KR101719790B1 - Compostion comprising Guibi-tang extract for treatment of lung cancer - Google Patents

Abstract

The present invention relates to a composition for treating lung cancer comprising an anticancer drug and a guibi-tang (mixed natural herbs) extract. According to the present invention, the composition significantly increases a treatment effect of the anticancer drug by administering the anticancer drug to an individual and administering the guibi-tang extract into the individual, and prevents and decreases side effects caused by various anticancer therapies.

Description

[0001] The present invention relates to a composition for treating lung cancer,

The present invention provides a composition for treating lung cancer comprising an anticancer agent and a natural water extract.

Lung cancer generally refers to primary lung cancer, and primary lung cancer refers to malignant tumors originating from the lungs. Cancer that has metastasized to the lungs is classified as metastatic cancer of the lungs, and it is reasonable to classify it as metastatic cancer of organs originating from lung cancer.

Lung cancer is divided into small cell lung cancer and non-small cell lung cancer according to the tissue type. The reason for this distinction is that small cell lung cancer is distinctive from other types of lung cancer in terms of treatment and prognosis. Therefore, lung cancer is very important for the histological diagnosis, ie, the result of biopsy, to determine the treatment policy.

Currently, gefitinib is mainly used for the treatment of lung cancer, especially non-small cell lung cancer. It has been reported that gefitinib is an oral anticancer agent for epidermal growth factor receptor (EGFR) inhibitor, which is frequently used for the treatment of various malignancies including breast cancer and lung cancer. However, its use is limited due to its toxicity and toxicity (Inoue et al., 2003; van Zandwijk, 2003; Rossi, 2004; Sipples, 2006).

Gui-ti is a combination of drugs (traditional medicine) and traditional medicine (补血) and anti-sinic medicine (晋神 行 气) in Oriental medicine. It is a representative prescription that is applied to the symptom of heart and spleen, heart failure, and non-infarction (splenic obstruction), which is a combined prescription consisting of 12 kinds of natural products.

The JiwiTiTang is made of Polygalae Radix, Zizyphi Fructus, Angelicae Gigantis Radix, Aucklandiae Radix, Longan Arillus, Glycyrrhizae Radix et Rhizoma, Zingiberis Rhizoma, Zizyphi Semen, Ginseng Radix Alba, Atractylodis Rhizoma Alba, Astragali Radix, and Hoelen.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a composition for treating lung cancer using the extract of Guizhoubang.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a composition for treating lung cancer comprising an anticancer agent and a natural vitamin extract.

In one embodiment of the present invention, the anticancer agent is gefitinib.

In another embodiment of the present invention, the Angelica gigantis Radix, Aucklandiae Radix, Longan Arillus, Glycyrrhizae Radix et Rhizoma, Polygalae Radix, Zizyphi Fructus, , Zingiberis Rhizoma, Zizyphi Semen, Ginseng Radix Alba, Atractylodis Rhizoma Alba, Astragali Radix, and Hoelen.

In still another embodiment of the present invention, the anticancer agent and the natural sweet pot extract may be pre-mixed and formulated or separately formulated.

In still another embodiment of the present invention, the anticancer agent and the natural water extract are parenterally, orally, locoregionally, or percutaneously administered.

In another embodiment of the present invention, the administration of the Guizhou tincture extract is started within 1 minute to 4 hours after administration of the anticancer agent.

The composition for treating lung cancer, which contains the extract of Guizhoubang, provided by the present invention as an active ingredient, is administered in combination with an anticancer agent, thereby enhancing the efficiency of lung cancer treatment and alleviating adverse side effects of the anticancer agent alone.

In particular, the therapeutic effect of gefitinib, which is used for the treatment of non-small cell lung cancer, and the effect of reducing side effects are excellent.

1 to 17 are views for the first embodiment.
1 is a schematic diagram showing an experimental method and an experiment group design of the first embodiment.
FIG. 2 is a graph showing the results of NCI-H520 cell survival rate according to Guizui administration.
FIG. 3 is a graph showing the results of the NCI-H520 cell survival rate change by administration of gefitinib. FIG.
FIG. 4 is a graph showing the results of observation of changes in body weight and weight gain according to each group.
FIG. 5 is a photograph showing a tumor volume observed according to each group. FIG.
FIG. 6 is a graph showing the results of observing changes in tumor volume according to each group. FIG.
FIG. 7 is a graph showing the results of observing changes in blood IL-6 and IFN-y contents according to each group.
8 is a graph showing the results of observing changes in NK cell activity according to each group.
FIG. 9 is a graph showing a decrease in tumor cell volume and a numerical increase in apoptotic cells according to each group (A = Tumor-bearing control, B = Gefitinib 120 mg / kg single mice in FIGS. = GBT 400 mg / kg single-treated mice, D = Gefitinib 120 mg / kg and GBT 400 mg / kg, co-administered mice within 5 min, E = Gefitinib 120 mg / kg and GBT 200 mg / within 5 min, F = Gefitinib 120 mg / kg and GBT 100 mg / kg, co-administered mice within 5 min).
FIG. 10 is a graph showing the degree of increase in the number of caspase-3 immunoreactive cells according to each group.
11 is a graph showing the degree of increase in the number of PARP-immunoreactive cells according to each group.
FIG. 12 is a graph showing the degree of numerical increase of intracellular COX-2 immunoreactive cells according to each group. FIG.
FIG. 13 is a graph showing the degree of increase in the number of iNOS-immunoreactive cells in the mass according to each group.
FIG. 14 is a graph showing the degree of increase in number of TNF-a immunoreactive cells in a mass according to each group.
FIG. 15 is a graph showing histopathological changes of spleen according to each group (A = Intact control, B = Tumor-bearing control, C = Gefitinib 120 mg / mice were treated with D = GBT 400 mg / kg single-treated mice, E = Gefitinib 120 mg / kg and GBT 400 mg / kg, co-administered mice within 5 min, F = Gefitinib 120 mg / kg and GBT 200 mg / -Administered mice within 5 min, G = Gefitinib 120 mg / kg and GBT 100 mg / kg, co-administered mice within 5 min).
16 is a diagram showing the results of observing histopathological changes of submandibular glands according to each group.
FIG. 17 shows histopathological changes of the ovarian fat in each group. FIG.
Figs. 18 to 26 are views for the second embodiment. Fig.
18 is a schematic diagram showing an experimental method and experimental group design of the second embodiment.
FIG. 19 is a graph showing the results of observing changes in body weight and weight gain according to each group. FIG.
FIG. 20 shows histopathological findings in liver according to each group.
FIG. 21 shows the histopathological findings of submandibular lymph nodes according to each group.
FIG. 22 shows the histopathological findings in the spleen red water quality according to each group.
FIG. 23 shows the histopathological findings of the lungs according to each group.
24 shows the histopathological findings on the body of each group.

The present inventors paid attention to herbal medicines in order to develop a composition capable of further increasing the anticancer effect while alleviating various side effects of administration of anticancer drugs for the treatment of lung cancer, It was confirmed that there was an effect and a side effect reduction effect, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a composition for treating lung cancer, which comprises an anti-cancer agent and a natural vitamin extract.

As used herein, the term " Guizui Extract " means an extract derived from 12 medicines. The twelve medicinal materials are Polygalae Radix, Zizyphi Fructus, Angelicae Gigantis Radix, Aucklandiae Radix, Longan Arillus, Glycyrrhizae Radix et Rhizoma, Zingiberis Rhizoma, It includes 12 medicines of Zizyphi Semen, Ginseng Radix Alba, Atractylodis Rhizoma Alba, Astragali Radix, and Hoelen.

In one embodiment of the present invention, the anticancer agent and the water extract of Guizhou can be formulated beforehand or may be separately formulated.

The period of administration of the water extract can be performed within 1 minute to 4 hours after administration of the anticancer drug, preferably within 3 minutes to 240 minutes, and most preferably within 5 minutes to 120 minutes , But may be administered within 5 minutes after the administration of the anticancer drug as in the examples of the present invention, but the present invention is not limited thereto.

The anticancer agent used in the present invention is gefitinib, but it is not limited thereto.

The anticancer agent and the water extract can be administered parenterally, orally, locoregionally, or percutaneously. The extract of Guizhoubang is preferably orally administered, but may be suitably selected by those skilled in the art depending on the condition and the weight of the patient, the degree of disease, and the period of time.

In the present invention, an 'individual' refers to a subject in need of treatment for diseases, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, And the like.

In addition, the present invention can provide a composition for treating lung cancer, which comprises a Guizhou tincture extract.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include, but is not limited to, physiological saline, polyethylene glycol, ethanol, vegetable oil, and isopropyl myristate.

In one embodiment of the present invention, the preferred dosage of the pharmaceutical composition varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. However, it is preferably administered at a daily dose of 0.001 to 300 mg / kg body weight, more preferably 0.01 to 200 mg / kg body weight.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. The method of administration is not limited and can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intra-cerebroventricular injection.

The composition for treatment of lung cancer including the Guizhou tincture extract of the present invention can increase the therapeutic effect of lung cancer and alleviate various side effects that have been caused when the anticancer agent is administered alone.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

In the examples, the absorption and excretion of gefitinib following the concomitant use of VIP water was confirmed, that is, the effect on the pharmacokinetics, and the co-administration method which did not affect the pharmacokinetics was selected, Evaluate synergistic effects on drug efficacy.

In order to evaluate the effect of Guipitinib on the pharmacokinetics of Guipitin in combination with Guizoti, it was confirmed that the Guizotin was administered in combination with oral gavage for 35 days or less within 5 minutes after the administration of gefitinib (Example 1) (Example 2). The effect of combination therapy on the anticancer drug efficacy was confirmed using NCI-H520, a human squamous cell lung carcinoma (NSCLC) non-small cell lung squamous cell carcinoma cell line.

The anticancer agent used in this example was selected from Suzhou Huihe Pharm Co., Ltd., Suzhou, China, and Guevitang (hereinafter referred to as GBT) was purchased from HanJing Pharm. 1.

