阅读说明：本技术 肾上腺素能β2受体激动剂R-对映体在治疗炎症性肠病及其肠外疾病的新应用 (New use of R-enantiomer of adrenergic beta 2 receptor agonist in treating inflammatory bowel disease and its diseases outside intestine ) 是由 谭文 于 2019-09-07 设计创作，主要内容包括：本发明公开了光学纯的肾上腺素能β2受体激动剂R-对映体(包括R-沙丁胺醇,R-特布他林,R-克伦特罗和R-班布特罗)治疗炎症性肠病及包括皮肤疾病的肠外疾病的新应用。(The invention discloses a new application of optically pure R-enantiomer of adrenergic beta 2 receptor agonist (including R-salbutamol, R-terbutaline, R-clenbuterol and R-bambuterol) in treating inflammatory bowel disease and parenteral diseases including skin diseases.)

1. A process for the preparation of a medicament for the treatment of inflammatory bowel disease and parenteral complications, characterized in that the medicament is the R or R' R enantiomer of an optically pure adrenergic beta 2 receptor agonist and pharmaceutically acceptable salts thereof, as well as a combined pharmaceutical preparation of the above isomeric medicament and an anti-inflammatory or immunosuppressive drug, an antibody, an antibiotic or a S1P receptor modulator.

2. The optically pure adrenergic β 2 receptor agonist according to claim 1, which is a short-acting β 2 receptor agonist, characterized by comprising: salbutamol, terbutaline, bitolterol, fenoterol, isoproterenol, metaproterenol or metaproterenol, procaterol and ritodrine.

3. The optically pure adrenergic β 2 receptor agonist according to claim 1, which is a long-acting β 2 receptor agonist, characterized by comprising: bambuterol, formoterol fumarate, salmeterol, and albuterol; also a super long acting β 2 agonist characterized by comprising: abediterol, carmoterol, indacaterol, oloterol, vilanterol, isoclenbuterol, mabuterol, and zilpaterol.

4. The optically pure R enantiomer according to claim 1, characterized in that the enantiomeric excess is greater than 80%.

5. Optical purity according to claim 1, characterized in that the enantiomeric excess is greater than 98.5% or preferably greater than 99%.

6. The inflammatory bowel disease according to claim 1 is crohn's disease and ulcerative colitis.

7. The Crohn's disease of claim 6 comprising perianal disorders: perianal erythema, abscesses, ulcerations, and perianal fissures or flaccidity canals.

8. The perianal condition of claim 7 comprising an inflammatory condition of hemorrhoids.

9. The Crohn's disease of claim 6 including fibrosis and intestinal stenosis.

10. The parenteral disorder of claim 1 which is arthritis, osteoarthritis or ankylosing spondylitis.

11. The parenteral disorder of claim 1 which is rhinitis or uveitis.

12. The parenteral disease of claim 1, which is oral Crohn's disease, amyloidosis, episcleritis, scleral malacia, corneal ulcer, primary sclerosing cholangitis, lupus and pulmonary inflammatory disease.

13. The parenteral disease of claim 1, which is an inflammatory skin disease selected from atypical dermatitis, psoriasis, rosacea, miliaria, acne, pyoderma gangrene, Sweet syndrome, intestinal associated skin-arthritis syndrome (BADAS), pyogenic dermatitis with increased value-suppurative stomatitis (PPV), and hypersensitivity vasculitis.

14. The parenteral disease of claim 1 which is an autoimmune skin disease including urticaria, vitiligo and alopecia areata.

15. The treatment of claim 1 comprising inhibiting the Jak2 and Stat2 signaling pathways.

16. The anti-inflammatory agent of claim 1, wherein the 5-aminosalicylate comprises sulfasalazine, mesalamine, olsalazine and the corticosteroid comprises prednisone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, dexamethasone.

17. The anti-inflammatory agent of claim 1 comprising 5-aminosalicylate, comprising sulfasalazine, mesalamine, balsalazide and oxaprazole.

18. The antibiotic of claim 1, which is an aminoglycoside antibiotic, ansamycin, carbacephem, carbapenem, cephalosporin, glycopeptide, lincosamide, lipopeptide, macrolide, monobactam, nitrofuran, oxazolone, penicillin, polypeptide, quaquinolones, sulfonamides, tetracycline, chloramphenicol, phosphonate antibiotic, and mycobacterial antibiotic.

19. The antibody of claim 1, which is infliximab, adalimumab, golimumab, vedolizumab, certolizumab ozogamicin, natalizumab, ustekab, and Bm-ca.

20. The S1P receptor modulator according to claim 1, being fingolimod, ponesimod, siponimod, ozapimod, amiselimod and GSK 2018682.

21. The treatment according to claim 1 comprising inhibiting macrophage activation and inducing metabolic remodeling of the cells.

22. The treatment of claim 1 comprising inhibiting the activation of gut resident lymphocytes and CD4, CD8T lymphocytes.

23. The treatment of claim 1 comprising metabolic remodeling of macrophages, resident lymphocytes and CD4, CD8T lymphocytes.

24. The pharmaceutically suitable salt according to claim 1, which is formed from a conventional pharmaceutically acceptable organic or inorganic acid, including hydrochloride, hydrobromide, sulfate, hydrogen sulfate, dihydrogen phosphate, sulfonate, bromide, potassium sulfate, acetate, oxalate, maleate, fumarate, succinate, 2-naphthalene-sulfonate, glycinate, gluconate, citrate, tartrate, lactate, isethionate, benzenesulfonate or polytoluenesulfonate of pyruvic acid.

25. The pharmaceutical combination according to claim 1, characterized by being administered orally, by inhalation, by nasal spray, by injection, externally, by eye drop, rectally or vaginally and administered to the patient in need thereof in solid form, in solution form, by spray, by aerosol form, by injection form, in ointment form, in skin patch form, in film form, in soft capsule form, in suppository form.

Technical Field

The present invention relates to a novel use of optically pure R-enantiomers of adrenergic beta 2 receptor agonists for the treatment of Inflammatory Bowel Disease (IBD) and parenteral diseases of inflammatory bowel disease. The exact cause of IBD is not clear, but IBD is the result of a deficiency in the immune system. IBD includes crohn's disease and ulcerative colitis, characterized by chronic inflammation of the gastrointestinal tract, persistent diarrhea, abdominal pain, bloody stool in the rectum, weight loss, and fatigue. IBD is associated with extra-intestinal symptoms such as liver problems, arthritis, skin symptoms and eye problems. There is currently no effective drug for the treatment of IBD. The primary therapeutic agents that control the symptoms of IBD include anti-inflammatory drugs such as sodium 5-aminosalicylate and corticosteroids, and immunosuppressive agents such as cyclosporine. The long-term use of these drugs often causes serious adverse reactions. IBD sometimes requires surgical treatment. Thus, there is an unmet medical need for a more effective and safer drug to help patients in need of treatment for IBD.

