CN1218983C - Folic acid-polydiol compound and its medicinal composition - Google Patents

Abstract

The present invention relates to folic acid-polydiol, particularly to a folic acid-polydiol compound, the combined composition of the folic acid-polydiol compound, iconography nuclide or an antitumor medicament and the application thereof in tumour target diagnosis and treatment, which belongs to the field of a chemical medicament. The experimental result of the present invention indicates that a folic acid-PEG compound with small molecular weight can be led into a lymphatic system when a tissue gap can not permeate a capillary blood vessel, and has the characteristics of a competition folic acid receptor and the targeting performance of tumour tissue. The folic acid-polydiol compound of the present invention, and the combined composition of the folic acid-polydiol compound, the iconography nuclide or the antitumor medicament can be used for preparing a tumour diagnosis medicament and an antitumor medicament. The present invention provides the target diagnosis and the treatment of a tumour for clinics, and particularly provides a material basis for the target diagnosis and the treatment of a constitutional or metastatic tumor of the lymphatic system.

Description

Folic acid-polyethylene glycols compound and medicinal composition thereof

Technical field

The invention belongs to the chemicals field, relate to folic acid-polyethylene glycols compound and with this compound and iconography nucleic or medicine bonded mixture, particularly folic acid-polyoxyethylene glycol compounds and with iconography nucleic or antitumor drug bonded mixture.The invention still further relates to respectively the application of mixture in preparation antitumor drug and diagnosing tumor medicine with folic acid-polyoxyethylene glycol compounds, folic acid-polyoxyethylene glycol compounds and iconography nucleic or antitumor drug, and be the pharmaceutical composition of activeconstituents with the above-mentioned substance, and their application in cancer target diagnosis and treatment, particularly in the targeting diagnosis of lymphsystem primary or metastatic tumo(u)r and the application in the treatment.

Background technology

Folic acid (Folic acid) is the small molecules VITAMIN that contains the petrin ring in a kind of molecular structure, by the carbon metabolism of eukaryotic cell list and nucleosides synthetic essential, its structure one end contains two carboxyls, and it is the passive less likely that is diffused into cytolemma under physiological temp and pH condition.Body initiatively absorbs the folic acid approach two kinds: first kind of mechanism is the folic acid-binding protein (k by low-affinity _D1-5 μ M), directly folic acid is imported endochylema.Second kind of mechanism is the specificity folic acid-binding protein (k by high-affinity _D～100pM), promptly (Folate Receptor, FR) the mediated cell internalization is taken in endochylema with folic acid to folacin receptor.

Along with most tumors cell surface FR expression number or activity are significantly higher than the Normocellular fact revealed gradually [Campbell et al:Cancer Res 1991,51:5329; Coney et al:Cancer Res 1991,51:6125; Weitman et al:CancerRes 1992,52:3396], had with the research of folate-mediated targeted tumour cell and to have developed rapidly.

The mixture that folic acid directly is formed by connecting by micro-molecule indirectly as FR part and radionuclide, show in the experimentation on animals result aspect the tumor imaging diagnosis, tumor locus target effect is [Low et al:WO96/36367 Nov.21,1996 and USA5688488Nov.18,1997 significantly; Wang S et al:Bioconjugate Chem 1996,7 (1): 56; Mathias CJ et al:Nucl Med Biol 1999,26 (1): 23; Wang S et al:Bioconjugate Chem 1997,8:673; Mathias CJ et al:J Nucl Med1998,39 (9): 1579; Mathias CJ et al:Nucl Med Biol1998,25 (6): 585; Wang S et al:Bioconjugate Chem 1997,8 (5): 673; Iigan S et al:Cancer Biother Radiopharmaceuticals1998,13 (6): 427; Guo W et al:J Nucl Med 1999,40 (9): 1563].

Folic acid combines the folic acid-polyoxyethylene glycol-liposome cell cultures and the experimentation on animals result that generate indirectly and shows with surface of liposome, its target tumor cell effect is significantly better than PEG-liposome or conventional liposome [Lee et al:J Biol Chem 1994,269 (5): 3198; Wanget al:Proc Natl Acad Sci USA 1995,92:3318; Lee et al:BiochimBiophys Acta 1995,1233:134; Vogel et al:J Am Chem Soc1996,118 (7): 1581; Thompson et al:WO97/31624 Sep.4,1997.RuiY et al:J A Chem Soc 1998,120 (44): 11213; Gabizon A et al:Bioconjugate Chem 1999,10 (2): 289; Shinoda T et al:J PharmSci 1998,87 (12): 1521; Lu Weiyue etc.: Shanghai Medical Univ's journal 2000,27 (1): 4].

Folic acid and polymer composite also can intactly pass polymer by FR and release to intracellular non-lyase somaplasm matter [Yingjuan Lu et al:Adv Drug Deliv Rev 2002,54:675]., horseradish peroxidase white with folic acid compound bovine serum albumin, ox immunity bladders, rnase, beans serinase inhibitor and anti-DNA oligonucleotide all can obviously import KB cell, HeLa cell and XC cell and bring into play its corresponding effect [Leamon et al:Proc NatlAcad Sci USA 1991,88:5572; Low et al:WO90/12096Oct.18,1990].Tumour cell and T cell or natural killer cell, monocyte, scavenger cell closely can be brought together together in conjunction with the anti-TXi Baoshouti monoclonal antibody of folic acid or anti-Fc acceptor monoclonal antibody, reach effective dissolving tumour cell purpose [David et al:WO96/34630 Nov.7,1996].[Semen Momordicae saponin (Momordin)-a kind of has only and arrives cytoplasm through rrna and just show Cytotoxic proteotoxin and have the toxin of arrestin complex functionality; Pseudomonas exotoxin fragment (LysPE38 and CysPE35)] with folic acid compound after, it suppresses growth of tumour cell ability and greatly strengthens [Leamon et al:JBiol Chem 1992,267:24966; 1993,268:24847].After the dextran that is used for blood volume expander of a kind of no anti-tumor activity and folic acid are compound, has obvious suppression tumor tissue growth effect (Chinese patent ZL00115752.3).

It is generally acknowledged that the particle diameter of pharmaceutical carrier or equivalent diameter after the tissue space injection, have good lymphsystem target between 9-100nm the time.Up to now, the material as lympha targeted pharmaceutical carrier research mainly comprises liposome, nanoparticle, micella, saccharan molecule etc., [Oussoren C et al:Adv Drug deliv Rev 2001,50:143; IllumL et al:Pharm Res 2001,18 (5): 640; Supersaxo A et al:PharmRes 1991,8 (10): 1286; Vera DR:J Nucl Med 2001,42 (6): 951; Lu Weiyue etc.: Shanghai Medical Univ's journal 1996,23 (suppl): 3], report is arranged, as antitumor drug carrier target lymphsystem, [Yan Zhongqin etc.: Chinese pharmaceutical chemistry magazine 1999,9 (4): 240].

The polyglycol compound is that a class has-O (CH ₂) _nO-or-(CH ₂) _{N '}O (CH ₂) _{N '}The linear polyethylene glycols macromolecular compound of-repeating structure, wherein n 〉=2 or n ' 〉=1.In the polyglycol compound modal be polyoxyethylene glycol (polyethylene glycol, PEG), its molecular composition is:

HOCH ₂CH ₂(OCH ₂CH ₂O) _nCH ₂CH ₂OH

Or HOCH ₂(CH ₂OCH ₂) _nCH ₂OH

PEG both had been dissolvable in water water, be dissolvable in water most organic solvents again, have good biocompatibility and blood compatibility, low [the Richter A W et al:Int ArchAllergy Appl Immunol 1984 of immunogenicity, 74:36], can be got rid of and do not produce any toxic side effects [Yamaoka T et al:J Pharm Sci 1994,83:601] rapidly by body.

