CN100366286C - Saposin C-DOPS: A new anticancer drug - Google Patents

Abstract

Compositions and methods for treating subjects with disorders characterized by hyper-proliferating cells such as tumors and cancers are provided. The compositions comprise agents that are combinations of saposin C (or prosaposin-related polypeptides) and dioleoylphosphatidylserine (or inner leaflet components). This anti-tumor agent is administered in the methods of the invention according to a dosing regimen. Administering an agent of the invention results in a positive therapeutic response in a subject with a tumor.

Description

Saposin C-DOPS: a kind of PTS

Cross reference with related application

Present patent application has precedence over and proposed on April 28th, 2003, application number is 60/466,166 U.S. Provisional Patent Application, and has the latter's interests, and includes the latter in present patent application as a whole.

The government-funded data

Patent of the present invention obtains the support of government, grant number R01DK57690.U.S. government has some right to patent of the present invention.

Invention field

The present invention studies the synthetic and the method for the volume size of regulating the diffusibility cancer, especially regulates the size of tumor and cancer.In addition, the present invention also studies the synthetic and the method for treatment cancer.

Background of invention

Saposins is gang's low profile thermal stabilizing sugar albumen (～80 aminoacid), and is indispensable for hydrolysing activity in the body on the glycosyl sphingolipid catabolic pathway for multiple lysozyme.(referring to (1990) Crit.Rev.Biochem.Mol.Biol.25:385-414 that the people showed such as Grabowski; (1992) Biochim.Biophys.Acta.1126:1-16 that the people showed such as Furst; (1992) J.Lipid Res.33:1255-1267 that the people showed such as Kishimoto).Four members (A, B, C and D) of saposin family be before the same precursor protein saposin (prosaposin) through Proteolytic enzyme (referring to (1985) Am.J.Hum.Genet that the people showed such as Fujibayashi, 37:741-748; (1988) Science 241:1098-1101 that the people showed such as O ' Brien; (1989) Genomics 5:486-492 that the people showed such as Rorman; (1989) Biochem. (Tokyo) 105:152-154 that the people showed such as Nakano; (1989) the J Mol.Neurosci that the people showed such as Reiner, 1:225-233; Include in by reference at this.Saposins A, B, the complete amino acid sequence of C and D have appeared in the newspapers and have led, the chromosome set structure of precursor protein and cDNA sequence also appeared in the newspapers and led (referring to (1985) Am.J.Hum.Genet that the people showed such as Fujibayashi, 37:741-748; (1988) Science 241:1098-1101 that the people showed such as O ' Brien; (1989) Genomics 5:486-492 that the people showed such as Rorman).

Saposin is defined as sphingolipid catalytic protein or coenzyme.On structure, saposins A, B, C and D have the similarity of about 50-60%, comprise six kinds of strict cysteine residue things (referring to (1992) Biochim.Biophys.Acta1126:1-16 that the people showed such as Furst) of preserving, these residues form disulphide bridges (referring to (1995) J.Biol.Chem.270:9953-9960 that the people showed such as Vaccaro) in three identical territories of placement location.All saposin all contain a glycosyl position, it preserves position half part in N-terminal sequence, but glycosyl is not essential (referring to (1995) J.Biol.Chem.270:30576-30580 that the people showed such as (1998) Biochemistry 37:11544-11554 that people such as Qi showed and Vaccaro, by reference it being included in as a whole at this) for the activity of saposin.

One " saposin pleat " all contained in all saposin and class saposin albumen and territory under dissolved state.This pleat is many alpha-helixs harness shape, has the disulfide structure and the several amphipathic polypeptide of three preservations.Although saposin and class saposin albumen all have the saposin pleat under dissolved state, the two strengthen lysozyme sphingolipid (SL) and glycosyl sphingolipid (GSL) by specific hydrolytic enzyme degraded on, have the interior biological function of different bodies.These effects of saposin make it to occupy middle cardiac status (referring to (1992) J.Lipid Res.33:1255-1267 that the people showed such as Kishimoto on control lysozyme sphingolipid and glycosyl sphingolipid metabolism; (1985) Am.J.Hum.Genet that the people showed such as Fujibayashi, 37:741-748; (1988) Science 241:1098-1101 that the people showed such as O ' Brien includes them in by reference at this).In addition, saposin C also participates in the fusion and the disturbance (referring to (1994) FEBS Letters 349:181-186 that the people showed such as Vaccaro, including [by reference it being included in as a whole at this] by reference at this) of acid phosphorus adipose capsule.

Acid beta-glucosidase (Gcase, EC 3.1.2.45) will be in vivo carries out best hydrolysis to glucosylceramide and need use saposin C with external.In addition, saposin C also makes fission lure to the Phosphatidylserine (referring to (1994) FEBS Letters 349:181-186 that the people showed such as Vaccaro, including in by reference at this) that contains capsule (under ultramicroscope as seen).And saposin C has the general property of adipose membrane in conjunction with activity or plasma membrane affinity.Saposin C is by imbedding the associating of exite and adipose membrane.H-1 and H-5 spiral are the indispensable parts of this process, show that the suitable interaction of saposin C and adipose membrane can influence its characteristic and activity.In addition, saposin C also brings out the variation of adipose membrane recurring structure.Interactional dynamic process once obtained real-time manifesting (referring to (2001) FEBS Lett.503:97-102 that the people showed such as (2001) J.Biol.Chem.276:27010-27017 that people such as Qi showed and You, including in by reference at this) by atomic force microscope between Saposin C and the plane phospholipid bilayer body.

Phospholipid is asymmetric to be a well-known feature of mammal plasma membrane.The exite of lipid bilayer body contains abundant phosphatidylcholine, and amino phospholipid is then preferentially selected endite (referring to (1998) Lupus Suppl.2:S126-S131 that the people showed such as Bevers).Phosphatidylserine (PS) and PHOSPHATIDYL ETHANOLAMINE (PE) almost reside at endite bar none, and lecithin (PC) and sphingomyelins are then very abundant in exite.Phospholipid is asymmetric may to be that the general property of all cells is (referring to (1999) Cell Calcium25 (4) that the people showed such as Woon: 313-320).Plasma membrane phospholipid is asymmetric to be maintained by various mechanism, and these mechanism comprise that amino phospholipid changes position and the random position of phospholipid (referring to Application No. 20020081698).

Generally speaking, tumor or cancerous cell are compared with common cell, and the speed of growth is faster.These abnormal cells are mainly by producing lactic acid or producing a large amount of protons by breathing (because accretion rate is fast) generation carbon dioxide during glycolysis.Therefore, acid acid high than the cell peripheral position of normal growth speed usually at position around these cells and the tissue.

Skin squamous cell carcinoma (SCC) is one of the most common skin carcinoma with very high cell transfer risk.(, including in by reference) at this referring to (2001) N.Engl.J.Med.344:975-983 that the people showed such as Alam.Skin carcinoma is divided into two classes: melanoma and non-melanoma (NMSC).Estimate that according to American Cancer Society the NMSC case load in U.S. every year is more than 1,000,000.SCC accounts for 20% of all skin tumour greatly, annual nearly 200,000 the new SSC cases of the U.S..SCC is modal malignant tumor from the cutaneous metastatic to the mucosa, and SCC also can directly betide (referring to (2002) Neuroendocrinology Letters23S2:48-51 that the people showed such as Boni) on the mucosa.Current treatment to SCC patient comprises electrodesiccation, scrapes division, excision method, cryotherapy, excision or Mohs operation.If electrodesiccation, scrape division, excision or cryotherapy and use properly, can eliminate little (diameter＜1 centimetre), clear-cut tumor, the neoplasm metastasis incidence rate is lower.Excision and Mohs operation are the highest to the cure rate with excessive risk constitutional or recidivity SSC patient.Yet these Therapeutic Method are costly, and hematoma, seroma, infection and wound cracking may take place.

Therefore, people wish to develop effectively and SCC Therapeutic Method low-cost and that have better appearance.In addition, the effectively and cheaply Therapeutic Method of the cancer (for example breast carcinoma, carcinoma of prostate and lymphatic cancer) of other type of exploitation treatment is also very important.

Abstract of invention

The invention provides the synthetic and the method for the interior exite component distributing of regulating plasma membrane.Medicine of the present invention comprises the endite composition polypeptide relevant with preceding saposin." endite composition " is meant molecule or its analog in any endite that is present in cytoplasma membrane naturally, and said here cell refers to zooblast especially, more outstanding finger mammalian cell.A concrete material of endite composition is Phosphatidylserine or its analog, and for example two oil base Phosphatidylserine (dioleoylphosphatidylserine) (DOPS).The aminoacid sequence of preceding saposin has detailed description in the SEQ of sequence list ID NO:1.At least have 80% homogeny and keep the plasma membrane affinity between aminoacid sequence shown in preceding saposin related polypeptide and the SEQID NO:1 or its fragment.A concrete material of preceding saposin related polypeptide is saposin C (the SEQ ID NO:2 in the sequence list) or saposin C related polypeptide.At least have 80% homogeny and keep the plasma membrane affinity between the aminoacid sequence shown in saposin C related polypeptide and the SEQ ID NO:2.The molar ratio scope of the polypeptide of medicine of the present invention and endite composition is between about 1: 1 to about 1: 50, but better ratio ranges is between about 1: 1 to about 1: 25, further ideal ratio ranges is between about 1: 1 to about 1: 10, and optimal ratio is about 1: 7 or 1: 3.A concrete medicine of this invention further comprises acceptable carrier on a kind of pharmacopedics.Medicine of the present invention can promote cell death, for example apoptosis.A concrete medicine of this invention preferentially brings out the apoptosis of super diffusibility cell, for example (but being not limited to) tumor and cancerous cell.Therefore, this invention concrete medicine is a cancer therapy drug.In one aspect of the invention, the invention medicine preferentially bring out cancerous cell apoptosis, for example (but being not limited to) sarcoma cell, neuroblast oncocyte and scale cancerous cell.

The invention provides the method for the endite component distributing in the cytoplasma membrane of regulating an experimental subject.These methods comprise to an experimental subject takes a kind of medicine that comprises endite composition and preceding saposin related polypeptide.These methods can change the distribution situation of the endite composition in pill taker's the exite of cytoplasma membrane.A concrete medicine of the present invention can make the endite constituent concentration in the exite be improved.The present invention can preferentially occur on pill taker's the super diffusibility cell a concrete medicament adjusting of endite component distributing.Preferential happening part is tumor cell or these super diffusibility cells of cancerous cell.Aspect of this invention, but the concentration inducing apoptosis of the endite composition of the exite of adjusting plasma membrane.

The method of the tumor size of regulating an experimental subject is provided.These methods comprise to an experimental subject takes a kind of medicine that comprises endite composition and preceding saposin related polypeptide.This medicine can further comprise acceptable carrier on a kind of pharmacopedics.The experimental subject that is fit to comprises the mammal that suffers from tumor, and is especially human.A concrete medicine of the present invention can promote the death of super diffusibility cell.Cell death can take place by apoptosis.Any tumor is potential target of the present invention.Target tumor comprises super diffusibility cell, for example tumor cell and cancerous cell.These target tumor include, but not limited to sarcoma, neuroblastoma and scale cancerous cell.A concrete medicine scalable tumor size of the present invention causes tumor to reduce.Can envision, super diffusibility cell immaturity is that death should cause tumor to reduce.

The method for cancer of an experimental subject of treatment is provided.These methods comprise to an experimental subject takes a kind of medicine that comprises endite composition and preceding saposin related polypeptide.This medicine can further comprise acceptable carrier on a kind of pharmacopedics.The experimental subject that is fit to comprises the mammal that suffers from cancer, and is especially human.A concrete medicine of the present invention can promote the death of super diffusibility cell.Cell death takes place by a process, and this process can be (but being not limited to) apoptosis.Any cancer is potential target of the present invention.The target cancer comprises super diffusibility cell, for example tumor and cancerous cell.These targeted cancerous cells include, but not limited to be born in the cell on sarcoma, neuroblastoma, breast carcinoma and the scale cancer.In one aspect of the invention, medication have through the intestines and stomach, without the intestines and stomach, through subcutaneous, through vein, through peritoneum or topical.Be the treatment cancer, can give the medicine of an experimental subject single dose or multiple dose.