Medicinal Scientific name Amount (g) Origin (Polygalae Radix) Polygala tenuifolia Willd . 0.67 Jujube (Zizyphi Fructus) Zizyphus jujuba there is . inermis (Bunge) Rehder 0.67 Angelicae Gigantis Radix Angelica gigas N. 0.67 Aucklandiae Radix Aucklandia lappa Decne. 0.33 Longan Arillus Dimocarpus longan Lour One Licorice (Glycyrrhizae Radix et Rhizoma) Glycyrrhiza uralensis Fisch 0.33 Health (Zingiberis Rhizoma) Zingiber officinale Roscoe 0.5 Zizyphi Semen Zizyphus jujuba Miller One Ginseng (Ginseng Radix Alba) Panax ginseng CAMeyer. One Atractylodis Rhizoma Alba Atractylodes ovate (Thunb.) DC. One Astragali Radix Astragalus membranaceus Bunge One Hoelen Poria cocos Wolf One Total 12 types 9.17

Example  One. Of Gefitinib  Lung Cancer Therapy VIP  Impact Assessment: VIP  Gefitinib Within 5 min 35 days Repeated Combined Oral Administration

In this Example 1, the effect of the guitinib on the anticancer effect of gefitinib as a part of the integrated medical research of gefitinib and guinea pig for lung cancer patients was compared with that of representative human squamous cell lung cancer cell line (human NSCLC, non-small cell lung squamous cell carcinoma cell line NCI-H520. In Example 1, the cytotoxicity of Navi-H520 cell line was evaluated by the general MTT method, and after 14 days of NCI-H520 lung cancer cell transplantation, 40, 200 and 100 mg kg body weight, tumor weight, immune organs (thymus and submandibular gland) weights, and interferon (serum) levels were measured by sacrificing all experimental animals after oral administration of gefitinib at a concentration of 120 mg / (TNF) -α, interleukin (IL) -1β, and IL-10 levels were significantly correlated with histopathologic changes in tumor and lymphoid organs And observed anti-cancer and immunological activity, respectively. In addition, to observe the effects on tumor cachexia, changes in the ovarian fat mass and blood IL-6 levels were observed. The changes in ovarian fat thickness and adipocyte diameter were examined histopathologically (COX-2), an inflammatory and angiogenic factor, and an inducible cytokine, which is a cytokine related to immune activity, in the formed mass, and the apoptotic markers caspase-3 and cleaved poly (ADP-ribose) Immunoreactivity of nitric oxide synthases (iNOS) and tumor necrosis factor (TNF) -a were observed by immunohistochemistry. The experimental results in this Example 1 were compared with that of the gefitinib 120 mg / kg alone group, and all the test materials were dissolved in sterilized distilled water, and the dose of 10 ml / kg, (Table 2, Fig. 1).

group Xenotransplantation Dose (mg / kg / day) Animal No. CIMI-14-02-05-02 G + GBT PD: Effect on NCI-H520 cell transplantation nude mice The control (control) Saline Vehicle 10 ml / kg M01 ~ M08 Control group NCI-H520 cells Vehicle 10 ml / kg M09 ~ M16 Reference NCI-H520 cells Gefitinib single formula (20 mg / kg) M17 to M24 Reference group NCI-H520 cells GBT single (400 mg / kg) M25 to M32 Experimental group (Active) NCI-H520 cells Gefitinib and GBT (20 and 400 mg / kg) M33 ~ M40 Experimental group NCI-H520 cells Gefitinib and GBT (20 and 200 mg / kg) M41 to M48 Experimental group NCI-H520 cells Gefitinib and GBT (20 and 100 mg / kg) M49 to M56

1.1. Preparation of experimental animals

In this example, a total of 100 SPF / VAF Hsd: Athymic Nude-Foxn1nu mice (6-week old female, Harlan Lab., Udine Italy) were obtained in the present example. Animals whose weights were constant after 13 days of purification were selected, . After transplantation, NCI-H520 cells were transplanted and transplanted mice with a tumor volume of 396.56-113.29 mm ³ (248.02 ~ 719.96 mm ³ ) were selected again, and 8 mice per group were used for this experiment. A total of 8 normal controls were also prepared based on body weight (body weight: normal group = 24.58 ± 1.36 g, 23.50 ~ 27.70 g; tumor graft group = 23.50 ± 2.24 g, 19.80 ~ 27.40 g).

(GIBCO, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) supplemented with NCI-H520 (American Type Culture Collection The cell suspension was maintained at 1.0 × 10 ⁸ cells / ml in a 5% CO ₂ incubator and 0.2 mL of NCI-H520 tumor cell suspension (2 × 10 ⁷ cells / ml) was injected subcutaneously into the dorsal hind paw of the mouse cell / mouse) were transplanted to form a solid mass.

Group separation (total 7 groups; 8 per group)

(1) Intact control group: Normal medium control group

(2) TB control: NCI-H520 tumor cell transplantation, sterile distilled water administration group

(3) G120: 120 mg / kg of gefitinib alone after tumor cell transplantation administration

(4) GBT400: VIP after injection of tumor cells 400 mg / kg single composition administration group

(5) G + GBT400: 120 mg / kg of gefitinib and 400 mg / kg of VIP water after tumor cell transplantation

(6) G + GBT200: 120 mg / kg of gefitinib and 200 mg / kg of adenovirus after tumor cell transplantation

(7) G + GBT100: 120 mg / kg of gefitinib and 100 mg / kg of VIP water after tumor cell transplantation

Observations

IC ₅₀ (cytotoxicity), which is a concentration that suppresses the survival rate of NCI-H520 cells by 50%, was evaluated by the general MTT method, and NCI-H520 lung cancer cell-transplanted mice were treated with anti-cancer and immunosuppressive activity and tumor- (Tables 2 and 3, Fig. 1).

The antiserum or screening kit (Antisera or detection) used was used according to Table 3 below.

Antisera or detection kits Code Source Dilution Primary antisera * Anti-cleaved caspase-3 (Asp175) polyclonal antibody 9661 Cell Signaling Technology Inc., Beverly, MA, USA 1: 400 Anti-cleaved PARP (Asp214) rat specific antibody 9545 Cell Signaling Technology Inc., Beverly, MA, USA 1: 100 Anti-tumor necrosis factor- (4E1) antibody sc-130349 Santa Cruz Biotechnology, Santa Cruz, CA, USA 1: 200 Anti-cyclooxygenase (murine) polyclonal antibody 160126 Cayman Chemical., Ann Arbor, MI, USA 1: 200 Anti-nitric oxide synthase 2 (N-20) polyclonal antibody sc-651 Santa Cruz Biotechnology, Santa Cruz, CA, USA 1: 100 Detection kits Vectastain Elite ABC Kit PK-6200 Vector Lab. Inc., Burlingame, CA, USA 1:50 Peroxidae substrate kit SK-4100 Vector Lab. Inc., Burlingame, CA, USA 1:50

* All serum is cultured in rabbits.

PARP = Cleaved poly (ADP-ribose) polymerase

ABC = Avidin-biotin-peroxidase

(1) Antitumor effect: Changes in tumor cell volume and apoptotic cell percentages in tumor volume, tumor weight, forming mass, changes in caspase-3, PARP, COX-2, iNOS and TNF-α immunoreactivity in mass

(2) Immunological activity: Changes in immunoglobulin (thymic and submandibular) weights, serum IFN-γ content, NK cell activity, splenic TNF-α, IL-1β and IL-10 content, , Changes in TNF-α immunoreactivity in the masses and subcutaneous lymph nodes,

(3) Tumor-related cachexia-inhibiting effects: Changes in body weight, ovarian fat mass, blood IL-6 content,

1.2. Cytotoxicity check

(1) Influence on the survival rate of NCI-H520 cells

A decrease in NCI-H520 cell survival rate (p < 0.01) as compared with the vehicle control group (0 mg / ml treated group) was recognized from the group treated with 0.5 mg / ml of VIP water and the IC ₅₀ was 1.15 + 0.73 mg / ml (Fig. 2).

In the treated group of 0.5, 1, 5, 10, 50, 100 and 500 mg / kg of VIP, the values of -39.20, -50.66, -65.04, -81.11 and -92.49 , -94.71, and -97.81% points, respectively.

(2) Effect of Gefitinib on NCI-H520 cell viability

The decrease in NCI-H520 cell viability was significantly (p <0.01) higher in the gefitinib 0.01 μM treated group compared to the vehicle control group (0 μM treated group) and the IC ₅₀ was 21.67 ± 4.45 μM (9.51 ± 1.96 μg / ml) (Fig. 3).

-0.87, -20.50, -27.62, -31.14, -37.45, -44.84 and -0.87, respectively, in the group treated with Gefitinib at the concentrations of 0.001, 0.01, 0.1, 61.14% point of NCI-H520 cell survival rate.

Cytotoxicity assays were carried out with the IC50 values of 1.15 ± 0.73 mg / ml and 21.67 ± 4.45 μM (9.51 ± 1.96 μg / ml) for NCI-H520 cells, respectively. And showed low cytotoxicity.

1.3. Weight and Weight gain  Confirm change

Significant changes in body weight compared to the normal medium control group were not observed during the entire experimental period in the tumor transplant control group but were significantly (p <0.01) lower than in the normal medium control group And a reduction in weight gain during the weight-based administration period, respectively. In the group treated with Gefitinib alone, a significant (p <0.01) decrease in body weight was observed from the 21st day after the administration of the tumor graft control, but no significant change in the actual weight and body weight was observed compared with the tumor graft control group. On the other hand, the actual weight gain and body weight gain were significantly (p <0.01) higher in the group treated with Gavitang alone, 120 mg / kg of gefitinib and 400, 200 and 100 mg / (p <0.01), respectively, compared to the group treated with gefitinib 120 mg / kg (Table 4, Fig. 4).

