Application of tanshinone IIA in carbonic anhydrase inhibitor and product

Abstract

The invention provides application of tanshinone IIA in a carbonic anhydrase inhibitor and a product, and belongs to the field of medicines. According to the application of the tanshinone IIA in the carbonic anhydrase inhibitor, the tanshinone IIA can be combined with carbonic anhydrase to inhibit the activity of the carbonic anhydrase, and the tanshinone IIA can be used in drugs for reducing intraocular pressure, inducing diuresis, correcting metabolic acid / alkali poisoning, increasing vascular oxygen flux, improving pulmonary ventilation, expanding cerebral vessels, inducing tumor cell apoptosis or inhibiting osteoclast bone resorption. The carbonic anhydrase inhibitor can be used for treating carbonic anhydrase-related cardiovascular diseases, altitude sickness, ocular diseases with intraocular hypertension as symptoms, osteoporosis or diseases caused by reduction of bone mineral density, inducing diuresis or correcting metabolic acid / alkali poisoning, and provides reference for design and drug research and development of novel carbonic anhydrase inhibitors. Carbonic anhydrase inhibition drugs with smaller side effects and better curative effects are expected to be developed.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to a carbonic anhydrase inhibitor, and in particular to the application of tanshinone IIA in carbonic anhydrase inhibitors and products. Background Art

[0002] Carbonic anhydrase (CA) is a family of zinc-containing metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. CA is widely distributed in human tissues and is involved in respiration, CO2 and HCO3 - Carbonic anhydrase is involved in many diseases, including glaucoma, cancer, epilepsy, etc.

[0003] The development of carbonic anhydrase inhibitors (CAI) has a long history. CAI, led by sulfonamide drugs such as acetazolamide, reduces H by inhibiting CA activity. + -Na + Exchange, HCO3 - It can improve metabolic acid / alkalosis, reduce intraocular pressure, and promote diuresis. It is commonly used in clinical practice to treat glaucoma, cardiogenic edema, and acute mountain sickness.

[0004] Currently, the only CAI approved by the FDA for marketing is acetazolamide, which is also the only drug approved by the FDA for the prevention and treatment of acute mountain sickness. However, acetazolamide can cause side effects such as frequent urination and numbness of the face, hands and feet.

[0005] Therefore, there is an urgent need to find carbonic anhydrase inhibitors with low side effects and high activity from natural products.

[0006] Salvia miltiorrhiza is a traditional Chinese medicine with the effects of promoting blood circulation and removing blood stasis, relieving pain and relieving pain, cooling blood and eliminating carbuncle. Salvia miltiorrhiza has anti-inflammatory, antioxidant, myocardial cell and blood vessel protection effects, and is often used to treat cardiovascular diseases such as atherosclerosis, coronary heart disease and hypertension.

[0007] The journal article (Liu Mingyue et al. Network pharmacology and fingerprint map study of the main pharmacological components of the Chuanxiong-Danshen drug pair [J]. Journal of Practical Clinical Medicine, 2023, 27(15):98-103) disclosed the "drug-ingredient-target-disease" network diagram of Chuanxiong-Danshen, and screened out target proteins with degree ≥ 10, including carbonic anhydrase 1 (CA1) and carbonic anhydrase 2 (CA2), indicating that the Chuanxiong-Danshen drug pair can regulate their expression and has therapeutic effects on body pain, cardiovascular disease, Alzheimer's disease, prostate cancer, brain damage, etc.

[0008] The components of Danshen mainly include water-soluble phenolic acids and fat-soluble diterpenoid tanshinones. Among them, tanshinone components mainly include tanshinone IIA, cryptotanshinone, dihydrotanshinone I, tanshinone I, etc. This type of component has the effects of anti-atherosclerosis, anti-myocardial ischemia, inhibition of vascular smooth muscle cell proliferation, anti-tumor, antioxidant and prevention of liver damage. Tanshinone IIA and cryptotanshinone are one of the main active ingredients of Danshen, which are concentrated in the cortex of Danshen root.

