JP4927535B2 - Particles encapsulating drugs that target tumors - Google Patents

Description

Related Information This patent application is filed on April 3, 2003, entitled “Paclitaxel-Loaded Particles for Enhancing Delivery of Therapeutic Agents to Tissue”. Claims priority of provisional application number 60 / 460,827. All patents, patent applications, and references cited herein are hereby incorporated by reference.

Government-sponsored research This study is supported in part by a grant from the United States Department of Health and Human Services (Grant No. R37CA49816).

SUMMARY OF THE INVENTION Successful cancer chemotherapy requires delivery of a sufficient concentration of active drug to tumor cells without causing intolerable toxicity in the patient. The present invention describes particles encapsulating two drugs intended for tumor targeting. One is a microparticle encapsulating a drug for treating peritoneal cancer, which is intraperitoneally administered into the abdominal cavity. Local administration of microparticles to other tumor-bearing organs is also possible. The other is nanoparticles encapsulating drugs for intravesical treatment of bladder cancer, which are administered intraperitoneally into the bladder cavity. Systemic administration of nanoparticles targeting the kidney is also possible.

Drug-encapsulated microparticles Several types of cancer are derived from organs located within the abdominal cavity, examples of such cancers being pancreatic cancer, liver cancer, colorectal cancer, and ovarian cancer. The abdominal cavity is also a site where cancer derived from extra-abdominal organs such as lung cancer metastasizes later in the disease. In the abdominal cavity, tumors are found on the pelvic and abdominal peritoneal surfaces, other peritoneal organs such as the mesentery, bladder, omentum, diaphragm, lymph nodes, liver. Occlusion of the diaphragm or abdominal lymphatic drainage by the tumor cells leads to a decrease in the peritoneal fluid efflux that causes carcinomatosis or ascites.

  Intraperitoneal chemotherapy has been used to treat peritoneal cancer, such as advanced ovarian and colon cancer, by instilling drugs directly into the abdominal cavity (Otto, SE, J Intraven. Nurs., 18: 170-176,1995 Collins, JM, J Clin Oncol, 2: 498-504, 1984). Intraperitoneal administration of platinum-binding compounds such as taxol (R) and cisplatin, which are commercially available paclitaxel formulations, has benefited patients and increased survival by about 20% (Gadducci, et al., Gynecol Oncol, 76: 157-162, 2000; Markman, et al., J Clin Oncol, 19: 1001-1007, 2001). However, intraperitoneal chemotherapy is limited in its use due to the following drawbacks. In the intraperitoneal treatment, 6 treatments are usually given every 3 weeks via an indwelling catheter. The two main side effects are infections associated with prolonged catheter use and abdominal pain caused by the presence of high drug concentrations in the peritoneal cavity (Francis, et al., J Clin Oncol, 13: 2961-2967, 1995). Furthermore, hospitalization is required for intraperitoneal administration and is quite expensive. For these reasons, the medical community has been reluctant to use intraperitoneal therapy, despite its survival benefits. The present invention overcomes these various imperfections.

  In a previous patent application (US patent application Ser. No. 09 / 547,825), the applicant has administered an anti-cancer agent (eg, paclitaxel, doxorubicin) outside of a solid tumor as is done during localized intraperitoneal treatment. When shown, the penetration of drugs into solid tumors was shown to be very slow and limited to the perimeter of the tumor. Applicants have also discovered a way to solve this penetration problem. This method expands the interstitial space within the tumor, thereby improving the penetration and distribution of simultaneously and subsequently administered drugs throughout the solid tumor, so as to induce apoptosis (eg, treatment with paclitaxel or doxorubicin) It consists of using. This method is referred to herein as the “tumor priming” method and requires two conditions. The first condition is that the drug concentration must be sufficient to induce apoptosis. Applicants have shown that 3 hours of treatment with 200 nM paclitaxel is sufficient to induce apoptosis in multiple human tumor cells (Au, et al., Cancer Res., 58: 2141-2148, 1998). The second condition is that the time interval between apoptosis induction pre-treatment and subsequent treatment should be sufficient for apoptosis to occur, eg 16-24 hours in the case of paclitaxel (Au, et al., Cancer Res., 1998; Jang, et al., J. Pharmacol. Exper. Therap., 296: 1035-1042, 2001). These conditions are cleared in the present invention.

  The applicant further discovered that a commercially available Taxole formulation disappears rapidly from the abdominal cavity after intraperitoneal administration due to rapid absorption from the peritoneum, a thin film that supports the abdominal cavity, and drainage from the lymphatic system. (See Examples 4 and 6 below).

  Based on these various considerations and discoveries, applicants can achieve therapeutically useful intravesical chemotherapy provided that the treatment meets some or most of the desired characteristics listed below. A conclusion was reached. Such desired properties are, firstly, that the drug has activity against the intended target cancer type. Secondly, treatment is easy to administer and does not require the use of an indwelling catheter over a long period of time, for example 1-2 days, and does not require frequent administration, for example once a week. Third, the drug should be such that the drug amount, and hence the drug concentration in the peritoneal cavity, is high enough to give an adequate control of the disease but at the same time low enough not to cause significant local toxicity. The speed at which it appears is to be optimized. Fourth, the drug or formulation must be able to penetrate the solid tumor and be widely distributed within the solid tumor. Fifth, the drug or formulation is to stay for a long time in the abdominal cavity where the tumor is localized. Sixth, the drug or formulation has a high affinity for tumor cells and is localized on the tumor surface or in the tumor mass. Seventh, the distribution of the drug or formulation is similar to the distribution or scattering of tumor cells within the abdominal cavity or organ. The microparticle encapsulating the drug of the present invention has most or all of these desired functions.

  Drug encapsulated microparticles are designed to release the drug at two rates. One is a rapid release to give a sufficiently high drug concentration to induce apoptosis and the other is a slower release to give a sustained drug delivery to the tumor. Induction of apoptosis promotes the penetration and distribution of the remaining microparticles and the remaining drug that are subsequently released from the sustained release particles. For example, slow drug release over a long period of days, weeks, or months provides patients with the convenience of a single dose, reduces the frequency of treatment, eliminates the need for hospitalization, and reduces health care costs Eliminates the need to use an indwelling intraperitoneal catheter, thereby reducing the risk of infection, improving the patient's quality of life, and administering the entire dose at once with all immediate release bolus High local concentrations occur, reducing local toxicity. Depending on their size and properties, these particles stay in the abdominal cavity, adhere to the tumor, and show a similar distribution within the abdominal cavity or organ.

  The particles may be a combination of two or more types of particles, but at least one is of a type that releases drug rapidly to induce apoptosis (fast release) and the rest are drugs A type that releases more slowly (sustained release). Although paclitaxel is widely used as an anti-cancer agent, a commercial paclitaxel formulation or taxol (registered trademark) in which paclitaxel is solubilized in Cremophor and ethanol by giving examples of microparticles encapsulating the paclitaxel. ) Demonstrates the usefulness of these particles in producing superior tumor targeting and antitumor activity in mice with peritoneal tumors.

  Applicants further disclose that other drugs can be formulated into the same microparticles for the purpose of treating peritoneal cancer.

  Applicants further describe the use of drug-encapsulated particles for the treatment of tumors located in organs or areas that are readily reachable by local or localized administration.

  The present invention uses biodegradable particles made of gelatin and PLGA polymers encapsulating therapeutic useful drugs. One drug formulated into gelatin and PLGA particles is paclitaxel. Various biodegradable polymer-bound paclitaxel formulations have been developed and shown to inhibit tumor growth and angiogenesis with minimal systemic toxicity in animal models, but the release kinetics of paclitaxel in these systems is It is in the range of about 10-25% of the drug released over about 50 days and is likely not optimal for clinical use (Patent Document 1). In addition, most of these previous studies aimed at achieving systemic drug delivery rather than localized. The advantages of biodegradable polymers as carriers for therapeutically useful drugs are recognized in the art (Patent Document 1) and include (1) complete biodegradation, i.e. drug No need for re-operation to remove the drug carrier that has been supplied, (2) tissue biocompatibility, (3) ease of administration, (4) sustained-release encapsulation during polymer hydrolysis Control of drug release, (5) Minimization or elimination of systemic toxicity such as neutropenia in localized use, (6) Convenience of biodegradable polymer system from the viewpoint of versatility included. Many of these benefits apply equally to gelatin drug release particles.

Intravesical chemotherapy is used to reduce the recurrence and / or progression of nanoparticle bladder cancer encapsulated with paclitaxel for intravesical treatment of bladder cancer (Kurth, KH, Semin. Urol. Oncol., 14: 30-35, 1996). Intravesical chemotherapy has the advantage of selectively delivering drugs at high concentrations to the tumor-bearing bladder while minimizing systemic exposure. Applicants have shown that unsuccessful treatment with superficial bladder cancer patients with chemotherapy such as mitomycin C or doxorubicin is due in part to poor drug delivery to tumors located in bladder tissue (Dalton, et al., Cancer Res., 51: 5144-5152, 1991; Schmittgen, et al., Cancer Res., 51: 3849-3856, 1991; Wientjes, et al., Pharm. Res., 8 : 168-173, 1991; Wientjes, et al., Cancer Res., 51: 4347-4354, 1991; Wientjes, et al., Cancer Res., 53: 3314-3320, 1993; Chai, et al., J Urol., 152: 374-378, 1994; Wientjes, et al., Cancer Chemother Pharmacol. 37: 539-546, 1996; Au, et al., J. Natl. Cancer Inst., 93: 597-604, 2001 ; Patent Document 2). The low drug delivery was also due to several reasons. First, only a portion of the mitomycin C or doxorubicin dose was able to penetrate the urothelium covering the inner surface of the bladder cavity, eg, -3% to -5%. Second, the concentration of drug was diluted by the presence of residual urine at the time of drug administration and by urine generated during the treatment interval (eg 2 hours). Third, because patients need to empty their bladder, total exposure of tumor cells to drugs such as mitomycin C was limited to the length of the treatment interval. Fourth, the residence of drugs such as mitomycin C in the bladder tissue is determined by the duration of treatment, and mostly ends within a few minutes after the patient empties the bladder.

  Based on these various considerations and discoveries, Applicant concludes that therapeutically useful intravesical chemotherapy is achievable if the treatment meets some or most of the desired properties listed below. Reached. First, the drug has activity against bladder cancer. Second, most of the drug present in the urine must be able to penetrate the urothelium. Third, the residence time of the drug in the bladder tissue does not exceed the duration of treatment. Fourth, the drug concentration in urine is not affected by urine volume. Paclitaxel is active against bladder cancer (Roth, B., J. Semin. Oncol., 22: 1-5, 1995) and, as Applicant has shown, for example 50% of the dose throughout the urothelium. It is easily distributed (Song, et al., Cancer Chemother. Pharmacol., 40: 285-292, 1997). Applicants have further noted that paclitaxel remains in tumor cells after removal of the drug from the extracellular matrix (Kuh, et al., J. Pharmacol. Exp. Ther., 293: 761-770, 2000), 2 We have discovered properties that provide an opportunity to prolong drug action beyond the treatment period of time. However, commercial paclitaxel formulations such as Taxol® are not useful because the cremophor used to solubilize paclitaxel reduces the rate of paclitaxel release by incorporating paclitaxel into micelles, As a result, the permeability of the drug to the bladder tissue is reduced (Knemeyer, et al., Cancer Chemother. Pharmacol., 44: 241-248, 1999).