Groups Body weights (g) Body weight gains (g) ± At first administration At sacrifice Actual body weights * Control group Intact 21.98 + 1.47 26.26 ± 1.52 26.26 ± 1.52   4.29 ± 1.20 TB (tumor-bearing) 20.78 ± 2.49 26.79 ± 1.72 15.99 ± 2.21 ^a -4.78 ± 2.35 ^a Single formula treated Gefitinib 20.59 ± 2.06 22.25 + 2.40 ^ac 15.68 ± 2.51 ^a -4.91 + 1.42 ^a GBT 20.60 ± 2.19 26.64 ± 1.92 ^d 19.71 ± 2.41 ^acd -0.89 ± 2.44 ^acd Gefitinib and GBT co-administered within 5 min 400 mg / kg 21.39 ± 2.02 26.84 ± 2.43 ^d 24.00 ± 2.65 ^bcd 2.61 ± 2.63 ^cd 200 mg / kg 22.24 + 1.81 27.53 + - 0.93 ^d 24.22 ± 1.39 ^cd 1.98 ± 2.22 ^bcd 100 mg / kg 21.30 ± 1.32 27.09 ± 1.85 ^d 22.53 + - 2.35 ^acd 1.23 + 1.84 ^acd

^a p <0.01 and ^b p <0.05 compared with intact control by LSD test

^c p <0.01 as compared with TB control by LSD test

^d p <0.01 compared with gefitinib single formula mice by LSD test

Actual body weight excluding tumor weight at the end of sacrifice showed -39.11% point change compared to the normal medium control group in the tumor-transplant control group, and gefitinib 120 mg / kg and VIP water 400 mg / kg alone, And 100 mg / kg and 120 mg / kg of gefitinib showed -1.96, 23.22, 50.09, 51.47, and 40.87% points of change compared with the tumor graft control group, respectively.

The body weight gain (35 days; actual body weight excluding the tumor weight at the end of the sacrifice - body weight at the start of the treatment) during the actual weight-based administration period was -211.58% point change in the tumor-transplant control group compared to the normal medium control group . In the group treated with gefitinib 120 mg / kg and VIP water 400 mg / kg alone, 400, 200 and 100 mg / kg, and gefitinib 120 mg / kg, respectively, compared with the tumor control group, -1.62, 82.29, 155.64 and 142.47 And 126.64% point, respectively.

1.3. Change in tumor volume

In the group treated with Gefitinib alone, the tumor volume was significantly decreased (p <0.01 or p <0.05) compared to the control group after 21 days from the start of administration, and the change in the tumor volume during the administration period was also significant (P <0.01 or p <0.05) in the group treated with 400 mg / kg of VIP alone, the tumor volume was significantly decreased (p <0.01 or p <0.05) (p <0.01) decreased compared to the control group. In addition, decrease in tumor volume was observed in the combined treatment group of 400, 200, 100 mg / kg and gefitinib 120 mg / kg for 7 days after the administration of VIP, compared to the tumor control group (p <0.01 or p <0.05) The tumor volume change during the administration period was also significantly (p <0.01) lower than that of the tumor graft control group. In particular, the decrease in tumor volume was significantly (p <0.01 or p <0.05) significantly lower than that of gefitinib alone (p <0.01 or p <0.05) from 14 days after administration of 400, 200 and 100 mg / kg and gefitinib 120 mg / And the change in tumor volume during the administration period was also significantly (p <0.01) lower than that of the gefitinib alone (Table 5, FIGS. 5 and 6).

Groups Tumor volume (mm ³ ) Changes (mm ³ )
[ BA ] First administration [A] Sacrifice [B] Control group TB   406.48 ± 150.77 7661.46 ± 2270.87 7404.86 ± 2794.07 Single formula treated Gefitinib   399.77 ± 110.18 4040.81 + - 917.86 ^a 3641.04 ± 897.39 ^a GBT   402.07 ± 95.01 4482.60 ± 1679.50 ^b 4080.52 + - 1623.45 ^b Gefitinib and GBT co-administered within 5 min 400 mg / kg   377.33 + - 152.25 2296.24 ± 586.19 ^ac 1918.91 + 479.68 ^ac 200 mg / kg   367.10 ± 185.41 2827.48 ± 591.10 ^ac 2460.38 ± 598.74 ^ac 100 mg / kg   395.97 + - 161.30 3014.49 + 472.76 ^ad 2618.52 + - 442.21 ^ac

Values are expressed mean ± S.D. of eight mice

^a p <0.01 and ^b p <0.05 as compared with TB control by MW test

^c p <0.01 and ^d p <0.05 compared with gefitinib single formula mice by MW test

The tumor volume during the drug administration period (5 weeks; tumor volume at the end of the sacrifice day - tumor volume at the start of the administration) was determined as gefitinib 120 mg / kg, VIP 400 mg / kg alone, VIP 400, 200 and 100 mg / kg And gefitinib (120 mg / kg) showed -50.83, -44.89, -74.09, -66.77 and -64.64%, respectively, as compared with the tumor-grafted control group.

1.4. Change in tumor weight

(P <0.01), respectively. In all groups, there was a significant decrease in tumor relative weight and absolute weight. In all groups, 400, 200 and 100 mg / kg of gefitinib and 120 mg / kg of gefitinib (p < 0.01) relative to the administration of mg / kg alone (Table 6 and 7, Fig. 5).

Groups Tumor mass Spleen 구mandibular lymph node Periovarian fat pad Control s Intact 0.093 + 0.010 0.020 ± 0.004 0.186 + 0.042 TB 10.797 ± 1.605 0.061 ± 0.009 ^a 0.007 ± 0.002 ^a 0.024 ± 0.009 ^e
Single formula treated Gefitinib 6.572 ± 0.865 ^f 0.054 ± 0.007 ^a 0.006 ± 0.001 ^a 0.024 ± 0.009 ^e GBT 6.993 ± 1.425 ^f 0.077 ± 0.006 ^acd 0.014 ± 0.002 ^acd 0.115 ± 0.013 ^efg
Gefitinib and GBT co-administered within 5 min 400 mg / kg 2.836 ± 0.878 ^fg 0.089 ± 0.006 ^cd 0.017 ± 0.003 ^bcd 0.139 0.016 ^efg 200 mg / kg 3.304 ± 0.892 ^fg 0.081 ± 0.009 ^acd 0.016 ± 0.002 ^acd 0.120 ± 0.010 ^efg 100 mg / kg 4.561 + - 0.804 ^fg 0.076 ± 0.007 ^acd 0.013 ± 0.002 ^acd 0.108 0.010 ^efg

^a p <0.01 and ^b p <0.05 compared with intact control by LSD test

^c p <0.01 as compared with TB control by LSD test

^d p <0.01 compared with gefitinib single formula mice by LSD test

^e p <0.01 as compared with intact control by MW test

^f p <0.01 as compared with TB control by MW test

^g p <0.01 compared with gefitinib single formula mice by MW test

Groups Tumor mass Spleen 구mandibular lymph node Periovarian fat pad Control s Intact 0.357 + 0.048 0.074 0.012 0.714 + 0.193 TB  40.419 + - 6.229 0.229 + 0.031 ^a 0.024 ± 0.009 ^a 0.091 0.037 ^f
Single formula treated Gefitinib 29.832 + 5.020 ^h 0.245 ± 0.029 ^a 0.028 ± 0.007 ^a 0.107 0.045 ^f GBT 26.140 + - 5.426 ^hj 0.290 ± 0.034 ^ace 0.054 ± 0.007 ^acd 0.433 + 0.064 ^fhi
Gefitinib and GBT co-administered within 5 min 400 mg / kg 10.675 ± 3.536 ^hi 0.332 ± 0.037 ^cd 0.063 0.014 ^bcd 0.522 + 0.069 ^ghi 200 mg / kg 12.030 占 3.314 ^hi 0.293 + 0.033 ^acd 0.056 ± 0.008 ^acd 0.436 ± 0.040 ^fhi 100 mg / kg 16.987 ± 3.604 ^hi 0.280 + 0.017 ^ace 0.049 ± 0.007 ^acd 0.400 0.046 ^fhi

^a p <0.01 and ^b p <0.05 compared with intact control by LSD test

^c p <0.01 as compared with TB control by LSD test

^d p <0.01 and ^e p <0.05 compared with gefitinib single formula mice by LSD test

^f p <0.01 and ^g p <0.05 compared with intact control by MW test

^h p <0.01 compared with TB control by MW test

ⁱ p <0.01 and ^j p <0.05 compared with gefitinib single formula mice by MW test

-39.13, -35.79, -73.73, -69.40, and -43.0, respectively, in the group treated with gefitinib 120 mg / kg and VIP water 400 mg / kg alone and in combination with VIP 400, 200 and 100 mg / kg and gefitinib 120 mg / -57.76% point, and the change in tumor relative weight at -26.19, -35.33, -73.59, -70.24, and -57.97% points, respectively.

1.5. Change in spleen weight

In the tumor-grafted control group, the spleen absolute and relative weight decreased significantly (p <0.01) compared to the normal control group. However, in the 400 mg / kg alone group and the VIP group, 400, 200 and 100 mg / kg and gefitinib 120 mg / (p <0.01), respectively. In particular, all of the VIP treatment group and gefitinib treatment group showed significant (p <0.01 or p <0.05) spleen compared to the gefitinib treatment group Increase of absolute and relative weight was recognized. On the other hand, in the case of gefitinib alone, the change in spleen absolute and relative weight was similar to that of the tumor transplant control (Tables 6 and 7).

The spleen absolute weights were -34.18% in the tumor control group compared with the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, 400, 200 and 100 mg / kg and gefitinib In the group treated with 120 mg / kg, the points of change were -11.61, 25.25, 44.20, 31.36 and 23.63%, respectively, compared with the control group.

The relative weight of spleen showed a change of -35.27% point compared to the normal medium control group in the tumor graft control group, and that of gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg and gefitinib In the group treated with 120 mg / kg, 6.76, 26.56, 44.75, 27.79 and 22.12% of the points were observed, respectively, compared with the control group.

