CN1703622A - Method of screening groups of radioactive molecules and applications thereof - Google Patents

Abstract

The invention relates to an amplification-free method of screening groups of radioactive molecules, the products from the molecule groups obtained and the application thereof in order to identify molecules that are capable of binding selectively to a tissue or a particular organ. The inventive method can be used for the development of novel therapeutic compounds and contrast agents for medical imaging and for the screening of medicaments. Said method of screening a group of molecules is characterised in that it comprises at least the following steps: (1) the group of molecules is administered to at least one animal, each molecule being marked first by a suitable radioactive isotope; (2) at least one of the animals is slaughtered and the tissue distribution of the radioactivity of the molecules administered is analysed from sections of tissue or organs taken using suitable imaging devices; (3) sections of tissue or organs in which a radioactivity signal is detected are selected; (4) radioactive fractions from the aforementioned sections of tissue or organs selected during step 3 are isolated using suitable techniques such as chromatography and/or extraction techniques; and (5) the molecule(s) from the radioactive fractions obtained in step 4 are characterised using suitable analysis techniques such as chromatography and/or mass spectrometry. According to one variation of the method, in step 2, where possible, the tissue distribution of the radioactivity of the molecules administered is analysed in vivo using suitable imaging devices from a biological sample which is pre-extracted and selected from the group containing cells and sections of tissue or organs. As a result, said variation does not require the animal to be slaughtered.

Description

The method and the application thereof of screening groups of radioactive molecules

The present invention relates to the non-amplification method in the library that a kind of screening is made up of different Geigers, relate to acquisition molecular library product and relate to described method in that differentiate can selective binding one particular organization or the molecule of certain organs, be used for developing the new treatment compound and the application of medical imaging contrast preparation and screening of medicaments.

In new drug development, obtain by chemosynthesis or in testing in vitro, estimate usually derived from the compound of organic sphere (natural materials).The major function of these screenings be differentiate can with interested any therapeutic target position (acceptor, enzyme etc.) interactional compound of high-affinity, perhaps separate relative non-activity but will enter compound in the optimizer.Can detect thousands of kinds of very original compounds although these screenings make, only to have a few part can become the object of zooscopy in them, and zooscopy is a unavoidable step that successfully obtains to be used for the product of health.In the test of all these in-vitro screenings, for the necessary parameter of result such as the metabolic stability or the Tissue distribution of the material of test in animal body and eliminate character and be not carried out consideration, illustrate that this strategy has very big defective.More effectively disclose the useful material demand of health is disclosed new screening technique.

The existing description of screening technique that comprises a step of selecting in vivo.For example, and R.PASQUALINI etc. (Nature, 1996,380,364-366) peptide library that produces by phage display for injected in mice has been described; Use this technology, the author of article can differentiate the part that combines with some organ specificity after being illustrated in amplification.Yet the success of method depends on the amplification possibility again of several bacteriophages of selecting in the body basically, with the peptide sequence that obtains to be presented by the protein of the bacteriophage of selecting in the body.

According to author's description, the method for describing in this document should be applicable to the library based on the principle outside the phage display, in this case, unique needs be its in conjunction with after differentiate to organize in the ability of compound; Yet PASQUALINI etc. point out in order to screen chemical molecular, need to exist a mark to carry out amplification step, and described mark is as a nucleotide sequence.

The technology that R.PASQUALINI etc. set forth (Nature, 1996, as above-mentioned) therefore can select can specificity in conjunction with the peptide of tumour, for example, when this class peptide and adriamycin coupling, they can improve this medicine to the effectiveness of tumour and reduce its toxicity.This article that is applied in W.Arap etc. (Science, 1998,279, described especially in 377-380).

The application people is in the PCT International Application No. WO 97/10507 of La Jolla NFCR, inventor E.RUOSLAHTI and R.PASQUALINI have described a kind of method of molecule of the organ or tissue that obtains can autospecific to be oriented to a kind of selection, and this method comprises the steps:

-give body one by one with a phage library,

-collect or reclaim selected organ or tissue sample,

-amplification bacteriophage, and

-discriminating can arrive a kind of molecule of selected organ or tissue.

It should be noted that described different molecule can be connected as a holder with a mark (tag).

In this PCT international application:

Term " library " is meant molecule, as the set of organic molecule, peptide, protein or nucleic acid.

The mark that is attached to the molecule of forming the library can be total mark or specific markers of some molecules.The mark of describing among the application is particularly: plastic beads (plastic microbeads), oligonucleotides, bacteriophage or molecule, and as biotin or hemagglutinin.

Arrive the discriminating of the molecule of its target organ and can be for example undertaken, also can carry out high performance liquid chroma-tography or can use the method for selective extraction target organ by independent mass spectrophotometry or mass spectrophotometry and gas chromatographic analysis combination.

For example, be pointed out that when described library comprises a group and divide the period of the day from 11 p.m. to 1 a.m, preferably from sample, remove genomic DNA to reduce " parasitic (parasitic) " PCR with the organic chemistry that can combine by the specific marker that the oligonucleotides that PCR differentiates is formed.

It is to be noted that also generally speaking the bacteriophage of enough numbers arrives target organ, this makes effectively and can differentiate them after amplification, differentiates described peptide sequence then.

More accurately, embodiment relates to the peptide that screening is carried the phage library of peptide and differentiated arrival brain, kidney or tumour.

Yet, be limited to peptide basically and have many shortcomings by the described method of E.RUOSLAHTI group.

Particularly:

-peptide has produced the restriction of space and conformation presenting of phage surface, and this must weaken the peptide sequence presented and the interaction between the potential target position.In addition, the ability that spreads in multiple tissue of bacteriophage is relatively poor and limited the phage display method considerably at the existence of purge mechanism in the body of this class particle;

-what screen the step discriminating in vivo is bacteriophage.Yet the combination of bacteriophage does not guarantee to have the peptide of the sequence that bacteriophage carries will preserve the distribution property of this bacteriophage.If consider the difference in size between bacteriophage and the independent peptide, then this situation is more possible.Therefore, although in fact can differentiate a large amount of bacteriophages, do not guarantee that single peptide will have the distribution property of bacteriophage by screening;

-after bacteriophage was extracted, they must increase by the transfection of bacterium again, and this restriction makes and add an extra step in described method;

-because bacteriophage produces many non-specific interactions, so the method that E.RUOSLAHTI group is described must comprise selection step (being generally 3 steps) in some bodies; This makes this method operate to require great effort very much and shortcoming is arranged that promptly screening comprises the changeability of using different animals and therefore may producing sizable deviation in the body;

-this method reckons without the metabolism of described compound and pharmacokinetics (distribute and remove) data because in the body screening step be carry out on the bacteriophage rather than on compound self, carry out;

-this method has been determined the selectivity of the Tissue distribution of selected bacteriophage rather than peptide self, and this is because E.RUOSLAHTI group uses the antibody of identification bacteriophage with due to definite its Tissue distribution; There is not other mode to monitor the Tissue distribution of peptide.

Therefore, consider these shortcomings, the described method of E.RUOSLAHTI group value in the useful molecule field in being disclosed in health is limited.

Also it was suggested some other method, they also use step in the body:

-US patent 5,770,455 has been described synthetic a kind of method of the product library that obtains by combinatorial chemistry.Need to prove that these methods have formed the strong instrument of the new part of quick discovery.

Some strategies (space-addressable strategy, fracture pearl (split-bead) strategy and reorganization strategy) that synthesize by combinatorial chemistry have been described, their synthesis condition difference: the control (time of the shape of reaction tube, the type of polymkeric substance, physical constant, temperature, solid phase or liquid phase are handled, the method for potpourri type and definite a plurality of members' in library structure).

The mark of proposing in that document is (" identifier (the identifier tag) ") that is specific to each product.

Therefore this method has the shortcoming of operation effort.

-PCT International Application No. WO 99/56789 has been described and has been used the library that obtains by combinatorial chemistry to select contrast preparation, and more accurately, each product in library all comprises a detectable label; Need to prove and only differentiated mark in vivo with desirable distribution plan or desirable removing figure.Described mark must can need not be taken a sample and not needed to put to death animal and detect; Suitable mark is chromophore, heavy atom, radioactive label and magnetic particle particularly; Yet molecular library must contain the mark that must be able to be distinguished from each other in a large number.No matter this instructions how, this PCT International Application No. WO 99/56789 admits that this is an ideal situation that is difficult to obtain, be recognized that especially the different members in library and the ratio of isolabeling not should be 1: 1 ideally, but can hang down as 10000: 1, described ratio is 1: 1-1: 1000,1: 1-1: 200,1: 1-1: 50 and 1: 1-1: between 25.In this case, promptly when the different members in library does not have the mark of a uniqueness entirely, propose to use a kind of technology of indirect detection mark to differentiate library member corresponding to desirable collection of illustrative plates.In addition, in that application, recommend the mark in the preferred detection biological fluids (blood, urine, cerebrospinal fluid, bile etc.); Described mark preferably can be amplified (oligonucleotides, bacteriophage), this means if each member in library all with a kind of unique mode mark, then this is best.Therefore this will get back to the technology and the shortcoming thereof of above announcement.In that application, described technology only can be effectively applied to the amplification unique tag or by being used in combination in the situation of indirect detection technology with the specificity of acceptor.

In aforesaid all technology, the compound of screening and mark coupling are carrying out discriminating subsequently, yet the existence of this mark can stop the compound and the interaction of its target position that is screened by spatial obstacle.

Therefore, the applicant provides a kind of method, and this method is compared with method of the prior art and satisfied actual needs better, and especially it makes the following possibility that becomes:

-from the potpourri that contains several thousand compounds, select can the oriented movable object tissue or the molecule of organ, the Tissue distribution of the compound self that screens by direct analysis is carried out, and

-differentiate molecule with special nature, described special nature is conceived to it at medical imaging or at the application in the therapeutic purposes.

According to the molecular library of being studied, should be with reference to different molecular library (for example obtaining) or biomolecule library (for example in the situation of peptide library) by combinatorial chemistry.