Brief Description Of Drawings

Fig. 1 shows normal immortalization keratinocyte (NIK) (A and B section) and scale cancerous cell (SCC) (C and D section).Cell in A and the C section is accepted placebo treatment.The cell acceptance of B and D section comprises the treatment of the medicine of the present invention of 8 μ M saposin C and 26 μ M DOPS.The detail of this experiment describes in other place of the application.

Fig. 2 shows the Mus lymphoma cell of taking from L5178Y-R cell line.Cell in the A section is accepted placebo treatment.Cell in the B section is accepted the treatment of 60 μ M DOPS.Cell in the C section is accepted 20 μ M saposin C Drug therapys.Cell acceptance in the D section comprises the treatment of the medicine of the present invention of 10 μ M saposin C and 30 μ M DOPS.

Fig. 3 shows that assessing the nude mice of carrying human scale cancerous cell xenograft is accepting placebo (phosphate buffer normal saline, represent with black triangle and dotted line) or medicine of the present invention (saposin C/DOPS, the former dosage is per kilogram of body weight 10mg, and latter's dosage is per kilogram of body weight 2mg) before the subcutaneous injection and the result that obtains of the size of average tumor afterwards.Error bars is represented standard error.The tumor size is with mm³Meter, the time calculates with tumor growth natural law.On behalf of Mus, A the section accept the result of two treatments.Represent with arrow with the date of injection for the second time for the first time.B section demonstration Mus is accepted the result of seance.Date of injection is represented with arrow.The detail of this experiment is introduced in other place of the application.

Fig. 4 shows the fluorescence micrograph of the human scale cancerous cell tumor tissues of taking from xenograft.Cell in the A section accepts to have the treatment of the mixture of the Phosphatidylserine of fluorized marking and DOPS (NBD-DOSP/DOPS).Cell in the B section accepts to have the treatment of mixture of Phosphatidylserine, DOPS and the saposin C of fluorized marking.The detail of this experiment is introduced in other place of the application.

Fig. 5 shows the microphotograph of the human scale cancerous cell tumor tissues of taking from xenograft.Tissue is taken from the tumor of accepting DOPS (per kilogram of body weight dosage 2mg, A and C section) or saposin C (per kilogram of body weight dosage 10mg) and DOPS (per kilogram of body weight dosage 2mg, B and D section) treatment.This tissue T UNEL colouring carry out painted or hematoxylin and eosin (C and D section).Arrow in the B section is represented apoptotic cell.Dark the zone of colour specification necrosis in the D section.

Detailed description of the invention

The present invention is relevant with synthetic and the method for endite component distributing in the adjusting plasma membrane. The present invention also with regulate super diffusivity cell, especially relevant with the disorder that involves super diffusivity cell, more particularly relevant with tumour and cancer, for example scale cancer cell and lymthoma. Synthetic is a kind of medicine that contains front saposin related polypeptide and endite composition that comprises, polypeptide exactly is saposin C, the endite composition exactly is phosphatidylserine or its analogue, or two oil base phosphatidylserines (dioleoylphosphatidylserine) are (DOPS) more precisely. The combination table of these two compounds reveals active anticancer, therefore is called as cancer therapy drug. " active anticancer " refers to reduce cellular invasion speed, thereby reduce the speed of growth of the tumour that has had tumour and during treating, occurred, and/or destroy and to have had knurl (tumour) cell or newly to have formed the knurl sexual cell, thereby during treating, reduce the size of population of tumour. Accept the two combined therapy of saposin C (or front saposin related polypeptide) and DOPS (or endite composition), can cause physiological reaction, and then regulate the distribution situation of the endite composition in the plasma membrane.

The active anticancer of medicine of the present invention is not limited to specific binding mode, and can play a role by various binding modes, comprising apoptosis. Environmental factor helps medicine preferential interaction of the present invention on tumour cell. The lipid that these environmental factors include, but not limited to change on the adventitia of flat, the further anoxia condition of tumour cell low sour water on every side and tumour cell shows. Anoxic is rear living factor, can stimulate expression and the release of the blood vessel endothelium growth factor (VEGF) of tumour cell. VEGF is known vascular permeability factor, in the disorder of the tumor tissues relative with the normal vessels system with leak in the vascular system and play a role. A concrete medicine saposin C-DOPS of the present invention preferentially penetrates tumor tissues after intravenous injection, but not health tissues.

The present invention includes protein or polypeptide synthetic isolated or that purify in fact. " isolated " or " purification " polypeptide or its biologically-active moiety be not subjected in fact or in essence those usually with naturally exist protein companion in the environment to go or the constraint of interactional composition. Therefore, a kind of protein that isolates or purify is not subjected in fact the constraint of other cellular material or culture medium when pressing method of gene recombination production, perhaps when it is combined, be not subjected in fact the constraint of precursor or other chemical substance. A kind of protein of cellular material constraint that is not subjected in fact comprises that impurity protein content (by dry weight basis) is lower than about protein formulation of 30%, 20%, 10%, 5% or 1%. When protein of the present invention or its biologically-active moiety were produced by method of gene recombination, the content (by dry weight basis) of precursor and impurity protein chemistry thing was lower than about 30%, 20%, 10%, 5% or 1% in the desirable culture medium.

At this, " front saposin related polypeptide " refers to have any polypeptide of front saposin amino acid sequence listed among the SEQ ID NO:1, listed amino acid sequence or a fragment of its proteolysis process have at least 80% to be identical among this amino acid sequence and the SEQ ID NO:1, and polypeptide wherein keeps plasma membrane affinity. Fragment and the variation of front saposin polypeptide (SEQ ID NO:1) are also included within the scope of the present invention. Front saposin becomes four kinds of saposin, i.e. saposin A, B, C and D after proteolytic treatment. At this, " saposin C related polypeptide " refer to have saposin C amino acid sequence listed among the SEQ ID NO:2 or with SEQ ID NO:2 in listed amino acid sequence have any polypeptide of the amino acid sequence of 80% homogeny at least, polypeptide wherein keeps plasma membrane affinity. Fragment and the variation of saposin C related polypeptide (SEQ ID NO:2) are also included within the scope of the present invention. Above-mentioned " fragment " thus refer to that amino acid sequence also is the part of protein. The biologically active of saposin before front saposin protein fragments keeps, thereby have plasma membrane affinity. Saposin C protein fragments keeps the biologically active of saposin C, thereby has plasma membrane affinity. At this, " plasma membrane affinity " refer to by static or hydrophobic effect and with the ability of phosphatide surface interaction.

A bioactive fragment of saposin polypeptide will encode at least 15 before the present invention, 25,30,35,40,45,50,55,60,65,70,75,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500, maximum 524 aminoacid before 510 and 520 adjacent amino acids or the code book invention in the saposin polypeptide.A fragment of the biologically-active moiety of saposin C polypeptide of the present invention is present at least 15,25,30,35,40,45,50,55,60,65,70,75 or 80 adjacent amino acids among the saposin C of the present invention with coding.

At this, " variation " is meant similar in fact sequence.For nucleotide sequence, conservative variation comprises the sequence that those are encoded to a kind of preceding saposin amino acid sequence of polypeptide of the present invention owing to the degeneracy of genetic code.Can treat as to using well-known molecular biotechnology as these abiogenous allelic variations, for example use sour polymerase chain reaction (PCR) and hybridization technique.The variation nucleotide sequence also comprises nucleotide sequence by being synthesized into, those sequences that generate by the mutagenesis that use the position guiding for example, the preceding saposin but mutagenesis is still encoded.

At this, " variation " protein is meant by deleting (so-called cutting off) or increase the protein that one or more aminoacid obtain at the N of crude protein end and/or C end; Or by in the one or more parts deletion of crude protein or increase the protein that one or more aminoacid obtain; Or the protein that obtains by the one or more aminoacid on the one or more positions that replace crude protein or by the synthetic polypeptide that produces with above-mentioned aminoacid sequence.The variant protein matter biologically active that the present invention comprised, promptly they continue to have the biological activity of our desired crude protein, i.e. the plasma membrane affinity of indication here.Above-mentioned variation can be handled and be produced by for example genetic polymorphism or the mankind.The variation of the biological activity of saposin native protein will have about at least 80%, 85% before the present invention, but better is to have about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% and more, and it would be desirable about at least 98%, 99% or more sequence identical with the aminoacid sequence of native protein, the aminoacid sequence of native protein is according to the sequence alignment program and use default parameters to determine, the alignment program is introduced in other place of the application.The proteinic biological activity variation of the present invention can have few difference to 1-15 aminoacid residue with this protein, even has only and lack to 1-10, and for example 6-10 is few to 5, few difference to 4,3,2 even 1 aminoacid residues.

Protein of the present invention changes by variety of way, comprises aminoacid replacement, deletes, cuts off and inserts.This processing method is generally known for the people in the present technique field.The proteinic variant amino acid sequence of for example preceding saposin can make by dna mutation.The method that sudden change and nucleotide sequence change is well-known technology.Can be referring to Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-492; People such as Kunkel (1987) Methods inEnzymol.154:367-382; U.S. Patent number 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan PublishingCompany, New York) and the document of wherein quoting.The relevant guidance that does not influence the proteinic bioactive suitable aminoacid replacement of gained, can referring to by people such as Dayhoff at (1978) Atlas of Protein Sequence and Structure (Natl.Biomed.Res.Found., Washington, (2001) J.Biol.Chem.276:27010-27017 that the people showed such as model of setting up D.C.) and Qi include in by reference at this).Can preferentially use conservative replacement, for example use a kind of aminoacid to replace another kind of aminoacid with like attribute.

Therefore, polypeptide of the present invention and aminoacid sequence comprise sequence and sudden change form, variation and the modification form thereof that nature exists.This kind variation will continue to have the plasma membrane affinity activity of our needs.Obviously, the sudden change that takes place in the deoxyribonucleic (DNA) of coding variation must not place sequence reads outside the frame, and comparatively ideal situation is not create the complementary region that can produce second messenger ribonucleic acid (mRNA) structure.Referring to EP patent application publication numbering 75,444.

Estimate can not make this proteinic characteristic generation essence to change to deletion, insertion and the replacement of the protein sequence that the present invention comprised.Yet, when the accurate result of prior prediction replacement, deletion or insertion is had any problem, will wish the result to be assessed by conventional screening test to the present technique veteran.That is to say that can use methods known in the art assessment plasma membrane affinity, these methods include, but not limited to fluorescence spectrophotometry, FRET (fluorescence resonance energy transfer) or circular two color measurement methods.For example, can include in by reference at this referring to (2001) J.Biol.Chem.276:27010-27017 that the people showed such as Qi }.

Variant protein matter comprises that also for example DNA confuses method by sudden change and reorganization program, and the protein that obtains.Use this program, can handle one or more different saposin C sequences, to create a new saposin C who has the attribute of being wanted.Like this, can from one group have correlated series, comprise sequence area with essence sequence homogeny and can be in vivo or the external polynucleotide that carry out homologous recombination generate the recombination of polynucleotide storehouse.For example, use this method, the sequence motif in the territory of wanting of coding can be before the present invention exchanges between saposin gene and other known, has the new gene code of protein of required improvement attribute (for example changing the plasma membrane affinity) with acquisition.This DNA confuses strategy for known to the present technique field.For example, referring to Stemmer (1994) Proc.Natl.Acad Sci.USA

91:10747-10751; Stemmer (1994) Nature 370:389-391; (1997) Nature Biotech.15:436-438 that the people showed such as Crameri; (1997) J.Mol.Biol.272:336-347 that the people showed such as Moore; (1997) Proc.Natl.Acad Sci.USA 94:4504-4509 that the people showed such as Zhang; (1998) Nature391:288-291 that the people showed such as Crameri; And U.S. Patent number 5,605,793 and 5,837,458.

