WO2013100500A1

WO2013100500A1 - Pharmaceutical composition for treating cancer, comprising peptide, 5-fluorouracil, and mature dendritic cells

Info

Publication number: WO2013100500A1
Application number: PCT/KR2012/011291
Authority: WO
Inventors: 배외식; 김상두
Original assignee: 성균관대학교산학협력단
Priority date: 2011-12-26
Filing date: 2012-12-21
Publication date: 2013-07-04
Also published as: KR101361445B1; KR20130074325A

Abstract

The present invention relates to a pharmaceutical composition for treating cancer, comprising the WKYMVm peptide having the sequence of SEQ ID NO:1, and containing an effective amount of 5-fluorouracil and/or mature dendritic cells. According to the present invention, the pharmaceutical composition enhances in vivo immune reaction, thereby showing an increase of anticancer effects, low side effects, a high suppression of metastasis, and the like.

Description

Peptides, 5-fluorouracil, and a pharmaceutical composition for treating cancer comprising mature dendritic cells

The present invention relates to a pharmaceutical composition for treating cancer, comprising a WKYMVm peptide having a sequence of SEQ ID NO: 1, and comprising 5-fluorouracil and / or mature dendritic cells.

Existing anticancer drugs intervene in various metabolic pathways of active cell division and inhibit the synthesis of nucleic acids or cause cytotoxicity, and most of them show anticancer activity. In particular, damage to tissue cells active in cell division causes serious side effects such as vomiting, gastrointestinal disorders, alopecia, leukopenia due to myelosuppression. In addition, there is a problem in obtaining resistance to anticancer drugs and recurrence due to metastatic capacity and genetic instability of cancer cells.

Recently, various studies have been actively conducted to develop effective anticancer drugs, such as the development of a target-oriented anticancer agent that can minimize damage to normal cells, a method of minimizing side effects by reducing the dose of anticancer drugs, and a method of enhancing the immune system of the body. It's going on. However, most anticancer drugs still have problems such as high side effects and recurrence risk.

In order to effectively treat cancer as described above, there is a demand for the development of an effective anticancer agent that has a high anticancer effect and can reduce side effects and the risk of recurrence.

The present invention has been made to solve the above problems in the prior art, comprising a WKYMVm peptide having a sequence of SEQ ID NO: 1, 5-fluorouracil and / or mature dendritic cells (mature dendritic) It is an object of the present invention to provide a pharmaceutical composition for treating cancer comprising an effective amount of cells).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for treating cancer comprising an effective amount of a WKYMVm peptide having a sequence of SEQ ID NO: 1 and 5-fluorouracil.

In another aspect, the present invention provides a pharmaceutical composition for treating cancer comprising an effective amount of the WKYMVm peptide having a sequence of SEQ ID NO: 1 and mature dendritic cells.

The present invention also provides a pharmaceutical composition comprising an effective amount of a WKYMVm peptide having a sequence of SEQ ID NO: 1, 5-fluorouracil, and mature dendritic cells.

In one embodiment of the present invention, the composition is characterized in that it is used to suppress cancer derived from colon cancer or lymph cells.

In another aspect, the present invention provides a method of increasing the anti-cancer treatment effect by using a combination of WKYMVm peptide having a sequence of SEQ ID NO: 1 and 5-fluorouracil.

In another aspect, the present invention provides a method of increasing the anti-cancer treatment effect by co-administration of WKYMVm peptide having a sequence of SEQ ID NO: 1 and mature dendritic cells.

In another aspect, the present invention provides a method of increasing the anti-cancer treatment effect by concurrently administering the WKYMVm peptide having a sequence of SEQ ID NO: 1, 5-fluorouracil and mature dendritic cells.

Concomitant administration of 5-fluorouracil and / or mature dendritic cells, comprising the WKYMVm peptide according to the present invention, can effectively reduce side effects and resistance caused by anticancer drugs because it can increase the immunity of the body and reduce the amount of anticancer drugs. In addition, it is expected to be applicable to the treatment of cancer because it can significantly increase the anticancer effect. In addition, it is expected that the type of anticancer agent can be freely used to treat various types of cancer.

