USE OF DIKETONE COMPOUND IN PHOTODYNAMIC THERAPY OR DIAGNOSIS

Abstract

The disclosure belongs to the technical field of medicine, and relates to use of a diketone compound in photodynamic therapy or diagnosis. Particularly, the disclosure relates to use of a compound of formula (I), pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, solvate or crystal form of the compound, metabolite of each of them, or any combination or mixture thereof in preparation of a drug or a reagent. The drug or the reagent is used for photodynamic therapy or photodynamic diagnosis of a disease or is used for skin beauty by means of photodynamic therapy.

##STR00001##

Claims

1. A photodynamic method of treating or diagnosing a disease or performing skin beauty in a subject in need thereof, comprising administering a compound of Formula (I), a pharmaceutically acceptable salt or ester, a prodrug, a stereoisomer, a hydrate, a solvate or a crystal form, a metabolite thereof, or any combination or mixture thereof, ##STR00017## wherein R.sup.1 and R.sup.2 are each independently a linear or branched alkyl with 1 to 6 carbon atoms, a halogenated linear or branched alkyl with 1 to 6 carbon atoms, a hydroxyl group, or the following groups optionally substituted with one or more same or different substituents comprising aryl with 6 to 14 carbon atoms, 5- to 6-membered heteroaryl, 5- to 6-membered heterocyclyl, and 3- to 6-membered cycloalkyl, wherein the substituents are each independently selected from a hydroxyl group, a carboxyl group, a sulfonic group, a halogen atom, an amino group, a mercapto group, a nitro group, —C(O)O-(linear or branched alkyl with 1 to 4 carbon atoms), —S(O).sub.2O-(linear or branched alkyl with 1 to 4 carbon atoms), or —O-(linear or branched alkyl with 1 to 4 carbon atoms).

2. The method according to claim 1, wherein R.sup.1 is selected from a linear or branched alkyl with 1 to 4 carbon atoms or an aryl with 6 to 14 carbon atoms; or wherein R.sup.2 is selected from a linear or branched alkyl with 1 to 4 carbon atoms, a halogenated linear or branched alkyl with 1 to 4 carbon atoms, or hydroxy.

3. The method according to claim 1, wherein the compound is characterized in that (1) R.sup.1 represents a linear or branched alkyl with 1 to 4 carbon atoms and R.sup.2 represents a linear or branched alkyl with 1 to 4 carbon atoms; (2) R.sup.1 represents a linear or branched alkyl with 1 to 4 carbon atoms or a phenyl and R.sup.2 represents a hydroxy; (3) R.sup.1 represents a linear or branched alkyl with 1 to 4 carbon atoms and R.sup.2 represents a halogenated linear or branched alkyl with 1 to 4 carbon atoms; (4) R.sup.1 and R.sup.2 each represent a phenyl, and the phenyl is optionally substituted with one or more substituents which are the same or different; or (5) R.sup.1 and R.sup.2 are each independently a nitrogen-containing 5- to 6-membered heteroaryl, and the nitrogen-containing 5- to 6-membered heteroaryl is optionally substituted with one or more substituents which are the same or different.

4. The method according to claim 1, wherein the compound is selected from the following: TABLE-US-00010 No. Structural formula BJMU-201 BJMU-202 BJMU-203 BJMU-204 BJMU-205 BJMU-206 BJMU-207 BJMU-208 BJMU-209 BJMU-210 BJMU-211 BJMU-212 BJMU-213 BJMU-214

5. The method according to claim 1, wherein the method further comprising irradiating the subject with a light after the administering, which is characterized by one or more of the following: (1) the light being a single-wavelength light or a mixed light; (2) at least part of the wavelength of the light being in a range of 10 nm to 1 mm; (3) irradiating for 1 s to 12 h; or (4) irradiating at a light density of 1 to 2000 mW/cm.sup.2.

6. The method according to claim 1, wherein the disease is selected from the group consisting of (i) a tumor and its concurrent disease; (ii) a disease related to a microorganism or parasite infection; and (iii) an immune-related disease; wherein the tumor and its concurrent disease is selected from the group consisting of breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer, kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectum cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer comprising squamous cell carcinoma or basal cell carcinoma, hemangioma, glioma and secondary complications of these diseases comprising pain, infection, pericardial effusion, and pleural effusion; wherein the microorganism is a bacterium comprising Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Bacillus pertussis, Bacillus diphtheria, Diplococcus meningitidis, Mycobacterium tuberculosis, Clostridium tetani, Bacillus leprosy, Group A hemolytic streptococcus, Brucella, Bacillus cholera, Bacillus typhi, Bacillus anthracis, Neisseria gonorrhoeae, Propionibacterium acnes, or Salmonella paratyphi A, B or C; wherein the microorganism is a virus comprising influenza virus, mumps virus, rubella virus, encephalitis B virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papilloma virus, polio virus, measles virus, varicella-zoster virus, hepatitis virus, new enterovirus type 70, Coxsackie virus A24 variant, or a human immunodeficiency virus; wherein the microorganism is a fungus comprising Candida albicans, Trichophyton rubrum, and Epidermophyton floccosum, a mycoplasma comprising Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, or Mycoplasma genitalium, a chlamydia comprising Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, or Chlamydia in livestock, a rickettsia comprising Rickettsia prowazekii, Rickettsia mooseri, Rickettsia ricketts, or Rickettsia tsutsugamushi, an actinomycete comprising Actinomyces Israel, or a spirochete comprising Leptospira or Treponema pallidum; wherein the parasite is selected from the group consisting of roundworm, hookworm, tapeworm, Trichomonas vaginalis, liver fluke, Paragonimus Westmans, Toxoplasma gondii, Swine cysticercosis, Trichinella spiralis, Amoeba, Leishmania donovani, Plasmodium, Schistosome, Filaria, Hydatid, Scabies mite, hairfollicle mite, lice, and flea; wherein the disease related to microorganism or parasite infection is selected from the group consisting of extra-intestinal infection or diarrhea caused by Escherichia coli, viral hepatitis, bacterial and amoebic dysentery, gonorrhea, syphilis, polio, Measles, pertussis, diphtheria, epidemic cerebrospinal meningitis, scarlet fever, epidemic hemorrhagic fever, rabies, leptospirosis, brucellosis, anthrax, epidemic encephalitis B, kala-azar, malaria, dengue fever, tuberculosis, schistosomiasis, filariasis, echinococcosis, leprosy, influenza, mumps, rubella, neonatal tetanus, acute hemorrhagic conjunctivitis, cholera, typhoid fever caused by typhoid bacilli, paratyphoid fever, mycoplasma pneumonia, non-gonococcal urethritis, mycoplasma cervicitis, trachoma, psittacosis, chlamydia pneumonia, epidemic typhus, endemic typhus, Rocky Mountain spotted fever, Rickettsialpox, Tsutsugamushi, spotted fever, trench fever, as tinea versicolor, black tinea palm, nodose trichomycosis, tinea pedis, tinea hand, tinea corporis, tinea cruris, onychomycosis, tinea capitis, Sporothriosis, Chromoblastomycosis, Candidiasis, Aspergillosis, Cryptococcosis, Zygomycosis, Penicillium marneffei, Condyloma Acuminatum, Herpes Zoster, AIDS, Pulmonary actinomycosis, acne, and infectious diarrheal diseases; or wherein the immune-related disease is selected from the group consisting of secondary immunodeficiency comprising infections caused by rubella, measles, leprosy, tuberculosis, cytomegalovirus infection, HIV infection, or coccidioidomycosis, protein loss related to nephrotic syndrome or protein-losing enteropathy, insufficient immunoglobulin synthesis, lymphocyte loss caused by drugs and/or systemic infection, diabetes, cirrhosis, and subacute sclerosing panencephalitis or secondary immunodeficiency caused by immunosuppressive therapy; and an autoimmune disease comprising systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, primary platelet purpura, autoimmune hemolytic anemia, ulcerative colitis, skin disease, or chronic liver disease.