1.6. Subconsciousness  Change in weight

In the tumor-grafted control group, absolute lymphocyte absolute and relative weight were significantly decreased (p <0.01) compared with the normal medium control group. However, in the case of the graft alone composition 400 mg / kg and VIP water 400, 200 or 100 mg / kg and gefitinib (P <0.01), respectively, compared to the control group, and those of 400, 200, and 100 mg / kg and gefitinib were significantly higher than those of the group treated with gefitinib alone (p <0.01 ) The increase of absolute and relative weight was recognized. In contrast, no significant change in the weight of submandibular glands was observed in the gefitinib 120 mg / kg alone group compared to the tumor-grafted control group (Tables 6 and 7).

The absolute and relative weights of the subcutaneous lymph nodes were -66.67 and -67.22%, respectively, in the transplantation group compared with the normal control group. The gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, And 100 mg / kg and gefitinib 120 mg / kg, respectively, showed a change in the absolute weight of the submandibular glands at the points of -3.65, 119.23, 157.69, 138.46 and 101.92%, respectively, compared to the tumor control group, and 16.19, 120.20, 159.91, 131.04 and 99.89% point, respectively.

1.7. Change of fat weight around ovary

In the tumor-grafted control group, there was a significant (p <0.01) decrease in the absolute and relative weight of the ovarian fat compared with the normal medium control group. However, in the group treated with Guizhou alone or in all three dose groups, gefitinib- (p <0.01), but there was a significant increase (p <0.01) in the ovarian fat weight, especially in the combination of 400, 200 and 100 mg / kg of gefitinib and 120 mg / kg of gefitinib An increase in weight was recognized. On the other hand, in the group treated with gefitinib 120 mg / kg alone, the absolute and relative weight changes of the fat around the ovary were similar to those of the control group (Table 6 and 7).

Absolute and relative weights of the ovarian follicles were -87.07 and -87.30%, respectively, in the transplantation control group compared with the normal control group. The gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, In the group treated with 200 and 100 mg / kg and 120 mg / kg combined with gefitinib, the absolute ovarian weight of the ovary was changed to -2.08, 377.60, 480.21, 399.48, and 349.48% points, respectively, compared with the control group, and 18.19, 377.22, 474.97 , 380.44, and 340.85% point, respectively.

1.8. Blood IL -6 and IFN -γ content change

In the tumor-grafted control group, serum IL-6 and IFN-γ levels were significantly decreased (p <0.01) compared to the normal control group. (P <0.01), respectively, and the increase of IFN-γ level was observed in the combined treatment group. In particular, all of the VIP treatment group and the gefitinib combination treatment group showed higher IFN- (P < 0.01) and decreased IFN-gamma and IL-6 levels. In the case of gefitinib alone, changes in blood IL-6 and IFN-γ levels were similar to those of the tumor-grafted control group (FIG. 7).

Serum IL-6 levels were 645.31% in the tumor control group compared with the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg In the group treated with gefitinib 120 mg / kg, 3.37, -38.40, -62.81, -47.74 and -33.61% of the points were changed, respectively.

Serum IFN-γ levels were -63.24% in the tumor-transplanted control group compared to the normal control group, and gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg And gefitinib 120 mg / kg, respectively, showed a change of -2.03, 71.82, 104.20, 87.19 and 48.01% points, respectively, compared to the control group.

1.9. NK cell  Change in activity

Significant (p <0.01) decrease in spleen and peritoneal NK cell activity was observed in the tumor transplantation control group compared to the normal medium control group, And peritoneal NK cell activity were increased. Especially, all of the VIP treatment group and gefitinib group showed significant increase (p <0.01) in spleen and peritoneal NK cell activity than gefitinib alone group. On the other hand, no significant change in spleen and peritoneal NK cell activity was observed in the group treated with gefitinib alone (FIG. 8).

The spleen NK cell activity was -67.00% in the tumor control group compared to the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg In the group treated with gefitinib 120 mg / kg, -1.99, 69.61, 98.95, 80.13 and 59.39% of the points were changed, respectively.

Peritoneal NK cell activity showed a -74.65% point change in the tumor-grafted control group compared with the normal medium control group. In addition, gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / In the group treated with gefitinib 120 mg / kg, the mice showed -3.74, 103.33, 180.64, 135.24 and 73.94% points of change compared to the tumor control group, respectively.

1.10. spleen cytokine  Change in content

In the tumor-grafted control, spleen TNF-α, IL-1β and IL-10 contents were significantly decreased (p <0.01) compared to the normal medium control group. (P <0.01), but the level of spleen cytokine was increased (p <0.01 or p <0.05). Especially, spleen TNF-α and IL- Lt; / RTI &gt; and &lt; RTI ID = 0.0 &gt; IL-10. On the other hand, no significant change in spleen cytokine levels was observed in the gefitinib-treated group compared to the tumor-grafted control group (Table 8).

Groups Tumor necrosis factor-alpha Interleukin-1? Interleukin-10 controls Intact 111.65 ± 33.50  46.04 + - 11.04 113.61 + - 46.56 TB 42.56 ± 12.38 ^a 8.63 ± 2.94 ^a 28.92 ± 12.34 ^a Single formula treated Gefitinib 40.96 + - 14.30 ^a 8.83 ± 4.02 ^a 29.99 ± 12.40 ^a GBT 62.05 ± 11.98 ^adf 25.55 ± 13.98 ^bce 53.45 ± 13.31 ^acce Gefitinib and GBT co-administered within 5 min 400 mg / kg 76.82 ± 20.18 ^bce 40.73 ± 14.64 ^ce 80.37 ± 15.96 ^ce 200 mg / kg 70.38 ± 12.01 ^acce 31.27 ± 14.71 ^ce 66.60 ± 12.33 ^bce 100 mg / kg 63.31 ± 11.48 ^aces 22.80 ± 8.97 ^ace 51.56 ± 11.40 ^ace

Values are expressed mean ± SD, pg / ml of eight mice

^a p <0.01 and ^b p <0.05 compared with intact control by MW test

^c p <0.01 and ^d p <0.05 compared with TB control by MW test

^e p <0.01 and ^f p <0.05 as compared with gefitinib single formula mice by MW test

The spleen TNF-α content was -61.88% in the tumor control group compared to the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, VIP water 400, 200 and 100 mg / kg And gefitinib 120 mg / kg, respectively, showed a change of -3.76, 45.79, 80.51, 65.38 and 48.77% points, respectively, compared to the tumor control group.

The spleen IL-1β content was -81.26% in the tumor control group compared to the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, VIP water 400, 200 and 100 mg / kg And gefitinib 120 mg / kg were 2.30, 196.00, 371.94, 262.31 and 164.14%, respectively, as compared with the control group.

The amount of splenic IL-10 in the tumor-transplanted control group was -74.55% higher than that in the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg And gefitinib 120 mg / kg, respectively, were 3.72, 84.83, 177.94, 130.31 and 78.30%, respectively, as compared with the control group.

1.11. Histological change

1.11.1. Histopathological change of mass

In the tumor-grafted control group, NCI-H520 lung cancer cells were relatively well-differentiated, and apoptosis-induced increase in cytoplasmic acidity and nuclear enrichment was observed in some cells, and mitosis was also frequently observed. On the other hand, apoptotic cells were significantly increased (p <0.01) in the group treated with gefitinib and Jiwi-tang alone, and in all three groups treated with adavitin and gefitinib, and the proportion of NCI-H520 cells was also significantly higher (p <0.01). Especially, the decrease of tumor cell volume and the number of apoptotic cells were significantly (p <0.01) lower in gefitinib alone group compared with 400, 200 and 100 mg / kg and gefitinib 120 mg / (Table 9, Fig. 9).

Groups Tumor cell volume (% / mm ² ) Apoptotic cell percentages (%) Immunore Experimental cell percentages (% / tumor cells) Caspase-3 PARP COX-2 iNOS TNF-a Control group TB 77.86 8.12 13.69 ± 6.16 10.91 ± 4.03 11.30 ± 3.58 41.53 + - 12.70 6.71 ± 1.45 4.79 ± 2.28 Single formula treated Gefitinib 52.49 ± 10.69 ^a 41.35 ± 11.34 ^a 37.72 ± 5.28 ^c 36.73 + - 14.63 ^c 44.86 ± 12.62 6.45 ± 2.33 4.80 ± 1.95 GBT 60.87 ± 13.26 ^a 34.93 ± 13.51 ^a 28.07 ± 8.24 ^ce 32.01 + - 7.13 ^c 23.10 ± 6.18 ^cd 19.95 ± 3.66 ^cd 26.05 ± 8.98 ^cd Gefitinib and GBT co-administered within 5 min 400 mg / kg 20.61 ± 5.66 ^ab 71.49 ± 11.49 ^ab 68.48 ± 12.74 ^cd 73.32 ± 12.21 ^cd 10.03 ± 1.80 ^cd 67.85 +/- 10.96 ^cd 71.92 + - 12.10 ^cd 200 mg / kg 28.30 ± 10.52 ^ab 64.96 ± 10.74 ^ab 58.62 ± 11.08 ^cd 67.41 ± 12.86 ^cd 16.29 ± 4.75 ^cd 49.16 ± 12.18 ^cd 59.14 ± 12.62 ^cd 100 mg / kg 35.11 ± 11.53 ^ab 56.84 ± 10.71 ^ab 51.01 + - 11.22 ^cd 56.75 ± 10.88 ^ce 21.46 ± 6.42 ^cd 45.71 ± 10.44 ^cd 39.52 ± 10.96 ^cd

PARP = cleaved poly (ADP-ribose) polymerase; COX-2 = Cyclooxygenase-2; iNOS = Inducible nitric oxide synthase; TNF = tumor necrosis factor

^a p <0.01 as compared with TB control by LSD test

^b p <0.01 compared with gefitinib single formula mice by LSD test

^c p <0.01 compared with TB Control by MW test

^d p <0.01 and ^e p <0.05 compared with gefitinib single formula mice by MW test

In addition, the number of caspase-3 and PARP-immunoreactive cells in the mass was significantly (p <0.01) significantly increased in all the treatment groups including 400 mg / kg of VIP, / kg and 120 mg / kg of gefitinib, respectively, were significantly increased (p <0.01 or p < 0.05) in the number of caspase-3 and PARP-immunoreactive cells than in the group treated with gefitinib alone (Table 9, ). The number of iNOS and TNF-α immunoreactive cells in the mass was significantly increased and the numbers of COX-2 immunoreactive cells were decreased significantly (p <0.01), compared with the tumor-grafted control group, The number of iNOS and TNF-α immunoreactive cells and the number of COX-2 immunoreactive cells were significantly (p <0.01) higher in gefitinib treated group compared with gefitinib alone group at 400, 200 and 100 mg / kg and gefitinib 120 mg / Decrease in numbers was recognized. On the other hand, the number of COX-2, iNOS and TNF-α immunoreactive cells in the mass was not significantly different from that of the transplant control group in the group administered with gefitinib 120 mg / kg alone (Table 9, Fig. 12-14).

