CN101134109A - Anticancer prior-medicine and method for preparing the same and use thereof - Google Patents
Anticancer prior-medicine and method for preparing the same and use thereof Download PDFInfo
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Abstract
The present invention is one kind of anticancer prodrug and its preparation process and use, and belongs to the field of medicine and chemical technology. The anticancer prodrug is macromolecular conjugate formed with lower ester pectin and anticarcinogen containing amino group or hydroxyl group and through amido bond or ester bond coupling. The present invention provides one new way for designing and developing new anticarcinogen and is scientifically significant for applying anticancer prodrug in treating tumor. The macromolecular conjugate is hopeful in developing lymph targeting anticancer prodrug, liver targeting anticancer prodrug and lung targeting anticancer prodrug.
Description
Technical field
The present invention relates to a kind of anticancer prodrug and its production and use, belong to anticancer pharmaceutical field.
Background technology
Traditional cancer therapy drug is by behind the administrations such as oral, intravenous injection, reaches certain blood drug level and is distributed in whole body and produces therapeutical effect.The defective of this Therapeutic Method maximum is to lack selectivity, and great majority cancer therapy drug molecular weight commonly used is low, and diffusion easily causes average relatively tissue distribution in vivo, produces bigger toxic and side effects, and the anticancer therapy that has a strong impact on these medicines is worth.By changing the structure of medicine, medicine can be assembled at target tissue, and reduce in the distribution of body other parts, can play the raising curative effect, reduce the effect of toxic and side effects.With cancer therapy drug and macromole coupling, form macromole cancer therapy drug conjugate, can change cancer therapy drug distribution in vivo, assemble at particular organization's organ, reach the targeted therapy effect.Study its targeting mechanism, both passive target can be arranged, for example macromole cancer therapy drug conjugate gathers in cancerous tissue easily, be not easy to discharge, what be cancerous tissue to macromole cancer therapy drug conjugate gathers retention effect (EPR effect), initiatively targeting also can be arranged, and for example the galactose structure in the conjugate can initiatively combine with the galactosylated acceptor of surface of hepatocytes.Like this, just macromole cancer therapy drug conjugate can be developed to targeted anticancer medicine at the particular target histoorgan.
In addition, macromole cancer therapy drug conjugate has the lymphsystem targeting.Transfer is one of most important biological characteristics of solid carcinoma, and lymph metastasis is very common in the transfer of solid carcinoma, and has a strong impact on therapeutic effect.The Therapeutic Method of solid carcinoma lymph metastasis mainly is an excision, but because the complexity of lymph metastasis, for example when the lymph node that shifts has the invasion and attack adhesion to normal vital structures such as trunks, operation can not be carried out or cause postoperative residual, and might cause the sequela and the dysfunction of varying degree.The existence that small lymphatic metastasis kitchen range and lymphatic vessel shift also easily makes removing not thorough.Though systemic chemotherapy and radiotherapy are effective, lymphatic metastasis kitchen range curative effect is lower than primary tumor, and because of the different untoward reaction of degree, the patient can not tolerate and treatment is failed.Therefore, research lymphsystem target administration is very important.
In recent years, the macromole targeted anticancer medicine becomes the emphasis to the chemotherapeutics research of lymph metastasis, this class conjugate has bigger molecular volume, behind local injection, can not see through the very narrow endothelium gap of blood capillary and enter blood, and enter partial draining lymph node than the endothelium gap that is easier to by the lymphatic capillary broad.In addition, this class conjugate can also take regional nodes to by by the mode of macrophage phagocytic, under the effect of lymph node endoenzyme, is hydrolyzed and discharges active substance (cancer therapy drug), plays the effect of killing metastasis cancer cell in the lymph node.Thereby this class macromolecular carrier targeted drug is suitable for the treatment that lymphatic cancer shifts most.
Pectin is commonly used for tablet, microsphere, the clothing layer of microcapsule and liposome etc., preparation drop pill and granule, embedding liquid or solid medicine, play the uncomfortable stink of slow releasing function and masking agents, it is natural macromolecular polysaccharide polymer, extensively be present in the cell wall of plant, acid macromolecular polysaccharide (the Hyunjo Kim that forms by α (1 → 4) D galactopyranose aldehydic acid unit, et al.International Journal of Pharmaceutics, 1998,161:149-159), its molecule constitutes a helicoidal structure by 3 units, and the pitch of its spiral is 1.34nm.Wherein, it is esterification forms that the carboxyl on the galacturonic acid has many, not the residual carboxyl of esterification then be free acid form or potassium, sodium, ammonium, calcium salt forms (Sun Yuanlin etc. food and machinery, 2004,20 (6): 60-63).In addition, also have acetyl group and the sugared side chain of other neutrality (many) in its structure, as existence such as rhamnose, galactose, arabinose xyloses; Pectin can be by strengthening mononuclear phagocyte system, and activating macrophage, T cell and B cell, NK cell and complement system promote cytokine secretion, strengthen raising host immune functions such as erythrocyte immune; Growth characteristics by changing the solid carcinoma cell membrane, influence signal pipeline in the solid carcinoma cell, anti-radical action, induce differentiation and apoptosis, the nucleic acid and the protein synthesis of inhibition solid carcinoma cell, influence the solid carcinoma cell ultrastructure, influence oncogene, antimutagenic effect, the vascularization of inhibition solid carcinoma are brought into play direct antitumaous effect (Chinese Chinese medicine information magazine, 1999.5:64).