7. The method according to claim 1, wherein the disease is a precancerous lesion or a skin disease; wherein the precancerous lesion is selected from the group consisting of cervical cancer precancerous lesions caused by papillomavirus infection and precancerous lesions of the cancer comprising breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer, kidney cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectum cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer comprising squamous cell carcinoma and basal cell carcinoma), hemangioma, or glioma; or wherein the skin disease is selected from the group consisting of condyloma acuminatum, acne related to microorganism infection, port wine stains, solar keratosis, skin cancer, precancerous lesions of skin cancer, and benign proliferative diseases of skin.

8. The method according to claim 1, wherein the skin beauty comprises removal of pigmented skin lesions, exfoliating, or relief of acne associated with microorganism infections.

9. The method of claim 1, wherein the skin beauty comprises removing wrinkles, eliminating skin sagging, removing pigmented skin lesions, removing skin growths, or removing keratin.

10. A method for inhibiting an activity of a tumor cell, a microorganism or a parasite, the method comprising: step i: applying a compound of Formula (I), a pharmaceutically acceptable salt or ester, a prodrug, a stereoisomer, a hydrate, a solvate or a crystal form, a metabolite thereof, or any combination or mixture thereof to the tumor cell, microorganism or parasite; and step ii: irradiating the tumor cell, microorganism or parasite with light. ##STR00032## wherein R.sup.1 and R.sup.2 are each independently a linear or branched alkyl with 1 to 6 carbon atoms, a halogenated linear or branched alkyl with 1 to 6 carbon atoms, a hydroxyl group, or the following groups optionally substituted with one or more same or different substituents comprising aryl with 6 to 14 carbon atoms, 5- to 6-membered heteroaryl, 5-to 6-membered heterocyclyl, and 3- to 6-membered cycloalkyl, wherein the substituents are each independently selected from a hydroxyl group, a carboxyl group, a sulfonic group, a halogen atom, an amino group, a mercapto group, a nitro group, —C(O)O-(linear or branched alkyl with 1 to 4 carbon atoms), —S(O).sub.2O-(linear or branched alkyl with 1 to 4 carbon atoms), or —O-(linear or branched alkyl with 1 to 4 carbon atoms).

11. The method according to claim 10, further characterized by one or more of the following: (1) the light in step ii being a single-wavelength light or a mixed light; (2) at least part of the wavelength of the light in step ii being in a range of 10 nm to 1 mm; (3) irradiating for 1 s to 12 h; or (4) irradiating at a light density of 1 to 2000 mW/cm.sup.2.

12. The method according to claim 10, wherein the tumor cell comprises breast cancer cell, melanoma cell, meningioma cell, soft tissue sarcoma cell, salivary gland tumor cell, primary liver cancer cell, intraspinal tumor cell, mediastinal tumor cell, brain cancer cell, bone cancer cell, penile cancer cell, osteosarcoma cell, intracranial tumor cell, tongue cancer cell, maxillary sinus cancer cell, thyroid cancer cell, malignant lymphoma cell, multiple myeloma cell, pituitary adenoma cell, testicular tumor cell, non-Hodgkin's lymphoma cell, bladder cancer cell, leukemia cell, gastric cancer cell, nasopharyngeal cancer cell, laryngeal cancer cell, oral cancer cell, esophageal cancer cell, lung cancer cell, kidney cancer cell, cervical cancer cell, choriocarcinoma cell, vulvar cancer cell, skin cancer cell, endometrial cancer cell, ovarian cancer cell, prostate cancer cell, pancreatic cancer cell, colon cancer cell, rectal cancer cell, colorectal cancer cell, Kaposi's sarcoma cell, non-melanoma skin cancer comprising squamous cell carcinoma or basal cell carcinoma cell, hemangioma cell, or glioma cell; wherein the microorganism comprises a bacterium comprising Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Bacillus pertussis, Bacillus diphtheria, Diplococcus meningitidis, Mycobacterium tuberculosis, Clostridium tetani, Bacillus leprosy, Group A hemolytic streptococcus, Brucella, Bacillus cholera, Bacillus typhi, Bacillus anthracis, Neisseria gonorrhoeae, Propionibacterium acnes, or Salmonella paratyphi A, B or C, a virus comprising influenza virus, mumps virus, rubella virus, encephalitis B virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papilloma virus, polio virus, measles virus, varicella-zoster virus, hepatitis virus, new enterovirus type 70, a Coxsackie virus A24 variant, or human immunodeficiency virus, a fungus comprising Candida albicans, Trichophyton rubrum, or Epidermophyton floccosum, a mycoplasma comprising Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, or Mycoplasma genitalium, a chlamydia comprising Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, or Chlamydia in livestock, a rickettsia comprising Rickettsia prowazekii, Rickettsia mooseri, Rickettsia ricketts, or Rickettsia tsutsugamushi, an actinomycete comprising Actinomyces Israel, or a spirochete comprising Leptospira and Treponema pallidum; or wherein the parasite is selected from the group consisting of roundworm, hookworm, tapeworm, Trichomonas vaginalis, liver fluke, Paragonimus Westmans, Toxoplasma gondii, Swine cysticercosis, Trichinella spiralis, Amoeba, Leishmania donovani, Plasmodium, Schistosome, Filaria, Hydatid, Scabies mite, hair follicle mite, lice, and flea.