  Applicants have therefore invented nanoparticles encapsulating paclitaxel that satisfy all of the desired properties identified in Applicants' discovery. These nanoparticles release a significant portion of the drug load within a 2 hour treatment interval so that the paclitaxel concentration in the tissue is sufficient to produce antitumor activity in human bladder tumors (eg, Example 8). ). The amount of paclitaxel released from the nanoparticles is limited by urine drug solubility. Therefore, the drug concentration in urine remains relatively constant and does not depend on the amount of urine (for example, Example 10). Paclitaxel released from the nanoparticles and penetrating the bladder also resides in the bladder tissue for a period beyond the 2 hour treatment period (eg, Example 8). Finally, Applicants have determined that nanoparticles are effective against bladder cancer that naturally occurs in domestic dogs (eg, Example 10).

  Applicant has also made it possible for the nanoparticles encapsulating the drug to remain in the bladder cavity or bladder tissue for a longer period of time, for example by coating the gelatin framework with a bioadhesive molecule. A method of modifying the encapsulated nanoparticles is disclosed (eg, Example 7).

  Applicants further disclose that other lipophilic compounds can be formulated in the same gelatin nanoparticles.

  Finally, Applicants disclose that intravenous administration of gelatin nanoparticles increases localization of drug content in the kidney (eg, Example 11). Also provided are methods and compositions for selective drug delivery to the kidney.

Definitions For purposes of clarity and consistency in understanding the invention, certain terms employed in the specification, examples, and claims are collected here.

  As used herein, the term “abnormal growth” refers to a cell phenotype that is different from the normal phenotype of the cell, particularly those that are directly or indirectly associated with diseases such as cancer.

  As used herein, the term “administering” refers to introducing a drug into a cell, such as in vitro, into a cell in an animal, ie in vivo, or into a cell that is returned to the animal (ie ex vivo). .

  In the present specification, the terms “drug”, “drug”, “compound”, “anticancer agent”, “chemotherapeutic”, “anticancer agent”, “antitumor agent” are used interchangeably, Refers to a drug that has the property of inhibiting or alleviating abnormal cell growth such as cancer (if not present). The above terms are intended to include cytotoxic agents, cell disrupting agents, or cytostatic agents. The term “drug” includes small molecules, macromolecules (eg, peptides, proteins, antibodies or antibody fragments), nucleic acids (eg, gene therapy constructs), recombinant viruses, nucleic acid fragments (eg, including synthetic nucleic acid fragments). Including.

  As used herein, the term “apoptosis” refers to non-necrotic, cell-regulated cell death as defined by criteria well established in the art.

  In the present specification, the terms “benign”, “before malignancy”, and “malignant” give meanings recognized in the art.

  As used herein, the terms “cancer”, “tumor cell”, “tumor”, “leukemia”, or “leukemia cell” are used interchangeably and any neoplasm (“new growth”), eg, Refers to cancer (derived from epithelial cells), adenocarcinoma (derived from gland cells), sarcoma (derived from connective tissue), lymphoma (derived from lymphoid tissue), or blood cancer (eg, leukemia or erythroleukemia). The term cancer or tumor cell is intended to include cancerous tissue or tumor mass to be interpreted as a cancer cell or a collection of tumor cells. Furthermore, the term cancer or tumor cell is intended to include cancers or cells that are either benign, pre-malignant or malignant. Usually, cancer or tumor cells exhibit various art-recognized salient features such as growth factor independence, lack of cell / cell contact growth inhibition and / or abnormal karyotype. In contrast, normal cells are usually only capable of being passaged in culture for a finite number of passages and / or significant features attributed to various art-recognized normal cells ( For example, growth factor dependence, contact inhibition and / or normal karyotype).

  In the present specification, the term “cell” refers to a eukaryotic cell such as a somatic cell or germline mammalian cell, or a cell line such as HeLa cell (human) or NIH3T3 cell (mouse), fetal stem cell and cell type. For example, hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, epithelial cells, and cell lines as described herein, for example.

  As used herein, the terms “peritoneal”, “intraperitoneal”, “peritoneally”, or “intraperitoneally” are used interchangeably and relate to the peritoneum or peritoneal cavity.

  As used herein, the terms “locally”, “locally”, and “systemically” each refer to “locally”, eg, treatment of a tumor mass, or “locally”, eg, suspected metastasis. Refers to the treatment of a general tumor field or region, or the treatment intended to be spread systemically throughout a subject, such as by “systemic”, eg, oral or intravenous administration.

  As used herein, the term “pharmaceutically acceptable carrier” is art-recognized and is a pharmaceutically acceptable substance or component suitable for administering a compound of the present invention to a mammal. Or media.

  As used herein, the term “pharmaceutical component” includes formulations suitable for administration to mammals such as humans. When the compound of the present invention is administered as a pharmaceutical to a mammal such as a human, irrespective of the other, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of an active ingredient (for example, Can be administered in combination with a pharmaceutically acceptable carrier as a pharmaceutical ingredient containing a therapeutically effective amount).

  In the present specification, the term “subject” is used with the intention of including humans and non-human animals (eg, mice, rats, rabbits, cats, dogs, domestic animals, primates). Suitable human animals include human patients having a disease as characterized by abnormal cell growth (eg, cancer).

  In the present specification, the term “microparticle” refers to a particle having a size of about 0.1 μm to about 100 μm, about 0.5 μm to about 50 μm, 0.5 μm to about 20 μm, and so that a higher effect can be obtained. Refers to particles of about 1 μm to about 10 μm, about 5 μm, or mixtures thereof. The microparticles can include, for example, a macromolecule, a gene therapy construct, or a chemotherapeutic agent. Usually, the microparticles can be administered, for example, locally or locally.

  As used herein, the term “nanoparticle” refers to particles having a size of about 0.1 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. , Refers to particles of about 600 to 800 nm, where higher effects are obtained. Nanoparticles can include macromolecules, gene therapy constructs, or chemotherapeutic agents. For example, usually nanoparticles are administered locally, locally, or systemically to a patient.

  As used herein, the term “particle” refers to a nanoparticle, a microparticle, or both a nanoparticle and a microparticle.

  As used herein, the term “formulation” refers to a composition recognized in the art, such as a dosage form of a therapeutically effective drug.

  In the present specification, the term “immediate release preparation” means that the drug content is preferably 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more Preferably, it refers to a drug formulation that releases 50% or more, more preferably 60% or more within one day. Examples of immediate release formulations include microparticle formulations and nanoparticle formulations. Methods for formulating these formulations are described in Examples 3 and 7.

  As used herein, the term “sustained release formulation” refers to a formulation of a drug that is delivered to a site of interest over a sustained period of time, preferably for several days, more preferably for several weeks. Or formulations that are maintained over time.

  As used herein, the term “tumor priming method” refers to penetration of a therapeutic agent by “priming” the tumor with an apoptosis-inducing agent to reduce tumor cell density and increase therapeutic agent reachability to the tumor mass. It refers to a method to increase sex. This method is sufficient to cause apoptosis in the tissue, thereby increasing the penetration of the tumor therapeutic agent (or simply “therapeutic agent”) into the tissue (eg, by creating a channel in the tissue). Is involved in the use of apoptosis-inducing drugs such as paclitaxel at various doses and periods. Thus, apoptosis inducers are used as a pretreatment before a therapeutic amount of the therapeutic agent is delivered to the tissue, and this pretreatment increases the penetration of the therapeutic agent into the tissue compared to the absence of pretreatment. Can be increased. Apoptosis inducers can also have therapeutic activity and therefore can also be used as therapeutic agents (ie, the same drug can be used as apoptosis inducers and therapeutic agents). Alternatively, apoptosis-inducing agents can be used to enhance delivery of other types of drugs or excipients to tissues for treatment (ie, apoptosis-inducing agents and therapeutic agents can be different drugs, or treatment The drug can be included in the nanoparticles or microparticles, where delivery of the nanoparticles or microparticles to the tumor tissue is improved compared to without pretreatment).

  In this specification, the term “PLGA” or “lactic acid glycolic acid copolymer” means a copolymer obtained by copolymerizing lactic acid or lactide (LA) and glycolic acid or glycolide (GA) at various ratios. Point to. Copolymers can have different average chain lengths, resulting in different internal viscosities and polymer properties. PLGA is used in the preparation of microparticles or nanoparticles that usually contain a therapeutic agent. The method used to dispense these particles is described in Example 3.

  As used herein, the term “gelatin” refers to a denatured form of connective tissue protein collagen. Gelatin aggregates are formed in solution but are stabilized by cross-linking protein chains. Gelatin is formed into gelatin nanoparticles using the preparation method of Example 7, but usually encapsulates paclitaxel. Gelatin with different protein chain lengths is available, indicated by different Bloom numbers. A higher bloom strength indicates a longer chain length.

As used herein, the term “IC ₅₀ ” refers to a drug concentration or dose that produces a 50% drug effect.

  As used herein, the term “burst release” refers to an art-recognized definition, that is, an early release of a portion of a drug load from a formulation, usually followed by a drug. The rest of the load is released more slowly.

  As used herein, “localize” and “concentrate” are used interchangeably and refer to a preferential distribution at a particular site, eg, tumor tissue.

  As used herein, the term “bioadhesive” refers to natural, synthetic or semi-synthetic substances that adhere to, preferably strongly adhere to, the surface of skin, mucous membranes, tumors and the like. Suitable bioadhesives include polylysine, fibrinogen, those prepared from partially esterified polyacrylic acid polymers, e.g. natural or synthetic polysaccharides such as polyacrylic acid polymers, cellulose derivatives, e.g. Examples include methyl cellulose, cellulose acetate, carboxymethyl cellulose, hydroxyethyl cellulose, pectin, a mixture of sulfated sucrose and aluminum hydroxide, and the like.

  As used herein, the term “interstitial cystitis” refers to a medical condition recognized in the art as a chronic painful inflammatory condition of the bladder wall.

  As used herein, the term “biodegradable” polymer or “bioerodible” polymer refers to degradation to low molecular weight compounds, commonly known to be involved in metabolic pathways. Refers to a possible polymer. Such polymers include a site of action that is a polymer system and can be subjected to destructive chemical action in a biological environment that affects the integrity of the system and possibly the polymer itself. Also included are polymer systems that provide fragments or other degradation byproducts that can be removed from, but not necessarily removed from the body.

SUMMARY OF THE INVENTION The present invention is for delivering a tumor therapeutic agent to a patient comprising an immediate release formulation of a tumor apoptosis inducer, a sustained release formulation of a tumor therapeutic agent, and a pharmaceutically acceptable carrier. A composition is provided. An apoptosis-inducing agent in a pharmaceutically acceptable carrier can be administered simultaneously with or prior to the composition. Nanoparticles or microparticles (eg, cross-linked gelatin) of a therapeutic agent (eg, paclitaxel) can also be used. Nanoparticles or microparticles can be coated with a bioadhesive coating agent. Microspheres that aggregate to block the lymphatic inlet of the bladder can also be used to delay clearance of the microparticles from the lymphatic system.

  The present invention also uses drug encapsulated gelatin and lactate glycolic acid copolymer (PLGA) nanoparticles and microparticles to target drug delivery to abdominal, bladder tissue, and kidney tumors.

US Pat. No. 6,447,796 US Pat. No. 6,286,513B1 US Pat. No. 6,132,759

  The present invention provides methods and compositions used therein for delivering therapeutic agents to tumors and kidney tissue. The methods of the present invention allow targeting of therapeutic agents and improved penetration into multi-layered tissues such as solid tissues and solid tumors.

