JP2003524624A - Methods and compositions for enhancing delivery of therapeutic agents to tissues - Google Patents

Abstract

  (57) [Summary] The present invention pertains, at least in part, to methods of enhancing the delivery of therapeutic agents, such as macromolecules and drugs, into tissues such as parenchymal tissues or tumors. The method uses an apoptosis-inducing agent, such as paclitaxel, at a dose that allows channels to be formed in the tissue and enhances penetration of the therapeutic agent into the tissue. Current tissue treatments are often not effective because the therapeutic agent is not delivered inside the tissue. By using the methods and compositions of the present invention, a therapeutic agent can be delivered inside a tissue.

Description

Detailed Description of the Invention

BACKGROUND Paclitaxel is one of the most important anti-cancer agents developed in the last two decades and is effective against multiple types of solid tumors in humans (Rowinsky EK (199
3) J Natl Cancer Inst Monogr 15: 25-37). Paclitaxel has pleiotropic effects. For example, it enhances tubulin polymerization, promotes microtubule assembly, binds to microtubules, stabilizes microtubule dynamics, induces mitotic arrest during metaphase / late transition, and undergoes apoptosis. Induce (Parness J and Horwitz SB (1981)
J Cell Biol 91: 479-489; Manfredi JJ et al. (1982) J Cell Biol. 94: 688-69.
6; Jordan MA, et al. (1993) Proc Natl Acad Sci USA 90: 9552-9556; Jordan
MA et al. (1996) Cancer Res 56: 816-825; Derry WB et al. (1995) Biochemis
try 34: 2203-2211). The intracellular concentration of paclitaxel is important for its pharmacological effect, and drug resistance in some resistance sublines correlates with low intracellular drug accumulation compared to susceptible parental cell lines (Lopes NM et al. . (1993) Cancer Chemothe
r Pharmacol 32: 235-242; Bhalla K et al. (1994) Leukemia 8: 465-475; Jekun
en AP et al. (1994) Br J Cancer 69: 299-306; Riou JF et al. (1994) Proc A
m Assoc Cancer Res 35: 160; Speicher LA et al. (1994) J Natl Cancer Inst
86: 688-694).

Doxorubicin is an anticancer drug with broad clinical activity. It has been used clinically to treat leukemias, lymphomas and solid tumors including breast, lung, prostate and ovarian cancers and sarcomas (Oesterling et al. (1997) In: Cancer: Pri
nciples and Practice of Oncology (Eds, DeVita, VT, Jr., Hellman, S., a
nd Rosenberg, SA, 1997); Doroshow, JH (1996) In: Cancer Chemothrapy
and Biotherapy: Principles and Practice (Eds, Chabner, BA and Longo, D
.L.)). Doxorubicin is one of the most effective agents for hormone-refractory prostate cancer (Smith, dC (1999) Urol. Clin. North Am. 26: 323-331).
In human prostate tumor tissue cultures, doxorubicin induces complete inhibition of tumor cell growth at an IC ₅₀ of 61 nM and complete tumor cell death at an LC ₅₀ of 2.1 μM. (Chen et al., (1998) Clin
Cancer Res. 4: 277-282, 1998).

To prevent tumor regrowth, it is necessary to deliver the drug to the nuclear part of the tumor (Erlans
on M et al. (1992) Cancer Chemother Pharmacol 29: 343-353; Durand RE (199
0) Cancer Chemother Pharmacol 26: 198-204), thus drug delivery is an important determinant of therapeutic effect (Jain RK (1996) Science 271: 1079-1080). After systemic intravenous injection, drug delivery to the tumor nucleus involves three processes: dispersion through the vascular space,
It involves transport across the microvascular wall and diffusion through the interstitial space of tumor tissue (Jain RK (1987) Cancer Res 47: 3039-3051). Intratumoral injection for superficial bladder cancer, intraperitoneal dialysis for ovarian cancer, intratumoral injection, or direct instillation into the peritumoral space Drug delivery is predominantly by diffusion through the interstitial space (Nativ O et al. (1997) Int J Canc
er 70: 297-301; Song D et al. (1997) Cancer Chemother Pharmacol 40: 285-29.
2; Markman M (1998) Semin Oncol 25: 356-360; Markman M et al. (1995) Semi
n Oncol 22: 84-87). Paclitaxel movement in the interstitial space may behave like a protein, despite its relatively low molecular weight (853 daltons), which is extensively bound to proteins in interstitial fluid. Depending on (
Baguley BC et al. (1995) Clin Exp Pharmacol Physiol 22: 825-828).

[0004] A recent study has shown that drug delivery to tissues during local treatment depends on the drug's ability to penetrate parenchymal tissue. This study showed that the distribution of paclitaxel in multicellular spheroids was marginal, but the barrier to paclitaxel permeation was unknown (Nicholson et al. (Nicholson et al.
1997) Eur. J. Cancer 33: 1291-1298). Therefore, there remains a need for methods of delivering therapeutic agents to tissues that allow the drugs to reach the tissues.

SUMMARY The present invention provides a method of delivering a therapeutic agent to a tissue, and a composition for use therein, which method allows the therapeutic agent to be introduced into a multi-layered tissue such as parenchymal tissue and tumor. The penetration of The method utilizes an apoptosis-inducing agent, such as paclitaxel, at a dose and for a period sufficient to cause apoptosis in the tissue to enhance penetration of the therapeutic agent into the tissue (eg, by forming channels in the tissue). Including steps. Thus, the apoptosis-inducing agent is used as a pretreatment prior to the delivery of a therapeutic dose of the therapeutic agent to the tissue, which preconditions the therapeutic agent into the tissue as compared to the absence of the pretreatment. The penetration of Further, the apoptosis-inducing agent may have therapeutic activity, and thus may be used as a therapeutic agent (
That is, the same drug may be used as an apoptosis inducer and a therapeutic drug). Alternatively, the apoptosis-inducing agent may be used (ie, the apoptosis-inducing agent and the therapeutic agent may be different agents) to enhance the delivery of other types of agents to the tissue.

Accordingly, in one embodiment, the present invention relates to a method of delivering a therapeutic agent to the tissue of a patient, eg, a mammal such as a human. The method comprises the steps of administering an apoptosis-inducing agent to a patient and allowing the apoptosis-inducing agent to pass for a time sufficient to induce apoptosis in the patient's tissue. The tissue can be, for example, liver, muscle (eg, heart muscle, smooth muscle, or skeletal muscle), nerve tissue, skin tissue or adipose tissue, or brain, breast, ovary, bladder, prostate, skin, colon, lung, liver,
Or it may be a tumor such as a tumor of the uterus. The apoptosis-inducing agent may be administered systemically, locally, locally, or both locally and locally (locally), or some combination thereof.

In a second embodiment, the invention relates to a method of delivering a therapeutic agent to a tumor (eg, a cancerous or benign tumor) in a patient. The method comprises the steps of administering a dose of an apoptosis-inducing agent to a patient, allowing the apoptosis-inducing agent to have sufficient time to induce apoptosis in the tumor, and administering a dose of a therapeutic agent to the patient. Delivering to. The tumor may be, for example, a tumor of the brain, breast, ovary, bladder, prostate, skin, colon, lung, liver, or uterus. Examples of apoptosis inducers are paclitaxel and doxorubicin.

In yet another embodiment, the invention features a method of delivering a chemotherapeutic agent to the tumor of a patient, such as a human (eg, a cancer patient). The method comprises the step of locally or locally administering to the patient a dose of an apoptosis-inducing agent, allowing the apoptosis-inducing agent to have sufficient time to induce apoptosis in the tumor, and Delivering the chemotherapeutic drug to said patient. In a further embodiment, the tumor is a cancerous tumor, for example a tumor of the brain, breast, ovary, bladder, prostate, colon, lung, liver, or uterus.

The present invention also relates to compositions for delivering therapeutic agents to a patient. The composition includes a rapid release formulation of the apoptosis-inducing agent, a sustained release formulation of the therapeutic agent, and a pharmaceutically acceptable carrier.

The present invention further includes microparticles and nanoparticles comprising therapeutic agents or apoptosis inducing agents. Further included is a method of treating a patient with the microparticles and nanoparticles.

The invention further relates to a kit for the treatment of tumors. The kit comprises an apoptosis-inducing agent in a pharmaceutically acceptable carrier, a therapeutic agent in a pharmaceutically acceptable carrier, a container, and when using the apoptosis-inducing agent and the therapeutic agent for treating a tumor. Instructions, and accommodate.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates, at least in part, to methods of enhancing the delivery of therapeutic agents, such as macromolecules and drugs, to the interior of multi-layered tissues such as parenchymal tissue or tumors. The method first uses an apoptosis-inducing agent such as paclitaxel in a dose such that channels are formed in the tissue and the penetration of the therapeutic agent into the tissue is enhanced. Current tissue therapies are often ineffective because the therapeutic agent is not delivered inside the tissue. By using the methods and compositions of the present invention, therapeutic agents can be delivered inside tissues.

The present invention relates, at least in part, to the penetration of therapeutic agents into the tissues and tumors of a patient by treating the tissue or tumor with an apoptosis-inducing agent such that the therapeutic agent permeability of the tissue or tumor is increased. Regarding The therapeutic agent may be a protein-binding agent, a chemotherapeutic agent, a gene therapy product, or other agent that may be beneficial to deliver inside a tissue such as a tumor.

In one embodiment, the invention relates to a method of delivering a therapeutic agent to the tissue of a patient. The method comprises the steps of administering an apoptosis-inducing agent to a patient, and allowing the apoptosis-inducing agent to pass for a time sufficient to induce apoptosis in the patient's tissue.

I Definitions Before describing the invention further, some terms used in the specification, examples and appended claims are collected here for convenience.

The term “apoptosis inducer” includes agents that induce apoptosis in cells such as tumor cells. It is possible to induce programmed cell death known as apoptosis in cells, including cancer cells. Apoptosis is characterized by programmed and selective destruction of cells into relatively small fragments of highly fragmented DNA (ie, the resulting fragment is typically less than about 200 bases). And During apoptosis, cell contraction and DNA cleavage between nucleosomes occur, resulting in DNA fragmentation. Eventually, the cells fall apart into smaller pieces. Examples of apoptosis-inducing agents include paclitaxel, doxorubicin, vincristine, vinblastine, vindesine, vinorelbine, taxotere (docetaxel), topotecan, camptothecin, irinotecan hydrochloride (CAMPTOSAR), etoposide, mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine , Epirubicin, mervalon, pyroxanthrone hydrochloride, 5-fluorouracil, methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, cytarabine (ARA-C), trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin, N- Phosphoacetyl-L-aspartate (PALA), pentostatin, 5-azacytidine, 5
-Aza-2'-deoxycytidine, adenosine arabinoside (ARA-A), cladribine, futrafer, UFT (a combination of uracil and futrafer), 5-fluoro-2- '
Deoxyuridine, 5-fluorouridine, 5'-deoxy-5-fluorouridine, hydroxyurea, dihydrolen chlorambucil, thiazofurin, cisplatin,
Carboplatin, oxaliplatin, mitomycin C, BCNU (carmustine),
Melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen-mustard, uracil mustard, pipobroman, 4-ipomeanol, dihydrolenperone, spiromustine, geldanamycin. , Cytochalasin, depsipeptide, lupron, ketoconazole, tamoxifen, goserelin (soldax), flutamide, 4'-cyano-3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3 '-(trifluoromethyl) Propionanilide, Herceptin, Anti-CD20 (Rituxan), Interferon alpha, Interferon beta, Interferon gamma, Interleukin 2, Interleukin 4, Interleukin 12, Tumor necrosis Child, and there is radiation.

The term “chemotherapeutic agent” includes anticancer agents such as paclitaxel and doxorubicin,
Agents such as agents that can be advantageously administered to tissues and other agents known to affect tumors are included. Further included are agents that modulate other conditions associated with tissue that can be permeabilized using the methods and compositions of the present invention. The chemotherapeutic agent may be, for example, a steroid, an antibiotic, or other pharmaceutical composition. Examples of chemotherapeutic agents include paclitaxel, doxorubicin, vincristine, vinblastine, vindesine, vinorelbine, taxotere (DOCETAXEL), topotecan, camptothecin, irinotecan hydrochloride (CAMPTOSAR), doxorubicin, etoposide, mitoxantrone, daudarubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunorubicin, daunubicin. , Amsacrine, Epirubicin, Melvalon,
Pyroxanthrone hydrochloride, 5-fluorouracil, methotrexate, 6-
Mercaptopurine, 6-thioguanine, fludarabine phosphate, cytarabine (
ARA-C), trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin, N-phospholeacetyl-L-aspartate (PALA), pentostatin, 5
-Azacytidine, 5-aza-2'-deoxycytidine, adenosine arabinoside (ARA-A
), Cladribine, futrafer, UFT (a combination of uracil and futrafer)
, 5-fluoro-2-'deoxyuridine, 5-fluorouridine, 5'-deoxy-5-fluorouridine, hydroxyurea, dihydrolenchlorambucil, thiazofurin, cisplatin, carboplatin, oxaliplatin, mitomycin C, BCNU (
Carmustine), melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen-mustard, uracil mustard, pipobroman, 4-ipomeanol, dihydrolenperone, spiromustine, Geldanamycin, cytochalasin, depsipeptide, lupron, ketoconazole, tamoxifen, goserelin (soldax), flutamide, 4'-cyano-3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3 '-(tri (Fluoromethyl) propionanilide, Herceptin, Anti-CD20 (Rituxan), Interferon alpha, Interferon beta, Interferon gamma, Interleukin 2, Interleukin 4, Interleukin 12, includes the action of tumor necrosis factor, and the like radiation.

The term “delivering” refers to making a therapeutic agent available to the interior of the tissue to be treated (eg, a tumor) so that the therapeutic agent can have a therapeutic effect on the interior of the tissue, eg Contacting the tissue with the therapeutic agent. The term "delivering" means that the therapeutic agent is administered to the patient as a separate dose (after administration of the apoptosis-inducing agent), or the therapeutic agent is administered to the patient together with the apoptosis-inducing agent (at the same time or at the same dose as the apoptosis-inducing agent). Is intended to encompass administration,
The formulation of the therapeutic agent is such that the tissue is in contact with the therapeutic agent after sufficient time for apoptosis to occur inside the tissue (ie, for the therapeutic agent to have sufficient time for apoptosis to occur). To be delivered to the tissue).

