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    • 专利摘要:
    The present invention provides the use of a combination of an mTOR inhibitor such as rapamycin and an antimetabolic antineoplastic agent such as gemcitabine or fluorouracil in the treatment of neoplasia.
    公开号:KR20040007491A
    申请号:KR10-2003-7013090
    申请日:2002-04-05
    公开日:2004-01-24
    发明作者:기봉스제임스조셉쥬니어；듀카트게리；프리슈유르겐헤르만에른스트
    申请人:와이어쓰；
    IPC主号:
    专利说明:

    Antioplastic combinations of rapamycin with gemcitabine or fluorouracil {Antineoplastic combinations such as rapamycin together with gemcitabine or fluorouracil}
    [1] FIELD OF THE INVENTION The present invention relates to anti-neoplastic combinations, more particularly in the treatment of neoplasia with ramipacin 42-esters with mTOR inhibitors (e.g. 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid (CCI- 779)) and the combination of anti-metabolic anti-neoplastic agents.
    [2] Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus, which has been found to have antifungal activity against Candida albicans, both in vitro and in vivo. [References: C. Vezina et al., J. Antibiot. 28, 721 (1975); S.N. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); US Patent No. 3,929,992; And US Pat. No. 3,993,749]. In addition, rapamycin alone (US Pat. No. 4,885,171) or in combination with fishvanyl (US Pat. No. 4,401,653) has been shown to have antitumor activity.
    [3] The immunosuppressive effects of rapamycin are described in FASEB 3, 3411 (1989). In addition, cyclosporin A and FK-506, and other macrocyclic molecules, are also effective as immunosuppressants and have been shown to be useful in preventing transplant rejection [Ref .: FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978); And US Patent 5,100,899]. [References to R. Martel et al., Can. J. Physiol. Pharmacol. 55, 48 (1977) include rapamycin in an experimental allergic encephalomyelitis model, multiple sclerosis model; It is described to be effective in adjuvant arthritis models, rheumatoid arthritis models, and to effectively inhibit the formation of IgE-like antibodies.
    [4] In addition, rapamycin can be used to treat skin diseases such as systemic lupus erythematosus [US Pat. No. 5,078,999], lung inflammation [US Pat. No. 5,080,899], insulin-dependent diabetes mellitus [US Pat. No. 5,321,009], and psoriasis [US Pat. , Intestinal disease [US Pat. No. 5,286,731], smooth muscle cell proliferation and endothelial hypertrophy after vascular injury [US Pat. Nos. 5,288,711 and 5,516,781], Adult T-cell leukemia / lymphoma [European Patent No. 525,960 A1], Ocular Useful for preventing or treating inflammation [US Pat. No. 5,387,589], malignant carcinoma [US Pat. No. 5,206,018], heart inflammatory disease [US Pat. No. 5,496,832], and anemia [US Pat. No. 5,561,138].
    [5] Rapamycin 42-ester (CCI-779) with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid has demonstrated a significant inhibitory effect on tumor proliferation in both in vitro and in vivo models. Ester. The preparation and use of hydroxyesters of rapamycin, including CCI-779, are described in US Pat. No. 5,362,718.
    [6] CCI-779 exhibits growth inhibition properties as opposed to cytotoxic properties and can delay tumor progression time or tumor recurrence time. CCI-779 is considered to have a mechanism of action similar to that of sirolimus. CCI-779 binds to the cytosolic protein FKBP to form a complex, which inhibits the enzyme mTOR (mammalian rapamycin target, also known as FKBP12-rapamycin bound protein [FRAP]). Inhibition of kinase activity of mTOR inhibits G1 signaling and inhibits various signal transduction pathways, including cytokine-stimulated cell proliferation, translation of mRNA for several important proteins that regulate the G1 phase of the cell cycle, and IL-2-induced transcription. Causes inhibition of the progression of the cell cycle from to S. The mechanism of action of CCI-779, which causes blocking of G1 to S phase, is novel in anticancer drugs.
    [7] In vitro, CCI-779 has been shown to inhibit the proliferation of several histologically diverse tumor cells. Central nervous system (CNS) cancer, leukemia (T-cell), breast cancer, prostate cancer, and melanoma were the most sensitive to CCI-779. The compound stopped cell proliferation on G1 of the cell cycle.
    [8] In vivo studies of nude mice have demonstrated that CCI-779 is active against various tumor types of human tumor xenografts. Gliomas were particularly sensitive to CCI-779, and the compounds were active in the orthotopic glioma model of nude mice. Proliferation factor (platelet-derived) -induced stimulation of human glioma cell line in vitro was markedly inhibited by CCI-779. Proliferation of several human pancreatic tumors in nude mice and one of two breast cancer lines studied in vivo was also inhibited by CCI-779.
    [9] The present invention provides the use of a combination of an mTOR inhibitor and an antimetabolic antineoplastic agent as an antineoplastic combination chemotherapy. In particular, these combinations include kidney cancer, soft tissue sarcoma, breast cancer, neuroendocrine tumors of the lung, cervical cancer, uterine cancer, head and neck cancer, glioma, non-small cell lung cell cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma, small cell It is useful for the treatment of lung cancer, ovarian cancer, colon cancer, esophageal cancer, gastric cancer, leukemia, colorectal cancer and unknown primary cancers. The present invention also provides a combination of an mTOR inhibitor and an antimetabolic antineoplastic agent for use as an antineoplastic combination chemotherapy wherein the dose of the mTOR inhibitor or the antimetabolic antineoplastic agent or both is administered It is used in less than pharmaceutically effective amount.