The present invention discloses novel uses of the R-enantiomer of a chiral adrenergic beta 2 receptor agonist in the treatment of IBD and parenteral diseases of IBD. The present invention also unexpectedly found that the S-enantiomer of a chiral adrenergic beta 2 receptor agonist is capable of causing exacerbation of IBD. Thus, the R-enantiomer of the β 2 receptor agonist is superior to its racemate in treating IBD, while avoiding the toxicity of the S-enantiomer. The present invention discloses the R-enantiomer of an adrenergic beta 2 receptor agonist useful for the treatment of IBD and its extra-intestinal diseases, with less side effects compared to its racemic and other marketed drugs. According to the invention, the R-enantiomers of adrenergic receptor agonists include short-acting beta 2 agonists (SABA), the eutomer R-salbutamol and long-acting beta 2 agonists (LABA), the eutomer R-bambuterol and R-clenbuterol. The present invention also discloses the use of at least one R-enantiomer of an adrenergic beta 2 receptor agonist in combination with at least one anti-inflammatory drug, such as a corticosteroid and at least one immunosuppressive agent, or with an antibody for IBD and an S1P receptor modulator. The invention discloses a new R-enantiomer of an adrenergic beta 2 receptor stimulant for treating the parenteral manifestations of IBD, including arthritis, uveitis, rhinitis, ankylosing spondylitis, vitiligo, psoriasis, amyloidosis, eczema, acne and primary sclerosing cholangitis.

Background

Inflammatory Bowel Disease (IBD) is a collective term for two diseases, crohn's disease and ulcerative colitis, characterized by chronic inflammation of the gastrointestinal tract resulting from autoimmune dysfunction. Prolonged inflammation leads to gastrointestinal damage. Some common symptoms are: persistent diarrhea, abdominal pain, rectal bleeding, hematochezia, weight loss and fatigue (Baumgart DC, & clipping SR inflammation bow disease: house and immunobiology. Lancet.369(9573):1627, May 2007). Related diseases include arthritis, pyoderma gangrenosum, primary sclerosing cholangitis, and non-thyroid disease syndrome (NTIS) (Liu S, et al, "non-systemic inequality syndrome: is it far away from Crohn' S disease. Second, crohn's disease and ulcerative colitis manifest as different degrees of extra-intestinal manifestations (e.g., liver disease, arthritis, skin symptoms, and eye symptoms) (extrinsic inflammation disorders of inflammation bowel disease, Levine JS et al Gastroenterology & hepatology Vol. &, Issue 4,235, April 2011).

The total annual IBD incidence is 396/100000. In 2013, a total of 5.1 million people died from IBD worldwide. Since world war ii, an increase in the incidence of IBD has been associated with an increase in meat consumption worldwide, supporting the statement that animal protein intake is associated with IBD. Inflammatory bowel disease is increasing in europe. The exact cause of IBD is not clear, but IBD is the result of a deficiency in the immune system. The normally functioning immune system attacks foreign organisms, such as viruses and bacteria, to protect the body. In IBD, the immune system responds incorrectly to the environment that causes inflammation of the gastrointestinal tract. There also appears to be a genetic factor.

There are several drugs that treat IBD: 5-aminosalicylic acid, corticosteroids (such as prednisone), immunomodulators, and drugs (biologicals) that are newly approved for IBD. There are also several vaccines recommended for use in preventing infection in patients with IBD. Patients with severe IBD may require surgical removal of damaged portions of the gastrointestinal tract, but advances in drug therapy mean that surgery is not as common as decades ago. Since crohn's disease and ulcerative colitis affect different parts of the gastrointestinal tract, the surgical approach for these two cases is different.

Steroids, such as the glucocorticoid prednisone, are often used to control disease onset and have been an acceptable maintenance drug. Biological treatments of inflammatory bowel disease, especially TNF inhibitors, are used in more severe or drug resistant crohn's disease patients, sometimes for ulcerative colitis.

Despite recent advances in therapy, there remains a need for a safe, well-tolerated, fast-acting therapeutic approach that enhances long-term remission. In addition, long-term use of drugs such as steroids can cause serious side effects. Therefore, there is an urgent need for better drugs for treating IBD to improve the therapeutic effect, reduce the adverse reactions and reduce the cost.

Disclosure of Invention

The IBD animal model is an important research tool for researching the pathogenesis of IBD, and provides a wide choice for researching various factors of IBD and evaluating different treatment schemes. Even though no model represents all aspects and stages of ulcerative colitis, that is, ulcerative colitis is mainly localized to the colonic mucosa, or crohn's disease affects the entire gastrointestinal tract by transmural inflammation (r.b. sartor and m.muehlbauer, "Microbial host interactions in IBD: pathologies for pathogenesis and therapy," Current Gastroenterology Reports, vol.9(6), pp.497, 2007).

The Dextran Sodium Sulfate (DSS) induced colitis model is currently a recognized and widely used model of inflammatory bowel disease. The DSS-induced colitis model has certain advantages over other animal colitis models. For example, by varying the administered concentration of DSS, acute, chronic or recurrent models can be readily established. In addition, abnormal proliferation that often occurs in clinical human enteritis is also often seen in DSS-induced chronic phase enteritis. In recent years, there has been a review of DSS-induced colitis-related cancer models. Furthermore, studies validating the DSS model by using different IBD treatment drugs showed that the DSS-induced mouse colitis model could be transformed into a relevant model of human disease (Perse M & Cerar A, Dextran sodium sulfate colitis mouse model: tracks and locks.J. of Biomedicine and Biotechnology, Vol.2012, ID 18617, Mar.2012). The present invention employs a DSS mouse model. In the prior art, R-salbutamol has been shown to be effective in the treatment of asthma. It is also a main medicine for treating discoid lupus erythematosus and other skin diseases. R-salbutamol was used in a randomized, controlled trial for the topical treatment of discoid lupus erythematosus (br.j. demortol.161(16):1365, dec.2009) to promote chronic wound healing in the skin. However, IBD is a disease completely different from asthma and discoid lupus (connective tissue disease) or chronic wound healing of damaged skin. The clinical manifestations and histological features of colitis described above are all significantly improved after treatment with R-salbutamol. DAI was significantly improved, weight gain, diarrhea decreased, fecal occult blood decreased. In pathological and histological examinations, the rate of colorectal shortening decreases after R-salbutamol treatment. Mucosal distortion or erosion is reduced. The inflammatory response of mucosa and submucosa is obviously reduced. A number of newly grown crypts appeared. The colonic mucosal intraepithelial layer was clearly restored. The lamina propria was intact and no significant inflammatory infiltration was seen. Compared with DSS-induced colitis mice, the histological score of R-salbutamol-treated colitis mice was significantly reduced. The invention discloses that R-salbutamol can be used for preventing and treating IBD. This report is novel and not predictable and inferred by those skilled in the art. IBD is generally not treated topically through the skin. IBD is a chronic disease whose pathogenesis is different from the clinical manifestations of the disease that can be treated with R-salbutamol or other β 2 agonists, and whether R-salbutamol or other similar β 2 agonists are used in the treatment of IBD has never been reported.

