CN105709227A - Anti-tumor medicine composition - Google Patents

Abstract

The invention discloses an anti-tumor drug composition, which comprises a two-week dosage of fibrin regulating drugs and tumor suppressors. The present invention combines fibrin-regulating drugs with tumor inhibitors, and controls the dosage of fibrin-regulating drugs, so as to improve the delivery effect of tumor inhibitors. On the premise of maintaining normal blood vessel functions, targeted drug delivery is possible as much as possible, so as to achieve Good antitumor effect.

Description

A kind of antitumor pharmaceutical composition

technical field

The invention belongs to the field of biomedicine, and more specifically relates to an antitumor drug composition.

Background technique

Tumor is a new organism formed by the body under the action of various tumorigenic factors, and the cells of local tissues lose their normal regulation of their growth at the gene level, resulting in abnormal proliferation and differentiation. Once a new organism is formed, it does not stop growing due to the elimination of the cause. Its growth is not regulated by normal body physiology, but destroys normal tissues and organs. This is especially obvious in malignant tumors. Compared with benign tumors, malignant tumors grow faster and show invasive growth, are prone to bleeding, necrosis, ulcers, etc., and often have distant metastasis, resulting in emaciation, weakness, anemia, loss of appetite, fever, and severe organ damage. Impairment of function, etc., eventually lead to death of the patient.

At present, anti-tumor drugs are research hotspots, and more effective anti-tumor drugs are still to be developed.

Contents of the invention

Aiming at the above deficiencies or improvement needs of the prior art, the present invention provides an anti-tumor drug composition, the purpose of which is to provide an anti-cancer effect and an Preferred targeted antineoplastic compositions.

In order to achieve the above object, according to one aspect of the present invention, an anti-tumor drug composition is provided, comprising a two-week dose of fibrin regulating drug and a tumor suppressor.

Preferably, in the antitumor pharmaceutical composition, the fibrin regulating drug is a thrombolytic drug and/or an anticoagulant drug.

Preferably, in the antitumor pharmaceutical composition, the thrombolytic drug is streptokinase, urokinase, tissue-type plasminogen activator, recombinant tissue-type plasminogen activator, single-chain urokinase-type plasmin Jue Hui activator, or acetylated plasmin-streptokinase activator complex.

Preferably, in the anti-tumor pharmaceutical composition, the anticoagulant drug is an anticoagulant drug for injection, an oral anticoagulant drug or a thrombin inhibitor; the anticoagulant drug for injection is preferably heparin, small molecule heparin; The coagulant drug is preferably warfarin, dicoumarin, and coumarin nitrate; the thrombin inhibitor is preferably hirudin or argatroban.

Preferably, in the anti-tumor pharmaceutical composition, the tumor suppressor is a free tumor suppressor and/or a nano-preparation of a tumor suppressor.

Preferably, in the antitumor pharmaceutical composition, the tumor inhibitor is paclitaxel, doxorubicin, doxorubicin or gene medicine.

Preferably, the nano-preparation of the anti-tumor pharmaceutical composition is a polyethylene glycol-polymer material nanoemulsion.

Preferably, the polymer material of the antitumor pharmaceutical composition is polylactic acid, polylactic acid-polyglycolic acid, polycaprolactone or distearoylphosphatidylethanolamine.

Generally speaking, compared with the prior art, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

The present invention combines fibrin-regulating drugs with tumor suppressors, and controls the dosage of fibrin-regulating drugs, thereby improving the delivery effect of tumor suppressors, and increasing the delivery of anti-tumor drugs by increasing the number of blood vessels with normal functions, thereby achieving Good antitumor effect.

Description of drawings

Fig. 1 is the experimental result of establishing a subcutaneous tumor model bearing A549 lung cancer in Example 1, and detecting the effect of rtPA administration on fibrin expression in the tumor; wherein Fig. 1A is the result of immunofluorescence experiment, and Fig. 1B is the result of Elisa.

Figure 2 shows the effect of rtPA on intratumoral blood perfusion in Example 2, compared with the normal saline group; Figure 2A is the result of immunofluorescence experiment, and Figure 2B is the semi-quantitative result of ImageJ statistics.

Fig. 3 is the characterization result of the nanoparticles of Example 2, Fig. A is the electron microscope result, and Fig. B is the particle size analyzer test result.

Fig. 4 shows the establishment of a subcutaneous tumor model bearing A549 lung cancer in Example 3, using the near-infrared dye DiR to label nanoparticles, and observing the difference in the distribution of nanoparticles in the tumors of the rtPA treatment group and the normal saline group.

Fig. 5 shows the establishment of a subcutaneous tumor model of A549 lung cancer in Example 4, using coumarin-6 to label nanoparticles, and observing the difference in the distribution of nanoparticles in the tumor slices of the rtPA treatment group and the normal saline group.

