Optical visualisation agents

FIELD: chemistry.

SUBSTANCE: visualisation agent contains a conjugate of formula (I) of benzopyrylium dye through a linker group with a 3-100-dimensional synthetic peptide which provides directed delivery to the biological target. Also disclosed is a pharmaceutical composition which contains said conjugate of formula (I), a set for preparing said pharmaceutical composition and methods for visualisation of a mammal body in vivo.

EFFECT: invention provides efficient visualisation of a mammal body in vivo while monitoring diseased state thereof.

24 cl, 5 tbl, 6 ex

The scope of the invention

The present invention relates to imaging agents suitable for optical imaging in vivo, which include conjugates benzopirilievyh dyes groups, providing targeted delivery to a biological target, such as peptides. Also disclosed are pharmaceutical compositions and kits and methods of visualization in vivo.

Prior art

In the US 6750346 disclosed compatible with laser marker dyes for near-infrared spectrum (NIR) of formula a, b or C:

,

,

,

where:

n is 1, 2 or 3;

R¹-R¹⁴are the same or different and selected from H, Cl, Br; aliphatic or mononuclear aromatic group of up to 12 carbon atoms, which may contain as the substituted group in addition to C and H to 4 oxygen atoms and 0, 1 or 2 nitrogen atom, or sulfur atom, or a sulfur atom and nitrogen; or can be amidofunctional group having a nitrogen atom with which it is bound, H or at least one Deputy, with up to 8 carbon atoms, with the specified Deputy selected from the group consisting of C, H and up to two sulfoxylate groups.

The dyes described in US 6750346 selected so that the pre is respectfully at least one of R ¹-R¹⁴contains solubilizers or an ionisable group. Believe that such groups include cyclodextrin, sugar,$$S{O}_3^{-},$$$$P{O}_3^{-2},$$$$C{O}_2^{-}$$or$$N{R}_3^{+}.$$In the US 6750346 indicated that the dyes, and also obtained from these systems (conjugates) can be used in optical, especially in fluorescent optical, methods of qualitative and quantitative definitions for the diagnostic properties of cells, biosensors (measurements in place to monitor the patient), in the study of the genome and in miniaturizing technology. Typically, these applications lie in the areas cytometry, sorting of cells, fluorescence correlation spectroscopy (FCS), technology ultravioletradiation screening (UHTS), multicolor fluorescence in situ hybridization (FISH) and micromission (gene and protein chips).

In the US 6924372 disclosed asymmetric polymethine dyes of formula D or E:

,

,

where:

n is 0, 1, 2 or 3;

R¹-R⁹are the same or different and may be H, the remains of alkyl, tert-alkyl, aryl-, carboxyethyl, dicarboxyethyl, heteroaryl, cycloalkyl, heteroseksualci, alkyloxy-, allylmercaptan- (where "alkyl" and "cycloalkyl also include the remains with olefinic bond), aryloxy-, allmerica, heteroaromatic-, heteroaromatic-, hydroxy-, nitro - or cyano and R¹and R², R²and R³, R³and R⁴, R⁵and R⁷can form one or more aliphatic, heteroaromatics or aromatic ring.

At least one of the substituents R¹-R⁹described in US 6924372, may possibly be solubilizers or ionisation Deputy (for example,$$S{O}_3^{-}$$,$$P{O}_3^{-2}$$, CO₂H, HE,$$N{R}_3^{+}$$the cyclodextrin or sugar) or may be a reactive group (for example, isothiocyanate, hydrazine, active ester, maleimide or iodine is Itemid), allowing to form a covalent bond between the dye and the other molecule. Believe that the dyes of formulas D and E is useful in the diagnosis characteristics of the cells or in biosensors, usually in cytometry and sorting of cells.

Lisy et al. (J. Biomed. Optics, 11(6), 064014 (2006)) disclosed a method for the diagnosis of peritonitis using optical imaging in the near infrared region of the spectrum and labeled monocytes or macrophages. Monocytes-macrophages may be too efficient in vitro dye DY-676 (Dyomics GmbH). The introduction of the dye DY-676 in an animal model of peritonitis in vivo resulted in increased fluorescence in the field of peritonitis. The authors concluded that the staining of monocytes-macrophages occurred in vivo.

In Lisy et al. (Invest. Radiol., 42(4), 235-241 (2007)) revealed a bimodal contrast agents for magnetic resonance imaging (MRI) and optical imaging, which include nanoparticles labeled with fluorescent magnetosomes.. Fluorescent magnetosome nanoparticles used for labeling of macrophages by phagocytosis. The dye used for labeling magnetosome was a DY-676.

Website Dyomics GmbH (www.dyomics.com contains a free copy of I.Hilger (FSU Jena), entitled "Visualisation of Arthritis in a Rat by Accumulation of DY-676 in Joints". No further details were not given.

In WO 2007/139815 disclosed are methods of imaging and therapeutic with the person, are involved in precursor cells. Disclosed are conjugates of the following formula:

A_B-X,

where:

A_Bincludes vitamin or analogue that binds to CD133⁺Flk1⁺endothelial cells predecessors;

X represents a quantifiable marker.

Quantifiable marker may represent, for example, radioactive probe or fluorescent probe. Argue that appropriate fluorescent probes are: fluorescein, rhodamine, Texas red (Texas Red), phycoerythrin, Oregon green (Oregon Green), Alexa Fluor 488..., SS3, So, Su and the like. In example 30 WO 2007/139815 disclosed only benzopyrylium dye (DyLight™ 680), conjugated to folate via the 5-dimensional peptide linker (Asp-Arg-Asp-Asp-Cys).

The present invention

According to the present invention proposed imaging agents suitable for optical imaging in vivo, which include a specific class benzopyrylium dye conjugated to the group, providing targeted delivery to a biological target (TMV). As part of such covalently-linked TMV-conjugates of the present invention identified from sulphonated benzopyrylium dyes suitable for applications in optical imaging in vivo.

Benzoperylene kr is the bearers (Bzp ^M) according to the present invention possess a combination of properties that make them useful for applications in optical imaging in vivo:

(1) the ability to conjugation to molecules, providing targeted delivery to a biological target (TMV);

(2) water solubility;

(3) absorption and emission in the red, far red or near infrared region of the electromagnetic spectrum;

(4) high extinction coefficients;

(5) low binding to plasma proteins;

(6) high photostability and brightness;

(7) the high stability of the dye and the dye conjugate-TMV in the blood;

(8) rapid elimination from the blood in vivo;

(9) the absence of potentially harmful metabolites (according Meteor/Derek analyses).

Detailed description of the invention

In the first aspect of the present invention proposed a pharmaceutical composition comprising a visualizing agent, suitable for optical imaging in vivo the body of a mammal, together with a biocompatible carrier, and the specified composition is in a form suitable for administration to a mammal, where the specified imaging agent comprises a conjugate of the formula I:

,

where:

TMV is a grouping, providing targeted delivery to a biological target;

n depict is to place an integer value of 0 or 1;

L represents a synthetic linker group of formula(A)_m-where m is an integer value from 1 to 20, and each And independently represents-CR₂-, -CR=CR-, -C≡C-, -CR₂CO₂-, -CO₂CR₂-, -NRCO-, -CONR-, -NR(C=O)NR-, -NR(C=S)NR-, -SO₂NR-, -NRSO₂-, -CR₂OCR₂-, -CR₂SCR₂-, -CR₂NRCR₂-With_4-8cyclohexanoltramadol group_4-8cycloalkenyl group_5-12Allenova group or_3-12heteroarenes group, an amino acid, a sugar or a monodisperse polietilenglikolya (PEG) building block; where each R is independently selected from H, C_1-4of alkyl, C_2-4alkenyl,_2-4the quinil,_1-4alkoxyalkyl or_1-4hydroxyalkyl;

Bzp^Mrepresents benzopyrylium dye of the formula II

,

where:

Y¹represents a group of formula Y^aor Y^b

,

R¹-R⁴and R⁹-R¹³independently selected from H, -SO₃M¹Hal, R^aor_3-12aryl, where each M¹independently represents N or B^cand B^cis a biocompatible cation;

R⁵represents H, C_1-4alkyl, C_1-6carboxyethyl,_3-12aryl-sulfonyl, Cl or R 5together with one of R⁶, R¹⁴, R¹⁵or R¹⁶it may possibly form a 5 - or 6-membered unsaturated aliphatic, unsaturated heteroaromatics or aromatic ring;

R⁶and R¹⁶independently represent groups of R^a;

R⁷and R⁸independently represent a_1-4alkyl, C_1-4sulfoalkyl or C_1-6hydroxyalkyl or possibly together with one or both of R⁹and/or R¹⁰may form a 5 - or 6-membered N-containing heterocyclic or heteroaryl ring;

X represents-CR¹⁴R¹⁵-, -O-, -S-, -Se-, -NR¹⁶- or-CH=CH-, where R¹⁴-R¹⁶independently represent groups of R^a;

R^arepresents a C_1-4alkyl, C_1-4sulfoalkyl, C_1-6carboxyethyl or C_1-6hydroxyalkyl;

w is 1 or 2;

J is a biocompatible anion;

provided that Bzp^Mcontains at least one sulfoxylates Deputy selected from the group R¹-R¹⁶.