  In a first aspect, the present invention provides a method for promoting penetration of a chemotherapeutic agent or particle into a tumor. This method relates to the use of apoptosis inducers, such as paclitaxel, at a dose and for a period sufficient to cause apoptosis in the tissue. Subsequent delivery of the therapeutic agent or particle to the tissue when substantial apoptosis occurs increases the permeability of the therapeutic agent or particle to the tissue. Thus, an apoptosis-inducing agent is used as a pretreatment before the therapeutic agent or particle is delivered to the tissue, and this pretreatment increases the permeability of the therapeutic agent or particle to the tissue compared to without pretreatment. Can do. Apoptosis inducers can also be used as therapeutic agents because they have therapeutic activity (ie, the same drug can be used as an apoptosis inducer or therapeutic agent). Alternatively, apoptosis-inducing agents can be used to enhance delivery of other types of drugs to the tissue (ie, apoptosis-inducing agents and therapeutic agents can be different drugs).

  In one embodiment, the present invention uses a formulation that allows the combination of an apoptosis inducer and a therapeutic agent. In this embodiment, the apoptosis-inducing agent is formulated as an immediate release formulation and the therapeutic agent is formulated in one or more sustained release formulations. After administration, the immediate release formulation will cause apoptosis in the tissue, and the sustained release formulation will allow the delivery of the majority of the entire therapeutic agent after apoptosis is substantially achieved. Become.

  In a related embodiment, the formulation is a single agent in which the apoptosis-inducing drug is burst released early and the remaining drug load that subsequently acts as a therapeutic agent is released more slowly.

  In a related embodiment, the formulation described above will be used for the treatment of tumors in the abdominal cavity and tissues adjacent to the abdominal cavity.

  In a further related embodiment, the above described mixed formulation will be used for the treatment of ascites tumors.

  In another further related embodiment, these formulations are used for the treatment of tumors that are metastatic from the source and grow in the abdominal cavity or in tissues adjacent to the abdominal cavity where the tumor protrudes into the abdominal cavity .

  In related embodiments, these formulations are readily accessible by direct administration, for example, to one or more tissues within or adjacent to the abdominal cavity, bladder tissue, brain tissue, prostate tissue, or lung tissue. Used to treat tumors located in organs or areas.

  In another embodiment, the tumor to be treated grows in tissue that is in or adjacent to the abdominal cavity of the subject.

  In a related embodiment, the tumor to be treated is located in an organ or region that is readily reachable by direct administration, eg, prostate, bladder, brain.

  In a further embodiment, the microparticles are delivered as a suspension by the intraperitoneal route.

  In another embodiment, the microparticles are delivered as a suspension by direct local injection into easily reachable organs or by direct local injection into organs adjacent to easily reachable organs. .

  In another further embodiment, the test subject is a mammal, preferably a human.

  In another embodiment, the patient to be treated is a pancreatic or ovarian cancer patient that has spread to the abdominal cavity.

  In a second aspect, the present invention provides a composition for use in the above method. These compositions consist of PLGA particles encapsulating various drugs that release the apoptosis-inducing agent and therapeutic agent over time, where the apoptosis-inducing agent is released quickly in the beginning, and then the therapeutic agent is released more slowly. It ’s like that.

  In certain embodiments, the formulation is preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at the content of apoptosis inducer. An immediate release component that releases 50% or more, more preferably 60% or more within one day and causes apoptosis in the tissue, and release of the therapeutic agent is preferably days, more preferably weeks It consists of 1 or several sustained release component maintained above.

  In a related embodiment, the composition can be a single agent of paclitaxel that is initially burst released and subsequently the remaining drug load is released more slowly.

  In certain embodiments, the composition may be a combination of two or more paclitaxel formulations, such that at least one formulation releases paclitaxel rapidly and at least one formulation releases paclitaxel more slowly. In a related embodiment, the mixed formulation provides better drug release control than that obtained from a single agent with an initial burst release, such as consisting of an initial rapid release followed by a delayed release.

  In another related embodiment, mixed formulations are particularly advantageous if different drugs are used as both apoptosis inducers and therapeutic agents.

  In yet another embodiment, the particles are PLGA particles formulated to contain an apoptosis-inducing or therapeutic agent. A typical formulation will contain at most 50%, preferably at most 30%, more preferably at most 20% of the total amount of the formulation in drug form.

  Sometimes it is advantageous to add a release enhancer such as Tween 20, Tween 80, isopropyl myristate, β-lactose, or diethyl phthalate to achieve the desired release rate for the application.

  In a preferred embodiment, the immediate release PLGA microparticles have a ratio of lactic acid to glycolic acid (LA: GA ratio) of 50:50, an average diameter of 3 to 5 μm, and a glass transition temperature lower than body temperature (eg 30 ° C.). The paclitaxel load is 4% or less and the drug release rate is 70% per day. An example of this composition is Batch 8 in Table 1.

  In a preferred embodiment, the sustained release PLGA microparticles have a ratio of lactic acid to glycolic acid of 50:50, an average diameter of 30-50 μm, and a glass transition temperature lower than body temperature (eg, 30 ° C.), With paclitaxel loading of 4% or less, release less than 20% at the initial burst drug release rate on day 1, followed by 70% of total cumulative release in 7 weeks with slower release. An example of the composition is Batch 7 in Table 1.

  In a preferred embodiment, the sustained release PLGA microparticles have a lactic acid to glycolic acid ratio of 75:25, an average diameter of 3 to 5 μm, and a glass transition temperature (eg 50 ° C.) higher than body temperature, With a paclitaxel load of 4% or less, release less than 5% at the initial burst drug release rate on day 1, followed by 30% of the total cumulative release in 7 weeks with slower release. An example of this composition is Batch 4 in Table 1.

  In a third aspect, the present invention provides a method for improving drug delivery to a tumor by administering drug-containing nanoparticles or microparticles locally to the location where the tumor is. At this time, the nanoparticles or microparticles selectively adhere to the tumor tissue, thereby producing high drug levels in the tumor compared to delivery in nanoparticles or microparticles that do not selectively adhere to the tumor.

  In one embodiment, the drug-containing nanoparticles or microparticles selectively adhere to tumor tissue and promote their penetration into the tumor through the release of apoptosis-inducing drugs. For example, the particles of Example 5 show extensive penetration into the tumor. This penetration was due to the release of the apoptosis-inducing agent, since equivalent particles that did not contain the apoptosis-inducing agent did not penetrate the tumor. The remaining drug load contained by the particles was released over time after penetrating the tumor, and the tumor tissue was intensively exposed to the released drug. The drug release rate will be slowed by the tissue environment, increasing the duration of tumor tissue exposure. Thus, the method of this embodiment using local administration of tumor adhesive particles that penetrates tumor tissue after initial drug release is an effective method of tumor treatment. Such embodiments are also not applicable to the use of immediate or sustained release therapeutics, but are applicable to the use of therapeutics to treat tumors because the particles contain apoptosis-inducing agents.

  In a fourth aspect, the present invention provides a composition for use in the above method.

  In one embodiment, these compositions consist of PLGA particles that have a glass transition temperature (Tg) below the patient's body temperature, thereby enhancing the selective adhesion of the particles to the tumor tissue.

  In a preferred embodiment, the immediate release PLGA microparticles are made of about 50% lactide and about 50% glycolide and have a glass transition temperature (eg, 30 ° C.) below body temperature. An example of such a composition is Batch 8 in Table 1.

  In certain embodiments, the particles are formulated in PLGA containing about 50% lactide and about 50% glycolide and encapsulating the drug. The total weight of the drug does not exceed 50% and preferably does not exceed 30% of the total weight of the formulation.

  In another embodiment, by cross-linking with polylysine to enhance the bioadhesive properties of the particle, or by coating the particle with fibrinogen, or by other art-recognized methods, Particles that adhere to the tumor tissue can be obtained. These particles can be gelatin nanoparticles or PLGA microparticles.

  In a fifth aspect, the invention focuses on tumors rather than those where the drug is delivered by a formulation that concentrates at the abdominal site where the metastatic tumor occurs most frequently, and not at the location where the metastatic tumor occurs most frequently. Methods are provided for improving drug delivery to tumors that have spread to the peritoneal cavity by administering intraperitoneal drug-containing microparticles that result in higher drug levels to be delivered.

  In a sixth aspect, the present invention is a composition for the above method, i.e., localized to the abdominal site where metastatic tumors occur most frequently when administered to the abdominal cavity, such as the omentum, mesentery, diaphragm, lower abdomen, etc. Such PLGA microparticles are provided.

  In a preferred embodiment, the PLGA microparticles have an average diameter of about 3-5 μm. These particles are preferentially localized near the omentum, mesentery, diaphragm and lower abdomen. Examples of these compositions are batches 4 and 8 in Table 1.

  In a preferred embodiment, the PLGA microparticles have an average diameter of about 30-50 μm. These particles are preferentially localized in the lower abdomen. Examples of these compositions are batches 2, 3, 5, 6, 7 in Table 1.

  In one embodiment, the microparticles are concentrated in the mesentery, omentum, diaphragm, and other sites that are the location of metastatic peritoneal tumor growth.

  In a related embodiment, the size of the particles is similar to the size of a single tumor cell or a small aggregate of tumor cells and is about 3 to about 30 μm in diameter.

  In a seventh aspect, the present invention provides a method for promoting the aggregation formation of drug-containing microparticles, thereby reducing the clearance of these particles by lymphatic drainage compared to microparticles having a Tg higher than body temperature. A method of achieving better retention of drug-containing microparticles in the peritoneal cavity is provided by formulating the drug in PLGA microparticles having a glass transition temperature (Tg) lower than the body temperature.

  In an eighth aspect, the present invention provides a composition for the above method, comprising a microparticle encapsulating a drug that delays lymphatic clearance.

  In a preferred embodiment, the PLGA microparticles are made of about 50% lactide and about 50% glycolide and have a glass transition temperature (eg, 30 ° C.) below body temperature. Examples of these compositions are batches 6, 7, and 8 in Table 1.

  In a ninth aspect, the present invention administers a drug without encapsulating the microparticle by formulating the drug in PLGA microparticles having a particle size large enough to reduce clearance of the drug by lymphatic drainage Compared to cases, a method is provided that achieves better retention of drug-containing microparticles in the peritoneal cavity.

  In a tenth aspect, the present invention provides a composition for the above method, comprising a microparticle encapsulating a drug having a size large enough to delay clearance by lymphatic drainage. .

  In a preferred embodiment, the PLGA microparticles have an average diameter of at least 10 μm. Examples of these compositions are batches 2, 3, 5, 6, 7 in Table 1.

  In an eleventh aspect, the invention provides for administering a drug without encapsulating the microparticles by formulating the drug in PLGA microparticles having a particle size that reduces clearance of the drug from the peritoneum by absorption from the peritoneum. Provides a way to achieve better retention of the drug-containing microparticles in the peritoneal cavity as compared to

  In a twelfth aspect, the present invention provides a composition comprising microparticles encapsulating a drug that delays absorption from the peritoneum for the above method.

  In a preferred embodiment, the PLGA microparticles have an average diameter of at least 10 μm. Examples of these compositions are batches 2, 3, 5, 6, 7 in Table 1.

  In a thirteenth aspect, the present invention provides the following desired properties: (1) provide rapid drug release followed by slow drug release, (2) selectively adhere to tumor tissue, (3) metastasis Provided is a composition having at least two of (4) low clearance by lymphatic drainage and (5) low absorption from the peritoneum, localized to the abdominal site where a sex tumor occurs most frequently.

