WO2018093029A1 - 육산화사비소의 결정다형을 포함하는 뇌암 예방 또는 치료용 약학 조성물 및 이의 제조방법 - Google Patents

육산화사비소의 결정다형을 포함하는 뇌암 예방 또는 치료용 약학 조성물 및 이의 제조방법 Download PDF

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Publication number
WO2018093029A1
WO2018093029A1 PCT/KR2017/009820 KR2017009820W WO2018093029A1 WO 2018093029 A1 WO2018093029 A1 WO 2018093029A1 KR 2017009820 W KR2017009820 W KR 2017009820W WO 2018093029 A1 WO2018093029 A1 WO 2018093029A1
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Prior art keywords
arsenic
pharmaceutical composition
brain cancer
oxide
hexaarsenic
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PCT/KR2017/009820
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English (en)
French (fr)
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배일주
연증림
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주식회사 케마스
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Priority to CA3043145A priority Critical patent/CA3043145C/en
Application filed by 주식회사 케마스 filed Critical 주식회사 케마스
Priority to US16/461,892 priority patent/US10493100B2/en
Priority to MX2019005737A priority patent/MX2019005737A/es
Priority to JP2019547051A priority patent/JP6824431B2/ja
Priority to BR112019009810A priority patent/BR112019009810A2/pt
Priority to EP17872515.6A priority patent/EP3542804B1/en
Priority to AU2017360407A priority patent/AU2017360407B2/en
Priority to MYPI2019002666A priority patent/MY196811A/en
Publication of WO2018093029A1 publication Critical patent/WO2018093029A1/ko
Priority to ZA201902611A priority patent/ZA201902611B/en
Priority to SA519401787A priority patent/SA519401787B1/ar
Priority to IL266660A priority patent/IL266660B/en
Priority to PH12019501110A priority patent/PH12019501110A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/285—Arsenic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G28/00—Compounds of arsenic
    • C01G28/005—Oxides; Hydroxides; Oxyacids
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/60—Compounds characterised by their crystallite size
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
    • C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2006/00—Physical properties of inorganic compounds
    • C01P2006/80—Compositional purity

Definitions

  • NAME OF THE INVENTION A pharmaceutical composition for the prevention or treatment of brain cancer, including a crystalline polymorph of hexaarsenic oxide, and a method of manufacturing the same
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of brain cancer, including the crystalline polymorph of hexaarsenic hexaoxide.
  • Cancer is characterized by uncontrolled cell growth, which is abnormal
  • Cancer affects all tissues and organs of the body with varying degrees of prevalence.
  • brain cancer malignant brain tumor
  • Conventional treatment for brain cancer is the most effective method of overexposure, but depending on the type and occurrence area of the brain cancer. Surgery is often impossible, and the risk of complications after complete excision is very high.
  • the brain has a brain-blood barrier (BBB) that inhibits the penetration of drugs.
  • BBB brain-blood barrier
  • Muscle arsenic purified by technology showed cancer metastasis suppression effect in animal experiments, and end stages such as allergic uterine cancer, bladder cancer, lung cancer, maxillary sinus cancer and kidney cancer
  • the method and other novel manufacturing methods allow the production of arsenic hexaarsenide with a polymorph a of 99% or more of arsenic arsenide, and the composition containing these arsenic arsenides has a particularly significant effect on the prevention or treatment of brain cancer.
  • the present invention has been completed.
  • the object of the present invention is to determine the polymorphic form of hexaarsenic oxide ( ⁇ 0 6 ) as an active ingredient.
  • Another object of the present invention is to provide a method for preparing a pharmaceutical composition for preventing or treating brain cancer, which includes a polymorph of hexaarsenic oxide (As 4 0 6 ) as an active ingredient.
  • Task solution includes a polymorph of hexaarsenic oxide (As 4 0 6 ) as an active ingredient.
  • the present invention yuksan bright arsenic (As 4 0 6) a pharmaceutical composition for preventing or treating brain tumors comprising the resolution jeongda type, yuksan crystals of a bright arsenic polymorphism comprising (As 4 0 6 -a) a 99% or more It is about pharmaceutical compositions.
  • the composition may contain less than 1% of polymorph b (As 4 O 6 -b) of arsenic hexaoxide.
  • the arsenic hexaoxide may be more than 99.9% pure.
  • the As 4 0 6 -a and As 4 0 6 -b may have the following characteristics (i) to (iii):
  • the As 4 0 6 -a is the peak represented by the velocity of Wmin (scan step 0.02 °) in the range of diffraction angle 2 ⁇ 10 ⁇ ⁇ 50 ⁇ at X-ray powder diffraction, when the light source wavelength is 1.5406A. It is a crystalline form characterized by 13.84, 27.88, 32.32, 35.3, 39.84, 42.38, 46.34, 48.6, and 49.34 (see Fig. 1). Also, the ratio of main peaks shown in 2 ⁇ values 13.8 and 27.9 is 1: 1.3. It is characterized by being
  • 0 6 -b is the peak represented by the velocity of Wmin (scan step 0.02 °) in the range of diffraction angle 2 ⁇ 10 ° ⁇ 50 ° when the light source wavelength is 1.5406 in X-ray powder diffraction. It is a crystalline form characterized by 13.86, 27.92, 32.36, 35.34, 39.9, 42.44, 46.4, 48.66, and 49.4 (see Fig. 1). In addition, the ratio of main peaks at 2 ⁇ 13.8 and 2 9 is 1: It is characterized by being 2.5.
  • the active ingredient of hexaarsenic oxide (As 4 0 6 )
  • compositions for inhibiting brain cancer metastasis containing crystalline polymorphs and for compositions for inhibiting brain cancer metastasis containing 99% or more of polymorph a of hexaarsenic oxide.
  • the active ingredient of hexaarsenic oxide (As 4 0 6 )
  • a method for preparing a brain cancer preventive or therapeutic pharmaceutical composition comprising a polymorph, comprising: a first step of heating sodium chloride at 100 to 800 ° C. and then cooling it;
  • a method for producing a pharmaceutical composition for preventing or treating brain cancer characterized in that the hexaarsenic crystal obtained in the fourth step contains 99% or more of the polymorph a (As 4 O 6-a) of arsenic hexaoxide. It's about.
  • the present invention is a composition for the prevention or treatment of brain cancer comprising arsenic hexaoxide (As 4 0 6 ) as an active ingredient, and comprises 99% or more of the crystalline polymorph a (As 4 0 6 -a) of arsenic hexaoxide. It is about the composition.
  • the method for producing a composition for the prevention or treatment of brain cancer of the present invention comprises sodium chloride.
  • Synthetic reaction mixture of kaolin material, and a clamp for mounting a filter on top of the synthesis reactor are prepared, and sodium chloride is placed during the synthesis reaction, and heating and cooling are carried out.
  • Sodium chloride is used in the manufacturing method of the present invention. This is because, in the second process, arsenic trioxide is placed on sodium chloride and heated to heat the arsenic compound: the heat is evenly transferred to help the arsenic compound sublimate and to remove impurities and moisture of the sodium chloride through the first process.
