KR101689542B1 - Nanocomplex of Gefitinib and Erlotinib for Enhanced Lung Cancer Therapy - Google Patents

Abstract

The present invention relates to a zetotinib or erlotinib nanocomposite for improving lung cancer treatment, and more particularly to a zetotinib or erlotinib nanocomposite for use in the treatment of lung cancer, wherein the zetitinib or erlotinib is attached to a nanomaterial Lt; / RTI > nanoparticles for improving lung cancer therapy.

Description

≪ Desc / Clms Page number 1 > SUMMARY OF THE INVENTION Nanocomplex of Gefitinib and Erlotinib for Enhanced Lung Cancer Therapy for Improving Lung Cancer Therapy [

The present invention relates to a zetytipine or erlotinib nanocomposite for improving lung cancer treatment, and it relates to a method for improving the efficacy of an anticancer drug in the treatment of lung cancer by adding nanomaterials to tyrosine kinase inhibitor Gefitinib or erlotinib Lt; / RTI >

As cancer incidence and mortality continue to increase globally, the importance of developing effective anti-cancer drugs is also increasing. The cancer drug market is the third largest market in the world, with cardiovascular and neurological diseases. Globally, about 10 million people have been diagnosed with cancer in 2000 and estimated to reach about 15 million by 2020 . The market size is estimated to be 35 trillion won in 2003 and to reach 60 trillion won in 2010. These findings demonstrate that research for the development of cancer drugs is being actively carried out globally.

According to the National Statistical Office, lung cancer is the first cancer-causing disease in Korea and the rate of increase is the fastest in the last 10 years compared to other cancers, and lung cancer incidence and mortality continue to increase in the future. More than doubled. The treatment of lung cancer can be divided into surgery, radiation therapy, and chemotherapy. In most cases, however, chemotherapy or chemotherapy is more effective in the treatment of advanced lung cancer. to be. Over the past decade, the quality of life and the survival rate have been greatly increased by the combination of platinum compounds and new anticancer drugs such as vinorelbine, gemcitabine, and taxane. Nevertheless, the 1-year survival rate of advanced non-small cell lung cancer is only 35% and the 2-year survival rate is only 15-20%. Molecular targeted therapy, a new concept of therapy, has recently been developed that aims to inhibit certain biological mechanisms and thereby increase the therapeutic effect and reduce side effects. Many drugs are also in clinical trials for the treatment of lung cancer. Among them, gefitinib; Some drugs, such as the product name, Iressa, have been approved by the US Food and Drug Administration (FDA) for molecular targeting in non-small cell lung cancer and have already been clinically applied to patients. However, since cancer develops into a very complicated pathway, such a molecular target therapeutic agent that selectively suppresses only a specific target factor has a problem in that it is resistant to long-term administration and has a disadvantage that it has a selective therapeutic effect depending on a mutation state of a specific gene Development of anticancer agents capable of overcoming these problems is urgently required.

On the other hand, epidermal growth factor receptor (EGFR) is EGFR (ErbB1), HER2 / neu (ErbB2). Is a member of closely related growth factor receptor tyrosine kinases including HER3 (ErbB3) and HER4 (ErbB4). Upon ligand binding, these receptors either homodimerize or heterodimerize to cause autophosphorylation and are then used to induce intracellular signaling cascades such as phosphoinositide 3-kinase (PI3K) / Akt, MAPK / Erk and Jak / Activation of the signaling pathway (which plays a major role in cell proliferation, survival and transformation and therapeutic resistance).

The downstream PI3K pathway of EGFR plays an important role in regulating cell viability and proliferation. The ErbB3 receptor plays a unique role in activating the PI3K pathway. ErbB3, with or without weak tyrosine kinase activity, is phosphorylated at the tyrosine residue upon heterodimerization with EGFR. Tyrosine-phosphorylated ErbB3 binds directly to PI3K and activates PI3K. Studies have shown that PI3K / Akt signaling is closely regulated by EGFR in TKI-sensitive NSCLC and that EGFR TKI downregulates (and also inhibits) growth of the PI3K / Akt pathway exclusively in its NSCLC cell line [ Engelman, JA et al., Proc Natl Acad Sci USA102, 3788-3793 (2005)).

Since EGFR is expressed in the majority of non-small cell lung carcinoma (NSCLC), it has become an attractive target for the development of therapeutic agents. Small molecule EGFR tyrosine kinase inhibitors (TKI), including zetitib and erlotinib, have been evaluated in clinical trials for patients with NSCLC. Both agents cause a partial response of 10% to 20% in all NSCLC patients.