In pharmaceutical industry, PEG useful as drug auxiliary material is to improve the various performances of medicine, as [Zalipsky S et al:Adv Drugdelivery Rev 1995,16:157] such as dispersiveness, film-forming properties, oilness, slow-releasings.In the synthetic and modification of novel biomaterial, PEG is as the part of material, can give new characteristic of material and function, as [Green wald R B et al:J Org Chem1995,60:333 such as wetting ability, flexibility, anticoagulant property, anti-macrophage phagocytics; Iphigenia L K et al:Int J Pharm 2000,157:103].

In the practical application of PEG, the end group decisive role.The PEG chain end is not only limited to hydroxyl, the functional group that other reactive behavior is stronger, as p-toluenesulfonic esters [SuzakiT et al:J Polym Sci Polym Lett Ed 1979,17:241], amino [GlassJD et al:J biopolym 1979,18:383], carboxyl [Fradet A et al:PolymBull 1981,4:205], aldehyde radical groups such as [Ciuffarin E et al:J Org Chem1981,46:3064] also can introduce two ends of PEG chain.The introducing of these functional groups has enlarged the range of application of PEG.

PEG modifies and comprises, the physical bond and the chemically modified of PEG and biomacromolecule class medicine, and it can change some character of biomacromolecule, as: the solubleness and the stability that 1. increase medicine; 2. weaken or eliminate immunogenicity, antigenicity and toxicity; 3. improve the interior pharmacokinetics behavior of body of medicine, the prolong drug transformation period in vivo; 4. improve medicine therapeutic index, enlarge clinical use etc.Most of at present research mainly concentrates on PEG and proteinic chemically composited aspect, as: [Wang YS et al:Adv Drug Deliv Rev 2002,54:547 such as Interferon, rabbit, superoxide-dismutase, granulocyte colony-stimulating factor, bovine hemoglobin, r-hirudin, tumour necrosis factor, Regular Insulin, interleukin-2, uriKoxidase adenosine desaminase, asparaginase; Bullick J et al:Anal Biochem 1997,254 (2): 254; Malic F et al:Exp Hematol 1992,20 (8): 1028; Conover CD et al:Artif CellsBlood Substit Immobil Biotechnol 1999,27 (2): 93; Fareed J etal:Thrombosis ﹠amp; Haemostasis 1991,65 (6): 1286; Tsutsumi Y etal:Br J Cancer 1995,71 (5): 963; Hinds K et al:Bionconjug Chem2000,11 (2): 195; Schiavon O et al:farmaco 2000,55 (4): 264; Goodson RJ et al:Biochem Biophysic Res Commun 1993,197 (1): 287; Inada Y et al:TIBTECH 1995,13:86; Jean-Francois J et al:Piotechnol Appl Biochem 1996,23 (Pt 3): 221].

In sum, tumor cell membrane surface FR is one and can will comprises that materials such as radionuclide, liposome and macromolecular compound import the effective way in the tumour cells by folic acid; PEG is the macromolecular compound that a class has broad prospect of application, and it mainly is used as the first-selected chemical modifier of biomacromolecules such as protein, polypeptide, nucleic acid, polysaccharide at biomedical sector.Therefore, the active target of folic acid combines as the premium properties of pharmaceutical carrier with PEG, may become the new diagnosing tumor with targeting or the pharmaceutical carrier of treatment.

Summary of the invention

The objective of the invention is to, particularly folic acid-polyoxyethylene glycol (PEG) compounds of a kind of folic acid-polyethylene glycols compound is provided, it has and can follow on the cytolemma folacin receptor approach and optionally accumulate in the tumour cell of overexpression folacin receptor, inducing apoptosis of tumour cell, suppress tumor tissue growth, do not damage the characteristic of healthy tissues simultaneously again.

Another object of the present invention provides with above-claimed cpd as targeting vector and iconography nucleic or antitumor drug bonded medicinal composition.

The further purpose of the present invention provide respectively with the mixture of folic acid-PEG compounds, folic acid-PEG compounds and iconography nucleic or with the mixture of antitumor drug be the pharmaceutical composition of activeconstituents.

The present invention also provide above-mentioned substance in preparation diagnosing tumor medicine and antitumor drug purposes and provide cancer target diagnosis and treatment, the particularly basic substance of lymphsystem primary or metastatic tumo(u)r targeting diagnosis and treatment for clinical.

Experimental result of the present invention shows, molecular-weight average only is 3,000 folic acid-PEG compounds can not see through capillary vessel and import lymphsystem in tissue space, the feature and the tumor tissues target that have the competition folacin receptor simultaneously, it is after entering lymphsystem, combine with the tumour cell of overexpression folacin receptor, its entrained drug main moving-target is to tumor tissues.

The folic acid that The present invention be more particularly directed to-polyoxyethylene glycol compounds comprises, after the terminal hydroxyl of polyoxyethylene glycol is replaced by amino again by the folic acid derivatize, the other end hydroxyl is by methoxyl group, amino, aldehyde radical, carboxyl substituted, or by the carboxymethyl derivatize, or by the compound that is generated by folic acid, many carboxyls molecule derivatize again after amino the replacement; The folic acid that relates to-polyoxyethylene glycol compounds and iconography nucleic or antitumor drug bonded mixture comprise that an end is by the folic acid-polyethylene glycol compound of many carboxyls derivatize and radionuclide ^99mTc and ¹¹¹In or Gd mr nucleic chelating and the mixture that generates, or one end be substituted with amino, aldehyde radical, carboxyl and sulfydryl, or derive many carboxyls or sulfonic folic acid-polyoxyethylene glycol compounds and methotrexate are arranged, mixture that antitumor drug covalent attachment such as anthracycline antibiotics, ametycin, Ismipur such as Zorubicin form.

The present invention adopts following technical scheme to realize the present invention and reaches the foregoing invention purpose.

1. prepare folic acid-polyethylene glycols compound

Folic acid or folic acid derivatives or other can be followed folacin receptor approach on the cytolemma enter the material of cell and different molecular weight, different types of polyethylene glycols compound, particularly PEG compounds, condensation generates the folic acid-polyethylene glycols compound of following formula,

X-Y

Wherein: on behalf of folic acid and derivative or other thereof, X can follow the material that folacin receptor approach on the cytolemma enters cell; Y represents polyglycol and derivative thereof.

Above-mentioned folic acid and derivative thereof or other can follow the material that folacin receptor approach on the cytolemma enters cell, it does not have obvious toxic-side effects to animal or human's somatocyte, and they comprise: folic acid, folinic acid, dihydrofolic acid, tetrahydrofolic acid (THFA), tetrahydrobiopterin, the many L-glutamic acid of pteroyl, 2-deaminize-and hydroxyl folic acid, 1-denitrification folic acid, 3-denitrification folic acid, 8-denitrification folic acid etc.Wherein, certain position " denitrification " is meant that this nitrogen-atoms is replaced by carbon atom in the folic acid.

Above-mentioned polyglycol and derivative thereof are the polyglycol ends, particularly the terminal hydroxyl of PEG is replaced by amino, carboxyl, methoxyl group, or by carboxymethyl and sulfonic group derivatize, or by the compound that is generated by folic acid, diethylenetriaminepeacidcetic acidcetic (DTPA), ethylene dinitrilotetra-acetic acid (EDTA) and carboxymethyl dextran derivatize again after amino the replacement.