The term “dose” refers to an amount of an apoptosis-inducing agent or therapeutic agent sufficient to perform its intended effect, eg, induce apoptosis and treat tissue, respectively. In one example, the dose of the apoptosis-inducing agent is about 0.
From about 0.01 nM to about 1000 nM over 01 to about 5.0 hours, from about 0.1 nM to about 500 nM over about 0.1 to about 4.0 hours, from about 1 nM to about 2 over about 0.5 to about 3.0 hours.
50 nM, about 10 nm to about 150 nM for about 0.5 to about 2.0 hours, about 30 nM to about 100 nM for about 0.75 to about 1.5 hours, about 40 nM to about 10 hours for about 1 hour.
It may be about 50 nM paclitaxel for between 70 nM and advantageously for about 1 hour. The dose of the therapeutic agent may be administered, for example, starting from 24 hours after administration of the apoptosis-inducing agent for 3 hours. The target concentration of therapeutic agent is, for example, about 50 nM.
May be In another embodiment, the dose of the apoptosis-inducing agent is about one-half of the usual clinical dose (135-225 mg / m ² ) for human patients, such as intravenous infusion over a period of about 3 hours. You may The dose of the therapeutic agent may be, for example, the remaining one-half the usual clinical dose, eg, 16 to 30 hours after administration or delivery of said dose of the apoptosis-inducing agent.

The term “gene therapy product” includes products useful for the purposes of gene therapy in the treatment of either genetic or acquired disorders, such as cancer. A general approach to gene therapy involves introducing a nucleic acid into a cell so that one or more gene products encoded by the transgene material are produced intracellularly to restore or enhance functional activity. . Anderson, EF for a review of gene therapy approaches
(1992) Science 256: 808-813; Miller, AD (1992) Nature 357: 455-460; Fri
edmann, T. (1989) Science 244: 1275-1281; and Cournoyer, D., et al. (1990
) Curr. Opin. Biotech. 1: 196-208.

Genes of particular interest for expression in cells of a subject for treatment of a genetic or acquired disease include adenosine deaminase, factor VIII, factor IX, dystrophin, β-globin, LDL receptor, CFTR, insulin, erythropoietin, anti-angiogenic factor, growth hormone, glucocerebrosidase, β-glucouronidase,
α1-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, ornithine transcarbamylase, arginosuccinate synthase, UDP-glucuronisyl transferase, apoA1, TNF, soluble TNF receptor,
Interleukins (eg IL-2), interferons (eg α- or γ-IFN)
And other cytokines and growth factors. Cell types that can be modified for purposes of gene therapy include tumor cells, hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, skin epithelial cells and respiratory epithelial cells. For further discussion of cell types, genes and methods for gene therapy, see, eg, Wilson, J. M et al.
. (1988) Proc. Natl. Acad. Sci. USA 85: 3014-3018; Armentano, D. et al.
1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Wolff, JA et al. (1990).
Science 247: 1465-1468; Chowdhury, JR et al. (1991) Science 254: 1802-
1805; Ferry, N. et al. (1991) Proc. Natl. Acad. Sci USA 88: 8377-8381; Wi
lson, JM et al. (1992) J. Biol. Chem. 267: 963-967; Quantin, B. et al.
(1992) Proc. Natl. Acad. Sci. USA 89: 2581-2584; Dai, et al. (1992) Proc.
Natl. Acad. Sci. USA 10892-10895; van Beuschem, VW et al. (1992) Proc
. Natl. Acad. Sci. USA 89: 7640-7644; Rosenfeld, MA et al. (1992) Cell
68: 143-155; Kay, MA et al. (1992) Human Gene Therapy 3: 641-647; Cristi
ano, RJ et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2122-2126; Hwu, P.
et al. (1993) J. Immunol. 150: 4104-4115; and Herz, J. and Gerard, RD.
(1993) Proc. Natl. Acad. Sci. USA 90: 2812-2816.

For gene therapy applications of particular interest in the treatment of cancer, tumor-infiltrating lymphocytes may be overexpressed with a cytokine gene (eg TNF-α) or tumor cells may be ectopically expressed with a cytokine. Method of inducing tumor immune response at tumor site, method of expressing enzyme capable of converting non-toxic substance into toxic substance in tumor cells, method of inducing tumor-specific antigen to induce anti-tumor immune response, tumor suppression Method for expressing gene (eg p53 or Rb) in tumor cells, multidrug resistance gene (eg MDR1 and / or MR)
P) is expressed in bone marrow cells to protect them from the toxicity of chemotherapy.

Gene therapy applications of particular interest in the treatment of viral diseases include, for example, trans-expressing inactive tat and rev mutants of HIV or trans-expressing non-expressing ICp4 mutants of HSV. Expression of active viral transactivating proteins (eg
Balboni, PG et al. (1993) J. Med. Virol. 41: 289-295; Liem, SE et al.
(1993) Hum. Gene Ther. 4: 625-634; Malim, MH et al. (1992) J. Exp. Med.
176: 1197-1201; Daly, TJ et al. (1993) Biochemistry 32: 8945-8954; and
Smith, CA et al. (1992) Virology 191: 581-588), HIV en
Expression of trans-expressing inactive envelope proteins such as v mutants (eg Steff
y, KR et al. (1993) J. Virol. 67: 1854-1859), intracellular expression of antibodies or fragments thereof directed against viral products ("internal immunization", eg Marasco, WA et al. al. (1993) Proc. Natl. Acad. Sci. USA 90: 7889-7893
) And soluble CD4 expression of soluble viral receptors, such as CD4. In addition, using the system of the present invention, suicide genes can be conditionally expressed in cells so that they can be eliminated after the cells perform their intended function.

The term “liposome” or “lipid vesicle” refers to a generally spherical structure composed of highly lipid-containing substances in which the lipids are organized in the form of lipid bilayers. Monolayer vesicles have a single lipid bilayer surrounding a central, amorphous cavity capable of encapsulating an aqueous volume. Monolayer vesicles can be made as either large unilamellar vesicles (LUV: diameter greater than about 1 μm) or small unilamellar vesicles (SUV: diameter less than about 0.2 μm). Multilamellar vesicles (MLV) have many onion-like shells with lipid bilayers. Owing to its high lipid content, MLV has applications for carrying certain small lipophilic molecules, but has a poor capacity for carrying aqueous substances. Paucilamellar vesicles (PLVs) are a series of approximately 2 to 10 bilayers in the shape of a generally spherical shell divided by an aqueous layer surrounding a central cavity without a lipid bilayer. is there. Since PLV can encapsulate both an aqueous substance and a hydrophobic substance, it can carry a wide range of substances. Unilamellar vesicles consist of a single bilayer of phospholipids and / or glycolipids and are the most commonly used lipid vesicles to mimic cell membrane structure because the phospholipids are also on the outer cell membrane. This is because it is a primary structural component of natural membranes. Liposomes (eg, phospholipid vesicles) can be used as carrier vehicles to deliver bioactive agents to tissues using the methods of the invention. For a review of transport of substances mediated by phospholipid vesicles, see Mannino, BioTechniques, 6: 682 (1988); Litzinger, DC Bioc.
See him. et Biophys. Acta, 1113: 201 (1992). Methods of making liposomes as carrier vesicles for delivering bioactive agents are known in the art (see, eg, US Pat. No. 4,522,811).

The term “topically” includes, for example, direct administration to the tissue to be treated, such as injection. Examples of local treatment include intratumoral injection and intralesional injection.

The term “topically and locally” includes both topical and topical administration. Also, for example, intraperitoneal treatment of ovarian cancer, instillation of drug into the bladder for treatment of the affected bladder, intraprostatic injection, intrahepatic infusion, perfusion of an excised organ (eg, lung), intrathecal treatment of brain tumor. , Intracerebral implantation of drug release devices for the treatment of brain cancer, and intralesional injection (eg, to skin lesions and tumors), eg, administering a compound into a fluid surrounding the tissue, You may inject directly into the inside. Topical local treatment may also be applied to other diseases such as viral or bacterial infections, interstitial cystitis and the like.

The term “microparticle” includes particles comprising apoptosis-inducing agents, therapeutic agents or other substances that can be advantageously delivered into tissues such as tumors using the methods of the invention. This term refers in size to about 0.1 μm to about 100 μm, about 0.5 μm to about 50 μm.
, 0.5 μm to about 20 μm, preferably about 1 μm to about 10 μm in size, about 5 μm in size, or a mixture thereof. Microparticles are, for example, macromolecules,
It may comprise a gene therapy product, a chemotherapeutic agent, or a protein binding agent. Typically, the microparticles can be administered, for example, topically, topically or locally.

The term “nanoparticles” includes particles comprising apoptosis-inducing agents, therapeutic agents or other substances that can be advantageously delivered using the methods of the invention inside tissues such as tumors. This term has a size of about 0.1 nm to about 1 μm, 1 nm to about 1 μm, about 10 nm to about 1 μm, about 50 nm to about 1 μm, about 100 nm to about 1 μm, about 250 to 900 nm, or preferably about 600 to 800 nm. Say particles. Nanoparticles may comprise, for example, macromolecules, gene therapy products, chemotherapeutic agents, or protein-binding agents. Typically, the nanoparticles can be administered to a patient by, for example, local, topical, local or systemic administration. In one example, the nanoparticles may comprise cross-linked gelatin.

The term “patient” includes animals that can be treated using the methods of the invention. Examples of animals are mice, rabbits, rats, horses, goats, dogs, cats, pigs,
Cows, sheep and primates (eg chimpanzees, gorillas, and preferably humans)
Such as mammals. In a further example, the patient is a cancer patient, eg, a human suffering from cancer.

The phrase “pharmaceutically acceptable carrier” is art-recognized and is a pharmaceutically acceptable substance, composition, or excipient suitable for administering a compound of the invention to a mammal. Including agents. Carriers include liquid or solid fillers, diluents, which are involved in carrying or transporting the drug from one organ or part of the body to another organ or part of the body,
Additives, solvents or encapsulants are included. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Examples of substances that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; celluloses such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate and derivatives thereof. Powdered tragacanth; Malt; Gelatin; Talc; Additives such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; glycerin , Polyols such as sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; Water without thermal substance; isotonic saline;
There are Ringer's solution; ethyl alcohol; phosphate buffered saline; and other non-toxic compatible substances used in pharmaceutical formulations. In one example, the pharmaceutically acceptable carrier is suitable for intravenous administration. In another example, the pharmaceutically acceptable carrier is suitable for topical local injection.

The term “pharmaceutically acceptable ester” refers to the relatively non-toxic, esterified products of compounds of the present invention. These esters may be made in situ during the final isolation and purification of the compounds, or the purified compounds may be reacted separately in the free acid or hydroxyl form with a suitable esterifying agent. You may produce by. The carboxylic acid can be converted to an ester by treating with an alcohol in the presence of a catalyst. Hydroxyl can be converted to an ester by treatment with an esterifying agent such as an alkanoyl halide. The term further includes the group of lower hydrocarbons that can be solvated under physiological conditions, such as alkyl esters, methyl esters, ethyl esters and propyl esters. (See, eg, Berge et al., Supra.)

The term “pharmaceutically acceptable salt” is art-recognized and comprises a compound of the invention:
Includes relatively non-toxic inorganic and organic acid addition salts. These salts may be made in situ during the final isolation and purification of the compounds of the invention, or the purified compounds of the invention in the form of the free base, into a suitable organic or inorganic acid. It may be made by reacting separately and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate,
Oleate, palmitate, stearate, laurylate, benzoate,
Lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptoate, lactobionate, and lauryl sulphate. Includes salts such as phonates. (For example, Berg
e et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

The phrase “pharmaceutical composition” includes formulations suitable for administration to mammals such as humans. When the compound of the invention is administered as a pharmaceutical to a mammal such as a human, it may be given alone or in a combination of 0.1 to 99.5% (more preferably 0.5 to 90%) with a pharmaceutically acceptable carrier. It may be given as a pharmaceutical composition containing the active ingredient.

The term “protein binding agent” includes agents that are bound to or capable of binding to proteins. Examples of protein binding agents are paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, vinca alkaloids, suramin, amitriptyline, amphotericin B, cefazoline, chlorothiazide, chlorpromazine, clindamycin, clofibrate, depsipeptide, desipramine, diazepam, Dicloxacillin, digitoxin, doxycycline, furosemide, heparin, indomethacin, lorazepam,
Nafcillin, nortriptyline, phenytoin, prazosin, prednisolone,
Propranolol, protriptyline, rifampin, sulfisoxazole,
Includes warfarin.

The term “rapid release formulation” means that the drug is in an already active form, or
Is a formulation of a drug that is delivered to a target site in a form that is effective in a relatively short period of time, such as within a few hours.
Includes formulations that can be released at doses of 50 nM and above. Examples of rapid release formulations include microparticle and nanoparticle formulations. The method of preparing these formulations is
This will be explained in Example 5.

The term “locally” refers to, for example, intraperitoneal administration to organs within the abdominal cavity (eg, bladder, ovary, etc.); intracranial administration to the brain; intraspinal administration to spinal column tissue; to cardiac tissue. Intravenous pericardial administration, etc., and administration to the local tissue location where the tissue to be treated is located.

The term “simultaneously” includes administration together. In one example, the therapeutic agent and the apoptosis inducing agent are formulated together. In such examples, the apoptosis-inducing agent is typically a rapid release formulation and the therapeutic agent is typically a sustained release agent,
For example, as a result, the therapeutic agent is released about 16 to 20 hours after the apoptosis inducer.

The term “sustained release agent” refers to a prescription of a drug that is delivered to a site of interest for a sustained period of time, after a sufficient time has elapsed for the apoptosis-inducing agent to induce apoptosis. Formulations which release the therapeutic agent from the. In one example, the sustained release agent comprises about 6 to 120 hours after administration, 6 to 96 hours after administration, about 6 to about 72 hours after administration, about 6 to about 48 hours after administration, about 6 to about 48 hours after administration. The therapeutic agent is released from 12 to about 36 hours, from about 12 to about 30 hours after administration, or, preferably, from 6 to about 24 hours after administration.

The term “parenchymal tissue cells” refers to cells containing solid tumors or tissues, including, but not limited to, “solid tumor cells”. These include outer or outer cell layers that can be treated with apoptosis-inducing agents so that therapeutic agents can be delivered to the interior of tumors or tissues, and the inner cell layers of multi-layered tissues. Solid tumor or tissue cells may be of epithelial cell lineage origin or may be of non-epithelial cell lineage origin. The term "solid tumor cells" includes cells comprising solid tumors, including cells in the outer or outer cell layers.

The term “sufficient time” means that the tissue is permeable to the therapeutic agent,
It includes the length of time required for the apoptosis inducer to induce apoptosis. For example, this sufficient time is sufficient for the density of epithelial cells, external or parenchymal tissue cells or tumor cells to be, for example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%. %, 35%, 40%, 45%, 5
It may be a time point when it decreases by 0% or more. In another embodiment, the sufficient time is
Parenchymal tissue cells, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12
It may be the time when apoptosis is induced by%, 15%, 17%, 20% or more. Example 3 showed that, in at least some situations, this sufficient time when an apoptosis-inducing agent such as paclitaxel was administered at a dose of 50 nM over 1 hour was about 16 to 24 hours. The sufficient time may vary depending on the type and dose of apoptosis-inducing agent and can be determined using the method as described in the examples.