Lung cancer with EGFR mutation and / or amplification appears to best diminish the response to EGFR inhibitors. Activating somatic mutations in the EGFR gene has been identified in the NSCLC patent. These "gain-of-function" mutations are substitutions, short in-frame deletions, or dense insertions around the region encoding the ATP-binding pocket of the tyrosine kinase domain of the receptor. In these cancers, EGFR is the major activator of important growth and survival signaling pathways, so these cancers are addicted to EGFR activity. When exposed to EGFR inhibitors, these important growth and survival signaling pathways are blocked, resulting in apoptosis and / or cell cycle arrest.

Recent data suggest that a novel therapeutic approach targeting signaling pathways involved in cell proliferation, apoptosis, angiogenesis and metastasis is being investigated. Among the many potential target pathways, Epidermal Growth Factor (EGF) receptor signaling pathway (EGFR) signaling pathway has been extensively studied, including EGFR overexpression in many solid tumors including 40% to 80% of non-small cell lung cancer (NSCLC) Because it was observed. As described above, the investigators tested agents that interfere with the epidermal growth factor receptor (EGFR). EGFR is partly expressed at abnormally high levels on the surface of many types of cancer cells, including non-small cell lung cancer. Examples of these experimental EGFR inhibitors include zetimidine (Iressa), and erlotinib (Tarceva).

Resistance to erlotinib or EGFR inhibition therapy has been clinically observed by activating mutations in the KRAS gene, an important downstream signaling component in the MAPK signaling pathway (Pao, W. et al., PLoS Med. 2, e73 (2005)]). Tolerance acquired for erlotinib therapy was associated with clinical use with secondary mutations in EGFR exon 20 (T790M).

In treating lung cancer with zetifinib or erlotinib, the ineffective increase in resistance was markedly increased. Accordingly, the development of a substance capable of improving lung cancer treatment in the treatment of lung cancer has been required.

Korea Patent Publication No. 2013-0057744 WO < RTI ID = 0.0 >

In order to solve the above problems, it is an object of the present invention to provide a substance that can further improve lung cancer therapeutic effect.

It is another object of the present invention to provide a substance that can be used as an anticancer agent by enhancing the ability to inhibit the activity of lung cancer cells.

In order to achieve the above object, the present invention provides a method for treating lung cancer, which comprises administering to a nano-substance a gefitinib or erlotinib used for the treatment of lung cancer, Nanocomposite.

Also, the present invention provides a pharmaceutical composition for treating lung cancer, which comprises the nanomaterial of the present invention is gold nanoparticles or diamond nanoparticles.

Also, the present invention provides a zetotinib or erlotinib nanocomposite for improvement of lung cancer treatment characterized in that Gefitinib or Erlotinib is adsorbed to gold nanoparticles or diamond nanoparticles .

The present invention also provides a zetotinib or erlotinib nanocomposite characterized in that the concentration of the zetotinib or erlotinib nanocomposite is 0.05 μM or more.

The present invention also provides a zetotinib or erlotinib nanocomposite characterized in that the inhalation of the zetotinib or erlotinib nanocomposite of the present invention into cells is inhaled by endocytosis.

Also, the present invention provides a zetotinib or erlotinib nanocomposite characterized in that the nanocomposite is subjected to pegylation capable of targeting cancer cells.

In addition, the present invention provides a zetotinib or erlotinib nanocomposite characterized by mixing DSPE-PEG when pegylation capable of targeting cancer cells to the nanocomposite is performed.

Also, the present invention provides a zetotinib or erlotinib nanocomposite characterized in that the concentration of the nanocomposite subjected to pegylation is 0.05 μM or more.

The zetotinib or erlotinib nanocomposite according to the present invention can be applied to drug-resistant lung cancer cells to improve lung cancer treatment.

In addition, the cell viability test was conducted by inhalation of the zetitip or erlotinib nanocomposite according to the present invention into lung cancer cells, and it was confirmed that the intracellular activity was lowered.