More accurately, one aspect of the present invention is a kind of method of screening molecular library, and it comprises:

(1) give at least a animal with described molecular library, wherein each molecule all is marks in advance, particularly but be not only atom in described molecule, existing with its radioactive isotope displacement,

(2) put to death at least one described animal, and use the tissue of therefrom sampling or the section of organ, utilize the radioactivity Tissue distribution of suitable imaging or radiophotography molecule that device analysis is used; β-imager that applied radiophotography device is especially selected according to the radioactive atom that is detected.For example, the emission β that can use β-imager to detect to exist on described tissue or the slices of organs ^--particle ( ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc) or the emission β ⁺-particle ( ¹⁸F) radio-labeled product,

(3) selection wherein detects the section of the tissue or the organ of radiated signal,

(4) by suitable technology, as extract and/or the section of chromatographic technique tissue or organ as described in step (3), select in separate radioactive segment, reach

(5) by suitable analysis techniques, molecule as described in the radioactive segment of acquisition in step (4) is identified as chromatography and/or mass spectrophotometry use.

In the time can changing, variation is that step (2) comprises and utilizes suitable imaging device, extracts from the cell of tissue or organ and section in advance and the biological sample analysis selected gives radioactive Tissue distribution of the molecule of at least a animal in advance in external use.Therefore this variation does not need to put to death animal, therefore also is applicable to human body under suitable condition.

With regard to purpose of the present invention, term " animal " comprises non-human animal and people according to the mode classification of generally acknowledging.

A preferred embodiment according to screening technique of the present invention, in step (2) before, described method comprises that a step (1 ') is to analyze the radioactivity Tissue distribution of the molecule that is given, this is by carrying out outside imaging with at least one animal, especially utilize the pick-up unit of character of particle that is suitable for detecting selected radioactive isotope emission with camera carry out ( ¹⁸F, ¹¹C, ⁶⁴Cu, ⁷⁶Br, ¹²⁴I,, ¹³N, ¹⁵O: positron emission transaxial tomography, PET; ^99mTc, ¹²³I: γ-camera).

Mean with screening in the body in the library of the radiolabeled compound of tritium and will put to death animal; In fact detect from the β-electronics of triton and only can the using-system section carry out because exceed several μ m then these electronics no longer can detect; This has explained why step (1 ') only can be carried out at some radioactive atom.

In the step (1) of method of the present invention, each library of Geigers is injected in one or more animal body preferred intraperitoneal injection (I.P.) or intravenous injection (I.V.); Observe the ability [step (1 ') and (2)] of directed tissue of different molecular or organ then by the radioactivity of detection in Different Organs or tissue.The pattern-dependent that detects depends on the character of the radioactive ray of radioactive tracer emission especially in the character of the selected radioactive atom of radio-labeled that carries out molecule.The time of observing is a variable element.

Step (1 ') has many advantages:

-its feasible data that can test and therefore obtain pharmacokinetic data and discriminating part to live animal, the bioavilability of described part, toxicity and metabolisming property are consistent with its application subsequently;

-therefore it is compared with the method for the outer step of occlusion body only has certain advantage, especially on cell culture;

-in addition, it makes and can manifest the distribution of the molecule that gives rapidly and can follow the tracks of its dynamics; Therefore can calculate the removing dynamics of Geigers, so that the definite Best Times that can observe and determine to carry out the described fabric analysis of step (2) until any point.As a result, the time of carrying out the described analysis Tissue distribution of step (2) is according to the character of the molecule of injection and life-span in vivo thereof and different.Therefore can between 48 hours, observe radioactivity at 10 minutes;

-it is based on the high energy radioactive ray that detect by some radioactive atom emission, particularly positron emission transaxial tomography (PET) can obtain the outside imaging that a kind of compound distributes in all compartments of animal, described animal such as rat or mouse, perhaps some primate.

The present invention includes the enforcement of described method:

-in it was multi-form, step (2) comprises utilized suitable imaging device to extract from tissue or the cell of organ and section in advance and radioactive Tissue distribution of the molecule that biological sample analysis gave selected in external use; And

-in it was multi-form, described method comprised a step (1 ') (body inner analysis).

In this regard, in human body, method of the present invention can be used for diagnosing when using with a molecular library, and described molecular library utilizes method of the present invention to select in advance in non-human animal's body at first.

Preferred embodiment of the process according to the invention, in step (2), radioactive Tissue distribution is to utilize the pick-up unit of the character with particle imaging device, that be suitable for detecting selected radioactive isotope emission to manifest.

Based on radioactive detection and the step of the Tissue distribution of analysis of compounds can be selected the library fast according to following condition:

A: have or do not exist the compound that can in biosome, distribute.

B: the standard of directional selectivity, if purpose is to differentiate energy a directed certain organs, for example compound of brain.

C: have the high toxicity compound; Described library is eliminated then or is synthetic again with more discrete form of mixtures, so that remove described toxic chemical fast.

Select step to reach more specifically step (1 ') in the body, have dual purpose:

-at first, and the number of the compound that remarkable minimizing is studied subsequently, in fact, all no longer are considered subsequently by the molecule that biosome is removed fast or tachymetabolism is removed then;

-secondly, with regard to keeping a few compounds in vivo, obtain to concentrate research as tumour about the information of its Tissue distribution and for example only at particular tissues.

Step (3)-(5) relate to the chemistry of the radioactive compound that exists in given tissue or the organ and differentiate.At first, these steps illustrate the radioactivity that observes only be since the molecule that in initial library, exists due to.

More particularly, step (4) can be separated and be contained active multiple part, especially uses suitable chromatography on the device of having equipped radiac and separates; Then these parts are carried out mass spectrophotometry (step (5)), to determine the chemical constitution of described radioactive product.This step (5) therefore can differentiate formally whether the product of separation is contained in the initial library.

In addition, (i) use a suitable imaging device with the step that detects radioactive compound in given tissue or the organ more sensitively and (ii) analytical procedure, especially by the step combination of mass spectrophotometry, compare the detection sensitivity that has significantly improved the inventive method with the method for differentiating described compound with only using the mass spectrophotometry detection.

In other words, before analyzing, particularly by mass spectrophotometry (step 3-5), the detection (particularly step 2) of combination radiated signal has significantly improved the sensitivity that detects, and ((fentomole) (10 flies to rub ^-15M)/mm ²Grade).Particularly a kind of β-imager of employed radiophotography device is selected according to detected radioactive atom.For example, the β-imager of Biospace company can detect flying of existing and rub/mm in the situation of tritium on histotomy ²The radiolabeled product of the emission beta particle of grade concentration; This radiophotography device can detect the emission beta particle, detection threshold is a little less than polytype radioactive atom of the detection threshold of tritium.

According to described radioactive atom, can limit following detection threshold (counting/minute or cpm):

³H：0.07cpm/mm ²

³⁵S、 ¹⁴C、 ³³P：0.01cpm/mm ²

³²P：0.1cpm/mm ²

Therefore, method of the present invention can be differentiated the compound with two kinds of fundamental propertys: metabolic stability and Tissue distribution.These steps can be extracted the compound that exists in the various tissues especially, and purpose is to identify its chemical constitution.It is vital using the selection of Geigers because radioactive monitoring formed a kind of very sensitive means with extract or even purification step during the monitoring compound of interest have a situation.

In case carried out multiple separating step (particularly extraction step), the chemical constitution of described compound is identified as HPLC by mass spectrophotometry or any other suitable analytical approach.

More accurately, described Geigers is injected with form of mixtures, can screen a large amount of compounds.In the given moment, the intensity of mark depends on the compatibility of compound to a special site, and this is regulated by its pharmacokinetics.Therefore, described screening experiment preferably carries out constantly in difference.Can set up pharmacokinetics like this and estimate these compounds application subsequently of envisioning.

According to the observation technology that has situation that is used for detecting organ or tissue's Geigers, can:

-live animal is analyzed: at the radioactive atom of emission positron, in the situation of technetium 99 as fluorine 18 or emission gamma-radiation, use the pick-up unit that combines camera that is suitable for detecting this type radioactive ray to carry out;

-or after putting to death animal, detect and radiological measuring is carried out in the using-system section.

In all situations,, put to death at least one animal for accurately analyzing.

Unrestricted about spendable type of animal.In fact, normally used all animals all can be research objects in pharmacology.

According to the present invention, in step (1) before, the library of the molecule of described mark prepares as follows:

-by a kind of potpourri (L-or D-peptide, false peptide, nucleic acid, lipid, organic compound) (combination is synthetic) of chemistry or the synthetic described molecule of enzymatic route, reach

The described molecule of-usefulness labelled with radioisotope, described radioactive isotope such as tritium ( ³H), carbon 14 ( ¹⁴C), iodine 131 ( ¹³¹I), iodine 125 ( ¹²⁵I), phosphorus 32 ( ³²P), fluorine 18 ( ¹⁸F), sulphur 35 ( ³⁵S), technetium 99 ( ⁹⁹Tc) or indium 113 ( ¹¹³In).

More accurately, in the situation of XC polymer or biomolecule (peptide, false peptide, nucleotide, the mixed polymkeric substance of nucleotide-peptide), the synthetic of a large amount of Geigers undertaken by making up to synthesize.In the situation of peptide, the method for deconvoluting of Miao Shuing (repeating the different molecular of fractionated (iterative fractionation) with separating mixture) is convenient to differentiate molecule (s) of interest in the literature.The non-peptide molecule that obtains by massive parallel solid phase synthesis (mass parallelsolid-phase synthesis) can be used for method of the present invention, and condition is that they are modified in order to mix a radioactive atom.

Because above-mentioned molecule is at solid phase synthesis, it is simple relatively therefore to import radioactive atom.For example in the situation of peptide, solid phase synthesis produces a kind of peptide, and its N terminal amine can be dissociated, and all reactive functionality of carrying on the side chain of these polymkeric substance are still protected.This character therefore can be very optionally simple acidylate by the N terminal amine of the polymkeric substance that still combines with solid support import the group that carries radioactive atom; especially use commercially available numerous radiolabeled tritium derivant to carry out; can acylated amine functional group as succinimide propionic ester or acetic anhydride, produce the compound that carries with the radiolabeled this amine functional group of tritium.Similar compound can import by acylated amine functional group ¹⁴C.With ¹⁸F or ^99mTc to the radioactive label of peptide can as carry out as described in for example A.HEPPELER etc. (Curr.Med.Chem., 2000,7,971-994).

