Method for reducing damage caused by free radicals

Abstract

The present invention features a method for reducing the damage caused by free radicals, the method comprises administering a pharmaceutical composition that comprises at least a metal or its ions, at least a drug or an antioxidant that is carrier-protected/modified, and a drug carrier to a subject. Said carrier helps to preserve the anti-oxidative activity, and hence, prevents the decrease of the effect of the antioxidant against free radicals produced in the environment or body fluids and prolongs protection, and be used for reducing damage caused by radiation and adverse effects induced by chemotherapeutic drugs.

Claims

1. A method for reducing damage caused by free radicals, comprising: administering a pharmaceutical composition to a subject, wherein the pharmaceutical composition comprises at least a ligand free metal or its ions; at least a drug or an antioxidant that is carrier-protected and modified; and a drug carrier, wherein the pharmaceutical composition containing the drug or the antioxidant is formed by self-assembly of the ligand free metal or its ions and the drug carrier via coordinate bonding and the center of the pharmaceutical composition consists the ligand free metal or its ions and is enclosed by the drug or antioxidant and the out layer is made of the drug carrier.

2. The method of claim 1, wherein the ligand free metal is selected from one of the following metals or their combinations: Fe, Cu, Ni, In, Ca, Co, Cr, Gd, Al, Sn, Zn, W, Sc, and Ti.

3. The method of claim 1, wherein the drug carrier is selected from one of the following groups or their combinations poly(ethylene glycol), poly(aspartic acid), poly(glutamic acid), polylysine, poly(acrylic acid), chitosan, polyethyleneimine, poly(methacrylic acid), hyaluronic acid, collagen, poly(N-isopropyl acrylamide), amylose, cellulose, poly hydroxybutyrate, poly(lactic acid), poly(butylene succinate), poly(caprolactone), carboxymethylcellulose, dextran, cyclodextrin, poly(ethylene glycol)-b-poly(glutamic acid) and phospholipid.

4. The method of claim 1, wherein the drug carrier is selected from one of the following groups or their combinations: liposome, micelle and dendrimer.

5. The method of claim 1, wherein the composition further contains one or more drugs which can be either bonded or not bonded with the metal and is selected from one of the following or their combinations: amifostine, WR-1065, ascorbic acid (Vitamin C), glutathione, melatonin, tocopherols, tocotrienol (Vitamin E), L-carnitine, carotenes, ubiquinol, lipoic acid, polyphenols, catecholamine, curcumin, resveratrol, piceid, acetylcysteine, tempo, asarone, aminoguanidine, tocopherol monoglucoside, glycyrrhizic acid, epicatechin, flavonoid, orientin, vicenin, MPG(2 -mercaptopropionyl glycine), and Mesna (2-mercaptoethanesulfonic acid).

6. The method of claim 1, wherein the subject comprises cells, tissues and organs that have damage caused by free radicals produced by ultraviolet light, radiation therapy, chemotherapeutic drugs or electromagnetic effects.

7. The method of claim 6, wherein the chemotherapeutic drugs comprise cisplatin.

8. The method of claim 1, wherein the drug is amifostine

9. The method of claim 1, wherein the pharmaceutical composition is injected intravenously into the subject.

10. A method for reducing the damage caused by free radicals, comprising administering a pharmaceutical composition to a subject, wherein the pharmaceutical composition consisting of iron ion, amifostine, and poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGA), and the center of the pharmaceutical composition consists the iron ions and is enclosed by amifostine and the out layer is made of poly(ethylene glycol)-b-poly(glutamic acid).

11. The method of claim 10, wherein the subject comprises cells, tissues and organs that have damage caused by free radicals produced by ultraviolet light, radiation therapy, chemotherapeutic drugs or electromagnetic effects.