1 is a schematic diagram of a method of administering the WKYMVm peptide, 5-fluorouracil, mature dendritic cells.

Figure 2 shows the results of measuring the anticancer effect of WKYMVm peptide, 5-fluorouracil, mature dendritic cells alone and in combination administration.

Figure 3 shows the results of measuring the effect of cancer cell death induced by WKYMVm peptide, 5-fluorouracil, mature dendritic cells alone and in combination.

Figure 4 shows the results of measuring the effect of inducing the migration of immune cells to tumor tissue by co-administration of WKYMVm peptide, 5-fluorouracil, mature dendritic cells.

5 shows the effect of anti-CD8 antibody, anti-CD4 antibody and anti-asialo-GM1 antibody on the effect of inducing the migration of immune cells into tumor tissue by coadministration of WKYMVm peptide, 5-fluorouracil, and mature dendritic cells. It is a figure which shows the result of having measured.

Figure 6 shows the results of measuring the anticancer effect of CD8 T lymphocytes and NK cells induced by the co-administration of WKYMVm peptide, 5-fluorouracil, and mature dendritic cells.

Fig. 7 shows the results of measuring the effect of the combined administration of WKYMVm peptide, 5-fluorouracil, and mature dendritic cells on the amount of cytokines in cancer tissues or peripheral blood.

Fig. 8 shows the results of measuring cancer metastasis inhibitory effect of WKYMVm peptide, 5-fluorouracil, and mature dendritic cell co-administration.

Fig. 9 shows the results of measuring anticancer effects of WKYMVm peptide, 5-fluorouracil, and mature dendritic cell co-administration.

Previously, the inventors have previously described peptide ligands (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ : SEQ ID NO: 1, hereinafter, “WKYMVm that increase leukocyte cells such as monocytes, neutrophils, etc.). Peptides ”(Bae et al., 1999, J. Leukoc. Biol. 65, 241-248). Therefore, the present inventors have completed the present invention by studying a method of increasing the anticancer effect while reducing the amount of the anticancer agent by simultaneously using an anticancer agent while promoting an immune response using the WKYMVm peptide.

One of the representative anticancer drugs, 5-fluorouracil (5-FU) has a structure similar to that of thymine, a precursor of DNA, which inhibits DNA synthesis of cancer cells and shows anticancer effects. Widely used in mechanical arms. However, there are limitations due to side effects such as bone marrow depression and diarrhea. In addition, dendritic cells have been suggested to be able to be used for immune chemotherapy by promoting the immune response in the body, many studies have been conducted, but the situation is still insufficient in the anti-cancer effect.

Therefore, the present inventors tried to use WKYMVm peptide and dendritic cells that can promote the immune response in the body and 5-fluorouracil, an anticancer agent, for use in anticancer treatment.

In one embodiment of the present invention, administration of 5-fluorouracil and / or mature dendritic cells in combination with WKYMVm peptide, 5-fluorouracil, or mature dendritic cells alone increases the volume of cancer. It was confirmed that it can be reduced by about 5 times (see Example 1), and in other examples, it was confirmed that the metastasis of cancer can be inhibited by 5 times or more through co-administration. It was confirmed that the survival rate can be significantly increased (see Examples 6 and 7).

From these results, co-administration of 5-fluorouracil and / or mature dendritic cells containing WKYMVm peptide having the sequence of SEQ ID NO. Not only can the anti-cancer effect be significantly increased, but also the side effects can be reduced and cancer metastasis can be suppressed. Accordingly, the present invention includes a WKYMVm peptide having a sequence of SEQ ID NO: 1, and provides a method of enhancing anti-cancer treatment effect by concurrently administering 5-fluorouracil and / or mature dendritic cells. do.

