CN108187151A - A kind of stent of medication coat - Google Patents

Abstract

The invention relates to a vascular stent containing a drug coating. The base of the vascular stent is bare metal or alloy, and the vascular stent is coated with a gefitinib drug coating. The gefitinib drug and the polylactic acid carrier form a coating together, and the mass ratio of the gefitinib drug to the polylactic acid carrier is 5 mg/g. Dissolve gefitinib and its carrier polylactic acid in 100WT% ethanol solution and heat to 40-80°C for more than 1 hour; fix the metal stent base and keep it at 60±10°C, drop the drug coating liquid Dry on the stent base, repeat 3-10 times until the coating thickness reaches 10-100 microns; or soak the metal stent in the drug coating liquid, and then dry to form a coating, repeat 3-10 times until needed coating thickness.

Description

A drug-coated stent

technical field

The invention relates to a medical device, which is a drug-loaded stent formed by loading a functional drug coating on a common stent base material composed of common CoPt, stainless steel and other alloy grids.

Background technique

US Patent No. 5697967 discloses such a drug-loaded stent. US Patent No. 9204982 discloses a method for injecting drugs into this type of stent. The application of rapamycin on bare stents in blood vessels can effectively inhibit the occurrence of restenosis. Most of the peripheral stents currently used clinically are bare metal stents, but stent implantation has long been limited by in-stent restenosis (ISR). In-stent restenosis (ISR) occurs in ~30% of patients, which has become an important problem in interventional therapy[1]. Studies have shown that drug-eluting stents (DES) can further reduce the incidence of ISR[2], but DES still faces late stent thrombosis (LST), very late stent thrombosis (very late stent thrombosis, VLST)[3] and other issues affect the efficacy of interventional therapy. There are also drug-coated stents that are studying drugs such as paclitaxel abroad. Paclitaxel DES, Siromlius and its derivative DES are commonly used vascular stents in clinical practice at present, and their action principle is to inhibit the proliferation and migration of vascular smooth muscle. Compared with traditional bare metal stents, they can inhibit intimal hyperplasia and reduce the incidence of ISR. However, the follow-up of patients implanted with these drug-eluting stents found that the incomplete coverage of advanced endothelial cells became the biggest disadvantage of this drug-eluting stent. Incomplete endothelialization (re-endothelialization, RE) is due to the non-specific anti-proliferative effect of eluting drugs. These drugs inhibit the growth of vascular endothelial cells while inhibiting the proliferation of vascular smooth muscle.

references

[1] F.Alfonso, M.J.Perez-Vizcayno, A.Cruz, J.Garcia, P.Jimenez-Quevedo, J.Escaned, R.Hernandez, Treatment of patients with in-stent restenosis, EuroIntervention 5 (Suppl.D) (2009) D70–D78.

[2] S.McGinty, S.McKee, R.M.Wadsworth, C.McCormick, Modeling drug-eluting Stents, Math.Med.Biol.28(1)(2011)1–29.

[3] Takano M, Yamamoto M, Inami S, et al. Long-Term Follow-Up Evaluation After Sirolimus-Eluting Stent Implantation by Optical Coherence Tomography [J]. Jacc, 2008(9): 968-969.

Contents of the invention

The object of the present invention is to propose a drug-coated stent, the drug-coated stent of the present invention comprises a stent and a drug coating coated on the stent, the drug coating is a polymer containing gefitinib, While basically not affecting the growth of endothelial cells, it effectively inhibits the proliferation of vascular smooth muscle cells, thereby achieving the effect of inhibiting restenosis in the stent.

The technical scheme of the present invention is: a stent containing drug coating, that is, a vascular stent functionalized with a novel composite drug coating, the vascular stent base is commonly used bare metal or alloy (or other stent materials), and the vascular stent metal The net is coated with gefitinib drug coating.

The gefitinib drug and the polylactic acid carrier form a coating together, and the mass ratio of the gefitinib drug to the polylactic acid carrier is 5 mg/g.

