CN101180080A - Nutritional composition comprising green tea polyphenols for treating osteosarcoma - Google Patents

Abstract

The present invention relates to the use of a composition comprising an ascorbic acid compound, a L-lysine compound, a L-proline compound and a polyphenol compound for the preparation of a pharmaceutical composition for the treatment of osteosarcoma. Furthermore, the invention relates to a method of treatment wherein said composition is administered to a subject suffering from osteosarcoma.

Description

Nutritional composition comprising green tea polyphenols for treating osteosarcoma

The present invention relates to the use of the composition comprising ascorbic acid compound, L-lysine compound, L-proline compound and polyphenol compound to prepare a pharmaceutical composition for treating osteosarcoma. Furthermore, the present invention also relates to methods of treatment wherein said composition is administered to a subject suffering from osteosarcoma.

Osteosarcoma, a primary malignant tumor of bone or soft parts arising from bone-forming mesenchymal cells, occurs primarily in the distal femur, proximal tibia, proximal humerus, and distal radius. Osteosarcoma typically exhibits aggressive, rapid growth with localized, "jump" metastases and a high risk of early, pulmonary metastases. It is the most prevalent bone cancer and the sixth most common cancer in children, and is more common in men than women. The vast majority of osteosarcomas are caused by non-genetic errors in the DNA of the growing bone cells. Because these errors occur randomly and unpredictably, there is currently no effective way to prevent this type of cancer [Miller, Dowshen et al (2002)].

For decades, the standard treatment for osteosarcoma has consisted of surgery (amputation or limb salvage surgery) and chemotherapy, which focuses on cancer cell destruction but does not address metastasis.

Radiotherapy and chemotherapy not only fail to achieve a curative effect, but also attack all cells indiscriminately-causing cell damage and damage to the body's connective tissue, thereby promoting cancer metastasis. For example, at the Anderson Cancer Center, patients with localized osteosarcoma [Jaffe, Carrasco et al (2002)] and treated with conventional chemotherapy [among them 3 patients were rescued with high-dose methotrexate and leucovorin and 28 patients with arterial In a study of 31 patients treated with internal cisplatin], only 3 patients did not experience local recurrence or lung metastasis during the follow-up period of 225+ months. Side effects of chemotherapy include: anemia, abnormal bleeding, increased risk of infection due to bone marrow destruction, liver and kidney damage, heart problems, and hearing loss. Approximately 20% of children diagnosed with osteosarcoma have advanced stages of osteosarcoma that has metastasized to the lung, brain and other bones [Miller, Dowshen et al (2002)]. It has even been reported that resection of the primary tumor can enhance the distant metastasis of osteosarcoma [Tsunemi, Nagoya et al (2003)].

Several nutritional compounds have been reported to exert anticancer activity. Ascorbic acid has been reported to have cytotoxic and anti-metastatic effects on malignant cell lines [Koh, Lee et al. (1998), Roomi, House et al. (1998), Naidu, Karl et al. (2003)]; Low levels of ascorbic acid have been reported in [Anthony and Schorah (1982), Nunez, Ortiz de Apodaca et al (1995), Kurbacher, Wagner et al (1996)]. ECGC is a potent anticancer agent that has been reported to have growth inhibitory effects on certain human cancer cell lines [Valcic, Timmerman et al. (1996), Mukhtar and Ahmed (2000), Yang, Liao et al. (1998) ]. However, the observed effect is rather weak and, therefore, not the basis for an appropriate therapeutic approach for rapidly growing and aggressive cancers. Other studies have shown that the synergistic anticancer effect of ascorbic acid and EGCG along with other compounds on several cancer cell lines in tissue culture studies is greater than that of the nutrients alone [Netke, Roomi et al. (2003)].

Clearly, safe and effective treatments are still needed to control the process of cancer metastasis, especially for invasive cancers such as osteosarcoma.

The technical problem underlying the present invention should therefore be seen as providing means and methods that meet the above requirements. This problem is solved by the embodiments characterized in the claims and hereinafter.

Therefore, the present invention involves

(a) ascorbic acid compounds,

(b) L-lysine compound,

(c) L-proline compounds, and

(d) polyphenolic compounds

Use of the composition for preparing a pharmaceutical composition for treating osteosarcoma.

