Pharmaceutical composition containing cryptotanshinone and omastolone as well as preparation method and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition, which is composed of cryptotanshinone and omastolone, and the weight ratio of the cryptotanshinone to the omastolone is (16.7-75): (25-83.3). The invention also provides an application of the pharmaceutical composition in preparation of an Nrf2 agonist. The invention also provides application of the pharmaceutical composition in preparation of medicines for treating lung injury or pulmonary fibrosis. The invention also provides a preparation method of the pharmaceutical composition. The composition disclosed by the invention is an Nrf2 agonist, has remarkable antioxidant and anti-inflammatory effects, and is particularly suitable for treating lung injury and pulmonary fibrosis caused by various factors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a pharmaceutical composition, specifically a pharmaceutical composition containing cryptotanshinone and omasolone, and a preparation method and use thereof. Background Art

[0002] Multiple organ injury (MOI), a severe complication of clinical critical illness, is commonly seen in pathological processes such as trauma, shock, infection, and major surgery. Its pathogenesis is complex, involving multiple pathological processes, including uncontrolled inflammatory responses, imbalanced oxidative stress, and abnormal apoptosis. It is characterized by high morbidity, mortality, and poor prognosis, and represents a major challenge facing modern medicine. Traditional treatments have primarily focused on supportive care and symptomatic management, but the lack of effective interventions targeting the core mechanisms of injury has hindered significant improvement in patient outcomes. In recent years, the nuclear factor E2-related factor 2 (Nrf2) pathway has become a hot topic in MOI treatment research due to its key role in regulating cellular redox homeostasis, anti-inflammatory activity, and anti-apoptosis. Activation of Nrf2 can induce the expression of phase II detoxification enzymes and antioxidant proteins (such as heme oxygenase-1 and glutathione synthetase), effectively scavenging excess reactive oxygen species, inhibiting the release of proinflammatory cytokines, and blocking apoptotic signaling pathways, thereby alleviating MOI. In-depth exploration of the endogenous protective mechanism mediated by Nrf2 will not only provide new therapeutic targets for multi-organ damage, but also bring hope for the development of safer and more effective intervention strategies.

[0003] Nrf2 is a key transcription factor that regulates the expression of numerous antioxidant and detoxification genes. Activation of Nrf2 can enhance cellular antioxidant capacity and mitigate oxidative stress damage, thereby playing a crucial role in the pathogenesis of various diseases. However, existing Nrf2 agonists still have several limitations in clinical application, such as insufficient efficacy and significant side effects. Therefore, the search for more effective and safer Nrf2 agonists is a current research hotspot.

[0004] Cryptotanshinone and omasorolone both have certain antioxidant and anti-inflammatory effects, but there have been no reports of combining these two ingredients to activate Nrf2. Therefore, developing a new combination to enhance Nrf2 activity has important scientific significance and clinical application value. Summary of the Invention

[0005] In response to the above-mentioned technical problems in the prior art, the present invention provides a pharmaceutical composition containing cryptotanshinone and omasorolone, a preparation method thereof, and use thereof. The pharmaceutical composition containing cryptotanshinone and omasorolone, a preparation method thereof, and use thereof are intended to solve the technical problem that the drugs in the prior art are not effective in treating lung injury or pulmonary fibrosis.

[0006] The present invention provides a pharmaceutical composition consisting of cryptotanshinone and omasolonone, wherein the weight ratio of cryptotanshinone to omasolonone is (16.7-75): (25-83.3).

[0007] Furthermore, the weight ratio of cryptotanshinone to omasorolone is 16.7:83.3.

[0008] Furthermore, the purity of the cryptotanshinone is ≥98%, and the purity of the omasorolone is ≥98%.

[0009] Furthermore, it also contains one or more pharmaceutically acceptable excipients.

[0010] Furthermore, relative to the total weight of the composition, the mass percentage of the cryptotanshinone is 5% to 30%, the mass percentage of the omasorolone is 1% to 10%, and the remainder is excipients.

[0011] Specifically, the excipients are lactose, microcrystalline cellulose, and magnesium stearate.

[0012] Furthermore, the composition is in the form of tablets, capsules, oral solutions, injections, dialysate, inhalants, syrups or nebulized solutions.