    [10] As used herein, the term "treatment" refers to mTOR inhibitors and anti-metabolism antineoplastics in mammals for the purpose of inhibiting neoplasia growth, eradication of neoplasms, or alleviating mammals of a mammal with a neoplastic disease. By providing an effective amount of the formulation, it is meant to treat the mammal.
    [11] As used in accordance with the present invention, the term "providing" in providing a formulation directly administers the formulation or administers a prodrug, derivative, or analog of one or both components, which forms an effective amount of the formulation in the body. Means.
    [12] mTOR is a rapamycin target in mammals and is also known as FKBP12-rapamycin bound protein [FRAP]. Inhibition of kinase activity of mTOR inhibits various signal transduction pathways, including cytokine-stimulated cell proliferation, translation of mRNA in several key proteins that regulate G1 phase of the cell cycle, and IL-2-induced transcription Causing inhibition of cell cycle progression from G1 to S.
    [13] mTOR regulates the activity of two or more proteins involved in the translation of specific cell cycle regulatory proteins [Burnett, P.E., PNAS 95: 1432 (1998) and Isotani, S., J. Biol. Chem. 274: 33493 (1999). One of these proteins, p70s6 kinase, is phosphorylated by mTOR on serine 389 and threonine 412. Such phosphorylation can be observed in proliferation factor treated cells by western blotting of whole cell extracts of these cells using antibodies specific for phosphoserine 389 residues.
    [14] As used in accordance with the present invention, “mTOR inhibitor” refers to a compound or ligand that inhibits cell replication by inhibiting the phosphorylation of serine 389 of p70s6 kinase by mTOR to block the progression of the cell cycle from G1 to S.
    [15] The following standard pharmacological test methods can be used to determine whether a compound is an mTOR inhibitor as defined herein. Treatment of growth factor stimulated cells with mTOR inhibitors such as rapamycin completely blocks the phosphorylation of serine 389 as demonstrated by Western blot, resulting in a good assay for mTOR inhibition. Whole cell lysates from cells stimulated by proliferation factors (eg IGF1) in culture in the presence of mTOR inhibitors should not show bands that can be labeled with antibodies specific for serine 389 of p70s6K on acrylamide gels. .
    [16] matter:
    [17] NuPAGE LDS Sample Buffers (Novex Cat # NP0007)
    [18] NuPAGE Sample Reducing Agent (Novex Cat # NP0004)
    [19] NuPAGE 4-12% Bis-Tris Gel (Novex Cat # NP0321)
    [20] NuPAGE MOPS SDS Expansion Buffer (Novex Cat # NP0001)
    [21] Nitrocellulose (Novex Cat # LC2001)
    [22] NuPAGE Transfer Buffer (Novex Cat # NP0006)
    [23] Hyperfilm ECL (Amersham Cat # RPN3114H)
    [24] ECL Western Blotting Detection Reagent (Amersham Cat # RPN2134)
    [25] Primary antibody: phospho-p70 S6 kinase (Thr389) (Cell Signaling Cat # 9205)
    [26] Secondary antibody: goat anti-rabbit IgG-HRP conjugate (Santa Cruz Cat # sc-2004)
    [27] Way:
    [28] A. Preparation of Cell Lysates
    [29] Cell lines were grown in optimal basal medium supplemented with 10% fetal bovine serum and penicillin / treptomycin. For phosphorylation studies, cells were subcultured in 6-well plates. After the cells were completely attached, they were serum-deficient. Treatment with the mTOR inhibitor ranged from 2-16 hours. After drug treatment, cells were washed once with PBS (phosphate buffered saline without Mg ++ and Ca ++) and then lysed in 150-200 μl NuPAGE LDS sample buffer per well. The lysates were briefly sonicated and then centrifuged for 15 minutes at 14000 rpm. The lysate was stored at -80 ° C until use.
    [30] After the cells are fully attached, the test procedure can also be progressed by culturing the cells overnight in proliferation medium. The results under both conditions should be the same for the mTOR inhibitor.
    [31] B. Western Blot Analysis
    [32] 1) Add 22.5 μl of lysate per tube to prepare a whole protein sample, and add 2.5 μl of NuPAGE sample reducing agent. The sample is heated at 70 ° C. for 10 minutes. Electrophoresis is performed using NuPAGE gels and NuPAGE SDS buffer.
    [33] 2) The gels are transferred to nitrocellulose membranes using NuPAGE transfer buffer. The membrane is blocked for 1 hour with blocking buffer (Tris buffered saline with 0.1% -Tween and 5% skim milk). The membrane is washed twice with wash buffer (Tris buffered saline with 0.1% -Tween).
    [34] 3) Blots / membranes are incubated overnight at 4 ° C. with P-p70 S6K (T389) primary antibody (1: 1000) in blocking buffer on a rotating plate.
    [35] 4) The blots are washed three times for 10 minutes each with wash buffer and incubated with secondary antibody (1: 2000) in blocking buffer for 1 hour at room temperature.
    [36] 5) After secondary antibody binding, blots were washed three times for 10 minutes each with wash buffer and twice each for 1 minute with tris-buffered saline, followed by chemiluminescence (ECL) detection followed by chemiluminescent film. Expose
    [37] As used in accordance with the present invention, the term "rapamycin" is defined as a specific class of immunosuppressive compounds containing the basic rapamycin nucleus (shown below). Rapamycin of the present invention can be modified chemically or biologically as a derivative of the rapamycin nucleus, but still retains immunosuppressive properties. Thus, the term “rapamycin” includes esters, ethers, oximes, hydrazones, and hydroxylamines of rapamycin, as well as rapamycin wherein functional groups on the rapamycin nucleus have been modified, for example, via reduction or oxidation. The term "rapamycin" also includes pharmaceutically acceptable salts of rapamycin, which can be formed by containing acidic or basic residues.