The synthetic of these XC polymer also can be designed so that accept a special radioactive atom after screening step in vivo.Therefore, can synthesize peptide library, wherein all elements all mix the tyrosine with the iodate of on-radiation iodine, and wherein the N end is with tritium-labeled.After the screening, molecule (s) of interest can synthesize again in vivo, but mixes radioiodine this moment in tyrosine.Because the tyrosine of iodate exists during the screening step, therefore final Geigers must be preserved all biologic activity.This example can be applicable to polytype XC polymer, mixes a synthon that carries the on-radiation atom in the described XC polymer, in case but screen then can be alternative with its radioactive isotope.This strategy is specially adapted to technetium 99 or fluorine 18.

The molecule of correct radiolabeled natural origin also can use the same procedure screening.Therefore can screen the extract of microorganism, wherein all molecules (are for example all used different radioactive isotopes ³H, ¹⁴C, ³²P, ³⁵S) radioactive label.When these microorganisms exist when cultivating under the radiolabeled nutraceutical condition, can obtain radiolabeled biomass.Natural extract is as venom (M.TAKEDA et al., Toxicon, 1974,12,633-641; E.KOCHVA et al., Toxicon, 1982,20,3,615-636) can obtain with radiolabeled form giving animal radioactivity amino acid so that it is incorporated under the condition in the toxin that produces in the venom gland.The strategy that can disclose radiolabeled natural products can be used for radiolabeled product in the screening technique of the present invention with a large amount of productions.

Advantageously, carry out simultaneously in the different several animals of step (1)-(5) amount of application in step (1), and the analysis of the increased radioactivity of the molecule of step (2) is to carry out in the different time according to animal.

Astoundingly, specificity between one or more molecule that contains in the potpourri that this method can be determined to be made up of molecular library and one or more specificity site in organ or tissue interacts, and need not specific marker, do not need the compound that increases in advance yet.

Method of the present invention has following advantage especially:

-its non-peptide sequence that is limited to can relate to all types of molecules, molecule, especially XC polymer or the biomolecule that obtains by combinatorial chemistry particularly, and non-peptide molecule and natural materials, particularly plant extracts;

-radio-labeled is not limited, because described in the document about the many radio-labeled methods by chemistry or the synthetic multiple family compound that obtains of enzymatic; In addition, in the situation of natural materials,, can set up the strategy that causes producing biosynthetic radioactive compound according to the type of the biosome that produces compound of interest.This strategy can enter the Geigers of the metabolic cycles of the biosome of being considered based on importing.For example the method for described radio-labeled peptides such as (also seeing above-mentioned) such as M.TAKEDA (seeing above-mentioned) or E.KOCHVA is mentioned;

-invade by promoting tissue, carry out the interior screening of body with the library of micromolecule (opposite) and can more effectively screen all acceptors that exist in the biosome with bacteriophage.Therefore use such screening can develop the molecular diversity that contains in the described library that is considered better.The elimination phenomenon of eliminating bacteriophage from biosome no longer becomes important restriction;

-in the different step of screening the chemistry of compound is differentiated it is identical, this is not the situation in the method that discloses of E.RUOSLHATI group as mentioned above;

-it can directly observe the Tissue distribution of the compound of screening;

-it avoids stoping the product that screened and the existence of the interactional mark of its target position by spatial obstacle;

-it does not need amplification step; And

-its can research institute the metabolism of compound of screening and pharmacokinetics (distribute and remove).

Astoundingly, the library by making up synthetic product or the radio-labeled of set, combinatorial chemistry, observation animal (difference according to circumstances, be inhuman or the people) radioactive technology and from organ or tissue, separate (for example extracting) molecule so that its method by mass spectrophotometry or other suitable analytical, can differentiate part effectively, its bioavilability, toxicity and metabolisming property will be consistent with application in its body subsequently, with prior art disclose different, comparing with radioactivity wherein that more complicated mark is considered to only is the effective mark of the molecule of certification mark.In fact, if very short radioactive emission thing with life-span of not jeopardizing animal of that use energetic species and half life period can observe a kind of radiolabeled product in live animal.Such tracer is compared therefore highly beneficial with the low energy tracer; Yet the short life of these tracers and external (ex vivo) analyze contradiction; In this case, have long-life tracer,, also prove very favorable, especially because processing more relative unrestriction aspect security of such tracer even they can not be used for the external observation of animal as tritium.

Different with the screening of carrying out on organ that separates or cell, in the scope of the modification of gene expression, the screening that is included in a step of carrying out on the live animal has guaranteed the functional completeness of the acceptor of part target in cell culture system.At last, and compare based on the targeted approach of using antibody, method of the present invention can be carried out (average 1000Da) with low-molecular-weight molecule.Compare with antibody, this character means better tissue intrusion and therefore develops the repertoire of selective expression's acceptor in tissue or organ better.

In addition, provide the size of the compound of research, they will not induce the immune response to the part host.

According to another advantageous embodiment of the inventive method, separating according to following steps of the Geigers that the described and described organ of step (4) is relevant carried out:

-remove and wherein detected radioactive organ,

-described the organ of grinding in suitable damping fluid,

-centrifugal every kind through grinding material and reclaim solution,

-filter described solution,

-adjusting pH,

-fractionated also reclaims radioactive segment.

More accurately, in the situation of peptide, this separating step can be included in and reclaim solution behind the centrifugal material through grinding and filter:

-acidifying filtrate,

-with described filtrate through a post that contains the silica that combines with hydrophobic group to carry out " anti-phase " elution technique; According to the situation that exists of plus or minus electric charge in the compound of library or set, can advantageously comprise the chromatography method of ion-exchange,

-collect eluate by fractionated, and

-selection contains the part of radiation material,

-concentrated described radioactive segment under vacuum,

-filter by keeping the filter membrane that molecular weight is higher than the product in those source document storehouses,

-concentrate described filter thing, then

-analyze described sample by high pressure chromatography (HPLC), use filling the post of anti-phase holder carry out; Use the existence of conventional planning detector detection of radioactive product in the outlet of separating column.

According to another advantageous embodiment of the inventive method, in step (5), the analysis of being undertaken by mass spectrometry is relevant with tandem mass spectrum analysis (MS/MS).

According to another advantageous embodiment of the present invention, the described library of step (1) molecule has following general formula: propyl group- ³H-NH-Yaa- _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂, wherein wherein there is the position of 20 natural amino acids in Yaa and Yaa ' representative.

Find that this method has many application, can mention particularly:

The new contrast preparation of medical imaging is carried out in-announcement; Because the compound that is screened just carries radioactive atom from beginning, therefore the molecule of selecting can be directly used in and carry out medical imaging or can add the contrast preparation that conforms to observation in the body by suitable technology modifying.In this respect, the library that contains the tritium molecule is particularly advantageous, and wherein all molecules all carry fluorine 19 atoms.In case by selecting in the body, then can synthesize the molecule of discriminating, mix fluorine 18 this moment, can use thus described molecule as contrast preparation to be used for based on imaging by suitable tomography device detection positron;

-be suitable for the generation of the compound that therapeutic uses; In fact, except its with ability that point-device site in the live organism combines, the molecule of some discriminatings can have special biologic activity and therefore as the basis of disclosing therapeutic compound;

-targeted drug or any other molecule (s) of interest (being delivered to the specificity site) by molecular chemistry coupling with medicine and a kind of selection.For with a kind of interested material target in special organization or organ, in fact can envision this interested material and the molecular chemistry coupling of differentiating by method of the present invention, and therefore it can be delivered to an organization space then to a specificity site;

-discriminating selective expression's in given tissue or organ novel receptor.

Method of the present invention can be differentiated the novel receptor of selective expression in given tissue or organ.Such information and discriminating are extremely important to disclosing new therapeutic strategy.In same step, identify that selective ligands and also unidentified relevant selective receptor thereof are to separate this acceptor most important by disclosing the affinity column that combines the part that is specific to this acceptor on it.This identical part is represented a kind of initial structure, can design other part based on it, especially for therapeutic purposes.

Therefore this method can also study multiple pathology.In fact, cause in normal individual, being difficult to or pathology that some acceptor of at all not expressing is crossed expression can easily detect by method of the present invention.Therefore, between the normal and infected animal confirmation of selected marker diagnosis, treatment, imaging and with drug targeting in being highly profitable aspect illing tissue or the organ.For example, this method relates to that differentiate without the normal structure of marking animals or organ can former of selective binding or the ability of the compound of secondary tumor.

In this regard, should notice that with identical library injection intact animal should be the means of identifying the expression of receptor that is specific to a pathology according to difference, and therefore also be for the same reason disclose be specific to the means of mark of given pathology.

Preferably, described molecule or part synthetic is to use technology known in the art to carry out in solid phase, for example uses the NH that contains the peptide that the tritium acetic anhydride acylation combines with solid support _2-Functional end-group and with a CT ₃-CT ₂-CO group imports the N terminal position.

A theme of the present invention relates to a kind of kit that carries out described screening technique of the present invention, it is characterized in that comprising:

The library of the molecule of-at least one mark, the equal mark in advance of each molecule wherein, particularly but do not get rid of the atom that exists with in the described molecule of one of its radioactive isotope displacement,

The method of the product of-detection of radioactive labels particularly is selected from the imaging device that is suitable for detecting the radioactive ray that detect in the section of organ or tissue,

The means of the radiolabeled product of-analyzing and testing are as HPLC and/or mass spectrophotometry.