  • the filter used at this time is 70-100 g / m 'of basic weight, 0.17 0.25 thickness, filtration speed 22-30 s / 100 ml, and retention rate 5-5. It is preferable to use 10.
  • the filter After the filter has been fitted and sealed, it is heated for 3 to 10 hours at 100 ° C to 1000 ° C. in the bottom of the synthetic reaction vessel and heated at the top center of the filter paper. The temperature is maintained at 150 ° C ⁇ 100 o C, and the arsenic hexaoxide is crystallized while passing through the filter paper, and then at room temperature for at least 5 hours, preferably
  • composition containing the crystalline polymorph of hexaarsenic hexaoxide of the present invention can be usefully used for the prevention or treatment of brain cancer.
  • compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods.
  • Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, textose, sucrose, sorbet, manny, xylly, erythritol, malty, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyridone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil
  • diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents and surfactants are usually used.
  • hexaarsenic hexaacetic oxide of the present invention for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid oral agents for oral use include suspensions, solvents, emulsions, and syrups. Excipients may include, for example, wetting agents, sweetening agents, fragrances, and preservatives.
  • sterile aqueous solutions for parenteral administration, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories may be used.
  • Vegetable oils such as propylene glycol, polyethylene glycol and olive oil and injectable esters such as ethyl oleate can be used.
  • Neunwi tepsol (witepsol), macrogol, teuwon (tween) 61, cacao butter, can be used as such as gelatin laurin, glycero-.
  • the dosage of the pharmaceutical composition is determined by the age, sex weight,
  • the determination of dosage based on these factors is within the level of the practitioner and generally the dosage is within 0.01 mg / kg / day. Of about 500mg / kg / day The preferred dosage is from 0.01 mg / kg / day to 100 mg / k g / day. The dosage may be administered once a day or may be divided several times. It is not intended to limit the scope of the invention.
  • the pharmaceutical composition can be administered in a variety of routes to mammals such as rats, cattle, and humans. All methods of administration can be expected, for example, oral, rectal or intravenous, muscular, subcutaneous, intrauterine mucosa. Or by intravascular cerebral injection.
  • the brain cancer preventive or therapeutic composition of the present invention has an excellent anticancer effect by containing more than 99% of the crystalline polymorph a of hexaarsenic oxide.
  • composition of the present invention inhibits brain cancer metastasis such as glioblastoma, in particular.
  • FIG. 2 shows the treatment of Examples 1 and Comparative Examples 1 to 3 with U-87MG cell line for 48 hours (FIG.
  • FIG. 5 is an MRI photograph showing the tumor size decreased after 2 months of administration and radiation treatment of Example 1 for patients with cerebral glioma of clinical trial-1.
  • FIG. 6 is a T2-weighted cross-sectional SE image showing the tumor almost disappeared as an MRI photograph after 3 months of administration and radiation treatment of Example 1 for a clinical trial-1 cerebral glioma.
  • FIG. 7 is a pre-treatment MRI photograph of a patient diagnosed with glioblastoma in the left parieto-temporal lobe (clinical trial-2).
  • FIG. 7A shows a T2 weighted axial SE image.
  • 7B are post-contrast Tl weighted axial SE images after contrast enhancement.
  • FIG. 8 is an MRI photograph after surgery and radiotherapy for glioblastoma patients in clinical trial-2
  • FIG. 8A is a T2 weighted SE & FLAIR images
  • FIG. 8B is a T2 highlighted cross-section SE. It is a video.
  • FIG. 9 shows administration of Example 1 for glioblastoma patients of clinical trial-2 (FIG. 9A),
  • FIG. 10A is a photograph (FIG. 10A) and a graph (FIG. 10B) showing tumor image signal suppression results after inoculation of a Ub-87MG cell line to a Balb / C female nude mouse.
  • the clamp is installed with a primary clamp 50 mm away from the synthesis reactor, 2 to 6 mm apart from the primary clamp, and a secondary to six clamp on top of each clamp.
  • the size is 210mm in diameter and 10mm in thickness.
  • ARSENIC CO., LTD. Put the 100g on the coarse salt in the synthesis reactor and collect the sublimated arsenic filter located on the top of the synthesizer so that the distance between each filter and the filter is 2 ⁇ 6 ⁇ .
  • the self-clamp is fitted.
  • the filter used is a basic weight of 70 to 100 g / m ! It is recommended to use a thickness of 0.17-0.25 ram, a filtration speed of 22-30 s / 100 ml, and a retention rate of 5-10 / m.
  • the outer part of the composite bottom is heated for 1 hour so that the temperature is about 350 ° C ⁇ 100 ° C, and then
  • DSC differential scanning thermal analysis
  • Powder X-ray diffraction of As 4 0 6 —a and As 4 0 6 -b is shown in FIG. 1, As 4 0 6 -a and As
  • As 4 0 6 -a is the ratio of the main peak at 2 ⁇ 13.8 and 27.9 is 1: 1.3, and As 4 0 6 -b is at 2 ⁇ 13.8 and 27.9. It is characterized by the ratio of the main peak appearing 1: 2.5.
  • DSC analysis, structure determination and X-ray diffraction analysis of the prepared compound were carried out in the following manner. :
  • Sample 20.0 mg was analyzed while increasing to 310 ° C. at a rate of 10 ° C./min.
  • the resultant crystals were pulverized, made into particles of 10 / m to 30 um (-325 mesh), filled into an X-ray diffraction analysis glass holder (20 mmxl6 mmxl mm), and then pressed with a glass slide or the like. Samples were prepared by smoothing the overholder side parallel. Using XRD's Cu ⁇ (1.54060 Person), the crystal's X-ray diffraction is plotted at a rate of Wmin (scan step 0.02 °) in the range of 2 ⁇ 10 ⁇ to 50 ⁇ .
  • ARSENIC CO., LTD. Put lOOg on the coarse salt in the synthesis reactor, and place the filter (filter paper) to collect the sublimed arsenic placed on the top of the synthesizer so that the space between each filter is 2 ⁇ 6 ⁇ .
  • the clamp is fitted with a basic weight of 70 to 100 g / m ! It is recommended to use a thickness of 0.17 ⁇ 0.25 ⁇ , filtration speed 22 ⁇ 30s / 100ml, and retention rate 5 ⁇ 10.
  • Example 1 composition having more than 99% of polymorphic As 4 0 6 -a
  • Comparative Example 1 composition having more than 99% of polymorphic As 4 0 6 -b
  • Examples 1 and Comparative Examples 1 to 3 were evaluated using an MTT assay.
  • the MTT assay is based on the ability of living cells to produce the insoluble dark blue formazan reactant to MTT. After trypsin-suspending the cells in the medium, they are collected at 96 densities of 4xl0 3 cells / well. -Dispensed in well culture dishes (Costar, Cambridge, MA, USA). After 24 hours, the cells containing medium containing 10% FBS were treated with 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 ⁇ M as Example 1 and Comparative Examples 1 to 3 respectively as treated samples. Cultures were added after addition.
