TWI245635B - Prodrug comprising vitamin k3 and polymers and method of preparing the same - Google Patents

Abstract

A prodrug including vitamin K3 and a polymer. The polymer provides high water solubility, low toxicity, and low immunoreaction in mammalian. The prodrug is hydrolyzed to release vitamin Kg in an acidic condition, protecting drugs from enzyme damage and increasing their retention time in a subject and permeability through cells thereby, inhibiting tumor cell growth in mammalian. The invention also provides a method of preparing the prodrug and applications of prodrug.

Description

1245635 V. Description of the invention (1) Field of the invention The present invention relates to a prodrug that combines vitamin κ3 and a high molecular polymer, and particularly relates to a medical composition containing a previous driving drug as a treatment for cancer. BACKGROUND OF THE INVENTION Vitamin K3 (2-methyl-l, 4-naphthoquinone; menadione) was discovered in 1929 as a yellow fat-soluble antihemorrhagic agent that is synthetic and cannot be saponified. Although it belongs to the vitamin κ family, it is different from vitamin L from dark green plants and vitamin I made in intestinal flora. In addition to its anticoagulant effect, it also has antitumor ability. Vitamin K3 and doxorubicin

Anticancer drugs such as daunorubicin, mitoxantrone, and mitomycin (mi tomycin C) have the same structure as quinone Uui none) (1-3). Due to this awake ring structure, nucleic acids, esters, hydrocarbons, etc. in the cell will generate semiquinone radicai with it, and through the reducing agent of the cell, an electron will be reduced. In the presence of oxygen, the semiquinone radical generates a spontaneous and oxidative oxidation of radicals to form peroxide radicals, which leads to the formation of h2O2. The quinone may have undergone one or two reductions to produce semi-awake and hydroquinone (h y d r 〇 q u i η ο n e). Half awake may be regenerated by oxygen knife or produce peroxide q2—. Similarly, hydrogen awakening can generate 〇 02—, and through the reaction with oxygen molecules to generate semiquinone, and enter an oxygen a = f. The cycle is redox cycling. The peroxides such as H2O2 and O2 produced in Deng Xunchen are directly consumed.

1245635 V. Description of the invention (2) Cytosine adenosine triphosphate (ATP) and intracellular cystathione (GSH) (4-7). Vitamin L also induces changes in the state of phosphory lat ion 'and changes the activity of p 3 4cdc2 kinase and protein-tyrosine phosphatase, which affects the cell growth cycle S / G 2 cycle, Inhibit cell growth (8). Although vitamin Ks has the effect of suppressing tumor cells, it is not as effective in vivo as in vitro cell testing. The probable cause is that when vitamins & are in the body, the catalytic enzymes or other enzymes contained in the serum can remove peroxides such as hydrogen peroxide produced by vitamin L, so the cytotoxicity is greatly reduced (9). In addition, the phosphate in the serum (including Ατρ, etc.) may also affect the ability of vitamin Ks to enter cells (10). In order to improve this shortcoming of vitamins and cooperate with the physiological mechanism of cancer cells, the present invention designs a macromolecule polymer to combine with vitamin Ks to protect the drug and achieve the drug target effect. This drug design concept was first developed by paul Enrlich's mag i c bullet. By 1975, Ring · Ringsdorf proposed a more complete standard water-soluble polymer model (11). Utilizing the EPR effect (enhance permeability and retention effect) (12), this polymer drug can stay in tumor tissues for a longer period of time, achieving the characteristics of natural targets. The macromolecules that can be applied to the EPR effect include polyethylene glycol (PEG), polypropylene glycol, etc. (see US Patent No. 4,179, 337 to Davis et al.), Which has been applied to many drugs and enzymes , Such as procaine, atropine, insulin, and superoxide dismutase. PEG has very high solubility in either the aqueous or organic phase. It has no creatures