[0013] The present invention also provides use of the above-mentioned pharmaceutical composition in preparing an Nrf2 agonist.

[0014] The present invention also provides use of the above-mentioned pharmaceutical composition in preparing a drug for treating lung injury or pulmonary fibrosis.

[0015] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition, comprising the following steps:

[0016] 1) Weigh cryptotanshinone and omasorolone according to the mass ratio;

[0017] 2) dissolving cryptotanshinone and omasorolone in appropriate amounts of solvents to obtain solution A and solution B, respectively;

[0018] 3) Mixing solution A and solution B, stirring evenly to obtain a mixed solution;

[0019] 4) Drying the mixed solution to obtain dry powder.

[0020] Furthermore, the dry powder is mixed with auxiliary materials to prepare a desired dosage form, which is a tablet, capsule, oral solution, injection, dialysate, inhalant, syrup or nebulized solution.

[0021] Compared with existing technologies, the present invention offers significant and effective technical benefits. It provides a cryptotanshinone-omasorolone combination, an Nrf2 agonist with significant antioxidant and anti-inflammatory effects. It is particularly suitable for treating lung injury and liver fibrosis caused by various causes. Furthermore, the composition exhibits significant therapeutic efficacy and minimal side effects. Furthermore, the present invention provides a simple and easy method for preparing the composition, which is suitable for industrial production and can be used to prepare a variety of pharmaceutical dosage forms. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used in this specification are intended to have the same meaning as that generally understood by a skilled expert in the art to which the invention belongs. Generally speaking, the nomenclature used in this specification is the nomenclature well known and commonly used in the art.

[0023] In the examples of the present invention, cryptotanshinone and omasorolone were mixed at different weight ratios and their DPPH free radical scavenging effects were measured. The results showed that the mixture exhibited a superior antioxidant effect compared to using either cryptotanshinone or omasorolone alone. Furthermore, cryptotanshinone and omasorolone were mixed at different weight ratios and their Nrf2 activation effects were measured. The results showed that the mixture significantly activated Nrf2, exhibiting good antioxidant and anti-inflammatory effects.

[0024] Therefore, one aspect of the present invention relates to a pharmaceutical composition containing cryptotanshinone and omasolon as active ingredients. The cryptotanshinone of the present invention has a specific chemical structure and can be synthesized using conventional methods in the art. Omasolone can also be obtained through commercial channels.

[0025] In the present invention, when cryptotanshinone and omasorolone are mixed, the antioxidant and anti-inflammatory effects of cryptotanshinone and omasorolone can be maximized while ensuring the stability of the composition.

[0026] Furthermore, the present invention is characterized in that the content of cryptotanshinone is 5% to 30% by weight of the total composition, and the content of omasorolone is 1% to 10% by weight of the total composition. If the sum of the two active ingredients is less than 6%, the inhibitory effect on oxidative stress and inflammation is weak; if it exceeds 40%, the composition may have solubility or stability issues.

[0027] The pharmaceutical composition of the present invention can be formulated into tablets, capsules, oral solutions, injections, dialysates, inhalants, syrups, nebulized solutions, and the like, but the dosage forms are not particularly limited thereto. Furthermore, the composition of the present invention may contain excipients commonly used in the pharmaceutical field, such as fillers, disintegrants, lubricants, wetting agents, flavoring agents, and preservatives. These excipients are added in amounts commonly used in the pharmaceutical field. The composition of the present invention may also contain substances that promote drug absorption to enhance the therapeutic effect.

[0028] In addition to the above-mentioned substances, the pharmaceutical compositions of the present invention may preferably contain other ingredients that can enhance the main effects, such as other antioxidants, anti-inflammatory agents, etc., within the range that does not affect the main effects. The mixing amounts of the above-mentioned ingredients can be easily selected by those skilled in the art within the range that does not affect the purpose and effects of the present invention. The mixing amounts are 0.01-5% by weight, preferably 0.01-3% by weight, relative to the total weight of the composition.

