Application of Mitoquinone mesylate in resisting mycobacterium tuberculosis infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of Mitoquinone mesylate in resisting mycobacterium tuberculosis infection. According to the application disclosed by the invention, the biological effect of the Mitoquinone mesylate in resisting mycobacterium tuberculosis infection is found, the MIC of the Mitoquinone mesylate to a mycobacterium tuberculosis standard strain can reach 1.25 mu M, and the MIC of the Mitoquinone mesylate to a drug-resistant mycobacterium tuberculosis clinical isolated strain is more concentrated to be 1.25 mu M. In conclusion, the autophagy regulation effect of the Mitoquinone mesylate is remarkable, and the Mitoquinone mesylate can become a new treatment medicine in the anti-mycobacterium tuberculosis infection process. The clinical guiding significance is realized; moreover, the Mitoquinone mesylate is easy to obtain and low in cost, the prepared medicine takes effect quickly, and the Mitoquinone mesylate has actual production and application prospects. The Mitoquinone mesylate is high in safety, small in side effect and good in patient compliance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of Mitoquinone mesylate in anti-Mycobacterium tuberculosis infection. Background Art

[0002] Mycobacterial infection is a disease caused by bacteria of the genus Mycobacterium, including Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Mycobacterial infection can affect multiple organ systems, including the lungs, skin, lymph nodes, bones, etc. Mycobacterium tuberculosis is the main pathogen causing tuberculosis and is mainly transmitted through the respiratory tract. After infection, patients may develop latent infection or active tuberculosis. Latent infection may turn into active disease under specific conditions (such as immune deficiency, diabetes, etc.) (Lunshan Lu. Diagnosis and prevention and control measures of Mycobacterium tuberculosis infection [J]. World Latest Medical Information Digest, 2016.).

[0003] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, mainly transmitted through the air, especially when patients cough, sneeze or spit out droplets containing the germs (SeunNurudeen Akorede, Ayodotun Edward Ajayi et al. “Knowledge of Tuberculosis among Tuberculosis Patients Attending Federal Medical Centre, Owo, Ondo State, Nigeria.” Unnes Journal of Public Health (2021)). Tuberculosis can affect multiple organs, but lung infection is the most common, known as pulmonary tuberculosis. Globally, tuberculosis remains a serious public health problem. According to the World Health Organization, there are approximately 10 million new cases each year, of which approximately 1.3 million people die from tuberculosis (Guoyin Shen. Will you definitely get tuberculosis if you have been in contact with tuberculosis patients? Food and Health, 2024).

[0004] The main method for treating tuberculosis is the use of anti-tuberculosis drugs, including isoniazid, rifampicin, ethambutol, and pyrazinamide, etc. (Anny Thuraidah, Rima Agnes Widya Astuti et al. “Anemia dan Lama KonsμMsiObat AntiTuberculosis.” Medical laboratory technology (2017).). The treatment is divided into the intensive phase and the consolidation phase. In the intensive phase, treatment is carried out twice a day to reduce the number of bacteria in the body, while the consolidation phase lasts longer but with a lower dose. However, due to the problem of drug resistance of Mycobacterium tuberculosis, the treatment has become complex and difficult (Xu Yin, Meng Xianmin, Zhang Yongxin. Research progress on the epidemic situation of drug-resistant tuberculosis and anti-tuberculosis drugs [J]. Shanghai Medical & Pharmaceutical Journal, 2013.). Drug-resistant tuberculosis (especially multi-drug resistant tuberculosis, MDR-TB) has become a major challenge to global public health (Shen Xin, Song Shenchao, Lei Shiguang. Research progress on the epidemic situation of drug-resistant tuberculosis and prevention and control countermeasures [J]. Occupation and Health, 2017.). Its treatment cycle is long, generally requiring 18 to 20 months. During this period, patients have poor compliance, and some treatment drugs have significant side effects, further increasing the difficulty of treatment. Moreover, the phenomenon of persister bacteria of Mycobacterium tuberculosis poses a major obstacle. Persister bacteria can survive under the action of antibiotics, which is a hidden danger for recurrence after treatment and the occurrence of latent infections. According to the report of the World Health Organization (WHO), about 3.3% of new tuberculosis cases and 18% of previously treated cases were multi-drug resistant or rifampicin-resistant tuberculosis globally in 2019. The treatment success rate of drug-resistant tuberculosis is much lower than that of non-drug-resistant tuberculosis. The global treatment success rate of multi-drug resistant tuberculosis is only 63%, while the cure rate of ordinary tuberculosis can reach 88%. In China, the treatment success rate of multi-drug resistant tuberculosis patients who started treatment in 2020 was only 51% by 2022.