The invention discloses that R-salbutamol can be used for preventing or treating IBD. In one embodiment, DSS is added to drinking water to induce IBD in mice for 7 days. The clinical manifestations of colitis, including weight loss, diarrhea, increased index of disease activity including Fecal Occult Blood (FOB) after several days (Cooper, S.et al, clinical pathology study of saline urine experimental community, Laboratory Investigation, vol.69, No.2, p.238-249,1993).

Mice were dissected and histologically characterized on day 10. The colorectal length is shortened. The histology of acute colitis is characterized by loss of mucus, degeneration of epithelial cells, necrosis leading to the disappearance of epithelial cells. With neutrophil infiltration and destruction of the lamina propria and submucosa, occult inflammation (transepithelial migration of neutrophils to the mucosal epithelium), and cellulitis of the mucosa and submucosa. Erosion, cellulite and alveoli change in the mucosa and submucosa, and disappearance of crypts. Each of the above histological features was scored individually and added together, collectively denoted as "histological score", for quantitative evaluation (Cooper, s.et al, above).

The clinical manifestations and histological features of colitis described above are all significantly improved after treatment with R-salbutamol. DAI was significantly improved. Weight gain, diarrhea and FOB were significantly reduced. In pathological and histological examinations, the rate of colorectal shortening decreases after R-salbutamol treatment. Mucosal abnormalities or erosion are reduced. The inflammatory response of mucosa and submucosa is obviously reduced. With a clear restoration of the colonic mucosal epithelial lining, a large number of neocrypts appear. The lamina propria was intact and no significant inflammatory infiltration was seen. Compared with DSS-induced colitis mice, the histological score of R-salbutamol-induced colitis mice is obviously reduced. The invention discloses that R-salbutamol can be used for preventing and treating IBD. This report is novel.

In other embodiments, the invention provides similar effects of the R-enantiomers of other short-acting β 2 agonists, including R-terbutaline, and long-acting β 2 agonists, including R-bambuterol and R-clenbuterol, in the prevention and treatment of DSS-induced IBD. The invention discloses a new application of an R enantiomer or an R enantiomer of an adrenergic beta 2 agonist in preventing and treating IBD. The present disclosure is novel.

Corticosteroids are common drugs for the treatment of IBD. In one embodiment, the invention provides an R-enantiomeric adrenergic agonist that is more effective than dexamethasone in ameliorating IBD.

Most chiral adrenergic β 2 agonists currently on the market are marketed in racemic form as a mixture of the R and S enantiomers. It is well known that one enantiomer may sometimes be inert and not have the same biological activity as the other. However, the toxic effect of the S-enantiomer on IBD has not been reported in the literature at present. In one example, S-salbutamol is used in place of R-salbutamol in IBD mice in the same manner as R-salbutamol. Surprisingly, S-salbutamol did not show improved or therapeutic effects of R-salbutamol, but, in contrast, S-salbutamol significantly worsened the clinical manifestations and inflammatory features of mice under DSS stimulation. DAI significantly worsened compared to untreated DSS mice. In particular, FOB was significantly worse than untreated DSS mice. The length of the colon was further shortened compared to untreated DSS mice. Compared to untreated DSS mice, mucosal erosion, epithelial cell damage and crypt loss were more severe in DSS mice. Compared to untreated DDS mice, there was increased inflammatory infiltration of the mucosa and submucosa, lamina propria, and muscle layers, and more severe destruction of the lamina propria. The histological score was significantly higher than that of untreated DSS mice. The invention discloses that S-salbutamol has toxic effect on intestinal tracts and can further aggravate clinical symptoms and inflammatory response of IBD. Furthermore, the removal of the S-enantiomer of salbutamol from racemic salbutamol, for example using the R-enantiomer of salbutamol instead of racemic salbutamol, may reduce the toxicity and unwanted adverse effects of the S-enantiomer of salbutamol. The disclosure of the present invention should be considered novel. The toxic effects of these S-enantiomer β 2 agonists were unexpected and unexpected.

In another embodiment, the other S-enantiomers of the beta 2 adrenergic agonists, including S-terbutaline and S-bambuterol, are used in the same manner in DSS mice as S-salbutamol. Similar toxic effects were observed. The Disease Activity Index (DAI) and histological score of DSS mice treated with S-enantiomer β 2 receptor agonists were significantly higher than those of untreated DSS mice. The present invention discloses that S-enantiomer β 2 agonists are toxic to the intestinal tract and may further exacerbate the clinical symptoms and inflammatory response of IBD. In addition, the invention also discloses a new application of the R-enantiomer beta 2 agonist in treating IBD, and the toxicity is reduced. The present invention teaches that the removal of the S-enantiomer from the racemate, for example, by replacing the racemic β 2 agonist with an R-enantiomer β 2 agonist, can reduce toxicity and adverse effects caused by the S-enantiomer. This report of the present invention should be regarded as novel. These toxicities were unexpected.

The invention also discloses the use of R-salbutamol in the treatment of IBD in the subacute or chronic phase. In one embodiment, DSS induces IBD, as described above, while R-salbutamol treatment is given, DSS is removed and replaced with drinking water for 2 weeks on day 7. Mice were tested periodically for Disease Activity Index (DAI). Mice were examined pathologically and histologically on day 23. In DSS-induced IBD mice, body weight and fecal occult blood were significantly reduced on days 14 and 22, and the overall colitis was severe, albeit improved compared to day 7. Compared with the control group, the colon length is obviously shortened, and the histological score is obviously increased. DSS-induced colitis mice were shown to have significant mononuclear cell infiltration, crypt structural disorders, epithelial cell degeneration, mucosal abnormalities. Mucosal lymphocytes proliferate, the lamina propria is destroyed, and transmural inflammation is sometimes seen.

