IMAGING AGENTS AND METHODS OF USE Field of Invention

The present invention relates to imaging agents, and methods of use thereof. In particular, the present invention relates to molecular imaging agents, pharmaceutical formulations containing them, method of their preparation, and their use in treatment, surgery and/or diagnosis. The molecular imaging agent can be used in imaging techniques such as (but not limited to) near infrared (NIR) imaging, optical (e.g., fluorescence) imaging and positron emission tomography (PET). For example, the imaging agents can be used to image compounds (such as particular enzymes) that are known or thought to have a role in particular disease processes. One such enzyme family is the transglutaminases, which form extensively cross-linked, generally insoluble protein polymers. These biological polymers are indispensable for an organism to create barriers and stable structures. One enzyme in this family is Factor XIII, and Factor Xllla in particular.

Background

Factor XIII is a plasma glycoprotein which is present in blood and certain tissues in a catalytically inactive (or zymogen) form. Factor XIII is transformed into its active form, Factor Xllla, by thrombin in the presence of calcium ions. Factor Xllla is also known as plasma transglutaminase, fibrinoligase or fibrin-stabilising factor.

Factor Xllla is a tetrameric transglutaminase and plays a role in several disease processes such as thrombosis, rheumatoid arthritis and

macrophage polarisation. In the coagulation pathway, Factor Xllla is an important mediator of fibrinolytic resistance: by crosslinking fibrin γ-chains and a-chains and by covalently binding molecules that impede plasmin activity (such as a2-antiplasmin), Factor XI I la increases the stability of thrombi. The final step in the formation of a blood clot is the covalent crosslinking of the fibrin which is formed by the proteolytic cleavage of fibrinogen by thrombin. Fibrin molecules align and Factor Xllla catalyses covalent crosslinking of the NH2 and CONH2 groups of lysyl and glutaminyl residues respectively giving structural rigidity to the blood clot. The crosslinking stabilises the fibrin clot structure and confers resistance to fibrinolysis. The crosslink formation is an important facet of normal blood coagulation and wound healing as well as pathological conditions such as thrombosis.

As atherothrombotic brain infarctions are a common sub-type of stroke, Factor Xllla substrates may allow diagnosis of stroke. It, and other transglutaminases, may also be implicated in atherosclerosis,

inflammatory processes, tumour growth and metastasis. WO 91/16931 discloses that radiolabeled analogues of Factor XIII (in which the active site has been inactivated by amino acid substitution) are useful as thrombus imaging radiopharmaceuticals. In addition, Factor Xllla is present both intracellular^ and at the cell surface of alveolar macrophages and within the extravascular alveolar space, potentially acting as an indicator of inflammation within the distal lung. Factor Xllla is also known to catalyse the incorporation of low molecular weight amines into the γ-glutamine sites of proteins. Similarly, Factor Xllla also catalyses the incorporation of low molecular weight glutamine analogues into lysyl residues. Thus, such low molecular weight amines (or glutamine analogues) function as competitive inhibitors of the

Factor Xllla-induced lysyl/glutaminyl crosslinking of proteins. A range of synthetic amines have been described, which are competitive inhibitors of the uptake of labelled putrescine (1 ,4-butanediamine) into N, N'-dimethylcasein catalysed by pig liver transglutaminase (L. Lorand et al., Biochem., 18, 1756 (1979)).

WO 89/00051 describes a method for targeting fibrin deposits using a labelled compound which is covalently bound to fibrin by Factor Xllla. The fibrin binding compound is stated to be "any peptide that is a substrate for the blood enzyme commonly known as Factor Xllla". Preferred peptides are said to include the tetrapeptide sequence Asn-Gln-Glu-Gln- [SEQ ID NO. 1 ] (or NQEQ in standard amino acid abbreviation notation). Also disclosed is the 12-mer peptide sequence from the N-terminus of the oc2- antiplasmin enzyme: NH2-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu- Lys-OH [SEQ ID NO. 2] together with a synthetic analogue: NH ₂ -Asn-Gln- Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys-OH [SEQ ID NO. 3], (denoted NQEQVSPLTLLK and NQEQVSPYTLLK respectively). The latter was radiolabeled with ¹²⁵ l and shown to be taken up in thrombin clots in vitro. WO 99/60018 discloses synthetic analogues of lysine and glutamine that are substrates for Factor Xllla and that are labelled with radiometals, in particular ^99m Tc, chelated to a chelate that is part of the structure.

Factor Xllla NIR fluorescence imaging agents and fluorescence imaging experiments have been described (F. Jaffer et al., Circulation, 1 10(2), 170 (2004)). The compounds described in Jaffer et al. have been found to be very hydrophobic.

Whilst several examples of Factor Xllla molecular imaging agents exist, it is believed that none are currently used in NIR, optical and/or PET imaging in a clinical context. Moreover, designing new compounds for use in NIR, optical and/or PET imaging and incorporating known Factor Xllla and transglutaminase substrates can lead to changes in solubility, which can create problematic pharmacokinetics. Also, it has been observed that the synthetic routes to many Factor Xllla molecular imaging agents are complex, and involve solubility problems in aqueous solvents in particular.

Therefore, it is an object of the present invention to obviate or mitigate at least some of the disadvantages of the prior art.

A further object of the invention is to provide alternative imaging agents that can be used to image one or more transglutaminase, in particular using N IR, optical and/or PET imaging. A still further object of the invention is to provide alternative imaging agents that can be used to image Factor Xllla, in particular using NIR, optical and/or PET imaging.