[0029] While the above details certain aspects of the present invention, it should be understood by those skilled in the art that these specific techniques are merely preferred embodiments and the scope of the present invention is not limited thereto. Therefore, the scope of the present invention is defined by the appended claims and their equivalents.

[0030] [Reference Example 1]

[0031] The cryptotanshinone used to test the effect of the composition of the present invention was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0032] [Reference Example 2]

[0033] Omasolone used to test the effect of the composition of the present invention was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0034] [Reference Example 3]

[0035] The Nrf2 luciferase reporter gene plasmid used to test the effect of the composition of the present invention was purchased from Jiman Biotechnology (Shanghai) Co., Ltd.

[0036] [Preparation of Examples 1-3 and Comparative Examples 1-3]

[0037] The cryptotanshinone of Reference Example 1 and the omasolon of Reference Example 2 were mixed at the weight ratios shown in Table 1 below, respectively, to prepare Examples 1-3 with the same total weight.

[0038] In addition, as comparative examples of the above Examples 1-3, cryptotanshinone in Reference Example 1 and omasolon in Reference Example 2 were mixed at the weight ratios shown in Table 1 below to prepare Comparative Examples 1-3 with the same total weight as Examples 1-3.

[0039]

Table 1

[0040] (Unit: weight %)

[0041] Composition Omasaurus Cryptotanshinone Example 1 25 75 Example 2 66.6 33.4 Example 3 83.3 16.7 Comparative Example 1 / 100 Comparative Example 2 100 / Comparative Example 3 71.4 28.6

[0042] [Test Example 1] Nrf2 activation test

[0043] In order to observe the Nrf2 activation effect of the mixture of cryptotanshinone and omasolon, Examples 1-3, the Nrf2 activation effects of cryptotanshinone alone, omasolon alone, and Comparative Examples 1-3 were measured and compared by the Nrf2 luciferase reporter gene method.

[0044] HEK293T cells stably transfected with the Nrf2 luciferase reporter gene plasmid were added to a 96-well microtiter plate containing DMEM medium containing 10% fetal bovine serum, with 10,000 cells per well, and cultured until the coverage reached about 90%. After culturing in serum-free DMEM medium for 24 hours, Examples 1-3 and Comparative Examples 1-3, dissolved in serum-free DMEM medium at the same 10 μM concentration, were treated for 24 hours. Luciferase expression was detected according to the requirements of the luciferase reporter gene test kit. The analysis results are shown in Table 2 below, where the induction fold represents the multiple of the luciferase expression level relative to the solvent control group.

[0045]

Table 2

[0046]

[0047] As can be seen from Table 2, the induction multiples of Examples 1-3 were all higher than that of the solvent control group, indicating that the mixture of cryptotanshinone and omasorolone can activate Nrf2. Specifically, the induction multiple of Example 1 was 5.9, the induction multiple of Example 2 was 8.6, and the induction multiple of Example 3 was 11.1, indicating that the mixture has a certain activation effect on Nrf2.

[0048] Looking at the comparative examples again, the induction fold of comparative example 1 is 1.3, the induction fold of comparative example 2 is 5.3, and the induction fold of comparative example 3 is 6.9. Compared with comparative examples 1-3, the induction folds of Examples 1 and 2 are higher than those of comparative examples 1 and 2, and the induction fold of Example 3 is higher than that of comparative example 1 and 2, and is similar to that of comparative example 3. This shows that the mixture of Examples 1-3 has certain advantages in activating Nrf2, especially in the case of using cryptotanshinone or omasorolone alone as represented by comparative examples 1 and 2, the activation effect of Examples 1-3 is more significant.

[0049] [Test Example 2] Antioxidant performance test

[0050] In order to verify the antioxidant effect of the mixture of cryptotanshinone and omasorolone (Examples 1-3), DPPH free radical scavenging experiment was used for testing and compared with Comparative Examples 1-3.

[0051] The experimental steps are as follows: 190 μL of 100 μM (in ethanol) DPPH solution was prepared with the same initial concentrations as Examples 1-3, Comparative Examples 1-3, and the synthetic antioxidant - water-soluble vitamin E (Trolox, positive control group), and diluted to final reaction concentrations of 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, and 15.63 μM, respectively. 10 μL was added to each of the reaction solutions. After reacting at 37°C for 30 minutes, the absorbance was measured at 540 nm. IC was calculated. 50 The value is the sample concentration required to reduce the absorbance of DPPH radical by 50%.