The DAI disease activity index after R-salbutamol treatment was significantly improved compared to untreated DSS animals. Body weight similar to control normal mice; the fecal occult blood of DSS mice was significantly reduced after R-salbutamol treatment. The histological index after treatment is also obviously improved. Crypt regeneration is essentially complete and the arrangement is normal. Normally growing epithelial cells form an intact mucosal surface. The natural layer is intact and basically normal. There is a small inflammatory infiltrate to either the mucosa or submucosa.

In one embodiment, similar to the study above, except that treatment started from day 7 until day 22, rather than starting from day 1 until day 22 as above. The results are similar to those described above, and R-salbutamol has obvious therapeutic effect on IBD mice caused by DSS.

The invention discloses that R-salbutamol can be used for preventing or treating subacute or chronic IBD. This report in the present invention is novel.

In one embodiment, the invention further discloses that R-albuterol and other R-enantiomer β 2 agonists have significant therapeutic effects in the treatment of TNBS-induced colitis in mice, a common animal model of crohn's disease. R-salbutamol has better curative effect on IBD than Infliximab (IFX). The male mouse colitis model is induced by rectal injection of ethanol and TNBS (trinitrobenzene sulfonic acid) in combination. Severe colitis in mice characterized by weight loss and diarrhea. Histologically, normal architecture loss, discrete epithelial granuloma, multiple erosive lesions, massive lymphocytic penetrating inflammatory infiltrates, goblet cell loss, are seen. However, the condition was significantly improved after oral administration of salbutamol or clenbuterol. There were no significant differences between R-salbutamol or R-clenbuterol treated mice and normal control mice in terms of weight loss, diarrhea, colon length. Lesions are obviously reduced, and inflammation infiltration with crypt growth is rarely generated, and the mucous epithelium is recovered. Furthermore, the therapeutic effect of infliximab is similar to that of salbutamol, however, IFX-treated animals have significant submucosal fibrosis and intestinal luminal narrowing, which is not the case for R-salbutamol and R-clenbuterol-treated animals. Furthermore, TNBS-induced colitis mice develop perianal symptoms: some mice develop erythema, abscesses, and ulceration, possibly leading to perianal fissure or fistula. However, TNBS induced a significant reduction of these perianal symptoms in mice following R-salbutamol or R-bambuterol treatment. In the prior reports, the therapeutic effects of R-salbutamol, R-clenbuterol and R-bambuterol on TNBS-induced symptoms of Crohn's disease have never been reported.

These perianal lesions are somewhat similar to the inflammatory symptoms of hemorrhoids. In one embodiment, the present invention discloses that the symptoms of inflammation of hemorrhoids can be significantly reduced by oral or topical administration of R-salbutamol, R-clenbuterol and R-bambuterol. The new beta 2 agonists have never been reported for the treatment of inflammatory symptoms of hemorrhoids.

In another embodiment, the invention reveals the presence of submucosal fibrosis and luminal narrowing of TNBS-induced crohn's disease, which may lead to surgery in IBD patients. R-salbutamol treatment significantly reduced intestinal fibrosis and stenosis. Infliximab treatment, on the other hand, enhanced submucosal fibrosis and luminal stenosis. These beneficial effects of R-salbutamol are novel.

In another embodiment, other R-enantiomers including the short-acting β 2 agonist, R-terbutaline or the long-acting β 2 agonists R-bambuterol and R-clenbuterol are used in subacute or chronic DSS mice, respectively, as described above. These drugs of R-enantiomer β 2 agonists significantly improved subacute or chronic clinical symptoms and pathological changes caused by DSS or TNBS. Disease activity index and histological score were significantly improved after R-enantiomer treatment with β 2 agonist compared to untreated DSS or TNBS-induced IBD mice. The invention discloses that R-enantiomer beta 2 agonists can be used for treating subacute or chronic IBD (including UC and Crohn's disease), and the treatment effect of the R-enantiomer beta 2 agonists is obviously better than that of the drugs on the market. The report of this invention is novel and not predictable and inferred by those skilled in the art.

The invention also discloses a group of new application of R-enantiomer beta 2 agonists in preventing and treating the IBD and related hemorrhoids symptoms as follows: short-acting β 2 agonists: bitolterol, fenoterol, isoproterenol, oxaprorine, or methamphetamine long-acting beta 2 agonists: formoterol, arformoterol, performist, salmeterol, Chumachiteluo; and ultra-long acting β 2 agonists: arbiterol, carmoterol, indacaterol, lodaterol, vilanterol, isosulpirine, maprotirol and zilpaterol.

In one embodiment, S-salbutamol and S-bambuterol are administered 8 days after DSS-induced IBD in the same manner as R-salbutamol mentioned above. DAI and histological scores were checked regularly over 22 days. Surprisingly, S-salbutamol and S-bambuterol showed no signs of improved symptoms or therapeutic effect compared to R-salbutamol, in contrast to DSS mice, both S-salbutamol and S-bambuterol worsened disease manifestations and inflammatory responses. DAI was markedly worse after S-salbutamol treatment compared to untreated DSS mice. In particular, mice treated with S-salbutamol and S-bambuterol weigh less, fecal occult is more severe on days 14 and 22, and colon length is shorter than untreated mice. Histologically, inflammatory infiltrates are found in the mucosa, lamina propria, submucosa and even muscularis. Disappearance of crypts, epithelial degeneration, mucosal erosion. The histological scores of S-salbutamol and S-bamterol treated DSS mice were significantly higher than untreated DSS mice.

The present invention reveals that, unlike the R-enantiomer, S-salbutamol and S-bambuterol are toxic, but not beneficial, to the intestinal tract of IBD. Treatment with S-salbutamol and S-bambuterol further exacerbates the clinical symptoms and inflammatory response of IBD. Furthermore, the removal of the S-enantiomer salbutamol and the S-enantiomer bambuterol from their racemic forms, e.g. using R-salbutamol instead of racemic salbutamol and bambuterol, can significantly reduce toxicity and adverse effects. The present invention therefore discloses a new use of R-salbutamol or R-bambuterol or other R enantiomer β 2 agonists for the treatment of subacute or chronic IBD to reduce adverse effects. The present report should be considered novel and involves inventive steps, as these toxic effects are unexpected.

In another embodiment, other S-enantiomers of β 2 adrenergic agonists, including S-terbutaline and S-clenbuterol, are used in DSS mice in the same manner as described above. Similar toxic effects were observed. Both Disease Activity Index (DAI) and histological score were much worse in S-terbutaline and S-clenbuterol treated DSS mice than untreated DSS mice. The present invention reveals that S-terbutaline and S-clenbuterol are toxic to the intestinal tract, rather than having therapeutic effect on the intestinal tract as the R-enantiomer.