At present do not see that pectin is used to prepare the report of lympha targeted medicine, hepatic targeting drug or lung targeted drug, do not see pectin and cancer therapy drug coupling, the report that is used for preparing the medicine of treatment solid carcinoma or the transfer of its draining lymph node or treats the purposes of hepatocarcinoma or lung cancer drugs yet.
Summary of the invention
The object of the present invention is to provide a kind of novel anticancer prodrug, provide a kind of new selection for clinical.
Anticancer prodrug provided by the invention is the macromole conjugate that is formed by pectin with excellent biological compatibility and cancer therapy drug coupling, wherein, usually high-esterpectin is obtained low methoxyl pectin through defat, contain the mode coupling of the cancer therapy drug of amino or hydroxyl and low methoxyl pectin with amido link.
Chinese scholars studies show that molecular weight can be used as plasma substitute less than 70,000 pectin, and molecular weight then can be accumulated at kidney greater than 70,000 pectin.Because pectin is not easy degraded in vivo, can only pass through renal excretion, therefore, it is 1000～70000 pectin and cancer therapy drug coupling that the present invention selects molecular weight, prepares the new type anticancer prodrug.The molecular weight that preferred scheme is a pectin is: 3000～35000, and more preferably the molecular weight of pectin is: 5000～25000.
Wherein the link coupled weight ratio of pectin and cancer therapy drug is: 1 part of pectin, 0.05～0.8 part of anticarcinogen.
Described cancer therapy drug is: amycin, epirubicin, zorubicin, aclarubicin, daunorubicin, bisantrene, mitomycin, mitoxantrone, bleomycin, methotrexate, camptothecine, paclitaxel or gemcitabine.
Above-mentioned anticancer prodrug adds that the auxiliary material of pharmaceutically accepting can be prepared into local injection drug-delivery preparation or intravenous formulations.Also can be 1 part of anticancer prodrug of the present invention, 1～30 part in lecithin, preferred anticancer prodrug be 1: 5～8 with the ratio of lecithin.Add that pharmaceutically acceptable auxiliary material is prepared into abdominal cavity or thoracic cavity local injection drug-delivery preparation or intravenous formulations.Described preparation is infusion solutions, freeze-dried powder, liposome, nanoparticle or microsphere.Above-mentioned local injection drug-delivery preparation mainly is meant abdominal cavity or thoracic cavity local injection drug-delivery preparation.
The present invention also provides the method for preparing above-mentioned anticancer prodrug, may further comprise the steps:
A, low methoxyl pectin are dissolved in water, in the mixture of then that cancer therapy drug is water-soluble and organic solvent;
B, with above-mentioned two solution mixing mixings, ethoxycarbonyl-2-ethyoxyl dihydroquinoline (EEDQ) be a dehydrant to add N-, 20～80 ℃ of temperature controls stir 3～24h, adding ethanol, centrifugalize;
C, with resolution of precipitate in water, transferring in the bag filter dialysis to detecting less than small-molecule drug, in bag filter, add ethanol in the liquid, centrifugal, the precipitation vacuum drying, the water solublity conjugate;
Above-mentioned organic solvent can be pyridine, triethylamine, oxolane or N, dinethylformamide, preferred N, dinethylformamide, and preferred water: N, dinethylformamide=1: 1;
Or
A, low methoxyl pectin is dissolved in a certain amount of water, then that cancer therapy drug is water-soluble and be added in the pectin solution, add dehydrant behind the stirring and evenly mixing, 20～80 ℃ of temperature controls stir 3～24h;
Described dehydrant is N, N-dicyclohexylcarbodiimide (DCC), N-cyclohexyl-N ＇ two-methylamine propyl group carbodiimide (CDC) or EDCHCl,
B, adding ethanol, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
Described low methoxyl pectin is that high-esterpectin obtains through the defat reaction, and crosses the pectin use that sephadex column is collected the desired molecule amount.Specifically can adopt following method to take off ester:
High-esterpectin is dissolved in water, with sodium hydroxide solution regulator solution pH to 10～13,15～80 ℃ of stirring reaction 0.5h～5h.Reaction is finished, and with hydrochloric acid adjusting pH to 2.8～3.5, adds organic solvent deposit, filters, and to no chloride ion, drying gets low methoxyl pectin with the aqueous solutions of organic solvent cleaning product;
Preferred scheme is: the dehydrant that preparation water solublity conjugate uses is EEDQ, 50 ℃ of reaction temperatures, and the response time is 8h; The dehydrant that the slightly solubility conjugate of preparation uses is EDCHCl, and reaction temperature is 50 ℃, and the response time is 8h.
The present invention also provides the purposes of above-mentioned anticancer prodrug in medicine, treatment hepatocarcinoma or the lung cancer drugs of preparation treatment solid carcinoma or the transfer of its draining lymph node.
Especially described anticancer prodrug is the medicine that is used for solid carcinoma and the administration of draining lymph node local injection thereof, be used for solid carcinoma causes the abdominal cavity of ascites or hydrothorax or the medicine of thoracic cavity drug administration by injection, or is used for the treatment of the purposes in the intravenous drug of hepatocarcinoma or pulmonary carcinoma.
The purposes of pectin in lympha targeted, liver targeting of preparation or lung targeted drug also is that the present invention proposes first.Owing to contain galactose residue on the pectin chain, and a large amount of galactosylated acceptors is arranged on the liver, as anti-cancer medicament carrier, cancer therapy drug initiatively can be directed to liver with pectin, for the targeted therapy of hepatocarcinoma provides a kind of new method.Test shows that after pectin and the amycin coupling, according to the difference of synthetic reaction, the water solublity difference after adjuvant is prepared into different preparations, has clear and definite targeting to lymph, liver or lung respectively.
