Use of furanodienone in the preparation of a medicament for the treatment of inflammatory bowel disease

Abstract

The present application relates to the technical field of medicine, and particularly relates to application of furan dienone in preparation of a medicine for treating inflammatory bowel disease. Through in-vivo animal experiments, it is found that furan dienone can effectively inhibit the disease degree of a DSS-induced C57BL / 6J mouse colonitis model, relieve colon shortening, mouse hematochezia, weight loss and other phenomena, effectively inhibit the activation of the NF-kappa B pathway in the mouse colon and the level of downstream inflammatory factors, and can relieve intestinal mucosal barrier damage caused by DSS. The present application separates and identifies PXR agonist-furan dienone from natural products, which is not only rich in raw materials, cheap, easy to prepare, has small side effects, and is suitable for clinical popularization and use, and can be used for preparing a medicine for treating inflammatory bowel disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of furandienone in the preparation of drugs for treating inflammatory bowel disease. Background Technology

[0002] In recent years, due to environmental and dietary factors, gut health has received significant attention. As the body's natural defense against pathogens, the gut is highly susceptible to inflammatory responses from various external stimuli. Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory bowel disease, in which abnormal immune responses and mucosal barrier disruption play crucial roles in its progression. With the incidence of IBD in my country rising annually, researching novel therapeutic drugs is a key breakthrough in this disease's research. Currently, animal models are primarily used to explore the pathogenesis and treatment of IBD. Dextran sulfate sodium (DSS) is a sulfated polysaccharide that increases intestinal permeability, disrupts the intestinal mucosal barrier, leads to gut microbiota dysbiosis, activates inflammatory signaling pathways, and thus induces colitis in mice. DSS-induced colitis in mice is similar to human IBD in histology, clinical manifestations and pathogenesis. Moreover, the modeling method is simple. Acute or chronic colitis models can be established by adjusting the concentration and administration time of DSS. It is one of the most widely used animal models for IBD research.

[0003] During the inflammatory response, nuclear receptors can inhibit the transduction of inflammatory signals. Studies have found that the expression of pregnane X receptor (PXR) is reduced in the intestines of IBD patients. PXR is a member of the nuclear receptor superfamily and is mainly expressed in structural tissues such as the kidneys, liver, and intestines. It can be activated by a large number of ligands, such as artemisinin, an effective component of traditional Chinese medicine, and galangal, which can act as ligands for PXR. Currently, the anti-inflammatory effect of PXR in IBD is widely recognized, and it is an effective drug intervention target for various inflammatory diseases. After PXR activation, it can inhibit the NF-κB pathway, thereby reducing the production of inflammatory factors and suppressing intestinal inflammation. Simultaneously, PXR activation can promote cell proliferation and migration, which is a key factor in the healing process of epithelial cell damage. Pregnenolone Carbonitrile (PCN) is a specific agonist of PXR in rodents and can be used as a positive control drug.

[0004] Chinese patent CN107007588A, published on August 4, 2017, discloses the application of furandienone in the preparation of drugs for treating colorectal cancer. Early studies reported that furandienone possesses anti-inflammatory activity; however, to date, no literature has reported the effects of furandienone on colitis or its molecular mechanism. Summary of the Invention

[0005] The first aspect of this invention aims to provide the use of furandienone or its derivatives in the preparation of products for treating inflammatory bowel disease.

[0006] A second aspect of the present invention is to provide a drug.

[0007] A second aspect of the present invention is to provide a method.

[0008] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0009] A first aspect of the invention provides the use of furandienone or its derivatives in the preparation of products for treating inflammatory bowel disease.

[0010] The English name of furandienone is (5E,9E)-3,6,10-trimethyl-8,11-dihydro-7H-cyclodeca[b]furan-4-one (FDN), and its structural formula is shown in Formula 1:

[0011]

[0012] Preferably, the furandienone or its derivative includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

[0013] Preferably, the pharmaceutically acceptable salt includes at least one of the following: metal salt, ammonium salt, salt formed with an inorganic acid, salt formed with an organic base, salt formed with an organic acid, salt formed with a basic amino acid, and salt formed with an acidic amino acid.