The invention therefore discloses a new use of R-terbutaline or R-clenbuterol and other R-enantiomer β 2 agonists for the treatment of subacute or chronic IBD to reduce adverse effects. Other R-enantiomer β 2 agonists according to the invention include: r or R' enantiomer of a short-acting β 2 agonist: bitolterol, fenoterol, isoproterenol, oxepriniline or meproterenol, bitbrerol, procaterol; long-acting β 2 agonists: salmeterol, traneniterol, arformoterol, formoterol, Perforomist, Chuangterol; and ultra-long acting β 2 agonists: arbiterol, carmoterol, indacaterol, lodaterol, vilanterol, isosulpirine, maprotirol and zilpaterol.

In one embodiment, the present invention discloses the therapeutic mechanism of R-enantiomer β 2 agonists in IBD and its associated symptoms, involving inhibition of macrophage polarization and inhibition of activation of gut resident lymphocytes, CD4 and CD8T lymphocytes, and induction of metabolism and recombination in these cells. On the other hand, S-enantiomer β 2 agonists play the opposite role. These findings are novel and have not been reported in the prior art.

Parenteral manifestations (EIMs) of Inflammatory Bowel Disease (IBD) are common in both Ulcerative Colitis (UC) and Crohn's Disease (CD). These manifestations can almost involve multiple organ systems including musculoskeletal, skin, hepato-pancreaticobiliary, ocular, renal, and lung. These all present significant challenges to physicians treating IBD patients. The results show that 31.4% of patients with UC and 40.4% of patients with CD have 1 of the 5 main manifestations, with a few patients having more than 1 of the main EIMs. Some data indicate that approximately one-third of patients develop symptomatic Primary Sclerosing Cholangitis (PSC) before being diagnosed with IBD. According to some small studies, 10-30% of arthritis patients associated with IBD develop symptoms of arthritis before IBD is diagnosed. (Levine et al, explicit management of information Bowell disease. gastroenterology & Heatology Volume 7, Issue 4,235 April.2011).

Skin problems affect about 15% of different types of IBD patients. Skin problems relate to Inflammatory Skin Diseases (ISDs) including mainly psoriasis, rosacea, and atopic dermatitis, among others (Kim et al, inflammation bounded disease associated with an increased risk of inflammation skin diseases, J Am ad dermatological 2017 Jan; 76 (1)). Also included are autoimmune skin diseases such as lupus, vitiligo and alopecia areata. Skin diseases associated with IBD also include: miliaria, pyoderma gangrenosum, Sweet syndrome, intestinal-related skin disease-arthritis syndrome (BADAS), vegetative pyoderma-pyoderma (PPV). Allergic vasculitis, acne and urticaria (Elaine K.Luo, and Ana Gotter,10 Skin Rashes Linked to ultrasonic diagnosis, medical review July 5,2017)

The invention discloses that the R-enantiomer of a β 2 agonist is useful for the treatment of extra-intestinal manifestations of inflammatory bowel disease (EMI), including arthritis, osteoarthritis, lupus, ankylosing spondylitis. Vitiligo, psoriasis, amyloidosis, erythema nodosum, pyoderma gangrenosum, primary sclerosing cholangitis, acne, eczema, urticaria, uveitis, iritis, episcleritis, scleromalacia, corneal ulcers, retinal vascular disease, rhinitis, and pulmonary inflammatory disease.

In one embodiment, knee joint damage is also present in DSS-induced IBD mice. The articular cartilage structure becomes thinner and partially destroyed. Cartilage cells are degenerated, reduced and aggregated. The meniscus is damaged, losing structural integrity. The X-ray result shows that the articular cartilage layer becomes thin and the bone structure is reduced. The above-mentioned lesions were significantly reduced in the R-salbutamol or R-bambuterol treated mice. The articular cartilage and bone structures gradually return to normal.

The invention provides a new application of R-salbutamol or R-bambuterol in treating IBD arthritis. In another example, using S-salbutamol and S-bambuterol in DSS-induced IBD mice, the damage to articular cartilage and meniscus was exacerbated compared to untreated DSS mice. The present invention discloses that S-salbutamol and S-bambuterol are detrimental to cartilage and meniscus. In addition, the invention discloses a new application of R-salbutamol or R-bambuterol in treating arthritis, and adverse reactions can be reduced. It is known in the art that the parenteral manifestations (EIMs) of IBD share a common pathogenesis. The invention also discloses the use of R-salbutamol or R-bambuterol for the treatment of other extra-intestinal manifestations (EIMs) of IBD as described above, including arthritis, ankylosing spondylitis. Oral crohn's disease, lupus, amyloid diseases, erythema nodosum and primary sclerosing cholangitis, ocular diseases, rhinitis and pulmonary inflammation.

In one embodiment, topical application of R-salbutamol, R-terbutaline and R-krebs significantly reduces the symptoms of acne, acne malignancy, urticaria and eczema. The oral administration of the medicine to rats can obviously improve psoriasis and eczema.

The invention provides a new application of R-salbutamol, R-terbutaline, R-bambuterol and R-clenbuterol in treating Inflammatory Skin Diseases (ISDs) and autoimmune skin diseases related to IBD, which mainly comprises the following steps: psoriasis, rosacea, atopic dermatitis, miliaria, acne, urticaria, pyoderma gangrenosum, Sweet's syndrome, bowel-related skin disease-arthritis syndrome (BADAS), pyodermas-suppurative stomatitis (PPV). Allergic vasculitis, vitiligo and alopecia areata. Other enantiomeric adrenergic beta 2 agonists have similar pharmacological effects. The present invention discloses the use of other R-enantiomer adrenergic beta 2 agonists for the treatment of arthritis and other extra-intestinal manifestations (EIM) of IBD as described above. Other chiral β 2 agonists according to the invention as described above include the R or R' enantiomer of the short-acting β 2 agonist: bitolterol, fenoterol, isoproterenol, oxepril or metaproterenol-pirbuterol, procaterol, ritodrine; and long-acting β 2 agonists: arformoterol, formoterol fumarate, salmeterol, tritelolone, and ultralong acting beta 2 agonists: abetidronol, carmoterol, indoxacarbirole, oloditerol, vilanterol, isosulpirine, mabuterol and zilpaterol.

The invention provides a new application of R-enantiomer adrenergic beta 2 agonist, which is used for treating IBD and EIMs of IBD and reducing adverse reaction. The disclosure of the present invention has never been reported in the prior art and was unexpected to one skilled in the art.

