CN102438646A - Treatment of metastatic tumors - Google Patents

Abstract

The present invention is directed to methods and methods for the treatment, inhibition and/or reduction, and detection of metastatic tumors. In some embodiments, the inventive methods include systemic (e.g., intravenous) administration of a chlorotoxin agent that may or may not be labeled. In some embodiments, the inventive methods allow treatment, inhibition and/or reduction, and detection of metastases in the brain. In some embodiments, neovascularization is inhibited and/or newly formed vessels are caused to regress.

Description

The treatment of metastatic tumo(u)r

Related application information

The application's case is advocated U.S. Provisional Application case the 61/053rd; No. 651 (application on May 15th, 2008), the 61/153rd; No. 273 (application on February 17th, 2009) and the 61/173rd; The priority and the rights and interests of No. 121 (applications on April 27th, 2009), the content of each application case all is incorporated herein by quoting in full.

Background technology

Cancerous cell diffusion or the ability that shifts are regarded as the most fatal aspect of cancer.Cancerous cell can break away from primary tumo(u)r, and advances to other part of health via blood flow and/or lymphsystem, forms metastasis.The distance that part is advanced from primary tumor site owing to the quantity and the transfer of the transfer that can form, the treatment of these metastatic tumo(u)rs and diagnosis become a challenge.Modal metastasis site comprises lung, skeleton, liver and brain.The problem that transfer caused that is positioned in the brain is different with the transfer that in other organ of health, forms, because the neuroprotective character of blood/brain barrier stops many effectively diagnostic agents and therapeutic agent delivery is arrived vascular system and nervous tissue.

Scorpion catilan (Chlorotoxin) is a kind of peptide of in huge Israel yellow scorpion (Giant Yellow Israeli scorpion) Israel kills a person the venom of scorpion (Leiurus Quinqestriatus), finding; Through research; At clinical previous crops is with the gliomatous material standed for of 131-iodine targeting (J.A. moral guest people such as (J.A.DeBin), U.S. physiology magazine (Am.J.Physiol.) (cytophysiology (Cell Physiol)), 1993; 264,33:C361-C369; L. rope sieve is just exerted people such as (L.Soroceanu), cancer research (Cancer Res.), 1998,58:4871-4879; S. Shen people such as (S.Shen), neural tumor is learned (Neuro-Oncol.), and 2005,71:113-119).(rope sieve such as just exerts at people, cancer research, 1998,58:4871-4879 to combine the ability of the tumor cell that neural outer embryo originates according to the scorpion catilan; Alder Ritchie people such as (Ullrich), neurological report (Neuroreport), 1996,7:1020-1024; People such as Alder Ritchie, U.S. physiology magazine, 1996,270:C1511-C1521); Developed diagnosis and the neural outer embryo tumor of treatment (for example glioma and meningioma) compositions (referring to United States Patent (USP) the 5th, 905, No. 027 and the 6th, 429; No. 187, the content of each patent all is incorporated herein by quoting in full) and method (referring to United States Patent (USP) the 6th, 028, No. 174 and the 6th; 319, No. 891, the content of each patent all is incorporated herein by quoting in full).

Summary of the invention

The present invention finds, but scorpion catilan targeting metastasis, the transfer that is included in the brain to be found.The present invention finds that also the scorpion catilan can suppress neovascularity and generate, and/or the blood vessel of existing new formation is degenerated.Do not hope to receive the constraint of any particular theory; The present inventor proposes; The scorpion catilan is because it can suppress the formation of neovascularity on the part at the effectiveness of treatment aspect the metastatic cancer at least, and/or the blood vessel of new formation is degenerated, and these neovascularity are considered to shift and are relied on.

On the one hand; The present invention is provided for treating the method for metastatic cancer; It comprises throws the scorpion catilan reagent that gives effective dose to the individuality of suffering from or be prone to suffer from least a transfer; So that said scorpion catilan reagent is incorporated into said at least a transfer, and wherein said transfer is caused by at least a primary tumo(u)r.In certain embodiments, scorpion catilan reagent is systemic delivery; In certain embodiments, scorpion catilan reagent is to send through intravenous.In certain embodiments, primary tumo(u)r is a melanoma, for example skin and/or intraocular melanoma.In certain embodiments, primary tumo(u)r is a glioma.

In another aspect; The present invention is provided for detecting the method for suffering from or once suffering from the existence of one or more transfers in the individual's body of at least a primary tumo(u)r; It comprises to said individual throw give effective dose through the scorpion catilan reagent of labelling and measure in the body of said individuality combination through the scorpion catilan reagent of labelling.In these areas, the combination level is higher than normal (no tumor) tissue in one or more zones in the body except that primary tumor site, with the existence of one or more transfers of indication.In certain embodiments; Throw the scorpion catilan reagent that gives second effective dose through labelling in second time period; Combination with the scorpion catilan reagent of said process labelling in the measure individuals body; To allow assessment bonded any change (for example combination degree and/or position), this can indicate progress, the stability of one or more transfers or degenerate.

Treating and/or detecting among some embodiment of the method that shifts, scorpion catilan reagent is systemic delivery.The whole body dispensing can comprise the intravenous dispensing.In certain embodiments, scorpion catilan reagent combines at least a neoplasm metastasis in the brain.In certain embodiments, neovascularity generates blood vessel (it can the support transfer) degeneration that is inhibited and/or makes new formation.

Description of drawings

Fig. 1 is the microphotograph that the melanoma cells of brain has been transferred in the combination of demonstration biotinylation scorpion catilan.The dyeing situation that microphotograph is described contiguous slices is following: " TM-601 ", also further use the section of C.I. 42590 counterstain with biotinylation scorpion catilan dyeing (the browning reaction product by DAB and biotin detects); " matched group " is only with the painted section of C.I. 42590; " H&E " is with the painted section of h and E.

Fig. 2 is the microphotograph that the melanoma tumor cell of lung has been transferred in the combination of demonstration biotinylation scorpion catilan.The dyeing situation that microphotograph is described contiguous slices is following: " TM-601 ", also further use the section of C.I. 42590 counterstain with biotinylation scorpion catilan dyeing (the browning reaction product by DAB and biotin detects); " matched group " is only with the painted section of C.I. 42590; " H&E " is with the painted section of h and E.

Fig. 3 is the microphotograph of the contiguous slices of following painted normal skin: " TM-601 ", also further use the section of C.I. 42590 counterstain with biotinylation scorpion catilan dyeing (the browning reaction product by DAB and biotin detects); " matched group " is only with the painted section of C.I. 42590; " H&E " is with the painted section of h and E.

Fig. 4 is the intravenous that in the patient who suffers from recurrent or healing property of refractory transitivity entity tumor, carries out ¹³¹The I phase of I-TM-601 form images and safety research in the dosage regimen used.

Fig. 5 suffers from patient's body of dissimilar entity tumors after the dispensing of general introduction intravenous ¹³¹The form that the tumour-specific of I-TM-601 absorbs.

Fig. 6 is presented at suffering from patient's intravenous injection that metastatic prostate cancer and known dispersivity bone shift ¹³¹The γ camera image of I-TM-601 (30mCi/0.6mg) back 3 hours, 24 hours and 7 days records.

Fig. 7 is presented at suffering from patient's intravenous injection of transitivity nonsmall-cell lung cancer ¹³¹The γ camera image of I-TM-601 (30mCi/0.6mg) back 3 hours, 24 hours and 48 hour records.

Fig. 8 (A) explicit declaration suffers from the MRI (left figure) before patient's the treatment of left side frontal lobe lesion of metastatic melanoma, and to this patient's intravenous injection ¹³¹The SPECT image (right figure) of I-TM-601 (30mCi/0.2mg) back record.Fig. 8 (B) explicit declaration suffers from the nuclear magnetic resonance (MRI) (left figure) before patient's the treatment of right side occipital lobe lesion of metastatic melanoma, and to this patient's intravenous injection ¹³¹The SPECT image (right figure) of I-TM-601 (30mCi/0.2mg) back record.

Fig. 9 explicit declaration suffers from the MRI (left figure) before patient's the treatment of left side frontal tumour of glioblastoma, and to this patient's intravenous injection ¹³¹The SPECT scanning (right figure) that obtained in 48 hours behind the I-TM-601.

Figure 10 describes treatment preceding (left figure) and systemic delivery potion 30mCi ¹³¹The MRI image that 3 weeks behind the I-TM-601 (right figure) are obtained from the glioma patient.This patient presents the obvious reduction of gross tumor volume and edema increase.

Figure 11 describes treatment preceding (last figure) and systemic delivery potion 30mCi ¹³¹The MRI image that 3 weeks (figure below) are obtained from another glioma patient behind the I-TM-601.This patient presents the obvious reduction of gross tumor volume and edema increase.

Figure 12 is presented at suffering from patient's intravenous injection of metastatic melanoma ¹³¹The γ camera image of back 24 hours of I-TM-601 (30mCi/0.6mg) and 48 hours (anteposition and position, back) records.Observe in the metastasis of brain, lung, liver and subcutaneous nodule known ¹³¹The absorption of I-TM-601.

Figure 13 describes that (choroidal neovascularization, CNV) test TM-601 suppresses the result that the experiment of angiopoietic ability obtains in the model by generating at mice choroid neovascularity.The gross area (the mm that shows neovascularity (NV) in the animal body that receives TM-601 or normal saline mediator ²* 10 ^-3).(Bruch ' s membrane) broke the same day and the 7th day at the Bu Luheshi film, in the animal of intraocular injection 50 μ g TM-601, observe the significant choroid neovascularity of statistics generate and reduce ( ^*P＜0.05).The 14th day, analyze choroidal injury.

Figure 14 describes by test TM-601 in mice CNV model and causes the result that the experiment of the ability that existing neovascularity is degenerated obtains.The gross area (the mm that shows neovascularity (NV) in the animal body that receives TM-601 or normal saline mediator ²* 10 ^-3)." baseline " is meant the measured value of obtaining in the time of the 7th day in (that is before, with the TM-601 processing) after the Bu Luheshi film rupture.The 7th day, inject within the eye observe in the animal of 50 μ g TM-601 the statistics that the choroid neovascularity generates degenerate significantly ( ^*P＜0.05).For " matched group " and " TM-601 " value, analyzed choroidal injury at the 14th day.

Figure 15 is presented at intravitreal injection TM-601 in the mice choroid neovascularity generation model to cause the hypovascular representative micro-image in vascular site place that produces at induced with laser.When the same day, TM-601 was given in throwing at induced with laser, neovascularity generated and is suppressed (last figure).When TM-601 was given in throwing in 7 days behind induced with laser, existing neovasculature degeneration (figure below).The 14th day, through fluorescein-labeled glucosan, preparation choroid plain film also utilized fluorescence microscopy to all mice perfusions.

Figure 16 be presented in the animal that receives 250 μ g TM-601 periocular injections liquid on the Bu Luheshi film rupture same day and the 7th day the significant choroid neovascularity of statistics generate and reduce ( ^*P＜0.05) experimental result.The 14th day, analyze choroidal injury.

Figure 17 is presented at research the 1st day and the 7th day periocular injections TM-601 suppress the generation of choroid neovascularity with the relevant mode of dosage experimental result.The 14th day, the analysis choroidal injury ( ^*P＜0.05).

Figure 18 is presented at the significant experimental result of degenerating of statistics of the animal median nexus film neovascularity generation that receives TM-601 intravenous injection liquid (3 times weekly, each 20mg/kg dosage).The 14th day, the analysis choroidal injury ( ^*P＜0.05).

The animal median nexus film neovascularity that Figure 19 is presented at intravenous local application TM-601 (3 eye drops every day) generates the experimental result that reduces.The 14th day, analyze choroidal injury.The eye that receives 0.75mg dosage TM-601 every day with receive the normal saline contrast between the difference of NV area reach 0.059 p value.

Figure 20 shows behind the intraocular injection TM-601 the localized frozen section micro-image of TM-601 in the CNV zone.The 7th day injection TM-601 after induced with laser Bu Luheshi film rupture.The 9th day to mice enforcement euthanasia.With rabbit anti--TM-601 (redness among A, B and the C) dyes to frozen section with GSA agglutinin (green among D, E and the F) so that endotheliocyte it is thus clear that.Inject the eye (B, E and H) of mediator and in the CNV zone, do not show the positive staining cell without the eye of injecting (C, F and an I).On the contrary, the hang oneself section (A, D and G) of the eye that TM-601 handles shows the obvious dyeing (A and D) that is directed against TM-601 in whole C NV zone.The delegation's exhibit red and the green painted registration (G, H and I) of bottom.Arrow indication CNV zone.

Figure 21 shows behind the periocular injections TM-601 the localized frozen section micro-image of TM-601 in the CNV zone.Injection TM-601 implements euthanasia to mice, and as shown in Figure 16 frozen section is dyeed.(also referring to material in the instance 6 and method).Utilize rabbit anti--dyeing that TM-601 carries out shown in the redness among A, B and the C, and utilize dyeing (being used for manifesting endotheliocyte) that the GSA agglutinin carries out shown in D, E and F green.Inject the eye (B, E and H) of mediator and in the CNV zone, do not show the positive staining cell without the eye of injecting (C, F and an I).On the contrary, the hang oneself section (A, D and G) of the eye that TM-601 handles shows the obvious dyeing (A and D) that is directed against TM-601 in whole C NV zone.The delegation's exhibit red and the green painted registration (G, H and I) of bottom.Arrow indication CNV zone.

Figure 22 shows the micro-image of intraocular injection TM-601 to the frozen section of the influence of CNV pathological changes inner cell apoptosis.Injection TM-601, and said like Figure 16 to mice enforcement euthanasia.(also referring to material in the instance 6 and method).Utilize nuclear staining (A, B) GSA (C, D) and TUNEL (E, F) to section statining.The delegation of bottom shows three kinds of painted registrations (G, H).There is TUNEL positive cell (E) in the middle discovery of eye (A, C, E and G) of injecting TM-601 within the eye in the CNV pathological changes.In the eye of injection mediator (B, D, F and H), do not observe the TUNEL positive cell.Arrow indication CNV zone.

Figure 23 shows the micro-image of periocular injections TM-601 to the frozen section of the influence of CNV pathological changes inner cell apoptosis.Injection TM-601, and said like Figure 16 to mice enforcement euthanasia.(also referring to material in the instance 6 and method).Utilize nuclear staining (A, B), GSA (C, D) and TUNEL (E, F) to section statining.The delegation of bottom shows three kinds of painted registrations (G, H).In the eye (A, C, E and G) of periocular injections TM-601, find in the CNV pathological changes, to exist TUNEL positive cell (E).In the eye of injection mediator (B, D, F and H), do not observe the TUNEL positive cell.Arrow indication CNV zone.

The result that the experiment of HUVEC cell proliferation obtained when Figure 24 described by measure in the presence of multiple TM-601 concentration maintenance 72 hours or 120 hours.Although low under low TM-601 concentration at cell proliferation speed ratio under the higher TM-601 concentration, its multiplication rate is not less than undressed control cells.

Figure 25 describes by the result who shows that choroidal neovasculature (being induced by the laser Bu Luheshi film that breaks) and the middle TM-601 of amphiblestroid neovasculature (ischemia by oxygen brings out is induced) and the common localized experiment of annexin A2 (Annexin A2) obtain.Intraocular injection TM-601 uses anti--TM-601 antibody and anti--annexin A2 antibody that tissue slice is carried out immunohistochemical analysis subsequently.

Figure 26 describes and shows that TM-601 suppresses the HUVEC cell migration and reduces the active experimental result of MMP-2.(A) utilize 50ng/ml VEGF to stimulate the HUVEC cell migration.Add TM-601 and suppress migration (as in Transwell analyzes, being assessed) by invasion and attack with the relevant mode of dosage.Use Ji's Albert'stain Albert liquid (Giemsa stain) to be apparent in the cell of the invasion and attack on the little chamber lower surface of Transwell.(B) calculating is moved by VEGF or bFGF (50ng/ml) institute stimulated cells by the visual cell counting, and through showing, 10 μ M TM-601 can make HUVEC invasion and attack inhibition about 50% in the Transwell cell.(C) MMP-2 that in culture medium, is obtained by warp cultivation HUVEC cell unprocessed, that handle or handle with 10 μ M TM-601 through bFGF through bFGF is active.Error bar indication standard error.

Figure 27 describes and shows that TM-601 reduces the secreted active experimental result of MMP-2 of U87 human glioma cell.Measure in the culture medium by unprocessed or add the active of 10 μ M TM-601 through cultivating the MMP-2 that the U87 cell obtains.

Figure 28 is presented at through Pegylation scorpion catilan (TM-601-PEG) in the non-cancer mice of intravenous injection and comparison without half-life of the TM-601 that modifies.Pegylation makes the half-life of TM601 increase to about 32 times.

Figure 29 is presented in the mice CNV model, and Pegylation TM-601 can be than realizing blood vessel formation against function without the low administration frequency of the TM-601 that modifies.Be plotted in the CNV model for figure without the microvessel density of the different dosing regimes of TM-601 that modifies or Pegylation TM-601.

The specific embodiment

Definition

To define several terms that this description is used in the following paragraph in the whole text.

Unless otherwise mentioned; Or from context, may be obvious that in addition, otherwise the term that this paper uses when mentioning numeral " (about) approximately " and " approximately (approximately) " are included in numeral in 20%, 10%, 5% or 1% scope of arbitrary direction (being greater than or less than said numeral) of said numeral (except that 100% the situation of said numeral above probable value) in this article.

The term " annexin A2 " that this paper uses be meant Entrez gene bank inventory ( Http:// www.ncbi.nlm.nih.gov) in the official code name be ANXA2 (in homo sapiens (Homo sapiens)) and official's full name protein (the multiple sequence of ANXA2 transcript can for example be shown in gene bank and go into to hide under registration number M62899, NM_001002857, NM_001002858, the NM_004039) for the gene of " annexin A2 ".Wherein, annexin A2 is also referred to as " annexin II " and lipocortin 2 (lipocortin 2).

Term " biological activity " is when being used to characterize polypeptide in this article; Be meant that a kind of molecule and parent polypeptide have enough consensus amino acid sequence property; (for example represent the characteristic similar or identical thus with said polypeptide; Specificity combines cancerous cell, and/or internalization entering cancerous cell, and/or the ability of kill cancer cell).

The term " cancer " that this paper uses is meant or describes the physiology's patient's condition that is grown to characteristic in the mammal usually with not modulated cell.The instance of cancer includes, but is not limited to carcinoma, lymphoma, blastoma, sarcoma and leukemia.Say that more specifically the instance of said cancer comprises pulmonary carcinoma, osteocarcinoma, hepatocarcinoma, cancer of pancreas, skin carcinoma, head or neck cancer, skin or intraocular melanoma, uterus carcinoma, ovarian cancer, rectal cancer, anal regions cancer, gastric cancer, colon cancer, breast carcinoma, uterus carcinoma, sexual organ and genitals's carcinoma, lymphogranulomatosis (Hodgkin ' s Disease), esophageal carcinoma, carcinoma of small intestine, hormonal system cancer, thyroid carcinoma, parathyroid gland cancer, adrenal carcinoma, soft tissue sarcoma, bladder cancer, renal carcinoma, renal cell carcinoma, renal pelvis carcinoma, central nervous system (CNS) tumor, neuroderm cancer, tumor of spine, glioma, meningioma and pituitary adenoma.

The term " cancerous cell " of this paper use is meant that the undesirable and not modulated cell growth of mammal (for example human) the interior experience of body perhaps retains unusually or the cell of abnormal structure's invasion and attack.External, it can unrestricted proliferating cells be that it is a kind of cell culture of being set up of permanent immortalization with not modulated mode that this term is also showed when giving suitable fresh culture and space.

The term " cancer patient " that this paper uses is meant to suffer from or cancer-prone individuality.The cancer patient can be through making a definite diagnosis or can suffering from cancer without making a definite diagnosis.This term also comprises the individuality of previous experience cancer therapy.

Term " chemotherapeutics " and " anticarcinogen or anticarcinogen " interchangeable in this article use.These terms are meant the medicament that is used to treat the cancer or the carcinous patient's condition.Anticarcinogen is classified as in one in the following group usually: alkylating agent class, purine antagonist class, pyrimidine antagonist class, plant bases, the property inserted antibiotics (intercalating antibiotics), aromatase inhibitor class, antimetabolite class, mitotic inhibitor class, growth factor receptor inhibitors class, cell cycle inhibitor class, enzyme, topoisomerase enzyme inhibitor class, BRM class, hormone antagonist class and antiandrogen class.The instance of said anticarcinogen includes, but is not limited to BCNU; Cisplatin (cisplatin); Gemcitabine (gemcitabine); Hydroxyurea (hydroxyurea); Paclitaxel (paclitaxel); Temozolomide (temozolomide); TPT (topotecan); Fluorouracil (fluorouracil); Vincristine (vincristine); Vincaleucoblastine (vinblastine); Procarbazine (procarbazine); Dacarbazine (decarbazine); Hexamethyl melamine (altretamine); Methotrexate (methotrexate); Purinethol (mercaptopurine); Thioguanine (thioguanine); Fludarabine phosphate (fludarabine phosphate); Cladribine (cladribine); Pentostatin (pentostatin); Cytosine arabinoside (cytarabine); Azacitidine (azacitidine); Etoposide (etoposide); Teniposide (teniposide); Irinotecan (irinotecan); Docetaxel (docetaxel); Doxorubicin (doxorubicin); Daunomycin (daunorubicin); Dactinomycin (dactinomycin); Darubicin (idarubicin); Plicamycin (plicamycin); Mitomycin (mitomycin); Bleomycin (bleomycin); Tamoxifen (tamoxifen); Flutamide (flutamide); Leuproside (leuprolide); Goserelin (goserelin); Aminoglutethimide (aminogluthimide); Anastrozole (anastrozole); Amsacrine (amsacrine); Asparaginase (asparaginase); Mitoxantrone (mitoxantrone); Mitotane (mitotane) and amifostine (amifostine).

The term " combination treatment " that this paper uses is meant that two or more different medicaments give with eclipsed scheme throwing, so that the person under inspection is exposed to the situation of both sides' medicament simultaneously.

