CN110856716A - Application of compound and pharmaceutical composition thereof in prevention and treatment of renal fibrosis - Google Patents

Abstract

The invention relates to application of dimethylamino michelia lactone fumarate in preparation of a medicament, belongs to the technical field of medicaments, and particularly relates to application of a compound shown in a formula (I) in prevention, treatment or adjuvant therapy of renal fibrosis and application of the compound in preparation of a medicament for prevention, treatment or adjuvant therapy of renal fibrosis.

Description

Application of compound and pharmaceutical composition thereof in prevention and treatment of renal fibrosis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of sesquiterpene lactone compounds and a pharmaceutical composition thereof in prevention, treatment or adjuvant treatment of Renal Fibrosis (RF).

Background

The prevalence rates of Chronic Kidney Disease (CKD) of adults in the world and our country are as high as about 10% and 10.8%, respectively, and with the improvement of living standard, the change of life style and the aging of population, the incidence rates of diabetes, hypertension, hyperuricemia and the like are increasing rapidly, so that CKD patients are increasing. Therefore, CKD is a worldwide public health problem that seriously harms human health.

RF is a common pathological return for the development of all CKD (including primary and secondary glomerular diseases, tubular, interstitial and vascular diseases, chronic rejection lesions of transplanted kidneys, etc.) to end-stage renal disease (ESRD), with massive deposition of extracellular matrix (ECM), tubular atrophy and glomerular sclerosis as the main manifestations, suggesting that the renal injury response enters the final pathway marked by reorganization of tissue structure and loss of organ function.

The mechanism of RF generation is quite complex and heretofore unknown. Except for etiology and diet therapy, an effective treatment method for RF is lacking clinically at present, and inhibition of activation of a renin-angiotensin-aldosterone system is the only treatment means which has evidence support and can delay the progress of the renin-angiotensin-aldosterone system, but not only has limited curative effect, but also easily induces hyperkalemia, acute kidney injury and the like, and cannot achieve satisfactory effect. Because of this, CKD patients inevitably progress toward ESRD once they develop RF, and eventually rely on dialysis treatment or kidney transplantation for survival, which imposes a great economic burden on the family and society. Obviously, how to effectively prevent and treat RF has become a common concern in the medical field, and the active research and development of new drugs for treating RF, which have the advantages of obvious curative effect, small toxic and side effects, controllable quality and convenient use, has very important significance.

At present, no report about the application of the sesquiterpene lactone compound or the pharmaceutical composition thereof in prevention, treatment or adjuvant therapy of RF is found.

Disclosure of Invention

The invention provides application of a compound (dimethylamino michelia lactone fumarate, code ACT001) in formula (I) in preparation of RF treatment or preparation of a medicament for adjuvant treatment of RF.

Chemical structural formula:

chemical name: (3R,3aS,9R,9aS,9bS) -3- ((dimethylamino) methyl) -9-hydroxy-6, 9-dimethyl-3, 3a,4,5,7,8,9,9 a-octahydroazuno [4,5-b ] furan-2 (9bH) -one fumarate.

The molecular formula is as follows: C17H27NO3 · C4H4O4, molecular weight: 409.47.

the application of the compound in the medicines for preventing, treating or assisting in treating RF.

The application of the compound in preparing the medicine for preventing, treating or adjunctively treating RF.

When the compound of the present invention is used as a medicament, it may be used as it is or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the invention, and the balance of pharmaceutically acceptable carriers and/or excipients which are nontoxic and inert to human beings and animals or drugs for preventing, treating or assisting in treating RF. The composition can be prepared into injection, tablets, capsules and the like.

The pharmaceutically acceptable carrier or excipient is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical composition of the present invention is used in the form of a dose per unit body weight. The medicine of the invention can be administered by two forms of injection and oral administration, such as intravenous injection and intramuscular injection, and the oral administration can be tablets and capsules.

