IMMUNOSUPPRESSIVE PHARMACEUTICAL COMPOSITION AND APPLICATION THEREOF
20220079915 · 2022-03-17
Assignee
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
A61K31/5575
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K31/201
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
A61K31/5575
HUMAN NECESSITIES
A61K31/352
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/573
HUMAN NECESSITIES
A61K9/0014
HUMAN NECESSITIES
A61K31/573
HUMAN NECESSITIES
International classification
A61K31/352
HUMAN NECESSITIES
A61K31/201
HUMAN NECESSITIES
A61K31/573
HUMAN NECESSITIES
Abstract
Provided are a drug, a pharmaceutical composition, or a pharmaceutical kit for immunosuppression, for the treatment of conditions such as psoriasis, comprising a forskolin derivative or a salt thereof and optionally a prostaglandin compound or a glucocorticoid compound. Further provided is a method for immunosuppression, comprising administration of a forskolin derivative or a salt thereof and optionally a prostaglandin compound or a glucocorticoid compound. Forskolin derivatives show promise as a class of small-molecule drugs for achieving immunosuppression using new mechanisms, for the treatment of conditions such as psoriasis, when used alone or in combination with other substances. Compared with first-line antibody drugs, forskolin derivatives have significant advantages in process and price, and have comparable efficacy and fewer side effects compared with the first-line drug calcipotriol betamethasone ointment.
Claims
1. A pharmaceutical composition comprising (1) a compound of formula I or a pharmaceutically acceptable salt thereof: ##STR00026## wherein: R.sup.3 is —CH═CH.sub.2, —CH.sub.2CH.sub.3, or cyclopropyl; one of R.sup.1 and R.sup.2 is —COCH.sub.2CH.sub.3, —CO.sub.2CH.sub.2CH.sub.3, —COCH.sub.2OCHO or group ##STR00027## wherein R.sup.4 and R.sup.5 are each independently hydrogen or lower alkyl, or R.sup.4 and R.sup.5 are combined to form a lower alkylene chain containing or not containing an oxygen atom or a nitrogen atom, and m is an integer from 1 to 5; the other of R.sup.1 and R.sup.2 is hydrogen or group CO(CH.sub.2).sub.nX, wherein X is hydrogen or group ##STR00028## wherein R.sup.6 and R.sup.7 are each independently hydrogen or lower alkyl, or R.sup.6 and R.sup.7 are combined to form a lower alkylene chain containing or not containing an oxygen atom or a nitrogen atom, and n is an integer between 1 to 5; or R.sup.1 is hydrogen or —COCH.sub.2CH.sub.2CO.sub.2H, and R.sup.2 is hydrogen, —COCH.sub.3, —COCH.sub.2CH.sub.2CH.sub.2CO.sub.2H or COCH(OH)CH.sub.2OH, with the proviso that when R.sup.1 is hydrogen, R.sup.2 is —COCH.sub.2CH.sub.2CH.sub.2CO.sub.2H or —COCH(OH)CH.sub.2OH, and (2) a prostaglandin compound or a glucocorticoid compound.
2-5. (canceled)
6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a compound of formula I selected from the group consisting of 6-(4-aminobutyryl)forskolin, 6-[4-(dimethylamino)butyryl]forskolin, 6-[3-aminopropionyl]forskolin, 6-[3-(methylamino)propionyl]forskolin, 6-[3-(dimethylamino)propionyl]forskolin, and 6-[(piperidino)acetyl]-7-desacetyl forskolin, or a pharmaceutically acceptable salt thereof.
7. (canceled)
8. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises 6-[3-(dimethylamino)propionyl]forskolin hydrochloride.
9-11. (canceled)
12. The pharmaceutical composition of claim 1, wherein the prostaglandin compound is selected from the group consisting of prostaglandin E2, dinoprost tromethamine, carboprost, carboprost tromethamine, prostaglandin E1, bimatoprost, iloprost, limaprost, limaprost a cyclodextrin, misoprostol, gemeprost, latanoprost, sulprostone, ornoprostil and pharmaceutically acceptable salts thereof.
13-14. (canceled)
15. The pharmaceutical composition of claim 1, wherein the glucocorticoid compound is selected from the group consisting of dexamethasone, hydrocortisone, prednisone, prednisolone, paramethasone, cortisone, betamethasone, meprednisone, fludrocortisone, triamcinolone acetonide and pharmaceutically acceptable salts thereof.
16. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and prostaglandin E2.
17. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and hydrocortisone.
18. The pharmaceutical composition of claim 1 for use in inducing immunosuppression in a subject.
19-20. (canceled)
21. The pharmaceutical composition of claim 18 for use in treating psoriasis in a subject.
22-25. (canceled)
26. A method of inducing immunosuppression in a subject, comprising administering to the subject a compound of formula I or a pharmaceutically acceptable salt thereof: ##STR00029## wherein: R.sup.3 is —CH═CH.sub.2, —CH.sub.2CH.sub.3, or cyclopropyl; one of R.sup.1 and R.sup.2 is —COCH.sub.2CH.sub.3, —CO.sub.2CH.sub.2CH.sub.3, —COCH.sub.2OCHO or group ##STR00030## wherein R.sup.4 and R.sup.5 are each independently hydrogen or lower alkyl, or R.sup.4 and R.sup.5 are combined to form a lower alkylene chain containing or not containing an oxygen atom or a nitrogen atom, and m is an integer from 1 to 5; the other of R.sup.1 and R.sup.2 is hydrogen or group CO(CH.sub.2).sub.nX, wherein X is hydrogen or group ##STR00031## wherein R.sup.6 and R.sup.7 are each independently hydrogen or lower alkyl, or R.sup.6 and R.sup.7 are combined to form a lower alkylene chain containing or not containing an oxygen atom or a nitrogen atom, and n is an integer between 1 to 5; or R.sup.1 is hydrogen or —COCH.sub.2CH.sub.2CO.sub.2H, and R.sup.2 is hydrogen, —COCH.sub.3, —COCH.sub.2CH.sub.2CH.sub.2CO.sub.2H or COCH(OH)CH.sub.2OH, with the proviso that when R.sup.1 is hydrogen, R.sup.2 is —COCH.sub.2CH.sub.2CH.sub.2CO.sub.2H or —COCH(OH)CH.sub.2OH.
27-30. (canceled)
31. The method of claim 26, wherein the method comprises administering to the subject a compound selected from the group consisting of 6-(4-aminobutyryl)forskolin, 6-[4-(dimethylamino)butyryl]forskolin, 6-[3-aminopropionyl]forskolin, 6-[3-(methylamino)propionyl]forskolin, 6-[3-(dimethylamino)propionyl]forskolin, and 6-[(piperidino)acetyl]-7-desacetyl forskolin, or a pharmaceutically acceptable salt thereof.
32. (canceled)
33. The method of claim 26, wherein the method comprises administrating 6-[3-(dimethylamino)propionyl]forskolin hydrochloride to the subject.
34. (canceled)
35. The method of claim 26, wherein the method comprises administrating to the subject in combination with a prostaglandin compound.