(1) polyoxyethylene glycol (NH that replaced by amino of molecule two terminal hydroxy group ₂-PEG-NH ₂) with folic acid (F) under carbodiimide dewatering agent and basic catalyst effect, carboxyl condensation among an end amino or two ends and F generation F-PEG-NH ₂Or F-PEG-F.

(2) F-PEG-NH ₂Generate F-PEG-DTPA with the condensation reaction of DTPA acid anhydrides.

(3) F-PEG-NH ₂With carboxymethyl dextran (CMDx) under carbodiimide dewatering agent and basic catalyst effect, the other end is amino to generate F-PEG-CMDx with the CMDx condensation.

(4) mono methoxy polyethylene glycol (NH that replaced by amino of molecule one terminal hydroxy group ₂-PEG-OCH ₃) with F under carbodiimide dewatering agent and basic catalyst effect, amino generate F-PEG-OCH with the F condensation ₃

2. the mixture for preparing folic acid-polyethylene glycols compound and iconography nucleic or antitumor drug

Folic acid-polyethylene glycols compound, particularly folic acid-polyoxyethylene glycol compounds and different images are learned nucleic or antitumor drug, reaction under certain condition generates the mixture of corresponding folic acid-polyethyleneglycol derivative and iconography nucleic or antitumor drug, and its general formula is as follows

X-Y-Z

Wherein: on behalf of folic acid and derivative or other thereof, X can follow the material that folacin receptor approach on the cytolemma enters cell; Y represents polyglycol and derivative thereof; Z is iconography nucleic or antitumor drug.

Above-mentioned folic acid and derivative thereof or other can follow the material that folacin receptor approach on the cytolemma enters cell, it does not have obvious toxic-side effects to animal or human's somatocyte, and they comprise: folic acid, folinic acid, dihydrofolic acid, tetrahydrofolic acid (THFA), tetrahydrochysene pterin, the many L-glutamic acid of pteroyl, 2-deaminize-and hydroxyl folic acid, 1-denitrification folic acid, 3-denitrification folic acid, 8-denitrification folic acid etc.Wherein, certain position " denitrification " is meant that this nitrogen-atoms is replaced by carbon atom in the folic acid.

Above-mentioned polyglycol and derivative thereof are the polyglycol ends, particularly the terminal hydroxyl of PEG is replaced by amino, carboxyl, aldehyde radical and sulfydryl, or by carboxymethyl and sulfonic group derivatize, or by the compound that is generated by diethylenetriaminepeacidcetic acidcetic (DTPA), ethylene dinitrilotetra-acetic acid (EDTA) and carboxymethyl dextran derivatize again after amino the replacement.

Above-mentioned iconography nucleic is meant can be by folic acid-polyethylene glycols compound, particularly can be by the radionuclide and the mr nucleic of folic acid-polyoxyethylene glycol compounds chelating.

Above-mentioned antitumor drug be meant have can with folic acid-polyethylene glycols compound, particularly have can with folic acid-polyoxyethylene glycol compounds one end amino, sulfydryl, aldehyde radical, carboxyl and the covalently bound antitumor drug that has carboxyl, amino or sulfydryl of sulfonic group.

(1) F-PEG-DTPA or F-PEG-CMDx are prepared into aqueous injection or lyophilized injectable powder with the reductive agent tin protochloride earlier under acidic conditions, again with fresh Na ^99mTcO ₄The physiological saline leacheate mixes, and room temperature condition reaction down generates F-PEG-DTPA- ^99mTc or F-PEG-CMDx- ^99mThe Tc mixture.

(2) F-PEG-DTPA or F-PEG-CMDx and GdCl ₃Under acid and room temperature condition, react, generate F-PEG-DTPA-Gd or F-PEG-CMDx-Gd mixture.

(3) F-PEG-DTPA or F-PEG-CMDx and ametycin (MMC) are under water-soluble carbodiimide, pH5.0-5.5 condition, and room temperature reaction generates F-PEG-DTPA-MMC or F-PEG-CMDx-MMC mixture.

(4) F-PEG-DTPA or F-PEG-CMDx and Zorubicin (Dox) are under carbodiimide and alkaline condition, and 40 ℃ of reactions generate F-PEG-DTPA-Dox or F-PEG-CMDx-Dox mixture.

(5) F-PEG-NH ₂With Rheumatrex (MTX) under carbodiimide and alkaline condition, 40 ℃ of reactions generate the F-PEG-MTX mixtures.

3. folic acid-PEG compounds is tested the tumor cell in vitro growth effect

Each phase DNA number change and apoptosis percentage and folic acid-PEG compounds are to the influence of growth of tumour cell in the tumour cell cycle after employing flow cytometer detection finite concentration folic acid-PEG compounds effect.

4. folic acid-PEG compounds follows the detection that the folacin receptor approach imports tumour cell

Adopt the radioligand-binding assay method, measure folic acid-PEG compounds competition and suppress ¹²⁵I-folic acid and tumor cell membrane surface folacin receptor combination degree are understood the specificity affinity of folic acid-PEG compounds to tumor cell membrane surface folacin receptor.

Adopt the radio-label method, detect the not variation of phase tumour cell endocytosis folic acid-PEG compounds amount simultaneously, confirm that folic acid-PEG compounds follows the folacin receptor approach and imports tumour cell.

5. folic acid-PEG compounds is in normal and the intravital behavior of animal pattern

Adopt the radio-label method, folic acid-PEG compounds is in the healthy tissues of normal mouse, rat and tumor bearing nude mice and the distribution in the tumor tissues in detecting simultaneously mutually, understands it by animal healthy tissues picked-up situation, pharmacokinetic parameter with to the target of tumor tissues.

6. folic acid-PEG compounds is to the detection of animal lymph system target

Adopt the radio-label method, detect not simultaneously phase folic acid-PEG compounds and to the observation of rabbit whole body dynamic imaging, understand it in conjunction with the SPECT instrument lymphoid target and drainage situation thereof in the tissue of normal rat and the distribution in the lymphoglandula.

Folic acid of the present invention-PEG compounds and can adopt supplementary product compatibilities such as the vehicle that allows on the pharmaceutics, binding property, suspending agent, disintegrating agent, thinner, lubricant, enteric-coating material, bioadhesive material, water-insoluble framework material with the mixture of iconography nucleic or antitumor drug is prepared into the relative medicine composite preparation according to the conventional of pharmaceutical field or with specific production method.Can adopt folic acid-PEG compounds and relevant reductive agent or pH regulator agent and corresponding radionuclide or mr nucleic compatibility, be prepared into the relative medicine composite preparation by the conventional of pharmaceutical field or with specific production method.

Described drug combination preparation comprises: in the tumour, by the tumour, vein or the folic acid-PEG compounds of tissue space injection and lyophilized injectable powder, aqueous injection, liposome, nanoparticle, the micella of iconography nucleic or antitumour drug complex thereof; The microspheres agent of folic acid for oral use-PEG compounds and antitumour drug complex thereof, enteric coated capsule etc.

Polyethylene glycols compound of the present invention, particularly polyoxyethylene glycol compounds, wherein the PEG average molecular weight range is 800-20,000.Polyoxyethylene glycol compounds and folic acid covalent attachment generate folic acid-PEG compounds, and selecting the PEG molecular-weight average is that folic acid-PEG compounds of 2000 carries out following test as model drug.