Drawings

FIG. 1 scheme for the preparation of the compounds of formula (I)

FIG. 2. scheme for the preparation of the compound of formula (I)

FIG. 3 is a graph of ACT001 improving UUO model renal fibrosis and TGF- β 1 induced tubular epithelial cell EMT process

FIG. 4 is a graph showing the process of promoting renal fibrosis and EMT of renal tubular epithelial cells by Mtdh

FIG. 5 is a graph showing that reduction of Mtdh expression inhibits TGF- β 1-induced EMT of tubular epithelial cells

FIG. 6 is a graph of the course of ACT001 inhibiting TGF- β 1-induced EMT in tubular epithelial cells by reverting to Mtdh levels

Detailed Description

The invention is further illustrated by the following examples for the understanding of the invention, which are not intended to limit the scope of the invention.

Example 1: process for the preparation of compounds of formula (I)

1.1 route to the preparation of the Compounds of formula (I) (FIG. 1)

(PTL parthenolide, MCL Sphaelactone, ACT001 dimethylamino Sphaelactone fumarate)

1.2 Process for the preparation of the Compound of formula (I) (FIG. 2)

(PTL parthenolide, MCL Sphaelactone, ACT001 dimethylamino Sphaelactone fumarate)

Example 2: anti-RF Activity testing of Compounds of formula (I)

In the initial stages of kidney injury, fibrotic matrix production is the mechanism of tissue damage repair, and after mild injury, this portion of fibrotic matrix is gradually absorbed without affecting the structure and function of the kidney. However, when CKD occurs, the fibrotic matrix is continuously deposited to escape regulatory compression, which eventually leads to disintegration of tissue and organ architecture, reduction of blood supply, reduction of organ function, and finally reduction of renal tissue repair function, resulting in renal failure. Therefore, RF is characterized primarily by excessive ECM deposition leading to scarring and by a pathological manifestation of proliferation of ECM-producing source cells such as fibroblasts and myofibroblasts, leading to excessive production and accumulation of collagen fibrils and fibronectin, progressive progression to glomerulosclerosis and renal interstitial fibrosis, and ultimately loss of kidney function.

The mechanisms by which the complex multifactorial regulation of RF occurs are not fully elucidated, and a variety of cellular mediators and growth factors are involved in this process, either directly or indirectly, including Transforming Growth Factor (TGF) - β, basic fibroblast growth factor, angiotensin II, hepatocyte growth factor, etc. injury to Renal Tubular Epithelial Cells (RTECs) is an early event in initiating the fibrotic response, it is currently believed that epithelial-mesenchymal transdifferentiation (EMT) of injured RTECs is an important mechanism leading to RF formation.

2.1 materials of the experiment

2.1.1 test drugs

The drug of the invention, prepared as described in example 1 above, is designated ACT 001. Telmisartan tablets (mecacacetin), manufactured by Shanghai Bo ringer Yiger pharmaceutical Co., Ltd., lot No. A22321, 80 mg/tablet.

2.1.2 Experimental animals

24C 57 mice 8 weeks old, SPF grade, provided by southern medical university, guangzhou laboratory animal center, certification number: yue Zheng Zi word NO. 44002100007603. The experimental preposed animal is adapted to the indoor environment for 7 days at the room temperature of 18-22 ℃ and the relative humidity of 65%. All mice were fed free of water and were fed with conventional feed (protein content 3.14 mg/g).

2.1.3 Experimental cell lines

Mouse-derived tubular epithelial cell immortalized strain was given by professor Jeffrey b.kopp, national institute of health.

2.2 Experimental methods

2.2.1 therapeutic Effect of ACT001 on Unilateral Ureteral Obstruction (UUO) renal fibrosis model

2.2.1.1 animal model creation, grouping

The method for constructing the UUO model comprises the following steps: under the anesthesia of intraperitoneal injection of 1% sodium pentobarbital, shearing and disinfecting the operation range of a median incision of an abdomen, finding a left ureter and dissociating an upper ureter after passing through the median incision of the abdomen, and performing complete double ligation of ureter obstruction by using 4-0 silk threads (UUO group), wherein a sham operation group (sham group) mouse exposes and dissociates the ureter by the same method but does not ligate. The abdominal incision is sutured layer by layer and disinfected with iodophor. Mice subjected to UUO surgery were randomly divided into a UUO group, a UUO + telmisartan group, and a UUO + ACT001 group, plus a sham surgery group (sham group), to give 4 groups of 6 mice each. The administration by gavage was started the next day after the operation, the sham group and the UUO group were given an equal volume of physiological saline as a control treatment, and the kidney tissue was collected 14 days after the operation. 2.2.1.2 drug dose setting, administration

