of PA-paclitaxel is about 16 days at room temperature.

(Effect of DTPA paclitaxel on in vitro growth of B16 mouse melanoma cells) Cells were plated in a 24-well plate at 2.5 × 10 ⁴ cells / ml.
And 10% fetal bovine serum.
In 0:50 Dulbecco's modified minimal essential medium (DEM) and F12 fold locations, 24 hours at 37 ° C., were grown in 97% humidified atmosphere of 5.5% CO _2. This medium is then
Replaced with fresh medium containing paclitaxel or DTPA paclitaxel at concentrations ranging from 5 × 10 ⁻⁹ M to 75 × 10 ⁻⁹ . After 40 hours, the cells were released by trypsinization and counted on a Coulter counter. The final concentration of DMSO (used to dissolve paclitaxel) and 0.05 M sodium bicarbonate (used to dissolve DTPA paclitaxel) in the cell medium was less than 0.01%. This amount of solvent had no effect on cell growth as determined by control studies.

The effect of paclitaxel on the growth of B16 melanoma cells is shown in FIG. After incubation at various concentrations for 40 hours, DTPA paclitaxel and paclitaxel were compared to cytotoxicity. IC for paclitaxel and DPTA paclitaxel
₅₀ is 15 nM and 7.5 nM, respectively.

(Anti-Tumor Effect on Breast Carcinoma (MCa-4) Tumor Model) Female C3Hf / Kam mice were inoculated into the right thigh muscle using breast carcinoma (MCa-4) (5).
× 10 ⁵ cells / mouse). When the tumors grew to 8 mm (about 2 weeks), a single dose of paclitaxel or DTPA paclitaxel was administered at 10, 20 and 40 mg.
It was given in equivalent paclitaxel / kg body weight. In a control study, saline and saline diluted (1: 4) absolute alcohol / Cremophor50 /
50 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 the tumor was approximately 15 mm.

The tumor growth curve is shown in FIG. Compared to controls, both paclitaxel and DTPA paclitaxel showed an antitumor effect at a dose of 40 mg / kg. This data was also analyzed to determine the average number of days for tumors reaching a diameter of 12 mm. Statistical analysis showed that DPTA paclitaxel at a dose of 40 mg / kg compared to saline-treated controls,
Tumor growth was significantly delayed (p <0.0
1). The average time for a tumor reaching a diameter of 12 mm is DT compared to 9.4 days for paclitaxel.
It was 12.1 days for PA paclitaxel (FIG. 4).

(Radiolabeling of DTPA Paclitaxel with ¹¹¹ In) In a 2 ml V vial, add 40 μl of 0.
6 M sodium acetate (pH 5.3) buffer, 40 μl 0.06 M sodium citrate buffer (pH 5.5),
20 μl DTPA paclitaxel solution in ethanol (2% w / v), and 20 μl ¹¹¹ InCl ₃ solution (1.0 mCi) in sodium acetate buffer (pH 5.5)
Was added continuously. After a 30 minute incubation period at room temperature, the mixture was washed with C18 Sep-Pac using saline and then ethanol as mobile phase.
¹¹¹ labeled by passing through a cartridge
In-DTPA paclitaxel was purified. Free ¹¹¹ I
n-DTPA (<3%) was removed by saline, while ¹¹¹ In-DTPA paclitaxel was recovered by washing with ethanol. The ethanol was evaporated under nitrogen gas and the labeled product was reconstituted in saline. Radiochemical yield: 84%.

[0121] ⁽¹¹¹ In-DTPA analysis of paclitaxel) using HPLC, and analysis of the reaction mixture and purity of ¹¹¹ an In-DTPA paclitaxel. This system is
An LDC binary pump consisted of a 100 × 8.0 mm (inner diameter) Waters column packed with ODS 5 μm silica gel. The column was run at a flow rate of 1 ml / min with a gradient mixture of water and methanol (0% over 15 minutes).
(Gradient of ~ 85% methanol). This gradient system was combined with a NaI ore detector and a Spectra-Ph
ysics UV / Vis detector. As demonstrated by HPLC analysis, Sep-
Purification with a Pak cartridge is ¹¹¹ In-DTPA
, Which had a retention time of 2.7 minutes. ¹¹¹ In-DTPA was probably derived from trace amounts of DTPA contaminants in DTPA paclitaxel. ¹¹¹
The radiochromatogram of In-DTPA paclitaxel correlated with its UV chromatogram, indicating that
The peak at 2.3 minutes indicates that it is indeed the target compound. Under the same chromatographic conditions, paclitaxel had a retention time of 17.1 minutes. The radiochemical purity of the final preparation was 90% as determined by HPLC analysis.

(Whole body scintigraphy) Female C3Hf
/ Kam mice were inoculated into the right thigh muscle using breast carcinoma (MCa-4) (5 × 10 ⁵ cells / mouse). When the tumors grew to 12 mm, the mice were split into two groups. In Group I, mice were injected by intraperitoneal injection of sodium pentobarbital followed by ¹¹¹ In
-DTPA paclitaxel (100-200 mCi) was anesthetized via the tail vein. A gamma camera with a medium energy collimator was placed over the mouse (1
3) per group. A series of 5 minute acquisitions, 5 minutes after injection, 3 minutes
Collected at 0, 60, 120, 240 and 24 hours. In Group II, the same procedure was followed except that mice were injected with ¹¹¹ In-DTPA as a control. FIG. 5 shows ¹¹¹ In-DTPA and ¹¹¹ In-DTPA.
Figure 4 shows a gamma scintigraph of an animal injected with In-DTPA paclitaxel. In the figure, arrows indicate tumors. ¹¹¹ In-
DTPA was characterized by rapid clearance from plasma, minimal retention in the kidneys and rapid and high excretion in urine with negligible retention in tumors, liver, intestines and other organs or body parts. In contrast, ¹¹¹ I
n-DTPA paclitaxel showed a pharmacological profile similar to brain (Eisemen et al., 1994). Radioactivity in the brain is negligible. Liver and kidney had the largest tissue: plasma ratio. Hepato-biliary elimination of radiolabeled DTPA paclitaxel or its metabolites is one of the major pathways for clearance of drugs from the blood. Unlike paclitaxel, significant amounts of ¹¹¹ In-DTPA paclitaxel are also excreted through the kidney, which only plays a minor role in paclitaxel clearance. The tumor is
^It did not have significant incorporation of ¹¹¹ In-DTPA paclitaxel. These results indicate that ¹¹¹ In-DTPA paclitaxel is capable of detecting specific tumors and quantifying ¹¹¹ In-DTPA uptake in tumors, which in turn selects patients for paclitaxel treatment. Demonstrate that can be assisted. In contrast, the smaller PG-TXL conjugate has a different distribution than DTPA paclitaxel and is partially localized to the liver and tumor of the test animal.