Disclosure of Invention

According to a first aspect of the invention there is provided a compound of formula:

where:

D is C=0 or -CR ₂ -;

X is selected from the group consisting of: -NRJ, -OR, -OJ, -

SR and -SJ;

Y is -(B)p(Z), each R is independently selected from the group consisting of: H, CI, C1-4 alkyl, C1-4 alkenyl, C1-4 alkynyl, C1-4 alkoxyalkyl and C1-4 hydroxyalkyl;

R ¹ is -(L)nM;

each L is independently selected from the group consisting of: -CR2-, -CR=CR-, -C≡C-, -NRCO-, -CONR-, -SO2NR-, -NRSO2-, - CR2OCR2-, -CR2SCR2-, -CR2NRCR2-, a C4-8 cycloheteroalkylene group, a C4-8 cycloalkylene group, a C5-12 arylene group, a C3-12 heteroarylene group, a polyalkyleneglycol moiety, a polylactic acid moiety, and a polyglycolic acid moiety;

each n is independently an integer of value 0 to 25;

each B is independently chosen from Q or A, and where (B) _p is a peptide or a pseudopeptide that is a substrate for one or more transglutaminase;

Q is a cyclic peptide;

each A is independently an amino acid residue or an amino acid analogue residue;

p is an integer of value 4 to 45;

J is R or R ¹ ;

Z is a protecting group;

m is 0 or 1 ; and

M is a detectable moiety or is configured to bind a detectable moiety. (B)p may be a peptide or a pseudopeptide that is a substrate for Factor Xllla.

Y may comprise one or more peptide fragments selected from the group consisting of: a2-antiplasmin, fibronectin, beta-casein, tetanus, amyloid, trappin, and polyglutamine residues, said peptide fragments comprising at least four amino acid residues.

The peptide fragment may be from a2-antiplasmin.

The amino acid in the 2-position from the peptide N-terminus may be glutamine.

X may be -NRJ, giving the formula:

R may be H. n may be an integer of value 0 to 8. Optionally n is 4. J may be R.

Z may be acetyl (Ac) and m may be 1 . p may be an integer of value 4 to 16, optionally an integer of value 8 to 12. Typically, p is 12.

The detectable moiety may be detectable by at least one of PET, optical imaging, NIR imaging, NIR tomography and optical tomography. The detectable moiety may be detectable by at least one of PET, fluorescence imaging and fluorescence tomography.

The detectable moiety may comprise at least one of a fluorescence emitter and a positron emitter.

The detectable moiety may comprise a positron emitter selected from the group consisting of: ¹⁸ F, ¹¹ C, ¹³ N, ¹⁵ 0, ⁶⁸ Ga, ⁸⁹ Zr, and ⁸² Rb. The detectable moiety may comprise ¹⁸ F.

The detectable moiety may comprise a fluorescence emitter selected from the group consisting of: cyanine dyes, indolenine based dyes,

benzoindolenine based dyes, phenoxazines, BODIPY dyes, rhodamines, Si-rhodamines, Alexa dyes, and derivatives thereof.

The detectable moiety may comprise a cyanine dye selected from the group consisting of: Cy5, Cy5.5, Cy7, Cy7.5, and derivatives thereof. The detectable moiety may comprise Cy5 or a derivative thereof.

The cyanine dye may comprise at least one hydrophilic group.

The cyanine dye may comprise at least one sulphonate group.

The cyanine dye may comprise at least two sulphonate groups. The compound may comprise the formula: where E is a peptide that is a substrate for at least one

transglutaminase, comprising the amino acid sequence:

(Z) _m -Asn-Gln-Glu-Gln-[Xaa ¹ ]r-Xaa ² - [SEQ ID NO. 4] where:

Xaa ¹ is any amino acid;

Xaa ² is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine;

r is an integer of value 0 to 41 ; and

Xaa ² further comprises at least one detectable moiety or comprises at least one moiety configured to bind at least one detectable moiety.

The compound may comprise the formula:

Xaa ² may be Lys, Tyr, or Cys. Typically, Xaa ² is Lys. r may be an integer of value 0 to 12. Optionally, r is an integer of value 4 to 8. Typically, r is 8.

Xaa ¹ may comprise the amino acid sequence:

-Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ - [SEQ ID NO. 5] where:

Xaa ³ is Val, Ala, lie, Leu, Met, Phe, Tyr, or Trp;

Xaa ⁴ is Ser, Thr, Asn, Gin, or Nle;

Xaa ⁵ is Pro or a pseudoproline derivative;

Xaa ⁶ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;

Xaa ⁷ is Thr, Ser, Asn or Glu;

Xaa ⁸ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; Xaa ⁹ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; and Xaa ¹⁰ is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.

Xaa ¹ may comprise the amino acid sequence:

-Val-Ser-Pro-Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ - [SEQ ID NO. 6] where:

Xaa ⁶ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; Xaa ⁷ is Thr, Ser, Asn or Glu;

Xaa ⁸ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; Xaa ⁹ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; and Xaa ¹⁰ is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.

Xaa ¹ may comprise the amino acid sequence:

-Val-Ser-Pro-Xaa ⁶ -Thr-Leu-Leu-Lys- [SEQ ID NO. 7] where:

Xaa ⁶ is Leu or Tyr.

Xaa ¹ may comprise the amino acid sequence:

-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys- [SEQ ID NO. 8].

E may be a peptide that is a substrate for Factor XI I la.

According to a second aspect of the invention there is provided a peptide comprising the amino acid sequence:

(Z) _m -Asn-Gln-Glu-Gln-[Xaa ¹ ]s-Xaa ² -(Z)m [SEQ ID NO. 4] where:

Xaa ¹ is any amino acid; Xaa ² is Lys, Tyr or Cys Homolysine, Ornithine,

Homocysteine, or Homotyrosine;

s is an integer of value 0 to 41 ; and

Xaa ² further comprises at least one detectable moiety.

The peptide may be a substrate for at least one transglutaminase. s may be an integer of value 0 to 12. Optionally, s is an integer of value 4 to 8. Typically, s is 8.

Xaa ² may be Lys, Tyr, or Cys. Typically, Xaa ² is Lys.

Xaa ¹ may comprise the amino acid sequence:

-Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ - [SEQ ID NO. 5] where:

Xaa ³ is Val, Ala, lie, Leu, Met, Phe, Tyr, or Trp; Xaa ⁴ is Ser, Thr, Asn, Gin, or Nle;

Xaa ⁵ is Pro or a pseudoproline derivative;

Xaa ⁶ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; Xaa ⁷ is Thr, Ser, Asn or Glu;

Xaa ⁸ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; Xaa ⁹ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; and

Xaa ¹⁰ is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.