    [38]
    [39] Rapamycin
    [40] Esters and ethers of rapamycin are esters and ethers of hydroxyl groups at the 42- and / or 31-positions of the rapamycin nucleus, esters and ethers of the hydroxyl groups at the 27-position (after chemical reduction of the 27-ketone) , Oxime, hydrazone and hydroxylamine are the oxime, hydrazone and hydroxylamine of the ketone (after oxidation of the 42-hydroxyl group) at the 42-position of the rapamycin nucleus, and of the 27-ketone of the rapamycin nucleus. Preference is given to oximes, hydrazones and hydroxylamines.
    [41] Preferred 42- and / or 31-esters and ethers of rapamycin are described in the following patents, all of which are incorporated herein by reference: alkyl esters of rapamycin (US Pat. No. 4,316,885); Aminoalkyl esters (US Pat. No. 4,650,803); Fluorinated esters (US Pat. No. 5,100,883); Amide esters (US Pat. No. 5,118,677); Carbamate esters (US Pat. No. 5,118,678); Silyl esters (US Pat. No. 5,120,842); Aminoesters (US Pat. No. 5,130,307); Acetal (US Pat. No. 5,51,413); Aminodiesters (US Pat. No. 5,162,333); Sulfonates and sulfate esters (US Pat. No. 5,177,203); Esters (US Pat. No. 5,221,670); Alkoxyesters (US Pat. No. 5,233,036); O-aryl, -alkyl, -alkenyl and -alkynyl ethers (US Pat. No. 5,258,389); Carbonate esters (US Pat. No. 5,260,300); Arylcarbonyl and alkoxycarbonyl carbamates (US Pat. No. 5,262,423); Carbamate (US Pat. No. 5,302,584); Hydroxyesters (US Pat. No. 5,362,718); Hindered esters (US Pat. No. 5,385,908); Heterocyclic esters (US Pat. No. 5,385,909); gem-disubstituted esters (US Pat. No. 5,385,910); Amino alkanoic acid esters (US Pat. No. 5,389,639); Phosphorylcarbamate esters (US Pat. No. 5,391,730); Carbamate esters (US Pat. No. 5,411,967); Carbamate esters (US Pat. No. 5,434,260); Amidino carbamate esters (US Pat. No. 5,463,048); Carbamate esters (US Pat. No. 5,480,988); Carbamate esters (US Pat. No. 5,480,989); Carbamate esters (US Pat. No. 5,489,680); Hindered N-oxide esters (US Pat. No. 5,491,231); Biotin esters (US Pat. No. 5,504,091); O-alkyl ethers (US Pat. No. 5,665,772); And PEG esters (US Pat. No. 5,780,462). The preparation of these esters and ethers is described in the patents listed above.
    [42] Preferred 27-esters and ethers of rapamycin are described in US Pat. No. 5,256,790, which is incorporated herein by reference. The preparation of these esters and ethers is described in the patents listed above.
    [43] Preferred oximes, hydrazones and hydroxylamines of rapamycin are described in US Pat. Nos. 5,373,014, 5,378,836, 5,023,264 and 5,563,145, which are incorporated herein by reference. The preparation of these oximes, hydrazones and hydroxylamines is described in the patents listed above. The preparation of 42-oxorapamycin is described in US Pat. No. 5,023,263, which is incorporated herein by reference.
    [44] Particularly preferred rapamycins include rapamycin [US Pat. No. 3,929,992], rapamycin 42-ester with CCI-779 [3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid; US Pat. No. 5,362,718], and 42-O- (2-hydroxy) ethyl rapamycin (US Pat. No. 5,665,772).
    [45] In application, pharmaceutically acceptable salts of rapamycin are organic and inorganic acids, such as acetic acid, propionic acid, lactic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mal, if rapamycin contains an appropriate basic moiety. Lonic acid, mandelic acid, malic acid, phthalic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphorsulfonic acid, and similar known acceptable acids Can be formed from. In addition, the salts are organic to inorganic bases such as alkali metal salts (eg sodium, lithium, or potassium salts), alkaline earth metal salts, ammonium salts, if the rapamycin contains an appropriate acidic moiety, from 1 to 1 for each alkyl group. Alkylammonium salts containing 6 carbon atoms or dialkylammonium salts containing 1 to 6 carbon atoms, and trialkylammonium salts containing 1 to 6 carbon atoms in each alkyl group.
    [46] The mTOR inhibitor used in the anti-neoplastic agent combinations of the present invention is preferably rapamycin, more preferably the mTOR inhibitor is rapamycin, CCI-779, or 42-O- (2-hydroxy) ethyl rapamycin.
    [47] As described herein, CCI-779 was evaluated as a representative mTOR inhibitor in a combination of an mTOR inhibitor of the invention with an anti metabolite.
    [48] The preparation of CCI-779 is described in US Pat. No. 5,362,718, which is incorporated herein by reference. When CCI-779 is used as an anti-neoplastic agent, the initial intravenous infusion dose is about 0.1 to 100 mg / m 2 when administered in a once-daily dose regimen (daily for five days, every two to three weeks), It is planned to be about 0.1 to 1000 mg / m 2 when administered in a once-weekly dose regimen. Oral or intravenous infusion is preferred as the route of administration, intravenous is more preferred.
