KR20240102304A - Novel gemcitabine conjugates and uses thereof - Google Patents

Abstract

Provided is a gemcitabine conjugate bound to sialyllactose. The gemcitabine conjugate can increase the penetration rate of gemcitabine into tissues, especially the brain and pancreas, and can be used to treat neoplastic diseases in those tissues. Additionally, to compensate for the low permeability, a high dose is administered, allowing gemcitabine, which has side effects such as toxicity, to be administered at a low dose.

Description

Novel gemcitabine conjugates and uses thereof {NOVEL GEMCITABINE CONJUGATES AND USES THEREOF}

The present invention relates to a novel gemcitabine conjugate with improved delivery to the brain and pancreas.

Gemcitabine is a nucleoside analog, used as a single agent or combined with other chemicals for the treatment of various solid tumors such as brain cancer, pancreatic cancer, testicular cancer, breast cancer, ovarian cancer, non-small cell lung cancer, and bladder cancer. It is used in combination with therapeutic agents. Gemcitabine is a powerful chemical treatment that can inhibit DNA chain elongation and act as a radiosensitizer that can increase tissue sensitivity to radiation. However, gemcitabine has a short half-life in plasma, has side effects such as toxicity when taken in high doses, and is reported to be less effective due to low penetration into tumor tissues such as the brain and pancreas. Therefore, if there is a way to increase the tissue delivery of gemcitabine, higher efficacy can be achieved even at low doses despite the short half-life.

Meanwhile, the blood-brain barrier (BBB) is a tight junction with a highly electrical resistance of more than 0.1 Ω·m between related pericytes and astrocytes and adjacent vascular endothelial cells. It is a cellular barrier composed of highly selective permeability that separates circulating blood from brain extracellular fluid in the central nervous system (CNS), and serves as a gateway to control the entry and exit of substances.

The blood-brain barrier blocks bacteria, pathogens, and potentially dangerous substances in the blood that can be transported through the blood from being delivered to the brain, but it also blocks most central nervous system drugs from being delivered to the brain, resulting in low drug efficiency, and to compensate for this. For example, as in the case of gemcitabine mentioned above, these drugs are administered in high doses, causing serious side effects in surrounding organs. Therefore, there is a need to discover an efficient drug delivery method that can penetrate the blood-brain barrier to prevent negative systemic effects and at the same time ensure the therapeutic effect of the drug. A brain shuttle has previously been developed using an antibody against the transferrin receptor expressed in cerebrovascular epithelial tissue. However, this is a polymer material and is much larger in size than small molecule substances such as small molecule compounds and peptides, so a single antibody and drug If only one is added, the drug delivery efficiency decreases. To solve these problems, research is being conducted on various drug delivery systems using small molecule compounds, peptides, and aptamers with molecular weights smaller than antibodies.

The present inventors confirmed through experiments that sialyllactose with a low molecular weight can increase the blood-brain barrier permeability of the substance when combined with other substances, and that gemcitabine combined with sialyllactose is well delivered not only to the brain but also to the pancreas. By confirming that this was the case, the present invention was completed.

The problem to be solved by the present invention is to provide a gemcitabine conjugate with improved penetration into tissues such as the brain and pancreas using sialyllactose.

The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above technical problem, one aspect of the present invention provides a gemcitabine conjugate containing sialyllactose and gemcitabine.

Here, the sialyllactose may be 2,3-sialyllactose or 2,6-sialyllactose.

The sialyllactose and gemcitabine may be connected by a linker. In one embodiment, the linker can be used without limitation as long as it has a structure that is structurally linked to both sialyllactose and gemcitabine. For example, the linker is from the group consisting of sialyllactose and -CONH-, -C(=O)-, -NH-, -0-, =N-, -SS- and -N(CH ₃ )- It can be selected and linked, and is selected from the group consisting of gemcitabine and -CONH-, -C(=O)-, -NH-, -0-, =N-, -SS- and -N(CH ₃ )- can be connected Between both connections -(CH ₂ )a-(NHCO)b-(CH ₂ )c-, -(CH ₂ )a-(CONH)b-(CH ₂ )c-, -(CH ₂ )a-( Structures of CO)b-(CH ₂ )c-, -(CH ₂ )a-(NH)b-(CH ₂ )c- and -(CH ₂ )a-(O)b-(CH ₂ )c- Can be selected from the group having. At this time, a may be an integer from 0 to 10, b may be an integer from 0 to 1, and c may be an integer from 0 to 10.