A theme of the present invention relates to a kind of method of using a kind of molecular library to differentiate and study selective expression's target molecule (or acceptor) in organ or tissue, and described method comprises:

(1) give described molecular library at least one animal, wherein the equal mark in advance of each molecule is not particularly got rid of an atom that exists with in the described molecule of one of its radioactive isotope displacement,

(2) put to death at least one described animal and utilize radioactive Tissue distribution of the molecule that above-mentioned suitable imaging device analysis given, by to the animal sampling and analyze a plurality of histotomies and carry out,

(3) selection wherein detects the section of radioactive tissue or organ,

(4) from the section of the tissue step (3), selected or organ, separate radioactive segment, undertaken by suitable analysis techniques, as extracting and/or chromatography,

(5) use the radioactive segment that in step (4), obtains to identify described molecule, undertaken by suitable analysis techniques, as chromatography and/or mass spectrophotometry,

(6) molecule that will obtain in step (5) contacts under certain condition with tissue or the organ samples selected according to step (2) and obtain, and described condition is to make in step (4) between the molecule that separates and the target molecule combine and form a kind of compound, reaches

(7) from described compound, separate described target molecule.

A kind of possible variation is the radioactivity Tissue distribution that step (2) comprises the molecule that analysis is used, and utilizes suitable imaging device to extract from tissue or the cell of organ and section in advance and the biological sample selected carries out in external use.Therefore this variation does not need to put to death animal.

According to an advantageous embodiment of the present invention, in step (2) before, described method comprises that step (1 ') is to analyze the radioactivity Tissue distribution of the molecule of being used, by being carried out outside imaging, at least one animal undertaken, particularly utilize and be suitable for detecting by the pick-up unit combining camera of the character of the ion of the radioactive isotope emission of selecting and undertaken ( ¹⁸F, ¹¹C, ⁶⁴Cu, ⁷⁶Br, ¹²⁴I, ¹³N, ¹⁵O: positron emission transaxial tomography, PET; ^99mTc, ¹²³I: γ-camera etc.).

The feasible especially character that can study the compound of having differentiated more accurately of the step of this method (7).Each molecule that synthetic once more, radioactive label and research are selected by screening in the body is undertaken by the same procedure that is used to screen with its Tissue distribution ability of illustration.

An aspect of of the present present invention be a kind of with a molecule (s) of interest target in the specific organization space and/or the carrier in site, be characterised in that it comprises the molecule of selecting by the method for screening molecular library as above-mentioned, and the Tissue distribution in described site has made discriminating in this way.

A theme of the present invention relates to a kind of locus specificity composition, be characterised in that it comprises a kind of molecule (s) of interest of (i) target in described site, itself and the molecule coupling that is specific to described site, the Tissue distribution of described molecule is differentiated by the method for above-mentioned screening molecular library, and (ii) at least a pharmaceutically-acceptable excipients.

A theme of the present invention relates to the application that is used for the composition of medical imaging by the molecule of the mark of selecting as the method for above-mentioned screening molecular library in preparation.

According to the advantageous embodiment of described application, the molecule of described mark is associated with a kind of suitable contrast preparation.

A theme of the present invention relates to the method for at least one target position (for example acceptor) of differentiating a molecule (s) of interest, is characterised in that it comprises at least:

(1) with molecule (s) of interest with as above-mentioned by the method for screening molecular library select be specific to as described in the radiolabeled molecule coupling of target position, obtaining a kind of radiolabeled conjugate,

(2) give at least one animal step (1) the middle conjugate that obtains, and

(3) use a kind of biological sample at least one target position of the described biological sample of analyzed in vitro and combining of the molecule (s) of interest that is coupled to described radiolabeled molecule.

A theme of the present invention relates to a kind of method of screening unlabelled molecule in vivo by competition, and this method is characterised in that and comprises:

(1) give the molecule of at least one a kind of mark of animal, the Tissue distribution of described molecule as the method for above-mentioned utilization screening molecular library differentiate,

(2) analyze the Tissue distribution of the Geigers that in step (1), gives the first time, by at least one animal carried out outside imaging, particularly utilize the pick-up unit combining camera be suitable for detecting by the character of the particle of selected radioactive isotope emission to carry out

(3) give the library of unmarked molecule,

(4) analyze for the second time the Tissue distribution of Geigers,, particularly utilize the pick-up unit combining camera of the character that is suitable for detecting the particle of launching by the radioactive isotope of selecting and carry out by at least one animal carried out outside imaging,

(5) by determining to replace the pharmacokinetics of the molecule of mark with analyzing the first time of the distribution of labeled molecule to compare by at least a unlabelled molecule, and

(6) detect at least a unlabelled molecule of the pharmacokinetics changed labeled molecule,, repeat molecule as described in fractionated (method of deconvoluting) and the evaluation as chromatography and/or mass spectrophotometry by suitable analysis techniques.

A kind of possible variation is the radioactivity Tissue distribution that step (2) and (4) comprise the molecule that analysis gives, and utilizes suitable imaging device to extract from tissue or the cell of organ and section in advance and the biological sample selected carries out in external use.

A theme of the present invention relates to a kind of compound of molecular library, general formula be propyl group- ³H-NH-Yaa- _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂, wherein Yaa represents Arg, reaches Yaa ' and represents Leu, and it can obtain by the method as above-mentioned screening molecular library.

Astoundingly, the general formula of selecting by method of the present invention is propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂This compound have following character especially:

-it is a kind of powerful inhibitor of zinc peptidase ACE (angiotensin I-changes enzyme) and NEP (neutral endopeptidase or enkephalinase (neprilysin)) in vivo, so finds to have using value in cardiovascular pathological changes;

-it can pass blood-brain barrier and therefore can have above-mentioned using value, particularly when it is labeled in following method: (1) is in medical imaging, (2) in the method for target molecule (s) of interest, by this molecule (s) of interest and this peptide are carried out in the brain coupling, (3) in screening in the method for the specific expressed molecule of brain.

Describe other and had phosphine peptide (phosphinic peptides) (the French patent application No.8914978 of different general formulas; French patent application No.9105403; French patent application No.9501328; French patentapplication No.9808464), some of them (seeing French patented claim No.9808464) present the inhibition activity to ACE; Yet, astoundingly, according to prior art, the chemical constitution of most compounds that shows as the mixed inhibitor of ACE and NEP has a free carboxylate groups at its C end, and it is considered to obtain the necessary group of strong interaction (Bralet et al., Tips between described inhibitor and this two kinds of peptases, 2001,22,3,106-109; Weber, The Lancet, 2001,358,1525-1532; Fink, Exp.Opin.Ther.Patents, 1996,6,11,1147-1164), compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Do not comprise this group, although it effectively suppresses this mixed activity.

Described peptide has above-mentioned application, mainly is:

-a kind of medicine;

The carrier of-a kind of target molecule (s) of interest is especially at brain;

-a kind of locus specificity composition in medical imaging;

-be used for differentiating the method for at least one target position (for example acceptor);

-be used for by competing direct method of screening unlabelled molecule in vivo.

More accurately:

A theme of the present invention relates to a kind of medicine, is characterised in that it comprises compound propyl group-NH-Arg-Phe (PO at least ₂-CH ₂) Leu-Ala-NH ₂And at least a medicine acceptable carrier.

A theme of the present invention relates to targeting vector or is used for a kind of carrier that target shifts a kind of medicine, is characterised in that it is by compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Form.

A theme of the present invention relates to as above-mentioned compound (propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂), its be selected from tritium ( ³H), carbon 14 ( ¹⁴C), carbon 11 ( ¹¹C) or phosphorus 32 ( ³²P) labelled with radioisotope.

An aspect of of the present present invention is a kind of composition that is used for medical imaging, is characterised in that it comprises as above-mentioned compound (propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂) and at least a pharmaceutically-acceptable excipients, described compound is also optional and another kind of compound coupling with labelled with radioisotope.

A theme of the present invention relates to the application that is used for the composition of medical imaging as the compound of above-mentioned mark in preparation.

A theme of the present invention relates to a kind of method as at least one target position of a kind of molecule (s) of interest of above-mentioned discriminating, is characterised in that described radiolabeled molecule is radiolabeled propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Peptide.

In this case, described target position is preferably placed at brain.

A theme of the present invention relates to a kind of method of screening unlabelled molecule in vivo by competition, and wherein radiolabeled molecule is radiolabeled propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Peptide.

Except afore mentioned rules, the present invention also comprises other regulation, and it manifests by the embodiment of reference the inventive method and the description of following accompanying drawing, wherein:

-Fig. 1 show the general formula propyl group- ³H-NH-Yaa- _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂The theoretical distribution of library quality, wherein the position of 20 natural amino acids appears in Yaa and Yaa ' representative, synthetic (if will owing to exist the diastereo-isomerism due to the asymmetric center to count then synthetic 1600 at Phe and Leu residue) that causes 400 different molecules;

-Fig. 2 represent the general formula propyl group- ³H-NH-Yaa- _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂The experimental mass spectrum in library;

-Fig. 3 representative phosphine peptide propyl group- ³H-Phe-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The chemical constitution example that in the MS/MS spectrum, observes during the fragmentation of (the selection quality is 595 compound);

-Fig. 4 show propyl group- ³H-Phe-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The MS/MS spectrum of peptide;

The autoradiograph of the whole sagittal section of the mouse that-Fig. 5 representative prepares on β-imager, described mouse be given contain corresponding to the general formula propyl group- ³H-NH-Yaa- _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂A kind of solution in library of phosphine peptide, put to death this animal in back 1 hour in injection;

-Fig. 6-8 representative is at the mass spectrum of Different Organs (kidney, lung and the heart) extract after the library of I.P. injection phosphine peptide;

-Fig. 9 and 10 the representative when with propyl group- ³H-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The fragment that this peptide obtains when carrying out the MS/MS spectrum;

-Figure 11-13 shows the fragmentation peak value that obtains from the extract of kidney, lung and the heart in the experiment of MS/MS fragmentation, the selection quality is 595 product.These figure show have product propyl group-NH-Arg-Phe (PO in these opzymes ₂-CH ₂) Leu-Ala-NH ₂

-Figure 14-19 show propyl group corresponding to purified form- ³H-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂(4 diastereo-isomerisms Figure 13) reach the spectrum of the HPLC in Different Organs (lung, the heart, liver, kidney and brain) extract then to peptide;

