阅读说明：本技术 一种预防和/或治疗脑瘤的药物及其应用 (Medicine for preventing and/or treating brain tumor and application thereof ) 是由 陶铸 丁文婷 程智鹏 冯银芳 秦莉 陈小平 于 2020-03-13 设计创作，主要内容包括：本发明涉及一种预防和/或治疗脑瘤的药物及其应用,所述药物包括疟原虫。本发明所涉及的药物对脑瘤具有显著的预防和/或治疗效果,能够抑制脑瘤的生长,延长患者的寿命；且无明显的毒副作用,安全性高；该药物丰富了脑瘤的治疗手段,由于脑部组织的特殊性和敏感性,化疗药物不容易穿过血脑屏障,不能发挥抗肿瘤效应,而本发明药物主要通过调节机体的免疫功能以及炎症相关因子,通过全面激活免疫能力突破血脑屏障,抑制脑瘤的生长,从而延长寿命。(The invention relates to a medicament for preventing and/or treating brain tumor and application thereof, wherein the medicament comprises plasmodium. The medicine has obvious effect of preventing and/or treating brain tumor, can inhibit the growth of the brain tumor and prolong the life of a patient; the traditional Chinese medicine composition has no obvious toxic or side effect and high safety; the medicine enriches the treatment means of brain tumor, and due to the particularity and sensitivity of brain tissues, chemotherapy medicine is not easy to pass through blood brain barrier and can not play the anti-tumor effect.)

1. A medicament for the prevention and/or treatment of brain tumors, said medicament comprising plasmodium.

2. The agent for the prophylaxis and/or treatment of a brain tumor according to claim 1, wherein the brain tumor comprises a primary brain tumor or a metastatic brain tumor.

3. The agent for the prophylaxis and/or treatment of brain tumor according to claim 1, wherein the brain tumor comprises glioma, meningioma, pituitary adenoma, or schwannoma, preferably glioma.

4. A medicament for the prophylaxis and/or treatment of brain tumors according to any one of claims 1 to 3, wherein the type of said plasmodium includes any one or a combination of at least two of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale or plasmodium knowlesi, preferably plasmodium vivax.

5. The agent for the prophylaxis and/or treatment of brain tumors according to any one of claims 1 to 4, wherein the Plasmodium is cryopreserved and/or revived Plasmodium, cryopreserved and/or revived Plasmodium sporozoites.

6. The agent for the prophylaxis and/or treatment of brain tumor according to any one of claims 1 to 5, wherein the agent is in any one of pharmaceutically acceptable dosage forms.

7. The agent for the prophylaxis and/or treatment of brain tumor according to any one of claims 1 to 6, wherein the agent is in the form of injection.

8. The medicament for preventing and/or treating brain tumor according to any one of claims 1 to 7, further comprising any one or a combination of at least two of pharmaceutically acceptable pharmaceutical excipients;

preferably, the pharmaceutical excipients include any one or a combination of at least two of a thickener, a diluent, a flavoring agent, a binder, or a filler.

9. The agent for the prophylaxis and/or treatment of brain tumor according to any one of claims 1 to 8, wherein the agent is an agent carried on a pharmaceutically acceptable carrier;

preferably, the pharmaceutically acceptable carrier comprises a liposome, micelle, dendrimer, microsphere or microcapsule.

10. Use of a medicament according to any one of claims 1 to 9 for the preparation of a medicament for the prophylaxis and/or treatment of brain tumors.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a medicine for preventing and/or treating brain tumor and application thereof.

Background

Brain tumors are clinically common tumors and seriously threaten the health and survival of human beings. Brain tumors are classified into primary brain tumors and metastatic brain tumors, which are more common than primary brain tumors. Among metastatic brain tumors, lung cancer is most common, followed by breast cancer and melanoma, accounting for approximately 67-80% of total brain metastases. The primary brain tumors are most commonly glioma, meningioma, pituitary adenoma and schwannoma sequentially, wherein the glioma accounts for 40% -50% of the primary tumor, and in adults, the glioma accounts for 75% of malignant primary brain tumors. According to a grading system established by WHO, the brain glioma is divided into 1 grade (lowest malignancy degree and best prognosis) to 4 grades (highest malignancy degree and worst prognosis), the glioblastoma and the 4 grade correspond to the malignant glioma, and the median survival time is only 14.6 months.