1. folic acid-PEG compounds is to the influence and the apoptosis induced effect of tumour cell cycle

Flow cytometer detects the influence degree of folic acid-PEG compounds to human cervical carcinoma cell HeLa229 and KB cell KB growth, and the result shows: F-PEG-OCH ₃, F-PEG-DTPA, F-PEG-NH ₂, F-PEG-F induces HeLa229 apoptosis degree to be followed successively by 43.14%, 36.48%, 14.37% and 10.95%, induces KB apoptosis degree to be followed successively by 52.09%, 32.76%, 15.14% and 3.81%.Two strain tumour cells show similar apoptosis induction effect, i.e. F-PEG-OCH ₃The highest, F-PEG-DTPA takes second place.

2. folic acid-PEG-DTPA is to tumor cell surface folacin receptor competitive capacity and internalization degree

F-PEG-DTPA with ¹²⁵The result of I-F competition HeLa229 cell surface folacin receptor shows that F-LY2-DTPA has the characteristic that combines folacin receptor with the similar competition of the folic acid that dissociates.The internalization experimental result of radio-label shows that KB cell internalizing F-PEG-DTPA amount is with prolonging duration of contact and increasing gradually, and KB cell intake reaches capacity substantially behind the 6h.

3. folic acid-PEG compounds is at normal and the intravital behavioral characteristic of animal pattern

The intact animal in-vivo tissue distributes and the radio-label result of pharmacokinetics shows: during 2h, F-PEG-DTPA mainly is distributed in kidney in the rat body, and always the plasma clearance transformation period is 7.5min, wherein t _{1/2 (α)}=1.85min, t _{(1/2) β}=17.84min, plasma clearance speed is fast; During 6h, F-PEG-CMDx mainly concentrates on kidney and lung in the rat body, and total plasma clearance transformation period is 37.44, wherein t _{(1/2) α}=10.69, t _{(1/2) β}=107.8, plasma clearance speed is relatively slow.

The tumor tissues target test-results of lotus knurl (SKOV3) nude mice shows that F-PEG-DTPA and F-PEG-CMDx all show certain tumor tissues target, and equal specific ionization folic acid target is good during 4h.Tumor bearing nude mice tail vein injection F-PEG-DTPA- ^99mBehind the Tc 1,4 and during 24h, tumour is respectively 2.97,3.68 and 3.59 times with the muscle ratio; Injection F-PEG-CMDx- ^99mBehind the Tc 4 and during 24h, tumour is respectively 2.09 and 5.64 times with the muscle ratio; Injection ^99mBehind the Tc-F 4 and during 24h, tumour is respectively 1.45 and 3.62 times with the muscle ratio.

4. folic acid-PEG-DTPA is to animal lymph system target degree

The lymphsystem target test-results of radio-label shows: rat and rabbit Interdigital Space injection F-PEG-DTPA- ^99mBehind the Tc, rat lymphoglandula and blood, liver, kidney and muscle ratio are respectively 25.86,31.78,1.13 and 33.10 during 2h, rabbit lymphsystem target is remarkable and rapid after the administration, after entering circulation of blood, intake is few in the liver, kidney is main distribution organ, and passes through the bladder rapid drainage through it, and the lymphoglandula image is desalinated gradually.Prompting F-PEG-DTPA has good lymphsystem target, and normal lymphoglandula does not have and obviously accumulates phenomenon.

Confirm that through above-mentioned experimental result the present invention has following characteristics: folic acid-PEG compounds can follow on the cytolemma folacin receptor approach and optionally accumulate in the tumour cell of overexpression folacin receptor, has the effect of inducing apoptosis of tumour cell; Folic acid-PEG-DTPA plasma clearance speed is fast, but after the tissue space administration, the lymphsystem target is good, in conjunction with its tumor-targeting, can be used as targeting vector and be combined into medicinal composition with iconography nucleic and antitumor drug respectively, be applied to lymphsystem primary or metastatic tumo(u)r targeting diagnosis and treatment.

Description of drawings

Fig. 1 shows: folic acid-PEG-DTPA competes inhibition ¹²⁵I-folic acid and HeLa229 cell bonded competition curve

Fig. 2 shows: the folic acid competition suppresses ¹²⁵I-folic acid and HeLa229 cell bonded competition curve

Fig. 3 shows: the KB cell not simultaneously mutually in to the internalization result of folic acid-PEG-DTPA

Fig. 4 shows: folic acid-PEG-DTPA distributes at the rat in-vivo tissue behind the tail vein injection

Fig. 5 shows: hind leg Interdigital Space injection back folic acid-PEG-DTPA distributes at the rat in-vivo tissue

Fig. 6 (A, B and C) shows: rabbit two hind leg Interdigital Spaces injection folic acid-PEG-DTPA- ^99mBehind the Tc, the normotopia scanned photograph of phase simultaneously not under SPECT

Embodiment

Below further describe the present invention by non-limiting example.

Embodiment 1 preparation folic acid-PEG compounds

1. prepare folic acid-PEG-NH ₂ And folic acid-PEG-folic acid

Get 30.9mg (0.15mmol) dicyclohexylcarbodiimide (DCC) and be dissolved in 5ml methyl-sulphoxide (DMSO), add 300mg NH ₂-PEG-NH ₂, 66.2mg F, 10 μ l pyridines, 40 ℃ of stirring reaction 12h, add 20ml distilled water termination reaction, have jelly to generate 20 ℃ of centrifugal 5min of 4000r/min, get supernatant liquor, after the dialysis of 5mM aqueous sodium carbonate, the water dialysis is carried out separation and purification with DEAE Sepharose FF ion exchange column and Sephadex G-15 post in conjunction with AKTAexplorer fast purifying system successively, freeze-drying gets xanchromatic F-PEG-NH respectively ₂With F-PEG-F solid 154mg and 55mg.

The preparation folic acid-PEG -DTPA

Get 200mg (0.0809mmol) F-PEG-NH ₂Slowly be dissolved among the 5ml DMSO, add 76.7mg (0.215mmol) DTPA acid anhydrides, reaction solution is the suspendible shape, room temperature reaction 30min, the reaction solution transparency improves, centrifugal, get supernatant liquor, splash in the 50ml acetone, get yellow mercury oxide, carry out separation and purification, suction filtration, vacuum-drying with DEAE Sepharose FF ion exchange column and Sephadex G-15 post in conjunction with AKTA explorer fast purifying system successively.Get xanchromatic F-PEG-DTPA solid 108.2mg.

The preparation folic acid-PEG -CMDx

Get 72mg carboxymethyl dextran (CMDx), add the 8ml methane amide, regulate pH to 5, add 300mg DCC with the vitriol oil, behind the room temperature activation 1h, centrifugal (8500rpm, 10 ℃, 10min), get supernatant liquor, add 48mg F-PEG-NH ₂, regulating pH to 9 with triethylamine, behind the room temperature reaction 1h, Sephadex G-100 post carries out separation and purification in conjunction with AKTA explorer fast purifying system.Get xanchromatic F-PEG-CMDx solid 54.1mg.

4. prepare folic acid-PEG-OCH ₃

Get 2000mg (1mmol) CH ₃O-PEG-NH ₂, 441.4mg (1mmol) F, 206mg (1mmol) DCC be dissolved in 25ml DMSO, adds 10 μ l than pyridine, behind the stirring at room reaction 24h, add 20ml distilled water termination reaction, centrifugal (8500rpm, 5min, 14 ℃), get supernatant liquor, behind 50mM sodium bicarbonate 1000ml dialysis 4h, use distill water dialysis instead, carry out separation and purification with DEAE Sepharose FF ion exchange column in conjunction with AKTA explorer fast purifying system, freeze-drying gets xanchromatic F-PEG-OCH ₃Solid 1280mg.