The term "imaging agent" understand the connection, suitable for optical imaging, areas of interest of the whole (i.e. intact) the body of a mammal in vivo. Preferably, the mammal is man. Visualization can be invasive (for example, conduct the th during surgery or endoscopic) or non-invasive. Visualization can be used to facilitate biopsy (for example, through a biopsy channel of an endoscopic instrument) or resection of tumor (eg, during intraoperative procedures by identifying the boundaries of the tumor).

The term "optical imaging" is understood as any method that forms an image for detection, staging or diagnosis of disease, monitoring the progression of the disease or to monitor the treatment of the disease, based on the interaction with light in the range from green to near-infrared region of the spectrum (wavelength 500-1200 nm). Optical visualization also includes all the ways, ranging from direct visualization without the use of any device, and including the use of devices such as various optical devices, catheters and equipment for optical imaging, for example, controlled by computer equipment for the tomographic images. The ways and methods of measurement include: fluorescent imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; visualization transmittance (transmittance imaging; visualization of transmittance with time resolution (time resolved transmittance imaging; confocal imaging; nonlinear microscopy; photoacoustic visualization; and is Ustica-optical visualization; spectroscopy; scattering spectroscopy; interferometry; coherence interferometry; diffuse optical tomography and indirect fluorescence diffuse optical tomography (continuous wave, time domain and frequency domain) and measurement of light scattering, absorption, polarization, luminescence, and the duration of the fluorescence quantum yield and extinction, but are not limited to. Additional details of these methods is presented in: Tuan Vo-Dinh (editor): "Biomedical Photonics Handbook" (2003), CRC Press LCC; Mycek & Pogue (editors): "Handbook of Biomedical Fluorescence" (2003), Marcel Dekker, Inc.; Splinter & Hopper: "An Introduction to Biomedical Optics" (2007), CRC Press LCC.

Light ranging from green to near-infrared region of the spectrum corresponds to a wavelength of 500-1200 nm, preferably a wavelength of 550-1000 nm, most preferably 600-800 nm. A method of optical imaging preferably is a fluorescent endoscopy. The body of a mammal, in the sixth aspect is preferably the human body. The preferred embodiments of the visualizing agent agents are described for the first aspect (above). In particular, it is preferable to use a dye Bzp^Mwas fluorescent.

The term "biocompatible carrier" understand the fluid, especially a liquid, in which the visualizing and the UNT can be suspended or dissolved so to the composition was physiologically acceptable, i.e. it can be entered in the body of a mammal without toxicity or undue discomfort. Biocompatible carrier is appropriately injectable liquid carrier, such as sterile, pyrogen-free water for injection; an aqueous solution, such as saline (which may preferably be balanced so that the final product for injection was isotonic); an aqueous solution of one or more regulatory toychest substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (for example sorbitol or mannitol), glycols (e.g., glycerol) or other non-ionic high molecular weight alcohols (for example, polyethylene glycols, propylene glycols and the like). Preferably, the biocompatible carrier is pyrogen-free water for injection or isotonic saline solution.

The term "conjugate" understand that TMV group (L)_nand dye Bzp^Mconnected by covalent bonds.

Although the conjugate of formula I suitable for imaging in vivo, it may also find application in vitro (for example, in the analysis of quantitative determination of TMV in biological samples or for visualization of TMV in tissue samples). Suppose the equipment, imaging agent used for imaging in vivo.

The term "sulfoxylates Deputy" understand Deputy formula-SO₃M¹where M¹represents N or B^cand B^cis a biocompatible cation. Deputy-SO₃M¹covalently linked to the carbon atom and the carbon atom may be aryl (i.e. solitarily, as for example, when R¹or R²represents-SO₃M¹) alkyl or (sulfoalkyl group). The term "biocompatible cation" (B^cunderstand positively charged counterion which forms a salt with ionized negatively charged group (in this case, the sulphonate group), where the specified positively charged counterion is also non-toxic and therefore suitable for introduction into the organism of a mammal, especially a human body. Examples of suitable biocompatible cations include the cations of alkali metals sodium or potassium; alkaline earth metals calcium and magnesium and ammonium ion. Preferred biocompatible cations are the cations of sodium and potassium, most preferably sodium.

The term "biocompatible anion" (J) understand the negatively charged counterion which forms a salt with ionized, positively uragannoj group (in this case: indolines group), where specified negatively charged counterion is also non-toxic and therefore suitable for introduction into the organism of a mammal, especially a human body. The counterion (J^-) represents an anion that is present in equimolar amounts, balancing, thus, the positive charge on the dye Bzp^M. Accordingly, the anion (J) is one - or multi-charged, provided that there is in balancing the charge quantity. Anion appropriately comes from inorganic or organic acids. Examples of suitable anions include halide ions such as chloride or bromide; sulfate; nitrate; citrate; acetate; phosphate and borate. The preferred anion is chloride.

The term "grouping, providing targeted delivery to a biological target" (TMV), understand the connection, which after the introduction into the organism of a mammal in vivo selectively absorbed or is located in a particular place of the body specified mammal. Such places are, for example, can be involved in a particular disease state and to be a good indication of the functioning of the body, or undergoes metabolic process. Grouping, providing targeted delivery to the biological target, preferably includes 3-100-dimensional peptides, peptide analogues, epeidi or peptide mimetics, which may be linear peptides or cyclic peptides, or combinations thereof; or substrates of enzymes, antagonists, enzymes or enzyme inhibitors; synthetic receptor-binding compounds, oligonucleotides or oligo-DNA or oligo-RNA fragments.

The term "peptide" refers to compounds containing two or more than two amino acids, as defined below, the United peptide bond (i.e. the amide bond connecting Amin one amino acid with carboxyla another). The term "peptide mimetic" or "mimetic" refers to biologically active compounds that mimic the biological activity of the peptide or protein, but are not peptide by their chemical nature, i.e. they do not contain any peptide bonds (i.e. amide bonds between amino acids). In this description, the term peptide mimetic is used in a broader sense to include molecules that are not fully peptide in nature, such as pseudopeptide, polypeptide and peptide. The term "peptide analog" refers to peptides containing one or more than one amino acid analog, as described below. See also "Synthesis of Peptides and Peptidomimetics", M.Goodman et al., Houben-Weyl E22c, Thieme.

The term "amino acid" is understood L - or D-amino acid, amino acid analog (e.g., NAF is jalanin) or amino acid mimetic, which can be natural or be purely synthetic origin and can be optically pure, i.e. it can be a single enantiomer and, therefore, to be chiral, or a mixture of enantiomers. In this description use the traditional three-letter or one-letter abbreviations for amino acids. Preferably, the amino acids of the present invention are optically pure. The term "amino acid mimetic" understand synthetic analogs of natural amino acids, which are isostere, that is developed to simulate the spatial and electronic structure of the natural compound. Such isostere well known to experts in the art and include depsipeptide, retroversive, thioamides, cycloalkanes or 1,5-disubstituted tetrazole (see M.Goodman, Biopolymers, 24, 137 (1985)), but are not limited to.

Suitable substrates, antagonists or inhibitors of enzymes include glucose and analogues of glucose, such as Tordesillas; fatty acids or elastase inhibitors, angiotensin II or metalloproteinases. Preferred ones antagonist of angiotensin II is losartan. Suitable synthetic receptor-binding compounds include estradiol, estrogen, progestin, progesterone and other steroid hormones; ligands for feminova D-1 or D-2 receptor or Transporter of dopamine, such as Trapani; and ligands for the serotonin receptor. If the receptor-binding compound is folate, the linker group preferably does not contain 5-dimensional peptide Asp-Arg-Asp-Asp-Cys. In the most preferred case, the receptor-binding compound is not a folate.

Benzopyrylium dye (Bzp^M) of formula II is a fluorescent dye or chromophore, which can be detected either directly or indirectly in the procedure optical imaging using light in the range from green to near-infrared region of the spectrum (wavelength 500-1200 nm, preferably 550-1000 nm, more preferably 600 to 800 nm). Preferably, Bzp^Mhas fluorescent properties.

It is envisaged that one of the roles of the linker group -(A)_min the formula I consists in distancing Bzp^Mfrom the binding site TMV. This is particularly important as Bzp^Mis relatively voluminous, therefore, possible adverse steric interactions. This can be achieved through a combination of flexibility (for example, simple alkyl chains)to Bzp^Mhad a degree of freedom for placement away from the binding site, and/or rigidity, for example, cycloalkyl or aryl spacer, which orients Bzp^Mthe side from the binding site. For modification of bearsdley visualizing agent you can also use the nature of the linker group. For example, the introduction of a linker simple ester groups will help to minimize binding to plasma proteins. If -(A)_m- includes polietilenglikolya (PEG) building block or a peptide chain of 1-10 amino acid residues, such linker group can function as modifying the pharmacokinetics and rate of clearance from the blood of the imaging agent in vivo. Such linker groups"biomodification" can accelerate the clearance visualizing agent of the background tissue, such as muscle or liver, and/or from the blood, thereby obtaining the best diagnostic image due to less background noise. The linker group-biomodification can also be used to promote specific ways of excretion, for example through the kidneys, in contrast excretion through the liver.