  • Examples 1 and Comparative Examples 1 to 3 were dissolved in 1 M sodium hydroxide as a stock solution at a concentration of 5 ⁇ 10 ⁇ 2 ⁇ M. After 48 and 72 hours of incubation, a ⁇ assay for cell proliferation was performed, the supernatant was removed at the end of each time interval, and 20 mg of 5 mg / mL MTT solution per well was added. Incubate at 37 ° C. for 4 hours to form. After incubation, the supernatant was removed again, DMSO was added 100 per every well, and mixed to completely dissolve dark dark crystals. All crystals were left at room temperature for 15 minutes. The solution was allowed to dissolve completely and the absorbance was measured with a micro-plate at 570 nm (A 570 nm ) wavelength.
  • the absorbance value of the untreated control group was calculated as 100, the absorbance value of the treated group treated with the sample compared to the 3 ⁇ 4 group was calibrated, and the inhibition of cell proliferation was calculated according to the following equation.
  • Example 1 After treatment, 48 hours and 72 hours of culture and MTT assay were shown in Fig. 2. All of the experimental results in which 48 hours (Figure 2A) and 72 hours (Figure 2B) were cultured after Example 1 was treated. In comparison with Example 1, it was confirmed that the rate of proliferation of cerebral glioblastoma cells was higher. In addition, Example 1 and Comparative Example 1 were found to be 4: 1. Compared with Comparative Example 2, Comparative Example 2 or 1: 1, it was confirmed that Example 1 has a higher proliferation inhibition rate of glioblastoma cells.
  • Example 1 After treatment, the results of 48 hours and 72 hours of culture and MTT assay were shown in FIG. 3. In the experimental results of 48 hours (3A) and 72 hours (FIG. 3B) of culture after Example 1 were treated. It was confirmed that the inhibition rate of glioblastoma cell proliferation was higher than that of Comparative Example 1. In addition, compared with Comparative Example 3, which is mixed with Example 1 and Comparative Example 1, and Comparative Example 2 or 1: 1, Example 1 was confirmed to have a high growth inhibition rate of brain glioblastoma cells.
  • Test Example 2 Phase 3 ⁇ 4 test
  • Example 1 The clinical test was carried out using the composition of Example 1 as follows.
  • Example 1 Brain biopsy at Seoul National University Hospital was diagnosed with gliomatosis cerebri (pathologic diagnosis is anaplastic astrocytomas), and the weakness of the arm and tremors appeared on the 10th day after the diagnosis. Since the onset, symptoms progressed rapidly.
  • Example 1 was administered 15 mg per day from the 17th day after the diagnosis date, and the movement weakness and tremor symptoms began to improve. Radiation therapy was performed for a period from the 27th to 75th day after diagnosis, and symptoms disappeared completely afterwards.
  • the MRI photograph before treatment of the patient is shown in FIG. 4, and 2 months and 3 months of radiation therapy with the administration of Example 1. Months later pictures were shown in 5 and 6, respectively.
  • Glioblastoma (left parieto-temporal lobe) was diagnosed as a left parieto-temporal lobe, and he underwent tumor removal on the third day after diagnosis, and histopathologic diagnosis was a glioblastoma of WHO grade IV IV. Confirmed, and during the period 45 days to 90 days after diagnosis,
  • Tremodal (61.2 Gy / 33fx) treatment was performed, and residual tumors were still identified on MRI after radiation and Tremodal treatment.
  • 5 mg of Example 1 was administered three times a day for 95 days after the diagnosis date. There was no subjective symptoms after 2 months of inoculation, and the size of the remaining tumor was confirmed to be reduced after the administration of Example 1.
  • the MRI photograph before treatment of this patient is shown in FIG. 7, and the MIR photograph after surgery and radiation treatment was shown in FIG.
  • MRI photographs of 4 months, 7 months, and 10 months after administration of Example 1 are shown in FIGS. 9A, 9B, and 9C.
  • composition of the present invention is a brain-blood barrier (brain-blood barrier;
  • BBB effectively prevents the growth of malignant brain tumors such as glioblastoma It was found to have an inhibitory effect.
  • Human glioblastoma cell line U-87MG (purchased from ATCC) was treated with 10% FBS, 100 U / mL penicillin, and 100 / g / mL streptomycin in incubators at 37 ° C, 5% C0 2 and 95% air. Culture was carried out using the included EMEM (Eagle's Minumum Essential Medium) medium. The medium was changed every 3 days.
  • EMEM Eagle's Minumum Essential Medium
  • Luciferase-labeled U-87MG cells were brain-inoculated in 15 brains of 15-19 g Balb / C female nude mice, and the cell inoculation was 5xlOV5uL. The animals were examined on the day 6 after the day of inoculation of the tumor cells which had grown slightly. The animals were divided into 2 groups of 4 animals in each group randomly according to the results of the imaging, and the test group Example 1 was divided into physiological saline (NaCl 0.9%). Solution was administered to the control group, and the control group was administered only physiological saline, which is the solvent used in the test group. The animals of the two groups were administered once daily with physiological saline or 2.25 mg / kg of Example 1, respectively.
  • the dose was set to lOuL / g of the test animal. During the administration, the animal's weight and health status were continuously examined and the imaging test was performed simultaneously. Euthanize and remove the tumor Formalin fixed, and proceed to the subsequent histological examination.
  • the control animals had a high intensity of the tumor signal from the 7th day of administration (13 days from the date of inoculation), and the tumor signal in the spine following the spinal metastasis of the tumor Compared with the distribution, the tumor signals of the test group animals are rarely identified in the spine and the intensity is much smaller.
  • FIG. 10B the biopsy results of the control and test groups on days 7 and 9 (day 13 and day 15 from the date of cell inoculation) were compared, resulting from tumor growth and metastasis in the control. It is confirmed that the luminescence signal intensity is much larger than the test group.

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Abstract

본 발명은 육산화사비소의 결정다형 a(As4O6-a)를 99% 이상 포함하는 뇌암 예방 또는 치료용 약학 조성물 또는 이의 제조방법에 관한 것으로서, 본 발명의 조성물은 우수한 암세포 증식 억제 및 전이 억제 효과를 나타내어 항암제로 유용하게 이용될 수 있다.

Description

명세서
발명의명칭:육산화사비소의결정다형을포함하는뇌암예방또는 치료용약학조성물및이의제조방법 기술분야
[1] 본발명은육산화사비소의결정다형을포함하는뇌암예방또는치료용약학 조성물에관한것이다.
배경기술
[2] 암은제어되지않은세포성장으로특징지어지며，이러한비정상적인
세포성장에의해종양 (tumor)이라고불리는세포덩어리가형성되어주위의 조직으로침투하고심한경우에는신체의다른기관으로전이되기도한다. 학문적으로는신생물 (neoplasia)이라고도불린다.암은다양한정도의유병률로 신체의모든조직및장기에영향을미친다.