36. The method of claim 35, wherein the prostaglandin compound is selected from the group consisting of prostaglandin E2, dinoprost tromethamine, carboprost, carboprost tromethamine, prostaglandin E1, bimatoprost, iloprost, limaprost, limaprost a cyclodextrin, misoprostol, gemeprost, latanoprost, sulprostone, ornoprostil and pharmaceutically acceptable salts thereof.
37. The method of claim 26, wherein the method comprises administrating to the subject in combination with a glucocorticoid compound.
38. The method of claim 37, wherein the glucocorticoid compound is selected from dexamethasone, hydrocortisone, prednisone, prednisolone, paramethasone, cortisone, betamethasone, meprednisone, fludrocortisone, triamcinolone acetonide and pharmaceutically acceptable salts thereof.
39. The method of claim 36, wherein the method comprises administrating 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and prostaglandin E2 to the subject.
40. The method of claim 38, wherein the method comprises administrating 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and hydrocortisone to the subject.
41-42. (canceled)
43. The method of claim 26, wherein the subject is a patient with autoimmune disease.
44. The method of claim 43, wherein the subject is a patient with psoriasis.
45-82. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0061]
[0062]
[0063]
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
[0080]
DETAILED DESCRIPTION OF THE INVENTION
[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.
[0082] Forskolin is a compound extracted from the roots of Coleus forskohlii of the Coleus genus in labiatae family in India in the 1970s. It acts as an adenylate cyclase activator and has cardiotonic action, hypotensive effect, etc. Its structural formula is shown in formula II:
##STR00006##
[0083] The PCT patent application with application number PCT/US84/00291 reported that a phenomenon was observed in 1985 that forskolin relieved the pathological symptoms of 4 patients with psoriasis. In the same peroid, there were fewer studies showing that forskolin affected the ratio of cAMP to cGMP in epidennal cells by activating cAMP, and further inhibited epidermal cell mitosis in epidermal hyperplasia symptoms such as psoriasis. It should be pointed out that psoriasis is an autoimmune disease, and, in addition to the proliferation of epidermal cells, immune cell invasion and repeated and continuous inflammation at the lesion sites are the most important pathological features. However, the above observations or studies always lacked the explanation and discovery of the mechanism of action (Mechanism of Action) of forskolin on the pathological inflammation of psoriasis. Moreover, because of the extremely limited number of 4 clinical patients, there is no obvious logical connection and sufficient data support. For this reason, the development and application of adenylate cyclase activators represented by Forskolin for the above-mentioned diseases have been stagnant for more than 30 years thereafter. There are many pharmacodynamic studie on Forskolin compound itself, but its poor water solubility limits its further development into medicine. The present invention chooses the structure-optimized adenylate cyclase agonist forskolin structural derivatives, and demonstrates for the first time that forskolin derivatives can specifically reduce the production of pro-inflammatory factor TNF-α in human monocyte macrophage THP-1 in vitro, and can specifically reduce the production of pro-inflammatory factor IL-17A during the induced differentiation of Naïve CD4+ T cells from mouse spleen to Th17. The above two inhibitory effects are synergistically enhanced when combined with prostaglandin E2 or hydrocortisone. At the same time, the present invention also demonstrates for the first time that the forskolin derivatives have an efficacy equivalent to that of the first-line clinical drug calcipotriol betamethasone in the imiquimod mouse psoriasis model, and have fewer side effects.
[0084] Based on a series of in vitro and in vivo pharmacodynamic evaluation studies, the present disclosure reveals for the first time that structural derivatives of the small molecule compound forskolin can effectively reduce the secretion level of tumor necrosis factor α in THP-1 human macrophages in pharmacodynamic experiments in vitro. When used alone, forskolin structure derivatives can reduce the basic level secretion of TNF-α by up to 75%, and show dose-effect dependence (Concentration response). When combined with prostaglandin E2 or hydrocortisone, they can further reduce the basic level secretion of TNF-α by 90%, showing a stronger pharmacodynamic activity. At the same time, the structural derivatives of the small molecule compound forskolin can effectively reduce the secretion level of interleukin 17A in the in vitro pharmacodynamic experiment of inducing the differentiation of Naïve CD4+ T cells from mouse spleen to Th17. When the forskolin derivatives are used alone, they can reduce the basic level secretion of IL-17A by up to 97%, and there is dose-effect dependence (Concentration response). When combined with prostaglandin E2 or hydrocortisone, they can further reduce the basic level secretion of IL-17A, showing astronger pharmacodynamic activity. Furthermore, in a mouse psoriasis model induced by Imiquimod, the structural derivatives of forskolin show the effect of slowing down a number of disease severity indexes. For example, psoriasis-like skin indiction score, inflammatory thickening of the ears of the model, histological change of inflamed skin of the model were changed correspondingly in high and low dose administration groups and combined administration groups. Among them, the efficacy in the groups administrated with PGE2 is comparable to the current first-line clinical external drug calcipotriol betamethasone, while inflammatory redness of the skin is weaker, and there is no side effect of calcipotriol betamethasone, such as, reduction of the body weights of mice. The above findings suggest that the structural derivatives of forskolin, when used alone or in combination, are expected to become a new class of drugs for the treatment of psoriasis and for immunosuppression, and have comparable efficacy and fewer side effects compared with current clinical first-line drugs.
[0085] The forskolin derivatives provided in the present disclosure can be represented by the following formula I:
##STR00007##
[0086] wherein: [0087] R.sup.3 is —CH═CH.sub.2, —CH.sub.2CH.sub.3, or cyclopropyl; [0088] one of R.sup.1 and R.sup.2 is —COCH.sub.2CH.sub.3, —CO.sub.2CH.sub.2CH.sub.3, —COCH.sub.2OCHO or group
##STR00008## [0089] wherein R.sup.4 and R.sup.5 are each independently hydrogen or lower alkyl, or R.sup.4 and R.sup.5 are combined to form a lower alkylene chain containing or not containing an oxygen atom or a nitrogen atom, and m is an integer from 1 to 5; the other of R.sup.1 and R.sup.2 is hydrogen or group CO(CH.sub.2).sub.nX, wherein X is hydrogen or group
##STR00009## [0090] wherein R.sup.6 and R.sup.7 are each independently hydrogen or lower alkyl, or R.sup.6 and R.sup.7 are combined to form a lower alkylene chain containing or not containing an oxygen atom or a nitrogen atom, and n is an integer between 1 to 5; or [0091] R.sup.1 is hydrogen or —COCH.sub.2CH.sub.2CO.sub.2H, and R.sup.2 is hydrogen, —COCH.sub.3, —COCH.sub.2CH.sub.2CH.sub.2CO.sub.2H or COCH(OH)CH.sub.2OH, with the proviso that when R.sup.1 is hydrogen, R.sup.2 is —COCH.sub.2CH.sub.2CH.sub.2CO.sub.2H or —COCH(OH)CH.sub.2OH.
[0092] In some embodiments, in formula I R.sup.1 is hydrogen or group
##STR00010##
wherein m, R.sup.4, and Rare as defined above.