Example 3: Polyethylene glycol-paclitaxel synthesis of polyethylene glycol-paclitaxel (PEG-paclitaxel) The synthesis was completed in two steps. First, 2'-succinyl-paclitaxel was converted to the reported procedure (Deutsch et al., 1989).
Was prepared according to the following procedure. Paclitaxel (200mg,
0.23 mmol) and succinic anhydride (288 mg,
2.22 mmol) in anhydrous pyridine (6 ml)
The reaction was performed at room temperature for 3 hours. The pyridine was then evaporated and the residue was treated with water, stirred for 20 minutes and filtered. This precipitate was dissolved in acetone, water was added slowly, and fine crystals were collected.
180 mg of 2'-succinyl-paclitaxel were obtained. PEG-paclitaxel was converted to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EED
Q) Synthesized by a mediated coupling reaction. 2'-
Succinyl-paclitaxel (160 mg, 0.18 m
mol) and methoxypolyoxyethyleneamine (P
EG-NH _2, molecular weight 5000,900mg, 0.18
mmol) in methylene chloride solution.
g, 0.72 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was chromatographed on silica gel using ethyl acetate followed by chloroform-methanol (10: 1). This gave 350 mg of the product. ¹ H NMR (CDC
l ₃ ) δ 2.76 (m, succinic acid, COCH ₂ CH ₂ C
O ₂ ), δ 3.63 (PEG, OCH ₂ CH ₂ O), δ
4.42 (C7-H) and δ 5.51 (C2′-
H). The UV absorption at 288 nm is maximal, which is also characteristic of paclitaxel. Attachment to PEG greatly improved the water solubility of paclitaxel (> 20).
mg equivalent paclitaxel / ml water).

(Hydrolysis stability of PEG-paclitaxel) PEG-paclitaxel was added to a phosphate buffer (0.0
1M) at various pHs at a concentration of 0.4 mM and the solution was incubated at 37 ° C. with gentle agitation. At selected time intervals, aliquots (200 μl) were removed and lyophilized. The obtained dry powder is subjected to gel permeation chromatography (GPC
Redissolved in methylene chloride for analysis). The GPC system includes 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.
It consisted of a detector and a data station. The eluate (methylene chloride) was flowed at 1.0 ml / min with a UV detector set at 288 nm. Retention times for PEG-paclitaxel and paclitaxel were 6.1 and 8.2 minutes, respectively. The peak area was quantified and the percentage of PEG-paclitaxel remaining and the percentage of paclitaxel released were calculated. pH
The half-life of PEG-paclitaxel determined by linear least squares at 7.4 was 54 minutes. The half-life at pH 9.0 was 7.6 minutes. PEG at pH 7.4
The release profile of paclitaxel from paclitaxel is shown in FIG. FIG. 8 shows the release profile (x) of paclitaxel from PEG-paclitaxel (open circles) at pH 7.4.

(Cytotoxicity study of PEG paclitaxel using B16 mouse melanoma cells in vitro) DTP
Following the procedure described in the cytotoxicity study with A-paclitaxel, melanoma cells are seeded in 24-well plates at a concentration of 2.5 × 10 ⁴ cells / ml and contain 10% fetal bovine serum 50: Grow in 50 Dulbecco's Modified Minimum Essential Medium (DEM) and F12 medium for 24 hours at 37 ° C. in a 97% humidified atmosphere of 5.5% CO ₂ . The medium was then added to 5 × 10 ⁻⁹ M to 75 × 10
Replaced with fresh medium containing paclitaxel or DTPA paclitaxel at concentrations ranging from ^-9 . Forty hours later,
The cells were released by trypsinization and counted on a Coulter counter. D in cell culture
The final concentrations of MSO (used to dissolve paclitaxel) and 0.05M sodium bicarbonate (used to dissolve DTPA paclitaxel) were 0.
It was less than 01%. This amount of solvent had no effect on cell growth as determined by control studies. Further, 5 × 10 ⁻⁹ M to 75 × 10
^The concentration range of PEG used to generate an equivalence paclitaxel concentration of ^-9 also did not affect cell growth.

(Anti-tumor effect of PEG-paclitaxel on MCa-4 tumors in mice) To evaluate the anti-tumor efficacy of PEG-paclitaxel on solid breast tumors, MCa-4 cells (5 × 10 ⁵ cells) were treated with female C3
Hf / Kam mice were injected into the right thigh. As described in Example 1 with DTPA paclitaxel, when the tumors grew to 8 mm (about 2 weeks), a single dose of paclitaxel or PEG-paclitaxel was added to 10, 2
It was given at 0 and 40 mg equivalent paclitaxel / kg body weight. Paclitaxel was first dissolved in absolute alcohol using an equal volume of Cremophor. This stock solution was further diluted (1: 4 by volume) with sterile physiological solution within 15 minutes of injection. PEG-paclitaxel was dissolved in saline (6 mg equivalent paclitaxel / ml) and sterilized filter (Mill).
(ipore, 4.5 μm). Saline, paclitaxel vehicle, anhydrous alcohol diluted with saline: Cremophor (1: 1) (1:
4) and PEG solution in saline (600 mg / kg
Weight) was used in the control study. Tumor growth,
It 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 tumor growth curve is shown in FIG. White square is PE
G in a single intravenous dose of saline solution (60 mg / m
1) shows the response to l), and the closed square is Cremophor.
/ Response to alcohol vehicle; open circles are 40
A single intravenous dose of paclitaxel in mg / kg body weight is shown, filled circles indicate PEG-paclitaxel (40 mg equivalent paclitaxel / kg body weight). At a dose of 40 mg / kg, both PEG-paclitaxel and paclitaxel effectively delayed tumor growth. Paclitaxel was more effective than PEG-paclitaxel, but this difference was not statistically significant. Tumors treated with paclitaxel were 9.4 until reaching a diameter of 12 mm.
Tumors treated with PEG-paclitaxel required 8.5 days, whereas days required 8.5 days. Statistically, these values were significant compared to their corresponding controls (6.7 days for paclitaxel vehicle and 6.5 days for saline solution of PEG (FIG. 4)) (p> 0.05).

