CN1403439A - Phytosphingosine derivate with antitumor activity - Google Patents
Phytosphingosine derivate with antitumor activity Download PDFInfo
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Abstract
The present invention relates to one kind of plant 4-sphingenine derivative with antineoplastic activity and it is used as the active component of antineoplastic agent.
Description
Technical field
The present invention relates to a kind of antineoplastic agent that contains phytosphingosine (phytosphingosine), concrete is, relates to a kind of antineoplastic agent, and it contains a kind of phytosphingosine, be the Phytosphingosine derivate with anti-tumor activity, its structural formula I is as a kind of active ingredient.
R wherein
1, R
2And R
3Represent a hydrogen atom or a C respectively
1-C
8Alkyl; X represents an atom, or an atomic group, and this atomic group is by a halogen atom, a hydroxyl, and an alkylsulfonate atomic group or an arylsulphonate atomic group are formed.
Background technology
The lipid that constitutes cytolemma is made up of phosphatide, glycolipid and sphingolipid usually.These lipids all are the amphipath materials, when they are dispersed in the water, just spontaneously produce the vesicles of complete closed, and this vesicles is similar to the cytolemma that is called as liposome.
Liposome can be by using a kind of or two or three kind of above-mentioned lipid preparation.Liposome is recognized that a kind of good carrier in the drug delivery system.When medicament was attached in the liposome, the hydrophilic segment of medicine was closed in the inside aqueous phase of liposome, and the hydrophobic part of medicine then is inserted between the bilayer of liposome.As a kind of pharmaceutical carrier, liposome can arrive sick position to a kind of useful for drug delivery of needs exactly, both has been that a spot of medicine also can be by the liposome transmission.Therefore, liposome can greatly reduce side effect, for example the multiple medicines thing resistance in heavy dose.Recently liposome has been widely applied to multiple field as a kind of pharmaceutical carrier, for example antigen, gene, medicine, comprise Zorubicin (a kind of antineoplastic agent), amphotericin B (a kind of anti-mycotic agent), other chemicalses and beauty treatment fields (M.Grunaug and partner thereof, Europe medical research magazine, 1988 the 21st phases, 13-19 page or leaf; D.S.Alberts, D.J.Garcia, medicine magazine, 1997 the 54th phases, 30-35 page or leaf; F.Braun and partner thereof, implantation method, 1988 the 30th phases, 1481-1483 page or leaf; V.Heinemann and partner thereof, anti-microbial agents chemotherapy, 1997 the 41st phases, 1275-1280 page or leaf; N.Weiner and partner thereof, pharmaceutical target magazine, 1994 the 2nd phases, 405-410 page or leaf).
The sphingolipid base is with phytosphingosine (PhytoS), and the form of ceramide (SPN) and dihydroxy ceramide is present in the person, and they are respectively the amino alcohols that contains 18 carbon atoms.These compounds have several stereocenters, are arranged with on position 3 that D-is erythrocytic to be found at occurring in nature.Ceramide and dihydroxy ceramide are to find in all tissues of human body, and phytosphingosine only is present in the stratum corneum of human skin.Just begun in early days broad research in the nineties (twentieth century), when finding that these compounds are the powerful inhibitor of protein kinase C (PKC), paces have been accelerated in their research ceramide and derivative thereof.In addition, find that ceramide both had been under lower concentration, also relevant (D.J.Bibel and partner thereof, clinical experiment dermatology, the 20th phase of nineteen ninety-five, 395-400 page or leaf with the most cells activity; D.J.Bibel, dermatitis research magazine, 1992 the 98th volumes, 269-273 page or leaf; Y.A.Hannun, science magazine, 1996 the 274th volumes, 1855-1859 page or leaf).These are active to display in stratum corneum mainly, and people have improved widely to the interest of phytosphingosine, yet they are very expensive, and to synthetic they derivative and their biological activity is known little about it.Particularly, the N that generally acknowledges, N-dimethyl ceramide (DMS) and N, N, N-halogenation trimethylammonium sphingosine (abbreviates as: TMShal), the derivative of-ceramide-be better than ceramide aspect their arrestin kinases, but also known they in vivo and in glass test tube, can both suppress the growth of various cancer cells.In addition, find that also TMShal has a kind of anti-tumor activity and antimetastatic activity in the B16/BL6 of mouse melanoma cell series, used liposome trimethylammonium sphingosine in this experiment, wherein Yelkin TTS (PC): cholesterol (chol): the mole ratio of TMShal is 4.5: 4.5: 1.But, need a large amount of relatively TMShal (for example about 0.1-0.3 milligram of each mouse) to show above-mentioned effect, this often causes side effect, for example: hemocytolysis, hemoglobinuria and inflammatory response.By using liposome technology to do very big effort to solving these toxicity, the result shows that the TMShal of liposome shows nontoxicity, and compare with the TMShal that does not use liposome, in vivo in the system, more effective (Y.S.Park, S.Hakomori aspect the growth of anticancer and anti-metastasis, S.Kawa, F.Ruan, and Y.Igarashi, cancer research, 1994 the 54th phases, the 2213-2217 page or leaf).