[0005] Facing such a severe drug-resistant situation, it has become an urgent task to develop new, highly effective, low-toxic and economically accessible tuberculosis inhibitors. The research and development of new drugs should not only target drug-resistant strains, but also overcome the problem of persister bacteria, and at the same time combine personalized and precise treatment plans to improve the cure rate, shorten the treatment course and reduce side effects. Only through continuous scientific research investment and policy support, and accelerating the research, development and application of new inhibitors, can we effectively respond to the challenge of drug-resistant tuberculosis, bring hope to patients and safeguard global public health security.

[0006] The molecular formula of Mitoquinone mesylate (MitoQ) is C 38 H 47 O 7PS, with a molecular weight of 678.81 and a CAS number of 84595-59-4, is an oily substance with a high purity (≥98%) and good solubility. It is soluble in solvents such as DMSO, methanol, and DMF, but insoluble in water. Mitoquinone mesylate is a mitochondria-targeted antioxidant based on TPP that targets mitochondria by covalently linking to a lipophilic triphenylphosphonium cation. Studies have shown that MitoQ has been proven to improve insulin resistance induced by a high-fat diet and activate the Nrf2 antioxidant system, thereby alleviating metabolic disorders; MitoQ has shown a protective effect against myocardial ischemia-reperfusion injury in animal experiments, and MitoQ shows an inhibitory effect on the proliferation of breast cancer and glioma cells by inhibiting the oxidative stress and apoptosis signaling pathways of cancer cells. There is currently no report on its activity against Mycobacterium tuberculosis infection. Summary of the Invention

[0007] The present invention discovers that Mitoquinone mesylate has the effect of inhibiting the activity of Mycobacterium tuberculosis. Based on this, the present invention is completed.

[0008] In a first aspect, the present invention provides a cleaning agent, and the cleaning agent includes Mitoquinone mesylate.

[0009] Furthermore, the cleaning agent inhibits the activity of Mycobacterium tuberculosis.

[0010] Even further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strain and / or Mycobacterium tuberculosis clinical isolate.

[0011] In a second aspect, the present invention provides a drug for treating Mycobacterium tuberculosis infection, and the drug includes Mitoquinone mesylate.

[0012] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strain and / or Mycobacterium tuberculosis clinical isolate.

[0013] Furthermore, one or more pharmaceutically acceptable carriers or excipients can be added to the drug.

[0014] Even further, the carrier material includes, but is not limited to, one or more of water-soluble carrier materials, poorly soluble carrier materials, and / or enteric-soluble carrier materials.

[0015] Furthermore, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acids.

[0016] Furthermore, the poorly soluble carrier material includes, but is not limited to, one or more of ethyl cellulose and / or cholesterol stearate.

[0017] Furthermore, the enteric carrier material includes, but is not limited to, one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0018] Furthermore, the drug can be formulated into various dosage forms, including, but not limited to, one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, buccal tablets, suppositories, and / or lyophilized powder injections.

[0019] Furthermore, the preparation can be one or more of an ordinary preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0020] Furthermore, various carriers well-known in the art can be widely used in the tablets, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters, and / or lubricants.

[0021] Furthermore, the diluents and absorbents include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0022] Furthermore, the wetting agents and binders include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and / or polyvinylpyrrolidone.

[0023] Furthermore, the disintegrants include, but are not limited to, one or more of dry starch, alginate, agar powder, laminarin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium dodecyl sulfate, methyl cellulose, and / or ethyl cellulose.

[0024] Furthermore, the disintegration inhibitors include, but are not limited to, sucrose, glyceryl tristearate, cocoa butter, and / or hydrogenated oils, etc.