The present invention has following advantage compared with the prior art:
Pectin is natural macromolecular, has excellent biological compatibility, and wherein molecular weight is lower than 35000 pectin, and the conjugate that forms with amycin, intravenous injection in the body after, can pass through renal excretion.Pectin amycin conjugate disclosed by the invention is a kind of macromolecular prodrug, and according to the difference of synthesis technique, water solublity has difference, and with after adjuvant is prepared into different preparations, its construction features makes them have lung targeted characteristic or hepatic targeting respectively respectively.Their construction features also makes them all possess lympha targeted characteristic, after local injection, can enter into injection site draining lymph node on every side specifically, and the carrier that can be used as the lympha targeted property new drug of design uses.Anticancer prodrug of the present invention can pass through the local injection administration, can be expelled in the solid carcinoma cancer, also can be expelled in the solid carcinoma draining lymph node on every side, conjugate enters behind the lymph node by huge has a liking for cytophagy, hydrolysis discharges cancer therapy drug under the effect of enzyme in lymph node, is used for the treatment of solid carcinoma (for example breast carcinoma, gastric cancer, esophageal carcinoma, incidence cancer, cervical cancer, endometrial carcinoma, colorectal cancer etc.) and draining lymph node thereof and shifts.
The pectin anticancer prodrug is expected to become an important channel of design and developing new drug, and the application for anticancer prodrug treatment tumor aspect has important scientific meaning.The present invention is carrier with pectin, and itself and cancer therapy drug are passed through the covalent bond coupling, and being expected to exploitation becomes lympha targeted anticancer prodrug, liver target anticancer prodrug or lung target anticancer prodrug, this synthetic route economical and effective.
Description of drawings
Fig. 1 water soluble pectin-amycin ultraviolet figure
Fig. 2 slightly solubility pectin-amycin ultraviolet figure
Wherein, curve representation among the figure: a, amycin; B, slightly solubility pectin amycin conjugate; C, pectin and amycin mixture; D pectin.
The infrared figure of Fig. 3 slightly solubility pectin-amycin
Wherein, curve representation among the figure: a, amycin; B, slightly solubility pectin amycin conjugate; C, pectin
Fig. 4 different pharmaceutical is to the influence of hepatocarcinoma tumor-bearing mice life span
Fig. 5 different pharmaceutical is to the influence of pulmonary carcinoma tumor-bearing mice life span
The specific embodiment
Below by the specific embodiment the present invention is further described, be limitation of the invention but should not be construed as.Those of ordinary skills can also make modification, replacement, the change of various ways according to technique scheme.All modification, replacement, changes of doing based on above-mentioned technological thought all belong to scope of the present invention.
Below by embodiment the present invention is further described.
Ester falls in embodiment one, high-esterpectin alkaline process
Take by weighing the 10g high-esterpectin, be dissolved in the 1000mL secondary water, regulate pH=10～13 with sodium hydroxide solution then, keep 10～60 ℃, difference stirring reaction 30,60,90,120,150 and 180 min, after reaction finished, concentrated hydrochloric acid was regulated pH=2.8～3.5, added 300ml ethanol, the centrifugal 15min of 4500r/min, precipitation is cleaned chloride ion with 60% alcoholic solution, and 100 ℃ of dryings get low methoxyl pectin.
Embodiment two, molecular weight segmentation
With handle well sephadex G 75 pour in the chromatographic column while stirring, natural subsidence 20min, put into the circular filter paper sheet that is slightly less than the chromatographic column internal diameter at last, getting the pectin solution for preparing carefully is added on the gel column, regulating the secondary water flow velocity with constant flow pump is 4ml/ pipe/6 minutes, GPC measures the molecular weight distribution of pectin, merges the pectin of certain molecular weight scope then, obtains molecular weight ranges and be 1000～70000 low methoxyl pectin.
The preparation one of embodiment three, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, the 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 30 ℃ with 10ml secondary water dissolution.Add 300mg EEDQ then, control temperature at 30 ℃, stirring reaction 24 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.The ultraviolet figure of pectin and amycin water solublity conjugate sees Fig. 1, the mixture uv absorption of pectin, amycin, pectin and amycin is seen Fig. 2, pectin does not have uv absorption, amycin has absorption at 479.5nm, pectin and amycin mixture have absorption at the 488.5nm place, water soluble pectin-amycin conjugate has absorption maximum at the 496nm place, and uv absorption generation red shift proves that covalent coupling has taken place for amycin and pectin.