The proportion of tumor cells in the tumor tissues was -32.59, -32.59, respectively, compared with the tumor-grafted control group in the combination therapy of gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg and gefitinib 120 mg / kg, -21.83, -73.53, -63.65, and -54.91%, respectively.

The proportion of apoptotic cells in tumor tissues was 202.02 and 155.11, respectively, compared with the grafted control group in the combined administration of gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg and gefitinib 120 mg / , 422.14, 374.49 and 315.17%, respectively.

The proportion of caspase-3 immunoreactive cells in tumor tissues was significantly higher in the group treated with gefitinib 120 mg / kg and VIP water 400 mg / kg alone compared with those treated with 400, 200, 100 mg / kg and gefitinib 120 mg / kg Respectively, at 245.66, 157.25, 527.62, 437.24, and 367.46%, respectively.

The proportion of PARP-immunoreactive cells in the tumor tissues was 225.04 compared to the tumor-grafted control group in the combined administration of gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg and gefitinib 120 mg / , 183.22, 548.79, 496.46 and 402.12% point, respectively.

The proportion of COX-2 immunoreactive cells in the tumor tissues was significantly higher in the group treated with gefitinib 120 mg / kg and VIP water 400 mg / kg alone compared with the control group treated with 400, 200 and 100 mg / kg of gefitinib and 120 mg / kg of gefitinib Respectively, and 8.03, -44.39, -75.85, -60.77 and -48.32% points, respectively.

The proportion of iNOS immunoreactive cells in the tumor tissues was significantly higher in the group treated with gefitinib 120 mg / kg and VIP water 400 mg / kg alone compared with the control group with 400, 200 and 100 mg / kg and gefitinib 120 mg / 3.95, 197.21, 910.58, 632.17 and 580.90%, respectively.

The proportion of TNF-α immunoreactive cells in the tumor tissues was significantly higher than that of the tumor-grafted control group in the combined administration of gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg and gefitinib 120 mg / Respectively, and the change was 0.37, 444.24, 1402.66, 1135.54, and 725.59% points, respectively.

1.11.2. Histopathological changes of the spleen

In the tumor-grafted control group, atrophy characterized by significant lymphocyte reduction in the spleen white water quality portion was recognized and a significant (p < 0.01) reduction in spleen thickness, white water quality diameter and number were recognized, respectively. On the other hand, significant increases in spleen thickness, white water diameter and number were observed in all groups compared to the control group, and in all groups treated with all the venom and gefitinib 120 mg / kg (p <0.01) of spleen thickness, white water quality diameter and number were found to be higher than those of gefitinib alone group. On the other hand, changes in spleen thickness, white water quality diameter and number similar to those of the tumor graft control were observed in the case of the gefitinib alone group (Table 10, Fig. 15).

Groups Total thickness
(mm / central regions) White pulp numbers (/ mm ² ) White pulp diameters (± m / white pulp) controls Intact    1816.11 ± 172.50      19.50 ± 2.45     611.06 + 149.04 TB 1193.23 ± 125.10 ^a 7.88 ± 1.25 ^a 353.32 ± 57.70 ^e
Single formula treated Gefitinib 1185.58 ± 153.55 ^a 8.25 + 2.31 ^a 354.09 ± 58.23 ^e GBT 1488.35 ± 161.28 ^acd 14.88 ± 2.03 ^acd 584.01 + - 30.38 ^fg
Gefitinib and GBT co-administered within 5 min 400 mg / kg 1661.14 ± 138.48 ^cd 17.25 ± 2.12 ^bcd 592.69 ± 45.24 ^fg 200 mg / kg 1609.26 ± 139.50 ^bcd 15.25 ± 2.12 ^acd 556.48 ± 49.19 ^fg 100 mg / kg 1573.55 ± 192.44 ^acd 12.63 + 1.41 ^acd 488.34 + - 85.94 ^fg

^a p <0.01 and ^b p <0.05 compared with intact control by LSD test

^c p <0.01 as compared with TB control by LSD test

^d p <0.01 compared with gefitinib single formula mice by LSD test

^e p <0.01 as compared with intact control by MW test

^f p <0.01 as compared with TB control by MW test

^g p <0.01 compared with gefitinib single formula mice by MW test

The total thickness of the spleen showed a change of -34.30% point in the tumor control group compared to the normal medium control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, 400, 200 and 100 mg / kg of gefitinib In the 120 mg / kg co-administered group, -0.64, 24.73, 39.21, 34.87, and 31.87% points were changed compared to the tumor transplant control group, respectively.

The number of spleen white water was -59.62% in the tumor control group compared with the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, 400, 200 and 100 mg / kg And gefitinib (120 mg / kg) showed 4.76, 88.89, 119.05, 93.65 and 60.32% points of change compared to the control group, respectively.

The diameter of spleen white water showed a -42.18% point change in the transplantation control group compared with the normal control group, and the gefitinib 120 mg / kg and the VIP water 400 mg / kg alone group, VIP water 400, 200 and 100 mg / kg And gefitinib 120 mg / kg, respectively, showed 0.22, 65.29, 67.75, 57.50 and 38.22% points of change compared to the tumor control group, respectively.

1.11.3. The Lymphatic  Histopathological change

In the tumor-grafted control group, significant atrophy of the lymph node was observed in the lymph node compared with the normal medium control, and significant decrease (p <0.01) in the total lymph node, cortical thickness and the number of follicles in the cortex were recognized. In addition, the total number of lymph nodes, cortical thickness, and the number of follicles in the cortex were found to be histopathologically significant (p <0.01). In particular, VIP, 400, 200 and 100 mg / kg and gefitinib 120 mg / kg were significantly (p <0.01) higher in the lymph node than in the gefitinib alone group and in the cortical thickness and the number of follicles in the cortex. On the other hand, in the group treated with gefitinib alone, there was no significant change in the lymph node thickness, cortical thickness and follicle number compared to the tumor graft control group (Table 11, Fig. 16).

Groups Total thickness
(μm / central regions) Cortex lymphoid cell follicle numbers (/ mm ² ) Cortex thickness (μm / lymph node) controls Intact    2013.40 ± 275.50      32.25 + - 11.91    1465.53 + - 375.67 TB 677.91 + - 132.33 ^a 12.25 ± 2.82 ^d 327.71 + - 80.05 ^d Single formula treated Gefitinib 632.44 + 156.03 ^a 12.00 ± 2.39 ^d 301.08 ± 74.94 ^d GBT 1340.37 ± 191.37 ^abc 20.00 ± ^1.85f 628.96 + 122.60 ^def Gefitinib and GBT co-administered within 5 min 400 mg / kg 1657.27 ± 162.00 ^abc 32.75 ± 5.85 ^ef 964.89 ± 135.64 ^def 200 mg / kg 1439.70 ± 167.26 ^abc 29.13 ± 6.38 ^f 793.36 ± 117.91 ^def 100 mg / kg 1042.79 ± 181.21 ^abc 27.63 ± 4.75 ^ef 618.33 ± 188.73 ^def

^a p <0.01 compared with intact control by LSD test

^b p <0.01 as compared with TB control by LSD test

^c p <0.01 compared with gefitinib single formula mice by LSD test

^d p <0.01 compared with intact control by MW test

^e p <0.01 as compared with TB control by MW test

^f p <0.01 as compared with gefitinib single formula mice by MW test

The total thickness of submandibular glands was -66.33% in the tumor control group compared to the normal medium control group. The gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, 400, 200 and 100 mg / In the group treated with gefitinib 120 mg / kg, -6.71, 97.72, 144.47, 112.37 and 53.82% of the points were changed, respectively, compared with the control group.

  The number of follicles in submandibular cortex was -62.02% compared to the normal control group in the transplantation control group. The gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, 400, 200 and 100 mg / kg and 120 mg / kg of gefitinib showed -2.04, 63.27, 167.35, 137.76, and 125.51% points of change compared to the tumor control group, respectively.

The subcortical cortical thickness of the transplantation group was -77.64% higher than that of the normal control group, and the gefitinib 120 mg / kg and VIP water 400 mg / kg alone, VIP 400, 200 and 100 mg / kg In the group treated with gefitinib 120 mg / kg, the points were -8.13, 91.93, 194.43, 142.09 and 88.68%, respectively, as compared with the control group.

1.11.4. Histopathological changes of the ovarian fat

In the tumor graft control group, atrophy characterized by a marked decrease in the size of white adipocytes was observed compared to the normal medium control group, and a significant (p < 0.01) accumulation fat thickness and a decrease in the average diameter of white adipocytes were recognized, respectively. On the other hand, the cumulative fat thickness and the mean diameter of white adipocytes were significantly (p <0.01) significantly higher in the group treated with Guizibang alone and 400, 200 and 100 mg / kg and gefitinib compared with the control group (P <0.01), especially in all three doses of VIP water and gefitinib, was significantly higher than that of gefitinib alone (p <0.01). On the other hand, the gefitinib alone showed similar changes in the ovarian cumulative adipose tissue thickness and white adipocyte mean diameter, similar to that of the tumor transplant control group (Table 12, Fig. 17).