The abbreviation " CTCAE " that this paper uses is meant common adverse events evaluation criterion (Common Terminology Criteria for Adverse Events); It is to be directed against the standard that adverse events (AE) is described and classification is developed by the American National ICR, is usually used in the clinical trial.

Term " cytotoxicity " refers to that a kind of part, chemical compound, medicine or reagent can suppress or stop the function of cell and/or cause cytoclasis when being used to characterize a kind of part, chemical compound, medicine or reagent in this article.

Term " dosage regimen " when being used for this paper, refer to through the some time section separately and independent the throwing give one group of UD (a common above dosage).One group of RD of particular agent (that is, dosage, time-histories, approach etc.) constitutes its dosage regimen.

The term " effective dose " that this paper uses and " effective dose " are meant is enough under acceptable benefit/risk ratio, realize the chemical compound of predetermined purpose (that is required biology or medical response among tissue or the person under inspection) or any amount or the dosage of compositions.Give an example, in certain embodiments of the invention, said purpose can be: specificity is incorporated into destination organization; Slow down or stop cancer symptoms progress, increase the weight of or worsen; Cancer symptoms is improved; And/or healing cancer.Relevant predetermined purpose can be objectively (that is, can measure through some tests or label) or subjective (that is, the person under inspection provides the indication of curative effect or feels curative effect).The general throwing by the dosage regimen that can comprise a plurality of UDs given the treatment effective dose.For any particular agent, treatment effective dose (and/or the suitable UD in effective dosage regimen) can for example depend on dosing way, change with the combination of other medicament.In certain embodiments, the concrete treatment effective dose (and/or UD) of any particular patient will depend on multiple factor, comprise the disease of being treated and the order of severity of disease; The activity of used particular agent; Used particular composition; Patient's age, body weight, general health situation, sex and diet; The dispensing time; Dosing way; And/or the drainage of used particular agent or metabolic rate; The time that treatment continues; And well-known similar factor in the medical domain.

The term " fluorogen " that this paper uses, " part fluoresces ", " fluorescent labeling ", " fluorescent dye " and " fluorescent labeling part " interchangeable in this article use.These terms are meant in solution and the molecule of emission light echo after with the optical excitation of appropriate wavelength.Many fluorescent dyes with multiple structure and characteristic are applicable to puts into practice the present invention.Similarly; The method and the material that become known for fluorescent labeling nucleic acid are (referring to the Glan (R.P.Haugland) suddenly of R.P. for example; " molecular probe: fluorescent probe and research chemical handbook 1992-1994 (Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals 1992-1994) ", the 5th edition, 1994; Molecular probe company (Molecular Probes, Inc.)).When selecting fluorogen; Usually need fluorescence molecule absorbing light and emitting fluorescence (that is) efficiently respectively with high molar absorption coefficient and fluorescence quantum yield, and be fast to light (promptly; After optical excitation, obvious degradation can not take place analyzing in the essential time in said fluorescence molecule).

The term " fusion rotein " that this paper uses is meant and comprises two or more protein or its fragment and utilize the covalent bond warp molecule of peptide main chain connection separately that said protein or its fragment most preferably are to produce through these proteinic polynucleotide molecules of gene expression coding.

The term " homology " (or " homology ") that this paper uses be meant between two peptide molecules or two nucleic acid molecules between the degree of consistency.When a position in two comparative sequences was occupied by identical base or amino acid monomer subunit, then individual molecule was in this position homology.Percent homology between two sequences equals the total coupling of two sequences or with the quantity of the source position quantity divided by the position of being compared, and multiply by 100.Generally speaking, when two sequences of aligning reach maximum homology, compare.The total consistent or similar amino acid residue of homologous amino acid sequence.Similar residue is that the conservative of corresponding amino acid residue in the canonical sequence replaces, or " point mutation of allowing "." the conservative replacement " of residue is physically or is similar to corresponding replacement with reference to residue on the function in the canonical sequence, and for example, it has similar size, shape, electric charge, chemical characteristic, comprises the ability of formation covalent bond or hydrogen bond etc.In certain embodiments; It is replacement (" protein sequence and structure atlas (Atlas of Protein Sequence and Structure) ", 1978, the nature-biomedical research basis (Nat.Biomed.Res.Foundation) of satisfying the standard of wearing artificial " acceptable point mutation " definition such as Hough (Dayhoff) that the used conservative of the present invention replaces; Washington (Washington; DC), the 3rd [, 22:354-352).

Term " individuality " and " person under inspection " can exchange use in this article.These terms are meant to suffer from disease or disease (for example cancer), perhaps easy human or another mammal (for example mice, rat, rabbit, Canis familiaris L., cat, cattle, pig, sheep, horse or primate) that suffers from but possibly have or not have said disease or disease.In many examples, the person under inspection is human.In many examples, the person under inspection is the patient.Unless otherwise mentioned, otherwise term " individuality " and " person under inspection " are not meant given age, contain adult, child and neonate thus.

The term " inhibition " that this paper uses is meant that the prevention something appearance of something generation takes place, delays and/or reduces degree or the probability that something takes place.Therefore, " suppress shift " is intended to contain prevention, the generation that delays to shift with " suppress shift formation " and/or reduces and shift the probability that takes place, and the quantity, growth rate, size etc. that reduce transfer.

The term that uses among this paper " initial " can be used for referring to the person under inspection who did not before receive medicament thrown first and gives this medicament when being used for dosage regimen.Perhaps or in addition, term " initial " can be used for referring to during patient's therapy, throwing the medicament that gives specific unit dosage.

Term " through labelling " and " but through detectable or part labelling " interchangeable in this article use, refering in particular to a kind of entity (for example scorpion catilan or scorpion catilan concatenator) for example can visual observations after being incorporated into another entity (for example neoplasm tissue).But preferred detectable or part be through selecting, make its produce can be measured signal, and signal intensity with the amount relevant (for example proportional) of bonded entity.The known multiple system that is used for labelling and/or detects protein and peptide in this technology.Through incorporating into or being linked to the labelling that spectroscope capable of using, photochemistry, biochemistry, immunochemistry, electricity, optics, chemistry or alternate manner detect, can make protein and peptide through labelling.Labelling or mark part can be that (that is, it can detect it without any need for other reaction or operation, for example in direct detection; Fluorogen can directly detect), or it can be that indirect detection is (that is, through detecting the entity reaction with another or combining; It can be detected; For example, hapten with comprise like reportorial suitable antibody responses such as fluorogens after, can detect through immunostaining).But the detectable that is fit to includes, but is not limited to radionuclide, fluorogen, chemical illuminating reagent, microgranule, enzyme, colourity labelling, magnetic mark, hapten, molecular beacon (Molecular Beacon), fit beacon etc.

The term " transfer " (being abbreviated as " mets " sometimes) that this paper uses is meant that tumor cell diffuses to another position from an organ or tissue.This term also refers to because of the tumor tissues of transfer in reposition formation." metastatic cancer " is the cancer from initial position or former position diffusion, and also can be described as " secondary cancer " or " secondary tumor ".Generally speaking, metastatic tumo(u)r is the tissue name with the primary tumo(u)r that is originated from.Therefore, be in the lung, also can be described as " transitivity breast carcinoma " even transfer to some cancerous cell of breast carcinoma of pulmonary.

The term " neovasculature " that this paper uses is meant as yet the blood vessel of not full ripe new formation, that is, it does not have the cell that is completed into and connects endotheliocyte liner or complete layer of smooth muscle cells on every side closely.The term " neovascularity " that this paper uses is used in reference to the blood vessel in the neovasculature.

Term " normally " and " health " can be exchanged use in this article.These terms are meant body one by one or the one group of individuality with tumor.Term " normally " also is used for limiting from healthy individuals isolated tissue sample in this article.

Term " medicament ", " therapeutic agent " and " medicine " interchangeable in this article use.These terms are meant effective treatment, inhibition and/or detect material, molecule, chemical compound, reagent, the factor or the compositions of disease, disease or the clinical patient's condition.

" pharmaceutical composition " is defined as the compositions of at least a active component that comprises effective dose (for example, but can through the scorpion catilan or the scorpion catilan concatenator of labelling or un-marked) and at least a pharmaceutically acceptable supporting agent in this article.

The term that uses among this paper " pharmaceutically acceptable supporting agent " be meant not can the interferon activity composition bioactive effectiveness and dispensing the time concentration can not produce the supporting agent medium of excessive toxicity to the host.This term comprises solvent, disperse medium, coating, antibacterium and antifungal, isotonic agent, absorption delay agent etc.The use that is suitable for this type of medium and the reagent of pharmaceutically active substance in this technology as everyone knows is (referring to for example " Lei Mingdunshi pharmacy complete works of (Remington ' s Pharmaceutical Sciences) "; E.W. Martin (E.W.Martin); The 18th edition, 1990, Merck publishing company (Mack Publishing Co.): Pennsylvania Easton (Easton; PA), it is incorporated herein by quoting in full).

The term " primary tumo(u)r " that this paper uses is meant that promptly, the other places person is relative with being diffused in the tumor in the initial initial site that produces of tumor.

Term " protein ", " polypeptide " and " peptide " interchangeable in this article use, and refer to the aminoacid sequence of all lengths, its (changing) form or salt form that can be neutral, and without modifying or through glycosylation, oxide side chain or phosphorylation modification.In certain embodiments, aminoacid sequence is the total length native protein.In other embodiments, aminoacid sequence be full length protein than small fragment.In other embodiments, aminoacid sequence is modified by extra substituent group that is connected to amino acid side chain (for example glycosyl units, lipid, or inorganic ions are like phosphate radical) and the modification that relates to the chemical conversion (for example, sulfhydryl oxidase) of chain.Therefore, the aminoacid sequence of the total length native protein that lives through the modification that does not change its particular characteristics planned to comprise in term " protein " (or its suitable term).Specifically, term " protein " is contained proteinic with the merit iso series, that is, and and by homologous genes coding, but pI or MW or variant that the two is different.Roughly the same the aminoacid sequence of merit iso series can different (for example being produced by substituting montage or limited proteolysis) for these, perhaps saidly can stem from differentiated post translational modification (for example glycosylation, acylated or phosphorylation) with the merit iso series.

The term " protein analogue " that this paper uses is meant that function is similar with parent polypeptide or consistent but may not comprises the aminoacid sequence similar or consistent with parent polypeptide, perhaps structure and the similar or consistent polypeptide of parent polypeptide.Preferably in context of the present invention, protein analogue has and the consistent aminoacid sequence of the aminoacid sequence 30% of parent polypeptide (more preferably at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%) at least at least at least at least at least at least at least at least at least at least at least at least at least at least.In addition, one of ordinary skill in the art should be understood that protein sequence is generally allowed and do not destroy active some replacement.Therefore; Term " protein analogue) in contain and keep the active of parent polypeptide and total at least about 30-40%, whole sequence identity usually above about 50%, 60%, 70% or 80% with parent polypeptide; and it is higher to comprise that in the zone of one or more high conservatives at least one and parent polypeptide have usually in addition, usually above any polypeptide in 90%, 96%, 97%, 98% or 99% conforming zone (contain usually at least 3 to 4 and reach 20 or 20 usually with upper amino acid).

The term " protein fragments " that this paper uses is meant the polypeptide of the aminoacid sequence that comprises at least 5 amino acid residues in second amino acid sequence of polypeptide.Proteinic fragment can have or can not have the functional activity of parent polypeptide.

Term " degeneration " when being used in reference to blood vessel and/or vascular system when (comprising neovasculature and/or neovascularity), is used in reference to contraction in this article, dwindles etc.

The term " micromolecule " that this paper uses comprises and can work and influence any chemistry or other part of bioprocess.Micromolecule can comprise current known and the numerous therapeutic agent that uses, and perhaps can be with the synthetic micromolecule of molecular library form from the purpose of screening biological function.Micromolecule is distinguished with macromole dimensionally mutually.Be applicable to that (dalton Da), preferably less than about 2,500Da, is more preferably less than 1, and 000Da is most preferably less than about 500Da less than about 5,000 dalton usually for micromolecular molecular weight among the present invention.

The term that this paper uses " is prone to trouble " and is meant for some things (promptly; Disease, disease or the patient's condition, for example metastatic cancer) risk and/or tendentiousness (normally based on hereditary inducement, environmental factors, individual medical history or its combination) be higher than viewed result in general groups.This term considers that the individuality of " be prone to suffer from " certain patient's condition possibly never make a definite diagnosis and suffer from this patient's condition.

The term " whole body dispensing " that this paper uses is meant that the dispensing of medicament makes a large amount of said medicaments extensively distribute in vivo, and in blood, has biological agent (for example its predetermined action) and/or arrive its predetermined action site through vascular system.Typical whole body dosing way comprises that through (1) medicament directly being introduced vascular system or (2) per os, lung or intramuscular throws to give and offer medicine; Under the situation of (2); Said medicament is absorbed, the intravasation system and via the blood carrying to one or more predetermined actions site.

Term " tissue " uses with its broad sense in this article.Tissue can be any biological entities that comprises (but nonessential) tumor cell.Under situation of the present invention, Considering Body is outer, body is interior and in vitro tissue.Therefore, tissue can be an individual part, perhaps can obtain (for example by biopsy) from individuality.Tissue can also comprise tissue slice, like the frozen section that obtains from histology's purpose; Perhaps known diagnosis, treatment and/or the file sample of medical history as a result.Term is organized and is also contained any material that derives through the processed group tissue samples.Deutero-material includes, but is not limited to the cell (or its filial generation) from separate tissue.The processing of tissue sample can relate to following one or more: filter, distillation, extraction, concentrate, the deactivation of interference component, the interpolation of reagent etc.

Term " treatment " is used for characterizing to following purpose method or process in this article: (1) delays or the outbreak of prevent disease, disease or the patient's condition; (2) slow down or stop one or more symptoms of disease, disease or the patient's condition progress, increase the weight of or worsen; (3) make the remission of disease, disease or the patient's condition; (4) order of severity or the sickness rate of reduction disease, disease or the patient's condition; Or (5) cure diseases, disease or the patient's condition.Treatment can be thrown before disease, disease or patient's condition outbreak and given, and realizes prevention or prevents effect.Perhaps or in addition, treatment can be thrown and given, realize therapeutical effect after disease, disease or the patient's condition are initial.

The specific embodiment

Once mentioned like preceding text, the present invention is directed to the method that is used to treat and/or detect neoplasm metastasis.But, the method that provides among this paper gives labelling or without the scorpion catilan reagent of test section labelling through the test section but comprising to throw substantially.In certain embodiments, scorpion catilan reagent is incorporated into neoplasm metastasis.In certain embodiments, scorpion catilan reagent suppresses and/or reduces the probability of new transfer formation.In certain embodiments, scorpion catilan reagent is that whole body (for example intravenous) throwing is given, and/or scorpion catilan reagent strides across blood/brain barrier.Therefore, in certain embodiments, the present invention provides treatment, suppresses and/or detect the method for the transfer that is arranged in brain.In certain embodiments, the formation of neovascularity is able to suppress and/or existing neovasculature is degenerated.

According to the present invention, can use conventional molecular biology, microbiology and recombinant DNA technology in this technology.These technology complete description all in document.Referring to for example Man Nitisi (Maniatis), Fu Leiqi (Fritsch) and Sa Brooker (Sambrook), " molecular cloning: laboratory manual (Molecular Cloning:A Laboratory Manual) ", 1982; " dna clone: hands-on approach (DNA Cloning:A Practical Approach), " I and II volume, D.N. lattice labor fragrance (D.N.Glover) (volume), 1985; " synthetic (the Oligonucleotide Synthesis) of PDT16 ", M.J. Gai Te (M.J.Gait) (volume), 1984; " nucleic acid hybridization (Nucleic Acid Hybridization) ", B.D. Hai Musi (B.D.Hames) and S.J. Hai Jisi (S.J.Higgins) (volume), 1985; " transcribe and translate (Transcription and Translation) ", B.D. Hai Musi and S.J. Hai Jisi (volume), 1984; " animal cell culture (Animal Cell Culture) ", R.I. Fu Leixili (R.I.Freshney) (volume), 1986; " fixed cell and enzyme (Immobilized Cells And Enzymes) ", IRL publishing house (IRL Press), 1986; B. wear precious (B.Perbal) " molecular cloning practice guideline (A Practical Guide To Molecular Cloning) ", 1984.

I. scorpion catilan reagent

Treatment of the present invention and/or detection method to individuality that needs are arranged (for example relate to; Suffer from least a transfer, once suffer from least a transfer, the risk of at least a transfer of development is arranged and/or be prone to suffer from the individuality of at least a transfer) throw at least a scorpion catilan reagent that gives effective dose so that said scorpion catilan reagent is incorporated into said at least a transfer.The term " scorpion catilan reagent " that this paper uses is meant the chemical compound that comprises at least one scorpion catilan part.In certain embodiments, scorpion catilan reagent comprises at least one scorpion catilan part and at least one treatment part (for example anticarcinogen) association.Scorpion catilan part (and/or treatment part) can be associated with at least one mark part.

A. scorpion catilan part

The term " scorpion catilan part " that this paper uses is meant scorpion catilan, biological activity scorpion catilan subunit or scorpion catilan derivant.

In certain embodiments; Term " scorpion catilan " is meant and derives from kill a person 36 amino acid whose total length natural polypeptides (the De Bin people such as (DeBin) that have of scorpion venom of Israel; U.S. physiology magazine (Am.J.Physiol), 1993,264:C361-369); It comprises the aminoacid sequence of the natural scorpion catilan described in No. 2003/101474 SEQ ID of international application case WO NO.1, and the content of said application case is incorporated herein by quoting.Term " scorpion catilan " comprises the polypeptide that comprises SEQ ID NO.1, and it is for synthetic or the reorganization generation, and for example United States Patent (USP) the 6th, 319, disclosed sequence in No. 891 (they are incorporated herein by quoting in full).

" biological activity scorpion catilan subunit " is to comprise in the scorpion catilan less than 36 aminoacid and keep the peptide of at least a characteristic of scorpion catilan or function." characteristic or the function " of the scorpion catilan that this paper uses includes, but is not limited to make the ability of abnormal cell growth stagnation; Compare normal cell, specificity is incorporated into the ability of lesion/cancer cell; Compare normal cell, specificity be incorporated into the lesion/cancer cell that shifting or shift in the ability of lesion/cancer cell; Ability in internalization to the lesion/cancer cell; The ability of kill tumor/cancerous cell; And/or the ability that suppresses neovascularization and/or neovascularity is degenerated.Said lesion/cancer cell can be in external, stripped, external, the part that shifts, from former generation separator, the cultured cells of individuality, or cell line.

The term " biological activity scorpion catilan derivant " that this paper uses is meant any in multiple derivant, analog, variant, polypeptide fragment and the analogies of scorpion catilan and related peptides, at least a characteristic or the function (as indicated above) of its reservation scorpion catilan.The instance of scorpion catilan derivant includes, but is not limited to the peptide variant of scorpion catilan; The fragments of peptides of scorpion catilan; For example comprise described in No. the 2003/101474th, the international application case among the SEQ ID No.1,2,3,4,5,6 or 7 adjacent 10 aggressiveness (10-mer) peptide or form, or comprise the residue 10-18 of SEQ ID No.1 described in No. 2003/101474, the international application case WO or the fragment of 21-30 by it; The core binding sequence; And peptide mimics.

The instance of scorpion catilan derivant comprise aminoacid sequence described in the SEQ ID No.1 with No. 2003/101474, international application case WO fragment, have at least about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30 or about 35 peptides with the active relevant adjacent amino acid residues of scorpion catilan.Said fragment can contain the functional areas of scorpion catilan peptide, and it is differentiated to be the aminoacid sequence district corresponding to the known peptide domain; And has an obvious hydrophilic zone.Said fragment can also comprise two core sequences that connect with any order each other, wherein inserts aminoacid and has been removed or has replaced through bridging agent.

Scorpion catilan derivant comprises the conservative or substituted polypeptide of non-conservation that when on to greatest extent, aiming at derivant sequence and scorpion catilan sequence, comprises at least one amino acid residue.Said replacement can strengthen at least a characteristic or the function of scorpion catilan; At least a characteristic or the function that suppress the scorpion catilan; Or it is neutral at least a characteristic or the function of scorpion catilan.

Be applicable to the case description (it is incorporated herein by quoting in full) in No. 2003/101474, international application case WO of putting into practice scorpion catilan derivant of the present invention.Particular instance comprises the polypeptide that comprises the SEQ ID NO.8 described in this international application case or SEQ ID NO.13 or be made up of said sequence, with and variant, analog and derivant.

Other instance of scorpion catilan derivant comprises and contains the predetermined sudden change that causes by means of for example homologous recombination, direct mutagenesis or PCR mutation, and the allele of peptide family or the polypeptide of other naturally occurring variant; And said peptide utilized except that natural exist part the aminoacid (but for example test section, like enzyme or radiosiotope) through replace, the derivant of chemistry, enzyme or other suitable mode covalent modification.

The scorpion catilan can use any preparation in the known several different methods in this technology with its peptide derivant, comprises standard solid-phase (or solution phase) peptide synthetic method.In addition, the nucleic acid of these peptides of encoding can use commercially available PDT16 synthetic instrument synthetic, and said protein can produce in the reorganization of use standard reorganization manufacturing system.

Other scorpion catilan derivant that is fit to comprises the peptide mimics of the three dimensional structure of simulation scorpion catilan.Said peptide mimics can have the obvious benefit that is superior to naturally occurring peptide, comprise for example make more economy, chemical stability is higher, pharmacological property (half-life, absorption, effectiveness, effect etc.) strengthens, specificity changes (for example broad-spectrum biological activity, antigenicity reduction etc.).

In certain embodiments, analogies are molecules of the element of simulation scorpion catilan peptide secondary structure.Proteinic peptide main chain is mainly used in directed in a certain way amino acid side chain, so that promote the interaction of molecule, for example antibody and antigenic interaction.The expection peptide mimics will allow and the similar interaction of molecules of natural molecule.Peptide analogues generally is used for pharmaceuticals industry, is similar to the non-peptide medicine of template peptide like property class.The chemical compound of these types be also referred to as peptide mimics (peptide mimetics) or intend peptide class (peptidomimetics) (referring to for example Fa Qieer (Fauchere), drug research progress (Adv.Drug Res.), 1986,15:29-69; Weber (Veber) and Fu Leidijie (Freidinger), 1985, neuroscience trend (Trends Neurosci.), 1985,8:392-396; Yi Wansi people such as (Evans), medical chemistry magazine (J.Med.Chem.), 1987,30:1229-1239), and develop by means of computer molecular simulation usually.

