Method for preventing blood coagulation and drug-eluting percutaneous coronary intervention therapy

Abstract

The present invention provides a method of preventing blood coagulation comprising administering to a subject in need thereof an effective amount of ginsenoside compound K (CK). The invention also provides an application of the ginsenoside compound K (CK) in preparation of a medicine for preventing blood coagulation. Further, the present invention provides a drug-eluting percutaneous coronary intervention therapy (PCI) comprising a vascular implant wherein a surface of the vascular implant is coated with a first layer comprising a ginsenoside compound K (CK) and a first bioresorbable polymer, wherein the first bioabsorbable polymer comprises poly-L-lactic acid (PLLA) and poly (L-lactide-co-epsilon-caprolactone) (PLCL) in a weight ratio of 60% to 80%: 20% to 40%.

Description

Background Art

[0001] A coronary stent is a medical device that helps treat patients with severe coronary artery disease. Stents are usually used in conjunction with balloon angioplasty. Briefly, balloon angioplasty is used to dilate a narrowed artery and a stent is placed in the blood vessel to provide support.

[0002] In the past, the incidences of stent thrombosis and in-stent restenosis within 1 month after the implantation of traditional coronary stents were 1% and However, after the invention of drug-eluting stents (DES) and drug-eluting balloons (DEB) that can inhibit endothelial cell growth, the incidence of in-stent restenosis has been significantly reduced to less than 1%. The drugs applied to DES and DEB include paclitaxel, sirolimus (also known as rapamycin), and limus family drugs (such as everolimus, zotarolimus, and biolimus). These drugs can help inhibit the proliferation of endothelial cells and, as immunosuppressants, can effectively reduce the risk of in-stent restenosis. Although current DES and DEB can solve the problem of restenosis, patients will need to take anticoagulant drugs for a long time and face the risk of late stent thrombosis. In addition, paclitaxel and sirolimus have been proven to have high toxicity and low lipid solubility, respectively.

[0003] On the other hand, the spraying temperature is an important issue. If the composition solvent is below 100 °C, high viscosity will occur and clog the chamber, but we know that CK has higher solubility to overcome this drawback.

[0004] Therefore, it is necessary to develop novel drugs for application to DES and DEB.

[0005] The prior art has not revealed the anticoagulant effect of ginsenoside compound K (CK) and its potential as an alternative drug for application to DES and DEB. In the present invention, CK will be used on DES and DEB to help overcome the drawbacks of stent thrombosis and restenosis caused by the prior art.

[0006] Ginsenosides are the main active components of ginseng and are known to have various pharmacological activities, such as anti-tumor, anti-fatigue, anti-allergy, and antioxidant activities. Ginsenosides have a common basic structure, consisting of a gonane steroid nucleus with 17 carbon atoms arranged in four rings. Ginsenosides are metallized in the body, and some recent studies have shown that ginsenoside metabolites, rather than naturally occurring ginsenosides, are more easily absorbed in the body and act as active ingredients. Among them, ginsenoside CK, also known as compound K (CK), is known to be a metabolite of protopanaxadiol-type ginsenosides processed by human intestinal bacteria via the gypenoside pathway. To date, no prior art literature has reported the anticoagulant effect of ginsenoside CK and its use in the preparation of coronary stents. Summary of the Invention

[0007] In the present technical solution, CK is uniformly distributed on the surfaces of the stent and the balloon (used for balloon angioplasty) by vacuum plasma spraying (VPS) technology. CK has unique low toxicity and anti-thrombotic properties; therefore, it will become a potential option for clinical use in preventing in-stent thrombosis and restenosis.

[0008] In one embodiment, the present invention provides a method for preventing blood coagulation, which comprises administering an effective amount of ginsenoside compound K (CK) to an individual in need.

[0009] In another embodiment, the present invention provides the use of ginsenoside compound K (CK) in the preparation of a drug for preventing blood coagulation.

[0010] In yet another embodiment, the present invention provides ginsenoside compound K (CK) in a method for preventing blood coagulation.

[0011] In yet another embodiment, the present invention provides a drug-eluting percutaneous coronary intervention (PCI) device, which comprises a vascular implant, wherein the surface of the vascular implant is coated with a first layer comprising ginsenoside compound K (CK) and a first bioabsorbable polymer.

[0012] In an embodiment of the drug-eluting PCI, the first bioabsorbable polymer comprises poly-L-lactic acid (PLLA) and poly(L-lactide-co-ε-caprolactone) (PLCL) in a weight ratio of 60% to 80%: 20% to 40%.