According to the results of the preliminary experiments, ACT001 was administered at 25 mg/kg-d. The maximum effective dose of telmisartan for adults is 160mg per day, and the equivalent dose of mice is calculated to be 10mg/kg.d (Chenqi. Chinese medicine pharmacology research methodology, 3 rd edition, Beijing: people's health publishing agency, 2011). ACT001 and telmisartan are prepared into solution by normal saline, and the solution is irrigated for 1 time and 2 weeks every day on the next postoperative day. The sham and UUO mice were drenched with the same amount of physiological saline every day for 2 weeks.

2.2.1.3 specimen Collection, processing

Kidney tissue was collected 2 weeks after the gavage treatment. Anesthetizing with sodium pentobarbital 1% by intraperitoneal injection at 35mg/kg, collecting eyeball, collecting blood, separating blood serum, and storing at-20 deg.C or-70 deg.C. Cutting abdomen, poking abdominal viscera with sterile gauze, perfusing cold PBS through heart until viscera turns white, collecting kidney tissue, weighing all left kidneys, splitting along midsagittal plane, washing with PBS, removing envelope, fixing in 10% formaldehyde neutral buffer solution, and performing pathological examination; the rest of the right kidney tissues are washed clean by PBS and stored by liquid nitrogen.

2.2.1.4 Observation index and determination method

2.2.1.4.1 renal pathology: and (3) observing pathological structures of the kidney and deposition conditions of collagen fibers by HE staining and Masson staining.

2.2.1.4.2 protein level detection, including isomucin (Mtdh, also known as stellate cell up-regulating factor 1, AEG-1), EMT related index E-cadherin, α -SMA, fibrosis phenotype related index Fn, Col-1.

2.2.2 Effect of ACT001 active ingredient MCL on TGF- β 1 induced EMT in tubular epithelial cells

2.2.2.1 preparation and grouping of cell models

A method for constructing a fibrotic cell model comprises the steps of adopting human recombinant TGF- β 1 cytokine (5 ng/. mu.l) to intervene renal tubular epithelial cells (mTEC) for 48 hours, and causing the mTEC to present fibroid change, increase in fibrotic phenotype expression (Fn, α -SMA, Col-1) and decrease in epithelial cell phenotype (E-cadherin). the active ingredient MCL intervention (1.25. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M) in ACT001 with different concentrations is added while TGF- β 1 is added, and the intervention is divided into 6 groups, namely a blank group, a TGF- β 1 model group and a TGF- β 1+ MCL dry pre-treatment group with different concentrations (1.25. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M).

2.2.2.2 Observation index and measuring method

And detecting the protein level of Mtdh, EMT related indexes E-cadherin, α -SMA, fibrosis phenotype related indexes Fn and Col-1.

2.2.3 exploration of the relationship of ACT001 to anti-fibrotic acting on Mtdh protein

2.2.3.1 construction of stably overexpressed Mtdh cell line

A lentiviral vector LV-Mtdh (with an empty vector as a negative control LV-NC) targeting an Mtdh sequence is constructed in Nanjing Gicko company, transfected into mTECs, screened out positive clones through puromycin (10 ng/. mu.l), and subjected to subsequent experiments after expanded culture and seed preservation.

2.2.3.2 construction of Mtdh cell strain with stable down-regulation expression

A lentivirus vector targeting an sh-Mtdh sequence (taking a random scrambled sequence as a negative control sh-NC) is constructed in Nanjing Gicko company, transfected into mTECs, screened out positive clones through puromycin (10 ng/mu l), expanded culture and seed preservation are carried out, then subsequent experiments are carried out, TGF- β 1 is given or not given to the sh-NC cell strain and the sh-Mtdh cell strain respectively, and cell proteins are collected for 48 hours for the following detection.

