Application of nelfinavir in preparing medicine for preventing and treating non-alcoholic steatohepatitis and/or anti-hepatic fibrosis
阅读说明：本技术 奈非那韦在制备防治非酒精性脂肪性肝炎和/或抗肝纤维化药物中的应用 (Application of nelfinavir in preparing medicine for preventing and treating non-alcoholic steatohepatitis and/or anti-hepatic fibrosis ) 是由 徐志建 李佳 臧奕 张勇 朱维良 孙丹丹 李波 谢荣荣 沈敬山 于 2020-04-16 设计创作，主要内容包括：本发明涉及奈非那韦在制备防治非酒精性脂肪性肝炎和/或抗肝纤维化药物中的应用。具体而言,本发明涉及奈非那韦及其药物组合物作为人肝星形细胞(HSC)活化的抑制剂在制备治疗和/或预防、缓解由各种急慢性肝损伤因素引起的非酒精性脂肪性肝炎和肝纤维化的药物中的用途。(The invention relates to application of nelfinavir in preparing a medicament for preventing and treating non-alcoholic steatohepatitis and/or anti-hepatic fibrosis. Specifically, the invention relates to application of nelfinavir and a pharmaceutical composition thereof as an inhibitor for activating human Hepatic Stellate Cells (HSC) in preparing a medicament for treating and/or preventing and relieving non-alcoholic steatohepatitis and hepatic fibrosis caused by various acute and chronic liver injury factors.)
1. Use (or pharmaceutical use) of an active ingredient or a formulation comprising said active ingredient, wherein said active ingredient is nelfinavir or a pharmaceutically acceptable salt thereof;
wherein said active ingredient or a formulation containing said active ingredient is used for the preparation of (a) an inhibitor of Hepatic Stellate Cell (HSC) activation; and/or (b) a medicament for treating and/or preventing, alleviating the related diseases caused by non-alcoholic steatohepatitis and/or hepatic fibrosis.
2. Use according to claim 1, wherein the active ingredient is selected from the group consisting of: nelfinavir, nelfinavir mesylate.
3. The use of claim 1, wherein the formulation or medicament comprises: oral and non-oral formulations.
4. A pharmaceutical composition for preventing and treating non-alcoholic steatohepatitis and/or anti-hepatic fibrosis, comprising:
(a) a first active ingredient selected from the group consisting of: nelfinavir, or a pharmaceutically acceptable salt thereof, or a combination thereof;
and (b) a pharmaceutically acceptable carrier.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition comprises:
(a1) a first active ingredient selected from the group consisting of: nelfinavir, or a pharmaceutically acceptable salt thereof; or a combination thereof;
(a2) a second active ingredient selected from the group consisting of:
(Y1) PPAR γ agonist (pioglitazone);
(Y2) FXR agonist (obeticholic acid);
(Y3) antioxidant (vitamin E);
(Y4) PPAR α/δ agonists (elafinigranor);
(Y5) any combination of the above Y1 to Y4;
and (b) a pharmaceutically acceptable carrier.
6. The pharmaceutical composition according to claim 4, wherein the concentration of the first active ingredient (single compound or sum of compounds) in the pharmaceutical composition is 0.01-100mg/ml, preferably 0.1-20mg/ml (liquid dosage form); and/or
The concentration of the first active ingredient (single compound or sum of compounds) in the pharmaceutical composition is 0.01-1000mg/g, preferably 0.1-50mg/g (solid dosage form).
7. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is in a form for oral administration or non-oral administration.
8. Use of a pharmaceutical composition according to claim 4 for the preparation of (a) an inhibitor of Hepatic Stellate Cell (HSC) activation; and/or (b) a medicament for treating and/or preventing, alleviating related diseases caused by non-alcoholic steatohepatitis and hepatic fibrosis.