Generally speaking, the similar of peptide mimics joins displacement but wherein one or more peptide bindings are optional through non-peptide bond in example polypeptide (paradigm polypeptide) (that is, having the polypeptide of biochemical characteristic or pharmacologically active).Through using combinatorial chemistry to produce drug reservoir, can strengthen the use of peptide mimics.Through differentiating the bonded amino acid mutation that increases or reduce peptide and for example tumor cell, can assist the design of peptide mimics.Methods availalbe comprise yeast two-hybrid method (yeast two hybrid method) (referring to for example strange grace (Chien) people of etc.ing, institute of NAS periodical (Proc.Natl.Acad.Sci.USA), 1991,88:9578-9582) and the use phage present method.Protein protein interaction among the said double cross method detectable enzyme mother (Field people such as (Field), nature (Nature), 1989,340:245-246).Phage appear method can detect fixing protein and as the interaction between the protein expressed on the phage surfaces such as λ and M13 (Amberg people such as (Amberg), the strategy (Strategies), 1993,6:2-4; Huo Gefei people such as (Hogrefe), gene (Gene), 1993,128:119-126).These methods allow just to select and bear and select peptide-protein interaction can measure these interactional sequences with differentiating.

In certain embodiments, scorpion catilan reagent comprises the polypeptide toxin that presents the active another kind of Scorpio similar or relevant with above-mentioned scorpion catilan.The term " activity similar or relevant with the scorpion catilan " that this paper uses refers in particular to the selectivity/specificity of lesion/cancer cell and combines.The instance of the relevant Scorpio toxin that is fit to includes, but is not limited to have with the scorpion catilan toxin or the related peptides in aminoacid and/or the conforming Scorpio of nucleotide sequence source.The instance of relevant Scorpio toxin includes, but is not limited to the CT neurotoxin (gene bank (GenBank) goes into to hide registration number AAD473730) from Sichuan scorpion (Mesobuthus martenssi); Neurotoxin BmK 41-2 (gene bank goes into to hide registration number A59356) from Scorpio (card is uncommon) (Buthus martensii karsch); Neurotoxin Bm12-b (gene bank goes into to hide registration number AAK16444) from Scorpio (Buthus martensii); From possible toxin (Probable Toxin) LGH 8/6 (gene bank goes into to hide registration number P55966) of the yellow scorpion of Israel (Leiurus quinquestriatus hebraeu) with from the little toxin (Small toxin) (gene bank goes into to hide registration number P15229) of Indian red scorpion (Mesubutus tamulus sindicus).

Be applicable to relevant Scorpio toxin among the present invention comprise the complete scorpion catilan sequence described in aminoacid sequence and No. 2003/101474 (it is incorporated herein by quoting in full) SEQ ID No.1 of international application case WO and have at least about 75%, at least about 85%, at least about 90%, at least about 95% or at least about the polypeptide of 99% sequence identity.In certain embodiments, relevant Scorpio toxin comprises SEQ ID NO.8 or homologous those Scorpio toxin of SEQ ID NO.13 of scorpion catilan described in No. 2003/101474, sequence and the international application case WO.

In certain embodiments, the scorpion catilan in scorpion catilan reagent partly passes through labelling.Described in the instance such as hereinafter of labeling method and mark part.

B. treat part

Once mentioned like preceding text, in certain embodiments, scorpion catilan reagent comprises at least one scorpion catilan part and at least one treatment part is associated.The treatment that is fit to partly comprises any in material, molecule, chemical compound, reagent or the factor of the multiple effective treatment disease or the clinical patient's condition.In certain embodiments, treatment part is chemotherapeutics (being anticarcinogen).The anticarcinogen that is fit to comprises multiple to direct or indirect any in poisonous or deleterious material, molecule, chemical compound, reagent or the factor of cancerous cell.

Those skilled in the art will understand, and the treatment part can be synthetic or native compound: the mixture of single molecule, different molecular or the complex of different molecular.The treatment part that is fit to can belong in the multiclass chemical compound arbitrary type, includes, but is not limited to micromolecule class, peptide class, protein-based, saccharide, steroid, antibody class (comprising its fragment and variant), fusion rotein class, antisense polymerized nucleoside acids, ribose enzyme, siRNA class, intends peptide class, radionuclide class etc.

When the treatment part comprises anticarcinogen; Among the kind anti-cancer drugs of number below anticarcinogen for example is found in: alkylating agent class, antimetabolite class, resisting mitosis antibiotics, alkaloid antitumor agent class, hormones and hormone antagonist class, interferons, non-steroidal anti-inflammatory drug class; And various other antitumor agent classes, for example inhibitors of kinases, proteasome inhibitor and NF-kB inhibitor.

The instance of anticarcinogen (for example for example includes, but is not limited to alkylating agent class medicine; Chlormethine (mechlorethamine), chlorambucil (chlorambucil), cyclophosphamide (cyclophosphamide), melphalan (melphalan), ifosfamide (ifosfamide), temozolomide etc.), the antimetabolite class (for example; Methotrexates etc.), purine antagonist class and pyrimidine antagonist class are (for example; Ismipur, 5-fluorouracil, cytosine arabinoside (cytraribine), gemcitabine etc.), the spindle poison class (for example; Vincaleucoblastine, vincristine, vinorelbine (vinorelbine), paclitaxel etc.), podophillotoxines (for example; Etoposide, irinotecan, TPT etc.), antibiotics (for example; Doxorubicin, bleomycin, mitomycin etc.), nitro ureas (for example, carmustine (carmustine), lomustine (lomustine), nimustine (nomustine) etc.), inorganic ions class (for example, cisplatin, carboplatin (carboplatin) etc.), enzyme (for example; Asparaginase etc.) and hormones (for example, tamoxifen, leuproside, flutamide, megestrol (megestrol) etc.).The more detailed argumentation of relevant up-to-date cancer therapy, referring to Http:// www.cancer.gov/The tumour medicine inventory of FDA approval Http:// www.fda.gov/cder/cancer/druglistframe.htmAnd Merck diagnosis and treatment handbook (The Merck Manual), the 17th edition, 1999, the complete content of said reference all is incorporated herein by quoting.

Some anticarcinogens are to work through blocking growth of cancer cells and/or duplicating.This type of medicine generally is classified as " cytostatic agent class ".In certain embodiments, treatment part comprises cytostatic agent.The instance of cytostatic agent class comprises that alkylating agent class, antimetabolite class, plant bases and terpenoid (comprise Changchun alkaloid (vinca alkaloid), podophyllotoxin (podophyllotoxin), taxane (taxane) etc.; VP-16 is the instance of plant alkaloid), topoisomerase enzyme inhibitor class, anti-tumour antibody class, hormones etc.

In certain embodiments, treatment part comprises cytotoxic agent.The instance of cytotoxic agent comprises toxin, other biological activity protein, conventional chemotherapy agent, enzyme and radiosiotope.

The instance of the cytotoxic agent that is fit to includes, but is not limited to antibacterial and phytotoxin, for example Rhizoma Melaleuca Viridiflora element, Ricin, Saponin, bacillus pyocyaneus (Pseudomonas) extracellular toxin, phytolacca american (pokeweed) antiviral protein, diphtheria toxin, diphtherotoxin etc.

The instance of the cytotoxicity biological activity protein that is fit to includes, but is not limited to the protein (or complement protein) of complement system.Complement system be help to remove the pathogen in the organism and promote healing the complex biochemical cascade (B.P. is root (B.P.Morgan) not, clinical laboratory's scientific verification property comment (Crit.Rev.Clin.Lab.Sci.), 1995,32:265).Complement system is formed by soluble cell is conjugated protein more than 35 kinds, and wherein 12 kinds directly relate to the complement path.

The instance of the cytotoxicity chemotherapeutics that is fit to includes, but is not limited to taxanes (for example Docetaxel, paclitaxel etc.), maytansine (maytansine) type, degree Ka-7038 (duocarmycin) class, CC-1065, Ao Ruitating (auristatin) type, Gary stop mycin (calicheamincin) type and other enediyne AGPM class.Other instance comprises anti-folic acid class (for example aminopterin-induced syndrome (aminopterin), methotrexate, pemetrexed (pemetrexed), thunder for Qu Sai (raltitrexed) etc.), Changchun alkaloids (for example vincristine, vincaleucoblastine, etoposide, vindesine, vinorelbine etc.) and anthracyclines (for example daunomycin, doxorubicin, epirubicin, darubicin, mitoxantrone, cut down soften than star (valrubicin) etc.).

The instance of the cytotoxicity enzyme that is fit to includes, but is not limited to nucleolytic enzyme.

The radioisotopic instance of cytotoxicity that is fit to comprises any α, β or the gamma emitter (S.E. Ward (S.E.Order) that when being positioned tumor locus, causes cytoclasis; " analysis, result and future prospect (Analysis; Results; and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy) that the treatment of radiolabeled antibody in cancer therapy used "; The monoclonal antibody (Monoclonal Antibodies for Cancer Detection and Therapy) that is used for cancer detection and therapy; R.W. blog article people's (volume) such as (R.W.Baldwin), academic press (Academic Press), 1985).This type of radioisotopic instance include, but is not limited to iodine-131 ( ¹³¹I), iodine-125 ( ¹²⁵I), bismuth-212 ( ²¹²Bi), bismuth-213 ( ²¹³Bi), astatine-211 ( ²¹¹At), rhenium-186 ( ¹⁸⁶Re), rhenium-186 ( ¹⁸⁸Re), phosphorus-32 ( ³²P), 90Y ( ⁹⁰Y), samarium-153 ( ¹⁵³Sm) and lutecium-177 ( ¹¹⁷Lu).

Perhaps or in addition; Be applicable to that the treatment part among the present invention can be that (USSN 60/954 for be entitled as " as the scorpion catilan (Chlorotoxins as Drug Carriers) of pharmaceutical carriers " applied on August 7th, 2007; 409) and on October 12nd, 2007 application be entitled as any treatment part described in " dispensing of scorpion catilan reagent whole body is used for diagnosis and treatment tumor (Systemic Administration of Chlorotoxin Agents for the Diagnosis and Treatment of Tumors) " total provisional application case (USSN60/), the complete content of said application case is incorporated herein by quoting in full.The instance of said treatment part classification includes, but is not limited to weak water-soluble anticancer agent, anticarcinogen, antisensenucleic acids, ribozyme, triplet reagent (triplex agent), short interfering rna (siRNA), photosensitizer, radiosensitizer, superantigen, prodrug activating enzymes and the anti-angiogenic agent relevant with drug resistance.

In certain embodiments, the therapeutic agent in scorpion catilan reagent (for example anticarcinogen) is a nucleic acid reagent.

Verified, multiple cancer is relevant with genetic damage in various degree with tumor, for example point mutation, gene delection or repetition (duplication).Developed the New Policy of many treatment cancers,, come expression (A. Carlotta people such as (A.Kalota), oncobiology and treatment (Cancer Biol.Ther.), 2004, the 3:4-12 of regulator gene like " antisense ", " anti-gene " and " RNA interference "; Y. middle field people such as (Y.Nakata), eukaryotic gene expression comment summary (Crit.Rev.Eukaryot.Gene Expr.), 2005,15:163-182; V. Wa Qike (V.Wacheck) and U. Ze Meisite-Wei Ke (U.Zangmeister-Wittke), oncology and hematology's comment (Crit.Rev.Oncol.Hematol.), 2006,59:65-73; A. Ke's Trotta people such as (A.Kolata), pharmacological experiment handbook (Handb.Exp.Pharmacol.), 2006,173:173-196).These methods are for example utilized antisensenucleic acids, ribozyme, triplet reagent or short interfering rna (siRNA); To shelter its DNA through the specific mRNA of the gene that covers over the object with antisensenucleic acids or with triplet reagent; Through with ribozyme cracking nucleotide sequence; Or, block transcribing or translating of said mRNA or DNA through relating to the interferential complex mechanism destruction of RNA mRNA.In all these strategies, PDT16 mainly is used as activating agent, but also once uses micromolecule and other structure.Have great potential although regulate strategy based on the gene expression of PDT16 for some treatment for cancer, the main obstruction that the pharmacology of PDT16 uses is, can't these chemical compounds effectively be delivered to its action site in cancerous cell.(P. He Deweijin people such as (P.Herdewijn), the exploitation of antisense nucleic acid medicament (Antisense Nucleic Acids Drug Dev.), 2000,10:297-310; Y. island (H.Nakashima) among village department (Y.Shoji) and the H., contemporary drug design (Curr.Charm.Des.), 2004,10:785-796; A.W. virgin people such as (A.W Tong), molecule therapy new development (Curr.Opin.Mol.Ther.), 2005,7:114-124).

The scorpion catilan pack that this paper provides contains toxin part (for example scorpion catilan part) and is suitable for the nucleic acid molecules of making therapeutic agent (for example anticarcinogen).The nucleic acid of number of chemical type and version goes for this type of strategy.It comprises (as limiting examples) DNA, comprises single stranded DNA (ssDNA) and double-stranded DNA (dsDNA); RNA includes, but is not limited to ssRNA, dsRNA, tRNA, mRNA, rRNA, ribozyme; RNA:DNA crossbred, triplet DNA (for example dsDNA associates with short PDT16) etc.

In some embodiments of the invention, the length of the nucleic acid reagent that exists in the scorpion catilan reagent is between about 5 and 2000 nucleotide.In certain embodiments, it is long that nucleic acid reagent is at least about 6,7,8,9,10,11,12,13,14,15,16,17,18,19 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or 50 above nucleotide.In certain embodiments, the length of nucleic acid reagent is less than about 2000,1900,1800,1700,1600,1500,1400,1300,1200,1100,1000,900,800,700,600,500,450,400,350,300,250,200,150,100,50,45,40,35,30,25,20 or 20 following nucleotide.

In certain embodiments, the nucleic acid reagent that exists in the scorpion catilan reagent of the present invention comprises the sequence of promoter and/or other regulatory transcription.In certain embodiments, the nucleic acid reagent that exists in the scorpion catilan reagent of the present invention comprises the sequence that origin of replication and/or other regulation and control are duplicated.In certain embodiments, the nucleic acid reagent that exists in the scorpion catilan reagent of the present invention does not comprise promoter and/or origin of replication.

Be applicable to and put into practice those medicaments that C12U of the present invention comprises the following gene of targeting: with the gene that tumor takes place with cell is grown or cell transformation is relevant (proto-oncogene for example; The splitted protein of its coding irritation cell), the gene of the gene of angiogenesis/angiogenesis inhibitor, tumor suppressor gene (its coding suppresses fissional protein), coding and tumor growth and/or tumor migration proteins associated matter; With the suicide gene (suicide gene) of cell death inducing or other form cell death, the strongest active suicide gene in rapid splitted cell especially.

The instance of the gene order relevant with tumor generation and/or cell transformation comprises MLL fusion gene, BCR-ABL, TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, Bcl-2, AML1-ETO, AML1-MTG8, Ras, Fos PDGF, RET, APC, NF-1, Rb, p53, MDM2 etc.; The sequence of overexpression is like the multidrug resistance gene; Cyclin; β-a chain of fibroin (beta-Catenin); Telomerase gene; C-myc, n-myc, Bcl-2, Erb-B1 and Erb-B2; And mutant nucleotide sequence, like Ras, Mos, Raf and Met.The instance of tumor suppressor gene includes, but is not limited to p53, p21, RB1, WT1, NF1, VHL, APC, DAP kinases, p16, ARF, neurofibromin (Neurofibromin) and PTEN.Can comprise coding and the tumor migration proteins associated matter gene of (as integrating element, selecting plain (selectin) and metalloproteases) by the instance of the gene that is applicable to the nucleic acid molecules targeting in the anti-cancer therapies; Coding promotes the protein of neovascularization (like VEGF (VEGF) or angiogenesis inhibitor gene VEGFr); Coding suppresses the angiogenesis inhibitor gene of the protein (like endotheliocyte blocker, angiogenesis blocker and VEGF-R2) of neovascularity generation; With the coding for example below proteinic gene: interleukin; Interferon; Fibroblast growth factor (α-FGF and β-FGF); Insulin like growth factor (for example IGF-1 and IGF-2); Platelet-derived growth factor (PDGF); Tumor necrosis factor (TNF); Transforming growth factor (for example TGF-α and TGF-β); Epidermal growth factor (EGF); Keratinocyte growth factor (KGF); Stem cell factor and its receptor c-Kit (SCF/c-Kit) part; CD40L/CD40; VLA-4VCAM-1; ICAM-1/LFA-1; Hyaluronic acid (hyalurin)/CD44 etc.One of ordinary skill in the art will recognize that previous examples is not exhaustive.

Nucleic acid in the scorpion catilan reagent of the present invention can have any in the various active, for example comprises as anticarcinogen or other therapeutic agent, probe, primer etc.Nucleic acid in the scorpion catilan reagent of the present invention can have enzymatic activity (for example ribozyme is active), gene expression suppresses active (for example as antisense reagent or siRNA reagent etc.) and/or other activity.Nucleic acid itself in the scorpion catilan reagent of the present invention can have activity, perhaps can be used as carrier delivery of active nucleic acid reagent (for example, duplicating and/or transcribing through institute's nucleic acid delivery).From the purpose of this description, if examples of such carriers nucleic acid coding or delivery treatments activating agent otherwise even itself does not have therapeutic activity so, also can be regarded as it " therapeutic agent ".

In certain embodiments, scorpion catilan reagent comprises the exonuclease treatment agent, and said exonuclease treatment agent comprises or encoding antisense platform thing.Term " antisense compounds or reagent ", " antisense oligomer ", " antisense PDT16 " and " antisense oligonucleotide analogues " interchangeable in this article use; And the sequence that refers to nucleotide base and subunit to subunit main chain; It allows antisense compounds through the hybridization of the target sequence among water Sen-Ke Like (Watson-Crick) base pairing and the RNA, in target sequence, forms RNA oligomer heteroduplex (heteroduplex) thus.Said oligomer can have the complementary or approaching complementarity of accurate sequence in target sequence.Said antisense oligomer is capable of blocking or suppress to contain the translation of the mRNA of target sequence, or suppressor gene is transcribed.The antisense oligomer can be incorporated into two strands or single stranded sequence.

Be applicable to that the instance of putting into practice antisense PDT16 of the present invention comprises those that mention in the for example following summary: the people such as (R.A Stahel) of R.A Si Dell, pulmonary carcinoma (Lung Cancer), 2003,41:S81-S88; K.F. Pai Erluo people such as (K.F.Pirollo), pharmacology and therapeutics (Pharmacol.Ther.), 2003,99:55-77; A.C. Shi Difensi (A.C.Stephens) and R.P. Rui Wensi (R.P.Rivers), molecular therapy new development (Curr.Opin.Mol.Ther.), 2003,5:118-122; N.M. Di's grace (N.M.Dean) and this nanotesla of C.F. (C.F.Bennett), oncogene (Oncogene), 2003,22:9087-9096; N. Si Qiweng people such as (N.Schiavone), contemporary drug design (Curr.Pharm.Des.), 2004,10:769-784; L. tie up Dare people such as (L.Vidal), European cancer magazine (Eur.J.Cancer), 2005,41:2812-2818; T. Ai Bo-Fa Dare (T.Aboul-Fadl), contemporary pharmaceutical chemistry (Curr.Med.Chem.), 2005,12:2193-2214; M.E. husband (M.E.Gleave) and B.P. cover Na (B.P.Monia) in the lattice, and cancer is commented on (Nat.Rev.Cancer), 2005,5:468-479 naturally; Y.S. (Y.S.Cho-Chung) in the Zhuo, contemporary drug design, 2005,11:2811-2823; E. thunder Berne people such as (E.Rayburn), drug design with find communication (Lett.Drug Design&Discov.), 2005,2:1-18; E.R. thunder Berne people such as (E.R.Rayburn), expert's viewpoint of emerging medicine (Expert Opin.Emerg.Drugs), 2006,11:337-352; I. safe nurse (I.Tamm) and M. Wagner (M.Wagner), molecular biotechnology (Mol.Biotechnol.), 2006,33:221-238 (being incorporated herein by quoting in full separately).

The instance of the antisense PDT16 that is fit to comprises that for example Ao Limosen sodium (olimerson sodium) (is also referred to as Genasense ^TMOr G31239, Ji Tena company (Genta, Inc.) exploitation; Berkeley sea, New Jersey thatch (Berkeley Heights; NJ)), thiophosphate (phosphorothioate) oligomer in the initiation codon subarea of promptly a kind of targeting bcl-2 mRNA, said bcl-2 mRNA is a kind of effective apoptosis inhibitor; And (the C.A. Si Taen people such as (C.A.Stein) of overexpression in the many cancers that comprise follicular lymphoma, breast carcinoma, colon cancer and carcinoma of prostate and moderate/height lymphoma; Oncology's collection of thesis (Semin.Oncol), 2005,32:563-573; S.R. Frankel (S.R.Frankel), oncology's collection of thesis, 2003,30:300-304).Other antisense PDT16 that is fit to comprise GEM-231 (HYB0165, extra large The Burton Corp. (Hybridon, Inc.); Cambridge, Massachusetts (Cambridge; MA)), it is mixed backbone PDT16 (S. Gao Er people such as (S.Goel), Clinical Cancer Research (Clin.Cancer Res.) to cAMP deopendent protein kinase A (PKA); 203,9:4069-4076); Affinitak (ISIS 3521 or A Pinuo Carson (aprinocarsen), and ISIS Pharmaceuticals Inc. (ISIS pharmaceuticals, Inc.), Carlsbad, California (Carlsbad, CA)), the antisense inhibitor of a kind of PKC-α; (Isis 112989 for OGX-011; Isis drugmaker (Isis Pharmaceuticals, Inc.)); A kind of 2 '-the antisense PDT16 to clusterin (clusterin) of methoxy ethyl modified; Said clusterin relates to the glycoprotein of cell cycle regulating, tissue reconstruction, lipid transportation and cell death, and overexpression in breast carcinoma, carcinoma of prostate and colon cancer; (Isis 112989 for ISIS 5132; Isis drugmaker), and 3 of a kind of and the c-raf-1 mRNA '-complementary thiophosphate PDT16 of untranslated region sequence (S.P. Henry (S.P.Henry) people of etc.ing, cancer therapy drug designs (Anticancer Drug Des.); 1997,12:409-420; B.P. Monia people such as (B.P.Monia), institute of NAS periodical (Proc.Natl.Acad.Sci.USA), 1996,93:15481-15484; C.M. Shandong fourth people such as (C.M.Rudin), Clinical Cancer Research (Clin.Cancer Res.), 2001,7:1214-1220); ISIS 2503 (Isis drugmaker), and the thiophosphate PDT16 antisense inhibitor that a kind of people H-ras mRNA expresses (J. Ku Leike (J.Kurreck), european journal of biological chemistry (Eur.J.Biochem.), 2003,270:1628-1644); The chain inhibitor of the X of targeted cells apoptotic proteins (X-linked inhibitor of apoptosis protein, XIAP; The quite most apoptosis of its blocking-up path) PDT16; (AEG 35156 like GEM 640; Ai Jina therapeutic agent company (Aegera Therapeutics Inc.) and extra large The Burton Corp. (Hybridon, Inc.)) or targeting survivin (survivin; A kind of apoptosis protein inhibitor (IAP)) PDT16, like ISIS 23722 (Isis drugmaker), a kind of 2 '-the chimeric PDT16 of O-methoxy ethyl; MG98, its targeting dnmt rna; And GTI-2040 ((the Lorus Therapeutics of Luo Lusi therapeutic agent company; Inc.), Toronto (Toronto, Canada)); A kind of 20 aggressiveness PDT16s, the coding region among the mRNA of itself and human ribonucleotide reductase R2 small subunit component is complementary.

