CN101513400A - Ampelopsin and basic amino acid solubilizing system - Google Patents
Ampelopsin and basic amino acid solubilizing system Download PDFInfo
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- CN101513400A CN101513400A CNA2008100337753A CN200810033775A CN101513400A CN 101513400 A CN101513400 A CN 101513400A CN A2008100337753 A CNA2008100337753 A CN A2008100337753A CN 200810033775 A CN200810033775 A CN 200810033775A CN 101513400 A CN101513400 A CN 101513400A
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Abstract
The invention provides an ampelopsin composition and a method for preparing the same. The composition comprises: a) ampelopsin and b) a basic amino acid, wherein the weight ratio the component a) to the component b) is between 1:1 and 1:10. The composition can also comprise: c) a pH regulator and or d) a pharmaceutically acceptable carrier and/or a diluting agent. The invention also relates to a medicamnet box containing the ampelopsin composition and carboplatin. The ampelopsin composition can provide improved solubility, stability and a proper pH range and is suitable for clinic use.
Description
Technical field
The present invention relates to field of pharmaceutical preparations.More specifically, the present invention relates to have the dissolubility and the stable ampelopsin preparation of raising.
Background technology
Ampelopsin (Ampelopsin AMP) is the extract of ampelopsis, belongs to polyphenol hydroxyl flavanone alcohols, relative molecular weight 356, and molecular formula is C
15H
12O
82H
2O, its pure product are faint yellow graininess.Contain 6 phenolic hydroxyl groups in the AMP molecule, have faintly acid, isoelectric point, IP near pH value about 5.0 (chief editor such as Sun Wenji, " and the simple and clear handbook of active skull cap components, Chinese Medicine Science Press, 1993:564).
AMP has that toxicity is low, wide material sources, advantage that extraction ratio is high, but its less stable, oxidation reaction easily takes place, be insoluble to chloroform, ether, be soluble in ethanol, methanol, dichloromethane and dimethyl sulfoxide (DMSO) (Liu Deyu etc., the preparation of ampelopsin microcapsule, Chinese crude drug, 2003,26 (5): 355-357.).AMP is slightly soluble in water, dissolubility in the time of 25 ℃ in aqueous solution only is 0.2mg/ml (Li-Ping Ruan etc., Improving the solubility of ampelopsin by solid dispersions and inclusioncomplexes (improving the dissolubility of ampelopsin by solid dispersion and embedding complex), Pharm BiomedAnal, 38 (2005): 457-464).
In recent years, find that ampelopsin has the effect (Liu Deyu etc. that suppress the growth of B16 melanoma in the mice body, the extraction of inermis middle ampelopsin and to melanomatous inhibitory action, Chinese Medical Sciences University's journal, 1999,20 (2): 127-129), can significantly suppress tumor cell people Folium Nicotianae preparatum cancer HK-1 external, human breast carcinoma MCF-7 cell proliferation (Liu Deyu etc., the antitumor action cancer of ampelopsin, Chinese Journal ofCancer, 2001,20 (12): 1372-1375), human promyelocytic leukemia HL-60, the K562 cell (Zhouning County is peaceful etc., ampelopsin extracorporeal anti-tumor function research in the no acupuncture root, Guangdong pharmacy, 2000,10 (4): 7-8).Ampelopsin has significant inhibitory effect to people's pulmonary carcinoma GLC-82 transplanted tumor in nude mice, prompting AMP may be a kind of broad-spectrum anti-cancer drug, and AMP is a kind of antitumor drug of low toxicity, have DEVELOPMENT PROSPECT (Ceng Sa etc. preferably, ampelopsin is to the inhibitory action of people's pulmonary carcinoma GLC282 transplanted tumor in nude mice, Chinese crude drug, 2004,27 (11): 842-844.).
Have extremely strong antitumor action though confirmed Caulis seu folium ampelopsis brevipedunculatae (Caulis Ampelopsis Brevipedunculae), it is soluble,very slightly in water, and it is clinical that this is used for its difficulty.How to improve AMP dissolubility and steady dissolution, preparation is suitable for the pharmaceutical dosage form of clinical practice, is the key issue that needs to be resolved hurrily in the present ampelopsin research.
Summary of the invention
The present invention provides a kind of stability and deliquescent ampelopsin injection and injectable powder with raising just at above-mentioned problem.
In a first aspect of the present invention, a kind of ampelopsin compositions is provided, it comprises following component:
A) ampelopsin; With
B) basic amino acid, it is selected from: L-arginine, L-lysine or their combination;
Wherein, component a) and components b) weight ratio be 1: 1～1: 10, preferred 1: 1～1: 8, more preferably 1: 1～1: 6.
In a preference, component a) and components b) mol ratio be 1: 2.5～1: 10, preferred 1: 3～1: 8, more preferably 1: 3～1: 6.
In one embodiment, described compositions is selected from ampelopsin injection or ampelopsin injectable sterile powder.
In a preference, described compositions is the ampelopsin injection, and wherein the volume in injection is a benchmark, and the concentration of ampelopsin is 0.01～500mg/ml, preferred 0.03～400mg/ml, more preferably 0.05～300mg/ml.
In another preference, the solvent in the described injection is water for injection, phosphate buffer, aqueous solution of propylene glycol, citrate buffer, acetate buffer solution or their combination.
In another preference, described compositions is the ampelopsin injectable sterile powder.
In another embodiment, described compositions also comprises amount of component b) the pH regulator agent.
In another embodiment, described amount of component b) be selected from: citric acid, hydrochloric acid, phosphate buffer, acetate buffer solution or their combination, thus making that the pH of composition solution is 5～9, preferred pH is 5.0～8.5.
In a preference, the pH of described solution is 5.5～8.0, and is preferred 6.0～7.5, more preferably 6.5～7.2.
In another embodiment, described amount of component b) be citric acid, and components b) basic amino acid and amount of component b) weight ratio of citric acid is 1: 0.05～1: 1, preferred 1: 0.1～1: 0.8, more preferably 1: 0.15～1: 0.5.
In a preference, described components b) basic amino acid and amount of component b) mol ratio of citric acid is 1: 0.08～1: 1, preferred 1: 0.1～1: 0.8, more preferably 1: 0.15～1: 0.5, most preferably 1: 0.2～1: 0.4.
In another embodiment, described compositions also comprises d) other pharmaceutically acceptable carrier and/or diluent except that the pH regulator agent.
In a preference, described pharmaceutically acceptable carrier and/or diluent are selected from: water for injection, phosphate buffer, aqueous solution of propylene glycol, lactose, citrate buffer, mannitol, sucrose or glucose.
Provide a kind of Serpentis grape promotor composition in a second aspect of the present invention, it comprises:
A) ampelopsin of 1-5 weight portion;
B) basic amino acid of 1-50 weight portion, it is selected from: L-arginine or L-lysine;
C) the pH regulator agent of 0.05-1 weight portion, it is selected from: citric acid, phosphate buffer, acetic acid or their combination; With
D) pharmaceutically acceptable carrier of other except that the pH regulator agent and/or diluent.
Ampelopsin concentration is 0.01～500mg/ml in the aqueous solution of described compositions, and pH is 5～9.
Provide ampelopsin preparation of compositions method of the present invention in a third aspect of the present invention, described method comprises step: blending ingredients is ampelopsin, components b a)) basic amino acid and optional amount of component b) pH regulator agent and component d) other pharmaceutically acceptable carrier and/or diluent except that the pH regulator agent.
In a preference, described step is carried out under aseptic condition.
In a preference, described method also comprises carries out lyophilization to obtain injectable sterile powder to the solution that obtains.
In one embodiment, described ampelopsin compositions is the ampelopsin injection, and this method comprises:
(i) a) ampelopsin of blending ingredients; B) basic amino acid; And d) pharmaceutically acceptable carrier of other except that the pH regulator agent and/or diluent are to form ampelopsin solution;
(ii) add c) the pH regulator agent, regulator solution pH to 5～9; With
(iii) to step (ii) the solution of gained remove pyrogen and sterilization, thereby obtain the ampelopsin injection, wherein the cumulative volume in injection is a benchmark, the concentration of ampelopsin is 0.01～300mg/ml.
In a preference, described ampelopsin compositions is the ampelopsin injectable sterile powder, and described method comprises:
(i) mix a) ampelopsin; B) basic amino acid; And d) pharmaceutically acceptable carrier of other except that the pH regulator agent and/or diluent form ampelopsin solution;
(ii) randomly add c) the pH regulator agent;
(iii) to step (ii) the solution of gained remove pyrogen and sterilization; With
(iv) step solution is (iii) carried out drying, thereby obtains the ampelopsin injectable sterile powder,
Wherein, can be formulated as pH before use be 5～9 injection to described sterilized powder.
