METHOD FOR PREVENTING OR TREATING VIRAL INFECTION AND TUMOR

Abstract

A method for preventing and treating viral infection and tumor using a drug composition having a benzophenone compound and analogues thereof having a symmetrical core structure and having same or different number of hydroxyl substitutions in benzene rings. The drug preparation for preventing and treating HIV, herpes virus and papillomavirus infection and the diseases induced thereby, wherein such viral infections include AIDS, genital warts, flat warts, common warts, herpes simplex, herpes zoster, vaginitis, cervicitis, cervical erosion and senile dementia, as well as cervical cancer, lung cancer, gastric cancer and colon cancer induced thereby. Preparation of hydroxy-substituted benzophenones and analogues thereof together with various compatible excipients into different medicaments or personal disinfected sanitary articles.

Claims

1. A method for preventing or treating viral infection and tumor, comprising administering to an individual to be treated a drug composition comprising 2,3,4,2,3,4-hexahydroxybenzophenone to prevent and treat viral infections.

2. The method according to claim 1, wherein the drug composition comprises an effective amount of the 2,3,4,2,3,4-hexahydroxybenzophenone compound as an active ingredient and excipients for formulating injections or oral preparations into powder injections, water injections, infusions, tablets, capsules, granules or solutions.

3. The method according to claim 1, comprising an effective amount of the 2,3,4,2,3,4-hexahydroxybenzophenone compound as an active ingredient and compatible excipients for formulating mucocutaneous preparations into gels.

4. The method according to claim 1, comprising an effective amount of the 2,3,4,2,3,4-hexahydroxybenzophenone compound as an active ingredient and compatible excipients for formulating anti-virus product, the anti-virus product is selected from one of the following personal care products: hand-washing lotions, bathing lotions, hand-washing soaps, bathing sponges, personal care wipers, facial tissues, intranasal sprays, mouthwashes, gynecological vaginal lotions, vaginal lubricants, contraceptives and combinations thereof.

5. (canceled)

6. The method according to claim 1, wherein the viral infection is herpes virus, HIV and human papillomavirus infected by humans or animals.

7. The method according to claim 1 for preventing and treating mucocutaneous warts.

8. The method according to claim 1 preventing and treating herpes virus-induced diseases.

9. The method according to claim 1 preventing and treating lips herpes, genital herpes and herpes zoster.

10. The method according to claim 1 for preventing and treating herpes virus-induced senile dementia, and does not include 3,4,5,2,3,4-hexahydroxybenzophenone.

11. The method according to claim 1 for removing or reducing human papillomavirus load and preventing the virus from inducing cancers.

12. The method according to claim 1 for preparation of drugs for removing or reducing human papillomavirus load and preventing the virus from inducing the cervical cancer.

13. The method according to claim 1 for preventing and treating AIDS.

14. (canceled)

15. (canceled)

16. (canceled)

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0064] The main materials (with contents of higher than 99%) adopted in the present invention and the sources thereof:

[0065] 3,4,5-trihydroxybenzoic acid and propyl 3,4,5-trihydroxybenzoate purchased from Long Yuan Natural Polyphenol Synthesis Plant of Nanjing; 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid purchased Zhong Da Chemical Co., Ltd. of Taizhou City; 2,2-dihydroxybenzophenone, 2,23-trihydroxybenzophenone, 2,4,2,4-tetrahydroxybenzophenone, 2,3,4-trihydroxybenzophenone, 2,3,4,2,3,4-hexahydroxybenzophenone, 2,3,4,2,4,5-hexahydroxybenzophenone and 3,4,5,2,3,4-hexahydroxybenzophenone purchased from Li Cheng Chemical Co., Ltd. of Shanghai.

Example 1 Preparation of Anti-HPV Microemulsion

[0066] 20 ml 1,2-propylene glycol, 15 ml Tween 80 and 5 ml ketone are mixed and added with sterilized distilled water to a total volume of 100 ml, so as to obtain an external preparation solution. 10 g 2,4,2,4-tetrahydroxybenzophenone is added to 50 ml of the external preparation solution, the pH thereof is adjusted to 5.5, and then the solution is added with the external preparation solution to 100 ml, so as to obtain 10% anti-HPV microemulsion of the present invention.