9. A method of inhibiting Hepatic Stellate Cell (HSC) activation comprising the steps of: contacting a first active ingredient or a formulation comprising said first active ingredient with a Hepatic Stellate Cell (HSC), thereby inhibiting the activity of said Hepatic Stellate Cell (HSC);
wherein, the first active ingredient is nelfinavir or pharmaceutically acceptable salt thereof.
10. A method of treating, preventing, and/or ameliorating a disease associated with nonalcoholic steatohepatitis and liver fibrosis, comprising the steps of: administering to a subject in need thereof a safe and effective amount of a first active ingredient or a formulation comprising a first active ingredient, wherein said first active ingredient is nelfinavir or a pharmaceutically acceptable salt thereof.
Technical Field
The invention relates to the field of medicines, in particular to application of nelfinavir in preparing a medicine for preventing and treating nonalcoholic steatohepatitis and anti-hepatic fibrosis.
Background
Liver fibrosis (Liver fibrosis) is a chronic injury repair reaction occurring in the Liver after various pathogenic factors are involved in the Liver, and the Liver fibrosis is possibly caused by viral hepatitis, chemical poison or drug-induced Liver disease, alcoholic/non-alcoholic fatty Liver disease, autoimmune Liver disease, congenital metabolic disease and the like. Nonalcoholic steatohepatitis (NASH) and hepatic fibrosis refer to the pathological changes caused by abnormal proliferation of connective tissues in the liver and chronic liver damage caused by various pathogenic factors, which are manifested by excessive abnormal deposition of extracellular interstitial components in the liver and influence on the function of the liver, and are the stages necessary for the chronic liver disease to progress to cirrhosis. Any liver injury has liver fibrosis in the process of liver repair and healing, and if the injury factor cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time. The number of the existing liver disease patients in China exceeds 2 hundred million, wherein about 9300 million hepatitis B virus carriers, about 4000 million hepatitis C virus carriers and about 1.2 million fatty liver patients. With the change of Chinese dietary structure and living habits, the incidence of fatty liver has increased dramatically in recent years, and the disease is gradually getting younger. The prevalence of fatty liver in children is as high as 2.6%, and young people are only 6 years old. 70% of chronic liver disease patients with long course of disease mostly have liver fibrosis, 25% of liver fibrosis in 10 years further develops into liver cirrhosis, and 5% of the patients can develop into liver cancer. The liver disease not only brings great pain to patients and families, but also causes no heavier economic burden to the society.
The pathogenesis of nonalcoholic steatohepatitis (NASH) and liver fibrosis is complex and is associated with multiple enzymes and receptors.
At present, hepatic fibrosis is considered to be a reversible pathological process which is unbalanced in the generation and degradation of extracellular matrixes (ECM) such as collagen in the liver and the like, so that abnormal deposition of fibrous connective tissues in the liver is caused, and a dynamic balance process of liver repair and scar formation is reflected. Although the pathogenesis of hepatic fibrosis is deepened, an effective treatment means for reversing hepatic fibrosis is still lacked so far, and the development of anti-hepatic fibrosis drugs is very slow.
Currently, there are no approved drugs based on true evidence in the clinic for the treatment of NASH, mainly by lifestyle intervention and weight loss surgery, and non-instructional medications (off-able) such as PPAR γ agonist (pioglitazone), antioxidants (vitamin E) to treat nonalcoholic steatohepatitis (NASH) and liver fibrosis. NASH is currently the fastest progressing drug in research as is obeticholic acid (OCA), a nuclear receptor family farnesoid X receptor FXR agonist. Obeticholic acid is rapidly approved by the FDA for marketing at 6 months in 2016 for the treatment of cholestatic cirrhosis. FXR, as an endogenous bile acid receptor, is widely involved in the regulation of bile acid, sugar, lipid metabolism, inflammation and other processes. After FXR is excited, the FXR can indirectly inhibit the gene expression of CYP7A1 and inhibit cholic acid synthesis so as to treat primary cholestatic cirrhosis, relieve symptoms such as hepatic ascites caused by hepatic portal hypertension and the like, and the therapeutic research of OCA on nonalcoholic steatohepatitis (NASH) is in the Pre-registration stage (Pre-Registered).