  In a preferred embodiment, the composition comprises immediate release and sustained release PLGA particles. The immediate release PLGA particles are made of 50% lactide and 50% glycolide, have an average diameter of 3 to 5 μm, a glass transition temperature lower than body temperature (for example, 30 ° C.), and have an apoptosis-inducing amount. Releases apoptosis inducer within hours. The sustained-release PLGA microparticles are made of 50% lactide and 50% glycolide, and have an average diameter of 30 to 50 μm and a glass transition temperature (for example, 30 ° C.) lower than body temperature. The release of the therapeutic agent releases preferably 30% or less, more preferably 20% or less of the drug load on the first day, followed by more than 60% of the total cumulative release in a few weeks due to slower release, Or, preferably 70% or more is released.

  In a more preferred embodiment, the apoptosis-inducing agent and the therapeutic agent are both paclitaxel.

  In a fourteenth aspect, the present invention provides a method for delivering a lipophilic drug to the bladder wall of a patient. This method uses gelatin nanoparticles in drug formulations that have several advantages over the administration of free drugs. By formulating the drug in gelatin nanoparticles, it is possible to administer a large number of low solubility drugs with a drug amount greater than would otherwise be possible. Furthermore, the drug formulated into gelatin nanoparticles can serve as a reservoir for continuous drug release, thereby maintaining a high drug concentration compared to administration of free drug. Furthermore, gelatin nanoparticles encapsulating the drug can remain in the bladder cavity even during urination, increasing the overall tumor exposure to the drug.

  In a preferred embodiment, the present invention provides a method of delivering paclitaxel to the bladder wall of a patient with superficial bladder cancer by intravesical instillation. Here, paclitaxel is not included in formulations that do not contain cremophor and is therefore more readily available for bladder wall penetration.

  In another embodiment, other highly lipophilic drugs for the treatment of superficial bladder cancer or interstitial cystitis can be administered using this method. Drugs that are expected to produce better therapeutic results using this method include, but are not limited to, docetaxel.

  In a fifteenth aspect, the present invention provides a composition for formulating paclitaxel in a delivery form suitable for intravesical instillation. Here, the delivery form is based on the following criteria: (1) a sufficient amount of paclitaxel can be contained in a small amount of instillation; (2) the delivery form allows for rapid and efficient paclitaxel on the bladder wall. (3) that a substantial and therapeutically active concentration of paclitaxel is achieved in the bladder wall.

  In one embodiment, the composition consists of crosslinked gelatin nanoparticles encapsulating paclitaxel.

  In a related embodiment, the gelatin nanoparticles are formulated to provide a limited release of paclitaxel solubility and therefore maintain a dissolved concentration of paclitaxel in urine. For this reason, paclitaxel is not dependent on processes that may cause dilution and reduction of drug concentration, including but not limited to residual urine at the time of instillation and urine production during or after treatment. The concentration will be kept constant and effective.

  In a preferred embodiment, a 175 bloom bloom strength, an average diameter of about 600 nm to about 1000 nm, 0.4% to 2.0% drug loading, more preferably 0.5% to 1.0% drug Gelatin nanoparticles are prepared from gelatin with loading. Gelatin cross-linking may be accomplished using glutaraldehyde as described in Example 7 or by other methods, for example, using oxidized polysaccharide molecules (Patent Document 3) as generally accepted in the art. You can get none. An example of these nanoparticles is shown in Table 3 and the properties are described in Example 7.

  In another related embodiment, the nanoparticle formulation will further comprise ingredients for increasing the solubility of paclitaxel and consequently increasing the concentration of paclitaxel available for tumor treatment. Examples of such compositions are cosolvents such as dimethyl sulfoxide or polyethylene glycol.

  In yet another related embodiment, hyperthermia can be applied to increase the solubility of paclitaxel in urine.

  In another embodiment, the particles adhere to normal bladder wall and / or inflammatory bladder wall often found in patients suffering from superficial bladder cancer, and / or tumor tissue exposed on the surface of the bladder wall. This adhesion is not limited, but the nanoparticle is coated with bioadhesive molecules including polylysine, fibrinogen, polyacrylic acid polymer, methylcellulose, cellulose acetate, carboxymethylcellulose, hydroxyethylcellulose, or pectin. Can be increased.

  In a related embodiment, the adhesion of the nanoparticles to the bladder wall allows maintenance of an effective drug concentration in the bladder cavity even after the patient empties the bladder.

  In certain embodiments, the particles that adhere to the bladder wall are gelatin nanoparticles encapsulated with a lipophilic therapeutic agent and coated with a bioadhesive molecule.

  In a preferred embodiment, the particles are gelatin nanoparticles encapsulated with paclitaxel and coated with polylysine.

  In a more preferred embodiment, a 175 Bloom bloom strength, an average diameter of about 600 nm to about 1000 nm, 0.4% to 2% drug loading, more preferably 0.5% to 1.0% drug A gelatin having a load, comprising 5% polylysine by weight, wherein the gelatin and polylysine molecules are formed using glutaraldehyde as described in Example 7 or by other art-recognized crosslinking methods. Gelatin nanoparticles are prepared from the gelatin to be crosslinked.

  In another embodiment, a delivery form suitable for intravesical introduction consists of immediate release PLGA microparticles.

  In another embodiment, the release rate of the therapeutic agent into the bladder contents is limited by the aqueous solubility of the drug.

  In a preferred embodiment, the drug is paclitaxel.

  In another embodiment, the rapid release PLGA microparticles have an average diameter of 100 nm to 6000 nm, preferably 500 to 5000 nm, more preferably 3000 to 4000 nm.

  In a preferred embodiment, the immediate release PLGA microparticles are made of 50% lactide and 50% glycolide and have an average diameter of 3000 to 5000 μm and a glass transition temperature below body temperature (eg 30 ° C.). And has a drug release rate that contains about 4% paclitaxel and releases about 70% of its drug content under sink conditions in one day.

  In another embodiment, PLGA microparticles are coated with bioadhesive molecules.

  In the sixteenth aspect, the present invention provides a method for increasing delivery of a drug to the kidney when the drug is administered by an intravenous route. This method is based on the distribution of gelatin nanoparticles to the kidney, which is selective compared to the distribution of drugs administered intravenously.

  In one embodiment, selective targeting of kidney tissue is achieved by intravenous administration of a drug formulated in gelatin nanoparticles.

  In a preferred embodiment, a method for selective targeting of the kidney is described in Example 11.

  In a more preferred embodiment, gelatin nanoparticles with an average size of about 600-900 nm encapsulating a therapeutically useful drug will be used to target the kidney of the subject.

  In another embodiment, the drug to be formulated into gelatin nanoparticles includes a chemotherapeutic agent for the treatment of renal cancer, a chemopreventive agent for the prevention of renal cancer, a gene for the treatment of kidney disease Therapeutic constructs include other drugs for which selective administration to the kidney is beneficial.

  In a seventeenth aspect, the present invention provides a composition for formulating paclitaxel or other drugs in a delivery form suitable for selective intravenous targeting of the kidney. The composition consists of crosslinked gelatin nanoparticles encapsulating the drug to be delivered.

  In a preferred embodiment, the drug is paclitaxel.

  In a more preferred embodiment, a 175 Bloom bloom strength, an average diameter of about 600 nm to about 1000 nm, 0.4% to 2% drug loading, more preferably 0.5% to 1.0% drug Gelatin nanoparticles are prepared from gelatin with loading. Gelatin cross-linking may be accomplished using glutaraldehyde as described in Example 7 or by other methods, for example, using oxidized polysaccharide molecules (Patent Document 3) as generally accepted in the art. You can get none. An example of these nanoparticles is shown in Table 3 and the properties are described in Example 7.

  In another embodiment, the drug is a gene product for selective delivery of gene constructs to kidney cells.

  In a seventeenth aspect, the present invention provides a method of delivering a drug to a patient's bladder wall. This method uses gelatin nanoparticles in drug formulations that have several advantages over the administration of free drugs. Formulating the drug in gelatin nanoparticles allows for the administration of a drug amount that is greater than would otherwise be possible.

  In a preferred embodiment, the drug formulated in gelatin nanoparticles can serve as a reservoir for continuous drug release, thereby maintaining a high drug concentration compared to administration of free drug. Furthermore, gelatin nanoparticles encapsulating the drug can remain in the bladder cavity even during urination, increasing the overall tumor exposure to the drug.

  In another embodiment, other highly lipophilic drugs for the treatment of superficial bladder cancer can be administered using this method. Drugs that are expected to produce better therapeutic results using this method include, but are not limited to, docetaxel.

  In another embodiment, other drugs for the treatment of superficial bladder cancer can be administered using this method. Such drugs include, but are not limited to, suramin, interferon (eg, interferon α, γ, ω), docetaxel, doxorubicin and other anthracyclines, thiotepa, mitomycin (eg, mitomycin C), calmet-gellan bacillus Cisplatin, methotrexate, vinblastine, 5-fluorouracil, leuprolide, flutamide, ethylstilbestrol, estramustine, megestrol acetate, cyproterone, flutamide, cyclophosphamide.

In another embodiment, a drug for the treatment of interstitial cystitis can be administered using this method. Drugs that are expected to produce better therapeutic results using this method include, but are not limited to, pentosan polysulfate and its sodium salts, antihistamines (eg, hydroxyzine and its salts, Cromolyn and its sodium salt), tricyclic antidepressants (eg amitriptyline, desipramine, nortriptyline, doxapine, imipramine), selective serotonin reuptake inhibitors (eg paroxetine), analgesics (eg gabapentin, clonazepam), Muscle relaxants (eg, diazepam, Baclofen), anticonvulsants (eg, gabapentin, clonazepam), opioid analgesics (eg, bicodin, percoset, oxycontin), other analgesics (eg, lidocaine hydrochloride, procaine hydrochloride, salicyl) Alcohol, tetrahydrochloride In, phenazopyridine hydrochloride, acetaminophen, acetylsalicylic acid, flufenisal, ibuprofen, indoprofen, indomethacin, naproxa, codeine, oxycodone, fentanyl citrate), antispasmodics (eg, Urimax) , Pyridium, Urised, flavoxate, dicyclomine, propanthelin), anticholinergic drugs (eg, detrol, ditropane, levsin, hyoscyamine), H ₂ blockers (eg, tagamet, zentac ( Zantac)), urine alkalizing agents, adrenergic antagonists (eg Cardura, Flomax, Hytrin), leukotriene antagonists (eg montelukast), and various actions Objects (e.g., dimethyl sulfoxide, heparin, oxychlorosene and its sodium salt, silver nitrate, Calmette - Guerin, sodium hyaluronate, resiniferatoxin, botulinum toxin) could include.

  In another embodiment, drugs such as antibiotics, antifungals, antiprotozoal drugs, antiviral drugs, and other antiinfectives can be used to treat bladder infection. Drugs suitable for the treatment of such infections include mitomycin, ciprofloxacin, norfloxacin, ofloxacin, methanamine, nitrofurantoin, ampicillin, amoxicillin, nafcillin, trimethoprim, sulfa, trimethoprimsulfamethoxazole, It includes erythromycin, doxycycline, metronidazole, tetracycline, kanamycin, penicillin, cephalosporin, aminoglycosides.