Xaa ¹ may comprise the amino acid sequence:

-Val-Ser-Pro-Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ - [SEQ ID NO. 6] where:

Xaa ⁶ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; Xaa ⁷ is Thr, Ser, Asn or Glu; Xaa ⁸ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;

Xaa ⁹ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; and

Xaa ¹⁰ is Lys, Tyr, Cys, Homolysine, Ornithine, Homocysteine, or Homotyrosine.

Xaa ¹ may comprise the amino acid sequence:

-Val-Ser-Pro-Xaa ⁶ -Thr-Leu-Leu-Lys- [SEQ ID NO. 7] where:

Xaa ⁶ is Leu or Tyr.

Xaa ¹ may comprise the amino acid sequence:

-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys- [SEQ ID NO. 8].

The peptide may be a substrate for Factor XI I la.

According to a third aspect of the invention there is provided a preparation for human or animal administration comprising the compound of the first aspect or the peptide of the second aspect. According to a fourth aspect of the invention there is provided a kit comprising the compound of the first aspect or the peptide of the second aspect useful in the preparation of a composition for human or animal administration. According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use as a medicament. According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use in diagnosis, surgery, treatment or therapy monitoring. According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use as an imaging agent.

According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use in the diagnosis, surgery, treatment or monitoring therapy of rheumatoid arthritis, atherosclerosis, fibrosis or cancer.

According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use in the diagnosis, surgery or treatment of sites of thrombosis, embolism or inflammation.

According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use in detecting tissue transglutaminse.

According a further aspect of the invention there is provided the compound of the first aspect or the peptide of the second aspect for use in detecting Factor XI I la activity.

According to a further aspect of the invention there is provided a method of diagnosis, surgery, treatment or therapy monitoring using the compound of the first aspect or the peptide of the second aspect. According to a further aspect of the invention there is provided a method of imaging using the compound of the first aspect or the peptide of the second aspect. According to a further aspect of the invention there is provided a method of diagnosing, treating or monitoring therapy of rheumatoid arthritis, atherosclerosis, fibrosis or cancer using the compound of the first aspect or the peptide of the second aspect. According to a further aspect of the invention there is provided a method of diagnosing, treating or monitoring therapy of sites of thrombosis, embolism or inflammation using the compound of the first aspect or the peptide of the second aspect. According to a further aspect of the invention there is provided a method of detecting tissue transglutaminse using the compound of the first aspect or the peptide of the second aspect.

According to a further aspect of the invention there is provided a method of detecting Factor XI I la activity using the compound of the first aspect or the peptide of the second aspect.

According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect.

According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect in diagnosis, surgery, treatment or therapy monitoring. According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect as an imaging agent. According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect in the diagnosis, surgery, treatment or therapy monitoring of rheumatoid arthritis, atherosclerosis, fibrosis or cancer. According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect in the diagnosis, treatment or therapy monitoring of sites of thrombosis, embolism or inflammation. According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect in the detection of tissue transglutaminse.

According to a further aspect of the invention there is provided the use of the compound of the first aspect or the peptide of the second aspect in the detection of Factor Xllla activity.

The invention also includes kits for the preparation of the above

compounds, and the use of these and related compounds in the diagnosis, therapy or therapy monitoring of thrombosis, embolism, atherosclerosis, inflammation, rheumatoid arthritis, fibrosis or cancer.

The alternative features and different embodiments as described apply to each and every aspect and each and every embodiment thereof mutatis mutandis. By the term "cyclic peptide" is meant a sequence of 5 to 15 amino acids in which the two terminal amino acids (or two other, non-terminal amino acids) are bonded together by a covalent bond which may be a peptide or disulphide bond or a synthetic non-peptide bond such as a thioether, phosphodiester, disiloxane or urethane bond.

By the term "amino acid" is meant an L-or D-amino acid, amino acid analogue or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e., a single enantiomer and hence chiral, or a mixture of enantiomers.

The amino acids of the present invention are optically pure. By the term "amino acid mimetic" is meant synthetic analogues of naturally occurring amino acids which are isosteres, i.e. have been designed to mimic the steric and electronic structure of the natural compound. Such

isosteres are well known to those skilled in the art and include but are not limited to depsipeptides, retro-inverso peptides, thioamides, cycloalkanes or 1 ,5-disubstituted tetrazoles [M. Goodman, Biopolymers, 24, 137, (1985)].

By the term "protecting group" is meant a biocompatible group which inhibits or suppresses in vivo metabolism of the peptide or amino acid at the amino or carboxyl terminus. Such groups are well known to those skilled in the art and are suitably chosen from, for the N (or amine) terminus: acetyl (Ac), Boc (where Boc is tert-butyloxycarbonyl), Fmoc (where Fmoc is fluorenyimethoxycarbonyl), benzyloxycarbonyl,

trifluoroacetyl, allyloxycarbonyl, Dde [i.e., 1 - (4,4-dimethyl-2,6- dioxocyclohexylidene) ethyl], or Npys (i. e. 3-nitro-2-pyridine sulfenyl); and for the C (or carboxyl) terminus: a carboxamide, tert-butyl ester, benzyl ester, cyclohexyl ester, or amino alcohol. Protecting groups also includes capping groups.

A "detectable moiety" is a moiety that emits a signal or is suitable for diagnostic imaging of the human body and may be a radioisotope for radiopharmaceutical imaging or therapy, a paramagnetic metal or species for MRI contrast imaging, a radiopaque group or metal for X-ray contrast imaging, a gas microbubble ultrasound contrast agent, or a suitable dye for detection by external or internal light imaging. In particular, a detectable moiety may be a moiety that is detectable by PET (e.g., a positron emitter), or a moiety that is detectable by NIR or optical imaging (e.g., a fluorescence emitter).