The preparation method of the vascular stent containing the drug coating, its typical compound drug coating liquid manufacturing process is to dissolve gefitinib and its carrier polylactic acid in 100WT% ethanol solution and heat to 40-80 Keep at ℃ for more than 1 hour; fix the metal stent base and keep it at 60±10°C, drop the drug coating liquid on the stent base and dry it, repeat the drops for 3-10 times until the coating thickness reaches 10-100 microns; or The metal stent is soaked in the drug coating liquid, and then dried to form a coating, repeated 3-10 times to the required coating thickness.

The metal stent is soaked in the composite drug coating liquid, and then dried to form a coating, and repeated 3-10 times to reach the required coating thickness. Prepare the gefitinib-coated stent GES of the present invention.

Gefitinib is an oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor, which is currently clinically used for chemotherapy of locally advanced or metastatic non-small cell lung cancer. In vitro experiments have shown that Gefitinib Compared with paclitaxel, nontinib can more specifically inhibit the proliferation of vascular smooth muscle cells (SMC), and has a lower cytotoxic effect on vascular endothelial cells (VEC) than paclitaxel. This study observed the preventive effect of gefitinib drug-coated stents on in-stent restenosis in a minipig femoral artery model, but there are no reports on this aspect at home and abroad.

The present invention is a vascular stent functionalized with a new type of drug coating. In vitro tests and animal tests show that the present invention can effectively inhibit the proliferation of vascular smooth muscle cells while basically not affecting the growth of endothelial cells, thereby achieving the purpose of inhibiting regeneration in the stent. Narrow effect.

The advent of DES is hailed as another "milestone" in the field of vascular intervention. DES is a stent with a drug-carrying carrier bound to its surface. After being placed in the lesion, it becomes a drug pool and continuously releases drugs to the lesion. It is targeted. Well, the effective treatment time is long, and the systemic side effects are small. The mechanism of in-stent restenosis has not been fully elucidated. Studies have shown that intimal hyperplasia is closely related to the occurrence of ISR. Multiple factors affect intimal hyperplasia, and smooth muscle cell proliferation and migration play an extremely important role [5]. Paclitaxel DES, Siromlius and its derivative DES are commonly used vascular stents in clinical practice at present, and their action principle is to inhibit the proliferation and migration of vascular smooth muscle. Compared with traditional bare metal stents, they can inhibit intimal hyperplasia and reduce the incidence of ISR. However, the follow-up of patients implanted with these drug-eluting stents found that the incomplete coverage of advanced endothelial cells became the biggest disadvantage of this drug-eluting stent. Incomplete endothelialization (re-endothelialization, RE) is due to the non-specific anti-proliferative effect of eluting drugs. These drugs inhibit the growth of vascular endothelial cells while inhibiting the proliferation of vascular smooth muscle. In this experiment, gefitinib was used to make DES, and it was confirmed from the perspectives of vascular color Doppler ultrasound, CTA, and histopathology that GES can effectively inhibit the occurrence of in-stent restenosis without affecting endothelialization.

In this study, through in vitro experiments, the optimal drug concentration was determined, so as to inhibit the proliferation of vascular smooth muscle cells and basically not affect the normal growth of vascular endothelial cells. After the GES was implanted into the femoral artery of the minipig, color Doppler ultrasound showed that the blood flow in the GES was smooth and the flow rate was normal one month after the operation. Three months after the operation, CTA showed that the stent was in place without obvious displacement and deformation, the diameter of the stent was normal, and the blood flow in it was smooth. At the same time, the blood vessel specimens with stents were taken out, and histopathological analysis showed that the GES group (n=10) had no significant difference in vessel diameter and injury score compared with the BMS group (n=10) (P>0.05), but the lumen area of the GES group Compared with the BES group, the intima thickness and neointimal area on the stent were significantly increased compared with the BES group. It can be seen that within 3 months after stent implantation, GES can effectively inhibit intimal hyperplasia and avoid the occurrence of in-stent restenosis. It provides a new idea for the drug selection of DES.