Have a about phytosphingosine---its structure be very similar to ceramide-report, disclosed because a kind of auxiliary lipid difference structurally, caused the KK-1 of system in vivo, DNA changes difference (T.Paukku and the partner thereof of efficiency of infection in COS-7 and the MSC-1 cell, the lipid of chemical physics therapy, 1997 the 87th phases, the 23-29 page or leaf).In addition, also the known plants sphingosine has excellent antimicrobial acivity for microorganism widely, and alleviates stimulation to skin by secreting leukocytes mesonium as a kind of inhibitor of protein kinase.N; N, N-halogenation trimethylammonium phytosphingosine (TMPhal) are a kind of derivatives of plant ammonia alcohol, and this is to announce (the untested patent No. 1999-78610 of Korea S) recently; derivative is wherein described as a kind of component of improving looks, and its purposes is confined to protect skin.Yet show that beyond example TMPhal is a kind of antineoplastic agent.
Summary of the invention
The present inventor has made the liposome of the derivative of the phytosphingosine that contains said structure formula I with various compositions, and has confirmed their anti-tumor activity and antimetastatic activity.Therefore, purpose of the present invention just provides a kind of anti-tumor activity that has, and as the Phytosphingosine derivate of structural formula I, also provides the antineoplastic agent that contains Phytosphingosine derivate simultaneously.
The object of the present invention is achieved like this, involved a kind of antineoplastic agent, and it contains a kind of Phytosphingosine derivate of as following structural formula (I) as active ingredient.
Among the superincumbent structural formula I, R
1, R
2And R
3Represent a hydrogen atom or a C respectively
1-C
8Alkyl; X represents an atom or an atomic group, and it contains a halogen atom, a hydroxyl, an alkylsulfonate atomic group or an arylsulphonate atomic group.
Antineoplastic agent of the present invention contains the above-mentioned Phytosphingosine derivate that exists with liposome or a kind of emulsion form, except Phytosphingosine derivate, also can contain other compositions, for example anti-angiogenic agent or Zorubicin ,-a kind ofly have a cytotoxic antineoplastic agent.
The present invention relates to a kind of antineoplastic agent, it contains a kind of Phytosphingosine derivate (being designated hereinafter simply as TMP) just like above structural formula (I), and this derivative is to make with the form of a kind of liposome or a kind of emulsifying agent.As a kind of TMP of the present invention, that selects the superior is to use N, N, and N-halogenation trimethylammonium phytosphingosine (TMPhal), that more selects the superior is to use N, N, N-iodate trimethylammonium phytosphingosine (TMPI).
Prepared the anti-metastasis liposome with various compositions in the present invention.The result shows that the DPPC/Chol/TMP of liposome or DPPC/Chol/PEGPE/TMP composition when being used in combination with a kind of anti-angiogenic agent, having excellent antimetastatic activity, and also have a kind of synergistic action effect in antimetastatic activity.The DPPC/Chol/TMP composition of liposome can not only suppress the transfer to lung, and they also suppress the growth of LLC (lewis lung cancer) cancer cells.
In the present invention, the cytotoxicity cancer therapy drug is used in combination with the anti-metastasis liposome, then the effect of anti-metastasis is strengthened.For example when Zorubicin uses with the TMP liposome, has stronger anti-metastasis effect than independent use Zorubicin.
In the present invention, used human liver oncocyte system and mouse melanoma cell series to check the cytotoxic effect of TMP liposome, the result shows that TMP has cytotoxicity in the human liver tumour cell, and in the mouse melanoma cells no cytotoxicity.In mouse, do acute toxicity test, do not observed toxic effect.
Antineoplastic agent of the present invention contains a kind of Phytosphingosine derivate as structural formula I as a kind of promoting agent, final preparation can be following form: powder, particle, capsule, and it and the acceptable carrier that makes up a prescription, the injection of getting up of a kind of vehicle and a kind of mixing diluents.Can oral administration medicine supplying, also can the parenteral dispensing, if medicament is made a kind of emulsion or the lipid build is offerd medicine later on, then biological effectiveness is more effective.
