KR102313068B1 - Pharmaceutical formulation for enhancing biodistribution of the lung or brain, including byakangelicin - Google Patents

Abstract

The present invention relates to a pharmaceutical formulation for enhancing the biodistribution of the lungs or brain containing Viacanjelysin, and more particularly, to a drug for treating lung or brain diseases, and biodistribution enhancement of a pharmaceutical formulation containing Viacanzelicin Regarding the effect, in the case of a pharmaceutical formulation comprising an active ingredient such as curcumin, umbelliferone or doxorubicin of the present invention and Viacanzelicin, the biodistribution is concentrated in the lungs and brain without affecting other organs. The effect can be confirmed and the distribution of the active ingredient can be directly analyzed by the method of fluorescence imaging, and the active ingredient can be analyzed without problems such as denaturation or concentration change of the active substance during the pre-treatment or extraction process of biological samples. , there is an effect that can easily and quickly see the effect of enhancing the biodistribution of the active substance in vivo.

Description

Pharmaceutical formulation for enhancing biodistribution of the lung or brain, including byakangelicin

The present invention relates to a pharmaceutical formulation for enhancing the biodistribution of the lungs or brain containing Viacanjelysin, and more particularly, to a drug for treating lung or brain diseases, and biodistribution enhancement of a pharmaceutical formulation containing Viacanzelicin It's about the effect.

A targeted drug delivery system that can selectively deliver a drug to a desired location is one of the most important tasks in the field of medicine and pharmaceuticals, and many researchers are still concentrating on the development of a more excellent selective drug delivery system. If a drug can be selectively delivered to a specific cell, organ or disease, many problems caused by current drug treatment can be immediately solved.

In general, most drugs, when administered systemically, express their toxicity not only in diseased cells but also in normal cells, causing many side effects. Due to these side effects, there are restrictions on the dose and frequency of administration of the drug, and thus there are cases in which the disease cannot be effectively treated. Representative side effects such as bone marrow decrease, gastrointestinal disorder, and hair loss that occur during chemotherapy by the administration of anticancer drugs may be mentioned.

Considering the importance of the drug delivery system and the potential for future development, the technology to image, evaluate, and analyze the in vivo behavior of the drug delivery system, such as in vivo distribution, metabolism, and disappearance, identifies the problems of the existing drug delivery system, and Its utility value is very high because it can accurately evaluate drug delivery systems more reliably and suggest a design direction for more efficient drug delivery systems.

As mentioned above, the pharmacological effect of a drug is expressed by the drug molecule reaching the site of action in the body, and side effects appear by the drug transferred to another site. Therefore, in order to effectively and safely perform drug treatment, it is necessary to selectively deliver the drug to the site of action as much as possible, and the optimal amount of the drug for the desired time, that is, the technology for efficiently evaluating the in vivo behavior of the drug is very important. .

Accordingly, the present inventors confirmed that the biodistribution of the drug in the brain and lung was significantly increased when the drug was treated with Byakangelicin than when the drug was treated alone, and in organs other than the brain or lung, The present invention was completed by confirming that there was no other influence.

Laid-open Patent Publication No. 10-2003-0051623

Elizabeth M. Ellis, Neuroprotective Effects of Umbelliferone and Esculetin in a Mouse Model of Parkinson's Disease, Journal of Neuroscience Research, 91, p453-461 (2013)

It is an object of the present invention to provide a pharmaceutical formulation for enhancing lung biodistribution.

Another object of the present invention is to provide a pharmaceutical formulation for enhancing brain biodistribution.

In order to achieve the above object,

The present invention is a drug for the treatment of lung diseases; And Via Angelicin (Byakangelicin); provides a pharmaceutical formulation for enhancing the biodistribution of the lungs, including.

In addition, the present invention is a drug for the treatment of brain diseases; And Via Angelicin (Byakangelicin); provides a pharmaceutical formulation for enhancing the biodistribution of the brain, including.

In the case of a pharmaceutical formulation comprising an active ingredient such as curcumin, umbelliferone, or doxorubicin of the present invention and Viacanzelicin, the biodistribution is concentrated in the lungs or brain without affecting other organs except the brain or lungs. The effect can be confirmed and the distribution of the active ingredient can be directly analyzed by the method of fluorescence imaging, and the active ingredient can be analyzed without problems such as denaturation and concentration change of the active substance during the pretreatment or extraction process of a biological sample, There is an effect that can easily and quickly see the effect of enhancing the biodistribution of the active substance in vivo.