[0014] Preferably, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0015] Preferably, the alkali metal salt includes at least one of sodium and potassium salts.

[0016] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0017] Preferably, the salt formed with the organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0018] Preferably, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0019] Preferably, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0020] Preferably, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0021] Preferably, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0022] Preferably, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

[0023] Preferably, the inflammatory bowel disease includes at least one of undefined inflammatory bowel disease, ulcerative colitis, Crohn's disease, undifferentiated colitis, acute colitis, and chronic colitis.

[0024] Preferably, the inflammatory bowel disease is ulcerative colitis.

[0025] Preferably, the products include pharmaceuticals.

[0026] Preferably, the drug comprises pharmaceutically acceptable excipients and / or any one or more other active ingredients.

[0027] Preferably, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, and carriers.

[0028] Furthermore, to facilitate medication, the active ingredient furandienone or its derivatives can be formulated into specific dosage forms with one or more pharmaceutically acceptable excipients. These excipients can be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base adjusters (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic adjusters (such as sodium chloride and glucose).

[0029] Preferably, the dosage form of the product includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0030] Preferably, the gastrointestinal dosage form includes at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0031] Furthermore, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0032] Preferably, the non-gastrointestinal dosage form includes at least one of the following: injectable dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0033] Furthermore, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0034] Preferably, the product is applied to mammals.

[0035] Preferably, the mammal includes humans.

[0036] In a first aspect of the invention, furanediones have been found to act as a natural PXR agonist, which binds to the PXR ligand-binding domain and effectively inhibits the pathological manifestations of DSS-induced colitis in C57BL / 6J mice, while in PXR... - / - There was no therapeutic effect in mice.

[0037] A second aspect of the present invention is to provide a medicament comprising a therapeutically effective amount of furanyldienone or a derivative thereof.

[0038] In some embodiments of the present invention, the therapeutically effective dose in mice is 1–30 mg / kg; preferably, it is 5–20 mg / kg; more preferably, it is 8–12 mg / kg.

[0039] A third aspect of the present invention aims to provide any one of methods 1) to 2), comprising the step of treating cells with furandienone or a derivative thereof.

[0040] 1) A method for activating pregnane X receptors in vitro without therapeutic target;

[0041] 2) An in vitro, non-therapeutic method for suppressing inflammation.

[0042] The beneficial effects of this invention are:

[0043] 1. Effective natural products that can bind to PXR, furandiones, were screened using mass spectrometry.

[0044] 2. Through in vivo animal experiments, this invention has found that furandienone can effectively inhibit the disease severity of DSS-induced C57BL / 6J mouse colitis model, and alleviate phenomena such as colonic shortening, bloody stools, and weight loss in mice.

[0045] 3. Through in vivo animal experiments, this invention has found that furandienone can effectively inhibit the activation of the NF-κB pathway and the level of its downstream inflammatory factors in the mouse colon.

[0046] 4. Through in vivo animal experiments, this invention has found that furandienone can alleviate intestinal mucosal barrier damage caused by DSS.

[0047] 5. This invention verifies, through in vivo animal experiments, that furanadienones act as PXR-specific agonists, in PXR... - / - In mice, it could not inhibit the disease progression of colitis.

[0048] 6. This invention isolates and identifies PXR agonists from natural products. These agonists are not only abundant and readily available from natural sources, but also inexpensive and easy to prepare, and have few side effects, making them suitable for clinical application. Attached Figure Description

[0049] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 For the binding analysis of FDN and PXR protein, including: (A) the binding level of FDN to the fusion protein (hPXR-LBD-SRC1); (B) a close-up view of the ligand binding bag of PXR and FDN; (C) the superposition of the structures of FDN and dabrafenib (DAB) binding to PXR (orange, PDB code 6HJ2).

[0051] Figure 2 To assess the effect of FDN on alleviating DSS-induced colitis in C57BL / 6J mice, the following factors were considered: (A) body weight change; (B) DAI index; (C) colon length; n=6, compared with the DSS group, ***p<0.001.