[0052]

Table 3

[0053]

[0054] It can be seen from the experimental data in Table 3 that this experiment aims to verify the antioxidant effect of the mixture of cryptotanshinone and omasorolone (Examples 1-3) through DPPH free radical scavenging experiment, and compare it with Comparative Examples 1-3.

[0055] The experimental results showed that the IC of water-soluble vitamin E (positive control group) 50 The value was 53.3 μM, which represents the level of a known strong antioxidant. 50 The values were 57.9 μM, 25.9 μM and 10.2 μM respectively. 50 The value is the lowest, indicating that it has the strongest antioxidant effect, even better than the known antioxidant water-soluble vitamin E.

[0056] Let’s look at the comparative example. The IC of comparative example 1 50 The value was 72.9 μM, and the IC of Comparative Example 2 50 The value was 65.0 μM, and the IC of Comparative Example 3 50 The value is 14.2μM. Compared with Comparative Examples 1-3, the IC values of Examples 2 and 3 are 50 The values are lower than those of Comparative Examples 1 and 2. The IC values of Example 3 are 50 The value is lower than that of Comparative Example 3. This shows that the mixture of Examples 1-3 has certain advantages in antioxidant properties, especially when cryptotanshinone or omasorolone is used alone as represented by Comparative Examples 1 and 2, the antioxidant effects of Examples 1-3 are more significant.

[0057] [Test Example 3] Pulmonary fibrosis therapeutic effect test

[0058] In order to observe the therapeutic effect of the mixture of cryptotanshinone and omasorolone - Examples 1-3 on pulmonary fibrosis, the protective ability of each was compared and measured with that of Comparative Examples 1-3.

[0059] (1) Cell culture and grouping: MLE-12 cells were cultured at 3.5×10 5 Cells were plated in 6-well plates and cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. The experiment was divided into three groups: a control group, cultured normally without TGF-β1 or drug; a model group, induced by the addition of 10 ng / mL TGF-β1; and a drug-pretreatment group, pretreated with various concentrations of drug for 24 hours before induction with TGF-β1.

[0060] (2) Drug pretreatment and TGF-β1 induction: After cells were cultured until adherent, they were treated according to group. The drug pretreatment group was treated with different concentrations of drugs for 24 hours. Subsequently, the control and model groups were treated with medium without drugs. The drug pretreatment group was treated with 10 ng / mL TGF-β1 in the original drug medium and cultured for another 24 hours.

[0061] (3) Detection of cell phenotypic markers (Western blot): Collect cells, extract total protein with RIPA lysis buffer, and determine protein concentration with a BCA kit. Take equal amounts of protein for SDS-PAGE electrophoresis, transfer to the membrane, and block with 5% skim milk powder for 1 hour. Add primary antibodies for E-cadherin, α-SMA, and vimentin, and incubate at 4°C overnight. Wash three times with TBST, add HRP-labeled secondary antibody, incubate at room temperature for 1 hour, wash three times with TBST, develop color with ECL reagent, and take pictures with a chemiluminescence imager. Quantitatively analyze the degree of decreased E-cadherin expression and increased α-SMA and vimentin expression by grayscale analysis.

[0062]

Table 4

[0063]

[0064]

[0065] The experimental results of Table 4 show that in the TGF-β1-induced lung injury model, the expression of epithelial cell marker E-cadherin is significantly reduced, while the expression of interstitial cell markers α-SMA and vimentin is significantly increased, which reflects the epithelial-mesenchymal transition (EMT) process of the cells. The experimental results show that the drug pretreatment of Examples 1-3 has a significant protective effect on E-cadherin and can effectively inhibit the expression of interstitial cell markers α-SMA and vimentin, especially the drug combination of Example 3 group has the best effect. In contrast, the drug treatment of Comparative Examples 1-3 is less effective in protecting E-cadherin expression and inhibiting interstitial cell marker expression, indicating that the therapeutic effect of the drug combination of cryptotanshinone or omasolon and Comparative Example 3 is limited. Specifically, Comparative Example 1 (using cryptotanshinone alone) had limited protective effects on E-cadherin and was not effective in inhibiting interstitial cell markers; Comparative Example 2 (using omasolone alone) also had limited effects on protecting E-cadherin and inhibiting interstitial cell markers; Comparative Example 3 (other drug combinations) also failed to effectively protect E-cadherin expression and inhibit the expression of interstitial cell markers.