Example 2 Preparation of Anti-HPV 2,3,4,2,4,5-hexahydroxybenzophenone Injection

[0067] 10 g 2,3,4,2,4,5-hexahydroxybenzophenone, 8.5 g sodium chloride, 10 ml 1,2-propanediol and 80 ml Tween 80 are mixed and added with the sterilized distilled water to be dissolved, then added with the sterilized distilled water to 100 ml, the pH is adjusted to 7.4, and then the solution is filtered, potted and sterilized at 100 C. for 30 mins. Consequently, 2,3,4,2,4,5-hexahydroxybenzophenone injection is obtained.

Example 3 Preparation of 2,3,4,2,4,5-hexahydroxybenzophenone Compound Effervescent Tablets

[0068] 0.25 g 2,3,4,2,4,5-hexahydroxybenzophenone, 0.01 g polyinoside and 0.45 g tartaric acid are screened separately by 80 mesh sieve, and prepared with anhydrous ethanol into damp mass, and then screened by 12 mesh sieve to obtain wet granules, then the wet granules are dried at 50 C. for use. Besides, 0.65 g sodium bicarbonate and 0.02 g dextrin in addition with the sterilized distilled water are prepared into damp mass, and are screened by 12 mesh sieve to obtain wet granules, then the wet granules are dried at 50 C. These granules are mixed with the above dry granules, and the mixed granules are granulated, added with an appropriated amount of sterilized distilled water, baked for a while, added with 0.01 g PEG6000, uniformly mixed, and finally compressed into tablets.

Example 4 Test of Sample Inhibition to HIV-1 Integrase

[0069] The test of sample inhibition to HIV-1 integrase is performed by the Chinese National Drug Screening Center.

[0070] I. Test Principle:

[0071] 30 synthetic oligonucleotides are used as donor substrates, 20 synthetic oligonucleotides are used as target substrates, purified HIV-1 integrase is added to donor substrates coated 96-well plate, ELISA reaction is performed, the product of the strand transfer of target DNA is detected, color development is conducted by biotin-labeled alkaline phosphatase system, and the OD value is measured by a microplate reader. The addition of samples to the reaction system can be used to screen inhibitors for the enzyme.

[0072] II. Materials and Method for Test:

[0073] 1. HIV-1 IN: it is extracted and saved by Chinese National New Drug Screening Center and Institute of Pharmaceutical Biotechnology under Chinese Academy of Medical Sciences.

[0074] 2. Sample Treatment: 10 samples A1-A10 dissolved into DMSO prior to use are prepared into suitable concentrations, and then diluted 5 times at 4 dilutions. Donor substrate and target substrate are from Shanghai Biochemical Synthesis.

[0075] 3. Method for Test: the samples after dilution are added into the donor substrate coated 96-well plate, then added to the reaction Buffer containing the genetically engineered target enzyme and the biotin-target substrate, incubated under optimal reaction conditions, and colored by biotin-labeled alkaline phosphatase system, and the OD value for absorbance value at 405 nm is measured.

[0076] Test Results:

TABLE-US-00001 TABLE 1 Preliminary Screening Report of HIV-1 IN Inhibition Experiment Initial Concentration IC.sub.50 Sample No. (g/ml) (g/ml) 2,2-dihydroxybenzophenone 109 15.32 2,23-trihydroxybenzophenone 109 10.46 2,4,2,4-tetrahydroxybenzophenone 109 11.23 2,3,4-trihydroxybenzophenone 109 12.11 3,4-dihydroxybenzoic acid 109 2,3-dihydroxybenzoic acid 109 2,3,4,2,3,4-hexahydroxybenzophenone 109 9.12 2,3,4,2,4,5-hexahydroxybenzophenone 109 10.51 3,4,5-trihydroxybenzoic acid 109 100.46 Note: in the table indicates that the inhibition activity to HIV-1IN for the sample at the initial concentration is less than 50%.