-Figure 20 shows the autoradiograph of a plurality of slices of organs that prepare from mouse, described mouse be given propyl group- ³H-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Peptide and behind the described product of injection, being condemned to death in 1 hour; And

-Figure 21 be illustrated in propyl group- ³H-NH-Tyr-Phe (PO ₂-CH ₂) Leu-Pro-NH ₂The autoradiograph of a plurality of slices of organs that prepare behind the peptide injection mouse.Described animal is condemned to death after 1 hour at the described product of injection;

-Figure 22 shows compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Inhibiting effect to ACE;

-Figure 23 shows compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Inhibiting effect to NEP;

It is propyl group-NH-Yaa-Phe (PO that-Figure 24 shows general formula ₂-CH ₂) Leu-Yaa '-NH ₂Sublibrary (sublibraries) to the inhibiting effect (concentration in each library is 500nM) of ACE;

It is propyl group-NH-Yaa-Phe (PO that-Figure 25 shows general formula ₂-CH ₂) Leu-Yaa '-NH ₂Sublibrary to the inhibiting effect (concentration in each library is 250nM) of NEP;

-Figure 26 shows a kind of scheme of separating described radiolabeled product by extracting;

-Figure 27,28 and 29 shows radiolabeled compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂(pro-Arg) Tissue distribution in kidney, lung and the brain of mouse respectively, this can be to this compound by combination radiophotography device (β-imager of Biospace company) and analytical technique of mass spectrum (Electrospray, Quatro, MicroMass) sensitivity of contrast phosphine peptide detection.

Embodiment 1: with the library or the set of the false peptide of the radiolabeled phosphine of tritium

Synthetic and the radioactive label in the library of the false peptide of-phosphine

A) radioactive label in library

The synthetic of the library of phosphine peptide carries out (A.Yiotakis et al., J.Org.Chem., 1996,61,19,6601-6605 based on the scheme of announcing; J.Jiracek et al., J.Biol.Chem., 1995,270,37,21701-21706; J.Jiracek et al., J.Biol.Chem., 1996,271,32,19606-19611; V.DIVE et al., PNAS, 1999,96,4330-4335).Rink-amide resin (J.Jiracek et al., J.Biol.Chem., 1996 and V.Dive et al., PNAS, 1999, above-mentioned) is used in described library on solid phase, utilize division ﹠amp; (J.Jiracek et al., J.Biol.Chem., 1995 and J.Jiracek et al., J.Biol.Chem., 1996, mentioned above) is synthetic for assembled scheme.When this synthetic schemes finished, the general structure of the peptide on the resin was: Fmoc-NH-Yaa-NH- _{(R, S)}Phe (PO (Oad)-CH ₂) _{(R, S)}Leu-Yaa '-CONH-R type.

Yaa and the position of Yaa ' representative with 20 different aminoacid replacement, R represents described resin.

Cracking Fmoc group makes radioreagent mix by acidylate N functional end-group to produce a free N end.Described phosphine peptide is by mixing N-succinimide-T5 propionic ester and radio-labeled on its free N functional end-group, N-succinimide-T5 propionic ester is that specific activity is a kind of compound of 97Ci/mmol.N-succinimide-T5 propionic ester/16.6 μ mol phosphine peptides (mean molecular weight is 607, is going to the protection back corresponding to the whole peptide of 10mg) of 10.3nmol are mixed in the described peptide, corresponding to mixing 1mCi radioactivity/library.

Described acylation reaction is finished with the N-succinimide propionic ester of excessive cooling.Behind the flushing resin, the phosphine peptide goes protection and the cracking of cracking scheme (J.Jiracek etal., J.Biol.Chem., 1996 and V.Dive et al., PNAS, 1999, above-mentioned) by routine.Described cracking solvent is removed by continuous vaporization cycle.With the DMSO of peptide at 50 μ l, the 1M NaHCO of 15 μ l ₃With carry out drying among the PBS of 50 μ l.The pH of this solution 1M NaHCO ₃Solution is adjusted into 7.With PBS final volume is adjusted into 500 μ l.This solution is used for being administered to animal through injection.The mass spectrum in this library (Fig. 2) and a plurality of MS/MS fragmentations experiment that carry out in this library is illustrated all compounds all have the extraordinary performance of expectation in theory.

B) evaluation in the library of Huo Deing

So obtained with the radiolabeled phosphine peptide library of tritium.General formula according to the synthetic library of such scheme is: propyl group- ³H-NH-Yaa _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂, wherein the position of 20 molar mixtures such as amino acid whose wherein appears in Yaa and Yaa ' representative.Therefore this library comprises 400 compounds, if consider two asymmetric centers that exist in these structures then formally have 1600.Described compound is mixed C at the N end ³H ₃ ^-C ³H ₂The containing the tritium propionic acid of-COOH type and radioactive label mixes 5 tritium atoms in the structure of described propionic acid.

It is propyl group-NH-Yaa that Fig. 1 shows general formula _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂The theoretical distribution of library quality.

Theoretical scope (envelope) corresponding to the quality of a plurality of phosphine peptides that contain in this library is represented in this drawing.Notice that this scope is not continuous, but form, reflected the distribution of each product quality group in this library by the peak of significantly not offering an explanation.

Fig. 2 shows general formula: propyl group-NH-Yaa _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂The experimental spectrum (ES-MS=Electro Spray MassSpectroscopy) in library.

When with theoretical scope (Fig. 1) with relatively the time, notice that gross data and experimental data are very identical corresponding to the experimental spectrum (Fig. 2) in library.Thisly consistently show that all phosphine peptides are all very well showed in this library.The MS/MS experiment of the peak value that observes being carried out fragmentation in the MS spectrum can be illustrated in the phosphine peptide that existence is expected in theory in this library.

By the evaluation of mass spectrophotometry to the phosphine peptide:

Find to select a CH ₃-CH ₂The protectiveness group of-CO type is useful in by mass spectrophotometry molecule being identified, described group is used for the different of chemistry of peptides with routine.Each phosphine peptide in this library is identified by the character of the amino acid residue that exists at N and C terminal position respectively.This library identifies that by there being paired peptide it has accurately identical quality and identical amino acid content, and just its sequence changes following illustration:

For example, following two peptides can not be distinguished according to its quality, and its quality is identical:

Propyl group-Asp-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂

Propyl group-Ala-Phe (PO ₂-CH ₂) Leu-Asp-NH ₂

Some phosphine peptide is carried out MS/MS fragmentation technology make and to infer the chemical constitution that to differentiate compound undoubtedly, even when at least two kinds of peptides identical in quality, also can differentiate.This character is because due to the fragmentation pattern of this type phosphine peptide, and this makes the character can differentiate residue in the N terminal position especially.

Fig. 3 shows the (PO as phosphine peptide propyl group-Phe-Phe ₂-CH ₂) Leu-Ala-NH ₂During by fragmentation, the chemical constitution example that in the MS/MS spectrum, observes.

Fig. 4 shows propyl group-Phe-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The MS/MS spectrum of peptide.

As Fig. 3 and 4 illustrations, the residue in the N terminal position always (pro-Phe-CO occurs with the entity form that carries the propoxide group ⁺, Fig. 3 and 4 D peak).In the MS/MS spectrum, observe the therefore feasible amino acid whose character that can determine at the N of described peptide terminal position in most applications of this entity, and therefore can determine the character of residue in the C terminal position, know the quality of peptide.Also systematically observe another kind (pro-Phe-Phe (PO ₂-CH ₂) LeuCO ⁺, Fig. 3 and 4 C peak); Described observed result has been proved conclusively at the character of the residue of this sequence N terminal position (the Phe residue in the example).

These characteristics of the fragmentation pattern of the phosphine peptide that exists in this library are from the importing of propoxide group.According to these characteristics, can also differentiate the chemical constitution of compound, even in some phosphine peptide situations identical in quality, also can differentiate.

Notice that when phosphine peptide during having quality in MS/MS composes is 120 peak (Fig. 3 and 4 E peak) by fragmentation.This peak is a kind of feature of phosphine peptide, observes this peak a kind of phosphine peptide that exists in the sense organ extract is highly profitable.

-library is injected into animal and observes the Tissue distribution that contains the tritium peptide

A) library that obtains is injected into animal

The mouse that is used for this experiment is female Balb/C mouse.The phosphine peptide (10mg, radioactivity is 0.8-1mCi) in described library is dissolved in the PBS solution that contains 10%DMSO.If consider to have 400 molecules in this library, then the injection magnitude of each product is 25 μ g/ animals, and expression weight is that the injected dose of the mouse of 20g is 1.25mg/kg.In the situation of the single product of injection, used dosage is 20mg/kg, and gross activity is 100-150 μ Ci.Inject at different time (5 minutes, 1 hour, 3 hours and 24 hours).

B) preparation of slices of organs

After putting to death animal, take out a plurality of organ or tissues immediately, in PBS solution, wash and be embedded in (eutectic mixture of heptane and dry ice) in the heptane that is cooled to-70 ℃.Utilize microtome to prepare frozen tissue section at-20 ℃, thickness is 25 μ m.After the drying, should cut into slices and in β-imager, analyze.

C) result

^*In β-imager, analyze section

According to such scheme the library of radiolabeled phosphine peptide is gone in the mouse through intraperitoneal injection.

After certain hour (1 hour or 15 minutes), put to death animal.The whole section of preparation is also analyzed by radioautograph on β-imager, and described imager is a kind of device that can detect the beta rays of tritium emission.These experiments show injecting described library after 15 minutes and 1 hour, all observe radioactivity in a plurality of organs; Fig. 5 shows the sagittal autoradiograph of the mouse integral body that produces on β-imager, this mouse has been applied a kind of solution of containing the phosphine peptide library and had been condemned to death in back 1 hour in injection.

Except liver and kidney, for example in the heart and lung, also observe radioactivity.In addition, this library did not have any compound that causes animal dead or toxicity during confirmation was being observed in the sacrificed subsequently experiment of described animal.Therefore can infer that at these experimental sessions the important function of animal is protected.This aspect is very important because its guaranteed the compound differentiated should with physiology on " acceptor " of expressing interact.