Figure 1 shows the Raman spectrum before and after the attachment of the gold nanomaterial to the zetotifen.
Figure 2 shows the Raman spectrum of erlotinib before and after gold nanomaterial attachment.
FIG. 3 is a view showing a state in which a zeta-nip nanocomposite according to an embodiment of the present invention is put into lung cancer cells.
FIG. 4 is a view showing an erlotinib nanocomposite according to an embodiment of the present invention when put into lung cancer cells.
FIG. 5 schematically shows a process for producing an erlotinib or a zetotin nanocomposite using a nano-diamond.
FIG. 6 is a graph showing the relationship between the pore diameter of the nano diamond (ND) (a), the pegylated nanodiamond (ND-PEG) (c), the pegylation using the erlotinib or zetti nip nanocomposite ND-PEG-GF (d)).
7 shows the activity of lung cancer cells in Examples 1 to 3 and Comparative Examples 1 to 3.
FIG. 8 shows the activity of lung cancer cells in Examples 4 to 6 and Comparative Examples 4 to 6. FIG.

Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings.

First, in the drawings, it is noted that the same components or parts are denoted by the same reference numerals as possible. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted so as to avoid obscuring the subject matter of the present invention.

The terms "about "," substantially ", etc. used to the extent that they are used herein are intended to be taken to mean an approximation of, or approximation to, the numerical values of manufacturing and material tolerances inherent in the meanings mentioned, It is used to prevent unauthorized exploitation by an unscrupulous infringer from disclosing the exact or absolute numerical value to help.

The present invention is characterized in that it is a nanocomposite having nanomaterial attached to Gefitinib or Erlotinib used for the treatment of lung cancer.

Figure 1 shows the Raman spectrum before and after the attachment of gold nanomaterials of the zetotifen.

The molecular structure of zetti nip is shown in the following chemical formula 1.

The molecular structure of the zetti nip is mainly composed of an aromatic quinazoline and a phenyl group, and it can be attached to the gold nanomaterial, which can be expected to adhere to the gold nanomaterial by adsorption have.

Referring to Chemical Formula (1), there is a nitrogen (N) atom of quinazoline in the zetotin, which is adsorbed to the gold nanomaterial by the nitrogen atom.

Six nitrogen atoms are bound to the nitrogen N1 and N3 of zetitibium, and sixteen gold atoms can be bonded in parallel with the zetotinib.

The bond energies and bond lengths of zetitibite and gold atoms are shown in Table 1 below.

The combined energy, bond length and number of gold atoms combined can be calculated using a polarized continuum model (PCM) model.

Referring to FIG. 1, it can be seen that there is a difference between before the attachment of the gold nanomaterial (FIG. 1 (a)) and after the gold nanomaterial (FIG. 1 (b)). There is a change in the spectrum after the gold nanomaterial is attached, and this change occurs at 1300 and 1425 cm ^-1 , which is due to the quinazoline group. In addition, the strongest band was formed at 1337 cm ^-1 before attachment of the gold nanomaterial, and the strongest band was formed at 1301 cm ^-1 after attachment of the gold nanomaterial.

Figure 2 shows the Raman spectrum of erlotinib before and after gold nanomaterial attachment.

The molecular structure of erlotinib is shown in the following formula (2).

The molecular structure of erythinib is composed mainly of two aromatic groups, quinazoline and phenylacetylene, and erlotinib can be attached to gold nanomaterials by adsorption of erlotinib to the gold nanomaterial Can be expected.

Referring to Formula 2, it can be seen that the adsorbed on gold nanomaterials by nitrogen (N) and acetylene groups in erlotinib.

Six gold atoms are bonded to the nitrogen N1 and N3 of the quinazoline of erlotinib, and six gold atoms can be bonded to the triple bonded carbon (C? C).

The bond energies and bond lengths of erlotinib and gold atoms are shown in Table 2 below.

The combined energy, bond length and number of gold atoms combined can be calculated using a polarized continuum model (PCM) model.

Referring to FIG. 2, it can be seen that there is a difference between before the attachment of the gold nanomaterial (FIG. 2 (a)) and after the attachment of the gold nanomaterial (FIG. 2 (b)). Gold for which a change in the spectrum after the nano-material is attached, such changes in the carbon bond 3 2111㎝ ^-1 (C≡C) band is seen that the 2008㎝ to go to ^-1 after the gold nano-material attached that forms . It can also be seen that the oscillation band of the phenyl group was shifted from 1617 cm ^-1 to 1599 cm ^-1 .

On the other hand, the nanocomposite of the present invention may be inactivated by lung cancer cells, thereby decreasing its activity.

FIG. 3 is a view showing a state in which a zeta-nip nanocomposite according to an embodiment of the present invention is put into lung cancer cells. FIG. 4 is a view showing an erlotinib nanocomposite according to an embodiment of the present invention when put into lung cancer cells.