[0013] In an embodiment of the drug-eluting PCI, the thickness range of the first layer is 0.5 to 2 μm, 0.5 to 1.5 μm, or 0.8 to 1 μm.

[0014] In one embodiment of drug-eluting PCI, the CK in the first bioabsorbable polymer is 0.1 to 5 μg / mm 2 、0.1 to 3 μg / mm 2 、0.1 to 1 μg / mm 2 、0.25 to 0.75 μg / mm 2 or 1 to 3 μg / mm 2 。

[0015] In one embodiment of drug-eluting PCI, the vascular implant is a stent.

[0016] In one embodiment of drug-eluting PCI, it further includes a second layer above the first layer, where the second layer contains a second bioabsorbable polymer, and the second bioabsorbable polymer contains 80% to 100% by weight of polyvinylpyrrolidone (PVP) based on the total weight of the second bioabsorbable polymer.

[0017] In one embodiment of drug-eluting PCI, the thickness range of the second layer is 0.5 to 3 μm, 1 to 3 μm, or 0.5 to 1.5 μm.

[0018] In one embodiment of drug-eluting PCI, it further includes a third layer between the first layer and the second layer, where the third layer contains the CK and a third bioabsorbable polymer, and the third bioabsorbable polymer contains PLLA and PLCL in a weight ratio of 25% to 35%: 65% to 85%.

[0019] In one embodiment of drug-eluting PCI, the thickness range of the third layer is 0.5 to 1.5 μm.

[0020] In one embodiment of drug-eluting PCI, the CK in the third bioabsorbable polymer is 0.1 to 5 μg / mm 2 、0.5 to 2 μg / mm 2 ,preferably 0.75 to 1.25 μg / mm 2 。

[0021] In one embodiment of drug-eluting PCI, the weight of CK applied on the vascular implant is greater than 100 μg and less than 1000 μg.

[0022] In one embodiment of drug-eluting PCI, it further includes a second layer below the first layer, where the second layer contains a second bioabsorbable polymer, and the second bioabsorbable polymer contains 80% to 100% by weight of polyvinylpyrrolidone (PVP).

[0023] In an embodiment of drug eluting PCI, the thickness of the second layer ranges from 0.5 to 3 μm, 1 to 3 μm, or 0.5 to 1.5 μm.

[0024] In one embodiment of drug eluting PCI, the first layer coated on the surface of the vascular implant is applied by vacuum plasma spraying (VPS). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 An exemplary three-layer coating of ginsenoside CK on a stent is shown.

[0026] Figure 2 Shown is the release kinetics of ginsenoside CK during the healing process. Nearly 80% of the drug is released within one month (initial burst). The remaining drug is scheduled to be released for 3 months. Designed to cover the entire period of arterial wound healing in real-world patients. After 90 days of drug release, the amount of residual drug in the stent is so low that it is below the detection / quantification limit.

[0027] Figure 3 An exemplary double-layer coating of ginsenoside CK on a balloon is shown.

[0028] Figure 4 Shown is the release kinetics of ginsenoside CK during the healing process. Nearly 80% of the drug is released within one month (initial burst). The remaining drug is scheduled to be released for 3 months. Designed to cover the entire period of arterial wound healing in real-world patients. After 90 days of drug release, the amount of residual drug in the stent is so low that it is below the detection / quantification limit.

[0029] Figure 5 Inhibition curves of Astemizole using the hERG FP assay are shown. Data points represent the average of three determinations.

[0030] Figure 6 Inhibition curves of CK using the hERG FP assay are shown. Data points represent the average of three determinations.

[0031] Figure 7 Shows the effect of CK on platelet aggregation.

[0032] Figure 8 HUVECs were shown to be incubated with CK alone (1 μM, 3 μM and 10 μM) for 24 hours and cell survival was determined by CCK-8 assay.

[0033] Figure 9Show the effects of ginsenoside CK on the expression of LPS-induced IL-6 and TNF-α in HUVEC. After HUVEC were treated with ginsenoside CK (1 μM, 3 μM, and 10 μM) for 1 hour, they were stimulated with LPS (1 μg / ml) for 24 hours, and the expression of (A) IL-6 and (B) TNF-α was detected. The values are the mean ± SEM of 3 independent experiments. Statistical significance was evaluated by one-way ANOVA analysis and Scheffe's post hoc test for multiple comparisons (***P < 0.01, *P < 0.05, compared with LPS).