FIG. 6 shows the animal model bearing A549 lung cancer subcutaneous tumor established in Example 5 and Example 6, and the evaluation of the treatment of pancreatic cancer by rtPA combined with nanomedicine. They were divided into four groups: normal saline group, rtPA group, normal saline+NP-PTX group and rtPA+NP-PTX group; Figure 6A shows the volume change of the tumor, Figure 6B shows the change of the animal body weight, and Figure 6C shows the stripping after the animal was sacrificed. Figure 6D is the weight of the stripped tumor, and Figure 6E is the TUNEI apoptosis staining of the stripped tumor after paraffin-embedded sections (magnifications are 100 times, respectively).

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The anti-tumor pharmaceutical composition provided by the invention comprises a two-week dose of fibrin regulation drug and tumor suppressor.

The fibrin regulating drug is a thrombolytic drug and/or an anticoagulant drug. Described thrombolytic drug is streptokinase, urokinase, tissue-type plasminogen activator (tPA), recombinant tissue-type plasminogen activator (rtPA), single-chain urokinase-type plasminogen activator (SCUPA), or acetylated plasmin-streptokinase activator complex (APSAC). The anticoagulant drugs are anticoagulant drugs for injection, oral anticoagulant drugs or thrombin inhibitors; the anticoagulant drugs for injection are preferably heparin, small molecule heparin; the oral anticoagulant drugs are preferably warfarin, dicoumarin Chlorine, coumarin nitrate; the thrombin inhibitor is preferably hirudin or argatroban.

The tumor suppressor is a free tumor suppressor and/or a nano-preparation of a tumor suppressor. The tumor suppressor is paclitaxel, doxorubicin, doxorubicin or gene medicine. Place

The nano-preparation of the tumor suppressor is preferably surface polyethylene glycol-modified liposomes, nanoparticles, polymer vesicles, polymer micelles, and solid lipid nanoparticles. The tumor suppressor is combined with the nano-carrier in the form of encapsulation or covalent connection, and the particle size of the nano-preparation is 10-300nm.

Said nano-preparation is further preferably polyethylene glycol-macromolecule material nano-emulsion. The polymer material is polylactic acid, polylactic acid-polyglycolic acid, polycaprolactone or distearoylphosphatidylethanolamine. The molecular weight of polyethylene glycol is 1000-20000Da, preferably 2000-5000Da, and the molecular weight of polylactic acid is 5000-50000Da, preferably 20000-4000Da.

The antitumor effect of the antitumor composition of the present invention is measured according to the following method:

Using PEG-PLA as carrier, nanoparticles were prepared by single emulsion method. Zeta/laser particle size analyzer was used to measure the average particle size and potential of nanoparticles, and the morphology was observed by transmission electron microscope.

Animal models of lung cancer subcutaneously loaded with A549 were established, and after regulating the formation of fibrin in the tumor, the changes of fibrin in the tumor were evaluated by immunofluorescence and Elisa techniques.

Immunofluorescence was used to evaluate the changes of intratumoral perfusion after regulating intratumoral fibrin.

After labeling the nanoparticles with the infrared dye DiR, the difference in the enrichment of the nanocarriers in the tumors of the experimental group and the control group was evaluated by the small animal in vivo imager after regulating fibrin in the tumor.

After the green fluorescent probe coumarin-6 was used to label the nanoparticles, the difference in the distribution of the nanocarriers in the tumors of the experimental group and the control group was observed after the regulation of fibrin in the tumor was observed by frozen section.

The inhibitory effect of the antitumor composition provided by the invention on A549 tumor was evaluated by tumor growth inhibition experiment.

The composition provided by the invention has good antitumor effect, possibly due to the following reasons:

Efficient hemoperfusion within tumors is the basis for tumor drug delivery. Due to the lack of pericytes and the incomplete basement membrane of tumor neovascularization, the function is not perfect, and the extracellular matrix squeezes the intratumoral blood vessels to a certain extent, and the blood perfusion in the tumor is often lower than that of the surrounding normal tissues, thus preventing the drug from being effective. Reaching the tumor thereby affecting drug delivery to the tumor. To increase intratumoral perfusion, strategies differ between tumors with rich blood vessels and tumors with rich stroma. For tumors with rich blood vessels, previous studies generally used the method of normalizing blood vessels. Since this method of promoting vascular normalization reduces the gap between vascular endothelial cells to a certain extent, it can generally only increase the delivery of small molecule drugs or nano-drugs (10-40nm) with a small particle size, but cannot increase the delivery of about 100nm delivery of nanomedicines.