Compared with other cancers of the nervous system, lung cancer, liver cancer, breast cancer and the like, brain tumors have specificity, and due to the fact that the diseased parts of the brain tumors are in the brain and the existence of blood brain barriers, the traditional three tumor treatment strategies, namely surgical resection, radiotherapy and chemotherapy, have certain limitations, not only affect the exertion of the therapy, but also cause serious toxic and side effects, and seriously affect the treatment of the brain tumors. The operation treatment is to completely remove brain tumor as far as possible, and also ensure that brain function is not damaged, malignant glioma is a type which is difficult to cure, tumor shows invasive growth, the boundary with normal brain tissue is not obvious, the operation is difficult to completely remove, the tumor is easy to relapse, complications of brain post-operation can exist, such as bleeding, blood clot, swelling and the like, for brain stem glioma and brain tumor at deep position of brain, the risk of operation treatment is higher, and the most commonly used treatment means at present is operation removal treatment matched with radiotherapy and chemotherapy. However, radiotherapy may damage and necrose normal brain tissue, and also risks inducing new tumors and stroke; some chemotherapy drugs can improve the efficacy of drugs by improving the permeability of the blood-brain barrier, but can cause side effects such as paraneoplastic edema, epilepsy, cognitive impairment and depression. Therefore, the prevention and treatment of brain tumors has been a scientific problem in the world.

Among them, the 5-year survival rate of patients with glioblastoma is only 3%, the continuous development of tumor immunotherapy provides a treatment strategy with high specificity and low toxic and side effects for brain tumors, and immunotherapy is now widely used in the research and treatment of brain tumor tests, and can enhance the immunity against tumors, but for malignant gliomas, the expectation of improving the quality of life is not yet achieved. Many early clinical trials have shown that glioma vaccines, while potentially achieving some therapeutic benefit, do not improve survival in randomized controlled trials. Clinical trials of immune checkpoint inhibitors on glioblastoma have also shown no significant effect on patient survival, and glioblastoma has no response to immune checkpoint inhibitors. Immunotherapy of brain tumors therefore requires further investigation.

Because the traditional three therapies have limited treatment effect on brain tumor, the existing immunotherapy, such as targeted therapy, immune checkpoint inhibitors and the like, has unsatisfactory clinical test results and can not improve survival. Therefore, it is very meaningful to develop a new therapeutic strategy that can achieve a significant anti-brain tumor effect, and a new brain tumor therapeutic concept can be provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a medicament for preventing and/or treating brain tumor and application thereof. The medicine can obviously inhibit the growth of brain tumor, obviously prolong the life cycle of a patient, break through the blood brain barrier to exert curative effect, has no obvious toxic or side effect and high safety, and provides a new strategy and thought for treating the brain tumor.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a medicament for preventing and/or treating brain tumors, the medicament comprising plasmodium.

The medicine related by the invention breaks through the blood brain barrier by comprehensively activating the immune system. Pathogen-associated pattern recognition molecules (PAMPs), which are danger signaling molecules released by plasmodium, include the known glycosylphosphatidylinositol anchor (GPI anchor), malarial pigment, plasmodium DNA, immunostimulatory nucleic acid motifs and other unknown molecules, which are recognized by Pattern Recognition Receptors (PRRs) of the host's immune cells. PRRs include toll-like receptors (TLRs) on the surface of the endosomal or cell membrane, RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) in the cytoplasm, and the PAMPs of plasmodium trigger different transcription programs and stimulate multiple downstream signaling pathways to induce a systemic immune response, release Th 1-type cytokines such as TNF- α, IL-1 β, IL-2, IL-6, IL-12, IFNs type I and II, activate NK cells, NKT cells, γ/T cells, macrophages and Dendritic Cells (DCs), and then deactivate CD4+ and CD8+ T cells. By down-regulating immunosuppressive factors such as TGF-beta, IL-10 and the like in tumor tissues, the number of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-related macrophages (TAMs) and the like in tumors is remarkably reduced, so that an immunosuppressive microenvironment in the tumors is remodeled into an immune-supported microenvironment, and finally, brain tumors can be transformed into an effective brain tumor vaccine. On the other hand, plasmodium infection Damages Associated Molecular Patterns (DAMPs), such as endogenous uric acid, microvesicles and plasmodium infected erythrocytes also induce similar immunological activity. Research shows that meningeal lymphatic vessels, pericerebrovascular gaps, lymphatic systems before lymphatic vessels and perineural lymphatic vessels play a vital role in lymphatic circulation of the central nervous system, and plasmodium comprehensively activates an immune system to break through a blood brain barrier through the above ways, so that growth of brain tumor is inhibited, and the effect of preventing and/or treating the brain tumor is achieved. Because the plasmodium can easily control the infection rate and thus toxic and side effects through the antimalarial drug, the plasmodium has limited toxic and side effects and is controllable and safe clinically, and the drug provides a new idea for preventing and/or treating brain tumor.

