Contrast agent aimed at urokinase plasminogen activator receptor

FIELD: chemistry.

SUBSTANCE: invention relates to contrast agents which contain a peptide vector linked with uPAR, marked with a visualising group.

EFFECT: obtaining a contrast agent for detecting urokinase plasminogen activator receptor.

6 cl, 6 ex

 

The scope of the invention

The invention relates to contrast agents for the detection of receptor urokinase plasminogen activator (uPAR). More specifically, the invention relates to contrast agents containing peptide vector, bound to uPAR, labeled rendered by the group. Contrast agents can be used to identify sites where uPAR is expressed, for the diagnosis of diseases associated with this receptor.

Prior art

The interaction of plasminogen activator urokinase type (Ira) with cell surface is mediated exclusively to its glycolipid-"anchored receptor (uPAR), which Ira is bound with high affinity.

uPAR localized in the outer layer of cell membranes by glycosyl-phosphatidylinositol communication (GPI anchor). It is a rich in cysteine heavily glycosylated protein consisting of three homologous domains. Ira consists of a catalytic C-terminal domain serine protease and modular N-terminal segment containing the domain-like growth factor (GFD; AK 1-49), and "kringle"-domain (AK 50-135). The interaction between the Ira and uPAR is mainly mediated by residues 19-31 GFD uPA.

Since it is established that the proteolytic degradation of the extracellular matrix plays a role in tumor invasion and metastasis the processes, uPAR is a potential target for diagnostic contrast agents. It turns out that uPAR has elevated levels of regulation in vivo in most human carcinomas studied to date, and specifically in the tumor cells, associated with tumor endothelial cells undergoing angiogenesis, and in macrophages. Overexpression of uPAR in patients with cancer is present when running the disease and correlates with poor prognosis in various human carcinoma. The fact that the expression of uPAR has elevated levels of regulation only under pathological conditions, are involved in remodeling the extracellular matrix and cell motility, such as cancer, makes it an attractive marker for the diagnosis.

In WO 01/25410 described labeled for the diagnosis or therapy of proteins and peptides aimed at uPAR. This peptide or protein contains at least 38 amino acid residues, including residues 13-30 uPAR-binding site of the Ira.

In the US 6277818 described cyclic peptide compounds aimed at uPAR, which can be konjugierte diagnostic label. These peptides based on the amino acid residues 20-30 uPA.

US 6514710 also directed cyclic peptides having affinity against uPAR. These peptides may carry a detectable label. The peptide contains the 11 amino acids, United connecting unit.

In Biochemistry (Ploug et al., 2001, 40, 12457-12168) described peptides aimed at uPAR, but not in the context of visualization, including amino acid sequences, which are described in this document.

For effective targeting and visualization of uPAR is required selective vector with high affinity, which is chemically robust and stable. These conditions correspond to the contrast agents according to the invention.

Summary of the invention

In light of the needs of the specific technical field the present invention proposed contrast agents for the detection of receptor urokinase plasminogen activator (uPAR). More specifically, the invention relates to a contrast agent containing the peptide sequence, binding to uPAR with high affinity labeled rendered by the group. Ploug disclosed, for example, peptide aimed at uPAR, with a sequence of X-Phe-X-X-Tyr-Leu-Trp-Ser, where used standard abbreviations for amino acids and where X is an amino acid selected from the group of 25 amino acids.

Detailed description of the invention

In the light of the first aspect of the invention proposed a contrast agent targeted to uPAR, formula Ia,

where

X0represents from 1 to 5 amino acids,

X1, X2 , X3, X4and X5independently represent one amino acid,

Phe represents phenylalanine,

Leu represents leucine,

Trp is a tryptophan,

X6represents from 0 to 5 amino acids or defined by the formula (Ib),

where the symbols are as defined for formula Ia,

and where

β-Ala represents β-alanine,

Lys represents lysine, and where

W1and W2are the same or different groups, and individually represent a spacer, biomodification or missing, and at least there is one Z1or Z2representing the rendered group capable of detection either directly or indirectly in the process of diagnostic imaging.

All amino acids X0X1, X2, X3, X4, X5and X6represent natural or unnatural amino acids, and preferably are natural, except for X1, which preferably is unnatural. All amino acids are either D-or L-form, where preference is given below.

X0-X1-Phe-X2-X3-X4-Leu-Trp-X5-X6agent component of the formula Ia has the affinity for uPAR and below the hereafter is Chen as the peptide vector. The peptide vector of contrast agent has homology domain of growth factor Ira, but differs from vectors aimed at uPAR, the prior art because it does not contain amino acid sequence identical to the amino acid sequence Ira man.

X0represents from 1 to 5 D - or L-amino acids. Preferably X0includes amino acids selected from the group of alanine (Ala), threonine (Thr), glycine (Gly), aspartic acid (Asp) and glutamic acid (Glu). Amino acids are preferably in the L-form, except for threonine, which preferably is in the D-form. More preferably X0is an L-Asp, D-Thr or Gly-Gly-Asp.

X1preferably represents a β-cycloalkylation, more preferably represents a β-cyclopentylamine, β-cyclohexylamine (Cha) or β-cycloheptylamine, and most preferably represents a β-cyclohexylamin.

X2preferably represents a serine (Ser) or alanine (Ala), more preferably serine, and most preferably D-serine.

X3preferably represents an arginine (Arg) or an arginine mimetic, such as N-methylarginine (mArg), tyrosine (Tight) or alanine, preferably D-arginine.