The peptide fragments comprise at least 4 and optionally 4 to 20 amino acid residues, typically 4 to 15 amino acid residues.

The amino acid sequences of a2-antiplasmin, fibronectin, beta-casein, fibrinogen and thrombospondin can be found in the following references: c(2-antiplasmin precursor [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)]; thrombospondin-1 precursor [V. Dixit et al, Proc. Natl. Acad. Sci., USA, 83, 5449, (1986)]; R. F.

Doolittle, Ann. Rev. Biochem., 53, 195, (1984). The amino acid sequence can be taken from the N-terminus of:

(i) c(2-antiplasmin: i.e.,

• NH ₂ -Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys-OH [SEQ ID NO. 2]

or variants of this in which one or more amino acids have been

exchanged, added or removed such as: • NH ₂ -Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys-Gly-O H [SEQ ID NO. 9],

• NH ₂ -Asn-Gln-Glu-Ala-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys-Gly-O H [SEQ ID NO. 10],

• NH ₂ -Asn-Gln-Glu-Gln-Val-Gly-OH [SEQ ID NO. 1 1 ]; or casein: i.e.,

• Ac-Leu-Gly-Pro-Gly-Gln-Ser-Lys-Val-lle-Gly [SEQ ID NO. 12].

According to a further aspect of the invention, there is provided compound of formula:

According to a further aspect of the invention, there is provided compound of formula:

NH ₂

Fluorine may be ¹⁸ F.

According to a further aspect of the invention, there is provided

compound of formula:

Fluorine may be ¹⁸ F. n may be 2, but can be any integer between 2 and 8.

Fluorine may be ¹⁸ F. n may be 2, but can be any integer between 2 and 8.

According to a further aspect of the invention, there is provided a compound of formula:

The alternative features and different embodiments as described apply to each and every aspect and each and every embodiment thereof mutatis mutandis.

Brief Description of the Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the drawings, in which:

Figure 1 is a plot of the RFU obtained using compound 1 in a platelet-rich plasma clot test using a Badimon chamber;

Figure 2 shows the imaging of ex-vivo formed human thrombus using Compound 1 ;

Figure 3 is the image of the binding of Compound 1 to in vivo murine model of arterial thrombosis

Figure 4 is the imaging of ex vivo binding experiment of ¹⁸ F- Compound 2 to thrombus;

Figure 5 is the imaging of ex vivo binding experiment of ¹⁸ F- Compound 2 to thrombus using microPET/CT;

Figure 6 is the in vivo microPET/CT (maximum intensity projection) imaging of mouse with active thrombus;

Figure 7 is a plot showing data obtained;

Figure 8 shows the biodistribution of ¹⁸ F-Compound 2 (30 seconds, 5 minutes, 10 minutes, 30 minutes, 60 minutes and 120 minutes; Figure 9 shows the specific accumulation of ¹⁸ F-Compound 2 in mice with active thrombus in contrast to the contralateral vein and the blockade animal;

Figure 10 is a plot of the standard uptake values obtained for mice with active thrombus and blockade animals (contralateral thrombus values also showed); and

Figure 1 1 is a plot of the standard uptake values obtained for mice with active thrombus and blockade animals (contralateral thrombus values also showed), expressed in g/ml_.

Detailed Description

Examples of Preparation of NIR, Optical and PET Imaging Agents There is described below the experimental procedure that was followed for the solid phase synthesis of two 13 amino acid peptides based around a common 12 amino acid peptide core, and their post synthetic

derivatisation to obtain a near infrared optical agent and a PET agent. The following references were used in the syntheses: Bioorganic &

Medicinal Chemistry Letters 16 (2006) 6190-6193; Nuclear Medicine and Biology 34 (2007), 315-323; Drug Metabolism and Disposition 35 (2007), 1979-1984; BioMed Research International (2014), Article ID 361329; Bioconjugate Chem. 25 (2014), 82-92. WO 99/60018 describes ^99m Tc-compounds that are radiotracers developed as an in vivo diagnostic markers of thromboembolic disease. The 12 amino acid peptide, (Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys [SEQ ID NO. 3]), is based on the N-terminal sequence of human 02- antiplasmin. The N-terminal sequence of a2-antiplasmin has been demonstrated to be a Factor XI I la substrate. HPLC methods

• Eluent A: 0.1 % trifluoroacetic acid in ultrapure water

• Eluent B: 0.09% trifluoroacetic acid in HPLC grade acetonitrile

Columns:

• Column A: Agilent™ Eclipse Plus C18, 3.5 μιτη, 4.6x50mm

• Column B: Agilent™ PrepHT SB-C18 Preparative, 7 μητι,

21 .1 x150mm

• Column C: Agilent™ Zorbax SB-C18 Semi-preparative, 5 pm,

9.4x100mm

Detection:

Detection was carried out at 21 Onm, 220 nm, 254nm, 280nm, 300nm and 647nm when relevant. Fluorescence detection was also performed (excitation: 647nm, emission 670nm) when relevant. Full DAD spectra were also recorded (190-500nm and 190-800 nm when relevant) for all runs.

Methods:

• Method 1 (23 min, eluents A and B, column A, 0.8 mL/min): 5 % B during 2.5 min, 5 % to 90 % B over 17 min, then 90% B for 1 .5 min, then 90 % to 5 % B over 0.5 min, then 5 % B for 1.5 min.

· Method 2 (23 min, eluents A and B, column A, 0.8 mL/min): 5 % B during 2.5 min, 5 % to 90 % B over 17 min, then 90% B for 1 .5 min, then 90 % to 5 % B over 0.5 min, then 5 % B for 1.5 min. UV detection 645 nm and fluorescence (excitation 647nm, emission 670nm). • Method 3 (26 min, eluents A and B, column B, 20 mL/min): 5 % B during 2.5 min, 5 % to 85 % B over 20 min, then 85% B for 0.5 min, then 85 % to 5 % B over 1 min, then 5 % B for 2 min. Fraction collection based on 645nm channel.