Current treatments for IBD include anti-inflammatory drugs, typically 5-aminosalicylates. Such drugs include sulfasalazine, mesalazine, balsalazide and olsalazine. On the other hand, corticosteroids may be used for moderate to severe ulcerative colitis, including prednisone and hydrocortisone, among others. Other drugs for treating IBD include immunosuppressive drugs, including azathioprine and mercaptopurine, with less use of cyclosporine. Antibodies are also used, including infliximab, adalimumab, and golimab, as well as wedelozumab, certolizumab, natalizumab, uitlizumab, and Bm-ca. However, the above drugs generally have serious side effects, and most are not suitable for long-term use. Chiral R-enantiomer β 2 agonists are less toxic and relatively safe in preclinical and clinical studies. The present invention discloses that R-salbutamol or R-bambuterol or one other R-or R 'R' enantiomer beta 2 agonist has a synergistic effect in combination with any anti-inflammatory agent (e.g. 5-aminosalicylate or corticosteroid) or allows a reduction in the dose and toxicity of commercially available anti-inflammatory agents.

The invention also discloses that one of R-salbutamol or R-bambuterol or other R-or R 'R' enantiomer beta 2 agonists has synergistic effect in combination with any one of the immunosuppressive drugs (e.g. azathioprine and mercaptopurine), or reduces the dose of the marketed immunosuppressive drug, as well as reduces the toxicity of the drug. The present invention discloses the use of at least one R or R' R-enantiomer β 2 agonist in combination with at least one anti-inflammatory agent (including at least one 5-glycine and a corticosteroid) or at least one immunosuppressive agent as described above, or the treatment of IBD and the parenteral manifestations of IBD with at least one immunomodulatory antibody as described above, or with at least one antibiotic.

Sphingosine-1-phosphate (S1P) receptors are drug targets in IBD. S1P regulates the efflux of lymphocytes from lymph nodes into the circulatory system and subsequently enhances the inflammatory response of the gut by producing pro-inflammatory cytokines. The present invention discloses the combined use of at least one R or R' R-enantiomer β 2 agonist and at least one S1P receptor modulator for the treatment of IBD and the parenteral manifestations of IBD.

Corticosteroids contemplated according to the present invention include budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, and dexamethasone.

Antibiotics to which the present invention relates include aminoglycosides, ansamycin, carbacephem, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactans, nitrofurans, oxazolidinones, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines, chloramphenicol, phosphonic acid antibiotics, and mycobacterial antibiotics.

The antibodies involved according to the present invention include antibodies to infliximab, adalimumab, golimab, wedelizumab, certolizumab, natalizumab, ubetazumab, and Bm-ca.

S1P receptor modulators involved according to the invention are fingolimod, beneximod, siponimod, ozanimod, amiserimod and GSK 2018682.

In an embodiment of the invention, there is provided a novel pharmaceutical composition comprising an effective amount of a β 2 agonist of R-salbutamol or R-bambuterol or other R or R' R-enantiomer and salts thereof for single or combined administration to a patient in need thereof by oral, oral film, inhalation, nasal spray, injection, topical, eye drop, rectal or vaginal administration. The dosage forms include solid preparation, solution, atomized aerosol, injection, ointment, skin patch, film, soft capsule and suppository.

The ee value or optical purity of the R-enantiomer of the β 2 agonist according to the invention is not less than 80% or preferably greater than 98% or more preferably greater than 99%.

Pharmaceutically acceptable salts of the R-enantiomer of the β 2 agonists according to the invention include those formed from conventional pharmaceutically acceptable inorganic or organic acids, for example: hydrochloride, hydrobromide, sulphate, hydrogen sulphate, dihydrogen phosphate, methanesulphonate, bromide, methyl sulphate, acetate, oxalate, maleate, fumarate, succinate, 2-naphthalenesulphonate, glycolate, gluconate, citrate, tartrate, lactic acid, pyruvic acid isothioester, benzenesulphonate or p-toluenesulphonate.

Examples

Example 1 Effect of R-and S-enantiomeric beta 2 agonists on DSS-induced acute colitis

Method

Animal and drug treatment

Male C57BL mice (8-10 weeks, 18-20g) were divided into control (drinking water only), DSS and DSS plus treatment (n-5). 2% (w/v) Dextran Sulfate Sodium (DSS) with molecular weight of 36-50 kda was added to the drinking water of mice for 7 days to cause acute colitis. At the end of day 7, DSS was removed and animals received drinking water for 2 days only. Animals were euthanized at day 10 after intake of DSS, and subjected to anatomical and histological examination.

R-salbutamol (R-S) (1mg/kg), S-salbutamol (S-S) (1mg/kg), R-terbutaline (R-T) (1mg/kg), S-terbutaline (S-T) (1mg/kg), R-bambuterol (R-B) (10mg/kg), S-bambuterol (S-B) (10mg/kg), R-clenbuterol (R-C) (1mg/kg), RS-clenbuterol (RS-C) (2mg/kg) were injected intravenously daily for 8 consecutive days starting on day 2 of DSS induction. Dexamethasone (Dex)1mg/kg was used as a control drug. The ee value of the enantiomer of each chiral drug is more than 98%, and the chemical purity is more than 99%.

Assessment of disease symptoms and histological changes

Disease Activity Index (DAI): animals were monitored daily for body weight, water/food consumption, morbidity, fecal consistency and fecal blood. And comprehensively calculating Disease Activity Indexes (DAI) according to various indexes such as weight loss, stool consistency, stool occult blood and the like, wherein the DAI range is 0-12. The score is defined as follows: the stool consistency is 0, no diarrhea is caused, 2, loose stool which does not stick to the anus is caused, and 4, liquid stool which sticks to the anus is caused; fecal occult blood 0 is no, 2 is moderate, 4 is major hemorrhage; weight loss, if body weight remains at 1% or higher of baseline, is assigned a value of 0, 1 indicating 1-5% weight loss, 2 indicating 5-10% weight loss, 3 indicating 10-15% weight loss, and 4 indicating more than 15% weight loss. Colon length was recorded as an index of inflammation. The entire colon was cut longitudinally and gently rinsed with sterile Phosphate Buffered Saline (PBS) to remove any fecal traces for pathological examination. The colon segment was fixed with 10% neutral formalin. Tissues were paraffin embedded, sectioned, and mounted on glass slides. H & E stained paraffin section images were collected. The pathology evaluation was performed by blind methods.

Histological scoring: epithelium, 0 ═ morphologically normal, 1 ═ goblet cell loss, 2 ═ large area goblet cell loss, 3 ═ crypt loss, 4 ═ large area crypt loss; inflammatory cell infiltration, 0 ═ no infiltration, 1 ═ pericrypt infiltration, 2 ═ infiltration to the muscularis mucosae, 3 ═ extensive infiltration to the muscularis mucosae, thickening of the mucosa with massive edema, and 4 ═ submucosal infiltration. Data are presented as mean ± Standard Deviation (SD).