Preferably, the brain tumor comprises a primary brain tumor or a metastatic brain tumor.

Preferably, the brain tumor comprises a glioma, a meningioma, a pituitary adenoma, or a schwannoma, preferably a glioma.

The medicament has good effect on preventing and/or treating brain tumor, particularly has good effect on glioma, and has the effect of resisting brain tumor by inducing strong anti-tumor immune response.

Preferably, the type of plasmodium includes any one or a combination of at least two of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale or plasmodium knowlesi, for example, a combination of plasmodium falciparum and plasmodium vivax, a combination of plasmodium malariae and plasmodium ovale, a combination of plasmodium falciparum and plasmodium vivax, and the like, and any combination thereof can be selected, and will not be described in detail herein. Plasmodium vivax is preferred.

Preferably, the plasmodium is cryopreserved and/or revived plasmodium, and cryopreserved and/or revived plasmodium sporozoites.

The cryopreservation of the resuscitation plasmodium comprises the following specific steps:

(1) cryopreservation of plasmodium

(1.1) centrifuging the plasmodium-containing whole blood for 5min at 300 Xg, and transferring the plasma to another 50mL centrifuge tube;

(1.2) sucking and removing leucocyte layers, adding 2 times of the culture medium 1640 by volume, uniformly mixing, and centrifuging for 5min at 300 Xg;

(1.3) slowly dripping the equal volume of 28% glycerol frozen stock solution into a 50mL centrifuge tube, shaking and uniformly mixing while adding, and incubating for 5min at room temperature;

(1.4) subpackaging the RBC-glycerol cryopreservation solution mixture into cryopreservation tubes, wherein each tube contains 1.0mL of the RBC-glycerol cryopreservation solution mixture;

(1.5) placing the mixture into a freezing storage box, and directly placing the mixture into liquid nitrogen for storage;

(1.6) freezing and storing quality control: adding appropriate amount of RBC-glycerol frozen stock solution mixture before freezing into appropriate amount of RPMI1640 medium at 37 deg.C under CO₂After 72 hours of incubation in the incubator, the color of the medium was observed to confirm the absence of turbidity.

(2) Recovery of plasmodium

(2.1) preparation: a water bath (37 ℃), a 50mL centrifuge tube, a 3.5% NaCl solution, sodium chloride injection, a centrifuge, a printing label and a table;

(2.2) checking and checking the label of the frozen tube;

(2.3) taking the frozen plasmodium blood, carrying out water bath at 37 ℃ for 1-3min, quickly mixing uniformly, and unfreezing;

(2.4) transferring to a 15mL centrifuge tube, slowly adding an equal volume of 3.5% NaCl solution along the tube wall, lightly blowing, uniformly mixing, standing at room temperature for 5min, and removing the supernatant;

(2.5) slowly adding 0.9% NaCL solution with 5 times volume of the solution along the tube wall, slightly blowing, beating and uniformly mixing, centrifuging at 300 Xg for 5min, and removing supernatant;

(2.6) adding physiological saline until the hematocrit is 50%, uniformly mixing, counting and recording the density of the red blood cells, temporarily storing at 4 ℃ for clinical inoculation;

(2.7) quality control of recovery of plasmodium: after completion of the inoculation, blood samples, blood smears, and the infection rate and protozoal status of the plasmodium before injection were observed by microscopy and recorded.