Use following term to describe sequence relation between two or more aminoacid sequences or the polypeptide: (a) " reference sequences ", (b) " comparison window ", (c) " sequence homogeny ", (d) " sequence homogeny percentage ratio " and (e) " essence is identical ".

(a) at this, " reference sequences " is defined as comparing as sequence the sequence of foundation.Reference sequences can be the subclass or the integral body of certain specified sequence; Overall length polypeptide or aminoacid sequence or complete peptide sequence fragment for example.

(b) at this, " comparison window " is with reference to the adjacent and specified fragment of an aminoacid sequence, wherein the aminoacid sequence in comparison window is compared with reference sequences (not comprising increases or deletion), and can comprise increases or deletion (promptly blank), makes two sequences realize best alignment.Generally speaking, the length of comparison window is at least the length of 20 adjacent amino acids, and selectable length is the length of 30,40,50,100 adjacent amino acids or longer.The present technique field has experience person to recognize, with reference sequences similarity is highly arranged owing to comprise blank in aminoacid sequence for avoiding, and can introduce a blank loss usually and deduct this blank loss in number of matches.

The method that aligned sequence compares is known in the present technique field.Therefore, can use mathematical algorithm to determine two sequence homogeny percentage ratios between the sequence.The non-limitative example of this mathematical algorithm preferentially is the algorithm that Myers and Miller propose in (1988) CABIOS4:11-17; The local clustalw algorithm that people such as Smith propose in (1981) Adv.Appl.Math.2:482; The homology alignment algorithm that Needleman and Wunsch propose in (1970) J.Mol.Biol.48:443-453; The search similarity method that Pearson and Lipman propose in (1988) Proc.Natl.Acad.Sci.85:2444-2448; Karlin and Altschul propose in (1990) Proc.Natl.Acad Sci.USA 87:2264, and the algorithm of revising in (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877.

The computer-implemented version of these mathematical algorithms can be used for carrying out sequence relatively, so that determine the sequence homogeny.For the present invention, should preferentially use GCG program GAP (10.00 editions or upgraded edition) and default parameters thereof or any equivalent program to carry out nucleotide or protein sequence comparison, to determine its sequence homogeny percentage ratio of comparing with the disclosed sequence of the application.At this, " equivalent program " is meant any sequence comparison program, and it can generate an alignment for any two sequences to be compared, this alignment is compared with corresponding alignment that priority routine generates, and has identical nucleotide or aminoacid residue coupling and identical sequence homogeny percentage ratio.

Sequence comparison program includes, but are not limited to: be included in the CLUSTAL in the PC/Gene program (the Intelligenetics company of California, USA Mountain View is on sale); (Genetics Computer Group (GCG) company of the U.S. is on sale to be included in ALIGN program (V2.0 version) among the V8 version Wisconsin Genetics Software Package and GAP, BESTFIT, BLAST, FASTA and TFASTA, address: 575 Science Drive, Madison, Wis., USA).The alignment of using these programs to carry out can be used default parameters.The detailed description of CLUSTAL program can be referring to (1988) Gene73:237-244 (1988) that the people showed such as Higgins; (1989) CABIOS 5:151-153 that the people showed such as Higgins; (1988) Nucleic Acids Res.16:10881-90 that the people showed such as Corpet; (1992) CABIOS 8:155-65 that the people showed such as Huang; And (1994) Meth.Mol.Biol.24:307-331 that the people showed such as Pearson.The ALIGN program is based on aforementioned Myers and Miller (1988) algorithm.When the comparing amino acid sequence, can use the residual table of PAM 120 weight, the loss of 12 space length and 4 blank losses to cooperate with the ALIGN program.The blast program that people such as Altschul deliver in (1990) J.Mol.Biol.215:403 is based on aforementioned Karlin and Altschul (1990) algorithm.The BLAST nucleotide search can use the BLASTN program, adopts score=100, wordlength=12 to finish, and can obtain to invent the homologous nucleotide sequence of proteinic nucleotide sequence with code book.The BLAST protein search can use the BLASTX program, adopts score=50, wordlength=3 to finish, and can obtain the aminoacid sequence with protein of the present invention or homologous peptide.In order to obtain the more required barren alignment of sequence, can use Gapped BLAST (being included among the BLAST 2.0), the detailed description of this program can be referring to (1997) Nucleic AcidsRes.25:3389. that the people showed such as Altschul in addition, can also use PSI-BLAST (being included among the BLAST 2.0) to carry out repeat search, the relation of becoming estranged between the detection molecules.The above-mentioned article of delivering in 1997 referring to people such as Altschul.When using BLAST, Gapped BLAST, PSI-BLAST, can use the default parameters of corresponding program (for example nucleotide sequence BLASTN program, PROTEIN B LASTX program).Referring to http://www.ncbi.nlm, the nih.gov website.Alignment can also be finished by the method for range estimation is manual.

(c) at this, " the sequence homogeny " or " homogeny " of two nucleic acid or peptide sequence with reference to the residue in these two sequences, and these residues are aligned on specifying comparison window length and when reaching the highest correspondence, are identical.When sequence homogeny percentage ratio and protein use relatively, we recognize, difference takes place because of conserved amino acid replaces in residue position inequality usually, wherein, the aminoacid residue is replaced by the aminoacid residue that other has similar chemical attribute (for example electric charge or hydrophobicity), does not therefore change the functional attributes of molecule.When sequence because of conservative replacement difference takes place, can adjust upward sequence homogeny percentage ratio, to revise the conservative that replaces.When sequence because of this conservative replacement difference took place, we said that it has " sequence similarity " or " similarity ".This method of adjustment is known by the experience person that has in present technique field.Usually, this method is included in to conservative when replacing scoring, it is decided to be part but not coupling fully, thereby improves sequence homogeny percentage ratio.Therefore, for example the mark that obtains when identical aminoacid is 1 minute, and the mark that non-conservative replacement obtains is 0 timesharing, one conservative replace the mark that obtains will be between 0 minute and 1 minute.The conservative mark that replaces gets as calculated, for example carries out the calculating in the PC/GENE program (the Intelligenetics company of California, USA Mountain View produces).

(d) at this, " sequence homogeny percentage ratio " is meant the value that compares the sequence of two best alignment and record in comparison window, wherein, in order to reach the best alignment of two sequences, polynucleotide sequence part in the comparison window is compared with reference sequences (not comprising increases or deletion), and can comprise increases or deletion (promptly blank).This percentage ratio calculates by the following method and gets: measure the positional number that identical nucleic acid base or aminoacid residue appear in two sequences, thereby obtain the matched position number, divided by the total number of positions in the comparison window, it is calling sequence homogeny percentage ratio that the merchant that obtains of being divided by multiply by 100 with the matched position number.

(e) (i) " essence is identical " this term of polynucleotide sequence is meant that polynucleotide comprise a sequence, this sequence is compared with reference sequences with canonical parameter by using one of above-mentioned alignment program, the sequence homogeny percentage ratio of the two should be 70% at least, betterly be at least 80%, further be desirably at least 90%, and be desirably at least 95% most.The experience person that has in present technique field will recognize, can carry out suitable adjustment to these numerical value, codon degeneracy, amino acid similarity be read confine position etc. and take into account, to determine by two two nucleotide sequence coded proteinic corresponding homogenies.With regard to these purposes, the essence homogeny of aminoacid sequence typically refers to the sequence homogeny and is at least 60%, betterly is at least 70%, 80%, 90%, and is desirably at least 95% most.

Show that another identical index of nucleotide sequence essence is, if two molecule hybridization each other under stringent condition.In general, selected stringent condition is the heat fusion joint (T of temperature than the particular sequence with definite ionic strength and pH value_m) low 5 ℃.Yet according to required strict degree, the temperature range that stringent condition comprises can be than T_mBetween low about 1 ℃ to 20 ℃, qualifications is discussed in other place of this paper.Under stringent condition, do not have the nucleic acid of hybridization each other, identical if their encoded polypeptide are essence, so they to remain essence identical.This may take place when the maximum codon degeneracy replicating nucleic acid that uses that gene code allowed.Show that two identical indexs of nucleotide sequence essence are, by having the immuning hybridization reactivity between the polypeptide of first nucleic acid coding and the polypeptide by second nucleic acid coding.

(e) (ii) for a kind of peptide, " essence is identical " this term represents that a kind of peptide comprises a sequence, this sequence is compared with reference sequences in specifying comparison window, the sequence homogeny of the two is at least 70%, betterly should be 80%, further ideal should be 85%, and the most desirablely should be at least 90% or 95%.The homology alignment algorithm that should preferentially use Needleman and Wunsch to propose in (1970) J.Mol.Biol.48:443-453 carries out best alignment.Show that two identical indexs of peptide sequence essence are, have immunoreactivity between the antibody of a peptide and anti-second peptide.Like this, it is identical that first peptide and second peptide come down to, and for example the difference between two peptides only is conservative a replacement." substantially similarity " peptide has above-mentioned sequence jointly, but residue position inequality may difference take place because of conserved amino acid changes.

Medicine of the present invention comprises preceding saposin peptide of class and endite composition." endite composition " is meant any molecule or its analog, and these molecules or its analog are present in the endite of cytoplasma membrane naturally, and said here cell refers to zooblast especially, more outstanding finger mammalian cell.In general, the concentration of the endite composition in the endite will be than the concentration height of the endite composition in the exite.It has been recognized that, in some cell perturbation process, for example apoptosis, necrosis and the growth of super reproductive ability, the normal composition of interior exite changes.Representational endite composition includes, but not limited to Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE and analog thereof.At this, " analog " of Phosphatidylserine is meant any anionic phospholipid or contains the acid lipid of electronegative head base, comprise, but be not limited to phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, palmitoleoyl Phosphatidylserine, Petiolus Trachycarpi elaidic acid Phosphatidylserine, Semen Myristicae oleic acid Phosphatidylserine, two inferior oil base Phosphatidylserine, the inferior oleoyl Phosphatidylserine of Petiolus Trachycarpi, defat acid Phosphatidylserine and two oil base Phosphatidylserine.

In concrete an application, synthetic of the present invention and method are absorbed in the distribution of regulating the endite composition in the plasma membrane.In another concrete application, synthetic of the present invention is absorbed in adjusting and treatment and the relevant disorder of super diffusibility cell (for example tumor and cancer) with method.At this, " adjusting " is meant that change, change, change, modification or sequence change at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.The distribution of regulating the endite composition in the plasma membrane is the quantity that changes endite composition in the plasma membrane, change the endite composition in endite relative position or change endite composition shared percentage ratio in the endite of plasma membrane and exite.This adjusting can cause the raising of the endite constituent concentration in the plasma membrane endite.Adjusting tumor size is change tumor size, changes the perhaps size of many tumor cells of at least one tumor cell.

The method of measuring the component distributing in the plasma membrane is known in the present technique field, it comprises, but be not limited to confocal microscope technology, atomic force microscope technology, FRET, fluorescence extinguishing, Electron Microscopy, circular dichromatic method, NMR MALDI-TOF, emission spectrographic analysis, light scattering technique, Electrospray Mass Spectrometry technology.Referring to (1978) Anal.Biochem.91:1331 that the people showed such as Chang; (1992) J.LipidResearch 33:1255-1267 that the people showed such as Kishimoto; (1995) J.Biol.Chem.270:9953-9960 that the people showed such as Vaccaro; And (1994) J Mol.Neurosci.5:59-67 that the people showed such as Fu, by reference they are included in as a whole at this.

The method of measuring the tumor size is known in the present technique field, it comprises, but be not limited to vernier caliper measurement, Plethysmometry, ultrasound wave, magnetic resonance imaging, enzyme-linked immunosorbent assay, physical examination, X-ray, positron emission X-ray tomography, bone scanning, resonance Raman spectroscopy technology, sense of touch imaging, computerized tomography art and cat scan.