In conclusion, there is no satisfactory drug that can effectively prevent and treat non-alcoholic steatohepatitis and/or hepatic fibrosis, and therefore there is an urgent need in the art to develop a new drug that can effectively prevent and treat non-alcoholic steatohepatitis and/or hepatic fibrosis.
Disclosure of Invention
The invention aims to provide a novel drug which can be effectively used for preventing and treating non-alcoholic steatohepatitis and/or anti-hepatic fibrosis and application thereof.
Specifically, the invention provides a new application of nelfinavir and a composition thereof in preventing and treating nonalcoholic steatohepatitis and/or anti-hepatic fibrosis.
In a first aspect of the invention, there is provided a use (or pharmaceutical use) of an active ingredient or a formulation containing said active ingredient, said active ingredient being nelfinavir or a pharmaceutically acceptable salt thereof;
wherein said active ingredient or a formulation containing said active ingredient is used for the preparation of (a) an inhibitor of Hepatic Stellate Cell (HSC) activation; and/or (b) a medicament for treating and/or preventing, alleviating the related diseases caused by non-alcoholic steatohepatitis and/or hepatic fibrosis.
In another preferred embodiment, the formulation further comprises one or more additional active ingredients selected from the group consisting of:
(Y1) PPAR γ agonist (pioglitazone);
(Y2) FXR agonist (obeticholic acid);
(Y3) antioxidant (vitamin E);
(Y4) PPAR α/δ agonists (Elafibranor).
In another preferred embodiment, the active ingredient is selected from the group consisting of: nelfinavir, nelfinavir mesylate.
In another preferred embodiment, the associated disease caused by non-alcoholic steatohepatitis and/or liver fibrosis is selected from the group consisting of: liver cirrhosis, liver cancer and complications thereof, or combinations thereof.
In another preferred embodiment, the formulation or medicament comprises: oral and non-oral formulations.
In another preferred embodiment, the formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet, or dripping pill.
In a second aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating non-alcoholic steatohepatitis and/or anti-hepatic fibrosis, the pharmaceutical composition comprising:
(a) a first active ingredient selected from the group consisting of: nelfinavir, or a pharmaceutically acceptable salt thereof, or a combination thereof;
and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition comprises:
(a1) a first active ingredient selected from the group consisting of: nelfinavir, or a pharmaceutically acceptable salt thereof; or a combination thereof;
(a2) a second active ingredient selected from the group consisting of:
(Y1) PPAR γ agonist (pioglitazone);
(Y2) FXR agonist (obeticholic acid);
(Y3) antioxidant (vitamin E);
(Y4) PPAR α/δ agonists (elafinigranor);
(Y5) any combination of the above Y1 to Y4;
and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the concentration of the first active ingredient (single compound or sum of compounds) in the pharmaceutical composition is 0.01-100mg/ml, preferably 0.1-20mg/ml (liquid dosage form); and/or
The concentration of the first active ingredient (single compound or sum of compounds) in the pharmaceutical composition is 0.01-1000mg/g, preferably 0.1-50mg/g (solid dosage form).
In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for inhibiting Hepatic Stellate Cell (HSC) activation.
In another preferred embodiment, the dosage form of the pharmaceutical composition is oral administration or non-oral administration.
In another preferred embodiment, the oral administration form is tablet, powder, granule or capsule, or emulsion or syrup.
In another preferred embodiment, the non-oral administration dosage form is injection or injection.
In another preferred embodiment, the pharmaceutical composition is used for the preparation of (a) an inhibitor of Hepatic Stellate Cell (HSC) activation; and/or (b) a medicament for treating and/or preventing, alleviating related diseases caused by non-alcoholic steatohepatitis and hepatic fibrosis.