2.2.3.3 Observation index and measuring method

And detecting the protein level of Mtdh, EMT related indexes E-cadherin, α -SMA, fibrosis phenotype related indexes Fn and Col-1.

2.2.4 treatment of Mtdh overexpression induced EMT phenotype with active ingredient MCL

2.2.4.1 model construction and grouping: a plasmid vector (Mtdh vector) containing an Mtdh sequence is constructed, an empty vector is used as a Negative Control (Negative Control), transient transfection is carried out on mTECs, 10 mu M MCL intervention is given, and cell protein is collected for 48 hours for subsequent experiments. The experiment was divided into 4 groups: a Negative Control group, an Mtdh vector group, a Negative Control + MCL group, and an Mtdhvvector + MCL group.

2.2.4.2 observation index and measuring method

And detecting the protein level of Mtdh, EMT related indexes E-cadherin, α -SMA, fibrosis phenotype related indexes Fn and Col-1.

2.3 results of the experiment

2.3.1 pathological changes in mouse Kidney tissue (FIG. 3)

FIG. 3 shows that ACT001 improves fibrosis of UUO model and TGF- β 1 induces EMT process of tubular epithelial cells (HE staining and Masson staining of kidney tissue of A.UUO model with scale of 50 μm and scale of 100 μm; B.ACT001 ACTs on immunohistochemical staining of kidney tissue of UUO model and detects expression of α -SMA, Fibronectin and E-cadherin; C & D. Western blot detects protein expression level and statistical chart of ACT001 acting on kidney tissue of α -SMA, Fibronectin and E-cadherin of UO model, E & F.Western blot detects protein expression level and statistical chart of different dose of MCL acting on kidney epithelial cells α -SMA, Fibronectin and E-cadherin induced by TGF- β 1; p 0.05, p 0.01; p 0.0001. 0.0001)

From A and B of FIG. 3, HE staining and Masson staining showed that the conditions of renal interstitial widening, extracellular matrix deposition, collagen fiber increase and inflammatory cell infiltration of UUO model mice were significantly reduced in the ACT001 treatment group, while immunohistochemical staining results of fibrosis indexes Fibronectin, α -SMA and E-cadherin showed very objectively that ACT001 treatment inhibited the expression of Fibronectin and α -SMA and that of epithelial cell marker E-cadherin was recalled in both D and F, the Wb bands in both D and F reflect that ACT001(MCL) could inhibit the progression of fibrosis at the in vivo and in vitro levels, and the difference has statistical significance (C-F) 2.3.2 001 ACT achieved kidney protection by regulating MTDH expression (FIG. 4, FIG. 5, FIG. 6)

FIG. 4 shows that Mtdh promotes fibrosis and EMT process of renal tubular epithelial cells (A & B. Mtdh efficiency of stably over-expressing cell strain; C & D. Western blot method detects protein expression level and statistical chart of α -SMA, Col-1, Fibronectin and E-cadherin of stably over-expressing Mtdh renal tubular epithelial cells, p is 0.05;, p is 0.01;. p is 0.0001)

Under the condition, a fibrosis index (C) is detected, the protein water level of fibrosis indexes fibrinectin, Col-1 and α -SMA is obviously increased on average and is most obviously increased by Col-1, and meanwhile, the expression of an epithelial cell marker E-cadherin is reduced, and the difference has statistical significance (D).

FIG. 5 shows that the reduction of the Mtdh expression can inhibit the process of TGF- β 1 inducing tubular epithelial cell EMT (A. Mtdh stably downregulating cell strain and control cell strain with or without TGF- β 1 inducing for 48 hours, collecting protein, detecting fibrosis by Western blot and expression of EMT phenotype protein; C-F. Western blot method detects Mtdh, Col-1, α -SMA, Fibronectin and E-cadherin of stably downregulating tubular epithelial cell of Mtdh, p is 0.05 v.ssh-NC; # p is 0.05v.ssh-NC + TGF- β 1; # p is 0.01v.ssh-NC + TGF- β 1)

