JP2003520255A - Magnetic resonance imaging using contrast agent prodrugs that are bioactivated by enzymatic cleavage - Google Patents

Abstract

(57) Abstract The present invention relates to a contrast agent for diagnostic magnetic resonance imaging. The compounds and compositions of the present application are in the form of targeted contrast agent prodrugs that contain cleavable chemical groups that reduce the binding of the contrast agent to the target molecule. The uncleaved prodrug has low affinity for the target protein and correspondingly exhibits low relaxivity. Activation of the prodrug occurs by enzymatic cleavage. The activated contrast agent binds to the target molecule and forms an active complex with the contrast agent and the target. This active contrast agent, after binding to the target protein, exhibits several times higher relaxivity than the uncleaved prodrug. This increased relaxivity improves contrast in MRI images.

Description

Detailed Description of the Invention

TECHNICAL FIELD OF THE INVENTION The present invention relates to contrast agents for diagnostic magnetic resonance imaging. In particular, the present invention provides a surprisingly improved relaxivity due to improved binding to amino acid targeting group proteins in the molecule after specific cleavage of the drug by peptidases.
y) relates to novel compounds. The present invention also includes these compounds and methods of using these compounds, as well as compositions for contrast enhancement during magnetic resonance imaging.

BACKGROUND OF THE INVENTION Diagnostic and therapeutic imaging techniques, such as magnetic resonance imaging (MRI), can utilize contrast agents to improve image contrast. These agents alter the intrinsic tissue response to the magnetic field and thus enhance the contrast between tissues in the image. The improved efficacy of contrast agents offers the promise of better sensitivity in detecting tissue or tissue defects.

Contrast agents utilize a variety of materials to improve the contrast of magnetic resonance images. For example, complexes between gadolinium or other paramagnetic metal ions and organic ligands are widely used to enhance and improve contrast. Gadolinium complexes enhance contrast by increasing the nuclear magnetic relaxation rate of protons found in water molecules available for contrast agents during MRI. (Car
avan, P.A. Ellison, J .; J. McMurry, T .; J. , And Lauffer, R .; B. (1999) Chem. Rev. 99: 2293
).

The relaxation rate of protons in these water molecules is increased compared to protons in other water molecules that are not available for contrast agents. This enhancement in relaxivity or relaxivity within a particular population of water protons results in the ability to collect additional image data in a given amount of time. This, in turn, results in an improved signal to noise ratio and an improved image contrast.

It has previously been established that in order to achieve the highest enhancement in relaxivity and, correspondingly, the optimal improvement in image contrast using this approach, the movement of the contrast agent must be limited. Was done. Thus, enhanced relaxivity can be achieved by limiting the tumbling motion of the entire molecule (Lauffer
R.K. B (1987) Chem. Rev. 87: 901-927).

One method that has been used to enhance the relaxivity of MRI contrast agents by limiting rotational movement utilizes macromolecules or rigid scaffolds to which multiple chelate ions are attached. (Shukla, R. et al., (1996) Mag. Reson. Med.
． 35: 928; Shukla, R .; B. Et al. (1997) Acta Radi
ol. 412: 121; Ranganathan, R .; S. Et al. (1998)
Invest. Radiol. 33: p. 779; Jacques, V .; ,
(1997) J. Alloys Cpd. 249: 173). Bound macromolecules slow the movement of molecules of the contrast agent and correspondingly enhance the relaxivity of the contrast agent. However, the images generated using these contrast agents suffer a high and unpleasant background due to the high signal and relaxivity of the contrast agent itself.

Another way to achieve this limitation of movement is by receptor-induced magnetization enhancement (Re
Cceptor Induced Magnification Enhancement
ment) (RIM). This method generated a new generation of gadolinium chelated imaging agents, where the drug has a reduced molecular motion when bound after administration. This RIM technique limits the movement of contrast agent molecules by binding them to target receptors or proteins.
This method has the advantage that the enhancement in signal intensity due to the enhanced relaxivity occurs only when it binds to the target protein, thus minimizing annoying background signals.