In a further embodiment, the sufficient time is, for example, 72 hours or more and 72 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less,
35 hours or less, 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 2
3 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18
Time or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

The term “therapeutic agent” is meant to encompass any agent capable of providing a therapeutic benefit to a patient, including gene therapy products, chemotherapeutic agents, antibiotics, macromolecules, and protein-binding agents. Including. The term further includes any agent that can be delivered inside a tissue using the methods described herein. In one example, the therapeutic agent is paclitaxel or doxorubicin, or an analog or derivative thereof. In one example, the therapeutic agent comprises the same active ingredient as the apoptosis-inducing agent. For example,
Both the apoptosis-inducing agent and the therapeutic agent may be compounds such as, but not limited to, paclitaxel or doxorubicin. The therapeutic agent may be formulated as microparticles or nanoparticles. Other examples of therapeutic agents include macromolecules such as liposomes, nanoparticles, plasmids, viral vectors, non-viral vectors, chemotherapeutic agents, and oligonucleotides.

The term “tissue” includes, for example, liver, muscle (eg heart muscle, skeletal muscle, or smooth muscle),
Includes normal mammalian tissues such as skin, nerve and adipose tissue, and both benign and cancerous tumors.

The term “tumor” refers to abnormally growing tissue of any tissue type and includes both benign and malignant tumors, eg, cancerous tumors. Examples of cancerous tumors include sarcoma,
There are carcinomas, adenocarcinomas, lymphomas, and leukemias. The cancerous tumor may include metastatic lesions. In addition, it includes other tumors that may be advantageously treated using the methods and compositions of the invention. Cancerous tumors include, for example, fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer , Squamous cell carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, biliary tract carcinoma, choriocarcinoma, seminoma, fetal cancer, Wilms Tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngeal tumor, ependymoma, pineal Somatoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma , Leukemia, lymphoma, or may be a Kaposi's sarcoma.

The term “tumor suppressor gene” includes genes known to suppress tumors, eg, tumors that can be treated using the methods of the invention. Examples of tumor suppressor genes include, for example, p53.

II. Methods of the Invention In one embodiment, the present invention relates to a method of delivering a therapeutic agent to a tissue (eg, within a multi-layered tissue) of a patient, eg, a mammal such as a human. The method comprises administering an apoptosis-inducing agent to a patient, allowing the apoptosis-inducing agent a sufficient time to induce apoptosis in the tissue of the patient, and delivering a therapeutic agent to the tissue. Including. The tissue can be, for example, liver, muscle (eg, myocardium, skeletal muscle, or smooth muscle), skin tissue, nerve tissue, or adipose tissue, or brain, breast, ovary, bladder, prostate, colon, lung, liver, or It may be a tumor such as a tumor of the uterus. The apoptosis-inducing agent may be administered systemically, locally, locally, or any combination thereof, such as both locally and locally (locally). In a further embodiment, the method further comprises obtaining the apoptosis-inducing agent prior to administration to the patient.

In a further embodiment, the apoptosis-inducing agent is paclitaxel or doxorubicin. The therapeutic agent may be a gene therapy product, a chemotherapeutic agent, a protein binding agent, or an antibiotic.

In a second further embodiment, the sufficient time and / or dose is sufficient to reduce the density of epithelial cells, exogenous or parenchymal cells in the tissue. For example, the density of parenchymal tissue cells is, for example, 32 hours or less, 30 hours or less, 27 hours or less, 2
4 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19
Time or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less , 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less, about 1%, 2%, 3%, 4%, 5%, 6%
, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%
, 30%, 35%, 40%, 45%, 50% or more.

In another embodiment, the density of parenchymal tissue cells is, for example, 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less,
15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 1
0 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less,
For less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour, about 0.
1% to about 50%, about 1% to about 45%, about 2% or about 45%, about 1% to about 40%, 5% to about 40%, 1
It may be reduced by 0% to about 40%, 20% or about 40%, 25% to about 40%, or about 30% to about 35%.

In another embodiment, the time is, for example, 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less,
9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less,
Approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% of parenchymal tissue cells over a period of 3 hours or less, 2 hours or less, or 1 hour or less , 10%, 11%, 12%, 13%, 14%, 15%
, 20%, 25% or more, sufficient to induce apoptosis.

In another embodiment, the time and / or dose is, for example, 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less,
Of the parenchymal tissue cells over 10 hours, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less About 1% to about 25%, or alternatively, about 1% to about 20%, about 1%
From about 17%, about 1% to about 15%, about 1% to about 14%, about 5% to about 13%, about 5% to about 12%,
Sufficient to induce apoptosis from about 1% to about 10%. Cell apoptosis and their densities can be measured using the techniques described in Examples 1, 2 and 3.

In a third further embodiment, the apoptosis-inducing agent is also a therapeutic agent. However, in this case, the therapeutic agent is typically administered in a separate dose or in a sustained release form.

In a fourth further embodiment, the apoptosis inducer and therapeutic agent are administered simultaneously.
In this example, the therapeutic agent is typically a sustained release agent that releases the therapeutic agent after a period of time sufficient for the apoptosis inducing agent to induce apoptosis in the tissue of the patient.

In yet another embodiment, the apoptotic agent and therapeutic agent are administered sequentially. In this method, the therapeutic agent is administered as another dose after a period of time sufficient for the apoptosis inducer to induce apoptosis in the patient's tissue.

In a second embodiment, the invention relates to a method of delivering a therapeutic agent to a tumor (eg a cancerous or benign tumor) in a patient. The method comprises administering a dose of an apoptosis-inducing agent to a patient, allowing the apoptosis-inducing agent to have sufficient time to induce apoptosis in the tumor, and delivering a dose of the therapeutic agent to the patient. And a step of performing. The tumor can be, for example, a brain, breast, ovary, bladder, prostate, colon, lung, liver, or uterine tumor. Examples of apoptosis inducers are paclitaxel and doxorubicin. In a further embodiment, the method further comprises obtaining an apoptosis-inducing agent.

In a further embodiment, the dose of the apoptosis-inducing agent is sufficient to reduce the density of epithelial cells, exogenous cells, or solid tumor cells so that the therapeutic agent can be delivered inside the tumor. . For example, the dose of the apoptosis-inducing agent depends on the cell density, for example, 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less. , 18 hours or less,
17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 1
2 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less Over about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%
, 15%, 20%, 25%, 30%, 35%, 40%, 45% or more, sufficient to reduce.

In yet another embodiment, the apoptosis-inducing agent is for 30 hours or less, 30 hours or less, for example.
Time less than 27 hours less than 24 hours less than 23 hours less than 22 hours less than 21 hours less than 20 hours less than 19 hours less than 18 hours less than 17 hours less than 16 hours less than 15 hours less than 14 hours , 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or About 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 2 of solid tissue cells over a period of less than 1 hour
Induces 0%, 25% or more apoptosis. In one particular case where the apoptosis-inducing agent is paclitaxel, said sufficient time is for example between about 16 and about 24 hours when it is administered at a dose of about 50 nM for about 1 hour.

In a second further embodiment, the apoptosis inducer is also a therapeutic agent. However, in this case, the therapeutic agent is administered as a separate dose or as a sustained release type apoptosis inducer. Examples of apoptosis inducers that are also effective as therapeutic agents include:
For example, paclitaxel and doxorubicin.

In a third further embodiment, the therapeutic agent comprises a gene therapy product, a protein binding agent, a chemotherapeutic agent, or an antibiotic.

In a fourth further example, the apoptosis-inducing agent and / or therapeutic agent can be administered systemically, locally, locally or locally, respectively. In an advantageous embodiment, the apoptosis-inducing agent and / or therapeutic agent is administered with one or more pharmaceutically acceptable carriers. For example, these agents can be formulated in a manner suitable for intravenous injection.

In yet another embodiment, the invention features a method of delivering a chemotherapeutic agent to a tumor of a patient, eg, a human (eg, a cancer patient). The method comprises the steps of administering a dose of an apoptosis-inducing agent to a patient locally or locally, allowing the apoptosis-inducing agent to have sufficient time to induce apoptosis in the tumor, and administering a dose of a chemical agent. Delivering a therapeutic agent to the patient. In a further embodiment, the tumor is a cancerous tumor, such as a brain, breast, ovary, bladder, prostate, colon, lung, liver, or uterine tumor. In a further embodiment, the method further comprises the step of obtaining an apoptosis-inducing agent.

In a second further embodiment, the apoptosis-inducing agent comprises paclitaxel, doxorubicin, or another effective apoptosis-inducing agent.

[0063] In a third further embodiment, the sufficient time is sufficient to reduce the density of parenchymal tissue cells so that the therapeutic agent can be delivered inside the tissue. For example, the sufficient time includes a cell density of 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less,
18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 1
3 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 It may be sufficient to reduce about 5%, 10%, 15%, 20%, 30%, 35% or more over time or less. In yet another further embodiment, the sufficient time is sufficient to induce apoptosis in parenchymal tissue cells so that the therapeutic agent can be delivered inside the tumor. For example, the sufficient time is, for example, 32 hours or less, 30 hours or less, 27 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less. Hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less,
11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less About 1% of tissue cells, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more,
May be sufficient to induce apoptosis of the.

In a fourth further embodiment, the apoptosis inducer is also a chemotherapeutic agent. However, in this case, the chemotherapeutic agent is administered as a separate dose or as a sustained release form.

In a fifth further embodiment, the apoptosis-inducing agent and / or therapeutic agent is a pharmaceutically acceptable carrier, such as, for example, systemic, topical, topical, or a carrier suitable for topical administration. Administer with. The carrier may be suitable for infusion.

In a sixth further embodiment, the apoptosis inducer and therapeutic agent are administered simultaneously. In this example, the therapeutic agent is formulated as a sustained release such that the apoptosis inducer is released after a time sufficient to induce apoptosis.

In a seventh further embodiment, the apoptosis inducer and therapeutic agent are administered sequentially. In this example, the therapeutic agent is administered after a time sufficient for the apoptosis inducer to induce apoptosis in the tissue of the patient.

In another embodiment, the invention features a method of delivering a dose of a therapeutic agent to a tissue of a patient.

The method comprises the step of administering to the patient a dose of an apoptosis-inducing agent, wherein the dose of the apoptosis-inducing agent does not comprise (i) the dose of the therapeutic agent, or (ii) ) Comprising said dose of said therapeutic agent in a sustained release formulation, and allowing said apoptosis inducer to have sufficient time to induce apoptosis in the tissue of the patient. (A) if the dose of the apoptosis-inducing agent does not comprise the dose of the therapeutic agent, the dose of the therapeutic agent is administered to the patient so that the dose of the therapeutic agent is delivered to the tissue of the patient. And (b) if the dose of the apoptosis-inducing agent comprises the dose of the therapeutic agent as a sustained release agent, the dose of the therapeutic agent is delivered to the tissue of the patient. Serial therapeutic agent; and a step of passage of sufficient time to be released at the tissue.

The invention further provides that a step of administering a dose of an apoptosis-inducing agent, such as paclitaxel or doxorubicin, to a patient locally, locally or locally, and the apoptosis-inducing agent is sufficient to induce apoptosis. For a period of time and delivering the therapeutic agent. In a further embodiment, the apoptosis inducer is paclitaxel, the dose is 50 nM over 1 hour, and the sufficient time is 16 to 24 hours.

The invention further provides that a dose of an apoptosis-inducing agent such as paclitaxel or doxorubicin is administered to a patient locally, locally, or locally, and the apoptosis-inducing agent is sufficient to induce apoptosis. For a period of time and delivering the therapeutic agent, the method of treating a breast cancer tumor. In a further embodiment, the apoptosis inducer is doxorubicin, the dose is 5: M over 1 to 4 hours, and the sufficient time is 16 to 30 hours.

III. Pharmaceutical Compositions of the Invention The present invention also relates to compositions for delivering therapeutic agents to a patient. The composition is
It includes rapid-release formulations of apoptosis-inducing agents, sustained-release formulations of therapeutic agents, and pharmaceutically acceptable carriers.

In a further embodiment, the apoptosis-inducing agent is paclitaxel. For example, one rapid release formulation advantageously releases about 50 nM paclitaxel over about 1 hour or less.

[0074]   In a second further embodiment, the apoptosis inducer is doxorubicin.

In a third further embodiment, the apoptosis-inducing agent is in an amount sufficient to reduce the density of solid tumor tumor cells by about 30% or more within about 16 to 24 hours after administration. provide. Further, the apoptosis inducer may be provided in an amount sufficient to induce apoptosis in 10% or more of the solid tumor tumor cells within about 16 to 30 hours after administration.

In a fourth further embodiment, the therapeutic agent is a gene delivery construct comprising paclitaxel or doxorubicin, a protein binding agent, a chemotherapeutic agent, an antibiotic, or a tumor suppressor gene, eg, p53. Gene delivery products such as.

In a fifth further embodiment, the pharmaceutical composition is suitable for intravenous injection. The composition may further be suitable for topical, topical, topical, or systemic administration.

In another embodiment, the pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers.

In yet another embodiment, the present invention relates to nanoparticles, said nanoparticles comprising cross-linked gelatin and a therapeutic agent such as paclitaxel or doxorubicin or an apoptosis inducer. In a further embodiment, the present invention relates to a composition containing said nanoparticles and a pharmaceutically acceptable carrier. The carrier may be suitable for systemic administration, topical administration, local topical administration, or topical administration, for example. In another example, the invention relates to a method of treating a patient, comprising administering the nanoparticles of the invention. In one embodiment, the nanoparticles have a diameter of about 500 to about 1 μm, or about 6
It is from 00 nm to about 800 nm.

The invention further relates to microparticles comprising a therapeutic agent such as paclitaxel or doxorubicin or an apoptosis inducer. In one embodiment, the microparticles have a diameter of about 500 nm to about 100 μm, about 500 nm to about 50 μm,
About 500 nm to about 25 μm, about 500 nm to about 20 μm, about 500 nm to about 15 μm, about 500 nm to about 10 μm, about 750 nm to about 10 μm, about 1
μm to about 10 μm, about 750 nm to about 7.5 μm, about 1 μm to about 7.5 μm
m, about 2 μm to about 7.5 μm, 3 μm to about 7 μm, or about 5 μm. In another embodiment, the invention relates to a composition comprising said microparticles and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be, for example, topical, topical,
Alternatively, it may be suitable for local, local administration to a patient. The invention further relates to a method of treating a patient, comprising the step of administering to the patient the microparticles of the invention and a pharmaceutically acceptable carrier. In a further embodiment, the administration may be local, local, or local.

In another embodiment, the invention features microparticles comprising paclitaxel suitable for topical, local, topical administration to a patient, said microparticles having a size of about 5 μm. Have a diameter. In another further embodiment, the invention features microparticles comprising doxorubicin suitable for topical, local, topical administration to a patient, said microparticles having a diameter of about 5 μm. Have.

