CN1235831A - Medicinal composition containing globoside and its use - Google Patents
Medicinal composition containing globoside and its use Download PDFInfo
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- CN1235831A CN1235831A CN99116073A CN99116073A CN1235831A CN 1235831 A CN1235831 A CN 1235831A CN 99116073 A CN99116073 A CN 99116073A CN 99116073 A CN99116073 A CN 99116073A CN 1235831 A CN1235831 A CN 1235831A
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Abstract
The medicinal composition may be solution-prepared through dissolving globoside into cosolvent and supersonic emulsification, or addition of excipient vehicle, then powder-prepared through freezing drying or tablet prepared through pressing the powder. It is used as medicine to treat cerebral gioma, kidney cancer, lung cancer and other tumor.
Description
The present invention relates to contain the pharmaceutical composition and the application thereof of globoside (Globoside), particularly can be used to treat the pharmaceutical composition and the application thereof of tumors such as cerebral glioma, renal carcinoma, pulmonary carcinoma.
Known glycosyl sphingolipid is positioned on the cell membrane mostly, and according to having or not ptyalin to be divided into ganglioside and neutral glycosyl sphingolipid in its structure, the former contains sialic acid.Glycosyl sphingolipid is made up of oligonucleotide chain and ceramide, stretches to the cell outside with the hydrophilic oligonucleotide chain that has, as one of composition of cell surface glycocalyx.According to above characteristics, through lot of experiments, commonly think that glycosyl sphingolipid participates in cell recognition, differentiation, signal transduction, interaction etc.Proposed since Hakomori since the relation of glycosyl sphingolipid and tumor, a large amount of when experimental studies have found that malignant change of cell, some variation of the disappearance of some glycosyl sphingolipid or generation can make cell forfeiture contact inhibition, thereby cause some life signal material disappearances, the sudden change material increases.The domestic and international at present research to glycosyl sphingolipid concentrates on acid glycosyl sphingolipid, and promptly ganglioside is less to the research of neutral glycosyl sphingolipid.Existing research concentrates on receptor, blood group antigen, tumor associated antigen, embryo's specific antigen of some cell verticillium toxin and microorganism etc., research to the relevant glycosyl sphingolipid of tumor then concentrates on the genesis mechanism aspect, does not see that as yet neutral glycosyl sphingolipid acts on the report of tumor pharmacother.
The purpose of this invention is to provide a kind of medicine that can be used to treat tumors such as cerebral glioma, renal carcinoma, pulmonary carcinoma.
Cerebral glioma is the modal primary tumor of brain, and its pathogeny and biological behaviour it be unclear that so far.The conventional general treatment measures such as operation, radiotherapy, change furuncle that adopt of the treatment of glioma over past ten years, all be difficult to obtain gratifying effect, and serious adverse effects has also limited the dosage and the course of treatment of traditional chemotherapeutics, add the influence of the specificity and the blood brain barrier of tumor, the curative effect of further having cut down medicine.The mechanism of therefore glioma generation, development becomes the focus of Recent study.
About the research of the relevant glycosyl sphingolipid of the cerebral tumor is to study comparatively deeply in the tumor glycosyl sphingolipid research field and a part of widely.Discover: 1. brain glue must tumor cell strain and the tumor tissue sample ganglioside of expressing mainly contain: GM2, GD2, GD3 and 3 '-isoLM, wherein 3 '-the isoLM specificity is stronger; 2. the composition of the neutral glycosyl sphingolipid of the cerebral glioma of the various histological type of discovery such as singh-LP has heterogeneity: wherein the content of total neutral glycosyl sphingolipid, CMH and the CDH of glioblastoma multiforme (AA) is higher, and the content of water is low.Do not contain CTH and Globoside; Total neutral glycosyl sphingolipid and the CMH content of neural germinal layer tumor (PNET) is low.But CTH and Globoside often appear; The astroglioma of low potential malignancy (LGA) water content height, total fat and CMH content are low; And the astroglioma of high malignancy (HGA), the composition of all neutral glycolipids all possesses, and content is medium; Mesoglioma CMH content height, water constituent content is low, less CTH and the Globoside of occurring; 3. the relative percentage composition of the content of glycosyl sphingolipid or joint glycosides fat is relevant with cerebral glioma patient's prognosis.