Groups Total thickness (mm / central regions) White adipocyte diameters (μm) controls Intact 2377.14 ± 583.37 63.65 + - 11.01 TB 923.83 + - 246.63 ^d 25.05 ± 5.91 ^a
Single formula treated Gefitinib 896.43 ± 156.87 ^d 24.34 + 3.99 ^a GBT 1522.07 ± 207.22 ^efg 38.97 7.70 ^abc
Gefitinib and GBT co-administered within 5 min 400 mg / kg 1785.84 ± 191.01 ^efg 49.21 ± 12.61 ^abc 200 mg / kg 1684.10 + - 216.45 ^efg 42.52 + - 11.47 ^abc 100 mg / kg 1558.78 ± 220.35 ^efg 37.50 + - 7.45 ^abc

^a p <0.01 compared with intact control by LSD test

^b p <0.01 as compared with TB control by LSD test

^c p <0.01 compared with gefitinib single formula mice by LSD test

^d p <0.01 and ^e p <0.05 compared with intact control by MW test

^f p <0.01 as compared with TB control by MW test

^g p <0.01 compared with gefitinib single formula mice by MW test

The cumulative ovarian fat thickness was -61.14% in the tumor control group compared with the normal control group. The gefitinib 120 mg / kg and VIP water 400 mg / kg alone group, 400, 200 and 100 mg / kg and gefitinib 120 mg / kg, respectively, showed a change of -2.97, 64.76, 93.31, 82.30 and 68.73% points, respectively, compared to the tumor control group.

The average diameter of ovarian white adipocytes was -60.64% in the transplant control group compared to the normal control group. The mean diameter of ovarian white adipocytes was 120 mg / kg of gefitinib and 400 mg / kg of VIP, 400, 200 and 100 In the combined group of mg / kg and 120 mg / kg of gefitinib, the points of -2.83, 55.58, 96.46, 69.75 and 49.70% were different from those of the control group, respectively.

As a result, the IC50 values of Navi-H520 cells were 1.15 ± 0.73 mg / ml and 21.67 ± 4.45 μM (9.51 ± 1.96 μg / ml), respectively, -H520 cell transplantation significantly decreased spleen and nodal lymph node mass, serum IFN-γ content, spleen TNF-α, IL-1β and IL-10 content, spleen cell and abdominal macrophage activity decreased lymphocytes And the reduction of body weight and body weight gain was also recognized. The increase in serum IL-6 level, the decrease of the ovarian fat mass, and the histopathologic change of the ovary Attenuation of peripheral fat tissue was recognized. Thus, after tumor transplantation, typical tumor-associated immunosuppression and cachexia are thought to have been induced. On the other hand, the reduction of mass volume and weight by gefitinib 120 mg / kg alone was confirmed by histopathological examination with a decrease in the proportion of tumor cells due to the increase of apoptosis cells in the mass, and the caspase-3 and PARP immunoreactivity (Change in body weight, ovarian fat and blood IL-6 content) and immunosuppression (spleen and subcutaneous lymph node weight, serum IFN levels), and tumor necrosis factor -γ content, NK cell activity, changes in splenic TNF-α, IL-1β and IL-10 content, and histological changes in immunological organs). On the other hand, in the group treated with Guigui alone, significant immunological activity and decrease of tumor - associated cachexia were observed compared with the tumor graft control group. However, the anticancer effect on the mass itself was relatively low as compared with the gefitinib group. In addition, all of the triple doses of adenovirus and gefitinib treated group showed significant inhibition of chemotherapy, immunological activity and tumor-associated cachexia compared to the tumor-grafted control group, especially in combination with all three doses of VIP, 400, 200 and 100 mg / kg and gefitinib Administration group showed a dose - dependent significant increase in anticancer effect compared with that of the gefitinib alone group, and a significant increase in dose - dependency of immune activity and cachexia - Therefore, as a result of the previous pharmacokinetic experiment [CIMI-13-02-02, 2014], it was observed that oral administration of guinea pig within 5 minutes did not affect the bioavailability of gefitinib. It is considered that gefitinib significantly enhances the anticancer effect of gefitinib and significantly inhibits tumor-associated cachexia by immunological activity without affecting the bioavailability of gefitinib. It is expected that it will provide a new treatment method which is very useful for the treatment. In addition, compared with gefitinib alone, the immunosuppressive effects of tumor-associated cachexia and the anticancer activity were remarkably increased in the group treated with 100 mg / kg of VIP and 100% of the group treated with gefitinib. And the control of tumor - associated cachexia could be controlled.

Therefore, it was observed that the combined administration of VIP of 400, 200, or 100 mg / kg in combination with 5 minutes interval significantly increased the anticancer effect of gefitinib through immunological activity, and particularly, the tumor-related cachexia phenomenon was also remarkably suppressed. Therefore, the 5-minute intraperitoneal administration of 100 mg / kg or more of VIP water significantly increases the anticancer effect of gefitinib in NCI-H520 cell transplanted mice by immunological activity without affecting the bioavailability of gefitinib, And it is expected that it will provide a new therapeutic method which is very useful for the treatment of both lung cancer patients and lung cancer patients.

Example  2. Of Gefitinib  Lung Cancer Therapy VIP  Identification of toxicity reduction effect: Guizu-tang and Gefitinib Oral administration for 28 days within 5 minutes

The same one as in Example 1 was used for the gefitinib and the VIP bottle.

2.1. Preparation of experimental animals

Male ICR mice and male mice (OrientBio, Seungnam, Korea) were cleaned for 7 days and grouped into 6 groups based on body weight (mean: 31.50 ± 1.20 g, 29.40 ~ 34.10 g) Respectively.

Group separation

Total 6 groups (including medium control group); (G3M) gefitinib 160 mg / kg alone, (G2M) alone, 400 mg / kg alone, and (G3M) gefitinib 160 mg / kg and (G4M), 160 mg / kg of gefitinib, 200 mg / kg of venom, and 160 mg / kg of gefitinib and 100 mg / kg of venom (Table 5).

group gender Dosage (mg / kg) Animal No. CIMI-14-02-05-01 G + GBT TX: Mouse repeated oral dose toxicity test Control Male Distilled water 10ml / kg M01 ~ M07 Reference Male Gefitinib single (160 mg / kg) M08 ~ M14 Reference Male GBT single (400 mg / kg) M15 to M21 Active Male Gefitinib and GBT (160 and 400 mg / kg) M22 to M28 Active Male Gefitinib and GBT (160 and 200 mg / kg) M29 to M35 Active Male Gefitinib and GBT (160 and 100 mg / kg) M36 to M42

2.2. Method of administration

In the manner of oral gavage (sterile distilled water used as a solvent and orally administered at a dose of 10 ml / kg once a day for 28 days), 400, 200 or 100 mg / kg of VIP water was administered to a mouse of 160 mg / kg of gefitinib Were administered once daily for 28 days at intervals of less than 5 minutes. In the single administration group, only the same volume of sterilized distilled water was administered to the dogs or gefitinib. In the medium control group, only sterile distilled water .

The dose of Gefitinib was set at 160 mg / kg, which is four times the minimum dose of 40 mg / kg [AstraZeneca Pharmaceuticals, 2005], which is known to show liver toxicity in rats for 28 consecutive days. Or 100 mg / kg and 5 min interval was established based on the existing pharmacokinetic combination study [CIMI-13-02-02, 2014]. In this experiment, Guizantin or gefitinib was dissolved in sterilized distilled water and orally administered for 28 days once a day at a dose of 10 ml / kg of rodent [Flecknell, 1996] within 5 minutes (Table 13 18).

2.3. Observation period and items

4 weeks; Mortality, clinical symptoms, weight change, and autopsy findings.

(14 items, Table 14) and blood chemistry (20 items; Table 15), histopathologic changes (23 organ: brain-cerebral, cerebellum and training) , The heart, thymus, lung, testis, epididymis, kidney, adrenal gland, liver, pancreas, gastrointestinal tract, esophagus, gastric fundus, duodenum, plant, ileum, cecum, colon and rectum, (GSH, CAT, and SOD).

Hematology Items Units Methods Abbreviations Full name 1. RBC Red blood cell count M / L Laser optical (Flow cytometry) 2. HGB Hemoglobin concentration g / dl Cyanmethemoglobin method 3. HCT Hematocrit % Calculated from Item 1 and 4 4. MCV Mean corpuscular volume fL Laser optical (Flow cytometry) 5. MCH Mean corpuscular hemoglobin pg Calculated from Item 1 and 2 6. MCHC Mean corpuscular hemoglobin concentration g / dL Calculated from Item 2 and 3 7. PLT Platelet count K / μL Laser optical (Flow cytometry) 8. RET Reticulocyte count ea / 1000 Laser optical with cytochemical reaction 9. WBC White blood cell count K / μL Laser optical with cytochemical reaction Differential counts of white blood cells 10. NEU% Percentages of neutrophils % Perox optical with chemical reaction 11. LYM% Percentages of lymphocytes % Perox optical with chemical reaction 12. MON% Percentages of monocytes % Perox optical with chemical reaction 13. EOS% Percentages of eosinophils % Perox optical with chemical reaction 14. BAS% Percentages of basophils % Perox optical with chemical reaction

Hematology Items Units Methods Abbreviations Full name 1. AST Aspartate aminotransferase IU / L UV-Rate method 2. ALT Alanine aminotransferase IU / L UV-Rate method 3. ALP Alkaline phosphatase IU / L P-NPP method 4. BUN Blood urea nitrogen mg / dL Urease-UV method 5. CRE Creatinine mg / dL Jaffe method 6. GLU Glucose mg / dL Enzyme method 7. CHO Total cholesterol mg / dL Enzyme method 8. PRO Total protein g / dL Biuret method 9. CPK Creatine phosphokinase IU / L UV-Rate method 10. ALB Albumin g / dL BCG method 11. BIL Total bilirubin mg / dL Jendrassik-cleghorn method 12. Globulin Globulin g / dL BCG method 13. A / G Albumin / globulin ratio Ratio Calculated from Item 10 and 12 14. IP Inorganic phosphorus mg / dL UV method 15. Ca Calcium mg / dL OCPC method 16. TG Triglyceride mg / dL Enzyme method 17. LDH  Lactate dehydrogenase IU / L UV-Rate method 18. Na Sodium mmol / L Electrode method 19. K Potassium mmol / L Electrode method 20. Cl Chloride mmol / L Electrode method

2.4. Mortality

The mortality associated with the administration of the test substance was not observed during the 28-day experimental period, and a final autopsy was performed on all the experimental animals (7/7; 100%) of all the experimental groups (Table 16).