13. The photodynamic method of claim 1, wherein the linear or branched alkyl with 1 to 6 carbon atoms is a linear or branched alkyl with 1 to 4 carbon atoms; wherein the aryl with 6 to 14 carbon atoms is a phenyl or a naphthyl; wherein the halogenated linear or branched alkyl with 1 to 6 carbon atoms is a fluoro-, chloro-, bromo- or iodo-linear or branched alkyl with 1 to 6 carbon atoms; wherein the 5- to 6-membered heteroaryl contains 1 to 3 ring atoms selected from nitrogen, oxygen, and sulfur; or wherein the 5- to 6-membered heterocyclyl contains 1 to 3 ring atoms selected from nitrogen, oxygen, and sulfur.

14. The photodynamic method of claim 1, wherein the linear or branched alkyl with 1 to 6 carbon atoms is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.

15. The photodynamic method according to claim 1, wherein R.sup.1 is methyl, ethyl or phenyl; or wherein R.sup.2 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, bromomethyl, bromoethyl, and hydroxy.

16. The photodynamic method according to claim 1, wherein the skin beauty comprises removal of melasma or freckles.

17. The photodynamic method according to claim 1, wherein the method for skin beauty further comprises using a red and blue light therapeutic apparatus.

18. The photodynamic method according to claim 1, wherein the skin beauty is photo-rejuvenation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0121] FIG. 1 shows size changes of tumor tissues in BALB/C mice during 2 weeks of treatment in Experimental Example 3. As shown in the figure, 14 days after administration, there is a very significant difference between a high-concentration treated group and a control group (p<0.001), and there is also a very significant difference between a low-concentration treated group and the control group (p<0.001). The tumor size is controlled and decreased, and this effect is more prominent in the high-concentration treated group. This experiment shows that the diketone compound has an inhibitory effect on the tumor.

[0122] FIG. 2 shows weight changes of tumor tissues in BALB/C mice after 2 weeks of treatment in Experimental Example 3. As shown in the figure, 14 days after administration of the diketone compound, there is a very significant difference between a high-concentration treated group and a control group (p<0.001), and there is also a very significant difference between a low-concentration treated group and the control group (p<0.001). Based on weight ratios of tumors, an obvious growth inhibition effect is shown in the treated groups and a better inhibitory effect on the tumor is shown in the high-concentration treated group. This experiment shows that the diketone compound has an inhibitory effect on the tumor.

[0123] FIG. 3 shows the number of B cells in tumor tissues of BALB/C mice after 2 weeks of treatment in Experimental Example 3. As shown in the figure, 14 days after administration of the diketone compound, there is a very significant difference between a high-concentration treated group and a control group (p<0.001), and there is also a very significant difference between a low-concentration treated group and the control group (p<0.01). The number of B cells in tumor tissues in the treated groups increases significantly, and a better immune enhancement effect is shown in the high-concentration treated group.

[0124] FIG. 4 shows the level of aspartate aminotransferase in blood of BALB/C mice after 2 weeks of treatment in Experimental Example 3.

[0125] FIG. 5 shows the level of urea nitrogen in blood of BALB/C mice after 2 weeks of treatment in Experimental Example 3.

[0126] FIG. 6 shows the level of creatine kinase in the blood of BALB/C mice after 2 weeks of treatment in Experimental Example 3.

[0127] FIG. 7 shows changes in body weights of BALB/C mice during 2 weeks of treatment in Experimental Example 4.

[0128] FIG. 8 shows the level of aspartate aminotransferase in blood of BALB/C mice after 2 weeks of treatment in Experimental Example 4.

[0129] FIG. 9 shows the level of urea nitrogen in blood of BALB/C mice after 2 weeks of treatment in Experimental Example 4.

[0130] FIG. 10 shows the level of creatine kinase in the blood of BALB/C mice after 2 weeks of treatment in Experimental Example 4.

[0131] FIG. 11 shows the toxicity of experimental photosensitizer groups at different concentrations to human cervical cancer Hela cells in the dark and under light in Experimental Example 5. Administration concentrations are 6×10.sup.−3 mol/L, 3×10.sup.−6 mol/L, 6×10.sup.−7 mol/L, 6×10.sup.−9 mol/L, 3×10.sup.−9 mol/L, and 6×10.sup.−10 mol/L. As shown in the figure, when the diketone compound is given in the dark, its cytotoxicity to Hela is relatively low, indicating that the diketone compound has good safety. When the diketone compound is given under light, its cytotoxicity to Hela is relatively high, and the toxicity is significantly different from that of the diketone compound in the dark, indicating that the diketone compound has significant inhibitory activity to the tumor cell under light.