[0009] Tanshinone IIA is orange-red needle-shaped crystals, mp 209-210℃, easily soluble in organic solvents such as ethanol, acetone, ether and benzene, slightly soluble or insoluble in water. Tanshinone IIA contains a quinone structure, active electronic behavior, easy to be oxidized and reduced, can participate in various biochemical reactions of the body and has a variety of biological activities.

[0010] The journal article (Li Xiaoxiao et al. Research progress on the pharmacological effects of Tanshinone IIA [J]. Journal of Biology, 2016, 33(6):91-94) reviewed the current status of research on the pharmacological effects and mechanisms of Tanshinone IIA. The pharmacological effects of Tanshinone IIA include: (1) Tanshinone IIA has anti-atherosclerotic, coronary artery dilation, increased coronary blood flow, myocardial protection, anti-myocardial hypertrophy and anti-platelet aggregation effects on cardiovascular and cerebrovascular diseases, and plays a significant role in the treatment of cardiovascular diseases. (2) Tanshinone IIA has a protective effect on ischemia-reperfusion injury, which includes myocardial ischemia-reperfusion, cerebral ischemia-reperfusion, limb ischemia-reperfusion and spinal cord ischemia-reperfusion. (3) Tanshinone IIA also has a significant anti-inflammatory effect, which can reduce cardiac dysfunction by inhibiting inflammation. It can also be used to treat chronic kidney disease and inhibit renal fibrosis and inflammatory response. (4) Tanshinone IIA also has anti-tumor effects. It can promote tumor cell apoptosis by inducing the expression of tumor suppressor genes and affecting the transduction of signal pathways. It can also achieve anti-tumor purposes by inhibiting the proliferation of tumor cells. (5) Tanshinone IIA also has neuroprotective effects. It can resist neuronal apoptosis and protect nerve cells. (6) Tanshinone IIA also has multiple biological activities such as anti-oxidation, anti-depression, and protection of liver fibrosis.

[0011] Journal literature (Wang Chunqing et al. Mechanism of high-altitude sleep disorders and their preventive and therapeutic drugs [J]. Medical Herald, 2022, 41(12): 1816-1821) disclosed that Tanshinone IIA inhibits the production of oxidized low-density lipoprotein independently of the concentrations of lipids, calcium ions, and active vitamin D in plasma. The preventive effect of Tanshinone IIA on high-altitude sleep disorders may mainly rely on vasodilation and anti-oxidation.

[0012] Journal literature (Xiao Fangjun, et al. Danshen intervention in osteoporosis: mechanism of action explained by network pharmacology [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(05): 772-778.) disclosed that Tanshinone IIA may promote bone remodeling by promoting osteoblast proliferation, differentiation and maturation.

[0013] Therefore, Tanshinone IIA has multiple pharmacological effects, and its more potential pharmacological effects still need further study. Summary of the invention

[0014] The present invention aims at the problems existing in the prior art and provides application of tanshinone IIA in carbonic anhydrase inhibitors.

[0015] Tanshinone IIA belongs to the fat-soluble diterpenoid compounds in Danshen, and is one of the main active ingredients of Danshen. It has anti-inflammatory, antioxidant, anti-platelet, nerve cell protection, and myocardial cell ischemia reperfusion effects. At present, the research on Tanshinone IIA mainly focuses on cardiovascular protection, such as inhibiting atherosclerosis and regulating vascular endothelial function, and its inhibitory activity on acute mountain sickness and CA has not been reported. The present invention applies Tanshinone IIA to carbonic anhydrase inhibitors, which has excellent carbonic anhydrase inhibitory activity.

[0016] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0017] In a first aspect, the present invention provides the use of tanshinone IIA in carbonic anhydrase inhibitors, wherein the structure of tanshinone IIA is shown in the following formula:

[0018]

[0019] Carbonic anhydrase inhibitors are drugs that inhibit the enzyme carbonic anhydrase.

[0020] Carbonic anhydrase exists in the human body in the form of 16 different isomers, including hCAI, hCAII, hCAIX and hCAXII, which are widely distributed in different tissues and organs and are related to a series of key physiological activities. Carbonic anhydrase inhibitors in natural products are mainly flavonoids, phenolic acids and fatty acids. This invention provides the inhibitory activity of diterpenoid compound Tanshinone IIA on carbonic anhydrase for the first time, which is applied to carbonic anhydrase inhibitors, providing a reference for the subsequent discovery and design of new CA inhibitors, and providing a basis for the development of new CA inhibitory drugs.