X4preferably represents a tyrosine, and the anin, leucine or cyclohexylamine, more preferably tyrosine, and most preferably L-tyrosine.

X5preferably represents a serine, histidine (His), alanine, tyrosine, or leucine, preferably L-serine or D-histidine.

X6preferably represents from 0 to 5 D - or L-amino acids.

Preferably X6includes amino acids selected from the group of glycine, aspartic acid, lysine, phenylalanine or β-alanine (β-Ala). Alternatively, X6contains a group of the formula (Ib)

where all symbols are as previously defined, and where β-Ala formula Ib is associated with X5formula Ia. Peptide chain in brackets connected by its X5with

the ε-amino group of lysine. In this embodiment, the contrast agent will contain dimer synthesized on the frame of the modified lysine, in which his

the α-amino group pre derivatization β-alanine with education, thus, pseudosymmetries dimer in relation to the α-carbon atom of lysine. In this embodiment, the dimer preferably is a glycosilated, where the peptide sequence in the two monomers are the same. Most preferably X6is missing.

Unexpectedly, it was found that tyrosine and serine, which Ploug uses in positions X 4and X5peptide vector, respectively, can be replaced by other amino acids, as set forth above.

W1and W2individually represent a group acting as a spacer, biomodification group or both of them or no, and preferably based on monodisperse polietilenglikoli (PEG) structural element containing from 1 to 10 units of said structural element. W1or W2also may constitute from 1 to 10 amino acid residues, preferably including glycine, lysine, aspartic acid, serine or aminohexanoic acid. More preferably W1or W2contains amino acid residues and the structure of PEG-based, for example 1-10 amino acid residues, in combination with the structure of PEG-based. Preferably either W1or W2is biomodification, and in the preferred embodiment at least one of W1or W2is a unit consisting of monodisperse structure of PEG-based, 17-amino-5-oxo-6-Aza-3,9,12,15-tetracosapentaenoic acid of formula (II)

,

where m is an integer from 1 to 10, and where the end is an amide or acid group. As biomodification W1or W 2has a function of modifying the pharmacokinetics and renal clearance of compounds. Biomodification provides less grip compounds in the tissue, i.e. muscle, liver and so on, giving thus the opportunity for the best diagnostic imaging due to less background interference. Secretion is carried out mainly by the kidneys, which is an additional advantage of biomodification.

As beatifically group W1or W2preferably is derived from glutaric and/or succinic acid and/or units based on PEG, for example, including a grouping of the formula II. In addition, W1or W2can act as a spacer properties biomodification linking rendered grouping Z1or Z2with a peptide vector. Other representative spacer elements elements include polysaccharides structural polysaccharides storing type, polyaminoamide and their methyl and ethyl esters, as well as polypeptides, oligosaccharides and oligonucleotides, which may contain or may not contain the sites of enzymatic cleavage. One of the roles of W1or W2as spacer elements grouping is distancing relative to the volume rendered groups from domain peptide, etc) is RA, binding the receptor. Alternatively, in the simplest form W1or W2represents a functional relationship or contains a functional group that enables easy conjugation rendered grouping with a peptide vector, and such groups include-NRa-, CO2, -N(C=S)-, -N(CO)-, -S -,- O-, -O-NH2and -- Cho, where Raindependently represents H, C1-10alkyl, C3-10alkylaryl,2-10alkoxyalkyl, C1-10hydroxyalkyl,1-10halogenoalkane and α-halogenoacetyl.

When Z1is absent, and W1present, W1preferably has a N-end with a free amino group or the amino group-containing group, inhibitory metabolism. The term group, inhibiting metabolism, mean biocompatible group which inhibits or suppresses the metabolism of the peptide or amino acids at the amino-end in vivo. Such groups are well known to experts in the art and suitably chosen from the group of acetyl, BOC (tert-butyloxycarbonyl), Fmoc (fluorenylmethoxycarbonyl), benzyloxycarbonyl, TRIFLUOROACETYL and allyloxycarbonyl. Preferred groups, any abscopal metabolism, are acetyl and benzyloxycarbonyl. When Z2and W2no, With the end of the peptide vector predpochtitel is but represents an amide or carboxyl group, and preferably represents an amide group. Also, when Z2is absent, and W2containing amino acids, is present, then W2preferably ends amide or carboxyl group; more preferably an amide group.

The contrast agent according to the invention preferably contains a linear peptide that means no bridges between any of the amino acids or between amino acids and other agent groups that form a cyclic structure. Therefore, preferably no sulfide, thioester bridges or other bridges, forming a cyclic peptide structure. Contrast agent preferably contains a maximum of 20 amino acids.

In the preferred embodiment the peptide vector is represented by X0-X1-Phe-X2-X3-X4-Leu-Trp-X5-X6formula Ia, contains the sequence: X0-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser, so that the contrast agent has the formula Z1-W1-X0-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-X6-W2-Z2where the symbols are as defined earlier.

More preferably the peptide vector contains the sequence:

Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser or

Thr-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser,

where italics are marked D-amino acids, where X6is missing.

One example of a contrast agent according to the invention is:

Z -W1-Gly-Gly-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-NH2,

where Z1and W1are as previously defined, and where X6, W2and Z2no, and where the end is an amide group.

At least one of Z1and Z2is present and represents rendered grouping. Z below is used to denote either Z1or Z2. Z can be any rendered grouping. The nature of Z will depend on the imaging method used in the diagnosis. A wide range of groups suitable for detection by in vivo imaging, known for example from WO 98/47541, the contents of which are incorporated herein by reference.