· Method 4 (34 min, eluents A and B, column C, 7 mL/min): 5 % B during 2.5 min, 5 % to 65 % B over 24 min, 65 % to 85 % B over 0.5 min, then 85% B for 2 min, then 85 % to 5 % B over 0.5 min, then 5 % B for 4.5 min. Fraction collection based on 210 nm channel.

· Method 5 (46 min, eluents A and B, column B, 20 mL/min): 5 % B during 2.5 min, 5 % to 85 % B over 40 min, then 85% B for 0.5 min, then 85 % to 5 % B over 1 min, then 5 % B for 2 min. Fraction collection based on 645nm channel.

Synthesis of Optical Agents

The first agent synthesised was the near infrared labelled peptide depicted below:

Compound 1

Synthesis of Peptide Substrate: A solid supported Ac-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys- Lys(Mmt) [SEQ ID NO. 13] peptide was prepared using Fmoc solid phase peptide synthesis protocol according to the following protocol:

• Synthetic scale: 0.1 mmol

• Resin: Rink amide chemMatrix, 0.5 mmol/g.

• Presynthetic washes: MeOH (2x1 min - 3 ml_), DMF (2x1 min - 3 ml_), DCM (3x1 min - 3 ml_), TFA/DCM 1 :99 (3x1 min - 3 mL), DIPEA/DCM 1 : 19 (3x1 min - 3 ml_), DCM (3x1 min - 3 ml_).

• Peptide couplings: Fmoc-AA-OH (3 equiv.) and HBTU (2.9 equiv.) were dissolved in 2 ml_ DMF. Sonication was used if needed to dissolve AA. DIPEA (6 equiv.) was added to the mixture. The coupling mixture was left for 1 min and poured onto the resin submitted to 75°C microwave irradiation for 5 minutes. The resin was then washed with DMF (5x0.5 min - 3 ml_) and DCM (5x0.5 min - 3 ml_). A Kaiser ninhydrin test was performed and in the case free amines were still present the coupling procedure was repeated.

• Fmoc deprotection: The resin was suspended in a solution of

piperidine in DMF (1 :4 v/v) - 4 ml_: 2x1 min, 2x10 min, 1x5 min. The resin was then washed with DMF (5x0.5 min - 3 ml_) and DCM (5x0.5 min - 3 ml_).

• Acetylation of N-terminal amine: DIPEA (20 equiv), AC2O (10 equiv.) in DMF were poured onto resin. After 45 minutes the resin was then washed with DMF (5x0.5 min - 3 ml_) and DCM (5x0.5 min - 3 ml_).

Near Infrared Labelling:

The key Lysine Mmt protecting group was then deprotected and labelled with Cy5 before cleaving off the compound from the solid support following the protocol below: Mmt deprotection: Protecting group was removed by treating the resin with TFA in CH2CI2 (1 :99 v/v) for 15 min. The deprotection step was repeated 3 times.

Coupling with sulfo-Cy5: The resin was swollen in DCM (2 ml_). Cy5.0 (1 .5 equiv.) in 2 ml_ DMF poured onto resin. DIPEA (4 equiv.) added. Pybop (1 .5 equiv.) in DMF (2 ml_) was then added to the resin and the mixture was shaken for 15h. The resin was then washed with DMF (5x0.5 min - 3 ml_) and DCM (5x0.5 min - 3 ml_). Deprotection/cleavage: TFA/TIS/H2O (90:5:5 v/v) 12 ml_, 1 h.

Solvents removed in vacuo. Cold Et20 precipitation was performed and the resulting crude peptide was centrifuged at 3260 rpm, -5°C, 5 min. Mcrude= 60 mg

Purification:

The final compound was purified by semi preparative HPLC using Method 3 (2 injections). The final compound was obtained in good yield (23.9 mg, 1 1 %).

Characterisation:

• HPLC (Method 2): t _R =10.5 min. P>98% (645

• MALDI: 2233 (M+)

A further near infrared labelled peptide as depicted below was prepared in accordance with the above methods and in accordance with the further details provided below:

Compound Name: EMI-R001-TB1-12

Compound name: IR Dye 800 CW NHS Ester

t

Compound 6

Reagents:

Table 1 : Reagents used in the amine deprotection of resin bound peptide.

Reagent Equi n M (g/mol) m C Supplier Lot v. (mmol) (mg) (M) ( L)

EMI-R001-TB1- 1.00 0.0029 0.209 13.68 0.03 - - EMI-R001-

12 mmol/g 0 TB1-12 IR Dye 800 CW 1.50 0.0043 1165.200 4.999 0.04 Li-cor C60215-04

NHS Ester

(CAS No.: 929- 70021)

DIPEA 1.50 0.0043 129.250 0.556 0.75 Sigma

(density 0.742 Aid rich

g/cm ³ )

(dry)

DMF 100 Sigma

(dry) Aid rich

Table 2: Reagents used in preparation of Compound 6.

Method:

NH2OH, HCI (70.044 mg, 1 .008 mmol, 144 equiv.) and Imidazole (51.468 mg, 0.756 mmol, 108 equiv.) were suspended in NMP (0.28 ml_).

Immediately before the reaction, 5 volumes of the solution were diluted with 1 volume of DCM (56 μΙ_). The solution was poured onto the resin EMI-R001 -TB1 -12, shaken for 4 hours and the reaction followed using the ninhydrin test to give a positive result.

The resin was washed with 20 % piperidine in DMF, DMF and DCM (5 times for 0.5 minutes). IR Dye 800 CW NHS Ester (5 mg, 0.0043 mmol, 1 .5 equiv.) in DMF (100 μΙ_) with DIPEA (0.75 μΙ_, 0.0043 mmol, 1 .5 equiv.) was added to the resin, and left for 5 hours with shaking. The labelled peptide was then cleaved, TFA/TIS/H2O (v/v 95:2.5:2.5) to give Compound 6 (EMI-R001 -TB1 -13) 1 .4 mg (10 %). Analytical HPLC: (Eclipse Plus C18, 3.5 μιη, 4.6 x 50 mm) sequence no: 1007, t _R: 8.9 min, P > 90 %. MALDI/TOF: m/z found; 2576.68.