12. The method of claim 11, wherein the chemotherapeutic drugs comprise cisplatin.

13. The method of claim 10, wherein the pharmaceutical composition is injected intravenously into the subject.

14. The method of claim 10, wherein a dosage of the amifostine is about 30 mg for a 60 kg body weight human.

15. The method of claim 10, wherein the pharmaceutical composition have a formulation weight ratio of PEG-b-PGA:FeCl.sub.2.4H.sub.2O:amifostine being 4:1:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the preferred embodiments shown.

[0019] FIG. 1 is a schematic diagram of the pharmaceutical composition with a metal core according to Example 1 of the present invention. ***P<0.001 significant differences from UV group. .sup.#P<0.05 significant differences from API 40×2 h group. .sup..box-tangle-solidup..box-tangle-solidup..box-tangle-solidup. P<0.001 significant differences from FePA 20×2 h group.

[0020] FIG. 2 is image of Comet Assay. CON is negative control, UV is UV irradiation without drug treatment as positive control, API 40×(2 hours), API 40×(30 mins), FeP 40×(2 hours), FePA 20×(2 hours) and FePA 20×(30 mins).

[0021] FIG. 3 is a quantitative diagram of Comet Assay DNA in tail(%). CON is negative control, UV is UV irradiation without drug treatment as positive control, API 40×(2 hours), API 40×(30 mins), FeP 40×(2 hours), FePA 20×(2 hours) and FePA 20×(30 mins).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0022] The present invention will now be described more specifically with reference to the following embodiments, which are provided for the purpose of demonstration rather than limitation.

EXAMPLE 1

[0023] Formulation of the Controlled-Release Drug Carrier of the Pharmaceutical Composition with a Metal Core

[0024] FIG. 1 is a schematic diagram of the pharmaceutical composition with a metal core. The center consists of a metal or its metal ions 120 and is enclosed by an antioxidant compound or other similar drug(s) 110, and the outer layer is made of drug carrier 100.

[0025] Active Substances

[0026] Amifostine is an antioxidant and a derivative of WR-1065 conjugating with phosphate groups, which exhibits antioxidative activity. Amifostine is currently approved by regulatory agencies in many countries for preventing radiation toxicity and adverse effects induced by cisplatin-like chemotherapeutic drugs.

[0027] The Drug Carrier for the Pharmaceutical Composition

[0028] Fifteen gram of γ-benzyl-L-glutamate and 7.5 g triphosgene were dissolved in tetrahydrofuran (THF) and stirred at 55° C. in the presence of N.sub.2 till the solution is clear. Following concentration of the solution, 400 mL n-hexane was used for precipitation. The monomer, N-carboxy-γ-benzyl-L-glutamate anhydride (BLG-NCA), is obtained after removal of n-hexane with 300 mL n-hexane/ethyl acetate (1/1) and crystallization. Fifteen grams of BLG-NCA and 2.1 g α-amino-ω-methoxy-poly(ethylene glycol) (PEG-NH.sub.2) were dissolved in 43 mL dimethyl sulfoxide (DMSO) and stirred at 40° C. for 72 hrs. The crude product was then subjected to precipitation using 215 mL diethyl ether followed by removal of diethyl ether, and 315 mL ethanol and 210 mL 1N NaOH were then added and the mixture stirred at 25° C. for 24 hrs. The pH value was adjusted to 7.0 by adding 35% HCl on ice, and then purified using MWCO 3500 membrane by dialysis. Poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGA) is then obtained after freeze-drying (lyophilization).

[0029] The present invention provides a material, and said material contains 206.44 mg Amifostine, 825.50 mg PEG-b-PGA and 206.44 mg Iron (II) chloride (FeCl.sub.2.4H.sub.2O). The obtained material was then added to 41.288 ml HEPES buffer 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) and stirred vigorously on a shaker at 200 rpm with the pH at 7.0 at 25 degree Celsius (° C.).

[0030] Formulation of FePA is based on the ratio of PEG-b-PGA:FeCl.sub.2.4H.sub.2O:amifostine=4:1:1 (w:w:w, according to the ratios of weight) with a preferred amifostine concentration at 5 mg/mL. Accordingly, the material containing amifostine is formed by self-assembly of ferrous ion (Fe.sup.2+) and PEG-b-PGA via coordinate bonding.