The linker may be characterized as a cleavable linker that promotes release after delivery of gemcitabine to tissues.

The linker may be characterized as binding to the hydroxy group (-OH) of the lactose portion of sialyllactose.

The gemcitabine conjugate has a structure represented by Formula 1 or Formula 2 below, and in the two formulas, L may be a linker:

[Formula 1]

[Formula 2]

The gemcitabine conjugate may have a structure represented by Formula 3 or Formula 4 below:

[Formula 3]

[Formula 4]

The gemcitabine conjugate may be characterized by improved brain or pancreas delivery rate compared to gemcitabine when administered in vivo.

Another aspect of the present invention provides a pharmaceutical composition for treating neoplastic diseases comprising the above-described gemcitabine conjugate.

The gemcitabine conjugate according to an embodiment of the present invention can effectively deliver gemcitabine, which has a low brain passage efficiency, to the brain and pancreas, and can therefore be used to treat neoplastic diseases of the brain or pancreas without toxicity problems caused by administration of high doses of the drug.

However, the effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 shows the results of an experiment comparing the brain penetration of Cy5.5 bound to lactose and sialyllactose.
Figure 2 shows the results of an experiment comparing the brain penetration of Cy5.5 bound to sialic acid and sialyllactose.
Figure 3 is a structural formula of a gemcitabine conjugate according to an embodiment of the present invention.
Figure 4 shows the results of an experiment comparing the cytotoxic effect of the gemcitabine conjugate according to an embodiment of the present invention with that of gemcitabine in different cell lines.
Figure 5 shows the results of an experiment confirming brain penetration of the gemcitabine conjugate in animals according to an embodiment of the present invention.

Hereinafter, the present invention will be described with reference to the attached drawings. However, the present invention may be implemented in various different forms and, therefore, is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts unrelated to the description are omitted, and similar parts are given similar reference numerals throughout the specification.

Throughout the specification, when a part is said to be “connected (connected, contacted, combined, attached)” to another part, this means not only “directly connected” but also “indirectly connected” with another member in between. Also includes cases where it is connected to. Additionally, when a part is said to “include” a certain component, this does not mean that other components are excluded, but that other components can be added, unless specifically stated to the contrary.

The terms used herein are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “include” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

A “linker” as used herein is a moiety that connects two parts of a compound and generally includes one or more direct covalent bonds, atoms, units, or chains of atoms. For example, atoms are oxygen (-O-) and sulfur (-S-), units are NR8, C(O), C(O)NH, SO, SO2 and SO2NH, and chains of atoms are substituted or unsubstituted. Unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl ), heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl (alkylarylalkynyl), alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylaryl Alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkyl teroarylalkenyl) , alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkyl, alkynylheteroarylalkynyl, alkynylheteroarylalkynyl, ), alkyl Heterocylheterocylalalkenyl, alkylhererocylalkylalkyl, alkenylheterocyclylkyl (alkenyl) TerocyclylLalkenyl), alkenylheterocyticyl alkinyl, alki alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, Alkylheteroaryl, alkenylheteroaryl and alkynylhereroaryl groups (one or more methylenes in the middle or end are O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, linked/substituted with a substituted or unsubstituted heterocyclic group; R8 may be selected from hydrogen, acyl, aliphatic or substituted aliphatic group, etc. The linker may preferably have 1-48 atoms, and more preferably 4-48 atoms.

In order to achieve the above technical problem, one aspect of the present invention provides a gemcitabine conjugate containing sialyllactose and gemcitabine.