Other antisense PDT16 that is fit to comprises that the antisense PDT16 just developed to Her-2/neu, c-Myb, c-Myc and c-Raf is (referring to A. for example than Luo Xi people such as (A.Biroccio), oncogene (Oncogene), 2003,22:6579-6588; Y. people such as (Y.Lee) Lee, cancer research (Cancer Res.), 2003,63:2802-2811; B. people such as (B.Lu) Lu, cancer research, 2004,64:2840-2845; K.F. Pi Ailuo people such as (K.F.Pirollo), materia medica and therapeutics (Pharmacol.Ther.), 2003,99:55-77; With A. Rui Te people such as (A.Rait), NYAS's yearbook (Ann.N.Y.Acad.Sci.), 2003,1002:78-89).

In certain embodiments, scorpion catilan reagent of the present invention comprises C12U, and said C12U comprises or coded interference RNA molecule.Term " RNA interfering " and " disturbance RNA molecule " interchangeable in this article use, and refer to and can for example disturb (RNAi) to suppress or down-regulation of gene expression through mediate rna, perhaps make the RNA molecule of gene silencing with the sequence-specific mode.It is a kind of sequence-specific of in evolution, guarding mechanism that is triggered by double-stranded RNA (dsRNA) that RNA disturbs (RNAi); It induces the degraded of complementary target strand mRNA and " silence " of its appropriate translation sequence (Mike graceful this (McManus) and Sharp (Sharp); 2002; The hereditism comments on (Nature Rev.Genet.) naturally, and 2002,3:737).RNAi through will than the long biological activity " short interfering rna " of 21 to 23 nucleotide of long dsRNA chain enzymatic cleavage written treaty (siRNA) sequence work (Ai Erbasa people such as (Elbashir), gene studies (Genes Dev.), 2001,15:188).RNA disturbs has become a kind of treatment of cancer approach that has much prospect.

Be applicable to that putting into practice RNA interfering of the present invention can provide by in the several forms any.For example, the RNA interfering that is provided can be one or more in separated short interfering rna (siRNA), double-stranded RNA (dsRNA), Microrna (miRNA) or the short hairpin RNA (shRNA).

The instance that is applicable to the disturbance RNA molecule among the present invention comprises that the iRNA:O. Mil who is mentioned in the for example following summary breathes out dimension people such as (O.Milhavet), pharmaceutical research (Pharmacol.Rev.), 2003,55:629-648; F. Billy people such as (F.Bi), contemporary gene therapy (Curr.Gene.Ther.), 2003,3:411-417; P.Y. people such as (P.Y.Lu) Lu, molecular therapy new development (Curr.Opin.Mol.Ther.), 2003,5:225-234; I. Fred Ritchie people such as (I.Friedrich), carcinobiology research literary composition volume (Semin.Cancer Biol), 2004,14:223-230; M. EZE Kui holder (M.Izquierdo), cancer gene therapy (Cancer Gene Ther.), 2005,12:217-227; P.Y. people such as (P.Y.Lu) Lu, the hereditism makes progress (Adv.Genet.), and 2005,54:117-142; G.R. wear dimension (G.R.Devi), cancer gene therapy, 2006,13:819-829; M.A. Bei Huoke (M.A.Behlke), molecular therapy (Mol.Ther.), 2006,13:644-670; With the general Bristol of L.N. people such as (L.N.Putral), medicine news and prospect (Drug News Perspect), 2006,19:317-324 (each document all is incorporated herein by quoting in full).

Other instance of the disturbance RNA molecule that is fit to includes, but is not limited to p53 RNA interfering (for example, T.R. Bu Lumeng Kemp people such as (T.R.Brummelkamp), science (Science), 2002,296:550-553; M.T. He Man people such as (M.T.Hemman), natural genetics (Nat.Genet.), 2003,33:396-400); The RNA interfering of targeting bcr-abl fusion gene; Said bcr-abl fusion gene relevant (for example, M. Si Qier people such as (M.Scherr), hematology (Blood) with the development of chronic lymphocytic leukemia and acute lymphoblastic leukemia; 2003,101:1566-1569; M.J. people such as (M.J.Li) Lee, PDT16 (Oligonucleotides), 2003,13:401-409); The RNA interfering that suppresses the expression of NPM-ALK; Said NPM-ALK is a kind of protein of in 75% primary cutaneous type, finding; It can cause with tumor and forms active kinase whose expression (the U. Rui Te people such as (U.Ritter) of relevant composition; PDT16,2003,13:365-373); The RNA interfering of targeting oncogene, said oncogene such as Raf-1 (T.F. building people such as (T.F.Lou), PDT16; 2003,13:313-324), K-Ras (people such as T.R. Bu Lumeng Kemp, cancerous cell (Cancer Cell); 2002,2:243-247), erbB-2 (G. poplar people such as (G.Yang), journal of biological chemistry (J.Biol.Chem.); 2004,279:4339-4345); The RNA interfering of a chain of fibroin of targeting b-; The overexpression of a chain of fibroin of said b-will cause the Transactivation (transactivation) of T-cytokine target gene; And this Transactivation is considered to main transformation event (Trane, the M. model Devi people such as (M.van de Wetering) in the colorectal carcinoma; EMBO reports (EMBO Rep.), 2003,4:609-615).

C. mark part

In certain embodiments, scorpion catilan reagent is through at least a mark part labelling.For example, can use mark part labelling scorpion catilan reagent interior one or more scorpion catilans parts and/or one or more treatment part.

The effect of mark part is the detection of convenient scorpion catilan reagent after being incorporated into tissue to be tested.Preferably, mark part is through selecting, and makes it produce the amount relevant (for example proportional) of signal and signal intensity and the diagnostic agent that is incorporated into tissue that can be measured.

Preferably, labelling can not disturb the biology of wanting or the pharmaceutically active of scorpion catilan reagent in fact.In certain embodiments, labelling relates to one or more mark parts is connected to or incorporates scorpion catilan part into, preferably is connected to or incorporates the non-interference position on the peptide sequence of scorpion catilan part into.Said non-interference position is not participate in scorpion catilan part and the bonded position of the specificity of tumor cell.

Mark part can be any entity that permission detects after scorpion catilan reagent is incorporated into linked groups or system.But any one all can be used as the mark part in the scorpion catilan reagent of the present invention in the multiple detectable.Mark part can directly detect or indirect detection.The instance of mark part includes, but is not limited to: various parts, radionuclide are (for example, ³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y, ^99mTc, ¹⁷⁷Lu etc.), fluorescent dye (relevant concrete exemplary fluorescent dye; Vide infra), distinguishable inorganic fluorescence semiconductor nanocrystal is (promptly on chemical illuminating reagent (for example dioxetane (stabilized dioxetane) of acridinium ester (acridinum ester), stabilisation etc.), bioluminescent reagents, the spectrum; Quantum dot), metal nanoparticle (for example; Gold, silver, copper, platinum etc.) nanocluster, paramagnetic metal ion, enzyme (instantiation of related enzymes vide infra), colourity labelling (for example dyestuff, gold colloidal etc.), biotin, digoxin (dioxigenin), hapten and antiserum is arranged or the available protein of monoclonal antibody.

In certain embodiments, mark part comprises fluorescent labeling.Many known fluorescent labelinies with number of chemical structure and physical characteristic partly are applicable to puts into practice diagnostic method of the present invention.The fluorescent dye that is fit to (for example includes, but is not limited to fluorescein (fluorescein) and fluorescein(e) dye; Fluorescein isothiocyanate or FITC, naphtho-fluorescein (naphthofluorescein), 4 '; 5 '-two chloro-2 '; 7 '-dimethoxy fluorescein, 6-CF 5(6)-Carboxyfluorescein or FAM etc.), carbocyanine (carbocyanine), merocyanine (merocyanine), styryl dye, oxonols (oxonol) dyestuff, phycoerythrin (phycoerythrin), erythrosine (erythrosin), Yihong (eosin), rhodamine (rhodamine) dyestuff (for example; Carboxyl tetramethyl-rhodamine or TAMRA, carboxyl rhodamine 6G, carboxyl-X-rhodamine (ROX), lissamine rhodamine B (lissamine rhodamine B), rhodamine 6G, rhodamine is green, rhodamine is red, tetramethyl rhodamine (TMR) etc.), coumarin (coumarin) and coumarine dye (for example; Methoxy coumarin, dialkyl amido coumarin, Hydroxycoumarin, aminomethyl coumarin (AMCA) etc.), red, the Dallas Pink-X of Oregon green (Oregon Green) dyestuff (for example, the Oregon is green 488, the Oregon is green 500, Oregon green 514 etc.), Dezhou (Texas), Spectrum Red ^TM, Spectrum Green ^TM, cyanines (cyanine) type dyestuff (for example, Cy-3 ^TM, Cy-5 ^TM, Cy-3.5 ^TM, Cy-5.5 ^TMDeng), Alexa Fluor series dyes (for example; Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680 etc.), the BODIPY dyestuff (for example; BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 etc.), IRD dyestuff (IRDyes) (for example, IRD40, IRD 700, IRD 800 etc.) etc.About the fluorescent dye that is fit to and with fluorescent dye and like more instances of the link coupled methods of other chemical entities such as protein and peptide; Referring to for example " fluorescent probe and research product handbook (The Handbook of Fluorescent Probes and Research Products) "; The 9th edition, and molecular probe company (Molecular Probes, Inc.); Eugene, Ore (Eugene, OR).The advantageous feature of fluorescent labeling reagent comprises high molar absorption coefficient, high-fluorescence quantum yield and light resistance.In certain embodiments, mark fluorescent group desirably is presented in spectrographic visible-range (that is, between 400nm and 750nm) but not absorption and emission wavelength in ultraviolet light range (that is, being lower than 400nm).

In certain embodiments, mark part comprises enzyme.The instance of the enzyme that is fit to includes, but is not limited to enzyme used among the ELISA, for example horseradish peroxidase, beta galactosidase, luciferase, alkali phosphatase etc.Other instance comprises beta-Glucuronidase, β-D-glucosidase, urase, glucoseoxidase etc.Can use to connect base,, enzyme is linked to scorpion catilan part like carbodiimides, vulcabond, glutaraldehyde etc.

In certain embodiments; Mark part comprises single photon emission computed body layer imaging (Single Photon Emission Computed Tomography capable of using; SPECT) or positron emission emission computer body layer imaging (Position Emission Tomography, the radiosiotope that PET) detects.The instance of this type of radionuclide include, but is not limited to iodine-131 ( ¹³¹I), iodine-125 ( ¹²⁵I), bismuth-212 ( ²¹²Bi), bismuth-213 ( ²¹³Bi), astatine-221 ( ²¹¹At), copper-67 ( ⁶⁷Cu), copper-64 ( ⁶⁴Cu), rhenium-186 ( ¹⁸⁶Re), rhenium-186 ( ¹⁸⁸Re), phosphorus-32 ( ³²P), samarium-153 ( ¹⁵³Sm), lutecium-177 ( ¹¹⁷Lu), technetium-99m ( ^99mTc), gallium-67 ( ⁶⁷Ga), indium-111 ( ¹¹¹In) and thallium-201 ( ²⁰¹T1).

In certain embodiments, mark part comprises the radiosiotope of γ phase machine testing capable of using.This type of radioisotopic instance include, but is not limited to iodine-131 ( ¹³¹I) and technetium-99m ( ^99mTc).

In certain embodiments, mark part comprises paramagnetic metal ion, and it is a good contrast-enhancing agent in the nuclear magnetic resonance (MRI).The instance of this type of paramagnetic metal ion includes, but is not limited to gadolinium III (Gd ³⁺), chromium III (Cr ³⁺), dysprosium III (Dy ³⁺), ferrum III (Fe ³⁺), manganese II (Mn ²⁺) and ytterbium III (Yb ³⁺).In certain embodiments, mark part comprises gadolinium III (Gd ³⁺).Gadolinium is the contrast medium that FDA approval is used for MRI, and it can accumulate in abnormal structure, makes these abnormal areas on MRI, become very bright (grow).Known gadolinium can make in the health zones of different, especially produces strong contrast between normal structure in the brain and the abnormal structure.

In certain embodiments, mark part comprises the stable paramagnetism isotope that NMR spectrum capable of using (MRS) detects.The isotopic instance of stable paramagnetism that is fit to include, but is not limited to carbon-13 ( ¹³C) and fluoro-19 ( ¹⁹F).

D. form scorpion catilan reagent

In certain embodiments, scorpion catilan reagent comprises at least one scorpion catilan part and at least one treatment part association.Therefore, scorpion catilan reagent is to be formed by at least two other molecular associations (for example combine, interaction, fusion or coupling).

Association in scorpion catilan reagent between scorpion catilan part and the treatment part can be covalently or non-covalently.No matter how are combining between scorpion catilan part and the treatment part, interaction or link coupled character; Association between the two preferably has selectivity, specificity; And enough strong so that scorpion catilan reagent can not transport/be delivered to tumor and getting in the tumor before or during dissociate.Association in scorpion catilan reagent between scorpion catilan part and the treatment part can use the known any chemistry of one of ordinary skill in the art, biochemistry, enzyme or gene coupling method to realize.

In certain embodiments, the association between scorpion catilan part and the treatment part is non-covalent.The instance of noncovalent interaction includes, but is not limited to hydrophobic interaction, electrostatic interaction, dipolar interaction, Van der Waals interaction (van der Waals interaction) and hydrogen bond.

In certain embodiments, the association between scorpion catilan part and the treatment part is a covalency.One of ordinary skill in the art will understand, said part can be each other directly or indirectly (for example through bridging agent, like the description of hereinafter institute) connection.

In certain embodiments, scorpion catilan part is directly covalently bound each other with the treatment part.Directly covalent bond can be through realizing like bindings such as amide, ester, carbon-to-carbon, disulfide bond, carbamate, ether, thioether, urea, amine or carbonic ester bindings.Covalent bond can be utilized scorpion catilan part and/or treatment part to go up the functional group that exists and realize.Perhaps, can be with the non-key aminoacid of another amino acid replacement that can introduce the useful group (amino, carboxyl or sulfydryl) that is suitable for the coupling purpose.Perhaps, can additional amino acid be added in the scorpion catilan part, be suitable for the useful group (amino, carboxyl or sulfydryl) of coupling purpose with introducing.Be applicable to that the functional group that each several part is linked together includes, but is not limited to amine, anhydride, hydroxyl, carboxyl, mercaptan etc.Can use and form direct binding like activators such as carbodiimides.Multiple activator is known and be applicable to and connect therapeutic agent and scorpion catilan part in this technology.

In other embodiments, scorpion catilan part is covalently bound indirectly each other via being connected base with the treatment part in scorpion catilan reagent.This can use, and well-known many stable difunctionality reagent (comprise same sense (homofunctional) reagent and assorted sense (heterofunctional) reagent in this technology; The instance of relevant said reagent is referring to for example Pierre's Si catalogue and handbook (Pierce Catalog and Handbook)) realize.Use difunctionality bridging agent and the difference of using activator to be that the former makes the coupling part be present in the gained scorpion catilan reagent, and two direct couplings of part that the feasible reaction of the latter relates to.The effect of difunctionality bridging agent can be to allow two to be in addition between the inert part and to react.Perhaps or in addition; Can be selected to the difunctionality bridging agent of the part of product; So that it (for example gives the conformation motility to a certain degree of scorpion catilan reagent; Said difunctionality bridging agent comprises the straight chained alkyl with several atoms, and for example said straight chained alkyl contains the carbon atom between 2 and 10).Perhaps or in addition, the difunctionality bridging agent can make that the binding that forms between scorpion catilan part and the treatment part is a cleavable through selecting; For example (instance of relevant this type of bridging agent is referring to for example United States Patent (USP) the 5th, 773 for hydrolyzable; No. 001, the 5th, 739, No. 116 and the 5th; 877, No. 296, each patent is incorporated herein by quoting in full).After this type of bridging agent for example is preferred for scorpion catilan part and treats concatenator hydrolysis partly, observe the situation of the greater activity of scorpion catilan part and/or treatment part.Can treatment part be included in hydrolysis under the acid pH of lysosome (amide (cis-aconitate-like amide) of hydrazone, acetal and type (Z)-Aconitic acid ester) from the cracked exemplary mechanism of scorpion catilan part, utilize the peptide cracking (cathepsin and other lysosomal enzymes) and the disulfide bond reduction of lysosomal enzyme.Another is used for treatment part is included in from the cracked mechanism of scorpion catilan reagent and carries out hydrolysis in extracellular or the cell under the physiological pH.This mechanism be applicable to be used for treatment part and the link coupled cross-linking agent of scorpion catilan part be like polydextran gel (polydextran) but etc. the situation of biodegradable/bio-digestion entity.

For example, can utilize the carbonyl of being introduced that predetermined release characteristics is provided, prepare the scorpion catilan reagent that contains hydrazone.Utilization comprises that an end has disulfide group and the other end has the bridging agent of the alkyl chain of hydrazine derivate, also can prepare scorpion catilan reagent.The bridging agent that contains the functional group except that hydrazone also might be by cracking in lysosomal sour environment.For example, can prepare scorpion catilan reagent by the thiol-reactive bridging agent, said thiol-reactive bridging agent contain except that hydrazone other can be in cell cracked group, like ester, amide and acetal/ketal.

The instance of another kind of pH sensitivity bridging agent is the (Z)-Aconitic acid esters, and its hydroxy-acid group and amide groups are adjacent.Carboxylic acid can quicken amide hydrolysis in acid lysosome.Also can use hydrolysis rate acceleration that can realize similar type and bridging agent with other type structure.

Another potential method for releasing of scorpion catilan reagent is to utilize lysosomal enzyme to carry out the enzymatic hydrolysis of peptide.In an example, the peptide toxin is connected to p-aminophenyl methanol via amido link, forms carbamate or carbonic ester between benzyl alcohol and the treatment part subsequently.Peptide cracking meeting makes aminobenzene methyl carbamate or carbonic ester fracture, and discharges the treatment part.In another example, phenol can the cracking through the fracture of bridging agent but not carbamate.In another version, use the initial fracture of disulfide bond reduction to sulfydryl benzyl carbamate or carbonic ester.

Treatment in scorpion catilan reagent partly is among the embodiment of protein, polypeptide or peptide, and scorpion catilan reagent can be fusion rotein.Once defined like preceding text, fusion rotein is a kind of molecule that comprises two or more protein or peptide and utilize covalent bond to connect through peptide main chain separately.Fusion rotein used in the inventive method can be by known any suitable method preparation in this technology.For example, it can use Peptide synthesizer, utilizes direct protein synthesis method preparation.Perhaps, can use anchor primer (anchor primer) to carry out the pcr amplification of genetic fragment, these anchor primers produce complementary outstanding sequence between two successive genetic fragments, and it can be annealed and amplification more subsequently, produces chimeric gene sequence thus.Fusion rotein can obtain by the standard weight group of methods (referring to for example Man Nitisi people such as (Maniatis); " molecular cloning: laboratory manual (Molecular Cloning:A Laboratory Manual) "; The 2nd edition, 1989, cold spring harbor laboratory (Cold Spring Harbor Laboratory); Cold spring city, New York (Cold Spring, N.Y.)).These methods generally comprise the nucleic acid molecules that (1) makes up the predetermined fusion rotein of coding; (2) said nucleic acid molecules is inserted in the recombinant expression carrier; (3) transform the host cell that is fit to said expression vector; (4) in host cell, express said fusion rotein.So institute is known in the technology, the fusion rotein for preparing by said method can be directly from the culture medium recovery with separate, or reclaim and separate through the dissolving of cell.Well-known many purification that are used for are by the method for protein that produces through transformed host cells in this technology.These methods include, but is not limited to deposition, centrifugal, gel filtration and (ion exchange, anti-phase and affine) column chromatography.Other purification process is also described (referring to for example De Qieer people such as (Deutscher) to some extent; " protein purification guide (Guide to Protein Purification) ", Enzymology method (Methods in Enzymology), 1990; The 182nd volume, academic press (Academic Press)).

One of ordinary skill in the art are easy to understand, and the scorpion catilan reagent that uses in the inventive method can comprise many scorpion catilan parts and many treatment parts, and it utilizes many different modes to associate each other.The design of concatenator will receive required properties influence in its predetermined purpose and the specific application environment.Scorpion catilan part is in one of ordinary skill in the art's the skill with the selection of treating the part association or combine to form scorpion catilan compositions and methods; And generally depend on chemical functional group's on the character of interactional character required between each several part (that is, covalency to non-covalent and/or cleavable to cleavable not), treatment part, the related part existence and character etc.