The extraction of-phosphine peptide

A) scheme

Putting to death 4 ℃ the PBS solution that exteriorizes rapidly and place behind the animal.After a few minutes, this organ moved in 4 ℃ the Potter homogenizer that contains 2ml PBS, grind with hand then.Material through grinding is moved in the centrifuge tube that contains 15ml PBS.After 4000rpm is centrifugal 30 minutes, separate with cell precipitation and through 0.45 μ m membrane filtration top.After will filtering the thing acidifying, in the cartridge case that contains 5g C18 phase (cartridge), this cartridge case washes in containing the aqueous solution of 0.1%TFA in advance with its application of sample.

The following composition of elution protocol: for the first time through containing the 20ml water of 0.1%TFA, then through containing the 30ml aqueous solution of 50% acetonitrile and 0.1%TFA, then through containing the 20ml aqueous solution of 80% acetonitrile and 0.1%TFA.

Partly collect the eluate that flows through in the C18 cartridge case with 2ml.Radiocounting makes can differentiate the part that contains radiolabeled phosphine peptide.

Radioactive segment is concentrated under vacuum and product is placed water, then with a plurality of solution through filter membranes, described filter membrane keeps the product of molecular weight greater than 5000Da.With the solution simmer down to 100 μ l volumes under vacuum that filter thus.

Then these samples are prepared to analyze, by efficient chromatography and in conjunction with the system of a detection of radioactive with observation radioactivity phosphine peptide, perhaps analyze by mass spectrophotometry.

The existence that this step discloses the phosphine-derivatives that comprises in the initial library can be distributed in some organ.In order to differentiate the phosphine-derivatives that is retained in these organs, use a plurality of schemes, with the situation that exists of the compound in library described in the step of monitoring extraction procedure as phosphine-derivatives as described in the above-mentioned radiological measuring method Test extraction.Use the fractionated scheme to make and by HPLC binding radioactivity detection method and to analyze these extracts by mass spectrophotometry.The optimization of mass spectrophotometry is used, and the sensitivity of especially described method is based on the character of the sample of analyzing.Therefore the scheme of preparation sample is extremely important in this step, but can change according to the described library entrained chemical functional group of product.For example, in the situation of the false peptide of phosphine, using the H that contains 0.1%TFA ₂After the O flushing, the false peptide of all phosphines in described library all can be used 50%CH ₃The CN wash-out, and at 80%CH ₃CN, many endogenous products of analysis subsequently that disturb are by wash-out.

B) derived from the mass spectrophotometry of injecting the opzyme of the animal that has given the phosphine peptide library by i.p.

The mass spectrophotometry of the multiple extract of preparation shows from a plurality of organs and tissue (kidney, liver, lung, the heart, brain, tumour), except kidney, does not have the signal that is specific to the phosphine peptide and occur in the mass spectrum of these extracts.

These results are illustration in Fig. 6-8.

Fig. 6 represents the mass spectrum of kidney extract, wherein observes the many characteristics peak (comparing with Fig. 2) that has the phosphine peptide.

Fig. 7 represents the mass spectrum of lung extract, wherein observes the characteristic peak of no phosphine peptide.

Fig. 8 represents the mass spectrum of heart extract, wherein observes the characteristic peak of no phosphine peptide.

Yet, find that be different by mass spectrophotometry with the result who obtains by HPLC; In fact, the existence at the characteristic peak of phosphine peptide is to be derived from lung and to be derived from the extract of the heart detected by HPLC.This difference is from such fact, and promptly in mass spectrophotometry, the mass peak of the interior source compound that is derived from the organ to be contained that may still be existed behind extraction step corresponding to the mass peak of the product of being sought is covered.Based on these results, the mass spectrophotometry of MS/MS fragmentation is connected with proof the existing of phosphine peptide in multiple extract by system.

The feasible fragment (spectral signature (spectralsignature)) that therefore a kind of peptide also only can be disclosed this peptide with given mass fragmentization in the MS/MS spectrum of this MS/MS mass spectrophotometry.Therefore prove that these experiments are more effective to the existence that proves a kind of product, even this product does not occur in the MS spectrum.

The feasible situation that exists that can differentiate lung and a kind of phosphine peptide in the heart of this MS/MS mass spectrum, described phosphine peptide is propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂, this peptide also is present in kidney and the liver; Yet, in these organs, also observe other phosphine peptide.Fig. 9 and 10 representatives are worked as propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The fragment that this peptide obtains when carrying out the MS/MS analysis of spectrum.In kidney (Figure 11), lung (Figure 12) and the heart (Figure 13) extract, observe propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The distinctive fragmentation of peptide peak makes can infer that this peptide is present in these organs.

Utilize this method of screening peptide library to select product propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Cause this peptide to prepare, to determine its Tissue distribution character in detail with pure form.

To radiolabeled propyl group-Arg-Phe (PO with pure form injection ₂-CH ₂) Leu-Ala-NH ₂Tissue distribution research of peptide illustrates (20mg/kg in a plurality of tissues that this compound can effectively be distributed in mouse, 100 μ Ci, after injecting described product, put to death this mouse in 1 hour through I.P.), a plurality of organ imagings that obtain by radioautography confirm; This product observes in the heart, brain, liver, lung and kidney.Highly beneficial ground observes this Toplink through blood-brain barrier, because it is observed in brain.Identical extraction and structural analysis program show that all radioactivity that observe by radioautograph are corresponding to the propyl group-Arg-Phe (PO that has complete form ₂-CH ₂) Leu-Ala-NH ₂Peptide (Figure 20).

Figure 14-19 represents the HPLC spectrum of detection of radioactive, corresponding to pure tritium product propyl group-Arg-Phe (PO that contains ₂-CH ₂) Leu-Ala-NH ₂, and corresponding to a plurality of opzymes that are derived from mouse, described mouse has been given propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Peptide (20mg/kg, 100 μ Ci) and execution in 1 hour behind this product of injection.

For showing propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Peptide at first target after one's own heart reaches the ability of secondly assembling with lung in some organ in these organs, use another kind of molecule as negative control under the same conditions, and this is the another kind of molecule in above-mentioned library, i.e. propyl group-Tyr-Phe (PO ₂-CH ₂) Leu-Pro-NH ₂

With compound propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Under the identical experiment condition of the condition of using its Tissue distribution research is illustrated, can be with compound propyl group-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Very clearly with propyl group-Tyr-Phe (PO ₂-CH ₂) Leu-Pro-NH ₂This peptide is distinguished mutually, according to its ability of assembling in the heart and lung and the ability that occurs in brain thereof.

The autoradiograph of a plurality of slices of organs that Figure 21 representative prepares from mouse, described mouse is given propyl group-Tyr-Phe (PO ₂-CH ₂) Leu-Pro-NH ₂Peptide (20mg/kg, 100 μ Ci) and execution in 1 hour behind this product of injection.

Embodiment 2: phosphine peptide propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The character and the discriminating of potential target position

Phosphine peptide propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Specific tissue distribute, especially in brain, such hypothesis is proposed, promptly this compound can be changed the zinc peptide interaction of enzyme (ACE) and/or neutral endopeptidase (NEP, enkephalinase) with angiotensin I-.In fact, generally speaking, the phosphine peptide has and zinc protease or the interactional ability of peptide enzyme spcificity (seeing that french patent application No.9808464 is described); In addition, known these two kinds of zinc peptidases have the (PO with phosphine product propyl group-NH-Arg-Phe ₂-CH ₂) Leu-Ala-NH ₂Corresponding to Tissue distribution (Cadwell et al., Science, 1975,191,1050-1051; Roques et al., Tips, 1990,11,245-249).

For this reason, estimate phosphine peptide propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Inhibition ability (according to Dive et al., PNAS, 1999,96, the described mensuration of 4330-4335 Ki value) to this two kinds of peptases of pure form.Shown in Figure 22 and 23, this peptide is to the IC of hypertensin conversion enzyme and NEP ₅₀Value equals 30nM and 100nM respectively, is respectively 15nM and 30nM corresponding to suppressing constant K i value.Therefore this compound shows as is the extremely strong power inhibitor of these two kinds of peptases.

Because these two kinds of peptases are considered to nonselective relatively, therefore shockingly occur only having differentiated propyl group-NH-Arg-Phe (PO after the screening in vivo by this screening technique ₂-CH ₂) Leu-Ala-NH ₂This peptide.

Contain general formula and be for better understanding the different molecular that contains in the initial library inhibition activity, estimating ACE and NEP:

Propyl group-NH-Aaa-Phe (PO ₂-CH ₂) Leu-Yaa-NH ₂The inhibition ability of sublibrary of discontinuous potpourri, wherein a special amino acid is contained in the position that indicates with Aaa, and a kind of potpourri of 20 natural amino acids occurs in the Yaa position.

Figure 24 shows according to the character at the residue of Aaa position, corresponding to the phosphine peptide mixer of the above-mentioned general formula inhibition number percent to ACE.Results reported shows that very clearly the many phosphine peptides that exist in these sublibraries look like the powerful inhibitor of ACE among this figure.Advantageously, making described compound as can be seen in the residue of Aaa position is His, Arg and Tyr to the residue of inhibition ability the best of ACE.

When NEP is tested identical sublibrary (Figure 25), find that also the many peptides that contain in this sublibrary should be the very powerful inhibitor of NEP.Attention can make enzyme-inhibitor interaction optimization at the amino acid of some types of Aaa position.

Based on these vitro datas, can understand the product propyl group-NH-Arg-Phe (PO that contains in the library ₂-CH ₂) Leu-Ala-NH ₂The most powerful external inhibitor that does not belong to these two kinds of peptides.

This is shown clearly in the advantage of the inventive method; In fact, only can not only select, for example compounds effective in fact based on in-vitro screening; Especially, the use testing in vitro can select to have the chemical compound lot as the library of inhibitor ability, and method of the present invention comprises step in the body, and is more effective in the compound scope of only selecting to act in vivo.