We discover that the expression of cerebral glioma, renal carcinoma, the neutral glycosyl sphingolipid of cancerous lung tissue exists the opposite sex, compare with normal person's respective organization, the expression of most of tumor cell CDH and CTH is more normally organized by force, they all do not contain neutral sheath sugar Globoside, though and the expression of the neutral glycosyl sphingolipid of normal structure also exists certain heterogeneity, but they all contain certain Globoside, and the shortage of prompting Globoside may be one of reason of cell carcinogenesis.
According to the present invention, we can be mixed with pharmaceutical composition with carrier (B component) in the various pharmacy with the Globoside (component A) that extracts from various zooblasts, thereby treatment brain glue is held tumors such as matter tumor, renal carcinoma and pulmonary carcinoma.
The source of component (A):
Fresh Medulla sus domestica, Medulla Bovis seu Bubali, Medulla caprae seuovis, animal hemocyte, normal person's hemocyte through homogenizer homogenate or Ultrasonic Pulverization, extract total fat with 2: 1,1: 2,1: 1 chloroform/methanol solution respectively, and cumulative volume spends the night under 4 ℃ for the last time for 20 times of tissue.Filter paper filtering, collection also merge filtrate three times, 39 ± 3 ℃ of following distilling under reduced pressure, obtain faint yellow oily thing, be total fat, with chloroform/methanol solution dissolving in 1: 1,-20 ℃ of preservations are standby: DEAE-Sephadex A-25 column chromatography: thing is got 2.2gDEAE-Sephadex A-25 and is put in the wide mouthed bottle, add 30mlB liquid (chloroform: methanol: the 0.8M sodium acetate is 60: 30: 8 mixture), vibrated 30 minutes, leave standstill, remove supernatant, so triplicate allows resin be immersed in the B liquid for the last time and spends the night under 4 ℃.B is fresh configuration.Inferior daily 30mlA liquid (chloroform: methanol: water is 60: 30: 8 mixture) is washed resin 3-4 time, be suspended at last and pour in the 10mlA liquid in the chromatographic column that external diameter is 14mm, put a little A liquid in the post earlier, allow the resin natural subsidence, solvent flows out, and reuse 60-80mlA liquid washs to remove whole sodium acetates.Total fat of various tissues is inserted slow upper prop in the A liquid, and speed is the 0.2ml/ branch, spends the night behind the upper prop total fat and resin are fully exchanged.Collect eluent again with A liquid 80ml eluting next day, and 39 ± 3 ℃ of following distilling under reduced pressure are neutral glycosyl sphingolipid crude extract; With quadrat method crude extract is crossed the Flossil chromatographic column, obtain the neutral glycosyl sphingolipid of purification.
The neutral glycosyl sphingolipid of purification dissolves with 1: 1 chloroform/methanol solution, mix silica gel H (10-40u) pressurized column chromatography,, be in charge of collection with variable concentrations chloroform/methanol solution gradient eluting, detect in TLC plate point sample, chloroform/methanol/water launched in 60: 35: 8, and iodine develops the color, and compared with the neutral glycosyl sphingolipid of standard, collect the Globoside of purification, speckle of TLC plate point sample, Rf value 0.45, evaporated under reduced pressure obtains white solid:
1NMR, FAB mass spectrograph are identified structure, are three～six glycosyl sphingosines.