[0132] FIG. 12 shows the toxicity of experimental photosensitizer groups to human immortalized keratinocyte (Hacat cells) in the dark and under blue light and the toxicity of control drug 5-α aminolevulinic acid (ALA) groups to human immortalized keratinocyte (Hacat cells) in the dark and under red light in Experimental Example 5. Administration concentrations are 6×10.sup.3 mol/L, 6×10.sup.4 mol/L, 6×10.sup.5 mol/L, 6×10.sup.−6 mol/L, 6×10.sup.−7 mol/L, 6×10.sup.−8 mol/L, and 6×10.sup.−9 mol/L. As shown in the figure, ALA is more toxic to normal cells under light. The toxicity of the diketone compound to normal cells under light is significantly different from the toxicity of ALA to normal cells under light. It indicates that, under light, the diketone compound is less toxic to normal cells and has high selectivity.

[0133] FIG. 13 shows that strong fluorescence is produced on the skin of mice in the experimental photosensitizer group under excitation of light at the moment of administration. Moreover, 4 hours after the administration, the fluorescence on the skin of mice decreases significantly. 8 hours after the administration, the fluorescence on the skin of mice is approaching zero. The above results indicate that the selected diketone compound of the disclosure can be used for photodynamic therapy or diagnosis on the skin. Moreover, the diketone compound metabolizes rapidly and does not stay in or out of the body for a long time, thus having good safety.

DETAILED DESCRIPTION

[0134] The embodiments of the disclosure will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the disclosure, not to limit the scope of the disclosure. Those without specific conditions in the examples are generally implemented under conventional conditions or conditions recommended by the manufacturers. The reagents or instruments used without specifying the manufacturers are all conventional products that can be purchased commercially.

[0135] Bioactivity Experiment

[0136] Cells, reagents, and instruments involved in in vitro experiments in the following experimental examples are as follows:

[0137] Drugs:

[0138] Compounds BJMU-201 to BJMU-214, all purchased from companies such asJ&K Scientific Ltd., Beijing Ouhe Technology Co. Ltd., or Beijing InnoChem Science & Technology Co., Ltd.

[0139] Cells:

[0140] SW480 colon cancer cells, Hela cells, and B16 melanoma cell, all provided by the ATCC cell bank.

[0141] Culture Solutions:

[0142] RPMI 1640 medium and fetal bovine serum (FBS); DMEM (containing 2 mL-glutamine and Earle's BSS, 1.5 g/L NaHCO.sub.3, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate) and FBS.

[0143] Cell Culture:

[0144] Under the environment conditions of 37° C., 5% CO.sub.2 and saturated humidity, the cells were incubated to a confluence of 80%, and then digested with 0.25% trypsin-EDTA.

[0145] Main Reagents and Instruments Involved:

[0146] DMEM high-sugar medium, 1640 medium, and trypsin (Gibco, Maryland, USA); FBS (PAN, Germany); low-temperature refrigerated high-speed centrifuge (Beijing DLAB Scientific, China); horizontal shaker (ZD-9550, Kylin Bell Lab Instruments, Jiangsu); ultra-clean workbench (Suzhou Antai Airtech, Suzhou); FlexStation 3 multifunctional microplate reader (Molecular Devices).

Experimental Example 1 Evaluation of Compound's Toxicity to Tumor Cells

[0147] In this experimental example, the toxicity of the compound to tumor cells was tested by an MTT method including the following specific steps.

[0148] (1) Logarithmic cells were collected, the concentration of a cell suspension was adjusted, the cell suspension was added to a cell culture plate in a dose of 100 μL per well, and the cells were then added to a cell culture plate so that the density of the cells to be tested was adjusted to 1000-10000 wells (edge wells were filled with sterile PBS).

[0149] (2) The cells were incubated at 37° C. in the presence of 5% CO.sub.2 until the cells in a single layer covered the bottom of each well (96-well flat-bottom plate), and a drug in concentration gradients was added. Generally, the drug in 5 to 7 gradients was added in a dose of 100 μL per well, and 3 to 5 multi-wells were set.

[0150] (3) The cells were randomly divided into treated groups under light and treated groups in the dark, incubated at 37° C. in the presence of 5% CO.sub.2 for 16 h to 48 h, and observed with an inverted microscope.

[0151] (4) The cells were irradiated for 900 s with a laser having a wavelength of 450 nm to 480 nm (at an intensity of 110 mW/cm.sup.2), and the final light dose was about 100 J/cm.sup.2. After light irradiation, the cells were washed with PBS.

[0152] (5) An MTT solution (5 mg/ml, 0.5% MTT) was added in a dose of 20 μL per well to further incubate the cells for 4 h. If the drug could react with the MTT, centrifuging was carried out first, and then the culture solution was discarded and the cells were carefully washed twice or three times with PBS, and then the culture solution containing MTT was added.

[0153] (6) The culture was terminated and the culture solution in the wells was carefully removed.

[0154] (7) Dimethyl sulfoxide was added in a dose of 150 μL per well, and the plate was then shaken on a shaker at a low speed for 10 min to fully dissolve crystals. The absorbance at OD 490 nm of each well was measured with enzyme-linked immunoassay.

[0155] (8) Zero adjustment wells (medium, MTT, dimethyl sulfoxide) and control wells (cells, dissolving media of the same concentration, culture solution, MTT, dimethyl sulfoxide) were set at the same time.

[0156] Table 1-1 and Table 1-2 show the toxicity of 14 tested compounds (BJMU-201 to BJMU-214) to three tumor cells under light. The results show that the selected diketone compounds of the disclosure have relatively high cytotoxicity to the SW480 cells, Hela cells and B16 cells when given under light. The above results indicate that the compound of the disclosure has significant inhibitory activity to the tumor cells.

