Application of physcion in preparation of analgesic product

Abstract

The invention discloses an application of physcion in preparation of analgesic products. The emodin monomethyl ether is used as a single active component, the pain relieving mechanism of the emodin monomethyl ether is deeply discussed, and it is proved that the emodin monomethyl ether can be used as a TRPV1 activity regulator, and expression of TRPV1 is effectively inhibited. Experiments find that the physcion can effectively reduce the twisting frequency of mice caused by acid, significantly increase the pain threshold value of the mice in a hot plate experiment and a CFA inflammatory pain experiment, show an effect of enhancing the pain resistance to heat and inflammation stimulation, and meanwhile, have a significant effect of relieving metastatic bone cancer pain of the mice. The physcion shows a remarkable analgesic effect on animals, and safety or toxic and side effects are not observed.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of physcion methyl ether in the preparation of analgesic products. Background Art

[0002] Pain is a complex physiological and emotional experience associated with actual or potential tissue damage, especially chronic pain, which significantly affects patients' quality of life and ability to work, and may even lead to long-term disability. Cancer-related pain is particularly severe, placing a huge physical and psychological burden on patients. Current drugs used in clinical pain management, especially opioid analgesics, are effective but often accompanied by addiction, tolerance and multiple side effects, which poses a challenge to long-term treatment. Therefore, the development of alternative therapies with fewer side effects, less addiction and higher safety has become an urgent need in pain research.

[0003] Natural products are biologically active metabolites extracted from animals, plants and microorganisms in nature. They have become a valuable resource for drug development due to their wide range of sources, low toxicity and side effects, and significant therapeutic effects. Many clinically used drugs are directly or indirectly derived from natural products. Natural compounds show great potential, especially in the treatment of complex diseases such as pain. Therefore, it is of great significance to quickly screen out compounds with analgesic activity from natural product libraries with the help of modern technology to promote pain treatment.

[0004] Transient receptor potential vanilloid receptor 1 (TRPV1) is an ion channel protein widely distributed in sensory neurons and is involved in temperature and pain perception. TRPV1 is highly sensitive to stimuli such as capsaicin, heat, and acidic environment. Its activation leads to calcium ion influx, which in turn triggers the transmission of pain signals. Due to the important role of TRPV1 in the mechanism of pain generation, research on antagonists of this receptor has received widespread attention. TRPV1 antagonists can effectively reduce pain transmission by blocking the activation of this channel and are considered potential drugs for the treatment of diseases such as neuropathic pain, arthritis, and cancer-related chronic pain. However, existing TRPV1 antagonists still have problems with safety and side effects, which limits their clinical application.

[0005] Therefore, developing a treatment method with few side effects, high safety and effective pain relief has become the focus of current research. By combining the diversity of natural products with modern screening technology, finding natural compounds or new antagonists targeting TRPV1 will provide a new breakthrough for the treatment of pain. Summary of the invention

[0006] In view of the defects in the prior art, the present invention proposes the use of physcion in the preparation of analgesic products.

[0007] The invention provides application of physcion methyl ether in preparing analgesic products.

[0008] In some embodiments, the application is specifically: application in the preparation of products for peripheral pain, acute pain, thermal stimulation pain, inflammatory pain or cancer pain caused by diseases.

[0009] In some embodiments, the disease comprises any one of herpes zoster, diabetic peripheral neuropathy, traumatic injury, postoperative pain, burns, arthritis, gout, infectious inflammation, muscle strain, herniated disc, and metastatic bone cancer pain.

[0010] In some embodiments, the product is a medicine or a functional food.

[0011] In some embodiments, the drug comprises a therapeutically effective dose of the physcion methyl ether of claim 1 and a pharmaceutically acceptable excipient.

[0012] In some embodiments, in the drug, physcion is the only active substance; physcion is the only active substance, which enables more precise dosage control and purification of analgesic products, reduces the risk of potential interactions or side effects in compound preparations, and the optimized dosage makes the analgesic effect more stable and predictable.