Description of drawings

Fig. 1 is the magnified observation diagram of the GES scanning electron microscope of the present invention.

Detailed ways

1.1 Experimental materials

1.1.1 Cell lines and main reagents: porcine bone marrow stem cells, fetal bovine serum, DMEM medium, EBM-2 medium, PDGF-BB growth factor, PBS, trypsin, FITC, DAPI nuclear fuel, MTT, DMSO.

1.1.2 Drugs, carriers and stents: gefitinib, polylactic acid, 100% ethanol, 3cm*2.5mm coronary balloon expansion stent (Buma)

1.1.3 Experimental animals: This experiment uses Bama miniature pigs, male or female, weighing 20-25kg, purchased from the Animal Experiment Center of Nanjing Drum Tower Hospital

1.2 Experimental method

1.2.1 Induction of vascular smooth muscle cells and vascular endothelial cells and identification by immunofluorescence: Normal-growing porcine bone marrow stem cells were cultured in DMEM medium and EBM-2 medium containing PDGF-BB growth factor respectively, and the parameters of the cell culture box were Adjust to 5% CO ₂ , and adjust the ambient temperature to 37°C. Induced to differentiate into vascular smooth muscle cells and vascular endothelial cells. Parallel immunofluorescence analysis. Among them, vascular smooth muscle cell-specific primary antibody was α-actin, and vascular endothelial cell-specific primary antibody was vWF and CD34.

1.2.2 Preparation and scanning electron microscope observation of drug-coated stents: In this experiment, dip-coating method was used to prepare stents. Sterilize the bare coronary stent for later use. Dissolve Gefitinib and its carrier polylactic acid in 100ml of 100% ethanol at a mass ratio of 0, 1mg/g, 2mg/g, 5mg/g, and 10mg/g, and immerse the scaffolds in ethanol containing gradient drugs at 37°C Dip coating for 24h. Each drug concentration gradient stent was taken out, fully air-dried, and a scanning electron microscope was used to observe the combination of the drug and the stent. All brackets were sterilized again for use.

1.2.3 MTT method to determine the appropriate drug concentration of gefitinib: stents with concentration gradients of 0, 1 mg/g, 2 mg/g, 5 mg/g, and 10 mg/g were placed in 50 ml of PBS, and stirred at 37 °C for 72 h , to make an extract. GES extracts with different concentration gradients were used as medium substrates to culture SMC and VEC respectively, and MTT test was carried out for four consecutive days. Cells in logarithmic growth phase were taken to make 1× ¹⁰⁴ cell suspension, and seeded in 96-well plates. 100 μl per well until the cells adhered to the wall, discard the old culture solution, add different concentration gradient extraction medium (, 1mg/g, 2mg/g, 5mg/g, 10mg/g), and continue to cultivate for 24, 48, 72 After 96 hours, add 20 μl of MTT solution to each well, continue to incubate for 4 hours, discard the culture solution in each well, add 150 μl of DMSO, shake on the shaker for 20 minutes, and measure the absorbance at OD490 of each group. Calculate the inhibition rate of each concentration gradient extract on MSC and EPC, and select the most appropriate drug concentration of gefitinib according to the MTT results.

1.2.4 Implantation of GES into porcine femoral artery: GES was prepared and sterilized according to the above-mentioned optimum concentration. Ten Bama miniature pigs were selected and anesthetized by intramuscular injection of ketamine (100 mg) and droperidocaine (5 mg), and the pigs were fixed on the surgical board in a supine position. After skin preparation and disinfection of both groins, a vertical incision was made in the groin, the muscle space was bluntly dissected, the bilateral carotid arteries were exposed, and the tape was wound for use. After whole-body heparinization (100U/kg), pug clips blocked the proximal and At the distal end, a transverse incision was made on the front wall of the blocked femoral artery, with a length of about 5 mm, and a 0.014 mm guide wire was inserted into the proximal end, and the GES to be released was sent to the pre-released blood vessel through the guide wire. The balloon is opened under atmospheric pressure, the stent is released into the lumen of the blood vessel, and the release balloon is withdrawn. After the operation, 8-0 prolene suture was used to continuously suture the transverse incision of the carotid artery. The blood flow was opened to observe whether the blood vessel was unobstructed and whether there was local stenosis. After careful hemostasis, the incision of the femoral artery was closed layer by layer. Before the completion of the operation, 800,000 U of penicillin was given intravenously, and after the operation, aspirin (300 mg/d) was given for antiplatelet.