The preparation two of embodiment four, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, the 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃ with 10ml secondary water dissolution.Add 300mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation three of embodiment five, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, the 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 70 ℃ with 10ml secondary water dissolution.Add 300mg EEDQ then, control temperature at 70 ℃, stirring reaction 6 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation four of embodiment six, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 6.9 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 220mg EEDQ then, control temperature at 50 ℃, stirring reaction 4 hours.It was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation five of embodiment seven, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 200mg EEDQ then, control temperature at 50 ℃, stirring reaction 6 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation six of embodiment eight, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7.1 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 240mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation seven of embodiment nine, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 6.8 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 200mg EEDQ then, control temperature at 50 ℃, stirring reaction 10 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation eight of embodiment ten, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7.2 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 240mg EEDQ then, control temperature at 50 ℃, stirring reaction 12 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation nine of embodiment 11, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 200mg EEDQ then, control temperature at 50 ℃, stirring reaction 16 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation ten of embodiment 12, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7.2 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 300mg EEDQ then, control temperature at 50 ℃, stirring reaction 20 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation 11 of embodiment 13, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7.0 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg doxorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 300mg EEDQ then, control temperature at 50 ℃, stirring reaction 24 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation 12 of embodiment 14, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 6.9 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 100mg doxorubicin hydrochloride is dissolved in 20ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 300mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation 13 of embodiment 15, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and 25 ℃ were stirred 60 minutes.Add 100mg EDCHCl then, 25 ℃ of stirring reactions 12 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
Its ultraviolet figure sees Fig. 2, and amycin has maximum absorption band at the 479.5nm place, and pectin and amycin mixture have maximum absorption band at the 488.5nm place, and the pectin amycin has maximum absorption band at the 498nm place, and pectin does not have absorption.Absorb red shift takes place, this explanation amycin and the coupling of pectin generation chemical bond, infrared spectrum is seen Fig. 3, Fig. 3 shows that compare with pectin, pectin one amycin is at 1620cm
-1The blended absorbent peak and the 1750cm of amide I band and amide II band appears in the place
-1The ester bond peak compare peak area ratio and significantly increase, and at 1100cm
-1And 1017cm
-1The anthracene nucleus characteristic absorption peak of tangible amycin, 1411.23cm appear in the place
-1The absworption peak of primary amide appears in the place, illustrates that pectin and amycin are bonded with the form of amido link.
The preparation 14 of embodiment 16, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and 25 ℃ were stirred 60 minutes.Add 110mg EDCHCl then, 25 ℃ of stirring reactions 24 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 15 of embodiment 17, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and 70 ℃ were stirred 60 minutes.Add 105mg EDCHCl then, 70 ℃ of stirring reactions 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 16 of embodiment 18, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 60 minutes at 50 ℃.Add 120mg EDCHCl then, control temperature at 50 ℃, stirring reaction 5 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 17 of embodiment 19, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 60 minutes at 50 ℃.Add 140mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 18 of embodiment 20, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 60 minutes at 50 ℃.Add 110mg EDCHCl then, control temperature at 50 ℃, stirring reaction 12 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 19 of embodiment 21, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 60 minutes at 50 ℃.Add 130mg EDCHCl then, control temperature at 50 ℃, stirring reaction 18 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 20 of embodiment 22, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 60 minutes at 50 ℃.Add 120mg EDCHCl then, control temperature at 50 ℃, stirring reaction 24 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 21 of embodiment 23, pectin-amycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 19mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 30 minutes at 50 ℃.Add 200mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 22 of embodiment 24, pectin-amycin conjugate
Take by weighing the 300mg low methoxyl pectin, with 30ml secondary water dissolution.The 36mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 30 minutes at 50 ℃.Add 40mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 23 of embodiment 25, pectin-amycin conjugate
Take by weighing the 300mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 57mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 30 minutes at 50 ℃.Add 60mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 24 of embodiment 26, pectin-amycin conjugate
Take by weighing the 300mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 80mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 30 minutes at 50 ℃.Add 65mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation 25 of embodiment 27, pectin-amycin conjugate
Take by weighing the 300mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg doxorubicin hydrochloride is dissolved in the 10ml secondary water, and temperature control stirred 30 minutes at 50 ℃.Add 50mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation one of embodiment 28, pectin-epirubicin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7.1 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg Farmorubine Hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 250mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation two of embodiment 29, pectin-epirubicin conjugate
Take by weighing the 100mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg Farmorubine Hydrochloride is dissolved in the 10ml secondary water stirring at room 30 minutes.Add 20mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation one of embodiment 30, pectin-daunorubicin conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg daunorubicin hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 200mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation two of embodiment hentriaconta-, pectin-daunorubicin conjugate
Take by weighing the 100mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg daunorubicin hydrochloride is dissolved in the 10ml secondary water stirring at room 30 minutes.Add 50mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation one of embodiment 32, pectin-mitoxantrone conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7.1 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 30mg mitoxantrone hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 210mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation two of embodiment 33, pectin-mitoxantrone conjugate
Take by weighing the 100mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg mitoxantrone hydrochloride is dissolved in the 10ml secondary water stirring at room 30 minutes.Add 50mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation one of embodiment 34, pectin-gemcitabine conjugate
Take by weighing the 100mg low methoxyl pectin, in the 100ml round-bottomed flask, regulate pH to 7 with 0.1mol/L NaOH solution then with 10ml secondary water dissolution.The 50mg gemcitabine hydrochloride is dissolved in 10ml dimethyl formamide/water, and (V/V=1: in the mixed solution 1), temperature control stirred 30 minutes at 50 ℃.Add 220mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation two of embodiment 35, pectin-gemcitabine conjugate
Take by weighing the 100mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg gemcitabine hydrochloride is dissolved in the 10ml secondary water stirring at room 30 minutes.Add 50mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation of embodiment 36, pectin-paclitaxel conjugate
Take by weighing the 100mg low methoxyl pectin, use 30ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg paclitaxel is dissolved among the 10mlDMF stirring at room 30 minutes.Add 50mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The preparation of embodiment 37, pectin-zorubicin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg zorubicin hydrochloride is dissolved in the mixture (DMF/ water=1/1) of 10ml DMF and water stirring at room 30 minutes.Add 60mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation of embodiment 38, pectin-aclarubicin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg aclarubicin hydrochloride is dissolved in the mixture (DMF/ water=1/1) of 10ml DMF and water stirring at room 30 minutes.Add 40mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation of embodiment 39, pectin-bisantrene conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg bisantrene hydrochloride is dissolved in the mixture (DMF/ water=1/1) of 10ml DMF and water stirring at room 30 minutes.Add 45mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation of embodiment 40, pectin-mitomycin conjugate
Take by weighing the 50mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 20mg silk is split in the mixture (DMF/ water=1/1) of mould 10mlDMF of being dissolved in and water stirring at room 30 minutes.Add 20mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation of embodiment 41, pectin-bleomycin conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.50mg hydrochloric acid bleomycin is dissolved in the mixture (DMF/ water=1/1) of 10ml DMF and water stirring at room 30 minutes.Add 50mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation of embodiment 42, pectin-methotrexate conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg methotrexate is dissolved in the mixture (DMF/ water=1/1) of 10ml DMF and water stirring at room 30 minutes.Add 55mg EEDQ then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, it was 5000 bag filter that reactant mixture is transferred to molecular cut off, with secondary water dialysis 24 hours.Add dehydrated alcohol 150ml in the liquid in bag filter, produce precipitation, centrifugal, precipitation is used absolute ethanol washing, and drying gets the water solublity conjugate.