[0093] In some embodiments, in formula I R.sup.1 is
##STR00011##
R.sup.2 is —CO(CH.sub.2).sub.nX, and R.sup.3 is —CH═CH.sub.2 or —CH.sub.2CH.sub.3, wherein R.sup.4, R.sup.5, in, n and X as defined above; or R.sup.1 is hydrogen or —COCH.sub.2CH.sub.2CO.sub.2H, R.sup.2 is —COCH(OH)CH.sub.2(OH), and R.sup.3 is —CH═CH.sub.2.
[0094] In some embodiments, in formula I R.sup.1 is —COCH.sub.2N(CH.sub.3).sub.2, —CO(CH.sub.2).sub.2N(CH.sub.3).sub.2, —CO(CH.sub.2).sub.3N(CH.sub.3).sub.2, or —CO(CH.sub.2).sub.3NH.sub.2, and R.sup.2 is —COCH.sub.3.
[0095] In some embodiments, in formula I R.sup.1 is hydrogen, R.sup.2 is —COCH.sub.2CH.sub.3, —CO.sub.2CH.sub.2CH.sub.3 or —COCH.sub.2OCHO, and R.sup.3 is —CH═CH.sub.2.
[0096] As used herein, “lower alkyl” refers to a straight or branched chain alkyl group containing 1 to 5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, etc.
[0097] In
##STR00012##
when R.sup.4 and R.sup.5 are combined to form a lower alkylene chain, it refers to that R.sup.4 and R.sup.5 and the nitrogen atom to which they are connected together form a five-membered, six-membered, or seven-membered ring, and the five-membered, six-membered, or seven-membered ring may additionally contain or not contain an oxygen atom or a nitrogen atom.
[0098] In
##STR00013##
when R.sup.6 and R.sup.7 are combined to form a lower alkylene chain, it refers to that R.sup.6 and R.sup.7 and the nitrogen atom to which they are connected together form a five-membered, six-membered, or seven-membered ring, and the five-membered, six-membered, or seven-membered ring may additionally contain or not contain an oxygen atom or a nitrogen atom.
[0099] In some preferred embodiments, the forskolin derivatives are 6-(4-aminobutyryl)forskolin, 6-[4-(dimethylamino)butyryl]forskolin, 6-[3-(dimethylamino)propiony]forskolin, 6-[3-(methylamino) butyryl]forskolin, 6-[3-aminobutyryl]forskolin, or 6-[(piperidino) acetyl]-7-7-deacetyl forskolin, etc, and their structural formulae are shown in Table 1.
[0100] It should also be pointed out that HIL568 is also a forskline derivative later developed by Hoechest for the treatment of glaucoma. It can be speculated that as an adenylate cyclase activator, it also has the effect of inhibiting TNF-α and IL-17A and it therefore has a certain value in the treatment field of the present invention. However, because there is no follow-up development report, it is speculated that the compound has defects such as druggability.
TABLE-US-00001 TABLE 1 Structures of preferred forskolin derivatives data of cAMP Compound names structures activation effect source 6-(4-aminobutyryl)- forskolin
[0101] In a particularly preferred embodiment, 6-[3-(dimethylamnino)propionyl]forslcolin hydrochloride (CAS number: 138605-00-2) of the following formula III is used:
##STR00025##
[0102] In some embodiments, the present disclosure provides a drug or pharmaceutical composition including a forskolin derivative or a pharmaceutically acceptable salt thereof.
[0103] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a forskolin derivative or a pharmaceutically acceptable salt thereof and a prostaglandin compound or a glucocorticoid compound.
[0104] In some embodiments, the present disclosure also considers a drug or a pharmaceutical composition comprising a prodrug of a forskolin derivative and optionally a prostaglandin compound or a glucocorticoid compound.
[0105] These drugs or pharmaceutical compositions can be used for inducing immunosuppression in a subject in need thereof.
[0106] “Pharmaceutically acceptable salts” herein refer to inorganic or organic acid addition salts that are basically harmless to animals or humans, such as hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, enanthate, fumarate, glutamate, hydroxyacetate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, p-toluenesulfonate, etc. In some embodiments, it is particularly advantageous that the salts of the forskolin derivatives are their hydrochloride. Such salts can be formed by methods well known to those skilled in the art.
[0107] “Prodrug of a forskolin derivative” herein includes a compound formed by modifying one or more reactive or derivatizable groups of the forskolin derivative. Of particular interest are compounds with modifications on the carboxyl, hydroxyl, or amino groups. Examples of particularly suitable prodrugs are the esters or amides of the forskolin derivative. These prodrugs are converted into the forskolin derivative or its salts in animals or humans, for example, under the action of enzymes.
[0108] In some embodiments, the present disclosure provides a method for inducing immunosuppression in a subject, which comprises administering to the subject a therapeutically effective amount of a forskolin derivative or a pharmaceutically acceptable salt thereof.
[0109] In some embodiments, the method includes administering a forskolin derivative or a pharmaceutically acceptable salt thereof in combination with a prostaglandin compound or a glucocorticoid compound.
[0110] The term “co-administration”, for example, for a pharmaceutical combination of a forskolin derivative and a prostaglandin compound, includes the forskolin derivative and the prostaglandin compound being administered sequentially and separately, for example, the prostaglandin compound is administered before or after the administration of the forskolin derivative; it also includes the simultaneous administration of the forskolin derivative and the prostaglandin compound in the same pharmaceutical preparation or in the form of separate pharmaceutical preparations. In the embodiment of sequential administration, usually the forskolin derivative and the prostaglandin compound coexist in the subject at least part of the time. In some embodiments of co-administration, the forskolin derivative and the prostaglandin compound may have a synergistic effect, for example, the amount of one compound is lower than the therapeutically effective amount when administered alone, or preferably, amounts of the two compounds are all lower than the therapeutically effective amounts when used alone.
[0111] In some embodiments, the present disclosure provides a pharmaceutical kit comprising a forskolin derivative or a pharmaceutically acceptable salt thereof, such as hydrochloride, and a prostaglandin compound or a glucocorticoid compound. The pharmaceutical kit can be used for inducing immunosuppression in a subject.
[0112] In some embodiments of the pharmaceutical kit, the forskolin derivative or a pharmaceutically acceptable salt thereof and the prostaglandin compound or the glucocorticoid compound can be formulated in a pharmaceutical composition, for example, they are mixed and coexist in the same dosage form or unit dosage form. In other embodiments of the pharmaceutical kit, the forskolin derivative or its pharmaceutically acceptable salt and the prostaglandin compound or glucocorticoid compound can be separately formulated and stored. For example, the forskolin derivative or its pharmaceutically acceptable salt and the prostaglandin compound are both in the form of a solution in different containers, or the forskolin derivative or its pharmaceutically acceptable salt is formulated as an injection, and the prostaglandin compound is formulated as an ointment.
[0113] In some embodiments, the pharmaceutical kit provided by the present disclosure may include at least two separate kits, one of which includes the forskolin derivative or a pharmaceutically acceptable salt thereof, and the other includes the prostaglandin compound or glucocorticoid compound. The pharmaceutical kit may also include instructions for simultaneously or sequentially administering the two kits to the subject.