[3] 특히뇌암 (악성뇌종양)의경우，뇌에큰손상을일으키며생존율이매우낮은 암이다.뇌암에대한기존의치료법으로는와과적인수술에의한적출이가장 효과적이지만뇌암의종류나발생부위에따라수술이불가능한경우가많으며, 완전적출시수술후합병증의위험성이매우크다.또한,뇌에는약물의침투를 억제하는뇌혈류장벽 (brain-blood barrier; BBB)이존재하므로,항암제를아용한 화학요법으로뇌암을치료하기위해서는다른암에비하여고농도의항암제 투여가필요하게되어신체의다른장기에심각한부작용을유발하게되는 문제점이있다
[4] 따라서 ,뇌암의치료와관련하여 BBB통과에어려움이없으면서항암효과가 우수한치료제가지속적으로요구되고있는실정이다.
[5] 한편,본발명자들은이미비소가함유된자연산신석으로부터분리정제
기술을통하여정제된육산화사비소가동물실험에서암전이억제효과를 나타냈으며，아을러자궁암，방광암，폐암,상악동암，신장암등의말기
암환자에게투여하였을때뛰어난항암치료효과가있다는기술적특징으로 특허등록 (한국특허등록제 272835호)을받은바있다.
[6] 본발명자들이비소에대한지속적인연구결과,상기특허등록에개시된
방법과다른신규한제조방법에의해，육산화사비소의결정다형 a가 99%이상인 육산화사비소를제조할수있고，이러한육산화사비소를포함하는조성물이 특히뇌암예방또는치료에현저한효과가있음을밝혀본발명을완성하게 되었다.
[7] 한편,종래육산화사비소가악성신경교종 (malignant glioma)의세포침습
억제를유도하는점이보고되었으나 (J Neurosurg 121: 1483-1491, 2014)，상기 문헌에는육산화사비소가 -어떤결정다형을갖는지에대해사개시된바없을 뿐만아니라，상기문헌에기재된육산화사비소보다，본발명의제조방법에 의해제조된，육산화사비소의결정다형 a가 99%이상인조성물이보다우수한 뇌암치료효과가있는것으로확인되었다.
발명의상세한설명
기술적과제
[8] 본발명의목적은유효성분으로서육산화사비소 (ᅀ 06)의결정다형을
포함하는뇌암예방또는치료용약학조성물을제공하는데있다.
[9] 본발명의다른목적은유효성분으로서육산화사비소 (As406)의결정다형을 포함하는뇌암예방또는치료용약학조성물의제조방법을제공하는데있다. 과제해결수단
[10] 본발명은육산화사비소 (As406)의결정다형을포함하는뇌암예방또는치료용 약학조성물로서，육산화사비소의결정다형 a(As406-a)를 99%이상포함하는 약학조성물에관한것이다.
[11] 상기조성물은육산화사비소의결정다형 b(As406-b)를 1%미만으로포함할수 있다.
[12] 상기육산화사비소는순도가 99.9%이상일수있다.
[13] 상기 As406-a및 As406-b는하기 (i)내지 (iii)의특성올갖는것일수있다:
[14] [표 1]
Figure imgf000003_0001
[15] 상기 As406-a는 X-선분말회절분광도에서,광원파장이 1.5406A일때,회절각 2Θ 10ο~50ο범위에서 Wmin의속도 (scan step 0.02°)로표시된피크가 13.84， 27.88, 32.32, 35.3, 39.84, 42.38, 46.34, 48.6,및 49.34로나타나는것을특징으로하는 결정형이다 (도 1참조).또한， 2Θ값 13.8과 27.9에서나타나는주피크의비율이 ： 1:1.3인것을특징으로한다.
[16] 상기 As406-b는 X-선분말회절분광도에서,광원파장이 1.5406人알때，회절각 2Θ 10°~50°범위에서 Wmin의속도 (scan step 0.02°)로표시된피크가 13.86, 27.92, 32.36, 35.34, 39.9， 42.44, 46.4, 48.66,및 49.4로나타나는것을특징으로하는 결정형이다 (도 1참조).또한, 2Θ값 13.8과 2그 9에서나타나는주피크의비율이 1:2.5인것을특징으로한다.
[17] 본발명의다른양태에따르면，유효성분으로서육산화사비소 (As406)의
결정다형을포함하는뇌암전이억제용약학조성물로서，육산화사비소의 결정다형 a를 99%이상포함하는뇌암전이억제용약학조성물에관한것이다.
[18] 본발명의다른양태에따르면，유효성분으로서육산화사비소 (As406)의 결정다형을포함하는뇌암예방또는치료용약학조성물의제조방법으로서 , [19] 염화나트륨을 100~800oC로가열한후냉각하는제 1공정;
[2이 염화나트륨위에삼산화이비소 (As203)를올려놓은후밀폐상태에서
100°C에서 1000°C까지가열한후냉각하는제 2공;정；
[21] 여과지에서결정화된결정을분리하는제 3공정;및
[22] 상기제 3공정에서얻은결정을제 2공정의삼산화이비소대신사용하여
저 12공정및제 3공정을 4~10회반복해서육산화사비소결정을얻는제 4공정;
[23] 을포함하는방법으로제조되며 ,
[24] 상기제 4공정에서얻은육산화사비소결정이육산화사비소의결정다형 a(As40 6-a)를 99%이상포함하는것을특징으로하는뇌암예방또는치료용약학 조성물의제조방법에관한것이다.
[25] 이하，본발명을더상세하게설명한다.
[26] 본발명은유효성분으로서육산화사비소 (As406)를포함하는뇌암예방또는 치료용조성물로서，육산화사비소의결정다형 a(As406-a)를 99%이상포함하는 조성물에관한것이다.
[27] 본발명의뇌암예방또는치료용조성물의제조방법은，염화나트륨을
100~800oC로가열한후냉각하는제 1공정；염화나트륨위에삼산화이비소 (As203 )를올려놓은후밀폐상태에서 100°C에서 1000°C까지가열한후냉각하는 저 12공정;여과지에서결정화된결정을분리하는제 3공정;및상기제 3공정에서 얻은결정을제 2공정의삼산화이비소대신사용하여제 2공정및제 3공정을 4~10회반복해서육산화사비소결정을얻는제 4공정;을포함한다.
[28] 고령토재질의합성반웅기，및합성반응기의상부에필터를장착할수있는 클램프를준비하고，합성반응가내에염화나트륨을올려.놓고가열및냉각을 진행한다.본발명의제조방법에서염화나트륨을사용하는이유는,제 2공정에서 삼산화이비소를염화나트륨위에올려놓고가열함으로써비소화합물에:열이 골고루전달되면서비소화합물이승화하는데에도움이되기때문이며， 제 1공정을통해이러한염화나트륨의불순물및수분을제거하기위해
100~800°C로 2~6시간동안가열한다.제 1공정에있어서염화나트륨을가열한후 실온에서 3~10시간동안넁각한다.