The Globoside in various sources, structure is as follows:
Globotriaosyl?cermide(Gb3)
GalNAc(betal-3)Gal(alphal-4)Gal(betal-4)Glc-cer
Gb5:Gal(betal→3)GalNAc(betal-3)Gal(alphal-4)Gal(betal-4)Glc-Cer
GL7:sialyl?Gal(betal→3)GalNAc(betal-3)Gal(alphal-4)Gal(betal-4)Glc-Cer
Globo?H:Fuc(alphal→2)Gal(betal→3)GalNAc(betal→3)Gal(alphal→4)Gal(betal→4)Glc-Cer
GalNAc:(N-acetylgalactosamine) and N-acetylamino galactosamine Gal:(galactose) galactose Glc:(9lucose) glucose Fuc:(fucose) fucose Cer:(ceramide) ceramide }
The structure of various source Globoside is respectively:
Medulla caprae seuovis cell extraction: three glycosyl sphingosines;
Medulla Bovis seu Bubali cell extraction: four and pentasaccharides base sphingosine;
Porcine mesencephalic cell extracts: four and pentasaccharides base sphingosine;
Human red blood cell is extracted: four and pentasaccharides base sphingosine;
Swine erythrocyte extracts: six glycosyl sphingosines; Component (B) is (1) cosolvent, comprises ethanol, dimethyl sulfoxide, water
(2) other excipient
Component (A) is dissolved in the cosolvent through ultrasonic emulsification or adds other excipient and is mixed with solution, also can adopt freeze-drying to make powder, or powder is recompressed into tablet.
The valid density 3～100% (V/V) of component (A) and component (B) preparation finished product.Optimum concentration range is 25～100% (V/V).
The pharmaceutical composition that the present invention proposes is the medicine that can be applicable to treat tumor, because of disturbing tumor cell mitosis and DNA, synthesizes Globoside, blocking-up during tumour cell cycle, signal conduction and growing multiplication suppress tumor cell gene expression, inducing apoptosis of tumour cell.
Describe embodiments of the invention in detail below in conjunction with accompanying drawing.
Accompanying drawing is cell growth curve figure of the present invention.
Experiment material:
(1) Globoside and cosolvent are mixed with 100% compositions.
(2) cell strain: external SWO-38, SK-RC-49, SPC-A-1 cell strain conventional cultivation the in the culture fluid of the RPMI-16-40 that contains 10% calf serum gone down to posterity.
(3) animal: the SCID nude mouse, male, body weight 20-26g is provided by Zhongshan Medical Univ.'s animal center, the quality certification numbers 96015.
(4) main agents: MTT (Sigma product)
Experimental technique:
MTT experiment: get index and give birth to tumor cell line in the phase such as SWO-38, SK-RC-49, SPC-A-l, with 2 * 10
2The hole is inoculated in 96 orifice plates, 5%CO
2, cultivate 24hr in 37 ℃ of incubators; The Globoside that adds the separate sources Concentraton gradient respectively, every kind of concentration is established 3 multiple holes, and sets up separate sources CDH and blank, continues to cultivate 48hr; The every hole of 4hr adds MTT (5mg/ml) liquid 10ul before the test, and every hole adds DMSO solution 200ml behind the 4hr, measures the OD value in every hole down in enzyme linked immunological instrument 570mm.Logarithm with concentration is mapped to suppression ratio, tries to achieve the IC of various Globoside
50Value is 23.4 ± 4.54.Separate sources Globeside is to the IC of SWO-38, SK-RC-49, SPC-A-1 cell strain
50
(unit: ug/ml)
Medulla Bovis seu Bubali Medulla sus domestica Medulla caprae seuovis human blood cell Sanguis sus domestica cell human leukocyte
SWO-38 21.3 26.4 29.2 16.5 27.6 367
SK-RC-49 24.5 23.1 28.6 18.4 26.7 358
SPC-A-1 23.6 22.4 26.0 17.4 26.9 396
Cell growth curve: get SWO-38, SK-RC-49, the SPC-A-1 of exponential phase of growth, cell strain, 1 * 10
5The hole is inoculated in 24 orifice plates, and adds the Globoside of separate sources, 5%CO in every hole
2, cultivate in 37 ℃ of incubators; Measure first group of cell strain sum in three holes next day, sequentially determining 7 days, be depicted as cell growth curve, as seen separate sources Globoside all can suppress SWO-38, SK-RC-49, the growth of SPC-A-1 cell (is seen accompanying drawing, abscissa is the concentration of Globoside, and vertical coordinate is suppression ratio %).
Globoside induces the glioma cell line apoptosis:
Flow cytometer detects: after Globoside acts on SWO-38, SK-RC-49, SPC-A-1 cell strain, collecting cell according to a conventional method, the up flow type cell instrument detects, and every routine sample is surveyed 10,000 nucleus, testing result is transported to computer and is for data processing, and special-purpose software is made cell cycle analysis.It is synthetic to found that Globoside can suppress tumor cell DNA, inducing apoptosis of tumour cell.