  In yet another embodiment, the drug can be used to treat urinary incontinence or inflammation. Suitable drugs include, among others, dicyclomine, desmopressin, oxybutynin, estrogen, terodiline, propantheline, doxapine, imipramine, flavoxate, phenylpropanolamine, terazosin, prazosin, pseudoephedrine, betanicol, anticholinergic, antispasmodic Drugs, antimuscarinic drugs, β-2 agonists, norepinephrine uptake inhibitors, serotonin uptake inhibitors, calcium channel antagonists, potassium channel openers, and muscle relaxants can also be used. Drugs suitable for the treatment of incontinence include oxybutynin, S-oxybutytin, emepronium, verapamil, imipramine, flavoxate, atropine, propantherine, tolterodine, rociverine, clenbuterol, darifenacin, terodiline, Trospium, hyoscyamine, propiverine, desmopressin, bamicamide (Fujisawa Pharmaceutical), YM-46303 (Yamanouchi Pharmaceutical), Lamperisone (Nippon Kayaku), NS-21 (Nippon Shinyaku, Orion, Iformenti (Formenti)) NC-1800 (Nippon Chemifa), ZD-6169 (Zeneca Co.), and stilonium iodide.

  In another embodiment, the drug could be used to improve bladder contractility or reduce urine retention in the bladder.

Example 1
Identification of Paclitaxel Concentration Active and Active for Human Tumor Cells and Duration of Treatment The present invention designs particles encapsulating paclitaxel to release cytotoxic levels of paclitaxel for a duration sufficient to produce cytotoxicity The method and composition of such particles are described. Example 1 identifies the effective paclitaxel concentration and treatment period for human tumor cells.

Applicants evaluated the cytotoxicity of paclitaxel (dissolved in water, 96 hours of treatment) in three human pancreatic cancer cells (PANC1, MIA-PaCa, Hs766T) and human bladder RT4 tumor cells. The drug effect was measured using an MTT (microtetrazolium dye reduction) assay. The concentrations required to produce 50% cytotoxicity (IC ₅₀ ) in these cells were 1.5, 0.7 and 0.7 nM, respectively. These _{IC 50} values human breast cancer cells MCF7 and paclitaxel _{IC 50} of the ovarian cancer cells SKOV3 (Au, et al, Cancer Res, 58:.. 2141-2148,1998) compared to. This data indicates that pancreatic cancer cells are highly sensitive to paclitaxel. Since patients with advanced pancreatic cancer did not show any visible activity after intravenous administration of Taxol (Paclitaxel dissolved in cremophor and ethanol) (Gebbia and Gebbia, Eur. J. Cancer, 32A: 1822-1823, 1996; Schnall and Macdonald. Semin. Oncol., 23: 220-228, 1996), this is surprising. The IC ₅₀ of paclitaxel in RT4 cells was 4.0 ± 0.4 nM (96 hours of treatment).

Example 2
Establishment of a human pancreatic and ovarian xenograft tumor model for examining the efficacy of microparticles encapsulating paclitaxel Example 2 illustrates the establishment of a peritoneal tumor model for examining the efficacy of microparticles encapsulating paclitaxel.

Applicants established orthotopic intraperitoneal tumors in athymic mice using human pancreatic Hs766T cells derived from lymph node metastases. For the orthotopic model, 2 × 10 ⁶ tumor cells were orthotopically transplanted into the body of the pancreas. Tumors were established after 2-3 weeks (diameter 1.5 cm or less to 2 cm or less). For intraperitoneal tumors, Applicants established metastatic sublineages by reimplantation of cells recovered from peritoneal lavage of mice that received intraperitoneal injection of Hs766T cells. Intraperitoneal reimplantation of metastatic Hs766T cells in 6-week-old female Balb / c mice revealed progressive tumors spread throughout the peritoneal cavity. Two to three weeks later, tumor nodules with a diameter of 1 cm or less to 1.5 cm or less were found in the net, and a plurality of nodules of 3 mm or less to 5 mm or less were found in the mesentery, lower abdomen, retroabdominal cavity, and diaphragm. There was also an ascites tumor. The animals died after 3-4 weeks.

  For the intraperitoneal ovarian tumor model, Applicants established a metastatic subline of human ovarian SKOV3 cells in the same manner as described for the Hs766T tumor. Re-transplantation of metastatic SKOV3 cells establishes tumors in the net in 2 weeks, and in the mesentery in 4 weeks, and then tumors that affect the parenchyma of internal organs such as the liver and kidney, and tumors that remain in the diaphragm Appears later (6 weeks later). The peritoneal protein concentration increased from 3% in normal mice to about 6% in tumor-bearing mice at 2 weeks. The volume of peritoneal fluid increased 7 to 10 times after 4 weeks and included tumor cell aggregation. The size of these ascites tumors ranged from 40 to several hundred μm. After 5-6 weeks, the animals showed significant weight loss (10-15%), but some mice showed peritoneal distention (20% increase in body circumference from 6.3 cm to 7.6 cm) and bowel obstruction. The animals died 7-9 weeks after tumor implantation.

  Disease progression in mice with pancreatic and ovarian tumors is similar to the disease progression reported in patients. For example, patients with ovarian cancer show similar tumor dissemination and progression in the abdominal cavity, with tumors appearing in the serosa, perihepatic and perisplenous ligaments, diaphragm, mesentery, and omentum. Ovarian cancer patients also show high protein concentrations in the peritoneal fluid (late 4.46 g / dl) due to leakage of serum proteins and / or the presence of ascites in the peritoneal cavity (Lee, et al ., Cancer, 70: 2057-2060, 1992). The patient's malignant ascites shows tumor cell aggregation of a size similar to that in SKOV3 ascites tumors (Tauchi, et al., Acta Cytol., 40: 429-436, 1996; Monte, et al., Acta Cytol. , 31: 448-452,1987).

Example 3
The present invention, which identifies the effect of PLGA microparticle properties on distribution and residence in the peritoneal cavity , discloses a composition of PLGA particles encapsulating a drug that targets peritoneal tumors. Example 3 shows the effect of different properties of PLGA microparticles on the distribution and residence of microparticles in the abdominal cavity to help identify PLGA microparticles having the desired residence and distribution characteristics. Examples 4 to 6 describe the preparation and application of PLGA microparticles encapsulating such drugs using paclitaxel, an anticancer agent that is widely used as an example.

Effect of PLGA microparticle size Applicants compared the distribution of two microparticle preparations with different diameters of 3 μm or less and 30 μm or less after intraperitoneal administration. These microparticles were encapsulated with acridine orange and could therefore be visualized under ultraviolet light. After 24 hours, the mice were euthanized and opened (3 mice per formulation). The results are shown in FIG. Small microparticles were found in the omentum, mesentery, diaphragm, and lower abdomen, while large microparticles were localized in the lower abdomen.

Effect of glass transition temperature on lymphatic clearance of microparticles from the abdominal cavity Applicants evaluated the effect of glass transition temperature (Tg) on clearance of microparticles from the abdominal cavity by lymph flow. A mixture of microparticles with a ratio of lactic acid to glycolic acid of 75:25 (ie, batch 1 in Table 1) and microparticles with a ratio of lactic acid to glycolic acid of 50:50 (ie, batch 8 in Table 1) was given to the peritoneal cavity. Injected. The 50:50 microparticles and the 75:25 microparticles had the same size (diameter 3 μm) but different Tg. Tg determines the polymer chain motion, that is, the motion that occurs at temperatures above Tg but not below Tg. The movement of the polymer segment causes agglomeration and adhesion of the microparticles. Thus, microparticles with a lactic acid to glycolic acid ratio of 50:50 and a Tg lower than body temperature (ie 30 ° C. to 37 ° C.) will form aggregates after being administered intraperitoneally, but the ratio of lactic acid to glycolic acid is Microparticles having a Tg higher than body temperature at 75:25 (ie 53 ° C.) do not form agglomerates. The effective diameter increases due to the aggregation of the fine particles. Microparticles with a ratio of lactic acid to glycolic acid of 75:25 contained acridine orange (green fluorescence), whereas microparticles with a ratio of lactic acid to glycolic acid of 50:50 contained rhodamine (red fluorescence).

After injection, the mice were euthanized and the diaphragm excised and rinsed with water. Half of the diaphragm was frozen and the frozen sections were examined by fluorescence microscopy. The other half was fixed with formalin and analyzed using a scanning electron microscope. According to both analyses, the microparticles in the lymphatic vessels inside the diaphragm were small at 1 hour but considerably increased at 24 hours. This indicates that microsphere drainage into the lymphatic system increases with time. In both 1-hour and 24-hour samples, microparticles with a 75:25 ratio of lactic acid to glycolic acid were present in lymphatic vessels about 3 times more than microparticles with a ratio of lactic acid to glycolic acid of 50:50. . A similar finding was found in the mediastinal lymph nodes, confirming that the lymphatic clearance of microparticles with a 50:50 ratio of lactic acid to glycolic acid was lower.

Using fluorescent microparticles encapsulating preferential retention rhodamine of PLGA microparticles in the peritoneal cavity, localization and arrangement of PLGA microparticles in the peritoneal cavity were examined after intraperitoneal administration. The configuration was compared with the configuration of rhodamine in solution. Rhodamine encapsulated microparticles (diameter 3 μm, ratio of lactic acid to glycolic acid 50:50) are distributed throughout the intratraperitoneal cavity early (eg after 15 minutes) and subsequently (eg 24 hours later) And at the time of 96 hours) It was localized in the mesentery, omentum and diaphragm. In contrast, rhodamine in solution was widely distributed throughout the peritoneal cavity at 15 minutes, but the fluorescence corresponding to rhodamine was no longer observed in the peritoneal cavity after 24 hours. This data indicates that the particles preferentially stayed in the abdominal cavity compared to the solution.

Preferential localization of PLGA microparticles in peritoneal tumor nodules Localization and placement of PLGA microparticles in the peritoneal cavity after intraperitoneal administration was investigated using fluorescent microparticles labeled with acridine orange. These particles had a diameter of 3 μm or less and a ratio of lactic acid to glycolic acid of 50:50. A cell suspension of human pancreatic Hs766T cells was implanted intraperitoneally into mice. On day 21 or when the disease was late (median survival was 24 days), a single dose of microparticles labeled with acridine orange was administered intraperitoneally. On day 24, the animals were anesthetized, the abdominal cavity was opened, and the microparticle distribution was assessed by fluorescence. The left panel of FIG. 1 shows the distribution of tumor nodules within the abdominal cavity. Tumors were mainly localized in the mesentery on the retina and diaphragm. The right panel of FIG. 1 shows the localization of green fluorescent isothiocyanate fluorescein (FITC) labeled microparticles in tumor nodules seeded in the peritoneum. Similar experiments were performed using rhodamine or acridine orange to label the same PLGA microparticles with similar results. In contrast, administration of these fluorescent dyes in solution (ie, not bound to microparticles) did not show localization in tumor tissue, which is a characteristic characteristic of microparticles It is shown that. This property is advantageous because it provides targeting of active tumors.

Based on these findings, the abstract applicants preferentially retain drugs encapsulated in PLGA microparticles in the peritoneal cavity compared to drugs in solution (for example, those not related to particles), the ratio of lactic acid to glycolic acid. 50:50 microparticles are slowly discharged from the abdominal cavity by lymphatic drainage, and small particles (diameter 3 μm or less) are more evenly distributed in the abdominal cavity than large particles (diameter 30 μm or less) PLGA particles with lower Tg are preferentially localized in the peritoneal cavity than PLGA particles with higher Tg, particle size determines clearance by lymph drainage and / or absorption from the peritoneum, It was concluded that PLGA microparticles (diameter 3 μm or less, ratio of lactic acid to glycolic acid 50:50) are preferentially localized in the tumor nodules.