Based on compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂The powerful in vivo ability that suppresses ACE and NEP peptase can expect that this product is used for cardiovascular pathological changes.

Compound propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂Metabolic stability and splendid Tissue distribution, particularly in the zone of these two kinds of zinc peptidases of known expression, cause expecting that this product has the splendid ability that suppresses these two kinds of peptases in vivo.Therefore, find that this product will have significant application value in the human cardiovascular disease becomes.

Embodiment 3: the importance of combination step in the inventive method (2) and (3)-(5)

The prompting of-the inventive method

Will be with tritium after radiolabeled library of compounds is injected into animal, method of the present invention comprises the radioactivity Tissue distribution of analyzing the molecule of using, use biological sample, the particularly therefrom tissue of sampling or the section of organ, the radiated signal that utilizes suitable radiophotography device to detect on described freezing tissue or the slices of organs carries out.

Only analyze the tissue present radiated signal or the organ homogeny with the compound that exists in the tissue of determining to be considered subsequently, the sensitivity that is used for the method for detection of radioactive signal plays an important role at this.

The contrast of-tritium radiophotography and the relative sensitivity of mass spectrophotometry

For carrying out this contrast, with the radiolabeled phosphine compound propyl group-Arg-Phe of 500 μ g tritiums (PO ₂-CH ₂) Ala-Ala-NH ₂(pro-Arg) (specific activity 68mCi/mmol) is injected in the mouse body.Behind this product of injection 1 hour, study its Tissue distribution by using frozen tissue section's detection of radioactive.To contain active organ handles with preparation and can carry out according to the extraction scheme of Figure 26 institute illustration by the tissue extract of mass spectrophotometry.Use the library of different Geigers to contrast the situation that exists that these two kinds of methods detections are injected into the interior phosphine compound of animal body, the step combination that method of the present invention can be shown can solve the detection and the discriminating problem of the radioactive compound that exists in the organ or tissue.

^*Kidney (Figure 27):

β-imager: radiograph shows and has radiolabeled compound (in these imagings, having represented radioactive minimum and maximum intensity, respectively with Dark grey and light grey expression) in this organ.

Based on the radioactive integration of the specificity of signal and product (68mCi/mmol), can determine every mm ²The product amount that nephridial tissue exists.

In section, the radiated signal that observes is 14.5pCi/mm ², represent 0.2pmol/mm ²Product (127pg/mm ²).By this amount is integrated mutually with the volume of whole kidney, can determine the product total amount that exists in the kidney 15% (75 μ g) corresponding to injected dose.

Quality: from the kidney material that is derived from the animal of handling under above-mentioned the same terms, extract product (extraction scheme as shown in Figure 26) through grinding, concentrating part places 100 μ l solvent (water/formic acid to drying and with product subsequently, 50: 50) in, if it is quantitative extracting, then the concentration of sample should be the 1.5mM rank in theory.Based on this hypothesis in working, solution (15 μ M) the expression 15pmol product that can estimate to inject this 100 times of dilutions of 1 μ l import in the mass spectrometer (Electrospray, Quatro, Micromass).

This amount is in full accord with the sensitivity of analytical technique of mass spectrum.

Figure 27 confirms in fact may observe fully the existence of described product in this sample, be characterised in that a mass peak corresponding to 596 Dalton molecular weights (M+H).On this mass spectrum, compare with other signal, have the highest intensity at the peak of 596Da, this makes the phosphine peptide can infer injection follow to compare with the endogenous product of this product co-extracted and is in the great majority.When changing described sample into the MS/MS pattern, when making this compound by fragmentation, the chemofacies same sex of described product is confirmed by observation Segment A, B and C.When described pure products observes identical peak A, B and C during by fragmentation.

Lung (Figure 28):

β-imager: autoradiograph is illustrated in more a spot of product in the lung: with radioactivity in kidney be 14.5pCi/mm ²Compare, radioactivity is 2.6pCi/mm in this organ ², promptly 2.2% of injected dose.

Quality: with theoretical concentration is that a kind of lung sample solution of 22 μ M carries out quality analysis.Compare with the experiment of in kidney, carrying out, inject this sample of 1 μ l and can hint a kind of extraordinary observed result product pro-Arg.The MS mass spectrum of Figure 28 shows that this is positioned at detection limit, is the noise rank that observes in this mass spectrum corresponding to the signal of pro-Arg.Yet, by fragmentation (MS/MS experiment), can infer that this product exists, in the MS/MS spectrum, clearly observe the fragment (A, B and C) of expection.

Brain (Figure 29):

β-imager: autoradiograph shows in the zone of the very limitation of brain very weak radioactive label: with the 14.5pCi/mm in kidney ²Comparing, is 0.35pCi/mm in brain ²Current owing to accurately do not determine to relate to radiolabeled brain volume, therefore more be difficult to estimate the described product total amount that exists in the brain.

Quality: after extracting described product, will have the whole parts that contain described product tendency all to be injected in the mass spectrometer, and fail to observe peak 596 from brain.The fragmentation experiment makes and can detect A, B and C peak, and therefore confirms the existence of complete product in the brain.Yet these experiments are in the limit of detection threshold.

The result of institute's illustration has proved the effect in the radiolabeled phosphine peptide in the detection tissue that is combined in of radiophotography device/mass spectrophotometry among Figure 27-29.In fact, in the situation of brain (Figure 29), it is limited to notice that independent mass spectrophotometry detects, and the existence of phosphine peptide discloses by the radiophotography device; These data illustrate need be used in combination the radiophotography device in organ or tissue the detection of radioactive product to detect the step of described radioactive product the most delicately, the step of analyzing by mass spectrophotometry makes and can determine to utilize the chemofacies same sex of radiophotography at the detected product of brain subsequently.

In fact, although mass spectrophotometry is very effective, it supposes contains very a large amount of endogenous products in a plurality of organs that according to the very small amount of product of the absolute purifying of character needs of tissue then this technology is very difficult to finish.In the mass range of the product of injecting (200-800Da), the existence of endogenous product has another very important influence to the relative sensitivity of mass spectrophotometry.The sensitivity of mass spectrophotometry in fact depends on the purity of the sample of being analyzed.Exist salt or other impurity can make the theoretical sensitivity of mass spectrophotometry reduce several magnitude in the sample of analyzing.

Utilize suitable radiophotography device, the step of detection of radioactive signal wishes that at separation the product of discriminating is without any need for processing, can detect these products in the best way like this, the step combination of the radioactive product that this step and explication de texte are selected thus makes the effort levels that obtains hope aspect the sensitivity that can detect described product in a plurality of organ or tissues.

Embodiment 4: by the analysis (have of the present invention step (1 ')) of positron emission transaxial tomography to carrying out with the Tissue distribution of the potpourri of the compound of fluorine 18 radiation part marks

Material and method:

Animal: this animal used as test is that weight is the Wistar rat (Janvier) of 200g, and random feeding of this rat and stable breeding be (controlled air, the day/night cycle is 12/12) in the conventional animal room.This rat is used for O in induction period ₂3.5% isoflurane (isoflurane) anesthesia in the gaseous mixture, and the anesthesia of maintenance 2% during whole collection.The injection of the potpourri (with regard to common injection) of different tracers is to be less than in time of 30 seconds with 300 μ l pill through intravenous injection at 4 animals.

PET: 4 rats are positioned (Siemens) in the EXACT HR+ camera (vertical 4.1mm, 1cm differentiates with two-dimensional approach from the distance center for 63 continuous cross sections of while, horizontal 4.5mm).Tissue and holder are proofreaied and correct the gamma-ray reduction of 511keV and are utilized ⁶⁸Ge- ⁶⁸The emission scan in Ga source obtains (15 minutes).Performance analysis behind the injection tracer utilized 102 frameworks (25 * 0.2 minutes; 19 * 0.3 minutes; 20 * 0.5 minutes; 38 * 1 minutes) the collection thing carry out.

Tracer: [ ¹⁸F] FDG is a kind of metabolic mark at glucose consumption; [ ¹⁸F] F-A85380 is a kind of α ₂β ₄The nicotine excitant.Be the potpourri (1: 1) that two rats give these two kinds of tracers, two rats only give one of these two kinds of tracers in addition.With regard to all animals, (pill) accumulated dose that gives by quick IV is in 400 μ l aqueous solution 1.4 * 10 ⁷Bq.

Data analysis: interesting areas utilizes CAPP software to follow the trail of, and radioactive concentration is with the expression of Bq/ml tissue.

The result:

After the potpourri with radioactive product is injected in the animal body positron emission transaxial tomography can search for fast can for example arrive in this potpourri brain or even the existence of the product of the heart; Based on this standard, the sorting potpourri is only to select to satisfy those of selected Tissue distribution standard fast.

This step, other step that makes up described method can detect the radiolabeled product with low-down detection threshold.

Claims (31)

1. method of screening molecular library is characterised in that it comprises following steps at least:

(1) give at least a animal with described molecular library, wherein each molecule is with a kind of suitable radioactive isotope mark in advance,

(2) put to death at least one animal, and the use sampling is organized or the radioactivity Tissue distribution of the molecule that the suitable imaging device analysis of section utilization of organ is given,

(3) selection detects the section of the tissue or the organ of radiated signal,

(4),, reach as extracting and/or chromatographic technique separates radioactive segment in tissue or the slices of organs as described in select step (3) by suitable technology

(5) by suitable analysis techniques, molecule as described in the radioactive segment of acquisition in step (4) is identified as chromatography and/or mass spectrophotometry use.

2. method of screening molecular library is characterised in that it comprises following steps at least:

(1) give at least a animal with described molecular library, wherein each molecule is with a kind of suitable radioactive isotope mark in advance,

(2) utilize suitable imaging device, from tissue or the cell of organ and section, extract in advance and the radioactivity Tissue distribution of the molecule that the biological sample analysis selected is given in external use,

(3) selection detects the cell or the section of the tissue or the organ of radiated signal,

(4) by suitable technology, as extract and/or the cell of chromatographic technique tissue or organ as described in step (3), select or section in separate radioactive segment, reach

(5) by suitable analysis techniques, molecule as described in the radioactive segment of acquisition in step (4) is identified as chromatography and/or mass spectrophotometry use.