[0021] In a second aspect, the present invention provides a carbonic anhydrase inhibitor, including tanshinone IIA. Tanshinone IIA in the carbonic anhydrase inhibitor may be the only component that inhibits carbonic anhydrase activity, and the carbonic anhydrase inhibitor may also include other components that inhibit carbonic anhydrase activity, which are not limited here.

[0022] In a third aspect, the present invention provides the use of Tanshinone IIA in the preparation of a medicament for treating or preventing carbonic anhydrase-related diseases.

[0023] The drug is a drug that reduces intraocular pressure, promotes diuresis, corrects metabolic acidosis / alkalosis, increases vascular oxygen flux, improves pulmonary ventilation, dilates cerebral blood vessels, induces tumor cell apoptosis or inhibits osteoclast bone resorption.

[0024] The carbonic anhydrase-related diseases include cardiovascular diseases, altitude sickness, eye diseases with high intraocular pressure as symptoms, osteoporosis or diseases caused by decreased bone density, edema or metabolic acid / alkalosis.

[0025] The altitude sickness is well known to those skilled in the art and is not limited here. It includes but is not limited to chronic altitude sickness, acute altitude sickness or altitude reaction caused by increasing altitude. The symptoms of altitude reaction include but are not limited to headache, dizziness, nausea, drowsiness, shortness of breath, palpitations, vomiting, fatigue, insomnia, numbness of hands and feet or arrhythmia.

[0026] The eye diseases with high intraocular pressure as a symptom are well known to those skilled in the art and are not limited here. In one embodiment, the carbonic anhydrase-related disease includes an eye disease with high intraocular pressure as a symptom, and the eye disease with high intraocular pressure as a symptom includes glaucoma, cataract or macular edema.

[0027] Tanshinone IIA can effectively lower intraocular pressure, promote diuresis, correct metabolic acid / alkalosis, increase vascular oxygen flux, improve pulmonary ventilation, dilate cerebral blood vessels, induce tumor cell apoptosis and inhibit osteoclastic bone resorption. It can be used to prevent and / or treat cardiovascular and cerebrovascular diseases, eye diseases with symptoms of high intraocular pressure, altitude sickness and discomfort caused by altitude sickness, and bone metabolic diseases caused by osteoporosis.

[0028] In one embodiment, the concentration of Tanshinone IIA in the preparation of a medicament for treating or preventing a carbonic anhydrase-related disease is 0.1-1000 μM.

[0029] In a fourth aspect, the present invention provides a medicine comprising tanshinone IIA and pharmaceutical excipients.

[0030] The pharmaceutical excipients include one or more of fillers, binders, wetting agents, absorption enhancers and surfactants. The fillers, binders, wetting agents, absorption enhancers and surfactants can be of any type known to those skilled in the art, and are not limited here. Preferably, the filler can be one or more of starch, sucrose and microcrystalline cellulose; the binder can be one or more of starch slurry, hydroxypropyl cellulose, gelatin and polyethylene glycol; the wetting agent can be one or more of magnesium stearate, micropowder silica gel and polyethylene glycol; the absorption enhancer can be one or more of polysorbate and lecithin; the surfactant can be one or more of poloxamer, fatty acid sorbitan and polysorbate. Preferably, the pharmaceutical excipients also include one or more of flavoring agents and sweeteners. The flavoring agents and sweeteners are of any type known to those skilled in the art, and are not limited here.

[0031] The dosage form of the drug is tablets, pills, powders, capsules, solutions, suspensions, emulsions, aerosols or suppositories. The tablets may be dispersible tablets; the capsules may be soft capsules or hard capsules; the solutions may be elixirs, syrups or injections; the emulsions may be oral emulsions, intravenous emulsions or liniments, etc. The drug may be prepared into conventional, immediate release or sustained release preparations.

[0032] The drug is administered orally, by nasal inhalation, by skin, by cavity or by injection, and the injection includes intramuscular injection, subcutaneous injection or intravenous injection.