The invention further relates to a kit for the treatment of tumors. The kit uses an apoptosis inducer in a pharmaceutically acceptable carrier, a therapeutic agent in a pharmaceutically acceptable carrier, a container, and the apoptosis inducer and the therapeutic agent to treat a tumor. And instructions for storing. For example, the kit of the present invention may comprise an apoptosis inducer and a therapeutic agent for subsequent intravenous injection. Further, the kit may provide an apoptosis-inducing agent and / or therapeutic agent formulated in a dose and a carrier suitable for topical, topical, or topical administration.

Pharmaceutical compositions comprising the compounds of the present invention include humectants such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, colorants, strippers, coatings, sweeteners, flavors and It may contain a fragrance and a preservative.

The formulations of the present invention include oral, nostril, topical, transdermal, buccal, sublingual, rectal, vaginal, and / or
Alternatively, those suitable for parenteral administration are included. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient which may be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. Generally, by uniformly and intimately bringing into association the compound of the present invention with the liquid carrier, or the finely divided solid carrier, or both, and then, if necessary, shaping the product. , Prepare the formulation.

Formulations of the present invention suitable for oral administration include capsules, cachets, pills, tablets, (usually sucrose and acacia gum or tragacanth) each containing a predetermined amount of a compound of the present invention as an active ingredient. Lozenges, powders, with perfumed bases that are
As a granule, or an aqueous or non-aqueous liquid solution or suspension, or as an oil-in-water or water-in-oil liquid suspension, or as an elixir or syrup (such as gelatin and glycerin or sucrose or acacia gum). It may be in the form of pastilles (using an inert base) and / or in the form of a mouthwash. Additionally, the compounds of the present invention may be administered as boluses, lozenges or pasta.

In the solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is, for example, sodium citrate or dicalcium phosphate, and / or Mixing with one or more pharmaceutically acceptable carriers such as any of the following: Fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, binders such as sucrose and / or acacia, glycerol. Wetting agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, specific silicates,
And disintegrators such as sodium carbonate, dissolution retarders such as paraffin, absorption enhancers such as quaternary ammonia compounds, wetting agents such as cetyl alcohol and glycerol monostearate, absorbents such as kaolin and bentonite clay, talc, Lubricants and colorants such as calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate, and mixtures thereof. In the case of capsules, tablets and pills, the pharmaceutical composition may further comprise buffering agents. Solid compositions of the same type may be utilized as fillers in soft and hard filled gelatin capsules with excipients such as lactose or lactose, high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets are binders (eg gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrating agents (eg sodium starch glycolate or crosslinked sodium carboxymethylcellulose), surfactants or dispersants. You may produce using. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets of the pharmaceutical compositions of the present invention, and other solid dosage forms such as dragees, capsules, pills and granules, may optionally be scored or enteric coated and It may be prepared with coatings and shells, such as other coatings known in the pharmaceutical industry. They further use various ratios such as hydroxypropylmethylcellulose to create the desired release curves, other polymeric matrices, liposomes and / or microspheres, to delay or control the release of the active ingredient therein. You may mix so that. They are for example filtered through a bacteria-retaining filter or by incorporating a sterilant in the form of a sterile solid composition which can be dissolved in sterile water, or some other sterile injectable medium immediately before use, It may be sterilized. Further, the composition may optionally contain an opacifying agent, and the active ingredient may be selected only in a specific part of the gastrointestinal tract, or selectively in a specific part, and in a delayed manner depending on the selection. The composition may be released. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in micro-encapsulated form, optionally with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms include, for example, water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3. -
Butylene glycol, oils and fats (especially cottonseed oil, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof, etc.,
May be included.

Oral compositions include, for example, wetting agents, emulsifying agents and suspending agents, in addition to inert diluents.
Adjuvants such as sweetening agents, flavoring agents, coloring agents, flavoring agents and preservatives may be included.

Suspending agents may be, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum deuteroxide, bentonite, agar and tragacanth, and mixtures thereof. A turbidity agent may be included.

Formulations of the pharmaceutical compositions of this invention for rectal or vaginal administration may be presented as suppositories, which also contain one or more compounds of the invention in one or more suitable formulations. It may be prepared by mixing with a non-irritating excipient or carrier which comprises cocoa butter, polyethylene glycol, suppository wax or salicylate, and Since the carrier is a solid at room temperature but a liquid at body temperature, it will melt in the rectal or vaginal cavity to release the active compound.

Formulations of the present invention suitable for vaginal administration further include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. .

Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments,
Includes pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants.

Ointments, pastes, creams and gels include, in addition to the active compounds of the invention, animal and vegetable fats, oils, waxes, paraffins, starches, tragacanths, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acids, Excipients such as talc and zinc oxide, or mixtures thereof may be included.

Powders and sprays contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Good. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. In addition, absorption enhancers can be used to increase the penetration of the compound through the skin. The rate of such permeation can be controlled by providing a rate controlling membrane or by dispersing the active compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of the present invention suitable for parenteral administration include one or more compounds of the present invention in one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions. ,
A dispersion, suspension or emulsion, or in combination with a sterile powder, which may be reconstituted into a sterile injectable solution or dispersion immediately before use. Possible solutions or dispersions may include buffers, bacteriostats, solvents that make the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention are water,
Ethanol, polyols (eg glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. A variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like, can be included to help control microbial activity. Additionally, it may be preferable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, for prolonged absorption of the injectable dosage form, substances which cause absorption may be included, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is preferable to delay absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by utilizing a liquid suspension of crystalline or amorphous material with poor water solubility. Thus, the rate of drug absorption depends on the rate of dissolution, which in turn depends on the size and shape of the crystals. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulation matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and depending on the nature of the particular polymer used, the rate of drug release can be adjusted. Examples of other biodegradable polymers include poly (
Orthoesters) and poly (anhydrides). Depot injectable formulations are also made by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

The formulations of the present invention may be given orally, parenterally, topically, rectally, intralesionally, intraorbitally, intracapsularly, by direct instillation into the cavity, or by inhalation.
They are, of course, provided in a form suitable for each administration route. For example, they are administered in tablets or capsules, by injection, inhalation, eye lotion, ointment, suppository, etc., administration by injection, infusion or inhalation, topical by lotion or ointment, and rectal administration by suppository.

These compounds can be administered to humans and other animals, including, for example, orally by spray, rectum by nose, powder, ointment or drops, vaginal, parenteral, vaginal and topical, as well as buccal and tongue. It may be administered for treatment by any suitable route of administration, including below.

Regardless of the route of administration chosen, the compounds of the invention may be used in a suitable hydrated form and / or the pharmaceutical compositions of the invention may be prepared by conventional methods known to those skilled in the art. It may be prepared in a pharmaceutically acceptable dosage form.

The actual dosage of active ingredients in the pharmaceutical compositions of the present invention will be effective to obtain the desired therapeutic response for a particular patient, composition, and route of administration without causing toxicity to the patient. It may be modified to obtain the amounts of the components.

The dosage chosen will depend on the activity of the particular compound of the invention, or ester, salt, or amide thereof, utilized, the route of administration, the time of administration, the rate of elimination of the particular compound used, the duration of treatment, A variety of other agents, compounds and / or materials used in combination with the specific compound, age, sex, weight, condition of the patient being treated, general health and previous medical history, and similar factors known in the medical arts. It will depend on various factors.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian may start the dose of the compound of the invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and obtain the desired effect. It may be possible to increase this dose progressively until given.

In general, a suitable dose of a compound of the invention will be that amount of the compound which is the lowest dose effective for producing a therapeutic effect, ie treating a condition in a subject such as cancer. Ah Such an effective amount will generally depend on the factors mentioned above.
Generally, an intravenous and subcutaneous dose of a compound of the invention to a single patient will be a dose of 0.0001 to about 100 mg / kg body weight, more preferably about 0.01 to about 10 mg / kg, even more preferably. Will range from about 0.10 to about 4 mg / kg. If necessary, the effective daily dose of the active compound may be divided into two, three, four, five, six, or more small doses at appropriate intervals over the whole day for selection. It may be administered in a unit dose form accordingly.

Where the compound of the present invention can be administered alone, it is preferable to administer the compound as a pharmaceutical composition.

As mentioned above, the compounds of some embodiments may contain a basic functional group such as, for example, an amino group or an alkylamino group, and thus may be combined with a pharmaceutically acceptable acid. They can be combined to form a pharmaceutically acceptable salt.

In another example, the compounds of the present invention may contain one or more acidic functional groups and, thus, are combined with a pharmaceutically acceptable base to form a pharmaceutically acceptable salt. Can be made.

EXAMPLES The present invention is further embodied by the examples below, but should not be construed as limiting.

Ingredients and Methods: Chemicals and Reagents: Paclitaxel and Doxorubicin are available from Bristol-Myers Squibb (Wallingford, CT), Pharmacia & Upjohn (Milan, Italy), and / or National Cancer Institute of the United States. (Bethesda, MD) and 3 "-[ ³ H] paclitaxel (specific activity 19.3 Ci / mmol) were obtained from the National Cancer Institute (Bethesda, MD). Cefotaxime sodium was from Hextorcel. (Somerville, NJ), Gentamicin from Solopac Laboratories (Franklin Park, IL), Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), RPMI
-1640, non-essential amino acids, L-glutamine and trypsin from Gibb Laboratories (Grand Island, NY), sterile pig skin collagen gel (Spongostane standard) from Health Design Institute (Rochester, NY), soluble tissue Gel solubilizers and atomlite scintillation fluids from DuPont Biotechnology Systems, Inc. (Boston, Mass.), Hyperfilm ³ H from Amersham Life Sciences (Arlington Heights, IL), autoradiograph supplies from Kodak (New York, Rochester), Cremophor EL from Sigma (St. Louis, Missouri), LKB2208 Ultrofilm from Leica, and autoradiograph supplies from Kodak (New York, NY). From Chester), cryotome buried polymer (OC
.T.) From Miles, Inc. (El Charter, Ind.), And monoclonal antibodies against Pgp (JSB-1) and polyclonal antibodies (ab-1) from Biogenex Inc. (San Ramon, Calif.) And Oncogene Inc., respectively. (Cambridge, Mass.).

Animals Male nu / nu balb / c mice weighing 18-21 g from the National Cancer Institute, Oscopene Hagen rats weighing 190-210 g from Harlan Biochemicals (Dolly, Ohio) and weighing 18-22 g. Male nu / nu balb / c mice were purchased from the National Cancer Institute. Animal care was provided by the Laboratory Animal Care Department of the Institute.

Tumor Acquisition Surgical specimens of patient tumors (ie prostate, head and neck, ovaries) were obtained from the Tumor Acquisition Service of the Ohio State University Comprehensive Cancer Center. Prostate tumor specimens were placed in MEM and head and neck and ovarian tumor specimens were placed in Hank's buffered saline for 10 to 30 minutes after surgical excision and stored on ice to prepare for culture within 1 hour of excision. .

Establishment of Human Xenograft Tumors in Mice Three human xenograft tumors, a human pharyngeal FaDu tumor, a human breast MCF7 tumor, and a human prostate PC3 tumor, were used in the following three cases. FaDu cells were obtained from the American Type Culture Collection (Rockville, MA). MCF7 cells were obtained from Dr. Kenneth Cowan, National Cancer Institute. Cells were harvested from subconfluent medium with trypsin and resuspended in fresh medium before plating. Cells with viability greater than 90% as determined by trypan blue exclusion were used for tumor implantation. Cells were centrifuged and resuspended in Matrigel (1: 1, v / v). Matrigel is a solubilized tissue basement membrane preparation extracted from Engelbreath-Holmsworm mouse tumors and has been shown to maintain human tumor growth in immunodeficient mice (Klienman H et al. (1990) Proc Am Assoc. Cancer Res 31: 490-4
91). Tumor colonization was performed with 10 ⁶ cells (0.1-0.2 ml) on the right and left upper backs using an 18 gauge needle.
) Was subcutaneously injected. Tumors were removed when they reached a size of 0.5 to 1 g and used for the experiment.

Establishing MCF7 tumors was performed as described for FaDu tumors, except that a sustained release pellet of 17-beta-estradiol was subcutaneously implanted in the posterior neck of mice.
Tumor collection was performed when tumor size reached approximately 0.5 in 2 weeks.

Androgen-independent human prostate PC3 cells were prepared using previously published methods (Pretlow
et al. 1993; Nagabhushan et al., 1996) and maintained in male nude mice as xenograft tumors. Briefly, finely chopped tumor tissue was mixed with an equal volume of Matrigel and 0.3 ml each was implanted on both flanks of nude mice. Tumor collection was performed approximately 1.5 to 2 months after transplantation when the amount reached 1 g.

Tissue Culture Patient tumors or xenograft tumor specimens were processed as previously described (K
uh, et al. (1999) J. Pharmcol. Exper. Therap. 290: 871-880). Specimens were washed 3 times with culture medium and cut into pieces of approximately 1 mm ³ under sterile conditions. The culture medium is
1: 1 mixture of MEM / DMEM for patient tumors, MEM for FaDu xenografts, and P
For C3 xenografts consists of RPMI1640, 9% heat inactivated FBS, 2mM glutamine, 0.1m
M non-essential amino acids (only for MEM / DMEM), 90: g / ml gentamicin, and 90: g / m
l Cefotaxime sodium was added. Five tissue culture sections were cultured on 1 cm ² pieces of presoaked collagen gel in 6 ml plates in 4 ml culture medium. 2 to 4
After a day, tumor tissue cultures were used for drug penetration kinetics studies.

Example 1: Determinants of Paclitaxel Penetration and Accumulation in Human Solid Tumors This study demonstrates that human patients had head and neck tumors and human pharyngeal Fa maintained in athymic mice.
It was performed using Du xenograft tumors. [ ³ H] paclitaxel was used in this study.
Paclitaxel permeation into tumor tissue cultures was visualized using autoradiography and drug concentration in tumor tissues was measured using liquid scintillation counting.