Page 5 1245635 5. Description of the invention (3) Decomposability, easy to be organic (1 3) and not easy to cause immune blood and tissue adsorption (1 5, molecular weight to various organisms higher than polyethylene glycol can reduce body fibrin in Between liposomes of organisms. Use of such polymerized enzymes or other enzymes to improve the purpose of the above-mentioned vitamins. Excretion from the body of the invention. It is recognized that ^ has a low toxicity in vivo ( 1 4). In addition to this, PEG does not cause coagulation. AA ^% chemically modified matrix from low molecular weight, even δ ^, particles are available. The various biological miscellaneous substances within the drug bond &, can be discriminated, and can reduce reticulum accumulation, such as liver, late Chu, bang, etc. In addition, it can also increase the biochemical properties of polymers during storage of microparticles and proteins, especially 1 ^^, in the blood, and eliminate the blood from the blood (17 19) to prevent enzymes from destroying the therapeutic effect of drugs. Kg in the body Disadvantages, this is the present invention. In view of this, the present invention provides a prodrug combining vitamin I and a high molecular polymer, including: a high molecular polymer, which has solubility, low toxicity, and is not easy in mammals. It has the characteristics of causing immune response and one-vitamin Ks. Among them, the previous flooding drug can be hydrolyzed in an acidic environment to release vitamin K3, thereby achieving the role of a drug target at the tumor site. Another aspect of the present invention is to provide a pharmaceutical composition for treating cancer in mammals, comprising: (i) an effective amount of a prodrug combining vitamin L and a high molecular polymer, or a pharmacological agent thereof Acceptable salts; and (ii) a physiologically acceptable carrier or excipient; wherein the high molecular polymer has the characteristics of high water solubility, low toxicity, and does not easily cause an immune response in mammals, and Prodrugs are water-soluble in acidic environments

Page 6 1245635 V. Description of Invention (4) Solution to release vitamin K3. Another aspect of the present invention is to provide a method for preparing a vitamin κ3 and a high molecular polymer precursor, including the following steps: (a) ketalization of vitamin K3; and (b) in three Coupling reaction of terminally aminated polymer with ketalized vitamin K3 in the presence of primary amine to obtain the precursor drug combining vitamin K3 and polymer; Molecular polymers have high water solubility, low toxicity, and are not easy to cause immune response in mammals. Brief description of the figure. Figure 1 is a cell growth curve diagram depicting in vitro experiments, where: 乂! (3, ^ 1? £ 0-20 0 0 41 (3 and? £ 〇- 34 0 0- ¥ 1 ( The concentration of 3 is 10 / ^, and the control group (control) is given an equal volume of buffer solution. Figure 2 depicts the cell growth curve of the in vitro test, in which VK3, MPEG-2 0 0 0 -VK3 and The concentration of PEG- 3 40 0-VK3 is 20 // M, and the control group (control) is given an equal volume of buffer solution. Figure 3 depicts the growth curve of tumor cells in mice, where 'tumor size is based on The formula of 1/2 a X b2 is estimated, where a is long and b is wide.

Figure 4 depicts the average survival time of mice administered different forms and doses of vitamins and a high-molecular polymer. Detailed description of the invention According to the combination of the first compound of the present invention, the obtained precursor drug state can combine vitamin K3 with a high molecular weight.

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V. Description of the invention (5) Characteristics' but take vitamin κ3 to the target position (for example, tumor site) 'to avoid the peroxide (such as' hydrogen peroxide) produced by vitamin L when it does not reach the site of action It is destroyed by catalytic enzymes in the body; and through this prodrug, it can be hydrolyzed in an acidic environment (PH 3 · 5 ~ 6) to release vitamin 1, thereby extending the residence time of vitamin K3 in the body. The high-molecular polymer used in the present invention must have the characteristics of high water solubility and toxicity, and it is not easy to cause immune response in mammals, thereby increasing water solubility and high permeability to cells, so as to reduce vitamins. & Take to the target area of action. The high-molecular polymer used is a straight-chain polymer. One or both ends of the polymer can be modified to couple with vitamin L to form the prodrug of the present invention. Suitable high-molecular polymers are unsubstituted, or polymers substituted with G Cs alkoxy or C ^ alkyl, such as polyethylene glycol (PEG) or methoxy polyethylene glycol (meth). Xy pEG; MPEG), wherein this polymer has molecules from about 1,500 to 5,000 canals, or 'N- (2-hydroxypropyl) fluorenylacrylamide (ΗρΜΑ), etc. In addition, the pre-drug drive of the present invention can be linked to an antibody, and this antibody can be an antibody against any tumor cell, whereby the pre-drug drive containing vitamin 1 can be more specifically delivered to the acting Target location.

According to the second aspect of the present invention, by driving a drug containing an effective amount of vitamin Kg to a patient, the polymer drug uses the EpR effect to accumulate in the vicinity of tumor tissue, and acts as a cell pellet. (endocytosis) or pinocytosis into the cells of the animal body. Under the low pH conditions of liposomes, the bond structure between the drug and the polymer is destroyed, and vitamin Ks is released.