In another aspect, the present invention provides a pharmaceutical composition for treating cancer comprising a WKYMVm peptide having a sequence of SEQ ID NO: 1, an effective amount of 5-fluorouracil and / or mature dendritic cells to provide. There is no restriction on the type of cancer to which the pharmaceutical composition for treating cancer of the present invention can be applied. Preferably, it can be used to suppress colon cancer or cancer derived from lymphoid cells.

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

Another aspect of the present invention provides a pharmaceutical composition comprising a WKYMVm peptide having a sequence of SEQ ID NO: 1, comprising 5-fluorouracil and / or mature dendritic cells as an active ingredient A method of treating cancer is provided by administering a pharmaceutically effective amount to a subject. In the present invention, an individual means a subject in need of treatment of a disease, and more specifically, a human, or a non-human primate, a mouse, a rat, a dog, a cat, a horse, a cow, and the like. Mean mammal. In addition, it will be apparent to those skilled in the art that the pharmaceutically effective amount in the present invention may be variously adjusted in the range depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. Do.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 100 mg / kg body weight per day, more preferably 0.01 to 30 mg / kg body weight. Administration may be administered once a day or may be divided several times. WKYMVm peptide, 5-fluorouracil, and / or mature dendritic cells having the sequence of SEQ ID NO: 1 of the present invention are preferably 0.0001 to 10% by weight, based on the total weight of the total composition, preferably May be present in an amount from 0.001 to 1% by weight.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. The method of administration is not limited, and may be administered by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dural, or intra cerbroventricular injection.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[실시예]EXAMPLE

실시예 1. WKYMVm 펩타이드, 5-FU, 성숙수지상세포의 항암효과 측정Example 1. Measurement of anticancer effect of WKYMVm peptide, 5-FU, mature dendritic cells

To determine whether the anti-cancer effect is obtained when WKYMVm peptide, 5-fluorouracil (5-FU), and mature dendritic cells (mDC) are used in combination, bone marrow cells were first added to DC medium (10%). Incubated for 5 days in RPMI 1640 medium containing fetal bovine serum, 2 mM L-glutamine, 50 uM beta-mercaptoethanol, and antibiotics, GM-CSF (1,000 U / mL) and IL-4 (500 U / mL) on

days

0, 2 and 4. mL) was treated. After 5 days, the cells were collected and transferred to a new DC medium, followed by incubation for 2 days by adding lipopolysaccharide (100ng / mL), CpG oligodeoxynucleotide 1826 (10μg / mL) and CT-26 cell lysate (100μg / mL). Mature dendritic cells were prepared. Subcutaneous injection of CT-26 cell line (5X10 ⁵ cells / 100μL phosphate buffered saline (PBS)), a colon cancer cell line, was induced in 6-8-week-old BALB / c male mice to induce cancer. -3 days). Subcutaneous injection of 100 μg / 100 μL of 5-FU on day 0 (0 day) and day 1 (1 day), respectively, and on the second day (2 day) WKYMVm peptide (100 μg / 100 μL) at 12 hour intervals Four subcutaneous injections and maturated dendritic cells (1 × 10 ⁶ cells / 100 μL) were injected into the tail vein twice at 12 hour intervals. Subsequently, 5-FU (100 μg / 100 μL) was injected subcutaneously at 4 days and 5 days. Thereafter, the mice were repeatedly dosed with WKYMVm peptide, 5-FU, and mature dendritic cells twice a week for 3 weeks. As a control group, mice treated with nothing, WKYMVm peptide, 5-FU, or mature dendritic cells alone or in combination of the two groups were divided and administered with the experimental group. And the volume of the cancer was quantified using the following formula. A schematic diagram of the experimental procedure is shown in FIG. 1, and the experimental results are shown in FIG. 2.

The volume of cancer (mm ³⁾ = length (mm) X width (mm) ^2/2

As shown in Figure 2, when treated with WKYMVm peptide, 5-FU, or mature dendritic cells, respectively, the volume of the cancer slightly reduced, while in the case of co-administration of WKYMVm peptide and mDC, or mDC and 5-FU The volume of the cancer was reduced. In addition, when the WKYMVm peptide, 5-FU, and mature dendritic cells were administered in combination, it was confirmed that the cancer volume was significantly reduced. Through the above results, the anti-cancer effect was increased even when the two doses in combination, it was confirmed that the anti-cancer effect is significantly increased when all three together.