In another preference, described ampelopsin compositions is the ampelopsin injectable sterile powder, and described method comprises:
(i) fully mix a) ampelopsin; B) basic amino acid; C) basic amino acid; And optional d) pH regulator agent;
(ii) under aseptic condition, step (i) gained mixture is made sterilized powder;
The (iii) described sterilized powder of packing,
Wherein, can be formulated as pH before use be 5～9 injection to described sterilized powder.
In a fourth aspect of the present invention, provide a kind of oncotherapy has been had synergistic medicine box, comprise in the described medicine box:
A) the ampelopsin compositions of the present invention of treatment effective dose;
B) carboplatin of treatment effective dose.
In a preference, the weight ratio of ampelopsin compositions and carboplatin is 1: 10～10: 1 in the described medicine box, preferred 1: 8～8: 1, and more preferably 1: 5～5: 1.
In another preference, go back operation instructions in the medicine box.
In another embodiment, provide ampelopsin compositions of the present invention to be used for the purposes of the medicine box of Synergistic treatment tumor, it is characterized in that described medicine box also comprises carboplatin in preparation.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Figure 1 shows that sour grapes element (AMP) and ampelopsin-arginine-citric acid (AAC) inhibitory action to human lung adenocarcinoma SPCA-1 cell proliferation.Wherein, abscissa is suppression ratio (IR%) for using concentration, vertical coordinate, and cell concentration is 5.5 * 10
4Individual cell/ml.
Figure 2 shows that ampelopsin-arginine-citric acid (AAC) and the synergism of carboplatin when the treatment lotus has the Balb/c nu.nu nude mice of people's pulmonary carcinoma GLC-82.
Figure 3 shows that the Balb/c nu.nu nude mice tumor photo that people's pulmonary carcinoma GLC-82 is arranged ampelopsin-arginine-citric acid (AAC) and the lotus under the carboplatin Synergistic treatment.Wherein, first group is matched group; Second group is carboplatin 40mg/kg group; The 3rd group is AAC 200mg/kg group; The 4th group is carboplatin 40mg/kg+ AAC200mg/kg group.
The specific embodiment
Caulis seu folium ampelopsis brevipedunculatae (Caulis Ampelopsis Brevipedunculae) have extremely strong antitumor action, but its dissolubility in water only is 0.2mg/ml, and this makes it be difficult to be applied to clinical.The inventor adopts the basic amino acid hydrotropy can improve the dissolubility of ampelopsin in water by long-term and deep discovering, and can make its solution keep certain dissolving and chemical stability.The pH regulator agent that the inventor also finds in the ampelopsin solution of basic amino acid hydrotropy further to add such as citric acid etc. not only can be adjusted to pH the scope that physiology is used that is more suitable for, and also can further improve the steady dissolution of ampelopsin.The applicant has finished the present invention on this basis.
Particularly, the objective of the invention is to: select effective cosolvent, and the consumption by adjusting effective cosolvent and the pH value of liquid, therefrom filter out suitable cosolvent prescription, thereby obtain dissolubility height, good stability and be suitable for the ampelopsin compositions of clinical application.
For this reason, the inventor adds the basic amino acid of different mol ratio example in the preparation of ampelopsin solution, observes AMP dissolubility and steady dissolution under the room temperature.AMP is all help solubilization to L-Arg and L-Lys by this experimental observation, and L-Arg is better than L-Lys to the hydrotropy effect of AMP.
The inventor also further selects the dissolubility height for use, the aminoacid of steady dissolution is as cosolvent, AMP-Freamine with the phosphate buffer compounding high concentration of different pH value, observe dissolubility, steady dissolution and pH value thereof, and with the dilution of the phosphate buffer of different pH value, observe dissolubility, steady dissolution and the pH value thereof of diluent.By showing that relatively the dissolubility of AMP-L-Arg of the present invention, steady dissolution and chemical stability are all owing to existing AMP-Na.
In addition, the inventor has also prepared ampelopsin-L-arginine-citric acid (AAC) solution, and observe its steady dissolution, measure its chemical composition stability with high-efficient liquid phase technique, determine the stability of its dissolving and drug effect, behaviour body and function medicine provides foundation, has obtained the preparation that final concentration and pH more meet animal experiment and clinical practice thus.
Tumor-inhibiting action in the cytotoxicity of ampelopsin compositions of the present invention and body have further been verified in the test of tumor models and model of nude mice bearing tumor, with and synergistic antitumor effect during with the carboplatin coupling.
Ampelopsin
Among the present invention, term " ampelopsin ", " active substance of the present invention " and " active medicine " are used interchangeably, and all refer to the ampelopsin chemical compound as active component in the compositions.
The ampelopsin that the present invention is used can extract from Ampelopsis or other contain the plant of this material and obtains, and also can synthesize by the method for chemosynthesis.
Basic amino acid
The inventor can be used in the hydrotropy of ampelopsin by discovering basic amino acid.Among the present invention, term " basic amino acid ", " cosolvent of the present invention " are used interchangeably, and its adding can improve the dissolubility and the stability of active substance in the ampelopsin solution.The used basic amino acid of the present invention includes but not limited to: L-arginine, L-lysine or their combination, preferred L-arginine.
Among the present invention, term " pharmaceutically acceptable " composition is meant and is applicable to people and/or animal and does not have the material that excessive bad side reaction (as toxicity, stimulation and allergy) promptly has rational benefit/risk ratio.
Though the present invention does not wish to be subject to theory, the hydrotropy mechanism of used cosolvent may be because the AMP isoelectric point, IP near pH value about 5.0, contains 6 hydroxyls in its molecule among the present invention, is faintly acid; And basic amino acid is an ampholyte, and the isoelectric point, IP of lysine is 9.74, and arginic isoelectric point, IP is 10.76, can be in acid solution and proton (H
+) be combined into positively charged cation (NH3
+).H in the AMP molecule on the hydroxyl
+Combine with basic amino acid, thereby make the dissolubility of ampelopsin increase.
Select basic amino acid for use, especially arginine has following advantage as cosolvent: 1. basic amino acid stable in properties, in water, dissolve not aerogenesis (CO
2); 2. the used basic amino acid of the present invention does not contain sodium ion, and uses safer to the patient who suffers from a heart complaint, needs restriction sodium ion such as hypertension, nephropathy, hepatic ascites are taken in; 3. basic amino acid, especially arginine are participated in a lot of physiological actions in human body: for example healing of the formation of the metabolism of ammonia, sperm, wound and immune keep etc. in the liver, and therefore replenish basic amino acid and also have relevant Nutrition.
The pH regulator agent
For the pH that makes the present composition more meets animal experiment and requirements for clinical application, can in the ampelopsin compositions of the present invention's usefulness basic amino acid hydrotropy, further add pharmaceutically acceptable pH regulator agent.
Among the present invention, term " pH regulator agent " and " acidic ph modifier " are used interchangeably.Described pH regulator agent is scalable pH not only preferably, also can further play the effect of hydrotropy and stabilizing solution.Can be used for pH regulator agent of the present invention includes but not limited to: citric acid, hydrochloric acid, phosphate, acetic acid or their mixture, preferably citric acid.
Select for use citric acid as the pH regulator agent, and play hydrotropy and Stabilization simultaneously, have lot of advantages: at first, the citric acid chemical property is stable, its liquid preparation is carried out high temperature sterilize also can not destroy its chemical constitution; Secondly, citric acid more easily dissolving in solvent for injection commonly used; In addition, when being used, also can quicken the dissolving of basic amino acid with basic amino acid.
The cosolvent combination
The present invention also provides a kind of " cosolvent combination ".Among the present invention, term " cosolvent combination " and " hydrotropy system " are used interchangeably, all refer to be made up by the hydrotropy that neutral and alkali aminoacid of the present invention and the acidic ph modifier with hydrotropy effect are formed, its solubilization-aid effect is better than single with basic amino acid or acidic ph modifier itself.
Do not wish to be subject to any theory, cosolvent of the present invention combination can obtain better solubilization-aid effect may be because: though two kinds of compositions in the cosolvent of the present invention combination have the hydrotropy effect separately, but list can cause pH value of solution higher with basic amino acid of the present invention, single then can cause pH value of solution on the low side, both are used in combination in hydrotropy, obtain to make active component to be stable at pH scope in the solution with acidic ph modifier; And acidic ph modifier of the present invention also can further promote the dissolving of basic amino acid, to form more stable solution system.