From A of attached figure 5, the expression of the sh-NC group cell baseline level is not high and is increased under the induction condition of TGF- β 1, the Mtdh level in the sh-Mtdh group cell is obviously reduced both at the baseline and under the induction condition of TGF- β 1, which shows that the construction of the stably down-regulated expression cell strain is successful, under the condition, the detection of fibrosis index (A) and the protein water average of fibrosis indexes Fibronectin, Col-1 and α -SMA of the sh-Mtdh group are obviously reduced, the increase degree is increased under the induction condition of TGF- β 1 and is far smaller than that of the NC-NC + TGF- β 1 group, the difference has statistical significance (C-F), and the above results show that under the condition of in vitro Mtdh deletion, the sensitivity of the renal tubular epithelial cell to TGF- β 1 induced EMT is reduced, and the EMT process of the renal tubular epithelial- β 1 induced renal tubular cell under the in vitro environment is proved by Mtdh.

FIG. 6 shows that ACT001 can suppress TGF- β 1 induced tubular epithelial cell EMT process by recovering Mtdh level (A.mTEC transiently transfects plasmid containing Mtdh sequence, blank vector is used as Control Negative Control, MCL intervention is given in ACT001 body, concentration is 10 μ M, protein is collected in 48 hours, Western blot is used to detect fibrosis and EMT phenotype protein expression, B-F.Western blot method is used to detect Mtdh, Col-1, α -SMA, Fibronectin and E-cadherin statistical chart of stably down-regulating Mtdh tubular epithelial cell, p <0.05v.s Negative Control;, p <0.01v.s Negative Control;, p <0.05v.sMtdh vector; #, p <0.001v.sMtdh vector #.

After MCL intervention, Mtdh, Col-1, α -SMA and Fibronectin in the Mtdh vector group are all reduced compared with the group without MCL intervention, and E-cadherin is recovered, and the difference is statistically different (B-F), which shows that Mtdh is a key molecule in the function of ACT001 to realize renal fibrosis, and ACT001 can inhibit the EMT process by regulating the expression of Mtdh.

2.4 discussion

2.4.1 animal models

At the end of the experiment, the animal model has thin renal cortex and obvious hydronephrosis, and belongs to a typical obstructive renal fibrosis model.

2.4.2 therapeutic Effect

Dimethylamino michelia lactone fumarate (code ACT001) can obviously improve kidney fibrosis change of UUO mice, inhibit synthesis of kidney interstitial matrix proteins Fibronectin, Col-1 and α -SMA, and recover the level of a renal tubular epithelial cell marker E-cadherin, so that the kidney protection function of inhibiting the renal tubular epithelial cells from generating EMT.ACT001 is mainly realized by inhibiting the expression of Mtdh, which shows that ACT001 can reduce the expression of Mtdh in vitro and in vivo and can inhibit the EMT process induced by the over-expression of Mtdh.

Example 3: injection solution

The compound prepared in example 1 is dissolved in a small amount of DMSO, and then injection water is added according to the conventional method, fine filtration is carried out, encapsulation and sterilization are carried out, thus preparing injection.

Example 4: tablet formulation

Example 1 the compound prepared and excipients were added at a weight ratio of 5:1 to form a granulate, which was compressed to form tablets.

Example 5: capsule

Example 1 the compound prepared and excipients were added in a weight ratio of 5:1 to make capsules.

The compounds, uses and methods of the invention have been described by specific examples. The invention can be used for other purposes by those skilled in the art by appropriately changing the raw materials, the process conditions and the like without departing from the content of the invention, and all similar substitutes and modifications obvious to those skilled in the art are deemed to be included in the scope of the invention.

Claims (3)

1. The application of dimethylamino michelia lactone fumarate of a compound shown as a formula (I) in preventing, treating or assisting in treating renal fibrosis.

2. The use of a compound as claimed in claim 1 for the preparation of a medicament for the prophylaxis, treatment or co-treatment of renal fibrosis.

3.A pharmaceutical composition for the prevention, treatment or co-treatment of renal fibrosis, comprising as an active ingredient at least one compound as claimed in claim 1 and a pharmaceutically acceptable carrier or other compound for the prevention, treatment or co-treatment of renal fibrosis.

Images