실시예 2. WKYMVm 펩타이드, 5-FU, 성숙수지상세포의 예정된 세포사멸 유도효과 측정Example 2 WKYMVm Peptide, 5-FU, Determination of Induced Apoptosis Induction Effect of Mature Dendritic Cells

To determine whether the anticancer effect of the combination of WKYMVm peptide, 5-fluorouracil (5-FU), and mature dendritic cell (mDC) is due to induction of apoptosis, the same method as in Example 1 39 days later, the cancer was removed from the mouse, fixed with 10% neutral phosphate buffered formalin (NBF), and then cut into several layers and stained with paraffin. For morphological analysis, stained with hematoxylin and eosin (H & E) and observed through light microscopy. For immunological analysis, anti-murine cleaved caspase-3 antibody or anti-murine FAS antibody was used. After secondary binding, anti-murine IgG bound to FITC or anti-murine IgG bound to Alexa 594 was used for secondary binding, followed by fluorescence microscopy. Also stained using TUNEL (Terminal deoxy-nucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling) staining method. The results are shown in FIG.

As shown in Figure 3, TUNEL staining, FAS and Caspase-3 staining all showed similar results. When treated with WKYMVm peptide or mature dendritic cells alone, the cells were almost dead, and when treated with 5-FU alone, some of the cells were dead. In addition, even when two were administered in combination, some cells were confirmed to be dead, but in combination with WKYMVm peptide, 5-FU, and mature dendritic cells, almost all of the cells were confirmed to be dead. Through the above results, co-administration of KYMVm peptide, 5-FU, and mature dendritic cells was confirmed to induce apoptosis and kill cancer cells.

실시예 3. WKYMVm 펩타이드, 5-FU, 성숙수지상세포의 항암면역반응 유도효과 측정Example 3. Determination of anti-cancer immune response effect of WKYMVm peptide, 5-FU, mature dendritic cells

Anti-murine CD3 conjugated with Fluorescein isothiocyanate (FITC) to determine whether co-administration of WKYMVm peptide, 5-fluorouracil (5-FU), and mature dendritic cells (mDC) induce anticancer immune responses Cancer was treated in the same manner as in Example 2 using an antibody, an anti-murine CD4 antibody bound with PE (Phycoerythrin), an anti-murine CD11b antibody bound with FITC, or an anti-murine DX5 antibody bound with PE. Stained and observed through a fluorescence microscope. The results are shown in FIG.

As shown in FIG. 4, co-administration of WKYMVm peptide, 5-fluorouracil (5-FU), and mature dendritic cells (mDC) induced CD8 T lymphocytes and NK (natural killer) cells significantly around cancer. It was confirmed that. In addition, CD4 T lymphocytes were found to gather weakly around the cancer.

In addition, rat anti-mouse CD8 antibodies, rat anti-mouse asialo-GM1 antibodies, or rat anti-mouse CD4 antibodies to confirm that CD8 T lymphocytes and natural killer (NK) cells do not influence each other to gather around cancer. 100 μg of each was intraperitoneal injection one day prior to subcutaneous injection of cancer into the mouse, the day of subcutaneous injection, and 5 days after subcutaneous injection. And stained in the same manner as above and observed through a fluorescence microscope. The results are shown in FIG.

As shown in FIG. 5, when the anti-CD8 antibody was treated, CD8 T lymphocytes were not induced around the cancer, but NK cells were confirmed to be collected around the cancer. In addition, anti-asialo-GM1 antibody treated NK cells did not induce cancer, but CD8 T lymphocytes gathered around cancer, and anti-CD4 antibody treated CD8 T lymphocytes and NK cells. It was confirmed that the induced around.