Z represents a group capable of detection either directly or indirectly in the process of diagnostic imaging in vivo, such as radio or mect - (single photon emission computed tomography), PET (positron emission tomography), Mr (magnetic resonance), x-ray, ultrasound or optical imaging. Z includes, for example, the group that emits detectable radiation, or which can cause the emission of detectable radiation (e.g., by radioactive decay, fluorescence excitation, excitation of spin resonance and so on), the grouping of which affect local electromagnetic fields (for example, paramagnetic, overpromising, ferrimagnetic or ferromagnetic type), a group that absorbs, emits or scatters the radiation energy (e.g., chromophores, particles (including bubbles containing the gas or liquid), heavy element and its compounds, and so forth), and a group that forms a detectable substance (e.g., generators, micro bubbles of gas).

Rendered the group Z can be represented by an object M and a group Y1where M represents metal ions, paramagnetic metals, radionuclides, metals, heavy metals and heavy metal oxides. Grouping Y1must be able to carry one or more groupings Meters carrying understand any form of Association between the grouping Y1and M, such as chemical bonding, such as covalent bond or electrovalent or ionic bonds, or the Association, through absorption or any other type of Association, and preferably M chelated chelating group, Y1.

Visualization techniques and visualized groups Z are described in more detail below.

In the first embodiment of this aspect of Z contains one or more than one group M, useful in the method of radio - or mect-visualization, such as an ion of a radioactive metal or gamma-emitting radio is active halogen, which may be borne by the grouping Y1. Preferably M is a gamma emitter with a low or absent alpha and beta emission with a half-life of more than one hour. Preferred groups M are the radionuclides67Ga111In123I125I131I81mKr99Mo,99mTc201TI and133Xe. Most preferred is99mVehicle.

In addition, M can be represented by the following isotopes or isotope pairs:47Sc21;141Ce58;188Re75;177Lu71;199Au79;47Sc21;131I53;67C29;131I53and123I53,188Re75and99mCu43,90Y39and87Y39;47Sc21and44Sc21,90Y39and123I53,146Sm62and153Sm62; and90Y39and111In49.

When M represents a radionuclide metal to radio or mect-visualization, then Y1preferably denotes a chelating agent suitable for the formation of a stable chelate with M Such chelating agents are well known in the prior art, and typical examples of such chelating agents are described in Table I of WO 01/77145.

In particular, preferred are chelating agents Y1formula (III)

img src="https://img.russianpatents.com/1056/10567952-s.jpg" height="55" width="65" />

where:

each R1, R2, R3and R4independently represents N or C1-10alkyl, C3-10alkylaryl,2-10alkoxyalkyl, C1-10hydroxyalkyl,1-10alkylamino, C1-10foralkyl, or 2 or more than 2 groups R together with the atoms to which they are attached, form a carbocyclic, heterocyclic, saturated or unsaturated ring.

More specifically, preferred are chelating agents Y1formula (III), where R1, R2and R3represent hydrogen or a methyl group, and R4represents an alkyl or alkylamino group. More preferably Y1is a chelate of the formula (IV), denoted here cPN216, and most preferably visualizing group M represents a99mThe TC. The sign " * " denotes a possible binding site.

Other preferred chelating agents have the formula (V)

,

where R1-R6 independently represent H, alkyl, aryl, or a combination, where the groups R1-R6 contain one or more than one functional group, such that the chelate can be konjugierte with W1or W2formula (Ia). Suitable functional groups are, for example, alkylamine, alkylsulfate, al is oxyalkylated, arylamine, allsolid or alpha halogenoacetyl.

In cases where Z is a gamma-emitting radioactive halogen, such as123I125I131I and77Br, where125I is preferred, it can be covalently linked W c1or W2through substitution reactions or accession, are well known in the prior art or, alternatively, with X0or X6if W1or W2no, either directly with X5if X6is missing.

In the second embodiment of the compound of formula (Ia) contains group Z, useful in the method of PET imaging. In this case, Z contains radioactive emitter with the properties of the emission of positrons, preferably a positron-emitting radioactive non-metal. Preferred groups Z include any of the positron emitters11C,18F,13N15O,77F,75Br,76Br and124I. Specifically,18F is preferred. Suitable metallic positron emitters are64Cu48V52Fe55Co.,94mTc68Ga or82Rb, and68Ga is preferred, which can be chelated chelating agent Y1.

Non-metallic radionuclides, such as containing18F, can be covalently associated with the group what irokoi W 1or W2when it is present, or, alternatively, with X0, X5or X6through substitution reactions or accession, well known from the prior art.

When Z represents a18F-labeled aldehyde, thiol or aminooxy-group, then W1or W2preferably has a functional group containing an alpha halogenoacetyl group, aldehyde or aminooxy group. Chemistry the combination of thiol and aldehyde chemistry or aminooxy-combination,18F-synthons and labeled peptides obtained using the chemistry described in WO 03/080544 and WO 04/080492, the contents of which are incorporated herein by reference. Alternative,18F you can enter through other groups, containing18F, such as, for example,18F-forbindelse. Then the binding site W1, W2X0X5or X6preferably should contain aminooxy group.

When M denotes a metallic positron emitter for PET imaging, then Y1denotes a chelating agent suitable for the formation of a stable chelate with M Such chelating agents are well known in the prior art, and typical examples of such chelating agents are described in Table I of WO 01/77145 in the previous embodiment relating to radio and mect-rendering.