The term "composition" includes both liquid and solid formulations of the aforementioned nutritional compounds and gels thereof. Solid preparations can be prepared in suitable forms, including tablets, capsules, powders, granules, tea preparations and the like. How to prepare liquid, gel and solid formulations mentioned herein is well known in the art. The compositions mentioned herein may be provided in the form of a mixture of compounds or a kit comprising the components separately, according to the use of the present invention. The components may be packaged in the kit as separate vials. The composition is also suitable for human or animal use. Preferably, the animal is a mammal, most preferably a dog, cat or horse.

The amino acid "proline" or "lysine" mentioned in the present invention is preferably an L-amino acid. "Proline" or "lysine" also includes its hydroxy derivatives hydroxyproline and hydroxylysine and salts thereof.

The term "ascorbic acid" preferably refers to ascorbate, ascorbic acid and salts thereof. Sometimes the ascorbic acid compound may be called vitamin C.

The term "polyphenolic compound" as used in the present invention preferably refers to a green tea herbal preparation comprising polyphenolic compounds, wherein said polyphenolic compounds are present in green tea. Polyphenolic compounds can make up as much as 30% of the dry weight of green tea. They include bioflavonoids such as flavanols, flavandiols, flavonoids and phenolic acids. Flavanols are the most abundant polyphenols in green tea and are commonly referred to as catechins. Most preferably, the catechin is EGCG, EG, ECG or EC. EGCG means (-)-epigallocatechin-3-gallate, EC means epicatechin which means (-)-epicatechin, ECG means epicatechin-3 - gallate, which means (-)-epicatechin-3-gallate, EGC means epigallocatechin, which means (-)-epigallocatechin. How such formulations can be obtained is well known in the art. Preferably, polyphenols are administered in an amount of 200 mg-5000 mg/day/subject.

The compounds mentioned for use according to the invention may be mixed in any suitable ratio or amount. Whether such ratios or amounts are appropriate can be determined by one skilled in the art by using the assays specified in the additional examples given below. Most preferably, the composition mentioned in the present invention provides vitamin C (ascorbic acid and ascorbic acid Mg, ascorbic acid Ca and ascorbyl palmitate) 700 mg; L-lysine 1000 mg; L-proline 750 mg; L-arginine 500 mg; Daily dose of N-acetyl cysteine 200 mg; standardized green tea extract (80% polyphenols) 1000 mg; selenium 30 mg; copper 2 mg; manganese 1 mg.

Pharmaceutical compositions administered according to the present invention may include a pharmaceutically acceptable carrier, diluent or excipient. Compositions for use according to the invention may be prepared by methods known in the art. The individual components can be formulated using common excipients, diluents or carriers, and formed into tablets, capsules, suspensions, powders and the like. Examples of excipients, diluents and carriers include: i) fillers and bulking agents such as starch, sugar, mannitol and silicic acid derivatives; ii) binders such as carboxymethylcellulose and other cellulose derivatives , alginate, gelatin and polyvinyl-pyrrolidone; iii) wetting agents such as glycerin; disintegrating agents such as calcium carbonate and sodium bicarbonate; delaying dissolution agents such as paraffin; iv) resorption accelerators such as quaternary ammonium compounds; v) Surfactants such as acetyl alcohol and glyceryl monostearate; v) adsorptive carriers such as kaolin and bentonite; and vi) lubricants such as talc, calcium and magnesium stearate and solid polyethylene glycol. The composition may also be formulated as an elixir or solution suitable for oral administration or as a solution suitable for parenteral administration, eg, by intramuscular, subcutaneous or intravenous routes. Ideally, the formulation is in the form of a pill, tablet, capsule, lozenge, liquid or similar dosage forms as mentioned above. The composition is well suited for formulation as sustained release dosage forms and the like.

The term "treating" as used herein refers to alleviating, inhibiting, attenuating or treating the syndromes associated with the pathological conditions referred to in the present invention. Treatment becomes apparent to the clinician by monitoring the symptoms accompanying the pathological condition. The symptoms are described in detail in standard textbooks such as Stedman or Pschyrembel. Treating preferably means substantially alleviating, inhibiting, attenuating or treating. Significance can be determined by standard methods of statistics, eg, Student's t-test, chi-square test.