실시예 4. WKYMVm 펩타이드, 5-FU, 및 성숙수지상세포의 병용투여에 의해 유도된 CD8 T lymphocyte 및 NK 세포의 항암효과 측정Example 4 Determination of Anticancer Effects of CD8 T Lymphocytes and NK Cells Induced by Combination of WKYMVm Peptide, 5-FU, and Mature Dendritic Cells

An anticancer immune response occurs when various types of leukocytes work together. Therefore, in order to determine whether CD8 T lymphocytes and NK cells induced by the co-administration of WKYMVm peptide, 5-FU, and mature dendritic cells induce an anticancer immune response, rat anti-mouse CD8 antibody, rat anti-mouse asialo- 100 μg of the GM1 antibody or the rat anti-mouse CD4 antibody was intraperitoneally injected in the same manner as in Example 3, and then the volume of the cancer was measured by date. The results are shown in FIG.

As shown in Figure 6, when administered in combination, the volume of the cancer was significantly reduced, but when administered with the antibody was confirmed that the volume of the cancer appears similar to the untreated control group. However, when the rat anti-mouse CD4 antibody was treated, it was confirmed that the cancer volume was the same as that of the co-administered mouse. Through the above results, it was confirmed that CD8 T lymphocytes and NK cells induced by co-administration of WKYMVm peptide, 5-FU, and mature dendritic cells play an important role in anticancer effects.

실시예 5. WKYMVm 펩타이드, 5-FU, 및 성숙수지상세포의 병용투여가 암세포의 사이토킨 분비에 미치는 효과 측정Example 5 Determination of Effect of WKYMVm Peptide, 5-FU, and Mature Dendritic Cells on Cytokine Secretion of Cancer Cells

To determine the effect of co-administration of WKYMVm peptide, 5-FU, and mature dendritic cells on cytokine secretion of cancer cells, 42 days after co-administration of WKYMVm peptide, 5-FU, and mature dendritic cells in mice Blood was extracted from mouse lysates of the mouse or the heart of the mouse and subjected to an enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay (ELISA). The results are shown in FIG.

As shown in FIGS. 7A and 7B, when the WKYMVm peptide, 5-FU, and mature dendritic cells were co-administered, interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) were rapidly increased. there was. In addition, even when only two WKYMVm peptides and mature dendritic cells were administered in combination, it was confirmed that interleukin-12 was rapidly increased.

In addition, administration of 100 μg of rat anti-mouse CD8 antibody, rat anti-mouse asialo-GM1 antibody, or rat anti-mouse CD4 antibody in the same manner as in Example 3 resulted in a sharp reduction in the amount of interferon-gamma and interleukin-12. I could confirm that. The results indicate that CD8 T lymphocytes and NK cells induced by co-administration of WKYMVm peptide, 5-FU, and mature dendritic cells produce interferon-gamma and interleukin-12.

실시예 6. WKYMVm 펩타이드, 5-FU, 및 성숙수지상세포 병용투여의 암전이 억제효과 측정Example 6 Determination of Cancer Metastasis Inhibition Effect of WKYMVm Peptide, 5-FU, and MEDG Combination

In order to prevent cancer from recurring, it was confirmed that co-administration of WKYMVm peptide, 5-FU, and mature dendritic cells suppresses cancer metastasis, since it is most important to prevent cancer metastasis. For natural lung metastasis experiments, cancer was induced in mice using CT-26 cell line as in Example 1, and WKYMVm peptide, 5-FU, and mature dendritic cells were co-administered for 3 weeks. And the number of cancer nodule on the lung surface of the mouse was measured on day 42. In addition, for artificial lung metastasis experiment, CT-26 cell line was injected into the tail of the mouse and WKYMVm peptide, 5-FU, and mature dendritic cells were co-administered for 2 weeks, followed by the same method as in Example 2. Lungs were stained and observed with hematoxylin and eosin (H & E). The results are shown in FIG.

As shown in FIG. 8A, when the CT-26 cell line was injected subcutaneously, the cancer naturally metastasized to lung tissue, and when the WKYMVm peptide, 5-FU, and mature dendritic cells were used in combination, it was confirmed that the cancer almost did not metastasize. Could.