Example 4
Example 4 of PLGA microparticles encapsulating paclitaxel shows a method for preparing PLGA microparticles encapsulating paclitaxel and the determination of the properties of the PLGA microparticles. PLGA is a copolymer obtained by copolymerizing lactic acid or lactide and glycolic acid or glycolide at various ratios. This example further demonstrates the effect of microparticle properties on drug release from the microparticles and helps to find PLGA microparticles that give the desired drug release rate. Paclitaxel was used as an example of a drug encapsulated in microparticles. Similar procedures can be used to encapsulate other drugs in these microparticles.

The Applicant prepared PLGA microparticles encapsulating paclitaxel using an emulsion / evaporation method. Briefly, in the water-in-oil-in-water (W / O / W) double emulsion method, PLGA and paclitaxel were codissolved in 5 ml of methylene chloride. . The solution was emulsified in 1 ml of water for 30 seconds by homogenization and 20 ml of 1% polyvinyl alcohol (PVA) solution was added. In the oil-in-water (O / W) emulsion method, the drug-polymer solution was added directly to 20 ml of 1% PVA solution. In either method, the resulting emulsion was diluted in 500 ml of 1% PVA solution preheated at 38 ° C. and continuously stirred until the solvent evaporation was complete. After 30 minutes, the microparticles were collected by centrifugation, washed 3 times with water and lyophilized.

  Microparticles were dissolved in acetonitrile to determine drug loading. The internal standard cephalomannine (100 μl of 20 ug / ml methanol) was added. The mixture was dried under a stream of air. The rest was reconstituted in 0.1 ml acetonitrile and 0.1 ml water was added. After centrifugation, the paclitaxel concentration of the supernatant was analyzed using high performance liquid chromatography. A reference sample consisting of a mixture of blank PLGA microspheres and a known amount of paclitaxel was similarly treated. The ratio of the paclitaxel concentration in the microparticle supernatant encapsulating the drug to the paclitaxel concentration in the reference sample indicated drug loading.

The surface morphology and internal structure of PLGA microparticles encapsulating characterization paclitaxel were examined using a scanning electron microscope. The microparticles were spherical and the surface was smooth. The microparticles prepared using the O / W method (batch 2) showed a packed internal structure of the same structure, whereas the microparticles prepared using the O / W / O method (batch 3) were porous multi-compartments. The internal structure is shown. Table 1 shows the properties of the fine particles encapsulating paclitaxel.

The specific applicant for PLGA microparticle properties that yields the desired drug release rate is the release and microparticle properties of paclitaxel in 0.1% Tween 80 phosphate buffered saline (pH 7.4) at 37 ° C. Evaluated the relationship. The results outlined below show that paclitaxel release rate and range from microparticles can be fine tuned by changing the properties of the microparticles.

The rate and range of effect release of PLGA particle size was inversely proportional to the size of the microparticles. For example, for microparticles prepared with a 50:50 molecular ratio of lactic acid to glycolic acid, Batch 8 with a diameter of 4 μm showed higher release than Batch 6 with a larger diameter of 30 μm. Similarly, batch 1, which differs only in size from batch 2 (diameter 3 μm or less and 60 μm or less), showed more rapid release. The smaller microparticles had a larger surface area to volume ratio than the larger microparticles. Increasing the surface area increased the exposure of paclitaxel to aqueous media and increased the initial burst. In addition, the smaller the microparticles, the shorter the diffusion path and the more rapid drug release and matrix degradation.

Effect of internal structure of PLGA microparticles Porous microparticles having a multi-compartment structure exhibited a faster release rate than microparticles having a packed structure. This is probably due to the shorter diffusion path length for microparticles partitioned into multiple compartments. For example, microparticles with a lactic acid / glycolic acid ratio of 50:50 as a carrier and a multi-compartment internal structure showed a greater initial release compared to microparticles with a packed internal structure (ie, Batch 7 and Batch 6). ). Similarly, the ratio of lactic acid to glycolic acid as a carrier is 75:25, and microparticles with multi-compartment internal structure release faster than microparticles with packed internal structure (ie, batch 3 and batch 2).

Effect of inherent viscosity of PLGA microparticles The inherent viscosity is determined by the polymer molecular weight but is inversely proportional to the release rate. For example, batch 3 and batch 7 were prepared using W / O / W, but the particle size was the same. Batch 3 showed higher inherent viscosity, but the release of paclitaxel was 3.5 times slower.

Effect of PLGA microparticle polymer composition Microparticles prepared with a 50:50 ratio of lactic acid to glycolic acid have a faster release rate than microparticles prepared with a lactic acid to glycolic acid ratio of 75:25 showed that. This is probably due to the fact that microparticles with a 50:50 ratio of lactic acid to glycolic acid are more non-crystalline (ie, less crystalline). Due to the low water solubility, fluid diffusion into and out of the microparticles plays a role in the release of paclitaxel from the microparticles. The decrease in crystallinity increases the diffusion of fluid into the microparticles. Enhanced fluid diffusion also accelerates polymer degradation and makes drug release faster (Alonso, etal., Vaccine, 12: 299-306, 1994).

Example 5
In Example 5 of PLGA microparticles encapsulating paclitaxel that penetrates the tumor, one formulation releases paclitaxel quickly (eg, 70% within 24 hours) to induce apoptosis, thereby causing PLGA microparticles to enter the tumor. The use of a mixture of microparticle formulations with different drug release profiles to enhance permeability is shown.

Permeation of microparticles in peritoneal tumors Human Hs766T pancreatic xenograft tumor cells were implanted into the peritoneal cavity of mice. When the intraperitoneal tumor was established (day 21), the animals were treated with a single microparticle of 3 μm or less in diameter and encapsulating paclitaxel labeled with acridine orange so that particle penetration into the tumor could be easily detected. These microparticles released paclitaxel quickly (70% at 24 hours), thereby inducing apoptosis, which also promoted microparticle penetration into the tumor. Another group of animals was treated with blank microparticles dispensed using the same method without paclitaxel. On day 24, net and adherent tumors were collected. The net is a mesenchyme that separates the abdominal and retroperitoneum and is the site of tumor concentration in mice and human patients. Tumors were frozen and cut. Particle penetration into the tumor at different depths was determined by fluorescence microscopy that visualized particles labeled with acridine orange. The results are shown in FIG. Particle penetration was extensive and the distribution was uniform across solid tumors. In contrast, in tumors from animals treated with blank microparticles that did not receive tumor priming treatment with paclitaxel, the microparticles remained around the tumor.

Paclitaxel Concentration in Peritoneal Tumors Applicants compared the concentration of paclitaxel in solid tumors located in the net after intraperitoneal instillation of microparticles encapsulating paclitaxel (Batch 8) or Taxol®. The peak concentration of paclitaxel derived from microparticles was significantly higher (13 μg / g) and reached a later time (ie 3 days) compared to the commercial taxol formulation (3.2 μg / g, reached in 24 hours). These concentrations are the sum of free drug and incorporated drug. The AUC showing total drug exposure was determined using the linear trapezoidal rule, but 16 times higher than the AUC for microparticles (82 vs. 5 μg-day / g), and thus significant tumor targeting of PLGA microparticles encapsulating paclitaxel It showed the advantages of directing.

Example 6
Biological Activity of PLGA Microparticles Encapsulating Paclitaxel This example shows that PLGA microparticles encapsulating paclitaxel are biologically active under in vitro and in vivo conditions. Biological activity against paclitaxel remains unchanged after encapsulation into microspheres. In addition, PLGA microspheres encapsulating paclitaxel showed superior in vivo efficacy to commercial Taxol® formulations due to their tumor targeting properties and persistent retention in the tumor.

In vitro bioactivity In vitro bioactivity was investigated using human ovarian SKOV3 cancer cells. Drug effects were measured using the sulforhodamine B assay (Au, et al., Cancer Chemother. Pharmacol., 41: 69-74, 1997). The study shows the concentration of free paclitaxel in aqueous solution (ie, paclitaxel dissolved in medium) and PLGA microparticle formulation encapsulating 4 paclitaxel (ie, batches 2, 4, 6, 8 in Table 1) after 96 hours of treatment − The reaction curves were compared. The order of bioactivity of the four formulations is the same as the order of drug release rate, and the more active is the immediate release formulation. For example, Batch 8 which released 81.5% in 96 hours is the most active, followed by Batch 6 which released 28.5% and Batch 4 which released 10.9%. Batch 2, which released 0.25%, had no cytotoxic effect. The concentrations required for 50% cytotoxicity were 5.0 nM, 18.5 nM, 108 nM, 1000 nM or higher, respectively.

In vivo bioactivity Applicant has compared the antitumor activity of paclitaxel PLGA microparticles with the antitumor activity of commercially available Taxol® formulations. The dose was 120 mg / kg paclitaxel equivalent for microparticles and 40 mg / kg for Taxol®. At these doses, these two formulations were equally toxic, resulting in about 10% weight loss in 2 days, but the animal's weight subsequently recovered. The microparticles consisted of three formulations, one that quickly released paclitaxel (70.5% batch 8 in one day) and two that slowly released paclitaxel (72.6% batch in 49 days). 28.7% batch at 6 and 49 days 4). The particle properties are described in detail in Example 2. The results show a significant survival advantage of the microparticles. Median survival was 47 days in the vehicle-treated control group, 85.5 days in the Taxol® group, and 115.5 days in the microparticle group (Taxol® and microparticles). Difference from particles, p <0.01). Note that this study was conducted in late stage disease (4 weeks after transplantation) and used a combination of two sustained release microparticles of different sizes (about 3-4 μm and about 30 μm).

  Similar results were obtained from animals with peritoneal advanced pancreatic cancer. Intraperitoneal human pancreatic Hs766T tumors were transplanted into mice. Treat the animal with saline, Taxol® (60 mg / kg), or paclitaxel particles (combination of immediate release formulation and two sustained release formulations) 10 days after tumor establishment and after tumor implantation did. Median survival was 24 days, 33 days, and 57 days, respectively. Blank microparticles showed data similar to the saline control.

Plasma and tissue concentrations after administration of the two formulations Paclitaxel concentrations in plasma and intraperitoneal tissues were determined using a high performance liquid chromatography assay, and AUC (drug blood from 0 to 168 hours using a linear trapezoidal rule. The area under the medium concentration-time curve) was determined. The results are shown in Table 2. It can be seen that administration of paclitaxel encapsulated in PLGA microparticles increases the concentration of peritoneal tissue by 2.5 to 6 times compared to Taxol (registered trademark). At the same time, the increase in plasma concentration is only 1.5 times, indicating preferential tissue targeting by using paclitaxel microparticles.

  In this study, only one dose of isotoxic microparticles and Taxol® was used. By this criterion, the dose equivalent to paclitaxel for microparticles was higher than the dose for Taxol®. This is because the sustained release of the drug reduced the dose intensity, thus reducing toxicity and increasing the maximum tolerated dose. In this group, repeated administration of Taxol® may have improved treatment outcome, but repeated administration was not necessary for microparticles encapsulating paclitaxel. The latter shows one advantage of microparticles that reduces the need for frequent administration.

Example 7
Preparation and characterization of gelatin nanoparticles encapsulating paclitaxel This example shows the preparation and characterization of gelatin nanoparticles encapsulating paclitaxel.