[0114] In the drugs, pharmaceutical compositions or pharmaceutical kits provided in the present disclosure, the forskolin derivative or a pharmaceutically acceptable salt thereof, as well as the prostaglandin compound and the glucocorticoid compound may be formulated with a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” herein refers to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered to animals or humans without excessive adverse side effects, and at the same time, it is suitable for maintaining the activity of the drugs or active agents therein. Depending on the route of administration, various carriers well known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils (such as castor oil), synthetic oil, polyol, alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water, etc. Suitable administration routes include, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or inhalation, transdermal, and other routes. Correspondingly, the forskolin derivative or a pharmaceutically acceptable salt thereof, as well as the prostaglandin compound and the glucocorticoid compound can be formulated together with these pharmaceutically acceptable carriers into any clinically acceptable dosage form, such as tablets, granules, powders, capsules, injection preparations, suppositories, drops, external plasters, ointments, medicated oils, or sprays, etc.
[0115] In some embodiments, the present disclosure provides the use of a forskolin derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inducing immunosuppression.
[0116] In some embodiments of this use, the drug is administered in combination with a prostaglandin compound or a glucocorticoid compound.
[0117] As used herein, “subject” refers to an individual (preferably human) who has or is suspected of suffering from a certain disease (such as psoriasis), or, for example, when predicting the risk of a disease, the “subject” may also include healthy individuals. This term can often be used interchangeably with “patient”, “test subject”, “treatment subject” and so on. As used herein, “therapeutically effective amount” refers to an amount sufficient to cause a biological or medical response expected by a clinician in the body of the subject, and it can usually be determined by those skilled in the art according to the route of administration, the weight, age, condition of the subject and other factors. For example, a typical daily dose may range from 0.01 mg to 100 mg of active ingredient per kg body weight. The present disclosure also considers other dosages.
[0118] In some specific embodiments, the present disclosure provides a drug comprising 6-[3-(dimethylamino)propionyl]forskolin hydrochloride itself or a pharmaceutical composition together with prostaglandin E2 or hydrocortisone, as well as methods or uses thereof for inducing immunosuppression in a subject.
[0119] In some embodiments, the present disclosure provides an immunosuppressive agent comprising a forskolin derivative or a pharmaceutically acceptable salt thereof, and optionally a prostaglandin compound or a glucocorticoid compound. In some embodiments, the immunosuppressive agent is a TNF-α inhibitor. In some embodiments, the immunosuppressive agent is an IL-17A inhibitor. In some embodiments, the immunosuppressant suppresses the expression or secretion of TNF-α and IL-17A at the same time.
[0120] In some embodiments, the prostaglandin compound is selected from the group consisting of prostaglandin E2 (PGE2), dinoprost tromethamine, carboprost, carboprost tromethamine, prostaglandin E1 (Alprostadil), bimatoprost, iloprost, limaprost, limaprost a cyclodextrin (Limnaprostalfadex), misoprostol, gemeprost, latanoprost, sulprostone, ornoprostil and pharmaceutically acceptable salts thereof.
[0121] In some embodiments, the glucocorticoid compound is selected from the group consisting of dexamethasone, hydrocortisone, prednisone, prednisolone, paramethasone, cortisone, betamethasone, meprednisone, fludrocortisone, triamcinolone acetonide and pharmaceutically acceptable salts thereof.
[0122] In some embodiments of the methods or uses of the present disclosure, the forskolin derivative or a pharmaceutically acceptable salt thereof acts by inhibiting the expression or secretion of TNF-α by immune cells, especially monocyte macrophages. In some embodiments of the methods or uses of the present disclosure, the forskolin derivative or a pharmaceutically acceptable salt thereof acts by inhibiting the secretion of IL-17A by immune cells, especially T lymphocytes. In some embodiments of the methods or uses of the present disclosure, the forskolin derivative or a pharmaceutically acceptable salt thereof acts by inhibiting the expression or secretion of TNF-α and IL-17A by immune cells.
[0123] “Immunesuppression” as used herein refers to the reduction of undesirable immune responses in a subject, including the production of some cytokines such as TNF-α or IL-17A. In some embodiments, the subject in need of immunosuppression is a patient with an autoimmune disease. In some embodiments, the subject in need of immunosuppression is a patient with psoriasis. In other embodiments, the subject in need of immunosuppression is a patient with inflammation.
[0124] In some embodiments, the drugs or pharmaceutical compositions provided in the present disclosure are used as anti-inflammatory drugs. Anti-inflammatory drugs are commonly used in medical practice to relieve or eliminate acute and chronic inflammation, such as the glucocorticoid hydrocortisone.
[0125] It should be understood that all drugs that have an anti-inflammatory effect by reducing the body's own immune response rather than inhibiting the activity of foreign pathogens (such as antibiotics) are essentially immunosuppressive drugs. In this case, the concept of anti-inflammatory drugs is equivalent to immunosuppressants and anti-inflammatory drugs are therefore also within the scope of this disclosure.
[0126] In summary, the forskolin derivatives involved in the present disclosure can have immunosuppressive functions by inhibiting the production of pro-inflammatory cytokines TNF-α and/or IL-17A, and have potential value in treating or alleviating inflammation and various autoimmune diseases. Based on a new molecular target and mechanism, the forskolin derivatives are expected to become a new type of immunosuppressant to make up for the shortage of current drugs.
[0127] The present invention reveals for the first time that structural derivatives of the small molecule compound forskolin have a dual-action mechanism of simultaneously reducing interleukin 17A and tumor necrosis factor α, and has multiple immunosuppressive effects compared with current single-action mechanism drugs on the market. At the same time, the compound itself has been fully optimized in structure and modified in properties, and possesses considerable druggability. It is also a rare small molecule chemical drug species in the drug market with similar action mechanisms and for related diseases. In summary, forskolin structural derivatives have great potential to effectively fill the gaps in the existing market in the field of psoriasis and immunosuppressive therapy.
[0128] The present invention is further illustrated with the following examplest.