The sialyllactose is a type of human milk oligosaccharide (HMO) most abundant in colostrum of breast milk, and is a form in which sialic acid is attached to lactose (milk sugar). Depending on the position where sialic acid binds to lactose, 2,3-sialyllactose represented by Formula 5 below (GCB100 in examples to be described later) and 2,6-sialyllactose represented by Formula 6 (examples to be described later) In GCB200), two types may exist.

[Formula 5]

[Formula 6]

The sialyllactose may be 2,3-sialyllactose or 2,6-sialyllactose.

The sialyllactose can be extracted from breast milk, synthesized chemically, or synthesized using an enzymatic reaction. Preferably, it can be mass-produced and synthesized using an enzymatic reaction that is environmentally friendly and safe because it does not use toxic catalysts or organic solvents.

The sialyllactose may be sialyllactose or a salt thereof, and the salt of sialyllactose may be, for example, a sodium salt, but is not limited thereto.

The sialyllactose and gemcitabine can be directly combined with each other. For example, the functional group of sialyllactose and the functional group of gemcitabine can be directly combined, and the direct binding does not inhibit the blood-brain barrier or pancreas penetration ability of sialyllactose and the physiological activity of gemcitabine, or allows the functional group to penetrate the blood-brain barrier or pancreas. If the bond is later broken down and sialyllactose and gemcitabine are separated, they can be combined directly without a linker. Preferably, the hydroxyl group (-OH) of the lactose portion of sialyllactose may be combined with a functional group that does not affect the physiological activity of gemcitabine.

The sialyllactose and gemcitabine may be connected by a linker. For example, the linker can bind to the functional group of sialyllactose and the functional group of gemcitabine, respectively, without inhibiting the blood-brain barrier or pancreatic permeability of sialyllactose and the physiological activity of gemcitabine. Preferably, the linker can bind to the hydroxyl group (-OH) of the lactose portion of sialyllactose and to a functional group that does not affect the physiological activity of gemcitabine.

The linker can be used without limitation as long as it has a structure that can be structurally linked to both sialyllactose and gemcitabine. For example, the linker is from the group consisting of sialyllactose and -CONH-, -C(=O)-, -NH-, -0-, =N-, -SS- and -N(CH ₃ )- It can be selected and linked, and is selected from the group consisting of gemcitabine and -CONH-, -C(=O)-, -NH-, -0-, =N-, -SS- and -N(CH ₃ )- can be connected Between both connections -(CH ₂ )a-(NHCO)b-(CH ₂ )c-, -(CH ₂ )a-(CONH)b-(CH ₂ )c-, -(CH ₂ )a-( Structures of CO)b-(CH ₂ )c-, -(CH ₂ )a-(NH)b-(CH ₂ )c- and -(CH ₂ )a-(O)b-(CH ₂ )c- Can be selected from the group having. At this time, a may be an integer from 0 to 10, b may be an integer from 0 to 1, and c may be an integer from 0 to 10.

The linker may be characterized as a cleavable linker that promotes release after delivery of gemcitabine to tissues. For example, pH sensitive linkers, peptidase-sensitive linkers, and photolabile linkers can be used. Preferably, a peptidase-sensitive linker that can be easily separated by peptidase present in the cell can be used. For example, one can choose from those that are susceptible to cleavage by peptidases, such as cathepsins B, C and D, which are peptidases preferentially expressed in tumor tissue.

The linker may be characterized as binding to the hydroxy group (-OH) of the lactose portion of sialyllactose. The linker comprises one or more alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, natural or unnatural amino acid, or a combination thereof, and the linker is -C(O)O- , ―OC(O)―, ―NHC(O)―, ―C(O)NH―, ―O―, ―NH―, ―S―, ―S―S―, ―C(O)―, ―OC (O)NH―, ―NHC(O)―O―, 〓NH―O―, 〓NH―NH― and 〓NH―N(CH3)―; Alternatively, it may be connected to the sialyllactose and the gemcitabine through any one selected from the group consisting of an amide and an ester, respectively.

The gemcitabine conjugate has a structure represented by Formula 1 or Formula 2 below,

[Formula 1]

[Formula 2]

In the above two chemical formulas, L may be a linker.