The cryopreservation resuscitation plasmodium sporozoites comprises the following specific steps:

(1) anopheles sanguinea feeding mosquito

(1.1) preparing in the early stage: monitoring the protozoan density and the gametophyte condition of a plasmodium infected volunteer, collecting 2mL of peripheral blood by veins, taking 24 hours in advance, and fasting anopheles (more than 300);

(1.2) before blood feeding, adjusting the temperature of a mosquito chamber to 26 ℃, preparing a membrane blood feeding system, adding collected peripheral blood into a membrane dead hole system, and feeding the blood for 30 min;

(1.3) sucking off the unopened saturated anopheles, writing the label, feeding the anopheles in an incubator at 26 ℃, and adding sugar water cotton containing 10% of glucose and 0.05% of PABA.

(2) Examination of oocysts

(2.1) examining oocysts 7-10 days after blood feeding infection, dissecting 10 eggs, and calculating the proportion of positive anopheles; note that: standard for oocyst infection, +: 1-10; ++: 11-100; +++: 101 or more;

(2.2) if the proportion above the oocyst infection degree "+ +" is less than 50%, 20 anopheles mosquitoes are rechecked.

(3) Examination of sporozoites

(3.1) examination of sporozoites on days 14-16 after blood feeding infection, dissecting 20 Anopheles, and calculating total sporozoites, the average amount of sporozoites per Anopheles. Note that: counting by using a cell counting plate, counting the number of all sporozoites in a large square, and following the principle of counting up, counting down, counting left and right; to minimize errors, the average of three counts was taken during counting.

(4) Sporozoite harvesting

(4.1) on days 14-20 after blood feeding infection, Anopheles mosquitoes were removed, sterilized with 75% ethanol 3 times for 5s each, and then flushed with insect physiological saline 3 times for 5s each. The tissue technician dissected the salivary glands and collected in an EP tube containing 200 μ L AB + human serum, which was always placed on ice; note that: if the inoculation is directly carried out, the dissected salivary gland is put into normal saline. Obtaining anopheles salivary gland, repeatedly blowing with 18G needle syringe for 30-40 times, crushing salivary gland and tissue, centrifuging at 4 deg.C and 1000rpm for 5min, and collecting supernatant containing sporozoite;

(4.2) notes: taking the complete salivary gland of anopheles moschata as much as possible during dissection, and avoiding collecting tissue fragments together, wherein the tissue fragments can influence the fragmentation of the salivary gland and the purification of sporozoites; sporozoite is obtained according to insect plants respectively, and marking is carried out, so that cross contamination among the insect plants in the sporozoite obtaining process is prevented; the process of obtaining the sporozoites is controlled within 1 hour;

(5) freezing storage of sporozoites

(5.1) taking a small amount of the supernatant containing the sporozoites, and counting the sporozoites by a blood counting chamber;

(5.2) centrifuging at 12000rpm and 4 deg.C for 10min, discarding supernatant, adding appropriate amount of AB type human serum containing 1% triantion, resuspending and precipitating, and adjusting sporozoite concentration to 2.5 × 10⁸/mL；

(5.3) subpackaging 200 mul/tube into a freezing tube, marking, quickly freezing in alcohol at-80 ℃, transferring to a liquid nitrogen tank after 3 h.

(6) Direct sporozoite inoculation

(6.1) preparing a 1mL syringe, 75% alcohol and a cotton swab;

(6.2) carrying out aseptic operation on the obtained sporozoite normal saline suspension, taking a small amount of sporozoites, counting the sporozoites by using a blood counting chamber, and adjusting the concentration of the sporozoites to the inoculation number;

(6.3) intravenous injection, inoculation, isolation and observation;

(6.4) reserving a small amount of sample before inoculation for carrying out a sterile test and making a record;

(6.5) 3 days after inoculation, blood smears were made daily for microscopic examination of Plasmodium.