[0013] In some embodiments, the pharmaceutically acceptable excipient is selected from any one of starch, dextrin, PEG-4000, PEG-6000, microcrystalline cellulose, lactose and magnesium stearate.

[0014] In some embodiments, the effective dose of physcion is not less than 1.7 mg / kg. When used to treat humans, the effective dose is 1.7-2 mg / kg. When used to treat animals such as mice, the effective dose is not less than 20 mg / kg.

[0015] In some embodiments, the medicament further comprises a second therapeutic agent;

[0016] The second therapeutic agent comprises one or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines;

[0017] The combination of physcion and other therapeutic agents can produce a synergistic effect, avoid the body's adaptation to a single drug, and thus improve the durability of the therapeutic effect. In addition, physcion has multiple biological activities. When combined with other therapeutic agents, it can improve the overall therapeutic effect by acting on multiple pain or inflammatory signaling pathways.

[0018] In some embodiments, the physcion methyl ether can be mixed with pharmaceutically acceptable excipients to prepare clinically acceptable injections, oral preparations, transdermal absorption preparations, and mucosal absorption preparations.

[0019] In some embodiments, the oral dosage form is a tablet, capsule, oral solution, granule, powder or pill.

[0020] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0021] 1. The present invention uses physcion as a single active ingredient to deeply explore its analgesic mechanism and proves that it can act as a regulator of TRPV1 activity and effectively inhibit the expression of TRPV1.

[0022] 2. The present invention has found through experiments that physcion can effectively reduce the number of acid-induced twisting of mice, significantly increase the pain threshold of mice in the hot plate test and CFA-induced inflammatory pain test, and show an enhanced effect of anti-pain to heat and inflammatory stimulation, and at the same time has a significant effect of alleviating metastatic bone cancer pain in mice. This drug shows a significant analgesic effect on animals, and no safety or toxic side effects are observed.

[0023] 2. The physcion methyl ether of the present invention can increase the threshold of CFA-induced inflammatory pain in mice, improve inflammatory response, and reduce the expression level of inflammatory factor genes in dorsal root ganglia.

[0024] 3. Compared with traditional compound formulas, single physaccharin methyl ether can reduce the adverse reactions that may be caused by other ingredients. Its relatively low toxicity and good tolerability can make it a safer analgesic.

[0025] 4. The present invention uses physcion as an analgesic drug, allowing for more precise dosage control and purification, reducing the risk of potential interactions or side effects in compound preparations. The optimized dosage makes the analgesic effect more stable and predictable. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 The chemical structure and parameter diagram of the small molecule compound physaccharin methyl ether of the present invention;

[0028] Figure 2The result diagram of the molecular docking of the emodin compounds and TRPV1 in Example 1 of the present invention; wherein, Figure 2 A is a two-dimensional diagram of the binding of aloe-emodin to TRPV1-related amino acids; Figure 2 B is a two-dimensional diagram of the binding of rheumatoid arthritis to TRPV1-related amino acids; Figure 2 C is a two-dimensional diagram of physaccharin methyl ether binding to TRPV1-related amino acids; Figure 2 D is a two-dimensional diagram of the binding of rhein to TRPV1-related amino acids;

[0029] Figure 3 The figure is a molecular dynamics simulation result diagram of the TRPV1 inhibitors capsaicin and rhamnolide compounds and TRPV1 according to Example 1 of the present invention; wherein Figure 3 A is the superposition of the trajectory of Lajiaoping during the simulation after molecular dynamics simulation; Figure 3 B is the superposition of the trajectory of aloe-emodin during the simulation after molecular dynamics simulation; Figure 3 C is the superposition of the trajectory of rhein during the simulation after molecular dynamics simulation; Figure 3 D is the superposition of the trajectory of physaccharin methyl ether during the simulation after molecular dynamics simulation; Figure 3 E is the superposition of the trajectory of rhein during the simulation after molecular dynamics simulation; Figure 3 F is the RMSD value of capsaicin over time after molecular dynamics simulation; Figure 3 G is the RMSD value of aloe-emodin changing with time after molecular dynamics simulation; Figure 3 H is the RMSD value of rhein changing with time after molecular dynamics simulation; Figure 3 I is the RMSD value of physaccharin methyl ether changing with time after molecular dynamics simulation; Figure 3 J is the RMSD value of rhein changing with time after molecular dynamics simulation;