    [49] As used in accordance with the present invention, the term "anti metabolite" refers to a substance that is structurally similar to a natural intermediate (metabolite) that is critical in the biochemical pathway leading to DNA or RNA synthesis, which is directed to the host in this pathway. But used to inhibit the completion of the pathway (ie DNA or RNA synthesis). More specifically, anti-metabolites typically (1) compete with metabolites for catalytic or regulatory sites of important enzymes in DNA or RNA synthesis, or (2) with metabolites that are generally incorporated into DNA or RNA. It acts by causing DNA or RNA to be substituted and not able to sustain replication. The main classes of antimetabolic products include (1) folic acid analogs, which are inhibitors of dihydrofolate reductase (DHFR); (2) purine analogs of natural purines (adenine or guanine) that are structurally different and that competitively or irreversibly inhibit nuclear processing of DNA or RNA; And (3) pyrimidine analogs that mimic natural pyrimidines (cytosine, thymidine, and uracil), but which are structurally different and competitively or irreversibly inhibit the nuclear process of DNA or RNA.
    [50] The following is a representative example of the anti metabolites of the present invention.
    [51] 5-fluorouracil (5-FU; 5-fluoro-2,4 (1H, 3H) -pyrimidinedione) is a topical cream (FLUOROPLEX or EFUDEX), a topical solution (FLUOROPLEX or EFUDEX), and 50 mg / mL It is commercially available as an injection (ADRUCIL or fluorouracil) containing 5-fluorouracil.
    [52] Phloxuradine (2'-deoxy-5-fluorouridine) is commercially available as an injection (FUDR or phloxuradine) containing 500 mg / vial of phloxuradine.
    [53] Thioguanine (2-amino-1,7-dihydro-6-H-purine-6-thione) is commercially available in 40 mg oral tablet (thioguanine).
    [54] Cytarabine (4-amino-1- (β) -D-arabinofuranosyl-2 (1H) -pyrimidinone) is a liposome injection (DEPOCYT) containing 10 mg / mL of cytarabine, or 1 mg to 1 g / Commercially available as vials or liquid injectables (cytarabine or CYTOSAR-U) containing 20 mg / mL.
    [55] Fludarabine (9-H-purin-6-amine, 2-fluoro-9- (5-O-phosphono- (β) -D-arabino-furanosyl) is a liquid containing 50 mg / vial It is commercially available as an injection (FLUDARA).
    [56] 6-mercaptopurine (1,7-dihydro-6H-purine-6-thione) is commercially available in 50 mg oral tablet (PURINETHOL).
    [57] Methotrexate (MTX; N- [4-[[((2,4-diamino-6-phthyridinyl) methyl] methylamino] -benzoyl] -L-glutamic acid) is 2.5 to 25 mg / mL and 20 mg to 1 g / bi Commercially available as a liquid injectable containing earl (methotrexate sodium or FOLEX) and 2.5 mg oral tablet (metotrexate sodium).
    [58] Gemcitabine (2′-deoxy-2 ′, 2′-difluorocytidine monohydrochloride ((β) -isomer)) is commercially available as a liquid injection (GEMZAR) containing 200 mg to 1 g / vial.
    [59] Capecitabine (5'-deoxy-5-fluoro-N-[(pentyloxy) carbonyl] -cytidine) is commercially available as a 150 or 500 mg oral tablet (XELODA).
    [60] Pentostatin ((R) -3- (2-deoxy- (β) -D-erythro-pentofuranosyl) -3,6,7,8-tetra-hydroimidazo [4,5-d] [1 , 3] diazepine-8-ol) is commercially available as a liquid injection (NIPENT) containing 10 mg / vial.
    [61] Trimetrexate (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino) methyl] -quinazolin mono-D-glucuronate) is 25 to 200 mg It is commercially available as a liquid injection (NEUTREXIN) containing / vial.
    [62] Cladribine (2-chloro-6-amino-9- (2-deoxy- (β) -D-erythropento-furanosyl) -purine) is commercially available as a liquid injection containing 1 mg / mL (LEUSTATIN). .
    [63] The table below briefly summarizes some recommended doses for the anti-metabolites listed above.
    [64] drug Volume feeding 5-fluorouracil 12 mg / kg oral 6 mg / kg oral 370-600 mg / m 2 intravenously No drugs on days 6, 8, 10, 12, 5, 7, 9, and 11 daily for 4 days; dose reduced in half when toxicity is observed daily for 5 days, every 3-4 weeks Phloxuradin (FUDR) 0.1-0.6mg / kg Daily with arterial injection Cytarabine (DEPOCYT) Cytarabine (injectable) 50 mg 100 mg / m 2 2-3 g / m 2 5 doses every 14 days during the induction period; Twice a day for 2-6 days every day for 7 days every 28 days thereafter Fludarabine (FLUDARA) 25 mg / m 2 30 minutes infusion for 5 consecutive days; Every 28 days 6-mercaptopurine (PURINETHOL) 2.5-5mg / kg1.5-2.5mg / kg Every day during judo Every day during maintenance Methotrexate 15-30mg oral Daily during the 5-day course; Repeat 3-5 times Gemcitabine (GEMZAR) 1000mg / m 2/30 bun 1000-1250mg / m 2/30 bun Single agent: Once weekly for 7 weeks, then 1 week off, once weekly for 3 weeks every 4 weeks Combination therapy: 1, 8, 15, or 21 days with 28-day cycles Caffecitabine (XELODA) 2500 mg / m 2 Daily rest for two weeks, followed by one week off Pentostatin (NIPENT) 4mg / m 2 Dilution as bolus injection or intravenous infusion; Biweekly Trimmetrecate (NEUTREXIN) 45mg / m 2 Intravenous injection once daily for 21 days Cladribine (LEUSTATIN) 0.09mg / kg / day 7 days continuous continuous infusion
    [65] The present invention also includes the use of a combination of mTOR and anti metabolites, wherein the biochemical modifier is part of chemotherapy feeding. The term “biochemical modifier” is well known and is understood by those skilled in the art as an agent provided as an adjuvant of anti-metabolite therapy, which not only acts to potentiate anti-neoplastic activity but also has side effects of anti-metabolites. Neutralize Luboboline and lebopolynate are typically used as biochemical modifiers for methotrexate and 5-FU therapy.