(7) Inoculation after sporozoite resuscitation

(7.1) preparing a 1mL syringe, 75% alcohol and a cotton swab;

(7.2) starting ultraviolet disinfection for more than 30 min;

(7.3) taking out the preserved sporozoites from the liquid nitrogen tank, and unfreezing the sporozoites in a water bath kettle at the temperature of 37 ℃ for 1-2 min;

(7.4) centrifuging at 12000rpm at 4 ℃ for 10min, discarding the supernatant, and adding 10 times volume of physiological saline for resuspension;

(7.5) centrifuging at 12000rpm at 4 ℃ for 10min, discarding the supernatant, and adding 200 mu L of physiological saline for resuspension;

(7.6) intravenous injection, inoculation, isolation and observation;

(7.7) reserving a small amount of sample before inoculation for carrying out sterile test and making a record;

(7.8) 3 days after inoculation, blood smears were made daily for microscopic examination of Plasmodium.

The cryopreservation and resuscitation method provided by the invention is more beneficial to infection of plasmodium, improves the infection efficiency, and improves the effect of plasmodium in preventing and/or treating brain tumor.

Preferably, the dosage form of the medicament is any one pharmaceutically acceptable dosage form. Such as tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or dropping pills and the like.

Preferably, the medicament is in the form of injection.

Preferably, the medicament also comprises any one or the combination of at least two of pharmaceutically acceptable pharmaceutical excipients.

The medicine related to the invention can be independently administrated or can be matched with auxiliary materials to be prepared into proper dosage forms for administration, and the auxiliary materials comprise any one or the combination of at least two of diluent, thickening agent, excipient, flavoring agent, filler, adhesive, wetting agent, disintegrating agent, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, pH regulator, antioxidant, bacteriostatic agent or buffering agent. Combinations of the at least two such as diluents and excipients, emulsifiers and co-solvents, fillers and binders and wetting agents, and the like.

Preferably, the drug is a drug loaded on a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier comprises a liposome, micelle, dendrimer, microsphere or microcapsule.

In another aspect, the invention provides the use of a medicament as described above in the manufacture of a medicament for the prevention and/or treatment of brain tumours.

In a further aspect, the present invention provides a method of treating brain tumors using a medicament as described above, the method comprising the steps of: the drug as described above is administered to a patient with brain tumor by a method that only requires the drug to be infused into the patient with brain tumor to successfully infect the malaria parasite, and those skilled in the art can select a method known in the art according to the actual situation, preferably an injection method. The plasmodium infection route may be through the natural transmission medium female anopheles bites humans or through blood transfusion into blood with plasmodium or using syringes or the like containing blood of plasmodium.

In the present invention, the number of plasmodium that can infect a patient with a brain tumor with plasmodium is feasible. The number of plasmodium which can be infected is different according to individual differences of patients, and the number of plasmodium can be adjusted by a person skilled in the art according to actual conditions, wherein the inoculation amount of the plasmodium is not less than 100 red blood cells infected by active plasmodium or not less than 5 sporozoites of the active plasmodium.

In the present invention, the plasmodium infection is a long-range plasmodium infection, the longer the infection period, the more obvious the inhibition effect on the brain tumor, the long-range plasmodium infection is that the plasmodium lasts to the chronic stage of the plasmodium infection or at least to the subacute stage, and is maintained for a period of time, and then the infection is stopped by administering the antimalarial drug. After an acute and subacute phase of infection of about 6-8 weeks after infection with plasmodium, a chronic phase is reached, in which only a small amount of plasmodium is detected in the peripheral blood, but clinical symptoms of the acute phase do not appear.

Because the infection of the human plasmodium can be reinfected after being cured and can also be repeatedly infected by different plasmodium, the repeated plasmodium infection state can be formed, the infection of the plasmodium can inhibit the growth of brain tumor cells, prevent the diffusion and the transfer of cancer cells, prolong the service life of patients, win longer treatment opportunity and immune environment of organisms for the brain tumor patients, and is beneficial to the long-term survival and the rehabilitation of the brain tumor patients.