Synthesis of PET Precursor and Cold PET Agents A further agent synthesised was the 'cold' PET agent depicted below:

Compound 2

Synthesis of Peptide Substrate:

A solid supported Ac-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys- Lys(Boc2AoA) [SEQ ID NO. 13] peptide was prepared using Fmoc solid phase peptide synthesis protocol and cleaved off the solid support according to the following protocol:

Synthetic scale: 0.25 mmol

Resin: Rink amide chemmatrix, 0.5 mmol/g.

Presynthetic washes: MeOH (2x1 min - 3 ml_), DMF (2x1 min - 3 ml_), DCM (3x1 min - 3 ml_), TFA/DCM 1 :99 (3x1 min - 3 mL), DIPEA/DCM 1 : 19 (3x1 min - 3 ml_), DCM (3x1 min - 3 ml_) Peptide couplings: Fmoc-AA-OH (3 equiv.) and HBTU (2.9 equiv.) were dissolved in 2 ml_ DMF. Sonication was used if needed to dissolve AA. DIPEA (6 equiv.) was added to the mixture. The coupling mixture was left for 1 min and poured onto the resin and shaken for 2h. The resin was then washed with DMF (5x0.5 min - ml_) and DCM (5x0.5 min - 8 ml_). A Kaiser ninhydrin test was performed and in the case free amines were still present the coupling procedure was repeated. Fmoc deprotection: The resin was suspended in a solution of piperidine in DMF (1 :4 v/v) - 8 mL: 2x1 min, 2x10 min, 1x5 min. The resin was then washed with DMF (5x0.5 min - 8 mL) and DCM

(5x0.5 min - 8 mL).

Acetylation of N-terminal amine: DIPEA (20 equiv), AC2O (10 equiv.) in DMF were poured onto resin. After 45 minutes the resin was then washed with DMF (5x0.5 min - 3 mL) and DCM (5x0.5 min - 3 mL). Deprotection/cleavage: TFA/TIS/H2O (90:5:5 v/v) 12 mL, 1 h.

Solvents removed in vacuo. Cold Et20 precipitation was performed and the resulting crude peptide was centrifuged at 3260 rpm, -5°C, 5min. Mcrude = 150 mg.

Purification:

The final compound was purified by semi preparative HPLC using Method 5 (4 injections). The final compound was obtained in moderate yield

(30mg, 4%). The structure of the peptide is presented below:

,

Compound 3

Characterisation:

• HPLC (Method 1 ): t _R =10.6 min, P>80% (254nm)

• ESI+/MS: 1889 (M+2TFA) ⁺ p-F benzaldehyde Labelling:

The aminooxyacetic acid functionality on one of the Lysine side chains was labelled with p-F benzaldehyde following the protocol below: To the oxyamine peptide (5 mg, 2.65 pmol) dissolved in H2O with 0.1 % TFA (500 u L) was added 4-fluorobenzaldehyde (0.57 uL, 2 equiv.). The reaction mixture was heated up to 70°C for 45 min.

This provided Compound 2 as depicted above.

Purification:

Compound 2 was purified by semi preparative HPLC using Method 4 (1 injection). The compound was obtained in moderate yield (0.9 mg, 41 %). Characterisation:

• HPLC (Method 1 ): 12.33 min, P>95% (254nm)

• ESI+/MS: 1897 (M+TFA+H ₂ 0) ⁺

Synthesis of further Cold PET Agents

Further 'cold' PET agents are depicted below:

Compound 4

Compound 5

In Compounds 4 and 5, n is 2, but can be any integer between 2 and 8.

The peptide substrate of Compounds 4 and 5 can be synthesised using the same synthetic route as for outlined for Compound 1 and 3. The introduction of the fluorinated PET emitting moiety is carried out by click chemistry as follows:

· selective deprotection of the ε amine on the lysine located near the

C-terminal;

• introduction of an alkyne or an azide moiety onto the lysine,

respectively by using propiolic acid or 3-azidopropanoic acid; and

• subsequent copper iodide (Cul) catalysed click chemistry,

respectively using an azidofluoroalkane or an alkynofluoroalkane

Synthesis of ¹⁸ F PET Agents

¹⁸ F versions of Compounds 2, 4 and 5 were prepared using a G

Healthcare FASTlab™ synthesiser to provide ¹⁸ F-Compound 2,

Compound 4 and ¹⁸ F-Compound 5 respectively.

Synthesis of Further PET Agents A further agent, this time designed to bind AI ¹⁸ F (i.e., configured to bind a detectable moiety), was prepared as follows:

Compound 7

Table 3: Reagents used in preparation of Compound 7.

FXIII resin (EMIR-002-TB1 -12, 20 mg, 4.22 mmol) was placed in a 1 mL solid phase extractor. Hydroxylamine hydrochloride (26 mg, 90 equiv.) and imidazole (19 mg, 67.5 equiv.) were dissolved in NMP (105 uL) and the solution diluted with CH2CI2 (20 μΙ_) and poured onto resin. The reaction was placed on an orbital shaker for 3 hours and subsequently washed with DMF (1 ml_, 5 x 30 seconds) and CH2CI2 (1 ml_, 5 x 30 seconds). A positive ninhydrin test was performed. DIPEA (3.67 μΙ_, 5 equiv.) in DMF (150 μΙ_) was added to the resin and NOTA-NHS (4.18 mg, 1 .5 equiv.) dissolved in dry DMF (75 μί) was also subsequently added. The resin was placed on an orbital shaker for 1 hour and was then washed with DMF (1 ml_, 5 x 30 seconds) and CH2CI2 (1 ml_, 5 x 30 seconds). A positive ninhydrin test was performed. A sample of resin (3 to 5 mg) was taken for test cleavage with TFA/TIS/H2O

(95:2.5:2.5 v/v, 100 μΙ_). Analytical HPLC showed two product peaks as well as some starting amine left, confirming ninhydrin test.