3. a method of using molecular library to differentiate and study selective expression's target molecule in an organ or tissue comprises

(1) give at least a animal with described molecular library, wherein each molecule is with a kind of suitable radioactive isotope mark in advance,

(2) put to death at least one animal, and use suitable imaging device, by the radioactivity Tissue distribution that the molecule that is given is analyzed in a plurality of sample of tissue and the analysis section of animal,

(3) selection detects the section of radioactive tissue or organ,

(4) by suitable technology, as extracting and/or separate radioactive segment in the section of tissue or organ as described in chromatographic technique is selected from step (3),

(5) pass through suitable analysis techniques, molecule as described in identifying as the radioactive segment that obtains in chromatography and/or the mass spectrophotometry use step (4),

(6) with described molecule that obtains in the step (5) and the tissue of selecting according to step (2) and obtaining or organ samples making the molecule that separates in the step (4) and described target molecule combination and forming under a kind of condition of compound contact, reach

(7) from described compound, separate described target molecule.

4. method of using molecular library discriminated union research selective expression's target molecule (or acceptor) in an organ or tissue comprises:

(1) give at least a animal with described molecular library, wherein each molecule is by the mark in advance with this atom that exists in the described molecule of one of radioactive isotope of atom displacement,

(2) utilize suitable imaging device, from tissue or the cell of organ and section, extract in advance and the radioactivity Tissue distribution of the molecule that the biological sample analysis selected is given in external use,

(3) selection detects the cell or the section of radioactive tissue or organ,

(4) by suitable technology, as extract and/or as described in chromatographic technique is selected from step (3) tissue or organ cell or cut into slices in separate radioactive segment,

(5) pass through suitable analysis techniques, molecule as described in identifying as the radioactive segment that obtains in chromatography and/or the mass spectrophotometry use step (4),

(6) with described molecule that obtains in the step (5) and the tissue of selecting according to step (2) and obtaining or organ samples making the molecule that separates in the step (4) and described target molecule combination and forming under a kind of condition of compound contact, reach

(7) from described compound, separate described target molecule.

5. each method of claim 1-4 is characterised in that in step (1), and the library of Geigers is injected in the animal body preferred intraperitoneal injection (I.P.) or intravenous injection (I.V.).

6. each method of claim 1-5, be characterised in that and carrying out step (2) before, described method comprises that a step (1 ') is to analyze the radioactivity Tissue distribution of the molecule that is given, this step is by carrying out outside imaging with at least one animal, particularly utilize the particle that is suitable for detecting selected radioactive isotope emission character the pick-up unit combining camera and carry out.

7. each method of claim 1-6, be characterised in that described radioactive isotope be selected from tritium ( ³H), carbon 14 ( ¹⁴C), carbon 11 ( ¹¹C), iodine 131 ( ¹³¹I), iodine 125 ( ¹²⁵I), iodine 124 ( ¹²⁴I), iodine 123 ( ¹²³I), phosphorus 32 ( ³²P), fluorine 18 ( ¹⁸F), sulphur 35 ( ³⁵S), technetium 99 ( ^99mTc), indium 113 ( ¹¹³In), copper 64 ( ⁶⁴Cu), bromine 76 ( ⁷⁶Br), nitrogen 13 ( ¹³N) and oxygen 15 ( ¹⁵O).

8. each method of claim 1-7 is characterised in that in step (2), and radioactive Tissue distribution utilization is suitable for detecting the pick-up unit of character of the particle of selected radioactive isotope emission and observes in conjunction with imaging device.

9. each method of claim 1-8 is characterised in that the form injection of described Geigers with a kind of potpourri.

10. each method of claim 1-9 is characterised in that the molecular library of described mark prepares by the following method in step (1) before:

-by a kind of potpourri of chemistry or the synthetic described molecule of enzymatic route, reach

-with the described molecule of described labelled with radioisotope.

11. each method of claim 1-10, be characterised in that step (1) to (5) carries out simultaneously on several animals, described animal is given the amount difference of molecular library in step (1), and the increased radioactivity of analyzing molecules is carried out at different time according to animal in the step (2).

12. each method of claim 1-11 is characterised in that and carries out separating according to following steps of Geigers relevant with described organ in the step (4):

-cutting-out detects radioactive tissue or organ,

-described tissue of grinding or organ in suitable damping fluid,

The material of-centrifugal every kind of grinding also reclaims solution,

This solution of-filtration,

-regulate pH, reach

-fractionated also reclaims radioactive segment.

13. each method of claim 1-12 is characterised in that in step (5), mass spectrophotometry and tandem mass spectrum analysis (MS/MS) combination.

14. each method of claim 1-13 is characterised in that the molecule in the library of step (1) has following general formula: propyl group- ³H-NH-Yaa- _{(R, S)}Phe (PO ₂-CH ₂) _{(R, S)}Leu-Yaa '-NH ₂, wherein the position of 20 natural amino acids appears in Yaa and Yaa ' representative.

15. one kind is carried out each the kit of method of claim 1-14, is characterised in that it comprises:

-at least a molecular library, wherein each molecule is all used a kind of suitable labelled with radioisotope in advance,

The means of the radiolabeled product of-detection,

The means of the radiolabeled product that-analysis is detected.

16. the product that can obtain by claim 1 or 2 method is characterised in that it is corresponding to the peptide with following formula: propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂

17. the peptide of claim 16 is characterised in that it uses a kind of labelled with radioisotope.

18. one kind with a molecule (s) of interest target in the carrier in a specificity site, be characterised in that it comprises the molecule of selecting by each the method for screening molecular library of claim 1-14, and the Tissue distribution in described specificity site made in this way differentiate.

19. the targeting vector in the claim 18 is characterised in that described molecule is the described peptide of claim 16: propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂

20. locus specificity composition, be characterised in that it comprises: (i) treat by a kind of molecule (s) of interest of target in described site, itself and a kind of molecule coupling that is specific to described site, its Tissue distribution is differentiated by each the method for screening molecular library of claim 1-14, and (ii) at least a pharmaceutically-acceptable excipients.

21. the composition of claim 20 is characterised in that the described molecule that is specific to described site is the described peptide of claim 16: propyl group-NH-Arg-Phe (PO ₂-CH ₂) Leu-Ala-NH ₂

22. the molecule of the mark of selecting through each the method for screening molecular library of claim 1-14 is used for the application of the composition of medical imaging in preparation.

23. the application of claim 22, the molecule that is characterised in that described mark are the described peptides of claim 17.

24. the application of claim 22 or 23 is characterised in that the molecule of described mark combines with a kind of suitable contrast preparation.

25. a method of differentiating at least one target position of molecule (s) of interest is characterised in that it comprises at least:

(1) with molecule (s) of interest and a kind of radiolabeled molecule coupling that is specific to described target position of selecting by each the method for screening molecular library of claim 1-14, obtain a kind of radioactivity conjugate,

(2) give the conjugate that at least a animal obtains in step (1), and

(3) use biological sample combining at the molecule (s) of interest of at least one target position of the described biological sample of analyzed in vitro and described radiolabeled molecule coupling.

26. the method for claim 25, the molecule that is characterised in that described mark are the described peptides of claim 17.

27. a method of screening unlabelled molecule in vivo by competition, this method is characterised in that it comprises:

(1) molecule with a kind of mark gives at least a animal, and the Tissue distribution of the molecule of this mark differentiates by each the method for screening molecular library of claim 1-14,

(2) analyze the Tissue distribution of the Geigers that in step (1), is given the first time, this is by carrying out outside imaging with at least one described animal, particularly utilize the pick-up unit combining camera of the character of the particle be suitable for detecting selected radioactive isotope radiation to carry out

(3) give the library of unlabelled molecule,

(4) analyze for the second time the Tissue distribution of described Geigers, this is by carrying out outside imaging with at least one described animal, particularly utilizes the pick-up unit combining camera of the character that is suitable for detecting the particle that selected radioactive isotope radiates to carry out,

(5) determine to reach dynamics by the molecule of at least a unlabelled molecular replacement mark by comparing with the distribution of analyzing for the first time described Geigers

(6) detect at least a unlabelled molecule that the dynamics make radiolabeled molecule changes, described detection is by suitable analysis techniques, as chromatography and/or mass spectrophotometry repeat fractionated and identify as described in molecule and carrying out.

28. a method of screening unlabelled molecule in vivo by competition, this method is characterised in that it comprises:

(1) molecule with a kind of mark gives at least a animal, and the Tissue distribution of the molecule of described mark differentiates by each the method for screening molecular library of claim 1-14,

(2) utilize suitable imaging device, from tissue or the cell of organ and section, extract in advance and the biological sample selected and analyze the Tissue distribution of the Geigers that in step (1), is given for the first time in external use,

(3) give the library of unlabelled molecule,

(4) utilize suitable imaging device, from tissue or the cell of organ and section, extract in advance and the biological sample selected and analyze the Tissue distribution of Geigers for the second time in external use,

(5) determine to reach dynamics by the molecule of at least a unlabelled molecular replacement mark by comparing with the distribution of analyzing for the first time Geigers

(6) detect at least a unlabelled molecule that the dynamics make radiolabeled molecule changes, this detection is by suitable analysis techniques, repeat fractionated as chromatography and/or mass spectrophotometry and identify as described in molecule and carrying out.

29. the method for claim 27 or 28, the molecule that is characterised in that the mark in the step (1) are the described peptides of claim 17.

30. a medicine is characterised in that it comprises the described propyl group-NH-Arg-Phe (PO of claim 16 at least ₂-CH ₂) Leu-Ala-NH ₂Peptide and at least a pharmaceutically-acceptable excipients.

31. the described propyl group-NH-Arg-Phe (PO of claim 16 ₂-CH ₂) Leu-Ala-NH ₂The application of peptide in the preparation drug for hypertension.

Images