[0077] Conclusion: the screening results of the experiment for in vitro drugs inhibition to HIV-1 IN show that the effective concentrations (IC.sub.50), in which the samples 2,2-dihydroxybenzophenone, 2,23-trihydroxybenzophenone, 2,4,2,4-Tetrahydroxybenzophenone, 2,3,4-trihydroxybenzophenone, 2,3,4,2,3,4-hexahydroxybenzophenone, and 2,3,4,2,4,5-hexahydroxybenzophenone inhibit half of HIV-1IN are 15.32 g/ml, 10.46 g/ml, 11.23 g/ml, 12.11 g/ml, 9.12m/ml and 10.51 g/ml respectively. Other samples have HIV-1IN inhibition activities of IC.sub.50>109 g/ml.

Example 5: Test of HPV Detection Via Fluorescence Quantitative PCR for Drugs Screening

[0078] The microemulsion obtained in example 1 is used as a test sample and saline for blank control.

[0079] HPV Virus Source:

[0080] The standard clinical genital warts specimens from a variety of patients after being cut off are washed with saline for removing bloods, and are cut into pieces under aseptic conditions, added with saline in 3 times of their volumes to be uniformly mixed, grinded into homogenate and placed at 40 C. for use.

[0081] Test Method:

[0082] Each test sample drug microemulsion and the saline of 50 l for each are respectively added with 50 l the genital warts tissue homogenate, and incubated at 37 C. for 24 hours; tests are performed according to the operation procedure provided by the HPV6, 11 and 16, 18 type FQ-PCR diagnostic kits provided by Da'an Gene Diagnostic Center. 0.2 ml of DNA extracted by the conventional alkaline lysis method is put into the thin-walled reaction tube, and simultaneously added with primers, F-PROBE, DNTP, DNA polymerase, buffers and the like in a certain concentration; ABI PRISM 7700 fluorescence quantitative PCR amplifier is used to repeat 40 circulations each with 45 s at 93 C. and 120 s at 55 C. after predegeneration at 93 C. for 2 mins; the quantitative results are automatically analyzed by the computer software to calculate the quantitative results of the initial copy number. The data are shown in Table 2 (completed by Da'an Gene Clinical Test Center).

TABLE-US-00002 TABLE 2 Test of HPV Detection via Fluorescence Quantitative PCR for Drugs Screening HPV 6/11 Virus HPV16/18 Tube No. (copy/ml) (copy/ml) Blank Control Tube 1.458 10.sup.6 1.40 10.sup.5 3,4,5-trihydroxybenzoic acid 0 0 Propyl gallate 0 0 2,2-dihydroxybenzophenone 0 0 2,23-trihydroxybenzophenone 0 0 2,4,2,4 tetrahydroxydiphenylketone 0 0 2,3,4-trihydroxybenzophenone 0 0 2,3,4,2,3,4-hexahydroxybenzophenone 0 0 2,3,4,2,4,5-hexahydroxybenzophenone 0 0 3,4-dihydroxybenzoic acid 1.90 10.sup.4 8.50 10.sup.4 2,3-dihydroxybenzoic acid 2.30 10.sup.4 3.20 10.sup.3

[0083] The results show that 24 hours later after the addition of test compounds 2,2-dihydroxybenzophenone, 2,23-trihydroxybenzophenone, 2,4,2,4tetrahydroxybenzophenone, 2,3,4-trihydroxybenzophenone, 2,3,4,2,3,4-hexahydroxybenzophenone and 2,3,4,2,4,5-hexahydroxybenzophenone into the tissue homogenate of genital warts, HPV 6/11 and HPV16/18 virus can no longer be detected by FQ-POVR. The groups of 3,4-dihydroxybenzoic acid and 2,3-dihydroxybenzene, however, have a high concentration HPV6/11 and HPV16/18 virus content, and the blank group also has a high HPV6/11 and HPV16/18 virus content, which shows that the compounds of the present invention have an ability of fast and effectively killing HPV virus in vitro.

Example 6: Test of Inhibition to Anti-Herpes Virus Type I, II (HSV-I, II)

[0084] I. Test Principle:

[0085] Vero cells (kidney cells of African green monkey) are used as viral hosts to test the inhibition degree of the samples to cytopathic effects of the Vero cell caused by herpes virus type I and type II.

[0086] II. Materials and Methods for Test

[0087] 1. Virus strains: HSV-I, VR733 strain, and HSV-II, SAV strain, which both are provided by ATCC.

[0088] 2. Sample treatment: the sample is dissolved in DMSO at an appropriate concentration before use, and diluted with the culture medium 3 times at a total of 8 dilutions for test.