Groups ^{Days of treatment Periods (Day 0 a} ~ 27) At termination (28 days of administration) Total * Vehicle control   Distilled water 0 0 0/7 (0%) Gefitinib single 160 mg / kg 0 0 0/7 (0%) GBT single   400 mg / kg 0 0 0/7 (0%) Gefitinib 160 mg / kg and GBT co-treated 400 mg / kg 0 0 0/7 (0%) 200 mg / kg 0 0 0/7 (0%) 100 mg / kg 0 0 0/7 (0%)

Values are expressed as number of died animals

GBT = Guibi - tang purchase from HANZUNG PHARM. CO. LTD. (Daejeon, Korea)

^a treatment day

* Total mortalities during 28 days of observation periods - died animals / total observed animals (percentages; seven mice per group)

2.5. Clinical symptoms

As a result of this experiment, the clinical symptoms associated with the administration of the test substance were not observed during the 28 day experimental period (Table 17).

Groups Normal appearance Clinical signs Any abnormal signs Vehicle control   Distilled water 7/7 (100%) 0/7 (0%) Gefitinib single 160 mg / kg 7/7 (100%) 0/7 (0%) GBT single   400 mg / kg 7/7 (100%) 0/7 (0%) Gefitinib 160 mg / kg and GBT co-treated 400 mg / kg 7/7 (100%) 0/7 (0%) 200 mg / kg 7/7 (100%) 0/7 (0%) 100 mg / kg 7/7 (100%) 0/7 (0%)

2.6. Weight and Weight gain  change

The change in body weight was significantly (p <0.05) higher than that of the vehicle control group, but not in all experimental groups, including 160 mg / kg of gefitinib alone, compared with the vehicle control group ) Body weight reduction during Day 14 ~ 27 was recognized. On the other hand, significant changes in body weight and body weight compared to gefitinib alone were not observed in all three doses of VIP water and gefitinib, and only 400 mg / kg of VIP water showed significant changes in body weight and weight gain (Table 18; Fig. 19).

Groups Intervals Day 0 * ~ Day 14 Day 14 ~ Day 27 Day 0 ~ Day 28 ** Vehicle control   Distilled water 5.89 ± 1.44 1.86 ± 0.66 3.26 ± 1.59 Gefitinib single 160 mg / kg 5.60 ± 1.42 1.59 ± 0.64 1.94 ± 1.21 GBT single   400 mg / kg 6.64 ± 2.41 1.84 0.61 3.50 + - 3.07 Gefitinib 160 mg / kg and GBT co-treated 400 mg / kg 7.21 ± 2.08 2.16 ± 0.86 3.60 1.55 200 mg / kg 5.86 ± 2.17 1.97 ± 1.16 2.54 + - 2.93 100 mg / kg 6.67 ± 1.70 0.86 + 0.81 ^a 2.66 ± 0.94

* Day of treatment after overnight fasted

** Days of sacrifice after overnight fasted

^a p <0.05 compared with vehicle control by LSD test

2.7. Change in long-term weight

In the case of Gefitinib 160 mg / kg alone, the absolute and relative weights of the spleen, liver and subcutaneous lymph nodes were significantly increased (p <0.01) compared to the vehicle control group. (P <0.01), respectively, compared to the group treated with 160 mg / kg of gefitinib alone. The absolute and relative weights of the spleen, liver and subcutaneous lymph nodes were decreased.

On the other hand, no significant change in relative and absolute organ weights was observed in the case of the VIP treatment 400 mg / kg alone group compared with the medium control group. In the group treated with gefitinib 160 mg / kg alone or in combination with all the VIP treatment and gefitinib, Changes in absolute and relative weights of significant lung, heart, thymus, kidney, adrenal, testis, pancreas, brain, and epididymis were not observed (Tables 19 and 20).

Values are expressed as mean ± SD. of seven mice, g

GBT = Guibi - tang purchase from HANZUNG PHARM. CO. LTD. (Daejeon, Korea)

L, left sides; S, splenic lobes; G, gland; LN, submandibular lymph node

^a p <0.01 and ^b p <0.05 compared with vehicle control by LSD test; ^c p <0.01 compared with gefitinib single-treated mice by LSD test

Values are expressed as mean ± SD. of seven mice,% of body weights at sacrifice

GBT = Guibi - tang purchase from HANZUNG PHARM. CO. LTD. (Daejeon, Korea)

L, left sides; S, splenic lobes; G, gland; LN, submandibular lymph node

^a p <0.01 and ^b p <0.05 compared with vehicle control by LSD test; ^c p <0.01 compared with gefitinib single-treated mice by LSD test

2.8. Hematological change

Fourteen hematologic tests showed that the reduction of RBC, HGB and HCT, as well as the increase in WBC, lymphocyte and monocyte ratios and the decrease in neutrophil leukocyte ratio associated with gefitinib alone were significant (p <0.01) (P <0.01 or p <0.05) compared with gefitinib alone (p <0.01 or p <0.05) compared with gefitinib alone group (100, 200 or 400 mg / kg and gefitinib) Decreased monocyte ratio and associated neutrophil leukocyte ratios were recognized. On the other hand, no significant hematologic changes were observed in the group treated with Guizibang 400 mg / kg alone compared to the control group, and significant differences in MCV, MCH, MCHC, PLT, RET, Changes in EOS% and BAS% were not observed, respectively (Table 21).

Values are expressed as mean ± SD. of seven mice

^a p <0.01 and ^b p <0.05 compared with vehicle control by LSD test; ^c p <0.01 and ^d p <0.05 compared with gefitinib single-treated mice by LSD test

^e p < 0.01 and ^f p < 0.05 as compared with vehicle control by MW test; ^g p <0.01 compared with gefitinib single-treated mice by MW test

2.9. Blood biochemical change

20 blood biochemical tests showed that albumin and A / G reduction were associated with an increase in AST, ALT, globulin, and LDH contents in the gefitinib 160 mg / kg alone group (p <0.01) (P <0.01 or p <0.05) compared with gefitinib alone, the albumin and A / G levels were increased with decreasing AST, ALT, globulin and LDH contents in the group treated with 100, 200 or 400 mg / kg and gefitinib Respectively. In addition, significant changes in blood biochemical changes were not observed in the case of VIP treatment 400 mg / kg alone compared with the medium control group. Significant levels of ALP, BUN, CRE, GLU, and CHO in gefitinib alone and all VIP treatment and gefitinib combination groups , PRO, CPK, T-BIL, TG, Ca, P, Na, K and Cl, respectively.

^a p <0.01 and ^b p <0.05 compared with vehicle control by LSD test; ^c p <0.01 and ^d p <0.05 compared with gefitinib single-treated mice by LSD test

^e p < 0.01 compared with vehicle control by MW test; ^f p <0.01 and ^g p <0.05 compared with gefitinib single-treated mice by MW test

2.10. Autopsy findings

In the group treated with Gefitinib 160 mg / kg alone, discolorization, spleen and sublingual lymph node enlargement were observed more frequently than the medium control group. However, gefitinib (100, 200 or 400 mg / Significant liver discoloration, spleen, and sublingual lymph node enlargement were observed less frequently than in the single dose group. Mild [1+] pulmonary hypertension, thymic atrophy, spleen atrophy, and subclinical lymphocytopenia were sporadically observed in all experimental groups including medium control (Table 23).

Values were expressed as observed animals / total observed animals (seven mice in each group)

^a) Bilateral submandibular lymph node

1+ = slight

2.11. Histopathological observation

Inflammatory cell infiltration (Fig. 20) and subclinical lymphadenopathy (Fig. 21) with slight hepatic necrosis were confined to the medium control group (1/7; 14.29% (Fig. 20), subcutaneous lymphadenopathy (Fig. 21), and spleen red watershed lymphocyte enlargement (Fig. 20) compared with the medium control group 22), and the increase in observation frequency was recognized, respectively. On the other hand, in the group of 400, 200 or 100 mg / kg and gefitinib combination treatment, the degree and frequency of local necrosis of the spleen and submandibular lymphocytes and inflammatory cell infiltration were significantly reduced compared to the group treated with gefitinib alone. Mild lung congestion (FIG. 23), subcutaneous lymphatic congestion (FIG. 21), and supratentorial cystocele (FIG. 24) were sporadically observed in all experimental groups including medium control (Table 24).

^a) Bilateral submandibular lymph node

± CG = congestions; rHP = hyperplasia of red pulp lymphoid cells; IF = focal inflammatory cell infiltrations; FN = focal cellular necrosis; dHP = diffused hyperplasia of lymphoid cells; fCY = focal cyst formation in the mucosa of the fundus; 1+ = slight; 2+ = moderate; 3+ = severe degrees.