For the scorpion catilan reagent through labelling, the association between scorpion catilan part (or treatment part) and the mark part can be covalently or non-covalently.Under the associating situation of covalency, scorpion catilan (or treatment) part can as indicated abovely directly or indirectly be connected with mark part each other.

In certain embodiments, the association between scorpion catilan part (or treatment part) and the mark part is non-covalent.Non-covalent associating instance includes, but is not limited to hydrophobic interaction, electrostatic interaction, dipolar interaction, Van der Waals interaction and hydrogen bond.For example, mark part can be through the non-covalent scorpion catilan part (or treatment part) (for example, metal isotope can with is connected (for example merging) to scorpion catilan poly histidine district (polyHis region) chelating partly) that is connected to of chelation.

In certain embodiments, scorpion catilan part (or treatment part) is through isotopic labeling (that is, wherein one or more atoms are different from usually the atomic mass found at nature or the atomic substitutions of mass number through atomic mass or mass number).Perhaps or in addition, isotope can be connected to scorpion catilan part and/or treatment part.

One of ordinary skill in the art are easy to understand, and the scorpion catilan reagent through labelling that uses in some method of the present invention can comprise many scorpion catilan parts, many treatments part and many mark parts, and it utilizes many different modes to associate each other.The design of the scorpion catilan reagent of process labelling will be influenced by characteristic required in its predetermined purpose, its applied environment and selected detection method.

E. modify

In certain embodiments, through modifying to macromole (like polymer) scorpion catilan reagent is covalently bound.Do not hope to receive any particular theory to fetter, the covalently bound antigenicity that can shelter scorpion catilan reagent of polymer for example, so that the bioavailability and/or the toleration of this reagent is improved in the animal body.Examples of such polymers is Polyethylene Glycol (PEG), and it is covalently bound N and/or C-terminal and/or cysteine to peptide and/or polypeptide usually." Pegylation (PEGylation) " is meant that the PEG covalency is added to a part.In certain embodiments, scorpion catilan reagent is not modified in any site.In certain embodiments, each molecule has a site through modifying (for example Pegylation) in the scorpion catilan reagent.In certain embodiments, each molecule has an above site through modifying (for example Pegylation) in the scorpion catilan reagent.

In certain embodiments, said modification increases the interior half-life of body of scorpion catilan reagent.Give an example, the half-life can be at least about 10 hours, at least about 16 hours etc. (referring to for example instances 9).In certain embodiments, the improvement of said bioavailability helps relating to the dosage regimen of low administration frequency.(dosage regimen such as this paper argumentation.)

II. treat and/or detection method

Therapeutic Method of the present invention comprise to the individuality that needs are arranged (for example suffer from least a neoplasm metastasis, once suffer from least a neoplasm metastasis, the risk of at least a neoplasm metastasis of development and/or the individuality of be prone to suffering from least a neoplasm metastasis are arranged) throw the scorpion catilan reagent or its pharmaceutical composition that give effective dose.Therefore, Therapeutic Method of the present invention can be used for reducing size and/or the quantity of neoplasm metastasis, growth and/or the formation that inhibition is shifted, and/or prolongs the time-to-live of the mammal (comprising the mankind) that suffers from the metastatic cancer and the metastatic cancer patient's condition.

Do not hope to receive the constraint of any particular theory, we notice that the formation of neovascularity (angiogenesis) is for development and/or keep shifting very important.Such as in the instance 4 confirmation, the scorpion catilan can suppress the formation of neovascularity.The scorpion catilan also can make the blood vessel (neovasculature) of new generation degenerate.In some embodiments of the invention, the neovasculature of at least a transfer is degenerated.In some embodiments of the invention, neovascularity generates and is inhibited.

A. indication

Use the convention of name metastatic tumo(u)r behind position, former initiation source in this description in the whole text.Therefore, for example " metastatic prostate cancer " is meant from prostate origin and has been diffused into the cancer of other organ, no matter transferring position how.Transfer can form in multiple organ, comprises for example brain, lung, skeleton, liver, lymph node, ovary etc.The tumor of some kind can be transferred to some organ usually.For instance, melanoma is often transferred to brain, and carcinoma of prostate is often transferred to skeleton, and women's gastric cancer is often transferred to ovary, and breast carcinoma is often transferred to skeleton, and colon cancer is often transferred to liver.The inventive method can be used for treating the transfer in the above-mentioned multiple organ, comprises the transfer away from primary tumor site.In addition, because scorpion catilan reagent can be striden blood/brain barrier (referring to for example instance 2 and 3), make the inventive method can be used for treating the transfer in the brain.

Primary tumo(u)r regular meeting is diffused into contiguous lymph node.The inventive method also can be used for controlling or eliminating the diffusion to lymph node.Cancer/tumor cell can break away from primary tumo(u)r, and through via blood flow or lymph row of passages and then shift.In certain embodiments of the invention, for example in some embodiment of systemic delivery scorpion catilan reagent, scorpion catilan reagent will combine and the cell of these transfers of targeting with its destruction.

In certain embodiments, Therapeutic Method of the present invention further is included in to throw and detects at least a transfer before giving scorpion catilan reagent.In some such embodiment, detect at least a transfer and comprise the scorpion catilan reagent that effective dose process labelling is given in throwing.

But, should be appreciated that the inventive method can be used for treating suffers from the individuality that one or more shift, once suffer from one or more transfers or the risk of suffering from one or more transfers is arranged, even the position of said one or more transfers or existence maybe be unknown.The patient possibly not be diagnosed as at all suffers from any transfer, perhaps has only a group to shift in patient's body and has obtained differentiating and/or the location.In some embodiments of the invention, systemic delivery scorpion catilan reagent makes scorpion catilan reagent in whole body, send.Therefore, there is no need the targeting of sending of scorpion catilan reagent is organized in a specific tissue or one group and realized scorpion catilan reagent sending to transfer.

The primary carcinoma disease of the metastatic cancer that can treat according to the present invention and the instance of the cancer patient's condition be can develop into and brain and central nerve neuroma (the for example tumor of meninges, brain, spinal cord, cranial nerve and other part of CNS, for example glioblastoma multiforme or medulloblastoma) included, but is not limited to; Head and/or neck cancer, mammary neoplasms, blood circulation tumor (for example, heart, mediastinum and pleura and other intrathoracic organ, vascular tumor, the vascular tissue relevant) with tumor; Blood and lymphsystem tumor (for example the leukemia of the sick lymphoma of lymphogranulomatosis, Fei Huoqijinshi, burkitt's lymphoma (Burkitt ' s lymphoma), AIDS dependency lymphoma, pernicious immunoproliferative disease, multiple myeloma and malignant plasma cell anything superfluous or useless, lymphoid leukemia, myelomatosis, urgency or chronic lymphocytic leukemia, monocytic leukemia, other particular cell types, do not indicate the leukemia of cell type; Unspecified malignant lymphatic property, hemopoietic and linked groups's anything superfluous or useless, for example dispersivity large celllymphoma, T-cell lymphoma or skin T-cell lymphoma); Excretory system (for example kidney, renal pelvis, ureter, bladder and other urinary organs) tumor; Gastrointestinal tract (for example esophagus, stomach, small intestinal, colon, knot rectum, proctosigmoid link (rectosigmoid junction), rectum, anus and anal canal) tumor; Relate to other part of liver and stones in intrahepatic bile duct, gallbladder and biliary tract, pancreas and other Alimentary tumor; Oral cavity (for example at the bottom of lip, tongue, gums, the oral cavity, palate, the parotid gland, salivary gland, tonsil, oropharynx, nasopharynx, pears shape nest, hypopharynx and other position, oral cavity) tumor; Reproductive system (for example vaginal orifice, vagina, cervix uteri, uterus, ovary and other position, Placenta Hominis, penis, prostate, testis and with male reproductive organ relevant other position relevant) tumor with the female reproductive organ; Respiratory tract neoplasms (for example nasal cavity, middle ear, paranasal sinus, larynx, trachea, bronchus and lung tumor, for example small cell lung cancer and nonsmall-cell lung cancer); Skeletal system (for example limbs skeleton and articular cartilage, bone articular cartilage and other position) tumor; Cutaneous tumor (for example the squamous cell carcinoma of the basal cell carcinoma of the malignant melanoma of skin, non-melanoma skin cancer, skin, skin, mesothelioma, Kaposi (Kaposi ' s sarcoma)); With the tumor that relates to other tissue that comprises peripheral nerve and autonomic nervous system, connective tissue and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine gland and dependency structure; The pernicious anything superfluous or useless of the unspecified Secondary cases of lymph node, breathing and the pernicious anything superfluous or useless of digestive system Secondary cases, and the pernicious anything superfluous or useless of Secondary cases at other position.

In certain embodiments of the invention, the present composition and method are used to treat the transitivity sarcoma.In certain embodiments, the compositions and methods of the invention are used to treat the metastatic cancer by following disease origin: bladder cancer, breast carcinoma, chronic lymphatic tumor leukemia, head and neck cancer, carcinoma of endometrium, non-Hodgkin lymphomas, nonsmall-cell lung cancer, ovarian cancer, cancer of pancreas and carcinoma of prostate.

In certain embodiments of the invention, compositions and method are used to treat neuroderm source property metastatic tumo(u)r.Any neuroderm source property metastatic tumo(u)r that exists in the human patients generally can use compositions/method of the present invention to treat.In certain embodiments, influence the member of patient's neuroderm source property metastatic tumo(u)r: the neuroderm source property metastatic tumo(u)r in glioma, meningioma, ependymoma, medulloblastoma, neuroblastoma, ganglioneuroma, pheochromocytoma, melanoma, the original neuroectodermal tumors of periphery, small cell lung cancer, ewing's sarcoma (Ewing ' s sarcoma) and the brain by the following group that forms.In certain embodiments, property metastatic tumo(u)r in neuroderm source is a melanoma.In some such embodiment, melanoma is skin or intraocular melanoma.

In certain embodiments, property metastatic tumo(u)r in neuroderm source influences patient's brain.In certain embodiments, the cerebral tumor is a glioma.Half is a glioma approximately in all former carbuncle in the occipital region tumors.Glioma has 4 kinds of main types: astrocytoma (in adult and child modal types of glioma in the two), ependymoma, oligodendroglioma and mixed nerve glioma.Glioma can be according to the classification of its position: under the canopy of the heavens on (infratentorial) (that is, be arranged in the Lower Half of brain, be mainly seen in child patient) or the canopy of the heavens (supratentorial) (that is, be arranged in the first half of brain, be mainly seen in adult patients.)

Also can further classify to glioma according to grade, said grade is to be confirmed by the pathological evaluation of tumor.(World Health Organization WHO) has developed a kind of hierarchy system, from tending to have minimum invasive I level glioma to tending to tool aggressive and virulent IV level glioma in World Health Organization (WHO).Rudimentary (being I level or II level) gliomatous instance includes, but is not limited to hair cell type astrocytoma (being also referred to as teenager hair cell type astrocytoma), fibrous astrocytoma, pleomorphic xanthoastrocytoma and dysontogenesis property neuroepithelioma.The higher nerve glioma contain III level glioma (anaplastic astrocytoma for example, AA) with IV level glioma (glioblastoma multiforme for example, GBM).The anaplastic astrocytoma is most commonly in 30 years old to 50 years old masculinity and femininity, and accounts for 4% of all cerebral tumors.Glioblastoma multiforme is one type of the strongest neuroglial tumor of aggressive, is most commonly in 50 years old to 70 years old masculinity and femininity, and accounts for 23% of all former carbuncle in the occipital region tumors.The gliomatous prognosis of IV level is the poorest, and mean survival time is 12 months.In certain embodiments, the inventive method is used to treat the higher nerve glioma.

Although adopted the aggressive treatment, glioma recurs usually, often appears more high-gradely, has different forms sometimes.Although relapse rate is different, IV level glioma always recurs.Therefore, in certain embodiments, the inventive method is used to treat transitivity recurrence glioma, especially recurrent higher nerve glioma.

Can use the metastatic tumo(u)r of the present composition and method treatment also to comprise with the intractable metastatic tumo(u)r of other chemotherapeutics treatment.The term that when this paper mentions tumor, uses " intractable " meaning refers to; When treating with the chemotherapeutics of at least a non-present composition; Tumor (and/or its shift) does not show or only shows that more weak antiproliferative reacts (that is, not suppressing or only more weak the tumor growth that suppresses) after with said chemotherapeutics treatment, that is to say; Tumor can not be treated fully with other (preferred standard) chemotherapeutics, maybe not satisfied result can only be obtained.When mentioning the treatment of intractable tumor etc.; The present invention is interpreted as not only containing (i) tumor that one or more chemotherapeutics have been failed during patient's treatment, and (ii) (for example biopsy and cultivation) demonstrates intractable tumor by alternate manner in the presence of chemotherapeutics but also contain.

The patient who receives treatment of the present invention generally comprises diagnosis or once diagnosed any patient who suffers from tumor.In certain embodiments, the patient is diagnosed as and suffers from one or more transfers.In certain embodiments, the patient is diagnosed as and suffers from the tumor that known generation is shifted.In certain embodiments, the patient is diagnosed as to suffer from and confirms the perhaps tumor in perhaps contingent residing stage of transfer.In certain embodiments, the patient once suffered from tumor, but no longer represented the symptom with primary tumo(u)r; In some these type of embodiment, the patient has transfer, its still available the inventive method treatment.One of ordinary skill in the art will recognize, can carry out different diagnostic methods with character according to the position of tumor and/or transfer, comprise imaging, biopsy etc.

B. dosage and dispensing

In Therapeutic Method of the present invention, scorpion catilan reagent or its pharmaceutical composition generally can be thought and realize that at least one predetermined result institute must or be enough to realize that the amount of at least one predetermined result and time throwing give.For example, the amount and the time of the scorpion catilan reagent that gives of throwing should make its kill cancer cell, reduce tumor size, reduce size, the inhibition of one or more transfers or postpone tumor growth or time-to-live of transfer, prolongation patient, or obtains clinical benefit in addition.

Treatment of the present invention can be made up of the multi-agent of single agent or lasting a period of time.Dispensing can every day, (or interval of some other a couple of days) one or many or carry out according to intermittent time-histories weekly.The exact amount of waiting to throw the scorpion catilan reagent that gives or its pharmaceutical composition will change with the person under inspection is different, and depend on several factors (vide infra).

Scorpion catilan reagent or its pharmaceutical composition can use any dosing way of effective realization predetermined treatment effect to throw and give.In certain embodiments of the invention, scorpion catilan reagent (or its pharmaceutical composition) is systemic delivery.Typical whole body dosing way includes, but is not limited to intramuscular, intravenous, pulmonary and oral route.Also can be for example through infusion or inject, or absorb (for example oral, mucosa, rectum and intestinal mucosa etc.) through epidermis or mucocutaneous internal layer, carry out the whole body dispensing.In certain embodiments, scorpion catilan reagent is thrown through intravenous and is given.Be described in the instance 2 and throw the exemplary program of giving scorpion catilan reagent in the human patients medium-sized vein.

Look dosing way and decide, can be according to body weight; Body surface area; Former organ/tumor size; And/or wait to treat quantity, size and/or the type that shifts in person under inspection's body, calculate effective dose.One of ordinary skill in the art can be easy to optimize optimal dose according to the pharmacokinetic data of in people's clinical trial, observing.The final dose scheme will be by the attending doctor according to the pharmaceutically-active factor decision of various improvement, like the concrete activity of medicine; The order of severity of patient's damage and reactivity; Patient's age, health condition, body weight, sex and diet; The order of severity of the infection of any existence; The dispensing time; The use of other therapies (or not using); And other clinical factor.When using scorpion catilan reagent to study, with other occurring about the information of optimal dose level with the treatment persistent period.

Typical doses comprises per kilogram of body weight 1.0pg to per kilogram of body weight 100mg.For example, for the whole body dispensing, dosage can be that per kilogram of body weight 100.0ng is to per kilogram of body weight 10.0mg.

More specifically say; Giving among some embodiment of scorpion catilan reagent through the intravenous throwing; The administration of said reagent can comprise to throw gives potion or multi-agent, and it comprises about 0.005mg/kg to about 5mg/kg, for example; About 0.005mg/kg arrives about 2mg/kg to about 4mg/kg, about 0.02mg/kg to about 3mg/kg, about 0.03mg/kg to about 5mg/kg, about 0.01mg/kg, or about 0.03mg/kg is to about 1.5mg/kg scorpion catilan.For example; In certain embodiments, throwing the potion or the multi-agent scorpion catilan reagent that give respectively contains the 0.03mg/kg that has an appointment, about 0.04mg/kg, about 0.05mg/kg, about 0.06mg/kg, about 0.07mg/kg, about 0.09mg/kg, about 1.0mg/kg or surpasses 1.0mg/kg scorpion catilan.In other embodiments, throwing the potion or the multi-agent scorpion catilan reagent that give respectively contains the 0.05mg/kg that has an appointment, about 0.10mg/kg, about 0.15mg/kg, about 0.20mg/kg, about 0.25mg/kg, about 0.30mg/kg, about 0.35mg/kg, about 0.40mg/kg, about 0.45mg/kg, about 0.50mg/kg, about 0.55mg/kg, about 0.60mg/kg, about 0.65mg/kg, about 0.70mg/kg, about 0.75mg/kg, about 0.80mg/kg, about 0.85mg/kg, about 0.90mg/kg, about 0.95mg/kg, about 1.0mg/kg or surpasses about 1mg/kg scorpion catilan.Again in other embodiments, throwing the potion or the multi-agent scorpion catilan reagent that give respectively contains 1.0mg/kg, about 1.05mg/kg, about 1.10mg/kg, about 1.15mg/kg, about 1.20mg/kg, about 1.25mg/kg, about 1.3mg/kg, about 1.35mg/kg, about 1.40mg/kg, about 1.45mg/kg, about 1.50mg/kg or surpasses about 1.50mg/kg scorpion catilan.In these embodiment, treatment can comprise to throw gives potion scorpion catilan reagent, or throws and give 2 doses, 3 doses, 4 doses, 5 doses, 6 doses or above 6 doses.Successive two doses can be at interval 1 day, 2 days at interval, 3 days at interval, 4 days at interval, 5 days at interval, 6 days at interval, 7 days or surpass 7 days at interval (for example 10 days, 2 week or 2 weeks of surpassing) throw and give at interval.

C. combination treatment

Should be appreciated that, Therapeutic Method of the present invention can use with the combination of extra therapy (that is, treatment of the present invention can with one or more required therapeutic agents or medical procedures simultaneously, before or after said therapeutic agent or medical procedures, throw and give).Be used for the required therapeutic agent of particular combination considered and/or the compatibility of program and the predetermined treatment effect to be achieved of the therapy (therapeutic agent or program) of said assembled scheme.

For example; Therapeutic Method of the present invention can use with other program; Depend on tumor to be treated, said other program comprises operation, X-ray therapy (for example γ-radiation, neutron beam X-ray therapy, electron beam X-ray therapy, proton therapy, closely therapy (brachytherapy), systemic radiosiotope), incretotherapy, high temperature (hyperthermia) and cryotherapy (cryotherapy).

In many metastatic brain tumor situations, treatment of the present invention is thrown after the exenterate primary tumo(u)r through regular meeting and is given.When the treatment cerebral tumor, the main target of operation is to realize complete excision (gross-total resection),, removes all visible primary tumo(u)rs that is.A difficult point that realizes this target is that these tumors are infiltrating, that is, it tends between the normal brain activity structure, pass in and out.The amount of the tumor of in addition, can be safely removing from patient's brain exists greatly and changes.If all or part of tumor is positioned at the zone of brain control key function, so generally can not remove.And, only unlikely or not-so-practical with the transfer of exenterate and/or destruction distal site.

In many metastatic brain tumor situations, treatment of the present invention through the combination of regular meeting and X-ray therapy (that is, with its simultaneously, before or after it) throw and give.In conventional therapy, X-ray therapy generally is after operation, to carry out.Radiation generally is to give with a series of treatments every day (be called and cut apart (fraction)) that last several weeks.This throwing is given radiating " partition type " method for destroying tumor cell to greatest extent and minimizing the side effect of adjacent normal brain activity very important.Thrown and given radiating zone (being called radiation field) care should be used to calculating, to avoid comprising more normal brain activity as far as possible.

Perhaps or in addition, Therapeutic Method of the present invention can be thrown with other therapeutic agent (as weakening the medicament of any ill effect, for example antiemetic etc.) and/or other granted chemotherapeutics combination and give.The instance of chemotherapeutics (for example includes, but is not limited to alkylating agent class medicine; Chlormethine, chlorambucil, cyclophosphamide, melphalan, ifosfamide etc.), the antimetabolite class (for example; Methotrexates etc.), purine antagonist class and pyrimidine antagonist class are (for example; Ismipur, 5-fluorouracil, cytosine arabinoside, gemcitabine etc.), the spindle poison class (for example; Vincaleucoblastine, vincristine, vinorelbine, paclitaxel etc.), podophillotoxines (for example, etoposide, irinotecan, TPT etc.), antibiotics (for example, doxorubicin, bleomycin, mitomycin etc.), nitro ureas (for example; Carmustine, lomustine, nimustine etc.), the inorganic ions class (for example; Cisplatin, carboplatin etc.), enzyme (for example, asparaginase etc.) and hormones (for example, tamoxifen, leuproside, flutamide, megestrol etc.) etc.The more detailed argumentation of relevant up-to-date cancer therapy, referring to Http:// www.cancer.gov/The tumour medicine inventory of FDA approval, Http:// www.fda.gov/cder/cancer/druglistframe.htmAnd Merck diagnosis and treatment handbook, the 17th edition, 1999, the complete content of said reference all is incorporated herein by quoting.