Formulation Example 1 (FePA)

[0031]

TABLE-US-00001 Amifostine 206.44 mg PEG-b-PGA 825.50 mg Iron(II) chloride 206.44 mg Total: 1238.38 mg 

[0032] In Vitro Antioxidative Activities of the Pharmaceutical Composition with a Metal Nucleus

[0033] UV irradiation of the cells was used in this experiment to simulate the effects of radiation on normal cells, and the results were evaluated by Comet Assay. Comet Assay is a fast, sensitive and convenient method for examining DNA damage and is widely used in studying DNA damage caused by radiation, examining DNA crosslink, evaluating genotoxicity of drugs and identifying cell apoptosis, etc. In total, eight groups were included in this experiment: [0034] API group received 1 mg/mL amifostine ; [0035] FePA group received 1 mg/mL FeCl.sub.2.4H.sub.2O, 4 mg/mL PEG-b-PGA and 1 mg/mL amifostine ; [0036] FeP group received 1 mg/mL FeCl.sub.2.4H.sub.2O and 4 mg/mL PEG-b-PGA [0037] The animals in each test group received 1 mL of the treatment drug(s) and the corresponding treatment methods are shown as follows:

TABLE-US-00002 TABLE 1 Group name Number of dish Treatment condition CON 2 No UV irradiation and no treatment UV 2 UV irradiation without treatment API 40x 2 Drug added 2 hr prior to UV irradiation API 40x 2 Drug added 30 min prior to UV irradiation FeP 40X 2 Drug added 2 hr prior to UV irradiation FePA 20x 2 Drug added 2 hr prior to UV irradiation FePA 20x 2 Drug added 30 min prior to UV irradiation CON: Negative Control UV: UV irradiation without drug treatment as Positive Control as Positive Control 40X: 40 folds dilution with PBS 20X: 20 folds dilution with PBS

[0038] The mouse embryonic liver cells (BNLCL.2) were inoculated onto a 35 mm culture dish at the density of 3*10.sup.5 cell/mL and cultured for at least 20 hours before subjected to the test. After removal of supernatant, fresh medium containing serum and various test compounds was added to the control and the test groups at different times according to the experimental design. The culture dish was then washed with PBS and irradiated with UVB (100 J.Math.m-2 UVB doses). Next, 2 mL fresh medium added, and the treated cells were cultured in the incubator for 4 hours to allow the drugs to take effects. The treated cells were then collected with a scraper, counted, and centrifuged at 1,200 rpm for 5 minutes. Followed by washing with PBS (Ca.sup.2+, Mg.sup.2+ free) once, the cell number was adjusted to 1*10.sup.5 cell/mL by adding PBS.

[0039] The bottle containing LM agarose was placed in 95° C. water for 5 minutes with loosed cap and then transferred to a 37° C. water bath for at least 20 minutes. The cells (1×10.sup.5/ml) and melted LM agarose were combined in the volume of 7 μL and 70 μL, respectively, at 37° C., and 60 μL mixture was immediately spread on a CometSlide ™ and allowed to set flat on ice for 10 minutes and protected from light. At the end of incubation, the slide was immersed in pre-cooled Lysis buffer and stored at 4° C. for 30 minutes. The excess buffer on the slide was removed by gently tapping, and then placed in freshly prepared Alkaline Unwinding Solution at room temperature for 60 minutes and protected from light. Later, 950 mL pre-cooled Alkaline Electrophoresis Solution was added to the electrophoresis tank followed by placement of the slide in the tank and covered with slide Tray Overlay. The electrophoresis conditions are 21 V for 30 minutes. At the end of electrophoresis, the solution was gently removed and the slide was immersed first in de-ionized water twice for 5 minutes each time; and then in 70% ethanol for additional 5 minutes. The slide sample was then air-dried in the hood to allow easy observation of the single plane view of the cells. The sample can be stored at room temperature at this step with desiccant or proceed to the next step directly. An aliquot of 100 μL diluted SYBR Green I was added to dried gel and stored at 4° C. for 5 minutes. Excess SYBR solution was removed from the slide by gently tapping and air-dried at room temperature and protected from light. The resulting images were analyzed using the epifluorescence microscope (the maximum excitation and emission wavelength of SYBR®Green I are 494 nm and 521 nm, respectively.) The fluorescence filter was also adjusted accordingly for efficient lighting) 200×.