Though adopt Caulis seu folium ampelopsis brevipedunculatae (Caulis Ampelopsis Brevipedunculae) usually to prove the excellent hydrotropy effect of hydrotropy system of the present invention among the present invention, it will be understood by those skilled in the art that this hydrotropy system also can be used in the hydrotropy of other similar activity composition.
In the cosolvent of the present invention combination, the ratio of basic amino acid and acidic ph modifier makes that preferably the pH of gained solution is 5.0～8.5, and is preferred 5.5～8.0, more preferably 6.0～7.5, most preferably 6.5～7.2.Wherein, preferably be combined as the combination of L-arginine or L-lysine and citric acid, its weight ratio is 1: 0.05～1: 1, preferred 1: 0.1～1: 0.8, and more preferably 1: 0.15～1: 0.5.
With regard to ampelopsin solution, though adopt basic amino acid of the present invention separately, the ginsenoside-Rd injection is played certain hydrotropy effect, but more preferably adopt the cosolvent combination of basic amino acid and acidic ph modifier.
Can in ampelopsin of the present invention, add basic amino acid or pH regulator agent in proportion earlier, or add this two kinds of materials simultaneously.
The ampelopsin compositions
The invention provides a kind of ampelopsin compositions, it comprises the active component ampelopsin and at least as the basic amino acid of cosolvent.Term among the present invention " ampelopsin compositions " and " compositions that contains ampelopsin " are used interchangeably.The weight ratio of active component and basic amino acid is 1: 1～1: 10, preferred 1: 1～1: 8, and more preferably 1: 1～1: 6.In another preferred embodiment, active component and basic amino acid mol ratio be 1: 2.5～1: 10, preferred 1: 3～1: 8, more preferably 1: 3～1: 6.
Ampelopsin compositions of the present invention also can comprise pH regulator agent mentioned above, is preferably the pH regulator agent with hydrotropy effect, most preferably citric acid, thereby make that the pH of composition solution is 5.0～8.5, preferred 5.5～8.0, more preferably 6.0～7.5, most preferably 6.5～7.2.The weight ratio of basic amino acid and pH regulator agent is 1: 0.05～1: 1, preferred 1: 0.1～1: 0.8, and more preferably 1: 0.15～1: 0.5.In another preferred embodiment, the mol ratio of basic amino acid and pH regulator agent is 1: 0.08～1: 1, preferred 1: 0.1～1: 0.8, and more preferably 1: 0.15～1: 0.5, most preferably 1: 0.2～1: 0.4.
Ampelopsin compositions of the present invention also can comprise pharmaceutically acceptable carrier and/or the diluent except that the pH regulator agent, include but not limited to: Chang Yong solvent pharmaceutically, as water, propylene glycol, ethanol, Polyethylene Glycol etc., preferred water, propylene glycol or their combination; Osmotic pressure regulator, for example glucose etc.; Antibacterial, for example benzyl alcohol, butyl hydroxybenzoate etc.; Antioxidant, for example thiourea etc." Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, Mack Pub.Co. can find discussing fully about pharmaceutically acceptable carrier in N.J.1991).It will be understood by those skilled in the art that described pharmaceutically acceptable carrier and/or diluent can not hinder the pharmaceutical active and the drug effect of active substance, and can select appropriate carriers and/or diluent, and determine their consumption according to concrete needs.
The concentration of ordinary dissolution of compositions of the present invention in water can be brought up to 50～500mg ampelopsin/ml solution, preferred 75～400mg ampelopsin/ml solution, more preferably 100～300mg ampelopsin/ml solution.Certainly, in use also can prepare the weak solution of ampelopsin as required, for example compound concentration is 0.01～50mg ampelopsin/ml solution, preferred 0.03～30mg ampelopsin/ml solution, more preferably the ampelopsin weak solution of 0.05～20mg ampelopsin/ml solution.
Before use, each component in the present composition can be separately or is placed different vessels in combination with one another, for example ampelopsin and basic amino acid are placed a container, and pH regulator agent and pharmaceutically acceptable diluent (as being mixed with solution with water) are placed another container.Those skilled in the art can be according to concrete needs and the concrete form and the distribution that should be used for determining compositions.Therefore, the present invention also comprises the medicine box that comprises each component of the present composition.
Compositions of the present invention can exist by dosage form known in the art, preferably exists with injection form and injectable sterile powder form.In a specific embodiment, described compositions is the ampelopsin injection, and wherein the volume in injection is a benchmark, the concentration of ampelopsin is 0.01～500mg/ml, preferred 0.03～400mg/ml, more preferably 0.05～300mg/ml, and pH is 5～9, preferred 6.0～7.5.
A kind of ampelopsin compositions is provided in preferred implementation of the present invention, comprising: the ampelopsin of 1-5 weight portion; The basic amino acid of 1-50 weight portion, it is selected from: L-arginine or L-lysine; 0.05-1 the pH regulator agent of weight portion for example is selected from: citric acid, phosphate buffer or their combination; With pharmaceutically acceptable carrier and/or diluent, ampelopsin concentration is 0.01～300mg/ml in the aqueous solution of described compositions, and pH is 5～9.
The present invention also further provides ampelopsin preparation of compositions method, and described method comprises mixes ampelopsin and basic amino acid and other optional component.The sequencing that those skilled in the art can select concrete preparation technology and step and each component to add according to concrete needs and preparation condition, this does not exceed scope of the present invention.
Ampelopsin compositions and carboplatin suppress the synergism of tumor
Ampelopsin compositions of the present invention has the dissolubility and the stability of raising, and suitable pH, can be advantageously used in the treatment of clinical tumor.In addition, the inventor finds also that by research ampelopsin compositions of the present invention and antineoplastic medicament carboplatin coupling can more effectively suppress tumor growth, and both have synergism.Thus, the present invention also provides ampelopsin compositions and the purposes of carboplatin in the therapeutic alliance tumor, and the purposes of these two kinds of medicines in the medicine box of preparation treatment tumor.
Also provide a kind of in an embodiment of the invention oncotherapy has been had synergistic medicine box, comprised in the described medicine box: the carboplatin of the ampelopsin compositions of the present invention of treatment effective dose and treatment effective dose.In a preferred implementation, the weight ratio of ampelopsin compositions and carboplatin is 1: 10～10: 1 in the described medicine box, preferred 1: 8～8: 1, and more preferably 1: 5～5: 1.Also can comprise description, container, buffer etc. in the described medicine box, this can be determined according to concrete purposes and needs by those skilled in the art.
In an embodiment of the invention, unite the therapeutic scheme that uses ampelopsin compositions and carboplatin and can be and give first dose of carboplatin and ampelopsin compositions of the present invention earlier, give every day then the ampelopsin compositions and the next day give carboplatin.Concrete therapeutic scheme can be determined according to patient's situation (for example age, body weight, condition of illness etc.), the needs of treatment etc. by the clinicist.
Advantage of the present invention
(1) provide to have and improved dissolubility and stability, and the ampelopsin compositions of appropriate pH scope, it can be used for tumor treatment as clinical application;
(2) provide a kind of hydrotropy system, described system comprises basic amino acid and acidic ph modifier, and it can be used for improving the dissolubility and the stability of ampelopsin or similar active substance, and obtains suitable pH;
(3) provide a kind of method and medicine box of clinical treatment tumour, wherein adopted ampelopsin compositions of the present invention and antineoplastic medicament carboplatin commonly used, obtained synergy.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight." room temperature " during the embodiment of the invention and preamble are described all refers to 20～30 ℃ temperature.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Medicine, reagent and instrument
(Ampelopsin AMP) provides purity 98.8% by the safe standing grain in Guangdong Pharmaceutical Technology Co., Ltd to ampelopsin.
Ampelopsin sodium (AMP-Na) is provided by the safe standing grain in Guangdong Pharmaceutical Technology Co., Ltd, and specification is that 1g/ props up, lot number 061218; 50mg/ props up, lot number 051115.
L-lysine (L-Lys) Kangda Amino-acid Factory of Shanghai product, lot number 8701.
L-arginine (L-Arg) is gone up seamount Pu chemical industry company limited product, lot number 20050302, and paper chromatography is qualified.
Citric acid is homemade analytical pure, and reagent one factory in Shanghai produces, lot number 80-06-06.
Carboplatin (0.1g/ props up) Qilu Pharmaceutical Co., Ltd. produces, lot number 5090061DA.
Na
2HP
42H
2O is homemade analytical pure, and Red Star chemical plant, Beijing produces, lot number 781225-1.
NaH
2P
412H
2O is homemade analytical pure, and chemical reagent factory in Xi'an produces, lot number 920216.