The preparation of embodiment 43, pectin-camptothecine conjugate
Take by weighing the 100mg low methoxyl pectin, use 10ml secondary water dissolution in the 100ml round-bottomed flask.The 50mg camptothecine is dissolved in the mixture (DMF/ water=1/1) of 10mlDMF and water stirring at room 30 minutes.Add 50mg EDCHCl then, control temperature at 50 ℃, stirring reaction 8 hours.During time to go, add organic solvent, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
The mensuration of embodiment 44, conjugate drug loading
The 10mg doxorubicin hydrochloride is dissolved in secondary water, and standardize solution becomes 100mL solution.Accurately pipette 1mL in the volumetric flask of 10mL, cause scale, obtain the amycin solution of 10 μ g/mL with the dilution of secondary water.With secondary water is blank, carries out UV scanning in the 200-600nm wave-length coverage, and the uv-absorption maximum wavelength of determining amycin is 480nm.Accurately compound concentration is the doxorubicin hydrochloride standard solution of 10.00,20.00,30.00,40.00,50.00 μ g/mL, measure absorbance in the maximum absorption wave strong point, concentration of standard solution C is carried out linear regression to its absorbance A, getting regression equation is that hence one can see that for A=0.0182C+0.0288 (R=0.9985), in 10-50 μ g/mL scope, linear relationship is good between doxorubicin concentration and absorbance.
The mensuration of sample drug loading: accurately take by weighing a certain amount of water soluble pectin-amycin, be dissolved in and be made into solution to be measured in the secondary water, measure absorbance.With the sample absorbance substitution regression equation of measuring, try to achieve concentration C (μ g/ml).The conjugate drug loading calculates according to following formula: drug loading=CV/1000M * 100%; V is a sample volume, unit/ml; M is a sample weighting amount, the mg of unit.
After slightly solubility pectin-amycin dripping hydrochloric acid makes its dissolving, with the dilution of secondary water, be mixed with finite concentration and measure drug loading then.
It is identical with pectin-amycin step that other prodrug drug loading are measured process.
Conjugate drug loading measurement result sees Table 1:
Table 1 conjugate drug loading measurement result
| Embodiment | Drug loading (%) |
| Embodiment three | 21.3 |
| Embodiment four | 16.8 |
| Embodiment five | 14.0 |
| Embodiment six | 17.2 |
| Embodiment seven | 18.5 |
| Embodiment eight | 20.9 |
| Embodiment nine | 21.2 |
| Embodiment ten | 21.5 |
| Embodiment 11 | 21.8 |
| Embodiment 12 | 22.0 |
| Embodiment 13 | 23.1 |
| Embodiment 14 | 38.0 |
| |
19.2 |
| Embodiment 16 | 20.1 |
| Embodiment 17 | 19.8 |
| Embodiment 18 | 17.4 |
| Embodiment 19 | 22.3 |
| |
22.5 |
| Embodiment 21 | 23.8 |
| Embodiment 22 | 11.4 |
| Embodiment 23 | 8.2 |
| Embodiment 24 | 7.5 |
| |
14.6 |
| Embodiment 26 | 15.2 |
| Embodiment 27 | 10.1 |
| Embodiment 28 | 21.3 |
| Embodiment 29 | 20.8 |
| |
19.5 |
| The embodiment hentriaconta- | 18.2 |
| Embodiment 32 | 21.0 |
| Embodiment 33 | 20.7 |
| Embodiment 34 | 19.8 |
| Embodiment 35 | 20.7 |
| Embodiment 36 | 17.9 |
| Embodiment 37 | 19.5 |
| Embodiment 38 | 21.2 |
| Embodiment 39 | 18.7 |
| Embodiment 40 | 22.5 |
| Embodiment 41 | 21.8 |
| Embodiment 42 | 18.9 |
| Embodiment 43 | 19.7 |
The preparation of embodiment 45, injection
1, medicine
Doxorubicin hydrochloride is available from Haizheng Medicine Stock Co., Ltd., Zhejiang Prov; Water soluble pectin-amycin conjugate and slightly solubility pectin-amycin conjugate are provided by synthetic chamber, Yaoyou Science and Technology Development Co., Ltd., Chendu City.