At this, " cancer ", " super diffusibility ", " tumor " and " tumor " these several terms are meant the cell of can independently grow (abnormality or the situation that promptly are grown to feature with the rapid diffusion sexual cell).Super diffusibility and tumor morbid state can be divided into pathologic, promptly characterize or constitute a kind of morbid state and non-pathologic, promptly derive but irrelevant with morbid state from normality.This term comprises all types of cancerous growths or oncogenic process, metastatic tissue or pernicious transformation cell, tissue or organ, and no matter histopathology type or intrusion stage." super diffusibility " this term further comprises the smooth muscle cell that produces rapid diffusion sexual cell growth (for example seeing some constitutional cardiomyopathy).It is the morbid state of feature that " the super diffusibility of pathologic " cell betides with the malignant growth.The example of the super diffusibility cell of non-pathologic comprises the cell proliferation relevant with wound repair.

The example of cell proliferation and/or diversity disorder comprises cancer, for example tumor, sarcoma, metastatic disorder, perhaps hemopoietic tumor sexual disorder, for example leukemia.Metastatic tumo(u)r can result from many kinds of primary tumo(u)rs, includes, but not limited to those and results from primary tumo(u)r on prostate, colon, lung, breast and the liver.

" cancer " or " tumor " these two terms comprise the pernicious of various organs, for example those influence the pernicious of lung, breast, thyroid, lymph, gastrointestinal or reproduction urethra, and adenocarcinoma, comprise pernicious, non-minicell cancer, carcinoma of small intestine and the esophageal carcinoma of for example most colon cancer, kidney cell cancer, carcinoma of prostate and/or testicular tumor, lung.

" diffusibility malignant tumor " is a technical term, means the pernicious of epithelium or endocrine tissue, comprises respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, carcinoma of testis, breast carcinoma, carcinoma of prostate, hormonal system cancer and melanoma.Typical diffusibility malignant tumor comprises the tumor that those form at the tissue of cervix uteri, pulmonary, prostate, breast, incidence, colon and ovary.This term also comprises carcinosarcoma, for example comprises the malignant tumor of being made up of carcinoma and sarcoma sample tissue.

" adenocarcinoma " be meant betide glandular tissue or wherein tumor cell constitute the diffusibility malignant tumor of discernible glandular structure.

" sarcoma " is a technical term, means the malignant tumor of mesenchyme derivant.

Cutaneous tumor and cancer include, but not limited to malignant melanoma, optimum epithelial tumor, include but not limited to seborrheic keratosis, acanthosis melanism, epithelium fibropolypus, epidermal cyst, keratoacanthoma, adnexa (adnexa) tumor; Include, but not limited to photochemical seborrheic keratosis, squama cell carcinoma, basaloma and Merkel cell cancer with pernicious epidermis tumor before the canceration; The corium tumor includes, but not limited to optimum dermatofibroma, dermatofibrosarcoma protuberans, xanthoma and skin vascular tumor; Cell migration to the tumor of skin includes, but not limited to histiocytosis X, cutaneous T cell lymphoma (cutaneous T cell lymphoma) and mastocytosis.

Medullary cell tumor and cancer include, but not limited to the disorder that caused by these cells.These disorders include, but are not limited to following disease: with the hematopoietic stem cell diseases associated; Typing lymph CFU-GM; Lymphocyte comprises B and T cell; The typing myeloid progenitor comprises mononuclear cell, granulocyte and megalokaryocyte; And typing ancestral erythrocyte.This includes, but not limited to leukemia and comprises B leukemic lymphoblastoid, T leukemic lymphoblastoid, do not break up leukemia; Erythroleukemia, megakaryoblast leukemia, mononuclear cell; [having differentiation and undifferentiated leukemia to include] interior; Chronic and acute lymphoblast leukemia, chronic and acute lymphoblastic ball leukemia, chronic and acute myeloblastic leukemia, lymphoma, myelodysplastic syndrome, chronic and acute myeloid leukemia, monocytic leukemia; Pernicious, for example teenager chronic lymphocytic leukemia of blood of chronic and acute myeloblastic leukemia, chronic and acute cellulous leukemia of bone marrow, chronic and acute promyelocytic leukemia, chronic and acute myeloblastic leukemia, mononuclear cell-macrophage system; Secondary cases acute myeloblastic leukemia, the disorder of precursor blood; The reactive skins Angioendotheliomatosis; Fibroid disorder, the disorder relevant with the expression that changes in the dendritic cell comprises system's sclerosis, E-M syndrome, popular malicious oiliness syndrome, eosinophil property fascitis local scleroderma type, keloid and fibroid colon disease; Hemangioma sample lump malignant fibrohistiocytoma; Cancer comprises constitutional head and neck squama cell carcinoma; Sarcoma comprises Kaposi sarcoma; Fibroadenoma and phyllodes tumor comprise the mammary gland fibroadenoma; Stromal tumor; Phyllodes tumor comprises histiocytoma; T cell lymphoma; And B cell lymphoma.

Cardiac tumor and cancer include, but not limited to primary cardiac tumor, for example the heart effect of myxoma, lipoma, papilla fiber Elastoma, rhabdomyoma, sarcoma and non-cardiac tumor.

Vascular tumor and cancer comprise, but be not limited to hemangioma, lymphangioma, glomus tumor, pulse tube expander, bacilus hemangioma and middle rank (criticality is rudimentary pernicious) tumor, for example Kaposi sarcoma and hemangioendothelioma, and malignant tumor, for example angiosarcoma and hemangiopericytoma.

B cell tumour and cancer include, but not limited to precursor B glucagonoma, for example lymphoblast leukemia/lymphoma.Periphery B glucagonoma comprises, but be not limited to chronic lymphocytic leukemia/small lymphocyte lymphoma, follicular lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, plasmocytoma, boniness myeloma and related entities, lymph chylema cell lymphoma (Walden Si Telun [Waldenstrom] macroglobulinemia), lymphoma mantle cell, marginal zone lymphoma (MALToma) and hairy cell leukemia.

Liver tumor and cancer include, but not limited to nodular hyperplasia, adenoma and malignant tumor, comprise primary hepatocarcinoma and metastatic tumo(u)r.

The cerebral tumor and cancer comprise, but be not limited to, glioma comprises astrocytoma, comprise fibroid (diffusivity) astrocytoma and glioblastoma multiforme, fibrous astrocytoma, pleomorphic xanthoastrocytoma and brain stem glioma, the other lump damage of oligodendroglioma and ependymoma and associated chamber, neural tumor, low differentiation tumor, comprise medulloblastoma and other brain parenchymal tumor, comprise constitutional brain lymphoma, germ cell tumor and pinus brain parenchymal tumor, meningioma, metastatic tumo(u)r, paraneoplastic syndrome, the peripheral nerve sheath tumor, comprise schwannoma, neurofibroma, pernicious peripheral nerve sheath tumour (malignant schwannoma) and neurocutaneous syndrome (phakomatosis), comprise neurofibroma, comprise I type neurofibroma (NF1) and II type neurofibroma (NF2), tuberous sclerosis and VonHippel-Lindau disease.

Ovarian tumor and cancer include, but not limited to oophoroma, for example coelomic epithelial tumor, serosity tumor, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, the optimum fibroepithelioma of ovary, superficial epithelium tumor; Germinoma, for example ripe (optimum) teratoma, cell monolayer teratoma, immaturity malignant teratoma, dysgerminoma, endodermal sinus tumor, choriocarcinoma; Sex cords stromal tumor, for example granulosa cell-thecoma, thecoma-fibroma and one-tenth male cell tumor, mountain glucagonoma and gonadoblastoma; And metastatic tumo(u)r, for example Crewe root Bao Shi tumor.

Tumor of kidney and cancer comprise, but be not limited to benign tumor, for example renal papillae adenoma, renal fibroma or hamartoma (renal medullary interstital cell tumor), renal angiomyolipoma and oncocyte tumor, and malignant tumor, comprise renal cell carcinoma (hypernephroma, renal adenocarcinoma), comprise renal pelvis urothelium cancer.

Rhabdomyoma and cancer include, but not limited to rhabdomyosarcoma.

Osteoblastoma and cancer comprise, but be not limited to, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibroid cortex are damaged, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma, endothelium sex myeloma, original neuroectodermal tumors, giant cell tumor and metastatic tumo(u)r.

Vipoma and cancer include, but not limited to cystic tumor and cancer of pancreas; The gland islet cell tumor includes, but not limited to insulinoma, gastrinoma and other rare gland islet cell tumor.

Mammary neoplasms and cancer include, but not limited to stromal tumor, for example fibroadenoma, lobate tumor and sarcoma, and epithelial tumor, for example big glandular tube papilloma; Breast carcinoma comprises original position (non-dispersive) tumor, comprise that interior tumor (comprising the Paget disease) of original position (non-dispersive) glandular tube and lobular carcinoma in situ and diffusibility (infiltration) cancer comprise, but be not limited to, tumor, diffusibility lobular carcinoma, hyloma, glue sample (mucus) cancer, tubular carcinoma and diffusibility papillary carcinoma in the non-special type diffusibility glandular tube, and other malignant tumor.

Male chest tumor and cancer include, but not limited to the diffusibility malignant tumor.

Tumor of prostate and cancer include, but not limited to the diffusibility malignant tumor.

Colon tumor and cancer include, but not limited to non-tumor polyp, adenoma, family's syndrome, rectum canceration, rectal cancer and carcinoid tumor.

Lung tumors and cancer include, but not limited to bronchogenic carcinoma, comprise paraneoplastic syndrome, bronchioloalveolar carcinoma, neuroendocrine tumor, for example carcinoid of bronchus, other tumor and metastatic tumo(u)r; Pleuroma comprises isolatism fibroma (pleura fibroma) and malignant mesothe.

Thymus neoplasms and cancer include, but not limited to thymoma, comprise germinoma, lymphoma, hodgkin's (Hodgkin) disease and carcinoid.Thymoma can comprise optimum or parcel property thymoma and malignant thymoma I type (diffusibility thymoma) or II type, appointment property thymic carcinoma.

Tonsil tumor and cancer include, but not limited to non-Hodgkin lymphomas B cell lymphoma.

The medicament a kind of of the present invention of saposin related polypeptide and endite composition before comprising (also claiming " active agents ") can synthesize the medicine that is fit to give a certain experimental subject use.These medicines generally comprise acceptable carrier on a kind of preceding saposin polypeptide, a kind of endite composition and a kind of pharmacopedics.In a concrete medicine, preceding saposin related polypeptide and endite composition constitute a kind of Na Pao.The diameter range of Na Pao is between 0.01 to 10 micron, more satisfactory should be between 0.1 to 1 micron, further ideal should be between 0.1 to 0.5 micron, and further ideal should be between 0.2 to 0.4 micron, and further ideal then should be between 0.2 to 0.3 micron again.The bulb diameter of typically receiving includes, but are not limited to 10 nanometers, 100 nanometers, 150 nanometers, 160 nanometers, 170 nanometers, 180 nanometers, 190 nanometers, 200 nanometers, 210 nanometers, 220 nanometers, 230 nanometers, 240 nanometers, 250 nanometers, 260 nanometers, 270 nanometers, 280 nanometers, 290 nanometers, 300 nanometers, 350 nanometers, 400 nanometers, 450 nanometers, 500 nanometers, 550 nanometers, 600 nanometers, 650 nanometers, 700 nanometers, 750 nanometers, 800 nanometers, 850 nanometers, 900 nanometers, 950 nanometers and 1000 nanometers.