    [66] Lucoboline (5-formyl-5,6,7,8-tetrahydrofolic acid) is a liquid injectable (lucoboline calcium or WELLCOVORIN) containing 5-10 mg / mL or 50-350 mg / vial, and 5--5 It is commercially available as a 25 mg oral tablet (Lucovorin calcium).
    [67] Levopolynate (pharmacologically active isomer of 5-formyltetrahydrofolic acid) is commercially available as an injection (ISOVORIN) containing 25 to 75 mg levopolynate, or as an oral tablet (ISOVORIN) of 2.5 to 7.5 mg.
    [68] Preferred combinations of the mTOR inhibitors and anti-metabolites of the present invention include CCI-779 and gemcitabine; CCI-779 and 5-fluorouracil; And CCI-779, 5-fluorouracil, and leuboborin. A combination of CCI-779 and gemcitabine is preferably used for the treatment of pancreatic cancer, and a combination of CCI-779 and 5-fluorouracil (with or without lucoboline) is preferably used for the treatment of colorectal cancer.
    [69] The anti-neoplastic activity of the combination of CCI-779 and antimetabolites was CCI-779 and gemcitabine; And in vitro and in vivo standard pharmacological test methods using a combination of CCI-779 and 5-fluorouracil as representative formulations of the present invention. The method used and the results obtained are briefly described below.
    [70] In vitro compounding studies of CCI-779 with anti-metabolite formulations were performed using human rhabdomyosarcoma Rh30 and Rh1 and human glioma strain SJ-GBM2. In vivo studies were carried out using human neuroblastoma (NB1643) and human colon GC3.
    [71] Dose response curves were measured for each of the desired drugs. Cell lines Rh30, Rh1 and SJ-G2 were plated in 6 well community plates at 6 × 10 3 , 5 × 10 3 and 2.5 × 10 4 cells / well, respectively. After a 24-hour incubation period, the drug was added with 10% FBS + RPMI for Rh30 and Rh1 or 15% FBS + DME for SJ-G2. After 7 days of exposure to the medium containing the drug, the cells were released by treating the cells with stock followed by detergent. The nuclei were then counted with a Coulter counter. Experimental results were graphed and IC 50 (drug concentration causing 50% proliferation inhibition) for each drug was determined by extrapolation. Since the IC 50 differed slightly from experiment to experiment, two values in parentheses enclosing the IC 50 of each drug were used in the interaction study. The point of maximum interaction between the two drugs appears when the isobols are in standard shape, when they are present in a ratio of 1: 1. Thus, each of the CCI-779 three IC 50 concentrations of approximately 3 and approximately the IC 50 each of gemcitabine or 5-FU 1: was mixed at a ratio of 1: 1. As a result, for CCI-779 and other drugs, 9 1: 1 combinations of the drug and the three IC 50 concentrations in each experiment were obtained. In general, this protocol resulted in one or more combinations for each drug containing IC 50 values. The 1: 1 combination of IC 50 concentrations for CCI-779 and each chemotherapeutic drug was then added using Berenbaum's formula: x / X 50 + y / Y 50, = 1, <1,> 1. Synergy, or antagonism was calculated. If three concentrations of CCI-779 tested alone did not result in ICs paired with any of the three ICs of other compounds tested alone, all 1 ICs were between the appropriate ICs of a single tested drug. : 1 The formulation was checked and confirmed. If they entered, they were considered to have an additive effect.
    [72] The results obtained in standard in vitro pharmacological test methods indicated that the combination did not in any case result in inhibition of proliferation of up to 50%, indicating that it was at least additive, and there was no evidence of antagonism.
    [73] Four-week-old female CBA / CaJ mice (Jackson Laboratories, Bar Harbor, ME) were immunized following thymicectomy followed by systemic irradiation (1200 cGy) using a 137 Cs source three weeks later. Mice received bone marrow cells with 3 × 10 6 nuclei within 6-8 hours of irradiation. Approximately 3 mm 3 of tumor pieces were implanted into the space of the dorsal outer flank of mice to initiate tumor proliferation. Tumor-bearing mice were randomized into seven groups prior to initiation of treatment. Mice bearing tumors each received drugs when the tumors were approximately 0.20-1 cm in diameter. Tumor size was measured at 7 day intervals using a digital Vernier caliper connected to a computer. Tumor volume was measured using the formula [(π / 6) × d 3 ] where d is the mean diameter, assuming the tumor was spherical. CCI-779 was given on 5 consecutive days for 2 weeks and this cycle was repeated every 21 days for 3 cycles. As a result, CCI-779 was 1-5, 8-12 (Cycle 1); 21-25, 28-32 (cycle 2); And 42-46, 49-53 (cycle 3) days. For each study, the schedule of other pharmacotherapy drugs is as follows:
    [74] Gemcitabine cycle 1, 4, 8 days only
    [75] The combination of CCI-779 and gemcitabine was evaluated by human colon (GC3) mouse xenograft test method. In this test method, CCI-779 is given five times a day for two consecutive weeks, every 21 days for three cycles, and gemcitabine is given on days 1, 4 and 8 only in the first cycle. The presence of CCI-779 did not enhance the tumor regression seen in the first cycle of gemcitabine treatment. However, the group treated with CCI-779 delayed the time required to reach two to three times the original pretreatment tumor volume (relative to gemcitabine alone), indicating that there was at least an additional benefit derived from combination treatment. .