[0033] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides for the first time the inhibitory activity of the diterpenoid compound tanshinone IIA on carbonic anhydrase, which can be used in drugs for lowering intraocular pressure, diuresis, correcting metabolic acid / alkalosis, increasing vascular oxygen flux, improving pulmonary ventilation, dilating cerebral blood vessels, inducing tumor cell apoptosis or inhibiting osteoclastic bone resorption, treating cardiovascular diseases related to carbonic anhydrase, altitude sickness, eye diseases with high intraocular pressure as symptoms, osteoporosis or diseases caused by decreased bone density, edema or metabolic acid / alkalosis, and applying tanshinone IIA to drugs for treating or preventing carbonic anhydrase-related diseases to obtain a new drug, which provides a basis for the subsequent development of new carbonic anhydrase inhibitors, and is expected to develop carbonic anhydrase inhibitors with fewer side effects and better efficacy, which can be used in drugs for preventing or treating carbonic anhydrase-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the liquid phase mass spectrometer of the sample eluate and the negative control group eluate;

[0035] Figure 2 It is an enlarged view of the extracted ion map of Tanshinone IIA in the sample eluate and the negative control group eluate;

[0036] Figure 3 It is an enlarged view of the extracted ion map of cryptotanshinone in the sample eluate and the negative control group eluate;

[0037] Figure 4 Graph showing the CA inhibitory activity of Tanshinone IIA, wherein A is a graph showing the CA inhibitory activity determination of Tanshinone IIA (200 μM) and Cryptotanshinone (200 μM), and B is a graph showing the inhibition rate of Tanshinone IIA at different concentrations on CA. DETAILED DESCRIPTION

[0038] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0039] The following sources of raw materials are provided for illustrative purposes:

[0040] Buffer: 3-morpholinepropanesulfonic acid (MOPS).

[0041] Example 1

[0042] A rapid discovery method for tanshinone IIA activity based on cross-linked enzyme aggregates

[0043] 1. Synthesis of carbonic anhydrase cross-linked enzyme aggregates (CA-CLEAs): CA was dissolved in a buffer solution to prepare a 1 mg / mL solution. 100 μL of CA solution was mixed with 900 μL of ethanol and shaken at 6°C for 2 hours. Glutaraldehyde solution was added to the mixture to a final concentration of 2 nM, and after mixing, the mixture was shaken at 25°C for 2.5 hours for cross-linking. The mixture was then centrifuged at 12,000 × g for 110 minutes, and the precipitate was washed with a buffer solution. This was repeated three times to obtain CA-CLEAs.

[0044] 2. Ligand fishing method based on cross-linked enzyme aggregates: After crushing the salvia miltiorrhiza, add 10%-90% ethanol to swell, percolate, and filter to collect the salvia miltiorrhiza extract. The ethanol is removed from the salvia miltiorrhiza extract by rotary evaporation, and the extract is allowed to stand overnight. The filter residue is collected after filtration, and it is repeatedly washed with pure water and freeze-dried to obtain the tanshinone component. CA-CLEAs are mixed with 0.1-20 mg / mL tanshinone component solution and incubated at 40°C for 1 hour. The incubation solution is centrifuged for 5-30 minutes, and the precipitate is rinsed with buffer and repeated three times. After washing, 400 μL of methanol for desorption is added to the precipitate, shaken for 1 hour, and centrifuged, and the supernatant is collected as the sample eluent.

[0045] The CA-CLEAs were placed in an oil bath and heated at 120°C for 0.5-5 hours to fully inactivate, thereby obtaining inactivated CA-CLEAs. The inactivated CA-CLEAs were mixed with 0.1-10 mg / mL salvianolic acid component solution and incubated at 40°C for 1 hour. The incubation solution was centrifuged for 5-30 minutes, and the precipitate was washed with a buffer solution, and repeated three times. After washing, 400 μL of desorption solution methanol was added to the precipitate for desorption, and the supernatant was collected as the eluate of the negative control group after shaking for 1 hour.