At this, " medication " speech has its implication the most widely, comprises any method with a certain experimental subject of introduction of medications of the present invention." experimental subject " is meant mammal, and be for example human, perhaps experiment or animal or disease model.Experimental subject also can be the non-human animal, for example, but is not limited to non-human primate, horse, cow, goat, pig, rabbit, Mus, Cavia porcellus, Canis familiaris L. or other domestic animal.In addition, medicine of the present invention can be used for the treatment of disorder described herein.Therefore, all treatments disorder relevant with super diffusibility cell (for example tumor or cancer) includes interior.At this, " treatment " speech is defined in a certain patient and goes up application or use medicament of the present invention, perhaps medicament of the present invention is applied to or offers medicine have on patient's a certain independent tissue or cell line of disease or disease symptoms in a certain name, purpose is to treat, cure, alleviate, alleviate, change, correct, improve, improve or influence the symptom of disease or disease.

At this, " acceptable carrier on the pharmacopedics " comprise any He all solvents, disperse medium, sugar-coat, antibiotic and antifungal medicine, etc. ooze or postpone to absorb medicament or the like the medicament compatible with the medicine medication.On active medicine, use this medium and medicament known in the present technique field.Unless any traditional sucrose or medicament and active medicine are incompatible, be among expecting otherwise in medicine, use this medium and medicament.The complementarity active agents also can be synthesized in the medicine.

A kind of anti-tumor agents of the present invention or medicine are after preparation, and be can route of administration predetermined with it compatible.The example of route of administration comprises without intestinal, for example intravenous injection, intradermal injection, subcutaneous injection, oral (for example sucking), percutaneous (external), per mucous membrane, abdominal cavity and the direct administration of rectum.Solution or suspension without intestinal, Intradermal or subcutaneous injection administration can comprise following ingredients: sterile diluent is water for injection, saline solution, fixing oil, Polyethylene Glycol, glycerol, propylene glycol or other synthetic for example; Antibacterial, for example benzyl alcohol or p-Hydroxybenzoate; Antioxidant is ascorbic acid usp/bp or sodium sulfite for example; Chelating agen is ethylenediaminetetraacetic acid for example; Buffer agent is acetate, citrate or phosphate and elasticity of muscle regulator for example, for example sodium chloride or glucose.Acid-base value can be regulated according to acid or alkali, for example hydrochloric acid or sodium hydride.In Enteral formulations can be packed ampoule, disposable syringe or multi-dose vials into, these containers can be glass or plastic material.

The medicine that is fit to injection comprises aseptic aqueous solution (if water soluble) or dispersion liquid and the sterilized powder that can temporarily be mixed with sterile injectable solution or dispersion liquid.For intravenous administration, suitable carrier comprises normal saline, bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS) (PBS).In all cases, medicine all must be aseptic and should have the flowability of easy injection.Medicine make and storage condition under must stablize and must have and prevent to be subjected to for example protective measure of antibacterial and fungal contamination of microorganism.Carrier can be solvent or disperse medium, and it can comprise for example suitable mixture of water, ethanol, polyhydric alcohol (for example glycerol, propylene glycol and liquid macrogol or the like) and these materials.Can keep suitable flowability by use sugar-coat (for example lecithin), by keeping the mode of required granularity (for dispersion liquid) and use surfactant.Prevent that action of microorganisms from can realize by using various antibiotic and antifungal (for example p-Hydroxybenzoate, chlorobutanol, phenol, ascorbic acid usp/bp, thiomersalate or the like).In many cases, preferably also comprise isotonic agent in the medicine, for example sugar, polyhydric alcohol for example mannitol, Sorbitol and sodium chloride.The medicament (for example monostearate aluminum and gel) that can comprise a kind of delayed absorption in medicine prolongs absorption.

The active agents (saposin related polypeptide and endite composition for example) that is dissolved in the requirement in the appropriate solvent can be synthesized with the above-mentioned a kind of composition of necessity or the combination of multiple composition, filter sterilization is mixed with sterile injectable solution then.In general, the compound method of dispersant is that active agents is fused in the sterile media, and sterile media comprises basic disperse medium and necessary above-mentioned other composition.For the sterilized powder that is used for preparing sterile injectable solution, the preferred approach of preparation powder is boulton process and freeze-drying, and these methods can be from before producing the active component powder and any other wanted the composition that obtains through the solution of disinfection filtering.

Oral drugs generally comprise inert diluent or edible carrier.They can be encapsulated in the gel capsule or be compressed into tablet.For oral therapy medication, active agents can merge with excipient, and uses with tablet or capsular form.Oral drugs can also use liquid-carrier to be mixed with collutory, and the medicament oral administration in the liquid-carrier also dashes the portion of gargling, and spue then or swallow.Pharmacopedics compatible adhesive and/or auxiliary material can be used as the part of medicine.Tablet, pill, capsule, tablet etc. can comprise the medicament of following any composition or similarity: binding agent is microcrystalline Cellulose, Tragacanth or gel for example; Excipient is for example alginic acid, Primogel or corn starch of starch or lactose, distintegrant for example; Lubricant is magnesium stearate or Sterotes for example; Slip agents is silicon dioxide colloid for example; Sweeting agent is sucrose or glucide for example; Perhaps for example Herba Menthae, methyl salicylate or the agent of orange flavor of flavoring agent.For inhalation, medicament is to be contained in the form administration of the aerosol spray in compression container or the dispenser, and this container comprises a kind of suitable propeller, for example carbon dioxide or nebulizer.

In a concrete medicine, active agents is with preventing that medicament is formulated by the quick carrier of discharging of health, and for example the sustained release prescription comprises and implanting and microcapsule encapsulation administration mechanism.Can use biodegradable, biocompatible polymer, for example ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen protein, poly-former phosphide and polylactic acid.The compound method of this prescription is conspicuous for having for the experience person of present technique field.Available Material also can be to AlzaCorporation and Nova Pharmaceuticals, and Inc. company buys.Liposome suspension (comprising that with the infection cell with the antigenic monoclonal antibody of antiviral be the liposome of target) also can be as acceptable carrier on the pharmacopedics.These materials can be prepared according to methods known in the art, for example use U.S. Patent No. 4,522,811 described methods.

It is especially favourable that oral or parenteral medicine is mixed with the dosage unit form, because can make things convenient for medication and guarantee the concordance of dosage.At this, " dosage unit form " is meant physically discrete unit, be fit to the unit dose of treatment experimental subject, per unit comprises the active agents of predetermined quantity, and predetermined quantity is can produce desired curative effect calculating according to active agents and required pharmacopedics carrier associating to get.The type and the order of severity according to disease, the predose of the medicament of selecting for use to patient of the present invention greatly about 1 microgram/kilogram between about 15 milligrams/kilogram (for example 0.1 to 20 milligram/kilogram), no matter be the method that adopts the individually dosed or continuous infusion of one or many.Typical every day, dosage can be between about 1 microgram/kilogram be to 100 milligrams/kilogram or high dose more, and specifically dosage will be decided according to above-mentioned factor.For many days or interior repeat administration of longer time, as the case may be, treatment can continue, till disease symptoms obtains suitable inhibition.Yet other dosage system also may be effectively.The progress of this therapy can obtain monitoring by traditional technology and chemical examination.A typical dosage system is open in WO 94/04188.Dosage unit form of the present invention depends on and directly depends on the peculiar property of active agents and the specific therapeutical that will obtain and compound this active agents in order to treat the inherent limitation of technology of individual patient.

System's medication can also be adopted the method for per mucous membrane or percutaneous.For per mucous membrane or percutaneous dosing, should in prescription, use the penetrating agent of the suitable barrier that will permeate.This penetrating agent is generally known for the people in the present technique field, and the penetrating agent that for example is used for transmucosal drug delivery has cleaning agent, bile salts and fusidic acid derivatives.Transmucosal drug delivery can use the method for nose with spray or suppository.For percutaneous dosing, active agents is formulated into ointment, ointment, colloid or cream, and these are well-known in the present technique field.Medicament can also be mixed with suppository (for example traditional suppository cupu oil and other glyceride) form or enema,retention is realized the rectum drug delivery.

Antitumor as herein described or anticancer agent can percutaneous dosings.Percutaneous dosing generally comprises and makes medicament enter experimental subject or patient's systemic circulation by the percutaneous passage.Skin part comprises the anatomical area of percutaneous dosing, comprises forearm, abdominal part, chest, back, hip, pars papillaris or the like.

The percutaneous drug delivery is exposed to medicament for a long time by the skin that makes the patient or drug source reaches the drug delivery purpose.The percutaneous plaster also has controlledly provides the advantage of medicament (to be shown referring to Hadgraftand Guy (eds) to healthTransdermal Drug Delivery:DevelopmentalIssues and Research InitiativesMarcel Dekker, Inc.; Robinson ﹠amp; (1987) that Lee (eds) is shownControlled Drug Delivery:FundamentalsAnd Applications, Marcel Dekker, Inc; And Kydonieas﹠amp; (1987) that Bemer (eds) is shownTransderrnal Delivery of DrugsVols 1-3, CRC Press includes in by reference at this).This dosage form can be fused to the conjugate of saposin C related polypeptide and two oil base Phosphatidylserine (dioleoylphosphatidylserine is called for short DOPS) in the suitable medium (for example elastomer matrix material) by dissolving, dispersion or alternate manner and be made.Absorption enhancer can also be used to improving the percutaneous flow of medicament.The speed of this flow can be by providing the speed controlling film or medicament being dispersed in polymer backbone or the gel and controlled.

Be applied in the described herein method of the percutaneous plaster of many types.For example, simple adhesive plaster plaster can use back lining materials and acrylates adhesive plaster to be made.Medicament and any reinforcing agent can be mixed with the colloidality cast-solution and can fully mix.This solution can direct pouring on back lining materials, casting solvent then evaporates in stove, stays glued membrane.Enclose the release liner, complete plaster.

Also can use the polyurethane skeleton plaster to carry out drug delivery.Each of this plaster layer comprises backing layer, polyurethane medicine/reinforcing agent casing play, rete, adhesive layer and discharges inner liner.Polyurethane skeleton uses the curing polyurethane at room temperature prepolymer formulated.After in this prepolymer, adding entry, ethanol and medicament and being mixed with solid viscous elastomer, can direct pouring on back lining materials.

A further concrete medicine of the present invention can use hydrogel skeleton plaster.The hydrogel skeleton generally comprises ethanol, water, medicine and several hydrophilic polymer.This hydrogel skeleton can be clipped between backing and the adhesive layer, is fused on the percutaneous plaster.

For passive drug delivery system, rate of release generally by be clipped in film between medicine storage and the skin, by from monolithic devices, spreading or by skin itself is controlled as the speed controlling barrier in the drug delivery system.(referring to United States Patent (USP), the patent No.: 4,816,258; 4,927,408; 4,904,475; 4,588,580; 4,788,062, include in by reference at this).Drug delivery speed will depend in part on the character of film.For example medicine passes the speed of film in vivo generally than the speed height that passes skin barrier.Medicament is to place a speed restriction film between medicine storage and skin from the most convenient control method that device is sent to the speed of film.When skin has sufficient permeability (being that percutaneous absorption exceeds the speed by film) for medicine, film will have the effect of control patient acceptable dose speed.

Having suitable infiltrative membrane material can select according to the character and the mechanical factor relevant with construction device of required permeability, medicament.Typical osmotic membranes material comprises nature and synthetic polymer widely, for example polydimethylsiloxane (silicone rubber), ethylene vinyl acetate copolymer (EVA), polyurethane, polyurethane-copolyether, polyethylene, polyamide, polrvinyl chloride (PVC), polypropylene, Merlon, polytetrafluoroethylene (PTFE), cellulosic material, for example cellulose triacetate and celluloid/cellulose acetate and hydrogel, for example 2-hydroxyl ethyl ester methacrylic acid (HEMA).

Device can contain other thing, for example can accept carrier on other pharmacopedics, specifically will see desired equipment energy characteristic and decides.For example, can also comprise one or more antiseptic or bacterial inhibitor, for example methyl hydroxybenzoate, nipasol, chlorocresol, alkyldimethylbenzylammonium chloride or the like according to compounding pharmaceutical of the present invention.These medicines can also comprise other active component, for example antimicrobial, especially antibiotic, anesthetis and pruritus.