The gemcitabine conjugate may have a structure represented by Formula 3 or Formula 4 below:

[Formula 3]

[Formula 4]

The gemcitabine conjugate may be characterized by improved brain or pancreas delivery rate compared to gemcitabine when administered in vivo. As confirmed in the examples to be described later, the conjugate according to an embodiment of the present invention can penetrate the blood-brain barrier at least twice, preferably at least three times, and more preferably at least five times compared to gemcitabine.

Another aspect of the present invention provides a pharmaceutical composition for treating neoplastic diseases comprising the above-described gemcitabine conjugate.

Here, neoplastic diseases are abnormal overgrowth of tissues, often forming lumps called tumors. Tumors are largely divided into benign tumors, which grow relatively slowly and locally, and malignant tumors, which grow relatively quickly and spread and metastasize to various parts of the body, called cancer.

The pharmaceutical composition may contain the gemcitabine conjugate alone, or may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients, or diluents.

The term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to cells or humans exposed to the composition.

Furthermore, the pharmaceutical composition can be provided by mixing it with a conventionally known agent for treating neoplastic diseases. That is, the pharmaceutical composition can be administered in parallel with a known compound that has a therapeutic effect on neoplastic diseases.

The term “administration” means introducing a predetermined substance into an individual by an appropriate method, and “individual” refers to all target animals such as rats, mice, and livestock, including humans, for the purpose of treating brain diseases. As a specific example, it may be a mammal, including humans.

The route of administration of the pharmaceutical composition is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. This is included.

The pharmaceutical composition can be administered orally or parenterally, and when administered parenterally, it is preferable to select an injection method for external application through the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. It is not limited to this.

When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogeratin, etc. can be used.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Blood-brain barrier penetration experiment of sialyllactose

To confirm the possibility of using sialyllactose as a blood-brain barrier penetrating conjugate, a brain penetration experiment of sialic acid, lactose, and sialic lactose was conducted.

First, the fluorescent substance Cy5.5 (Genechem, Cat no. GCCD0064) was attached to lactose, 2,3-sialyllactose (GCB100), and 2,6-sialyllactose (GCB200), and each substance was dissolved in 10% DMSO. Samples were prepared by dissolving and diluting (final DMSO concentration was approximately 2% of the total volume). After administering each sample at 1 μmol/kg into the vein of a BALB/c nude mouse, the brain of the nude mouse was analyzed over time (1, 2, 4, 6, 8, 24 hours) using IVIS spectrum CT from Perkin Elmer. Fluorescent signals were confirmed (Figure 1A), and 24 hours after administration, nude mice were perfused with saline solution, brains were extracted, and fluorescence present in the brain was measured (Figure 1B). As a result, it was shown that Cy5.5 with two sialyllactoses attached was better delivered to the brain than lactose.

Next, the fluorescent substance Cy5.5 (Genechem, Cat no. GCCD0064) was attached to sialic acid, 2,3-sialyllactose (GCB100), and 2,6-sialyllactose (GCB200), and each substance was treated with 10 Samples were prepared by dissolving in % DMSO and then diluting (final DMSO concentration was approximately 2% of the total volume). Each sample was administered into the vein of BALB/c nude mice at 1 μmol/kg, and then analyzed over time (10 minutes, 30 minutes, 1, 2, 4, 6, 8, 24 hours) using IVIS spectrum CT from Perkin Elmer. The fluorescence signal in the brain of the nude mouse was confirmed (Figure 2A), and 24 hours after administration, the nude mouse was perfused with saline solution, the brain was removed, and the fluorescence present in the brain was measured (Figure 2B). As a result, Cy5.5 (control) was found to barely penetrate the blood-brain barrier, and it was confirmed that brain penetration was at a similar level even when sialic acid was attached. In comparison, Cy5.5 with two sialyllactoses attached was found to be better delivered to the brain than sialic acid.

In summary, Cy5.5, which has a low brain permeability, can better pass through the blood-brain barrier when sialylaltose is attached. In particular, Cy5.5 with sialylaltose attached has a higher brain permeability than sialic acid or lactose, which are part of sialyllactose. It was confirmed that this was significantly higher.