The term "sugar" understand mono-, di - or trisaccharide. Suitable sugars include glucose, galactose, maltose, mannose and lactose. In sugar may be introduced functional groups for easy combination with amino acids. For example, glucosamine derived amino acids can be konjugierte with other amino acids through peptide bonds. One is m example of this is glucosamine derived aspartic acid (commercially available from NovaBiochem):

Formula I means that the group -(L)_n[Bzp^M] may be attached at any suitable position TMV. Such appropriate provisions for the group -(L)_n[Bzp^M] is selected in such a way that they are located away from parts of TMV, which is responsible for binding to the active site in vivo. Grouping [BTM]-(L)_nin the formula I can be attached in any suitable position Bzp^Mformula II. Grouping [TMV]-(L)_nor takes the place of the existing substituent (for example, one of the groups R¹-R¹⁶), or covalently attached to an existing in Bzp^Mthe Deputy. Grouping [BTM]-(L)_n- preferably attached via carboxialkilnuyu Deputy in Bzp^M.

Suitable imaging agents according to the invention are agents that have Bzp^Mhas the formula IIa or IIb:

,

,

where X, w, J and R¹-R¹³are as defined for formula II.

When R⁵together with one of R⁶/R¹⁴-R¹⁶forms a 5 - or 6-membered unsaturated aliphatic, unsaturated heteroaromatics or aromatic ring, such suitable aromatic rings include phenyl, furan, thiazole, peregrinae, pyrrole or iresolve rings. Suitable unsaturated ring containing at least C=C, which is attached R⁵.

When R⁷and/or R⁸together with one or both of R⁹and/or R¹⁰form a 5 - or 6-membered N-containing heterocyclic or heteroaryl ring, such suitable rings include thiazole, peregrinae, pyrrole or pyrazole nucleus ring or partially hydrogenated version, preferably peregrinae or dihydropyridine.

In an alternative embodiment, the dyes of formula IIb may be selected so that at least one of R¹-R⁴represents F or -(CF₂)_f-F, where f is an integer value from 1 to 4.

The pharmaceutical composition is delivered in suitable vials or vessels, which include a hermetically sealed container that can maintain a sterile clean, plus, possibly, an inert filling space gas (e.g. nitrogen or argon), at the same time allowing for the addition and selection of solutions by syringe or cannula. Therefore, the preferred container is a bottle with a sealing gasket, in which the gas-tight cover is compressed an additional capping agent (usually aluminium). The cover is suitable for single or multiple puncture needle for under the one injection (for example, compressed lid with sealing gasket) while maintaining the sterile cleanliness. Such containers have the added advantage that the cover can withstand a vacuum (for example, to replace the filling space of the gas or degassing of solutions) and can withstand pressure changes, for example, reduce pressure without penetration of gases from the external environment, such as oxygen or water vapor.

Preferred containers for multiple doses include single large bottle (for example, a volume of from 10 to 30 cm³), which contains multiple intended for patient dose, which intended for single patient dose can thus be selected in syringes clinical stamps with different intervals of time during the shelf life of the drug in accordance with the clinical situation. Pre-filled syringes are designed to enable a single dose or standard dose" and therefore preferably represent a disposable syringe or other needle suitable for clinical application. The pharmaceutical compositions of the present invention preferably have a dosage that is appropriate for one patient, and provided p is chodashim the syringe or container, as explained above.

The pharmaceutical composition may contain additional excipients, such as an antimicrobial preservative, pH-adjusting agent, a filler, a stabilizer or regulatory osmollnosti agent. The term "antimicrobial preservative" refers to an agent that inhibits the growth of potentially harmful microorganisms, such as bacteria, yeast or fungi. Antimicrobial preservative may also demonstrate some bactericidal properties depending on the dosage. The primary role of the antimicrobial preservative(s) of the present invention consists in the inhibition of growth of any such microorganism in the pharmaceutical composition. However, antimicrobial preservative may also may be used to inhibit the growth of potentially harmful microorganisms in one or more components of the kits used for the preparation of specified composition prior to introduction. These sets are described in the second aspect (below). Suitable(e) antimicrobial(s) preservative(s) include(s): parabens, that is, methyl-, ethyl-, propyl - or butylparaben, or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservatives are parabens.

The term "pH-regulating agent" means a compound or mixture of compounds useful for finding the pH of the composition within, acceptable (approximately pH from 4.0 to 10.5) for the introduction of a human or mammal. Such suitable pH-adjusting agents include pharmaceutically acceptable buffers, such as tricin, phosphate or TRIS (i.e. Tris(hydroxymethyl)aminomethane), and pharmaceutically acceptable bases, such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition is used in the form of a kit, pH-regulating agent can be presented in a separate vial or container, so that the user can set to regulate the pH as part of multistage procedure.

The term "filler" is understood pharmaceutically acceptable which increases the amount of agent that can facilitate the work with the material in the process of obtaining and lyophilization. Suitable fillers include inorganic salts such as sodium chloride, and water-soluble sugar or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

The pharmaceutical compositions according to the first aspect can be prepared in an aseptic manufacturing environment (clean room) to give the desired sterile, pyrogen-free product. Preferably, the key components, especially associated reagents, plus those parts of the equipment that is brought into contact with the imaging agent (e.g. the measures the vials were sterile. Components and reagents can be sterilized by methods known in the art, including: sterile filtration, the final sterilisation using e.g. gamma-irradiation, autoclaving, dry processing with heat or chemical treatment (e.g. with ethylene oxide). Some components are preferably sterilized in advance in order to perform the minimal number of manipulations. However, as a precaution, it is preferable to include at least the stage of sterile filtration as the final stage of the preparation of pharmaceutical compositions.

The pharmaceutical composition according to the first aspect preferably prepared from a set as described below for the second aspect.

Preferred indications

Convenient to the molecular mass of the visualizing agent amounted to 30,000 daltons, inclusive. Molecular weight is preferably in the range from 1000 to 20000 daltons, most preferably from 2,000 to 18,000 daltons, and especially preferred is the range from 2500 to 16000 daltons.

TMV can be of synthetic or natural origin, but is preferably synthetic. The term "synthetic" has its conventional meaning, that is made by man unlike isolated from p. radnih sources, for example, from the body of a mammal. The advantage of these compounds is that their manufacture and impurity profile can completely control. Thus, monoclonal antibodies and their fragments of natural origin are outside the scope of the term "synthetic"as used in this description.

TMV is preferably selected from: 3-100-dimensional peptide substrate of the enzyme, the antagonist of the enzyme or enzyme inhibitor. Most preferably TMV is a 3-100-dimensional peptide or peptide analogue. If TMV is a peptide, preferably represents 4-30-dimensional peptide and most preferably 5-28-dimensional peptide.

Grouping [BTM]-(L)_nin the formula I is preferably attached to Bzp^Mformula II according to the provisions of R⁵, R⁶, R¹⁴, R¹⁵or R¹⁶more preferably R⁶, R¹⁴, R¹⁵or R¹⁶most preferably R⁶, R¹⁴or R¹⁵. To facilitate attaching the relevant Deputy R⁵, R⁶, R¹⁴, R¹⁵or R¹⁶preferably represents C_1-6carboxyethyl, more preferably_3-6carboxyethyl, carboxypropyl used as an active complex of ether.

Benzopyrylium dye (Bzp^M) preferably have the t at least 2 sulfoxylate Deputy, more preferably 2-6 sulfoxylate substituent, most preferably 2-4 sulfoxylate Deputy. Preferably, at least one of sulfoxylate substituents is_1-4sulfoalkyl group. Such sulfoalkyl groups are preferably located at positions R⁶, R⁷, R⁸, R¹⁴, R¹⁵or R¹⁶; more preferably R⁶, R⁷, R⁸, R¹⁴or R¹⁵; most preferably R⁶together with one or both of R⁷and R⁸of formula II. Sulfoalkyl group of the formula II preferably have the formula -(CH₂)_kSO₃M¹where M¹represents N or B^c, k is an integer value from 1 to 4, and B^cis a biocompatible cation (as defined above). k is preferably equal to 3 or 4.

In the formula II preferably w is 1. R⁵preferably represents N or C_1-4carboxyethyl and most preferably represents N. X preferably represents-CR¹⁴R¹⁵- or-NR¹⁶and most preferably represents-CR¹⁴R¹⁵-.

Preferred dyes Bzp^Mhave the formula III:

,

where Y¹, R¹-R⁴, R⁶, R¹⁴, R¹⁵and J are such as defined for formula II.

Suitable dyes of formula III have the formula IIIa or IIIb:

,

.

Preferred groups R¹-R⁴and R⁶-R¹³from formulas III, IIIa and IIIb are the groups described above for formula IIa and IIb. In formulas III, IIIa and IIIb R¹⁴and R¹⁵preferably selected such that one represents a group R^band the other represents a group R^c. R^brepresents a C_1-2alkyl, most preferably methyl. R^crepresents a C_1-4alkyl, C_1-6carboxyethyl or_1-4sulfoalkyl, preferably_3-6carboxylic or -(CH₂)_kSO₃M¹where k is chosen equal to 3 or 4.