Another aspect of the present invention is also stipulated the external drug delivery of medicament of the present invention.This treatment system is fit to system's medication of anti-tumor agents, also is fit to local treatment, promptly directly delivers to pathologic or ill tissue.

In general, the external used medicine prescription comprises directly to be delivered to medicament infection site, comprises the preparation of medicine, and the drug concentrations scope is generally between about 0.001% to 10%; Comparatively ideal scope is between about 0.01 to about 10%; Further ideal scope is between about 0.1 to about 5%; And optimal scope is between about 1 to about 5%, this concentration comprise acceptable external carrier on non-toxicity, the pharmacopedics (Barry (eds).Dermatological Formulations:Percutaneous Absorption(1983) Marcel Dekker, Inc; The standard dose of relevant traditional medicament can be referring to for example, Physicians Desk Reference (1992 editions); And (1992) Drug Evaluations Subscriptions of AMA).

External preparation can combine formulated by medicament is done carrier commonly used in matter, liquid, white matter and the aerosol prescription with conventional medicament diluent and external.Unguentum and cream can (for example) use water base or oil base adds that suitable denseization agent and/or gum material are formulated.These bases can comprise water and/or oil, for example liquid paraffin, or vegetable oil, for example Oleum Arachidis hypogaeae semen or Oleum Ricini.The denseization agent of using according to the character of base can comprise soft paraffin, aluminium stearate, 16 mixed alcohols, propylene glycol, Polyethylene Glycol, lanoline, hydrogenated lanolin, Cera Flava or the like.Lotion can use water base or oil base is formulated, and generally also comprises following one or more: stabilization agent, emulsifying agent, dispersant, suspending agent, denseization agent, coloring agent, essence or the like.Powder can use any suitable base (for example Talcum, lactose, starch or the like) to be made.Drop can use water base or non-water base formulated, and these bases can also comprise one or more dispersants, suspending agent, solubilizing agent or the like.

The topical dose form of medicament of the present invention comprises powder, spray, ointment, ointment, cream, lotion, colloid, solution, plaster and inhalant.Active agents can be accepted carrier, any antiseptic, buffer agent or necessary propeller and mix on aseptic condition and pharmacopedics.

Ointment, ointment, cream and colloid also can comprise excipient, for example the mixture of animal and plant fat, oil, wax, paraffin, starch, tragacanth, cellulose derivative, Polyethylene Glycol, silicones, bentonite, Talcum and zinc oxide or above material.Powder and spray also can comprise excipient, for example the mixture of lactose, Talcum, aluminium hydroxide, calcium silicates and polyamide powder or above material.Spray also can comprise habitual propeller, and for example chloride fluorine hydroxyl and volatility be substituted hydrocarbons not, for example butane and propane.

Method of the present invention also is applicable to by the mucosa drug delivery, for example gastrointestinal, Sublingual, oral cavity, nose, lung, vagina, cornea, eye mask (referring to (1991) Adv.Drug Del.Rev.7:313-338 that the people showed such as Mackay).

For through the oral cavity or Sublingual film drug delivery, can use typical formula of oral for example buccal tablet, tablet or capsule.The method of making these prescriptions for known to the present technique field, includes, but not limited to add medicament in prefabricated tablet; Cold compression inert filler, binding agent and encapsulated.

Another formula of oral can apply on oral mucosa with binding agent (for example cellulose derivative, hyprolose), and as U.S. Patent No. 4,940,587 descriptions are included in by reference at this.When applying on oral mucosa, the bonding prescription in this oral cavity can be controlled medicament and be discharged in the mouth and pass oral mucosa.

To nose and/or lung film, can adopt typical aerosol formulations for drug delivery." aerosol " this term comprises that any gas of medicament of the present invention carries suspended phase, can be inhaled in bronchioles or the nasal meatus.Particularly, aerosol comprises the conductance suspension of present invention medicament microdroplet, for example is contained in metered dose inhaler or the aerosol apparatus.Aerosol can also comprise the medicament dry-powder medicament that is suspended in air or other carrier gas, can be by the method drug delivery that sucks from inhaler device.

Medicine of the present invention has the effect of treatment disorder described herein.Medicine provides by curative effect quantity.Here " curative effect quantity " is meant the quantity that is enough to regulate the reaction that will reach.According to this definition, the scope of the curative effect quantity of protein in the medicament or polypeptide (being effective dose) is between about 0.001 to 30 milligram/kg body weight, comparatively ideal scope is between about 0.01 to 25 milligram/kg body weight, further ideal scope is between about 0.1 to 20 milligram/kg body weight, and further ideal scope is between about 1 to 15 milligram/kg body weight.The scope of the endite composition curative effect quantity (being effective dose) of medicament of the present invention is between about 0.001 to 30 milligram/kg body weight, comparatively ideal scope about 0.01 between about 30 milligrams/kg body weight, further ideal scope about 0.01 between about 20 milligrams/kg body weight, further ideal scope is between 0.01 to 10 milligram/kg body weight, and further more ideal scope is at about 0.1 to 9 milligram/kilogram, 0.1 to 8 milligrams/kilogram, 0.1 to 7 milligrams/kilogram, 0.1 to 6 milligrams/kilogram, 0.1 to 5 milligrams/kilogram, 0.1 between 4 milligrams/kilogram or 0.1 to the 3 milligram/kg body weight.

The molar ratio scope of polypeptide and endite composition is between about 1: 1 to about 1: 50 in the medicament of the present invention, comparatively ideal scope is between about 1: 1 to about 1: 25, further ideal scope is between about 1: 1 to 1: 10, and further more ideal ratio is about 1: 7 or about 1: 3.Suitable ratio treasure-house, but be not limited to 1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9,1: 10,1: 11,1: 12,1: 13,1: 14,1: 15,1: 16,1: 17,1: 18,1: 19,1: 20,1: 21,1: 22,1: 23,1: 24,1: 25,1: 30,1: 35,1: 40,1: 45 and 1: 50.The quality ratio scope of polypeptide in the medicament of the present invention and endite composition is between about 15: 1 to 3: 10, comparatively ideal scope is between about 15: 1 to about 3: 5, further ideal scope is between about 15: 2 to about 3: 0, and optimal ratio is about 15: 7 or about 5: 1.See the influence that the polypeptide that draws in the medicament of the present invention and the preferential ratio between the endite composition may be subjected to some factor, for example, but be not limited to the target cell type.

[0100] experienced technical staff sees that drawing some factor may influence the required dosage of a certain experimental subject of effective treatment, comprise, but be not limited to the general health and/or age of the disease or the disorderly order of severity, previous treatment, experimental subject and whether have other disease.And, use a certain experimental subject of protein, polypeptide or Antybody therapy of curative effect quantity can comprise single therapy or preferably comprise a series of treatments.In a preferred example, use a certain experimental subject of pharmaceutical treatment of curative effect quantity weekly, administration time is between about one thoughtful ten weeks, and more satisfactory is between two thoughtful eight weeks, further be desirably for about three thoughtful seven weeks, further be desirably approximately around, five week or six weeks.See also that in addition the effective dose that draws treatment used antibody, protein or polypeptide may or increase or subtract in concrete therapeutic process.The dosage change may be because and may obviously be because of due to diagnosis verification result described herein.

[0101] when a certain experimental subject in the treatment has the partial reaction performance or recurs after symptom disappears for a long time, can use medicament of the present invention as successive treatment.Therefore, first treatment cycle (may comprise single dose system or multiple dose system) afterwards at interval after a period of time, a certain experimental subject can be accepted one or more additional procedures cycles, and the additional procedures cycle can comprise single dose system or multiple dose system.At this, be meant withdrawal time the blanking time between two treatment cycle.See to draw that withdrawal time length depends on the tumor response degree that reaches in the treatment cycle of any previous use anti-tumor agents of the present invention.

[0102] medicine can use container, dress bag or Bistributor package, and the medication instruction of enclosing.

[0103] a kind of treatment for cancer result can adopt any method known in the art to examine and determine, comprise, but be not limited to health check-up, experiment, atom and electrography (being computerized laminaghaphy and/or mr imaging technique), ultrasound wave and other method.

[0104] at this, " cell death " is meant by any mechanism and comprises that apoptosis, necrosis and dissolving etc. lose one's life cell.At this, " apoptosis " or " inside the plan cell death " is meant the normal physiological processes of the modulation metabolic activity that needs dying cell, and its common feature is that cellular contraction, chromatin concentrate and/or disappearance, DNA division and/or the plasma membrane infringement of nuclear fission, film or bubble.

[0105] following example is an illustrative purposes only, is not to limit the invention.

Experiment

Example one: reorganization saposinCPurification

[0106] adopts isopropyl-1-sulfo--β-when the D-galactoside is induced the pET system, reorganization saposin C in the E.coli cell by overexpression (, by reference they being included in as a whole) at this referring to (1994) J.Biol Chem.269:16746-16753 that the people showed such as Qi.By polypeptide expressed with His-tag elution from the nickel post.After the dialysis, polypeptide is further purified by following HPLC high performance liquid chromatography.C4 reversed-phase column and 0.1% trifluoroacetic acid (TFA) keep balance to reach 10 minutes.Protein was pressed linearity (0-100%) gradient elution of 0.1%TFA more than 60 minutes in acetonitrile.Main protein peak all is collected and lyophilizing.Protein concentration is measured (referring to (1994) J.Biol.Chem.269:16746-16753 that the people showed such as Qi) as stated above.

Example two: the sonication of saposin C and two oil base Phosphatidylserine is bathed

[0107] two oil base Phosphatidylserine (DOPS) can be bought to Avanti Polar Lipids (Alabaster AL) company.Evacuation becomes adipose membrane to have 20 to 30 micromolar DOPS to be dried also under N2 in the chloroform.Five to ten micromole saposin C polypeptide are added on the desciccator diaphragm and are added in the 50 microlitre Mcllvanine buffer agents (pH4.7) makes suspension.Adding cell culture medium or phosphate buffered saline (PBS) (PBS) then makes the suspension volume reach 1 milliliter (referring to (2002) Current Protocols inMolecular Biology.John Wiley ﹠amp that the people showed such as Ausubel; Sons, New York, New York includes in by reference at this).Above mixture in bathing method sonication device about 20 minutes through sonication.As needs, add an amount of ice, prevent that sample is overheated.

Example three: conditions of tissue culture

[0108] squama cell carcinoma (SCC) cell and L5178Y cell are cultivated in being added with the DEME medium (Gibco) of 10%FBA.Normal immortalization keratinocyte (NIK) is grown in 50%Cascade medium 154 (Cascade Biologics) and 50% keratinocyte-SFM medium (Gibco).

Example four: analyzed in vitro saposinC-DOPSSuppressSCCThe effect of cell

[0109] squama cell carcinoma (SCC) cell and control cells (NIK cell) are all grown in medium, and this medium is introduced in this paper other places.NIK cell in contrast, be because show that SCC is (referring to (2002) J.Med.Invest.49:111-117 that the people showed such as Kubo) that grows by a multistep process in the human skin keratinocyte.From the NIK that set up and SCC cell plate, remove culture medium.Culture medium is left intact, and adds saposin C, DOPS or 8 μ M saposin C+26 μ M DOPS in NIK that has set up and SCC cell plate.These cells were checked after treatment in 48-72 hour.Fig. 1 shows an above-mentioned result of experiment.

[0110] squama cell carcinoma (SCC) the cell medium of growing is introduced in this paper elsewhere.Culture medium is removed from the SCC cell plate of having set up.Culture medium is left intact or adds the medicament that contains 10 μ M saposin C+30 μ M DOPS in the SCC cell plate of having set up.Allow the cell hatching after 24 hours, colloid electrophoresis or use anti-agglomerating agent albumen painted by TUNEL, chromosome set DNA are that annexin V carries out hybridization assays, analyze these cells, and analytical data does not show.