1245635 V. Description of the invention (6) Tumor cells ’and avoid drugs obliquely fine

It destroys the structure of the drug and deactivates it within 7 days. By linking an antibody, it is more specific, which can increase the anti-cancer effect. Before the invention, the drug expulsion or its pharmacological method was administered alone or in the presence of pharmacy 'alone or with other pharmaceutically acceptable drugs such as hydrochloric acid, hydrogen acid, sulfuric acid and phosphoric acid, cis Carboxylic acid, tartaric acid, such as: arginine, aspartic acid, and amine carrier or excipients include any lubricants, absorption delaying agents, etc. It can be delivered to human or animal body by intra-arterial, intra-arterial, subcutaneous, intraperitoneal, orthotopic injection (i n s i t u). Cytotoxicity, and reduce yeast in the body. In addition, before the present invention, the drug can be acted on specific tumor sites, and the acceptable salts can be injected into the patient with a carrier drug or an excipient cancer drug at the same time. The salts ' include inorganic acids, such as salts of acids; organic acids, such as: salts of methanesulfonic acid; amino acids, salts of acids. Pharmaceutically acceptable vehicles, disintegrating agents, binding agents, pharmacology well known in the art, suitable methods of administration include intravenous intracranial or intramuscular administration, or 'primarily' intravenous injection According to the third aspect of the present invention, the formula is combined with vitamin K3 and high-complex rexalox, which can be prepared by the following method: Vitamin Ks undergoes a ketalization reaction, and the reaction flow is as follows: Second, the hydroxyl groups (one or both ends) of the high molecular polymer (for example, polyethylene glycols) are modified (including substitution, substitution, or alteration) by any method known in the art to produce terminal amines. Basic polymer. Terminally aminated polymers are commercially available, or

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’You can use trinitro acid (TNBS) test to synthesize it yourself; if you use the latter method to synthesize the content of the amine group produced by the trinitrobenzene sulfonic method.

Dissolving the two reactants obtained in the above mentioned sub-addition of a tertiary amine, taking polyethylene glycol as an example, the secondary amines that can be used in reaction scheme two include, but are not limited to, for example, triamines; Suitable organic solvents include, but are not limited to, for example, didiamine (DMF) or any similar organic solvent. After the reaction is completed, purification steps such as rod shrinkage, precipitation, and dialysis are performed, and the precursors for the combined vitamin Ks and polymer polymers are analyzed by fast liquid chromatography (FP ^ C). The above-mentioned prodrugs combining vitamin L and high molecular weight polymers can be linked to an anti-tumor cell antibody, so that the previous drive can reach the site of action more specifically. In order to make the above and other objects, features, and advantages of the present invention more comprehensible, the following exemplifies preferred embodiments, which are described in detail below, but these embodiments are merely examples of the present invention, and are not intended to be used as examples. To limit the scope of the invention. Example f Example 1: Reduction of Vitamin Kq 40 g of vitamin K3 (menadione; VK3), 30 ml of 3-chlorochun-1,2-propanediol, and 50 ml of triethyl orthoformat e) and 225 mg of p-toluenesulfonic acid (P-toluenesulfonic acid) in 200 ml of dichloromethane. Stir at room temperature for 10 days, then add 6 ml of triethylamine

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5. Description of the invention (8) (triethylamine) Terminates the reaction. Extract with saturated aqueous NaHC03 solution, retain the organic phase, and extract a total of 3 times. The organic phase was then dehydrated with MgS04. After filtration and removal of M g S 04, monochloromethane was concentrated to 1/4 of the original volume, and then purified by silica gel colloidal column using dichloromethane as a mobile solvent. The physical data are as follows: IR, 600-700, 1000-1300, 1500, 1600, 1 6 70-1 780,280 0- 290 0 (: 111 ^^^ 1 ^ (01 ^ 0-1) 53.4-3. 8 (2H ), 4.2-4.4 (2H), 4.0-4.2-2 (1H), 2.0-2.2-2 (3H). Example 2: MPEG-20 00-N. Synthesis of Γ9ί 20 grams of MPEG-2 0 0 0 -〇Η (0.01 mol) and 0.778 ml of pyridine (0.01 mol) were dissolved in 100 ml of toluene. The reaction temperature was 120 ° C, and a reflux tube was set up. The evaporated toluene was refluxed. Then, freshly distilled thionium chloride (thi ο ny 1 ch 1 〇ride) was added dropwise over 30 minutes, and maintained at about 0.3 mol (2.2 ml). The temperature was reacted for 4 hours. After the by-product salts were filtered off, MgS04 was added to remove water, and then the water removing agent was filtered off. The filtrate was concentrated to dryness. Crystallization was carried out with a mixed solution of methane and ether. The product was obtained.