The inventive method also can combine use with one or more other the cytotoxic agent as the part of therapeutic scheme, and wherein said other cytotoxic agent combination is selected from: CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone (prednisone) and procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone); COP (cyclophosphamide, vincristine and prednisone); CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine and prednisone); M-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, Dexamethasone (dexamethasone) and folinic acid (leucovorin)); ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, folinic acid, chlormethine (mechloethamine), vincristine, prednisone and procarbazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, folinic acid, cytosine arabinoside, bleomycin and vincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and folinic acid); MOPP (chlormethine, vincristine, prednisone and procarbazine); ABVD (amycin (adriamycin)/doxorubicin, bleomycin, vincaleucoblastine and dacarbazine); MOPP (chlormethine, vincristine, prednisone and procarbazine) and ABV (amycin/doxorubicin, bleomycin and vincaleucoblastine) replace; MOPP (chlormethine, vincristine, prednisone and procarbazine) and ABVD (amycin/doxorubicin, bleomycin, vincaleucoblastine and dacarbazine) replace; ChlVPP (chlorambucil, vincaleucoblastine, procarbazine and prednisone); IMVP-16 (ifosfamide, methotrexate and etoposide); MIME (methyl-GAG (methyl-gag), ifosfamide, methotrexate and etoposide); DHAP (Dexamethasone, high dose cytosine arabinoside and cisplatin); ESHAP (etoposide, methylprednisolone (methylpredisolone), high dose cytosine arabinoside and cisplatin); CEPP (B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin); CAMP (lomustine, mitoxantrone, cytosine arabinoside and prednisone); CVP-1 (cyclophosphamide, vincristine and prednisone), ESHOP (etoposide, methylprednisolone, high dose cytosine arabinoside, vincristine and cisplatin); (etoposide, vincristine and doxorubicin continue 96 hours to EPOCH; And bolus dose cyclophosphamide and oral prednisone), ICE (ifosfamide, cyclophosphamide and etoposide), CEPP (B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin), CHOP-B (cyclophosphamide, doxorubicin, vincristine, prednisone and bleomycin), CEPP-B (cyclophosphamide, etoposide, procarbazine and bleomycin), and P/DOCE (epirubicin or doxorubicin, vincristine, cyclophosphamide and prednisone).

One of ordinary skill in the art will understand, and treat that making up the selection of throwing one or more therapeutic agents that give with Therapeutic Method of the present invention will depend on metastatic tumo(u)r to be treated.

For example; The chemotherapeutic that is used for cerebral tumor prescription includes, but is not limited to temozolomide (Temodar

), procarbazine (Matulane ) and lomustine (CCNU), and these medicines are oral; Vincristine (Oncovin

or Vincasar PFS

), cisplatin (Platinol

), carmustine (BCNU, BiCNU) and carboplatin (Paraplatin

), these are intravenous dispensings; And methotrexate (Rheumatrex or Trexall

); But (that is, being injected directly in the spinal fluid) thrown and given in this medicine per os, intravenous or the sheath.BCNU can also give with polymer disk implant (Giadel

disk) form at intra-operative.A kind of the most frequently used prescription combination treatment for the cerebral tumor is PCV (procarbazine, CCNU and a vincristine), and it gives once usually in per 6 weeks.

In tumor to be treated is among the embodiment of the neuroderm source property cerebral tumor, and the inventive method can be used with the medicament combination of handling like symptoms such as spasm and cerebral edemas.After dosing successfully controlled with brain tumor-related spasticity anticonvulsant agents include (but are not limited to) phenytoin (phenytoin) (Dilantin

), carbamazepine (Carbamazepine) (Tegretol ) and divalproex sodium (divalproex? sodium) (Depakote

).Brain swelling can be used steroid (like Dexamethasone (Decadron )) treatment.

D. pharmaceutical composition

As mentioned above, treatment of the present invention, suppress and/or reduce and/or detection method comprises throwing and gives scorpion catilan reagent itself or its pharmaceutical compositions.Pharmaceutical composition generally comprises at least a scorpion catilan reagent and at least a pharmaceutically acceptable supporting agent or the excipient of effective dose.

Pharmaceutical composition can use well-known conventional method preparation in this technology.Best visual dosing way of pharmaceutical preparation and required dosage and change.Said preparation possibly influence the interior rate of release of physical state, stability, body and the interior clearance rate of body of the chemical compound that gives of throwing.Preparation can produce solid, liquid or semiliquid pharmaceutical composition.

Even for facility dispensing and dosage, pharmaceutical composition can be mixed with dosage unit form.The statement " unit dosage forms " that this paper uses is meant the physics discrete unit that is used to wait treat patient's scorpion catilan reagent.Each unit contains the active substance that can produce the scheduled volume of required therapeutic effect through calculating.Yet, should be appreciated that the accumulated dose of compositions will be confirmed by the attending doctor in the scope that rational medicine is judged.

As mentioned above, in certain embodiments, scorpion catilan reagent is thrown through intravenous through injection or infusion and is given.Be suitable for throwing the pharmaceutical composition that gives and use the dispersant or wetting agent and the suspending agent preparation that are fit to according to known technology through injection or infusion.Pharmaceutical composition also can be sterile injectable solution, suspension or the emulsion in nontoxic diluent or solvent, for example in 2, and the solution in the 3-butanediol.Operable acceptable mediator and solvent have water, Ringer's mixture (Ringer ' s solution), U.S.P. and isotonic sodium chlorrde solution.In addition, usually sterile non-volatile oils is used as dissolving or suspension media.For this reason, the nonvolatile oil of any gentleness be can use, synthetic monoglyceride or Diglyceride comprised.Also can be used for like fatty acids such as oleic acid in the preparation of injectable formulation.

Can for example hold back filter, or, come injectable formulation is sterilized through incorporating the biocide that can be dissolved or dispersed in the aseptic solid composite form in sterilized water or other sterile injectable medium before use into through filtering antibacterial.

Be the effect of prolong drug, the absorption of the medicine of the injection of need slowing down usually.This can realize through active component is dissolved or is suspended in the oily mediator.Make injectable through the microcapsule substrate that forms medicine with biodegradable polymer (like polylactide-gather Acetic acid, hydroxy-, bimol. cyclic ester) and accumulate form.Depend on the character of medicine, speed that can control drug release to polymer ratio and used particular polymers.Accumulating the formula injectable formulation also can prepare through pharmaceutical pack being embedded in liposome compatible with bodily tissue or the microemulsion.

III. detection method

A. dispensing

In another aspect, the present invention provides the method that detects neoplasm metastasis in vivo.For example, possibly need to use method of the present invention to detect the individual intravital neoplasm metastasis of suffering from or once suffered from least a primary tumo(u)r.Said method comprises throws scorpion catilan reagent or its pharmaceutical composition through labelling described herein that gives patient's effective dose, and the scorpion catilan reagent of consequently said process labelling with the cell in primary tumo(u)r tissue and/or the metastatic tumo(u)r tissue specificity can take place and combine.

Generally speaking, will depend on like consideration such as the body region of patient's age, sex and body weight, examine and dosing way through the dosage of the scorpion catilan reagent of labelling and change.Also factor adjustment such as considered such as contraindication, concomitant therapy and other variable wait to throw the dosage of the scorpion catilan reagent of the process labelling that gives.But, this can easily be accomplished by trained doctor.Generally speaking, the suitable dosage of the scorpion catilan reagent of process labelling is corresponding to the medicament minimum flow that is enough to allow to detect the intravital neoplasm tissue of patient.

Give an example, using ¹³¹I labelling scorpion catilan reagent and in the embodiment of intravenous dispensing can comprise to throw through the administration of the scorpion catilan reagent of labelling and give potion or multi-agent, and it respectively comprises about 5mCi to about 50mCi, and for example about 5mCi arrives about 40mCi, or about 10mCi arrives about 30mCi ¹³¹I.For example, throw potion or the multi-agent warp that gives ¹³¹The radiolabeled scorpion catilan of I reagent can respectively contain the 10mCi that has an appointment, about 20mCi or about 30mCi ¹³¹I.In these embodiment, diagnotor can comprise throwing and give single agent warp ¹³¹The radiolabeled scorpion catilan of I reagent, or throw and give multi-agent, for example 2 doses, 3 doses or 4 doses.Successive two doses can be at interval 1 day, 2 days at interval, 3 days at interval, 4 days at interval, 5 days at interval, 6 days at interval, 7 days or surpass 7 days at interval and throw and give at interval.

Using warp ¹³¹Among the embodiment of the radiolabeled scorpion catilan of I reagent, can throwing give through ¹³¹(for example, treating according to the present invention preceding 1 day, 2 days or 3 days) before the radiolabeled scorpion catilan of I, the patient is being thrown give oversaturated potassium iodide.Throwing is given oversaturated potassium iodide and is stoped thyroid to absorb ¹³¹I, thus prevention is like patient's condition such as hypothyroidism.

Throwing is given through after the scorpion catilan reagent of labelling, and specificity taking place after bonded enough past time, detects through bonded scorpion catilan reagent through labelling.

In certain embodiments, throw the scorpion catilan reagent that passes through labelling that gives second effective dose in second time period, and the said combination of passing through the scorpion catilan reagent of labelling in the measure individuals body.Can allow assessment to combine any change of (for example combination degree and/or position) for throwing to give through bonded measurement behind the scorpion catilan reagent of labelling for the second time, this can indicate progress, the stability of one or more transfers and/or degenerate.In the later time section, (for the third time promptly, the 4th is inferior) throws the scorpion catilan reagent through labelling that gives effective dose and can obtain extra measurement result again.This possibly be progress, the stability of (for example) one or more transfers of assessment in one long period and/or degenerate needed.

In certain embodiments, the time span between the continuous dispensing of the scorpion catilan reagent of the process labelling of effective dose can change.In certain embodiments, throw the scorpion catilan reagent that gives through labelling with regular time intervals roughly.In certain embodiments, the time-histories of the dosage regimen of dispensing time-histories and patient experience treatment matches or is corresponding.

The detection and the location of B. shifting

One of ordinary skill in the art will recognize; Can be undertaken by in the several different methods any through the bonded detection of the scorpion catilan reagent of labelling and linked groups, said method includes, but is not limited to spectrum, photochemistry, biochemistry, immunochemistry, electricity, optics or chemical mode.The selection of detection method generally is based on the character of the mark part (that is, fluorescence part, radionuclide, paramagnetic metal ion etc.) of said reagent.In certain embodiments, the detection and the location of one or more transfers is to use imaging technique to carry out in patient's body.

Can use different imaging techniques according to the character of mark part.For example, if mark part comprises paramagnetic metal ion (Gd for example ³⁺), can use nuclear magnetic resonance method (MRI) to detect so and combine.If mark part (for example comprises radiosiotope ¹³¹I etc.), can use single photon emission computed body layer imaging (SPECT) and/or positron emission emission computer body layer imaging (PET) to combine to detect so.Other imaging technique comprises the γ camera imaging.

According to monitoring method of the present invention; If the scorpion catilan reagent of comparing with the combination level of linked groups through labelling through the scorpion catilan reagent of labelling raises with the level that combines of normal structure to some extent, will differentiate to shifting except that the tissue the primary tumo(u)r tissue so.Once mentioned like preceding text, normal structure is defined as non-superfluous natural disposition tissue in this article.For example; When carrying out said method in vivo, can the combination level of in a certain zone of relevant organ (for example brain), measuring through the scorpion catilan reagent that passes through labelling measured in the combination level of the scorpion catilan reagent of labelling and the normal region at same organ be compared.

In certain embodiments, if measured combination level is higher than the level that combines with normal structure, linked groups is differentiated to be superfluous natural disposition tissue so.For example, said combination level possibly be with normal structure combine level at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about more than 200 times or 200 times.

Instance

Following case description forms and puts into practice patterns more of the present invention.Yet, should be appreciated that these instances are for purposes of illustration, and do not plan to limit scope of the present invention.In addition, only if the description in the instance part provides with past tense, otherwise text is as this description remainder, and being not intended to the hint experiment and being actual that carry out or data is actual acquisitions.

Instance 1: the scorpion catilan combines to transfer to the melanoma of brain and lung

The experiment of describing in this instance shows that in slicer, the scorpion catilan is incorporated into the melanoma of transferring to brain and/or lung.

Materials and methods

Utilize chemosynthesis type scorpion catilan (TM-601) that the freezing or paraffin section of people's biopsy is carried out histochemical stain, said scorpion catilan contains the detectable biotin group that chemically is attached to N-terminal.People's tissue sample is from two kinds of sexes and different age and races.Most of sample all is human brain tissue storehouse (the Human Brain Tissue Bank) acquisition through tissue sampling mechanism (Tissue Procurement), UAB hospital and the Canadian London of human tissue cooperation net (Cooperative Human Tissue Network), UAB (University of Alabama of Birmingham (University of Alabama at Birmingham)).Urgency in the freezing gel is frozen tissue and flesh tissue is cut into 8 microns thin slice with being embedded in, and picks on the positively charged microscope slide.Subsequently, according to the dyeing scheme, fixing these sections in 4% paraformaldehyde or Milloniqs solution (buffer solution of sodium phosphate by containing 4% formaldehyde, 0.4%NaOH and 7% methanol constitutes).Paraffin mass is cut into slices, and prepare according to standardization program.

The blocking-up biopsy slice is 1 hour in the PBS that contains 10% normal goats serum, and spends the night with the diluent processing of biotinylation scorpion catilan down at 4 ℃.Behind cleaning down, by avidin-biotin composite (avidin-biotin complex, ABC) system's (Vectastain Elite ABC test kit; Wei Kete experiment (the Vector Laboratories of company from the California Burlinton; Burlignton, California)) dyeing is manifested, and utilize DAB (3; 3 '-diaminobenzidine, Wei Kete tests company) observe with the chrominance response of ABC complex.

With C.I. 42590 (a kind of nuclear dyestuff) biopsy slice is carried out counterstain, to observe undyed cell.Because valid density, organization factors or the duration of the reaction of labelling change with experiment, make non-specific context marker to change.Therefore, utilize C.I. 42590 but need not biotinylated scorpion catilan, identical dyeing is carried out in the contrast section.Scorpion catilan labelling exceeds and is defined as positive cell dyeing outside the background when comparing with adjacent contrast section.The cell that contains a large amount of endogenous peroxidase represents darker background dyeing because of the reaction of DAB and peroxidase in matched group.

With hematoxylin (pair cell nuclear staining) and Yihong (dyeing of pair cell matter) the two section statining adjacent to third phase.Therefore, analyze tissue, three the adjacent sections of all dyeing for each.

The result

Fig. 1 shows that biotinylation scorpion catilan makes the painted microphotograph of the melanoma of transferring to brain.11 all was that TM-601 is positive during 11 melanoma vertigo moved, and in 5 plain struma tumors of former blackout 5 all be positive.In addition, the scorpion catilan also combines to transfer to the melanoma (Fig. 2) of lung.On the other hand, normal skin to TM-601 Fails To Respond (6/6 all is negative) (Fig. 3), although even have the dyeing of part background in the melanocyte in matched group.

Instance 2: in patient's medium-sized vein of suffering from recurrent or refractory gonosome and/or brain transitivity entity tumor ¹³¹The I phase of I-TM-601 forms images and safety research.

This case description is tested the PRELIMINARY RESULTS that obtains by the I phase of carrying out at 5 clinical scenes.In this clinical trial, TM-601 is given in 48 patient's intravenouss throwings.This multicenter, opening, derandominzation " in the person under inspection " gain studies continuously comprise the patient who suffers from the pernicious constitutional entity tumor of confirming through the histology (recurrent or intractable), and it has shown the clear and definite evidence with the detected MET that does not receive the standard treatment effect.

The purpose that the said I phase is studied is: a) estimate intravenous ¹³¹Whether I-TM-601 can locate by tumour-specific in the patient who suffers from recurrent or intractable transitivity (comprising that vertigo moves) entity tumor; B) confirm what the intravenous throwing was given ¹³¹The distribution of I-TM-601 and dosimetry; And c) confirms what the intravenous throwing was given ¹³¹The safety of I-TM-601 and toleration.

Patient and therapeutic scheme

48 persons under inspection of registration in this research.These persons under inspection are used the scheme of hereinafter description.The person under inspection is experienced increase progressively for 1 to 2 time intravenous dosages ¹³¹I-TM-601, a series of subsequently body scans are to confirm ¹³¹Whether I-TM-601 has been positioned target tumor, and is showing ¹³¹After the tumour-specific of I-TM-601 absorbs, promptly give an intravenous therapy dosage ¹³¹I-TM-601.Graphic extension administration flow process among Fig. 4.

The research patient receives nearly 3 doses through intravenous (IV) infusion ¹³¹I-TM-601 (scope is that 10mCi/0.2mg is to 30mCi/0.6mg).Only to give in throwing 10 or 20mCi dosage after showed by imaging in 24 hours ¹³¹30mCi dosage is given in patient's throwing that the tumour-specific of I-TM-601 absorbs ¹³¹I-TM-601.

¹³¹The preparation of I-TM-601

Final TM-601 medicine is to pack into the aseptic white in the vial of stopper jam-pack to the canescence lyophilized powder.The imaging and the therapeutic dose that use in this test all are the dosage through radiolabeled TM-601.

Final TM-601 medicine is restored in 0.56mL radioactive label buffer, obtain the 1mg/mL warp ¹³¹The radiolabeled solution of I, and it is delivered to clinical scene.Contain the 4mL infusion solution of having an appointment in the syringe, and carry out roughly labelling according to inclusions and exit dose.After receiving at the scene, radiation safety official or other field personnel who is fit to promptly confirm ¹³¹The radiation counter of I-TM-601 is in the specification standards of prescription.Shielding contains the syringe of the radiolabeled medicine of final process, subsequently it is transferred to suitable hospital area, so that the patient is offerd medicine.Under 2-8 ℃, keep in Dark Place ¹³¹I-TM-601 solution also shields for use.Using ¹³¹Behind the I radioactive label, be recommended in and use this product in 24 hours.

¹³¹The dispensing of I-TM-601 and imaging research

Make all receive radiolabeled proof load ¹³¹The supersaturation potassium iodide of the oral reception of the patient of I-TM-601 300mg dosage every day (supersaturated potassium iodide, SSKI), radiolabeled from infusion ¹³¹I-TM-601 was about to begin begin before the infusion same day, continued minimum 3 days, absorbed to stop thyroid and other organ ¹³¹I.Before the research medicine is given in throwing, SSKI is distributed to the patient, to the patient the relevant explanation that correctly makes medicament not in clinic/hospital the time is provided simultaneously.

To contain ¹³¹The syringe of I-TM-601 is inserted in the infusion orifice in 6 inches venous detaining needle/conduits with " back of the body carries (piggy-back) " mode.With in 100mL infusion 0.9% sodium chloride per hour, threw and give product by " slowly intravenous push (slow IV push) " mode through about 5 to 10 minutes.If observe following arbitrary situation, just stop ¹³¹I-TM-601 infusion: (1) systolic pressure reduction＞25mmHg; (2) the tangible respiratory distress that proves by research worker; (3) body temperature＞102 ℉; (4) spasm; (5) outbreak of level of consciousness change or new nerve injury, or other reason, for example clinician's judgement or patient's requirement.

Give in throwing ¹³¹Behind the M-TM-601 24 hours, by the γ camera and in some cases by the SPECT imaging, to confirm to receive 30mCi dosage ¹³¹The location of I-TM-601 and qualification.

Safety results

End 22 serious adverse events of 17 patient's report footings in May, 2008.Research worker judges, all SAE " unlikely " cause by the research medicine or with research medicine " uncorrelated ", and have 4 to be to agree not continue reception in these incidents ¹³¹Take place among two patients after the I-TM-601 research medicine.The 5th incident is to take place among the patient before receiving the research medicine, and it is after a while by successful administration.

Efficacy outcomes

After the intravenous dispensing, in the kinds of tumors type, observe tumour-specific and absorb, comprise 7 among 8 glioblastoma patients; All, comprise that 1 dispersivity bone shifts the patient among 2 metastatic prostate cancer patients; Among 5 transitivity nonsmall-cell lung cancer patients 3; Among 8 metastatic melanoma patients 7; Among 8 transitivity colon cancer patients 6; In 3 transitivity Pancreas cancer patients 2; In 4 transitivity patients with mastocarcinoma 1; 1 transitivity transitional cell carcinoma patient; 1 transitivity pheochromocytoma patient; With 1 pleomorphic xanthoastrocytoma patient (such as among Fig. 5 general introduction; Also referring to Fig. 6-9).

All patients are through the 10mCi of intravenous acceptance test dosage (0.2mg peptide) ¹³¹I-TM-601.In injection ¹³¹(smaller or equal to 60 minutes), 3 hours, 24 hours, 48-72 hour and obtained 5 successive whole body γ camera images in 168 hours immediately behind the I-TM-601 are to carry out tumor-localizing and dosimetry analysis.After 1 week, make and utilize γ camera or SPECT imaging method to show that the patient of tumor-localizing receives the 30mCi (0.6mg peptide) of second therapeutic dose ¹³¹I-TM-601.After 1 week, with 20mCi (0.4mg peptide) ¹³¹I-TM-601 treats and does not show the patient who absorbs, under higher dosage, to confirm possible location.

When estimating in the 28th day, in 8 glioma patients, see tumor response (the volume reduction that is strengthened focus by gadolinium defines) in 2 the nuclear magnetic resonance (MRI), show that gross tumor volume is by the measurable reduction of baseline (referring to Figure 10 and 11).

The scorpion catilan reagent that these results have shown systemic delivery in vivo (for example ¹³¹I-TM-601) therapeutical effect.These results show that also the intravenous throwing is given ¹³¹I-TM-601 will stride blood brain barrier, and can cause the improvement of inoperable glioma patient's MRI imaging.In addition, in suffering from the patient of metastatic cancer, intravenous is sent ¹³¹I-TM-601 can targeting transfer (for example referring to Figure 12) at a distance.

Instance 3: in metastatic melanoma patient medium-sized vein ¹³¹I-scorpion catilan ( ¹³¹I-TM-601) tumour-specific targeting

In the clinical trial formerly, will ¹³¹The local throwing of I-scorpion catilan given in the patient's who suffers from the recurrent glioblastoma multiforme the tumor resection chamber.In this example, inspection intravenous (IV) throwing is given ¹³¹The distribution of I-scorpion catilan, whether feasible and can cause absorption in the tumor to confirm the intravenous dosing way for the patient who suffers from metastatic melanoma (comprising the transfer that gets into CNS).The experiment of describing in this instance shows that intravenous is sent ¹³¹I-scorpion catilan is positioned the tumor locus of whole body and the transfer in the brain.Therefore, the intravenous throwing is given ¹³¹I-scorpion catilan is striden blood brain barrier, and can be used for targeting transfer at a distance.