In another preferred embodiment, the related diseases caused by non-alcoholic steatohepatitis and liver fibrosis are selected from the group consisting of: liver cirrhosis, liver cancer and complications thereof, or combinations thereof.
In a third aspect of the invention, there is provided a method of inhibiting Hepatic Stellate Cell (HSC) activation comprising the steps of: contacting a first active ingredient or a formulation comprising said first active ingredient with a Hepatic Stellate Cell (HSC), thereby inhibiting the activity of said Hepatic Stellate Cell (HSC);
wherein, the first active ingredient is nelfinavir or pharmaceutically acceptable salt thereof.
In another preferred embodiment, the first active ingredient is selected from the group consisting of: nelfinavir, nelfinavir mesylate.
In another preferred embodiment, the inhibition method is an in vitro method and is a non-therapeutic and non-diagnostic method.
In another preferred embodiment, the method of inhibition is an in vivo method and is a therapeutic method.
In another preferred embodiment, the method is non-therapeutic and non-diagnostic.
In another preferred embodiment, the method is in vitro.
In a fourth aspect of the present invention, there is provided a method for treating, preventing, and/or alleviating a disease associated with nonalcoholic steatohepatitis and liver fibrosis, comprising the steps of: administering to a subject in need thereof a safe and effective amount of a first active ingredient or a formulation comprising a first active ingredient, wherein said first active ingredient is nelfinavir or a pharmaceutically acceptable salt thereof.
In another preferred example, the method further comprises: administering a safe and effective amount of a second active ingredient to a subject in need thereof, wherein
The second active ingredient is selected from the group consisting of:
(Y1) PPAR γ agonist (pioglitazone);
(Y2) FXR agonist (obeticholic acid);
(Y3) antioxidant (vitamin E);
(Y4) PPAR α/δ agonists (elafinigranor);
(Y5) any combination of the above Y1 to Y4.
In another preferred embodiment, the subject is a mammal, preferably a primate, more preferably a human.
In another preferred embodiment, the first active ingredient is selected from the group consisting of: nelfinavir, or nelfinavir mesylate.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIGS. 1 and 2 (Gray-scan quantification) show the results of a phenotypic screening experiment of Nelfinavir Mesylate (DRN788) on hepatic stellate cells cultured in vitro.
Detailed Description
The inventors have made extensive and intensive studies and, for the first time, have unexpectedly developed a class of active ingredients effective in inhibiting the activation of hepatic stellate cells. Experiments show that the active ingredient (nelfinavir or pharmaceutically acceptable salt thereof) can effectively inhibit the activation of hepatic stellate cells, thereby achieving the effects of preventing and treating non-alcoholic fatty liver and/or resisting hepatic fibrosis. The present invention has been completed based on this finding.
Specifically, the invention discloses application of nelfinavir and a composition thereof in preventing and treating non-alcoholic fatty liver and/or resisting liver fibrosis. The nelfinavir and the composition thereof can obviously inhibit the activation of hepatic stellate cells under clinically accessible concentration, and have good clinical application prospect.
Term(s) for
As used herein, "active ingredient of the present invention", "active compound of the present invention inhibiting hepatic stellate cell activation" are used interchangeably and refer to a compound having excellent inhibitory activity against hepatic stellate cell activation, especially nelfinavir or a pharmaceutically acceptable salt thereof, or a combination thereof.
As used herein, "formulation of the invention" refers to a formulation containing an active compound of the invention.
As used herein, the term "comprising" or variations thereof, such as "comprises" or "comprising," etc., are understood to imply the inclusion of stated elements or components but not the exclusion of any other elements or components.
Hepatic stellate cell
Hepatic Stellate Cells (HSCs) are key cells involved in the process of liver fibrosis. Studies have shown that when the liver is subjected to acute or chronic injury factors, such as in the course of becoming non-alcoholic steatohepatitis, HSCs in a quiescent state become activated, differentiate from storage cells into myofibroblasts, eventually leading to liver fibrosis.