[0052] Figure 3 The effect of FDN on the expression of NF-κB pathway proteins p-p65 and p-IκB.

[0053] Figure 4 The results show the inhibition of FDN on colonic inflammatory factors (A) IL-1β, (B) IL-6, and (C) TNF-α in C57BL / 6J mice; compared with the DSS group, ***p<0.001, *p<0.05.

[0054] Figure 5 The effect of FDN on the pathological changes of colon tissue in C57BL / 6J mice (hematoxylin-eosin staining).

[0055] Figure 6 Effects of FDN on the intestinal mucosal proteins Occludin and ZO-1 in C57BL / 6J mice (immunofluorescence).

[0056] Figure 7 The effect of FDN on DSS-induced colitis in PXR- / - mice was evaluated, including: (A) body weight change; (B) DAI index; and (C) colon length. n = 6. Compared with the DSS group, ***p < 0.001; ns: no significant difference.

[0057] Figure 8The results show the inhibition of FDN on the colonic inflammatory factors (A) IL-1β, (B) IL-6, and (C) TNF-α in PXR- / - mice; compared with the DSS group, **p<0.01, *p<0.05, ns: no significant difference.

[0058] Figure 9 The effect of FDN on the pathological changes of colon tissue in PXR- / - mice (hematoxylin-eosin staining). Detailed Implementation

[0059] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0060] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0061] The main sources of reagents and materials are as follows:

[0062] Reagents: Methylcellulose (M112869, Aladdin); furanyldienone (HF029913, Herbest); PCN (P911021, Macklin); dimethyl sulfoxide (DMSO, Sigma); DSS (MP Biomedical); ZO-1 (CY3) and occludin (488) fluorescent antibodies (Thermo Fisher Scientific); p-p65 and p-IκB antibodies (Proteintech); HRP-labeled anti-rabbit IgG (Abcam); ELISA kit for mouse IL-6, IL-1β and TNF-α (BioLegend); HE staining solution (Wuhan Boerfu Biotechnology Co., Ltd.); DAB colorimetric kit (K3468, DAKO); DAPI (Wuhan Boerfu Biotechnology Co., Ltd.).

[0063] Mice: Male C57BL / 6J mice were obtained from SeBiona Bio-Tech. PXR - / - Male mice (Nr1i2-ko) were obtained from Cyagen (Suzhou) Biotechnology Co., Ltd. All mice were SPF grade and maintained under a strict 12-hour light cycle.

[0064] Instruments and equipment: Microplate reader (SpectraMax M2); Microscope (Nikon Eclipse CI, Nikon, Japan); Fluorescence microscope (80i, Nikon, Japan); MicroCal Auto-iTC200 isothermal titration calorimeter.

[0065] Example 1: Analysis of the binding of the natural compound FDN to PXR protein

[0066] 1. Experimental Methods

[0067] (1) Isothermal titration calorimetry (ITC)

[0068] The binding effect of FDN to PXR was investigated using the human PXR receptor ligand-binding domain (hPXR-LBD, residues 130-434). To enhance the stability of the PXR protein, it was co-expressed with a steroid receptor coactivator-1 (SRC-1, 623-710) fragment. Specifically, the PXR-LBD gene was cloned into the pRSET-A vector (with a His6 tag at the N-terminus), and the SRC-1 fragment gene was inserted into the pACYC184 vector. hPXR-LBD-SRC1 protein was then expressed in *E. coli* BL21(DE3) cells. Purified hPXR-LBD-SRC1 protein (25 μM) was dissolved in 400 μL of PBS containing 0.5% (v / v) DMSO, and 200 μM FDN was used. Titration of the FDN solution with the His-tagged hPXR-LBD-SRC1 fusion protein resulted in an exothermic binding event. Microcal Origin 7 software was used to analyze integrated thermal effects.