Preferably, the dyes of the formula III have the C_1-6carboxialkilnuyu Deputy to facilitate covalent joining TMV.

In the formula II or III, when R⁷and/or R⁸together with one or both of R⁹and/or R¹⁰form a 5 - or 6-membered N-containing heterocyclic or heteroaryl ring, such preferred rings are peregrinae or dihydropyridine. A preferred group Y¹in which R⁸cyklinowanie with R¹⁰has the formula Y^c:

.

Before Occitania group Y ¹in which, as a group, R⁷and R⁸cyklinowanie, has the formula Y^d:

,

where:

R⁷, R⁹and R¹¹-R¹³are as defined above;

each X¹independently represents N or C_1-4alkyl.

In the formula Y^cpreferably, when:

each X¹represents CH₃;

R⁹is an R¹¹that represents N;

R¹²represents N;

R¹²represents CH₃or-C(CH₃)₃more preferably- (CH₃)₃.

In the formula Y^dpreferably, when:

R⁹represents N;

R¹²represents N;

R¹²preferably represents CH₃or-C(CH₃)₃more preferably- (CH₃)₃.

Preferably, the group-NR⁷R⁸of formula III either:

1) existed in the form of an open circuit, i.e. the groups of R⁷/R⁸were not cyklinowanie with one or both of R⁹/R¹⁰. Such preferred groups R⁷and R⁸independently selected from C_1-4the alkyl or C_1-4sulfoalkyl, most preferably ethyl or_3-4sulfoalkyl;

2) was cyklinowanie obtaining cyclic substituent Y¹forms the crystals Y ^cor Y^dmore preferably the formula Y^c.

Form an open circuit (1) is most preferable.

Especially preferred dyes of the formula III are of formula IIIc, IIId or IIIe:

,

,

,

where:

M¹and J are as defined above;

R¹⁷and R¹⁸independently selected from C_1-4the alkyl or C_1-4sulfoalkyl;

R¹⁹represents N or C_1-4alkyl;

R²⁰represents a C_1-4alkyl, C_1-4sulfoalkyl or C_1-6carboxyethyl;

R²¹represents a C_1-4sulfoalkyl or C_1-6carboxyethyl;

R²²represents a C_1-4alkyl, C_1-4sulfoalkyl or C_1-6carboxyethyl;

X²X³and X⁴independently represent N or C_1-4alkyl.

The dyes of formula IIId, IIIe and IIIf are preferably chosen in such a way that one or more than one of R²⁰-R²²represents a C_1-4sulfoalkyl.

Specific preferred dyes of formula IIId are DY-631 and DY-633:

,

.

Specific preferred dye of formula IIIe is DY-652:

.

Concrete is predpochtitelnye dyes are DY-631 and DY-652, and DY-652 is most preferred.

When TMV is a peptide, such preferred peptides include:

- somatostatin, octreotide and analogues;

peptides that bind to the receptor ST, where ST means thermostable toxin produced by E. coli and other microorganisms;

- laminine fragments, for example YIGSR, PDSGR, IKVAV, LRE and KCQAGTFALRGDPQG;

- N-formyl-containing peptides for targeted delivery to the sites of accumulation of leukocytes;

platelet factor 4 (PF4) and its fragments;

- RGD(Arg-Gly-Asp)-containing peptides, which may, for example, to have a target angiogenesis (R.Pasqualini et al., Nat. Biotechnol. 1997 Jun, 15(6): 542-6; .Ruoslahti, Kidney Int. 1997 May, 51(5): 1413-7);

peptide fragments α₂-antiplasmin, fibronectin or beta-casein, fibrinogen or thrombospondin. Amino acid sequence of α₂-antiplasmin, fibronectin, beta-casein, fibrinogen and thrombospondin can be found in the following references: the precursor α₂-antiplasmin (M.Tone et al., J. Biochem, 102, 1033, (1987)); beta-casein (L.Hansson et al., Gene, 139, 193, (1994)); fibronectin (A.Gutman et al., FEBS Lett., 207, 145, (1996)); the predecessor of thrombospondin-1 (V.Dixit et al., Proc. Natl. Acad. Sci., USA, 83, 5449, (1986); R.F.Doolittle, Ann. Rev. Biochem., 53, 195, (1984));

peptides that are substrates or inhibitors of angiotensin, for example:

angiotensin II: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (..Jorgensen et al., J. Med. Chem., 1979, Vol.22, 9, 1038-1044),

[Sar,Ile]-angiotensin II: Sar-Arg-Val-Tyr-Ile-His-Pro-Ile (R.K.Turker et al., Science, 1972, 177, 1203);

angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu.

When TMV is a peptide, one or both ends of the peptide, preferably both, conjugated with inhibiting the metabolism group (M^IG). Both ends of the peptide is protected in this way, important for applications in imaging in vivo, because otherwise you can expect a fast metabolism with subsequent loss of affinity and selective binding to peptide TMV. The term "inhibiting the metabolism group (M^IGunderstand biocompatible group which inhibits or suppresses the enzymatic, especially peptidases, such as carboxypeptidases, metabolism of peptide TMV or amino end or carboxy-Termini. In particular, such groups are important for applications in vivo, they are well known to experts in the art, and appropriately chosen for the amino end of the peptide from:

N-acylated groups,- NH(C=O)R^Gwhere the acyl group is -(C=O)R^Ghas R^Gselected from C_1-6alkyl, C_3-10aryl groups, or contains polietilenglikolya (PEG) building block. Suitable PEG groups described for the linker group (L) below. Such preferred PEG groups are biomodification formulas Bio1 or Bio2 (below). Such preferred aminocore the groups M ^IGare acetyl, benzyloxycarbonyl or TRIFLUOROACETYL, most preferably acetyl.

Suitable inhibiting the metabolism of the group for the carboxyl end of the peptide include: carboxamid, complex tert-butyl ether, complex benzyl ether complex cyclohexyloxy ether, aminoplast or polietilenglikolya (PEG) building block. A suitable group of M^IGfor carboxykinase amino acid residue peptide TMV is a group, where the terminal amine of the amino acid residue is N-alkylated With_1-4alkyl group, preferably methyl group. Such preferred groups M^IGare carboxamid or PEG, most preferably such groups is carboxamid.

If one or both ends of the peptide is protected by a group of M^IGthe group -(L)_n[Bzp^M] perhaps can be attached to the group of M^IG. Preferably, at least one end of the peptide had no group M^IGin order to join the group -(L)_n[Bzp^M] this position was given to compounds of the formula IVa or IVb, respectively:

;

;

where:

Z¹attached to the N-end of the peptide TMV and represents N or M^IG;

Z²attached to the C-end of the peptide TMV and is a HE, OB^{c/sup> or MIG,}

where B^cis a biocompatible cation (as defined above).

In formulas IVa and IVb Z¹and Z²preferably both independently represent M^IG. Such preferred groups M^IGfor Z¹and Z²are the groups described above for the ends of the peptide. While inhibition of the metabolism of the peptide TMV on either end of the peptide can also be achieved by joining the group -(L)_n[Bzp^M], the group -(L)_n[Bzp^M] is outside the definition of M^IGaccording to the present invention.

Peptide TMV may possibly contain at least one additional amino acid residue having a side chain suitable to facilitate conjugation with Bzp^Mand forming part of the residue And of the linker group (L). Such suitable amino acid residues include residues Asp or Glu for conjugation with amine-functionalized dyes Bzp^Mor Lys residue for conjugation with carboxy-functionalized dye Bzp^Mor dye Bzp^M, functionalized active complex ether. Additional(e) amino acid(s) residue(s) for conjugation with Bzp^Mrespectively is(s) away from the binding site of the peptide TMV and is preferably(s) or at the C-or N-end. Pre is respectfully, amino acid residue for conjugation is a Lys residue.

In cases where there is a synthetic linker group (L), it preferably contains a terminal functional groups that facilitate conjugation with [TMV] and Bzp^M. Such suitable group (Q^a) described below. When L contains a peptide chain of 1-10 amino acid residues, these amino acid residues preferably selected from glycine, lysine, arginine, aspartic acid, glutamic acid or serine. When L contains PEG-group, it preferably contains units derived from the oligomerization of monodisperse PEG-like structures of formulas Bio1 or Bio2:

,

17-amino-5-oxo-6-Aza-3,9,12,15-tetraoxalate acid of the formula Bio1, where p is an integer from 1 to 10. Alternatively, you can use the PEG-like structure on the basis of the propionic acid derivative of the formula Bio2:

,

where p is as defined for formula Bio1, and q is an integer from 3 to 15.

In the formula Bio2 preferably p is 1 or 2 and q is preferably from 5 to 12.

When the linker group does not contain a PEG or peptide chain, the preferred groups L have a main chain of linked atoms that form the group -(A)_m- from 2 to 10 atoms, most predpochtitel is about 2-5 atoms, particularly preferably from 2 or 3 atoms. Minimum main chain of the linker group of the 2 atoms provides the advantage that Bzp^Mwell separated, so that any unwanted interaction is minimized.

Peptides TMV that there are no commercially available, can be synthesized by solid phase peptide synthesis, as described in .Lloyd-Williams, F.Albericio and E.Girald; Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997.

Imaging agents can be obtained as follows.