[29] 다음으로,염화나트륨위에삼산화이비소 (As203)를올려놓은후밀폐상태에서 100°C에서 1000°C까지가열하고，이후넁각하는제 2공정을진행한다.여기서, 삼산화이비소를올려놓은후클램프에승화되는비소를포집할수있는 필터 (여과지)를각필터와필터사이의간격이 2mm~6mm가되도록 3~6개:
장착한다.이때사용되는필터는,평량 (basic weight) 70~100g/m',두께 (thickness) 0.17 0.25匪，여과속도 (filtration speed) 22~30s/100ml,및보류입자경 (retention rate) 5~10 인것을사용하는것이바람직하다.
[30] 필터를장착하고나서밀폐시킨후합성반웅기하부에 100oC에서 1000°C까지 단계적으로올려주면서 3시간 ~10시간동안가열해서여과지의최상부중심부의 온도는 150°C±100oC유지되도록하며，여과지를통과하면서육산화사비소가 결정화되도록한다.그리고나서상온에서 5시간이상，바람직하게는
5시간 ~10시간동안냉각한다.
[31] 다음으로，이격하여적층으로설치된 3~6개의여과지에포집된백색결정을 분리하는제 3공정을수행한다.
[32] 합성반웅기의염화나트륨위에잔류하는미량의삼산화이비소를제거한후 상기포집된백색결정을을려놓은후제 2공정및제 3공정을동일조건으로
4~10회반복해서최종적으로육산화사비소결정을수득한다.본발명의 제조방법에따라얻어진결정구조를확인한결과대부분 As406-a였으며， As406
-a가 99<¾이상인것으로확인되었다.
[33] 본발명의육산화사비소의결정다형을포함하는약학조성물은뇌암의예방 또는치료에유용하게사용될수있다.
[34] 본발명의약학조성물은각각통상의방법에따라산제，과립제，정제,캡슐제， 현탁액，에멀젼, :시럽,에어로졸등의경구형제형，외용제，좌제및멸균 주사용액의형태로제형화하여사용될수있다.상기약학조성물에포함될수 있는담체,부형제및희석제로는.락토즈，텍스트로즈，수크로스,솔비를，만니를， 자일리를,에리스리롤，말티를，전분，아카시아고무，알지네이트，젤라틴，칼슘 포스페이트，칼슘실리케이트,셀를로스，메틸셀를로스，미정질셀를로스， 폴리비닐피를리돈,물，메틸히드록시벤조에이트，프로필히드록시벤조에이트， 탈크,마그네슘스테아레이트및광물유를들수있다.제제화할경우에는보통 사용하는충진제，증량제，결합제，습윤제，붕해제，계면활성제등의희석제또는 부형제를사용하여조제된다.경구투여를위한고형제제에는정제，환제，산제， 과립제，캡술제등이포함되며,이러한고형제제는본발명의육산화사비소에 적어도하나이상의부형제예를들면,전분，탄산칼슘，수크로스또는락토즈, 젤라틴등을섞어조제된다.또한단순한부형제이외에마그네슘스테아레이트, 탈크같은윤활제들도사용된다.경구를위한액상제제로는현탁제 ,내용액제， 유제，시럽제등이해당되는데흔히사용되는단순회석제인물,리퀴드파라핀 이외에여러가지부형제，예를들면습윤제，감미제,방향제，보존제둥이포함될 수있다.비경구투여를위한제제에는멸균된수용액,비수성용제，현탁제，유제， 동결건조제제，좌제가포함된다.비수성용제,현탁제로는프로필렌글리콜， 폴리에틸렌글리콜，올리브오일과같은식물성기름，에틸올레이트와같은주사 가능한에스테르등이사용될수있다.좌제의기제로는위텝솔 (witepsol), 마크로골，트원 (tween) 61,카카오지，라우린지，글리세로젤라틴등이사용될수 있다.
[35] 상기약학조성물의투여량은치료받을대상의연령，성별 체중과,치료할
특정질환또는병리상태,질환또는병리상태의심각도,투여경로및처방자의 판단에따라달라질것이다.이러한인자에기초한투여량결정은당업자의수준 내에있으며，일반적으로투여량은 0.01mg/kg/일내지대략 500mg/kg/일의 범위이다.더바람직한투여량은 0. 1 mg/kg/일내지 100mg/kg/일이다.투여는 하루에한번투여할수도있고,수회나누어투여할수도있다.상기투여량은 어떠한면으로든본발명의범위를한정하는것은아니다.
[36] 상기약학조성물은쥐，가축,인간등의포유동물에다양한경로로투여될수 있다.투여의모든방식은예상될수있는데，예를들면，경구，직장또는정맥， 근육，피하，자궁내점막또는뇌혈관내주사에의해투여될수있다.
발명의효과
[37] 본발명의뇌암예방또는치료용조성물은，육산화사비소의결정다형 a를 99% 이상포함함으로써，우수한항암효과를갖는다.
[38] 또한，본발명의조성물은，특히뇌교모세포종등의뇌암전이를억제하는
효과가우수한것으로확인되었다.
도면의간단한설명
[39] 도 1은 As406-a및 As406-b의 X-선분말회절분광도이다.
[40] 도 2는 U-87MG세포주에실시예 1，및비교예 1내지 3를처리하고 48시간 (도
2A)및 72시간 (도 2B)배양한후 MTT어세이에의해세포증식 억제효과를 평가한결과그래프이다.
[41] 도 3은 U- U 8MG세포주에실시예 1，및비교예 1내지 3를처리하고 48시간 (도
3A)및 72시간 (도 3B)배양한후 MTT어세이에의해세포증식 억제효과를 평가한결과그래프이다.
[42] 도 4는대뇌신경교종증으로진단된환자 (임상시험 -1 )의치료전 MRI
사진으로서 T2강조횡단면 SE영상 (T2 weighted axial SE images)이다.
[43] 도 5는임상시험 -1의대뇌신경교종증환자에대한실시예 1의투여및방사선 치료 2개월후종양크기가감소되었음을보여주는 MRI사진으로서 T2강조 횡단면 SE영상이다.
[44] 도 6은임상시험 - 1의대뇌신경교종증환자에대한실시예 1의투여및방사선 치료 3개월후 MRI사진으로서종양이거의사라졌음을보여주는 T2강조 횡단면 SE영상이다.
[45] 도 7은좌측두정측두엽 (left parieto-temporal lobe)에교모세포종이있는것으로 진단된환자 (임상시험 -2)의치료전 MRI사진으로서，도 7A는 T2강조횡단면 SE 영상 (T2 weighted axial SE images)이고，도 7B는조영증강후 T1강조횡단면 SE 영상 (Post-contrast Tl weighted axial SE images)이다.
[46] 도 8은임상시험 -2의교모세포종환자에대한수술및방사선치료후의 MRI 사진으로서，도 8A는 T2증강 SE & FLAIR영상 (T2 weighted SE & FLAIR images)이고，도 8B는 T2강조횡단면 SE영상이다.
[47] 도 9는임상시험 -2의교모세포종환자에대한실시예 1의투여 4개월 (도 9A),
7개월 (도 9B)및 10개월 (도 9C)후각각의 MRI사진으로서 T1강조횡단면 SE 영상이다ᅳ " [48] 도 10은 U-87MG세포주를 Balb/C암컷누드마우스에접종한후종양영상신호 억제결과를나타낸사진 (도 10A)및그래프 (도 10B)이다.