[0030] Figure 4 Different doses of physaccharin methyl ether in Example 1 of the present invention inhibit the calcium ion fluorescence of human TRPV1-transfected HEK293 cells; wherein, Figure 4 A is the graph showing the inhibition of calcium ion fluorescence by physcion methyl ether at different concentrations; Figure 4 B is a statistical graph showing the inhibition of calcium ion fluorescence over time by physcion methyl ether at different concentrations;

[0031] Figure 5 This is a statistical graph of the number of writhing times induced by acetic acid in mice according to Example 2 of the present invention;

[0032] Figure 6 This is a statistical graph of the first paw licking time induced by the hot plate in mice according to Example 3 of the present invention;

[0033] Figure 7 This is a graph showing the pain threshold of the Von Frey test of CFA-induced inflammatory pain in mice according to Example 4 of the present invention;

[0034] Figure 8 This is a statistical graph of the relative mRNA expression of inflammation-related IL-1β, IL-6, CXCL10, IL-1 and calcium ion-related TRPV1, PKC, CAMK2A and CAMK2B in mice with CFA-induced inflammatory pain after treatment with physcion in Example 4 of the present invention; wherein, Figure 8 A is the statistical chart of the relative expression of IL-1β mRNA; Figure 8 B is the statistical chart of the relative expression of IL-6 mRNA; Figure 8 C is the statistical graph of the relative expression of CXCL10 mRNA; Figure 8 D is the statistical graph of the relative expression of IL-11 mRNA; Figure 8 E is the statistical graph of the relative expression of TRPV1 mRNA; Figure 8 F is the statistical graph of the relative expression of PKC mRNA; Figure 8 G is the statistical graph of the relative expression of CAMK2A mRNA; Figure 8 H is the statistical graph of the relative expression of CAMK2B mRNA; * represents p < 0.05, ** represents p < 0.01, n = 5-7;

[0035] Fig. 9 This is a graph of the pain threshold in the VonFrey test of the Lewis lung cancer cell-induced femoral cancer pain model in mice according to Example 5 of the present invention;

[0036] Fig.10 This is a statistical graph of the first paw licking time in the hot plate test of Lewis lung cancer cells-induced femoral cancer pain in mice according to Example 5 of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0038] In the first part of the present invention, a small part of the natural product database was used, TRPV1 was taken as the target protein, and small molecules and TRPV1 were virtually screened using the molecular docking software AutoDock Vina 1.2.3 to obtain the screened small molecules and complexes, and molecular dynamics simulation was performed. The compound physaccharin methyl ether that can inhibit TRPV1 was preliminarily predicted, and calcium ion fluorescence imaging of HEK293 cells transfected with TRPV1 was used to find that physaccharin methyl ether can significantly inhibit the flow of calcium ions.

[0039] The second part of the present invention verifies physcion by in vivo experiments, and detects the analgesic effect of physcion on abdominal pain symptoms caused by abdominal inflammation in animals, pain caused by hot plate method, inflammatory pain and metastatic bone cancer pain. The results show that physcion can significantly relieve various pain symptoms in mice and play an anti-inflammatory and analgesic effect. Through the changes in the expression levels of TRPV1-related factors, it is further verified that the mechanism of action of physcion in relieving inflammatory pain is related to the inhibition of TRPV1-related genes and inflammatory reactions.

[0040] The above indicates that physcion can be widely used as an analgesic product in peripheral pain, acute pain, thermal stimulation pain and inflammatory pain caused by diseases to relieve patients' pain symptoms.