The dosage of antineoplastic agent of the present invention can change according to the variation of health specific absorption, body weight, age, sex, healthy state, diet, dosing interval, medication administration method, excretion rate, disease severity and analogue.Dosage according to qualifications is about per kilogram of body weight 0.5-1 milligram.The preparation of antineoplastic agent should be considered the useful range of dosage, Zhi Bei unit formulation can be offerd medicine several times in certain time interval like this, or according to an expert's decision, with a kind of special dosage method preparation, this expert is the requirement according to curee (or subject), the supervision of responsible medical treatment and observation.
Although have the scheme according to qualifications of singularity to a certain degree describes the present invention in conjunction with the present invention, just can obtain product according to qualifications of the present invention but the people who is good at technology should understand the method for having only by following example, and can take multiple variation and not leave the spirit and scope of the present invention in the structure of composition, details aspect the combination and permutation.
Description of drawings
Fig. 1 illustrates according to N, N, the content of N-trimethylammonium iodate ceramide (abbreviating TMPI as), the photo of the antimetastatic activity of TMPI liposome on a melanoma cells.
Fig. 2 illustrates the photo of the antimetastatic activity of TMPI liposome and TMPI emulsion, and TMPI liposome wherein and TMPI emulsion all contain the compound of angiogenesis inhibitor.
Fig. 3 illustrates the photo of the relation between TMPI liposome and a kind of antineoplastic agent-Zorubicin.
Fig. 4 is a cytotoxic figure that the cationic-liposome of a kind of TMPI of containing is shown.
Fig. 5 is a figure who is illustrated in the stability of the emulsion that contains the TMP derivative that stores under 4 ℃ the temperature and various liposomes.
Embodiment
Below in conjunction with accompanying drawing the present invention is done more detailed narration.
Example 1:N, N, N-iodate trimethylammonium phytosphingosine (TMPI) synthetic
In 3 ml methanol, add 0.30 gram phytosphingosine (0.946 milliliter), 0.523 gram K
2CO
3(3.79 millimole) and 0.298 milliliter of methyl iodide (4.73 millimole), stirred reaction mixture reaches 4 hours under 50 ℃ temperature.Under reduced pressure solvent is evaporated, and in resulting mixture, add 4 ml distilled waters.Solution is extracted dry then (Na with 8 milliliters of ethyl acetate
2SO
4), filter.The evaporation of acetic acid ethyl ester obtains 0.26 gram white solid product.
Yield rate: 76%
Fusing point: 210-213 ℃ IR (KBr) ν maximum: 3309 (OH), 2918,2850 (C-H) cm
-11H NMR (600MHz, DMSO-d
6): δ 3.95 (dd, 1H, CH
2O, J=14.4Hz), 3.89 (dd, 1H, CH
2O, J=14.4Hz), 3.76 (d, 1H, J=8.7Hz), 3.6 (dd, 1H), 3.11 (s, 9H, N
+CH3), 1.68 (m, 1H, CH
2), 1.48 (m, 1H, CH
2), 1.23 (s, 24H, CH
2), 0.84 (t, 3H, CH
3) ppm
13C NMR (600MHz, DMSO-d
6): δ 76.80,71.01,55.69,52.18,33.21,31.21,30.60,29.15,29.03,28.99,28.93,28.62,24.87,22.00,13.84ppmMS (FAB, glycerine, m/z): 361 (M
+).
Example 2:N, N, N-trimethylammonium phytosphingosine is synthetic to monomethyl sulfonate
In 5 ml methanol, add 0.5 gram phytosphingosine (1.575 millimole), 1.612 gram K
2CO3 (9.449 millimole) and 1.188 milliliters of methyl are to a tosylate (to a toluenesulphonic acids methyl esters) (7.874 millimole), and stirring the mixture under 50 ℃ temperature reaches 3 hours.Under reduced pressure solvent is evaporated, in resulting mixture, add 5 ml distilled waters.With 10 milliliters of ethyl acetate extraction solution, dry then (Na
2SO
4), filter.Evaporate ethyl acetate, obtain 0.46 gram white solid product.