The term "osteosarcoma" refers to a type of bone cancer. Symptoms associated with such diseases or conditions are well known in the art and described in detail in medical texts such as Stedman or Pschyrembel. Thus, clinicians can determine whether a patient has osteosarcoma without much trouble. The term "prevention" means that the nutritional composition can also be administered in order to avoid the occurrence of osteosarcoma.

The results of the studies on which the present invention is based surprisingly demonstrated that by supplementing 0.5% of a nutrient mixture containing ascorbic acid, lysine, proline and epigallocatechin gallate, immunocompromised (athymic) Osteosarcoma tumor growth was significantly inhibited in male nude mice. In addition, nutrient supplementation resulted in a reduction in mitotic index compared to control mice, see appended examples. Current treatments for osteosarcoma are associated with a poor prognosis, especially due to the increased risk of developing other cancers with chemotherapy. Therefore, new, safe and effective therapeutic strategies are needed. According to the present invention, the synergistic effect of a nutrient mixture (NS) of lysine, proline, arginine, ascorbic acid and epigallocatechin gallate on the growth of human osteosarcoma xenografts in athymic nude mice has been investigated . Furthermore, the effect of NS on the human osteosarcoma cell line MNNG-HOS was investigated in vitro by measuring cytotoxicity, regulation of MMP-2 and -9, cancer cell invasive potential, and angiogenesis. One week after isolation, 5-6 week old athymic male nude mice (n=12) were inoculated with 3×10 ⁶ osteosarcoma cells MNNG-HOS. After injection, the mice were randomly divided into two subgroups: group A fed a regular diet and group B fed a regular diet supplemented with 0.5% nutrient mixture. After 4 weeks, mice were sacrificed and their tumors were excised, weighed, and processed for histological studies. Cell proliferation was assessed by MTT assay, MMP expression by gelatinase zymography, and invasion by Matrigel. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) to study enhancement of MMP expression. The results showed that the nutrient mix (NS) inhibited growth and reduced tumor size in nude mice. Furthermore, the mitotic index was decreased in the supplemented group (4-5) compared to the control group (12-15). The invasion of osteosarcoma MNNG-HOS cells assessed by Matrigel was significantly reduced in a dose-dependent manner, and the invasion of MNNG cells was 100% inhibited at a concentration of nutrient mixture of 50 μg/ml. No significant antiproliferative effect was seen with MNNG cells. Zymography showed dose-dependent inhibition of MMP secretion by MNNG in the presence of NS. Nutrients synergistically inhibited tumor growth strongly without causing any side effects in nude mice, suggesting the potential of this nutrient combination as an anticancer agent. In vitro studies demonstrated inhibition of cancer cell invasion and MMP secretion—key parameters for cancer control and prevention.

Thanks to the present invention, osteosarcoma can be treated without the harmful side effects of conventional therapies such as surgery and/or radiotherapy.

The above definitions and explanations of terms are applicable to other embodiments of the present invention disclosed hereinafter, mutatis mutandis in detail.

In a preferred embodiment of the use of the present invention, the ascorbic acid compound is selected from ascorbic acid, pharmaceutically acceptable ascorbic acid salts, ascorbic acid esters, more preferably ascorbyl palmitate and/or mixtures of the foregoing compounds. More preferably, the pharmaceutically acceptable salt of ascorbate is selected from calcium ascorbate and magnesium ascorbate. Suitable amounts of the compounds used to prepare the compositions of the present invention are disclosed in WO 03/057201 A2, the details of which are incorporated herein by reference. Preferably, the ascorbic acid compound is ascorbic acid in an amount from 25 mg to 5000 mg and/or calcium ascorbate and/or magnesium ascorbate and/or ascorbyl palmitate in the same amount. All amounts are calculated per subject per day.

In another preferred embodiment of the use of the present invention, the L-lysine compound is selected from the group consisting of L-lysine hydrochloride, L-lysine and pharmaceutically acceptable L-lysine salts Group. Suitable amounts of the compounds used to prepare the compositions of the present invention are disclosed in WO 03/057201A2, the details of which are incorporated herein by reference. Preferably, the L-lysine compound may also be a mixture of at least two chemicals from the aforementioned group. Preferably, the L-lysine compound is L-lysine in an amount ranging from 50 mg to 5000 mg/day/subject.