[0041] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0042] Main reagents:

[0043] Acetic acid: McLean, F14201;

[0044] Physcion: MacLean, P828305;

[0045] Freund's complete adjuvant: Sigma-Aldrich, F5881-10ML;

[0046] Reagents: Invitrogen;

[0047] Reverse transcriptase: SuperScript TM II reverse transcriptase, Invitrogen, 18064014;

[0048] Power SYBR TM Green PCR Master Mix, Life Technologies, 4367659.

[0049] Example 1 Virtual screening of small molecule compounds that can inhibit TRPV1

[0050] Gysum-dl was used to minimize the energy of 267 small molecules of natural products and then converted into PDB files, and then prepare_ligand4.py in AutoDock 4 was used to convert the file into PDBQT. The structure of TRPV1 protein was downloaded from the Protein Data Bank (PDB) website (https: / / www.rcsb.org / ), the specific PDB ID is 5IS0, the ligand was extracted using PyMOL software, and the binding position of the extracted ligand was used as a potential active site for molecular docking studies. These small molecules were virtually screened using AutoDock Vina 1.2.3 to obtain the 10 small molecules with the highest scores, and 4 of them were found to be rhubarb derivatives. The complex of the small molecule and TRPV1 was then imported into Discovery Studio 2021 to analyze the binding position and binding amino acid of the small molecule and the complex. The structure of rhubarb methyl ether is shown in the figure. Figure 1 As shown in Figure 2, the binding of TRPV1 inhibitors capsaicin and rhamnolide compounds to TRPV1 is shown in Figure 2. Figure 2 shown.

[0051] Molecular dynamics simulation of the complex: The small molecule and TRPV1 protein complex was uploaded to the CHARMM-GUI platform and processed through the High-Throughput Simulator module (URL: https: / / charmm-gui.org / ?doc=input / hts), and the distance between the protein and the simulation box was set to 1.5 nanometers. Afterwards, the membrane protein was constructed using POPC lipids, and 150mM NaCl was added to simulate the physiological environment. After downloading the file from CHARMM-GUI, 100ps of NVT (canonical ensemble) and NPT (isothermal and isobaric ensemble) equilibrium were performed, and then the GROMACS2022.5 software was used to perform a 100ns molecular dynamics simulation of the complex. The motion trajectory of the complex, as well as the trajectory overlay of the small molecule, RMSD changes, and the number of hydrogen bonds were obtained through simulation. The relevant data are as follows: Figure 3 shown.

[0052] In vitro TRPV1 calcium fluorescence imaging: HEK293 cells were purchased from ATCC. The culture dishes, centrifuge tubes, 24-well plates, and pipettes used in the experiment were all sterilized by steam. The sterile operating table and pipette were wiped with 75% alcohol before use and then sterilized under ultraviolet light for 30 minutes.

[0053] Cell culture: The plasmid encoding human TRPV1 was transfected into HEK293 cells. After several generations of culture, 5×10 5The cells were seeded in 24-well plates and incubated for 24 hours to allow the cells to adhere.

[0054] Drug treatment: Different concentrations (12.5, 17.5, 25 μM) of physcion and blank solvent were added, and after 4 hours of incubation, the cells were washed three times with HEPES to remove unbound drugs.

[0055] Calcium ion probe treatment: Add Fluo-4 calcium ion probe, incubate for 30 minutes, and then wash with HEPES.

[0056] Fluorescence microscope observation: Place the 24-well plate in an inverted fluorescence microscope, use a 494nm light source for excitation, and a 516nm light wavelength for receiving light. Immediately use a fluorescence microscope to observe and record the video, add 10nM capsaicin for stimulation, and record the changes in cell fluorescence.

[0057] The results are as follows Figure 4 As shown in the figure, after adding different concentrations of physophanol methyl ether to cells, the fluorescence intensity was significantly reduced when stimulated by capsaicin compared with the control group, and the activity of TRPV1 was inhibited. Moreover, as the concentration of physophanol methyl ether increased, the fluorescence intensity gradually decreased, indicating that physophanol methyl ether had a dose-dependent inhibitory effect on TRPV1 activity.