Example 5 Poly (L-glutamic acid)-
Paclitaxel (PG-TXL) and the pharmacological properties of paclitaxel The purpose of this study was to compare the pharmacological properties of PG-TXL and paclitaxel in the following abilities: to facilitate the in vitro assembly of tubulin Ability to treat, rat mammary adenocarcinoma cell line 13762F
And the ability to inhibit cell growth against human breast tumor cell lines, induce p53 protein, and rescue paclitaxel-dependent mutant cell lines. The release of paclitaxel from PG-TXL in vivo was measured to determine whether the mechanism of action of PG-TXL could be attributed to free paclitaxel.

Microtubule polymerization using poly-glutamic acid-paclitaxel (PG-TXL) and paclitaxel Tests whether intact PG-TXL has any inherent biological activity in promoting tubulin polymerization. Paclitaxel, PG-TX
L and "aged" PG-TXL were compared for their relative ability to facilitate in vitro assembly of purified bovine brain tubulin. The tubulin assembly reaction was performed at 32 ° C. in PEM buffer (80 m
M PIPES buffer, 1 mM EGTA, 1 mM M
1 mg / ml tubulin (bovine brain, Cytoskeleton Inc., in gCl ₂ , pH 6.9).
Boulder, CO) concentration of drug (1.0 μm)
(M equivalent paclitaxel) and 1.0 mM guanosine 5'-triphosphate (GTP). “Aging” PG-TXL, PG-TXL in PBS
(PH 7.4) at 37 ° C. for 3 days. Tubulin polymerization was followed by measuring the absorbance of the solution at 340 nm over time. Addition of 1 μM paclitaxel to the tubulin solution in assembly buffer caused a clear increase in absorbance due to the increase in light scattering resulting from polymerization of tubulin into microtubules. In contrast, 10 μM paclitaxel equivalent of PG-T
XL had no significant effect on the polymerization. A solution of the conjugate that was "aged" for 3 days in PBS (pH 7.4) at 37 ° C. showed enhanced activity, but the ability to promote tubulin polymerization was also significantly less than paclitaxel. (FIG. 10). In FIG. 10, open squares indicate paclitaxel (1.0 μM), and open triangles indicate PBS (pH
7.4) PG-T incubated at 37 ° C. for 3 days
XL (10 μM equivalent paclitaxel) and open circles indicate freshly dissolved PG-TXL.

(Effect of polyglutamic acid-paclitaxel (PG-TXL) on in vitro growth of rat and human tumor cell lines) PG- observed in animals
To assess whether the superior anti-tumor activity of TXL was due to increased cytotoxicity, PG-TXL and paclitaxel were added to an established rat mammary adenocarcinoma cell line 13
762F were compared for their ability to inhibit cell growth. The effect of PG-TXL on cell proliferation was tested by a plating efficiency assay. Rat 137
62F cells were grown in growth medium (5% fetal calf serum, 50 U /
Seed in a 60 mm dish containing drug concentrations varying from 0 to 200 nM in 200 ml of penicillin and alpha modified minimal essential medium (α-MEM) containing 50 μg / ml streptomycin (200 cells). Six days after growth, the cells were stained with 0.1% methylene blue solution and colonies were counted. Then 50% of the colony formation
The drug concentration producing inhibition (IC ₅₀ ) was calculated. Approximate IC ₅₀ values after 6 days of continuous exposure are as follows: paclitaxel (2nM), PG-TXL ( 100nM),
"Aging" (see below) PG-TXL (50n
M). PG-TXL is about 30 to about paclitaxel itself.
It was 50 times weaker. PG-TXL was placed in phosphate buffered saline (PBS, pH 7.4) at 37 ° C for 3 hours.
When incubated for days to obtain an "aging" solution, only about 10% of paclitaxel was released. Since the "aging" solution is more potent than freshly dissolved PG-TXL, in vitro degradation of PG-TXL or release of the active drug is important for PG-TXL to exert this biological activity. It seems to be. However, even after "aging", PG-TXL is also 25 times less potent than paclitaxel.

In a similar study, the effect of PG-TXL on cell growth of a human breast cancer cell line was tested by MIT assay 3 days after continuous exposure. PG-TXL
Was 8 and 30 times more potent than paclitaxel on the MDA330 and MDA-MB453 cell lines, whereas PG-TXL was MCF7 / her-
Paclitaxel 2 and MCF7 cell lines
One-third and one-third the strength. These results suggest that PG-TXL and paclitaxel have different activities on different cell lines. PG-
TXL may be a product with distinct pharmacological properties different from those of paclitaxel.

(Paclitaxel polyglutamate (PG
-TXL) Ability to support paclitaxel-dependent cell lines in vitro The present inventors studied the ability of PG-TXL to rescue paclitaxel-dependent mutant cell lines. Tax18, a CHO cell line selected for resistance to paclitaxel, is a well-characterized mutant that has been found to require a continuous presence of paclitaxel for cell division. In the absence of drug, a functional spindle device cannot form (Cabral et al., 198
3). The mitotic phase of the cell cycle is extended by subsequent failure to segregate chromosomes and split into daughter cells. Nevertheless, cells continue to progress through the cell cycle and replicate their DNA, resulting in the formation of large polypoid cells that eventually die due to genomic instability (Cabras and Barlo).
w, 1991). Low concentrations of paclitaxel may rescue the mutant phenotype by allowing microtubule assembly and the formation of sufficient spindle fibers. Thus, these cells provide a convenient bioassay for agents that promote microtubule assembly. Expansion of paclitaxel-dependent CHO mutant Tax-18 cells was performed on 24-well tissue culture dishes. Approximately 100 cells were added to wells containing growth medium and an equal concentration of paclitaxel varying from 0 to 1.0 μM. After 6 days of incubation at 37 ° C., the medium was removed and the cells were stained with methylene blue.