To facilitate conjugation Bzp^Mwith TMV to Bzp^Mproperly attach the reactive functional group (Q^a). Group Q^ait is intended to interact with complementary functional group TMV, resulting in a covalent bond between Bzp^Mand TMV. Complementary functional group TMV can be an integral part of the TMV can be introduced as a result of obtaining derived using a bifunctional group, as is known in the art. Table 1 shows examples of reactive groups and their complementary counterparts.

The term "activated ester" or "active ester" is understood ester carboxylic acid derivative, which is designed to be the best leaving group and thus the nutrient, to allow for easier interaction with the nucleophile, such as amines. Examples of suitable active esters are N-hydroxysuccinimidyl (NHS), pentafluorophenol, pentafluorothiophenol, para-nitrophenols and hydroxybenzotriazole. Preferred active esters are N-hydroxysuccinimidyl or pentafluorophenol esters.

Examples of functional groups present in TMV, such as proteins, peptides, nucleic acids, carbohydrates and the like, include hydroxy, amino, sulfhydryl, carbonyl including aldehyde and ketone) and thiophosphate. Suitable groups Q^acan be selected from carboxyl; activated esters; isothiocyanate; maleimide; halogenated; hydrazide; vinylsulfonic, dichlorotriazines and phosphoramidite. Preferably, Q^ais an activated ester of carboxylic acid, isothiocyanate, maleimide or halogenoacetyl.

When the complementary group is an amine or hydroxyl, and Q^apreferably represents an activated ester; preferred esters are the same as described above. Such preferred substituents on Bzp^Mis activated ester 5-carboxypentyl group. When the complementary group is particularly the thiol, Q^apreferably represents maleimido or iodated group.

General methods of conjugation of dyes with biological molecules described Licha et al. (Topics Curr. Chem., 222, 1-29 (2002); Adv. Drug Deliv. Rev., 57, 1087-1108 (2005)). Peptide, protein and oligonucleotide substrates for use in this invention can be in the state at the end position or, alternatively, one or more internal positions. Reviews and examples of labeling of proteins using reagents labeling, representing the fluorescent dyes described in "Non-Radioactive Labelling, a Practical Introduction", Garman, A.J. Academic Press, 1997; "Bioconjugation - Protein Coupling Techniques for the Biomedical Sciences", Aslam, M. and Dent, A., Macmillan Reference Ltd, (1998). Available protocols achieve site-specific labeling of the synthesized peptide is shown, for example, in Hermanson, G.T, "Bioconjugate Techniques", Academic Press (1996).

Preferably, the method of obtaining imaging agent includes or:

(1) the interaction of amine functional groups in TMV with the compound of the formula J¹-(L)_n-[Bzp^M]; or

(2) the interaction between the functional group of carboxylic acid or activated complex ester in TMV with the compound of the formula J²-(L)_n-[Bzp^M];

(3) the interaction Tilney group in TMV with the compound of the formula:

,

where WM, M^IG, L, n and Bzp^Mare as defined above, and

J¹is a group of carboxylic acids, activated complex ether, isothiocyanato or thiocyanato group;

J²is an amine group;

J³represents maleimido group.

J²preferably represents a primary or secondary amine group, most preferably a primary amine group. At stage (3) Tolna group in TMV preferably represents a cysteine residue.

At stages (1)-(3) TMV possibly can have other functional groups that are potentially able to interact with Bzp derivatives^Mprotected by suitable protective groups, so that a chemical reaction occurs selectively only in the desired place. The term "protective group" means a group which inhibits or suppresses undesired chemical reactions, but which is designed to be sufficiently reactive that it can be chipped off from the considered functional group under conditions sufficiently mild so as not to modify the rest of the molecule. After removal of the protecting obtain the target product. Amine protective groups are well known to experts in the art, and their appropriately selected from: Boc (where Boc is a tert-butyloxycarbonyl), Fmoc (where Fmoc p is ecstasy fluorenylmethoxycarbonyl), the TRIFLUOROACETYL, allyloxycarbonyl, Dde (1-(4,4-dimethyl-2,6-dioxocyclohex)ethyl) or Npys (i.e. 3-nitro-2-pyridylsulfonyl). Suitable thiol-protective groups are Trt (trityl), Acm (atsetamidometil), tert-Bu (tert-butyl), tert-butylthio, methoxybenzyl, methylbenzyl or Npys (3-nitro-2-pyridine sulfenyl). The use of additional protective groups described in "Protective Groups in Organic Synthesis", Theodora W.Greene and Peter G.M.Wuts (John Wiley & Sons, 1991). Preferred amino protective groups are Boc and Fmoc, most preferably Boc. Preferred thiol-protective groups are Trt and Acm.

Benzopyrylium dyes (Bzp^M), functionalityand appropriate for conjugation with TMV, available commercially from Dyomics (Dyomics GmbH, Winzerlaer Str. 2A, D-07745 Jena, Germany; www.dyomics.com), in which the reactive functional group (Q^a) is a complex NHS-ester, maleimide, amino or carboxylic acid. Precursors suitable for the synthesis of benzopirilievyh dyes, can also be obtained as described in US 5405976. Methods of conjugation optical reporter dyes with amino acids and peptides described Licha (see above)and Flanagan et al. (Bioconj. Chem., 8, 751-756 (1997)); Lin et al. (ibid, 13, 605-610 (2002)) and Zaheer (Mol. Imaging, 1(4), 354-364 (2002)). Methods of conjugation of the linker group (L) with TMV include a similar chemical reaction, and that with the actual color is s (see above), and they are known in this technical field.

In the second aspect of the present invention proposed a kit for the preparation of pharmaceutical compositions according to the first aspect, where the specified set contains the conjugate of formula I in sterile, solid form, such that after the restoration of sterile source biocompatible carrier is the dissolution of obtaining the desired pharmaceutical composition. "Conjugate" and "biocompatible carrier" along with their preferred embodiments are as described in the first aspect.

As for the set, then the conjugate, together with other possible eccipienti that described above, can be represented in the form of dried powder in a suitable vial or container. The powder is designed to further restore the desired biocompatible carrier with obtaining a pharmaceutical composition in a sterile pyrogen-free form ready for administration to a mammal.

The preferred sterile solid form of the conjugate is liofilizirovannoe solid. Sterile solid form preferably supplied in the container of a pharmaceutical brand, as described for the pharmaceutical composition (above). When the set is liofilizirovannam, the composition may contain cryoprotector selected from a saccharide, preferred is entrusted mannitol, maltose or trizina.

In the third aspect of the present invention proposed a conjugate of the formula I:

,

where L and n are as defined for the first aspect, and Bzp^Mhas the formula II as defined above; TMV' is a TMV that is defined in the first aspect, which is selected from synthetic and:

(1) 3-100-dimensional peptide;

(2) the substrate of the enzyme, the antagonist of the enzyme or enzyme inhibitor;

(3) the receptor-binding compounds;

(4) the oligonucleotide;

(5) oligo-DNA or oligo-RNA fragment.

The term "synthetic" has the definition given above. The preferred embodiments of Bzp^Mformula II in the conjugate are described above in the first aspect. Preferred aspects of TMV' (1)to(5) are such that described above in the first aspect for these types of TMV. TMV' preferably represents 3-100-dimensional peptide.

The conjugates according to the third aspect are useful in the preparation of pharmaceutical compositions imaging agent according to the invention. The conjugates can be obtained as described in the first aspect.

In the fourth aspect of the present invention, a method of optical imaging in vivo the body of a mammal comprising applying the pharmaceutical composition according to the first aspect of getting out the interests of the places of localization of TMV in vivo.

The term "optical imaging" is as defined in the first aspect (above).

In the method according to the fourth aspect preferably, the pharmaceutical composition visualizing agent was previously introduced into the body of the specified mammal. Under "pre-introduction" understand that stage, including the participation of the Clinician when the patient is given a visualizing agent, for example in the form of intravenous injections done before rendering. This embodiment includes the use of a conjugate as defined in the first aspect, in the manufacture of the diagnostic agent for optical imaging in vivo of painful conditions of the body of a mammal are involved in TMV.

The preferred method for optical imaging according to the fourth aspect of the visualization is based on the reflection fluorescence (FRI). In FRI-method of imaging agent of the present invention is administered diagnosed subject and then the tissue surface of the subject is irradiated with excitation light by the excitation of a continuous wave (CW). The light excites the dye Bzp^Mvisualizing agent. Fluorescence from the imaging agent, which is generated under the action of the exciting light, detected using a fluorescence detector. The reflected light is preferably Phil is trout for separating fluorescence component (exclusively or partially). From fluorescent lights to form an image. Usually spend a minimal amount of processing (do not use any processor to calculate the optical parameters, such as duration, quantum yield, and so on), and the image displays a picture of the fluorescence intensity. Imaging agent designed to concentrate in the area of the disease in order to give a higher intensity of fluorescence. Thus, the scope of the disease gives a positive contrast in the picture fluorescence intensity. The image is preferably obtained using a CCD camera (camera charge-coupled) or a CCD element (element charge-coupled), to be able to visualize in real-time.