TABLE-US-00002 TABLE 1-1 Cytotoxicity of diketone compounds to different tumor cells under light IC.sub.50(μM) Compound SW480 Hela B16 BJMU-201 63.9 ± 10.5 59.9 ± 9.9  64.4 ± 10.6 BJMU-202 2.5 ± 0.5 2.6 ± 0.6 2.9 ± 0.1 BJMU-203 3.1 ± 0.5 2.8 ± 1.1 2.7 ± 0.9 BJMU-204  0.6 ± 0.05  0.6 ± 0.02  0.5 ± 0.03 BJMU-205 6.4 ± 1.9 5.7 ± 1.7 6.6 ± 1.8 BJMU-206 143.8 ± 31.9  141.1 ± 37.4  144.2 ± 32.5  BJMU-207 99.2 ± 25.7 93.3 ± 20.3 96.6 ± 26.3 BJMU-208 172.9 ± 10.8   162 ± 10.1 174.3 ± 10.9 

TABLE-US-00003 TABLE 1-2 Cytotoxicity of diketone compounds to different tumor cells under light IC.sub.50(mM) Compound SW480 Hela B16 BJMU-209 7.8 ± 0.48 8.2 ± 0.53 8.1 ± 0.45 BJMU-210 9.6 ± 0.6  9.9 ± 0.7  9.8 ± 0.7  BJMU-211 3.2 ± 0.23 3.8 ± 0.29 3.6 ± 0.26 BJMU-212 2.8 ± 0.21 2.7 ± 0.17   3 ± 0.27 BJMU-213 6.6 ± 0.37 6.4 ± 0.4  6.9 ± 0.41 BJMU-214 8.9 ± 0.5  8.8 ± 0.48   9 ± 0.51

[0157] Table 2 shows the toxicity of 14 tested compounds (BJMU-201 to BJMU-214) to three tumor cells in the dark. The results show that the selected diketone compounds of the disclosure have relatively low cytotoxicity to the SW480 cells, Hela cells and B16 cells when given in the dark. The above results indicate that the compound of the disclosure has good safety.

TABLE-US-00004 TABLE 2 Cytotoxicity of diketone compounds to different tumor cells in the dark IC.sub.50(mM) Compound SW480 Hela B16 BJMU-201 207.3 ± 55.6 194.2 ± 52.1 208.9 ± 56   BJMU-202 246.7 ± 12.2 205.7 ± 11.4 251.7 ± 12.3 BJMU-203 131.4 ± 5.5  197.7 ± 5.2  135.5 ± 5.6  BJMU-204 263.9 ± 39.6 253.5 ± 37.1 265.2 ± 39.9 BJMU-205 371.2 ± 29   360.4 ± 27.2 372.5 ± 29.3 BJMU-206 172.9 ± 10.8   162 ± 10.1 174.3 ± 10.9 BJMU-207 113.8 ± 49.7   175 ± 46.5 118.6 ± 50   BJMU-208 204.1 ± 73.5 253.2 ± 68.9 210.4 ± 74.1 BJMU-209 138.3 ± 28.6   139 ± 29.1 143.8 ± 30.7 BJMU-210 185.1 ± 42.1 179.9 ± 48.4 200.4 ± 50.2 BJMU-211   95 ± 21.3   99 ± 19.8   99 ± 22.5 BJMU-212   99 ± 15.8   98 ± 16.3   96 ± 14.9 BJMU-213   136 ± 35.1   142 ± 37.2   139 ± 42.0 BJMU-214   105 ± 23.8   109 ± 26.7   108 ± 25.2

Experimental Example 2 Evaluation of Compound's Inhibitory Effect on Bacteria

[0158] A single Escherichia coli colony was transferred from a solid Luria Bertani (LB) agar plate to 5 ml liquid LB medium and incubated at 37° C. for 12 h. Bacteria were collected by centrifugation (7000 rpm, 1 min) and washed with PBS buffer three times. The supernatant was discarded, and the remaining Escherichia coli cells were resuspended in the PBS buffer. The bacterial suspension was adjusted to make its optical density (OD600) to 1.0. The suspension was then diluted (5 times) with PBS buffer. The diluted Escherichia coli cell suspension was incubated in the dark at 37° C. for 15 min in the presence of a photosensitizer (diketone compound) solution with a concentration of 60 mM, and then irradiated for 200 s under a 15 mW/cm.sup.2 laser light (with a wavelength of 450 nm to 480 nm and in the final light dose of about 3 J cm.sup.2), and after the irradiation the bacterial suspension was serially diluted (10.sup.4 times) with PBS buffer. 100 μL of the diluted bacterial Escherichia coli cell was dispersed on a solid LB agar plate and incubated at 37° C. for 12 h to 16 h, and colonies formed were counted. In the meanwhile, a treated group in the dark and an untreated group in the dark were set; the inhibition rate was determined by dividing the number of colony forming units (cfu) killed in the treated group under light or the treated group in the dark by the number of colony forming units (cfu) killed in the untreated group in the dark.

[0159] The results are shown in Table 3. The diketone compounds have a significant inhibitory effect on the growth of Escherichia coli under light. The results indicate that the compound of the disclosure has significant antibacterial activity. The diketone compounds have a slight inhibitory effect on the growth of Escherichia coli in the dark. The results indicate that the compound of the disclosure has good safety.

TABLE-US-00005 TABLE 3 Inhibition rates of diketone compounds to Escherichia coli under light and in the dark Inhibition rate Inhibition rate Compound under light in the dark BJMU-201 58% ± 7% 4.9% ± 1%   BJMU-202    90 ± 3% 8.6% ± 1.7% BJMU-203 93% ± 9% 4.5% ± 0.9% BJMU-204 96% ± 1% 10.3% ± 2.1%  BJMU-205 80% ± 2%   2% ± 0.4% BJMU-206 46% ± 5% 11.5% ± 2.3%  BJMU-207 64% ± 4% 7.3% ± 1.5% BJMU-208 45% ± 7% 8% ± 3% BJMU-209 26% ± 3% 9.5% ± 2.1% BJMU-210 24% ± 4% 6.4% ± 3.2% BJMU-211 31% ± 2% 7.2% ± 1.3% BJMU-212 33% ± 7% 3.6% ± 2.5% BJMU-213    28 ± 5% 6.1% ± 3.3% BJMU-214 23% ± 3% 5.6% ± 2.1%

Experimental Example 3 Evaluation of Compound's Inhibitory Effect on Tumor (Animal Experiment)

[0160] 1. Photodynamic therapy experiments on tumors were carried out by using BJMU-204 as a photosensitizer and BALB/C mice as experimental animals.