[0069] (2) Molecular docking

[0070] Download the structure of the FDN ligand from PubChem, and then use... Preparation using the LigPrep utility in the kit. The ligand structure was optimized in implicit water using the Prime Macrocycle conformational search tool in conjunction with the OPLS3e force field. Docking was then performed. The Glide XP (Extra Precision) included in kit 2019-3 was used. The PXR structure mesh was set using default parameters, with the bonding frame centered on the centroid of the FDN crystal ligand. The re-docking of FDN within its crystal structure resulted in a good bonding orientation (from a crystal structure perspective). The XP score (-8.962 kcal mol⁻¹) confirmed the method's ability to predict the interaction of the ligand within the binding site.

[0071] 2. Experimental Results

[0072] likeFigure 1 As shown in Figure A, analysis of the total thermal changes with increasing PXR-LBD-SRC1 protein concentration indicates a binding stoichiometry of approximately 2:1. Curve fitting analysis yielded a dissociation constant (Kd) of 4.5 μM. Figure 1 As shown in Figure B, FDN is a relatively small compound that can interact with a small group of eight PXR LBP residues. (Distance cutoff). In contrast, the potent PXR agonist and anticancer drug Dabrafenib (DAB) occupies a larger volume and has more contact with LBP residues ( Figure 1 This is consistent with its higher affinity for the receptor (EC50 = 87 nM). Two main anchoring sites of FDN include (1) multiple contacts between its ten-membered ring and the so-called PXR π-trap composed of amino acids F288, W299, and Y306, and (2) a hydrogen bond between the carbonyl moiety of FDN and Q285. Furthermore, the furan ring of FDN is in close contact with M243, M246, S247, and F288.

[0073] Example 2: Study on the efficacy of natural compounds in improving DSS-induced colitis in C57BL / 6J mice

[0074] 1. Experimental Methods

[0075] (1) Establishment and pharmacodynamic evaluation of an acute colitis model induced by DSS in C57BL / 6J mice

[0076] Forty SPF-grade, 6-8 week old male C57BL / 6J mice were acclimatized for 7 days and then randomly divided into four groups (n=6 per group): control group, model group (DSS), positive control group (PCN 10 mg / kg), and FDN group (10 mg / kg). The DSS group received oral administration of 2.8% DSS (w / v) aqueous solution for 7 days (days 4-10). FDN and PCN were administered by gavage for days 1-10 (dissolved in 3% DMSO and 1% methylcellulose). Mouse weight was recorded daily, and the Disease Activity Index (DAI) was scored based on weight loss, fecal viscosity, and fecal bleeding. Scores for each index ranged from 0 to 4. Mice were sacrificed on day 11, and colonic tissue was collected for analysis.

[0077] (2) Histopathological analysis

[0078] Colonic tissue was histologically analyzed by hematoxylin and eosin (H&E) staining. Fixed colon sections were embedded in paraffin and then sectioned. After hematoxylin-eosin staining, the sections were dehydrated, air-dried, mounted with neutral resin, and then observed under a microscope. For immunohistochemistry, tissue sections were incubated with p-p65 and p-IκB antibodies (Proteintech) and HRP-labeled anti-rabbit IgG (abcam), stained with hematoxylin and DAB, mounted with neutral resin, and observed under a microscope (Nikon Eclipse CI, Nikon). For immunofluorescence, tissue sections were incubated with ZO-1 (CY3) and occludin (488) fluorescent antibody (Thermo Fisher Scientific), stained with DAPI, mounted with an anti-fluorescence quencher, stored at 4°C in the dark, and observed under a fluorescence microscope.

[0079] (3) Measurement of inflammatory factors in colon tissue

[0080] Colon tissue was ground, centrifuged, and the supernatant was collected for ELISA assay and BCA protein quantification. The inflammatory factors TNF-α, IL-6, and IL-1β were detected and their levels analyzed according to the ELISA kit instructions (Biolegend; IL-6 (#431315), IL-1β (#432615), and TNF-α (#430915)).