[0025] Furthermore, the absorption promoters include, but are not limited to, one or more of quaternary ammonium salts and / or sodium dodecyl sulfate.

[0026] Furthermore, the lubricants include, but are not limited to, one or more of talc, silica, corn starch, stearates, boric acid, liquid paraffin, and / or polyethylene glycol.

[0027] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets, and multi-layer tablets.

[0028] Furthermore, various carriers well-known in the art can be widely used in the pills, including diluents and absorbents, binders, and / or disintegrants.

[0029] Furthermore, the diluents and absorbents include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, and / or talc.

[0030] Furthermore, the binders include, but are not limited to, one or more of gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, and / or batter.

[0031] Furthermore, the disintegrants include, but are not limited to, one or more of agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and / or ethylcellulose.

[0032] Furthermore, various carriers well-known in the art can be widely used in the suppositories, including, but not limited to, one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and / or semi-synthetic glycerides.

[0033] Furthermore, the injectable preparations include, but are not limited to, one or more of solutions, freeze-dried powder injections, and / or suspensions.

[0034] Furthermore, all diluents commonly used in the art can be used in the injectable preparations, including, but not limited to, one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, and / or polyoxyethylene sorbitan fatty acid esters.

[0035] Furthermore, in order to prepare an isotonic injection solution for the injectable preparations, an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional solubilizers, buffers, and / or pH regulators can be added to the injectable preparations.

[0036] Furthermore, coloring agents, preservatives, fragrances, flavoring agents, sweeteners, or other materials can also be added to the pharmaceutical preparations if needed for the various preparations.

[0037] In a third aspect, the present invention provides the use of Mitoquinone mesylate in the preparation of a drug for treating Mycobacterium tuberculosis infection, and the drug has at least one of the following uses:

[0038] Treating Mycobacterium tuberculosis infection;

[0039] Preventing and / or treating tuberculosis.

[0040] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis clinical isolates.

[0041] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the anti - Mycobacterium tuberculosis infection drug.

[0042] Furthermore, the carrier is selected from water - soluble carriers, poorly soluble carriers or enteric - soluble carriers.

[0043] Furthermore, the dosage form of the anti - Mycobacterium tuberculosis infection drug is selected from tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, suppositories or lyophilized powder injections.

[0044] Furthermore, the anti - Mycobacterium tuberculosis infection drug is selected from one or more of conventional preparations, sustained - release preparations, controlled - release preparations and / or various particulate drug delivery systems.

[0045] Furthermore, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents or other materials may be added to the pharmaceutical preparation for various preparations.

[0046] Furthermore, the anti - Mycobacterium tuberculosis infection drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissues by injection, penetration, absorption, physical or chemical - mediated methods; or it can be mixed or encapsulated with other substances and then introduced into the body.

[0047] Beneficial effects

[0048] The present invention discovers the biological role of Mitoquinone mesylate in anti - Mycobacterium tuberculosis infection. The MIC of Mitoquinone mesylate against the standard strain of Mycobacterium tuberculosis can reach 1.25 μM, and the MIC of Mitoquinone mesylate against clinically isolated strains of drug - resistant Mycobacterium tuberculosis mostly concentrates on 1.25 μM. In summary, the autophagy - regulating effect of Mitoquinone mesylate is significant. In anti - Mycobacterium tuberculosis infection, Mitoquinone mesylate can become a new therapeutic drug, which has guiding significance for clinical practice; moreover, Mitoquinone mesylate is simple to obtain, low in cost, has a quick effect when made into a drug, and has a prospect of practical production application. Mitoquinone mesylate has high safety, few side effects and good patient compliance. Brief description of the drawings

[0049] Figure 1 It is the detection result of the antibacterial activity of Mitoquinone mesylate against clinically isolated strains of multi - drug - resistant Mycobacterium tuberculosis.

[0050] Figure 2Results of the cytotoxicity test of Mitoquinone mesylate on THP-1 cells. Detailed implementation manners

[0051] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is used to help understand the present invention, but does not constitute a limitation on the present invention. In addition, the technical features involved in the following described implementation manners can be combined with each other as long as they do not conflict with each other.