Materials and methods

The whole body of in instance 2, discussing is thrown and is given ¹³¹The patient of 7 metastatic melanomas of registration in the prospective clinical trical of I-scorpion catilan.This instance is discussed by these 7 results that the patient obtains in more detail.All patients are through the 10mCi of intravenous acceptance test dosage (0.2mg peptide) ¹³¹I-scorpion catilan.In injection ¹³¹Behind the I-scorpion catilan (smaller or equal to 60 minutes) and 3 hours, 24 hours, 48-72 hour immediately and obtained 5 successive whole body γ camera images in 168 hours, to carry out tumor-localizing and dosimetry analysis.After 1 week, make and utilize γ camera or SPECT imaging method to show that the patient of tumor-localizing receives the 30mCi (0.6mg peptide) of second therapeutic dose ¹³¹I-scorpion catilan.After 1 week, with 20mCi (0.4mg peptide) ¹³¹I-scorpion catilan is treated and is not shown the patient who absorbs, under higher dosage, to confirm possible location.

The result

Give in the intravenous throwing ¹³¹Behind the I-scorpion catilan, there are 6 in γ camera of following the tracks of or SPECT imaging, to show the tumour-specific location in 7 melanoma patients of registration.Tumor-localizing is observed at the position in the central nervous system and outside the cranium.(instance is referring to Figure 13.) from research, withdraw from 1 remaining patient in the proof load first time (10mCi/0.20mg peptide) back, and think that it can not be estimated.Do not observe dose-limiting toxicity.3 patients that University of Alabama in Birmingham received treatment carry out dosimetry analysis completely.Average radiation dosage to whole body is about 0.24cGy/mCi (scope is about 0.21 to about 0.27cGy/mCi); And the average radiation dosage to tumor is about 2.56cGy/mCi (scope is about 1.36 to about 4.43cGy/mCi), and the treatment ratio that wherein calculates is about 10 (tumor dose/whole-body dose).

The result shows that the intravenous throwing is given ¹³¹I-scorpion catilan is striden blood brain barrier, and produces the selectively targeted rate of tumors of higher.Therefore, can use ¹³¹The transfer at a distance of I-scorpion catilan targeting comprises the transfer in the brain.Following clinical trial will be estimated in kinds of tumors type medium-sized vein throwing and give higher dosage ¹³¹The safety and the effect of I-scorpion catilan.

The inhibition that instance 4:TM-601 generates the choroid neovascularity and make it to degenerate

The formation of neovascularity (angiogenesis) and these blood vessels keep the important elements that is considered to shift.In this example, use the choroid neovascularity to generate and analyze, estimate the ability that the scorpion catilan suppresses angiogenesis and/or causes the blood vessel degeneration of existing new formation.About the time that induction of vascular forms, begin to throw when giving TM-601, TM-601 obviously reduces neovascularization.Form back a couple of days throwing at induction of vascular and give in the experiment example of TM-601, TM-601 makes the choroid neovascularity generate obviously degeneration.

Materials and methods

Coagulate art by means of light, utilize 530nm laser, induce the choroid neovascularity to generate (CNV).Utmost point portion 9 points, 12 and 3 positions produce 3 places burn behind each retina.If when induced with laser, produce bubble, judge that so the Bu Luheshi film successfully breaks.Include only the burn of observing bubble in this research.

In first experiment; Behind laser photocoagulation; In an eye, be dissolved in solution (n=17 the animal that obtains in the normal saline through intravitreal injection 1 μ L 50mg/mL TM-601; The quantifiable burn in 49 places), injection 1 μ L normal saline (n=17 animal, the quantifiable burn in 44 places) and in to branch hole.After 7 days, duplicate injection.Study the 14th day, (mean molecule quantity is 2 * 10 through fluorescein-labeled glucosan to the mice perfusion ⁶, Sigma company (Sigma)), preparation choroid plain film also utilizes fluorescence microscopy.

In second experiment, carried out laser photocoagulation in the 1st day.For the baseline measures of handling the preceding CNV of beginning,, one group of 10 mice (the quantifiable burn in 30 places) are poured into through fluorescein-labeled glucosan at the 7th day.Eye of residue mice receives 1 μ L 50mg/mL TM-601 solution (n=12 animal, the quantifiable burn in 34 places) of intraocular injection, and branch hole is received normal saline (n=13 animal, the quantifiable burn in 32 places).The 14th day, through fluorescein-labeled glucosan, preparation choroid plain film also utilized fluorescence microscopy to all residue mice perfusions.

Measure the size that choroid plain film median nexus film neovascularity generates damage.Perfusion is taken out eyes behind fluorescein-labeled glucosan, and in 10% formalin (formalin) buffer, fixes 1 hour.Remove cornea and crystalline lens, and from eye socket, downcut whole retina.(equator) makes radial slot from the edge to the equator in choroid, and the tiling eye socket.Utilize the fluorescence microscopy plain film, and with image digitazation.Use Image-Pro Plus software (Mai Dengsaibonike company (Media Cybernetics)) to measure the gross area that the choroid neovascularity relevant with each burn generates.

The result

In mice, cause that the choroid neovascularity generates (CNV), many situation of the CNV that its simulation is taken place in the patient who suffers from neovascularity property AMD with the laser photocoagulation Bu Luheshi film that breaks.In order to confirm whether TM-601 influences the formation of neovascularity in this model, the laser photocoagulation same day (the 1st day) and the 7th day, through intravitreal injection 50 μ g TM-601.At identical time point, normal saline is injected the contrast eye.After the Bu Luheshi film rupture 14 days, analyze each choroid plain film.When discovery utilized ophthalmic 50 μ g dosage TM-601, TM-601 handled the formation (Figure 13 and Figure 15) that significantly reduces neovascularity.

In order to assess the effect of TM-601, postpone processing, after the Bu Luheshi film rupture 7 days through intraocular injection 50 μ g TM-601 to the neovasculature that is pre-existing in this model.At this moment, existed bigger neovascularity to generate position (referring to the baseline among Figure 14).When measuring in the 14th day, the formation of neovascularity is not had influence (matched group among Figure 14), and single injection TM-601 makes CNV significantly degenerate (Figure 14 and Figure 15) at the 7th day single injection normal saline.

Discussion/conclusion

This instance shows that the local TM-601 that gives that throws can significantly suppress CNV, and CNV is degenerated.The CNV mouse model is simulated the morbid state of the degeneration of macula of moist form.Dosing way is relevant clinically in the vitreous body that uses in this research, because it is to be used for throwing Lucentis approach of (approval is used for the therapy of degeneration of macula clinically) that gives.

Instance 5: the TM-601 via various route of delivery suppresses the generation of mice CNV model medium vessels

The experiment of describing in this instance is the ability that is used to estimate the TM-601 angiogenesis inhibitor of sending by various dosing ways.Use the choroid neovascularity to generate the growth of neovascularity around the position of analyzing the Bu Luheshi film rupture of measuring, and definite part or whole body are thrown and are given TM-601 and whether cause that angiogenesis reduces at induced with laser.Three new dosing ways have been tested: (be also referred to as under the conjunctiva) near the eyes, intravenous and part (eye drop).

Materials and methods

Light by means of utilizing 530nm laser coagulates art, and inducing mouse median nexus film neovascularity generates (CNV).Utmost point portion 9 points, 12 and 3 positions produce 3 places burn behind each retina.If when induced with laser, produce bubble, the Bu Luheshi film obviously successfully breaks so.Include only the burn of observing bubble in this research.

Administration

Periocular injections 5 μ L TM-601 solution, the concentration that wherein is dissolved in the TM-601 in the normal saline are 2,10,50 and 200mg/mL.After carrying out laser photocoagulation, injection 5 μ L normal saline in to branch hole.After 7 days, duplicate injection.Study the 14th day, (mean molecule quantity is 2 * 10 through fluorescein-labeled glucosan to the mice perfusion ⁶, Sigma company), preparation choroid plain film also utilizes fluorescence microscopy.

Pass through tail vein injection 20mg/kg dosage TM-601 3 times weekly, carry out intravenous administration.

Through using the local application that eye drop is realized TM-601 3 times every day, each eye drop contains the TM-601 of 10 μ L volumes.TM-601 is dissolved in the artificial tear (Artificial Tears) of nonprescription drugs (Rui Teai medicine company (Rite Aide) made in Germany), and it contains 70% glucosan and 0.3% hypromellose as active component.Non-active ingredient comprises 0.1% benzalkonium (benzalkonium), disodiumedetate, potassium chloride, pure water and sodium chloride.The ultimate density of TM-601 is 1,5 and 25mg/mL, obtains per 10 μ L eye drops single dose of 10,50 and 250 μ g respectively.

Histology and imaging

Measure the size that choroid plain film median nexus film neovascularity generates damage.Perfusion is taken out eyes behind fluorescein-labeled glucosan, and in 10% formalin buffer, fixes 1 hour.Remove cornea and crystalline lens, and from eye socket, downcut whole retina.In choroid, from the edge to the equator, make radial slot, and the tiling eye socket.Utilize the fluorescence microscopy plain film, and with image digitazation.Use Image-Pro Plus software (Mai Dengsaibonike company) to measure the gross area that the choroid neovascularity relevant with each burn generates.

The result

In mice, cause that the choroid neovascularity generates (CNV), many situation of the CNV that its simulation is taken place in the patient who suffers from neovascularity property AMD with the laser photocoagulation Bu Luheshi film that breaks.Use this model, the present inventor confirmed before that intraocular injection TM-601 obviously reduced neovascularity and generates, but also neovascularity is degenerated.(referring to instance 4).

In current research, check other dosing way (intravenous and part near the eyes).In first is studied near the eyes, in the laser photocoagulation same day (the 1st day) and the 7th day periocular injections 250 μ g TM-601.At identical time point, normal saline is injected the contrast eye.After the Bu Luheshi film rupture 14 days, analyze each choroid plain film.When find utilizing the TM601 of 250 μ g dosage near the eyes, TM-601 handles the formation (Figure 16) that significantly reduces neovascularity.

In order to confirm dose response, inject the TM-601 of 10 μ g, 50 μ g, 250 μ g or 1000 μ g dosage for periocular injections.10 μ g dosage can not obviously reduce the choroid neovascularity and generate, but 50 μ g or more high dose make CNV reduce to similar degree (Figure 17).What is interesting is, receive periocular injections animal branch hole is also represented the minimizing (green curve among Figure 17) of CNV.

For whether the TM-601 that confirms systemic injection also can infiltrate in the choroid and suppress angiogenesis, 3 TM-601 through tail vein injection 20mg/kg dosage weekly continue the search time in two weeks.When utilizing intravenous injection, find that TM-601 makes CNV obviously reduce (Figure 18).

Also test topical eye drops and use, the minimum approach of aggressive in these three kinds of dosing ways that are in this instance to be checked.In nonprescription drugs eye drop lubricant, and every day, 3 warps, 10 μ L eye drops were used with the TM-601 resuspending.When sending (every 0.25mg, every day 3 times), observe CNV and reduce, but do not have significant difference (Figure 19) with the normal saline matched group with maximum dose level.

Discussion/conclusion

The CNV mouse model is simulated the morbid state of the degeneration of macula of moist form.Shown in instance 4, dosing way can significantly reduce the generation of choroid neovascularity in the vitreous body.This is to give Lucentis (approval is used for the therapy of degeneration of macula clinically) the identical approach that uses with throwing.Yet the present inventor recognizes, the less delivery modality of aggressive maybe be useful, and tested TM-601 near the eyes, intravenous and local delivery.Show all that in all cases TM-601 reduces CNV, but according to observations, utilize the seen reduction of local dropping liquid not reach statistical significance.Periocular injections TM-601 needing possibly be superior to the medicine (for example, Lucentis ) of intraocular delivery.

Observed in Figure 17 " to branch hole effect (fellow-eye effect) " possibly cause concern.The TM-601 of periocular injections significantly reduces the CNV in the adjacent eye (not receiving the injector).Do not hope to receive the constraint of any particular theory, to the possible explanation of this phenomenon be, the material of injection gets into systemic circulation, causes drug exposure in to branch hole thus.Utilize topical eye drops not observe similarly to the branch hole effect.

These results show that the TM-601 that sends by different dosing ways demonstrates blood vessel formation against function, as the effectiveness of the therapeutic agent of metastatic cancer further support are provided for the scorpion catilan thus.

Instance 6:TM-601 is positioned neovascularity and the inductive neovascularity endothelial cell apoptosis of TM-601

Experiment described in the instance 4 and 5 shows, generates in (CNV) model at mice choroid neovascularity, and the scorpion catilan can suppress angiogenesis, and neovascularity is degenerated.The experiment of describing in this instance is intended to understand the mechanism like the scorpion catilan blood vessel formation against function that is shown in instance 4 and 5.Result in this instance shows that in mice CNV model, TM-601 is positioned in the neovascularity, and induces the apoptosis of neovascularity endotheliocyte.In addition, the result in this instance also shows, generates in the zone at CNV model and the retinopathy model neovascularity in the two, and TM-601 and annexin A2 locate altogether, have observed annexin A2 during the present inventor formerly studies and have been incorporated into TM-601.

Although show, TM-601 can suppress the choroid neovascularity and generate and make the blood vessel of new formation to degenerate in eye, and is previous and do not know that TM-601 is the particular cell types that directly is incorporated in the eye, still presents pharmacological activity with non-specific mode.In intravenous injection ¹³¹Behind the I-TM-601, in the plane picture of whole body, do not observe and generally the combining of vascular system.Therefore, under the situation of not hoping to receive any particular theory constraint, suppose a small set of cell of TM-601 selective binding in the activatory of neovascularization position or propagation.

In mice CNV model, induce neovascularization, and in vitreous body or after the subconjunctival injection, confirm the position of TM-601 in this model.In addition, use TUNEL to analyze to confirm that whether the degeneration of neovascularity is that endothelial cell apoptosis by choroid neovascularity generation position causes.

In order further to understand the mechanism of the blood vessel formation against function of TM-601, by the common location of immunohistochemical method inspection possible cell receptor annexin A2 of TM-601 and scorpion catilan in the zone that neovascularity generates.Observe according to the present inventor, annexin A2 is incorporated into TM-601.Annexin A2 expresses on endothelial cell surface, and overexpression in some tumor type.Annexin A2 has been characterized as being the dock (docking station) of short angiogenic proteins of regulation and control and/or anti-angiogenic proteins (for example plasminogen and fibrinolysin) conversion.

Materials and methods

Light by means of utilizing 530nm laser coagulates art, and inducing mouse median nexus film neovascularity generates (CNV).Utmost point portion 9 points, 12 and 3 positions produce 3 places burn behind each retina.If when induced with laser, produce bubble, the Bu Luheshi film obviously successfully breaks so.Include only the burn of observing bubble in this research.

Administration

Damaged the back the 7th day at CNV, intraocular injection 1 μ L TM-601 (50 μ g/ μ L are dissolved in the normal saline).The 7th day and the 8th day, use the 10 μ g/ μ L TM-601 concentration that are dissolved in the normal saline to carry out the subconjunctival injection of 5 μ L.Branch hole is injected.The 9th day, put to death animal, take out eyes, and cut out section through the CNV damage.As mentioned below, frozen section is dyeed.

Immunohistochemistry

In the TM-601 Position Research, use the anti-TM-601 of rabbit (redness among Figure 20 A, B and C and Figure 21 A, B and the C) primary antibody.Use fluorescently-labeled anti-rabbit igg secondary antibodies to carry out fluoroscopic examination.Also utilize fluorescently-labeled GSA agglutinin to section statining (green among Figure 20 D, E and F and Figure 21 D, E and the F) to differentiate endotheliocyte.For the detection of apoptotic cell, (Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling is TUNEL) to section statining to terminal deoxynucleotidyl transferase dUTP otch end labelling.This analysis is used for detecting the nucleus that represents dna segmentization (sign of apoptotic cell death).Also utilize the nuclear staining agent to section statining.With the common Position Research of annexin A2 in, also utilize anti--annexin A2 to section statining.

Endothelial cell proliferation is analyzed

Use is from the single solution cell proliferation detecting kit of CellTiter 96

Aqueous (the Aqueous One Solution Cell Proliferation Kit of Pu Luomaige company (Promega); Catalog number (Cat.No.) #G3582) carries out the endothelial cell proliferation analysis.Briefly, be coated with 4,000 cells (analyzing in 72 hours) or 1,000 cell (analyzing in 120 hours) spends the night at each hole middle berths of 96 porose discs.Next day, TM-601 or diluent are added in the hole.Subsequently 37 ℃ of cultured cells 72 hours or 120 hours.According to the test kit scheme,, measure cell quantity through with the dyeing of MTS tetrazolium nitrogen (tetrazolium) chemical compound.Amount by the measured De Jia of the absorbance under 490nm Za (formazan) product is directly proportional with the quantity of living cells in the culture.

The result

In order to study the mechanism of the blood vessel formation against function of TM-601 in mice CNV model, coagulate postoperative the 7th day intraocular injection TM-601 or in little rathole through light at the 7th day and the 8th day periocular injections TM-601 at induced with laser, study the location of TM-601.Study the 9th day, eye is carried out immunostaining to TM-601.When utilizing two kinds of dosing ways, find in the endotheliocyte of TM-601 specific localization in choroid (Figure 20 and 21).Do not observe the blood vessel association that detectable TM-601 and layer of retina below is pre-existing in, show TM-601 selective binding new blood vessel that forms in choroid.

Described in instance 4, cause a week after the neovascularization by laser photocoagulation, single intraocular injection TM-601 obviously degenerates CNV.Yet the mechanism of this effect is still unknown.In order to test the apoptosis whether in this model TM-601 can cause the cell that helps newly to form vascular system, to the section statining of TUNEL to cutting out through the CNV damage.Apoptotic cells (such as by discriminatings of positive TUNEL dyeing institute) is located with the endothelium stain altogether, is illustrated in ophthalmic or near the eyes after the TM-601 treatment, the endotheliocyte in the zone of choroid neovascularity generation experiences apoptosis (Figure 22 and 23).In the eye of injecting normal saline, do not detect apoptosis.Because at the external processing no cytotoxicity on multiple TM-601 concentration that utilizes TM-601 to endothelial cells cultured, so do not reckon with the apoptosis (Figure 24) that occurs endotheliocyte in the treated in vivo eye.

For further understanding the mechanism of the blood vessel formation against function of TM-601, utilize the possible cell receptor annexin A2 of TM-601 and scorpion catilan to carry out common Position Research.In two kinds of different models, in eye, induce neovascularity to generate: to break that the inductive choroid neovascularity of Bu Luheshi film institute generates and generate by the inductive retinopathy neovascularity of oxygen inductive ischemia institute by laser.Said like this paper about similar experiment, intraocular injection TM-601.In two neovascularity generation models, TM-601 and annexin A2 locate (Figure 25) altogether.

Discussion/conclusion

Several important discoveries that the support as a result that appears in this instance is relevant with the blood vessel formation against function of scorpion catilan.The first, after induced with laser Bu Luheshi film rupture, the TM-601 selective binding of ophthalmic or periocular injections is the endotheliocyte in the new vascular system that forms in choroid layer.TM-601 is not incorporated into the ripe blood vessel that is pre-existing in.These results with observe ¹³¹I-TM-601 can not be incorporated into indistinguishably after intravenous injection that all blood vessels conform in the body.But, because of tumour-specific and/or the combination of neovascularity specificity, mainly in tumor region, detect radioactive label.The second, TM-601 is incorporated into endotheliocyte and causes the neovascularity selective apoptotic that forms at the damage from laser position on every side, but the blood vessel that is pre-existing in can not.The 3rd; TM-601 and annexin A2 locate altogether; Annexin A2 relates to the regulation and control of short angiogenic proteins and/or anti-angiogenic proteins (for example plasminogen and fibrinolysin) conversion, has improved the scorpion catilan thus and has brought into play its probability of part blood vessel formation against function at least via annexin A2.

Instance 7: in the influence of external TM-601 to tumor cell migration

Experiment described in this instance is intended to illustrate the effect of scorpion catilan on cell migration, and cell migration plays an important role in transfer process.Like what assessed by Transwell invasion and attack analysis, the TM-601 on cell migration demonstrates the dosage correlation inhibitory action.

Materials and methods

In triplicate, utilize about 5 * 10 ⁴The cell of individual serum starvation carries out Human umbilical vein endothelial cells, and (Human Umbilical Vein Endothelial Cell HUVEC) strides the migration of Transwell (Corning Incorporated (Corning), 8 μ m).In containing the culture medium of 0.4%FBS, the chemical inhibitor in base apertures comprises VEGF or bFGF (50ng/ml).Under the room temperature, 10 micro-molar concentration TM-601 were cultivated 30 minutes with cell, subsequently cell is loaded in the Transwell cell.At 37 ℃ after following 22 hours, use the Q-tip cotton swab to remove on the upper surface the not cell of migration.In methanol, and utilize Ji's Albert'stain Albert liquid to observe the cell fixation of migration through film.Through Transwell is stacked on the hematimeter, and calculate in 5 zones each cell quantity, the quantitative counting of the cell of attacking.

The result

Shown in Figure 26 A, TM-601 suppresses cell migration with the relevant mode of dosage.In the presence of TM-601, no matter be with VEGF or bFGF irritation cell, about cell below 50% moves (Figure 26 B).

Instance 8:TM-601 is to the active influence of MMP2

Discuss like preamble, cell migration plays a major role in transfer process.Degradation of extracellular matrix promotes that cell moves, and is the committed step of cell migration.In this example, utilize two kinds of different cell type research TM-601 to matrix metalloproteinase 2 (MMP2; A kind of enzyme of degradation of cell epimatrix) influence: HUVEC and U87 glioma cell line.These result of experiment show that it is active that TM-601 suppresses MMP2.

By unprocessed, handle or in the culture medium that the HUVEC cell of the cultivation that bFGF handles with 10 μ M TM-601 is obtained, to measure MMP-2 active through bFGF.(described in instance 7, cultivate the HUVEC cell.) described in Figure 26 C, handle intimate active the increasing of eliminating by the inductive MMP2 of bFGF institute with TM-601.