발명의실시를위한최선의형태
[49] 이하본발명의바람직한실시예를상세히설명한다.하지만본발명은여기서 설명되는실시예에한정되지않으며，다른형태로구체화될수도있다ᅳ오히려， 여기서소개되는내용이철저하고완전해지며，당업자에게본발명의사상을 충분히전달하기위해제공하는것이다.
[50] 심시예 1:본발명의육산화사비소제조
[51] 고령토로특수제작된합성반웅기 (높이 lOOmn^지름 190mm)와, 3~6개의
필터를장착할수있는클램프를준비한다.클램프는합성반응기로부터 50mm 떨어져서 1차클램프를설치하고，상기 1차클램프로부터 2~6mm간격으로그 상부에 2차 ~6차클램프를설치하며，이때각클램프의규격은지름 210mm및 두께 10mm이다.
[52] 칭량한굵은소금 (수분함량 10%이하) 400g~600g을합성반웅기에투입하여 20mm내외로두께를고르게펴서다져주고 100°C~800°C에서 3시간동안열을 서서히가열하면서반웅기내부의소금표면온도가 290±30oC가되도록연속 가열하여수분및불순물을제거하고실온에서 5시간동안냉각시킨다. ;
[53] 원료물질인 As203(순도 98%이상，제조사 YUNNAN WENSHAN JINCHI
ARSENIC CO., LTD.) 100g을합성반웅기내의굵은소금위에넣어주고 합성기의상부에위치한승화되는비소를포집할수있는필터 (여과지 )를각 필터와필터사이의간격이 2~6匪가되도록 3~6개클램프를장착한다.이때 사용되는필터는,평량 (basic weight) 70~100g/m!,두께 (thickness) 0.17-0.25 ram, 여과속도 (filtration speed) 22~30s/ 100ml,및보류입자경 (retention rate) 5~10 /m인 것을사용하는것이바람직하다.
[54] 클램프을이용하여필터를고정시키고합성반웅기하부에열을가하여
100oC에서 1,000°C까지단계적으로온도를올려준다.먼저합성반웅기하부의 외부은도를 350°C±100°C정도가되도록 1시간동안가열하고,이후에
합성반웅기하부의외부온도를 600~650°C,및 700~1,000°C정도가되도록 가열하여，총 5시간에서 10시간동안가열하여필터의최상부여과지의 중심부온도가 150°C±100°C를유지하도록한후상온에서 5~7시간정도 냉각한다.이과정에서합성반웅기의소금위에올려진분말 As203는합성반웅기 내부에서기체로변하며상승하다가,합성반웅기외부의상부온도가
상대적으로낮기때문에액체로변하고이후고체로결정화되어필터상에 백색결정체가생성된다.
[55] 채취한백색결정체를합성반웅기의굵은소금위에넣고다시가열및
냉각하고,결정을채취하는공정을다시 4희반복해서최종적으로결정 12.0g을 얻었다.얻어진비소화합물결정의구조를확인한결과대부분 As406-a였으며 As 406-a가 99중량 «¾이상이며 As406-b가 1중량 %미만인것으로확인되었다.
[56] DSC (시차주사열분석)값이승온속도가 10°C/min일때， As406-a는 282.670C인 점에서흡열피크 (융점)가나타나는것으로확인되었으며 , As406-b는 286.77°C인 점에서흡열피크 (융점)가나타나는것으로확인되었다.
[57] As406—a및 As406-b의분말 X선회절분광도를도 1에 £시하였고， As406-a및 As
406-b의회절데이터는다음의표 1과같다. ：
[58] [표 2]
Figure imgf000008_0001
[59] 도 1및표 1에서확인되는바와같이， As406-a는 2Θ값 13.8과 27.9에서나타나는 주피크의비율이 1: 1.3이고, As406-b는 2Θ값 13.8과 27.9에서나타나는주피크의 비율이 1:2.5인것을특징으로한다.제조된화합물의 DSC분석，구조결정및 X선 회절분석은다음과같은방법으로수행하였다. ：
[60] (l) DSC분석
[61] DSC장비 (SDT Q600 V20.9 Build 20)에서， N2 lOOmL/min을흘려주면서
10°C/min승온속도로 310°C까지상승시키면서시료 20.0mg을분석하였다.
[62] (2) X선결정학
[63] 육산화사비소 (As406, MW=395.6)의단결정을 glass fiber위에올린후, X-ray beam를 2여회절되어나오는사진무늬와회절데이터의체계성의:유무를 관찰하여공간군 (space group)을결정하고，단위세포상수 (cell parameter)를 결정한다.회절세기는 10ο<2θ<50°범위에서수집한다.이데이터를구조결정 프로그램 (SHELXTL프로그램)을사용하여중원자방법 (Patterson방법)으로 As40 6의결정구조를확인한다.
[64] (2) X선회절분석법
[65] 수득한결정올분쇄하여 10 /m~30 um (-325메시 )의입자로만들고 X-선회절 분석용유리홀더 (20 mmxl6 mmxl mm)에충진시킨후,유리슬라이드등으로 압착시키고검체면과홀더면이평행을이루도록매끈하게하여시료를 준비하였다. XRD의 Cu Κα, (1.54060人)을이용하여 2Θ 10ο~50ο범위에서 Wmin의 속도 (scan step 0.02°)로결정의 X-선회절분광도를그린다.
[66] 비교예 1:육산화사비소제조：
[67] 고령토로특수제작된할성반웅기 (높이 100mm,지름 190mm)와， 3~6개의 필터를장착할수있는클램프를준비한다.클램프는합성반웅기로부터 50mm 떨어져서 1차클램프를설치하고，상기 1차클램프로부터 2~6mm간격으로그 상부에 2차 ~6차클램프를설치하며，이때각클램프의규격은지름 210mm및 두께 10mm이다ᅳ
[68] 칭량한굵은소금 (수분함량 10%이하) 400g~600g을합성반응기에투입하여 20mm내외로두께를고르게펴서다져주고 100oC~800oC 3시간동안열을 서서히가열하면서반웅기내부의소금표면온도가 290±30oC가되도록연속 가열하여수분및불순물을제거하고실온에서 5시간동안냉각시킨다.
[69] 원료물질인 As203(순도 98%이상，제조사 YUNNAN WENSHAN JINCHI
ARSENIC CO., LTD.) lOOg을합성반응기내의굵은소금위에넣어주고， 합성기의상부에위치한승화되는비소를포집할수있는필터 (여과지 )를각 필터와필터사이의간격이 2~6誦가되도록 3~6개클램프를장착한다.이때 사윷되는필터는,평량 (basic weight) 70~100g/m!,두께 (thickness) 0.17~0.25薩， 여과속도 (filtration speed) 22~30s/100ml,및보류입자경 (retention rate) 5~10 인 것을사용하는것이바람직하다.