Yield rate: 55%
Fusing point: 185-186 ℃ IR (KBr) ν maximum: 3326 (OH), 2920,2852 (C-H) cm
-11H NMR (500MHz, CD
3OD): δ 7.70 (d, 2H, arom H, J=8.2Hz), 7.23 (d, 2H, arom H, J=8.0Hz), 4.16 (dd, 1H, CH
2O, J=14.4Hz), 4.09 (dd, 1H, CH
2O, J=14.4Hz), 3.89 (d, 1H, J=8.7Hz), 3.73 (dd, 1H), 3.44 (t, 1H), 3.21 (s, 9H, N
+CH3), 2.37 (s, 3H, PhCH3), 1.81 (m, 1H, CH
2), 1.58 (m, 1H, CH
2), 1.29 (s, 24H, CH
2), 0.90 (t, 3H, CH
3) ppm
13C NMR (500MHz, CD
3OD): δ 142.07,140.16,128.31,125.45,76.73,71.56,56.09,52.21,33.25,31.56,29.28,29.26,28.96,25.03,22.22,19.81,12.93ppm
Example 3: the preparation of liposome
(1) preparation of multilamellar vesicle (MLV) and small-sized unilamellar vesicle (SUV)
Phosphatide is joined in the vial, and with organic solvent (CHCl
3) dissolving.Use a kind of nitrogen or a kind of rotatory evaporator that solvent is removed fully, in vial, form a kind of lipid membrane.Add phosphate buffered saline (PBS) (PBS) then, at room temperature shake lightly,, rotate mixture (making it into swirl shape) then tempestuously,, finally form multilamellar vesicle (MLV) so that make the lipid membrane diffusion of phosphatide so that reach enough hydration levels.
By using a ultrasonic degradation device that the MLV that obtains is transformed into small-sized unilamellar vesicle.In addition, use a forcing machine (also will use in the experiment afterwards of this machine), under high pressure make SUV pass through a suitable PC membrane filter, can make the liposome of required specification.
(2) preparation of anti-metastasis liposome
Preparation contains various phosphatide and TMP-a kind of anti-metastasis compound as follows---liposome.
The natural mixtinite of TMP and DOPE-a kind of-mix with the ratio of 1: 1 (w/w), be dissolved in 20 milliliters the vial that organic solvent is housed, then, exist under the situation of nitrogen, under reduced pressure evaporate, when generating lipid membrane, the film complete drying, glucose with distilled water or 5% carries out hydration, prepares cationic-liposome by rotation or ultrasonic degradation.TMP joined contain 70% Yelkin TTS (PC); The mixture of 100% Yelkin TTS of a kind of 1: 1 mole ratios and cholesterol (Chol); The mixture of DPPC of a kind of 1: 1 mole ratios (DPPC) and cholesterol (Chol); A kind of mole ratios is 5: 5: 1 DPPC, in the mixed composition that the mixture of Chol and PE-PEG (phosphatidylethanolamine-polyoxyethylene glycol) is formed, be dissolved in a kind of organic solvent, by using a rotatory evaporator to remove organic solvent fully, in vial, form a kind of lipid membrane.Add phosphate buffered saline (PBS) (PBS) then, at room temperature make film hydration sufficiently, make the film diffusion of phosphatide, obtain the liposome of anti-metastasis then by rotation or ultrasonic degradation.
Example 4: have the preparation of emulsion of antimetastatic activity and the measurement of physical properties thereof.
(1) preparation of emulsion
Yelkin TTS 70% and TMP are diffused in the sweet oil, add glycerol and a little tween 20 (polysorbas20), add distilled water, and use ultrasonic degradation, obtain a kind of emulsion.The membrane filter of the emulsion that obtains like this, stand-by by one 0.2 micron.
(2) experiment of liposome and emulsion stability
Prepare the liposome and the emulsion of the various compositions that contain TMP, and under 4 ℃ temperature, store.By using sizer to measure the variation of liposome specification, and measure the stability of liposome and emulsion according to the component of phosphatide.
Example 5: the analysis of anti-metastasis in the body
The direct lung that uses the B16F10 melanoma cells to observe in the system in vivo a C57/BL6 mouse on one's body shifts.Inject melanoma cells (phosphate buffered saline (PBS), 2 * 10 of various concentration from the vein of a mouse afterbody
4, 2 * 10
5With 2 * 10
6), so that determine the concentration of suitable melanoma cells that mouse is thrown in.Its lung is taken out in after injection 15 days after anesthesia, be checked through the bacterium colony that exists cancer in lung.