0.2mol/L the phosphate buffer of pH 6.0,6.8 of pH 5.0 phosphate buffers, 0.1mol/L: press 2005 editions two (chemical publishing houses of Pharmacopoeia of People's Republic of China, appendix XV D buffer, 2005.1) and " medical agent and handbook commonly used (Jilin science tech publishing house, 1985 March the 1st edition) preparation.
Citric acid-sodium hydrogen phosphate buffer (pH4.0), phosphate buffer (pH2.0): press 2005 editions two ones of Pharmacopoeias of People's Republic of China (chemical publishing house, appendix XV D buffer, 2005.1) (the same) preparation.
Delta 320 pH meters, Mettler-Toledo Instrument's product.
High performance liquid chromatograph, Tianjin, island LC-10AT of company.
The research of embodiment 1. ampelopsin basic amino acid hydrotropy systems
Test objective
By relatively L-lysine (L-Lys) and L-arginine (L-Arg) to the hydrotropy effect of AMP and generate the dissolubility and the steady dissolution of solution, to determine a kind ofly AMP fully to be dissolved and the preparation method of the AMP preparation of former anti-tumor activity is stablized and kept to dissolubility.
Test method
The AMP isoelectric point, IP contains 6 hydroxyls near pH value about 5.0 in its molecule, be faintly acid; Simultaneously L-Lys and L-Arg are basic amino acid, the clinical human body that can be used for, and safety is reliable, is ampholyte, and isoelectric point, IP is respectively 9.74 and 10.76, can be in acid solution and proton (H
+) be combined into positively charged cation (NH3
+).The H on the hydroxyl combines with L-Lys or L-Arg in the AMP molecule in order to make, and AMP is pressed different molar concentration rate (AMP: L-Lys=1: 1,1: 2,1: 3,1: 4 and 1: 5 with L-Lys or L-Arg; Or AMP: L-Arg=1: 1,1: 2 and 1: 3) mix, add isopyknic distilled water, the vortex concussion makes abundant dissolving.Observation under different preservation conditions, the steady dissolution and the pH value of prepared AMP solution.
Buffer salt (PBS) preparation AMP-L-Arg solution with different pH on the basis of above-mentioned test is observed its steady dissolution and pH, or adopts hydrochloric acid Arg preparation AMP-arginine hydrochloride solution, observes its water miscible variation.
Result of the test
1. calculate the consumption of AMP with 50mg/ml, after the L-Lys mixed preparing with AMP and different molar concentration rates, the gained preparation room temperature and 4 ℃ of dissolubilities down with stable as shown in following table 1:
Table 1. variable concentrations L-Lys is to the hydrotropy effect of 50mg/ml AMP and the steady dissolution of solution
Table 1 shows: AMP calculates consumption with 50mg/ml, and AMP and L-Lys can not dissolve under the room temperature condition fully by the preparation in 1: 1,1: 2 of mole concentration ratio; AMP and L-Lys are by the preparation in 1: 3 of mole concentration ratio, and room temperature condition needs 1.5h to dissolve down, and room temperature and 4 ℃ of preservations are easy to separate out crystallization; When AMP and L-Lys prepared by the mole concentration ratio at 1: 4,20min dissolved fully under the room temperature condition; AMP and L-Lys are by the preparation in 1: 5 of mole concentration ratio, and 20min dissolves fully under the room temperature condition, room temperature and 4 ℃ of preservations, and its dissolubility is stable.
With AMP and L-Lys by 1: 4 mixed preparing of mole concentration ratio, making in the solution AMP concentration is 100,200 or 300mg/ml, the gained preparation is in the following dissolubility of room temperature and 4 ℃ and stable as shown in following table 2:
Table 2.L-Lys is to the hydrotropy effect of variable concentrations AMP and the steady dissolution of solution
Table 2 shows: when AMP with L-Lys during by the preparation in 1: 4 of mole concentration ratio, the concentration of AMP is 100mg/ml, 200mg/ml in the solution, 20min dissolves fully under the room temperature condition, room temperature and 4 ℃ of preservations, its dissolubility is stablized; The concentration of AMP is 300mg/ml in the solution, and room temperature condition needs 20min to dissolve fully down, and room temperature and 4 ℃ of preservations are easy to separate out crystallization.
3. calculate the AMP consumption with 50mg/ml, after the L-Arg mixed preparing with AMP and different molar concentration rates, the gained preparation room temperature and 4 ℃ of dissolubilities down with stable as shown in following table 3:
Table 3. variable concentrations L-Arg is to the hydrotropy effect of 50mg/ml AMP and the steady dissolution of solution
Table 3 shows: when AMP and L-Arg press mol ratio preparation in 1: 1,1: 2, can not dissolve fully under the room temperature condition; AMP and L-Arg are by the preparation in 1: 3 of mole concentration ratio, and its dissolubility was stable when room temperature condition was preserved down, and the gained pH value of solution is 8.89.
With AMP and L-Arg by mixed in molar ratio preparation in 1: 3 after, AMP concentration is 50,100 or 200mg/ml in the solution, this solution is in the following dissolubility of room temperature and 4 ℃ and stable as shown in following table 2:
Table 4.L-Arg is to the hydrotropy effect of variable concentrations AMP and the steady dissolution of solution
Table 4 shows: AMP and L-Arg by 1: 3 mol ratio preparation after, the concentration of AMP is 50,100 and 200mg/ml in the solution, 20min dissolves fully under the room temperature condition, room temperature condition is preserved down, its dissolubility is stablized.
5. prepare the AMP of 1: 3 mol ratio: L-Arg with the PBS of different pH value, the steady dissolution and the pH value of gained solution are as shown in table 5:
Table 5. is with the steady dissolution and the pH of the AMP of the PBS of different pH preparation: L-Arg (M/M)=1: 3 solution
* the concentration of AMP is 200mg/ml in the solution, and "-" for not adding this composition, pH is 25 ℃ of mensuration.
By table 5 as seen: the concentration of AMP is 200mg/ml in the solution, and 20min dissolves fully under the room temperature condition, and room temperature condition is preserved down, and its dissolubility is stable.The phosphate buffer of different pH does not have obvious influence to its dissolubility, and the pH in the time of 25 ℃ is about 9.0.
6. the mol ratio of preparation AMP: hydrochloric acid-Arg is 1: 3 a solution, and to the water miscible influence of 100mg/ml AMP, the result is as shown in table 6 with research hydrochloric acid-Arg:
Table 6 hydrochloric acid-Arg is to the water miscible influence of 100mg/ml AMP
The result shows under the room temperature condition, and AMP is at AMP: hydrochloric acid-Arg (M: M)=do not dissolve in 1: 3 the solution (concentration of AMP is 100mg/ml).
Interpretation of result and discussion
Contain 6 phenolic hydroxyl groups in ampelopsin (AMP) molecule, has faintly acid, AMP is slightly soluble in water, dissolubility in aqueous solution is 0.2mg/ml only, studies show that the dissolubility of ampelopsin in alkaline solution is bigger, and dissolubility is less in water, this may be because have phenolic hydroxyl group in its structure, show faintly acid, can form salt, thereby increase dissolubility with alkali.
The inventor utilizes the alkaline nature of basic amino acid L-Lys, L-Arg, studies its hydrotropy effect to AMP, and the result shows that AMP and L-Arg are by the preparation in 1: 3 of mole concentration ratio, AMP concentration can reach 200mg/ml, 20min dissolves fully under the room temperature condition, and room temperature condition is preserved down, and its dissolubility is stable; AMP and L-Lys are by the preparation in 1: 3 of mole concentration ratio, and AMP concentration reaches 50mg/ml, and room temperature condition needs 1.5h to dissolve down, and room temperature and 4 ℃ of preservations are easy to separate out crystallization.But AMP and L-Lys are pressed the preparation in 1: 4 of mole concentration ratio, and when AMP concentration reached 200mg/ml, the AMP dissolving was complete and gained solution storage back dissolubility is stable.
As seen by above-mentioned, L-Arg and L-Lys all help solubilization to AMP, but comparatively speaking, L-Arg is better than L-Lys to the hydrotropy effect of AMP.
The steady dissolution test objective of the basic amino acid hydrotropy ampelopsin solution of the different pH buffer dilutions of embodiment 2. usefulness
The pH value of injection can not surpass people's physiological tolerance scope, and general pH can be 4～9.The a small amount of intravenous fluid, because blood has cushioning effect, pH can suitably relax, generally can be between 3～10.And when importing in a large number, cross alkali as peracid, will cause acid alkali poisoning, so to be advisable near blood pH (7.4).