Methods for Tissue Culture Drug Uptake and Elimination Studies Tumor tissue cultures were cultured with 4 ml of culture medium containing a mixture of radiolabeled and unlabeled paclitaxel of 12 to 12,000 nM. The final concentration of [ ³ H] paclitaxel was 2.6 nM at 0.05 mCi / ml or 5.2 nM at 0.1 mCi / ml. These paclitaxel concentrations are within the range of clinically attainable plasma concentrations (ie up to 13,000 nM, Kearns et al. (1995) Sem Oncol 22 (Suppl.6): 16-23).
. For excretion studies, tumor tissue cultures were cultured with paclitaxel for a maximum of 24 hours before substantial apoptosis occurred (Au JLS et al. (1998) Cancer Res.
58: 2141-2148), and then transferred to a new plate and maintained in a drug-free medium. After a certain period of time, 100 ml of medium was collected from each well and the tissue culture was taken out from the plate. Tissue cultures were blotted dry on filter paper and weighed. Mix 100 ml of medium or tumor sample with 0.5 ml of soluble tissue / gel lysate and oven
After overnight culture at 50 ° C, total radioactivity was analyzed using liquid scintillation counting. In a preliminary study, the method described previously (Royer I et al. (1995) Rapid Commun
Mass Spectrom 9: 495-502) and high pressure liquid chromatographic fractions revealed that 95% of the radioactivity in the medium represented paclitaxel and the epimerization product 7-epitaxol. The ratio of 7-epitaxol to paclitaxel in the culture medium with FaDu cells was influenced by the culture time and the drug concentration in the medium, from 2% after 3 hours to 7% after 24 hours and 100% after 24 hours. 7% at nM
From 5,000 nM to 25%. 7-Epitaxol has similar microtubule binding affinity and cytotoxicity to paclitaxel (Ringe I et al. (1987) J Phar
macol Exp Ther 242: 692-298), total radioactivity was expressed in paclitaxel equivalents. The drug concentration in the tissue was expressed in mol by dividing (drug amount) by (tissue weight).

For each tumor classification, namely patient head and neck tumor, patient ovarian tumor, and FaDu xenograft tumor, three tumors were used per experiment, with 30 to 35 tumor tissue cultures at each concentration and time point. I used a thing. The study design of the experiment with patient tumors was dictated by the size of the specimen. In some cases, a sample from a single patient was sufficient for studying drug uptake and excretion at one or more drug concentrations but insufficient for studies at all concentrations. A total of 7 head and neck tumors and 3 ovarian tumors were used. For FaDu xenograft tumors, specimens from each animal were large enough.
Each tumor was used for uptake and elimination studies at drug concentrations across all species.

Paclitaxel Accumulation in Tumor Tissue Cultures Elevated paclitaxel concentrations in patient tumor tissue cultures (head and neck and ovary) and in xenograft tumors are shown in FIG. 1 and Table 1. Drug concentration in tumor tissue cultures increased over time in all three tumors, reaching quasi-steady state between 48 and 72 hours, with less than 5% increase over the next 24 to 48 hours It was During this time, the drug concentration in the medium decreased by about 25%. An analysis of the mass balance suggests that it accounted for about 90% of the dose. The steady-state tumor-to-medium concentration ratio was 20 to 120, suggesting a significant drug accumulation in the tumor.

[0127] Figure 1 and Table 1 are the results of an experiment designed to examine the uptake of paclitaxel into tissue cultures of FaDu xenograft tumors and patient tumors. As shown in FIG. 1, the concentration-time profile of paclitaxel in the tumor tissue cultures obtained after culturing at different initial drug concentrations (C _medium ) in the culture _medium showed that the tumor concentration was at the pseudo-steady state level (T _The time to reach half of _{1/2, uptake} ) and the tumor-medium concentration at steady state were analyzed. Statistically significant differences in patient tumors (head and neck and ovary, n = 6) and FaDu tumors (n = 3) at equal initial media concentrations were analyzed by unpaired two-tailed Student's t-test.

(Table 1)

While steady-state paclitaxel concentrations in tumors increased, the steady-state tumor-to-medium concentration ratio decreased with the initial drug concentration in the culture medium, but the relationship was not linear. In general, the time T _{1/2, uptake} required to reach 50% of the quasi-steady state level is
It decreased as the initial medium concentration increased (P <0.01, regression analysis). These data suggest that drug accumulation may be partially saturated, indicating that higher initial extracellular drug concentrations reach steady state faster.

Paclitaxel Excretion from Tissue Cultures Figure 2 and Table 2 compare the kinetics of drug elimination from patients and xenograft tumors.
Drug concentrations decreased to quasi-steady-state levels in 48 hours in all three tumors. The rate of excretion also depends on the initial concentration and within the first 24 hours, 29% to 60% at 120 nM
At 200 nM, the range was 41% to 81%. The rate of decrease in drug concentration over the next 48 hours was several times smaller, ranging from 1% to 12% at 120 nM and 3% to 13% at 1200 nM. The time to reach 50% of the quasi-steady state level, T _{1/2, efflux} , ranged from 3 to 7.5 hours. Tumor concentration decreased with increasing medium concentration. Tumor to media concentration ratio
It ranged from 400 to 4,000 for 24 hours and 250 to 2,700 for 72 hours. These ratios are the steady-state tumor-to-medium concentration ratios observed during uptake studies (ie 20 to 1
20) 8 to 38 times more, suggesting that the inhalation conditions were maintained during the emission study. Therefore, this high steady-state tumor-to-medium concentration ratio suggests that drug retention in tumors is significant, i.e. 19% of the initial drug concentration.
To 71% were maintained up to 24 hours and 16 to 72% were maintained up to 72 hours. Overall, the percentage maintained and the tumor-to-medium concentration ratio reached at low initial medium concentrations of 120 nM were significantly higher than at high initial concentrations of 1,200 nM (p <0.05, unpaired two-tailed Student's t-test) . However _, the difference between the apparent T _{1/2 and efflux at} these two initial medium concentrations was not statistically significant. Thus, these data suggest significant tumor drug retention and an inverse relationship between retention and drug concentration rather than excretion rate.

FIG. 2 and Table 2 show the results of experiments designed for the measurement of paclitaxel excretion from FaDu xenografts and patient tumor tissue cultures. Tumors were incubated with paclitaxel for 24 hours. After replacing the drug-containing medium with drug-free medium, 24 hours and 72 hours after the treatment.
The drug concentration remaining in the tissue culture over time was analyzed to determine the time taken to reach 50% of the quasi-steady state level (T _{1/2, efflux} ). Statistically significant differences between patients (head and neck and ovary, n = 6) and FaDu (n = 3) tumors at equal initial media concentrations were analyzed using unpaired t-test. Different units were used for media concentration and tumor concentration.

(Table 2)

Differences Between Patients and Xenograft Tumors Among patient tumors, head and neck tumors have higher accumulation (ie, higher steady state tumor to medium concentration ratio) and slower uptake rates compared to ovarian tumors. (Longer apparent T _{1/2, uptake} ) tended (Tables 1 and 2). However, the differences are not statistically significant due to the large variability between individual tumors. On the contrary, there is a significant difference between the drug uptake rate, drug accumulation range and drug maintenance range of patient tumors and xenograft tumors (Tables 1 and 2). Compared to patient tumors, xenograft tumors have a slow uptake rate and a large accumulation range when the initial drug concentration is 120 nM or higher, but not when the medium concentration is 12 nM, which is low. Xenograft tumors also 3 compared to patient tumors
Showed 4-fold greater drug retention. Differences in drug uptake and accumulation between patients and xenograft tumors were used to study determinants of paclitaxel uptake and elimination in solid tumors.

Determinants of Paclitaxel Uptake and Excretion The rate of paclitaxel uptake in tumors involves multiple kinetic processes, from the medium to the collagen gel matrix, to the tumor tissue culture,
And then translocation through the interstitial space into cells and binding to tubulin and microtubulin and / or other macromolecules (Jordan MA et al.
(1993) Proc Natl Acad Sci USA 90: 9552-9556; Manfredi JJ et al. (1982) J
Cell Biol 94: 688-696). Binding to macromolecules determines the extent of drug accumulation, and the plateau drug accumulation reached at high drug concentrations in the culture medium reflects saturation of the binding site (Jordan MA et al. (1996). ) Cancer Res 56: 816-825; Kang et
al. (1997) Proc Am Assoc Cancer Res 38: 604). During excretion, free drug, including drug dissociated from the binding site, diffuses and / or is transported by the drug-extracting mdrl p-glycoprotein into the intercellular space, into the collagen gel matrix, and into the culture medium periphery. And move. Tumor drug retention is determined by binding to macromolecules and elimination rate by dissociation of drug from the binding site. The quasi-steady state reached during excretion reflects the slow rate of dissociation of paclitaxel from the binding site.

Three studies were conducted to reveal the major determinants of paclitaxel uptake and elimination in solid tumors. These studies included (a) evaluation of drug diffusion from the culture medium into tissue culture, (b) evaluation of the role of drug efflux mdrl p-glycoprotein, and (c) evaluation of the role of cellularity and apoptosis. Is included.

Diffusion of paclitaxel from culture medium into three-dimensional tumor tissue cultures In this study, slow drug diffusion was determined to be the full drug by determining the rate of drug diffusion from the culture medium to the collagen gel matrix that maintains the tissue culture. It was evaluated whether it contributed to the slow penetration into the primary tumor. 1 cm ^{2 of} collagen gel pieces were presoaked and placed in the wells of a 6-well plate with 4 ml of complete culture medium. No tumor was added. After culturing for 3-4 days, the medium was replaced with a medium containing 4 ml of 120 nM [ ³ H] paclitaxel,
It was cultured at 7 ° C for 24 hours. After a certain period of time, 100: 1 medium was removed from each well. In order to collect the medium captured by the porous collagen gel, one piece of the collagen gel was transferred to a new plate, and the gel was squeezed with forceps to collect the medium. These operations took less than 20 seconds. The radioactivity of the medium was measured. The results show that immediately after adding the drug solution to the medium (ie, less than 12 seconds), the drug concentration in the culture medium entrapped in the collagen gel matrix (C _gel ) is about the initial drug concentration in the culture medium.
It shows that it was 50%. Since the rate of increase of C _gel was faster than the increase of drug concentration in the tissue culture, the drug diffusion from the medium through the collagen gel matrix showed that the drug was diffused into the tumor tissue culture within the first 24 hours. It suggests that it is not the rate-determining factor for slow penetration.

Effect of mdr1 p-Glycoprotein (Pgp) Expression on Drug Accumulation in Patients and Xenograft Tumors Differences in Pgp expression in tumors could be one factor that might be related to differences in drug accumulation. there were. The measurement of Pgp expression was performed by immunohistochemical methods using the method previously described (Toth K et al. (1994) Am J Pathol 144: 227-236). Briefly, tissue pieces were dewaxed and rehydrated with xylene, ethanol and water in that order. The tissue pieces were placed in 0.1 M citrate buffer pH 6.0, boiled in the microwave, then cooled and washed with phosphate buffered saline (PBS). The tissue fragment was blocked with a Dako blocking solution for 10 minutes, and then the following antibody solutions, namely mouse anti-human Pgp antibody (JSB-1, 1: 200 dilution) and rabbit anti-human Pgp polyclonal antibody (ab-1, 1 :). The cells were sequentially cultured with 100 dilution). JSB-1 does not cross-react with MDR3 (
Schinkel AH et al. (1991) Cancer Res 51: 2628-2635). The culture was performed in a humidified chamber at room temperature. The antibody was diluted with 5 mg / ml of bovine serum albumin containing PBS. Mouse IgG was used as the primary antibody for the negative control. High P as a positive control
Human adrenal gland expressing gp was used (Pavelic ZP et al. (1993) Arch Otolaryngol H
ead Neck Surg 119: 753-757). After washing with PBS, the tissue fragments were covered with linker solution and then with peroxidase-conjugated streptavidin solution. After washing twice with PBS,
Tissue fragments were incubated with diaminobenzidine for 5-7 minutes and counterstained with hematoxylin.

Only tumors that stained with two different Pgp antibodies and showed Pgp protein in at least two-thirds of the tissue cultures were considered Pgp positive. By these criteria, xenograft tumors, 3 head and neck tumors, 2 ovarian tumors were Pgp positive, 4 head and neck tumors, and 1 ovarian tumor were Pgp negative. Paclitaxel accumulation in these tumors was compared in the two cases with initial concentrations of 120 and 12,000 nM. The results are shown in Table 3. Statistically significant differences between groups were analyzed by unpaired two-tailed Student's t-test. Table 3 also shows groups having a statistically significant difference.

(Table 3)

Xenograft tumors showed higher accumulation than Pgp positive patient tumors. Among patient tumors, Pgp expression did not always result in low drug accumulation. For example, Pgp-positive patient tumors tended to show lower drug accumulation compared to Pgp-negative patient tumors with drug concentrations as high as 12,000 nM, but the difference was small (ie, <25% on average) and statistically significant. Was not significant. Furthermore, at the low drug concentration of 120 nM, no difference between the two groups was observed. From these data, Pgp expression may contribute to low drug accumulation in some tumors, but is not a major determinant of drug accumulation, and differences in drug accumulation between patients and xenograft tumors range from 50% to 100%. I didn't fully explain why it was%.

Role of Cellularity and Apoptosis in Tumor Paclitaxel Penetration, Accumulation, and Maintenance Tumor [ ³ H] paclitaxel permeation rates and spatial relationships of drug penetration, tumor structure, and tumor cell distribution were autoradiographed. It was evaluated using graph technology and image analysis. The autoradiographic method has been described in the past (Lesser GJ
et al. (1995) Cancer Chemother Pharmacol 37: 173-178). After culturing with [ ³ H] paclitaxel (corresponding to 0.231 and 2.31 mCi / ml, 12 and 120 nM) for 1 hour to 3 days, tumor tissue cultures were collected, immersed in ice-cold drug-free medium and washed twice. Tissue samples were mounted on a cryostat chuck with embedded substrate (Miles, Ind. OCT compound) and cut into 10 mm thick pieces in a -20 ° C. cryostat.
The fragments were thawed, placed on glass slides, and fixed by heating for 15 minutes with a slide warmer. Slides with tissue sections were exposed to a tritium sensitive film (Ultrofilm) against an X-ray cassette and exposed at room temperature for 1-2 weeks. Develop the film at room temperature for 3 to 5 minutes (D-19 developer) and place in development stop solution for 30 seconds,
It was immersed in the fixer for 3 minutes and then exposed to running water at room temperature for 15 minutes. The film was then washed with Photo-Flow 200 and air dried. Separately, tissue slides were stained with hematoxylin and eosin.

Image analysis was then performed on autoradiographic images (granular dots indicated the location of the radiolabeled drug) and histological images of histological slides stained with hematoxylin and eosin (showing histology and tumor cell distribution. It was used to capture The autoradiographic image threshold was adjusted to minimize background signal. Autoradiograph images were overlaid on histological images visualizing [ ³ H] paclitaxel distribution in tumor tissue culture.

Head and neck tumors and xenograft tumors were treated with 120 nM paclitaxel. The drug uptake rate in xenograft tumors was about 50% to 80% slower than in patient tumors, and the accumulation was twice that in patient tumors (Table 1). In xenograft tumors, paclitaxel permeated only a few layers of surrounding cells after 4 hours, but permeated 10 to 15 layers of cells after 24 hours, and was evenly distributed throughout the tumor (cell layer thickness (More than 80)
It was after 48 hours. These data suggest that the drug uptake rate rises sharply after 24 hours. Penetration of the drug into the patient's tumor is more rapid, 4
Half of the tumor tissue culture was reached at time and evenly distributed at 24 hours. In both xenograft and patient tumors, a comparison of radioactivity in areas with high and low cell densities suggests that radioactivity is highly intracellularly localized compared to the interstitial space. There is. In addition, there was a rapid distribution of radioactivity in low-density epithelial cells at early time points, with a low tumor cell fraction (ie 51 ± 18% of tissue culture).
Drug Penetration of Patient Head and Neck Tumors with% Cell Tumors) Compared to FaDu Xenograft Tumors with Higher Cell Fraction (ie 79 ± 7.0% of Tissue Culture Corresponds to Tumor Cells) And was observed to be more rapid, suggesting that decreased cellularity is consistent with more rapid drug penetration.