In the present invention, fever caused by infection with plasmodium may promote death of brain tumor cells. Plasmodium hemorrhages are necessary to effectively inhibit brain tumor growth, however in mice, plasmodium causes only short-term infection without fever, and repeated plasmodium infections are difficult to observe in murine models. In humans lacking effective anti-malarial therapy, plasmodium infection can cause long-term parasitemia, accompanied by acute phase hyperthermia, which syndrome can be repeated many times throughout life. Therefore, plasmodium obtained by biting anopheles mosquito naturally or injecting sporozoite artificially can generate liver stage and blood stage infection, continuously stimulate immune system to convert brain tumor into effective brain tumor vaccine, and play a multi-channel and multi-target therapeutic role on brain tumor together with acute stage fever and tumor angiogenesis inhibition effect thereof. In the medical literature, febrile infections are associated with spontaneous regression of tumors, malaria being the typical febrile infection.

Compared with the prior art, the invention has the following beneficial effects:

(1) the medicine has obvious effect of preventing and/or treating brain tumor, can inhibit the growth of the brain tumor and prolong the life of a patient;

(2) the medicine has limited toxic and side effects and high safety; the plasmodium immunotherapy mainly comprises intermittent cold and heat attacks, can use antimalarial drugs to control the infection rate, avoids severe cold and heat attacks, ensures safety, and does not harm the normal functions of the brain and the functions of other organ tissues;

(3) the medicine related by the invention enriches the treatment means of brain tumor, and due to the particularity and sensitivity of brain tissues, the chemotherapeutic medicine is not easy to pass through the blood brain barrier and can not exert the anti-tumor effect of the chemotherapeutic medicine; many targeted drugs cannot enter brain tumors to exert direct anti-tumor effects; radiotherapy requires radiation to the brain; the medicine of the invention inhibits the growth of brain tumor mainly by regulating the immunologic function of the organism and factors related to inflammation, thereby prolonging the life of patients;

(4) the long-range plasmodium infection has better treatment effect on the brain tumor, and the plasmodium immunotherapy does not take the fever time as the treatment course standard when treating the brain tumor, but prolongs the plasmodium infection time until the progress of the tumor can be controlled on the premise of protecting the organ function and life safety of a patient;

(5) the medicament and the plasmodium immunotherapy using the medicament are relatively economical, have relatively few side effects, only need simple symptomatic treatment and regular monitoring of blood routine and liver and kidney functions, do not need additional other expenses for patients, can perform treatment or terminate treatment at any time according to the change of the disease condition, and have manually controllable treatment course; not only can reduce the burden of patients, but also integrates the advantages of immunotherapy, fever therapy and anti-angiogenesis therapy.

Drawings

FIG. 1 is a graph showing the effect of Plasmodium infection on glioma growth in tumor-bearing mice in a subcutaneous inoculation model;

FIG. 2 is a graph showing the effect of Plasmodium infection on the survival time of tumor-bearing mice in a subcutaneous inoculation model;

FIG. 3 is a graph showing the results of the effect of Plasmodium infection on body weight of tumor-bearing mice in a subcutaneous inoculation model;

FIG. 4 is a graph showing the trend of the infection rate of Plasmodium in the treatment group of Plasmodium in the subcutaneous inoculation model;

FIG. 5 is a schematic representation of pWPXld-mcherry-F2A-Luciferase-Puro lentiviral vector;

FIG. 6 is a field of view for positive rate detection of a single clone of GL261.mcherry-Luc cell line;

FIG. 7 is a graph of the statistical results of Luciferase activity of the GL261.mcherry-Luc cell line;

FIG. 8 is a graph of in vivo imaging results of Plasmodium infection on glioma growth in tumor-bearing mice in an intracranial in situ vaccination model;

FIG. 9 is a graph showing the effect of Plasmodium infection on glioma growth in tumor-bearing mice in an intracranial in situ vaccination model;

FIG. 10 is a graph showing the effect of Plasmodium infection on the survival time of tumor-bearing mice in an intracranial in situ vaccination model;

FIG. 11 is a graph showing the effect of Plasmodium infection on body weight of tumor-bearing mice in an intracranial in situ vaccination model;

FIG. 12 is a graph showing the trend of the infection rate of Plasmodium in the treatment group of Plasmodium in the intracranial in situ inoculation model.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.