The zetifinib nanocomposite with gold nanomaterials can be inhaled into the cells. The result of inhalation into the lung cancer cell A549 is shown by dark-field microscopy (DFM) and transmission electron microscope (TEM) in FIG.

Inhalation of the zetitib or erlotinib nanocomposite into A549 cells can be inhaled by endocytosis.

The zetotinib or erlotinib nanocomposite is in an aqueous solution state and sucked into lung cancer cells to lower the degree of activation. The appropriate concentration is preferably 0.05 μM or more, more preferably 0.05 μM to 0.5 μM.

Although the present invention has been described with respect to nanocomposites attached to gold nanomaterials, it is applicable to diamond nanomaterials as well as gold nanomaterials.

FIG. 5 schematically shows a process for preparing an erlotinib or a zetotin nanocomposite using a nano-diamond. FIG. 6 shows a process for producing a nanodiamond (ND) (a), a pegylated nanodiamond (ND- (c) ND-PEG-EL (c) ND-PEG-GF (d) material using erlotinib or zetti nip nanocomposite.

It is also possible to prepare a zetotinib or erlotinib nanocomposite using nanodiamonds, as shown in FIG. 5, after attaching zetidipine or erlotinib to the nanodiamonds, followed by PEGylation with PEG May be inhaled into lung cancer cells.

The process of attaching zetotinib or erlotinib to nanodiamonds, that is, preparation of nanocomposites, is as follows.

A nanodiamond solution (about 1000 ppm) was prepared and mixed with 10 μL of zetytipine (or erlotinib) per 1 mL and mixed through a centrifuge to adsorb zetifinib (or erlotinib) on the nanodiamonds. In this case, the nanocomposite may be prepared by self-assembly or self-assembly, and the mixture may be prepared by mixing the mixture at 10000 rpm for about 5 minutes through a centrifuge.

Next, pegylation can be performed on the nanocomposite in which the zetotinib or erlotinib is attached to the nanodiamond, and the process is as follows.

Glycero-3-phosphoethanolamine-N-methoxy (polyethyleneglycol) -2000 was added to an aqueous solution of a mixture of nano-diamonds with zetytipib or erlotinib-attached nanocomposite (ND-GF or ND- ] (DSPE-PEG) are mixed and maintained at ambient temperature overnight. Mixing ratio is 10 μL of DSPE-PEG per mL of ND-GF or ND-EL. Whereby pegylation (ND-PEG-EL or ND-PEG-GF) material using an erlotinib or zetti nip nanocomposite can be formed.

6 (c) and 6 (d) show ND-EL and ND-GF pegylated materials (ND-PEG-EL c), and ND-PEG-GF (d).

6 (a) shows a nanodiamond (ND), which is slightly aggregated during the drying process, and DSPE-PEG is more useful for maintaining the size of the nanodiamond (ND) . 6 (a) and 6 (b), DSPE-PEG was used in the pegylation process, and it was confirmed that aggregation formation in the nanodiamonds after pegylation was further reduced. It can be seen that DSPE-PEG keeps it in a size suitable for living body.

Hereinafter, embodiments of the present invention will be described in detail.

Example 1

Manufacture of nanocomposites

A gold nanomaterial solution having a concentration of about 9 x 10 < ^-9 > M was prepared, and 10 l of erlotinib was mixed per 1 ml of the gold nanomaterial solution and mixed through a centrifuge to adsorb erlotinib to the gold nanomaterial. In this case, they were manufactured by self-assembly or self-assembly, and the mixture was mixed through a centrifuge at 10000 rpm for about 5 minutes.

In preparing nanocomposites, they can be prepared by self-assembly or self-assembly.

Nanocomposite inhalation in lung cancer cells

The nanocomposite prepared above can be sucked into cells. The method is as follows.

Inhalation of nanocomplexes into cells is inhaled by endocytosis. At this time, the concentration of the nanocomposite was varied, and the concentration of the zetotin nanocomposite was 0.005, 0.01, 0.05, 0.1 μM.

A549 cells were used for lung cancer cells, and cell activity was observed after 48 hours.

Comparative Example 1

Lung cancer cells were inoculated with pure zephitinib in A549 cells to observe cell activity. The concentrations of zetytipine in the cells were 0.005, 0.01, 0.05 and 0.1 μM, respectively.

Examples 2 and 3

The same procedure as in Example 1 was carried out except that lung cancer cells were treated with NCl-H460 (Example 2) and NCl-H1975 (Example 2).