Alpha-smooth muscle actin (alpha-SMA) is a hallmark of HSC activation. The expression level of alpha-SMA in the HSC in a static state is low, and the expression level of alpha-SMA in the HSC in an activated state is high.
Hepatic fibrosis (Liver fibrosis)
As used herein, the term "liver fibrosis" refers to a reversible pathological process in which the production and degradation of extracellular matrix (ECM) such as collagen in the liver is unbalanced, thereby resulting in abnormal deposition of fibrous connective tissue in the liver.
Research shows that various pathogenic factors affect a chronic injury repair reaction in the liver after the liver is affected, and the chronic injury repair reaction comprises viral hepatitis, chemical poison or drug-induced liver disease, alcoholic/non-alcoholic fatty liver disease, autoimmune liver disease, congenital metabolic disease and the like which are all likely to cause liver fibrosis.
Nonalcoholic steatohepatitis (NASH)
Non-alcoholic fatty liver disease (NAFLD) refers to a series of clinical pathological syndromes with the main characteristic of excessive accumulation of liver cell fat, except for excessive drinking and other factors definitely causing liver injury. NAFLD is classified according to its histological changes into simple non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH) to cirrhosis. As used herein, the term "nonalcoholic steatohepatitis" refers to a disease that manifests as a fatty degeneration of hepatocytes of greater than 5% with inflammation within the leaflets and ballooning degeneration of hepatocytes. The early NASH is not combined with hepatic fibrosis or only has mild fibrosis (F0-1); combining significant hepatic fibrosis or septal fibrosis (F2-3) into fibrotic NASH; the combined liver cirrhosis (F4) is NASH liver cirrhosis. NASH is closely related to metabolic disorders such as obesity, insulin resistance, type 2 diabetes, hyperlipidemia, etc. There is currently no approved therapy.
Active Compounds and active ingredients of the invention
In the present invention, an active ingredient effective in inhibiting the activation of hepatic stellate cells is provided. The active ingredient is selected from the group consisting of: nelfinavir, or a pharmaceutically acceptable salt thereof, or a crystal thereof, or a solvate thereof.
The structural formula of Nelfinavir (Nelfinavir) is:
the molecular formula of the compound is C32H45N3O4S, white powder, molecular weight 567.78, CAS number 159989-64-7. The generic name nelfinavir, an orally bioavailable human immunodeficiency virus HIV-1 protease inhibitor (Ki ═ 2nM), is the fourth FDA-approved drug for aids treatment following saquinavir, ritonavir and indinavir.
One preferred pharmaceutically acceptable salt of nelfinavir is its mesylate salt (i.e., nelfinavir mesylate):
the molecular formula of nelfinavir mesylate is C33H49N3O7S2White powder, molecular weight 663.89, CAS number 159989-65-8. The compound has the general name Nelfinavir mesylate (Nelfinavir mesylate).
Tests show that the active ingredient can effectively inhibit the activation of hepatic stellate cells, so as to prevent, treat and/or relieve non-alcoholic steatohepatitis and hepatic fibrosis related diseases.
As used herein, "the active compound of the present invention", "the active compound of the present invention inhibiting the activation of hepatic stellate cells", which are used interchangeably, refer to a compound having an excellent inhibitory activity of the activation of hepatic stellate cells, including nelfinavir, or a pharmaceutically acceptable salt thereof, or a crystal thereof, or a solvate thereof.
It is to be understood that the active ingredient of the present invention includes the active compound of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate thereof, or a prodrug thereof, which inhibits activation of hepatic stellate cells. It is to be understood that the active ingredients of the present invention also include crystalline, amorphous, and deuterated forms of the active compounds of the present invention.
The "pharmaceutically acceptable salts" are conventional non-toxic salts formed by the reaction of the active compounds of the present invention with inorganic or organic acids. For example, conventional non-toxic salts can be prepared by reacting the active compounds of the present invention with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, or organic acids including citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like; or sodium, potassium, calcium, aluminum or ammonium salts of the active compounds of the invention which are esterified with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid and then with an inorganic base; or the corresponding inorganic acid salt formed by the active compound of the invention and lysine, arginine and ornithine after forming ester and then hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or the corresponding organic acid salt formed by the active compound of the invention and formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.