The above observations suggest that drug penetration may be increased by loss of cellularity and subsequent apoptosis induced by paclitaxel treatment, for example, resulting in a lag time of 16 to 24 hours. (Saunders DE et al. (1997) Int J
Cancer 70: 214-220). This hypothesis was confirmed by assessing the time course of tissue composition of xenograft tumors. Xenograft tumors were treated with two concentrations of paclitaxel, 120 nM which caused significant apoptosis and 12 nM which did not cause significant apoptosis. The fraction of stromal tissue and tumor cells in each tissue culture was measured using image analysis. Briefly, stromal and tumor cells were outlined with a computer mouse in a 100x field of view. The size of each of these regions was determined by counting the number of pixels in the region and performing image analysis. To determine the fraction of apoptotic cells in the tumor, tumor cell density and apoptotic cell fraction were determined by counting the total number of tumor cells and apoptotic cells in the 400-fold expanded non-necrotic area. Apoptotic cells were determined on the basis of morphological changes in tumor cells, such as chromatin condensation and marginal prolapse, nucleolar loss, membrane vesicle formation, apoptotic bodies and / or cell shrinkage (Kerr JFR et al. (1994) Cancer
73: 2013). This method uses terminal deoxynucleotidyl transferase-mediated dUT
P nick end labeling (TUNEL) method (Gan Y et al. (1996) Cancer Res 56: 2086;
Gold R et al. (1994) Lab Invest 71: 219-225) showed similar results. For each tumor piece, 50-100 images were processed and the tumor fraction represented by tumor cells, stromal tissue and interstitial space was calculated.

When xenograft tumor tissue cultures were treated with 120 nM paclitaxel, tumor cell density was significantly reduced and apoptotic cells were increased. This effect was first detected at 24 hours and progressively increased over time. The percentage of apoptotic cells in treated tumors was ~ 30% at 24 hours and increased to ~ 50% at 72 hours, whereas less than 5% apoptotic cells were seen in untreated controls . The onset of apoptosis and the expansion of the interstitial space at 24 hours resulted in a rapid increase in drug penetration, ie the drug penetrated 15 layers of cells in the first 24 hours, while
This was consistent with permeation of more than 80 layers of cells at 24 hours. When tumors were treated with 12 nM paclitaxel, there was no significant decrease in cellularity or increase in apoptotic cells, and the fraction of apoptotic cells remained relatively constant below 7%. Under these conditions, drug penetration was marginal. These results suggest that drug-induced apoptosis causes a decrease in tumor cell density and, consequently, enhanced drug penetration into the solid tumor lining.

Summary In summary, this example shows that (a) paclitaxel penetration into the tumor is more rapid, but accumulation is lower, as epithelial tumor cell density is reduced, (b) drug-induced apoptosis of solid tumors. Promoting drug penetration into the inner cell layer, (c) concentration-dependent drug penetration rate is associated with concentration-dependent apoptotic effect, and (d) time-dependent drug penetration is associated with apoptosis kinetics. Shows.

Example 2 Effect of Apoptosis-Inducing Pretreatment on Drug Penetration and Accumulation When Paclitaxel is Used as an Apoptosis-Inducing Agent This example consists of in vitro and in vivo studies. In vitro studies were performed using human pharyngeal FaDu xenograft tumors maintained in athymic mice. In vivo studies were performed using syngeneic tumors, MAT-LyLu prostate tumors maintained in Copenhagen rats.

Drug Treatment-In Vitro Studies Drug treatment was as described above. Tissue cultures were transferred to collagen gels presoaked in drug-containing medium for at least 8 hours. Presoaking was to ensure that the drug concentration in the medium inside the collagen gel was in equilibrium and to eliminate the delay of drug transport from the medium through the collagen gel. Drug treatment was terminated by transferring the tissue culture to collagen gel presoaked in drug-free medium. The tissue culture was moved using barbed tweezers, taking care not to pinch or crush.

Effect of Apoptosis-Inducing Pretreatment on Tumor Drug Delivery—In Vitro Studies [ ³ H] paclitaxel spatial distribution in tissue cultures was determined using autoradiography and imaging techniques as described in Example 1. Researched. Total drug concentration in tumor tissue culture was measured as described in Example 1. Briefly, 4 ml of culture medium containing a mixture of radiolabeled and non-radiolabeled paclitaxel (specific activity 0.301: Ci / ml
), The tissue culture was collected and analyzed for total radioactivity using a liquid scintillation counter. 30 to 35 tumor tissue cultures were used at each time point.

Two studies were conducted. The first study used two groups of tissue cultures at the same concentration of 10
Alternatively, the cells were cultured with 50 nM [ ³ H] paclitaxel for 72 hours. These two groups were exposed to the same amount of radiolabeled paclitaxel, except that the pretreatment group received an additional 3000 nM.
Exposure of hr to non-radiolabeled paclitaxel (ie unlabeled paclitaxel 1000
nM for 3 hours) was started 24 hours before the start of radiolabel exposure. The drug concentration and timing of pretreatment were found in a preliminary study in which significant apoptosis (~ 30%) was produced at 24 hours. The second study showed the same total drug exposure, namely 1,20
It was performed at 0 nM · hr, but was performed at a different schedule as described later. In both studies, tissue cultures were treated with [ ³ H] paclitaxel and collected at certain time intervals for drug permeation analysis. The first study was conducted to quantitatively demonstrate the effect of pretreatment on the rate of drug penetration into solid tumors. Drug accumulation was assessed only in the second study, which showed that dilution of [ ³ H] paclitaxel with non-radiolabeled drug changed the specific activity of [ ³ H] paclitaxel in the first study, resulting in accurate data. This is because it was thought to affect the degree.

The results show the effect of pretreatment with 1: M non-radiolabeled paclitaxel on [ ³ H] paclitaxel in FaDu tumor tissue cultures. Non-radiolabeled paclitaxel was not detected by autoradiography and did not interfere with the detection of [ ³ H] paclitaxel. Two concentrations of [ ³ H] paclitaxel with different abilities to induce apoptosis and reduce tumor cell density were used as examples, with a concentration of 50 nM being more effective than a concentration of 10 nM (Table 4). Tumor penetration of 10 nM [ ³ H] paclitaxel without pretreatment was restricted to the margins of tissue culture within 72 h, whereas at 50 nM, penetration was restricted to the periphery for the first 24 h. , Then increased with increasing apoptotic fraction and decreasing cell density, and by 72 hours the drug was evenly dispersed. Paclitaxel pretreatment prior to [ ³ H] paclitaxel treatment induces apoptosis,
Reduced cell density. When pretreated, the penetration of [ ³ H] paclitaxel is
It was elevated at both 10 nM and 50 nM and reached an even distribution in the tumor.
It was at 72 and 24 hours. These findings show that split-dose administration at time intervals capable of causing apoptosis is associated with a larger drug dose than a sequential treatment schedule with the same total drug exposure (ie concentration-time product, CxT). It is suggested that this results in penetration and accumulation.

Table 4 shows the experimental results when tumor tissue cultures were treated with different concentrations of [ ³ H] paclitaxel with and without pretreatment with 1: M non-radiolabeled paclitaxel. The apoptotic cell fraction and tumor cell density were determined by counting the total and apoptotic cell numbers in a 400x microscope field (3 fields per tumor).

(Table 4)

To evaluate the effect of treatment schedule on intratumoral paclitaxel delivery, a second study investigated drug penetration and accumulation in two groups similarly treated with the two schedules. The pretreatment group received split doses. That is, the cells were cultured in 600 nM [ ³ H] paclitaxel for 1 hour and then further cultured in the drug-free culture medium for 23 hours to induce apoptosis, and then treated with 50 nM [ ³ H] paclitaxel for 12 hours. The control group was incubated with 50 nM [ ³ H] paclitaxel for 24 hours continuously. In the control group, paclitaxel permeation was restricted to 5-10 layers of cells in the margins of the tissue culture in the first 24 hours, at which point drug-induced apoptosis began to develop. During the next 12 hours, drug penetration began to rise concurrently with increasing apoptotic fraction and decreasing cell density, while drug penetration remained confined to less than the marginal 20 layers of cells. In comparison, the pretreatment group had a high percentage of apoptosis, low cell density, faster permeation, and even distribution of drug throughout the tissue culture at 36 hours (Table 5). The comparison of drug accumulation in the two groups showed that the accumulation of the pretreatment group at the end of drug treatment (ie, 36 hours after pretreatment group versus 24 hours after control group; Table 5). Is 40%
It was high and was consistent with the broader drug penetration shown by autoradiography. The results of this study confirm that treatments divided in time intervals that cause drug-induced apoptosis and reduce cell density will result in greater drug penetration and accumulation in tumors than a series of treatments. There is.

Table 5 below shows tumor tissue cultures equivalent to CxT [ ³ H] paclitaxel, ie, 1,200.
Shown are the experimental results of treatment with two different schedules at nM · hr.
One schedule used 50% of total CxT to induce apoptosis and the other schedule used continuous treatment. [ ³ H] paclitaxel concentration in tumor tissue cultures was determined by liquid scintillation counting. Apoptotic cells and cell densities were determined by counting total and apoptotic tumor cell numbers in a 400x microscopic field (3 fields per tumor).

(Table 5)

In summary, these results show that under in vitro conditions, (a) apoptosis-inducing pretreatment increases drug penetration during subsequent treatments, and (b) induces measurable apoptosis in tumor cells. It is suggested that treatment with high drug concentration, which reduces the density, caused more rapid drug penetration compared to treatment with low concentration.

Effect of Apoptosis-Inducing Pretreatment on Drug Accumulation in Tumors-In Vivo Studies Rat MAT-LyLu tumor cells were first obtained from Dr. J. Isaac (John-Hopkins University, Baltimore, MD). % Heat inactive FBS, L-glutamine 2m
M, R supplemented with gentamicin 90: g / ml and cefotaxime sodium 90: g / ml
Maintained in PMI-1640 medium. Tumor cells (5x10 ⁶ cells in 0.1 ml medium, viability> 90%
, Measured by trypan blue exclusion) was subcutaneously injected into the left and right upper dorsal regions of Oscopene Hagen rats with a 21G needle. The experiment started when the tumor was 0.3 to 0.5 g in size.

Tumor maintenance rats under mild ether anesthesia were catheterized with polyethylene tubing (PE-50 Becton Dickinson, Sparks, MD) into the jugular vein and carotid artery for drug administration and blood sampling, respectively. I passed it. Each catheter is placed on the back side of the neck, and a second polyethylene tube (PE-50
) Was attached. The catheter and tubing were covered with metal coil tubing. 4 animals
It was allowed to recover for -5 hours, after which intravenous infusion of paclitaxel was performed using an infusion pump (Harvard Appalatas, Inc., South Natick, Mass.). Paclitaxel was dissolved in Cremophor EL / ethanol (1: 1, v / v) and
Diluted with .9% NaCl. The total injection volume was 2-3 ml and the final Cremophor EL concentration in the dosing solution was 10-15%. Five groups of animals were treated as described in the results. Blood samples (0.12 or 0.22 ml) were taken at a certain time point. Plasma protein fractions were obtained by centrifugation at 2000g for 3 minutes and stored at -70 ° C for HPLC analysis. Tumors were collected at the end of the experiment. One quarter of the tumors were fixed in 10% neutralized formalin solution and processed for histomorphological evaluation. Tumor cell density and apoptotic cell fraction were determined by counting the number of tumor cells and apoptotic cells in a non-necrotic area expanded 400 times (5 microscopic fields / tumor). The rest of the tumor was stored at -70 ° C for HPLC analysis of drug concentration.

Non-radiolabeled paclitaxel concentrations in plasma and tumors were determined by an HPLC assay using a column exchange method previously reported by us (Song D et al. (1995) J Chromatogr B Biome.
d Appl. 663: 337). Pre-weighed tumors were used as internal standard
Homogenized by mixing with 100: 1 cephalomannine (10: g / ml in methanol). HPLC stationary phase is washed column (Milford, Waters Associates Inc. Novapak C _8, 75x3.9 mm ID, particle size 4: m) and analytical column (Phillipsburg, NJ, JT Baker Co. base over carbon Dooctadecyl 250 x 4.6 mm ID, particle size 5: m). The sample was injected onto the wash column and eluted with the wash mobile phase (37.5% acetonitrile in water) at 1 ml / min. at the same time,
The analytical mobile phase (49% acetonitrile in water) was passed through the analytical column at a flow rate of 1.2 ml / min.
Fractions 5 to 12 minutes with paclitaxel and cephalomannine were transferred from the wash column to the analytical column. Detection of paclitaxel and cephalomannine was performed at 229 nm with a detection limit of 1 ng paclitaxel / injection.

Pharmacokinetic and histomorphological studies were performed to reveal potential doses and times of drug administration. The results listed in Table 6 indicate that a pretreatment dose of 5 mg / kg infused over 1 hour is sufficient to induce apoptosis and reduce tumor cell density. These changes in tissue composition are sufficient to increase drug and even drug penetration into the tumor, as shown in the in vitro studies above. Pharmacokinetic data further indicate that paclitaxel has a major half-life of 1.
1 hour, indicating that plasma and tumor drug concentrations would theoretically be within 97% of steady state (ie, half-lives of 5 or more) after 6 hours or more of infusion. Suggested. The latter is confirmed by the plasma concentration-time profile, with concentrations ranging from 6
It was shown to approach the plateau value at the end of the 12 hour infusion (13% difference).

Table 6 shows the results (Tx) of the experiments in which the animals were given the treatment to be adapted. Paclitaxel concentrations in tumor and plasma were measured by HPLC. The apoptotic cell fraction and cell density were determined by counting the total and the number of apoptotic cells in a 400 × microscope field (5 fields / tumor). Mean ± standard deviation.