Comparative Example 2 and Comparative Example 3

And lung cancer cells were treated with NCl-H460 (Comparative Example 2) and NCI-H1975 (Comparative Example 2).

7 shows the activity of lung cancer cells in Examples 1 to 3 and Comparative Examples 1 to 3.

Example 1 and Comparative Example 1 are shown in FIG. 7 (a), Example 2 and Comparative Example 2 are shown in FIG. 7 (b), and Example 3 and Comparative Example 3 are shown in FIG. 7 (c).

Referring to FIG. 7, it can be seen that the activity of lung cancer cells is significantly lowered when the concentration of the zetotifen nanocomposite is 0.05 μM or more.

Example 4

Manufacture of nanocomposites (diamond nanocomposite production)

The process of attaching zetotinib or erlotinib to nanodiamonds, that is, preparation of nanocomposites, is as follows.

A nanodiamond solution (about 1000 ppm) was prepared and mixed with 10 μL of zetytipine (or erlotinib) per 1 mL and mixed through a centrifuge to adsorb zetifinib (or erlotinib) on the nanodiamonds. In this case, the nanocomposite was prepared by self-assembly or self-assembly, and the mixture was mixed through a centrifuge at 10000 rpm for about 5 minutes to prepare a nanocomposite.

Pegylation

Glycero-3-phosphoethanolamine-N-methoxy (polyethyleneglycol) -2000 was added to a mixed aqueous solution of a nanocomposite (ND-GF or ND-EL) ] (DSPE-PEG) are mixed and maintained at ambient temperature overnight. Mixing ratio is 10 μL of DSPE-PEG per mL of ND-GF or ND-EL. Thereby forming a pegylation (ND-PEG-EL or ND-PEG-GF) material using the erlotinib or zetti nip nanocomposite.

Nanocomposite inhalation in lung cancer cells

The diamond nanocomposite prepared above can be sucked into cells. The method is as follows.

Inhalation of nanocomplexes into cells is inhaled by endocytosis. At this time, the concentration of the nanocomposite was inhaled into the cell, and the concentration of the nanocomposite was 0.005, 0.01, 0.05, and 0.1 μM.

A549 cells were used for lung cancer cells, and cell activity was observed after 48 hours.

Comparative Example 4

Pure zetytibe, pure erlotinib and nano-diamond pegylated material (ND-PEG) are prepared.

Lung cancer cells were inhaled into A549 cells and their cell activity was observed. Pure zetifinib, pure erlotinib, and ND-PEG concentrations were 0.005, 0.01, 0.05 and 0.1 μM, respectively, when inhaled into the cells.

Examples 5 and 6

The same procedure as in Example 4 was carried out except that lung cancer cells were treated with NCl-H460 (Example 5) and NCl-H1975 (Example 6).

Comparative Example 5 and Comparative Example 6

And lung cancer cells were treated with NCl-H460 (Comparative Example 5) and NCl-H1975 (Comparative Example 6).

FIG. 8 shows the activity of lung cancer cells in Examples 4 to 6 and Comparative Examples 4 to 6. FIG.

Example 4 and Comparative Example 4 are shown in FIG. 8 (a), Example 5 and Comparative Example 5 are shown in FIG. 8 (b), and Example 6 and Comparative Example 6 are shown in FIG. 8 (c).

8, when the concentrations of the respective substances were 0.005, 0.01, 0.05, and 0.1 μM, the activity of lung cancer cells was examined in lung cancer cells in which ND-PEG-EL and ND-PEG- The activity is remarkably reduced.

Thus, it can be seen that the use of zetotinib or erlotinib nanocomposite, which is not only using zetotinib or erlotinib, as an anticancer agent, significantly improves cell deterrence.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. It will be clear to those who have knowledge of.

Claims (8)

Gefitinib or erlotinib, used for the treatment of lung cancer, is mixed with a nanomaterial solution to produce an aqueous nanocomposite solution,
A pegylation process is performed in which 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethyleneglycol) -2000] (DSPE-PEG) is mixed with an aqueous solution of the nanocomposite,
The nanomaterial is a diamond nanoparticle,
Wherein the zetotinib or erythritip is adsorbed on the diamond nanoparticles during the production of the nanocomposite aqueous solution,
Wherein the concentration of the nanocomposite aqueous solution is 0.05 μM or more and 0.5 μM or less.
delete delete delete The method according to claim 1,
Wherein the inhalation of the zetifinib or erlotinib nanocomposite into the cells is inhaled by endocytosis. ≪ Desc / Clms Page number 15 >
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