[0034] Figure 10 Show the effects of ginsenoside CK on the expression of LPS-induced ICAM-1 and VCAM-1 mRNA in HUVEC. After HUVEC were treated with ginsenoside CK (1 μM, 3 μM, and 10 μM) for 1 hour, they were stimulated with LPS (1 μg / ml) for 24 hours, and the expression of (A) ICAM-1 and (B) VCAM-1 mRNA was detected. The values are the mean ± SEM of 3 independent experiments. Statistical significance was evaluated by one-way ANOVA analysis and Scheffe's post hoc test for multiple comparisons (***P < 0.01, *P < 0.05, compared with LPS). Detailed implementation manners

[0035] The foregoing and other embodiments of the present invention will now be described in more detail with other examples described herein. It should be understood that the present invention can be implemented in different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

[0036] The terms used herein to describe the present invention are only for describing specific embodiments and are not intended to limit the present invention. As used in the description of the present invention and the appended claims for patents, the singular forms "a", "an", and "the" are also intended to include the plural forms unless the context clearly indicates otherwise.

[0037] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing" or any other variant thereof are intended to cover non-exclusive inclusion, subject to any limitations expressly indicated. For example, a composition, mixture, process, or method that includes a list of elements need not be limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0038] As used herein, the term "about" means a value that includes, for example, variations inherent in a measuring device, a method for determining the value, or differences among subjects being studied. Generally, the term is intended to cover variations of about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the circumstances.

[0039] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. However, the present invention supports definitions that refer to alternatives only and "and / or".

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent disclosures, patent cases and other references cited herein are incorporated by reference in their entirety for the purpose of disclosing sentences and / or paragraphs related to the references.

[0041] The following representative examples and embodiments illustrate various features and embodiments of the present invention, which are intended to be illustrative and not restrictive. Those skilled in the art will readily understand that the specific examples are merely illustrative of the present invention, which is more fully described in the claims. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each of the embodiments included therein.

[0042] 1. Materials and Methods

[0043] 1.1 Research Drugs

[0044] a. Ginsenoside compound K (CK) powder was provided by Wellhead Biological Technology Corp, purity: 95%

[0045] b. Collagen (1x2ml) (Helena Laboratories, Shang Li Instruments Co., Ltd)

[0046] c. Human platelets were isolated from blood samples before testing

[0047] d. Dimethyl sulfoxide (Lot number RNBH8393, Sigma)

[0048] e. 10X Phosphate Buffered Saline (Lot number AC13074270, HyClone)

[0049] f. Predictor TMThe hERG fluorescence polarization detection kit (PV 5365, Invitrogen, CA, USA) was purchased from Invitrogen (Carlsbad, CA, USA).

[0050] g. 384-well polypropylene plates (Corning #3657) and black 384-well assay plates (Corning #3677) were purchased from Corning (Lowell, MA, USA).

[0051] 1.2 Experimental equipment

[0052] a. 2.7 ml blood collection tubes (containing 3.2% sodium citrate)

[0053] b. Safety blood collection kits with 21G Luer connectors

[0054] c. Alcohol cotton balls

[0055] d. Tourniquets

[0056] e. 15 ml centrifuge tubes and 1.5 ml microcentrifuge tubes

[0057] f. 200- and 1000-μl plastic tips

[0058] g. 96-well microplates

[0059] h. Adjustable volume pipettes

[0060] 1.3 Experimental devices

[0061] a. Biosensors Technology - Automatic pipette

[0062] b. Epoch TM Microplate spectrophotometer system (BioTek)

[0063] c. AggRAM analyzer (Helena Laboratories)

[0064] d. Evolution TM Spectrophotometer (Thermo Scientific TM )

[0065] e. Tecan equipped with a fluorescence polarization module The F200 microplate reader was purchased from TECAN Group Ltd (Switzerland). The multi-channel handheld disposable-tip pipette (eight tips) was purchased from Axygen (CA, USA).

[0066] 2. Experimental operations and procedures

[0067] CK Coating on the Stent

[0068] 2.1 Preparation of CK-Coating on Superflex Cruz

[0069] Mix the composition of PLLA and PLCL fully with chloroform under water heating, then add DMSO at 25°C, and add CK Control the temperature of the spraying chamber below 100°C to avoid deformation of the polymer at high temperatures.