EXAMPLES
Example 1 In Vitro Cytology Experiment: Regulatory Effect of 6-[3-(dimethylamino)propionyl]Forskolin Hydrochloride on TNF-α
1.1 Experimental Materials and Main Equipments are Shown in Table 2 Below.
[0129]
TABLE-US-00002 TABLE 2 Materials and equipments used in the in vitro cytology experiment materials brands catalog No. RPMI1640 medium Thermo 11835030 Fetal Bovine Serum Thermo 10099133 phosphate buffer (DPBS) Thermo 14190144 Double antibiotics Thermo 15070063 human monocyte macrophage (THP-1) BeNa Culture BNCC337680 lipopolysaccharide (LPS) Sigma L6529-1MG phorbol ester (PMA) Sigma P8139-1MG human TNF-α ELISA kit Abcam ab181421 cell viability assay kit (MTT) Beyotime C0009 prostaglandin E2 (PGE2) MCE HY-101952 Forskolin Sigma F6886 NKH 477 Sigma N3290 Hydrocortisone MCE HY-N0583 microplate reader TECAN infinite M1000 cell incubator Thermo Forma 371 ultra-clean worktable BIOBASE BBS-V800 invert microscope Novel NIB-100 centrifuge Sigma 2-5 96-well culture plate Costar T3603 T75 culture flask Corning 430641
1.2 Experimental Steps
1.2.1 Cell Culture, Drug Treatment and Cell Viability Assay:
[0130] 1) Recoveried and expanded THP-1 cells in a T75 culture flask. The growth medium was 10 milliliters of 10% fetal bovine serun/RPMI1640 (already containing 2 mM glutamine)/1% double antibiotics in each flask. Observed the cells under an invert microscope, and, when the cells were in the logarithmic growth phase, started the following drug treatment experiments;
[0131] 2) Cultured 2*10e5/mL THP-1 cells in a 96-well culture plate with 100 microliters of growth medium (10% fetal bovine serum/RPMI640/1% double antibiotics) per well, and added 100 ng/ml PMA to incubate for 24 hour;
[0132] 3) Eluted the non-adherent cells once with 300 microliters of fresh growth medium of 10% fetal bovine serum/RPMI1640/1% double antibiotics;
[0133] 4) Added 200 microliters of growth medium of 10% fetal bovine serum/RPMI1640/1% double antibiotics, with/without test compound, to pre-incubate the cells at 37° C. for 2 hours;
[0134] 5) Then added 100 ng/mL lipopolysaccharide (LPS) and incubated at 37° C. for 4 hours:
[0135] 6) Collected 50 microliters of cell culture supernatant for the detection of the levels of related cytokines by enzyme-linked immunoassay, or stored it in a refrigerator at minus 80 degrees Celsius for future detection;
[0136] 7) Added 10 microliters of 5 mg/ml MTT solution to each cell, and incubated for 4 hours in the cell incubator;
[0137] 8) Added 100 microliters of Formazan dissolving solution to the cells of each well, and continued to incubate in the cell incubator for about 4 hours, until it was observed under a common optical microscope that Formazan crystals are completely dissolved;
[0138] 9) Detected the absorbance at 570 nm with a microplate reader.
1.2.2 Detecting Cytokine Expression Levels by Enzyme-Linked Immunoassay:
[0139] 1) Prepared 1× elution buffer for enzyme-linked immunoassay. Prepared it with ultrapure water: 10× elution buffer mother solution=9:1 in volume ratio for use;
[0140] 2) Prepared the antibody cocktail for enzyme-linked immunoassay. Prepared it with the antibody diluent (provided by Ab221825):antibody 1:antibody 2=18:1:1 in volume ratio for use. Antibody 1 was a TNF-α capture antibody and Antibody 2 was a TNF-α detector antibody. They were all supplied by kit Ab221825;
[0141] 3) Prepared a series of concentration gradient samples of TNF-α standard with ultrapure water for the plotting of reaction standard curve and determining the linear range of detection signal;
[0142] 4) Added 50 microliters of cell culture supernatant/standard and 50 microliters of the above antibody cocktail to a 96-well plate used for enzymatic reaction, sealed it with a film, placed it on a horizontal shaker, and incubated at 400 rpm for 1 hour at room temperature to allow the antibody-antigen complex was fully bound and coupled at the bottom of the well plate;
[0143] 5) Discarded the supernatant, eluted with 350 microliters of 1× elution buffer/well for 3 times. In the last elution, put the well plate upside down on an absorbent paper and sucked and washed it thoroughly;
[0144] 6) Added 100 microliters of TMB substrate solution (supplied by Ab221825) to each well, placed them on a horizontal shaker and incubated in the dark at room temperature for 5 minutes at a speed of 400 rpm;
[0145] 7) Added 100 microliters of reaction stop solution (provided by Ab221825), placed on a horizontal shaker and mix at 400 rpm for 1 minute;
[0146] 8) Detected the absorbance at 450 nm with a microplate reader.
1.3 Experimental results
1.3.1 6-[3-(dimethylamino) propionyl]forskolin hydrochloride Reduced the Expression of TNF-α in Monocyte Macrophages
[0147] The compound 6-[3-(dimethylamino) propionyl]forskolin hydrochloride (Colforsin daropate hydrochdeloride) was able to concentration-dependently reduce the expression level of TNF-α in human monocyte macrophage THP-1 cultured in vitro after stimulation by lipopolysaccharide (LPS), and the reduction of TNF-α is not due to the compound's influence on the cell viability of THP-1.
[0148] As shown in
1.3.2 6-[3-(dimethylamino) propionyl]forskolin hydrochloride Combined with Prostaglandin E2 (PGE2) Reduced the Expression of TNF-α in Monocyte Macrophages
[0149] When prostaglandin E2 (PGE2) was used in combination with 6-[3-(dimethylamino) propionyl]forskolin hydrochloride, it could strengthen the effect of 6[3-(dimethylamino) propionyl]forskolin hydrochloride in reducing the level of TNF-α.
[0150] As shown in
[0151] In order to further clarify that the combined effect of 6-[3-(dimethylamino) propionyl]forskolin hydrochloride and PGE2 was a synergistic effect rather than a purely additive effect, according to the judgment principle for synergistic effect of compounds in reference [9-11]: expected value of combined treatment with A and B=(value of treatment with A alone/control group value)*(value of treatment with B alone/control group value)*control group value, and the ratio of expected value of combined treatment with A and B/ratio of actual value of combined treatment with A and B is the Combination Index. If the Combination Index is greater than 1, then the compounds A and B have a synergistic effect, otherwise, it is a purely additive effect.
[0152] According to this principle and the data in Table 3 below, it was obtained through calculation that the combined effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and PGE2 was a synergistic effect.
TABLE-US-00003 TABLE 3 Results of the combined use 6[3-(dimethylamino)propionyl]- forskolin hydrochloride and PGE2 A + B in combina- TNF-α A B combination tion idex Treat- 6-[3-(dimethyl- prosta- 6-[3-(dimethyl- A * B/ ment amino)propionyl]- glandin amino)propionyl]- (A + B in group forskolin E2 (PGE2) forskolin combina- hydrochloride hydrochloride + tion) PGE2 treat- 33% 46% 10% 1.52 ment value
[0153] Similarly, we added the same amount of MTT solution as mentioned before at the end point of the drug stimulation in the above experiment. The data showed that cell viability was not affected by the drug treatment, and there was no significant difference between the groups (
1.3.3 6-[3-(dimethylamino)propionyl]forskolin hydrochloride Combined with Hydrocortisone (HC) Reduced the Expression of TNF-α in Monocyte Macrophages
[0154] When hydrocortisone was used in combination with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride, it could strengthen the compound's effect in reducing TNF-α.