[70] 클램프을이용하여필터를고정시키고합성반웅기하부에열을가하여
100oC에서 l,000oC까지단계적으로온도를올려준다.먼저합성반웅기하부의 외부온도를 350°C±100oC정도가되도록 1시간동안가열하고,이후에
합성반웅기하부의외부은도를 600~650oC,및 700~1,000°C정도가되도록 가열하여,총 5시간에서 10시간동안가열하여필터의최상부여과지의 증심부은도가 150°C±100°C를유지하도록한후상온에서 5~7시간정도 냉각한다.이과정에서합성반웅기의소금위에올려진분말 As203는합성반응기 내부에서기체로변하여상승하다가,합성반웅가외부의상부온도가
상대적으로낮기때문에액체로변하고이후고체로결정화되어필터상에 백색결정체가생성된다.필터로부터 48.5g의결정을채취하였다.채취한 비소화합물의결정구조를확인한결과 99%이상대부분 As406-b인것으로 확인되었다.
[71] 비교예 2내지 4:육산화사비소제조
[72] 실시예 1(결정다형 As406-a가 99%이상인조성물)과비교예 1(결정다형 As406 -b가 99%이상인조성물)을각각 4: 1,및 1:1로흔합하여，각각비교예 2및비교예 3으로하였다.
[73] 심험예 1:이간 ^1경교종 nmman glioma)세포증식억제효과심 ¾
[74] 1-1.세포증식어세이 (MTT어세이 )
[75] (1)실험재료및세포배양
[76] 소태아혈청 (FBS)과세포배양배지를준비하였고 (Hyclone사)，디메틸
설폭사이드 (DMSO),
3-(4,5-디메틸-티아졸 -2일) -2,5-디페닐테트라졸륨브로마이드 (3-(4,5-dimetyl-thiaz ol-2yl)-2,5-diphenyltetrazolium bromide, MTT)를준비하였다 (Amresco LLC, USC). [77] 인간의암세포주로서，인간뇌교모세포종세포주 (human brain star glioblastoma cells)인 U-87MG및 U-118MG세포주를중국과학아카데미의상하이세포 은행으로부터입수하였고,세포배양조건에따라 10% FBS, 100U/mL페니실린, 및 lOC^g/mL스트렙토마이신이：포함된 EMEM(Eagle's Minumum Essential Medium)배지를이용하여 5% C02및 95%공기와습기가있는상태의
배양기에서세포를배양하였다.배지는 3일마다교체하였다.
[78] (2)세포:증식어세이 (MTT어세이)
[79] 실시예 1，및비교예 1내지 3의세포증식에대한효과를 MTT어세이를이용해 평가하였다. MTT어세이는 MTT에대해살아있는세포가불용성암청색포마잔 반응물을생성하는능력에기반한다.트립신처리에의해세포들을배지에 부유시켜모은후, 4xl03세포 /웰 (cells/well)의밀도로 96-웰배양디쉬 (Costar, Cambridge, MA, USA)에분주된다. 24시간후에，처리시료로서실시예 1，및 비교예 1내지 3각각을 0, 0.625, 1.25， 2.5, 5, 10, 20, 40또는 80μΜ아되도록 10% FBS를포함하는배지가들어가있는세포에첨가한후배양하였다.실시예 1,및 비교예 1내지 3은 5χ10-2Μ농도로저장용액으로서 1M수산화나트륨에 용해하였다. 48시간및 72시간배양하고，세포증식에대한 ΜΤΤ어세이를 수행하였고,각시간간격의마지막에상등액을제거하고웰 (well)당 5mg/mL의 MTT용액을 20 씩첨가하고나서포마잔결정을형성하기위해 37°C에서 4시간동안인큐베이션하였다.인큐베이션후에，상등액을다시제거하고， DMSO를모든웰당 100 씩첨가하고,암창색결정을완전히용해하도록 흔합하였다.실온에서 15분동안방치하여모든결정이완전히용해되도록하고， 570nm(A570nm)파장에서마이크로-플레이트기로흡광도를측정하였다.
[8이 (3)통계분석
[81] 시료를처리하지않은대조군의흡광도값을 100으로계산하고，상기대 ¾군 대비시료를처리한처리군의흡광도값을교정 (calibration)하였고，세포증식 억제율을하기식에따라계산하였다.
[82] 세포증식억제율 (%) = ((대조군세포의평균흡광도 -처리군세포의평균
흡광도 )/대조군세포의평균흡광도) X 100
[83] 모든데이터는평균土표준오차 (means士 SEM)으로표현된다.일원배치
분산분석 (one-way analysis of variance, ANOVA)후던넷의사후검증 (Dunnett's post-test)에의해다중비교를수행하였다.유의수준은 p<0.05였고，각실험은 3번씩반복:되었다.
[84] (4) U-87MG세포주이용실험결과
[85] 실시예 1，및비교예 1내지 3을인간뇌교모세포종세포주인 U-87MG에
처리하고나서 , 48시간및 72시간배양하고 MTT어세이를수행한결과는,도 2에 나타내었다.실시예 1을처리한후 48시간 (도 2A)및 72시간 (도 2B)배양한 실험결과모두에서,비교예 1을처리한경우에비해뇌교모세포종세포의증식 억제율이높은것으로확인되었다.또한，실시예 1과비교예 1이 4:1로흔합된 비교예 2나， 1: 1로흔합된비교예 3에비해，실시예 1이뇌교모세포종세포의 증식억제율이높은것으로확인되었다.
[86] (5) U-118MG세포이용실험결과
[87] 실시예 1,및비교예 1내지 3을인간뇌교모세포종세포주인 U-118MG에
처리하고나서， 48시간및 72시간배양하고 MTT어세이를수행한결과는,도 3에 나타내었다.실시예 1을처리한후 48시간 (3A)및 72시간 (도 3B)배양한 실험결과모두에서,비교예 1에비해교모세포종세포의증식억제율이높은 것으로확인되었다.또한，실시예 1과비교예 1이 4:1로흔합된비교예 2나， 1 :1로 혼합된비교예 3에비해，실시예 1이뇌교모세포종세포의증식억제율이높은 것으로확인되었다.
[88] 심험예 2: ¾상시험
[89] 실시예 1의조성물을이용하여다음과같이임상시험을시행하였다.
[90] (1)임상시험 -1: 51세여성환자
[91] 자각증상은없었으나건강검진에서뇌병변이발견된여성환자로서
서울대학병원에서행한뇌조직검사에서대뇌신경교종증 (gliomatosis cerebri; 병리적진단은역형성성상세포증 (anaplastic astrocytomas)임 )진단을받았고， 진단일후 10일째되는날부터두팔의운동성약화및떨림증상이나타나기 시작된이후증상이급속하게진행되었다.진단일후 17일째되는날부터하루에 15mg씩실시예 1을투여하였고，운동성약화및떨람증상이개선되기시작했다. 진단일후 27일째〜 75일째까지의기간동안방사선치료를병행하였고，이후에 증상이완전히사라졌다.이환자의치료전 MRI사진은도 4에나타내었고, 실시예 1의투여와함께방사선치료 2개월및 3개월후의사진은각각도 5및도 6에나타내었다.