1 is a result of confirming the biodistribution of umbelliferone in each organ.
2 is a result of quantifying the amount of umbelliferone distributed in the brain and lungs.
3 is a result of confirming the biodistribution of curcumin in each organ.
4 is a result of confirming the biodistribution of doxorubicin in each organ.
5 is a result of confirming the biodistribution of Evan's blue in each organ.

Hereinafter, the present invention will be described in detail.

Pharmaceutical formulations for enhancing lung or brain biodistribution

The present invention is a drug for the treatment of lung or brain diseases; and Byakangelicin; It provides a pharmaceutical formulation for enhancing the biodistribution of the lung or brain comprising a.

In one embodiment of the present invention, the lung disease may be one or more selected from the group consisting of chronic cough, asthma, bronchiectasis, chronic obstructive pulmonary disease (COPD), acute bronchitis and lung cancer.

In addition, in one embodiment of the present invention, the brain disease is dementia, Alzheimer's disease, multiple sclerosis, Parkinson's disease, stroke and ischemia, Jacob -Jacob-Creutzfeldt disease, Huntington's disease, nieman-pick disease, Epilepsy, Depressive Disorder, Anxiety Disorders and brain tumor tumor) may be one or more selected from the group consisting of.

In one embodiment of the present invention, the pharmaceutical formulation is formulated in various forms such as granules, tablets, suspensions, emulsions, syrups, soft or hard gelatin capsules and aerosols according to conventional methods for each purpose of use. and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Examples of suitable carriers, excipients or diluents that may be included in such formulations are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical formulation of the present invention may further include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical formulation, for example, starch, calcium carbonate, sucrose, lactose, gelatin. It is formulated by mixing, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Oral liquid formulations may include suspensions, solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. can

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized agents, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Conventional additives may be included.

The pharmaceutical preparation of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The agent of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, in the pharmaceutical formulation of the present invention, the effective amount may vary depending on the age, sex, and weight of the patient, and in general, 0.01 to 100 mg, preferably 0.1 to 10 mg per kg of body weight, is administered daily or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical formulation of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical formulation of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the pharmaceutical formulation of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

In the present invention, "subject" means an animal, including, but not limited to, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, guinea pigs or guinea pigs, In one embodiment a mammal, in another embodiment a human.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

<Preparation example>

Using DMSO, an organic solvent, umbelliferone (Umb) was dissolved at 100 mg/ml, curcumin (Cur) was 2.66 mg/ml, and doxorubicin (Dox) was dissolved at 5.4 mg/ml, and the desired concentration It was diluted so as to be used.

<Experimental Example 1> Measurement of biodistribution of Umbelliferone (Umb)

After dissolving umbelliferone (Umb) at 100 mg/ml using DMSO, an organic solvent, diluted with PBS containing 15% of Cremophor and DMSO, respectively, ICR mouse Injected into the tail vein (4 mg/kg).

At this time, the final umbelliferone concentration was fixed at 0.5 mg/ml, and the concentrations of other polyphenols (Decursin ;Dec, Nodakenin; Nod, Byakangelicin; Byn) were 0 and 10. By varying the mg/ml, the mass ratio was 1:0, 1:20, and 200 μl of the prepared solution was injected into the tail vein of the ICR (female, 6 weeks).

In order to observe the in vivo distribution of umbelliferone 2 minutes after administration of umbelliferone, organs from euthanized mice were removed and washed with PBS. Each organ was measured at excitation 360 nm and emission 490 nm using Lago-X (Spectral Instruments Imaging, Tucson, AZ, USA).

The total flux of each organ was analyzed using Lago-X, and in order to exclude errors due to autofluorescence, the total flux of the general organ was subtracted from the total flux of the organ to which Umbelliferone was administered. The total flux of only peron (Umbelliferone) was obtained, and the organs were stored at -70°C until the next mass analysis.

As shown in FIG. 1 , the fluorescence of Umb measured in the brain was 8.9 ± 4.7, and when mixed with Dec and Nod, there was no difference in fluorescence even when the mixing ratio increased.

However, when Byn was mixed at 1:20 (w/r), it was confirmed that it was 37.4 ± 0.1, which was 4.2 times higher. The fluorescence of Umb measured in the lungs was 8.1 ± 9.1, and when Byn was mixed and delivered at 1:20 (w/r), it was measured 7.2 times higher (58.0 ± 29.5), confirming a significant difference. Even when the mixing ratio of Byn was increased to 1:20 (w/r), the fluorescence of Umb measured in the pancreas decreased from 8.2 ± 6.2 to 1.1 ± 3.8, and the fluorescence measured in the liver and spleen showed no significant difference.