    [76] Based on the results of these standard pharmacological test methods, combinations of mTOR inhibitors and anti-metabolite chemotherapeutic agents are useful as anti-neoplastic therapies. More particularly, these combinations include renal carcinoma, soft tissue sarcoma, breast cancer, neuroendocrine tumors of the lung, cervical cancer, uterine cancer, head and neck cancer, glioma, non-small cell lung cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma, small cell lung cancer It is useful for the treatment of ovarian cancer, colon cancer, esophageal cancer, gastric cancer, leukemia, colorectal cancer, and unknown primary cancers. Since these combinations contain two or more active anti-neoplastic agents, the use of such combinations is also provided for use of the combination of each agent in which one or both agents are used in less than therapeutically effective amounts, thereby ensuring toxicity associated with individual chemotherapeutic agents. Decreases.
    [77] In providing chemotherapy, many agents with different modalities of action are typically used as part of the chemotherapy "cocktail". The combination of the present invention is expected to be used as part of a chemotherapeutic cocktail which may contain one or more additional anti-neoplastic agents depending on the nature of the neoplasm to be treated. For example, the present invention also relates to alkylating agents (ie cisplatin, carboplatin, streptazoin, melphalan, chlorambucil, carmustine, metchloretamine, lomustine, bisulfan, thiotepa, ifopamide or Cyclophosphamide); Hormonal agents (ie, estradiomustine, tamoxifen, toremifene, anastrozole, or letrozole); Antibiotics (ie, plicamycin, bleomycin, mitoxantrone, idarubicin, dactinomycin, mitomycin, doxorubicin or daunorubicin); Immunomodulators (ie interferon, IL-2 or BCG); Antimitotic agents (ie vinblastine, vincristine, teniposide or vinorelbine); Isomerase inhibitors (ie, topotecan, irinotecan or etoposide); And mTOR inhibitors used with other chemotherapeutic agents such as other agents (ie hydroxyurea, trastuzumab, altretamine, retuximab, paclitaxel, docetaxel, L-asparaginase or gemtuzumab ozogamicin). Includes use of anti-metabolite combinations.
    [78] As used herein, the combination diet may be provided at the same time, or may be provided in a differential regime in which the mTOR inhibitor is provided at a different time than the antimetabolic product during the chemotherapy process. This time difference may range from minutes, hours, days, weeks, or more between administrations of the two agents. Thus, the term combination does not necessarily mean being administered simultaneously or in one dose, but rather that each component is administered during the desired period of administration. The formulations can also be administered by different routes. For example, in a combination of an mTOR inhibitor and an antimetabolic product, the mTOR inhibitor may be administered orally or parenterally, preferably parenterally, while the antimetabolic agent may be administered parenterally, orally or in another acceptable way. It is expected to be. In the combination of gemcitabine and CCI-779, gemcitabine is preferably administered parenterally. In the combination of 5-FU and leuko borin with CCI-779, 5-FU and leuco borin are preferably administered parenterally. These combinations can be administered daily, weekly or once a month. Typically for chemotherapy feeding, the chemotherapy process can be repeated after several weeks, following the same time interval for administration of the two agents, or modified based on patient response.
    [79] Accordingly, the present invention also provides a product comprising an mTOR inhibitor and an anti-metabolite anti-neoplastic as a combined formulation for simultaneous, separate or sequential use in the treatment of neoplasia in mammals.
    [80] As typical of chemotherapy, dose intake is directed to the attending physician on the basis of a number of factors, including the severity of the disease, the responsiveness of the disease, any treatment associated with toxicity, the patient's age, health, and other concurrent diseases or treatments. Are closely monitored.
    [81] Based on the results obtained with a combination of representative CCI-779 and anti metabolites, an initial intravenous infusion dose of the mTOR inhibitor is planned to be about 0.1 to 100 mg / m 2 , preferably about 2.5 to 70 mg / m 2 . . In addition, the mTOR inhibitor is typically administered intravenously over a 30 minute period, preferably about once a week. The initial dose of the anti metabolite component will depend on the component used and will be based on the experience of the attending physician with the initially selected formulation.
    [82] Based on the results obtained with the combination of CCI-779 and the anti metabolites, for the combination of the mTOR inhibitor and gemcitabine, the initial intravenous infusion dose of the mTOR inhibitor is about 0.1 to 100 mg / m 2 , preferably About 2.5 to 70 mg / m 2, and the initial intravenous infusion dose of gemcitabine is planned to be about 400 to 1500 mg / m 2 , preferably 800 to 1000 mg / m 2 . Initially, the patient is scheduled to receive an intravenous infusion of gemcitabine immediately after or 30 minutes prior to receiving the mTOR inhibitor for 30 minutes by intravenous infusion on days 1 and 8 of the 21 day treatment cycle. After one or more treatment cycles, the dose may be adjusted up or down depending on the results obtained and the side effects observed.