[0161] Modeling mode: BALB/C mice were divided into 5 groups in total, 11-12 mice in each group. 4T1 cells (breast cancer cells in mice) were resuscitated and subcultured to a better cell state for tumorigenesis inoculation. BALB/C mice were locally depilated and disinfected, and tumor cells were injected into the mouse mammary fat pad in situ. Tumors were formed within 7 to 10 days.

[0162] Administration time: drug intervention was given to mice after tumor formation, and photodynamic therapy was given by intratumoral injection every other day under the irradiation of laser with a wavelength of 450 nm to 480 nm at a light intensity of 200 mW/cm.sup.2.

[0163] Administration mode: as shown in the table below, the photosensitizer was dissolved in physiological saline and injected in mice in groups 1-3 intratumorally, and the groups receiving photodynamic therapy were irradiated with light immediately after the injection.

TABLE-US-00006 No. Group Mode of administration Group 1 Photodynamic High-concentration (3%) intratumoral therapy group injection + light irradiation for 10 min at the (High- tumor site concentration group) Group 2 Photodynamic Low-concentration (0.5%) intratumoral therapy group injection + light irradiation for 10 min at the (Low- tumor site concentration group) Group 3 Control group-1 Intratumoral injection at a concentration of 3% Group 4 Control group-2 Light irradiation for 10 min at the tumor site Group 5 Control group-3 No administration, no light irradiation at the tumor site

[0164] 2. Mice in each experimental group were subjected to blood collection for serum separation and necropsy, and tumors were taken out and measured to obtain their wet weights; tumor tissues were fixed, pathological examinations were performed, and H. E. staining was used to observe the changes of tumors of the mice under an optical microscope; blood routine indicators (including white blood cell count (WBC), red blood cell count (RBC), lymphocyte count (LY), platelet count (PLT) and many other blood routine indicators) and blood biochemical indicators (including routine blood biochemical indicators) were tested and analyzed in the Laboratory of Peking University Third Hospital.

[0165] 3. Tumors were taken out of mice in each experimental group, a piece of tumor tissue about 1 cm*1 cm*0.5 cm in size was cut up and placed in a 10 ml EP tube. 5 ml of pancreatin was added for digestion. 30 min later, the digestion was ended. The digestion solution and the tumor tissue were sieved through a single cell sieve to obtain a tumor tissue single-cell suspension. B220 antibody (for flow cytometry) was added to the suspension and the resulting solution was stained with a PI staining solution; 30 min later, the staining was ended and the staining solution was removed; next, re-suspending was carried out with a PBS solution for test with a flow cytometry.

[0166] FIG. 1 shows size changes of tumor tissues in BALB/C mice during 2 weeks of treatment. As shown in the figure, 14 days after treatment, there is a very significant difference between a high-concentration treated group and a control group (p<0.001), and there is also a very significant difference between a low-concentration treated group and the control group (p<0.001). The tumor size is controlled and decreased, and this effect is more prominent in the high-concentration treated group. This experiment shows that the diketone compound has an inhibitory effect on the tumor.

[0167] FIG. 2 shows weight changes of tumor tissues in BALB/C mice after 2 weeks of treatment. As shown in the figure, 14 days after treatment, there is a very significant difference between a high-concentration treated group and a control group (p<0.001), and there is also a very significant difference between a low-concentration treated group and the control group (p<0.001). Based on weight ratios of tumors, an obvious growth inhibition effect is shown in the treated groups and a better inhibitory effect on the tumor is shown in the high-concentration treated group. This experiment shows that the diketone compound has an inhibitory effect on the tumor.

[0168] FIG. 3 shows the number of B cells in tumor tissues of BALB/C mice after 2 weeks of treatment. As shown in the figure, 14 days after treatment, there is a very significant difference between a high-concentration treated group and a control group (p<0.001), and there is also a very significant difference between a low-concentration treated group and the control group (p<0.01). The number of B cells in tumor tissues in the treated groups increases significantly, and a better immune enhancement effect is shown in the high-concentration treated group.

[0169] Table 4 shows the results of blood routine tests of BALB/C mice after 2 weeks of treatment. The number of platelets and white blood cells in the blood of mice decreases, and there is a very significant difference between the treated groups and the control groups (p<0.001). The number of platelets and white blood cells decreases in the treated groups, indicating that the immune function in the treated groups is enhanced. This experiment shows that photodynamic therapy involving the diketone compounds enhance immunity.

[0170] FIG. 4 to FIG. 6 show the blood biochemical data of BALB/C mice after 2 weeks of treatment. FIG. 4, FIG. 5, and FIG. 6 respectively correspond to the levels of aspartate aminotransferase, urea nitrogen, and creatine kinase.

[0171] The blood routine data (except LY and PLT) of BALB/C mice in Table 4 and the blood biochemical data in FIG. 4 to FIG. 6 show that diketone compounds will not cause liver, kidney and blood toxicity after continuous treatment for 14 days at high or low concentration therapeutic doses.