[0058] Example 2 Effect of Physcion on Acetic Acid-Induced Writhing in Mice

[0059] The detailed description of experimental animals and main reagents is as follows:

[0060] The experimental animals were male C57BL / 6 mice that met the SPF standard, were 8 weeks old, weighed (20±2) g, and were kept in a clean place with free access to water and food, room temperature (19±2)°C, humidity (50±5)%, and day and night cycle light (12 h / 12 ​​h);

[0061] The main reagent is acetic acid (produced by McLean, product number F14201).

[0062] Drug groups: blank group (injection of normal saline), model group (injection of acetic acid) and physcion group (injection of 20 mg / kg physcion + acetic acid).

[0063] The mice in different groups were first injected with normal saline and physcion methyl ether intraperitoneally, and then adapted in acrylic cages for 1 hour. Subsequently, except for the blank group injected with normal saline, the other groups were injected with 0.6% acetic acid at a dose of 10 ml per kg body weight intraperitoneally. After a 5-minute adaptation period, the writhing reactions of the mice, such as abdominal concavity, extension of the trunk and hind legs, and high hips, were recorded in the next 20 minutes. Finally, the number of writhing reactions between the model group and the drug-treated group was compared.

[0064] like Figure 5 As shown in the figure, after intraperitoneal injection of acetic acid, the number of writhing of mice increased rapidly. In contrast, the injection of physalis methyl ether in advance can reduce the number of writhing, which has a significant analgesic effect.

[0065] Example 3 Effect of Physcion on Paw Licking on Hot Plate in Mice

[0066] The experimental animals used in this example are the same as those in Example 2.

[0067] The animals were divided into two groups: a model group (intraperitoneal injection of normal saline) and a physcion group (intraperitoneal injection of 20 mg / kg physcion).

[0068] After placing the mice in the cages for 1 hour, they were placed on a plate at 56°C to observe the mice's reactions after the heat stimulation, such as licking their toes or jumping up, and the corresponding time was recorded. This operation was repeated three times, and the analgesic effect of the small molecule drug was evaluated by comparing the time of the model group and the drug group.

[0069] like Figure 6 As shown in the figure, compared with the control group, injection of physcion can significantly increase the time of the first licking of the mice's feet, indicating that physcion can increase the pain threshold of mice sensitive to heat.

[0070] Example 4 Effect of Physcion on CFA-induced Inflammatory Pain in Mice

[0071] Main reagents: Freund's complete adjuvant (CFA, Sigma-Aldrich, F5881-10ML).

[0072] The animals were divided into a blank group (injected with an equal volume of saline), a model group (CFA), a low-dose physcion group (CFA + 10 mg / kg physcion), a high-dose physcion group (CFA + 20 mg / kg physcion) and a high-dose physcion group (without CFA + 20 mg / kg physcion).

[0073] (1) Von Frey test to evaluate CFA-induced inflammatory pain in mice

[0074] The mice were placed in a transparent acrylic cage and placed on a mechanical pain stimulation device. The right hind paw of the mice was stimulated with a VonFrey fiber with a strength of 0.16 g. The reaction behavior of the mice was recorded, the stimulation strength was adjusted according to the reaction, and 6 consecutive tests were performed to determine the pain threshold. Subsequently, CFA (20 μL) was injected into the right hind paw of the mice, and the pain threshold was re-measured 3 days later as the benchmark of the model group. The drug was then intraperitoneally injected, and the pain threshold was measured again 1 hour later. Finally, the difference in pain threshold between the drug-treated group and the model group was compared.