As a result:

beta 2 receptor agonists ameliorate DSS-induced acute colitis.

The mice developed symptoms of colitis including weight loss, diarrhea, hematochezia after oral administration of 2% DSS7 days. The Disease Activity Index (DAI) of DSS mice was significantly increased. Beta 2 receptor agonist treatment significantly improved symptoms, reduced DAI values and histological scores. The therapeutic effect of the R-enantiomer, a β 2 receptor agonist, was superior to that of the control drug dexamethasone (table 1).

TABLE 1 observation of the efficacy of different treatment methods for DSS acute colitis

DAI: disease activity index, FOB: fecal occult blood, Col: colon, Hist: histology.

FIG. 1 DSS-induced colitis and treatment with RS (R-salbutamol), RB (R-bambuterol), RT (R-terbutaline), RC (R-clenbuterol), S-S (S-salbutamol), SB (S-bambuterol), S-T (S-terbutaline), RS-C (racemic clenbuterol) and dexamethasone (dexamethasone) for one week.

Histological analysis of control H & E stained colon specimens (fig. 1) showed that the colonic mucosal epithelium remained intact, continuous, regularly arranged crypts, clear structures, and without inflammatory cell infiltration and ulceration. The DSS model group has the defects of mucosa losing structure, discontinuous mucosa epithelium, wide ulcer lesion, crypt disappearance, inherent layer destruction, inflammation expansion to mucosa and submucosa and large inflammatory cell infiltration. In the R-enantiomer treatment group (DSS + R-A or R-B or R-T or R-C), the histological integrity was greatly improved and the laminA propriA was intact. The crypts are regenerated, the mucous epithelium is recovered clearly, and inflammatory infiltration is obviously reduced. The mucosa and submucosa of the animals treated with DSS + DEX had inflammatory infiltrates, the lamina propria was destroyed, but there were a few crypts and mucosal epithelium that did not grow completely. In general, there was some improvement in DSS + DEX treatment, but less improvement compared to R-enantiomer β 2 agonist treatment.

There was no evidence of improvement in S-enantiomer treated DSS mice (DSS + S-S or S-B or S-T) compared to untreated DSS mice. In contrast, inflammatory infiltrates are more severe in the mucosa, submucosa and muscularis (DSS + S-B), with complete destruction of the lamina propria (DSS + S-S). The histopathology is obviously changed. S-enantiomer treated DSS mice had a higher histological score than untreated DSS mice and was much higher than R-enantiomer treated DSS mice.

When the racemate RS-clenbuterol was used, the histological score was better than that of mice not treated with DSS, but worse than that of mice treated with the R-enantiomer. Treatment of DSS mice with clenbuterol hydrochloride resulted in disappearance of the mucosal epithelium, destruction of the lamina propria, inflammatory infiltration of the mucosa, submucosa and muscularis, but a small regeneration of crypts. The overall histology and DAI performance of RS-clenbuterol mice was much less than that of DSS mice treated with R-clenbuterol.

Example 2 Effect of R-and S-enantiomeric beta 2 agonists on DSS-induced subacute colitis

Method

In the same manner as in example 1,

1) the study period was three weeks with one week of treatment.

At the end of week 1, DSS and all treatments were removed and replaced with drinking water. Mice were then given free water and food for 2 weeks. Evaluation and examination were performed on day 23 according to example 1.

2) Only R-salbutamol (R-S) (1mg/kg), S-salbutamol (S-S) (1mg/kg), R-Bambutrol (R-B,10mg/kg), S-bambuterol (S-B) (10mg/kg) were used.

As a result:

effect of R-bambuterol on disease activity index and histological score.

In DSS-induced IBD mice, there was still a significant decrease in body weight and fecal occult blood at day 14 and day 22 after one week of DSS cessation, but a slight improvement compared to day 7. The DAI increased significantly. Disease Activity Index (DAI) was significantly improved after R-salbutamol treatment compared to untreated DSS animals. The body weight of the mice is similar to that of the normal control group, and the fecal occult blood is obviously reduced after the treatment of the R-salbutamol and the R-bambuterol compared with the DSS mice. (Table 2)

In DSS-induced colitis mice, colon length was significantly shortened, with a significantly higher histological score than the control group. Has obvious mononuclear leukocyte infiltration, small amount of newly grown crypt structural disorder, epithelial denudation, mucosa abnormality, inherent layer destruction and granuloma formation. The histological index of the R-salbutamol and the R-bambuterol after the administration is also obviously improved. The mucosa is regenerated completely, the crypt arrangement is normal, and the epithelium grows normally. The lamina propria is normal. Inflammatory infiltration was not seen in both mucosa and submucosa.

TABLE 2 Observation of the efficacy of different treatment methods for DSS colitis

(DAI) discrete active index, fob local occlusion blood, col, last. historical DAI: disease activity index, FOB: fecal occult blood, Col: colon, Hist: histology.

Example 3 Effect of extended treatment in DSS-induced subacute colitis

Method

1) Two weeks of treatment in a three week experimental period

At the end of the first week, the DSS aqueous solution was removed. The treatment was administered for 14 consecutive days starting on the first day. In the third week, only purified water is drunk. Mice were sacrificed on day 23 and their colons evaluated and examined according to the method of example 1. The therapeutic agents used include R-salbutamol (R-S) (1mg/kg), S-salbutamol (S-S) (1mg/kg), R-bambutamol (R-B) (10mg/kg), S-bambutamol (S-B) (10 mg/kg).

As a result:

despite some recovery of disease status during the acute phase, DAI and histological scores were significantly elevated in untreated DSS group mice. In DSS mice treated with R-salbutamol (DSS + R-S) or R-bambuterol (DSS + R-B), both DAI and histological score returned to normal. The mucosa, epithelium and crypts all appeared normal, and after two weeks of R-B or R-S treatment, the mucosa, submucosa and lamina propria were normal without inflammatory infiltration (FIG. 2, DSS + R-B and DSS + R-S). In contrast, in S-S and S-B treated DSS mice, colitis symptoms worsened, with both DAI and histologic scores significantly higher than those in the DSS group. Ulceration and lesions of the mucosal epithelium, disorganization of crypt structures, destruction of lamina propria and muscularis mucosae, inflammatory infiltration and abnormal structural status of each layer (figure 2, DSS + S-S, DSS + S-B). Overall, in DSS-induced chronic colitis, S-S and S-B treatment resulted in severe exacerbation of the disease compared to the model group.