[0089] 3. Positive control drug: acyclovir (ACV) produced by the Hubei Ke Yi pharmaceutical factory.

[0090] 4. Test method: Vero cells are planted on a 96-well culture plate, and after 24 hours, infected with herpes virus type I 10 (50 times of TCID.sub.50 infection) and herpes virus type II 10 (10 times of TCID.sub.50 infection), and absorbed for 2 hours; the virus solution is discarded; the samples and positive control drugs are added according to the above dilution; simultaneously the cell control wells and the virus control wells are set; the cytopathic effects (CPE) are observed for 48 hours; and the half inhibitory concentrations (IC.sub.50) of the samples against the herpes virus type I and type II are respectively calculated by the Reed-Muench method.

[0091] III. Test Results:

TABLE-US-00003 TABLE 3 Screening Report of Anti-herpes Virus Type I and II (HSV-I, II) Activity Against HSV-1 Against HSV-II TC.sub.50 IC.sub.50 IC.sub.50 Sample No. (g/ml) (g/ml) SI (g/ml) SI Blank 2,2- 64.15 28.74 2.18 28.74 2.23 dihydroxybenzophenone 2,3,4- 53.42 28.74 2.66 28.74 1.86 trihydroxybenzophenone 2,4,2,4 tetra- 111.11 28.74 2.22 28.74 2.11 hydroxybenzophenone 2,3,4,2,4,5-hexa 86.23 28.74 3.33 28.74 3.00 hydroxybenzophenone 2,3,4,2,3,4-hexa 12.35 28.74 1.26 10.85 1.14 hydroxybenzophenone ACV >100 1.092 >91.57 1.23 >81.30 Note: (1) in the table indicates there's no anti-herpes virus activity for the samples at the maximum non-toxic dose. (2) TC.sub.50: half toxic concentration; IC.sub.50: half inhibitory concentration to virus; SI: selectivity index, SI = TC.sub.50/IC.sub.50.

[0092] The results show that the above mentioned various hydroxy-substituted benzophenones have different inhibitory rates on both HSVI and HSV-II after the addition of the test compounds.

[0093] V. Clinical Observation of Treatment of Lips Herpes:

[0094] 1. Materials:

[0095] 20 ml glycerol, 20 ml 1,2-propylene glycol, 15 ml Tween 80 and 5 ml ketone are mixed and added with sterilized distilled water to a total volume of 100 ml for obtaining the external preparation solution. 10 g 2,3,4,2,4,5-hexahydroxybenzophenone is added with 50 ml external preparation solution with pH thereof adjusted to 5.5, and then is added with the external preparation solution to 100 ml, for obtaining a anti-HPV microemulsion containing 10% of the compound of the present invention, which is referred to as A; acyclovir ointment sold in pharmacy market (produced by Shanghai General Pharmaceutical Co., Ltd.) is referred to as the B.

[0096] 2. Methods and Results:

[0097] 10 patients with lips herpes were randomly divided into group A and group B;

[0098] Group A: 5 patients are smeared with A, 4 times a day, and all 5 patients scab at the next day;

[0099] Group B: 5 patients are smeared with B, 4 times a day, 2 patients scab at the third day, and 3 patients scab at the fifth day;

[0100] The efficacy of group A is significantly better than that of group B;

[0101] This shows that 2,3,4,2,4,5-hexahydroxybenzophenone has an anti-herpes function and can be used to treat herpes infection.

Example 7 Test of Compounds Resistant to Tumor In Vitro

[0102] I. Materials for Test

[0103] 1. Tumor cell lines: human glioma cell line (U251), human colon cancer cell line (LOVO), human hepatoma cell line (HepG2), human lung cancer cell line (PC84045), human endometrial carcinoma cell line (JEC), human renal carcinoma (GRC), human gastric cancer cell line (MCG804), human cervical cancer cell line (HeLa), human hepatocellular carcinoma cell line (BEL-7402) and human umbilical vein endothelial cell line (ECV304) purchased from Animal Experimental Center of Zhongshan Medical College, and preserved by the present laboratory.