MPEG-2 000-CK17 grams). Dissolve 15.2 grams of MPEG-2 0 0 0-C1 and 4.16 grams of sodium azide in 80 ml of dimethylformamide (DMF) and stir at 120 ° C for 3 hours. After it was cooled, the filtrate was filtered and concentrated to completely remove DM F. Then add a small amount of dichloromethane (about 30 milliliters) > after disintegration 'and then filter to remove unreacted sodium azide. The filtered filtrate was dropped into the pre-cooled ethyl acetate to precipitate the product MPEG-2 000-N3. 13 g of product were obtained after filtration and drying. Take 5 g of MPEG-2000-N3 and dissolve it in 120 ml of absolute alcohol, then add 0.3 g of 10% Pd / C, first pass nitrogen to remove the solvent and the oxygen in the reaction tank, and

Page 11 1245635 V. Description of the invention (9) After that, the hydrogenation reaction is performed in a Parr low pressure hydrogenation apparatus for 24 hours. Fresh hydrogen was continuously supplied during the reaction. After the reaction was completed, the catalyst was removed by filtration (10%

Pd / C), and then precipitated with pre-cooled ether to obtain 4.2 g of the product MPEG-2 0 0 0-NH2. The reaction flow is as follows: The physical data are as follows: IR MPEG-2 0 0 0 -C1 1110, 663-670 cm-1; MPEG-2 000-N3 2 1 0 0 -2 1 1 0, 1110 cm i; MPEG-2 0 00-NH2 1110 cm1. Example 3: Trinitrobenzene sulfonic acid method test 11 is the 9.3 heart 407 buffer solution, and 75.07 mg of glycine was dissolved in 100 ml of the above buffer solution, and then diluted to 0, 2, 4, 6, 8, 10 X 10-5 Μ 2 ml of glycine solution. Add 0.004 ml of a solution of 0.032 M trinitrobenzenesulfonic acid (TNBS) separately and react at room temperature for 30 minutes. The UV / Vis wavelength was set to 4200 nm, and the absorbance of glycine at different concentrations was measured to make a calibration curve. Then take a certain amount of MPEG-2000-NH2 dissolved in 2 ml of buffer solution, and then add 0.004 ml of 0.032 M TNBS to react for 30 minutes, observe the absorption of MPEG-200 0-Off 2 with the same absorption peak, and The interpolation calibration line is used to estimate the amino group content of MPEG-2000-NQ2. As a result, its amine group content was 81.8%. Embodiment 4: MPEG-20 00-VK. Synthesis: Dissolve MPEG-200 0-NH2 in DMF, add VK3-C1 and triethylamine to stir uniformly, and react at 50 ° C for 2 days. After the reaction was completed, DMF was concentrated with a rotary vaporizer, and then precipitated with ether. This precipitate

Page 12 1245635 V. Description of the invention (10) After the ether is dried and removed, an appropriate amount of pure water is added to dissolve it, and acidified to pH 7.2. Dialysis is performed on a dialysis membrane with a molecular weight sieve (MWCO) of 6000 to 8000 for about 4 to 6 days. The solution in the dialysis membrane was collected and concentrated, and then freeze-dried to obtain the product. Take 5 mg of MPEG-2 0 0 0-VK3 and dissolve it in 1 ml of 0.1 M tris-methyl methylcarbamate buffer (Tris buffer), put it into the FPLC superose 12 column, and use 0 · 1 M Tris buffer is the eluent, and the UV / Vis absorption is set to 280 run. Observe the RI and UV absorption patterns in MPEG-2 0 0-VK3 KFPLC. Example 5: Synthesis of MPEG-34 00-VK, 34 grams of PEG-34 00-〇Η (0.01 mol) and 1.6 ml of pyridine (0.01 mol) were dissolved in 100 ml of toluene. The reaction temperature was 120 ° C, and a reflux tube was set to reflux the evaporated toluene. Then, about 30 minutes (0.4 ml) of freshly distilled thiosulfinyl chloride was added dropwise over 30 minutes, and the temperature was maintained for 4 hours. After the by-product salts were filtered off, MgS04 was added to remove water, and the water removing agent was filtered off, and the filtrate was concentrated to dryness. A mixed solution of CH2C12 and ether was used for crystallization. The product PEG-3400-CU33 g was obtained). Dissolve 24 g of PEG-3400-C1 and 7.6 g of sodium azide in 150 ml of DMF. 5 Stir and react at 120 ° C for 3 hours. After it was cooled, the filtrate was filtered and concentrated to completely remove DMF. Then, a small amount of CH2C12 (about 30 ml) was added to dissolve, and then filtered to remove unreacted sodium azide. The filtered filtrate was dropped into ^ pre-cooled ether to precipitate, which is the product PEG-3 400-N3. After filtering and drying, 21 g of the product was obtained. Take 15 g of PEG-3400-N3 and dissolve it in 200 ml of anhydrous alcohol, and then add 0.5 g of 10% Pd / C. Nitrogen was first introduced to remove the solvent and the oxygen in the reaction tank, and then a low-pressure hydrogenation reactor (Parr low