Example five: analyzed in vitro saposin C-DOPS is to the curative effect of Mus lymphoma cell

[0111] tissue culture's plate kind has Mus L5178Y-R lymphoma cell.Culture is removed culture medium and is cleaned cell after setting up.Cover outside the DEME+10%FBA on these cells, perhaps do not have medication or additional 60 μ M DOPS, 20 μ M saposin C or 10 μ Msaposin C and 30 μ M DOPS.Culture hatching 24-48 hour.Check culture after incubation period.Fig. 2 shows an above-mentioned result of experiment.

Example six: body inner analysis saposin C-DOPS is to the influence of tumor size

[0112] nude mice is fed by the regulation of the relevant experimental mouse treatment of (U.S.) Cincinnati child study foundation (CincinnatiChildren ' s Research Foundation).Respectively two groups of each five nude mices are carried out 2 * 10⁶The last back of SCC subcutaneous injection makes it to take place tumor growth.Every Mus grows two tumors respectively.Allow tumor growth 21 days.At the 21st day, the tumor locus of these animals was accepted pure PBS diluent or is comprised saposin C (10 milligrams/kg body weight) and the medicament subcutaneous injection of DOPS (2 milligrams/kg body weight).At the 27th day, the tumor locus of these animals was accepted for the second time pure PBS diluent or is comprised saposinC (10 milligrams/kg body weight) and the medicament subcutaneous injection of DOPS (2 milligrams/kg body weight).Every other day use kind of calliper tumor size, gross tumor volume is V=(π/4) LW by formula²Estimate.The A section of Fig. 3 shows an above-mentioned result of experiment.

[0113] in one group of independent experiment, the regulation that nude mice is taken care of by the relevant experimental mouse of (U.S.) Cincinnati child study foundation (Cincinnati Children ' s Research Foundation) is fed.Respectively two groups of each five nude mices are carried out 2 * 10⁶The last back of SCC subcutaneous injection makes it to take place tumor growth.Every Mus grows two tumors respectively.Allow tumor growth 6 days.At the 6th day, the tumor locus of these animals was accepted pure PBS diluent or is comprised saposin C (10 milligrams/kg body weight) and the medicament subcutaneous injection of DOPS (2 milligrams/kg body weight).

Every other day use kind of calliper tumor size, gross tumor volume by formula V=(π/4) LW2 is estimated.The B section of Fig. 3 shows an above-mentioned result of experiment.

Example seven: saposin C-DOPS is to the curative effect of human squama cell carcinoma tumor tissues

[0114] use the SCC tumor to prepare the Mus xenograft by methods known in the art.Fluorized marking nitro benzodiazole (NBD) and Phosphatidylserine be connected and prepare the mixture of NBD-PS and DOPS.Use NBD-DOPS to prepare fluorized marking NBD-PS/DOPS/saposin C medicament.At the dosage injection NBD-PS/DOPS of tumor locus by 0.1 milligram of NBD-PS/ kg body weight and 2 milligrams of DOPS/ kg body weight.At the dosage injection NBD-PS/DOPS/saposin C of tumor locus by 0.1 milligram of NBD-PS/ kg body weight, 2 milligrams of DOPS/ kg body weight and 10 milligrams of saposin C/ kg body weight.Tumor was killed after medication in 24 hours.Whether the microscopic section of checking tumor has fluorescence.Fig. 4 shows an above-mentioned result of experiment.

Example eight: analyzed in vitro saposin C-DOPS is to human cell's curative effect

[0115] cell of taking from human cancer tissue and health tissues is all grown in the medium that is fit to this cell line.The cell of taking from following cancerous tissue and health tissues cell line is analyzed: the MCF-7 of breast carcinoma: MCF-7, the negative caspase 9 of transfection dominance, MCF-7, the BT-549 of transfection vector control; Head and neck: SCC-25, FaDu; Melanoma: MeWo, Sk-Mel-28; Leukemia: K-562, HL60; Cervical cancer: Hela; PA1, the PA1-E6 of ovarian cancer: PA1, the negative caspase9 of transfection dominance; SK-OV3; Carcinoma of prostate: DU145, PC3; Neuroblastoma: SK-N-SH, SK-SY-5Y, CHLA-79; Ewing sarcoma: 5838; T cell lymphoma; RoduT; GCT; Pulmonary carcinoma: A549, H441; Hepatocarcinoma: HepG2; Healthy breast: MCF-10A; And healthy keratinocyte: NIK.The flat tissue culture of 96 wells ware (Falcon, Becton-Dickson Labware, Franklin Lakes NJ) is planted by the density of 10000 cells of every well.The cell ware is put into the complete medium that contains or do not contain medicament of the present invention respectively.

[0116] (4, the 5-dimethylthiazole-2-yl)-2,5-hexichol tetrazolium bromide (can buy to Sigma) changes first product (18992) but estimates survivaling cell density to use 3-.After the cell culture 72 hours, in each well, add 3-(4,5-dimethylthiazole-2-yl)-2,5-hexichol tetrazolium bromide (MTT) reaches ultimate density and is till 0.25 mg/ml.The cell ware was hatched 3 hours under 37 ℃ of temperature in the dark.The reaction that is risen is stopped by the isopropyl alcohol that adding contains 0.04N HCI.The cell ware is thoroughly mixed, and on little ELISA plate reader (by SpectraMax Plus, Molecular Devices, Sunnyvale CA manufacturing), do 570 nanometer analyses.Use PharmTools Pro computer software (The McCaryGroup, Elkins Park, PA product) to carry out growth curve and regression analysis.

The human cell	Cell death (%)		The known defective
				Cancerous cell line	There is not treatment	Treatment
Breast: MCF-7	6.8±3.5	96.1±1.0					Caspase-3 zero
			MCF-7 (transfection)	7.5±3.5	18.9±6.8	Caspases-9DN
MCF-7 (transfection)	10.7±3.7	94.0±0.7					Vector control
			BT-549	6.9±2.8	33.6±6.6	The p53 sudden change
Head and neck: SCC-25	7.4±2.8	57.8±4.6					P53 zero
			FaDu	14.6±5.7	91.0±0.2
Melanoma: MeWo	8.3±2.6	81.9±5.4					The p53 sudden change
			SK-Mel-28	7.7±3.2	76.2±2.0	The Apaf-1﹠p53 sudden change
Leukemia: K-562	7.0±2.8	58.0±3.4					Apaf-1﹠p53 zero
			HL-60	21.2±5.4	44.2±3.6	P53 zero
Cervix uteri: Hela	3.8±1.1	47.3±4.4					The p53 sudden change
			Ovary: PA1	7.1±1.9	89.9±0.2
PA1 (transfection)	8.3±3.0	54.1±2.5					Caspase-9DN
			PA1-E6	10.6±3.9	71.2±2.7	The p53 sudden change
SK-OV3	11,8±5.9	47.4 soil 12.1
			Prostate DU145	4.0±1.2	37.0±2.8
PC3	9.0±2.6	48.9±16.8
			Neuroblastoma SK-N-SH	12.4±4.6	51.7 soil 19.1	The Caspase-8 sudden change
SK-SY-5Y	3.7±0.6	68.0±12.3					The Caspase-8 sudden change
			CHLA-79	3.6±0.6	52.5±6.5
Ewing sarcoma: 5838	12.8±3.7	72.1±3.9
			T cell lymphoma	12.1±2.0	77.2±5.8
Rodu T	6.1±2.9	22.2±8.5
			GCT	3.4±1.4	26.6±1.4
Lung A549	5.0±0.8	25.2±13.7					The p53 sudden change
			H441	10.1±4.0	25.4±2.2	The p53 sudden change
Liver: HepG2	9.4±3.1	22.0±10.2
			Normal cell
Breast: MCF-10A	12.8±5.2	19.5±5.9
			Keratinocyte NIK	16.1±8.4	18.2±6.7

Example nine: assessment saposin C/DOPS IC₅₀

[0117] preparation saposin C and DOPS molar ratio is respectively the mixture of 1: 7,1: 3 and 1: 10.Prepare the polypeptide formed by multistage saposin C protein as stated above (referring to (2003) Arch.Biochem.﹠amp that the people showed such as Wang; Biophys.415:45-53 includes them in by reference as a whole at this).Sudden change saposinC polypeptide is as follows: HNSC is made up of aminoacid residue 1-40; H1 is made up of residue 4-20; H-2 then is made up of aminoacid residue 24-40.

[0118] human SK-Mel-28 melanoma cells is cultivated in the flat tissue culture of 96 wells ware.Overwrite media on the cell, this medium contain saposin C, DOPS, the HNSC of various concentration: DOPS (1: 3), H-1: DOPS 1: 3; H-2: DOPS 1: 3; Perhaps total length saposin C and DOPS are by the mixture of 1: 7,1: 3 or 1: 10 mixed.Respectively four SK-Mel-28 cell wares are used each treatment.Use MTT conversion algoscopy (being introduced) to analyze the inhibition effect of pair cell in this paper elsewhere.Use PharmTools Pro computer software (The McCary Group, Elkins Park, PA company product) that data are carried out the substantially linear regression analysis.Analysis result is presented in the table 2.

Table 2SK-Mel-28 melanoma
			IC50
Sample	saposin C	DOPS
			saposin C∶DOPS(1∶7)	19.5±11.0	136.64±78.0
saposin C∶DOPS(1∶3)	99.8±13.0	299.3±39.0
			saposin C∶DOPS(1∶10)	81.3±13.2	813.4±132.3
Only contain sapos in C	786.0±25.4
			Only contain DOPS		14193.4±1886.0
HNSC(saposin C(1-40))∶DOPS(1∶3)	211±32	633±96.0
			H-1 (saposin C spiral-1): DOPS (1: 3)	327±36	981±108.0
H-2 (saposin C spiral-2): DOPS (1: 3)	243±20	729±60.0

Example ten: body inner analysis saposin C-DOPS is to the curative effect of tumor cell

[0119] nude mice is fed by the regulation of the relevant experimental mouse treatment of (U.S.) Cincinnati child study foundation (CincinnatiChildren ' s Research Foundation).Respectively experimental mouse is carried out 2 * 10⁶The last back of SCC subcutaneous injection makes it to take place tumor growth.Allow tumor growth.These animals are used DOPS (2 milligrams/body weight) respectively or contain saposinC (10 milligrams/kilogram) and the medicament of DOPS (2 milligrams/kg body weight) is treated.Treatment 48 hours afterwards, tumor is killed.

[0120] preparation tumor tissue section and make in all sorts of ways and check.

[0121] the deoxyuridine triphosphate salt breach end sign (TUNEL) that uses the terminal deoxynucleotidyl transferase mediation is assessed the apoptosis situation to the tissue slice inspection.(an above-mentioned result of experiment shows in A and the B section of Fig. 5.)

[0122] tissue slice uses hematoxylin and eosin to carry out painted.(an above-mentioned result of experiment shows in C and the D section of Fig. 5.)

[0123] all publications, patent and the patent application of mentioning in the description shows those have experience person to field of the present invention level.All publications, patent and patent application are included in by reference at this, as quoting each publication or patent application particularly, they are included in respectively generally.

[0124] although in order to understand the sake of clarity, by the diagram and the method for giving an example specifying to a certain degree done in aforementioned invention, obviously, in the claim of enclosing, can do some variation and modification.