Fibrin, as the end product of the coagulation cascade, is not only enriched at the site of endothelial injury, but also deposited outside the tumor cells as a component of the tumor stroma, and is in a cross-linked state, similar to the structure of fibrin at the site of thrombus and injury. Fibrin in the tumor stroma is mainly formed by the leakage of tumor blood vessels and tissue factor on the surface of tumor cells. Normal tissue does not express fibrin due to the integrity of the vascular endothelium. In addition to being highly tumor specific, fibrin also has a certain position specificity in its intratumoral distribution. For tumors with rich blood vessels, the fibrin in the tumor is mainly distributed near the blood vessels. This is due to the conversion of fibrinogen into fibrin by the coagulation cascade initiated by the overexpressed tissue factor in the tumor after it penetrates into the blood vessels. A large amount of fibrin may squeeze the blood vessels in the tumor to a certain extent, thereby reducing the blood perfusion in the tumor and further weakening the drug delivery of the tumor.

The present invention combines fibrin-regulating drugs with tumor inhibitors, and controls the dosage of fibrin-regulating drugs, so as to improve the delivery effect of tumor suppressors, and increase drug delivery by increasing the number of blood vessels with normal functions, so as to achieve a good anti-tumor effect. tumor effect.

The following are examples:

Example 1

Frozen sections were prepared after intraperitoneal injection of rtPA in A549 lung cancer model mice for two consecutive weeks (at a dose of 25 mg/kg/d). The results of immunofluorescence staining showed that the fibrin in the saline group was distributed around the blood vessels in the form of cords, while there was little distribution far away from the blood vessels (Fig. 1A, B). However, after rtPA treatment, the fibrin fragments in the tumor were scattered (Fig. 1C, D), indicating that rtPA can effectively degrade the fibrin in the tumor. Further quantitative analysis by Elisa found that D-dimer, a specific degradation product of fibrin, was about 2.3 times that of the control group in the rtPA treatment group, which once again proved the effective destruction of fibrin in tumors by rtPA.

Example 2

Tail vein administration after rtPA administration 488 labeled lectins ( 488-lectin) at a dose of 5 mg/kg. After 1 hour, the animals were sacrificed by carbon dioxide asphyxiation, and quickly perfused with PBS and 4% paraformaldehyde solution, and frozen sections were prepared. After CD31 immunofluorescence staining, confocal microscopy analysis of functionalized blood vessels ( 488+CD31+) accounted for the proportion of all blood vessels (CD31+). The results showed that after rtPA treatment, the functionalized blood vessels in the tumor increased significantly, from 38.3±3.9% in the normal saline group to 75.5±7.0%, which greatly improved the intratumoral blood flow perfusion (Figure 2). We speculate that the main reason is that rtPA destroys the fibrin near the tumor blood vessels and releases the extrusion of the fibrin on the tumor blood vessels, so that some of the originally squeezed blood vessels can regain perfusion after the extrusion is released and become functional blood vessels. Efficient delivery of nanomedicines establishes the conditions.

Example 3

Nanoparticles were prepared by a single emulsification method. 24mg of polyethylene glycol-polylactic acid (MPEG-PLA) was dissolved in 1mL of dichloromethane and added to 5mL of 0.6% sodium cholate solution, followed by 5s/5s pulse ultrasound for 15 times in an ice-water bath with a power of 200W. Unmodified nanoparticles (NP) were obtained by rotary evaporating to remove dichloromethane and then concentrating to an appropriate concentration. The results of the particle size/potential measuring instrument showed that the particle size of the nanoparticles was 115.5±0.7nm (Figure 3A), and the potential was 11.5±0.6mv. Under the electron microscope, the nanoparticles were uniform in size, well dispersed, and the surface was smooth and regular spherical (Figure 3B).

Example 4

After the administration of rtPA, the nanoparticles were labeled with the near-infrared dye DiR, and 24 hours after the tail vein administration at a dose of 10 μg/kg, the aggregation degree of the nanoparticles in the tumor site was detected by a small animal in vivo imager. The granules were cohesive in the tumor (left side of Figure 4A: rtPA, right side: normal saline group), isolated tissue imaging and semi-quantitative results confirmed that the average fluorescence intensity in the tumor tissue of the rtPA treatment group was about 2.0 times that of the normal saline group ( Figure 4B and C), while the mean fluorescence values in normal organs were not significantly different between the experimental group and the control group (Figure 4D and E). It shows that the treatment of rtPA can effectively increase the distribution of nanoparticles in tumor tissues, but has no effect on the distribution of nanoparticles in normal organs.

Example 5

After the administration of rtPA, the green fluorescent probe coumarin-6 was used to label the nanoparticles, and 4 hours after the tail vein administration at a dose of 0.05 mg/kg, it was perfused and frozen sections were prepared. The confocal microscope observation results showed that the nanoparticles in the rtPA-treated group could be widely distributed inside the tumor and reach tumor sites far away from blood vessels (Fig. 5, C, D). In normal saline, the main part of the nanoparticles was near blood vessels, and the fluorescence was weaker than that of the rtPA group (Fig. 5, A, B). We speculate that the effect of rtPA destroys the fibrin near blood vessels, relieves blood vessel extrusion and weakens the possible penetration resistance of nano-drugs, so that nano-drugs can be effectively delivered to the tumor tissue and distributed more uniformly throughout the tumor tissue middle.