Little or no increase in cell number was seen in the absence of drug, but 0.05
Paclitaxel at a concentration of ０．２0.2 μM clearly supported the growth of this cell line. Higher concentrations of paclitaxel are probably toxic to cells. This is because microtubule over-stabilization is observed for normal cells. On the other hand, freshly prepared PG-TXL, even at the highest paclitaxel equivalent concentration tested (1 μM),
Showed little ability to rescue -18 cell proliferation. When PG-TXL was “aged” by incubating it at 37 ° C. for 6 days in PBS, Tax
Its ability to support -18 cell growth was partially restored. These data indicate that PG-TXL did not promote microtubule assembly, and that the in vitro biological activity of "aged" OG-TXL was associated with paclitaxel released from polyglutamic acid-paclitaxel (PG-TXL). Indicates a distribution.

[0134] (in vivo of ^[3 H] paclitaxel PG- ^[3 H] release TXL) to evaluate the pharmacokinetics and release characteristics of paclitaxel in vivo, normal female C3Hf / Kam mice (25-30 g ), 2
0 mg equivalent of [ ³ H] paclitaxel or PG-
One dose of [ ³ H] paclitaxel was injected intravenously into the tail vein. Each mouse received 6 μCi of radiolabeled drug. [ ³ H] Paclitaxel was added to Cremoph
or EL® vehicle, while PG-
[ ³ H] paclitaxel was dissolved in physiological saline. The volume injected into each mouse was 0.2-0.3 ml. 0 minutes, 5 minutes, 15 minutes, 30 minutes, and 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, 4 hours after injection
At 8 hours, animals were sacrificed and blood samples were collected (4-5 mice per time point). Total radioactivity in plasma was determined by liquid scintillation counting (Beckm
anModel LS 6500, Fullerto
n, CA) using 10 μl aliquots of plasma. Up to 200 μl of plasma is
Extracted with 3 volumes of ethyl acetate according to Kker et al. (1987). The extraction efficiency for paclitaxel was 8
It was 0%. This sample is 2,500 rpm for 5 minutes.
And the supernatant was separated and dried. The dried extract was combined with 195 μl of HPLC
Reconstitute in mobile phase and reconstitute 5 μl cold paclitaxel (0.2
mg / ml) and 100 μl was injected into the HPLC for determination of free paclitaxel radioactivity. Pharmacokinetic parameters were analyzed by the non-compartmental model using the WinNonlin software package. Each data point generated was the average of 4-5 mice.

FIG. 11 shows the clearance of both drugs from plasma.
Shown in In FIG. 11, open squares indicate 6 μCi of radiolabeled P.
Shows the radioactivity of PG-TXL after injection of G-[< ³ > H] paclitaxel (20 mg equivalent paclitaxel / kg);
Indicates 6 μCi of radiolabeled [ ³ H] paclitaxel (20 m
(g equivalent paclitaxel / kg) shows the radioactivity of paclitaxel after injection, and the open circles show the “paclitaxel” radioactivity released from the injected PG- [ ³ H] paclitaxel. Paclitaxel has a very short half-life (t _1/2 , 29 minutes) in mouse plasma, whereas PG-T
The apparent half-life of XL is extended (t _1/2 , 317
Minutes). The slower clearance of PG-TXL from the blood was a design feature of polymer-drug conjugates with the goal of improving tumor uptake. Surprisingly, the rate of conversion of PG-TXL in plasma to paclitaxel is slow, with less than 0.1% of the radioactivity from PG- [ ³ H] TXL within 144 hours after drug infusion [ ³ H]. It was recovered as paclitaxel (FIG. 11).

In another study, mice bearing OCA-1 tumors were prepared as described above. 50 tumors
When 0 mm ³ is reached, animals are given 20 mg equivalent of paclitaxel / kg of [ ³ H] paclitaxel or PG-
One dose of [ ³ H] TXL was injected into the tail vein. Animals,
2 hours, 5 hours, 9 hours, 24 hours, 48 hours after injection,
And killed at 144 hours. Remove the tumor, weigh it,
Then, it was homogenized with 3 volumes of PBS (w / v).
The percentage of injected dose per gram of tissue is calculated based on the total radioactivity associated with the tumor, which was determined by counting aliquots of the prepared tissue homogenate. An aliquot of the tissue homogenate is mixed with a tissue solubilizer, followed by the addition of scintillation solvent,
Then the total radioactivity was counted. The efficiencies counted were verified by standard addition methods. Alternatively, an aliquot of the tissue homogenate is extracted with ethyl acetate and HP
Free paclitaxel was analyzed by LC. This HP
The LC system is 150 x 3.9 mm Nova-Pa
k columns (Waters, Milford, M
A), liquid chromatography pump (Waters model 510), UV / Vis detector set at 228 nm (Waters model 486), flow scintillation analyzer (Packard model 500TR, Do)
wners Grove, IL) and Packard radiometry (radiomat) for data analysis.
ic) Consists of software. The elution solvent (methanol: water = 2: 1) was flowed at 1.0 ml / min. OCA
-1 tumor uptake as a percentage of dose administered per gram of tissue, and association of radioactivity in oca-1 tumors as free paclitaxel as dpm per gram of tissue. did.

Quantitative assessment of tumor uptake in C3Hf / Kam mice indicates that relatively high levels of radioactivity from radiolabeled PG-TXL were slowly injected into tumor tissue after injection, as compared to radiolabeled paclitaxel. Appearing (FIG. 12A). However, only a small amount of radioactivity in the tumor tissue is due to the release of free paclitaxel (FIG. 12B). The data are shown in FIGS. 12A and 12B as the mean ± SD from three mice per time point. In FIG. 12A, the white bar indicates 6 μCi of radiolabeled PG.
-Shows the radioactivity of PG-TXL after injection of [ ³ H] paclitaxel (20 mg equivalent paclitaxel / kg), black bars indicate 6 μCi of radiolabeled [ ³ H] paclitaxel (2
2 shows the radioactivity of paclitaxel after injection of 0 mg equivalent paclitaxel / kg). In FIG. 12B, the total radioactivity measured after injection of 6 μCi of radiolabeled PG- [ ³ H] paclitaxel is indicated by a white bar and is derived from “paclitaxel” released from injected PG- [ ³ H] paclitaxel. Radioactivity is indicated by black bars. The percentage of radioactivity in tumor tissue due to paclitaxel did not increase appreciably over time, suggesting that PG-TXL is not merely a prodrug for the gradual release of paclitaxel.