[0104] 2. Main instruments and reagents for test: CO.sub.2 humidification incubator of Sheldon company; bechtop of Suzhou Purification Equipment Factory; microplate reader of Biored Company; trypsin of Sigma Company; RPMI-1640 medium of Gibco Company; 96-well culture plate of Corning Company; newborn bovine serum of Hangzhou Sijiqing Company.

[0105] II. Method for Test

[0106] 1. Cell Culture:

[0107] The tumor cell lines are cultured in RPMI-1640 medium containing 15% of the newborn bovine serum in a conventional manner, placed in the humidification incubator containing 5% of CO.sub.2 for being cultured at 37 C., and are observed for growth by an inverted microscope. A subculture is conducted every about 3-5 days, the cells in logarithmic growth phase are adopted to be digested with 0.25% of trypsin, and the serum-free RPMI-1640 medium is used for culturing the cell suspension; the blood counting plate makes counting, the trypan blue exclusion tests the cell activity, and the cell viability>95% is used for formal tests [1].

[0108] 2. MTT Method

[0109] Liquid preparation: the compounds are labeled with the codes respectively, DMSO is used to dissolve each compound, the bacterial filter is used to remove bacteria, and each compound is prepared into 10.sup.4 g/ml, 10.sup.5 g/ml, 10.sup.6 g/ml and preserved at 4 C. for use.

[0110] MTT method: a bottle of 4-5 days old cells in the exponential growth phase are adopted to be cultured, and added with an appropriate amount of Trypsin-EDTA solution, so as to enable the adherent cells to fall off, and are prepared with 10 ml of RPMI-1640 medium containing 15% newborn bovine serum into suspensions; after stained with trypan blue, the cell number is counted on the blood counting plate to ensure that the live cells is more than 97%; the cell suspensions are diluted by the complete medium so that each 100 ml of solution contains 5000-400000 cells; the 96-well plate is used with 200 l of cell suspensions added into each. The 96-well plate is placed at 37 C. in 5% of CO.sub.2 containing incubator for 24 hours; the test compounds are diluted at 3 dilutions with 5 wells in parallel for each concentration; the serum-free RPMI1640 is used as the control group. The 96-well plate is incubated in incubators having 5% of CO.sub.2 and 100% humidity at 37 C. for 3 days; MTT is prepared to 1 mg/ml solution with serum-free RPMI 1640 medium, and each well is added with 50 l thereof and incubated at 37 C. for 4 hours, so as to reduce the MTT to formazan; the supernatant is aspirated, 200 l of DMSO is added to dissolve the formazan and the obtained solution is shaken with a plate shaker for 5 minutes to be uniform; the microplate reader is used to measure the light absorbance value, measures the absorbance value of each well at a wavelength of 570 nm with a reference wavelength of 630 nm; the obtained data is processed by SPSS11.0 statistic software to calculate the inhibitory rate of the cells.

Inhibitory rate %=(1average absorbance value of sample group/average absorbance value of solvent group)100%

[0111] 3. Determination of Results

[0112] Inhibitory rate>50% means sensitive;

[0113] 30%50% means moderately sensitive;

[0114] <30% means insensitive.