DIPEA (10 μΙ_, 13 equiv.) in DMF (150 μΙ_) was added to NOTA-NHS (4.18 mg, 1 .5 equiv.) dissolved in dry DMF (150 μΙ_) and was subsequently added to the resin in dry DMF (150 μΙ_). The resin was placed on an orbital shaker for 1 hour and then washed with DMF (1 ml_, 5 x 30 seconds) and CH2CI2 (1 ml_, 5 x 30 seconds). A negative ninhydrin test was performed. The product was cleaved off with TFA/TIS/H2O

(95:2.5:2.5 v/v, 1 ml_). Analytical HPLC of the crude mixture showed two peaks. MALDI of the crude mixture failed to conclude to the presence of the product in the crude.

The crude product was dissolved in 300 μΙ_ water (0.1 % TFA), and purified by semi preparative HPLC (TFA95 AcN5%, 1 % per minute, 7 mL per min, column Zorbax C18 9.4 x 250mm, 1 injection, three fractions, tRi=26.5 min, tR2=26.8 min, tR3=27.9 min). Fraction 1 was considered insignificant due to its very low volume. Compound 7 (fraction 2) was obtained in undetermined yield (too little compound) but excellent purity (>97%, 254 nm). HPLC (Compound 7 - 2.1x150mm col) - Trace files 1066 and 1067 (QC) †R starting amine: 27.0 min tR product 1 - peak B: 28.0 min tR product 2 - peak B: 29.7 min.

MALDI/TOF: Product 1 (peak 2): 1875.55; Product 2 (peak 3): 1801 .6. ESI+/MS: Product 1 (peak 2): 1874.47; Product 2 (peak 3): 1800.8.

ESI-/MS: Product 1 (peak 2): 1872.6; Product 2 (peak 3): 1803.6; 1798.6.

General Structure for Cyanine Dyes

The cyanine dyes used with the optical and NIR imaging agents described herein can be, but are not limited to, derivatives of the following general structure referred to as Cy5:

Each R ² is typically independently selected from the group consisting of: H, CI, Ci-4 alkyl, Ci- ₄ alkenyl, Ci _-4 alkynyl, Ci _-4 alkoxyalkyl and Ci _-4 hydroxyalkyl. R ² may also be the attachment point for a peptide or a substrate for a transglutaminase, or for a moiety that links Cy5 to a peptide or a substrate for a transglutaminase.

R ³ can be a hydrophilic group such as, for example, sulphonate. In one example, Cy5 has the following structure:

Other cyanine dyes that can be used include Cy5.5, Cy7 and Cy7.5 and derivatives of such dyes, general structures for which known in the art.

Imaging Experiments

The compounds as described above were studied in the following experiments.

Compound 1

A platelet-rich plasma (PRP) clot test was carried our using a Badimon chamber (ex vivo model of thrombosis that mimics flow conditions within the coronary circulation of man). These ex vivo experiments carried out on human thrombus using compound 1 are shown in graph form in Figure 1 .

Clots were labelled by compound 1 (referred to on the graph as

EMI07678), but not the Cy5 dye alone (referred to on the graph and in the discussion of these results as Cy5) and not in the presence of an inhibitor (referred to on the graph as EM I07678 + IAA). This illustrates that compound 1 is useful as an imaging agent. Compound 1 and Cy5 were added at 100 nM. After incubation clots where washed to remove nonspecific unbound compounds. The results are also summarised in Table 4 below.

Table 4: Binding of EMI07678 to ex vivo human thrombi: specificity shown by inhibition of FXIIIa (with iodoacetamide, IAA) and Cy5 control Figure 2 illustrates the same ex vivo experiments carried out on ex vivo formed human thrombus using Compound 1 and that show specific binding of compound 1 to ex vivo human thrombus.

Compound 1 (referred to on the image as EM07678) conjugated with Cy5 fluorescent probe readily binds to thrombus (B and C) and this is inhibited by Factor Xllla inhibition (D) . No non-specific binding to thrombus by Cy5 (E) or auto fluorescence (F) was observed and accounted to determine specific binding. Quantification of binding demonstrates discrimination by 2 to 3 orders of magnitude (see Table 1 ). in vivo experiments were carried out as follows. Compound 1 (80 g/kg i.v.) was injected 5 min after application of FeCl3 (10%) to induce thrombosis in the mouse left femoral artery. Fluorescence was detected in vivo using Fluorescence Molecular Tomography (Visen FMT2500) at intervals following probe injection. The results are shown in Figure 3. The in vivo result shows that Compound 1 is actively accumulated into areas of active clot thrombosis, from 5 min though to 60 min,

demonstrating that thrombus can be imaged against background tissues. The positive image of thrombi indicates that clinical imaging of thrombi and areas of activated transglutaminase (e.g., Factor Xllla) is possible.

Positive accumulation and imaging also gives an indication of the stability of Compound 1 .

Compound 2

The following ex vivo experiments were carried out using ¹⁸ F-Compound 2 as well as Compound 2 (blockade experiments).

Figure 4 shows on the left side the total binding (incubation with just the radiotracer) and on the right side non-specific binding (incubation with radiotracer and inhibitor, iodoacetamide).

Figure 5 shows PET and CT data for the total binding (incubation with just the radiotracer) and on the right side non-specific binding (incubation with radiotracer and inhibitor, iodoacetamide). Total and nonspecific binding are shown side to side.

In vivo studies were also carried out. Micro PET/CT and limited organ bio-distribution was performed on naive control mice and mice with active thrombi (after application of FeCl3 (10%) to induce thrombosis in the mouse left femoral artery). Imaging studies were performed in control mice and mice with active thrombi, with imaging data taken at 30 seconds, 5 minutes, 10 minutes, 30 minutes and 60 minutes post injection, and summed images at 1 hour and 2 hours post imaging (Figures 6 and 7).