In addition, as shown in FIG. 8B, even when artificially metastasized cancer to lung tissue, the cancer was almost metastatic when WKYMVm peptide, 5-FU, and mature dendritic cells were administered in combination.

실시예 7. WKYMVm 펩타이드, 5-FU, 및 성숙수지상세포 병용투여의 항암효과 측정Example 7 Determination of Anticancer Effect of WKYMVm Peptide, 5-FU, and MDF

In order to confirm the substantial anticancer effect of the co-administration of the WKYMVm peptide, 5-FU, and mature dendritic cells, cancers were induced in mice in the same manner as in Example 1, followed by the WKYMVm peptide, 5-FU, and mature dendritic cells alone. Or co-administered and the survival rate of the mice was measured. The results are shown in Figure 9A.

As shown in FIG. 9A, when the WKYMVm peptide, 5-FU, and mature dendritic cells were coadministered, more than 80% of mice survived after 80 days.

In addition, in order to determine whether the antitumor effect when co-administered with CD8 T lymphocytes and / or NK cells, the CT-26 cell line was raised to a concentration of 1 × 10 ⁶ cells, and then administered to mice. The results are shown in Figure 9B.

As shown in FIG. 9B, when the concentration of CT-26 cell line was increased, survival rate was reduced by 40% at 80 days even when KYMVm peptide, 5-FU, and mature dendritic cells were co-administered. In addition, the survival rate was slightly increased when CD8 T lymphocytes or NK cells were used alone with KYMVm peptide, 5-FU, and mature dendritic cells, but 80 days after CD8 T lymphocytes and NK cells were coadministered. Survival rate was found to be more than 80%.

Through the above results, the combination of WKYMVm peptide, 5-FU, and mature dendritic cells was found to significantly increase the anti-cancer effect compared to the treatment alone, and the combination of CD8 T lymphocytes and NK cells. It was confirmed that the anticancer effect was further increased.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Since the pharmaceutical composition for cancer treatment comprising the WKYMVm peptide according to the present invention and containing an effective amount of 5-fluorouracil and / or mature dendritic cells can reduce the amount of anticancer agent by enhancing the body's immunity, Side effects and resistance can be effectively reduced. In addition, when combined with various types of anticancer drugs can significantly increase the anticancer effect, it can be used as a pharmaceutical composition for treating various types of cancer.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY

<120> Composition comprising peptide, 5-fluorouracil, and mature

dendritic cells for cancer treatment

<130> PCT01606

<150> KR 10-2011-0142340

<151> 2011-12-26

<160> 1

<170> KopatentIn 2.0

<210> 1

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> WKYMVm peptide

<400> 1

Trp Lys Tyr Met Val Met

1 5

Claims

A pharmaceutical composition for treating cancer, comprising an effective amount of a WKYMVm peptide having the sequence set forth in SEQ ID NO: 1, and 5-fluorouracil.
A pharmaceutical composition for treating cancer, comprising an effective amount of a WKYMVm peptide having a sequence set forth in SEQ ID NO: 1, and a mature dendritic cell.
A pharmaceutical composition comprising an effective amount of a WKYMVm peptide having a sequence set forth in SEQ ID NO: 1, 5-fluorouracil, and mature dendritic cells.
The method according to any one of claims 1 to 3,

The composition is characterized in that it is used to inhibit cancers derived from colon cancer or lymph cells.
A method of enhancing the anticancer therapeutic effect by co-administration of WKYMVm peptide having the sequence set forth in SEQ ID NO: 1, and 5-fluorouracil.
A method of enhancing the anticancer treatment effect by concurrently administering a WKYMVm peptide having the sequence set forth in SEQ ID NO: 1 and a mature dendritic cell.
A method of enhancing the anticancer treatment effect by concurrently administering a WKYMVm peptide having a sequence as set forth in SEQ ID NO: 1, 5-fluorouracil, and mature dendritic cells.