In the preferred embodiment of Y 1is a chelating agent DOTA and M represents a68Ga, which can easily enter in the chelate using the methods of microwave chemistry.

In the third embodiment Z contains the grouping Y1bearing one or more than one group M, is useful for MRI visualization method. M here denotes a paramagnetic metal; suitable ions of such metals include: Gd(III), Mn(II), Cu(II), Cr(III), Fe(III), Co(II), Er(II), Ni(II), Eu(III) or Dy(III). In particular, preferred are Gd(III), Dy(III), Fe(III) and Mn(II). Y1denotes a chelating agent, in particular such a chelating agent, as well as acyclic or cyclic polyaminocarboxylic (e.g., DTPA, DTPA-BMA, DOTA and DO3A), as described, for example, in US 4647447 and WO 86/02841. Also, M may denote metal oxides, such as oxides of metals super-paramagnetic, ferrimagnetic or ferromagnetic type adsorbed Y1or contact Y1for example, so that Y1functioned as coatings for metal oxide. Metal oxides for use as Mr contrast agents are described, for example, in U.S. patent 6230777, which is incorporated herein by reference.

In the fourth embodiment Z represents rendered grouping that are useful for x-ray visualization method. Z here contains a heavy metal, such as tungsten, evil is and bismuth, preferably in the form of oxides. Also Z can be represented ladirovannye aryl derivatives, in particular, is well known as x-ray contrast agents, for example, Lopamiron™ and Omnipaque™. These agents may be linked by means of acid or amine functional groups of the peptide vector formula (Ia), possibly through W1or W2.

In the following embodiment of the compound of formula (Ia) contains Z in the form of micro bubbles filled with gas. Such agents ultrasonic imaging can be used when rendering receptors, when they, for example, functionalized to bind to the peptide, as described in the prior art, for example, in WO 98/18500.

In the sixth and preferred embodiment of the present invention, the group Z of formula (Ia) can be any group capable of detection either directly or indirectly in the process of optical imaging. Detectable group can be svetorasseivateley (for example, painted or unpainted particle), setpagetitle or light emitter. More preferably Z is represented by a dye, such as a chromophore or a fluorescent compound. The group Z may be any dye that interacts with light in the electromagnetic spectrum with wavelengths from ultravio ecologo light to near infrared light. In a preferred embodiment, Z has fluorescent properties.

A preferred group of organic dyes include groups having an extensive system of delocalized electrons, such as cyanine, merocianine dyes, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethane, porphyrins, prelievi dyes, thiapyrilium dyes, squarewave dyes, croconaw dyes, asplenietea dyes, ingoonline, benzobisoxazole dyes, benzodioxathiepin dyes, anthraquinones, naftochinona, endocrine, phthaloylation, triperoxonane, azo dyes, dyes with intramolecular and intermolecular charge-transfer complexes and dyes, trophy, tetrazine, bis(dithiolene)new complexes, bis(benzene-dithiolate)ing complexes, stanilova dyes and bis(S O-dithiolan)type complexes. Of fluorescent dyes, the group of cyanine dyes is preferred. Even more preferred are groups carbocyanines, oxazinone, dialanine and aslanidou. Group Su and Su-dyes are most preferred fluorescent dyes, and they can be associated with a peptide vector by N-hydroxysuccinimide of ester (NHS-ether complex). Fluorescent proteins such as green fluorescent protein (GFP) and modification of GFP, is the quiet have different absorption/emission properties, are also useful. In certain cases, apply some complexes of rare earth metals (e.g., europium, samarium, terbium or dysprosium), and fluorescent nanocrystals (quantum dots).

Preferred dyes selected from the group of carbocyanines; even more preferred are carbocyanine dyes indole type. Preferred dyes of this type are illustrated by formula VI:

where the group Q1are the same or different and represent substituted or unsubstituted lower alkyl group, for example, C1-C6alkyl groups, which are possibly substituted. Alkyl group substituted, for example, carboxypropyl, groups, sulfonic acid, amino, ammonium or ester groups such as heterocyclic ester groups (for example, NHS-ester). Group Q2are the same or different and represent lower alkyl groups, such as C1-C6alkali, preferably a methyl group, possibly substituted, for example, carboxypropyl or sulfonic acid groups. Possible aromatic group is indicated by the dashed lines, to include structures containing condensed Pensacola and condensed nafeco is ICA. Any of the rings are substituted or unsubstituted. These rings can be substituted by a group V selected from sulfonic acid groups, carboxyl groups, hydroxyl groups, alkyl(sulfoalkyl)amino group, bis(sulfoalkyl)-amino group, sulfoalkyl, sulfoalkylation groups, alkyl or substituted alkyl, or sulfoalkylation. p represents a positive integer 1, 2, 3 or 4. Preferably cyanine dye is intimately or heptamethine dye carbon bridges 5 and 7 carbon atoms, respectively.

Q1, Q2and V represent potential binding sites for binding of the dye with a peptide vector, possibly through W1and/or W2and group Q1and V are the preferred binding sites. In a preferred aspect of one group of Q1linked to a peptide vector, while another group of Q1represents a possibly substituted lower alkyl group.

In the light of the second aspect of the proposed invention the compounds of formula (VII), aimed at uPAR:

,

where the symbols are as defined for formula Ia, and where at least one of W1and W2is present and represents biomodification, as is isane. The compounds of formula VII may be associated with the rendered group, as described in the first aspect, or may have other applications, such as in therapy.