In yet another preferred embodiment of the use of the present invention, the L-proline compound is selected from the group consisting of L-proline hydrochloride, L-proline and pharmaceutically acceptable L-proline salts Group. Suitable amounts of the compounds used to prepare the compositions of the present invention are disclosed in WO 03/057201A2, the details of which are incorporated herein by reference. Preferably, the L-proline compound can also be a mixture of at least two chemicals from the aforementioned group. Preferably, the L-proline compound is L-proline in an amount ranging from 25 mg to 3000 mg/day/subject.

In yet another preferred embodiment of the use according to the invention, said composition further comprises trace elements selected from the group consisting of selenium, manganese, magnesium, calcium and copper. Suitable amounts of the compounds used to prepare the compositions of the present invention are disclosed in WO 03/057201 A2, the details of which are incorporated herein by reference. Preferably, the composition comprises at least two, at least three or more trace elements of the aforementioned group. Preferably, the trace elements may be administered together, separately or in any combination in the following amounts per day per subject: Selenium: 1 μg-200 μg; Copper: 20 μg-9000 μg; Manganese: 50 μg-10000 μg; Calcium: 300 mg- 600mg; Magnesium: 300mg-600mg.

In yet another preferred embodiment of the use according to the invention, said composition further comprises an L-arginine compound. More preferably, the L-arginine compound is selected from the group consisting of L-arginine hydrochloride, L-arginine and pharmaceutically acceptable L-arginine salts. Suitable amounts of the compounds used to prepare the compositions of the present invention are disclosed in WO 03/057201 A2, the details of which are incorporated herein by reference. Preferably, the L-arginine compound may also be a mixture of at least two chemicals from the aforementioned group. Preferably, the L-arginine compound is L-arginine in an amount ranging from 50 mg to 3000 mg/day/subject.

In yet another preferred embodiment of the use according to the invention, the composition may also comprise N-acetylcysteine. "N-acetylcysteine" as used herein includes cysteine or cystine (a dimer of cysteine) and cysteine salts thereof. Suitable amounts of the compounds used to prepare the compositions of the present invention are disclosed in WO 03/057201 A2, the details of which are incorporated herein by reference. Preferably, N-acetylcysteine is administered in an amount ranging from 10 mg to 1500 mg/day/subject.

In a preferred embodiment of the use according to the invention, said composition is administered orally, parenterally, subcutaneously, intraarterially or intravenously.

The present invention also relates to a method for treating osteosarcoma in a subject comprising administering to a subject suffering from osteosarcoma a therapeutically effective amount of a composition as defined above. How to administer such compositions to a subject is well known to those skilled in the art. A particularly preferred technique is described in detail in WO 03/05 7201 A2, which is incorporated herein by reference. In addition, all embodiments of the use of the present invention are applicable to the aforementioned method for treating osteosarcoma, and necessary modifications can be made in the details.

In preferred embodiments of this method, said subject is a human.

In another preferred embodiment of the method, said composition is administered orally, parenterally, subcutaneously, intraarterially or intravenously.

Full bibliographic information for the references cited above and below can be found below. The disclosures of these references are incorporated herein by reference.

references

1. H.M.Anthony, C.J.Schorah(1982). Severe hypovitaminosis C in lung-cancer pa-tients: The utilization of vitamin C in surgical repair and lymphocyte related host re-sistance. Br J Cancer.46: 354-367

2. N. Jaffe, H. Carrasco, K. Raymond, A. Ayala, F. Eftekhari (2002). Can cure in pa-tients with osteosarcoma be achieved exclusively with chemotherapy and abrogation of surgery? Cancer. 10:2202-10.

3. W.S.Koh, S.J.Lee, H.Lee, C.Park, M.H.Park, W.S.Kim, S.S.Yoon, K.Park, S.I.Hong, M.H.Chung, C.H.Park (1998). Differential effects and transport kinetics of ascorbate derivatives in leukemic cell lines. Anticancer Res8: 2487-2493.

4. C.M.Kurbacher, U.Wagner, B.Kolster, P.E.Andreotti, D.Krebs, H.W.Bruckner (1996). Ascorbic acid (vitamin C) improves the antineoplastic activity doxorubicin, cisplatin and paclitaxel in human breast careinoma cells. Cancer in vitro Lett. 103(2): 183-189.