2. Gemcitabine combined with sialyllactose in vivo and in vitro Experiment

2-1) Preparation of gemcitabine conjugated with sialyllactose

First, to prepare the 2,3-sialyllactose-gemcitabine conjugate, 25 ml of dimethylformamide and 0.89 ml of triethylamine were added to 1.4 g of gemcitabine, stirred, and then added with 0.94 g of imidazole and 1.44 g of TBDMS-Cl. was added and stirred for 4 hours. This was concentrated and purified using silica gel to obtain 1.9 g of material. 12.5 ml of pyridine and 0.6 g of 4-dimethylaminopyridine were added thereto, stirred, and then 1.39 ml of tert-butyl dicarbonate was slowly added. This was stirred, concentrated, fractionated with 0.5 M citric acid aqueous solution and methylene chloride, treated with sodium sulfate, filtered through a glass filter, concentrated, and purified, and 1.2 g of the obtained material was dissolved in 8 ml of pyridine. 0.03 g of 4-dimethylaminopyridine and 0.327 g of succinic anhydride were added thereto, stirred, concentrated, and fractionated with 0.5 M citric acid aqueous solution and methylene chloride. This was treated with sodium sulfate, filtered through a glass filter, concentrated, and purified, and 0.6 g of the obtained material and 0.013 g of 4-dimethylaminopyridine were dissolved in 6 ml of pyridine and 0.42 ml of triethylamine. 0.248 g of 4-nitrophenyl chloroformate was added thereto and stirred for 4 hours. After the reaction was completed, the mixture was partitioned into 0.5 M aqueous citric acid solution and 100 ml of methylene chloride. From this, 6 ml of dimethylformamide and 0.62 ml of triethylamine were added to 0.6 g of the material obtained by receiving the methylene chloride layer, treatment with sodium sulfate, filtering and concentrating, and then adding 0.5 g of Aminohexyl-3'SL and stirring. did. After concentrating this and purifying it with silica gel, 5 ml of distilled water and 0.025 ml of acetic acid were added to 0.25 g of the material, stirred at 60 degrees for 24 hours, concentrated, and purified with silica gel to obtain Gemcitabine-succinate-aminohexyl-3'SL (GCB033). .

Next, to prepare the 2,6-sialyllactose-gemcitabine conjugate, 41 ml of dimethylformamide and 1.45 ml of triethylamine were added to 2.28 g of gemcitabine, stirred, and then added with 1.53 g of imidazole and 2.35 mL of TBDMS-Cl. g was added. After stirring for 4 hours, concentration, and purification with silica gel, 1.9 g of material was obtained. To 3.75 g of the material obtained in this way, 50 ml of pyridine and 1.2 g of 4-dimethylaminopyridine were added, and after stirring, 2.7 ml of ditertbutyl dicarbonate was slowly added. After stirring, the mixture was concentrated and fractionated with 0.5 M citric acid aqueous solution and methylene chloride. After treatment with sodium sulfate, concentration by filtration through a glass filter, 3.2 g of purified material was dissolved in 21 ml of pyridine, 0.082 g of 4-dimethylaminopyridine and 0.87 g of succinic anhydride were added, stirred, and concentrated to obtain 0.5 M citric acid. It was fractionated into aqueous solution and methylene chloride. After treatment with sodium sulfate, it was filtered through a glass filter, concentrated, and purified to obtain 2.9 g of material, and 0.061 g of 4-dimethylaminopyridine was dissolved in 29 ml of pyridine and 2.5 ml of triethylamine, and then dissolved in 1.21 g of 4-nitrophenyl chloroformate. g was added and stirred for 4 hours. After the reaction was completed, it was fractionated into 0.5 M aqueous citric acid solution and 100 ml of methylene chloride. The methylene chloride layer was treated with sodium sulfate, filtered through a glass filter, and concentrated. 29 ml of dimethylformamide and 3 ml of triethylamine were added to 2.9 g of the material and stirred. Add 2.4 g of Aminohexyl-6'SL and stir, then concentrate and purify with silica gel. Add 24 ml of distilled water and 0.12 ml of acetic acid to 1.2 g of the material, stir at 60 degrees for 24 hours, and concentrate and purify with silica gel. Gemcitabine-succinate-aminohexyl-6'SL (GCB034) was obtained.