Preparation of gelatin nanoparticles encapsulating paclitaxel Using several formulations of gelatin with different bloom strengths (75-100 bloom, 175 bloom, 300 bloom), nanoparticles were prepared using a desolvation method (Oppenheim, RC , Int. J. Pharm., 8: 217-234, 1981). Gelatin (200 mg) was dissolved in 10 ml water containing 2% Tween 20. The solution was heated to 40 ° C. with constant stirring at 300 rpm. To this solution was slowly added 2 ml of a 20% aqueous solution of sodium sulfate, and 1 ml of isopropanol containing 2 mg of paclitaxel was added. A second aliquot of sodium sulfate solution (5.5-6 ml) was added until the solution turned cloudy indicating the formation of gelatin aggregates. Thereafter, about 1 ml of distilled water was added until the solution became clear. An aqueous solution of glutaraldehyde (25%, 0.4 ml) was added to crosslink the gelatin. Sodium metabisulfite solution (12%, 5 ml) was added after 5 minutes to stop the crosslinking process. After 1 hour, the crude product was purified on a Sephadex G-50 column. The nanoparticle-containing fraction was lyophilized for 48 hours in a vacuum lyophilizer.

Preparation of gelatin nanoparticles coated with polylysine and encapsulating paclitaxel Nanoparticles were prepared using a method similar to nanoparticles without coating . At a later stage of crosslinking after nanoparticle formation, a weight of polylysine equal to about 5% to about 10% of the weight of gelatin was added, resulting in nanoparticles coated with polylysine. The purification step is the same as that used for the nanoparticles without the coating described above.

Determination of paclitaxel loading in gelatin nanoparticles Nanoparticles encapsulating 2 mg paclitaxel were dispersed in 0.5 ml phosphate buffered saline (PBS) and metabolized by 0.5 ml pronase (1 mg / ml in PBS) Digest at 37 ° C. in a shaker. After about 1 hour or when a clear solution was obtained, an internal standard substance cephalomanine (50 μl of 20 μg / ml of methanol) was added, and each was extracted with 2 volumes of 3 ml of ethyl acetate. The ethyl acetate layers were pooled, dried under a stream of air and reconstituted in acetonitrile. The paclitaxel concentration of the extract was compared with the concentration of the reference sample to determine paclitaxel loading. A reference sample consisting of a mixture of blank gelatin nanoparticles and a known amount of paclitaxel was processed as described for nanoparticles.

The physical properties of different formulations of paclitaxel nanoparticles are shown in Table 3. The yield of nanoparticles ranges from 40 to 82% and decreases with increasing molecular weight of gelatin. The actual drug load was 33-78% of the theoretical load.

  Nanoparticles prepared using high molecular weight gelatin formed large aggregates, with aggregate diameters ranging from 10 to 30 μm. When these aggregates were removed during the column chromatography purification step, the yield and drug uptake efficiency were low. Low uptake efficiency was also seen in nanoparticles prepared using low molecular weight gelatin. Optimal and higher yield and uptake efficiency were achieved by using medium molecular weight gelatin (175 bloom). In a later study, nanoparticles encapsulating paclitaxel were prepared using 175 bloom gelatin.

Characterization of nanoparticles encapsulating paclitaxel A mixture of gelatin nanoparticles and distilled water (less than 50 μl) is placed on foil paper, dried, coated with gold, Philips XL30 scanning electron microscope (SEM) Observed with. Over 1000 nanoparticles were examined using SEM images taken from 4 to 6 fields for particle size distribution. The yield of production was calculated from the weight of lyophilized gelatin nanoparticles and expressed as a percentage of the starting weight of gelatin. SEM results indicated that the nanoparticles were hemispherical and the average size was 600 to 1,000 nm.

  A wide-angle X-ray of pure paclitaxel, a mixture of paclitaxel (2% by weight) and blank gelatin nanoparticles, and nanoparticles encapsulating paclitaxel (1.62% loading) using a Scintag PAD-V diffractometer A diffraction (WAXD) spectrum was obtained. The sample was scanned from 5 ° to 60 ° at a scan rate of 1 ° per minute. The WAXD results showed a sharp peak in the X-ray diffraction spectrum for free paclitaxel and for the mixture of free paclitaxel and blank gelatin nanoparticles, but not for gelatin nanoparticles encapsulating paclitaxel. There wasn't. This indicates that paclitaxel incorporated into the nanoparticles is in an amorphous state rather than a crystalline state. Since the amorphous state has a high dissolution rate, the amorphous state is desirable.

Release of paclitaxel from gelatin nanoparticles Paclitaxel nanoparticles (12 mg) were dispersed in 100 ml PBS and incubated at 37 ° C. A continuous sample (1 ml) was removed and centrifuged at 40,000 rpm for 15 minutes using a Beckman L-70 ultracentrifuge. 400 μl of the nanoparticle-free supernatant was removed and extracted twice with 3 ml of ethyl acetate. The paclitaxel concentration of the ethyl acetate extract was analyzed by high performance liquid chromatography.

  The results of the absorption study indicated that about 4.5% of the total amount of paclitaxel encapsulated in the nanoparticles was absorbed on the nanoparticles. Release of paclitaxel from the nanoparticles to PBS was rapid, with 55%, 87%, and 92% being released after 15 minutes, 2 hours, and 3 hours at 37 ° C, respectively.

Human RT4 bladder transitional bladder cancer cells were obtained from the bioactive ATCC of nanoparticles encapsulating paclitaxel (the American Type Culture Collection, Rockville, MD), 9% fetal bovine serum, 2 mM L-glutamine, 90 μg The cells were cultured at 37 ° C. in a humidified atmosphere of 5% CO ₂ in cultured air in McCoy's medium supplemented with / ml gentamicin and 90 μg / ml cefotaxime sodium. Cells were harvested from subconfluent cultures using trypsin and resuspended in fresh medium. The viable cell rate was determined by the trypan blue dye exclusion method, and cells with a viable cell rate of 90% or more were used. Cells were seeded in 96-well microtiter plates (2,000 cells / well or less) to allow cells to adhere to the plate surface for 24 hours. Applicants have already shown that paclitaxel produces immediate and delayed cytotoxicity (Au, et al., Cancer Res., 58: 2141-2148,1998). To assess immediate effects, cells were incubated for 48 and 96 hours at the same paclitaxel dose with 0.2 ml medium containing paclitaxel in water (called free paclitaxel) or nanoparticles encapsulating paclitaxel. . Drug effect was measured immediately after treatment. To assess the delayed effect, the cells were similarly treated for 15 minutes and 2 hours, washed once with PBS and then incubated in drug-free medium for a total of 96 hours, at which time the drug effect was measured.

For free drug, a stock solution of paclitaxel was prepared by first dissolving paclitaxel in ethanol and then serially diluting with media. The final ethanol concentration was 0.1% or less. The number of cells remaining after treatment was determined using the sulforhodamine B assay (Au, et al., Cancer Chemother. Pharmacol., 41: 69-74, 1997). Sigmoidal concentration-response curves were analyzed using non-linear regression analysis to obtain drug concentrations that produced 50% inhibition (IC ₅₀ ).

With regard to immediate effects, treatment with either free paclitaxel or paclitaxel incorporated into nanoparticles gave a maximum inhibition of 60% at 48 hours and 84% at 96 hours. The increase in maximal paclitaxel cytotoxicity with treatment time is consistent with the applicant's previous observations (Au, et al., Cancer Res., 58: 2141-2148, 1998). For 48 hours and 96 hours of treatment, the IC ₅₀ value of free paclitaxel is 11.0 ± 0.4 nM and 4.0 ± 0.4 nM, respectively, and the IC ₅₀ value of nanoparticles encapsulating paclitaxel is 9.6 respectively. There was ± 1.1 nM, 4.0 ± 0.3 nM paclitaxel equivalent (both formulations Mean ± SD of 3 experiments). There was no significant difference in IC ₅₀ values for these two formulations (unpaired t-test statistics; p = 0.15 at 48 hours, p = 0.71 at 96 hours).

For late effects (ie, effects measured at 96 hours), 15 minutes and 2 hours of treatment with either free paclitaxel or paclitaxel incorporated into nanoparticles gave a maximum inhibition of 74% to 85%. It was. For the 15 minute and 2 hour treatments, the IC ₅₀ value of free paclitaxel is 156.7 ± 6.6 nM, 33.0 ± 4.8 nM, respectively, and the IC ₅₀ value of nanoparticles encapsulating paclitaxel is 165.7 ± 33, respectively. The paclitaxel equivalent of 5 nM, 31.4 ± 1.8 nM (both formulations Mean ± SD of 3 experiments). There was also no significant difference in the corresponding IC ₅₀ values for the two formulations (unpaired t-test statistics; p = 0.70 for 15 minutes, p = 0.64 for 2 hours). This data shows the rapid release of paclitaxel from the nanoparticles and that nanoparticles encapsulating paclitaxel are as effective as free paclitaxel dissolved in ethanol and water (in the absence of cremophor) .

Example 8
Example 8 of delivery of high paclitaxel concentration to the bladder wall after intravesical instillation of gelatin nanoparticles encapsulating paclitaxel is a case where gelatin nanoparticles encapsulating paclitaxel is highly concentrated in the bladder tissue when instilled into the bladder cavity To deliver.

  The determination of delivery of paclitaxel to the bladder tissue has been described by other literature (Wientjes, et al., Cancer Res., 51: 4347-4354,1991; Song, D., after instillation of gelatin nanoparticles encapsulating paclitaxel. et al., Cancer Chemother. Pharmacol., 40: 285-292, 1997). Briefly, a solution of gelatin nanoparticles encapsulating paclitaxel (containing 1 mg paclitaxel in a total weight of 250 mg) was instilled into the anesthetized dog bladder over 2 hours. The bladder was then excised, snap frozen, and cut into 40 μm sections parallel to the urothelial surface using a cryotome. After washing, the paclitaxel concentration of the frozen tissue sample was analyzed using a high performance liquid chromatography assay as described above (Song and Au, J. Chromatogr., 663: 337-344, 1995). The results show that the concentration of paclitaxel in the bladder wall declines from about 50 μg / g (approximately equal to 60 μg / g) on the urothelium, the inner surface of the bladder, to about 1 μg / g at a depth of 500 μm away from the urothelium. This indicates that the tissue depth of 500 μm or more was kept relatively constant at about 0.5 μg / g. The urothelium concentration was approximately 20 times higher than the unbound concentration in urine, indicating good penetration of paclitaxel into the bladder wall. These tissue concentrations also exceeded paclitaxel concentrations that produced cytotoxicity in human bladder RT4 cancer cells (ie -33 nM with 2 hours of treatment, see Example 1).

  In other dogs, the tissue retention of paclitaxel in gelatin nanoparticles after administration was analyzed for bladder tissue concentration 22 hours after the end of 2 hours of treatment and after draining the solution (ie 24 hours after dosing). Examined. The paclitaxel concentration was 0.14 μg / g (about 165 nM) in the urothelium and slowly declined to 0.01 μg / g at a depth of 500 μm. This data indicates a significant retention of paclitaxel in bladder tissue. The half-life of drug clearance was estimated to be 150 minutes, which is more than 30 times longer than the half-life of mitomycin C in human bladder tissue of 5 minutes (Wientjes, et al., Cancer Res., 53: 3314-3320 , 1993).

Example 9
Efficacy of paclitaxel-encapsulated gelatin nanoparticles in tissue cultures from diseased canine bladder tumors Bladder tumors were obtained by bladder transurethral resection from 3 affected dogs diagnosed with bladder cancer. Tumors were cut to 1 mm and cultured on collagen gel as tissue culture (Au, et al., Cancer Chemother. Pharmacol., 41: 69-74, 1997) and treated with nanoparticles encapsulating paclitaxel for 2 hours. did. IC ₅₀ values for inhibition of bromodeoxyuridine labeling expressed as paclitaxel equivalents were 2.2 μM for tumors from one dog and 10 μM or more for tumors from the remaining two dogs.