Animal models indicated that ¹⁸ F-Compound 2 was cleared rapidly from the blood and the majority of clearance of the agent was via the renal system, indicated by radioactivity in the kidneys and bladder (Figure 8). This data indicates that ¹⁸ F-Compound 2 has suitable imaging

characteristics for the imaging of thrombi, lung disorders, and other cardiovascular diseases. Specific accumulation of ¹⁸ F-Compound 2 was shown in mice with active thrombi, with increased uptake seen in the thrombus vs the control contralateral femoral vein, and specific accumulation was also shown by blockade studies (pre-injection of excess Compound 2) by the reduction of the uptake of ¹⁸ F-Compound 2 into clots (Figures 9, 10 and 1 1 )

Various modifications and variations to the described embodiments of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be

understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

The following abbreviations are used herein:

HPLC: High Performance Liquid Chromatography

TFA: Trifluoroacetic Acid

AcN: Acetonitrile

PET: Positron Emission Tomography

CT: Computed Tomography

FL: Fluorescence Level

FMT: Fluorescence Molecular Tomography

RFU: Relative Fluorescence Units

IAA: lodoacetamide

PRP: Platelet-Rich Plasma

ESI: Electrospray lonisatiom

MS: Mass spectrometry

PCTMIN: Percent per minute

MLMIN: mL per minute

p-F: para-fluoro

Et ₂ 0: ditehyl ether

DIPEA: diisopropylethylamine

DMF: Λ/,/V-dimethylform amide

DCM: dichloromethane

TIS: triisopropylsilane

AA: aminoacid

Fmoc: 9-fluorenylmethoxycarbonyl

Boc: fe/f-butylcarbonyl

Dde: 1 - (4,4-dimethyl-2,6- dioxocyclohexylidene

Npys: 3-nitro-2-pyridine sulfenyl

Ac: acetyl

HBTU: 2-(1 H-benzotriazol-1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate MeOH: methanol

AoA: aminooxyacetic acid

MALDI: matrix assisted laser desorption ionisation

Mmt: Monomethoxytrityl

DAD: diode array detector

NIR: near infrared

SUV: standardized uptake value

MIP: maximum intensity projection

NMP: N-Methyl-2-pyrrolidone

NOTA-NHS: 2,2'-(7-(2-((2,5-dioxopyrrolidin-1 -yl)oxy)-2-oxoethyl)-1 ,4,7- triazonane-1 ,4-diyl)diacetic acid

Sequence Listing Free Text

SEQ ID NO. 1

Asn-Gln-Glu-Gln Tetrapeptide seguence that is a substrate for the blood enzyme commonly known as Factor Xllla.

SEQ ID NO. 2

Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys

12-mer peptide seguence from the N-terminus of the a2-antiplasmin enzyme.

SEQ ID NO. 3

NH ₂ -Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys-OH

Synthetic analogue of 12-mer peptide seguence from the N-terminus of the a2-antiplasmin enzyme. SEQ ID NO. 4

Asn-Gln-Glu-Gln-[Xaa ¹ ]r-Xaa ²

Xaa ¹ is any amino acid;

Xaa ² is Lys, Tyr, Cys, Homolysine, Orn, Homocysteine, or Homotyrosine; and

r is an integer of value 0 to 41 .

Peptide seguence, or synthetic analogue peptide seguence, that is a substrate for the blood enzyme commonly known as Factor Xllla. SEQ ID NO. 5

Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰

Xaa ³ is Val, Ala, lie, Leu, Met, Phe, Tyr, or Trp;

Xaa ⁴ is Ser, Thr, Asn, Gin, or Nle;

Xaa ⁵ is Pro or a pseudoproline derivative;

Xaa ⁶ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;

Xaa ⁷ is Thr, Ser, Asn or Glu;

Xaa ⁸ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;

Xaa ⁹ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; and

Xaa ¹⁰ is Lys, Tyr, Cys, Homolysine, Orn, Homocysteine, or Homotyrosine. Peptide sequence, or synthetic analogue peptide sequence, that is a substrate for the blood enzyme commonly known as Factor Xllla. SEQ ID NO. 6

Val-Ser-Pro-Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰

Xaa ⁶ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;

Xaa ⁷ is Thr, Ser, Asn or Glu;

Xaa ⁸ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp;

Xaa ⁹ is Leu, Tyr, Val, Ala, lie, Met, Phe, or Trp; and

Xaa ¹⁰ is Lys, Tyr, Cys, Homolysine, Orn, Homocysteine, or Homotyrosine.

Peptide sequence, or synthetic analogue peptide sequence, that is a substrate for the blood enzyme commonly known as Factor Xllla.

SEQ ID NO. 7

Val-Ser-Pro-Xaa ⁶ -Thr-Leu-Leu-Lys

Xaa ⁶ is Leu or Tyr. Peptide sequence, or synthetic analogue peptide sequence, that is a substrate for the blood enzyme commonly known as Factor Xllla.

SEQ ID NO. 8

Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys

Peptide sequence, or synthetic analogue peptide sequence, that is a substrate for the blood enzyme commonly known as Factor Xllla.

SEQ ID NO. 9

Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys-Gly

Synthetic analogue peptide sequence of the a2-antiplasmin enzyme. SEQ ID NO. 10

Asn-Gln-Glu-Ala-Val-Ser-Pro-Leu-Thr-Leu-Leu-Lys-Gly

Synthetic analogue peptide sequence of the a2-antiplasmin enzyme.

SEQ ID NO. 1 1

Asn-Gln-Glu-Gln-Val-Gly

Synthetic analogue peptide sequence of the a2-antiplasmin enzyme. SEQ ID NO. 12

Leu-Gly-Pro-Gly-Gln-Ser-Lys-Val-lle-Gly Casein sequence. SEQ ID NO. 13

Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Leu-Lys-Lys Synthetic analogue peptide sequence of the a2-antiplasmin enzyme.