The wavelength of excitation varies depending on the specific dye Bzp^Mbut usually is in the range of 500-1200 nm for dyes of the present invention. Apparatus for generating excitation light can be a traditional source of exciting light such as a laser (e.g., ion laser, laser dyes or semiconductor laser); halogen light source or a xenon light source. For optimal wavelength of excitation may be used in various optical filters. Predpochtitel the hydrated FRI-the method includes the following stages:

(1) the exposure of interest of a tissue surface of a body of a mammal exciting light;

(2) detecting fluorescence from the imaging agent, which is generated by excitation of Bzp^Musing fluorescence detector;

(3) may filter the light detected by the fluorescence detector, for separating a component of fluorescence;

(4) the formation of the image of interest specified tissue surface as a result of fluorescent light from the stages (2) or (3).

Exciting light from the stage (1) are inherently preferably is a continuous wave (CW). At stage (3) of the detected light is preferably filtered. Especially preferred FRI-method is fluorescence endoscopy.

In an alternative visualization method according to the sixth aspect of using FDPM (migration of photons in the frequency range). This method has advantages in comparison with the methods of continuous wave (CW) in cases where the depth of detection of the dye within the fabric (Sevick-Muraca et al, Curr. Opin. Chem. Biol., 6, 642-650 (2002)). For this type of imaging using frequency/time domain favorably, if Bzp^Mhas fluorescent properties that can be modulated depending on the depth of tissue damage, the image which is to receive, and the type of measuring equipment.

FDPM-method consists in the following:

(a) the exposure of the light-scattering biological tissues specified mammal having a heterogeneous composition, light from the light source with a predefined time-varying intensity for excitation imaging agent, and the fabric repeatedly scatters exciting light;

(b) detecting the emission of multiply scattered light cloth in response to the specified exposure effects;

(C) quantification of the fluorescence characteristics in all tissues as a result of emissions by establishing a range of values using a processor, each of which corresponds to the level of fluorescence characteristics in different locations within the tissue, and the level of fluorescence characteristics varies from heterogeneous tissue composition; and

(d) generating an image of tissue by mapping the heterogeneous tissue composition in accordance with the values of stage (b).

The characteristic fluorescence from step (C) preferably corresponds to the absorption imaging agent, and preferably further includes the mapping of a number of quantitative characteristics corresponding to the absorption coefficient and RA is sowing tissue before the introduction of the visualizing agent. The characteristic fluorescence from step (C) preferably corresponds to at least one of: the duration of the fluorescence quantum efficiency of fluorescence, fluorescence yield and uptake of imaging agent. The characteristic fluorescence preferably not depends on the intensity of the issue and does not depend on the concentration of the imaging agent.

Quantitative determination of stage (C) preferably includes: (1) determination of the estimated values, (2) determining the calculated emission as a function of the estimated values, (3) comparison of calculated emissions with emissions resulting from the specified detection, error detection, and (4) establishing the modified estimates of the characteristics of the fluorescence as a function of the error. Quantitative determination preferably includes determining values based on mathematical relationships, modeling multiple light scattering behavior of the tissue. The method according to the first embodiment preferably further includes monitoring the metabolic properties of tissue in vivo by detecting changes specified characteristic fluorescence.

Optical visualization according to the fourth aspect preferably used to facilitate regulation (management) is a painful condition of the body of the mammalian the future. The term "regulation" to understand the use in: detection, staging, diagnosis, monitoring of disease progression or treatment monitoring. Painful condition accordingly represents a state in which involved TMV visualizing agent. Imaging preferably include surface rendering using the camera, endoscopy and surgical indications. Additional details of suitable methods of optical imaging are listed in the overview Sevick-Muraca et al. (Curr. Opin. Chem. Biol., 6, 642-650 (2002)).

In the fifth aspect of the present invention, a method of detection, staging, diagnosis, monitoring of disease progression or monitoring the treatment of painful conditions of the body of a mammal, including a method of optical imaging in vivo according to the fourth aspect.

The present invention is illustrated non-limiting Examples are described in detail below. Example 1 describes the synthesis of a peptide, providing targeted delivery to the biological target, (Peptide 1), which binds to cMet. Example 2 describes the methods of conjugation of dyes Bzp^Maccording to the invention with peptides, in particular, with Peptide 1. In Example 3, the above data demonstrate that the peptide conjugates of Peptide 1 in the image is the shadow retain the affinity against cMet, that is, the conjugated dye does not preclude biological binding and selectivity. Demonstrated acceptable low binding to albumin human serum and high stability in plasma. In Example 4, it is shown that the peptide conjugates of the invention exhibit useful ratio tumor: background on animal models of colorectal cancer. Example 5 describes the use of prediction software for dyes according to the invention and shown that the dyes according to the invention there are no potentially dangerous in vivo metabolites. Example 6 describes the testing of the toxicity of Compound 6, showing that the expected clinical dose was well tolerated and did not cause any related drug adverse effects.

where R^drepresents -(CH₂)₃SO₃H, R^erepresents -(CH₂)₃CO₂H and R^frepresents -(CH₂)₅CO₂H.

DY-752 has the same ring and pattern of substitution, and DY-652, but has intemational communication (i.e. w = 2 and R⁵represents N) instead trimethylol communication DY-652.

Reduction

Use traditional trehu the public-and one-letter abbreviations of amino acids.

Example 1. The synthesis of Peptide 1

Used a 26-dimensional bicyclic peptide containing 2 Cys-Cys communication (Cys4-16 and 6-14), having the following sequence:

Ac-Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂("Peptide 1").

Stage (a): synthesis of the protected linear precursor Peptide 1

The linear peptide, the precursor has the following sequence:

Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂.

Spent the Assembly peptidyl resin H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-Glu(OtBu)-Thr(ψ^Me,Mepro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(BOC)-polymer on a peptide synthesizer, Applied Biosystems 433A using Fmoc-chemistry, starting with 0.1 mmol of resin Rink Amide Novagel. On stage combination was used surplus pre-activated AMI is ocelot 1 mmol (using HBTU). In the sequence have introduced Glu-Thr-pseudoproline (Novabiochem 05-20-1122). The resin was transferred into the apparatus with nitrogen bubbling and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol) in DCM (5 ml) for 60 minutes a Solution of anhydride was removed by filtration, the resin washed with DCM and dried in a stream of nitrogen.

Simultaneous removal of the protective groups of the side chains and cleavage of the peptide from the resin was carried out in TFA (10 ml)containing 2.5% TIS (triisopropylsilane), 2.5% of 4-thiocresol and 2.5% water for 2 hours and 30 minutes the Resin was removed by filtration, TFA was removed in vacuo and to the residue was added diethyl ether. The precipitate was washed with diethyl ether and dried in the air, which allowed us to obtain 264 mg of the crude peptide.

Purification preparative HPLC (gradient: 20-30% over 40 min where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 10 ml/min, column: C18 (2) (5 μm; 250×21,20 mm Phenomenex Luna, detection: UV 214 nm, retention time of product: 30 min) of the crude peptide was possible to obtain 100 mg of pure linear precursor Peptide 1. Pure product was analyzed by analytical HPLC (gradient: 10-40% b over 10 min where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 0.3 ml/min, column: C18 (2) (3 μm; 50×2 mm) Phenomenex Luna, detection: UV 214 nm, retention time of product: 6,54 min). Removing additional product features implemented with IP is the use of mass spectrometry with elektrorazpredelenie ( $$M{H}_2^{2+},$$calculated: 1464,6;$$M{H}_2^{2+},$$found: 1465,1).

Stage (b): formation of a disulfide bridge Cys4-16

Cys4-16; Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂.

The linear precursor from step (a) (100 mg) was dissolved in a mixture of 5% DMSO/water (200 ml) and the solution pH was brought to 6 with the use of ammonia. The reaction mixture was stirred for 5 days. Then the solution pH was brought to 2 with TFA and most of the solvent was removed by evaporation in a vacuum. Cleaning product residue (40 ml) portions were injected into the column for preparative HPLC.

Purification preparative HPLC (gradient: 0% b for 10 min, then 0-40% b over 40 min where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 10 ml/min, column: C18 (2) (5 μm; 250×21,20 mm Phenomenex Luna, detection: UV 214 nm, retention time of product: 44 min) residue allowed to obtain 72 mg of pure monocyclic precursor Peptide 1.

Pure product (as a mixture of isomers P1-P3) were analyzed by analytical HPLC (gradient: 10-40% b over 10 min where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 0.3 ml/min, column: C18 (2) (3 μm; 50× mm) Phenomenex Luna, detection: UV 214 nm, retention time of product: lower than the 5.37 min (P1); 5,61 min (P2); 6,05 min (P3)). Removing additional product features was performed using mass spectrometry with elektrorazpredelenie ($$M{H}_2^{2+},$$calculated: 1463,6;$$M{H}_2^{2+},$$found: 1464,1 (P1); 1464,4 (P2); 1464,3 (P3)).