[0046] 3. Confirmation of active ingredients: Inject the two groups of eluents into the liquid chromatography-mass spectrometer for analysis, and compare the peak areas of each peak in the total ion flow diagram of the two groups of samples. If the difference in the peak area of ​​the corresponding peak in the two groups of samples is greater than 10%, it can be confirmed that the compound represented by the mass-to-charge ratio of the peak is a potential active ingredient. If the difference in the peak area is greater than 10%, further compare the peak area of ​​the extracted ion diagram corresponding to the mass-to-charge ratio shown in one of the primary mass spectra.

[0047] The chromatographic conditions of the liquid chromatography-mass spectrometry were as follows: Acquity UPLC HSS T3 column (2.1×100 mm, 1.8 μm); mobile phase A (0.1% formic acid in water)-B (acetonitrile); gradient elution: 0-6 min, 0%-5% B; 6-7 min, 5%-70% B; 7-10 min, 70%-100% B; flow rate 0.3 mL / min; column temperature 35° C., injection volume 5 μL. The mass spectrometry conditions of the liquid chromatography-mass spectrometer are as follows: ESI ion source, positive ion mode scanning, ion scanning range of m / z 100-1000; fragmentation voltage: step, 75m / z-90V, 250m / z-100V, 450m / z-110V, 750m / z-120V, 1000m / z-130V; scan / dwell time 563ms; dryer flow rate 11.0L / min, dryer temperature 325℃; nebulizer gas pressure 50.0psi; capillary voltage 3.5kV (positive ion mode), -3.5kV (negative ion mode); capillary current 5nA, chamber current 0.04μA.

[0048] The liquid chromatography mass spectrometry detection spectra of the eluate after the tanshinone components were incubated with active CA-CLEAs and inactivated CA-CLEAs are shown in Figure 2. Figure 1 As shown, Figure 1 The black curve is the liquid phase mass spectrum of the eluent of the negative control group, and the red curve is the liquid phase mass spectrum of the eluent of the sample. The horizontal axis is the retention time, and the vertical axis is the intensity. After calculating the peak area, it was found that the peak area of ​​the extracted ion map of Tanshinone IIA changed by more than 10%, such as Figure 2 As shown, Figure 2The figure is an enlarged view of the extracted ion map of Tanshinone IIA in the sample eluate and the negative control group eluate, where the black curve is the liquid phase mass spectrum of the negative control group eluate, and the red curve is the liquid phase mass spectrum of the sample eluate. There is also a non-specific binding diterpenoid component - cryptotanshinone, and its extracted ion map is shown in Figure 3 , Figure 3 It is an enlarged view of the extracted ion map of cryptotanshinone in the sample eluate and the negative control group eluate, wherein the black curve is the liquid phase mass spectrum of the negative control group eluate, and the red curve is the liquid phase mass spectrum of the sample eluate.

[0049] Figure 1-Figure 3 The results showed that among the tanshinone components, only the extracted ion map peak area change of tanshinone IIA was greater than 10%. Tanshinone IIA bound to active CA-CLEAs, which suggested that tanshinone IIA could act on CA, while cryptotanshinone hardly bound to active CA-CLEAs, which suggested that cryptotanshinone had almost no inhibitory effect on CA.

[0050] Example 2

[0051] Verification of Tanshinone IIA activity

[0052] Tanshinone IIA and cryptotanshinone were weighed separately and dissolved in dimethyl sulfoxide (DMSO) to prepare a 50mM stock solution. The stock solution was then diluted with buffer to obtain 200μM Tanshinone IIA and cryptotanshinone solutions. The buffer was further used to dilute to obtain 100μM, 50μM, 25μM, 12.5μM, and 6.25μM Tanshinone IIA and cryptotanshinone solutions, which were the test sample solutions for activity determination.

[0053] 40 μL of the test solution was added to a 96-well plate, mixed with 50 μL of p-nitrophenyl acetate (PNPA, 6 mM) solution, and then 10 μL of carbonic anhydrase solution (100 nM) was added to start the reaction. The well plate was placed in a constant temperature mixer at 37 ° C and 600 r / min for 20 min to obtain a mixed sample, set control samples 1-3, and use an ELISA reader to detect and record the absorbance value of each well at 405 nm.