Even contrast agents with single chelated ions can be effectively immobilized by non-covalently binding to the target protein using the RIME principle. This non-covalent bond serves to specifically enhance the relaxivity of the contrast agent upon binding to the target protein. This approach is based on the first gadolinium-based blood pool contrast agent, M
Guided the discovery of S-325, which at the time of this application was in a phase III clinical trial for non-invasive angiography (Lauffer, RB; Parmel.
ee, D.E. J. Dunham, S .; Ouellet, H .; S. ; Dolan
R.K. P. Witt S .; McMurry, T .; J. ; Walovich
R.K. C. (1998) Radiology 207: 529). In the bloodstream,
This contrast agent does not covalently bind human serum albumin (HSA). This interaction enhances the relaxivity of the protein-bound form of the contrast agent by 7-8 fold compared to unbound contrast agent in aqueous solution by slowing the rotation of the molecule. Another advantage is that extravasation from blood vessels into the surrounding tissue is greatly reduced. MS-
325 is described in detail in International Patent Application WO 96/23526, which is incorporated herein by reference in its entirety.

However, there is still a need for an improved mechanism for the effective control of enhancement in vivo at specific times and locations in relaxivity upon binding of contrast agents, which is specific to contrast agents. Consider activation. The more mechanisms and more effective mechanisms to control the activation of a contrast agent, the greater the potential application range in which this contrast agent can be used. Contrast agents that can be activated at specific locations in the body and at specific times at the right time have the advantage that unwanted background signals are reduced or eliminated.

One inventor has discovered a contrast agent that is not targeted but can be activated with physiological targets (WO 96/381 invented by Thomas Meade).
84, and hereafter referred to as "Meade"). But Me
The ade activation method is based on a principle completely different from the method of the present invention. Meade
The blocking moieties of the invention inhibit the interaction of hydroprotons with coordination sites on the metal ion.
Agents of the Meade invention that include a blocking moiety do not contain available coordination sites (or partially available sites as a result of dynamic equilibrium) due to interaction with the water protons. Therefore, activation occurs only if the blocking site is removed, making the water molecule more readily accessible to the internal coordination site on the metal complex. The increased exchange at these coordination sites allows the drug to enhance the contrast of tissues near the hydroproton.

The masking polypeptides of the present invention reduce the protein binding affinity of the prodrug as compared to its bioactivated contrast agent. An important feature of the contrast agents of the invention is the increased relaxivity is correlated with the non-covalent binding of the contrast agent to the target. Furthermore, binding to the target is specific and can only occur if the masking polypeptide is removed by peptidases. This cleavage converts the prodrug into an active contrast agent that binds to the target and exhibits increased relaxivity after binding.

Similarly, WO 97/36619 (incorporated herein by reference in its entirety), invented by Randy Laufer and others, and hereinafter referred to as “Lauffer”, Disclosed are bioactivated MRI contrast agents. However, Lauffer compounds are distinguished because the compounds include a masking polypeptide that can be cleaved by a peptidase and a targeting group masking polypeptide that is a peptide or amino acid. That is, the present contrast agents have the significant advantage that a significant portion of any molecule can be synthesized by an automated solid phase polypeptide synthesizer. This provides ease of synthesis and provides a general synthetic scheme that can be adapted for the synthesis of many different contrast agents.

DETAILED DESCRIPTION OF THE INVENTION The compounds and compositions of the present application are in the form of targeted prodrugs of contrast agents that include cleavable chemical groups that reduce the binding of the contrast agent to the target molecule. is there.
The uncleaved prodrug has a low affinity for the target protein and correspondingly a low relaxivity. Activation of the prodrug occurs by enzymatic cleavage. The activated contrast agent binds to the target molecule and forms an active complex with the contrast agent and the target. This active contrast agent shows several times higher relaxivity than the uncleaved prodrug after binding to the target protein. This increased relaxivity is due to MRI
Improves contrast in the image. In an example of a preferred embodiment, the target protein is human serum albumin (HSA), the linking group is a substituted phenyl, and the cleaving group is polylysine. After cleavage of polylysine, the contrast agent binds tightly with HSA, and the activated contrast agent and the tight binding complex of the target show increased relaxivity, which results in improved signal and image contrast. Cause

In the present invention, the relaxivity (r ₁ ) of the uncleaved contrast agent is preferably 80% or less than the r ₁ of the activated contrast agent. More preferably, the r ₁ relaxivity is 50% or less of the r ₁ relaxivity of the activating agent, and even more preferably 20.
% Or less, and most preferably 10% or less.

I. Definitions The term “alkyl” used herein alone or as part of another group.
Represents a linear and / or branched saturated hydrocarbon, which is optionally substituted (eg methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t
ert-Butyl and other groups well known in the art).