Compounds of the present invention can be synthesized using known methods of chemical synthesis, but is particularly useful solid-phase methodology on Merrifield using an automated peptide synthesizer (J. Am. Chem. Soc, 85: 2149 (1964)). Usually using solid-phase peptide synthesis collect the target sequence. Standard methods synthetic strategy used in the examples of this invention described E. Atherton & R.C. Sheppard in "Solid phase peptide synthesis: a practical approach', 1989, IRL Press, Oxford.

For example, use of a resin with a variety of colorability linker groups, which will give the peptides with C-terminal(th) acid, amine or Amida. Then remove the amino-protective group, and, using a suitable condensation reagent, combine the second amino acid in the sequence. Use of amino acids with polystability (semipermanent) amino-protective groups and stable protective groups for functional side chains. Then the cycles of removal of the amino-protective group, and combinations repeat in alternating stages up until not collect the sequence of interest.

Alternatively, the peptides can Interoute methods peptide synthesis in solution, well known in the art, or stepped manner from the carboxyl end and/or by applying methods of segment condensation or crosslinking using protection strategy in full or minimal protection. You can also apply a combined approach to condensation segments "solution - solid phase".

In General, the present reactive groups of the side chains (for example, amino, hydroxyl, guanidino and carboxyl groups) will be protected in the process of synthesis, as described above. Known for a wide range of protective groups for amino acids (see, for example, Greene, T.W. & Wuts, P.G.M. (1991) Protective groups in organic synthesis, John Wiley & Sons, New York). Amino-protective group, which can be used include 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Vos). Protective groups of the side chains, which can be used include tert-butyl (tBu), trityl (Trt), Vos and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc). It is obvious that in this field of technology is known for a wide range of other such groups.

Finally, a stable protective groups of the side chains are removed, and the peptide otscheplaut from the resin, usually simultaneously by treatment with a suitable acidic reagent, for example triperoxonane acid (TFA).

W1and/or W2you can konjugierte with a peptide vector, using known methods is imicheskogo synthesis. Especially useful is the direct conjugation of W1and/or W2with a peptide vector through the formation of amide linkages, both to obtain the peptide vector. Alternatively, you can use the reaction of nucleophilic substitution, where the leaving group on the peptide N-end is replaced by a nucleophilic group W1and/or W2. Such a leaving group may be bromide, attached in alpha-position to a carbonyl group, and therefore nucleophile may be a nitrogen atom.

Z can be konjugierte directly with the peptide, using the same methods as for the conjugation of W1and/or W2with a peptide vector. In the case when Z is attached to the peptide via a W1and/or W2any methods of chemical synthesis can be used in conjugation Z and W1or W2. Particularly useful is the automatic combination of Z with education, for example, amide bond between the peptide and rendered by the group. How to bind reporter group with a peptide are well known in the art, and will depend on the selected reporter and used spacers.

The peptide vector and the contrast agent can be cleaned using high-performance liquid chromatography (HPLC) and characterized using mass spectrometry and analytical HPLC.

Proc. of the requirements for the compounds according to the invention in functioning as effective contrast agents uPA/uPAR are that, on the one hand, the peptide vector should have a high affinity to the receptor, and on the other hand, the vector should "stay" on the receptor as long as it is necessary. Thus, the system of contrast agent/uPAR preferably should exhibit slow kinetics of dissociation (the so-called "dissociation rate" ("off-rate")), which is conveniently expressed as the rate constants of dissociation of Kdiss. Found that the contrast agents according to the invention have significantly improved the kinetics of binding of the receptor. On this basis, the contrast agents according to the invention which are particularly preferred are the rate constant of dissociation (Kdiss) relative to the domain of uPA growth factor (GFD) is preferably less than 50, more preferably less than 20, even more preferably less than 10, and most preferably less than 1.

Contrast agents according to the invention is preferably capable of inhibiting the binding of uPA to uPAR on the cell surface. The contrast agent according to the invention preferably is tantamount inaktivirovannye diisopropylfluorophosphate Ira. Parameter IC50gives the concentration where 50% of the usability replaced. Preferred contrast agents according to the invention have the IC50less than 50 nm, preferably less than 25 nm, more preferably less than 10 nm, and even more FAV is preferably less than 3 nm.

Contrast agents according to the invention is preferably used for identifying sites where uPAR is expressed, for the diagnosis of diseases associated with a high level of regulation of the receptor. Preferably the receptor is presented in the diseased tissue by more than 50% compared with the surrounding tissue. More preferably, the receptor is more than two times more represented in diseased tissue than in surrounding tissue. Even more preferably, the receptor was at least 5 times more represented in diseased tissue than in surrounding tissue.

In the light of the next aspect of the invention, a method for detecting the presence of uPAR on the cell surface, tissue, organ or biological sample suspected to overexpression of uPAR due to pathological conditions. This method involves the following stages:

a) bringing into contact the cell, tissue, organ or biological sample with a contrast agent according to the invention and

b) detecting the presence of the rendered group associated with the cell, tissue, organ or sample. In this way bringing into contact, and the detection can be performed in vitro; the alternative of bringing into contact is conducted in vivo, and the detection in vitro, preferably bringing into contact and the detection is conducted in vivo.

The preferred method includes the t image of the human or animal by means of diagnostic imaging, including introduction of contrast agent, as described in the specified organism, for example in the vascular system, and obtaining an image of at least part of the specified organism in which distributed the specified contrast agent.