A tripartite comparison of IC ₅₀ values, drug concentrations in bladder tissue (see Example 8), and clinical outcomes of these three dogs (see Example 10) show that the most sensitive tumor (ie, the lowest IC _50). Value) would have been administered enough drug to respond well to treatment with nanoparticles encapsulating paclitaxel and produce a therapeutic benefit when the tumor size was reduced by 50% or more It is shown that. In contrast, the IC ₅₀ values of the remaining two dogs exceeded the reachable bladder tissue concentration, and in these animals the tumors showed progressive disease (ie an increase in size of more than 50%) (Helfand, et al , J. Am. Anim. Hosp. Assoc., 30: 270-275, 1994).

This example demonstrates the correlation between bladder tumor sensitivity to paclitaxel formulated in gelatin nanoparticles and in vitro and in vivo results. Paclitaxel's IC ₅₀ value in human superficial bladder tumors is lower than in dogs, and bladder tumors in human patients who are candidates for direct intravesical instillation are superficial and beyond the mucosal layer of the bladder wall It is worth noting that it does not penetrate. The IC ₅₀ value for the human bladder tumor previously obtained by the applicant is 1.2 μM for the T0 and T1 bladder tumor stages obtained from human patients (Au, et al., Cancer Chemother. Pharmacol., 41: 69-74,1997). This suggests that clinical results in human patients are better than those in dogs.

Example 10
Efficacy of paclitaxel-encapsulated gelatin nanoparticles in dogs with bladder cancer Dogs with transitional cell carcinoma of the bladder (TCC) and no evidence of metastasis are eligible. Under general anesthesia, gelatin nanoparticles encapsulating 1 mg of paclitaxel dissolved in 20 ml of physiological saline suspension were administered into the bladder once a week for 3 weeks via a Foley catheter. The dosage was 1 mg paclitaxel in 250 mg gelatin. All patients received prophylactic antibiotics and deracoxib. Blood and urine samples were taken before and during the 2 hour treatment period. Urine and tissue paclitaxel concentrations were analyzed by column switching HPLC. Tumor response was monitored using abdominal ultrasonography.

  Six dogs were treated, four of which had not been previously treated. Plasma concentrations were below the HPLC detection limit at all time points. Median urine initial and final concentrations were 27.51 ± 8.59 g / ml (n = 16) and 11.16 ± 8.63 g / ml (n = 15), respectively. The concentration of unbound paclitaxel remained constant at 0.8-1 μg / ml, which is approximately the maximum value of paclitaxel water solubility at body temperature. The total response was 2 partial responses, 2 stable diseases (ie not partial response but not progressive disease) and 2 progressive disease. The definition of clinical outcome or outcome is described in Example 9. There was no evidence of systemic drug absorption or toxicity. The objective response rate of affected dogs is 2/6 (33%), which is higher than the response rate reported in the literature (12.5%) for intravesical treatment with other chemotherapeutic drugs (Range 0-20%, average 12.5%; Mutsaers, et al., J. Vet. Intern. Med., 17: 136-144, 2003).

Example 11
We examined the benefits of kidney targeting with gelatin nanoparticles by comparing the distribution of gelatin nanoparticles encapsulating kidney paclitaxel with gelatin nanoparticles after intravenous administration and the distribution of commercially available Taxol® formulations . Gelatin nanoparticles were prepared and administered intravenously to mice over 1 minute from the tail vein. The paclitaxel dose was 10 mg / kg. The animals were euthanized after 24 hours. Organs were excised, homogenized, extracted and analyzed for paclitaxel concentration using high performance liquid chromatography. These procedures were performed as described in Example 7. Nanoparticles (diameter 664 nm, load 4%) released paclitaxel rapidly (70% at 24 hours at 37 ° C.). The total blood concentration of paclitaxel (free and bound) decreased with a major half-life of 14 hours. Paclitaxel was distributed and retained in the organs at the highest levels in the liver, small intestine and kidney (Table 4). The ratio of tissue to blood concentration was liver> small intestine> kidney >> large intestine> spleen = stomach>lung> heart. The tissue distribution of Taxol (registered trademark) is in the order of liver> small intestine> large intestine>stomach>lung>kidney>spleen> heart, and the distribution is different. Selective retention was determined as a ratio of post-administration tissue concentration in nanoparticles or as taxol after normalization to plasma concentration (Table 4). Selective retention in the kidney was 7.86 times, highest in all organs. After administration in nanoparticles, the terminal half-life of paclitaxel in the kidney was 13.7 hours. The administration as Taxol® was 1.94 hours. This data indicates that gelatin nanoparticles preferentially stay in the kidney.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are to be construed as being included within the scope of the claims.

Tumor targeting with PLGA microparticles Intraperitoneal human Hs766T pancreatic xenograft tumors were transplanted into mice. After tumor establishment (day 21), one dose of fluorescein isothiocyanate (FITC) labeled particles was administered intraperitoneally. FITC exhibits green fluorescence under ultraviolet light. On day 24, the animals were anesthetized to expose the abdominal cavity. The left panel shows a picture under room lighting. Note the multiple tumors that appear in the peritoneum as white or yellowish irregularly shaped nodules. The right panel shows a photograph under ultraviolet light. Note the localization of the particles on the tumor nodules, indicated by green fluorescence. Tumor priming with paclitaxel promoted microparticle penetration and distribution in solid tumors Intraperitoneal human SKOV3 ovarian xenograft tumors were transplanted into mice. After tumor establishment (day 42), PLGA microparticles (diameter 3 μm or less, ratio of lactic acid to glycolic acid 50:50) were injected intraperitoneally into animals. These particles are labeled with a green fluorescent compound (acridine orange). Animals were given either microparticles encapsulating immediate release paclitaxel containing a 10 mg / kg amount of paclitaxel or blank microparticles without paclitaxel. Three days later, the animals were anesthetized and the tumor attached to the net was frozen and cut. A fluorescence microscope image showing the position of the particles was superimposed on a phase contrast microscope image in which the outline of the tumor was drawn. The left panel shows a representative section of a tumor obtained from an animal that received tumor priming with paclitaxel, and the right panel shows a section of a tumor obtained from an animal given blank microparticles without paclitaxel. Show. Microparticles can be identified as green fluorescent circles. Note that the distribution of microparticles is more uniform and the amount of microparticles is greater throughout the tumor treated with tumor-primed paclitaxel treatment. In contrast, in tumors that were not treated with paclitaxel, the microparticles were confined around the tumor. The photo is magnified 200 times. The white line indicates a distance of 100 μm.

Claims (22)

A composition for intraperitoneally delivering an antitumor drug to a patient with a peritoneal tumor ,
(A) Lactic acid glycolic acid copolymer (PLGA) microparticles having a particle size of 3 to 5 μm and a glass transition temperature lower than the patient's body temperature, containing paclitaxel and forming a rapid release formulation of paclitaxel;
(B) a microparticle or nanoparticle containing an antitumor drug and forming a sustained-release preparation of the antitumor drug;
(C) a composition comprising a pharmaceutically acceptable carrier.   2. The composition of claim 1, wherein the PLGA microparticles release at least 50 nM paclitaxel over 1 hour or less.   The composition of claim 1, wherein the paclitaxel is provided in an amount sufficient to reduce the density of tumor epithelial cells by 20% or more.   The composition of claim 1, wherein the paclitaxel is provided in an amount sufficient to reduce the density of tumor epithelial cells by 20% or more within 16 to 24 hours after administration.   The composition of claim 1, wherein the paclitaxel is provided in an amount sufficient to induce apoptosis in 10% or more of the tumor epithelial cells.   2. The composition of claim 1, wherein the paclitaxel is provided in an amount sufficient to induce apoptosis in 10% or more of tumor epithelial cells within 16 to 24 hours after administration.   The composition according to claim 1, wherein the antitumor agent is one or more of paclitaxel or doxorubicin.   The composition according to claim 1, wherein the anti-tumor drug is one or more of a chemotherapeutic drug or an antibiotic. A kit for the treatment of peritoneal tumors,
(A) Lactic acid glycolic acid copolymer having a particle diameter of 3 to 5 μm and a glass transition temperature lower than body temperature in a pharmaceutically acceptable carrier, containing paclitaxel, and forming an immediate-release preparation of paclitaxel (PLGA) microparticles;
(B) a microparticle or nanoparticle comprising an antitumor drug in a pharmaceutically acceptable carrier and forming a sustained release formulation of the antitumor drug;
(C) A kit comprising the immediate-release preparation and the instructions for use of the sustained-release preparation for tumor treatment. 10. The kit of claim 9 , wherein the PLGA microparticles release at least 50 nM paclitaxel over 1 hour. The kit according to claim 9 , wherein the single or plural nanoparticle or microparticle preparation contains both the immediate release preparation (a) and the sustained release preparation (b). The kit according to claim 9 , wherein the antitumor agent is one or more of paclitaxel or doxorubicin. Adapted to deliver paclitaxel and antineoplastic drugs to patients with intraperitoneal tumors,
The one or more nanoparticles that include one or more nanoparticles or microparticles that selectively adhere to the tumor to deliver the antineoplastic agent in the vicinity of the tumor, thereby not selectively adhering to the tumor 2. The composition of claim 1, wherein more of the therapeutic agent accumulates in the tumor as compared to delivery by microparticles. 14. The composition according to claim 13 , wherein the one or more nanoparticles or fine particles are coated with a bioadhesive coating agent to increase tissue adhesion. The bioadhesive coating agent is one or more of polylysine, fibrinogen, partially esterified polyacrylic acid polymer, polysaccharide, pectin, or a mixture of sulfated sucrose and aluminum hydroxide. The composition according to claim 14 . The composition according to claim 1, wherein the PLGA microparticles are particles that adhere to a tumor. Microparticles or nanoparticles forming a sustained-release preparation of the antitumor agent ,
(1) provide drug release followed by slow drug release,
(2) selectively adheres to tumor tissue;
(3) concentrate on the abdominal cavity,
The composition according to claim 1, which has the following characteristics. The composition according to claim 1, wherein the microparticles or nanoparticles constituting the sustained-release preparation of the antitumor drug are bioadhesive and coated. The microparticles or nanoparticles that form the sustained-release preparation of the antitumor agent are:
A PLGA microparticles, and the ratio of lactic acid and glycolic acid is about 50:50, has an average diameter of 30 [mu] m, and a glass transition temperature lower than body temperature of the subject, and containing 4% paclitaxel, The composition according to claim 1 , characterized in that it releases 20% at the initial burst drug release rate on day 1 followed by 70% of the total cumulative release in 7 weeks with a slower release. The microparticles or nanoparticles that form the sustained-release preparation of the antitumor agent are:
A PLGA microparticles, and the ratio of lactic acid and glycolic acid is about 75:25, has an average diameter of 3 to 5 [mu] m, and a high glass transition temperature than the body temperature of the subject, containing 4% of paclitaxel 2. The composition of claim 1 , wherein 5% is released at an initial burst drug release rate on day 1, followed by 30% of the total cumulative release in 7 weeks by slower release. object. The microparticles or nanoparticles that form the sustained-release preparation of the antitumor agent are:
A PLGA microparticles, and the ratio of lactic acid and glycolic acid is about 50:50, and containing 4% paclitaxel, to claim 1, wherein the drug release rate is 70% in one day The composition as described . 22. A composition according to any of claims 19 to 21 , further comprising a release enhancer such as one or more of Tween 20, Tween 80, isopropyl myristate, beta-lactose, or diethyl phthalate.

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