In addition, the active ingredient is particularly suitable for being combined with other medicines for preventing and treating non-alcoholic steatohepatitis and hepatic fibrosis. Representative other antiviral drugs include (but are not limited to): a PPAR γ agonist, an FXR agonist, an antioxidant, a PPAR α/δ agonist, or a combination thereof.
The active ingredients of the present invention can inhibit hepatic stellate cell activation. Therefore, when the active ingredient of the invention is applied or administered in treatment, the activation of hepatic stellate cells can be inhibited, and the effects of preventing and treating non-alcoholic steatohepatitis and hepatic fibrosis can be achieved.
Pharmaceutical composition and application
The invention also provides application of the active compound for inhibiting the activation of hepatic stellate cells, or pharmaceutically acceptable salt, prodrug, extract or mixture of one or more of medicinal materials thereof as an active ingredient in preparing a medicament for treating and/or preventing and relieving non-alcoholic steatohepatitis and hepatic fibrosis related diseases.
The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of active ingredient, preferably 0.1-90 wt% or 1-50 wt% of active compound of the present invention as active ingredient, the rest being pharmaceutically acceptable carrier, diluent or solution or salt solution.
If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.
The compounds and pharmaceutical compositions provided herein may be in a variety of forms such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and the like, and may be presented in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or instillation.
Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dose of the formulation generally comprises 0.05 to 400mg of active compound according to the invention, preferably 1 to 500mg of active compound according to the invention.
The compounds and pharmaceutical compositions of the present invention may be administered to mammals in the clinical setting, including humans and animals, by oral, dermal, or gastrointestinal routes of administration. Most preferably oral. Most preferably, the daily dose is 0.01-400mg/kg body weight, and is administered once or in portions of 0.01-200mg/kg body weight. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found.
The drug or inhibitor of the present invention can be administered by a variety of different means, e.g., by injection, spray, nasal drop, eye drop, osmotic, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed with other materials or encapsulated and introduced into body.
The main advantages of the invention include:
(a) the active compound can effectively inhibit the activation of hepatic stellate cells, can reduce the expression of alpha-SMA at 1 mu M, and can obviously reduce the expression of the alpha-SMA at 10 mu M better than that of a positive drug obeticholic acid (OCA). .
(b) The active compound of the invention has low toxic and side effects and good drug property. Nelfinavir is an anti-AIDS drug with high safety, and for adults and adolescents 13 years old and older, the recommended dose is 1250mg (BID) twice a day or 750mg (TID) three times a day. The recommended dose for children 2-13 years of age is 25-35mg/kg 3 times daily or 45-55 mg/kg twice daily. This suggests that it has good medicinal prospect in preventing and treating non-alcoholic steatohepatitis and anti-fibrosis.
(c) The maximum plasma concentration of the active compound of the invention was 4.0mg/L (7.04. mu.M) and the minimum plasma concentration was 2.2mg/L (3.87. mu.M) at the clinically recommended dose (1250mg BID) (FDA Package Insert, Reference ID: 3986049). At a dose of 1875mg BID, the maximum plasma concentration was 13.3 μ M and the minimum plasma concentration was about 5.5 μ M (Haematologica 2016; 101(3): 346-55). In rat tissue profiling experiments, the highest concentration of the active compounds of the invention in the liver, 16 times the blood concentration (Antirhizob Agents Chemother 1996; 40(1):110-4), is indicative of a good efficacy in the liver of the patient.
(d) The active compounds of the invention are very little hepatotoxic and are very safe even in hepatitis patients.
(e) The active compounds of the invention are safe in patients with moderate liver damage.