Further, the suitable melanoma cells concentration of determining from top experiment is injected in the tail vein of C57/BL6 mouse, so that check the anti-metastasis effect of anti-metastasis liposome and the emulsion of the above-mentioned TMP of containing.After injection tumour cell 60-90 minute, the liposome that contains 250 microgram TMP of preparation is thrown to each mouse, then, carry out the 2nd time and the 3rd dispensing the 3rd day and the 6th day behind the injection melanoma cells respectively.In needs, carried out the 4th and offer medicine in the 9th day after injection.After 15 days, lung is taken out, check out the cancer bacterium colony of lung.
Example 6: the toxic measurement of TMP liposome in cytotoxicity and the body
Use human liver neoplasm clone SNU398 and the melanoma cell series B16F10 of mouse to check the cytotoxic effect of TMP liposome to cancer cells.SNU398 and B16F10 are carried out trysinization, use the medium (RPMI-1640) of serum-free to clean then.With the trypanosome orchid they are dyeed again, then with 1 * 10
5The concentration of individual cells/ml is coated on the plate that 48 pits are arranged, and handles with the cationic-liposome that contains TMP of various concentration then.After 3 days, once more they are dyeed with trypanosome is blue, check viable cell to reduce.
In order to check the toxicity in vivo of TMP liposome, the method by intravenous injection or peritoneal injection is injected into them on one's body the mouse, checks the lethality rate of mouse then.
Example 7: the analysis that anticancer is grown in vivo
Use lewis lung cancer (LLC) cell to check in the system in vivo inhibition on one's body to growth of cancer cells the BDF1 mouse.The concentration of employed lewis lung cancer cell is such: give 1,000,000 cancer cells of each mouse subcutaneous injection, so that bring out cancer.Injected 100 milliliters of TMP liposomes (TMP100 microgram) by the method for intravenous injection and peritoneal injection respectively in the 1st day, the 3rd day, the 6th day and the 9th day behind the above-mentioned LLC cell of injection.As a kind of positive regulation, make AG3340 (U.S. A Gulong medicine company limited product)-known a kind of MMP-2 inhibitor (matrix metaloproteinasse-2)-be suspended in the mixed solution of 0.2% tween/0.5% carboxymethyl cellulose, obtain 2 milligrams of suspension liquids and carry out peritoneal injection every day.Kill each mouse behind injection LLC cell 21 days with the method for cervical vertebra dislocation, check the variation that corpus carcinosus is long-pending then, and take pictures.
Example 8: the preparation of tablet
Active ingredient ... 1 gram
Lactose ... 7 grams
Crystal fibre ... 1.5 gram
Magnesium Stearate ... 0.5 gram
Amount to ... 10 grams
Above-mentioned component is smash fragmentate after, again they are mixed, by the method for directly making tablet they are made tablet, every total amount is 500 milligrams, wherein active ingredient is 50 milligrams.
Example 9: the preparation of powder (powder)
Active ingredient ... 1 gram
W-Gum ... 5 grams
Carboxylated fiber ... 4 grams
Amount to ... 10 grams
They are smash fragmentate after, again they are mixed, make pulverous powder then, 500 milligrams of powder are packed in the soft capsule, just can be made into capsule preparations.Experimental example 1
At first, check the antimetastatic activity of TMPI in the system in vivo.Check to cancer metastasis is to carry out in 4 groups of different mouse, and these 4 groups of mouse have different B16F10 melanoma cells concentration, are respectively 2 * 10
4, 2 * 10
5, 2 * 10
6And PBS (phosphate buffered saline (PBS)), so that determine to be suitable for observing the cancer cells number of metastasis of cancer.In the tail vein of mouse after the injection 15 days are taken out the lung of mouse and are checked.Found that from injecting 2 * 10
6The size of the lung that one group of mouse of B16F10 melanoma cells concentration obtains has on one's body been grown up, and has formed a large amount of bacterium colonies at last.On the contrary, from 2 * 10
4The lung that obtains in handle with PBS one group does not demonstrate obvious variation dimensionally, does not demonstrate the generation of cancer bacterium colony simultaneously yet, and from 2 * 10
5One group of lung that obtains handling demonstrates a spot of bacterium colony and exists, and the number of bacterium colony increases constantly, after the 21st day after handling till.Therefore, for the experiment of TMPI anti-metastasis, the suitable concentration of melanoma cells is defined as 2 * 10
5
Threw the TMPI derivative of 300 micrograms for respectively the mouse of every group of experiment in the 1st day, the 3rd day and the 6th day after injection B16F10.The lung that obtained in the 15th day after injection is littler than the lung of the mouse in the control group, and colony number also reduces (table 1) significantly.