Page 13 1245635 V. Description of the invention (11) Pressure hydrogenation apparatus) The hydrogenation reaction is performed for 48 hours. Fresh hydrogen was continuously supplied during the reaction. After the reaction was completed, the catalyst (10% Pd / C) was removed by filtration, and precipitation was performed with pre-cooled ether. This gives 14.3 g of product PEG-3400-NH2. Then the content of amine group was measured by TNBS. Then PEG-34 00 -NH2 was dissolved in DMF, and VK3-C1 and triethylamine were added to stir uniformly, and the reaction was carried out at 50 ° C for 2 days. After the reaction was completed, DMF was concentrated with rotavapor and then precipitated with ether. After the precipitate is dried to remove the scale B, an appropriate amount of pure water is added to dissolve it, and it is acidified to pΗ7 · 2, and the dialysis membrane with a molecular weight of 6000 to 8000 is subjected to dialysis for about 4 to 6 days. The solution in the dialysis membrane was collected, concentrated, and then cold-dried to obtain the product. Take 5 mg of PEG-340 0-VK3 and dissolve it in 1 ml of 0.1 M Tris buffer, insert it into the FPLC superose 12 column, use 0.1 M Tris buffer as the eluent, and set UV The absorption of / Vis is 280 nm. Observe the RI and UV absorption patterns of PEG-340 0-VK3 in FPLC. The physical data are as follows: IR PEG-340 0-C1 1110, 663 -67 0 cnr1; PEG-340 0-N, 2100-2110, 1110 cm-i; PEG-3400-NH2 1110 cm-1 〇 TNBS detection of amine content 92.9%. f Example 6: MPEG- 50 00-VK, 50 g of MPEG-5 0 00-〇Η (0.01 mole) and 0.79 ml of pyridine (0.01 mole) were dissolved in 100 ml of toluene in. The reaction temperature was 120 ° C, and a reflux tube was set to reflux the evaporated toluene. Then, freshly distilled sulfite gas of about 0.3 mol (2.2 ml) was added dropwise over 30 minutes, and the reaction was maintained at this temperature for 4 hours. After the by-product salts were filtered off, MgS04 was added to remove water, and the water removing agent was filtered off, and the filtrate was concentrated to dryness. Mixed with CH2C 12 and ether

Page 14 1245635 V. Description of the invention (12) The liquid is crystallized. 39 g of the product MPEG-50O-C1 was obtained. 36 g of MPEG-5 0 0-C1 and 4.0 g of sodium azide were dissolved in 76 ml of DMF, and the reaction was stirred at 120 ° C for 3 hours. After it was cooled, the filtrate was concentrated and concentrated until DMF was completely removed. Then a small amount of CH2C12 (about 30 ml) was added to dissolve, and then filtered to remove unreacted sodium azide. The filtered filtrate was dropped into the pre-cooled ether to precipitate the product MPEG-5 0 0-N3. 34 g of product was obtained after filtration and drying. Take 10.3 g of MPEG-500 0-N3 and dissolve it in 150 ml of absolute alcohol, and then add 0.245 g of 10% Pd / C. Nitrogen was first introduced to remove the solvent and the oxygen in the reaction tank, and then a hydrogenation reaction was performed in a Parr low pressure hydrogenation apparatus for 48 hours. Fresh hydrogen was continuously supplied during the reaction. After the reaction was completed, the catalyst (10% Pd / C) was removed by filtration, and then immersed in pre-cooled ether. The product MPEG-500 0-NH2 was obtained in an amount of 8.2 g. Then the content of amine group was measured by TNBS. Then take MPEG-5 00 0-NH2 and dissolve it in DMF, add VK3-C1 and triethylamine to stir evenly, and react at 50 ° C for 2 days. After the reaction was completed, DMF was concentrated with rotavapor and then precipitated with ether. After the precipitate is dried to remove the scale B, an appropriate amount of pure water is added to dissolve it, and it is acidified to pΗ7 · 2, and the dialysis membrane with a molecular weight of 6000 to 8000 is subjected to dialysis for about 4 to 6 days. The solution in the dialysis membrane was collected and concentrated, and then freeze-dried to obtain the product. Take 5 mg of MPEG-500 0-VK3 and dissolve it in 1 ml of 0.1 M Tris buffer, put it into the FPLC superose 12 column, use 0.1 M Tris buffer as the eluent, and set the UV / Vis The absorption is 280 nm. Observe the RI and UV absorption patterns of MPEG-5 0 0-VK3 in FPLC. The physical data are as follows: I R M P E G-5 0 0 0-C 1 1 1 1 0, 6 6 3-6 7 0 c nr1;