2, the preparation of injection
2.1 the preparation of water soluble pectin-amycin conjugate injection
Taking by weighing drug loading is 20.9% water soluble pectin amycin conjugate 100mg, adds normal saline, and stirring and dissolving is diluted to 10ml with normal saline then, gets water soluble pectin-amycin conjugate injection (being equivalent to amycin 2mg/ml).
2.2 the preparation of slightly solubility pectin-amycin conjugate injection
Take by weighing drug loading and be slightly solubility pectin-amycin 100mg of 20.1% and place the 10ml normal saline, stirred 2 hours under the room temperature, make the abundant mixing of medicine, slightly solubility pectin-amycin conjugate injection (being equivalent to amycin 2mg/ml).
2.3 the doxorubicin hydrochloride physiological saline solution is mixed with and contains amycin 2mg/ml.
The preparation of embodiment 46, slightly solubility pectin-amycin conjugate lecithin injection
A certain amount of soybean lecithin is dissolved with distilled water, stirred under the room temperature 2-4 hour, form soybean lecithin colloid solution; Take by weighing drug loading and be 20.1% slightly solubility pectin amycin, add soybean lecithin solution, be stirred to the complete mixing of medicine under the room temperature, slightly solubility pectin-amycin conjugate lecithin injection (being equivalent to amycin 2mg/ml).
The preparation of the lecithin injection of various proportioning preparations sees Table 2:
The preparation prescription of table 2 slightly solubility pectin-amycin lecithin injection
| Slightly solubility pectin-amycin (mg) | Soybean lecithin concentration | Soybean lecithin volume (ml) | Anticancer prodrug/soybean lecithin |
| 100 | 1％ | 10 | 1∶1 |
| 100 | 2％ | 10 | 1∶2 |
| 100 | 5％ | 10 | 1∶5 |
| 100 | 8％ | 10 | 1∶8 |
| 100 | 15％ | 10 | 1∶15 |
| 100 | 30％ | 10 | 1∶30 |
Embodiment 47, the water soluble pectin-lympha targeted property of amycin conjugate experiment
1, animal
The SD rat, female entirely, body weight 230～250g is provided by Chengdu Baikang Pharmaceutical Industry Pharmacological and toxicological Rearch Institute, the production facility licence: No. 06, the real moving Guan Zhidi in river.
2, method
2.1 chromatographic condition
Mobile phase: methanol: acetonitrile: phosphate buffer (25mM NH
4H
2PO
4-30mM H
3PO
4, pH=5)=5: 2: 3, flow velocity 1.0ml/min, sample size 20 μ l, wavelength 480nm.
2.2 medication and sample collection
Choose 112 of rats, be divided into amycin group and pectin-amycin group at random, fasting 12h before the experiment.Injection among the left and right callosity subcutaneous injection of the two treated animals embodiment 45 (0.02ml/ only) respectively at 0,10,30,60,90,120,180,240,300min puts to death animal, wins Zuo You popliteal nest portion lymph node, peels off fatty tissue, is weighed.
2.3 sample treatment
Add 50%ACN homogenate by weight proportion, 3000 rev/mins centrifugal 10 minutes, get supernatant, 0.45 μ m membrane filtration is got the filtrate sample introduction.
2.4 the preparation of standard curve
Get blank lymph node homogenate, it is 0.04,0.08,0.12 μ g/ml that the adding amycin makes concentration, and by 3.3 processing sample introductions, record chromatogram, peak area A carry out linear regression with A to concentration.
3, result
Doxorubicin concentration sees Table 3 in the different time points Da Shu popliteal nest portion lymph node.
Doxorubicin concentration (n=7) in the table 3 different time points lymph node
| Group | Time (min) | ||||||||
| 0 | 10 | 30 | 60 | 90 | 120 | 180 | 240 | 300 | |
| Amycin group (10 -6mmol/kg) | 0 | 15.0 | 18.3 | 17.7 | 15.1 | 10.2 | 1.2 | 0.2 | |
| Pectin-amycin group (10 -6mmol/kg) | 0 | 5.7 | 11.3 | 26.3 | 34.7 | 38.0 | 16.5 | 13.2 | 8.7 |
4, conclusion
Pectin-amycin and amycin behind drug administration by injection in 30 minutes the drug level of amycin group be higher than pectin-amycin group; 60,90,120,180,240,300 minutes concentration ratio amycin group height, after 300 minutes the drug level of amycin group detected less than.Above result shows that pectin-amycin has lympha targeted property, and has slow releasing function in lymph node, can keep higher concentration for a long time, plays persistent therapeutical effect.
After embodiment 48, the pectin-amycin conjugate whole body administration in the intravital distribution test of mice
1, animal
Kunming mouse, male and female half and half, body weight 18～25g is available from Sichuan University's West China medical college animal center.
2, method
2.1 medication
Choose 50 mices, be divided into 5 groups at random, 10 every group.Give the slightly solubility pectin amycin lecithin injection (anticancer prodrug/soybean lecithin=1: 2) of the doxorubicin injection that contains amycin 2mg/ml, water soluble pectin doxorubicin injection, slightly solubility pectin-doxorubicin injection, embodiment 46 preparations of tail vein injection embodiment 45 preparation and normal saline 0.1ml/ respectively.