The structures of GCB033 (conjugate of 2,3-sialyllactose-gemcitabine) and GCB034 (conjugate of 2,6-sialyllactose-gemcitabine) prepared as above are shown in Figure 3.

2-2) Anticancer effect experiment of gemcitabine combined with sialyllactose

To determine the effect of combination with sialyllactose on the anticancer activity of gemcitabine, which is used as an anticancer drug, human pancreatic cancer cell line (BxPC-3) was treated with gemcitabine conjugate (GCB034) and gemcitabine at different concentrations (5, 10, 20, 40 nM), then cell viability was confirmed after 72 and 96 hours (Figures 4A and 4B), and after 72 hours, the pro forms of PARP and Caspase-3, which are apoptosis markers, and their cleaved products were examined. The amount was confirmed (Figure 4C). As a result, it was confirmed that the gemcitabine conjugate could not only induce cancer cell death at all treated concentrations, but also exhibited a higher anticancer effect compared to gemcitabine. Corresponding to these results, a decrease in the pro form of PARP and an increase in Caspase-3 cleavage were detected more when cell death progressed in a concentration-dependent manner with the gemcitabine conjugate.

In addition, to confirm the cytotoxic effect on cell lines other than pancreatic cancer cell lines, the same concentration of gemcitabine as before was applied to normal myoblast cell line (C2C12, normal mouse myoblast) and normal lung fibroblast cell line (MRC-5, normal human lung fibroblast). The conjugate (GCB034) was treated, and cell survival rate was confirmed after 24 and 30 hours in that order (gemcitabine is known to exhibit cytotoxicity by inhibiting DNA synthesis and repair, so the time elapsed after treatment is the doubling time for each cell line. (referred to as a later point in time). As a result, it was confirmed that the gemcitabine conjugate according to the present invention had little effect on the survival rate of myoblasts and lung fibroblasts, which are normal cell lines, at a concentration that showed cytotoxicity in pancreatic cancer cells like gemcitabine (Figures 4D and 4E). .

In summary, combining sialyllactose with gemcitabine did not inhibit the anti-cancer effect of gemcitabine, but rather increased its anti-cancer activity. From the results of comparing the cytotoxicity after doubling time with normal cells and other tissue cells, the gemcitabine conjugate according to the present invention can exhibit a high cytotoxic effect specifically on pancreatic cancer cells, and it acts specifically on pancreatic cells or cancer cells. This can be interpreted as the results shown.

2-3) Brain penetration experiment of gemcitabine conjugated with sialyllactose

To confirm the blood-brain barrier penetration of the gemcitabine conjugate, a blood-brain barrier penetration experiment was performed by attaching Cy5.5 to GCB033 and GCB034, respectively. At this time, Cy5.5 alone and gemcitabine with only Cy5.5 attached were used as controls. First, samples were prepared by dissolving each material in 10% DMSO and then diluting (final DMSO concentration was approximately 2% of the total volume), and each sample was administered into the vein of BALB/c nude mice at 1 μmol/kg. Later, the fluorescence signal intensity of the brain of the nude mouse was measured at different times (10 minutes, 30 minutes, 1, 2, 4, 6, 8, and 24 hours) using Perkin Elmer's IVIS spectrum CT (Figure 5A), and 24 hours. Later, the nude mouse was perfused with saline solution, the brain was extracted, and the intensity of the fluorescence signal present in the brain was measured (Figure 5B).

As a result, when gemcitabine conjugated with sialylaltose was administered compared to gemcitabine, a stronger fluorescence signal appeared in the brain, confirming that the brain penetration rate of the gemcitabine conjugate according to the present invention was high.