1245635 V. Description of the invention (13) MPEG-5 0 00-N3 2 1 0 0 -2 1 1 0, 1 1 1 0 cm1; MPEG-5 0 00 -NH2 1110 cnr1 ° TNBS detection amine content is 71.4 %.瘦. Lean example 7 _: _ in vitro test I. Cell line culture: H A 2 2 T (liver cancer cell line) was cultured in a 37 C 'incubator containing 5% -carbon monoxide. The culture medium was MEM (minimun essential medium), which contained 10% fetal bovine serum (FBs), 2 millimoles / liter of glutamine, and 1% of non-essential amino acids. After several days of cultivation, 24 hours before the cell survival experiment, 5 ml of HA22T cells at a concentration of 4 × 104 / ml were seeded in 25 ml culture flasks. I I. Cell Health County Curve: First, take a 25 ml culture flask as described above, trypsin was separated, and trypan blue was added to stain the cells to calculate the number of cells (Day 0, Dq). Add the remaining culture flasks to 0M; 10 // M, 20 // M of VK3; 10, 20 // M of MPEG-20 0 0-VK3 and =, 20 of PEG-3 400 -VK3 after 24 hours of culture Then, trypsin was used to separate the cells. Then trypan blue was added to stain the cells, and the cell number (D1) was calculated. After another 24 hours, the number of cells was calculated in the same procedure. Plot the number of cells versus time. II I. Results: The human liver cancer cell line HA22TgMEM was cultured in a 25-ml branch mouth culture bottle, and two experimental doses of 丨 〇 # and 〇v Ｍ were added to the experimental group and a group of non-medicated drugs. In the control group, the number of cells was calculated daily for two days, and the number of cells was plotted against time (Figure i and Figure 2). Figure] Join

1245635 V. Description of the invention (14) ------ 1 0 // Μ drug concentration and cell growth chart without drug, add v ^, PEG-34 0 0-VK3 and MPEG- 20 0 0-VK3 Cells of drugs enter the cell with the effect of cytotoxicity, because the polymer drug itself is limited to = 1, which is larger and the drug concentration is lower, so it is less effective at inhibiting cell growth. VKS is obvious. This is normal because in vitro cell experiments "the catalytic enzyme did not destroy the peroxides produced by VKS, and VKS was able to exhibit its cytotoxic properties. However, it can be found on Day 2 that the growth of cells added with polymer drugs is slower than that with VKS, which should be the result of Vi KDay 1 being exhausted and the polymer drugs still continuing to act. Figure 2 is a graph of cell growth with and without drug concentration at 20 // M. It can be seen from Figure 2 that at higher drug concentrations, PEG-34OO-VK3 and MPEG-2 0 0 0-^ 3 polymer drugs have the same cell growth inhibitory effect as VKs, while on Day 2 it can be seen from 3 to 9 MPEG-20 00-VK3 has a longer effect. Although the polymer is less likely to enter the cell, it is also relatively difficult to excrete it 'and the destruction of the bond structure between the drug and the polymer takes some time, so the purpose of slow drug release is achieved. Implementation Example 8: In vivo test I. Cell line culture: 1 ME A · 7R. 1 (a cell line that can cause extensive cancer in mice) was cultured in a 37 C 'incubator containing 5% carbon dioxide In the medium, the culture solution was MEM (Dullecco's modified eagie medium), which contained 10% fetal bovine serum and 2 millimoles / liter of glutamine. After several days of culture, after the number of cells reaches a certain amount, wash the cells with phosphate buffered saline (PBS), then trypsin to separate them, collect them by centrifugation, and discard the upper layer.

1245635 V. Description of the invention (15), add DMEM without any additives, so that it has 5 X 105 cells per ml. I I. Survival of animals The above collected cells were implanted into the experiment by subcutaneous injection.