TABLE-US-00007 TABLE 4 12 h mouse blood routine results of mice after 2 weeks of treatment in BALB/C mice Photodynamic Photodynamic therapy group therapy group (High-concentration (Low-concentration group) group) Control group-1 Control group-2 Control group-3 Hematocrit (HCT)  32.4 ± 12.48 32.64 ± 12.12 28.8 ± 5.76 28.44 ± 5.88  27.6 ± 1.68 Hemoglobin (HGB)  123 ± 40.8 141.6 ± 42.72   117 ± 26.64   132 ± 16.92 132 ± 0  Mean corpuscular hemoglobin (MCH) 18.43 ± 1.93  20.1 ± 1.84 18.43 ± 0.67  21.47 ± 2.2  22.1 ± 1.84 Mean corpuscular hemoglobin 387.5 ± 40.38   447 ± 48.54 404.5 ± 26.01 456.5 ± 32.02  479 ± 29.7 concentration (MCHC) Mean corpuscular volume (MCV) 47.5 ± 1.95 45.04 ± 1.21  45.65 ± 1.35  45.57 ± 1.75  46.05 ± 1.06  Mean platelet volume (MPV) 6.63 ± 0.76 6.23 ± 0.61 6.1 ± 0   6.55 ± 0.51 6.3 ± 0   Plateletocrit (PCT) 0.24 ± 0.12 0.48 ± 0.24 0.24 ± 0    0.6 ± 0.24 0.36 ± 0   Platelet distribution width (PDW) 15.13 ± 0.76  13.37 ± 1.47  16.2 ± 0   14.58 ± 0.99  14.2 ± 0   Platelet count (PLT)   267 ± 167.28 494.4 ± 387.6   312 ± 125.04 766.32 ± 340.56   360 ± 339.36 Red blood count (RBC) 6.81 ± 2.51 7.22 ± 2.62 6.33 ± 1.36 6.24 ± 1.24   6 ± 0.51 Red blood cell volume distribution 14.58 ± 0.84  13.78 ± 0.59  14.18 ± 0.75  14.06 ± 0.91  13.95 ± 0.07  width (RDW) White blood cell count (WBC) 136.8 ± 31.45 193.5 ± 70   352.8 ± 45.95  370.2 ± 133.97  472.2 ± 138.31

Experimental Example 4 Evaluation of Compound's Phototoxicity to Healthy Mice (Animal Experiment)

[0172] 1. Phototoxicity Evaluation Experiments were Carried Out by Using BJMU-204 as a Photosensitizer and BALB/C Mice as Experimental Animals.

[0173] BALB/C mice were divided into 2 groups in total, 9 mice in each group. The mice were fed adaptively for 2 days. Mice in a control group were fed normally, and mice in an experimental group were given photodynamic therapy by subcutaneous injection every other day under the irradiation of laser with a wavelength of 450 nm to 480 nm at a light intensity of 200 mW/cm.sup.2.

[0174] Administration mode: as shown in the table below, the photosensitizer was dissolved in physiological saline and subcutaneously injected in mice in the experimental group, and the group receiving photodynamic therapy was irradiated with light immediately after the injection.

TABLE-US-00008 No. Mode of administration Experimental High-concentration (3%) subcutaneous injection + light group irradiation for 10 min Control Normal feeding group

[0175] 2. The mice in the experimental group and the control group were weighed every other day. At the end of the experiment, the mice were subjected to blood collection for serum separation and necropsy, and blood routine indicators (including WBC, RBC, LY, PLT and many other blood routine indicators) and blood biochemical indicators (including routine blood biochemical indicators) were tested and analyzed in the Laboratory of Peking University Third Hospital.

[0176] Table 5 shows the blood routine data of BALB/C mice.

TABLE-US-00009 TABLE 5 12 h mouse blood routine results of mice after 2 weeks of treatment for BALB/C mice Experimental Control group group Hematocrit (HCT) 55.88 ± 16.52 45.22 ± 2.83  Hemoglobin (HGB) 187.63 ± 42.41  160.22 ± 12.07  Mean corpuscular hemoglobin (MCH) 16.73 ± 0.56  16.18 ± 0.34  Mean corpuscular hemoglobin 363.13 ± 12.52  354.11 ± 8.19  concentration (MCHC) Mean corpuscular volume (MCV) 46.07 ± 0.53  45.71 ± 0.6  Mean platelet volume (MPV) 6.33 ± 0.49 6.23 ± 0.37 Plateletocrit (PCT) 0.21 ± 0.08 0.17 ± 0.03 Platelet distribution width (PDW) 14.3 ± 0.91 14.84 ± 0.6  Platelet count (PLT) 321.50 ± 124.76   290 ± 50.23 Red blood count (RBC) 10.52 ± 1.45   9.9 ± 0.72 Red blood cell volume distribution 13.16 ± 1.33  12.67 ± 0.54  width (RDW) White blood cell count (WBC) 7.03 ± 1.45 6.56 ± 0.54

[0177] FIG. 7 shows changes of body weight of BALB/C mice during 2 weeks of treatment. As shown in the figure, 14 days after treatment, there is no significant difference in body weight between the control group and the experimental group (p<0.05), and there is no significant change in the body weight of the experimental group during the treatment period. This experiment shows that the diketone compound has little phototoxicity to normal mice.

[0178] FIG. 8 to FIG. 10 show the blood biochemical data of BALB/C mice after 2 weeks of treatment. FIG. 8, FIG. 9, and FIG. 10 respectively correspond to the levels of aspartate aminotransferase, urea nitrogen, and creatine kinase.

[0179] The blood routine data of BALB/C mice in Table 5 and the blood biochemical data in FIG. 8 to FIG. 10 show that diketone compounds will not cause liver, kidney and blood toxicity after continuous treatment to healthy mice for 14 days at high concentration therapeutic doses.

Experimental Example 5 Evaluation on Compound's Cytotoxicity

[0180] In this experimental example, the toxicity of the compound BJMU-204 to cells was tested by an MTT method including the following specific steps.

[0181] (1) Logarithmic phase cells were collected, the concentration of a cell suspension was adjusted, the cell suspension was added to a cell culture plate in a dose of 100 μL per well, and the cells were then added to a cell culture plate so that the density of the cells to be tested was adjusted to 1000-10000 wells (edge wells were filled with sterile PBS).

[0182] (2) The cells were incubated at 37° C. in the presence of 5% CO.sub.2 until the cells in a single layer covered the bottom of each well (96-well flat-bottom plate), and a drug in concentration gradients was added. Generally, the drug in 5 to 7 gradients was added in a dose of 100 μL per well, and 3 to 5 wells were set for each concentration of a drug.

[0183] (3) The cells were randomly divided into treated groups under light and treated groups in the dark, then incubated at 37° C. in the presence of 5% CO.sub.2 for 16-48 h, and observed with an inverted microscope. Cells in the experimental photosensitizer-light irradiation group were incubated for 15 min and then irradiated with light. Cells in the control 5-α aminolevulinic acid (ALA)-light irradiation group were incubated for 3 h and then irradiated with light (the control drug ALA produced porphyrins for light absorption only after 3 h of incubation). MTT solution was added directly after the mice in the experimental photosensitizer-no light irradiation group were incubated for 15 min or after the mice in the control ALA-no light irradiation group were incubated for 3 h.