In the light of one of the following aspect of the invention, a method for obtaining improved images of the human or animal by rendering, in which previously entered the composition of the contrast agent containing a contrast agent, as defined, which receive an image of at least part of the specified organism.

New contrast agents according to the invention can be used as contrast agents using any method of visualization depending on the selected rendered grouping. The use of contrast agents in diagnostic imaging, therefore, is an aspect of the invention. A preferred aspect of the amount of contrast agents, as described, for use in imaging and diagnosis of various forms of cancer and metastasis, such as cancer of the breast, skin, colon and rectum, pancreas, prostate, lung or ovarian cancer. Alternative contrast agent can be used for detection of diseases, which are activated macrophages, such as sensitive the plaques in atherosclerosis.

The present invention also proposed a pharmaceutical composition comprising an effective amount, such amount, effective for improved image contrast when imaging in vivo contrast agent according to the invention or salts thereof together with one or more pharmaceutically acceptable adjuvants, excipients or diluents.

In the light of the next aspect of the invention proposed the use of a contrast agent according to the invention for the manufacture of an agent, which improves the contrast for use in the diagnostic method, comprising the introduction of this agent, which improves the contrast, in the human or animal and obtaining an image of at least part of the specified organism.

The present invention hereinafter will be further illustrated with the help of non-limiting examples in which the following abbreviations:

BAEEG: bis(aminoethyl)glycol

BOC: tert-butyloxycarbonyl

Cy: cyanin

DEG: diethylene glycol

DMF: N,N-dimethylformamide

Fmoc: 9-fluorenylmethoxycarbonyl

HATU: hexaphosphate 2-(7-Aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

HPLC: high performance liquid chromatography

Krytofix 222: 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo(8,8,8)hexacosane

LC: liquid chromatography

MS: mass spectrometry

NHS: N-hydroxysuccinimide

NMM: N-methylmorpholine

PEG: polyethylene glycol

FB: phosphate buffer

Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl

Roar: hexaflurophosphate (7-asobancaria-1 yloxy)three-pyrrolidinone

Resin Rink Amide MBHA: 4-methylbenzhydrylamine associated with the polystyrene matrix

RP-HPLC: HPLC with reversed phase

CT: room temperature

Sep-Pak: the separation column with C18 resin

TFA: triperoxonane acid

TIS:triisopropylsilane

Example 1:

Cy5(bis-SO3)-Thr-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH (2).

Synthesis of H-Thr-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH (1).

The peptide corresponding to the above sequence, was synthesized by standard methods of solid phase peptide chemistry.

Algae Su(bis-SO3)-mono-NS ether complex with peptide (1).

Su(bis-SO3)-mono-NS ester (1,18 mg, 0,0017 mmol) was dissolved in DMF and to the solution was added to the peptide (1) (2 mg, 0,0015 mmol) as a solid substance, followed by addition of NMM (0,55 μl, of 0.005 mmol). The reaction vessel was wrapped in foil and placed in the instrument for shaking for 16 hours. The target product was confirmed by mass spectrometry (MS) with elektrorazpredelenie: [M+H]+product expected when 1850,85 m/z, found at 1850,9 m/z.

Example 2:

Cy5(bis-SO3)-Gly-Gly-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-NH2(3).

/p>

Synthesis Su(bis-SO3)-Gly-Gly-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-NH2(3).

Standard methods of solid-phase peptide chemistry collected amino acids in the above sequence. Then DMF was dissolved Cu(bis-SO3)-mono-NS ester (0.5 EQ.) and added to H-Gly-Gly-Asp(OtBu)-Cha-Phe-Ser(tBu)-Arg(Pmc)-Tyr(tBu)-Leu-Trp(Boc)-Ser(tBu)-Rink Amide MBHA resin (1 EQ.) with the subsequent addition of NMM (3 EQ.).

The reaction vessel was wrapped in foil and placed in the instrument for shaking for 16 hours. With peptide conjugated to dye So, filmed protection and by treatment with TFA containing 2.5% water and 2.5% TIS, within 1 hour tsalala it from the resin. The target product was confirmed by MS elektrorazpredelenie: [M+H]+product expected when 1977,88 m/z, found at 1977,8 m/z.

Example 3:

Cy5(bis-SO3)-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-βAla-Lys(Cy5(bis-SO3)-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser)-NH2(5).

Synthesis of H-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-βAla-Lys(H-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser)-NH2(4).

The peptide corresponding to the above sequence, collected by standard methods of solid phase peptide chemistry.

Algae Su(bis-SO3)-mono-NS ether complex with the peptide (4).

Su(bis-SO3)-mono-NS ester (2.2 EQ.) dissolved in DMF and to the solution was added the peptide (4) (1 EQ.) in the form of a solid substance, followed by addition of NMM (3 EQ.). Reacts the vessel was wrapped in foil and placed in a device for shaking for 24 hours to give the desired bis-conjugated product. Product analyze RP-HPLC and MS elektrorazpredelenie.

Example 4

N-(4-18F-formanilide)aminooxyacetic-PEG(4)-digicolor-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-NH2(8).

Synthesis of N-BOC-aminooxyacetic-PEG(4)-digicolor-sp-h-h-Sr-Arg-Tyr-Leu-Trp-Ser-NH2(7).