2.2 sample collecting and processing
2 hours excision eyeballs are got blood after the administration, put to death mice.Get an amount of heart, liver, spleen, lung and renal tissue with homogenate (60% ethanol that contains 0.3mol/L hydrochloric acid) by weight volume ratio be made into tissue homogenate at 1: 9.The centrifugal 10min of 3000rpm after the homogenate.Get the 0.1ml supernatant and carry out fluoroscopic examination.The centrifugal 10min of blood sample 3000rpm.Get the 0.1ml supernatant and carry out fluoroscopic examination.
3, result
The fluorophotometric value of amycin sees Table 4 in the different tissues sample.
The fluorophotometric value (n=10) of amycin in 2 hours different tissues samples after table 4 administration
| Project | Heart | Liver | Spleen | Lung | Kidney | Blood |
| A B C D E | 166.63±22.16 353.07±65.27 291.86±30.84 397.41±35.26 343.5±16.69 | 177.62±34.11 303.48±27.83 569.88±75.63 483.15±69.38 887.99±103.62 | 71.03±12.04 248.9±47.51 224.67±35.87 277.13±20.14 248.15±26.93 | 51.45±18.20 480.78±25.89 523.58±42.35 570.81±42.13 288.89±25.71 | 132.19±14.24 273.42±30.14 283.4±59.87 467.43±63.52 411.89±15.69 | 7.79±1.635 10.42±2.519 13.504±2.634 14.301±0.213 14.297±0.568 |
Annotate: A. normal saline group; B. amycin normal saline group; C. water soluble pectin-amycin group; D. slightly solubility pectin-amycin normal saline group; E. slightly solubility pectin-amycin lecithin group
4, conclusion
Different doxorubicin formulations are not quite similar in the intravital distribution of mice.Water soluble pectin-amycin and slightly solubility pectin-amycin all have the trend of enrichment in liver, distinguish to some extent with the preparation difference, but concentration in the blood all.Illustrate that the pectin amycin has certain liver property that becomes.
The experimentation of embodiment 49, the anti-rat liver cancer of pectin-amycin
1. animal and cell strain
The C57BL/6J mice, female, 18～22g, 4～6 ages in week are available from Sichuan University's Experimental Animal Center.
Rat liver cancer cell line (Hepa) is purchased in ATCC (American Type Culture Collection), and this laboratory is protected and planted.
2. method
2.1 liver cancer model preparation
Use the chloral hydrate anesthesia mice, be fixed in and dissect on the plate, under the aseptic condition, open-the long transverse incision of 0.5cm on the right side under xiphoid-process, exposes the abdominal cavity, extrudes leftlobe of liver and fix, thrust 2～3mm under the Glisson's capsule with syringe (No. 7 pins), injection Hepa cell suspension 50 μ l (contain cell 10
6Individual), close abdomen, sterilization skin is with prevention infection.
2.2 medication
30 mices are divided into 3 groups at random, every group 10, the slightly solubility pectin amycin lecithin injection (anticancer prodrug/soybean lecithin=1: 2) (being equivalent to amycin dosage 10mg/kg) of matched group (normal saline), amycin group (dosage 10mg/kg), embodiment 46 preparations.3 days begin treatments behind inoculated tumour, tail vein injection, volume injected 100 μ 1, per 5 days 1 time, totally 3～4 times.
3. result
Different pharmaceutical is seen Fig. 4 to the influence of tumor-bearing mice life span.
4. conclusion
Compare with matched group, the visible tumor-bearing mice life span of doxorubicin hydrochloride group and pectin-amycin group obviously prolongs, and difference has statistical significance.And pectin-amycin group effect is best, is better than the doxorubicin hydrochloride group, can prolong the life span of hepatocarcinoma tumor-bearing mice.
The experimentation of anti-mouse lung metastatic tumor of embodiment 50 pectin-amycin and pulmonary carcinoma
1. animal and cell strain
The C57BL/6J mice, female, 18～22g, 4～6 ages in week are available from Sichuan University's Experimental Animal Center.
Mice Bearing Lewis Lung Cancer cell line (LL/2) is purchased in ATCC (American Type Culture Collection), and this laboratory is protected and planted.
2. lung cancer model preparation and treatment
The lung cancer model preparation: collect the LL/2 cell of exponential phase, the centrifugal 3min of 1500rpm, cell precipitation washs 1 time with the culture medium of serum-free, antibiotic-free, and adjusting cell concentration with serum-free, antibiotic-free culture medium after the counting cells quantity is 1 * 10
7/ ml.Every mice is in tail vein injection 1 * 10
6Individual (0.1ml) tumor cell, 3 days begin treatments after the kind tumor.
Treatment: divide three groups, matched group (normal saline), amycin group (dosage 10mg/kg), water soluble pectin-amycin group (being equivalent to amycin dosage 10mg/kg), tail vein injection, volume injected 100l, per 5 days 1 time, totally 3～4 times.
3. result
Different pharmaceutical is seen Fig. 5 to the influence of pulmonary carcinoma tumor-bearing mice life span; Each is organized medicine and pulmonary carcinoma tumor-bearing mice pulmonary is shifted what the influence of joint footing sees Table 5.
Table 5 different pharmaceutical shifts what the influence (10) of a joint footing to pulmonary carcinoma tumor-bearing mice pulmonary
| The blank group | The doxorubicin hydrochloride group | Pectin amycin group | |
| Pulmonary 〉=3mm saves footing | 103±15.6 | 65±23.0 | 50±14.9 |
* compare P＜0.01 with matched group.
4. conclusion
Pectin-amycin is compared the life span that all can prolong the pulmonary carcinoma tumor-bearing mice with the doxorubicin hydrochloride group with matched group, suppress the growth of cancer joint knot in pulmonary.Pectin-amycin effect is more obvious.