In contrast to paclitaxel, studies using PG-TXL in vitro have shown that this complex is a strong cell, whether prepared as a fresh solution or after "aging" in buffer. It clearly showed that it was not a damaging species. It does not strongly support neither tubulin polymerization nor the growth and survival of paclitaxel-dependent CHO cell lines. Furthermore, the data obtained from pharmacokinetic studies indicate that both the presence in plasma and the release rate are very slow (144
(Less than 0.1% in time). While the uptake of the PG-TXL substance is about 5-fold greater than that achieved with paclitaxel when using an equivalent anti-tumor dose, the substance that gains tissue invasion is shown to be not free paclitaxel. Predominantly in the organization in the form.

Example 6: Effect of polymer structure on the activity of water-soluble polyamino acid-paclitaxel conjugate In this study, the structure of the polyamino acid used for the drug conjugate was determined to be the antitumor of the polymer-paclitaxel conjugate. It was evaluated whether it affected the activity. Paclitaxel was converted to poly (L-glutamic acid), poly (D-glutamic acid), and poly (L
-Aspartic acid). These polyamino acid-paclitaxel conjugates had similar paclitaxel content, water solubility, and molecular weight (30-40K). Mouse OCA-1 ovarian cancer (500 at the time of treatment
mm ³⁾ in C3Hf / Kam mice with, 80 mg
A single i.p. of poly (L-glutamic acid) -paclitaxel at equivalent paclitaxel / kg body weight. v. The injection resulted in a 21-day tumor growth delay relative to 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) is OCA-1
Completely inactive against the tumor. In a separate study, the antitumor activity of polymer-paclitaxel conjugates of different molecular weights (1K, 13K, and 36K) was compared. The lower molecular weight conjugate was not significantly more effective than the higher molecular weight conjugate. Higher molecular weights above 50,000 were too viscous.

Example 7: Polyglutamic acid-paclitaxel (PG-TXL) induces less apoptosis compared to paclitaxel. To evaluate the mechanism of PG-TXL related antitumor activity, paclitaxel and PG-TXL Tissue sections of OCA-1 tumors excised from treated mice were examined. OCA-1 tumor-bearing mice were
Prepared as previously described. Tumor volume is 500mm ³
When the animal reaches paclitaxel (80 mg / k
g) or PG-TXL (160 mg equivalent paclitaxel / kg) was injected. 0-144 after treatment
At different time points in the time range, tumors were histologically analyzed to quantify mitotic and apoptotic activities according to Milas et al. (1995). Mice were sacrificed by cervical dislocation and 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 evaluated on numbered slides by microscopy at 400 ×. Five fields of non-necrotic area were randomly selected in each tissue specimen and the number of apoptotic nuclei and cells undergoing cell division in each field was determined by the
Values were recorded as per number and expressed as a percentage.
This value was obtained from 150 mice from 3 mice per time point.
Based on 0 nucleus evaluation.

The changes observed in paclitaxel-treated mice were the same as those previously described (Milas et al., 199).
It was quantitatively similar to 5). Tumor cells showed significant nuclear fragmentation with the formation of apoptotic bodies. this is,
In particular, it was remarkable on the first day (FIG. 13). FIG.
-160 mg equivalent paclitaxel of TXL (MTD) /
kg and 80 mg / kg paclitaxel (MT
D) is a graph showing the kinetics of apoptosis in OCA-1 tumors after a single intravenous dose. The white square is
PG-TXL (160 mg equivalent paclitaxel / kg,
MTD), the response to a single intravenous dose of paclitaxel (80 mg paclitaxel / kg).
MTD). Visible tumor cell clumps with normal mitosis were still present in these tumors for up to 144 hours, indicating that these tumors did in fact regrow. Treatment with PG-TXL
Probably due to the small amount of free paclitaxel released from PG-TXL, only a modest increase in mitotically arrested and apoptotic cells occurred (FIG. 13). By 96 hours, tumors from PG-TXL-treated mice had developed extensive edema and necrosis, and only margins of visible tumor cells remained. 144
By time, the remaining tumor clumps, as compared to the control, were composed of larger, more polymorphic, and less mitotically active cells.

These data indicate that the water-soluble PG-
This suggests that the TXL conjugate has superior anti-tumor 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 this drug is used to solubilize paclitaxel and contributes to the overall antitumor activity of this remarkable new complex. is there. These data indicate that PG-TXL has the ability to produce cell death in a manner separate from, and in addition to, apoptosis caused by free paclitaxel released.

Example 8: Synthesis of polyglutamic acid-camptothecin (PG-CPT) conjugate The synthesis of PG-CPT followed a similar reaction as previously described for the synthesis of PG-TXL. To 80 mg of PG polymer in 2.5 ml of dry DMF was added 20 mg of CPT (Han
de Tech. ), 34 mg of DCC and traces of DMAP were added as catalyst. After stirring overnight at room temperature, the reaction mixture was poured into chloroform and the precipitate was collected. The dried precipitate was redissolved in sodium carbonate solution, dialyzed against 0.05 M phosphate buffer (pH 4.5), filtered and lyophilized. The CPT content in the polymer conjugate was measured at an emission wavelength of 430 nm and 37
A fluorescence spectrometer (Perki) using an excitation wavelength of 0 nm
n Elmer Model MPF-44A). Content: 2% -5% (w / w), soluble:>
200 mg conjugate / ml.

Example 9: Poly-lysine (PL) TXL
Synthesis of Conjugate (PL-TXL)) Polylysine (MW> 3)
0,000, Sigma) by converting polylysine to succinic anhydride, glutaric anhydride,
Alternatively, it is converted to a carboxylic acid functional group by reacting with DTPA. Remaining unreacted NH _{2 in} polylysine
The groups are blocked by reacting the modified polymer with acetic anhydride. TXL, desekitaxel, other taxoids, etoposide (etopsi)
de), teniposide, camptothecin, epothilone (e
pothilone) or other anti-tumor drug
-Conjugate to the resulting polymer according to the procedure previously described for the synthesis of TXL.

Example 10 Synthesis of Other Polyamino Acids Used to Conjugate TXL The polyamino acid containing glutamic acid was converted to the N-amino acid of the corresponding amino acid using γ-benzyl-L-glutamic acid NCA. It can be synthesized by copolymerization of carboxyanhydride (NCA). The resulting benzylglutamic acid-containing copolymer is converted to a glutamic acid-containing copolymer by removing the benzyl protecting group (FIG. 15). TXL, docetaxel, other taxoids, etoposide, teniposide, camptothecin, epothilone, or other antineoplastic agent,
-Conjugate to the resulting polymer according to the procedure previously described for the synthesis of TXL and PG-CPT.