5. folic acid-PEG-NH ₂ Middle folic acid and amino mol ratio detect

The F-PEG-NH that method for preparing is obtained ₂, with ninhydrin reaction and folic acid determination of quantitative analysis NH ₂-PEG-NH ₂Last folic acid combination degree.

Amino typical curve is formulated

14 test tubes are divided into two groups, add 0.5mmol/l NH respectively ₂-PEG-NH ₂(reference substance) solution 0,0.2,0.4,0.6,0.8,1.0ml water is supplied 1ml, adds 1ml triketohydrindene hydrate colour developing liquid and mixes, and heats 15min in 100 ℃ of water-baths, 5-10min is placed in the cooling back, add 3ml 60% alcohol dilution, fully shake up, measure the A value in the 570nm place, with 1ml H ₂O is as blank.Get two groups of mean values of measuring, with NH ₂-PEG-NH ₂Concentration is X-coordinate, and absorption value A is an ordinate zou, gets typical curve to be

A＝0.3326C-0.0085(R ²＝0.9998)

In the formula: A is the optical density value of measuring at the 570nm place, and C is amino volumetric molar concentration (mmol/l)

The folic acid typical curve is formulated

Precision takes by weighing 10mg F, places the 1000ml volumetric flask, adds the dissolving of 1% sodium hydroxide solution, and be settled to 1000ml, get 1,1.5 respectively, 2,2.5,3,3.5, the F solution of 4ml100 μ g/ml places the volumetric flask of 10ml, adds 1%NaOH to the scale constant volume, measures its optical density in the 365nm place, the line linearity of going forward side by side returns, and resulting typical curve equation is

A＝0.0190C+0.0051(R ²＝0.9999)

In the formula: A is the ultraviolet absorptivity value at F 365nm place in 0.1M NaOH; C is folic acid concentration (μ g/ml)

Folic acid and primary amino mol ratio are measured

Precision takes by weighing the F-PEG-NH behind the purifying ₂12.21mg, be dissolved in the 10ml distilled water, getting 1ml concentration is the 0.5mmol/l sample solution, adds 1ml triketohydrindene hydrate colour developing liquid and mixes, subsequent operations is the same.Measure its optical density A value at the 570nm place.As a result, 0.5mmol/lF-PEG-NH ₂Sample liquid is 0.1569 in this wavelength place optical density value, can be got 0.5mmol/l F-PEG-NH by amino typical curve ₂Middle amino volumetric molar concentration is 0.4975mmol/l.

At wavelength is the F-PEG-NH that the 366nm place measures 0.5mmol/l ₂The optical density of sodium hydroxide solution, A value are 4.4630, can be got 0.5mmol/l F-PEG-NH by the folic acid typical curve ₂The volumetric molar concentration of middle folic acid is 0.4980mmol/l.

Be F-PEG-NH ₂Middle amino is approximately 1 with folic acid mol ratio (0.4975/0.4980).

6. the detection of folic acid-PEG compounds

Folic acid-PEG compounds with method for preparing obtains carries out quantitative analysis with the HPLC method to its purity, with ultraviolet, infrared, nucleus magnetic resonance its structure is differentiated, its result is as follows respectively:

The HPLC method detects the purity result

With the F-PEG-NH behind the purifying ₂, F-PEG-F, F-PEG-DTPA, F-PEG-CMDx, F-PEG-OCH ₃HPLC detect collection of illustrative plates, adopt area normalization method to record purity and be respectively 100%, 99.18%, 98.97%, 97.67% and 100%.

The UV scanning collection of illustrative plates

Show F-PEG-NH by the uv-vis spectra scanning spectra ₂, F-PEG-F, F-PEG-DTPA, F-PEG-CMDx, F-PEG-OCH ₃With 365nm wavelength place the absorption peak identical with folic acid arranged all 283.

Infared spectrum

With F-PEG-NH ₂, F-PEG-F, F-PEG-DTPA, F-PEG-CMDx, F-PEG-OCH ₃Infared spectrum and folic acid relatively all have the contained group characteristic peak of folic acid, as 1693cm in above-mentioned folic acid-PEG compounds collection of illustrative plates ^-1The amide group characteristic peak at place, 1600cm ^-1Near carbonyl characteristic peak; The characteristic peak that also has simultaneously the contained group of PEG is as 1100cm ^-1Near aliphatic ether characteristic peak.

Nuclear magnetic resonance map

With F-PEG-NH ₂, F-PEG-F, F-PEG-DTPA, F-PEG-CMDx, F-PEG-OCH ₃HNMR collection of illustrative plates and folic acid, NH ₂-PEG-NH ₂, CH ₃O-PEG-NH ₂, DTPA, CMDx relatively, all have the displacement peak similar: δ 8.67 to folic acid (s, 1H, C7-H), 7.66 (d, 2H, Ar), 6.63 (d, 2H, Ar), 4.34 (dd, C19-H), 2.0-2.4 (m, 2H C21-H2), also has the displacement peak similar to PEG: δ 3.1-4.0 (m ,-OCH ₂CH ₂); F-PEG-OCH ₃Have and CH ₃O-PEG-NH ₂Similar displacement peak: δ 3.36 (s ,-OCH ₃); F-PEG-DTPA have the displacement peak similar: δ 3.29 to DTPA (t, C5, C7-2H), 3.42 (t, C4, C8-2H); F-PEG-CMDx have to CMDx in the similar displacement multimodal in δ 3.4-4.5 place.

Embodiment 2 preparation folic acid-PEG compounds with ^99mThe mixture of Tc

1. prepare folic acid-PEG-DTPA- ^99m Tc

After getting PBS (0.01mol/l) dissolving that 10mg F-PEG-DTPA adds 1ml pH 7.4, after adding 10 μ l concentration and being the 0.15N hydrochloric acid soln of 10 μ g/ μ l tin protochlorides, add the fresh Na of 1-2ml again ^99mTcO ₄Solution (1-5mCi), mixing is placed 10min under the room temperature, promptly gets F-PEG-DTPA- ^99mThe Tc injection liquid.With Sephadex G-15 post, 0.01MPBS with pH 7.4 is the moving phase wash-out, it is carried out purifying, collect yellow region band elutriant, (stationary phase is Xinhua's filter paper to detect its radiochemical purity with Paper Chromatography, developping agent is 85% aqueous ethanolic solution, launches the back and measure each section radiocounting on gamma counter, calculates F-PEG-DTPA- ^99mThe Tc radiocounting accounts for the percentage ratio of gross activity counting).F-PEG-DTPA- ^99mTc radiochemical purity＞95%.

2. prepare folic acid-PEG-CMDx- ^99m Tc

Get the same method reaction of 10mg F-PEG-CMDx and purifying, get F-PEG-CMDx- ^99mThe Tc injection liquid, its radiochemical purity＞95%.

The mixture of embodiment 3 preparation folic acid-PEG compounds and Gd

Get F-PEG-DTPA 100mg and be dissolved in 6ml water for injection, add 1.5ml 50mmol/lGdCl ₃0.1N HCl solution, stirring at room reaction 24h, reaction solution carries out separation and purification with Sephadex G-15 post in conjunction with AKTA explorer fast purifying system, collection has the flow point of folic acid characteristic absorption wavelength section, lyophilize, get xanchromatic F-PEG-DTPA-Gd solid 184mg, add injection water 4.2ml again, by freeze-drying after every bottle of 1ml packing.