[0052] Unless otherwise specified, the test methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples can all be obtained through conventional commercial channels.

[0053] Term explanation

[0054] The "Mycobacterium tuberculosis standard strain" mentioned in the present invention is H37Rv (ATCC27294), which is purchased from the American Type Culture Collection (ATCC).

[0055] The "human blood-derived monocyte THP1 cell line" mentioned in the present invention is purchased from the Cell Bank of the Chinese Academy of Sciences.

[0056] The "Mitoquinone mesylate", "mitoxantrone mesylate" and "MitoQ" mentioned in the present invention: The molecular formula is C 38 H 47 O 7 PS; the molecular weight is 678.82 g / mol; the CAS number is 845959-50-4, and its structural formula is as follows:

[0057]

[0058] CCK-8 method: Used to determine the cell viability. The CCK-8 method uses a reagent containing WST-8 [(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which can be reduced by dehydrogenases in the mitochondria of living cells to a water-soluble orange-yellow formazan dye under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate. The amount of formazan produced is proportional to the number of living cells.

[0059] THP-1 cells: A cell line derived from acute monocytic leukemia, initially isolated and cultured by Japanese scientists from the blood of a patient with acute monocytic leukemia. This cell line has typical monocyte characteristics, including surface expression of Fc receptors and C3b receptors, but lacks surface or cytoplasmic immunoglobulins. THP-1 cells are widely used in in vitro experiments to study biological processes such as immune regulation, inflammatory responses, and cell differentiation.

[0060] Example 1 Detection of the antibacterial activity of standard strains of Mycobacterium tuberculosis

[0061] A. Preparation of a suspension of standard strains of Mycobacterium tuberculosis

[0062] Culture Mycobacterium tuberculosis (MycobacteriμM tuberculosis, ATCC27294) to the logarithmic phase, scrape the strains in the logarithmic phase, place the colonies in a grinding bottle and grind evenly, dilute the bacterial suspension with 7H9 medium and adjust the turbidity to 1 McFarland, and then add it to 7H9 liquid medium at a ratio of 1:20 and mix well to obtain a Mycobacterium tuberculosis suspension.

[0063] B. Minimum inhibitory concentration (MIC)

[0064] (1) Add 7H9 liquid medium, and then add the Mycobacterium tuberculosis suspension so that the final volume in each well is 200 μl, and the final concentration of the bacterial suspension is 4×10 5 CFU / mL. The specific final drug concentrations in each well are shown in Table 1;

[0065] (2) Incubate the culture plate for 7 days;

[0066] (3) Add the chromogenic solution and observe the color change of the plate;

[0067] (4) Read the minimum inhibitory concentration (Minimal inhibitory concentration, MIC).

[0068] Table 1. Drug concentrations of Mitoquinone mesylate

[0069] 1 2 3 4 5 6 7 8 9 10 11 12 A Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM B Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM C Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM D Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM E Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM F Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM G Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM H Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM

[0070] Method for reading MIC: MIC is the drug concentration that can inhibit the growth of 90% of the colonies. The blue wells indicate no bacterial growth, and the pink wells indicate bacterial growth. The lowest drug concentration that prevents the color from changing from blue to pink is recorded as the MIC that can inhibit bacterial growth.

[0071] The negative control is the culture medium without drugs and bacterial liquid, i.e., the first column. The positive control is the bacterial-containing culture medium without drugs, i.e., the second column; when the positive control is pink and the negative control is blue, the MIC data measured in this batch are valid.

[0072] C. Test results

[0073] The MIC of Mitoquinone mesylate against the standard strain of Mycobacterium tuberculosis is 1.25.

[0074] Example 2 Detection of antibacterial activity of clinically isolated multidrug-resistant Mycobacterium tuberculosis strains

[0075] A. Test method

[0076] According to the method in Example 1, the in vitro antibacterial activity of Mitoquinone mesylate against 12 clinically isolated strains of Mycobacterium tuberculosis was detected. The specific final drug concentrations in each well are shown in Table 2.