FIG. 2 pathological graphs of DSS-induced colitis and R-S, S-S, R-B, S-B treatment for 2 weeks

Example therapeutic Effect of tetra R-Salbutamol on psoriasis (extra-intestinal manifestations of IBD)

Psoriasis is one of the major parenteral manifestations of IBD. Since psoriasis occurs in the present invention only in a fraction of DSS-or TNBS-induced IBD mice. A recognized mouse model of psoriasis was therefore used: IMQ-induced psoriasis-like skin lesions for subsequent study.

The method comprises the following steps:

mice were divided into control, psoriasis and psoriasis plus treatment groups (n-8) and applied to the back skin with imiquimod cream (62.5mg) for 7 consecutive days to induce psoriasis. The control group used an equal amount of petrolatum. Set 4 treatment groups: 3 doses of R-salbutamol (R-Sal) (0.5mg, 1mg, 2mg/kg), dexamethasone (1mg/kg) were used as positive controls. The control group was gavaged with an equal amount of physiological saline. In some experiments, R-bambuterol (a long-acting β 2 agonist) was used as an additional treatment group (n ═ 3). The severity index of the psoriatic region was used for evaluation. Each score for erythema, scaling and skin thickness was measured separately and then combined to calculate scores (fig. 2).

As a result:

(1) skin performance and psoriasis regional severity score

Typical psoriatic lesions can be induced with imiquimod cream. The focus was significantly improved after drug treatment (fig. 3). Psoriasis (PSO) group psoriasis area severity score was highest. Score decreased significantly after treatment. There is a dose dependence on R-salbutamol. The effect of high doses of salbutamol (2.0mg/kg) on each score was best, similar to that of dexamethasone (Dex) (FIG. 2).

FIG. 1 Effect of R-salbutamol (R-Sal) and dexamethasone (Dex) on IMQ-induced psoriatic dorsal skin lesions after 8 days of treatment

Figure 2 IMQ-induced psoriatic zone severity scores of psoriatic dorsal lesions following treatment with dexamethasone (Dex), 0.5mg/kg (R1), 1.0mg/kg (R2), 2.0mg/kg (R3), respectively.

(2) Histological changes

Figure 3 shows the histological changes after treatment of psoriasis with R-salbutamol, S-salbutamol and R-bambuterol. After IMQ-induced psoriasis, the skin is severely damaged. The lesions were substantially recovered after treatment with R-salbutamol and R-bambuterol. It is noted that treatment with S-salbutamol worsens the histological changes of psoriasis, widens the lesions, thickens the epidermis (acanthosis), stretches the reticulate pattern, and presents inflammatory infiltrates on the epidermis/dermis. The exacerbating effects of S-salbutamol on psoriasis have never been disclosed in the prior art.

FIG. 3 shows the results of HE staining of dorsal skin of IMQ-induced psoriasis mice treated with salbutamol (R-Sal), bambuterol (R-BMB) and s-salbutamol.

(3) Signal path

Compared with psoriasis mouse plasma, the mRNA expression of non-receptor tyrosine kinase Janus kinase 2(JAK2) and a signal converter and activator of transcription protein (STAT3) is enhanced, and the JAK2 and STAT3 proteins are suggested to be increased. The results show that R-salbutamol inhibits the overexpression of mRNA of JAK2 and STAT3 in a dose-dependent manner (left and right fig. 3).

The invention discloses a therapeutic effect of an R-salbutamol R-beta 2 agonist on psoriasis, which relates to JAK2 and STAT3 signal pathways, and the JAK2 and STAT3 signal pathways are also considered to be closely related to IBD diseases. The present disclosure is novel and unexpected to those skilled in the art.

Example therapeutic Effect of pentaR-Salbutamol on eczema (an extra-intestinal manifestation of IBD)

Eczema (atopic dermatitis) is one of the major intestinal manifestations of IBD. Since only a fraction of the IBD mice induced with DSS or TNBS produced eczema, a commonly accepted mouse model of atopic dermatitis, 2,4-Dinitrochlorobenzene (DNCB) -induced allergic contact dermatitis, was used in subsequent studies (Ku et al. the prevention of 2,4-dinitrochlorobenzene-induced allergic contact dermatitis in BALB/c microwave by Jawooongo, BMC comparative and Alternative Medicine (2018)18: 215).

The method comprises the following steps: model of DNCB-induced allergic contact dermatitis (eczema)

The mouse abdominal cavity was shaved, DNCB (100. mu.L 0.5%) was dissolved, and the resultant solution was applied to the shaved skin to induce atopic dermatitis for 2 consecutive days. On day 8, DNCB was applied to the inner and outer ear surfaces, respectively, to induce eczema. Treatment with R-albuterol and indomethacin was administered beginning on the first day with gavage for 8 consecutive days. Mice were dissected and evaluated accordingly on day 9.

Animal and treatment

Mice were divided into 4 groups (n-8), group a being a control group; group B is model group (DNCB); group C is the treatment group of R salbutamol 0.05, 0.1 or 0.2 mg/kg; and the D group is an indometacin treatment group with the concentration of 5 mg/kg. Inflammation scores were based on binaural swelling, erythema, scratching, thickness, etc.

Scoring criteria

As a result:

(1) inflammation score changes

The binaural thickness and erythema integral served as inflammatory indicators of eczema, and the model group had a significant increase in binaural thickness and erythema integral compared to the control group. After treatment with different doses of R-salbutamol, both the thickness and erythema were significantly reduced, while the therapeutic effect of indomethacin was less.

(2) Histological changes

After DNCB modeling, serious eczema can be generated, and the treatment of R-salbutamol can obviously improve the histological change of eczema, reduce inflammatory infiltration and show dose dependence. While indomethacin had less therapeutic effect on eczema (panels a-F).

Figure effect of R-salbutamol treatment on DNCB-induced eczematous mouse ear skin histopathology (400X).

(A) A control group; (B) and (3) eczema group: (a) monocyte infiltration and (b) epidermal keratinization; (C) r-salbutamol (0.05mg/kg) treatment group: the influence on eczema is small; (D) r-salbutamol (0.1mg/kg) treatment group: monocyte infiltration is significantly reduced; (E) r-salbutamol (0.1mg/kg) treatment group: monocyte infiltration and epidermal keratinization are further reduced; (F) indometacin treatment group (5mg/kg/day)

Example therapeutic Effect of hexaR-salbutamol on urticaria (an extra-intestinal manifestation of IBD)

A healthy female has urticaria on her face and eyelid area for one week. Symptoms were significantly alleviated in the morning on day 2 by topical application of R-salbutamol (R-sal) cream (5%) overnight (lower panel).