2.12. Changes in liver lipid peroxidation and antioxidant defense systems

In the group treated with Gefitinib 160 mg / kg alone, there was a significant (p <0.01) increase in liver lipid peroxidation and a decrease in endogenous antioxidant or related enzymes GSH, SOD and CAT compared with the medium control group. (p <0.01 or p <0.05) in the combination group of gefitinib / kg and gefitinib compared to the group treated with 160 mg / kg of gefitinib alone, the increase of GSH content or SOD and CAT activity was found to be dose dependent . On the other hand, no significant change in the lipid peroxidation and antioxidant defense system was observed in the VIP treatment group alone (400 mg / kg) (Table 26).

Items
Unit
Groups LPO Antioxidative Defense Systems MDA levels
(nM / mg tissue) Glutathione
(μM / mg tissue) CAT
(U / mg tissue) SOD
(U / mg tissue) Vehicle control 18.42 + - 2.30 30.64 + - 6.93 16.69 ± 2.29 2.47 ± 0.95 Gefitinib single 160 mg / kg 30.20 ± 2.16 ^a 14.65 + 2.04 ^a 9.97 ± 1.46 ^a 0.66 + 0.14 ^d GBT single   400 mg / kg 18.79 ± 2.04 ^b 31.34 + 4.77 ^b 17.08 ± 1.98 ^b 2.51 ± 0.47 ^e Gefitinib 160 mg / kg and GBT co-treated 400 mg / kg 23.77 + - 3.48 ^ab 23.54 + - 2.46 ^ab 15.41 ± 2.82 ^b 1.52 + 0.45 ^e 200 mg / kg 25.01 + - 2.51 ^ab 21.26 ± 2.11 ^ab 13.11 ± 1.52 ^ab 1.30 ± 0.37 ^de 100 mg / kg 26.38 ± 2.09 ^ab 19.93 ± 2.09 ^ac 12.68 +/- 1.48 ^ac 0.95 + 0.15 ^de

Values are expressed as mean ± SD of seven mice

GBT = Guibi - tang purchase from HANZUNG PHARM. CO. LTD. (Daejeon, Korea)

LPO = lipid peroxidation; MDA = malondialdehyde;

SOD = superoxide dismutase; CAT = catalase

^a p <0.01 as compared with vehicle control by LSD test

^b p <0.01 and ^c p <0.05 compared with gefitinib single treated group by LSD test

^d p <0.01 as compared with vehicle control by MW test

^e p <0.01 as compared with gefitinib single treated group by MW test

The liver lipid peroxidation showed a change of 63.94% point compared to the vehicle control group at 160 mg / kg of gefitinib alone. In the group treated with 400 mg / kg of VIP alone, 400, 200 or 100 mg / kg of VIP, and 160 mg of gefitinib / kg, respectively, compared to the control group.

GSH content in hepatic tissues was -52.17% in the gefitinib 160 mg / kg group compared to the vehicle control group. In the group treated with 400 mg / kg alone, VIP 400, 200 or 100 mg / kg and gefitinib compared with gefitinib 160 mg / kg alone, 113.88, 60.66, 45.10 and 36.00%, respectively.

The intracavernous CAT activity showed a -40.26% point change in the gefitinib 160 mg / kg alone group compared with the medium control group. In the group treated with 400 mg / kg alone, VIP 400, 200 or 100 mg / kg and gefitinib and 71.33, 54.51, 31.50 and 27.16%, respectively, compared to the gefitinib 160 mg / kg alone group.

SOD activity in hepatic tissues was -73.35% compared to the control group at 160 mg / kg of gefitinib alone. In the group treated with 400 mg / kg alone, VIP 400, 200 or 100 mg / kg and gefitinib compared with gefitinib 160 mg / kg alone, 281.52, 131.96, 98.48 and 45.22%, respectively.

In this Example 2, a gefitinib toxicity reduction effect of Guibitin was evaluated using a mouse as a part of the integrated medical study of gefitinib and guibitin for lung cancer patients.

As a result, mortality and clinical symptoms related to the administration of gefitinib or guinea pig were not observed during the experimental period. However, in the case of gefitinib 160 mg / kg alone, the weight gain of spleen, liver and subcutaneous lymph node, decrease of RBC, HGB and HCT, And ALB and A / G were decreased. In addition, there was a significant decrease in ALB and A / G levels, especially in the presence of splenic red water And lymph node enlargement of submandibular gland, and spleen and submandibular gland carcinoma due to local necrotic lesions in association with inflammatory cell infiltration. In addition, increased liver lipid peroxidation and decreased contents or activity of endogenous antioxidants and related enzymes (GSH, SOD and CAT) were respectively recognized.

In addition, the anemia and hepatotoxicity of these gefitinibs and the associated secondary spleen and lymph node enlargement were significantly inhibited by the combined use of 100, 200 or 400 mg / kg of VIP, and significant lipid peroxidation And antioxidant defensive system, respectively. In conclusion, the combination of guitin and gefitinib in patients with lung cancer significantly reduced the general toxic symptoms of gefitinib (anemia and liver toxicity) without affecting the bioavailability of gefitinib. It is expected that it will provide a new treatment method which is very useful for the treatment. On the other hand, in the case of VIP alone 400 mg / kg, significant weight, clinical symptoms, hematology, blood biochemistry, gross autopsy and histopathological changes were not observed compared with the medium control group.

The mice of the group administered with the medium control, gefitinib, and VIP water alone group, 400, 200 or 100 mg / kg of all three doses of VIP water, and 5 minutes of the gefitinib 160 mg / kg used in Example 2 were divided into weight categories [Plata and Murphy, 1972; Yamaguchi et al., 1983], respectively. On the other hand, the decrease in the body weight gain during the day 14 to 27 days (p <0.05), which was confirmed to be confined to the group treated with Guizui 100 mg / kg and gefitinib compared to the vehicle control group, It is difficult to consider it as a symptom.

The histopathological examination of the hepatic discoloration findings in the case of gefitinib 160 mg // kg alone in this Example 2 was observed due to local necrosis accompanied by inflammatory cell infiltration in the liver, . Reduced activity of antioxidant enzymes SOD and CAT [Cheeseman and Slater, 1993] was also recognized, along with a decrease in the content of endogenous antioxidant GSH [Odabasoglu et al., 2006] as well as an increase in lipid peroxidation in the liver [Comporti, 1985] , An increase in serum AST, ALT and LDH levels and a decrease in albumin content due to liver damage, which are blood chemistries indicating liver damage [Sodikoff, 1995]. Thus, previous studies [Cohen et al., 2004; Festuccia et al., 2005; Li et al., 2009; Sylvester, 2012], it appears that the administration of gefitinib resulted in liver toxicity due to damage to the liver antioxidant defense system. In addition, there was a significant increase in serum globulin content and associated A / G, splenic and subclinical lymphocyte accumulation and associated spleen and subclinical lymph node enlargement and weight gain, increased WBC, lymphocyte and monocyte ratio and associated neutrophil leukocyte ratio (Sodikoff, 1995), which is a secondary change due to chronic inflammation due to hepatic injury caused by gefitinib administration. In addition, changes in the lymphatic system, spleen, and submandibular glands due to gefitinib were significantly inhibited by the combined administration of 400, 200, and 100 mg / kg of VIP water. In particular, gefitinib 160 mg / kg alone (P <0.01 or p <0.05), respectively. The increase of GSH content and the increase of SOD and CAT activity were observed in the group treated with 400, 200 or 100 mg / kg of geuvitin and gefitinib. Therefore, it is considered that Guibitang significantly inhibits the hepatic damage of gefitinib by activating the hepatic antioxidant defense system.

Reductions in RBC, HGB and HCT in hematologic tests indicate anemia [Sodikoff, 1995], and the possibility of anemia induced by gefitinib administration is well known [Norman, 2001; Yoshimura et al., 2004; Barni et al., 2012]. In Example 2, a significant decrease in RBC, HGB and HCT was observed in the case of gefitinib alone, but the increase in RBC, HGB and HCT in all three doses of VIP was more than that in the case of only 160 mg / kg of gefitinib, Concomitant use of guinea pigs also significantly inhibited gefitinib anemia.

On the other hand, slight pulmonary congestion, thrombocytopenia, spleen atrophy and subcutaneous lymphocytopenia observed at the time of gross autopsy, mild pulmonary hyperemia observed at histopathologic examination, subcutaneous lymphocytopenia and supratentorial cystic findings were observed in all experimental groups including medium control It is sporadically observed and is thought to be an accidental lesion, not a toxic symptom due to the administration of the test substance. These symptoms are rarely seen in normal mice.

Through the above Example 2, the administration of Juvitang 400, 200, or 100 mg / kg for 5 minutes or less can significantly inhibit hepatic damage due to gefitinib-induced anemia and antioxidant defense system through immune regulation and antioxidative effect .

Therefore, it was concluded that the combined administration of the VIP water of 100 mg / kg or more within 5 minutes did not affect the bioavailability of gefitinib but immunosuppression and hepatic damage by gefitinib were significantly reduced by immunological activity. The combination of gefitinib and gujitinib is expected to provide a new treatment method that is very useful in the treatment and management of oriental herbal medicine.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

A composition for the treatment of lung cancer comprising gefitinib and guinea pig extract as an active ingredient, wherein said Guizui extract is selected from the group consisting of Polygala tenuifolia Willd., Zizyphus jujuba var. Inermis (Bunge) Rehder, Angelica gigas N.), Aucklandia lappa Decne., Dimocarpus longan Lour, Glycyrrhiza uralensis Fisch, Zingiber officinale Roscoe, Zizyphus jujuba Miller, Panax ginseng CAMeyer. Wherein the composition comprises atractylodes ovata (Thunb.) DC., Astragalus membranaceus Bunge, and Poria cocos Wolf.
delete delete The method according to claim 1,
Wherein the gefitinib and guinea pig extract are formulated prior to mixing or separately formulated.
The method according to claim 1,
Wherein the gefitinib and guinea pig extract are administered parenterally, orally, locoregionally, or transdermally.
The method according to claim 1,
Wherein said Guizotui extract administration starts between 1 minute and 4 hours after administration of said gefitinib.

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