[0155] Hydrocortisone is a glucocorticoid drug commonly used clinically to treat psoriasis and for immunosuppression. We also investigated whether it had a synergistic effect with forskolin (or its derivatives) pharmacodynamically. As shown in
TABLE-US-00004 TABLE 4 Results of the combined use of 6[3-(dimethylamino)propionyl]- forskolin hydrochloride and hydrocortisone A + B in combina- TNF-α A B combination tion idex Treat- 6-[3- hydro- 6-[3- A * B/ ment (dimethylamino)- cortisone (dimethylamino)- (A + B in group propionyl]- (HC) propionyl]forskolin combina- forskolin hydrochloride + tion) hydrochloride hydrocortisone treat- 58% 200% 12% 9.67 ment value
[0156] We also added the same amount of MTT solution as mention before at the end point of the drug stimulation in the above-mentioned experiment. The data results ruled out the possibility that cell viability was affected by drug treatment (
Example 2 In Vitro Cytology Experiment: Regulatory Effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride on IL-17A
2.1 the Experimental Materials and Main Equipments are Shown in Table 5 Below.
[0157]
TABLE-US-00005 TABLE 5 Materials and equipments used in the in vitro cytology experiment materials brands catalog Nos. RPMI 1640 medium Gibco 11875-093 FBS Biological Industries 04-002-1A phosphate buffer (DPBS) Biosera LM-S2041/500 double antibiotics Gibco 15140122 mTGF-β R&D 7666-MB mIL-6 Peprotech 216-16 mIL-23 R&D 1887-ML mTNF-α Peprotech 315-01A mIL-lβ Peprotech 211-11B anti-mouse IL-4 Biolegend 504102 anti-mouse IFN-r Biolegend 505702 anti-mouse CD3e BD Biosciences 553057 anti-mouse CD28 BD Biosciences 553294 Stimulation cocktail eBioscience 00-4970-93 RoboSep ™ Buffer Stemcell 20104 Mouse Naïve CD4+ T Cell STEMCELL 19765A Isolation Kit prostaglandin E2 (PGE2) MCE HY-101952 NKH 477 Sigma N3290 Hydrocortisone MCE HY-N0583 96-well flat-bottom cell Corning 3599 culture plate Mouse IL-17 Duo Set ELISA R&D DY421 CytoTox-one Homogeneous Promega G7891 Membrane Integrity Assay Kit
Animal strain: adult C57BL/6 female mice, 8-12 weeks old.
2.2 Experimental Steps
2.2.1 Isolation and Culture of Mouse Primary T Cells, Th17 Cell Differentiation and Compound Treatment
[0158] 1) Pre-coated the 96-well plate with 2 μg/mL anti-mouse CD3e and incubated overnight at 4 degrees Celsius:
[0159] 2) Separated fresh spleens from adult C57BL/6 female mice (8-12 weeks old), ground and filtered through a 70 μM nylon cell strainer to obtain a single cell suspension in pre-cooled DPBS;
[0160] 3) Washed the spleen cells again with DPBS, and resuspended in RoboSep™ buffer at a density of 1×10.sup.8/mL;
[0161] 4) Separated primary CD4+ T cells with mouse primary CD4+ T cell isolation kit as required by the kit;
[0162] 5) Resuspended the isolated CD4+ T cells in complete medium (RPMI 1640 medium+10% inactivated FBS+1% double antibiotics) at a density of 1×10.sup.6/mL;
[0163] 6) Washed the 96-well plate pre-coated with anti-mouse CD3e twice with DPBS, added 50 μL of cytokine cocktail, the composition and final concentration of which are shown in Table 6 below, and then added 50 μL of compound solution (the final concentration of DMSO was 0.1%). Added 100 μL of cell suspension to each well to allow the final cell number per well to be 1×10.sup.5/100 μL;
TABLE-US-00006 TABLE 6 Composition of the cytokine cocktail Anti- Anti- Anti- Anti- mCD3e mCD28 composition mTGF-β mIL-6 mIL-4 IFN-γ mIL-23 mTNF-α mIL-1β (Pre-coat) (Soluble) final 5 50 10 10 10 10 10 2 2 concentration ng/mL ng/mL μg/mL μg/mL ng/mL ng/mL ng/mL μg/mL μg/mL
[0164] 7) Cultured the cells for 7 days under the above conditions. The culture conditions were 37 degrees Celsius, 5% CO.sub.2 concentration;
[0165] 8) After 7 days, added 1× stimulation cocktail (eBioscience 500X) to the culture medium for 4 hours, and then collected cell culture supernatant for detection with IL-17A enzyme-linked immunoassay kit;
[0166] 9) Completed the detection of supernatant IL-17A concentration with mouse IL-17 DuoSet ELISA kit;
[0167] 10) Read the OD450 nm absorbance value with a microplate reader, and generated the standard curve by using the 4-parameter logistic fitting (4-PL) method.
2.2.2 Detecting Expression Levels of Cytokine IL-17A by Enzyme-Linked Immunoassay
[0168] 1) Diluted the capture antibody (provided in the kit) to a working concentration in PBS without carrier protein, that is, a 96-well plate can be coated with 100 microliters per well. Sealed and incubated the plate overnight at room temperature;
[0169] 2) Aspirated the coating buffer of the well plate, and eluted with 400 microliters of eluent (provided in the kit) for three times, aspirating as much liquid as possible each time;
[0170] 3) Added 300 microliters of diluent (provided in the kit) to each well, and equilibrated for 1 hour at room temperature;
[0171] 4) Eluted the well plate as in the above step 2) to be ready for the addition of sample;
[0172] ) Added 100 microliters of sample or standard to each well containing the added diluent, and sealed and incubated the well plate for 2 hours at room temperature;
[0173] 6) Eluted the well plate as in the above step 2);
[0174] 7) Added 100 microliters of detector antibody (provided in the kit) to each well, and sealed and incubated the well plate for 2 hours at room temperature.
[0175] 8) Eluted the well plate as in the above step 2);
[0176] 9) Added 100 microliters of Streptavidin-HRP working solution (provided in the kit) to each well, and sealed and incubated the well plate in dark for 20 minutes at room temperature.
[0177] 10) Eluted the well plate as in the above step 2);
[0178] 11) Added 100 microliters of reaction substrate solution (provided in the kit) to each well, and incubated at room temperature for 20 minutes, protected from light:
[0179] 12) Added 50 microliters of stop solution (provided in the kit) to each well, and tapped the well plate to ensure that the liquid is evenly mixed;
[0180] 13) Detected the optical density value of each well at 450 m. The values could be corrected by deducting the values at 540 nm or 570 nm.
2.2.3 Compound Cytotoxicity Detection
[0181] 1) Separated fresh spleens from adult C57BL/6 female mice (8-12 weeks old), and ground and filtered through a 70 μM nylon cell strainer to obtain a single cell suspension in pre-cooled PBS;
[0182] 2) Seeded primary CD4+ T cells in 96-well plate with a density of 1×10.sup.5/90 μL. The medium was 1640 medium (serum-free);
[0183] 3) Added the serially diluted compounds formulated in a volume of 10 μL to designated wells;
[0184] 4) Incubated the cells for 4 hours at 37 degrees Celsius and 5% CO.sub.2;
[0185] 5) Detected lactate dehydrogenase (LDH) release according to CytoTox-one Homogeneous Membrane Integrity Assay Kit instructions.