Example 11 PG-TXL in Human
(Introduction) Poly-L-glutamic acid-paclitaxel (P
G-TXL) is a conjugate of poly-L-glutamic acid and paclitaxel. This compound is water soluble,
Based on previous animal studies, it appears that the compound can be administered as a short-term (ie, few minutes) intravenous injection. Based on in vitro and early animal studies, this compound appears to be at least as active against cancer as monomeric paclitaxel in Cremophor and may have slight side effects. Based on these observations, the drug will be studied in humans. This study first requires that the compound be formulated in a solvent commonly used for intravenous infusion. We have:
It was expected that either normal saline, 5% dextrose in water or sterile water would be used as the solvent. The PG-TXL formulation is then administered to a test animal (eg,
Rats and dogs) to determine the toxicity of the drug in these animals and to determine the dose of the drug, which is then the lowest starting dose for the first phase of human studies Can be provided as This phase 1 human study will define the dose of PG-TXL, which can subsequently be used in phase 2 studies in patients. Phase II studies are performed on several tumor types to determine the activity of PG-TXL in various cancers. One skilled in the art will appreciate that administration, selection of animal models, and modifications of administration regimes can be made in the methods disclosed in the Examples below, and that such modifications are included in the present invention.

Animal Studies These studies were performed in rats and beagle dogs, three in each study with each dose level of drug. This level is increased until life-threatening toxicity is indicated. The animals are blood tested and necropsied,
The dog's susceptible organ systems are determined to predict side effects in human studies. Once the dose at which 10% of the animals die is determined, then 1 /
10 equivalents are recommended as starting dose for human studies. This is usually recommended by the Food and Drug Administration (FDA) as the starting dose for human phase I studies.

(Phase 1 Study) A Phase 1 study of this drug
This is done using the starting dose specified in animal studies.
The drug may be injected intravenously by syringe over several minutes, or it may be administered for up to about 10-15 minutes.
It can be injected as a short infusion. Solvent doses range from 10 ml to about 100, depending on which of the two intravenous injection approaches used.
It is in the range of ml. Drugs are administered every three weeks. This schedule is based on previous animal studies and the scheme used for paclitaxel in Cremophor. Three
Human patients are treated with an injection of PG-TXL, starting at the lowest dose prescribed by animal studies. Blood tests are performed at baseline and blood counts are evaluated weekly; liver and kidney function tests are performed every three weeks. Numeric and physiological parameters were fully collected from PG-TXL,
It is expected that the next cycle of treatment will be resumed three weeks after the previous study. Then, in this case, the treatment is repeated every three weeks. If the first cohort of these patients tolerates the drug for three weeks, then they will be given increased capacity by a given scheme commonly used in phase 1 studies. Once three patients have accepted the first cycle, the next three cohorts are started for the next higher dose level. This escalating dose level process continues until at least two of the three patients at the dose level have side effects that are so severe that they refuse to continue to administer the drug. In such a situation, the dose level immediately before excessive toxicity would be the drug level administered in a subsequent study. Six to ten patients will be treated at dose levels recommended for a phase 2 study to confirm this tolerability. Once an appropriate dose has been defined and acute drug toxic side effects have been evaluated, a phase 2 study is initiated.

Phase II Studies Phase II studies of PG-TXL are performed on several tumor types. Each study is designed in a standard standard Phase 2 study format according to either the Gahan or Simon design. Briefly, about 14 patients of a given tumor type are treated first, if no anti-cancer activity is demonstrated in each tumor type, then PG
-Further study of TXL will be discontinued. However, if at least one patient had a clinical benefit (defined as at least a 50% reduction in the sum of products of the vertical cross-sectional diameter of the tumor), then patients with this tumor type treated with PG-TXL Increase the number of to 30 people. These studies allow us to define the activity of PG-TXL in various cancers and to refine information on drug side effects. The tumor types of particular interest for PG-TXL are those that have shown a good response to paclitaxel and docetaxel. These include ovarian tumors, breast cancer, and lung cancer. A study comparing polyglutamic acid-paclitaxel with paclitaxel in tumors responding to PG-TXL is performed. Such a study is called a phase 3 study.

Phase III Study of PG Paclitaxel Based on the activity of paclitaxel in ovarian, breast and lung cancers, these are the tumor types in which PG-TXL was compared to paclitaxel. Given the large number of patients required for such a randomized study, we predict that a multicenter study will be needed. The present inventors have developed a joint regional cancer program (Cooperati).
ve Community Oncology Pro
Gram) (CDDP) and many other multicenter trial groups have institutional tools. P for paclitaxel
In addition to the potential clinical benefits of G-TXL, it is appropriate to assess the economic impact of two drugs. PG-TXL
It is anticipated that short-term infusions of can result in less expensive treatments. And, therefore, it is expected that PG-TXL may be cost-effective in proportion to paclitaxel monotherapy. Because not only is the infusion shorter, but also considering the absence of cremophor, patients are expected to experience fewer side effects, so premedication regimens including steroids and intravenous H2 and H1 blockers are Can no longer be needed. All of these factors result in reduced cost of treatment.

(Summary) It is expected that the first animal toxicology assessment will require up to 6 months. If drug prescriptions are available, the subsequent human Phase 1 study can be completed in an additional 6-9 months. Once these are completed, phase 2 trials on various tumor types can take an additional 6-9 months. At that point, we have a good idea about the efficacy of this drug. A targeted Phase 3 study can be designed and started. Phase 2 studies may show sufficient clinical activity to omit phase 3 studies, or phase 3 studies may not be necessary.

Example 12: Enhancement of the tumor radioactivity response of poly (L-glutamic acid) -paclitaxel conjugate of mouse ovarian carcinoma (Introduction) The combination of chemotherapy and radiotherapy in the treatment of various tumors It resulted in a complete response and a substantial improvement in viability (Rotman, 1992). In both in vitro and in vivo studies, it was shown that paclitaxel could potently enhance tumor radiation responsiveness. In animal experiments, the potentiators were 1.2-2. Depending on tumor type, drug concentration, and dosing schedule.
It is a range exceeding 0. In this study, the radiosensitizing effect of poly (L-glutamic acid) -paclitaxel (PG-TXL) was investigated.