[0077] The activity of Mitoquinone mesylate against clinically isolated multidrug-resistant strains was determined by the microdilution method in a 96-well plate. The drug-resistant strain samples were MDR-1, MDR-2, MDR-3, MDR-4, MDR-5, MDR-6, MDR-7, MDR-8, MDR-9, MDR-10, MDR-11, and MDR-12.

[0078] Table 2. Distribution table of each concentration of Mitoquinone mesylate in a 96-well plate

[0079]

[0080] B. Test results

[0081] As Figure 1 and shown in Table 3, for the MIC results of Mitoquinone mesylate against 12 clinically multi-drug resistant bacteria, the MIC of most strains concentrated at 1.25 μM; Mitoquinone mesylate has a good inhibitory effect on Mycobacterium tuberculosis in vitro.

[0082] Table 3. MIC results of Mitoquinone mesylate against 12 clinically multi-drug resistant bacteria

[0083] Serial number Strain number Mitoquinone mesylate MIC (μM) 1 4 1.25 2 5 1.25 3 36 1.25 4 64 5 5 80 1.25 6 112 0.625 7 130 2.5 8 154 2.5 9 178 0.3125 10 180 1.25 11 185 1.25 12 205 0.625

[0084] Example 3 Mitoquinone mesylate inhibition of Mycobacterium tuberculosis cytotoxicity test

[0085] A. Test method

[0086] (1) Culture THP-1 cells in RPMI 1640 complete medium and passage them every 2 days.

[0087] (2) Take cells in good growth state, add PMA, and inoculate and culture them at 1×10 5 cells / well to induce adherent macrophages.

[0088] (3) Aspirate and discard the old medium, add medium to the culture plate to dilute Mitoquinone mesylate to final concentrations of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM; the negative control wells do not contain the drug, and incubate the cell plate.

[0089] (4) Then aspirate and discard the medium, and operate according to the instructions of the CCK-8 cell proliferation / cytotoxicity kit to detect the cytotoxicity of the drug.

[0090] (5) Calculate the survival rate (%) of THP1 cells after stimulation with Mitoquinone mesylate = (OD450nm [experimental group] - OD450nm [negative control group] / OD450nm [positive control group] - OD450nm [negative control group]) × 100%.

[0091] B. Test results

[0092] As Figure 2 shown, after treating THP-1 with 5 μM of Mitoquinone mesylatemesylate for 24 h, the cell survival rate was 104.75 ± 3.96%, indicating its low cytotoxicity ( Figure 2 ).

[0093] In summary, the test results show that Mitoquinone mesylate exhibits good antibacterial activity against Mycobacterium tuberculosis and can significantly inhibit the growth of Mycobacterium tuberculosis. In addition, Mitoquinone mesylate has a high survival rate for THP-1 cells. Therefore, Mitoquinone mesylate can be used as a drug for preparing anti-Mycobacterium tuberculosis agents.

[0094] Through a series of tests, it was found that not all antioxidants and similar compounds have the effect of anti-Mycobacterium tuberculosis.

Claims

A cleaning agent comprising Mitoquinone mesylate.

2. The cleaning agent according to claim 1, which has the function of inhibiting the activity of Mycobacterium tuberculosis.

3. The cleaning agent according to claim 2, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis and / or a clinical isolate of Mycobacterium tuberculosis.

4. A drug for preventing Mycobacterium tuberculosis infection, comprising Mitoquinone mesylate.

5. The drug according to claim 4, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis and / or a clinical isolate of Mycobacterium tuberculosis.

6. The drug according to claim 4, comprising one or more pharmaceutically acceptable carriers or excipients.

7. The drug according to claim 4, wherein the drug preparation is selected from one or more of ordinary preparations, sustained-release preparations, controlled-release preparations and / or various microparticle delivery systems.

8. Use of Mitoquinone mesylate in the preparation of a drug for resisting Mycobacterium tuberculosis infection, wherein the drug comprises at least one of the following uses: Against Mycobacterium tuberculosis infection; Prevent and / or treat tuberculosis.

9. The use according to claim 8, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis and / or a clinical isolate of Mycobacterium tuberculosis.

10. The use according to claim 8, wherein the drug for preventing Mycobacterium tuberculosis infection is introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or is introduced into the body after being mixed or wrapped with other substances.

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