[0040] The results are shown in FIG. 23. Compared with the positive control group (UV), the average percentage of damaged DNA in the tail of the Comet (% TDNA) of the FePA20×, API 40×, FeP 40× or group were all significantly reduced when the treatment was given either 30 minutes or 2 hr before UV irradiation. However, the in-vitro experiment was not with sensitivities enough to show the different between drug carrier and API. Thus, administration of FePA indeed protected cells from damage caused by UV irradiation and the treated cells were almost morphologically identical to normal cells without UV irradiation.

[0041] Other in Vivo Results Showing Improvements After Radiation Toxicity and Chemotherapy-Induced Adverse Effects

[0042] Efficacy Result: In animal study, 30-day old NMRI mice weighing 20 to 30 g were tested for acute radiation protection. For radiation control group, mice were given 1, 4, and 8 Gy irradiation in 10 minutes, and for the standard treatment group, FDA-approved amifostine (6.25 mg/kg) was injected intravenously, and 30 minutes later the animals were given 8 Gy irradiation in 10 minutes. The 30 mins duration design is based on the short half-life of amifostine. A-01 indicates FePA with a formulation ratio of PEG-b-PGA:FeCl.sub.2.4H.sub.2O:amifostine=4:1:1 (w:w:w). The animals in the test group A-01 were administered intravenously with 37.5 mg/kg A-01 (containing the same amount of amifostine as used in the standard treatment group), and 120 minutes post injection, the animals were given 8 Gy radiation in 10 minutes. WBC number and survival rates were analyzed 30 days after treatment.

[0043] As shown in Table 2, WBC numbers in A-01 group were 3 to 4 times more than radiation control group, which suggests that drug

[0044] A-01 can protect hematopoietic system from damage for at least 2 hr and can significantly reduce infection induced by radiation. Additionally, A-01 contains 6.25 mg/kg amifostine, which suggests that about 30 mg amifostine (equivalent to 180 mg of the said pharmaceutical composition) is effective in providing radiation protection for a 60 kg body weight human. Amifostine, approved by the FDA and used in radiation therapy for head and neck tumor, must be administered 3 minutes prior to radiation therapy and only provides obvious protection for salivary gland. The dosage of amifostine required 200 mg/m.sup.2 translates to 320 mg amifostine for a 60 kg human. Thus, A-01 drug can provide better protection against radiation toxicity by selective accumulation and slow release.

TABLE-US-00003 TABLE 2 WBC 1000/cm.sup.3 1Gy 4Gy 8Gy Normal Control 1.90-1.92 1.44-1.84 0.54-0.68 2.72-3.54 Amifostine (−30 min) — — 3.04-3.80 — — A-01(−2 hrs) — — 1.66-2.08 — — (— indicates radiation dosage is not enough to produce severe damage in the hematopoietic system; therefore, no drug is administered) (— — group indicates no adverse effects were observed in either rats or mice in the acute toxicity test and the results are within the normal range, hence WBC counts were not shown)

[0045] In conclusion, the present invention provides a metal ion based micelle technology to encapsulate amifostine or other antioxidants so as to reduce the damage caused by radiation and adverse effects induced by chemotherapy. Many changes and modifications in the above described embodiments of the invention can, evidently, be carried out to better control both the release and selective distribution of amifostine in vivo, and thus enhance the drug effects. Accordingly, to promote the progress in science and useful arts, the invention disclosed and the scope of the appended claims are submitted for approval.