AMP and L-Lys are by the preparation in 1: 4 of mole concentration ratio, and L-Arg is by the preparation in 1: 3 of mole concentration ratio, and AMP concentration can reach 200mg/ml, pH value is about 9.0, basicity is higher, in order more to meet zoopery and clinical practice desired concn and suitable pH value, can dilute it.Therefore, steady dissolution and the pH value of solution in the phosphate buffer of different pH value that by comparison L-lysine (L-Lys) and L-arginine (L-Arg) the AMP hydrotropy is generated in the present embodiment is to determine more to meet the AMP preparation of zoopery needs and clinical practice.
Test method
The solution that AMP is formed under L-Lys or the effect of L-Arg hydrotropy with the phosphate buffer dilution of pH value 5.0,6.0,6.8, is observed and is preserved under different condition, the pH value of the steady dissolution of diluent and 25 ℃.
Result of the test
1. adopt AMP: L-Lys=1: 3 or the solution of 1: 4 proportioning, steady dissolution after pH5.0,6.0 or 6.8 phosphate buffer dilution and pH value are shown in table 7 and table 8:
Table 7.AMP: L-Lys=1: steady dissolution and the pH value of the AMP solution of 3 (M/M) after dilution
AMP is subjected to test solution concentration 50mg/ml, and the AMP final concentration is 5mg/ml
Table 8.AMP: L-Lys=1: steady dissolution and the pH value of the AMP solution of 4 (M/M) after dilution
AMP is subjected to test solution concentration 50mg/ml, and the AMP final concentration is 5mg/ml
The result of table 7 shows: the solution of AMP: L-Lys (M/M)=preparation in 1: 3, in pH value 5.0,6.0,6.8 buffer, dilute, and be easy to separate out crystallization.Table 8 is demonstration then: AMP: the solution of L-Lys (M/M)=1: 4 preparation, in the phosphate buffer of pH value 5.0,6.0, be easy to separate out crystallization, and steady dissolution in the buffer of pH value 6.8, and the pH 25 ℃ the time is 7.27.
2. with AMP and L-Arg mol ratio be 1: 3 hydrotropy solution with pH5.0,6.0 or 6.8 buffer dilution after, the steady dissolution and the pH value of the solution of gained final concentration 5mg/ml or 10mg/ml are as shown in table 9:
The steady dissolution and the pH value of the AMP solution of the L-Arg hydrotropy after the dilution of table 9. different pH buffer
Table 9 shows: AMP and L-Arg are by the preparation in 1: 3 of mole concentration ratio, the concentration of AMP is 200mg/ml, dilution is AMP final concentration 5mg/ml in the phosphate buffer of pH value 5.0,6.0,6.8, can steady dissolution 3 hours at pH value 5.0,6.0 phosphate buffers, pH value is 5.9～6.4 (25 ℃), the energy steady dissolution is 5 hours in pH value 6.8 phosphate buffers, and pH value is 7.26 (25 ℃).And the AMP final concentration is when being 10mg/ml, can steady dissolution 3 hours at pH value 5.0,6.0 phosphate buffers, and pH value 6.1～6.8 (25 ℃) can steady dissolution in pH value 6.8 phosphate buffers 5 hours, pH value 7.91 (25 ℃).
Interpretation of result and discussion
AMP: L-Lys (M/M)=1: 4 obtain solution, AMP are subjected to test solution concentration 50mg/ml, dilute in pH value 6.8 buffer, the AMP final concentration is 5mg/ml, steady dissolution in pH value 6.8 buffer, pH7.27 (25 ℃), very suitable zoopery and clinical practice.AMP and L-Arg are by the preparation in 1: 3 of mole concentration ratio, the concentration of AMP is 200mg/ml, dilutes in pH value 5.0,6.0,6.8 phosphate buffers, and the AMP final concentration is 5mg/ml, can steady dissolution 3 hours at pH value 5.0,6.0 phosphate buffers, pH value 5.9～6.4 (25 ℃); The energy steady dissolution is 5 hours in pH value 6.8 phosphate buffers, and pH value 7.26 (25 ℃) is fit to clinical and zoopery requirement; The AMP final concentration is 10mg/ml, can steady dissolution 3 hours at pH value 5.0,6.0 phosphate buffers, and pH value 6.1～6.8 (25 ℃); Be fit to zoopery and clinical practice requirement.
L-Arg compares with L-Lys, the solution that AMP and L-Arg, L-Lys all prepare by the mole concentration ratio at 1: 3, in pH value 5.0,6.0,6.8 phosphate buffers, dilute, when the AMP final concentration was 5mg/ml, AMP-L-Arg solution more met zoopery and requirements for clinical application than AMP-L-Lys solution.
Weak point is that the dilute solution that L-Arg makes the AMP hydrotropy is AMP 5mg/ml, only stable in a few hours, if want long preservation, then the stock solution of high concentration can be made lyophilized formulations, facing with preceding the dilution with pH value 5.0,6.0,6.8 phosphate buffers is desired concn.
The comparison of embodiment 3.AMP-L-Arg and AMP-Na dissolubility, steady dissolution and chemical stability
Test objective
By comparing the solution (AMP-L-Arg that ampelopsin forms at L-arginine hydrotropy, AA) with dissolubility, steady dissolution and the chemical composition stability of ampelopsin sodium (AMP-Na), so that find a kind of dissolubility better, the preparation method of the better AMP preparation of steady dissolution makes that AMP is suitable to carry out zoopery and for instructing clinical application that foundation is provided.
Test method
1.AMP-L-Arg comparison with AMP-Na dissolubility, steady dissolution
AMP and L-Arg separately weighed by a mole concentration ratio at 1: 3 mix, in the PBS of pH value 5.0, dissolve AMP-L-Arg (AA) solution, with the PBS dissolving of AMP-Na, compare both dissolubilities, steady dissolution with pH value 5.0.
2.AMP-L-Arg compare with the steady dissolution and the pH value of AMP-Na diluent
By AMP concentration is 200mg/ml, AMP: L-Arg (M/M)=1: 3, add pH 4.0 citric acid-sodium hydrogen phosphate buffer 1.5ml dissolving, again by L-Arg: citric acid (M/M)=add citron adjust pH get 200mg/ml ampelopsin-L-arginine-citric acid (AAC) solution at 1: 0.2, dilute AAC solution and the 200mg/ml AMP-Na solution (in the PBS of pH value 5.0, dissolving) of 200mg/ml respectively with NS, relatively steady dissolution and the pH value under both diluent room temperature conditions.
3.AMP-L-Arg chemical composition stability with AMP-Na
By AMP concentration is 200mg/ml, AMP: L-Arg (M/M)=1: 3, add pH 4.0 citric acid-sodium hydrogen phosphate buffer 1.5ml dissolving, again by L-Arg: citric acid (M/M)=adding citric acid adjust pH was 7.2～7.4 in 1: 0.2, and it is 2mg/ml with NS 3960 μ l dilution that gained AAC solution is got 40 μ l; Getting AMP-Na1, to prop up (50mg) be 8mg/ml with the PBS of pH6.8 dilution, gets PBS that 1ml adds 1ml pH6.8 again and add 2ml NS again and get 2mg/ml.Measure chemical composition stability with high-efficient liquid phase technique.
Result of the test
1.AA dissolubility, steady dissolution with AMP-Na
Dissolubility, the steady dissolution of table 10.AA and AMP-Na
The result of table 1 shows: AMP and L-Arg are by the PBS dissolving of 1: 3 usefulness pH value 5.0 of mole concentration ratio, and AMP concentration can reach 200mg/ml, and room temperature condition is preserved down, and the 48h dissolubility is stable; AMP-Na is dissolved as under the 50mg/ml room temperature condition with the PBS of pH value 5.0 and preserves, and the 24h dissolubility is stable, and 100,200mg/ml dissolves during preparation immediately, but room temperature condition preserves down, and 30min promptly has medicine to separate out.
2.AAC steady dissolution and pH value with the AMP-Na diluent
Table 11.AMP-Na steady dissolution and pH value
Table 12.AAC steady dissolution and pH value
Shown in table 11 and 12: the AAC 24h steady dissolution of 200mg/ml, pH value 7.37,200mg/mlAMP-Na dissolved at that time immediately with pH value 5.0 PBS dissolving, pH value 8.59, but the 0.5h medicine is separated out under the room temperature condition; 8h steady dissolution under the 40mg/ml AAC room temperature condition, pH value 7.13,24h steady dissolution under the 30mg/ml AMP-Na room temperature condition, pH value 8.55; AAC that 30mg/ml is following and the diluent of AMP-Na, room temperature condition are protected the 24h dissolubility down and are all stablized; AAC diluent and AMP-Na diluent identical or close concentration compare, and the pH value of AAC diluent is starkly lower than the pH value of AMP-Na diluent.