The mixture of embodiment 4 preparation folic acid-PEG compounds and antitumor drug

1. prepare folic acid-PEG-DTPA-ametycin

Get F-PEG-DTPA 83mg and be dissolved in 10ml water for injection, after adding ametycin (MMC) 15mg dissolving, under agitation add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 83mg, keep stirring at room reaction 24h between the pH 5.0-5.5.Under 4 ℃ of conditions, reaction solution is dialysed to water for injection behind the 24h, carry out separation and purification with DEAE Sepharose FF ion exchange column and Sephadex G-15 post in conjunction with AKTA explorer fast purifying system successively, collect the flow point that has MMC and folic acid characteristic absorption wavelength section simultaneously, lyophilize, the purplish F-PEG-DTPA-MMC solid of De Huangzhong 61mg.

2. prepare folic acid-PEG-DTPA-Zorubicin

Get F-PEG-DTPA 83mg and be dissolved in 5ml DMSO, under agitation add DCC 18mg, under 40 ℃ of conditions, activate 30min, add Zorubicin (Dox) 20mg and triethylamine 10 μ l, stirring reaction 24h, add 20ml distilled water termination reaction, 4000r/min is centrifugal under the room temperature, get supernatant liquor, carry out separation and purification with DEAE Sepharose FF ion exchange column and Sephadex G-15 post in conjunction with AKTA explorer fast purifying system successively, collect the flow point that has Dox and folic acid characteristic absorption wavelength section simultaneously, lyophilize gets orange red F-PEG-DTPA-Dox solid 67mg.

3. prepare folic acid-PEG-DTPA-Rheumatrex

Get Rheumatrex (MTX) 22mg and be dissolved in 5ml DMSO, under agitation add DCC20mg, under 40 ℃ of conditions, activate 30min, F-PEG-NH ₂60mg and pyridine 10 μ l, stirring reaction 24h, add 20ml distilled water termination reaction, 4000r/min is centrifugal under the room temperature, get supernatant liquor, carry out separation and purification with DEAE Sepharose FF ion exchange column and Sephadex G-15 post in conjunction with AKTA explorer fast purifying system successively, collect the flow point that has MTX and folic acid characteristic absorption wavelength section simultaneously, lyophilize gets yellow F-PEG-MTX solid 46mg.

Embodiment 5 folic acid-PEG compounds is to the influence of vitro human cancer cells cycle dna and apoptosis

With the no folic acid RPMI1640 nutrient solution that contains 10% calf serum (BCS), (37 ℃, 5%CO in CO2gas incubator ₂, saturated humidity) continuously HeLa229, KB cell are cultured to q.s.Made it adherent in one day with continuing after the packing of some amount cell to cultivate, add the F-PEG compounds respectively, making ultimate density is 10 μ g/ml, twice of cultured continuously 72h.With 0.02%EDTA and 0.25% tryptic digestion and disperse HeLa229, KB cell, make single cell suspension, centrifugal, abandoning supernatant is suspended among the PBS again, adjusts cell concn to 10 ⁶Cell/ml.Isopyknic cell suspension and propidium iodide staining fluid are mixed, place 20-30min for 4 ℃, after 300 order nylon membranes filter, detect in 488nm excitation wave strong point, investigate F-PEG-NH with flow cytometer ₂, F-PEG-F, F-PEG-DTPA, F-PEG-OCH ₃Influence to HeLa229, each phase DNA quantitative changeization of KB cell cycle and apoptosis thereof.The result shows that the F-PEG compounds has tangible inducing apoptosis of tumour cell effect, and its apoptosis-induced degree is followed successively by: F-LY2-OMe＞F-LY2-DTPA＞F-LY2-NH ₂＞LY2-F ₂(HeLa229 cell); F-LY2-Ome＞F-LY2-DTPA＞LY2-F ₂＞F-LY2-NH ₂(KB cell).

Table 1 is the apoptosis influence of folic acid (F)-PEG class folic acid compound to Hela229 and KB cell.

Table 1

Cell type	Experimental group	Dna content			Apoptosis rate (%)
						G0-G1	G2-M	S
		KB	Contrast PEG-F 2 F-PEG-OCH 3 F-PEG-NH 2 F-PEG-DTPA	91.82 64.77 70.20 64.72 82.03					6.41 6.12 28.13 1.61 4.60	1.77 29.10 1.67 33.67 13.37	1.02 15.14 52.09 3.81 32.76
HeLa229	Contrast PEG-F 2 F-PEG-OCH 3 F-PEG-NH 2 F-PEG-DTPA				96.81 59.42 88.99 65.42 94.63	0.00 0.41 0.06 0.35 4.99	5.96 40.16 10.95 34.23 0.38	5.96 10.95 43.14 14.37 36.48

The evaluation of embodiment 6 folic acid-PEG-DTPA and folacin receptor binding characteristic

1. folic acid-PEG-DTPA- ^99m Tc to the competition of HeLa 229 folacin receptors in conjunction with test

The HeLa229 cell no folic acid RPMI1640 nutrient solution that contains 10% calf serum (BCS), (37 ℃, 5%CO in CO2gas incubator ₂, saturated humidity) cultured continuously to q.s, the preparation into about containing 2.2 * 10 ⁵The cell suspension of cells/ml.50 μ l will be added in this cell suspension ¹²⁵I-F and different concns F-PEG-DTPA or different concns folic acid, in the 0.01mol/l of pH 7.4 phosphate buffered saline buffer, behind 4 ℃ of reaction 20h, the centrifugal 10min of 4000r/min, abandoning supernatant is measured throw out (cell binding substances) radiocounting (CPM) with gamma counter.With the radiocounting of cell bonded folic acid, F-PEG-DTPA concentration are mapped respectively, get the combination of HeLa229 cell ¹²⁵The inhibition curve of I-F.The result shows that F-PEG-DTPA and F competition suppress ¹²⁵I-F and tumor cell surface folacin receptor bonded feature similarity, but F-PEG-DTPA with ¹²⁵I-F competition folacin receptor ability is weaker than F, and both suppress the tumour cell combination ¹²⁵Concentration during I-F 50% is respectively 11.2 and 1.58fmol

2.KB cell internalizing picked-up folic acid-PEG-DTPA test

The KB cell in vegetative period of taking the logarithm by ordinary method digestion, is made single cell suspension, and it is inoculated in four 12 orifice plates, and (every porocyte number is every hole 2ml: 6.25 * 10 ⁴Cells).After four days nutrient solution in the plate is replaced with the no folic acid RPMI-1640 that does not contain serum, every hole 2ml (washing, 1ml * 2 time).Change nutrient solution next day, every hole 1ml does not contain the no folic acid RPMI-1640 of serum, and subsequent disposal is operated according to the following steps.

Add F-PEG-DTPA solution mixing in the non-specific combination pipe (NBS) earlier, in non-specific combination pipe, specific combination pipe and internalization pipe, respectively add 100 μ lF-PEG-DTPA-then respectively ^99mTc draws not simultaneously supernatant liquor in the phase hole.With after the 0.01M PBS of pH 7.4 washing (1ml * 2 time), 0.25% tryptic digestion 5min firmly blows and beats attached cell respectively for non-specific combination pipe and specific combination pipe, and sucking-off is added to measuring tube; The internalization pipe adds the acetum of pH 3, make the dissociation of ligand that is combined in the cell surface folacin receptor after, again by specific combination pipe treatment process operation.Measure radiocounting in non-specific combination pipe, specific combination pipe and the internalization pipe after the processing respectively with gamma counter.Results suggest, KB cell endocytic picked-up F-PEG-DTPA amount is with prolonging duration of contact and increasing gradually, and KB cell intake reaches capacity substantially behind the 6h.