5. J.T.Masman (1983). Rapid colorimetric assay for cellular growth and survival: Ap-plication to proliferation and cytotoxicity assay. J Immunol Methods 65: 55-57

6. R. Miller, S. Dowshen, and M. Trigg, Childhood Cancer: Osteosarcoma (May 2002) The Nemours Foundation: Kids Health http://kidshealth.org/parent/medical/cancer/cancer osteosarcoma p1-3.html

7.H.Mukhtar and N.Ahmed(2000).Tea polypheonols: prevention of cancer and opti-mizing health.Am J Clin Nutr.71:1698S-1720S.

8. K.A.Naidu, R.C.Karl, K.A.Naidu, D.Coppola D(2003).Antiproliferative and proapoptotic effect of ascorbyl stearate in human pancreatic cancer cells: association with decreased expression of insulin-like growth factor.Sci 8 lig receptor( ): 230-7.

9.S.P.Netke, M.W.Roomi, V.Ivanov, A.Niedzwiecki, and M.Rath.A specific com-bination of ascorbic acid, lysine, proline and epigallocatechingallate inhibits prolif-eratio nand extracellular matrix invasion of various human cancer lines. cell Research Communications in Pharmacology and Toxicology: Emerging Drugs. 2, 37-50.

10.C.Nunez, Y.Ortiz de Apodaca, A.Ruiz(1995).Ascorbic acid in the plasma and blood cells of women with breast cancer.The effect of consumption of food with an elevated content of this vitamin.Nutr Hosp.10: 68-372.

11.M.Rath and L.Pauling(1992).Plasmin-induced proteolysis and the role of apopro-tein(a),lysine and synthetic analogs.Orthomolecular Medicine.7:17-23.

12. M.W.Roomi, D.House, M.Eckert-Maksic, Z.B.Maksic, C.S.Tsao(1998).Growthsuppression of malignant leukemia cell line in vitro by ascorbic acid(vitamin C)and its derivatives.Cancer Letters 122:93-99.

13. T. Tsunemi, S. Nagoya, M. Kaya, S. Kawaguchi, T. Wada, T. Yamashita, S. Ishii (2003) Postoperative progression of pulmonary metastasis in osteosarcoma. Clin Or-thop. 407: 159-66 .

14.S.Valcic, B.N.Timmerman, D.S.Alberts, G.A.Wachter, M.Krutzch, J.Wymer, J.M.Guillen(1996).Inhibitory effects of six green tea catechins and caffeine on the growth of four selected human tumor cell lines. .7: 461-468.

15. G.Y.Yang, J.Liao, K.Kim, E.J.Yurkow, C.S.Yang(1998).Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols.Carcinogenesis.19:611-616.

Description of drawings:

Figure 1: (A) Effect of NS on the total weight of osteosarcoma MNNG xenografts in male nude mice; (B) histology of tumor tissues in supplemented (Suppl) and control mice; (C) tumors in control and supplemented nude mice Photo.

Figure 2: (A) the effect of nutrient mixture (NS) and PMA on the proliferation of human osteosarcoma cell MNNG-HOS; (B) the effect of contacting with nutrient composition (NS) on the expression of MMP-2 caused by human osteosarcoma MNNG HOS cells .

Figure 3: Effect of Nutrient Mixture (NS) on Matrigel invasion and migration induced by human osteosarcoma cells MNNG-HOS.

The invention is illustrated by the following examples. However, these examples should not be used to limit the scope of the present invention.