[0070] The detailed steps are as follows:

[0071] CK DES has a conformal coating. It has three layers of coating, as Figure 1 shown, including the following layers:

[0072] Top layer: with a thickness of This layer is a drug-free polymer layer added with hydrophilic PVP (polyvinylpyrrolidone). It prevents light, moisture, and premature release of drugs, and provides lubrication during stent implantation.

[0073] Middle layer: with a thickness of Same as the base layer, it contains a mixture of PLLA (poly-L-lactic acid), PLCL (poly(L-lactide-co-ε-caprolactone)), and CK drug Designed to provide a sufficient amount of drug after stent implantation.

[0074] Base layer: with a thickness of This layer contains a mixture of PLLA (poly L-lactic acid), PLCL (poly(L-lactide-co-ε-caprolactone)), and CK drug Designed for slow drug release.

[0075] 2.2 Healing process is as Figure 2 shown.

[0076] The kinetic curve is as shown by the arrow.

[0077] 2.3 Coating process

[0078] 2.4 Polymer and drug preparation:

[0079]

[0080] * The total CK DES drug concentration is

[0081] * The total CK DES drug for each stent (3.0mm X 20mm) is

[0082] CK coating on the balloon

[0083] Preparation of CK coating on 2.5 balloon

[0084] CK DEB has an abluminal coating. It has a double-layer coating, as Figure 3 shown:

[0085] Top layer: with a thickness of This layer contains a mixture of PLLA (poly-L-lactic acid), PLCL (poly(L-lactide-co-ε-caprolactone)) and CK drug (2 μg / mm 2 ). It is designed to provide a sufficient amount of drug after DEB implantation.

[0086] Base layer: with a thickness of This layer is a drug-free polymer layer added with hydrophilic PVP (polyvinylpyrrolidone). It is designed to provide a boundary for the top layer on the balloon surface and peel off after DEB implantation.

[0087] 2.6 The healing process is as follows.

[0088] Drug release kinetics: It can cover the 120-day vascular healing process, as Figure 4 shown. The kinetic curve is as shown by the arrow.

[0089] 2.7 Coating process

[0090] Polymer and drug preparation:

[0091]

[0092] * The total CK DEB drug concentration is

[0093] * The total CK DEB drug for each balloon (3.0 mm X 20 mm) is

[0094] hERG test

[0095] 2.8 Preparation of chemical reagents and solutions

[0096] a. Serial dilute the 100% DMSO stock solution of 3 mM CK and astemizole 3-fold in the same solvent to obtain 16 different concentrations in the range from 3 mM to 210 pM. These solutions are further diluted 25-fold with FP (fluorescence polarization) assay buffer and then transferred to the assay plate.

[0097] b. Each test solution in the assay plate was further diluted 4-fold with FP assay buffer. Predictor TM The hERG membrane preparation was thawed at room temperature and sonicated. Predictor was diluted to 4 nM with hERG FP assay buffer. TM hERGTracer Red (250 nM). All experiments were conducted in FP assay buffer containing 5% DMSO.

[0098] 2.9 Experimental procedure

[0099] a. FP assay was performed using Predictor TM hERG fluorescence polarization detection kit. The binding assay was performed according to the manufacturer's recommended protocol with some modifications. Aliquots (5 μL) of each concentration of the reference test substance were transferred into the appropriate wells of a black 384-well microplate containing 10 μL of hERG membrane and 5 μL of 4 nM fluorescent tracer, and the plate lid was placed to protect the reagents from light exposure.

[0100] b. Experiments were performed in triplicate for each concentration. After incubation for 3 hours at room temperature, the microplate was read on a Tecan

[0101] F200 plate reader using a polarized excitation filter set at 535 nm and an emission filter set at 590 nm (25 and 20 nm bandwidths, respectively). The assay robustness was evaluated by measuring 16 replicates of the positive control wells (containing 30 μM E-4031, a known hERG channel blocker, in the FP assay mixture) and negative control wells (without hERG channel blocker in the FP assay mixture). F200 plate reader using a polarized excitation filter set at 535 nm and an emission filter set at 590 nm (25 and 20 nm bandwidths, respectively). The assay robustness was evaluated by measuring 16 replicates of the positive control wells (containing 30 μM E-4031, a known hERG channel blocker, in the FP assay mixture) and negative control wells (without hERG channel blocker in the FP assay mixture).