[0184] (4) For the experimental photosensitizer-light irradiation group, the cells were irradiated for 900 s with a laser having a wavelength of 450 nm to 480 nm (at an intensity of 110 mW/cm.sup.2), and the final light dose was about 100 J/cm.sup.2. After light irradiation, the cells were washed with PBS.

[0185] For the control ALA-light irradiation group, the cells were irradiated for 900 s with a laser having a wavelength of 630 nm to 650 nm (at an intensity of 110 mW/cm.sup.2), and the final light dose was about 100 J/cm.sup.2. After light irradiation, the cells were washed with PBS.

[0186] (5) MTT solution (5 mg/ml, 0.5% MTT) was added in a dose of 20 μL per well to further incubate the cells for 4 h. If the drug could react with the MTT, centrifuging was carried out first, and then the culture solution was discarded and the cells were carefully washed twice or three times with PBS, and then the culture solution containing MTT was added.

[0187] (6) The culture was terminated and the culture solution in the wells was carefully removed.

[0188] (7) Dimethyl sulfoxide was added in a dose of 150 μL per well, and the plate was then shaken on a shaker at a low speed for 10 min to fully dissolve crystals. The absorbance at OD 490 nm of each well was measured with enzyme-linked immunoassay.

[0189] (8) Zero adjustment wells (medium, MTT, dimethyl sulfoxide) and control wells (cells, dissolving media of the same concentration, culture solution, MTT, dimethyl sulfoxide) were set at the same time.

[0190] FIG. 11 shows the toxicity of experimental photosensitizer groups to human cervical cancer Hela cells in the dark and under light. Administration concentrations are 6×10.sup.3 mol/L, 3×10.sup.−6 mol/L, 6×10.sup.7 mol/L, 6×10.sup.−9 mol/L, 3×10.sup.4 mol/L, and 6×10.sup.−10 mol/L. The results show that the selected diketone compound of the disclosure has relatively low cytotoxicity to the human cervical cancer Hela cells when given in the dark. The above results indicate that the compound of the disclosure has good safety. The results show that the selected diketone compound of the disclosure has relatively high cytotoxicity to the human cervical cancer Hela cells when given under blue light. The above results indicate that the compound of the disclosure has significant inhibitory activity to the tumor cell under light.

[0191] FIG. 12 shows the toxicity of experimental photosensitizer groups to human immortalized keratinocyte (Hacat cells) in the dark and under blue light and the toxicity of control drug ALA groups to human immortalized keratinocyte (Hacat cells) in the dark and under red light. Administration concentrations are 6×10.sup.−3 mol/L, 6×10.sup.−4 mol/L, 6×10.sup.−5 mol/L, 6×10.sup.−6 mol/L, 6×10.sup.−7 mol/L, 6×10.sup.−8 mol/L, and 6×10.sup.−9 mol/L. The results show that the selected diketone compound of the disclosure has relatively low cytotoxicity to the human immortalized keratinocyte (Hacat cells) when given in the dark and under blue light. The control drug ALA has relatively high cytotoxicity to the human immortalized keratinocyte (Hacat cells) when given under red light. The toxicity of the selected diketone compound of the disclosure to normal cells under light is significantly different from the toxicity of ALA to normal cells under light. The above result indicates that, under light, the compound of the disclosure has low toxicity to normal cells and has high selectivity.

Experimental Example 6 Evaluation of Compound's Fluorescence on Mouse Skin

[0192] This experimental example evaluates the potential of compound BJMU-204 as a photosensitizer administrated to skin. Specific steps are as follows.

[0193] The BALB/C mice were depilated, and the skin was thoroughly cleaned, and then the mice were kept from light for more than 4 h. A small animal live imager IVIS SPECTRUM was used to take pictures of the backs of the mice and the excitation was carried out for 2 s at wavelengths of 480 nm and 520 nm. The mice not administered with the photosensitizer were photographed as a control. The mice with 100 μL of a 30% compound administrated to the skin (with a diameter of 10 mm) of the back were photographed and the fluorescence intensity was recorded, and then pictures were taken at 2 h, 4 h, 8 h, and 12 h after the administration of the compound. Each image was processed using Living Image 4.3.1, and average fluorescence in a circular field of view with a diameter of 10 mm on the skin was measured. Ambient light and the background fluorescence of mouse skin were corrected on the basis of fluorescence values of the backs of the mice without administration of the compound.

[0194] FIG. 13 shows that strong fluorescence is produced on the skin of mice in the experimental photosensitizer group under excitation of light at the moment of administration. The results indicate that the selected diketone compound of the disclosure can be used for photodynamic therapy or diagnosis on the skin. The diketone compound quickly produces fluorescence after being excited by light, and there is no need to wait for a long time. Moreover, 4 hours after the administration, the fluorescence on the skin of mice decreases significantly. 8 hours after the administration, the fluorescence on the skin of mice is approaching zero. The above results indicate that the compound of the disclosure is rapidly metabolized, does not stay in or out of the body for a long time, and has good safety.

[0195] The above experiments show that the selected diketone compound of the disclosure exhibits a significant killing activity to tumor cells under light, and exhibits a significant inhibitory activity to the growth of bacteria under light; in the dark, the diketone compound does not exhibit the two activities and has low toxicity. The selected diketone compound of the disclosure also significantly inhibits the activity of tumor cells in mice, and significantly enhances immune activity. In summary, the selected diketone compound of the disclosure can be used in photodynamic therapy to achieve the purpose of treating cancers, microorganism infections and related complications, and can also be used in the treatment of immune-related diseases.

[0196] While the specific embodiments of the disclosure have been described in detail, those skilled in the art will understand that according to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the disclosure. The full scope of the disclosure is given by the appended claims and any equivalents thereof.