1.2.5 Review color Doppler ultrasound at one month after operation: One month after operation, intramuscular injection of ketamine (100 mg) and droperidocaine (5 mg) was performed for anesthesia and skin preparation. All animals underwent bilateral femoral artery color Doppler ultrasound examination.

1.2.6 CTA examination three months after operation: Three months after operation, after basic anesthesia, 50ml of iodophorol was injected into the marginal ear vein of miniature pigs, CT scanning of both forelimb arteries and three-dimensional reconstruction.

1.2.7 Histological examination: Three months after the operation, the femoral artery segment implanted with the stent was exposed, and the patency and appearance of the artery were observed with the naked eye. The femoral artery specimen containing the stent segment was cut and divided into 3 segments on average. The first segment was fixed in 10% formalin solution, embedded in methyl methacrylate, sliced to 50 μm with a hard tissue microtome, and stained with hematoxylin-eosin. The second segment of the stent specimen was cut longitudinally, and the stent was spread flat, soaked in 1.6% glutaraldehyde, and prepared for scanning electron microscopy to observe the growth of endothelial cells. Immunohistochemical analysis of the third scaffold specimen

2. Results

2.1 Immunofluorescence identification results

The porcine vascular smooth muscle cells and endothelial cells were in good growth condition. Immunofluorescence results indicated that the smooth muscle cells were positive for α-actin, and the endothelial cells were positive for CD34 and vWF. The above results confirmed that the two types of cells were consistent with the phenotype.

2.2 Preparation of GES and SEM results

The drug-coated stent was successfully prepared according to the above method, and the scanning electron microscopy showed that the drug particles were evenly distributed on the surface of the BMS, and the surface of the BMS was still relatively smooth, which was not affected by the preparation process (Figure 1).

2.3 MTT results

In this experiment, the MTT method was used to explore the killing effect of different drug concentrations of GES on VSMC and VEC, so as to determine the appropriate drug concentration. The inhibitory rate of GES at each concentration gradient on the two kinds of cells shows that Gefitinib can significantly inhibit the growth of VSMC, but the growth inhibitory effect on VEC is weak. The inhibitory effect of Gefitinib on VSMC is positively correlated with the drug concentration and action time. However, when the drug concentration is too high (10mg/g), it has obvious inhibitory effect on VEC, and the 96h inhibition rate is 15%. Comprehensively considering the effect of drugs on VSMC and VEC, 5 mg/g was selected as the drug concentration for subsequent animal experiments.

2.4 Ultrasound after operation

B-ultrasound was rechecked 1 month after the operation, and it can be seen that the blood flow in the GES stent is smooth and the flow rate is normal.

Claims (3)

1. A vascular stent containing a drug coating is characterized in that the vascular stent base is bare metal or alloy, and the vascular stent is coated with a gefitinib drug coating. 2. the drug-coated vascular stent according to claim 1 is characterized in that the gefitinib drug and the polylactic acid carrier form a coating together, and the mass ratio of the gefitinib drug and the polylactic acid carrier is 5 mg/g . 3. the preparation method of the vascular stent containing drug coating according to claim 1 is characterized in that its typical composite drug coating liquid manufacturing process is that gefitinib and carrier polylactic acid thereof are dissolved in 100WT% in ethanol solution and heated to 40-80°C for more than 1 hour; fix the metal stent base and keep it at 60±10°C, drop the drug coating liquid on the stent base and dry it, repeat 3-10 times until it is coated The thickness of the layer reaches 10-100 microns; or the metal stent is soaked in the drug coating liquid, and then dried to form a coating, repeated 3-10 times to the required coating thickness.