Vos-aminooxyacetic-PEG(4)-digitalia acid can be obtained by an expert in the field of machinery using active complex ester BOC-aminooxyacetic acid, which is subjected to interaction with the corresponding amino-PEG(4)-diglycolic acid. The resulting product Vos-aminooxyacetic-PEG(4)-diglycolic acid (1.4 EQ.) and Roar (1.2 EQ.) dissolved in DMF. Added NMM (2 EQ., 200 µl)and the mixture stirred for 5 minutes, Add a solution of the peptide (6) (1 EQ.) and NMM (4 equiv.) in DMF, and the reaction mixture is stirred for 30 minutes DMF is evaporated under vacuum and the product purified using preparative RP-HPLC. The fraction containing the target product was adjusted to pH 5 using a mixture of 0.3% ammonia (NH3)/water before lyophilization to prevent removal of the BOC-protective group. Product analyze RP-HPLC and MS elektrorazpredelenie.

Tagging peptide (7) 18F.

18F-Fluoride azeotrope dried in the presence of Kryptofix 222 (5 mg in 0.5 ml ACN) and potassium carbonate (50 ál of 0.1 M solution) by heating to 110°C in a stream of nitrogen over their 20 minutes During this time add and remove 3×0.5 ml ACN. The mixture is cooled to a temperature less than 40°C and add trimethylammonium benzaldehyde, triplet synthesized according to the procedure described Haka et al. in J. Labelled Cpds. & Radiopharms 1989 27(7) 823, (1 mg in 0.4 ml of dimethyl sulfoxide). The reaction vessel is sealed and heated to 90°C for 15 min to implement tagging. Then a solution of crude 4-18F-forventelige cooled to room temperature. Meanwhile peptide (7) (6 mg) is treated with 5% water in TFA (200 L) for 5 min at room temperature to remove the BOC-protective group. The solvents are removed in vacuo. The peptide with the remote protection Vos re-dissolved in 0.1 M solution of ammonium acetate (pH 4, 0.4 ml) and combined with a solution of crude 4-18F-forventelige in the reaction vessel. The vessel is sealed and heated to 70°C for 15 min for the implementation of the conjugation. After cooling to room temperature the crude conjugate is purified preparative HPLC. The fraction containing the target conjugate, diluted with 10 ml of water and applied on 18 Sep-Pak (pre-prepared by sequential washing with 10 ml of ethanol and 20 ml of water). Sep-Pak rinsed with 10 ml water, then elute with 2 ml of ethanol. The ethanol is removed in vacuo and prepare the drug product (8) in FB.

Example 5

Acetyl-PEG(4)-digicolor-sp-h-h-Sr-Arg-Tyr-Lu-Tr-Ser-Gly-BAEEG-Glut-cPN216 (10).

Synthesis aimil-PEG(4)-digicolor-s-h-h-Sr-Arg-Tight-Lu-Thr-Sr-Gly-BAEEG-NH2(9).

The peptidyl-resin corresponding to the above sequence, are synthesized by standard methods of solid phase peptide chemistry. Acetyl-PEG(4)-diglycolic acid (5 EQ.) and Roar (4.5 EQ.) dissolved in DMF. Added NMM (10 EQ., 200 µl)and the mixture stirred for 5 minutes and Then the mixture is added to the peptide resin, pre-swollen in DMF, and the reaction continued overnight. Then with peptide remove protection and using TFA containing 2.5% water and 2.5% TIS, within 1 hour otscheplaut it from the resin. Product analyze RP-HPLC and MS elektrorazpredelenie.

Algae CPN216 with peptide (9).

Peptide (9) (1 EQ.) dissolved in DMF and added cPN216-glutaryl-tetrafluorothiophenol ester (2 EQ.), and then NMM (3 EQ.). After stirring over night the reaction mixture is treated by removal of the solvent under reduced pressure, and the product (10) purified preparative RP-HPLC. Product analyze RP-HPLC and MS elektrorazpredelenie.

Staining of peptide (10)99mVehicle.

Peptide (10) (0.1 mg) restore in saline solution or in methanol (0.1 ml) and transferred into a freeze-dried set of excipients Toolbox. Set Toolbox designed to provide the General conditions for the active labeling for chelates based on amine, contained dehydrat chloride divalent tin (16 μg), methylenediphosphonic acid (25 mg), sodium bicarbonate (4500 g), sodium carbonate (600 g), para-aminobenzoic sodium (200 mg), pH set = 9,2. Then by injection add pertechnetate sodium (99mTc) (2,1 GBq) in saline solution (3 ml), the set is turned several times to dissolve the content, and then leave incubated at room temperature for 15-20 minutes Sample immediately analyzed by HPLC and ITLC (instant thin-layer chromatography, rapid thin-layer chromatography), and labeled 99 MTS peptide (11) it is necessary to introduce the subject to the subject between 1-3 hours after recovery set.

Example 6

The complex of gadolinium(III) acetyl-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-Gly-BAEG-Glut-DOTA (15).

Synthesis of Acetyl-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-Glv-DEG-NH2(12).

The peptide corresponding to the above sequence, are synthesized by standard methods of solid phase peptide chemistry.

Synthesis of Three-tBu-DOTA (13).