It is active also in the culture medium that is obtained by the U87 cell of the cultivation of handling through 10 μ M TM-601 or not handling with TM-601, to measure MMP-2.As shown in Figure 27, TM-601 makes the active reduction of the secreted MMP-2 of U87 human glioma cell.

These results show that TM-601 is inhibited to the MMP-2 activity.

The discussion of instance 6 to 8:

Generally speaking, the data that appear in the instance 6 to 8 provide the better understanding to the blood vessel formation against function of TM-601.In cell culture, show that TM-601 is incorporated into the endotheliocyte of propagation, and stop cell migration (referring to instance 7), and cell migration is the committed step of neovascularization.Do not hope to receive the constraint of any particular theory, can find out, TM-601 is incorporated into the cell activation that endotheliocyte needs certain form in vivo, because have only the blood vessel of new formation to combine TM-601, and the akinete in the ripe vascular system can not.In addition, the HUVEC cell of the propagation in the culture can not simulated activated endothelial cells in the neovasculature, because TM-601 does not reduce the propagation (as desired when the external generation apoptosis) of HUVEC cell.Therefore, these data show that TM-601 is through stoping endothelial cell migration to suppress neovascularization to an effect of the vascular system of new formation.According to observations, TM-601 brings into play second effect in the CNV model: the new formation blood vessel experience apoptosis that combines TM-601.

Summary is got up, and external evidence shows that TM-601 stops endothelial cell migration, prevents the generation of the crucial early stage step of angiogenesis thus.In addition, show that in the CNV model, TM-601 causes the blood vessel degeneration of new formation via the apoptosis effect.

The result who describes in these instances helps to explain the mechanism of the angiogenesis inhibitor characteristic of scorpion catilan, and these characteristics are attractive especially quality for the scorpion catilan as the effectiveness to the therapeutic agent of metastatic tumo(u)r.

Instance 9: the TM-601 of the un-marked that intravenous is sent is to the influence of the tumor in the human patients

The ability of the TM-601 treatment cancer of the un-marked that the people's clinical data check intravenous that appears in this instance is sent.Discuss as previous, vitro data shows that TM-601 is incorporated into vascular endothelial cell, and stops endothelial cell migration.The present inventor has also obtained data in the body, and these data show that intravenous infusion TM-601 reduces neovascularity and generates.These observed results impel the startup I phase to test, to estimate the purposes of in having the invasive cancer glioblastoma medium-sized vein of known many blood vessels (hypervascularity), sending the TM-601 of un-marked.The main purpose of this research is: a) confirm safety and the toleration of TM-601 in the adult patient of suffering from the recurrent glioblastoma; B) target of confirming TM-601 when changing the intravenous dispensing according to magnetic resonance (MR) Perfusion Imaging is recommended II phase dosage and biological activity dosage; And c) confirms the pharmacokinetics of TM-601 under each dosage level.

Patient and therapeutic scheme

By intravenous (IV) infusion 10mCi/0.2mg is given in the recurrent glioblastoma patient throwing that meets this study condition ¹³¹I-TM-601 is to show tumour-specific location (imaging dosage).Only make under study for action in brain SPECT scanning and show ¹³¹The patient that the tumour-specific of I-TM-601 absorbs receives the treatment of the TM-601 that utilizes un-marked.Throwing give the imaging dosage after 1 week; In the cycle in 4 weeks; 1 TM-601 to a kind of un-marked in research 6 kinds of dosage levels of patient's intravenous infusion (0.04mg/kg, 0.08mg/kg, 0.16mg/kg, 0.3mg/kg, 0.6mg/kg and 1.2mg/kg) continued for 3 weeks weekly.In the ensuing cycle, throw the TM-601 give un-marked, short of PD sign, and also the patient does not experience dose-limiting toxicity and gets final product.In the 4th week in each cycle, by the dynamic responsive contrast MRI evaluate patient of routine, with assess perfusion.

End in January, 2009, registered 6 recurrent glioblastoma patients in this research, and its imaging data can be used for analyzing.Aspect safety, have one be considered to maybe be relevant with therapy tumor internal hemorrhage incident.Also have three to be considered to comprise PD in the therapeutic process, Hip Fracture and have the patient of renal calculus medical history renal calculus to occur with the irrelevant serious adverse events of therapy.In this first administration group, among 6 patients 2 show relative cerebral blood flow rates (rCBF) and/or relatively cerebral blood volume (rCBV) compare baseline before the treatment and reduce and surpass 25%.Such as a plurality of treatment cycle demonstration, 2 patients that the perfusion MRI parameter is improved have the reaction of expansion to intravenous TM-601, and do not have the tumour progression sign.

These results show that the scorpion catilan of un-marked demonstrates as the prospect to the therapeutic agent with possible invasive cancer of strong transfer (being glioblastoma in this example).

Instance 10: the bioavailability and the blood vessel formation against function of Pegylation scorpion catilan

In this example, whether research Pegylation scorpion catilan can increase with the half-life in the body of confirming the scorpion catilan.Also checked the blood vessel formation against function of Pegylation scorpion catilan.

Materials and methods

Pegylation

Use polydispersity, linear 40kDa PEG-propionic aldehyde (Dao Er drugmaker (DowPharma)), via the N-terminal Pegylation of reduction amination with the TM-601 peptide.

The measurement of TM-601 half-life

Through the single tail vein injection, with TM-601 (dosage is about 2mg/kg) through intravenous injection in no tumor C57BL/6 mice.At the different time points taking blood sample, and, use the content of anti--TM-601 TPPA TM-601 by ELISA.

Mice substrate plug

Under 4 ℃, (BD biotechnology company) mixed with 100ng/ml VEGF, 100ng/ml bFGF and 3ng/ml heparin with high concentration type matrigel substrate.The female C57BL/6 mice in 8 ages in week is assigned randomly in each group every group of 6 mices.Each mice receives 2 part of 500 μ L substrate plug, is expelled in the subcutaneous tissue through bilateral.In order to form circular stopper, after the subcutaneous insertion program of routine, the move left and right needle point forms a wide subcutaneous capsule bag.Utilize the 21-25G pin to inject rapidly, be delivered in the stopper to guarantee whole inclusions.Study the 0th day and implant the substrate plug, and began to handle at the 1st day.To animal intravenous injection mediator (normal saline), TM-601 or Pegylation TM-601.Use three kinds of dosage regimens: 1 time weekly, lasting two weeks (the 1st day 1 time and the 8th day 1 time; " Q7Dx2 "); 2 times weekly, continue (the 1st day, the 4th day, the 8th day and the 11st day 2 weeks; " Q3Dx2/2 "); 5 times weekly, continue (the 1st day, the 2nd day, the 3rd day, the 4th day, the 5th day, the 8th day, the 9th day, the 10th day, the 11st day and the 12nd day 2 weeks; " Q1Dx5/2 ").Collect stopper after 14 days.Mice is implemented euthanasia, and the skin of stopper top is pulled back.Downcut stopper, fixing, and be embedded in the paraffin, for histologic analysis.Utilize CD31 antibody that but 35 μ m slabs from each valuation stopper are carried out immunostaining, and carry out counterstain with haematoxylin and Yihong.At microscopically, analyze the quantity of the cross-sectional area medium vessels of each substrate plug.

Result/discussion

Shown in figure 28, with the TM-601 comparison without modification, the half-life increases in the body of Pegylation TM-601.Pegylation makes the half-life of TM-601 increase about 32 times, that is, about 25 minutes (TM-601) was to about 16 hours (TM-601-PEG).

Half-life is increased in and changes in the angiogenesis model with low frequency more the ability of animals administer.In mice substrate plug is analyzed,, give animal TM-601 or Pegylation TM-601 (TM-601-PEG) according to multiple time-histories.Measure microvessel density, and the reduction of explaining said density is with the expression blood vessel formation against function.

The administration time-histories that two frequencies that utilization is tested are the highest (2 times weekly, continued for 2 weeks, " Q3DX2/2 "; 5 times weekly, continued for 2 weeks, " Q1Dx5/2 ") time, TM-601 and TM-601-PEG have blood vessel formation against function (Figure 29).When utilizing the minimum administration time-histories (1 time weekly, continued for 2 weeks, " Q7DX2 ") of the frequency tested, TM-601 does not show any blood vessel formation against function, and when utilizing this administration time-histories, causes that with the processing of TM-601-PEG microvessel density significantly reduces (Figure 29).

Not hoping to receive the constraint of any particular theory, possibly be because the time ratio TM-601 capable of using of TM-601-PEG is long with low frequency more to the ability of animals administer.This utilization rate increase possibly make the time that is exposed to the neovascularization position elongated, thereby allows more persistent effect.These characteristics (for example utilization rate increases, effect prolongs etc.) can be of value to the treatment metastatic tumo(u)r.

The influence that instance 11:TM-601 shifts lung

In this example, use mouse model (in mice, injecting melanoma cells) research TM-601 to suppress the ability that lung shifts.The calculating quantity that consequent lung shifts in the mice body under the situation of using or treating without TM-601.

B16/F10 mouse black-in tumor cell system obtains from ATCC, and cultivates according to the standard of recommending.Contain tumor cell suspension (1 * 10 through intravenous with 0.2mL ⁵Individual cell/mice) each mice of 0.9%NaCl solution inoculation.

According to following table 1, throw simultaneously and give medicaments injection and tumor cell.Be described in table 1 below, animal receives the test article of multiple dosage.Make for 5 times animal receive dosage every day weekly, continued for 2 weeks.Aseptic PBS is as the mediator reference substance.TM-601 is restored in aseptic PBS solution.Behind the begin treatment, note the measured body weight value of mice weekly for 2 times, and totally observe every day at least for 1 time.The 14th day (or after confirming moribund condition) put to death all mices of all groups, and calculates the quantity of B16/F10 lung group.Utilization is through the bag Yin Shi of tracheal instillation fixative (Bouin fixative solution), and the group of counting is shown as in expansible lung visible black lung tuberosity on the visible yellow background.

Table 1: the evaluation of pulmonary's relations of metastasis of the TM-601 of single medicine type and B16/F10 mouse black-in lymphoma

^*The 0th day, throw simultaneously and give first dose and tumor cell.

Other embodiment

Consider the explanation or the practice of the invention that this paper discloses, other embodiment of the present invention is clearly to one of ordinary skill in the art.Said explanation and instance should only be regarded as exemplary, and true scope of the present invention will be by claims explanation of enclosing.

Claims (71)

1. a treatment suffers from or is prone to suffer from the method for the individuality of at least a transfer; Wherein said transfer is to be caused by at least a primary tumo(u)r, and said method comprises gives the scorpion catilan reagent of effective dose so that the step that said scorpion catilan reagent is incorporated into said at least a transfer to said individual throwing.

2. method according to claim 1, wherein said scorpion catilan reagent are that the whole body throwing is given.

3. method according to claim 2, wherein said scorpion catilan reagent are thrown through intravenous and are given.

4. method according to claim 1, wherein said scorpion catilan reagent is incorporated at least a neoplasm metastasis that is arranged in brain.

5. method according to claim 1, wherein said scorpion catilan reagent selectivity target cancer cell but not normal cell.

6. method according to claim 1, wherein said scorpion catilan reagent comprises the scorpion catilan part that is selected from by the following group that forms: scorpion catilan, biological activity scorpion catilan subunit and scorpion catilan derivant.

7. method according to claim 1, wherein said scorpion catilan reagent comprise scorpion catilan part and associate with at least one treatment part.

8. method according to claim 7, wherein said scorpion catilan part is associated with the direct covalency of treatment part.

9. method according to claim 7, wherein said scorpion catilan part forms fusion rotein with the treatment partial fusion.

10. method according to claim 7, wherein said scorpion catilan part is associated via the bridging agent covalency with the treatment part.

11. method according to claim 7, wherein said treatment partly comprises anticarcinogen.

12. method according to claim 7, wherein said treatment partly comprises cytostatic agent.

13. method according to claim 7, wherein said treatment partly comprises cytotoxic agent.

14. method according to claim 13, wherein said cytotoxic agent is selected from the group that is made up of following: toxin, biological activity protein, chemotherapy are with antibiotic, nucleolytic enzyme and radiosiotope.

15. method according to claim 14, wherein said cytotoxic agent comprises radiosiotope.

16. method according to claim 15, wherein said radiosiotope comprise iodine-131 ( ¹³¹I).

17. method according to claim 11, wherein said anticarcinogen is selected from the group that is made up of following: alkylating agent class, purine antagonist class, pyrimidine antagonist class, plant bases, the property inserted antibiotics, aromatase inhibitor class, antimetabolite class, mitotic inhibitor class, growth factor receptor inhibitors class, cell cycle inhibitor class, enzyme, topoisomerase enzyme inhibitor class, BRM class, hormone antagonist class and antiandrogen class.

18. method according to claim 11, wherein said anticarcinogen comprise the nucleic acid reagent of length between about 5 and 2000 nucleotide.

19. method according to claim 18, wherein said nucleic acid reagent comprises antisense reagent.

20. method according to claim 18, wherein said nucleic acid reagent encoding antisense reagent.

21. method according to claim 18, wherein said nucleic acid reagent serves as carrier, sends antisense reagent with in introducing cell the time.

22. method according to claim 18, wherein said nucleic acid reagent comprises inhibitory RNA.

23. method according to claim 18, wherein said nucleic acid reagent coding inhibitory RNA.

24. method according to claim 18, wherein said nucleic acid reagent comprises carrier, sends inhibitory RNA with in introducing cell the time.

25. method according to claim 1, wherein said primary tumo(u)r is an entity tumor.

26. method according to claim 1, wherein said primary tumo(u)r are intractable tumors.

27. method according to claim 1, wherein said primary tumo(u)r is a recurrent tumor.

28. method according to claim 1, wherein said primary tumo(u)r are by the member of the following group that forms: pulmonary carcinoma, osteocarcinoma, hepatocarcinoma, cancer of pancreas, skin carcinoma, head or neck cancer, skin or intraocular melanoma, uterus carcinoma, ovarian cancer, rectal cancer, anal regions cancer, gastric cancer, colon cancer, breast carcinoma, uterus carcinoma, sexual organ or genitals's carcinoma, lymphogranulomatosis (Hodgkin ' s Disease), esophageal carcinoma, carcinoma of small intestine, hormonal system cancer, thyroid carcinoma, parathyroid gland cancer, adrenal carcinoma, soft tissue sarcoma, carcinoma of urethra, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal carcinoma, renal cell carcinoma, central nervous system (CNS) tumor, neuroderm cancer, tumor of spine, glioma, meningioma and pituitary adenoma.

29. method according to claim 28, wherein said primary tumo(u)r are skin or intraocular melanoma.

30. method according to claim 1, wherein said primary tumo(u)r are neuroderm source property tumors.

31. method according to claim 30, wherein said neuroderm source property tumor are by the member of the following group that forms: glioma, meningioma, ependymoma, medulloblastoma, neuroblastoma, ganglioneuroma, pheochromocytoma, melanoma, the original neuroectodermal tumors of periphery, small cell lung cancer and ewing's sarcoma (Ewing ' s sarcoma).

32. method according to claim 31, wherein said neuroderm source property tumor is a glioma.

Throw the scorpion catilan reagent that gives at least one dosage 33. method according to claim 1, wherein said dispensing step comprise, wherein said dosage is between about 0.01mg/kg and about 5mg/kg.

34. method according to claim 1, it further is included in said individual the throwing is given the step that said scorpion catilan reagent detects at least a transfer before.

35. method according to claim 34 wherein detects said at least a transfer and comprises following steps:

To the said individual scorpion catilan reagent that gives effective dose of throwing through labelling; With

Measure the combination of the scorpion catilan reagent of said process labelling in the said body at least one position in said individual's body except that the position of said at least a primary tumo(u)r, wherein combine level to raise and indicate the existence of one or more transfers with respect to normal structure.

36. being the whole body throwings, method according to claim 35, wherein said scorpion catilan reagent through labelling give.

37. method according to claim 36, wherein said scorpion catilan reagent through labelling is thrown through intravenous and is given.

38. method according to claim 35, wherein said scorpion catilan reagent through labelling is selected from the mark part labelling by the following group that forms through at least one: fluorogen, radiosiotope and paramagnetic metal ion.

39. according to the described method of claim 38, wherein said mark part comprise iodine-131 ( ¹³¹I) or iodine-125 ( ¹²⁵I).

40. according to the described method of claim 38, wherein said mark part comprise technetium-99m ( ^99mTc).

41. according to the described method of claim 38, wherein said mark part comprise copper-64 ( ⁶⁴Cu).

42. according to the described method of claim 38, wherein said measurement is said to be to use the technology that is selected from by the following group that forms to carry out through the scorpion catilan reagent of labelling and the bonded step of tissue: the fluorescent spectrometry of induced with laser, γ camera, single photon emission computed body layer imaging (SPECT) and positron emission emission computer body layer imaging (PET).

43. method according to claim 1, it further comprises said individual the throwing is given chemotherapeutics.

44. according to the described method of claim 43, wherein said chemotherapeutics is selected from the group that is made up of following: alkylating agent class, purine antagonist class, pyrimidine antagonist class, plant bases, the property inserted antibiotics, aromatase inhibitor class, antimetabolite class, mitotic inhibitor class, growth factor receptor inhibitors class, cell cycle inhibitor class, enzyme, topoisomerase enzyme inhibitor class, BRM class, hormone antagonist class and antiandrogen class.

45. method according to claim 1, the neovasculature of wherein said at least a transfer is degenerated.

46. method according to claim 1, wherein neovascularity generates and is inhibited.

47. method according to claim 1, the migration of at least one cell is inhibited in the wherein said primary tumo(u)r.

48. method according to claim 1, wherein said scorpion catilan reagent is covalently bound to polymer.

49. according to the described method of claim 48, wherein said polymer is Polyethylene Glycol (PEG).

50. a detection suffers from or once suffered from the method for the existence of one or more transfers in the individuality of at least a primary tumo(u)r, said method comprises following steps:

To the said individual scorpion catilan reagent that gives effective dose of throwing through labelling; With

Measure the combination of the scorpion catilan reagent of said process labelling at least one position in said individual's body except that the position of said at least a primary tumo(u)r, wherein combine level to raise and indicate the existence of one or more transfers with respect to normal structure.

51. according to the described method of claim 50, wherein said scorpion catilan reagent through labelling is that the whole body throwing is given.

52. according to the described method of claim 51, wherein said scorpion catilan reagent through labelling is thrown through intravenous and is given.

53. according to the described method of claim 50, wherein said scorpion catilan reagent is incorporated at least a neoplasm metastasis that is arranged in brain.

54. according to the described method of claim 50, wherein said scorpion catilan reagent selectivity target cancer cell but not normal cell through labelling.

55. according to the described method of claim 50, wherein said scorpion catilan reagent through labelling comprises the scorpion catilan part that is selected from by the following group that forms: scorpion catilan, biological activity scorpion catilan subunit and scorpion catilan derivant.

56. according to the described method of claim 50, the scorpion catilan reagent of wherein said process labelling is through at least one mark part labelling, wherein said mark part is selected from the group that is made up of fluorogen, radiosiotope and paramagnetic metal ion.

57. according to the described method of claim 56, wherein said mark part comprise iodine-131 ( ¹³¹I) or iodine-125 ( ¹²⁵I).

58. according to the described method of claim 56, wherein said mark part comprise technetium-99m ( ^99mTc).

59. according to the described method of claim 56, wherein said mark part comprise copper-64 ( ⁶⁴Cu).

60. according to the described method of claim 56, wherein said measurement is said to be to use the technology that is selected from by the following group that forms to carry out through the scorpion catilan reagent of labelling and the bonded step of tissue: the fluorescent spectrometry of induced with laser, γ camera, single photon emission computed body layer imaging (SPECT) and positron emission emission computer body layer imaging (PET).

61. according to the described method of claim 50, wherein said at least a primary tumo(u)r is an entity tumor.

62. according to the described method of claim 50, wherein said at least a primary tumo(u)r is intractable tumor.

63. according to the described method of claim 50, wherein said at least a primary tumo(u)r is a recurrent tumor.

64. according to the described method of claim 50, wherein said at least a primary tumo(u)r is the tumor that is selected from by the following group that forms: pulmonary carcinoma, osteocarcinoma, hepatocarcinoma, cancer of pancreas, skin carcinoma, head or neck cancer, skin or intraocular melanoma, uterus carcinoma, ovarian cancer, rectal cancer, anal regions cancer, gastric cancer, colon cancer, breast carcinoma, uterus carcinoma, sexual organ or genitals's carcinoma, lymphogranulomatosis, esophageal carcinoma, carcinoma of small intestine, hormonal system cancer, thyroid carcinoma, parathyroid gland cancer, adrenal carcinoma, soft tissue sarcoma, carcinoma of urethra, lymphocytic lymphoma, bladder cancer, renal carcinoma, renal cell carcinoma, central nervous system (CNS) tumor, neuroderm cancer, tumor of spine, glioma, meningioma and pituitary adenoma.

65. according to the described method of claim 50, wherein said at least a primary tumo(u)r is skin or intraocular melanoma.

66. according to the described method of claim 50, wherein said at least a primary tumo(u)r is a neuroderm source property tumor.

67. according to the described method of claim 66, wherein said neuroderm source property tumor is by the member of the following group that forms: neuroderm source property metastatic tumo(u)r in glioma, meningioma, ependymoma, medulloblastoma, neuroblastoma, ganglioneuroma, pheochromocytoma, melanoma, the original neuroectodermal tumors of periphery, small cell lung cancer, ewing's sarcoma and the brain.

68. according to the described method of claim 67, wherein said neuroderm source property tumor is a glioma.

69. according to the described method of claim 50, wherein said dispensing step comprises whole body and throws the scorpion catilan reagent through labelling that gives at least one dosage.

70. according to the described method of claim 69, wherein said dispensing step comprises whole body and throws the scorpion catilan reagent through labelling that gives first dosage and second dosage, wherein said second dosage is higher than said first dosage.

71. according to the described method of claim 69; Wherein said dispensing step comprises whole body and throws the scorpion catilan reagent through labelling that gives first dosage, second dosage and the 3rd dosage; Wherein said second dosage is higher than said first dosage, and said the 3rd dosage is higher than said second dosage.

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