[92] (2)임상시험 -2: 16세여성환자
[93] 두통，구토，복시 (diplopia)및시야장애증상을보인환자로서
좌측두정측두엽 (left parieto-temporal lobe)에교모세포종 (glioblastoma, left parieto-temporal lobe)이있는것으로진단되었다.진단일후 3일째되는날종양 제거수술을받았고병리학적진단결과 WHO등급 IV IV의교모세포종인 것으로확인되어，진단일후 45일째 ~90일째까지와기간동안방사선과
Tremodal(61.2Gy/33fx)치료를하였고,방사선및 Tremodal치료후여전히 MRI상 잔존종양이확인되었다.진단일후 95일째부터하루에 3회씩 5mg의실시예 1을 Π개월동안투약하였다.실시예 1을투껴받은지 2개월이후부터자각증상이 없었고，잔존종양의크기가실시예 1의복용후감소한것으로확인되었다.이 환자의치료전 MRI사진은도 7에나타내었고，수술및방사선치료후의 MIR 사진은도 8에나타내었으며，실시예 1의투여 4개월, 7개월및 10개월후각각의 MRI사진은도 9A,도 9B,및 9C로나타내었다.
[94] 임상시험결과를통해,본발명의조성물은뇌혈류장벽 (brain-blood barrier;
BBB)을통과해서교모세포종과같은악성뇌종양의증식을효과적으로 억제하는효과가있는것으로확인되었다.
[95] 심험예 3:이간뇌교모종세포종이식동물모템에서의 체내약효시험
[96] (1)실험방법
[97] 인간뇌교모종세포주 U-87MG(ATCC에서구입)를온도 37°C, 5% C02및 95% 공기조건하의배양기에서 10% FBS, 100U/mL페니실린，및 100/ g/mL 스트렙토마이신이포함된 EMEM(Eagle's Minumum Essential Medium)배지를 이용하여배양하였다.배지는 3일마다교체하였다.
[98] 루시페라제로표기한 U-87MG세포를체중 16~19g의 Balb/C암컷누드마우스 15마리의뇌안에뇌원발부위접종을진행하며세포접종양은 5xlOV5uL으로 하였다.동물의수술부위가회복되고종양세포가조금성장한접종일로부터 6일째되는날동물에대하여영상검사를실시하며，영상결과에따라랜덤으로 각그룹별 4마리씩 2그룹으로나누고,시험군에는실시예 1을생리식염수 (NaCl 0.9%)에용해한용액을투여하고，대조군에는시험군에사용된용매인 생리식염수만을투여하는것으로하였다.두그룹의동물은각각매일 1회씩 생리식염수또는 2.25mg/kg의실시예 1을투여하고,투여용량은실험동물의 체증 lOuL/g으로하였다.투여과정에동물체중과건강상태를계속검사하며 동시에영상검사를진행한다.체중이과도 (：〉 20%)감소혹은건강상태가좋지 않은동물에대하여안락사시키고종양을제거하여포르말린고정을하고，이후 조직학검사를진행한다.
[99] (2)체중측정결과
[100] 약물투여후 9일경과시다음과같이시험군동물의체중은거의변화가
없었지만，대조군동물의체중감소가보다크게나타났다.
[101] [표 3]
Figure imgf000012_0001
[102] 또한,실험과정에서대조군동물은세포접종 15일이경과하면서체증이
현저하게하강하였으며 17~19일정도에동물이사망하기시작했다.
[103] (3)종양영상신호억제결과
[104] 또한，종양영상신호억제결과를확인한결과,도 10A에서와같이투약 7일 째 (접종일로부터 13일째)부터대조군동물은종양신호의강도가컸고,종양의 척추전이에따라척추에서종양신호가분포하는것으로나타난것에비하여 , 시험군동물의종양신호는척추에서거의확인되지않고그강도도훨씬적은 것으로나타났다.도 10B에서확인되는바와같이，투약 7일째및 9일째 (세포 접종일로부터 13일째및 15일째)의대조군및시험군의생체영상결과를비교해 보면,대조군에서종양의증식및전이로인한발광신호강도가시험군에비해 훨씬크게나타나는점이확인된다.

Claims

청구범위
[청구항 1] 유효성분으로서육산화사비소 (As406)를포함하는뇌암예방또는치료용 약학조성물로서 ,
상기육산화사비소는하기 ω내지 (no의특성을갖는육산화사비소의 결정다형 a가 99중량 %이상인것을특징으로하는뇌암예방또는치료용 약학조성물.
(i)단위세포상수:
a = b = c = 11.0734 A
α = β = γ = 90°
V = 1357 82人 3
( ") As-0결합길이: 1.786 A
(iii) O-As-O결합각： 98.36°
[청구항 2] 제 1항에있어서，
상기육산화사비소는순도가 99.9%이상인것을특징으로하는뇌암예방 또는치료용약학조성물.
[청구항 3] 제 1항에있어서，
상기결정다형 a는 X-선분말회절분광도에서,광원파장이 1.5406A일때， 회절각 2Θ 10°~50°범위에서 l0/min의속도 (scan step 0.02°)로표시된 피크가 13.84, 27.88, 32.32, 35.3, 39.84, 42.38, 46.34, 48.6,및 49.34로 나타나는것을특징으로하는뇌암예방또는치료용약학조성물.
[청구항 4] 유효성분으로서육산화사비소 (As406)를포함하는뇌암전이억제용약학 조성물로서,
상기육산화사비소는하기 (i)내지 (lii)의특성을갖는육산화사비소의 결정다형 a가 99중량 %이상인것을특징으로하는뇌암전이억제용약학 조성물. .
(i)단위세포상수:
a = b = c = 11.0734 A
α = β = γ = 90°
V = 1357.82 A3
(") As-0결합길이: 1.786 A
(iii) O-As-0결합각: 98.36°
[청구항 5] 제 1항내지제 3항중어느한항에기재된육산화사비소 (As406)를
포함하는뇌암예방또는치료용약학조성물의제조방법으로서 , 염화나트륨을 100~800°C로가열한후냉각하는제 1공정 ; 염화나트륨위에삼산화이비소 (As203)를올려놓은후밀폐상태에서 100oC에서 1000oC까지가열한후냉각하는제 2공정;
여과지에서결정화된결정을분리하는제 3공정;및 상기제 3공정에서얻은결정을계 2공정의삼산화이비소대신사용하여 제 2공정및제 3공정을 4~10회반복해서육산화사비소결정을얻는 제 4공정 ;을포함하는방법으로제조되며，
상기제 4공정에서얻은육산화사비소결정은육산화사비소의결정다형 a(As406-a)가 99증량 %이상인것을특징으로하는뇌암예방또는치료용 약학조성물의쎄조방법 .
PCT/KR2017/009820 2016-11-18 2017-09-07 육산화사비소의 결정다형을 포함하는 뇌암 예방 또는 치료용 약학 조성물 및 이의 제조방법 WO2018093029A1 (ko)

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