[0081] 2. Experimental Results

[0082] The results showed that, compared with the control group mice, the body weight of mice in both the model group and the treatment group gradually decreased after DSS-induced ulcerative colitis. Figure 2 (A), and the Disease Activity Index (DAI) score is elevated ( Figure 2 (B) The length of the colon is significantly shortened. Figure 2 (C) Figure 2 The diagram in Figure D represents a representative colon. The administration of the natural PXR agonist FDN and PCN increased colon length and decreased DAI scores in DSS-group mice; analysis of IHC staining results, such as... Figure 3 As shown, FDN can significantly inhibit the expression levels of p-p65 and p-IκB proteins in DSS-induced colonic tissue, and can also inhibit downstream inflammatory factors related to the NF-κB signaling pathway, such as IL-6, IL-1β, and TNF-α. Figure 4 H&E colon pathology analysis, such as Figure 5As shown, the DSS-induced colitis mice exhibited abnormal overall intestinal tissue structure, significant local mucosal necrosis, and near-complete loss of normal intestinal structure. Numerous inflammatory cells were present throughout the entire tissue layer, along with densely clustered areas of inflammatory cells. The drug-treated group showed improved histological microstructure, reduced inflammatory cell infiltration, and the absence of inflammatory cell clusters. It alleviated the morphological changes in colonic crypts in the DSS-treated mice, protected goblet cells, and immunofluorescence results showed that, compared to the model group, FDN effectively maintained the tight distribution of occludin and ZO-1 in the inflammatory colon, reduced the decrease in tight junction proteins, and protected the integrity of the intestinal barrier. Figure 6 Therefore, it can be seen that the natural PXR agonist of the present invention can effectively improve colonic pathological damage in mice.

[0083] Example 3: Study on the efficacy of natural compounds against DSS-induced colitis in PXR- / - mice

[0084] 1. Experimental Methods

[0085] Animal modeling, inflammatory factor measurement, and H&E staining methods were all consistent with those in Example 2.

[0086] 2. Experimental Results

[0087] like Figure 7 As shown, the natural PXR agonist FDN of this invention has no therapeutic effect on DSS-induced PXR- / - colitis in mice, including weight loss, colon shortening, and increased DAI index, and has no inhibitory effect on the increase in colonic inflammatory factors IL-6, IL-1β, and TNF-α levels. Figure 8 ), and also could not improve the pathological damage of the colon group ( Figure 9 ).

[0088] The above are some of the experimental results from the applicant's extensive research, but they are sufficient to demonstrate that the natural PXR agonist of this invention activates PXR signaling by binding to the PXR target, inhibiting the release of downstream NF-κB signaling pathways and inflammatory factors, thereby alleviating DSS-induced weight loss, colon shortening, and increased DAI index in mice. It can also reduce inflammatory cell infiltration in the colon and the secretion of IL-6, IL-1β, and TNF-α inflammatory factors, increase occludin and ZO-1 protein expression, maintain the intestinal barrier, and has good anti-inflammatory activity, providing a scientific basis for its application in the preparation of drugs for treating colitis.

Claims

1. The use of furandienones or pharmaceutically acceptable salts thereof in the preparation of drugs for treating inflammatory bowel disease; The inflammatory bowel disease mentioned is ulcerative colitis; The structural formula of the furanyl diketone is shown in formula (I); Equation (I).

2. The application according to claim 1, characterized in that: The pharmaceutically acceptable salt is at least one of the following: metal salt, ammonium salt, salt formed with an inorganic acid, salt formed with an organic base, and salt formed with an organic acid.

3. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

4. The application according to claim 3, characterized in that: The pharmaceutically acceptable excipients are at least one of the following: solvents, propellants, solubilizers, cosolvents, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, flow aids, flavoring agents, preservatives, suspending agents, anti-adhesion agents, integrators, buffers, plasticizers, defoamers, thickeners, encapsulating agents, humectants, absorbents, flocculants, anti-flocculation agents, filter aids, and release inhibitors.

5. The application according to claim 1, characterized in that: The dosage forms of the drug include those administered via the gastrointestinal tract or those administered outside the gastrointestinal tract; The gastrointestinal dosage form is one of the following: powder, tablet, granule, capsule, solution, emulsion, or suspension; The non-gastrointestinal dosage form is one of the following: injection dosage form, respiratory dosage form, skin dosage form, and mucosal dosage form.

Images