Three-tBu-DO3A (tri-tert-butyl ester 1,4,7,10-tetraazacyclododecane-N,N',N"-trioxanes acid, 10 mmol) and bromoxynil acid (10 mmol) was dissolved in Meon (50 ml). Added K2CO3(30 mmol)dissolved in water (50 ml) (pH 11 in a mixture Meon/water)and the reaction mixture was stirred for 24 hours and then was heated at 40 g is adosh in the next 24 hours. The crude product was purified RP-HPLC (Phenomenex Luna 5 micron, C18, 250×21,2 mm, gradient, 5-50% b for 40 min at 10 ml/min). The target product was confirmed by MS elektrorazpredelenie ([M+Na]+product expected when 595,4 m/z found [M+Na]+595,3 m/z), and1H-NMR at 80 degrees.

Synthesis D-conjugated peptide (14).

Three-tBu-DOTA (13) (tri-tert-butyl ester 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraoxane acid, 1 EQ.) activated by HATU (1 EQ.) in DMF in the presence and NMM (3 EQ.) within 5 minutes the Mixture was added to the peptide (12), and the reaction continued overnight. The solvent was removed in vacuo, and the product was subjected to removal of the tBu-protection in TFA containing 5% water. The product was purified RP-HPLC and analyzed by RP-HPLC and MS elektrorazpredelenie.

The formation of the complex with Gd(III).

DOTA-conjugated peptide (14) (1 EQ.) were subjected to interaction with GdCl3(1 EQ.) in aqueous solution at pH 6.5 and KG. Gd(III), unreacted education complex was removed by centrifugation of the solution at alkaline pH. The product (15) was isolated by lyophilization.

1. The agent visualization of receptor urokinase plasminogen activator (uPAR) formulas And

where Cha is cyclohexylamine,
Phe represents phenylalanine,
Ser is a serine,
Arg represents an arginine,
Tyr is a t the Rozin,
Leu represents leucine,
Trp is a tryptophan,
X6represents from 0 to 5 amino acids selected from the group of glycine, aspartic acid, lysine, phenylalanine or β-alanine, or defined by the formula (In)

where the peptide chain, indicated in parentheses, are connected with the ε-amino group of lysine through a serine residue (Ser),
β-Ala represents β-alanine,
Lys represents lysine,
terminal Ser formulas associated with W2Formula A, and where
each X0represents from 1 to 5 amino acids selected from the group of alanine, threonine, glycine, aspartic acid and glutamic acid,
W1and W2each represent the same or different group or absent, where at least one of W1and W2contains from 1 to 10 units of monodisperse polietilenglikoli (PEG) building block, or from 1 to 10 amino acid residues, and at least there is one Z1or Z2and represents the rendered group capable of detection either directly or indirectly in the process of diagnostic imaging, for use in the diagnosis in vivo, where this visualization includes radio, mect- (single photon emission computed tomography), PET(positron emission is ortografia), Mr (magnetic resonance) or optical imaging.

2. Agent imaging according to claim 1, where one or both of Z1and Z2contain the grouping of M representing a gamma-emitting group for radio or mect-visualization representing99mTc.

3. Agent imaging according to claim 1, where one or both of Z1and Z2contain the grouping of M representing a radioactive emitter from the emission of positrons for PET imaging, representing18F.

4. Agent imaging according to claim 1, where one or both of Z1and Z2contain the grouping of M representing a paramagnetic metal selected from the group of Gd(III), Mn(II), Cu(II), Cr(III), Fe(III), Co(II), Er(II), Ni(II), Eu(III) or Dy(III), or a metal oxide, such as a metal oxide super-paramagnetic, ferrimagnetic or ferromagnetic type, for Mr imaging.

5. Agent imaging according to claim 1, where at least any one of Z1and Z2is a cyanine dye for optical imaging.

6. Pharmaceutical composition for binding to uPAR, containing the agent imaging according to any one of claims 1 to 5 in an effective amount together with one or more pharmaceutically acceptable adjuvants, excipients or diluents.



 

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Peptide compounds // 2393167

FIELD: chemistry.

SUBSTANCE: invention relates to novel peptide compounds and their use in diagnostic optical visualisation techniques. More specifically, the present invention pertains to use of such peptide compounds as targeted vectors which are related to receptors associated with angiogenesis. The compounds are labelled using at least one cyanine reporter dye.

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9 cl, 5 ex

FIELD: chemistry; biochemistry.

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4 dwg, 2 ex

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7 cl, 3 ex, 2 dwg

FIELD: chemistry.

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31 cl, 2 tbl, 82 ex

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20 cl, 8 dwg, 4 tbl, 4 ex

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2 cl, 12 dwg, 2 tbl, 30 ex

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4 dwg, 2 ex

FIELD: chemistry.

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5 dwg, 2 ex

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25 cl, 15 ex

FIELD: medicine.

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21 cl, 10 ex, 7 tbl, 5 dwg

FIELD: medicine, pharmaceutics.

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11 cl, 13 ex, 4 tbl, 20 dwg

FIELD: medicine, pharmaceutics.

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11 cl, 13 ex, 4 tbl, 20 dwg

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19 cl, 2 tbl, 6 ex

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60 cl, 42 dwg, 100 ex

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FIELD: chemistry.

SUBSTANCE: invention relates to novel peptide compounds and their use in diagnostic optical visualisation techniques. More specifically, the present invention pertains to use of such peptide compounds as targeted vectors which are related to receptors associated with angiogenesis. The compounds are labelled using at least one cyanine reporter dye.

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9 cl, 5 ex

FIELD: medicine.

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1 ex

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5 tbl, 3 ex

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3 dwg, 5 ex

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10 cl, 31 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention refers to the conjugates of formula (V)

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EFFECT: use as radiopharmaceuticals.

25 cl, 15 ex

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