    [83] Based on the results obtained, when CCI-779 is used in combination with 5-FU and leuboborin, the initial intravenous infusion dose of the mTOR inhibitor in a combined diet of mTOR inhibitor and 5-FU and leuboborin is about 0.1 To 100 mg / m 2 , preferably about 2.5 to 70 mg / m 2 ; The initial intravenous infusion dose of leuboline is about 50-500 mg / m 2 , preferably about 200 mg / m 2 ; The initial intravenous infusion dose of 5-FU is planned to be about 500 to 7500 mg / m 2 , preferably about 1000 to 5000 mg / m 2 . Initially, the combination is planned to be administered according to the following diet: patients receive intravenous 1 hour intravenous injection of leubovirin each week for 6 week treatment cycles; Immediately after each lucoborin administration, 5-FU is administered by 24 hours continuous intravenous infusion. The mTOR inhibitor is first administered on day 8 of Cycle 1 and will be given once a week as a 30 minute intravenous infusion. Each six-week treatment cycle rests one week before beginning the next six-week treatment cycle. After one or more treatment cycles, the dose may be adjusted up or down depending on the results obtained and the side effects observed.
    [84] For commercially available anti metabolites, existing dosage forms can be used in divided doses as needed. Alternatively, the formulations or anti metabolites which are not commercially available may be formulated according to standard pharmaceutical methods. Oral formulations containing the active compounds of the present invention may include any commonly used oral forms, including tablets, capsules, oral forms, troches, lozenges and oral liquids, suspensions or solutions. have. Capsules are inert such as active compound (s) and powdered celluloses such as pharmaceutically acceptable starch (e.g. corn, potato or tapioca starch), sugars, artificial sweeteners, crystalline and microcrystalline cellulose, flour, gelatin, gums and the like. It may contain a mixture with fillers and / or diluents. Useful tablet formulations can be prepared by conventional compression, wet granulation, or anhydrous granulation methods, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone , Gelatin, alginic acid, acacia gum, xan gum, sodium citrate, complex silicate, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, anhydrous starch and Pharmaceutically acceptable diluents, binders, lubricants, disintegrants, interfacial modifiers (including surfactants), suspensions or stabilizers can be used, including but not limited to powdered sugars. Preferred surface modifiers include nonionic and anionic surface modifiers. Representative examples of surface modifiers include poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan ester, colloidol silicon dioxide, phosphate, sodium dodecyl sulfate, magnesium aluminum silicate And triethanolamine. Oral formulations herein can use standard sustained release or sustained release formulations to alter the absorption of the active compound (s). Oral formulations may also consist of administering the active ingredient in water or fruit juice containing an appropriate solubilizer or emulsifier, if necessary.
    [85] In some cases, it may be desirable to administer the compound directly into the airways in aerosol form.
    [86] In addition, the compounds may be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as free bases or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersants can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent microbial growth.
    [87] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersants and sterile powders for the instant preparation of sterile injectable solutions or dispersants. In all cases, the form must be sterile and must be in an amount in which there is easy injectability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols), suitable mixtures thereof, and vegetable oils.
    [88] For the purposes of the present description, transdermal administration is understood to include all administrations through the body surface and the inner layer of the body passages including epithelial and mucosal tissue. Such administration can be carried out using the present compounds, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions and suppositories (rectal and vaginal).
    [89] Transdermal administration can be achieved through the use of a transdermal patch that contains a carrier that is inert to the active compound and the active compound, nontoxic to the skin, and can move the agent for systemic absorption through the skin into the bloodstream. The carrier may take any of a number of forms such as creams and ointments, pastes, gels and sealing devices. Creams and ointments may be viscous liquids or semisolid emulsions in oil-in-water or water-in-oil form. Pastes made of absorbent powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. Many sealing devices can be used to release the active ingredient into a semi-permeable membrane comprising a reservoir containing the active ingredient, with or without a carrier, or a matrix containing the active ingredient. Other sealing devices are known in the literature.
    [90] Suppository formulations can be prepared from traditional materials, including cocoa butter, and glycerin, with or without wax that changes the melting point of the suppository. Water-soluble suppository bases such as polyethylene glycol of various molecular weights can also be used.
    权利要求:
    Claims (31)
    [1" claim-type="Currently amended] A method of treating neoplasia of a mammal comprising providing an effective amount of the combination comprising an mTOR inhibitor and an antimetabolic anti-neoplastic agent to a mammal in need of neoplasia.
    [2" claim-type="Currently amended] The method of claim 1, wherein the mTOR inhibitor, antimetabolic product, or both are provided in a therapeutically effective amount.
    [3" claim-type="Currently amended] The method of claim 2, wherein the mTOR inhibitor is provided in a therapeutically effective amount.
    [4" claim-type="Currently amended] The method of claim 2 or 3, wherein the anti metabolites are provided in a therapeutically effective amount.
    [5" claim-type="Currently amended] 5. The method of claim 1, wherein the neoplasia is kidney cancer; Soft tissue sarcoma; Breast cancer; Neuroendocrine tumors of the lung; Cervical cancer; Uterine cancer; Head and neck cancer; Glioma; Non-small cell lung cancer; Prostate cancer; Pancreatic cancer; Lymphoma; Melanoma; Small cell lung cancer; Ovarian cancer; Colon cancer; Esophageal cancer; Stomach cancer; leukemia; Colorectal cancer; Or one of the unknown primary cancers.
    [6" claim-type="Currently amended] 6. The method of claim 1, wherein the combination further comprises a biochemical modifier. 7.
    [7" claim-type="Currently amended] The method of claim 6, wherein the biochemical modifier is leuboline or lebopolynate.