[0102] 2.10 Determination of assay Z'-value

[0103] a. To evaluate the robustness of the hERG assay, the signal-to-noise ratio was quantified by calculating the Z'-factor for each assay. In statistics, the Z'-factor provides a method to evaluate the quality of an assay. A Z-value greater than 0.5 is generally considered good assay performance, while a value of 1 represents a theoretically ideal assay with no variability. The Z'-factor value was determined using the formula of Zhang et al. [1], subtracting the polarization values of the blank from the 16 wells of the negative and positive control groups.

[0104]

[0105] where μ NC and σ NC are the mean and standard deviation of the negative control group (pre-dose signal exposed to vehicle), respectively, and μ PC and σPC They are the mean and standard deviation of the positive control group (post - dosing signal exposed to 30 μM positive inhibitor), respectively.

[0106] 2.11 Data analysis

[0107] All data analysis and presentation were performed using (Redmond, WA, USA) Office Excel 2010 and SigmaPlot version 12 (SPSS, Inc., Chicago, IL). For concentration - response curve analysis, inhibition data were fitted to a four - parameter logistic equation:

[0108]

[0109] E = fluorescence polarization response (binding ligand in the presence of an inhibitor concentration)

[0110] E max = maximum response of fluorescence polarization

[0111] E min = minimum response of fluorescence polarization

[0112] x = inhibitor concentration

[0113] IC 50 = drug concentration required to inhibit 50% of the current

[0114] Hill slope = slope of the curve identified at the mid - point

[0115] The following table lists the risk classification of the proposed hERG blocker potency:

[0116]

[0117] Cytochrome P450 inhibition in human liver microsomes was analyzed using LC - MS / MS

[0118] In the IC 50 shift experiment, 8 different concentrations of CK (0.2 μL, prepared in DMSO) were combined with pooled human liver microsomes ( (depending on the enzyme to be evaluated), pre-incubated for 30 minutes at 37 °C in 100 μL of potassium phosphate buffer (100 mM, pH 7.4) and MgCl2 (2.5 mM) in the absence or presence of 1 mM NADPH. After pre-incubation, 100 μL of a substrate mixture of potassium phosphate buffer (100 mM, pH 7.4), MgCl2 (2.5 mM), and NADPH (1 mM) containing the probe substrate was added to measure the corresponding P450 activity. The incubation was terminated by adding 200 μL of methanol. The samples were then extracted and centrifuged, and the supernatant was subjected to validated LC-MS / MS analysis.

[0119] Anticoagulant test

[0120] 2.12 Preparation of chemical reagents and solutions

[0121] a. Prepare a 20 mM CK stock solution using DMSO and dilute it to different working concentrations (0, 62.5, 125, 250, 500, 1000, and 2000 μM) with 1X PBS before the experiment.

[0122] 2.13 Experimental procedure

[0123] a. Blood sample collection

[0124] Donors should rest and fast for 12 hours before blood collection and should not smoke. No medications such as antihistamines, antibiotics, aspirin, and anti-inflammatory drugs are allowed 24 hours before the examination as they may affect platelet function. To ensure non-coagulation, blood samples should be thoroughly mixed with an anticoagulant and stored at 24 to 27 °C. It is recommended to perform platelet aggregation tests within

[0125] minutes after blood sample collection. minutes.

[0126] b. Preparation of platelet-rich plasma (PRP)

[0127] Gently invert the blood collection tube times to resuspend the blood cells. Centrifuge the sample at 170 x g for 7 minutes, then transfer the supernatant to a new tube. The sample should be used within minutes after separation; otherwise, the sample should be stored at 4 °C to avoid agglutination.

[0128] c. Preparation of platelet-poor plasma (PPP)

[0129] Gently invert the blood collection tube times to resuspend the blood cells. Centrifuge the sample at 2,400 x g for 10 minutes, then transfer the supernatant to a new tube. The sample should be tested within 5 minutes after separation from whole blood; otherwise, the sample should be stored at 4 °C.

[0130] d. Platelet count

[0131] Dilute the concentration of PRP to 3x10 8 cells / ml with PPP.