(Experimental method) PG-TXL was converted to poly (L-
Glutamic acid) (Sigma, viscosity molecular weight: 31K) was synthesized as described herein. The conjugate contained 20% paclitaxel (w / w), which was coupled to PG via an ester linkage.
5 × 10 ⁵ ovarian tumors O in female C3Hf / Kam mice
I.Ca-1 carcinoma cells in the right hind leg. m. Inoculated. When the tumors reached 8 mm in diameter, the mice were randomly divided into 12 groups, each group consisting of 6-12 mice. Group 1
~ 5 mice received only saline, 14 Gy irradiation,
Alternatively, PG-TXL alone was given at a dose of 47, 80, or 120 mg equivalent paclitaxel / kg. Group 6 ~
Nine mice received 47 mg equivalent paclitaxel / kg of PG-TXL and received 2,24,4 of PG-TXL treatment.
14 Gy local irradiation was given after 8, and 144 hours. Group 10 received 80 mg equivalent paclitaxel / kg P
G-TXL was given and 1 hour after PG-TXL treatment
A 4 Gy irradiation was given. Groups 11 and 12 are 120 m
PG-TXL was given in g equivalent paclitaxel / kg,
14 Gy of irradiation was given 24 hours before or after G-TXL treatment. PG-TXL was given as a single intravenous injection. Topical gamma irradiation to the tumor was delivered from a ¹³⁷ Cs irradiator at a dose rate of 7 Gy per minute. Tumor growth delay was determined by measuring the orthogonal tumor diameter in three directions until the tumor reached 14 mm in diameter.

(Results and Discussion) The radiosensitizing effect of PG-TXL was dose-dependent. At a lower PG-TXL dose of 47 mg equivalent paclitaxel / kg,
A secondary effect was observed. The average enhancement factor varied between 0.54 and 0.75, depending on the timing of the delivery of the irradiation. However, additional effects were observed at higher doses of PG-TXL. The mean enhancer was given when PG-TXL was given 24 hours prior to irradiation and the PG-TXL dose was 47-80.
And from 0.75 to 1.8 and 4.2 when increased to 120 mg equivalent paclitaxel / kg (Table 7). The side effects of actinic radiation observed at low doses of PG-TXL may be due to inappropriate cell killing and rapid regrowth of viable cells. At higher doses, PG-
TXL may have a sufficient effect on populations of circulating tumor cells and / or tumor reoxygenation, and may produce a sufficiently enhanced radiosensitizing effect. Interestingly, when tumors were irradiated at 14 Gy before treatment with PG-TXL at 120 mg equivalent paclitaxel / kg, a side effect with a potentiator of 4.3 was observed (Table 7). This result contrasts with previous observations that paclitaxel induces irradiation resistance when given after irradiation (Ingram and Re).
dpath, 1997).

[0155]

[Table 7]

A. Absolute growth delay was 8 to 8 tumors in the treatment group.
It is defined as the number of days that the tumor in the 8- to saline-treated group is subtracted from the number of days that the tumor grows to 8 to 14 mm from the number of days that grow to 14 mm. b. Normalized growth delay was the number of days that tumors in mice treated with the combination of PG-TXL and irradiation grew to 8-14 mm minus the number of days tumors in mice treated with PG-TXL alone grew to 8-14 mm. Defined as days. c. Enhancement factors are obtained by dividing the normalized tumor growth delay of mice treated with a combination of PG-TXL and irradiation by the absolute growth delay of mice treated with irradiation alone. d. Irradiation was given 24 hours prior to PG-TXL treatment. All other combination groups: irradiation was given 24 hours after PG-TXL treatment. e. Data is based on tumors regrown on day 120.
Tumors in two of the six mice in both groups were 12%
Measurement was still possible on day 0.

Conclusions The results of this study indicate that PG-TXL in combination with radiation therapy could be used effectively before or after irradiation to enhance radiosensitization. These results further suggest that conjugation of radiosensitizers and anti-tumor drugs to water-soluble polymer carriers can provide enhanced radiosensitizing effects. In view of the present disclosure, one skilled in the art will recognize doses of PG-TXL and other compositions disclosed herein, as well as radiation administered either externally or internally (ie, an external radiation source). It is recognized that the dose of radiation administered or by systemic administration, for example, by injection or ingestion of a radioactive material, such as a radioisotope containing formulation, can vary. Treatment schedule and dosing will be based on the patient and will include factors such as, for example, patient weight and age, type of tumor being treated, severity of disease state, previous and / or current therapeutic intervention, mode of administration, and the like. And may vary from patient to patient, and can be readily determined by one skilled in the art. For example, a preferred range of doses of PG-TXL is TXL at an equivalent paclitaxel dose.
About 0.5 to 2 times the maximum tolerated dose of The amount of PG-TXL administered may be spread over the course of radiation therapy due to sub-dose. The preferred range of irradiation is about 5
It is also contemplated that from about 50 to about 70 Gray (Gy) administered over about 7 weeks, or 10 Gray per week. A preferred dosing schedule is about 1 to about 2 days before irradiation, or about 1 to about 2 days after irradiation.
Administering G-TXL. The schedule of administration of PG-TXL and other polymer-anti-tumor drug or chelator-anti-tumor drug compositions will, of course, vary and can be determined by one of skill in the art for maximum benefit of each patient. And / or repeated.

Although the compositions and methods of the present invention have been described with reference to preferred embodiments, various modifications may be made without departing from the spirit, scope and spirit of the invention. It will be apparent to those skilled in the art that the method can be applied during, during, and between steps of a method. More specifically, certain agents that are both chemically and physiologically related, as well as other water-soluble polymer-drug conjugates, can be replaced by the agents described herein, with identical or similar results. It is clear that it can be obtained. All such similar substitutions and modifications apparent to those skilled in the art are
It is deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES The following references are incorporated herein by reference in detail to the extent that they provide exemplary procedures or other explanations that assist in the description herein.

[0160]

[Table 8]

[0161]

The composition of the present invention provides a water-soluble taxoid which overcomes the disadvantages associated with the insolubility of the drug itself. The compositions of the present invention also provide the advantage of improved efficacy and controlled release as shown herein that tumors are eradicated in animal models after a single intravenous administration.