3.AAC with the AMP-Na chemical composition stability
Table 13.AMP-Na, AAC solution chemistry stability is (x ± S) relatively
**P＜0.01
Table 14. high-efficient liquid phase technique is measured AMP, AAC appearance time (min)
Shown in table 13 and 14: AMP content is that 2mg/ml AAC solution, AMP-Na 2mg/ml are degraded to 50.25% and 37.37% of 0h respectively behind the high-efficient liquid phase technique mensuration 24h, chemical composition stability obviously reduces, but AMP content is AAC solution and the AMP-Na 2mg/ml comparison AMP-Na 2mg/ml decline degree bigger (P＜0.01) of 2mg/ml; Measure the AAC of 0h, 24h and the appearance time of AMP-Na through high-efficient liquid phase technique, AAC and AMP-Na compare, no significant change (P＞0.05).
Interpretation of result and discussion
The inventor successfully is prepared into AMP sodium salt (AMP-Na) in early stage, and the dissolubility of AMP is obviously improved, and dissolving can reach 25mg/ml (24 ℃), and room temperature and 4 ℃ of conditions are preserved dissolubility and stablized.By the research of AMP basic amino acid hydrotropy system, find that AMP under the effect of basic amino acid hydrotropy, can improve the dissolubility of AMP greatly, dissolving can reach 200mg/ml.PBS with pH 5.0 dissolves AMP-L-Arg and AMP-Na respectively, and the result shows AMP under the hydrotropy effect of L-Arg, and dissolubility obviously improves, and can reach 200mg/ml, and dissolubility is stable, can only dissolve 50mg/ml and the AMP-Na dissolubility is stable.By the relatively steady dissolution and the pH value of the NS diluent of AAC and AMP-Na, the result shows that both do not have significant difference at steady dissolution, but the pH value of the AAC diluent of or close concentration identical with the AMP-Na diluent is starkly lower than the pH value of AMP-Na diluent, AAC variable concentrations diluent pH value scope is 7.04～7.37, meet zoopery and requirements for clinical application, AMP-Na variable concentrations diluent pH value scope is 7.99～8.59, acid-base value is higher, does not very meet zoopery and requirements for clinical application.
Preparation as zoopery and clinical practice, also need the active chemical of medicine stable, therefore 2mg/ml AAC solution and 2mg/ml AMP-Na solution are contrasted, the result shows that both 24h are degraded to 50.25% and 37.37% of 0h respectively, chemical composition stability all obviously reduces, but AMP-Na 2mg/ml decline degree is bigger, overcome the unsettled deficiency of solution chemistry by AMP-Na being made freeze-dried formulation, similarly, AAC can be prepared into freeze-dried formulation and overcome this weak point.
The stability of embodiment 4. ampelopsins-L-arginine-citric acid (AAC) solution
Test objective
The pH value of AMP-L-Arg (AA) solution (25 ℃) about 9.0 by add citric acid in AMP-L-Arg solution, makes the pH value (25 ℃) about 7.2～7.4 of the AAC solution of formation, meets zoopery and requirements for clinical application.Observe its steady dissolution again, measure its chemical composition stability, determining the stability of its dissolving and drug effect, thereby provide foundation for people's body and function medicine with high-efficient liquid phase technique.
Test method
1. the variable concentrations citric acid is to the influence of AMP-L-Arg (AA) stability of solution and pH
AMP and L-Arg separately weighed by a mole concentration ratio at 1: 3 mixes, dissolve, obtain AMP-L-Arg (AA) solution at citric acid-phosphate sodium dihydrogen buffer solution of pH 4.0; Add not commensurability citric acid then therein, the pH value of AAC solution that makes formation is observed its steady dissolution under the room temperature about 7.2～7.4, with normal saline NS dilution, observe diluent stability and pH value (25 ℃) under the room temperature.
2. the steady dissolution and the pH of variable concentrations ampelopsin-L-arginine-citric acid (AAC) solution are 200mg/ml by AMP concentration, AMP: L-Arg (M/M)=1: 3, add pH4.0 citric acid-sodium hydrogen phosphate buffer 1.5ml dissolving, press L-Arg again: citric acid (M/M)=1: 0.2 adding citric acid 71.7mg adjust pH, gained AMP concentration is the compound method of the AAC solution of 200mg/ml, take by weighing AMP20mg in proportion, L-arginase 12 9.4mg, add 1.5ml citric acid-20 times of diluents of sodium hydrogen phosphate buffer (pH 4.0), get 1.5ml after the dissolving, add citric acid 7.17mg again, add 0.5ml NS solution, get 2ml AAC solution, AMP concentration is 10mg/ml, be diluted to variable concentrations with NS and preserve at ambient temperature, observe its steady dissolution and pH value (25 ℃).
3. measure its chemical composition stability with high performance liquid chromatogram.
Chromatographic column is Tianjin, island VP-ODS C18 (150 * 4.6mm, 5 μ m) post, and mobile phase is acetonitrile-2% acetic acid (1: 9), and flow velocity 1.0ml/min detects wavelength 290nm, 25 ℃ of column temperatures, room temperature 22-23 ℃, sample size 10 μ l.
4. high concentration ACC is to the zest of rat muscle
ACC and the contrast NS of 200mg/ml are expelled to respectively in the rat muscle, and perusal and microscopically are observed muscle after a period of time has no abnormal.
Result of the test
1. the variable concentrations citric acid is to the influence of AMP-L-Arg (AA) stability of solution and pH
AMP content is 200mg/ml, AMP: L-Arg (M/M)=1: 3, add pH 4.0 citric acid-sodium hydrogen phosphate buffer dissolving, press L-Arg again: citric acid (M/M)=1: 0.2 adding citric acid adjust pH, gained contains the AAC solution of AMP 200mg/ml and preserves at ambient temperature, its dissolubility is stable, and pH value is 7.2～7.4 (seeing Table 15).
The citric acid of table 15. variable concentrations is to the influence of AA solution pH value
2. the steady dissolution and the pH of variable concentrations ampelopsin-L-arginine-citric acid (AAC) solution
The AAC solution that contains AMP 200mg/ml is diluted to AAC aqueous solution below the AMP 2mg/ml with NS, preserves at ambient temperature, and its dissolubility is stable, and pH value is 7.05～7.06 (25 ℃); The AAC aqueous solution of AMP 40mg/ml, AMP 10mg/ml is preserved at ambient temperature, easily separates out crystallization (seeing Table 16) respectively behind 8h and 24h.
The steady dissolution and the pH value of table 16. variable concentrations AAC solution
By the method for joining of the AAC solution that contains AMP 200mg/ml, prepare AMP 10mg/ml AAC aqueous solution in proportion, room temperature condition preservation 2h dissolubility down is stable, is to preserve the 4h steady dissolution under the AMP 5mg/ml room temperature condition with the NS dilution; And dilution is preserved down for the following room temperature condition of AMP 2mg/ml (comprising 2mg/ml), and its dissolubility is stable, and pH value is 6.44～6.53 (25 ℃) (seeing Table 17).
Table 17.AMP 10mg/ml AAC solution dilution is variable concentrations solution steady dissolution and pH value
3.AAC the chemical composition stability of aqueous solution
High-efficient liquid phase technique is measured, and AMP content is 40mg/ml AAC solution chemical composition stability in 6h.See (table 18).
The chemical composition stability of table 18.AAC aqueous solution
AMP content dropped to 50.25% of 0h for 2mg/ml AAC after high-efficient liquid phase technique was measured 24h, obviously descended with the more stable property of AMP 2mg/ml, and there was a significant difference (P＜0.01) (seeing Table 19)
Table 19.AMP, AAC solution chemistry stability is (x ± S) relatively
**P＜0.01
The appearance time of AMP and AAC when high-efficient liquid phase technique is measured 0h, 24h, AAC and AMP compare, and difference does not have significance (P＞0.05) (seeing Table 20).
Table 20. high-efficient liquid phase technique is measured AMP, AAC appearance time (min)
4. high concentration ACC is to the zest of rat muscle
The perusal result: the rat muscle of injection NS is no abnormal.And in the injection 200mg/ml AAC rat muscle pigmentation being arranged, part muscle is black.
The microscopically observed result: the rat muscle muscle bundle rule of injection NS contrast, close proximity, nucleus is fusiformis, is positioned at cell edges.And the rat muscle bundle of injection 200mg/ml AAC is irregular, and interstice's edema, broadening have a small amount of inflammatory cell.
Results suggest high concentration AAC has zest to muscle, should not carry out intramuscular injection with it.