[0054] The difference between control sample 1 and the mixed sample is that control sample 1 uses buffer to replace the carbonic anhydrase solution. The specific preparation method of control sample 1 is: 40 μL of the test sample solution is added to a 96-well plate, mixed with 50 μL of p-nitrophenyl acetate (6 mM) solution, and then 10 μL of buffer is added to react to obtain control sample 1.

[0055] The difference between control sample 2 and the mixed sample is that control sample 2 uses buffer to replace the test sample solution. The preparation method of control sample 2 is as follows: 40 μL of buffer is added to a 96-well plate, mixed with 50 μL of p-nitrophenyl acetate (6 mM) solution, and then 10 μL of carbonic anhydrase solution (100 nM) is added to start the reaction to obtain control sample 2.

[0056] The difference between control sample 3 and the mixed sample is that control sample 3 uses buffer to replace the carbonic anhydrase solution and the test sample solution. The preparation method of control sample 3 is as follows: 40 μL of buffer is added to a 96-well plate, mixed with 50 μL of p-nitrophenyl acetate (6 mM) solution, and then 10 μL of buffer is added to react to obtain control sample 3.

[0057] The inhibition rate of the test product was calculated according to the following inhibition rate calculation formula:

[0058] Inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100%

[0059] Among them, A1 is the absorbance of the well of the mixed sample; A2 is the absorbance of the well of the control sample 1; A3 is the absorbance of the well of the control sample 2; A4 is the absorbance of the well of the control sample 3.

[0060] The inhibition rate data were used to calculate the half inhibitory concentration (IC 50 ).

[0061] The results are as follows Figure 4 As shown, Figure 4 Figure 1 is a graph of the CA inhibitory activity of Tanshinone IIA, where A is a graph of the CA inhibitory activity of Tanshinone IIA (200 μM) and Cryptotanshinone (200 μM), and B is a graph of the inhibition rate of Tanshinone IIA at different concentrations on CA. The results showed that at the same concentration, the inhibition rate of Tanshinone IIA on CA was 51.7±2.9%, while Cryptotanshinone had a lower inhibitory effect on CA, with an inhibition rate of 1.7±2.5%. IC of Tanshinone IIA on CA 50 It is 210.4±15.4μM. Tanshinone IIA has inhibitory activity on CA and can be used in carbonic anhydrase inhibitors for the prevention and / or treatment of altitude sickness, the prevention and / or treatment of eye diseases with symptoms of high intraocular pressure, edema or metabolic acid / alkalosis.

[0062] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. The use of tanshinone IIA in carbonic anhydrase inhibitors, characterized in that: The structure of Tanshinone IIA is shown in the following formula:

2. A carbonic anhydrase inhibitor, characterized in that Including Tanshinone IIA.

3. Application of Tanshinone IIA in the preparation of drugs for treating or preventing carbonic anhydrase-related diseases.

4. The use according to claim 3, characterized in that: The drug is a drug that reduces intraocular pressure, promotes diuresis, corrects metabolic acidosis / alkalosis, increases vascular oxygen flux, improves pulmonary ventilation, dilates cerebral blood vessels, induces tumor cell apoptosis or inhibits osteoclast bone resorption.

5. The application according to claim 3, characterized in that: The carbonic anhydrase-related diseases include cardiovascular diseases, altitude sickness, eye diseases with high intraocular pressure as symptoms, osteoporosis or diseases caused by decreased bone density, edema or metabolic acid / alkalosis.

6. The use according to claim 5, characterized in that: The carbonic anhydrase-related diseases include ocular diseases with high intraocular pressure as a symptom, and the ocular diseases with high intraocular pressure as a symptom include glaucoma, cataract or macular edema.

7. A drug, characterized in that Comprising tanshinone IIA and pharmaceutical excipients.

8. The drug according to claim 7, characterized in that: The pharmaceutical excipients include one or more of fillers, binders, wetting agents, absorption enhancers and surfactants.

9. The drug according to claim 7, characterized in that: The dosage form of the drug is tablet, pill, powder, capsule, solution, suspension, emulsion, aerosol or suppository.

10. The drug according to claim 7, characterized in that: The drug can be administered orally, by nasal inhalation, by skin, by cavity or by injection.

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