<Experimental Example 2> Quantitative analysis of umbelliferone (Umb) in organs

To quantify the amount of umbelliferone distributed in the brain and lungs, PBS (pH 7.4, organ: PBS = 1:3, w/v) was added and organs were homogenized.

After mixing 200 μl of 10% SDS and 500 μl of ethyl acetate in 500 μl of the homogenate, vortexing was performed for 10 minutes. The supernatant obtained by centrifuging this mixture at 12,000 rpm for 3 minutes was dried at room temperature, and the final product obtained was dissolved using DMSO and then Excitation 325 nm and Emission 495 nm were measured.

In order to quantify the umbelliferone present in the organ, umbelliferone dissolved in DMSO was serially diluted (serial dilution, 0-500 ng/ml) and used as a standard.

In order to compare the results obtained through the imaging of FIG. 1 , the fluorescence of Umb extracted from each organ to which the polyphenol mixture was administered in proportions was analyzed based on the fluorescence of Umb extracted from the brain and lung administered with Umb alone.

As a result, it was confirmed that the results extracted from organs as shown in FIG. 2 showed a similar trend to the results of FIG. 1 .

<Experimental Example 3> Measurement of biodistribution of curcumin (Curcumin; Cur)

Curcumin (Cur) was used as a tracing molecule of the polyphenol mixture to check whether the biodistribution was changed by other polyphenol Byn. 2.66 mg/ml of curcumin dissolved in DMSO and 0, 1.33, 2.66, 5.32, 53.2, or 133 mg/ml of another polyphenol (Dec, or Byn) were mixed in a volume ratio of 1:1, and a mass ratio of 1:0, 1 :0.5, 1:2, 1:20, 1:50.

A PBS solution containing 15% each of DMSO and Cremophor in the mixture was prepared, and 200 μl of the solution was injected into the tail vein of an ICR (female, 6 weeks) mouse (curcumin standard 1.6 mg/kg).

In order to observe the in vivo distribution of curcumin (Curcumin) 2 minutes after administration of curcumin (Curcumin, Cur), the organs of euthanized mice were excised and washed with PBS. Each organ was measured at Excitation 430 and Emission 509 nm using the In Vivo Imaging System (IVIS, Caliper Life Sciences Lumina Ⅱ, Massachusetts, USA). The total flux of each organ is analyzed using IVIS, and in order to exclude autofluorescence, the total flux of a general organ is subtracted from the total flux of the organ to which curcumin is administered to obtain the total flux of curcumin alone, and the organ is subjected to the next analysis. Stored at -70°C until

As shown in FIG. 3 , when curcumin was administered alone, fluorescence measured in brain, lung, pancreas, liver, and spleen was 3.2 ± 1.6, 5.9 ± 3.8, 3.6 ± 1.2, 11.2 ± 11.3, 0.3 ± 0.2, and when administered by mixing Decersin (Dec) and 1:50 (w/r), the fluorescence was 5.7 ± 1.7, 6.8 ± 2.8, 4.0 ± 1.0, 8.8 ± 1.9, 0.3 ± 0.1 of curcumin. (cur) showed a similar tendency to umberryferon as it did not show a significant difference compared to the case of alone. When Viacanjelysin (Byn) was mixed with curcumin (Cur) and delivered, the fluorescence measured in the brain increased 2.7-fold to 8.6 ± 2.1 when treated with 1:50 (w/r), and curcumin delivered to the lungs ( Cur) fluorescence was observed to be 46.6 ± 2.0 at 1:50 (w/r), indicating that the amount of curcumin (Cur) delivered to the brain and lungs significantly increased as the mixing ratio of Viacanjelysin (Byn) increased. Confirmed. However, the fluorescence of curcumin (Cur) measured in the liver showed a tendency to decrease to 3.7 ± 2.5, and the fluorescence of the pancreas and spleen was 2.7 ± 2.9 and 0.2 ± 0.2, showing no significant difference from the pre-mixing.

<Experimental Example 4> Measurement of biodistribution of doxorubicin (Dox)

In order to confirm the effect of Biacanjelicin on the biodistribution of chemical drugs, doxorubicin, which is widely used as an anticancer agent, was selected. Doxorubicin (Dox) is administered intravenously in clinical practice and has fluorescence, so it is a drug suitable for this research system to check biodistribution through imaging after intravenous administration. Using DMSO, doxorubicin was dissolved at 5.4 mg/ml, and decursin and byakangelicin were each dissolved at 300 mg/ml. The concentration of doxorubicin is 4.17 mg/ml, and the concentration of decursin or byakangelicin is 0, 20.85, 41.7 mg/ml. Byakangelicin and weight ratio were 1:0, 1:5, 1:10.