Discussion of results and analysis
Research by ampelopsin basic amino acid hydrotropy system, find that AMP and L-Arg are by the preparation in 1: 3 of mole concentration ratio, AMP concentration can reach 200mg/ml, 20min dissolves fully under the room temperature condition, room temperature condition is preserved down, and its dissolubility is stable, and distilled water, phosphate buffer do not have obvious influence to its dissolubility, pH value is about 9.0 (25 ℃) all, can not satisfy zoopery and requirements for clinical application well.
By AMP content is 200mg/ml, AMP: L-Arg (M/M)=1: 3, with pH4.0 citric acid-sodium hydrogen phosphate buffer dissolving, press L-Arg again: citric acid (M/M)=1: 0.2 adding citric acid, gained AMP content is the AAC solution of 200mg/ml, pH value meets clinical and zooperal requirement at 7.2～7.4 (25 ℃).
Is 40mg/ml with the AAC solution of 200mg/ml with the NS dilution, measure chemical analysis stability with high-efficient liquid phase technique, the result is presented at its chemical composition stability in the 6h, pH value 7.13 (25 ℃), the requirement of concentration and pH value when meeting clinical practice and zoopery administered intramuscular.
Studies show that high concentration anticancer experiment in vitro and clinical infusion solutions need the low concentration medicine, therefore the AAC solution with 200mg/ml is 2mg/ml with the NS dilution.Room temperature condition is preserved down, this dilution dissolubility is stable, pH value 6.4～6.5 (25 ℃), meet anticancer experiment in vitro and clinical infusion solutions application requirements, but 50.25% when measuring its chemical analysis 24h and be degraded to 0h with high-efficient liquid phase technique, suggestion is made lyophilized formulations with the AAC solution of 200mg/ml, and facing the time spent is that the 2mg/ml use can overcome this weak point with the NS dilution.
The comparison of embodiment 5. ampelopsins (AMP) and ampelopsin-L-arginine-citric acid (AAC) solution extracorporeal anti-tumor function
Test objective
Relatively AAC and AMP tentatively inquire into AAC and AMP the SPCA-1 cell proliferation are had obvious inhibiting cell concentration the inhibited proliferation of human lung adenocarcinoma SPCA-1 cell, have or not significant change to investigate AMP its anti-tumor activity after being prepared into AAC.
Test method
1. medicine preparation
The AMP preparation: the AMP powder is with 5% DMSO dissolving and be made into application liquid concentration.
AAC preparation: take by weighing AMP 20mg, L-arginase 12 9.4mg, add 1.5ml citric acid-20 times of diluents of sodium hydrogen phosphate buffer (pH 4.0), get 1.5ml after the dissolving, add citric acid 7.17mg again, add 0.5ml NS solution and get 2ml, AMP concentration is 10mg/ml, is diluted to NS and uses liquid concentration.
2. cell and cultivation
Human lung adenocarcinoma SPCA-1 cell is available from Chinese Academy of Sciences's Shanghai cell biological institute cell bank.Cell derives from people's primary pulmonary adenocarcinoma, is that to cultivate and built in 1980 be that form is a multiform epithelium sample, about 30 hours of population doubling time.Cell is put 37 ℃, 5%CO with the RPMI RPMI-1640 that contains 10% calf serum and each 100U/ml of penicillin and streptomycin
2Incubator cultivate.
3. comparative test
Adopt mtt assay comparison same dose AAC and AMP inhibitory action to human lung adenocarcinoma SPCA-1 cell proliferation.Negative control group, blank group, AMP group, AMP blank group, AAC group, AAC blank group are established in experiment.Each concentration in every group is established 3 multiple holes.Each group is treated to negative control group: add cell and do not add medicine; Blank group: add RPMI 1640 and do not add cell; AMP group: cell+AMP; AMP blank group: RPMI 1640+AMP; AAC group: cell+AAC; AAC blank group: RPMI 1640+AAC.The take the logarithm SPCA-1 cell of trophophase is adjusted cell concentration to 5.5 * 10 with the RPMI RPMI-1640
4Individual/ml (5000/hole) add cell suspension with 90 μ l/ holes in 96 well culture plates, 10 μ l/ holes add medicine, cultivate 48h.4h adds MTT (10 μ l/ hole) before experiment stops, and adds 10%SDS (100 μ l/ hole) when experiment stops, and places 12h, shakes 10 minutes, measures light absorption value in wavelength 570nm place.Calculate suppression ratio (IR) according to following formula, relatively AMP and AAC are to the IR and the IC of SPCA-1 cell
50Value.
Each is organized data and is measurement data, represents with x ± S, adopts the t-check to analyze, and adds up with Excel-2003.
Result of the test
Cell concentration is 5.5 * 10
4Individual/ml, wait dosage AMP and AAC to the inhibitory action result of human lung adenocarcinoma SPCA-1 cell proliferation shown in Fig. 1 and table 21.
Table 21.AMP and AAC to the inhibitory action of people's pulmonary carcinoma SPCA-1 cell proliferation (48h, n=3)
The result shows: AMP and AAC all have inhibited proliferation significantly to the SPCA-1 cell, and the processing time, inhibitory action was the most obvious during 100 μ g/ml concentration when being 48h, and AMP and AAC are respectively 59.98%, 61.86%, IC to the IR% of SPCA-1 cell
50Be respectively 96.18 and 86.74 μ g/ml, and in concentration is the dosage range of 25～100 μ g/ml, be tangible dose dependent.The AMP of same dose and AAC compare no significant difference (p＜0.05) at the light absorption value at 570nm place.
Interpretation of result and discussion
Investigated in the present embodiment AMP made AAC after, whether its anti-tumor activity significant change takes place, the result shows that the SPC-A-1 cell concentration is 5.5 * 10
4Individual/as during ml, when AMP, AAC concentration are 100 μ g/ml, the propagation of SPC-A-1 cell to be had the obvious suppression effect, it is 59.98% that AMP uses IR separately, IC
50Be 96.18 μ g/ml, AMP is after being prepared into AAC, and IR is 61.68%, IC
50Be 86.74 μ g/ml, its antineoplastic pharmacologically active does not have significant change, and the AAC in 25～100 μ g/ml dosage ranges uses separately, to the inhibited proliferation amount effect relationship of SPC-A-1 cell.
Antitumor action research in embodiment 6. ampelopsins-L-arginine-citric acid (AAC) solution body
Test objective
Research AAC uses separately and share inhibitory action to people's pulmonary carcinoma GLC-82 cell nude mice subcutaneous transplantation tumor with chemotherapeutic, determine the independent medication of AAC and with the anti-tumor in vivo effect of chemotherapeutic use in conjunction.
Test method
1. transplanted tumor plantation: collect the GLC-82 cell (available from Chinese Academy of Sciences's Shanghai cell biological institute cell bank) that is in exponential phase, adjust cell concentration to 1.0 * 10 with the RPMI RPMI-1640 that does not contain serum
7Individual/ml, by 1.0 * 10
6Individual cell/only, in nude mice (Balb/c nu.nu nude mice, female, body weight: 17～23g cultures factory available from this laboratory animal of last Hai'an) back, right side subcutaneous vaccination (0.1ml/ is only).
2. medicine preparation
The preparation of 200mg/ml AAC: take by weighing AMP 200mg, L-arginase 12 94mg gets 1ml after adding 0.75ml citric acid-sodium hydrogen phosphate buffer (pH 4.0) dissolving, adds citric acid 71.7mg again, and pH value is at 7.2-7.4 (25 ℃).
The preparation of 40mg/ml AAC: get 200mg/ml AAC 1ml+4ml NS, 0.22 μ m micropore filter filtration sterilization, pH value 7.13 (25 ℃).
The preparation of carboplatin: get 1 of carboplatin for inj, add NS to 25ml, i.e. 4.0mg/ml.
The preparation of contrast buffer: take by weighing L-arginase 12 94mg, add 0.75ml citric acid-sodium hydrogen phosphate buffer (pH 4.0), add citric acid 107.5mg (L-arginine: citric acid M::M=1: 0.3) add 4ml NS after the dissolving again, 0.22 the filtration sterilization of μ m micropore filter, pH value about 7.0 (25 ℃).
3. grouping and administration
The PBS matched group is established in experiment, and carboplatin 40mg/kg (0.1ml/10g) group is single with AAC 200mg/kg group and AAC 200mg/kg and carboplatin 40mg/kg, 0.1ml/10g drug combination group.Random packet and give Drug therapy when nude mouse grows to 100-300mg in inoculation posterior tuberosity volume averaging, medication be singly to use AAC 200mg/kg, 0.05ml/10g, im; During drug combination, give chemotherapeutic carboplatin 40mg/kg, 0.1ml10g-1 gave AAC 200mgkg-1,0.05ml10g-1, im, the administration next day of carboplatin, AAC administration every day in 30 minutes again behind the ip.Weighed 1 time every 2 days.Use in the AAC preparation back 2h, observe nude mice simultaneously and have or not abnormal response.