Behavior test in the animal body of embodiment 7 folic acid-PEG compounds

1. folic acid-PEG-DTPA is in normal rat body inner blood kinetics and distribution thereof

Rat vena femoralis injection 0.1ml concentration is about the F-PEG-DTPA-of 10mg/ml ^99mTc, respectively at the injection after not simultaneously phase tail vein get blood 50 μ l, on gamma counter, measure radiocounting (CPM).Use MicroMath PKAnalyst Statistics Software and press the two-compartment model processing, pharmacokinetic parameters is: t _{1/2 (E)}=7.5min, t _{1/2 (α)}=1.85min, t _{1/2 (β)}=17.84min.The result shows that F-PEG-DTPA blood removing speed is very fast.

Rat tail vein injection 0.1ml concentration is about the F-DTPA-PEG-of 10mg/ml ^99mTc, sacrificed by decapitation behind 2h is got blood, the heart, liver,spleen,kidney, lung, large intestine, small intestine, stomach, muscle, and radiocounting (CPM) is measured in weighing respectively on gamma counter.The radiocounting of calculating every gram tissue accounts for the percentage ratio (%ID/g) of total injection volume radiocounting.Results suggest, F-DTPA-PEG- ^99mTc mainly is distributed in rat kidney when 2h, and the amount in other internal organs such as liver is much smaller than kidney.

2. folic acid-PEG-CMDx is in normal rat body inner blood kinetics and distribution thereof

6 of rats are divided into two groups, and every femoral vein is injected the F-PEG-CMDx-that 0.1ml concentration is about 5mg/ml respectively ^99mTc or ^99mTc-CMDx, in the injection after not simultaneously phase tail vein respectively get blood 50 μ l, on gamma counter, measure radiocounting (CPM).Use MicroMath PKAnalystStatistics Software and press the two-compartment model processing, the pharmacokinetic parameters of F-PEG-CMDx and CMDx is respectively: t _{1/2 (E)}=37.44min, t _{1/2 (α)}=10.69min, t _{1/2 (β)}=107.8min and t _{1/2 (E)}=53.57min, t _{1/2 (α)}=22.71min, t _{1/2 (β)}=202.3min.Results suggest, F-PEGization have shortened CMDx residence time in blood, and F-PEG has then been prolonged the blood circulation time of F-PEG compounds by the CMDx derivatize.

Sacrificed by decapitation behind the injection 6h is cored, lung, liver,spleen,kidney, and radiocounting (CPM) is measured in weighing respectively on gamma counter.The radiocounting of calculating every gram tissue accounts for the percentage ratio (%ID/g) of total injection volume radiocounting.The result shows that behind the administration 6h, F-PEG-CMDx and the CMDx all distribution in liver are less, mainly concentrate on kidney and lung, but the former intake in tissue is starkly lower than the latter.Table 2 is F-PEG-CMDx-behind the rat vena femoralis injection 6h ^99mWith ^99mThe tissue distribution of Tc-CMDx.

Table 2.

Tissue	％ID/g
			F-PEG-CMDx- 99mTc	99mTc-CMDx
	Conscience lung spleen kidney	0.31±0.04 0.46±0.04 2.55±0.18 0.58±0.28 9.88±6.24			0.30±0.06 0.75±0.05 4.22±0.78 2.16±0.19 12.49±5.72

3. folic acid-PEG-DTPA distributes at the tumor animal in-vivo tissue

10 of lotus knurl (SKOV3) nude mices after three days, are divided into three groups with low folic acid forage feed, remove F-PEG-DTPA- ^99mWhen Tc group is established 1 mutually outside, F-PEG-CMDx- ^99mTc, ^99mPhase when Tc-F is 2.The tail vein is injected the F-PEG-DTPA-of 0.1ml concentration 10mg/ml respectively ^99mTc, F-PEG-CMDx- ^99mTc, ^99mBehind the Tc-F, F-PEG-DTPA- ^99mTc organize in two groups of 4h, back respectively at 4 and 24h put to death, get blood, the heart, liver,spleen,kidney, lung, large intestine, small intestine, stomach, muscle, tumour, radiocounting (CPM) is measured in weighing respectively on gamma counter.Calculate the percentage ratio (%ID/g) that every gram tissue radiation counting accounts for total injection volume radiocounting.

Other gets 14 of (SKOV3) tumor bearing nude mices, after three days, organizes (4,5,5) with low folic acid forage feed when being divided into 3 mutually, and the tail vein is injected folic acid-PEG-DTPA-that 0.1ml concentration is 7mg/ml respectively ^99mTc ^99mBehind the Tc-DTPA-LY2-F, in 1,4,24h sacrificed by decapitation respectively, subsequent operations is the same.Calculate the percentage ratio (%ID/g) that every gram tissue radiation counting accounts for total injection volume radiocounting.

The result shows, behind intravenous injection 1,4 and the 24h, F-PEG-DTPA in tumor tissues abundance than high respectively 2.97,3.68 and 3.59 times of muscle; Behind intravenous injection 4 and the 24h, F-PEG-CMDx in tumor tissues abundance than high respectively 2.09 and 5.64 times of muscle; Behind intravenous injection 4 and the 24h, folic acid in tumor tissues abundance than muscle respectively high 1.45 and 3.62.Prompting F-PEG-DTPA, F-PEG-CMDx have certain tumor tissues target, and all specific ionization folic acid target is good in the 4h after administration.

Embodiment 8 F-PEG-DTPA lymphsystem target tests

1.F-PEG-DTPA to the lymph gland targeted test of rat

Each injects rat two hind leg Interdigital Spaces a bit, and every 60 μ l concentration is about the F-PEG-DTPA-of 10mg/ml ^99mTc in injection back 2h sacrificed by decapitation, gets blood, the heart, liver,spleen,kidney, lung, large intestine, small intestine, stomach, muscle, state's lymphoglandula, and radiocounting (CPM) is measured in weighing respectively on gamma counter.Calculate the percentage ratio (%ID/g) that every gram tissue radiation counting accounts for total injection volume radiocounting.The result shows, the amount of F-PEG-DTPA in lymphoglandula be far above blood, the heart, spleen, lung, liver, large intestine, small intestine, stomach, and is suitable with amount in the kidney, and prompting F-PEG-DTPA has lympha targeted preferably property.

2.F-PEG-DTPA rabbit lymphsystem target is tested

Each injects rabbit two hind leg Interdigital Spaces a bit, and every 0.1ml concentration is the F-PEG-DTPA-of 10mg/ml ^99mTc carries out total body opacification respectively at injection back 5,15 and 120min and observes and take the photograph the sheet record under the SPECT instrument.Dynamic image is the result show, F-PEG-DTPA at tissue space target lymphsystem rapidly and significantly, whole lower limb lymphsystem imports and finishes in 5min, except that kidney and bladder, other internal organs or tissue there is no obviously and accumulate in the 2h, but the lymphsystem picked-up F-PEG-DTPA amount that comprises lymphoglandula prolongs in time and significantly successively decreases.In addition, accumulation increases in time in the rabbit bladder, and accumulation reduces in time in the kidney, and the prompting kidney may only be the F-PEG-DTPA excretion pathway.