Subcutaneously (BAL-BALB / cByJ, 8-week-old male rat, National Laboratory Animal Breeding and Research Center of the National Academy of Sciences), after 10 to 14 days, after the tumor grows to 0.4 X 0.4 cm , Divided it into u group, each group 4 ~ 6, respectively for the Zhao group (without injection); VK3 (salts): 200 mg / kg (drug content per kg of mouse) (LD5G); PEG -34〇〇-Vk3: 400 mg / kg (2 L); PEG-3400-VK3: 80 0 mg / kg (4 LD50); MpEG-2000- ', κ 3 · 2 0 0 ¾ g / kg (LD5 ()) and MpEG_20000-Vk3: 400 mg / kg (2 LD, all by intraperitoneal injection, observe and record tumor size every 2 to 3 days =. Time to tumor size (Tumor size) mapping, tumor size calculation method is 1 / 2axb2, where a is long and b is wide. ° II I. Results: Different doses of drugs were injected into the abdominal cavity of mice, and mouse cancer cells ^ The long-term situation is compared with the tumor size of the uninjected drug (control group). As can be seen from Figure 3, the tumor growth of the mice injected with the polymer drug is relatively correct. Slow, this is because the polymer material protects the drug. After the drug enters the cell, the drug is released in the endosomes (lysosomes) or lysosomes with a lower "value. Avoid the catalytic enzyme in the blood to destroy the hydrogen peroxide produced by it, so that the drug can completely affect the cancer cells. In addition, the tumor tissue will exclude the surrounding lymphatic system and form a system. When the polymer drug enters the tumor tissue Cannot

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(qu i none Bad cancer cells (d0x〇rub effect will reduce binding, increase content and increase by living polymer accumulation and knowledge, high fatality. Especially although it is used to limit the spirit and range of the original lymphatic tissue excreted from the body , And stay in the body for a longer time, making the drug release time longer. Especially at a dose of 400 mg / kg injected j ΥΕ6 " " 2ί) () () —νΚ3 (2ΙΛ〇), the tumor growth is the slowest Figure 4 shows the old and average survival time. From Figure 4, it can be seen that the average survival rate of the mice injected with polymer drugs is longer than that of the control group and the VK3 injection group. The effect is directly proportional to the average survival. However, when 80,000 kg of PEG-340-VKs is injected, the excessive dose of the drug may cause the mice to die without being too strong and cause the mice to die after the injection, making them average. Survival time is short. ^ As an anticancer drug, the most important is that its quinone structure produces oxidation and reduction, and continuously produces peroxide to stone. The cytotoxicity it produces is comparable to that of adriamycin ici η). However, when it is present in the body alone, the enzyme reduces its cytotoxicity. Therefore, the present invention utilizes the effect of polymers and drugs to protect drugs, and also increases the accumulation of vitamin 1 into the cells and the strong peroxide in the cell, so that the in vitro experiment of cytotoxicity (invitr 〇) can be observed. Drugs MPEG-20 0 0 -VK3 and PEG-3400-VK3 have slow release characteristics in cells ^. In vivo experiments (in vivo) can reduce the growth of cancer cells and prolong the life of animals. The polymer drug MPEG-200 0-VKS has the most outstanding performance. The present invention has been disclosed in the preferred embodiment as above, but the second aspect of the present invention is that any person skilled in the art can make changes and retouches within the scope of the present invention.

Page 19 1245635 V. Description of the invention (17) The patent protection scope of the present invention shall be determined by the scope of the attached patent application scope. References 1. Begleiter A.? Cytocidal action of the quinone group and its relationship to antitumor activity. Cancer Res, 1983; 43: 481-484. 2. Rotilio G ·, Mavelli I ·, Rossi L ·, and Ciriolo MR · ,

Biochemical mechanism of oxidation damage by redoxcycling drug, Environ Health Perspect, 1985; 64: 259-264. 3. Bachur NR.? Gordon SL.? And Gee MV.? A general mechanism for microsomal activation of quinone anticancer agents to free redicals, Cancer Res., 1978; 38: 1748-1750. 4. Thor H.? Smith M.? Hartzell P.? Bellomo G.? Jewell and Orrenius S.? The metabolism of menadione (2-methyl-l? 4-naphthoquinone ) by isolated hepatocytes, J. Biol. Chem., 1982; 257: 12419-12425. 5. Di Monte D.? Ross D.? Bellomo G.? Eklow L.? and Orrenius S.? Alternations in intracellular thiol homeostasis during the metabolism of menadione by islated rat hepatocytes?

Page 20 1245635 V. Description of the invention (18)

Arch. Biochem. Biophys. ^ 1984; 235: 334-342. 6. Ross D.? Thor H.? Threadgill M.? Sandy M.? Smith M.?