[0066] In summary, the drug combination of Examples 1-3 (a mixture of cryptotanshinone and omasorolone) showed significant synergistic therapeutic effects in protecting E-cadherin expression and inhibiting the expression of interstitial cell markers α-SMA and vimentin, which was superior to the use of cryptotanshinone or omasorolone alone and other drug combinations, and provided new ideas and methods for the treatment of pulmonary fibrosis.

[0067] As described above, the combination of cryptotanshinone and omasolone exhibited significant synergistic therapeutic effects in activating Nrf2, anti-oxidation, protecting E-cadherin expression, and inhibiting the expression of mesenchymal cell markers α-SMA and vimentin, which was superior to the use of either alone.

[0068] It will be understood by those skilled in the art that this synergistic effect is superior to that of cryptotanshinone or omasorolone alone. However, the scope of the present invention is not limited thereto. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0069] The pharmaceutical compositions of the present invention can be used in a variety of dosage forms, including but not limited to tablets, capsules, oral solutions, injections, dialysates, inhalants, syrups and nebulized solutions, etc. The essential scope of the present invention should be determined according to the claims and their equivalents.

[0070] The specific ratios of various dosage forms are as follows:

[0071]

Dosage Form Example 1

[0072]

[0073]

Dosage Form Example 2

[0074]

[0075] [Dosage Form Example 3] Oral solution (taking 100 mL as an example)

[0076]

[0077] [Dosage Form Example 4] Injection (taking 10 mL as an example)

[0078]

[0079] [Dosage Form Example 5] Dialysis fluid (taking 1L as an example)

[0080]

[0081] [Dosage Form Example 6] Inhalation (taking 10 mL as an example)

[0082]

[0083] [Dosage Form Example 7] Syrup (taking 100 mL as an example)

[0084]

[0085] [Dosage Form Example 8] Nebulized solution (taking 10 mL as an example)

[0086]

Claims

1. A pharmaceutical composition according to claim 1, characterized in that The weight ratio of cryptotanshinone to omasorolone is (16.7-75): (25-83.3).

2. A pharmaceutical composition according to claim 1, characterized in that The weight ratio of cryptotanshinone to omasorolone is 16.7:83.

3.

3. A pharmaceutical composition according to claim 1, characterized in that The purity of the cryptotanshinone is ≥98%, and the purity of the omasorolone is ≥98%.

4. A pharmaceutical composition according to claim 1, characterized in that It also contains one or more pharmaceutically acceptable excipients.

5. A pharmaceutical composition according to claim 3, characterized in that Relative to the total weight of the composition, the mass percentage of the cryptotanshinone is 5% to 30%, and the mass percentage of the omasorolone is 1% to 10%.

6. A pharmaceutical composition according to claim 1, characterized in that The composition is in the form of tablets, capsules, oral solutions, injections, dialysates, inhalants, syrups or nebulized solutions.

7. Use of the pharmaceutical composition according to claim 1 in the preparation of an Nrf2 agonist.

8. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating lung injury or pulmonary fibrosis.

9. The method for preparing the pharmaceutical composition according to claim 1, characterized in that The following steps are involved: 1) Weigh cryptotanshinone and omasorolone according to the mass ratio; 2) dissolving cryptotanshinone and omasorolone in appropriate amounts of solvents to obtain solution A and solution B, respectively; 3) Mixing solution A and solution B, stirring evenly to obtain a mixed solution; 4) Drying the mixed solution to obtain dry powder.

10. The method for preparing a pharmaceutical composition according to claim 8, characterized in that: The dry powder is mixed with auxiliary materials to prepare the desired dosage form, which is tablets, capsules, oral liquids, injections, dialysate, inhalants, syrups or nebulized solutions.

Images