DNA electrophoresis detection apoptosis trapezoid belt: Globoside acts on the situation of change of gene expressions such as cell bcl-2, c-fos behind SWO-38, SK-RC-49, the SPC-A-1 cell, c-myc.It is anti-that SWO-38 after the Globoside effect, SK-RC-49, SPC-A-1 cell add mouse-anti people one successively, and after fluorescently-labeled sheep anti mouse two resisted, the up flow type cell instrument detected, and finds a little less than the said gene expression decreased.
Influence research to lotus people mice with tumor:
(1) foundation of bearing mouse model: get some, exponential phase growth, vigor at the SWO-38 more than 95%, SK-RC-49, SPC-A-1 cell, be inoculated in nude mice right hind femoribus internus muscle successively, about 1 * 10
5Individual oncocyte;
(2) administrated method: after the tumour transplatation the 4th day, can touch and transplant the part when slightly protuberance being arranged, nude mice is divided into 4 groups at random, Globoside group and normal saline group, respectively 12.Experimental group gives tumor body local injection or oral Globoside10mg/kg, matched group corresponding site injecting normal saline or oral vehicle respectively;
(3) draw materials and film-making: after the tumour transplatation 14 days, each group is put to death 4, gets tumor body, the other lymph of homonymy ventral aorta, axillary gland, and the tumor body is with scales/electronic balance weighing, and lymph node is measured length and width, volume calculated.Organize all with formalin fixed specimens paraffin embedding slices, HE dyeing microscopic examination;
(4) result: experimental group tumor body weight on average alleviates than matched group, has statistical significance; Experimental group lymph volume reduces than matched group, has statistical significance.
The Globoside of separate sources is to the influence of lotus people mice with tumor:
Gross examination of skeletal muscle: can touch the tumor knot on the 4th day behind the mice-transplanted tumor, later tumor propagation is rapid, suffers from the limb function limitation.All be the state of becoming thin before the execution.
Medulla Bovis seu Bubali Medulla sus domestica Medulla caprae seuovis human blood cell Sanguis sus domestica cell physiological saline group tumor weight g 1.54 1.69 1.82 1.70 1.63 5.16 lymph nodes size 0.01 0.01 0.01 0.01 0.01 0.036
P＜0.05
Toxicological experiment:
When (1) Globoside of separate sources acts on tumor cell line, measure it to the leukocytic IC of normal person
50Value, the IC of human leukocyte as a result
50Value is higher than tumor cell far away.As seen Globoside is low to the toxicity of normal plasma cell;
(2) observe the effect of Globoside to normal mouse brain tissue:
The Balb/c mice, male and female have concurrently, and body weight 23-259, is divided into 9 groups, 11 every group at random by totally 99; Give following medicine or reagent respectively: the Globoside of normal saline, solvent, excipient, animal origin and normal person's hemocyte Globoside, tween is adopted in contrast;
With mice with etherization after, open the Globoside that cranium or vein are annotated the various sources of body;
Observe mouse breathing, heart rate, consciousness, limb activity situation respectively and have or not tic, vomiting, body weight change etc.Afterwards, in the 2nd, 4,6 day every group get and get cerebral tissue after 2 mices are put to death and do pathological section, after the 7th day all mices are put to death and do cerebral tissue and important organ pathology:
Separate sources Globoside is to the influence of mice vital sign and cerebral tissue:
Unusual performance and the 7th day average body weight (g) of death condition
Medulla Bovis seu Bubali does not have 24.5
Medulla sus domestica does not have 25.9
Medulla caprae seuovis does not have drowsiness 23.1 human red blood cell of 1 Mus of 25.8 swine erythrocytes and does not have 24.6 normal saline groups and do not have 23.8 group of solvents not have 1 Mus activeness of 24.8 vehicle group poor, do not have dead 25.1 tween matched groups generally take food poor, dead 9 14.2
P＜0.05
(3) long term toxicity test: tried the long-term oral or intravenous injection separate sources Globoside of mice, observe damage, and set up the BCNU matched group to tract.