The result of table 1.DOPE/TMP liposome antimetastatic activity
Experimental example 2
| Classification | The dosage of TMPI (microgram) | Colony number |
| ?????????PBS | ????????- | ???200±20 |
| DOPE/TMPI liposome (1: 1 weight ratio) | ???????300 | ???30±15 |
Checked the antimetastatic activity of TMPI according to different components.TMPI is joined in the mixture of 70% Yelkin TTS and 100% Yelkin TTS/cholesterol (Chol) (1: 1 mole ratio), be used for the anti-metastasis experiment.Inject 2 * 10 from the tail vein of C57BL mouse
5The B16F10 melanoma cells of concentration.Mouse is handled with the liposome that 250 micrograms contain TMPI injection behind the melanoma cells 1 hour, the liposome that after for the second time injecting melanoma cells the 3rd day contains TMPI with 250 micrograms is handled mouse, and the TMPI that behind the 3rd injection melanoma cells the 7th day contains liposome with 250 micrograms handles mouse.After for the first time injecting melanoma cells the 15th day by the method for the cervical vertebra of mouse dislocation is killed mouse, taken out lung on one's body from every group of mouse then, and comparison colony number (table 2).Table 2. contains the result of 70% Yelkin TTS and the antimetastatic activity of the TMPI liposome that contains 100% Yelkin TTS
Experimental example 3
| Classification | The dosage of TMPI (microgram) | Colony number |
| ????????????PBS | ?????????- | ???200±20 |
| 70%PC/TMPI liposome (10: 1 weight ratios) | ????????250 | ???35±15 |
| 100%PC/Chol/TMPI liposome (5: 5: 1 mole ratios) | ????????250 | ???30±10 |
Use contains the antimetastatic activity of the liposome component check TMPI concentration of 70% Yelkin TTS.Except 70% Yelkin TTS, also added cholesterol, so that increase the stability of liposome.Carry out the experiment of antimetastatic activity with the liposome that contains 70% Yelkin TTS, cholesterol and TMPI mixture.
The result shows that the liposome that contains 50 microgram TMPI has the antimetastatic activity (Fig. 1 and table 3) more than 60%.On the contrary, the conventional TMS liposome that contains 250 microgram TMS (trimethylammonium ceramide) demonstrates about 50% antimetastatic activity (Y.S.Park and partner thereof, cancer research, 1994 the 54th phases, 2213-2217 page or leaf).Like this, the result proves that the anti-metastasis effect that contains the TMPI liposome is better than the TMS liposome.
The antimetastatic activity that table 3. is determined according to different TMPI content
Experimental example 4
| Classification | The dosage of TMPI (microgram) | Colony number |
| ??????????PBS | ???＞250 | |
| 70% Yelkin TTS/cholesterol/TMPI liposome (4: 4: 1 weight ratios) | ??????50 | ???80±30 |
| ??????100 | ???30±10 | |
| ??????200 | ???35±25 |
Check the antimetastatic activity of various anti-metastasis liposomes according to different components, and checked the antimetastatic activity (table 4) of the liposome that adds anti-angiogenic agent (AG3340, U.S. A Gulong pharmaceuticals product).Make the control liposome by weight ratio melting lecithin/cholesterol/phytosphingosine with 4: 4: 1.Lipid and the ratio of medicine remain on 20: 1 weight ratio.The component of liposome shown in the table 4 below and effect.The TMPI of dispensing and the concentration of medicine (angiogenesis inhibitor medicine) all fix on 100 micrograms.In given liposome, no matter the existence of medicine (angiogenesis inhibitor medicine) whether, it is identical that the concentration of TMPI all keeps.
Except in injection the 1st hour after the melanoma cells, outside the 3rd day, the 6th day and 4 throwings of the 9th natural gift liposome, the carrying out of experiment is identical with method in the example 5.The result shows that colony number has minimizing slightly in the control group of the liposome that contains phytosphingosine, yet effect is also not obvious.When the DPPC liposome that comes into operation adds a kind of medicine (a kind of angiogenesis inhibitor medicine), demonstrate almost very little anti-metastasis effect.But when the liposome that contains TMPI when coming into operation added a kind of medicine (a kind of angiogenesis inhibitor medicine), colony number reduced to significantly and is less than 15 countings.When coming into operation (TMPI adds PEG) liposome, this lipid is 10% PEG-PE to be joined in the liposome that contains TMPI make, and coming into operation has like this increased the residence time of liposome in blood, has observed similar superiority (Fig. 2).