[0132] e. Platelet aggregation test

[0133] To test the effect of CK on platelet aggregation function, different concentrations of CK (0, 2.5, 5, 10, 20, 40, and 80 μM) were added to 0.25 ml of PRP. The mixture samples were incubated at 37 °C for 2 minutes and immediately treated with 5 μg / ml collagen (100 μg / ml stock solution). The PRP - DMSO - distilled water mixture served as the non - aggregated control group. For the blank control group, (1) original PPP, (2) PPP - DMSO - distilled water mixture, (3) PPP - DMSO - collagen mixture, and (4) PPP - CK - collagen mixture were prepared. All mixture samples and control groups were incubated at 37 °C on an orbital shaker at 200 rpm for 5 minutes. Then the samples were centrifuged at 170 x g for 7 minutes, and 200 μl of the supernatant was transferred to a 96 - well microplate. The OD value was measured at 650 nm with a spectrophotometer, and the value was converted into the aggregation percentage.

[0134]

[0135] 2.14 Data analysis

[0136] The platelet aggregation percentage was calculated as follows:

[0137] a.

[0138] b.

[0139] c.

[0140] 3. Results and discussion

[0141] hERG assay

[0142] Based on three repeated measurements, the hERG IC 50 value of astemizole (reference compound) was 0.004 μM. Figure 5 Show the log concentration - response curve of the inhibitor astemizole. The IC 50 value of this astemizole was in good agreement with the values in the literature using this method. CK showed a slight inhibition in the fluorescence polarization assay ( Figure 6)。CK showed a slight blocking effect on the binding of the tracer to hERG membrane protein at a concentration of 30 μM (from three repeated measurements), and the reported IC 50 value of CK > 30 μM. CK can be classified as a weak ligand of the hERG channel (IC 50 > 1 μM) (Table 1).

[0143] Table 1. Summary of IC 50 values of CK and astemizole

[0144]

[0145] * The IC 50 value of astemizole is in good agreement with the IC 50 value reported in the literature.

[0146] Inhibition of cytochrome P450 in human liver microsomes using LC-MS / MS analysis

[0147] The mean IC 50 values of CK as an inhibitor of eight CYP-specific probe substrates of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 in pooled human liver microsomes were >50, 14.84, >50, >50, >50, >50, 9.76 (using midazolam as the CYP3A4 substrate) and 13.86 (using testosterone as the CYP3A4 substrate) μM (Table 2). For the CYP2B6, 3A4 (using midazolam as the CYP3A4 substrate) and 3A4 (using testosterone as the CYP3A4 substrate) inhibition assays, after pre-incubation for 30 minutes, the IC 50 shifts of CK were 1.08, 0.98 and 1.06-fold, respectively. After pre-incubation for 30 minutes with or without NADPH, CK at a concentration of 50 μM did not inhibit CYP1A2, 2C8, 2C9, 2C19 and 2D6 (Table 3); therefore, meaningful IC 50 shift values for these isoforms could not be calculated.

[0148] Table 2. IC 50 values of CK as an inhibitor of seven human cytochrome P450 enzymes in pooled human liver microsomes

[0149]

[0150] * When the IC 50 is greater than 50 μM, the inhibition percentage value at 50 μM is included.

[0151] Table 3. IC of CK for human P450 enzymes after pre-incubation with human liver microsomes for 30 minutes in the absence and presence of NADPH 50 value

[0152]

[0153] a Ratio of IC of pre-incubation without NADPH 50 to IC of pre-incubation with NADPH 50

[0154] b When IC 50 is greater than 50 μM, the inhibition percentage value at 50 μM is included

[0155] c ND, not detected

[0156] Anticoagulant test

[0157] This experiment was designed to find out the effect of CK on platelet aggregation. After treating platelets with CK, the degree of aggregation decreased. In addition, the platelet aggregation level in Group 9 was approximately 7-fold lower than that in Group 4. These results suggest that CK can act as an anticoagulant for platelets ( Figure 7 ).

[0158] Effect of ginsenoside CK on the expression of LPS-induced IL-6 and TNF-α in HUVEC

[0159] The results showed that treatment with different concentrations (1 μM, 3 μM, and 10 μM) of ginsenoside CK alone for 24 hours did not affect cell viability, indicating that ginsenoside CK is non-toxic ( Figure 8 ). Treatment of HUVEC with LPS (1 μg / ml) increased the expression of IL-6 and TNF-α. To determine whether the expression of LPS-induced IL-6 and TNF-α was affected by ginsenoside CK, human ginsenoside CK (1 μM, 3 μM, and 10 μM) was used to treat HUVEC for 1 hour, and then stimulated with LPS (1 μg / ml) for 24 hours. Ginsenoside CK significantly decreased the expression of LPS-induced IL-6 ( Figure 9 , Part A) and TNF-α ( Figure 9 , Part B) in a concentration-dependent manner.