The result: compare with positive controls, mice prolonged application Globoside does not see obvious organ injury.
Embodiment 2:
The Globoside of various separate sources and cosolvent be mixed with 3% compositions, carry out (1) MTT experiment, (2) cell growth curve, (3) Globoside according to the method for embodiment 1 then and induce struma cell strain apoptosis, (4) influence research, (5) toxicological experiment lotus people mice with tumor, can draw to draw a conclusion, 3% Globoside compositions can suppress growth of tumour cell, and toxic and side effects is little.
Embodiment 3:
The Globoside of various separate sources and cosolvent be mixed with 50% compositions, carry out (1) MTT experiment, (2) cell growth curve, (3) Globoside inducing tumor cell strain apoptosis, (4) influence research, (5) toxicological experiment to lotus people mice with tumor according to the method for embodiment 1 then, can draw to draw a conclusion: growth and toxic and side effects that 50% Globoside compositions can suppress tumor cell are little.
Claims (5)
1, a kind of pharmaceutical composition contains Globoside (globoside) component (A) and pharmacy acceptable carrier
(B): it is characterized in that:
Component (A) is that the Globoside structure of various structures is as follows:
globotraosyl?ceramide(Gb3)
GalNAc(betal-3)Gal(alphal-4)Gal(betal-4)Glc-Cer
Gb5:Gal(betal→3)GalNAc(betal-3Gal(alphal-4)Gal(betal-4)G1c-Cer
Globo?H:Fuc(alphal→2)Gal(betal→3)GalNAc(betal→3)Gal(alphal→4)Gal(betal→4)Glc-Cer
GalNAc:(N-acetylgalactosamine) and N-acetylamino galactosamine Gak:(galactose) galactose Glc:(glucose) glucose Fuc:(fucose) fucose Cer:(ceramide) ceramide }
Component (B) is (1) cosolvent, comprises ethanol, dimethyl sulfoxide, water
(2) other excipient
Component (A) is dissolved in the cosolvent through ultrasonic emulsification or adds other excipient and is mixed with solution, also can adopt freeze-drying to make powder, or powder is recompressed into tablet.
2,, it is characterized in that the valid density 3～100% (V/V) of component (A) and component (B) preparation finished product according to the described compositions of claim 1.
3,, it is characterized in that the optimum concentration range of component (A) and component (B) preparation finished product is 25～100% (V/V) according to the described compositions of claim 1.
4, according to the described compositions of claim 1, it is characterized in that; Globoside is three～six glycosyl sphingosines, and comes from different zooblasts, is respectively:
Medulla caprae seuovis cell extraction: three glycosyl sphingosines;
Medulla Bovis seu Bubali cell extraction: four and pentasaccharides base sphingosine;
Porcine mesencephalic cell extracts: four and pentasaccharides base sphingosine;
Human red blood cell is extracted: four and pentasaccharides base sphingosine;
Swine erythrocyte extracts: six glycosyl sphingosines;
5, according to the application of any compositions described in the claim 1 to 4 as tumor medicines such as treatment cerebral glioma, renal carcinoma, pulmonary carcinoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99116073A CN1235831A (en) | 1999-03-02 | 1999-03-02 | Medicinal composition containing globoside and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99116073A CN1235831A (en) | 1999-03-02 | 1999-03-02 | Medicinal composition containing globoside and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1235831A true CN1235831A (en) | 1999-11-24 |
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ID=5278924
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99116073A Pending CN1235831A (en) | 1999-03-02 | 1999-03-02 | Medicinal composition containing globoside and its use |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102066940A (en) * | 2008-06-16 | 2011-05-18 | 中央研究院 | Cancer diagnosis based on levels of antibodies against Globo H and its fragments |
-
1999
- 1999-03-02 CN CN99116073A patent/CN1235831A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102066940A (en) * | 2008-06-16 | 2011-05-18 | 中央研究院 | Cancer diagnosis based on levels of antibodies against Globo H and its fragments |
| CN102066940B (en) * | 2008-06-16 | 2015-05-13 | 中央研究院 | Cancer diagnosis based on levels of antibodies against Globo H and its fragments |
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