Sequence table

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＜120〉Saposin C-DOPS PTS

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Gly Pro Val Leu Gly Leu Lys Glu Cys Thr Arg Gly Ser Ala Val Trp

20 25 30

Cys Gln Asn Val Lys Thr Ala Ser Asp Cys Gly Ala Val Lys His Cys

35 40 45

Leu Gln Thr Val Trp Asn Lys Pro Thr Val Lys Ser Leu Pro Cys Asp

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Ile Cys Lys Asp Val Val Thr Ala Ala Gly Asp Met Leu Lys Asp Asn

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Ala Thr Glu Glu Glu Ile Leu Val Tyr Leu Glu Lys Thr Cys Asp Trp

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Leu Pro Lys Pro Asn Met Ser Ala Ser Cys Lys Glu Ile Val Asp Ser

100 105 110

Tyr Leu Pro Val Ile Leu Asp Ile Ile Lys Gly Glu Met Ser Arg Pro

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Gly Glu Val Cys Ser Ala Leu Asn Leu Cys Glu Ser Leu Gln Lys His

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Leu Ala Glu Leu Asn His Gln Lys Gln Leu Glu Ser Asn Lys Ile Pro

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Glu Leu Asp Met Thr Glu Val Val Ala Pro Phe Met Ala Asn Ile Pro

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195 200 205

Gln Thr Ala Val Arg Thr Asn Ser Thr Phe Val Gln Ala Leu Val Glu

210 215 220

His Val Lys Glu Glu Cys Asp Arg Leu Gly Pro Gly Met Ala Asp Ile

225 230 235 240

Cys Lys Asn Tyr Ile Ser Gln Tyr Ser Glu Ile Ala Ile Gln Met Met

245 250 255

Met His Met Gln Pro Lys Glu Ile Cys Ala Leu Val Gly Phe Cys Asp

260 265 270

Glu Val Lys Glu Met Pro Met Gln Thr Leu Val Pro Ala Lys Val Ala

275 280 285

Ser Lys Asn Val Ile Pro Ala Leu Glu Leu Val Glu Pro Ile Lys Lys

290 295 300

His Glu Val Pro Ala Lys Ser Asp Val Tyr Cys Glu Val Cys Glu Phe

305 310 315 320

Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys

325 330 335

Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro Lys Ser

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Leu Ser Glu Glu Cys Gln Glu Val Val AspThr Tyr Gly Ser Ser Ile

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Leu Ser Ile Leu Leu Glu Glu Val Ser Pro Glu Leu Val Cys Ser Met

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Leu His Leu Cys Ser Gly Thr Arg Leu Pro Ala Leu Thr Val His Val

385 390 395 400

Thr Gln Pro Lys Asp Gly Gly Phe Cys Glu Val Cys Lys Lys Leu Val

405 410 415

Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser Thr Lys Gln Glu Ile

420 425 430

Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe Leu Pro Asp Pro Tyr Gln

435 440 445

Lys Gln Cys Asp Gln Phe Val Ala Glu Tyr Glu Pro Val Leu Ile Glu

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Ile Leu Val Glu Val Met Asp Pro Ser Phe Val Cys Leu Lys Ile Gly

465 470 475 480

Ala Cys Pro Ser Ala His Lys Pro Leu Leu Gly Thr Glu Lys Cys Ile

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Trp Gly Pro Ser Tyr Trp Cys Gln Asn Thr Glu Thr Ala Ala Gln Cys

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Asn Ala Val Glu His Cys Lys Arg His Val Trp Asn

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Asp Lys Met Cys Ser Lys Leu Pro Lys Ser Leu Ser Glu Glu Cys Gln

35 40 45

Glu Val Val Asp Thr Tyr Gly Ser Ser Ile Leu Ser Ile Leu Leu Glu

50 55 60

Glu Val Ser Pro Glu Leu Val Cys Ser Met Leu His Leu Cys Ser Gly

65 70 75 80

Claims (24)

Translated fromChinese

1.一种用于制备可促进超扩散性细胞死亡的药物的组合物，其特征是该组合物包含二油基磷脂酰丝氨酸和saposin C相关多肽，其中saposin C相关多肽具有一个氨基酸序列，系选自由下列氨基酸序列组成的群组：1. A composition for preparing a medicine that can promote superdiffusion cell death, characterized in that the composition comprises dioleyl phosphatidylserine and saposin C-related polypeptides, wherein saposin C-related polypeptides have an amino acid sequence, which is selected from the group consisting of the following amino acid sequences:(a)SEQ ID NO：2中所列的氨基酸序列；(a) the amino acid sequence set forth in SEQ ID NO: 2;(b)与SEQ ID NO：2中所列的氨基酸序列的1-40氨基酸残基相同的氨基酸序列；(b) an amino acid sequence identical to 1-40 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2;(c)与SEQ ID NO：2中所列的氨基酸序列的4-20氨基酸残基相同的氨基酸序列；(c) an amino acid sequence identical to 4-20 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2;(d)与SEQ ID NO：2中所列的氨基酸序列的24-40氨基酸残基相同的氨基酸序列；以及(d) an amino acid sequence identical to 24-40 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2; and(e)包含有上述(a)-(d)任一氨基酸序列的氨基酸序列。(e) An amino acid sequence comprising any one of the above-mentioned (a)-(d) amino acid sequences.2.权利要求1所述的组合物，其中多肽与二油基磷脂酰丝氨酸的摩尔比率的范围在大约1∶1至大约1∶50之间。2. The composition of claim 1, wherein the molar ratio of polypeptide to dioleylphosphatidylserine ranges from about 1:1 to about 1:50.3.权利要求2所述的组合物，其中，多肽与二油基磷脂酰丝氨酸的摩尔比率的范围在大约1∶1至大约1∶10之间。3. The composition of claim 2, wherein the molar ratio of polypeptide to dioleylphosphatidylserine ranges from about 1:1 to about 1:10.4.权利要求1中所述的组合物，其特征在于多肽与二油基磷脂酰丝氨酸的质量比率范围在大约15∶1至大约3∶10之间。4. The composition of claim 1, wherein the mass ratio of polypeptide to dioleylphosphatidylserine ranges from about 15:1 to about 3:10.5.权利要求4中所述的组合物，其特征在于多肽与二油基磷脂酰丝氨酸的质量比率大约为5∶1。5. The composition as claimed in claim 4, characterized in that the mass ratio of polypeptide to dioleylphosphatidylserine is about 5:1.6.权利要求4中所述的组合物，其特征在于多肽与二油基磷脂酰丝氨酸的质量比率大约为15∶7。6. The composition of claim 4, wherein the mass ratio of polypeptide to dioleylphosphatidylserine is about 15:7.7.权利要求1中所述的组合物，其特征在于其包含大约10μM的多肽和大约30μM的二油基磷脂酰丝氨酸。7. The composition as claimed in claim 1, characterized in that it comprises about 10 [mu]M polypeptide and about 30 [mu]M dioleylphosphatidylserine.8.权利要求1中所述的组合物，其特征在于其包含大约10μM的多肽和大约70μM的二油基磷脂酰丝氨酸。8. The composition as claimed in claim 1, characterized in that it comprises about 10 [mu]M polypeptide and about 70 [mu]M dioleylphosphatidylserine.9.权利要求1-8任一权利要求所述的组合物，其可以进一步包含一种制药学上可接受的载体。9. The composition of any one of claims 1-8, which may further comprise a pharmaceutically acceptable carrier.10.权利要求1-8任一权利要求所述的组合物，其中所述的促进超扩散性细胞死亡是指促进肿瘤细胞死亡。10. The composition of any one of claims 1-8, wherein said promotion of super-diffusion cell death refers to promotion of tumor cell death.11.权利要求9所述的组合物，其中所述的促进超扩散性细胞死亡是指促进肿瘤细胞死亡。11. The composition of claim 9, wherein said promotion of hyperdiffusion cell death refers to promotion of tumor cell death.12.权利要求1-8任一权利要求所述的组合物，其所述的促进超扩散性细胞死亡是指促进癌症细胞死亡。12. The composition according to any one of claims 1-8, wherein said promotion of super-diffusion cell death refers to promotion of cancer cell death.13.权利要求9所述的组合物，其所述的促进超扩散性细胞死亡是指促进癌症细胞死亡。13. The composition of claim 9, wherein said promotion of super-diffusion cell death refers to promotion of cancer cell death.14.一种组合物在制备促进超扩散性细胞死亡的药物中的应用，其特征在于该组合物包含二油基磷脂酰丝氨酸和saposin C相关多肽，其中saposin C相关多肽具有一个氨基酸序列，系选自由下列氨基酸序列组成的群组：14. The application of a composition in the preparation of a drug for promoting superdiffusion cell death, characterized in that the composition comprises dioleyl phosphatidylserine and a saposin C-related polypeptide, wherein the saposin C-related polypeptide has an amino acid sequence, which is selected from the group consisting of the following amino acid sequences:(a)SEQ ID NO：2中所列的氨基酸序列；(a) the amino acid sequence set forth in SEQ ID NO: 2;(b)与SEQ ID NO：2中所列的氨基酸序列的1-40氨基酸残基相同的氨基酸序列；(b) an amino acid sequence identical to 1-40 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2;(c)与SEQ ID NO：2中所列的氨基酸序列的4-20氨基酸残基相同的氨基酸序列；(c) an amino acid sequence identical to 4-20 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2;(d)与SEQ ID NO：2中所列的氨基酸序列的24-40氨基酸残基相同的氨基酸序列；以及(d) an amino acid sequence identical to 24-40 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2; and(e)包含有上述(a)-(d)任一氨基酸序列的氨基酸序列。(e) An amino acid sequence comprising any one of the above-mentioned (a)-(d) amino acid sequences.15.权利要求14所述的应用，其特征在于其中提到的组合物中含有的saposin C相关多肽和二油基磷脂酰丝氨酸的摩尔比率为1∶1至1∶50之间。15. The application of claim 14, characterized in that the molar ratio of the saposin C-related polypeptide and dioleyl phosphatidylserine contained in the composition mentioned therein is between 1: 1 and 1: 50.16.权利要求14所述的应用，其特征在于其中提到的组合物中含有的saposin C相关多肽和二油基磷脂酰丝氨酸的摩尔比是1∶1至1∶25之间。16. The application according to claim 14, characterized in that the mol ratio of the saposin C-related polypeptide and dioleyl phosphatidylserine contained in the composition mentioned therein is between 1:1 and 1:25.17.权利要求14所述的应用，其特征在于其中提到的组合物中含有的saposin C相关多肽和二油基磷脂酰丝氨酸的摩尔比是1∶1至1∶10之间。17. The application of claim 14, characterized in that the mol ratio of the saposin C-related polypeptide and dioleyl phosphatidylserine contained in the composition mentioned therein is between 1:1 and 1:10.18.权利要求14所述的应用，其特征在于其中提到的组合物中含有的saposin C相关多肽和二油基磷脂酰丝氨酸的摩尔比是1∶10或1∶7或1∶3。18. The application of claim 14, characterized in that the mol ratio of the saposin C-related polypeptide and dioleyl phosphatidylserine contained in the composition mentioned therein is 1:10 or 1:7 or 1:3.19.权利要求14-18任一权利要求所述的应用，其特征在于其中提到的组合物还可以包含有医学上可以接受的载体。19. The use according to any one of claims 14-18, characterized in that the composition mentioned therein may also contain a medically acceptable carrier.20.权利要求14-18任一权利要求所述的应用，其所述的促进超扩散性细胞死亡是指促进肿瘤细胞死亡。20. The use according to any one of claims 14-18, wherein said promotion of superdiffusion cell death refers to promotion of tumor cell death.21.权利要求19所述的应用，其所述的促进超扩散性细胞死亡是指促进肿瘤细胞死亡。21. The use according to claim 19, wherein said promotion of superdiffusion cell death refers to promotion of tumor cell death.22.权利要求14-18任一权利要求所述的应用，其所述的促进超扩散性细胞死亡是指促进癌症细胞死亡。22. The use according to any one of claims 14-18, wherein said promotion of super-diffusion cell death refers to promotion of cancer cell death.23.权利要求19所述的应用，其所述的促进超扩散性细胞死亡是指促进癌症细胞死亡。23. The use according to claim 19, wherein said promotion of super-diffusion cell death refers to promotion of cancer cell death.24.权利要求14-18中任一项的应用，其中所述药物用于治疗肿瘤。24. The use according to any one of claims 14-18, wherein the medicament is for the treatment of tumors.

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