Embodiment 1: the treatment of nude mouse osteosarcoma

Human osteosarcoma cells MNNG-HOS obtained from ATCC (American Type Culture Collection, Rockville, MD) were maintained in MEM culture supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Media and serum used were obtained from ATCC, and antibiotics (penicillin and streptomycin) were obtained from Gibco BRL, Long Island, NY. At near confluence, cultured cells were detached by trypsinization, washed with PBS, and diluted and emulsified to a concentration of 3 x ¹⁰⁶ cells in 0.2 ml PBS and 0.1 ml Matrigel (BD Bioscience, Bedford, MA). , for inoculation. Approximately 6-week-old male athymic nude mice (NCr-nu/nu) were purchased from Simonsen Laboratories, Gilroy, CA and housed in micro-insulated cages on a 12 hr light/12 hr dark cycle in the absence of pathogens Arranged for a total of 1 week. All animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals. After 1 week in cage, mice were inoculated with 3 × ¹⁰⁶ human osteosarcoma MNNG-HOS cells dissolved in 0.2 ml PBS and 0.1 ml Matrigel. After injection, the mice were randomly divided into two groups, A and B. Six mice were assigned to each group. From the first day, mice in group A were fed a regular diet, while mice in group B were fed a regular diet supplemented with 0.5% NS. After 4 weeks, mice were sacrificed, tumors were excised, weighed, fixed in 10% (v/v) buffered formalin and processed for histological studies. The results showed that the nutrient-supplemented nude mice developed significantly smaller tumors (53% smaller, p=0.0001) and fewer blood vessels than the control nude mice (FIG. 1A). In addition, histological examination revealed an average of 12-15 mitotic figures per high-power field in the control group; in contrast, nutrient-supplemented rats developed tumors with lower mitotic figures (4-5 per high-power field) (Fig. 1B). Tumors located in the subcutaneous layer were expansive, demonstrating peripheral invasion. Neoplasms consist of spindle-shaped or irregularly round cells with large, irregularly round to oval, hyperchromatic nuclei and scant cytoplasm with indistinct borders. Irregular areas of tumor necrosis comprised about 70% of the tumor mass.

The stock solution (total weight 4.4 g) of the nutrient mixture used for the experiment consisted of the following: vitamin C (ascorbic acid and ascorbic acid Mg, ascorbic acid Ca and ascorbyl palmitate) 700 mg; L-lysine 1000 mg; L-proline 750 mg ; L-Arginine 500 mg; N-Acetyl Cysteine 200 mg; Standardized Green Tea Extract (80% Polyphenols) 1000 mg; Selenium 30 mg; Copper 2 mg; Manganese 1 mg.

The results are expressed as the mean ± SD of each group. Data were analyzed by "t" test for independent samples.

Example 2: Effect of Nutrient Composition on Isolated MNNG-HOS Cells from Nude Mice in Culture

Human osteosarcoma cells MNNG-HOS obtained from ATCC (American Type Culture Collection, Rockville, MD) were maintained in MEM culture supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Media and serum used were obtained from ATCC, and antibiotics (penicillin and streptomycin) were obtained from Gibco BRL, Long Island, NY. At near confluence, cultured cells were detached by trypsinization, washed with PBS, and diluted and emulsified to a concentration of 3 x ¹⁰⁶ cells in 0.2 ml PBS and 0.1 ml Matrigel (BD Bioscience, Bedford, MA). , for inoculation.

Approximately 6-week-old male athymic nude mice (NCr-nu/nu) were purchased from SimonsenLaboratories, Gilroy, CA and housed in micro-insulated cages on a 12-h light/12-h dark schedule in the absence of pathogens 1 week in total. All animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals. After 1 week in cage, mice were inoculated with 3 × ¹⁰⁶ human osteosarcoma MNNG-HOS cells dissolved in 0.2 ml PBS and 0.1 ml Matrigel. After injection, the mice were randomly divided into two groups, A and B. Six mice were assigned to each group. From the first day, mice in group A were fed a regular diet, while mice in group B were fed a regular diet supplemented with 0.5% NS. After 4 weeks, mice were sacrificed, tumors were excised, weighed, fixed in 10% (v/v) buffered formalin and processed for histological studies.

Human osteosarcoma cells were grown in MEM in 24-well tissue culture plates (Costar, Cambridge, MA). Cell cultures were supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 mg/ml). Incubate cells with 1 ml of medium in a tissue culture incubator equilibrated with 95% air and 5% _CO2 at 37 °C. At near confluence, cells were treated with nutrient mix (NS) in culture medium and tested in triplicate at doses of 0, 10, 100, 500, and 1000 μg/ml. And one group of cells was treated with PMA 200ng/ml. The plates were then returned to the incubator. Cell proliferation was assessed 24 hours after incubation with test reagents.