    [8" claim-type="Currently amended] The method according to any one of claims 1 to 5, wherein the anti metabolite is gemcitabine.
    [9" claim-type="Currently amended] The method of claim 8, wherein the neoplasm is pancreatic cancer.
    [10" claim-type="Currently amended] 6. The method of claim 1, wherein the anti metabolites are 5-fluorouracil. 7.
    [11" claim-type="Currently amended] The method of claim 10, wherein the combination further comprises a biochemical modifier.
    [12" claim-type="Currently amended] 6. The method of claim 1, comprising providing an effective amount combination of an mTOR inhibitor, 5-fluorouracil, and leuboborin to a mammal in need of neoplastic treatment. 7.
    [13" claim-type="Currently amended] 13. The method of any one of claims 10-12, wherein the neoplasm is colorectal cancer.
    [14" claim-type="Currently amended] The method of any one of claims 1-13, wherein the mTOR inhibitor is rapamycin.
    [15" claim-type="Currently amended] The method of claim 14, wherein the rapamycin is rapamycin.
    [16" claim-type="Currently amended] The method of claim 14, wherein the rapamycin is 42-O- (2-hydroxy) ethyl rapamycin.
    [17" claim-type="Currently amended] The method of claim 14, wherein the rapamycin is rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid.
    [18" claim-type="Currently amended] An anti-neoplastic combination comprising an effective amount of an mTOR inhibitor and an anti-metabolic anti-neoplastic agent.
    [19" claim-type="Currently amended] The combination of claim 18, further comprising a biochemical modifier.
    [20" claim-type="Currently amended] The combination of claim 19, wherein the mTOR inhibitor is rapamycin.
    [21" claim-type="Currently amended] The combination of claim 19, wherein the rapamycin is rapamycin.
    [22" claim-type="Currently amended] The combination of claim 19, wherein the rapamycin is 42-O- (2-hydroxy) ethyl rapamycin.
    [23" claim-type="Currently amended] 20. The combination of claim 19, wherein the rapamycin is rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid.
    [24" claim-type="Currently amended] A product comprising an mTOR inhibitor and an antimetabolic anti-neoplastic agent as a formulation formulated for simultaneous, separate or sequential use in the treatment of neoplasia in a mammal.
    [25" claim-type="Currently amended] The product of claim 24, further comprising a biochemical modifier.
    [26" claim-type="Currently amended] 26. The method of claim 24 or 25, wherein the neoplasm is kidney cancer; Soft tissue sarcoma; Breast cancer; Neuroendocrine tumors of the lung; Cervical cancer; Uterine cancer; Head and neck cancer; Glioma; Non-small cell lung cancer; Prostate cancer; Pancreatic cancer; Lymphoma; Melanoma; Small cell lung cancer; Ovarian cancer; Colon cancer; Esophageal cancer; Stomach cancer; leukemia; Colorectal cancer; Or a product that is one of unknown primary cancers.
    [27" claim-type="Currently amended] 27. The product of any one of claims 24 to 26, wherein the mTOR inhibitor is rapamycin.
    [28" claim-type="Currently amended] The product of claim 27, wherein the rapamycin is rapamycin.
    [29" claim-type="Currently amended] The product of claim 27, wherein the rapamycin is 42-O- (2-hydroxy) ethyl rapamycin.
    [30" claim-type="Currently amended] The product of claim 27, wherein the rapamycin is rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid.
    [31" claim-type="Currently amended] 31. The product of any one of claims 24-30, wherein the anti metabolites are gemcitabine or 5-fluorouracil.
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    同族专利:
    公开号 | 公开日
    PL363991A1|2004-11-29|
    AT406892T|2008-09-15|
    HU0304093A3|2008-08-28|
    KR100862178B1|2008-10-09|
    IL157898D0|2004-03-28|
    HU0304093A2|2004-04-28|
    JP2004525950A|2004-08-26|
    CA2442849A1|2002-10-17|
    EA200301091A1|2004-02-26|
    NO20034433L|2003-11-25|
    SI1385551T1|2008-12-31|
    AU2007242958A1|2008-01-10|
    MXPA03009092A|2004-02-12|
    EP1385551B1|2008-09-03|
    NZ540047A|2007-01-26|
    CN1309421C|2007-04-11|
    BR0208627A|2004-03-09|
    WO2002080975A1|2002-10-17|
    NO20034433D0|2003-10-03|
    DE60228699D1|2008-10-16|
    EP1385551A1|2004-02-04|
    CN1545419A|2004-11-10|
    EA010184B1|2008-06-30|
    SG152906A1|2009-06-29|
    PT1385551E|2008-11-03|
    HK1060062A1|2008-11-14|
    ES2312568T3|2009-03-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-04-06|Priority to US28238501P
    2001-04-06|Priority to US28238801P
    2001-04-06|Priority to US60/282,385
    2001-04-06|Priority to US60/282,388
    2002-04-05|Application filed by 와이어쓰
    2002-04-05|Priority to PCT/US2002/010912
    2004-01-24|Publication of KR20040007491A
    2008-10-09|Application granted
    2008-10-09|Publication of KR100862178B1
    优先权:
    申请号 | 申请日 | 专利标题
    US28238501P| true| 2001-04-06|2001-04-06|
    US28238801P| true| 2001-04-06|2001-04-06|
    US60/282,385|2001-04-06|
    US60/282,388|2001-04-06|
    PCT/US2002/010912|WO2002080975A1|2001-04-06|2002-04-05|Antineoplastic combinations such as rapamycin together with gemcitabine or fluorouracil|
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