Table 4. contains the check that the TMPI liposome adds the collaborative antimetastatic activity of anti-angiogenic agent
Experimental example 5
| Classification | The dosage of TMPI (microgram) | Anti-angiogenic agent (microgram) | Colony number |
| ??PBS | ??????- | ??????- | ??＞250 |
| The control liposome 1) | ??????- | ??????- | ??200±30 |
| The DPPC liposome 2) | ??????- | ?????100 | ??210±40 |
| The TMPI liposome 3) | ?????100 | ?????100 | ??15±5 |
| The TMPI liposome 3) | ?????100 | ??????- | ??35±10 |
| The TMPI+PEG liposome 4) | ?????100 | ?????100 | ??15±5 |
| 1) DPPC/Chol/PEG-PE/TMPI (5: 5: 1 mole ratios) DPPC/Chol/TMPI (5: 5: 1 mole ratios) 4 DPPC/Chol (1: 1 mole ratio) 3 70%PC/Chol/ phytosphingosine (4: 4: 1 weight ratios) 2))) | |||
Use a kind of emulsion to measure antimetastatic activity, this emulsion is to be made by a kind of TMPI derivative with antimetastatic activity.Except the anti-metastasis emulsion is that experiment is undertaken by the method for experimental example 4 with the intraperitoneal dispensing.Compare with those lungs from the lung that undressed a group (a control group) obtains through the mouse of TMPI emulsion processing.The colony number that the results are shown in the mouse in the control group is 250, and the colony number of the group of handling with the TMPI emulsion is 70 ± 20.The implication of this value is that antimetastatic activity is greater than 70% (Fig. 2).Experimental example 6
(DPPC/Chol/TMPI=5: mole ratio was thrown to the BDF1 mouse that has inoculated lewis lung cancer (LLC) cell in 5: 1, had observed their inhibition effects to the tumor growth the TMPI liposome that contains transfer activity.The concentration of employed LLC cancer cells is 1,000,000 cancer cells of each mouse, by the subcutaneous injection induced tumor.Each mouse intravenous injection and peritoneal injection TMPI liposome (TMPI content: 100 micrograms) are given in after the subcutaneous injection the 1st day.After subcutaneous injection the 3rd day, the 6th day and the 9th day, give the identical dosage of each mouse subcutaneous injection repeatedly, the 21st day after injection with killing each mouse to the method for mouse cervical vertebra dislocation, with the volume of following formula measurement cancer, the result who measures shown in the table 5 below.[formula 1]
(major axis of cancer) * (minor axis of cancer)
2Dosing way and the dosage number of/2 table 5.TMPI liposomes and AG3340 (a kind of positive regulation),
And to the inhibition result of cancer growth
| Classification | Dosage (microgram) | Dosing way/dosage number | Volume (the mm of cancer 3) | Inhibiting rate (%) |
| Control | ?1587±400 | ????0 | ||
| ??AG3340 | ????2000 | Peritoneal injection/20 | ?1100±250 | ????31 |
| ??TMP·I | ????100 | Peritoneal injection/4 | ?220±50 | ????87 |
| ??TMP·I | ????100 | Intravenous injection/4 | ?540±150 | ????66 |
As above shown in the table, in a control group, the volume of cancer growth is very big, and a kind of active vasculogenesis process is arranged around demonstrating in the cancer district.In the group of peritoneal injection TMPI liposome, the volume of cancer reduces significantly, and the process of vasculogenesis also worsens widely.Simultaneously, in another group, the AG3340 of 2000 micrograms-a kind of MMP-2 inhibitor-be suspended in tween/carboxymethyl cellulose mixed solution, in 20 days, all inject to mouse every day then, and the volume of cancer does not dwindle greatly; That is to say, work as AG3340, a kind of positive regulation, when doing peritoneal injection, the volume of cancer has only reduced about 30%.On the contrary, when the TMP liposome is done belly when injection, it is about 85% that the volume of cancer reduces, and when the TMP liposome done intravenous injection, the volume of cancer reduced about 60%.Experimental example 7
Use the B16F10 melanoma cells, the conventional antineoplastic agent of check TMPI liposome and Zorubicin-have cytotoxic agent-to the effect of mouse cancer metastasis.Give each mouse intravenous injection 2 * 10
5Individual melanoma cells, after injection, the 33 microgram Zorubicins that come into operation immediately, and behind the intravenous injection melanoma cells the 3rd day, the 6th day and also dropped into the Zorubicin of same dose on the 9th day respectively, the 25 microgram Zorubicins that come into operation in another group add the TMPI liposome.The result shows that the TMPI liposome that comes into operation adds Zorubicin than the Zorubicin that comes into operation separately more effective aspect the anti-metastasis, even some also are (Fig. 3) like this to the amount of the Zorubicin that uses less
Experimental example 8
Prepare two kinds of dissimilar TMPI liposomes, so that check toxicity in vivo and cytotoxicity.Two kinds contain the TMPI sun and are used for checking their cytotoxic effects to cancer cells from liposome-DOPE/TMP and DPPC/Chol/TMP liposome.Employed liver neoplasm clone is human liver neoplasm clone SNU398 and mouse melanoma cell series B16F10.Go out cationic-liposome (TMPI: DOPE=1: 1 weight ratio), checked their cytotoxic effects (Fig. 4) then with different prepared at concentrations to cancer cells.The result shows do not observe cytotoxic effect (up to 200 micrograms) in the mouse melanoma cell series, when liver neoplasm clone after the TMPI of 12.5 micrograms liposome-treated, observe the death of some cancer cells, after the TMPI liposome-treated with 100 micrograms, nearly all cancer cells is all dead.Under the situation of SNU cancer cells, LD50 is 25 micrograms (Fig. 4).