(Table 6)

Three in vivo studies have been performed. The first study examined the effect of pretreatment on drug delivery on tumor tissue in three groups of animals. Group 1 is 5 mg / kg
Was given over 1 hour and at 24 hours a second dose was infused at a slower rate (ie 5 mg / kg over 6 hours). Group 2 received only pretreatment and Group 3 received only the second treatment to make baseline measurements of drug delivery to the tumor at each time point of the two doses. If drug delivery is not promoted due to apoptosis, the drug concentration in Group 1 should be lower than the combined concentration in Group 2 and Group 3. Because the concentration of pretreatment group 1 was measured at the end of the total treatment time of 30 hours, the concentration continued to decrease with time, so the group measured immediately after 1 hour from pretreatment This is because the concentration should be lower than 2. Conversely, the concentration of Group 1 would exceed the combined concentration of Groups 2 and 3 if drug delivery was substantially enhanced due to apoptosis. The results show that the tumor concentration at 30 hours after treatment in Group 1 or at the end of treatment was 50% compared with the sum of the concentration at 24 hours in Group 2 and the concentration at 6 hours in Group 3.
It is shown to be elevated (Table 6), thus supporting our hypothesis that drug delivery is enhanced due to pretreatment-induced apoptosis.

In the second study, the group 1 tissue morphology and tissue concentration and the same infusion schedule as Group 1 were similarly given two doses, but the interval between the two doses was 23 for Group 1. The tissue morphology and tissue concentration of Group 4, which differed in time but only 10 minutes (the time required to replace and reset the infusion syringe and pump), were compared. As mentioned above, 24 hours was the duration required for apoptosis and cell density reduction to occur. At the end of treatment (30 hours in Group 1 and 7.2 hours in Group 4) Group 4 had 60% lower apoptosis, 30% higher cell density and 18% lower drug concentration compared to Group 1 (Table 6). ). These results suggest the importance of drug-induced changes in tissue morphology and the timing of drug delivery.

The third study received Group 1 histomorphology and tumor concentrations and the same total dose as Group 1, but the administration was slower and continuous over a longer duration of 12 hours. In comparison, the tissue morphology and tumor concentration of Group 5 differing in that At the end of treatment (30 hours in Group 1 and 12 hours in Group 5), Group 5 had 35% lower apoptosis, 25% higher cells and Group 5 had higher drug concentration at 12 hours compared to Group 1. 25 from the concentration after 30 hours of 1
% Was low (Table 6).

Another tumor permeation assay for tumors is the tumor to plasma concentration ratio at the end of drug treatment when the plasma concentrations of Groups 1, 4 and 5 are apparently stable. The comparison of tumor-to-plasma concentration ratio in these three groups was 130% comparing Group 1 and Group 4.
, And a 40% higher ratio comparing Group 1 and Group 5, respectively (Table 6), demonstrating higher drug delivery in the group receiving the apoptosis-inducing pretreatment.

In summary, the results of the in vivo studies substantiate the findings of tumor tissue cultures and further
It is suggested that under vo conditions more than 10% increase in apoptosis and more than 25% decrease in cell density are required to promote drug delivery to tumors.

Example 3 Effect of Apoptosis-Inducing Pretreatment on Tissue Drug Penetration and Accumulation when Doxorubicin is Used as an Apoptosis-Inducing Agent In this example, permeation of doxorubicin into multi-layered tissues was collected from human cancer patients. And were monitored with human prostate PC3 xenograft tumors maintained in athymic mice. Doxorubicin was monitored by fluorescence.

Doxorubicin Accumulation in Tumor Tissue Culture Tumor tissue cultures were cultured with 1, 5 or 20: M doxorubicin as described in Example 1. These concentrations were selected on the basis of previously published data, which show that 0.4: M doxorubicin is sufficient to suppress tumor cell growth by 90% in patient prostate tumors, 2.1: M and 4.2: M doxorubicin has been demonstrated to be sufficient to cause 50% and 90% cell death, respectively (Chen
et al., (1998) Clin. Cancer Res. 4: 277-282, 1998). 4, 12, 24, 36, 48 and
At 72 hours, tissue cultures were harvested and processed as described in Example 1. The fluorescence emitted by doxorubicin was visualized using a fluorescence microscope. Excitation and emission wavelengths were 546 nm and 565 nm, respectively. Charge-coupled device (
CCD) camera is used to analyze the obtained image.
(Silver Spring, Maryland) was used. Doxorubicin concentration in tissues was quantified using a standard curve (each curve contains 6 data points) as established below. Samples for the standard curve were prepared by applying a known amount of doxorubicin in solution to microscopic sections of empty dog prostate tumors. Standard doxorubicin solution (2
: 1) was pipetted onto 10: m thick empty tissue pieces and carefully spread over a surface area of about 1.5 -2 cm ² . Standard slides were scanned using the same conditions as the samples. The average fluorescence intensity per unit area was measured. The plot of fluorescence readings vs. coated doxorubicin concentration in the standard curve sample was the standard curve and was used to quantify doxorubicin in the actual sample. In the latter, at least 3 fragments were used to determine the average value of one tumor, and at least 3 fragments were used to determine the average value at each time point.

FIG. 3 summarizes doxorubicin accumulation in tumor tissues expressed as a ratio of tissue culture medium concentration as a function of time and initial extracellular drug concentration. The results show that the drug concentration at the tumor margin and at the extreme end of the tumor (ie 325 mm from the tumor margin that is in contact with the culture medium) increased more rapidly in patient tumors than in PC3 xenograft tumors. ing. The ratio reached at the 72 h result increased with extracellular drug concentration, but the degree of increase was not linear with concentration. The latter is believed to be due to a non-linear drug binding that saturates at higher drug concentrations, as described for paclitaxel in Example 1. At drug concentrations as low as 1 mM, the drug concentration at the extreme end of the tumor remained several times lower than that at the margins. On the contrary, at the higher drug concentrations of 5 and 20 mM, the concentration at the end of the tumor was close to that at the margin of the tumor.
Thus, these data suggest that the drug accumulation rate and extent of solid tumors is time and initial drug concentration dependent.

Table 7 shows the patient tumors and PC3 graft tumors at 72 doxorubicin concentrations of 1, 5 and 20 mM.
The result of doxorubicin accumulation after time culture is shown.

(Table 7)

Both doxorubicin concentrations in patient tumors and PC3 xenograft tumors increased with culture time and initial drug concentration in the culture medium. The findings for doxorubicin accumulation rate and extent in patients and xenograft tumors are similar to those for paclitaxel described in Example 1. For example, drug accumulation in xenograft tumors is slow compared to patient tumors, but the difference is not statistically significant (Figure 3 and Table 7). Tumor cell density in xenograft tumors without drug is 1.3 compared to patient tumors
Twice as expensive. Altogether, these data suggest that the slower drug uptake and higher drug accumulation in PC3 xenograft tumors compared to patient tumors is due to the higher tumor cell density in xenograft tumors. is doing.

Doxorubicin Penetration Rates into Tumors Doxorubicin penetration rates in patients and PC3 xenograft tumors were monitored by fluorescence microscopy. Fluorescence refers to the spatial distribution of doxorubicin in tumor tissue as the presence of fluorescence is consistent with the presence of doxorubicin. The findings for doxorubicin penetration and distribution in tumor tissue are similar to those for paclitaxel described in Example 1. For example, patients and xenograft tumors showed different doxorubicin penetration rates and patterns, and drug penetration rates were also drug concentration dependent. In patient tumors treated with 5 or 20 mM, the fluorescence signal was restricted to the margin (about 75 mm thick) for the first 12 hours, but after 24 hours it was evenly distributed throughout the tissue culture (about 325 mm thickness). It was spreading. For xenograft tumors treated with the same drug concentration (ie 5 or 20 mm), the fluorescence signal was marginal for the first 24 hours but was evenly distributed after 36 hours. At drug concentrations as low as 1 mM, it took 72 hours for the drug to disperse evenly throughout the patient's tumor, whereas in xenografts it remained marginal at 72 hours.

FIG. 4 summarizes the drug concentration in tumor tissue expressed as tissue media concentration ratio as a function of distance from the tumor tissue culture margin. The results show that the drug concentration at the extreme end of the tumor (ie 325 mm from the tumor margin in contact with the culture medium) and the tumor margin (ie 75 m
It shows that whether the drug concentration of m) reaches the equilibrium state depends on the initial drug concentration of the culture medium. At drug concentrations as low as 1 mM, the drug concentration at the end of the tumor did not reach equilibrium with the concentration at the margin. At higher concentrations of 5 and 20 mM, equilibrium was reached at 36 hours.

In summary, these results suggest that the rate and extent of doxorubicin permeation into the tumor are dependent on treatment time and extracellular drug concentration. These data are similar to the findings for paclitaxel described in Example 1, suggesting that doxorubicin-induced apoptosis increases the rate and extent of drug penetration into tumors.

Role of Cell Death in Drug Penetration Tumor cell density was monitored as described in Example 1. The cell death and consequent reduction in cell density induced by doxorubicin treatment was dependent on the initial drug concentration. For example, the cell density at the margins of PC3 xenograft tumors (ie, about 75 mm thick) decreased with time after treatment with 5 or 20 mM doxorubicin, but did not change after treatment with 1 mM doxorubicin. Figure 5 shows that there is an inverse correlation between mean drug concentration of PC3 xenograft tumors and tumor margin tumor cell density after treatment with 20 mM doxorubicin.

Summary In summary, this example shows that (a) as epithelial tumor cell density decreases, doxorubicin penetration into tumors is more rapid, but accumulation is lower (b) drug-induced apoptosis of solid tumors. Show that (c) concentration-dependent drug permeation rate is associated with concentration-dependent apoptotic effect, and (d) time-dependent drug permeation is associated with apoptotic kinetics, facilitating drug penetration into the inner cell layer. There is. These findings are quantitatively identical to those for paclitaxel (described in Example 1), suggesting that apoptosis-inducing pretreatment is a generally applicable principle for promoting drug penetration into multilayered tissues. There is.

Example 4 Apoptosis-Inducing Pretreatment Enhances Macroparticle Penetration into Multilayered Tissues Two experiments were performed to evaluate the ability of apoptosis-inducing pretreatment to enhance macromolecule penetration into multilayered tissue. It was In the first study, fluorescent tagging (ie FITC
Labeled nanoparticles with a diameter of 600-800 nm were used. Nanoparticle penetration was monitored by fluorescence microscopy. In the second study, microparticles (diameter about
5 micron) was used. Microparticle penetration was monitored by scanning electron microscopy.

The multilayered tissue model of the two studies was a human breast xenograft tumor derived from MCF7 cell subcutaneous implants. Tumors were removed from mice, fragmented and grown as tissue culture on collagen gel substrates. One group was pretreated with 100 nM paclitaxel for 24 hours. The tissue culture was then transferred to a second culture flask and further with paclitaxel-free culture medium containing FITC nanoparticles or microparticles.
Cultured for 48 hours. A control group was treated in the same way but no paclitaxel was added to the culture medium. Samples of tissue culture were taken at 0, 4, 6, 12, 24 and 48 hours after contact with nanoparticles or microparticles. The tissue culture was frozen and cut into 10 micron sections. Frozen sections were mounted on microscope slides and microscopically verified.

The results showed that in the control group, when the tumor tissue cultures were infiltrated with nanoparticles and microparticles at a contact time of 48 hours, penetration was limited to the margins. In the pretreatment group, nanoparticles and microparticles penetrated to the center of the tissue culture at 24 hours and were evenly distributed throughout the tissue culture at 48 hours.

Overall In summary, apoptosis-inducing pretreatment promotes the penetration of particles approximately 5 microns in diameter into multi-layered tissues.

Example 5 Preparation of Nanoparticles and Microparticles Poly (lactide-co-glycolide) (PLGA) microspheres were prepared using a standard oil-in-water emulsion solvent evaporation method (Thies C., 1991, In Donbrow. M. (Ed.) Microca
psules and Nanoparticles in Medicine and Pharmacy, CRC Press, Ann Arbor,
pp. 47-71). In the case of doxorubicin-containing microspheres, the free base form of doxorubicin and PLGA was dissolved in a 1: 6 mixture of methanol and methylene chloride.
The polymer and drug solution were treated with 1% (w / v) polyvinyl alcohol and 10 nM boric acid, pH 8.8.
Was emulsified by occasionally stirring. Then the emulsion was diluted and the organic solvent was evaporated. Microspheres were collected by filtration and freeze dried. For paclitaxel microspheres, PLGA and paclitaxel were dissolved in methylene chloride. The solution was emulsified with a glycerin / water mixture containing Tween 80. Microspheres were collected by centrifugation and freeze dried. Average particle size is 1 mm
Met.

Paclitaxel-containing nanoparticles were prepared by the phase separation method. The aqueous gelatin solution and Tween 20 were heated and sodium sulfate was added until a suspension was observed. Then, the paclitaxel-containing isopropanol solution was added until the suspension disappeared. Glutaraldehyde and potassium metabisulfite were added sequentially. The nanoparticles obtained were collected by centrifugation and freeze-dried. The average particle size was 620 nm.

Paclitaxel-containing nanocapsules were prepared by the oil-in-oil-in-water method. Tween 20
Was added to 10 acres (acres) gelatin solution. Paclitaxel was dissolved in methylene chloride. The two solutions were emulsified to form an oil-in-water emulsion. Mineral oil containing glutaraldehyde was then added. Teaching a mixture (original: teaching
After that, potassium metabisulfite was added. The nanoparticles obtained were collected by centrifugation and freeze-dried. The average particle size was 13 mm.

Paclitaxel-containing liposomes were prepared by mixing triasterin, paclitaxel and Tween 80. Phosphate buffered saline was added during stirring to produce an emulsion.
The lipospheres were collected by centrifugation. Addition of egg phosphatidylcholine to the mixture improves the smoothness of the particle surface. Average particle size is 10 mm
Met.

INCORPORATION BY REFERENCE The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated by reference in their entirety.

Equivalents One of ordinary skill in the art will recognize, or be able to ascertain numerous equivalents to the particular embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be covered by the following claims.

[Brief description of drawings]

FIG. 1 is a line graph showing the kinetics of paclitaxel uptake in patient and xenograft tumor tissue cultures. Patient head and neck tumors are represented by ●, ovarian tumors by patients are represented by □, and FaDu xenograft tumors are represented by ○.

FIG. 2 is a line graph showing the kinetics of paclitaxel excretion in patient and xenograft tumor tissue cultures. Patient head and neck tumors are represented by ●, ovarian tumors by patients are represented by □, and FaDu xenograft tumors are represented by ○.

FIG. 3 is a graph showing the accumulation of doxorubicin in tumor tissues as a function of tissue to medium concentration ratio as a function of time and initial extracellular drug concentration. Around the tumor (ie 75 μm) and the furthest edge from the tumor (ie 325 μ from the tumor periphery in contact with medium
The mean drug concentration in m,) is shown. (A) Upper panel: Patient's prostate tumor. (B) Lower panel: PC3 xenograft tumor. Tumors treated with 1 μm doxorubicin are indicated by ▼. Tumors treated with 5 μm doxorubicin are represented by ◯. 20 μm
Tumors treated with Doxorubicin are represented by ●.