A PBS solution containing 15% each of DMSO and Cremophor was made, and 200 μl was injected into the tail vein of ICR (female, 6 weeks) rats (doxorubicin standard 5 mg/kg).

To observe the in vivo distribution of doxorubicin 2 minutes after doxorubicin (Dox) administration, organs from euthanized mice were extracted and washed with PBS. Each organ was measured at excitation 465 and emission 583 nm using the In Vivo Imaging System (IVIS, Caliper Life Sciences Lumina Ⅱ, Massachusetts, USA). The total flux of each organ is analyzed using IVIS, and in order to exclude autofluorescence, the total flux of a general organ is subtracted from the total flux of the organ to which doxorubicin has been administered to obtain the total flux of only doxorubicin. Stored at -70°C until next analysis.

As shown in FIG. 4 , when doxorubicin (Dox) was administered alone, fluorescence measured in brain, lung, pancreas, liver, and spleen was 2.3 ± 0.6, 12.9 ± 2.9, 8.0 ± 6.5, 5.2 ± 10.5, 0.6 ± 0.5 There was no difference between 2.9 ± 0.6, 11.6 ± 0.7, 6.1 ± 0.7, 14.0 ± 1.6, 0.8 ± 0.3 when mixed with nodakenin (Nod), but when mixed with deckercin (Dec), the brain, lung and liver showed a tendency to increase to 9.0 ± 6.5, 20.6 ± 7.7, and 18.3 ± 9.3, but the pancreas and spleen showed no difference at 11.9 ± 7.6 and 0.8 ± 0.9. In addition, in the case of mixing viacanjelysin (Byn), brain, lung and liver increased to 15.0 ± 6.8, 21.9 ± 4.8, and 14.6 ± 2.4, and the pancreas and spleen showed a tendency to decrease to 2.3 ± 1.4 and 0.3 ± 0.3. It was confirmed that, as in the previous results, it was Decersin (Dec) and Viacanzelicin (Byn) that increased the delivery of the active ingredient to the brain and lungs.

<Experimental Example 5> Measurement of biodistribution of Evan's blue

A tracing molecule (tracing molecule) to determine whether Viacanjelysin (Byn), a substance other than a polyphenol, also increases the amount delivered to the lungs, and whether the increase in the amount delivered to the brain is caused by damage to the blood-brain barrier (BBB). ) as Evan's blue (EB) was used.

Biacanjelysin (Byn) 0, 0.665, 1.33, 2.66, 26.6, or 66.5 mg/ml was dissolved in DMSO to prepare a PBS solution containing 15% each of DMSO and Cremophor. At this time, Evan's blue was dissolved in PBS at 57.14 μg/ml and used. 200 μl of the prepared solution was injected into the tail vein of ICR (female, 6 weeks) rats (1.6 mg/kg based on Ivans blue).

In order to observe the biodistribution of Evan's blue 2 minutes after administration of Evan's blue, organs from euthanized mice were removed and washed with PBS, and each organ was treated with the In Vivo Imaging System (IVIS, Caliper). Life Sciences Lumina II, Massachusetts, USA) was used to measure Excitation 535 and Emission 705 nm. The total flux of each organ is analyzed using IVIS, and the total flux of the general organ is subtracted from the total flux of the organ to which Evan's blue has been administered to exclude autofluorescence. The flux was obtained, and the organs were stored at -70°C until the next analysis.

As shown in Figure 5, the fluorescence measured in the brain, lung, pancreas, liver, and spleen administered with Ivans Blue alone was 1.6 ± 0.1, 5.3 ± 1.3, 2.0 ± 0.7, 2.7 ± 0.9, 1.0 ± 0.4, Although the mixing ratio of Biacanjelysin was increased to 1:50 (w/r), the fluorescence measured in each organ showed no difference as 1.5 ± 0.1, 5.1 ± 0.8, 1.3 ± 0.4, 1.6 ± 0.2, or -0.0 ± 0.2. Based on the above results, it was confirmed that the change in the brain distribution of substances caused by Viacanjelysin was not caused by BBB damage.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than in the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (6)

A composition for drug delivery to brain tissue comprising Byakangelicin:
The drug is at least one of Umbelliferone, Curcumin, Doxorubicin, and acceptable salts thereof.
delete According to claim 1,
The composition for drug delivery to brain tissue is one type of formulation selected from the group consisting of powders, granules, tablets, suspensions, emulsions, syrups, soft or hard gelatin capsules and aerosols. Drug delivery to brain tissue for composition.
delete delete delete

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