[0075] (2) Real-time fluorescence quantitative PCR

[0076] 700 μL was used Reagent (Invitrogen) was used to lyse mouse dorsal root ganglion (DRG) tissue and grind it into a homogenate using steel beads. TM cDNA was synthesized from total RNA (1000 ng per sample) using Power SYBR TM Green PCR Master Mix (Life Technologies, 4367659) was used to perform real-time quantitative PCR (qRT-PCR) on the samples to determine the expression levels of inflammatory factors (IL-1β, IL-6, CXCL10, and IL-11) and TRPV1-related genes (TRPV1, PKC, CAMK2A, and CAMK2B) in different groups of mice. Each individual transcript was normalized using the level of the mouse ribosomal 18S gene and 2 -△△ct The method was used to calculate the samples in each medication group.

[0077] (3) Experimental results

[0078] like Figure 7 As shown, injection of a high dose of 20 mg / kg of physaccharin methyl ether can significantly increase the pain threshold of mice, indicating that physaccharin methyl ether has a significant analgesic effect and is safe and has no toxic side effects on mice.

[0079] like Figure 8 As shown in the figure, after treatment with physcion, the transcription levels of inflammatory factors IL-1β, IL-6, CXCL10 and IL-11 decreased, and the transcription levels of TRPV1-related factors TRPV1, PKC, CAMK2A and CAMK2B also decreased. The above results indicate that the mechanism of physcion in relieving inflammatory pain and the inhibition of TRPV1-related genes are related to inflammatory factors.

[0080] Example 5 Effect of Physcion on Femoral Cancer Pain Induced by Lewis Mouse Lung Cancer Cells

[0081] The animals were divided into a blank group (injected with the same volume of normal saline), a model group (injected with Lewis mouse lung cancer cells), and a physcion group (injected with Lewis lung cancer cells + 20 mg / kg physcion).

[0082] Lewis mouse lung cancer cells were purchased from ATCC. The culture dishes, centrifuge tubes, 24-well plates, and pipettes used in the experiment were all sterilized by steam. The sterile operating table and pipette were wiped with 75% alcohol before use and then sterilized under ultraviolet light for 30 minutes.

[0083] The cultured lung cancer cells were digested with trypsin, centrifuged and then resuspended in PBS to prepare 2×10 7 100 cells / mL were inoculated into the femoral bone marrow cavity of male C57BL / 6 mice. The mice were observed every other day starting from 7 days after surgery. The pain sensitivity of the mice was then tested using Von Frey fibers as in Example 4, and the heat sensitivity of the mice was tested using a hot plate as in Example 3.

[0084] The results are as follows Figures 9-10 As shown, under the influence of bone cancer pain, the mechanical pain and thermal sensitive pain thresholds of mice were greatly reduced, and intraperitoneal injection of 20 mg / kg physoxaline methyl ether could increase the effects of mechanical pain and thermal sensitive pain on mice.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of physcion in the preparation of analgesic products.

2. The use according to claim 1, characterized in that: The application is specifically: application in the preparation of products for peripheral pain, acute pain, thermal stimulation pain, inflammatory pain or cancer pain caused by diseases.

3. The use according to claim 2, characterized in that: The disease includes any one of herpes zoster, diabetic peripheral neuropathy, traumatic injury, postoperative pain, burns, arthritis, gout, infectious inflammation, muscle strain, herniated disc, and metastatic bone cancer pain.

4. The use according to claim 1, characterized in that: The product is a medicine or a functional food.

5. The use according to claim 4, characterized in that: The medicine comprises a therapeutically effective dose of the physcion methyl ether as claimed in claim 1 and pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: In the drug, physcion is the only active ingredient.

7. The use according to claim 5, characterized in that: The pharmaceutically acceptable excipient is selected from any one of starch, dextrin, PEG-4000, PEG-6000, microcrystalline cellulose, lactose and magnesium stearate.

8. The use according to claim 5, characterized in that: The effective dose of the physaccharin methyl ether is not less than 1.7 mg / kg.

9. The use according to claim 5, characterized in that: The medicament further comprises a second therapeutic agent; The second therapeutic agent comprises one or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

10. The use according to claim 5, characterized in that: The physcion methyl ether can be mixed with pharmaceutically acceptable adjuvants to prepare clinically acceptable injections, oral preparations, transdermal absorption preparations, and mucosal absorption preparations.

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