4. observation index and effectively criterion
The calculating of tumour inhibiting rate (IR) and drug interaction index (CDI): weigh next day after the last administration, puts to death animal, dissects and strip the tumor piece, claims tumor heavy.Be calculated as follows tumour inhibiting rate and drug interaction index (CDI).
In the formula, AB is two medicine drug combination groups and the heavy ratio of matched group tumor, and A or B are private medicine group of each prescription and the heavy ratio of matched group tumor.When CDI＜1, two medicines have synergism; When CDI＜0.7, the synergism highly significant.Tumour inhibiting rate＜30% is invalid; Tumour inhibiting rate＞30% is also learned by statistics and handled p＜0.05 is effective.
Each is organized data and is measurement data, represents with x ± S, adopts the t-check to analyze, and adds up with Excel-2003.
Result of the test
The anti-tumor in vivo effect result of study of AAC is seen Fig. 2, Fig. 3 and table 22:
Synergism when treating Balb/c nu.nu lotus people pulmonary carcinoma GLC-82 of table 22.AAC and carboplatin (x ± S, n=7)
With the matched group ratio
*P＜0.05, with carboplatin than Δ Δ P＜0.01.
The result shows: the independent medication group of AAC 200mg/kg tumour inhibiting rate is 15.84%, with the matched group ratio tumor weight is alleviated, but difference does not have significance (p＞0.05).The tumour inhibiting rate of carboplatin group is 47.78%, and AAC and carboplatin use in conjunction group tumour inhibiting rate are 70.78%, and AAC and carboplatin use in conjunction group and matched group comparison can make tumor weight obviously alleviate (p＜0.05); Compare with the carboplatin group and can make tumor weight alleviate (p＜0.01), invalid according to the independent medication of criterion AAC 200mgkg-1, but AAC 200mgkg-1 and carboplatin use in conjunction have obvious synergistic antitumor effect, and CDI is 0.66.
Existing result of study shows that ampelopsin has significant inhibitory effect to people's pulmonary carcinoma GLC-82 transplanted tumor in nude mice.Present embodiment has been studied that AAC uses separately and has been share inhibitory action to people's pulmonary carcinoma GLC-82 cell nude mice subcutaneous transplantation tumor with chemotherapeutic, the result shows that the independent medication of AAC 200mg/kg can make tumor weight alleviate, tumour inhibiting rate is 15.84%, according to criterion, tumour inhibiting rate＜30% is invalid, but AAC 200mg/kg and carboplatin use in conjunction can make tumor weight obviously alleviate, tumour inhibiting rate is 70.78%, CDI is 0.66, point out the two drug combination to have obvious synergistic antitumor effect, AAC can be united use with other antineoplastic agents as antineoplastic agent in zoopery and clinical practice.
The preparation of embodiment 7. ampelopsins-L-arginine (AMP-L-Arg) and ampelopsin-L-arginine-citric acid (AAC) injection and lyophilized formulations
Prescription:
AMP?200mg
L-arginase 12 94mg
Add 0.75ml citric acid-sodium hydrogen phosphate buffer (pH 4.0)
Get 1ml after the dissolving
Add citric acid 71.7mg, pH value is at 7.2-7.4
The settled solution that makes according to above prescription, through the filtering with microporous membrane degerming, with 1 milliliter be a loading amount unit, be sub-packed in volume and be 5 milliliters glass tube vial, place freezer dryer to make processed.Freeze-drying time is 24-32 hour.Medicine sealing after lyophilizing is preserved, and directly uses with sterilized water dissolving back before the injection.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. ampelopsin compositions, it comprises following component:
A) ampelopsin; With
B) basic amino acid, it is selected from: L-arginine, L-lysine or their combination;
Wherein, component a) and components b) weight ratio be 1: 1～1: 10, preferred 1: 1～1: 8, more preferably 1: 1～1: 6.
2. ampelopsin compositions as claimed in claim 1 is characterized in that, described compositions is selected from ampelopsin injection or ampelopsin injectable sterile powder.
3. ampelopsin compositions as claimed in claim 1 is characterized in that described compositions also comprises amount of component b) the pH regulator agent.
4. ampelopsin compositions as claimed in claim 3 is characterized in that, described amount of component b) be selected from: citric acid, hydrochloric acid, phosphate buffer, acetate buffer solution or their combination, thus making that the pH of composition solution is 5～9, preferred pH is 5.0-8.5.
5. ampelopsin compositions as claimed in claim 3, it is characterized in that described amount of component b) be citric acid, and components b) basic amino acid and amount of component b) weight ratio of citric acid is 1: 0.05～1: 1, preferred 1: 0.1～1: 0.8, more preferably 1: 0.15～1: 0.5.
6. ampelopsin compositions as claimed in claim 1 is characterized in that described compositions also comprises d) other pharmaceutically acceptable carrier and/or diluent except that the pH regulator agent.
7. ampelopsin compositions, it comprises:
A) ampelopsin of 1-5 weight portion;
B) basic amino acid of 1-50 weight portion, it is selected from: L-arginine or L-lysine;
C) the pH regulator agent of 0.05-1 weight portion, it is selected from: citric acid, phosphate buffer, acetic acid or their combination; With
D) pharmaceutically acceptable carrier of other except that the pH regulator agent and/or diluent,
Ampelopsin concentration is 0.01～500mg/ml in the aqueous solution of described compositions, and pH is 5～9.
8. each described ampelopsin preparation of compositions method among the claim 1-7, described method comprises step: blending ingredients is ampelopsin, components b a)) basic amino acid and optional amount of component b) pH regulator agent and component d) other pharmaceutically acceptable carrier and/or diluent except that the pH regulator agent.
9. method as claimed in claim 8 is characterized in that, described ampelopsin compositions is the ampelopsin injection, and this method comprises:
(i) a) ampelopsin of blending ingredients; B) basic amino acid; And d) pharmaceutically acceptable carrier of other except that the pH regulator agent and/or diluent are to form ampelopsin solution;
(ii) add c) the pH regulator agent, regulator solution pH to 5～9; With
(iii) to step (ii) the solution of gained remove pyrogen and sterilization, thereby obtain the ampelopsin injection, wherein the cumulative volume in injection is a benchmark, the concentration of ampelopsin is 0.01～300mg/ml.
10. one kind has synergistic medicine box to oncotherapy, comprises in the described medicine box:
A) each described ampelopsin compositions among the claim 1-7 of treatment effective dose;
B) carboplatin of treatment effective dose.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102133212A (en) * | 2011-01-17 | 2011-07-27 | 北京世纪博康医药科技有限公司 | Asarone medical composition |
| CN101555241B (en) * | 2009-05-20 | 2012-05-02 | 福建卫生职业技术学院 | Ampelopsin pro-dug and preparing method and application thereof |
| CN106750272A (en) * | 2016-12-05 | 2017-05-31 | 福建卫生职业技术学院 | A kind of water-soluble ampelopsin polymer |
| CN107365330A (en) * | 2017-07-10 | 2017-11-21 | 石家庄学院 | Dihydromyricetin two banks mono-sodium salt derivative and its preparation method and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1857295A (en) * | 2006-03-22 | 2006-11-08 | 四川国康药业有限公司 | Complex salt or composition medicine of breviscapine and basic amino acid for treating cardiac and cerebral vascular diseases |
| CN101045721B (en) * | 2006-03-31 | 2011-01-12 | 兰州大学 | Preparation of porcelain vine element unsaturated sodium salt and its application |
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2008
- 2008-02-22 CN CN2008100337753A patent/CN101513400B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555241B (en) * | 2009-05-20 | 2012-05-02 | 福建卫生职业技术学院 | Ampelopsin pro-dug and preparing method and application thereof |
| CN102133212A (en) * | 2011-01-17 | 2011-07-27 | 北京世纪博康医药科技有限公司 | Asarone medical composition |
| CN102133212B (en) * | 2011-01-17 | 2012-10-31 | 北京世纪博康医药科技有限公司 | Asarone medical composition |
| CN106750272A (en) * | 2016-12-05 | 2017-05-31 | 福建卫生职业技术学院 | A kind of water-soluble ampelopsin polymer |
| CN106750272B (en) * | 2016-12-05 | 2019-05-28 | 福建卫生职业技术学院 | A kind of water solubility ampelopsin polymer |
| CN107365330A (en) * | 2017-07-10 | 2017-11-21 | 石家庄学院 | Dihydromyricetin two banks mono-sodium salt derivative and its preparation method and application |
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| CN101513400B (en) | 2012-05-16 |
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