When to mouse peritoneal injection 2000 micrograms contain the TMPI cationic-liposome, and after injection for the first time the 3rd day and when the 6th day repeatedly carries out identical injection respectively, tested, and observed mouse and still live in the 15th day after injecting for the first time.In addition, when doing intravenous injection with the DPPC/Chol/TMPI liposome of 1000 micrograms to mouse, tested their also still live (tables 6) in the 15th day after injection for the first time by top mode.
Table 6. contains the toxicity in vivo experiment of TMPI liposome
Experimental example 9
| Classification | Dosage (microgram) | Dosing way | The dosage number | Lethality rate (%) |
| ??DOPE/TMP·I | ????2000 | Peritoneal injection | ????3 | ??0/5(0) |
| ??DPPC/Chol/TMP·I | ????1000 | Intravenous injection | ????3 | ??0/5(0) |
Use a platform sizer 3000,, checked the anti-metastasis liposome that remains on 4 ℃ and the stability of anti-metastasis emulsion according to the variation of the specification of liposome and emulsion.It is stable in the glucose solution of distilled water and 5% that the result illustrates positively charged ion TMPI liposome.If the liposome of PC base, the liposome that contains DPPC and PEG-PE is the most stable, and emulsion also shows stable (Fig. 5) in during bimestrial.
As implied above, the invention provides the multi-functional liposome that contains TMP, as a kind of Phytosphingosine derivate of structural formula I, transfer and growth that it can not only anticancer, and when being used in combination, can also make the anti-metastasis effect reach optimum extent with another kind of antineoplastic agent.Different with the liposome of routine is that these anti-metastasis liposomes can show antimetastatic activity separately, thereby they will be very useful in the transfer system of antineoplastic agent, and can reduce the dosage level of given antineoplastic agent.
Claims (4)
1. Phytosphingosine derivate with anti-tumor activity, the Phytosphingosine derivate that contains a kind of structural formula (I) is as a kind of active ingredient.
R wherein
1, R
2And R
3Represent a hydrogen atom or a C respectively
1-C
8Alkyl; X represents an atom or an atomic group, and this atomic group contains a halogen atom, a hydroxyl, an alkylsulfonate atomic group or an arylsulphonate atomic group.
2. according to the described Phytosphingosine derivate of claim 1, it is characterized in that this Phytosphingosine derivate is N, N, N-halogenation trimethylammonium phytosphingosine.
3. according to the described Phytosphingosine derivate of claim 1, it is characterized in that this Phytosphingosine derivate is contained in liposome or the emulsion.
4. according to the described Phytosphingosine derivate of claim 1, it is characterized in that the material or a kind of cytotoxicity cancer drug that contain a kind of anti-angiogenesis activity add this Phytosphingosine derivate.
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| CN107382965A (en) * | 2017-08-14 | 2017-11-24 | 河南科技大学第附属医院 | The synthetic method of the receptor stimulating agent drug molecules of new S1P 1 with antitumor activity |
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| CN107382965A (en) * | 2017-08-14 | 2017-11-24 | 河南科技大学第附属医院 | The synthetic method of the receptor stimulating agent drug molecules of new S1P 1 with antitumor activity |
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