[0160] Effect of ginsenoside CK on the expression of LPS-induced ICAM-1 and VCAM-1 mRNA in HUVEC

[0161] ​Treatment of HUVEC with LPS (1 μg / ml) increased the expression of ICAM-1 and VCAM-1 mRNAs. At 24 hours after LPS treatment, ICAM-1 and VCAM-1 reached their maximum expression. To determine whether the expression of LPS-stimulated ICAM-1 and VCAM-1 mRNAs was affected by ginsenoside CK, HUVEC were treated with ginsenoside CK (1 μM, 3 μM, and 10 μM) for 1 hour, followed by stimulation with LPS (1 μg / ml) for 24 hours. Ginsenoside CK significantly inhibited LPS-stimulated ICAM-1 ( Figure 10 , part A) and VCAM-1 ( Figure 10 , part B) mRNA expression in a concentration-dependent manner.

[0162] The present invention is described and further illustrated by the following examples:

[0163] Example 1. A drug-eluting percutaneous coronary intervention (PCI) device, which comprises a vascular implant

[0164] wherein the surface of the vascular implant is coated with a first layer comprising ginsenoside compound K (CK) and a first bioabsorbable polymer.

[0165] Example 2. The drug-eluting PCI according to Example 1, wherein the first bioabsorbable polymer comprises poly-L-lactic acid (PLLA) and poly(L-lactide-co-ε-caprolactone) (PLCL) in a weight ratio of 60% to 80%: 20% to 40%.

[0166] Example 3. The drug-eluting PCI according to Example 1 or 2, wherein the thickness of the first layer ranges from 0.5 to 2 μm, 0.5 to 1.5 μm, or 0.8 to 1 μm.

[0167] Example 4. The drug-eluting PCI according to any one of Examples 1 to 3, wherein the CK in the first bioabsorbable polymer is 0.1 to 5 μg / mm 2 , 0.1 to 3 μg / mm 2 , 0.1 to 1 μg / mm 2 , 0.25 to 0.75 μg / mm 2 or 1 to 3 μg / mm 2 .

[0168] Example 5. The drug-eluting PCI according to any one of Examples 1 to 4, wherein the vascular implant is a stent.

[0169] Example 6. The drug-eluting PCI according to any one of Examples 1 to 5, further comprising a second layer on top of the first layer,

[0170] Preferably, the second layer comprises a second bioabsorbable polymer. More preferably, the second bioabsorbable polymer comprises 80% to 100% by weight of polyvinylpyrrolidone (PVP), based on the total weight of the second bioabsorbable polymer.

[0171] Example 7. The drug-eluting PCI according to any one of Examples 1 to 6, wherein the thickness of the second layer ranges from 0.5 to 3 μm, 1 to 3 μm, or 0.5 to 1.5 μm.

[0172] Example 8. The drug-eluting PCI according to any one of Examples 1 to 7, further comprising a third layer between the first layer and the second layer.

[0173] Preferably, the third layer comprises the CK and a third bioabsorbable polymer. More preferably, the third bioabsorbable polymer comprises PLLA and PLCL in a weight ratio of 25% to 35%: 65% to 85%.

[0174] Example 9. The drug-eluting PCI according to any one of Examples 1 to 8, wherein the thickness of the third layer ranges from 0.5 to 1.5 μm.

[0175] Example 10. The drug-eluting PCI according to any one of Examples 1 to 9, wherein the CK in the third bioabsorbable polymer is 0.1 to 5 μg / mm 2 、0.5 to 2 μg / mm 2 , preferably 0.75 to 1.25 μg / mm 2 .

[0176] Example 11. The drug-eluting PCI according to any one of Examples 1 to 10, wherein the weight of CK applied to the vascular implant is greater than 100 μg and less than 1000 μg.

[0177] Example 12. The drug-eluting PCI according to any one of Examples 1 to 11, wherein the vascular implant is a balloon.

[0178] Example 13. The drug-eluting PCI according to any one of Examples 1 to 12, further comprising a second layer below the first layer.

[0179] Preferably, the second layer comprises a second bioabsorbable polymer. More preferably, the second bioabsorbable polymer comprises 80% to 100% by weight of polyvinylpyrrolidone (PVP).

[0180] Example 14. The drug-eluting PCI according to Example 13, wherein the thickness of the second layer ranges from 0.5 to 3 μm, 1 to 3 μm, or 0.5 to 1.5 μm.