Moldeus P., and Orrenius S_, The role of oxidative processes in the cytotoxicity of substituted 1.4-naphthoquinone in isolated hepatocytes, Arch. Biochem. Biophys., 1984; 248: 460-466. 7. Rossi L_, Moore G., Orrenius S ·, and O'Brien P ·, Quinone toxicity in hepatocytes without oxidative stress, Arch. Biochem. Biophys., 1986; 251: 25-35. 8_Juan C. -C ·, and Wu F · Y · -H ·, Vitamin K3 inhibits growth of human hepatoma HepG2 cell by decreasing activities of both p34cdc2 kinase and phosphatase, Biochem · Biophys. Res. Commun., 1993; 190: 907-913. 9. Noto V.? Aper HS.? Jiang YH.? Jassens J., Bonte J.? And

Loecker W., Effects of sodium ascorbate (Vitamin C) and 2-methyl-1? 4-naphthoquinone (Vitamin K3) treatment on human tumor cell growth in vitro, Cancer, 1989; 63: 901-906. 10. Dendy PP. ? Further studies on the uptake of synkavit and radioactive analogue into tumor cells in tissue culture, Br.

Page 21 1245635 V. Description of the invention (19) J. Cancer ^ 1970; 817-825. 11. Ringsdorf H., Structure and properties of pharmacologically active polymers, J. Polymer Sci. Symp., 1975; 51: 135-153 12. Matsumura Y_, and Maeda H_, A new concept for macromolecular therapeutics in cancer therapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS, Cancer Res.y 1986; 46: 6387-6392. M 13.Sinko P · , And Kohn J ·, Polymeric drug delivery systems, in: M. El Nokali, DM Piatt and BA Charpantier (Eds.)

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Claims (1)

1245635 Case No. 89103653% s Amendment / Π VI. Patent application scope 1. A prodrug combining vitamin 1 (3 and a high molecular polymer, including: a high molecular polymer, which has high water solubility, low toxicity, and It is not easy to cause immune response in mammals, wherein the high molecular polymer is unsubstituted or a polymer substituted with G-C5 alkoxy or G-C5 alkynyl and has a molecular weight of about 1.0. Molecular weight of 0 to 5,000 ear puffs; and one vitamin κ3, wherein the prodrug is hydrolyzed in an acidic environment to release vitamin K3. 2. Combined with vitamins as described in item 1 of the scope of patent application A prodrug of κ3 and a high molecular polymer, wherein the high molecular polymer is polyethylene glycol. 3. The prodrug of combining vitamin K3 and a high molecular polymer as described in item 1 of the scope of patent application, wherein the The acidic environment is an environment in which ρ Η is between 3 and 6.5. 4. The prodrug that combines vitamin κ3 and a polymer as described in item 1 of the scope of the patent application, and further includes an antibody that binds to the prodrug 5 -A pharmaceutical composition for treating mammalian cancer, comprising: (i) an effective amount of a prodrug combining vitamin K3 and a high molecular polymer, or a pharmaceutically acceptable salt thereof; and (ii) A physiologically acceptable carrier or excipient, wherein the high molecular polymer has high water solubility, low toxicity, and does not easily cause immune response in mammals. The high molecular polymer is an unsubstituted 5 or Is a polymer substituted with a G-C5 alkoxy group or a q-C5 alkynyl group and has a molecular weight from about 10,000 to 5,000 channels, and the prodrug is hydrolyzable in an acidic environment, To release vitamin K3. 0643-5100TWFl; david.ptc Page 24 1245635 Case No. 89103653 Month Amendment 6. Scope of Patent Application 6. The pharmaceutical composition according to item 5 of the scope of patent application, wherein the polymer is polyethylene glycol. 7. The pharmaceutical composition according to item 5 of the scope of the patent application, wherein the prodrug releases vitamin K3 in the environment of a mammalian body. 8. The pharmaceutical composition according to item 5 of the scope of patent application, further comprising an antibody bound to the prodrug. 9. A method for preparing a precursor drug combining vitamin K3 and a high molecular polymer, comprising the following steps: (a) ketalizing vitamin K3; and (b) making a terminal amine in the presence of a tertiary amine A coupling reaction between the polymerized base polymer and the ketalized vitamin K3 is performed to obtain the prodrug that combines the vitamin K3 and a polymer polymer, wherein the polymer polymer has high water solubility and low toxicity. And it is not easy to cause immune response in mammals. The polymer is unsubstituted 'or a polymer substituted with G-C5 alkoxy or G-C5 alkynyl and has a molecular weight of about 1,0 0. Molecular weight of 0 to 5, 0 0 ear swallows. I 0. The method according to item 9 of the scope of the patent application, wherein the high molecular weight polymer is polyethylene glycol. II. The method according to item 9 of the scope of the patent application, wherein the tertiary amine is triethylamine. 12. The method according to item 9 of the scope of patent application, further comprising the step of binding an antibody to the prodrug. 0643-5100TWFl; david.ptc Page 25