[0115] III. Test Results and Analysis

TABLE-US-00004 TABLE 4 Report of In Vitro Inhibition of Compounds to Tumor Cells Inhibitory rate % 2,3,4,2,4,5- 2,3,3,4,4,5- 2,3,4,2,3,4- hexahydroxybenzophenone hexahydroxybenzophenone hexahydroxybenzophenone 10.sup.4 10.sup.5 10.sup.6 10.sup.4 10.sup.5 10.sup.6 10.sup.4 10.sup.5 10.sup.6 Cell lines g/ml g/ml g/ml g/ml g/ml g/ml g/ml g/ml g/ml U251 68.82 31.35 insensitive 64.27 insensitive insensitive 83.26 30.87 insensitive (sensitive) (moderately (sensitive) (sensitive) (moderately sensitive) sensitive) LOVO 51.19 insensitive insensitive 75.61 insensitive insensitive insensitive insensitive insensitive (sensitive) (sensitive) HepG2 85.12 51.57 insensitive 77.03 55.49 insensitive 86.15 75.54 insensitive (sensitive) (sensitive) (sensitive) (sensitive( (sensitive) (sensitive) PC84045 82.49 70.97 49.70 80.43 70.86 41.369 84.60 72.34 insensitive (sensitive) (sensitive) (moderately (sensitive) (sensitive) (moderately (sensitive) (sensitive) sensitive) sensitive) JEC 83.16 42.57 insensitive 79.84 32.36 insensitive 85.63 42.79 insensitive (sensitive) (moderately (sensitive) (moderately (sensitive) (moderately sensitive) sensitive) sensitive) MCF7 73.19 35.48 insensitive 65.36 insensitive insensitive 76.37 43.52 insensitive (sensitive) (moderately (sensitive) (sensitive) (moderately sensitive) sensitive) MCG804 83.48 72.89 insensitive 71.91 insensitive insensitive 80.93 30.12 insensitive (sensitive) (sensitive) (sensitive) (sensitive) (moderately sensitive) HeLa 60.92 insensitive insensitive 58.94 insensitive insensitive 61.12 insensitive insensitive (sensitive) (sensitive) (sensitive) ECV304 72.41 53.62 30.75 70.81 43.47 insensitive 79.37 insensitive insensitive (sensitive) (sensitive) (moderately (sensitive) (moderately (sensitive) sensitive) sensitive) Inhibitory rate % 2,3,4-tridyroxybenzophenone 2,4-dihydroxybenzophenone 10.sup.4 10.sup.5 10.sup.6 10.sup.4 10.sup.5 10.sup.6 Cell lines g/ml g/ml g/ml g/ml g/ml g/ml 2,4,2,4tetrahydroxybenzophenone U251 85.42 43.57 insensitive 85.31 67.25 31.21 (sensitive) (moderately (sensitive) (sensitive) (moderately sensitive) sensitive) LOVO 90.02 46.78 insensitive 93.80 insensitive insensitive 53.29 insensitive insensitive (sensitive) (moderately (sensitive) (sensitive) sensitive) HepG2 93.40 58.05 insensitive 94.78 66.74 58.63 74.85 insensitive insensitive (sensitive) (sensitive) (sensitive) (sensitive) (sensitive) (sensitive) PC84045 85.75 32.79 insensitive 92.65 89.88 44.39 53.40 insensitive insensitive (sensitive) (moderately (sensitive) (sensitive) (moderately (sensitive) sensitive) sensitive) JEC 86.79 40.97 insensitive 93.58 62.30 32.56 46.70 insensitive insensitive (sensitive) (moderately (sensitive) (sensitive) (moderately (moderately sensitive) sensitive) sensitive) MCF7 78.59 38.60 insensitive 87.60 68.62 28.57 45.73 insensitive insensitive (sensitive) (moderately (sensitive) (sensitive) (moderately (moderately sensitive) sensitive) sensitive) MCG804 87.14 62.75 insensitive 92.54 86.35 41.35 38.95 insensitive insensitive (sensitive) (sensitive) (sensitive) (sensitive) (moderately (moderately sensitive) sensitive) HeLa 89.81 34.53 insensitive 95.65 48.96 37.32 31.05 insensitive insensitive (sensitive) (moderately (sensitive) (moderately (moderately (moderately sensitive) sensitive) sensitive) sensitive) ECV304 89.63 45.81 insensitive 96.17 73.12 45.37 42.13 insensitive insensitive (sensitive) (moderately (sensitive) (sensitive) (moderately (moderately sensitive) sensitive) sensitive)

[0116] The results show that the above-mentioned various hydroxy-substituted benzophenones have different inhibitory rates on various tumor cells after 3 days of addition of the test compounds, and significantly inhibit human lung cancer cell line (PC84045), human endometrial carcinoma cell line (JEC), human renal carcinoma (GRC), human cervical cancer cell line (HeLa), and human hepatocellular carcinoma cell line (BEL-7402). The results demonstrate that the hydroxy-substituted benzophenone compound of the present invention and its analogues are effective in inhibiting tumor cells.

INDUSTRIAL UTILITY

[0117] The present invention uses hydroxybenzophenone in the preparation of antiviral and antitumor drugs which can prevent and treat the infection of HIV, herpes virus, papillomavirus and the diseases induced thereby. The hydroxy-substituted benzophenone and its analogues can be prepared together with a variety of compatible excipients into different medicaments or personal disinfected sanitary articles.