[0181] Example 15. The drug-eluting PCI according to any one of Examples 1 to 14, wherein the first layer coated on the surface of the vascular implant is coated by vacuum plasma spraying (VPS).

[0182] Example 16. A method for preventing blood coagulation, comprising administering an effective amount of ginsenoside compound K (CK) to an individual in need thereof.

[0183] Example 17. Use of ginsenoside compound K (CK) for the preparation of a medicament for preventing blood coagulation.

[0184] Example 18. Ginsenoside compound K (CK) in a method for preventing blood coagulation.

[0185] References

[0186] [1] Zhang, J.H., Chung, T.D., and Oldenburg, K.R. (1999) A simple statistical parameter for use in evaluation and validation of high-throughput screening assays. J. Biomol. Screen. 4: 67-73.

Claims

1. A drug-eluting percutaneous coronary intervention (PCI) device, which comprises a vascular implant, wherein the surface of the vascular implant is coated with a first layer comprising ginsenoside compound K (CK) and a first bioabsorbable polymer.

2. The drug-eluting PCI according to claim 1, wherein the first bioabsorbable polymer comprises poly-L-lactic acid (PLLA) and poly(L-lactide-co-ε-caprolactone) (PLCL) in a weight ratio of 60% to 80%: 20% to 40%.

3. The drug-eluting PCI according to claim 1 or 2, wherein the thickness of the first layer ranges from 0.5 to 2 μm, 0.5 to 1.5 μm, or 0.8 to 1 μm.

4. The drug-eluting PCI according to any one of claims 1 to 3, wherein the CK in the first bioabsorbable polymer is 0.1 to 5 μg / mm 2 , 0.1 to 3 μg / mm 2 , 0.1 to 1 μg / mm 2 , 0.25 to 0.75 μg / mm 2 or 1 to 3 μg / mm 2 .

5. The drug-eluting PCI according to any one of claims 1 to 4, wherein the vascular implant is a stent.

6. The drug-eluting PCI according to any one of claims 1 to 5, further comprising a second layer on top of the first layer, preferably the second layer comprises a second bioabsorbable polymer, and more preferably the second bioabsorbable polymer comprises polyvinylpyrrolidone (PVP) in a weight ratio of 80% to 100% based on the total weight of the second bioabsorbable polymer.

7. The drug-eluting PCI according to any one of claims 1 to 6, wherein the thickness of the second layer ranges from 0.5 to 3 μm, 1 to 3 μm, 0.5 to 1.5 μm.

8. The drug-eluting PCI according to any one of claims 1 to 7, further comprising a third layer between the first layer and the second layer, preferably the third layer comprises the CK and a third bioabsorbable polymer, and more preferably the third bioabsorbable polymer comprises PLLA and PLCL in a weight ratio of 25% to 35%: 65% to 85%.

9. The drug-eluting PCI according to any one of claims 1 to 8, wherein the thickness of the third layer ranges from 0.5 to 1.5 μm.

10. The drug-eluting PCI according to any one of claims 1 to 9, wherein the CK in the third bioabsorbable polymer is 0.1 to 5 μg / mm 2 , 0.5 to 2 μg / mm 2 , preferably 0.75 to 1.25 μg / mm 2 .

11. The drug-eluting PCI according to any one of claims 1 to 10, wherein the weight of CK applied on the vascular implant is greater than 100 μg and less than 1000 μg.

12. The drug-eluting PCI according to any one of claims 1 to 11, wherein the vascular implant is a balloon.

13. The drug-eluting PCI according to any one of claims 1 to 12, further comprising a second layer below the first layer, preferably the second layer comprises a second bioabsorbable polymer, and more preferably the second bioabsorbable polymer comprises polyvinylpyrrolidone (PVP) in a weight ratio of 80% to 100%.

14. The drug-eluting PCI according to claim 13, wherein the thickness of the second layer ranges from 0.5 to 3 μm, 1 to 3 μm, or 0.5 to 1.5 μm.

15. The drug-eluting PCI according to any one of claims 1 to 14, wherein the first layer coated on the surface of the vascular implant is coated by vacuum plasma spraying (VPS).

16. A method for preventing blood coagulation, comprising administering an effective amount of ginsenoside compound K (CK) to an individual in need.

17. Use of ginsenoside compound K (CK) for preparing a medicament for preventing blood coagulation.

18. Ginsenoside compound K (CK) in a method for preventing blood coagulation.

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