Example 6

When the tumor diameter reached 4-5 mm, 24 tumor-bearing nude mice were divided into four groups on average, with 6 mice in each group. a, control group (intraperitoneal injection of normal saline for two weeks, saline in the tail vein from the second week); b, rtPA group (intraperitoneal injection of rtPA in two weeks, saline in the tail vein from the second week); c, NP -PTX group (peritoneal injection of normal saline for two weeks, NP-PTX administered by tail vein from the second week); d, rtPA+NP-PTX group (peritoneal injection of rtPA for two weeks, NP-PTX administered by tail vein from the second week) ). The beginning of tail vein administration was counted as day 0, and the dose of PTX was 8 mg/kg, administered once every two days, and the size of the tumor and the body weight of the tumor-bearing mice were recorded every two days during the experiment. After the end of administration, continue to monitor for 4 days, then kill the animals by carbon dioxide asphyxiation, take tumor tissues, weigh them and prepare frozen sections, perform apoptosis staining according to the instructions of the TUNEL kit, stain with 1 μg/ml Hochest33342 at room temperature for 5 minutes, wash with PBS three times The apoptosis of tumor cells in the tumor tissues of different treatment groups was observed under a fluorescence microscope. Based on the recorded weights of nude mice, the body weight change curve of the mice was drawn, and the tumor growth inhibition rate was calculated. The curve of tumor volume change showed that the tumor in the normal saline group continued to grow, and the tumor growth in the rtPA only group was similar to that in the normal saline group. However, only NP-PTX group can inhibit the growth of tumor to a certain extent. On the basis of administration of rtPA, NP-PTX can inhibit tumor growth more significantly than that of the NP-PTX single administration group (Fig. 6A, C, D). The tumor weight inhibition rates of the NP-PTX and rtPA+NP-PTX groups on tumor growth were 35.7% and 73.7%, and the inhibition rates on tumor volume were 42.8% and 71.7%, respectively. During the whole experiment period, there was no significant difference in body weight of tumor-bearing mice in each group (Fig. 6B), indicating that the dose of PTX was within the acceptable range of mice and did not cause obvious side effects. The results of TUNEL staining showed that the normal saline group and the rtPA group had only scattered single cell apoptosis, while the NP-PTX group could induce slightly more tumor cell apoptosis, and the rtPA+NP-PTX group could induce extensive apoptosis in the tumor tissue. (Fig. 6E) (from left to right are the control group, rtPA group, NP-PTX group and rtPA+NP-PTX group), and this result is consistent with the tumor volume change curve reflecting the tumor growth.

Those skilled in the art can easily understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (8)

1. an antineoplastic pharmaceutical compositions, it is characterised in that include fibrin regulating medicine and the tumor inhibitor of two weeks dosages.

2. antineoplastic pharmaceutical compositions as claimed in claim 1, it is characterised in that described fibrin regulating medicine is thrombolytic drug and/or anticoagulant.

3. antineoplastic pharmaceutical compositions as claimed in claim 2; it is characterized in that, described thrombolytic drug is that agent is lived or acetylation fibrinolysin is fainted-streptokinase activator complex in streptokinase, urokinase, tissue-type plasminogen activator, rt-PA, single chain urokinase type plasminogen activator fiber type plasmin emblem of fainting.

4. antineoplastic pharmaceutical compositions as claimed in claim 2, it is characterised in that described anticoagulant is injection anticoagulant, oral anticoagulation thing or thrombin inhibitor；The described preferred heparin of injection anticoagulant, low molecular weight；The described preferred warfarin of oral anticoagulation thing, dicoumarol, nitric acid coumarin；The preferred hirudin of described thrombin inhibitor or argatroban.

5. antineoplastic pharmaceutical compositions as claimed in claim 1, it is characterised in that described tumor inhibitor is free tumor inhibitor and/or tumor inhibitor nanometer formulation.

6. antineoplastic pharmaceutical compositions as claimed in claim 5, it is characterised in that described tumor inhibitor is paclitaxel, amycin, doxorubicin or genomic medicine.

7. antineoplastic pharmaceutical compositions as claimed in claim 5, it is characterised in that described nanometer formulation is Polyethylene Glycol-macromolecular material nano-emulsion.

8. antineoplastic pharmaceutical compositions as claimed in claim 7, it is characterised in that described macromolecular material is polylactic acid, polyglycolic-polylactic acid, polycaprolactone or DSPE.