Cell proliferation was assessed based on the MTT assay. The MTT assay [Masman JT, (1983)] is a colorimetric assay based on the reduction of the soluble yellow tetrazolium salt [3-(4,5-dimethylthiazole] by living cells through their mitochondrial succinate dehydrogenase activity. -2-yl) 2,5-diphenyltetrazolium bromide] (MTT) has the ability to form blue formazan crystals. After addition of MTT (0.5 mg/ml), the plates were covered and returned to the 37°C incubator for 2 hours, the optimal time for formazan product formation. After incubation, the supernatant in the wells was carefully removed, the formazan product was dissolved in 1 ml DMSO, and the absorbance was measured at 570 nm in a Bio Spec 1601, Shimadzu spectrometer. The _OD570 of the DMSO solution in each well was considered to be proportional to the number of cells. The _OD570 of the control group (not treated with supplements) was considered as 100%. MMP expression in conditioned media was determined by gelatinase zymography. Gelatinase zymography was performed on 10% polyacrylamide precast Novex gels (Invitrogen) in the presence of 0.1% gelatin. Medium (20 μl) was loaded and subjected to SDS-PAGE using Tris-glycine SDS buffer. After electrophoresis, the gel was washed with 5% Trion X-100 for 30 minutes. In the presence of 50 mM Tris-HCl, 5 mM CaCl ₂ , 5 μM ZnCl ₂ , pH 7.5, the gel was incubated at 37°C for 24 hours and stained with Coomassie blue R0.5% for 30 minutes, then destained. Simultaneously run protein standards and determine approximate molecular weights.

Invasion studies were performed in 24-well plates using Matrigel ^™ (Becton Dickinson) inserts. Suspended in medium, osteosarcoma cells were supplemented with nutrients and seeded on inserts in wells as detailed in the experimental design. Therefore, the media on the inserts and in the wells contained the same supplements. Incubate the plate with the insert in an incubator _equilibrated with 95% air and 5% CO. After incubation, the medium in the wells was removed. Use a cotton swab to gently wipe off the cells on the upper surface of the insert. Cells that permeated the Matrigel membrane and migrated onto the lower surface of the Matrigel were stained with hematoxylin and eosin and counted visually under a microscope.

The stock solution of the nutrient mix was as described above. The results are expressed as the mean ± SD of each group. Data were analyzed by "t" test for independent samples.

Claims (15)

1. comprise

(a) ascorbic acid compound,

(b) L-lysine chemical compound,

(c) the L-proline compounds and

(d) polyphenolic substance

Preparation of compositions be used for the treatment of the purposes of osteosarcomatous pharmaceutical composition.

2. the purposes of claim 1, wherein said ascorbic acid compound is selected from the mixture of ascorbic acid, pharmaceutically acceptable Ascorbate, acid ascorbyl ester and/or aforesaid compound.

3. the purposes of claim 2, wherein said pharmaceutically acceptable Ascorbate is selected from ascorbic acid calcium salt and ascorbic acid magnesium salt.

4. the purposes of claim 1, wherein said ascorbic acid compound is an acid ascorbyl ester.

5. the purposes of claim 4, wherein said acid ascorbyl ester is an ascorbic palmitate.

6. any one purposes of claim 1-5, wherein said L-lysine chemical compound is selected from L-lysine hydrochloride, L-lysine and pharmaceutically acceptable L-lysinate.

7. any one purposes of claim 1-6, wherein said L-proline compounds is selected from L-proline hydrochlorate, L-proline and pharmaceutically acceptable L-proline salt.

8. any one purposes of claim 1-7, wherein said compositions also comprises the trace element that is selected from selenium, manganese, magnesium, calcium and copper.

9. any one purposes of claim 1-8, wherein said compositions also comprises the L-arginine compounds.

10. the purposes of claim 9, wherein said L-arginine compounds is selected from L-arginine monohydrochloride, L-arginine and pharmaceutically acceptable L-arginine salt.

11. the purposes that claim 1-10 is any, wherein said compositions also comprises the N-acetylcysteine.

12. the purposes that claim 1-11 is any, wherein said compositions gives with oral, non-intestinal, subcutaneous, intra-arterial or intravenous form.

13. osteosarcomatous method among the treatment curee comprises and suffers from the step of compositions that osteosarcomatous curee treats any qualification of claim 1-12 of effective dose.

14. the method for claim 13, wherein said curee is the people.

15. the method for claim 13 or 14, wherein said compositions gives with oral, non-intestinal, subcutaneous, intra-arterial or intravenous mode.

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