(f) The active compound of the invention has no toxicity under the clinical dosage of 4250mg BID and has good safety.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Example 1: phenotypic screening assay for nelfinavir against hepatic stellate cells
1.1 Experimental consumables
Name (R)
Batch number
Suppliers of goods
Period of validity up to
50ml centrifuge tube
430828
CORNING
N/A
24-hole plate
3524
CORNING
N/A
Transparent bottom white plate
3903
CORNING
N/A
1.2 cells for experiments
Cell name
Cell type
Culture medium
Additive component
LX2
Adherent cells
DMEM
2% serum
1.3 dosing of solutions
1.4 Experimental procedures
a) Culturing LX2 cells in a DMEM + 2% FBS medium for passage according to a ratio of 1: 2;
b) collecting cells when the growth density of LX2 is 80-90%, adding 1.5ml of pancreatin for 3 minutes, adding 3ml of MEM culture medium for termination, and collecting the cells in a 15ml centrifuge tube;
c) uniformly mixing 20 ul of cell liquid and 20 ul of trypan blue according to a ratio of 1:1, counting cells, and recording data; centrifuging a 15ml centrifuge tube for 5 minutes at 300 g;
d) resuspending the cells to 15000 cells/ml with DMEM, mixing well, and adding 500. mu.l/well to a # 352424 well plate with the gel;
e) after the cells are attached to the wall, starving for 12h by changing DMEM and 0.5% FBS;
f) preparing 10ng/ml of TGF-beta by using a DMEM medium, and preparing a compound by using a prepared TGF-beta solution;
g) adding the prepared compound (NC is negative control, OCA is positive compound, nelfinavir mesylate is test compound) into a 24-well plate, mixing uniformly, placing in an incubator at 37 ℃, and incubating for 24 hours;
h) the cells were washed twice with PBS buffer, 100. mu.l of 1 × loading buffer was added to each well, and the cells were collected in 1.5ml EP tubes;
i) boiling the sample at 100 ℃ for 10 minutes, centrifuging and putting the sample into a refrigerator at-20 ℃ for later use;
j) cellular immunoblotting (western blot).
1.5 results
As shown in fig. 1 and 2, the results show that:
(a) TGF-beta can obviously activate hepatic stellate cells, and the activated LX2 expresses alpha-SMA.
(b) Both 1 μ M and 10 μ M nelfinavir mesylate (DRN788) were able to significantly reduce α -SMA expression to varying degrees, thereby inhibiting activation of LX 2;
(c) compared with the positive compound OCA (10 mu M), the 10 mu M nelfinavir mesylate can significantly reduce the expression of the alpha-SMA.
The results show that 1-10 mu M nelfinavir mesylate has obvious effect of inhibiting the activation of hepatic stellate cells in vitro, and can be obviously superior to a positive compound obeticholic acid (OCA) to reduce the expression of alpha-SMA at 10 mu M.
Discussion of the related Art
In 2018, it was discovered that nelfinavir can inhibit pulmonary fibrosis by inhibiting TGF beta 1 pathway, and in addition, nelfinavir has certain effect on myocardial fibrosis and fibrosis of skin (Arthritis Rheumatotol 2018; 70(1): 115-26). Hepatic stellate cells are essentially different from the fibroblasts in pulmonary fibrosis. Hepatic stellate cells are unique in many aspects such as metabolism, differentiation ability, lipid storage, source, etc., and thus cannot be deduced from the results of fibrosis of the lung, heart and skin.
Prior to the present invention, the inhibitory effect of nelfinavir or its pharmaceutically acceptable salts on hepatic stellate cell activation has not been reported.
In the invention, the inventor firstly discloses that the nelfinavir or the pharmaceutically acceptable salt thereof has obvious inhibition effect on the activation of hepatic stellate cells, and is stronger than the positive drug obeticholic acid. Therefore, the nelfinavir or the pharmaceutically acceptable salt thereof has excellent activity of preventing and treating the non-alcoholic steatohepatitis and hepatic fibrosis, and has good clinical application prospect.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.