Claims (23)

1. folic acid-polyethylene glycols compound is characterized in that it has following general formula,

X-Y

Described X can follow the material that folacin receptor approach on the cytolemma enters cell, and it comprises that folic acid, folinic acid, dihydrofolic acid, tetrahydrofolic acid (THFA), tetrahydrochysene pterin, the many L-glutamic acid of the acyl of talking endlessly, 2-deaminize-hydroxyl folic acid, 1-denitrification folic acid, 3-denitrification folic acid and 8-denitrification folic acid; Described Y is for having-O (CH ₂) _nO-or-(CH ₂) _{N '}O (CH ₂) _{N '}The polyglycol of-repeating structure and derivative thereof, wherein: n in the repeating structure 〉=2 or n ' 〉=1, the polyglycol molecular-weight average is 800-20,000, polyglycol and derivative thereof are meant the polyglycol terminal hydroxyl or are replaced by amino, carboxyl, methoxyl group, or by carboxymethyl and sulfonic group derivatize, or by after amino the replacement again by folic acid, diethylenetriaminepeacidcetic acidcetic, ethylene dinitrilotetra-acetic acid and carboxymethyl dextran derivatize.

2. by the described folic acid of claim 1-polyethylene glycols compound, it is characterized in that described polyglycol and derivative thereof are polyoxyethylene glycol and derivative thereof, wherein, the polyoxyethylene glycol molecular-weight average is 800-20,000; Polyethyleneglycol derivative is meant the polyoxyethylene glycol terminal hydroxyl or by methoxyl group, amino, carboxyl substituted, or by carboxymethyl and sulfonic group derivatize, or by after amino the replacement again by folic acid, diethylenetriaminepeacidcetic acidcetic, ethylene dinitrilotetra-acetic acid or carboxymethyl dextran derivatize.

3. by the described folic acid of claim 2-polyethylene glycols compound, it is characterized in that described polyoxyethylene glycol molecular-weight average is 2,000-20,000.

4. by the described folic acid of claim 3-polyethylene glycols compound, it is characterized in that described polyoxyethylene glycol molecular-weight average is 2,000.

5. press the application of folic acid-polyethylene glycols compound in the preparation antitumor drug of claim 1 or 2 or 3 or 4.

6. one kind is used for antitumor medicine composition, it is characterized in that being mixed and made into by the folic acid-polyethylene glycols compound and the pharmaceutical excipient of claim 2, and its preparation is small-volume injection, lyophilized injectable powder, microspheres agent, nanoparticle and enteric coated capsule.

7. by the described antitumor medicine composition that is used for of claim 6, it is characterized in that described excipient substance is vehicle, tackiness agent, suspending agent, disintegrating agent, thinner, lubricant, enteric-coating material, bioadhesive material and the water-insoluble framework material that allows on the pharmaceutics.

8. the mixture of folic acid-polyethylene glycols compound and iconography nucleic or antitumor drug is characterized in that having following general formula

X-Y-Z

Described X can follow the material that folacin receptor approach on the cytolemma enters cell, and it comprises that folic acid, folinic acid, dihydrofolic acid, tetrahydrofolic acid (THFA), tetrahydrochysene pterin, the many L-glutamic acid of the acyl of talking endlessly, 2-deaminize-hydroxyl folic acid, 1-denitrification folic acid, 3-denitrification folic acid and 8-denitrification folic acid;

Described Y is for having-O (CH ₂) _nO-or-(CH ₂) _{N '}O (CH ₂) _{N '}The polyglycol of-repeating structure and derivative thereof, wherein: n in the repeating structure 〉=2 or n ' 〉=1, the polyglycol molecular-weight average is 800-20,000, the polyglycol derivative is meant the polyglycol terminal hydroxyl or is replaced by amino, aldehyde radical, carboxyl or sulfydryl, or by carboxymethyl and sulfonic group derivatize, or by after amino the replacement again by diethylenetriaminepeacidcetic acidcetic, ethylene dinitrilotetra-acetic acid and carboxymethyl dextran derivatize; Described Z is iconography nucleic or antitumor drug.

9. press the mixture of the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug, it is characterized in that the polyethylene glycols compound is a polyethyleneglycol derivative in described folic acid-polyethylene glycols compound, wherein, the polyoxyethylene glycol molecular-weight average is 800-20,000, polyethyleneglycol derivative is meant that the polyoxyethylene glycol terminal hydroxyl is replaced by amino, aldehyde radical, carboxyl or sulfydryl, or by carboxymethyl and sulfonic group derivatize, or by after amino the replacement again by diethylenetriaminepeacidcetic acidcetic, ethylene dinitrilotetra-acetic acid and carboxymethyl dextran derivatize.

10. by the mixture of the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug, it is characterized in that described iconography nucleic is can be by the radionuclide and the mr nucleic of folic acid-polyethylene glycols compound chelating.

11. by the mixture of the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug, it is characterized in that described antitumor drug be have can with folic acid-polyethylene glycols compound one end amino, sulfydryl, aldehyde radical, carboxyl or the covalently bound antitumor drug that has carboxyl, amino, sulfydryl of sulfonic group.

12. the mixture by the described folic acid of claim 9-polyethylene glycols compound and iconography nucleic or antitumor drug is characterized in that described polyoxyethylene glycol molecular-weight average is 2,000-20,000.

13. the mixture by the described folic acid of claim 12-polyethylene glycols compound and iconography nucleic or antitumor drug is characterized in that described polyoxyethylene glycol molecular-weight average is 2,000.

14. mixture by the described folic acid of claim 9-polyethylene glycols compound and iconography nucleic or antitumor drug, it is characterized in that the amino replacement of described polyoxyethylene glycol terminal hydroxyl or quilt, or by the carboxymethyl derivatize, or by after amino the replacement again by diethylenetriaminepeacidcetic acidcetic or carboxymethyl dextran derivatize.

15. the mixture by the described folic acid of claim 10-polyethylene glycols compound and iconography nucleic or antitumor drug is characterized in that described radionuclide is ^99mTc and ¹¹¹In, described mr nucleic is Gd.

16. the mixture by the described folic acid of claim 11-polyethylene glycols compound and iconography nucleic or antitumor drug is characterized in that described antitumor drug is Rheumatrex or anthracycline antibiotics or ametycin.

17. the mixture by the described folic acid of claim 16-polyethylene glycols compound and iconography nucleic or antitumor drug is characterized in that described anthracycline antibiotics is a Zorubicin.

18. the purposes of mixture in preparation diagnosing tumor medicine by the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug.

19. by the purposes of mixture in preparation diagnosing tumor medicine of the described folic acid of claim 18-polyethylene glycols compound and iconography nucleic or antitumor drug, wherein said diagnosing tumor medicine is lymphsystem primary or metastatic tumo(u)r diagnostic medicine.

20. the purposes of mixture in preparation neoplasm targeted therapy medicine by the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug.

21. the purposes of mixture in preparation lymphsystem primary or metastatic tumo(u)r medicine by the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug.

22. pharmaceutical composition that is used for neoplasm targeted therapy, it is characterized in that being mixed and made into by the mixture and the pharmaceutical excipient of the described folic acid of claim 8-polyethylene glycols compound and iconography nucleic or antitumor drug, its preparation is small-volume injection, lyophilized injectable powder, microspheres agent, nanoparticle and enteric coated capsule.

23., it is characterized in that described excipient substance is vehicle, tackiness agent, suspending agent, disintegrating agent, reductive agent, thinner, lubricant, enteric-coating material, bioadhesive material and the water-insoluble framework material that allows on the pharmaceutics by the described pharmaceutical composition that is used for neoplasm targeted therapy of claim 22.

Images