2-4) PK experiment of gemcitabine conjugated with sialyllactose

To check the distribution and dissociation of the gemcitabine conjugate in tissues, GCB034 was administered and the presence of GCB034 and its dissociate, linker-6'SL (L-6'SL) and linker, were checked in the brain and pancreas over time. In detail, GCB034 was dissolved in 10% DMSO and then diluted to prepare a sample (final DMSO concentration was approximately 2% of the total volume), and the sample was administered intravenously (IV) at a concentration of 82 mg/kg or 246 mg/kg. After oral administration (PO), anesthesia was performed at different times (0, 0.5, 2, and 4 hours), heart blood was collected, and saline solution was perfused, and the brain and pancreas were removed. Each extracted tissue was homogenized in 50mM Tris-HCl buffer (pH 7.5) at a ratio of 1:5 and centrifuged at 3,000 rpm and 4°C for 20 minutes to obtain the supernatant, which was analyzed by mass spectrometry (SCIEX Triple Quad). 3500 LC-MS/MS system).

(ng/ml) pancreas brain GCB034 L-6'SL linker GCB034 L-6'SL linker Vehicle G1 101 N/D N/D N/D N/D N/D N/D 102 N/D N/D N/D N/D N/D N/D 103 N/D N/D N/D N/D N/D N/D GCB034
(82 mg/kg, IV) 0.5h G2 201 785.00 N/D N/D N/D N/D N/D 202 668.69 N/D N/D 137.90 5.42 N/D 203 688.03 N/D N/D N/D N/D N/D 2h G3 301 197.69 N/D N/D N/D N/D N/D 302 1972.06 N/D N/D 62.21 N/D N/D 303 806.31 N/D N/D 66.96 5.49 N/D 4h G4 401 620.25 N/D N/D N/D N/D N/D 402 111.18 N/D N/D N/D N/D N/D 403 433.72 N/D N/D N/D N/D N/D GCB034
(246 mg/kg, PO) 0.5h G5 501 122.23 N/D N/D N/D N/D N/D 502 238.48 N/D N/D N/D N/D N/D 2h G6 601 N/D N/D N/D N/D N/D N/D 602 N/D N/D N/D N/D N/D N/D 4h G7 701 115.78 N/D N/D N/D N/D N/D 702 N/D N/D N/D N/D N/D N/D

As shown in Table 1, when GCB034 was administered intravenously, the substance passed through the BBB and some dissociated substances (brain) were detected in the brain and pancreatic tissue. When GCB034 was administered orally, the substance was detected in the pancreatic tissue. It was detected within the body, confirming that GCB034 can be delivered to the brain and pancreas when administered in vivo. In addition to these results, the anticancer effect of the gemcitabine conjugate on pancreatic cancer cell lines was confirmed in Example 2-2 without affecting normal cell lines, and this improved effect appears to be the result of better delivery into the relevant tissues and cells. It can be seen that this can be done. In summary, the gemcitabine conjugate according to the present invention is better delivered to the above two tissues and can produce an improved anticancer effect compared to gemcitabine.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (9)

A gemcitabine conjugate comprising sialyllactose and gemcitabine.
According to paragraph 1,
A gemcitabine conjugate, wherein the sialyllactose is 2,3-sialyllactose or 2,6-sialyllactose.
According to paragraph 1,
A gemcitabine conjugate in which the sialyllactose and gemcitabine are connected by a linker.
According to paragraph 3,
A gemcitabine conjugate, wherein the linker is a cleavable linker that promotes release after delivering gemcitabine to a tissue.
According to paragraph 1,
The linker is a gemcitabine conjugate characterized in that it binds to the hydroxy group (-OH) of the lactose portion of sialyllactose.
According to paragraph 1,
The gemcitabine conjugate has a structure represented by Formula 1 or Formula 2 below,
[Formula 1]

[Formula 2]

In the above two formulas, L is a linker, gemcitabine conjugate.
According to paragraph 1,
The gemcitabine conjugate has a structure represented by Formula 3 or Formula 4 below:
[Formula 3]

[Formula 4]
.
According to paragraph 1,
The gemcitabine conjugate is characterized in that the delivery rate to the brain or pancreas is improved compared to gemcitabine when administered in the body.
A pharmaceutical composition for treating neoplastic diseases comprising the gemcitabine conjugate of claim 1.