Stage (C): the formation of a disulfide bridge Cys6-14 (Peptide 1)

Monocyclic precursor from step (b) (72 mg) was dissolved in a mixture of 75% Asón/water (72 ml) in a protective atmosphere of nitrogen. Added 1 M HCl (7.2 ml) and 0.05 M I₂in the Asón (4.8 ml) in that order and the mixture was stirred for 45 minutes was Added 1 M ascorbic acid (1 ml), which made the mixture is colorless. A large part of the solvents evaporated in vacuo, the residue (18 ml) was diluted with a mixture of water/0.1% of TFA (4 ml) and the product was purified using preparative HPLC. Purification preparative HPLC (gradient: 0% b for 10 min, then 20-30% over 40 min where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 10 ml/min, column: C18 (2) (5 μm; 250×21,20 mm Phenomenex Luna, detection: UV 214 nm, retention time of product: 43-53 min) residue allowed to obtain 52 mg of pure Peptide 1. Clean p is oduct were analyzed by analytical HPLC (gradient: 10-40% b for 10 min, where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 0.3 ml/min, column: C18 (2) (3 μm; 50×2 mm) Phenomenex Luna, detection: UV 214 nm, retention time of product: 6,54 min). Removing additional product features was performed using mass spectrometry with elektrorazpredelenie (MH₂²⁺calculated: 1391,5;$$M{H}_2^{2+},$$found: 1392,5).

Example 2. Synthesis of peptide conjugates with benzopyrylium dyes

A common way conjugation

To a solution of Peptide 1 (Example 1; 4 mg; 1.4 mmol) in DMF (0.5 ml) was added a solution of NHS-ester Bzp^M(1 mg; 1 mmol) and SIM.-kallidin (8 ml; 60 mmol) in DMF (0.5 ml). The reaction mixture was heated (microwave radiation) at 60°C for 1 h, then kept at RT (room temperature) overnight. The reaction mixture was then diluted with a mixture of 20% ACN/water/0.1% of TFA (7 ml) and the product was purified using preparative HPLC.

Cleaning and removal characteristics

Purification preparative HPLC (gradient: 20 to 40% over 40 min where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 10 ml/min, column: C18 (2) (5 μm; 250×of 21.2 mm Phenomenex Luna, detection: UV 214 nm) of the crude peptide was possible to obtain pure conjugate [Peptide 1]-Bzp^M. The pure product was analyzed analysis is coy HPLC (gradient: 10-40% b for 5 min, where A = H₂O/0.1% of TFA and B = ACN/0.1% of TFA, flow rate: 0.6 ml/min, column: C18 (2) (3 μm; 20×2 mm) Phenomenex Luna, detection: UV 214 nm). Removing the additional characteristics of the product was performed using mass spectrometry with elektrorazpredelenie.

The compounds obtained are shown in Table 3.

Example 3. Analysis of the polarization of fluorescence in vitro

Analysis of the polarization of fluorescence was used to study the affinity of binding of the imaging agent to the target cMet and binding properties in relation to plasma proteins. The principle of the polarization of fluorescence may briefly be described as follows.

Monochromatic light passes through a horizontal polarizing filter and excites the fluorescent molecules in the sample. Only those molecules that orientation properly in the vertical plane polarized (vertically polarized plane) absorb the light, become excited and subsequently emit light. The emitted light is measured in both the horizontal and vertical planes. The magnitude of the anisotropy (A) is the ratio of light intensities corresponding to the equation:

$$A=\frac{\mathrm{and}nten\mathrm{with\; a}\mathrm{and}\mathrm{in}n\mathrm{about}\mathrm{with\; a}tb\text{with Horizont is determined as being the polarizer, the intensity with vertical polarizer}}{\mathrm{and}nten\mathrm{with\; a}\mathrm{and}\mathrm{in}n\mathrm{about}\mathrm{with\; a}tb\text{with gorizontalnym polarizer}+\text{2* the intensity with vertical polarizer}}.$$

Measurement of the anisotropy of fluorescence was performed in a 384-well microplate in a volume of 10 μl in a buffer for binding (PBS; 0.01% tween-20; pH 7.5) using a tablet reader polarized fluorescence Tecan Safire (Tecan, US) for excitation at 635 nm and emission at 678 nm. The concentration of dye-labeled peptide kept constant (5 nm)and the concentration of chimeric constructs of c-Met/Fc person (R&D Systems) was varied in the range of 0-250 nm. For the implementation of the binding mixture was balanced in the microplate for 10 min at 30°C. the Observed change of the anisotropy was approximatively to the equation:

$$r_{obs}=r_{free}+(r_{bound}-r_{free})\frac{(K_D+cMet+P)-\sqrt{(K_D+cMet+P)2-4\cdot cMet\cdot P}}{2\cdot P}$$,

where r_obsrepresents the observed anisotropy, r_freerepresents the anisotropy of the free peptide, r_boundrepresents the anisotropy of the bound peptide, K_Drepresents the dissociation constant, cMet is the sum of the concentration of c-Met and P is the total concentration of dye-labeled peptide. According to the equation assumes that the synthetic peptide and receptor form a reversible complex in solution with a stoichiometry of 1:1. The data fitting was performed by the method of nonlinear regression, using the software SigmaPlot to obtain values of K_D(linking in one center).

Compounds 1-6 were tested in respect of binding to c-Met person (chimeric construct with Fc). The results (see Table 4) showed that K_Dall tested compounds due to binding to c-Met man lie in the field of nanomolar concentrations.

The change in the value of polarization was used to assess binding of the compounds with albumin human serum, because a small change in the magnitude of polarization associated with Abim binding, suitable for use in vivo. Binding to plasma proteins (eres) was confirmed by measurements on a Biacore. The stability of the visualizing agent in the plasma was confirmed by measuring the amount of compound remaining after incubation in mouse plasma for 2 hours at 37°C.

Example 4. Testing of compounds 2-6 in vivo

(a) Animal model

The study used female Nude mice with BALB/A (Bom). The use of animals was approved by the local ethics Committee of behavior. Nude mice with BALB/And represent a line of inbred mice with weakened immune systems and with high frequency in human tumors compared with other lines in Nude mice. The age of the mice upon receipt was 8 weeks, and body weight at baseline was approximately 20 grams. Animals were housed in individual ventilated cages (IVC, Scanbur BK) filtered through a HEPA filter air. The animals were given access without restriction to feed the Rat and Mouse nr. 3 Breeding" (Scanbur BK) and tap water, acidified by adding HCl to the molar concentration of 1 mm (pH 3.0).

HT-29 Cell cancer of the colon comes from carcinoma of the colon of a person, and it is known that expresses c-Met (Zeng et al., Clin. Exp. Metastasis, 21, 409-417 (2004)). It is proved that this cell line is cancero the military after subcutaneous inoculation of Nude mice (Flatmark et al., Eur. J. Cancer 40, 1593-1598 (2004)).

Cells HT-29 were grown in medium McCoy's 5A (Sigma, No. L), supplemented with 10%fetal calf serum and penicillin/streptomycin. Concentrated preparations were prepared from passage number four (P4) and frozen for storage in liquid nitrogen in an amount of 10⁷cells/vial in the appropriate culture media containing 5% DMSO. On the day of transplantation, the cells were rapidly thawed in a water bath at 37°C (approximately 2 min), washed and resuspendable in a mixture of PBS/2% serum (centrifugation at 1200 rpm for 10 min). Thorough mixing of the cells in vials provided each time the cells were sucked into the dosing syringe. The cell suspension volume of 0.1 ml was administered via subcutaneous (s.c.) injections in the shoulder and back, using a needle with a thin channel (25 G). After that, animals were returned to their cells and tumors were given the opportunity to grow within 13-17 days. The animals were provided a period of acclimatization for at least 5 days before inoculation.

(b) Procedure

All tested substances were restored using PBS from lyophilized powder. To obtain planar images, which was used to correct inhomogeneities lighting, used a small stack of white paper for your printer.

To obtd IGITI the animal during the procedure, optical imaging, animals were anestesiologi with isoflurane (typically 1.3 to 2%), using the coaxial open mask, to ensure easy surgery under anaesthesia, using oxygen as the carrier gas. A small piece of skin (3-5 mm) were removed over parts of the tumor and the surrounding muscles, using forceps and fine scissors, while the animal was under anesthesia. This was done in order to measure the signal from the tumor and muscle without interference from lying on top of skin tissue. The wound was closed using liquid afluorescent spray dressing (bandage spray) (3M, MN, USA).

Breathing and body temperature of the animal was recorded using the system BioVet (m2m Imaging Corp., NJ, USA), using the pneumatic sensor under animals and rectal temperature probe. The BioVet system also allowed for external heating using a heating Mat with a temperature setting of 40°C to maintain normal body temperature in the continuation of imaging procedures (2 hours). The tail vein was put a Venflon catheter for injection of contrast agent. Each animal did one injection of contrast agent. The volume of injected by the injection of the tested compounds was 0.1 ml, followed immediately carried out washing, 0.2 ml of saline. Picture fluorescence was recorded directly before and what Yecla (ri.) and then every 30 seconds within 2 hours.

(C) Visualization

Visualization was carried out by means of clinical laparoscope, adapted for use with a light source in order to initiate a reporter connection and filtering systems for extracting components of fluorescence. For excitation of the reporter molecule used the 635 nm laser. As detector we used a CCD camera Hamamatsu ORCA ERG. The camera was operated in a mode binning 2×2 gain 0. The standard exposure time for imaging of the colon was 4 C. the Distribution of intensities in the image corrected for inhomogeneities in the illumination using the calibration data of the system. I calculated the ratio of target to background for interest areas located over the exposed tumor and normal background muscle tissue.