2.3 Experimental Results
[0186] 2.3.1 6-[3-(dimethylamino)propionyl]forskolin hydrochloride Reduced the Expression of IL-17A in Mouse Th17 Cells Induced to Differentiate
[0187] Compound 6-[3-(dimethylamino)propionyl]forskolin hydrochloride (Colforsin daropate hydrochdeloride) could concentration-dependently reduce the secretion and expression of IL-17A in mouse primary CD4+ T cells induced to differentiate under the combined action of cytokines TGF-β, IL-6, IL-23, etc., and the reduction in IL-17A was not due to the toxicity of the compound to Th17 cells.
[0188] As shown in
2.3.2 6-[3-(dimethylamino)propionyl]forskolin hydrochloride Combined with Prostaglandin E2 (PGE2) Reduced the Expression of IL-17A in Mouse Th17 Cells Induced to Differentiate
[0189] When prostaglandin E2 (PGE2) was used in combination with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride, it could strengthen the action of 6[3-(dimethylamino)propionyl]forskolin hydrochloride in reducing IL-17A levels.
[0190] As shown in
[0191] Similarly, in order to further clarify that the combined effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and PGE2 in inhibiting the secretion of IL-17A was a synergistic effect rather than a purely additive effect, we similarly calculated the relevant combination index as shown in the table below. According to the foregoing principles and the data in Table 7 below, it was obtained through calculation that the combined effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and PGE2 in reducing IL-17A was a synergistic effect.
TABLE-US-00007 TABLE 7 Results of the combined use 6[3-(dimethylamino)propionyl]- forskolin hydrochloride and PGE2 A + B in combina- IL-17A A B combination tion idex Treat- 6-[3-(dimethyl- prosta- 6-[3-(dimethyl- A * B/ ment amino)propionyl]- glandin amino)propionyl]- (A + B in group forskolin E2 forskolin combin- hydrochloride (PGE2) hydrochloride + ation) PGE2 treat- 88% 17% 2% 7.48 ment value
[0192] Similarly, we tested the toxic effect of the compounds on mouse CD4+ T cells under the aforementioned experimental dosing conditions. The compounds were incubated with cells for 4 hours to detect the concentration of lactate dehydrogenase released in the supernatant. The data showed that cell viability was not affected by drug treatment and there was no significant difference between groups (
2.3.3 6-[3-(dimethylamino)propionyl]forskolin hydrochloride Combined with Hydrocortisone (HC) Reduced the Expression of IL-17A in Mouse Th17 Cells Induced to Differentiate
[0193] When hydrocortisone was used in combination with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride, it could strengthen the action of 6[3-(dimethylamino)propionyl]forskolin hydrochloride in reducing IL-17A levels.
[0194] Hydrocortisone is a glucocorticoid drug commonly used clinically to treat psoriasis and for immunosuppression. We also investigated whether it had a synergistic effect with forskolin derivatives pharmacodynamically. As shown in
[0195] Similarly, we calculated and analyzed in Table 8 below to clarify that the combined effect of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and hydrocortisone in reducing IL-17A secretion was a synergistic effect.
TABLE-US-00008 TABLE 8 Results of the combined use 6[3-(dimethylamino)propionyl]- forskolin hydrochloride and hydrocortisone A + B in combina- IL17-A A B combination tion idex Treat- (dimethylamino)- hydro- (dimethylamino)- A * B/ ment propionyl]- cortisone propionyl]- (A + B in group forskolin (HC) forskolin combina- hydrochloride hydrochloride + tion) hydrocortisone treat- 88% 62% 39% 1.40 ment value
[0196] Similarly, we tested the toxic effect of the compounds on mouse CD4+ T cells under the aforementioned experimental dosing conditions. The compounds were incubated with cells for 4 hours to detect the concentration of lactate dehydrogenase released in the supernatant. The data showed that cell viability was not affected by drug treatment and there was no significant difference between groups (
Example 3 In Vivo Disease Model Experiment
3.1 the Experimental Materials and Main Equipments are Shown in Table 9.
[0197]
TABLE-US-00009 TABLE 9 Materials and equipments used in the in vivo disease model experiment. catalog Nos. (or drug materials brands registration Nos.) 5% imiquimod cream Aldara H20160079 calcipotriol betamethasone LEO H20160204 ointment phosphate buffer (DPBS) Thermo 14190144 prostaglandin E2 (PGE2) MCE HY-101952 75% ethanol Greagent G73537N 4% paraformaldehyde fixative Beyotime P0099-100ML digital display micrometer SYNTEK CLXL005 thickness gauge electric mouse hair scraper Zhongke life 3303
[0198] Animal strain: 28 male BALB/c mice, 6-8 weeks old, SPF environment, 12 hours light and dark cycle, 24-26 degrees Celsius.
3.2 Experiment Groups:
[0199] Blank group: no treatment, n=3;
[0200] Positive drug group: calcipotriol betamethasone ointment, n=5;
[0201] Model group: 5% imiquimod ointment, n=5;
[0202] High-dose compound test group: 3.5 mg/kg drug intraperitoneal injection+5% imiquimod ointment, n=5;
[0203] Low-dose compound test group: 0.8 mg/kg drug intraperitoneal injection+5% imiquimod ointment, n=5;
[0204] Combined drugs test group: 0.8 mg/kg drug intraperitoneal injection+0.0025% PGE2 (administered epidermally in 75% ethanol)+5% imiquimod ointment, n=5;
3.3 Experimental Steps:
Blank Group:
[0205] Day 0: anesthetized the mouse, shaved the back with an area about 2*3 cm;
[0206] Day1-7: every other day, measured the mouse body weight, left and right ear thickness, and observed the skin to obtain a score;
[0207] Day7: killed the mouse, sampled the back skin and fixed in 4% PFA solution for H&E staining;
Model Group Induced with Imiquimod:
[0208] Day 0: anesthetized the mouse, shaved the back with an area about 2*3 cm;
[0209] Day1-7: every other day, measured the mouse body weight, left and right ear thickness, and observed the skin to obtain a score;
[0210] Day1-7: at a fixed time each day, applied 100 μL and 10 μL of 75% ethanol on the back and right ear on model sites respectively, and massaged briefly until the liquid evaporated off;
[0211] Day1-7: then injected 200 μl sterile DPBS intraperitoneally as a drug solvent control;
[0212] Day1-7: then applied 62.5 mg of imiquimod ointment on the shaved area on the back of each mouse, applied 250 μg of imiquimod ointment to the inside and outside of the ear on the right ear, and massaged against the direction of hair to help absorption;
[0213] Day7: killed the mouse, sampled the back skin and fixed in 4% PFA solution for H&E staining;
Positive Drug Group:
[0214] Day 0: anesthetized the mouse, shaved the back with an area about 2*3 cm;
[0215] Day1-7: every other day, measured the mouse body weight, left and right ear thickness, and observed the skin to obtain a score;
[0216] Day1-7: At a fixed time each day, applied 18 mg calcipotriol betamethasone ointment on the shaved area on the back of each mouse, and 1.8 mg calcipotriol betamethasone ointment on the inside and outside of the right ear. Massaged moderately against the direction of the hair to help absorption;