[Brief description of the drawings]

FIG. 1A: Paclitaxel, PEG-paclitaxel,
FIG. 2 shows the chemical structure of DTPA-paclitaxel.

FIG. 1B shows the chemical structure and reaction scheme for the production of PG-TXL.

FIG. 2 is a graph showing the effect of paclitaxel, PEG-paclitaxel, and DTPA-paclitaxel on the growth of B16 melanoma cells.

FIG. 3 is a graph showing the antitumor effect of DTPA-paclitaxel on MCa-4 mammalian tumors.

FIG. 4. Paclitaxel, DTPA-paclitaxel,
Figure 7 is a graph showing the median time (days) to reach a tumor diameter of 12 mm after treatment with PEG-paclitaxel.

FIG. 5. ¹¹¹ In-DTPA-paclitaxel and ¹¹¹
FIG. 2 shows a γ-ray scintigraph of a mouse with MCa-4 tumor after intravenous injection of In-DTPA.

FIG. 6 is a graph showing PG-TXL hydrolysis measured in PBS as a function of time at different pH levels.

FIG. 7A. Syngeneic OCA in female C3Hf / Kam mice.
1 is a graph showing the antitumor effect of PG-TXL on ovarian cancer tumors.

FIG. 7B is a graph showing the anti-tumor effect of PG-TXL on 13762F tumor in female rats.

FIG. 7C is a graph showing the antitumor effect of PG-TXL in mice bearing MCa-4 mammalian cancer tumors.

FIG. 7D: Soft tissue sarcoma tumors in mice (FSa-
It is a graph which shows the antitumor effect of PG-TXL with respect to II).

FIG. 7E shows syngeneic liver cancer tumors (HCa-
It is a graph which shows the antitumor effect of PG-TXL with respect to I).

FIG. 8 is a graph showing the release profile of paclitaxel from PEG-paclitaxel in phosphate buffer (pH 7.4).

FIG. 9 is a graph showing the antitumor effect of PEG-paclitaxel on MCa-4 mammalian tumors.

FIG. 10: 1.0 mM GTP and 1.0 mg / ml
FIG. 5 is a graph showing a tubulin polymerization assay performed at 32 ° C. in the presence of tubulin.

FIG. 11. PG- in C3Hf / Kam mice.
FIG. 2 is a graph showing plasma clearance of radioactivity after intravenous injection of [ ³ H] paclitaxel and [ ³ H] paclitaxel.

Figure 12A is a graph showing the time-dependent OCA-1 tumors content after injection of either radioactivity in mice PG- ^[3 H] paclitaxel and ^[3 H] paclitaxel.

FIG. 12B is a graph showing the conversion of PG- [ ³ H] paclitaxel to [ ³ H] paclitaxel in OCA-1 tumors.

FIG. 13 is a graph showing the kinetics of apoptosis in OCA-1 tumors after a single intravenous dose of 160 mg equivalent paclitaxel / kg of PG-TXL (MTD) and 80 mg / kg paclitaxel (MTD).

FIG. 14 is a graph showing the survival of nude mice with human ovarian cancer cells (SKOV3ip1) treated with PG-TXL.

FIG. 15 shows the chemical structure and reaction scheme for the production of a glutamic acid-containing polyamino acid.

 ──────────────────────────────────────────────────の Continued on the front page (71) Applicant 500165359 3324 Pittsburgh Street, Houston, Texas 77005, United States of America (72) Inventor Li, Chun United States of America Texas 77459, Missouri City, Buckeye Place 811 (72) Inventor Wallace, Sydney United States Texas 77005, Houston, Pittsburgh Street 3324 (72) Inventor Yu, Don Juan United States Texas 77062, Houston, Beacast Drive 15002 (72) Inventor Jan, David United States Texas 77878, Sugar Land, Spinnaker Way 1123 F-term (reference) 4C076 AA12 BB01 BB11 CC37 EE41E FF15 4C088 AB03 AC05 AC06 BA08 BA13 CA03 MA05 NA05 ZB26

Claims (14)

[Claims] 1. A method of enhancing the response of a tumor to irradiation, comprising the steps of: a) administering paclitaxel, docetaxel conjugated to a water-soluble polyamino acid polymer to a patient in need of such treatment; , Etoposide, teniposide, camptothecin,
Or administering a radiosensitizing amount of a pharmaceutical composition comprising epothilone and a pharmaceutically acceptable carrier; b) irradiating the tumor;
The conjugated paclitaxel or docetaxel is
A method that increases water solubility, potency, and intratumoral accumulation as compared to a corresponding unconjugated drug. 2. The method of claim 1, wherein said polymer is selected from polyglutamic acid, polyaspartic acid, or polylysine. 3. The method of claim 1 wherein said polymer is from about 5,000 to about 100.
3. The method of claim 2, having a molecular weight of 000 daltons. 4. The method according to claim 1, wherein the polymer comprises from about 20,000 to about 80.
3. The method of claim 2, having a molecular weight of 000 daltons. 5. The method of claim 5, wherein the polymer is from about 25,000 to about 50.
3. The method of claim 2, having a molecular weight of 000 daltons. 6. The method according to claim 1, wherein said polymer is polyglutamic acid. 7. The method of claim 6, wherein the conjugate comprises about 2% to about 35% by weight of paclitaxel or docetaxel. 8. The method of claim 1, wherein step (b) is performed by administering gamma radiation to the tumor. 9. The method of claim 8, wherein said composition is administered prior to irradiation. 10. The method of claim 8, wherein said composition is administered after irradiation. 11. The method of claim 1, wherein the radiation dose is about 10 Grays per week, and the composition is
9. The method of claim 8, wherein the method is administered within ~ 2 days. 12. The method of claim 8, wherein the cancer is breast, ovarian, malignant melanoma, lung, stomach, prostate, colon, head and neck cancer, leukemia or Kaposi's sarcoma. . 13. A method of treating cancer comprising the steps of: a) providing paclitaxel, docetaxel, etoposide, teniposide, camptothecin, or epothilone conjugated to a polyglutamic acid polymer to a patient in need of such treatment; Administering a radiosensitizing amount of a pharmaceutical composition, comprising: and a pharmaceutically acceptable carrier; and b) irradiating the tumor. 14. The method of claim 13, wherein the amount of the composition is about 0.5 to about 2 times the maximum tolerated amount of paclitaxel at an equivalent paclitaxel dose.