FIG. 4 is a graph showing the concentration of doxorubicin in tumor tissue as a function of the concentration ratio of tissue to medium as a function of distance from the tumor edge in contact with medium. (A) Upper panel: Patient's prostate tumor. (B) Lower panel: PC3 xenograft tumor. Tumors 1m (right panel), 5m (middle panel), and 20 (left panel)
m treated with doxorubicin. The processing time is as described in the graph.

FIG. 5 is a graph showing the effect of cell density around the tumor (ie, 100 μm distance from the edge in contact with the medium). The percent cell density is the ratio between the cell density in treated tumors and that in untreated control samples.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/711 A61K 31/711 48/00 48/00 A61P 35/00 A61P 35/00 43/00 43 43 / 00 105 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, R, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (71) Applicant Wintee's M Jurome United States 43235 Colombus Palm Leaf Court, Ohio 2287 2287 Palmleaf Court, Columbus, OH 43235 U.S.A. S. A. (72) Inventor Oh Jesse L.S. USA 43235 Colombus Palm Leaf Court 2287, Ohio 2287 (72) Inventor Wintees Jurome United States 43235 Colombus Palm Leaf Court, Ohio 2287 F Term (Reference) 4C076 AA22 AA61 AA65 AA67 BB11 CC27 DD63H EE30H EE42H 4C084 AA13 MA02 MA23 MA38 MA66 NA12 ZB212 ZB262 4C086 AA01 AA02 BA02 EA10 EA16 MA01 MA02 MA04 MA23 MA38 MA66 NA12 ZB21 ZB26

Claims (108)

[Claims] 1. A method of delivering a therapeutic agent to a tissue of a patient, the method comprising: administering an apoptosis-inducing agent to the patient, the apoptosis-inducing agent being a tissue of the patient so that the therapeutic agent is delivered to the tissue of the patient. At a time sufficient to induce apoptosis. 2. The method of claim 1, wherein the tissue is a tumor. 3. The method according to claim 1, wherein the tissue is liver tissue, nerve tissue, muscle tissue, skin tissue or adipose tissue. 4. The tumor is brain, breast, ovary, bladder, prostate, colon, lung, liver,
The method according to claim 2, which is a tumor of the uterus. 5. The method according to claim 1, wherein the apoptosis inducer is paclitaxel or doxorubicin. 6. The method of claim 1, wherein the sufficient time is sufficient to reduce the density of parenchymal cells in the tissue by about 30% or more. 7. The method of claim 1, wherein the sufficient time is sufficient to induce apoptosis in 10% or more of parenchymal cells of the tissue. 8. The method according to claim 1, wherein the apoptosis-inducing agent is locally administered. 9. The method according to claim 1, wherein the apoptosis-inducing agent is locally administered. 10. The method of claim 1, wherein said apoptosis-inducing agent is also said therapeutic agent, but said therapeutic agent comprises another dose or a sustained release type of apoptosis-inducing agent. 11. The method of claim 1, wherein the therapeutic agent is a gene therapy product, a chemotherapeutic agent, a protein binding agent or an antibiotic. 12. The method of claim 1, wherein the patient is a human. 13. The apoptosis-inducing agent and the therapeutic agent are simultaneously administered,
2. The therapeutic agent is a sustained release formulation that releases the therapeutic agent after a time sufficient for the apoptosis inducing agent to induce apoptosis in the patient's tissue.
The method described in. 14. The apoptosis-inducing agent and the therapeutic agent are sequentially administered, and the therapeutic agent is administered after a time sufficient for the apoptosis-inducing agent to induce apoptosis in the tissue of the patient. The method described in. 15. A method of delivering a therapeutic agent to a tissue of a patient, comprising: obtaining an apoptosis inducer; administering the apoptosis inducer to the patient; and the apoptosis inducer induces apoptosis in the tissue of the patient. A method comprising: allowing sufficient time to induce and delivering the therapeutic agent to the tissue of the patient such that the therapeutic agent is delivered to the tissue of the patient. 16. A method of delivering a therapeutic agent to a tumor of a patient, the method comprising administering to the patient a dose of an apoptosis-inducing agent, the apoptosis-inducing agent having sufficient time to induce apoptosis in the tumor. A method comprising the steps of: delivering a dose of the therapeutic agent to the patient such that the therapeutic agent is delivered to the tumor. 17. The method of claim 16, wherein the tumor is cancerous. 18. The method of claim 16, wherein the tumor is a benign tumor. 19. The cancerous tumor is brain, breast, ovary, bladder, prostate, colon, lung,
18. The method of claim 17, which is a tumor of the liver or uterus. 20. The apoptosis-inducing agent is paclitaxel.
The method according to 6. 21. The apoptosis-inducing agent is doxorubicin.
The method according to 6. 22. The method of claim 16, wherein the dose of the apoptosis-inducing agent is sufficient to reduce the density of solid tumor cells. 23. The method of claim 22, wherein the decrease in solid tumor cell density is about 10% or greater. 24. The method of claim 23, wherein the density of solid tumor cells is reduced by about 20% or more. 25. The method of claim 24, wherein the decrease in solid tumor cell density is about 30% or greater. 26. The method of claim 25, wherein the decrease in solid tumor cell density is about 35% or greater. 27. The dose of the apoptosis-inducing agent is about the amount of solid tumor cells.
The method according to claim 16, which induces apoptosis by 5% or more. 28. The dose of the apoptosis-inducing agent is about the amount of solid tumor cells.
The method according to claim 27, which induces apoptosis by 10% or more. 29. The dose of the apoptosis-inducing agent is about the amount of solid tumor cells.
29. The method of claim 28, which induces apoptosis by 15% or more. 30. The method of claim 29, wherein the sufficient time is sufficient to reduce the density of solid tumor cells by 20% or more. 31. The method of claim 20, wherein the sufficient time is sufficient to reduce the density of solid tumor cells by 30% or more. 32. The method of claim 16, wherein the sufficient time is sufficient to induce apoptosis in more than 5% of the solid tumor cells. 33. The method of claim 31, wherein the sufficient time is sufficient to induce apoptosis in more than 10% of the solid tumor cells. 34. The method of claim 20, wherein the sufficient time is between about 16 and about 24 hours. 35. The method of claim 20, wherein the dose of the apoptosis-inducing agent is about 50 nM for about 1 hour. 36. The method of claim 16, wherein the patient is a human. 37. The apoptosis inducer is also the therapeutic agent, but the therapeutic agent comprises another dose or a sustained release form of the apoptosis inducer.
The method described in. 38. The method of claim 37, wherein the therapeutic agent and the apoptosis inducer comprise paclitaxel. 39. The method of claim 37, wherein the therapeutic agent and the apoptosis inducer comprise doxorubicin. 40. The method of claim 16, wherein the therapeutic agent comprises a gene therapy product, a protein binding agent, a chemotherapeutic agent, or an antibiotic. 41. The method of claim 40, wherein said therapeutic agent is a gene therapy construct and said construct comprises a tumor suppressor gene. 42. The method of claim 41, wherein the tumor suppressor gene is p53. 43. The method of claim 40, wherein the therapeutic agent is a protein binding agent. 44. The method of claim 16, wherein the apoptosis-inducing agent is systemically administered. 45. The method of claim 16, wherein the apoptosis-inducing agent is locally administered. 46. The method according to claim 1, wherein the apoptosis-inducing agent is locally administered locally.
The method according to 6. 47. The method of claim 16, wherein the apoptosis-inducing agent is locally administered. 48. The method of claim 16, wherein the therapeutic agent is administered systemically. 49. The method of claim 16, wherein the therapeutic agent is administered locally. 50. The method of claim 16, wherein the therapeutic agent is administered locally topically. 51. The method of claim 16, wherein the therapeutic agent is administered locally. 52. The method of claim 16, wherein the apoptosis-inducing agent or the therapeutic agent is administered with a pharmaceutically acceptable carrier. 53. The method of claim 52, wherein the pharmaceutically acceptable carrier is suitable for intravenous injection. 54. The method of claim 16, wherein the apoptosis-inducing agent is formulated as microparticles or nanoparticles. 55. The method of claim 17, wherein the therapeutic agent is formulated as microparticles or nanoparticles. 56. A method of delivering a therapeutic agent to a tumor of a patient, comprising: obtaining an apoptosis inducer; administering a dose of the apoptosis inducer to the patient; said apoptosis inducer causing apoptosis in the tumor. A method comprising: allowing sufficient time to induce and delivering a dose of a therapeutic agent to said patient such that the therapeutic agent is delivered to the tumor. 57. A method of delivering a chemotherapeutic agent to a tumor in a patient, comprising the step of locally or locally administering to the patient a dose of an apoptosis-inducing agent, the apoptosis-inducing agent inducing apoptosis in the tumor. A method comprising allowing sufficient time to pass, and delivering a dose of the chemotherapeutic agent to the patient so that the chemotherapeutic agent is delivered to the tumor. 58. The method of claim 57, wherein the tumor is cancerous. 59. The cancerous tumor is brain, breast, ovary, bladder, prostate, colon, lung,
59. The method of claim 58, which is a liver or uterine tumor. 60. The apoptosis-inducing agent is paclitaxel.
7. The method according to 7. 61. The apoptosis-inducing agent is doxorubicin.
7. The method according to 7. 62. The method of claim 57, wherein the decrease in solid tumor cell density is about 30% or greater. 63. The method of claim 62, wherein the decrease in solid tumor cell density is about 35% or greater. 64. The method of claim 63, wherein the sufficient time is sufficient to reduce the density of solid tumor cells by 20% or more. 65. The method of claim 64, wherein the sufficient time is sufficient to reduce the density of solid tumor cells by 30% or more. 66. The method of claim 57, wherein the sufficient time is sufficient to induce apoptosis in more than 5% of the solid tumor cells. 67. The method of claim 66, wherein the sufficient time is sufficient to induce apoptosis in more than 10% of the solid tumor cells. 68. The method of claim 60, wherein the sufficient time is between about 16 and about 24 hours. 69. The method of claim 60, wherein the dose of the apoptosis-inducing agent is about 50 nM for about 1 hour. 70. The method of claim 57, wherein the patient is a human. 71. The method of claim 57, wherein the apoptosis-inducing agent is also the chemotherapeutic agent, but the chemotherapeutic agent comprises another dose or a sustained release form of the apoptosis-inducing agent. 72. The method of claim 57, wherein the chemotherapeutic agent and the apoptosis inducer comprise paclitaxel. 73. The method of claim 57, wherein the therapeutic agent and the apoptosis inducer comprise doxorubicin. 74. The method of claim 57, wherein the apoptosis-inducing agent is formulated as microparticles or nanoparticles. 75. The method of claim 57, wherein the therapeutic agent is formulated as microparticles or nanoparticles. 76. The method of claim 57, wherein the apoptosis-inducing agent or the therapeutic agent is administered with a pharmaceutically acceptable carrier. 77. The method of claim 76, wherein the pharmaceutically acceptable carrier is suitable for intravenous injection. 78. The apoptosis inducer and the therapeutic agent are administered simultaneously,
6. The therapeutic agent is a sustained release formulation that releases the therapeutic agent after a time sufficient for the apoptosis inducer to induce apoptosis in a patient's tissue.
7. The method according to 7. 79. The apoptosis-inducing agent and the therapeutic agent are sequentially administered, and the therapeutic-agent is administered after a time sufficient for the apoptosis-inducing agent to induce apoptosis in the tissue of the patient. The method described in. 80. The method of claim 57, wherein the patient is a human. 81. A method of delivering a chemotherapeutic agent to a tumor of a patient, comprising: obtaining an apoptosis-inducing agent; administering a dose of the apoptosis-inducing agent locally or locally to the patient; A method comprising: allowing sufficient time to induce apoptosis in a tumor, and delivering a dose of the chemotherapeutic agent to the patient such that the chemotherapeutic agent is delivered to the tumor. 82. A rapid release preparation of an apoptosis inducer,   Sustained-release preparation of therapeutic agent, and   A pharmaceutically acceptable carrier, A composition for delivering a therapeutic agent to a patient, comprising: 83. The apoptosis inducer is paclitaxel.
The composition according to 2. 84. The composition of claim 82, wherein the rapid release formulation releases about 50 nM paclitaxel over about 1 hour or less. 85. The method of claim 8, wherein the apoptosis inducer is doxorubicin.
The composition according to 2. 86. The apoptosis inducer is provided in an amount sufficient to reduce the density of solid tumor cells by about 30% or more within about 16 to 24 hours after administration.
A composition according to claim 82. 87. An amount of the apoptosis inducer sufficient to induce apoptosis in 10% or more of solid tumor cells within about 16 to 24 hours after administration,
83. The composition of claim 82, provided. 88. The composition of claim 82, wherein the therapeutic agent is paclitaxel or doxorubicin. 89. The composition of claim 82, wherein said therapeutic agent is a protein binding agent, chemotherapeutic agent, antibiotic or gene delivery construct. 90. The composition of claim 89, wherein said gene delivery construct comprises a tumor suppressor gene. 91. The composition of claim 82, wherein the pharmaceutical composition is suitable for intravenous injection. 92. The composition of claim 82, wherein the pharmaceutical composition is suitable for topical administration. 93. The composition of claim 82, wherein said pharmaceutical composition is suitable for topical administration. 94. The composition of claim 82, wherein the apoptosis-inducing agent is formulated as microparticles or nanoparticles. 95. The composition of claim 82, wherein the therapeutic agent is formulated as microparticles or nanoparticles. 96. An apoptosis inducer in a pharmaceutically acceptable carrier, a therapeutic agent in a pharmaceutically acceptable carrier, a container, and the use of the apoptosis inducer and the therapeutic agent for the treatment of a tumor. A kit for the treatment of tumors, comprising the instructions. 97. Nanoparticles comprising crosslinked gelatin and a therapeutic or apoptosis inducing agent. 98. The nanoparticles are from about 500 nm to about 1 μm in diameter.
7. The nanoparticles according to 7. 99. The nanoparticles of claim 98, wherein the therapeutic agent or the apoptosis inducer is paclitaxel. 100. The nanoparticles of claim 97, wherein said therapeutic agent or said apoptosis inducer is doxorubicin. 101. Microparticles comprising a therapeutic agent or an inducer of apoptosis and suitable for topical, topical or topical administration to a patient. 102. The microparticle of claim 101, having a diameter of about 1 μm to about 10 μm. 103. The microparticle of claim 102, having a diameter of about 3 μm to about 7 μm. 104. The microparticle of claim 103, which has a diameter of about 5 μm. 105. The microparticle of claim 101, wherein the therapeutic agent or the apoptosis inducer is paclitaxel. 106. The microparticle of claim 101, wherein the therapeutic agent or the apoptosis inducer is doxorubicin. 107. Microparticles comprising paclitaxel suitable for topical, topical or topical administration to a patient, said microparticles having a diameter of about 5 μm. 108. Microparticles comprising doxorubicin suitable for topical, topical or topical administration to a patient, said microparticles having a diameter of about 5 μm.