[0217] Day1-7: After calcipotriol betamethasone ointment was administered for 1 hour and the complete absorption was observed by naked eyes, applied 62.5 mg imiquimod ointment on the shaved area on the back of each mouse, and 250 μg imiquimod on the inside and outside of the right ear. Massaged moderately against the direction of the hair to help absorption;
[0218] Day7: killed the mouse, sampled the back skin and fixed in 4% PFA solution for H&E staining;
High-Dose Compound Group:
[0219] Day 0: anesthetized the mouse, shaved the back with an area about 2*3 cm;
[0220] Day1-7: every other day, measured the mouse body weight, left and right ear thickness, and observed the skin to obtain a score;
[0221] Day1-7: at a fixed time each day, applied 100 μL and 10 μL of 75% ethanol on the back and right ear on model sites respectively, and massaged briefly until the liquid evaporated off;
[0222] Day1-7: then injected intraperitoneally 3.5 mg/kg in 200 μL sterile DPBS;
[0223] Day1-7: then applied 62.5 mg of imiquimod ointment on the shaved area on the back of each mouse, applied 250 μg of imiquimod ointment on the inside and outside of the right ear, and massaged moderately against the direction of hair to help absorption;
[0224] Day7: killed the mouse, sampled the back skin and fixed in 4% PFA solution for H&E staining;
Low-Dose Compound Group:
[0225] Day 0: anesthetized the mouse, shaved the back with an area about 2*3 cm;
[0226] Day1-7: every other day, measured the mouse body weight, left and right ear thickness, and observed the skin to obtain a score;
[0227] Day1-7: at a fixed time each day, applied 100 μL and 10 μL of 75% ethanol on the back and right ear on model sites respectively, and massaged briefly until the liquid evaporates off;
[0228] Day1-7: then injected intraperitoneally 0.8 mg/kg in 200 μL sterile DPBS;
[0229] Day1-7: then applied 62.5 mg of imiquimod ointment on the shaved area on the back of each mouse, applied 250 μg of imiquimod ointment on the inside and outside of the right ear, and massaged moderately against the direction of hair to help absorption;
[0230] Day7: killed the mouse, sampled the back skin and fixed in 4% PFA solution for H&E staining;
Group Treated with Compound in Combination with PGE2:
[0231] Day 0: anesthetized the mouse, shaved the back with an area about 2*3 cm;
[0232] Day1-7: every other day, measured the mouse body weight, left and right ear thickness, and observed the skin to obtain a score;
[0233] Day1-7: At a fixed time each day, applied 100 μL and 10 μL of 0.0025% PGE2 (2.5 μg on the back of each mouse, in 75% ethanol) on the back and ear model sites respectively, and massaged briefly until the liquid evaporates off;
[0234] Day-7: then injected intraperitoneally 0.8 mg/kg in 200 μL sterile DPBS;
[0235] Day1-7: one hour after PGE2 administration, applied 62.5 mg of imiquimod ointment on the shaved area on the back of each mouse, and applied 250 μg of imiquimod ointment on the inside and outside of the right ear. Massaged moderately against the direction of hair to help absorption:
[0236] Day7: killed the mouse, sampled the back skin and fixed in 4% PFA solution for H&E staining;
3.4 Experimental Results
[0237] The experimental indicator used was the psoriasis-like index score:
[0238] Four aspects of lesion skin area, plaque, redness and rash. The area was fixed in the disease model and was not considered, and the other three aspects were quantified as follows: 0=asymptomatic, 1=mild, 2=moderate, 3=significant, 4=very significant;
[0239] Changes of the thickening of the ear;
[0240] Body weight;
[0241] Skin histological changes (H&E staining).
[0242] In the experiment of mouse psoriasis model induced with imiquimod, we used the groups with 6-[3-(dimethylamino)propionyl]forskolin hydrochloride at a high dose of 3.5 mg/kg and a low dose of 0.8 mg/kg, and at the same time designed a combined administration group of 0.8 mg/kg 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and 0.0025% prostaglandin E2 (PGE2). The positive treatment group was the current first-line clinical topical combination medicine calcipotriol betamethasone ointment. During 7 consecutive days of administration, we observed that the groups with the high and low doses of 6-[3-(dimethylamino)propionyl]forskolin hydrochloride and the combined administration group with PGE2 could all show an effect of slow down various psoriasis-like indicators in a certain period of time. Moreover, compared with the positive control first-line medicine, the combined administration group had better effects as to indicators such as avoiding mouse weight loss, skin inflammation and redness.
[0243] As shown in
[0244] The model group was manifested by the severity of skin scurf, redness and rash (
[0245] In addition, as shown in
[0246]
[0247] We also analyzed the body weight changes of the mice during the administration period (
[0248] The histological analysis of the skin of the back model area (
[0249] The above examples are only used to illustrate the technical solutions of the present invention, not to limit them; those of ordinary skill in the art should understand that the technical solutions described in the foregoing examples can be modified, or some or all of the technical features can be equivalently replaced; these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the examples of the present invention, and they should all be covered in the scope of the specification of the present invention.
REFERENCES
[0250] 1. Patent: EP 0222413, Novel forskolin derivatives. [0251] 2. Tochiro Tatee, et al. (1996) Forskolin Derivatives. I. Synthesis, and Cardiovascular and Adenylate Cyclase-Stimulating Activities of Water-Soluble Forskolins. Chem. Pharm. Bull. 44, 2274-2279. [0252] 3. Shunichi Kametani et al. (1995) The Pharmacodynamics of 6-(3-Dimethylaminopropionyl)forskolin and a Possible Metabolite in Beagles. J of Pharmaceutical Sciences 85, 377-380. [0253] 4. Mutsuhito Kirura et al. (2004) Pharmacokinetics and a simulation model of colforsin daropate, new forskolin derivative inotropic vasodilator, in patients undergoing coronary artery bypass grafting. Pharmacological Research 49, 275-281. [0254] 5. Y. Khandelwal et al. (1988) Cardiovascular Effect of New Water-Soluble Derivatives of Forskolin. J. Med. Chem. 31, 1872-1879. [0255] 6. A. Laurenza et al. (1987) Stimulation of Adenylate Cyclase by Water-Soluble Analogues of Forsklin. Molecular Pharmacology, 32: 133-139. [0256] 7. Bansi Lal et al. (1998) Hydroxyacyl Derivatives of Forskolin—their Positive Inotropic Activity. Bioorganic & Medicinal Chemistry 6, 2061-2073. [0257] 8. K. B. Seamon, et al. (1983) Structure-Activity Relationship for Activation of Adenylate Cyclase by the Diterpene Forskolin and Its Derivatives. J. Med. Chem. 26, 436-442. [0258] 9. Chou, T. C. et al. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22, 27-55. [0259] 10. Yokoyama, Y. et al. (2000) Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth. Cancer Res., 60, 2190-2196. [0260] 11. Zhou, J. R. et al. (2004) Combined inhibition of estrogen-dependent human breast carcinoma by soy and tea bioactive components in mice. Int. J. Cancer. 108, 8-14.