(g) the Results

The tested Compounds were characterized by the following average ratios of the tumor: muscle (table 5).

Example 5. Prediction of metabolism and toxicity

Tools software Derek and Meteor received from Lhasa Ltd (22-23 Blenheim Terrace, Leeds LS2 9HD, UK). Derek used for prediction of the toxicity of new chemical compounds based on the known dependence of toxicity from structure. Similarly, using Meteor predict the possible metabolites of new chemical compounds. Both software tools are based on published and unpublished (but confirmed) data for chemical compounds. Have introduced the chemical structure of the dye DY-652. Any potentially dangerous metabolite in vivo was not predicted.

Example 6. Testing the toxicity of compound 6

Conducted a study of the dose acute toxicity to study tolerance to the compound 6 in a dose 100 times higher than the dose used in preclinical imaging (50 nmol/kg body weight).

The compound was administered to male rats by intravenous injection and the animals were killed on the 1st, 14th, 21st and 28th day after injection (ri.). At autopsy carried out and inspection of the main authorities on macroscopic pathology and kidney were placed in buffered to neutral values formalin for subsequent histological evaluation. Directly after injection was observed weak blue staining of the skin and mild blue coloration of the urine, which disappeared within 1 day ri. Kidneys at autopsy were diffuse green on the 1st day ri. Light microscopy showed no connection-related 6 changes. Other observed minor changes were minor and common for young laboratory rats. Strong staining with a fluorescent substance of the blood vessels of the kidney were observed on the 1st day ri. Staining was decreased to 14-day ri. and became indistinguishable from the control at 21 days ri.

No evidence of degeneration, necrosis or inflammation was not observed in none of the treated animals, which confirms the low nephrotoxicity connection. It was concluded that a single intravenous administration of compound 6 to male rats at a dose of 100 times greater than the applied clinical dose was well tolerated and did not cause any side effects associated with the drug.

1. Pharmaceutical composition containing a visualizing agent, suitable for optical imaging in vivo the body of a mammal, together with a biocompatible carrier, and the specified composition is in a form suitable for administration to a mammal, where the specified imaging Agay the t includes a conjugate of formula I:

where VTM is a 3-100-dimensional synthetic peptide;
n represents an integer of value 0 or 1;
L represents a synthetic linker group of formula(A)_m-where m is an integer value from 1 to 20, and each And independently represents-CR₂-, -CR=CR-, -C≡C-, -CR₂CO₂-, -CO₂CR₂-, -NRCO-, -CONR-, -NR(C=O)NR-, -NR(C=S)NR-, -SO₂NR-, -NRSO₂-, -CR₂OCR₂-, -CR₂SCR₂-, -CR₂NRCR₂-With_4-8cyclohexanoltramadol group_4-8cycloalkenyl group_5-12Allenova group or_3-12heteroarenes group, an amino acid, a sugar or a monodisperse polietilenglikolya (PEG) building block; where each R is independently selected from H, C_1-4of alkyl, C_2-4alkenyl,_2-4the quinil,_1-4alkoxyalkyl or_1-4hydroxyalkyl;
Bzp^Mrepresents benzopyrylium dye of formula II:

where Y¹represents a group of formula Y^aor Y^b

R¹-R⁴and R⁹-R¹³independently selected from H, -SO₃M¹Hal, R^aor_3-12aryl, where each M¹independently represents N or B^cand B^cis the BIOS is compatible cation;
R⁵represents H, C_1-4alkyl, C_1-6carboxyethyl,_3-12arylsulfonyl, Cl, or R⁵together with one of R⁶, R¹⁴or R¹⁵it may possibly form a 5 - or 6-membered unsaturated aliphatic, unsaturated heteroaromatics or aromatic ring;
R⁶represents a group R³;
R⁷and R⁸independently represent a_1-4alkyl, C_1-4sulfoalkyl or C_1-6hydroxyalkyl or possibly together with one or both of R⁹and/or R¹⁰may form a 5 - or 6-membered N-containing heterocyclic or heteroaryl ring;
X represents-CR¹⁴R¹⁵-where R¹⁴and R¹⁵independently represent groups of R^a;
R^arepresents a C_1-4alkyl, C_1-4sulfoalkyl, C_1-6carboxyethyl or C_1-6hydroxyalkyl;
w is 1 or 2;
J is a biocompatible anion;
provided that Bzp^Mcontains at least one sulfoxylates Deputy selected from the group R¹-R⁴and of groups R⁶-R¹⁵.

2. The composition according to claim 1, where Bzp^Mhas the formula IIa:

3. The composition according to claim 1, where Bzp^Mhas the formula IIb:

4. The composition according to claim 1, where Bzp^Mcontains from 2 to 4 sulfates is now Deputy.

5. The composition according to claim 1, where Bzp^Mcontains at least one_1-4sulfoalkyl Deputy.

6. The composition according to claim 5, where sulfoalkyl substituent has the formula
-(CH₂)_kSO₃M¹where M¹represents N or B^cand k is an integer value from 1 to 4.

7. The composition according to claim 1, where w is 1.

8. The composition according to claim 1, where R⁵represents N.

9. The composition according to claim 1, where Bzp^Mhas the formula III:

where Y¹, R¹-R⁴, R⁶, R¹⁴, R¹⁵and J are as defined in claim 1.

10. The composition according to claim 9, which contains the formula IIIc, IIId or IIIe:



where M¹is such as defined in claim 1;
R¹⁷and R¹⁸independently selected from C_1-4the alkyl or C_1-4sulfoalkyl;
R¹⁹represents N or C_1-4alkyl;
R²⁰represents a C_1-4alkyl, C_1-4sulfoalkyl or C_1-6carboxyethyl;
R²¹represents a C_1-4sulfoalkyl or C_1-6carboxyethyl;
R²²represents a C_1-4alkyl, C_1-4sulfoalkyl or C_1-6carboxyethyl;
X²X³and X⁴independently represent N or C_1-4Alki is.

11. The composition according to claim 1, which contains the formula IVa or IVb:
;
;
where Z¹attached to the N-end of the peptide TMV and represents N or M^1G;
Z²attached to the C-end of the peptide TMV and is a HE, OB^cor M^1G,
where B^cis such as defined in claim 1, and
M^1Grepresents inhibitory metabolism group, representing a biocompatible group which inhibits or suppresses the enzymatic metabolism of peptide TMV.

12. The composition according to claim 11, where each of the Z¹and Z²independently represents M^1G.

13. Composition according to any one of claims 1 to 12, which has a dosage that is appropriate for one patient, and provided in a suitable syringe or container.

14. Kit for the preparation of a pharmaceutical composition according to any one of claims 1 to 13, containing a conjugate of formula I, as defined in claims 1 to 12, in sterile, solid form, such that after the restoration of sterile source biocompatible carrier is the dissolution of obtaining the desired pharmaceutical composition.

15. Set in 14, where sterile solid form is a liofilizirovannoe solid.

16. The conjugate of formula I:

where L and n are as defined in claim 1, Bzp^{M is as defined in any one of claims 1 to 10, and TMV' is a TMV as defined in claim 1.}

^{17. The method of optical imaging in vivo the body of a mammal comprising applying the pharmaceutical composition according to any one of claims 1 to 13 to obtain images of sites of localization of TMV in vivo.}

^{18. The method according to 17, where the pharmaceutical composition is previously introduced into the body of the specified mammal.}

^{19. The method according to p, which includes stages:
(1) the exposure of interest of a tissue surface of a body of a mammal exciting light;
(2) detecting fluorescence from the imaging agent, which is generated by excitation of BzpMusing fluorescence detector;
(3) may filter the light detected by the fluorescence detector, for separating fluorescence component;
(4) the formation of the image of interest specified tissue surface as a result of fluorescent light from the stages (2) or (3).}

20. The method according to claim 19, where the exciting light from the stage (1) by its nature is a continuous wave (CW).

21. The method according to p, including:
(a) the exposure of the light-scattering biological tissues specified mammal having a heterogeneous composition, light from the light source with a predefined esmeraude the SJ in time intensity for excitation imaging agent, moreover, the fabric repeatedly scatters exciting light;
(b) detection of the emission of multiply scattered light cloth in response to the specified exposure effects;
(C) quantification of the fluorescence characteristics in all tissues as a result of emissions by establishing a range of values using a processor, each of which corresponds to the level of fluorescence characteristics in different locations within the tissue, and the level of fluorescence characteristics varies from heterogeneous tissue composition; and
(g) generating image tissue by mapping the heterogeneous tissue composition in accordance with the values of stage (b).

22. The method according to 17, where the means of optical imaging is a fluorescence endoscopy.

23. The method according to any of PP-22, where optical imaging in vivo is used to assist in the detection, staging, diagnosis, monitoring of disease progression or monitoring the treatment of painful conditions of the body of a mammal.

24. The method of regulation of painful conditions of the body of a mammal selected from the group comprising a method of detection, staging, diagnosis, monitoring of disease progression or monitoring the treatment of painful conditions of the body of melicope the surrounding, including the method of optical imaging in vivo according to any one of PP-23.