The pernicious behavior of malignant cell, as the blood phase transfer, cell surface saccharide complex synthetic, adhesion molecule in glycan structures and cell surface and the substrate and receptor thereof all have the ten minutes confidential relation with sugar.The glycan structures of skin covering of the surface has significant change in the malignant change of cell process, and synthetic and excretory glycoprotein of tumor cell and sugar chain structure also have similar variation, and the glycolipid in tumor cell enters body is formed also and normally marked difference.Therefore research and develop the medicine on directly effect and the film water horizontal target point, certainly will help to find efficient specificity antineoplastic medicine.
There is galactose residue in the pectin surface, and there is a large amount of galactosylated acceptors in liver, factor based on above-mentioned two aspects, use principle of hybridization, with cancer therapy drug and pectin coupling, preparation pectin anticancer prodrug, by both addition complementations, purpose is to improve the targeting and the bioavailability thereof of chemical compound, and searching curative effect height, the new type anticancer medicine that toxic and side effects is low are in the hope of the clinical new type anticancer medicine that provides is provided.Those skilled in the art can other cancer therapy drug of reasonable prediction according to technological thought of the present invention, adopt the inventive method, become prodrug with the pectin coupling, for the design and developing new drug a new way is provided, application for anticancer prodrug treatment tumor aspect has important scientific meaning and good application prospects.
Claims (12)
1. anticancer prodrug is characterized in that: be the macromole conjugate that is formed with the mode coupling of amido link by low methoxyl pectin and cancer therapy drug; Wherein, described cancer therapy drug contains amino or hydroxyl, and the number-average molecular weight of described low methoxyl pectin is 1000～70000.
2. anticancer prodrug according to claim 1 is characterized in that: the number-average molecular weight of described low methoxyl pectin is: 3000～35000.
3. anticancer prodrug according to claim 1 is characterized in that: the number-average molecular weight of described low methoxyl pectin is: 5000～25000.
4. according to each described anticancer prodrug of claim 1～3, it is characterized in that: the link coupled weight ratio of low methoxyl pectin and cancer therapy drug is: 1 part of low methoxyl pectin, 0.05～0.8 part of anticarcinogen.
5. anticancer prodrug according to claim 4 is characterized in that: described cancer therapy drug is: amycin, epirubicin, zorubicin, aclarubicin, daunorubicin, bisantrene, mitomycin, mitoxantrone, bleomycin, methotrexate, camptothecine, paclitaxel or gemcitabine.
6. anticancer prodrug preparation; it is characterized in that: it is to add local injection drug-delivery preparation or the intravenous formulations that pharmaceutically acceptable auxiliary material is prepared from by each described anticancer prodrug of claim 1～5, and the dosage form of described preparation is infusion solutions, freeze-dried powder, liposome, nanoparticle or microsphere.
7. anticancer prodrug preparation according to claim 6 is characterized in that: also contain lecithin, the weight proportion of anticancer prodrug and lecithin is 1: 1～30 parts.
8. anticancer prodrug preparation according to claim 7 is characterized in that: the weight proportion of anticancer prodrug and lecithin is 1: 5～8 parts.
9. prepare the method for the described anticancer prodrug of claim 1, it is characterized in that: may further comprise the steps:
A, low methoxyl pectin are dissolved in water, in the mixture of cancer therapy drug is water-soluble and organic solvent;
B, with above-mentioned two kinds of solution mix homogeneously, adding EEDQ is a dehydrant, 20～80 ℃ of temperature controls stir 3～24h, add ethanol, centrifugalize;
C, with resolution of precipitate in water, transferring in the bag filter dialysis to detecting less than small-molecule drug, in bag filter, add ethanol in the liquid, centrifugal, the precipitation vacuum drying, the water solublity conjugate;
Above-mentioned organic solvent can be pyridine, triethylamine, oxolane or N, dinethylformamide, preferred N, dinethylformamide;
Or
A, low methoxyl pectin is dissolved in water, then that cancer therapy drug is water-soluble and be added in the pectin solution, add dehydrant behind the stirring and evenly mixing, 20～80 ℃ of temperature controls stir 3～24h, and described dehydrant is DCC, CDC or EDCHCl;
B, adding ethanol, centrifugalize washes with water and is precipitated to no small-molecule drug, and the precipitation vacuum drying gets the slightly solubility conjugate.
10. according to the described method for preparing anticancer prodrug of claim 9, it is characterized in that: the reaction temperature of preparation water solublity conjugate is 50 ℃, and the response time is 8h; The reaction temperature of preparation slightly solubility conjugate is 50 ℃, and the response time is 8h.
11. each described anticancer prodrug of claim 1～5 is used for the treatment of the intravenous drug of hepatocarcinoma or pulmonary carcinoma in preparation, is used for the medicine of solid carcinoma and the administration of draining lymph node local injection thereof or is used for the purposes that solid carcinoma causes the medicine of the abdominal cavity of ascites or hydrothorax or thoracic cavity drug administration by injection.
12. the purposes of pectin in lympha targeted, liver targeting of preparation or lung targeted drug.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100588429C (en) * | 2008-12-23 | 2010-02-10 | 重庆莱美药业股份有限公司 | PH sensitive type anticancer pro-drug, preparation method and uses thereof |
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| WO2011072510A1 (en) * | 2009-12-18 | 2011-06-23 | 重庆莱美药业股份有限公司 | Passive solid tumor targeting anticancer prodrug and preparation method thereof |
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