The term “cycloalkyl” used alone or as part of another group herein, refers to an optionally substituted saturated cyclic hydrocarbon ring system (eg, cyclopropyl). , Cyclobutyl, cyclopentyl, and other groups well known in the art).

The compounds and compositions of the present invention include their pharmaceutically acceptable derivatives.
By "pharmaceutically acceptable" is meant that the compound or composition can be administered to an animal without unacceptable adverse effects. "Pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, salt of ester, or other derivative of a compound of the invention (when administered to a recipient, this Means (directly or indirectly) a compound of the invention or its active inhibitory metabolite or active inhibitory residue.
Produce). Particularly preferred derivatives are compounds of the invention when such compounds are administered to a mammal (eg, by allowing the orally administered compound to be more readily absorbed into the blood). Or a derivative that enhances delivery of the derivative of the parent compound to a biological compartment (eg, brain or lymphatic system) as compared to other parts of the body.

The pharmaceutically acceptable salts of the compounds of the present invention also include pharmaceutically acceptable inorganic and organic bases and cations and anions derived from inorganic and organic acids, as are known in the art. .

Relaxability R ₁ and R ₂ (defined as the increase in 1 / T ₁ or 1 / T ₂ per mM of metal ion, respectively) enhances the relaxation rate of spectroscopic or imaging nuclei. It is defined as a measure of the ability of a contrast agent to induce. The relaxivity unit is mM ⁻¹ s ⁻¹ .

The compounds of the present invention contain one or more asymmetric carbon atoms and can therefore occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each asymmetric carbon can be in the R or S configuration.

The combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that do not deteriorate during manufacture and / or for a sufficient period of time thereafter. Accordingly, such compounds are suitable for the purposes detailed herein (e.g., therapeutic, diagnostic, or prophylactic administration to animals, or use in affinity chromatography applications). for
). Typically, such compounds are stable at temperatures below 40 ° C. for at least 1 week in the absence of moisture or other chemical reaction conditions.

It is also to be understood that the compounds of the present invention can be in various conformational and ionic forms in solution, in pharmaceutical compositions and in vivo. Although the description herein of particularly preferred compounds of the present invention refers to particular configurations and ionic forms, the present disclosure is not so limited.

II. Contrast Agent Structure The compounds of the present invention include at least three domains: (a) a paramagnetic metal and a chelating ligand scaffold, (b) an optional linker, (c) Amino acid targeting groups (preferably directed against protein targets),
And (d) a covalently cleavable polypeptide that prevents binding to this target. This cleavable group is cleaved after administration of the drug (either after administration of the endogenous protease or after administration of the exogenous protease).

According to one embodiment, the present invention provides a compound comprising: (a) a paramagnetic metal chelate backbone structure comprising a chelating ligand and a paramagnetic metal ion, wherein the chelating ligand is diethylenetriamine. 5 acetic acid (DTP
A), 1,4,7,10-tetraazacyclododecane-4acetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), and 1,4,7,10-tetraazacyclododecane-1,4,7-3. Selected from the group consisting of acetic acid (DO3A); and wherein the chelating ligand is atomic number 13, 21-34, 39-42, 4;
Forms a complex with one or more paramagnetic metal ions selected from the group consisting of metal ions having 4 to 50 and 57 to 83); (b) an optional linker; (c) a protein containing an amino acid. A linking group (wherein this amino acid side chain is 1-3
Phenyl rings are included, where each phenyl group is optionally substituted with up to 5 substituents selected from the Z group; where Z is halogen, CN, NO ₂ , CF ₃ , OCF ₃ , OH, S (C ₁ -C ₄ )-
Alkyl, SO (C ₁ -C ₄ ) -alkyl, SO ₂ (C ₁ -C ₄ ) -alkyl,
_{NH 2, NH (C 1 ~C} 4) - _{alkyl, N ((C 1 ~C 4} ) - _alkyl) 2,
COOH, C (O) O (C ₁ -C ₄ ) -alkyl, O (C ₁ -C ₄ ) -alkyl, (C ₁ -C ₆ ) -alkyl, (C ₂ -C ₆ ) -alkenyl, ( C ₃ ~C ₆₎
- alkynyl, and _(C 3 -C ₇₎ - cycloalkyl), and (d) covalently bound cleavable shielding polypeptide (wherein the shielding polypeptide comprises 1 to 10 amino acid residues, And here, this cleavage site is a peptide bond).

Particularly preferred chelating ligands include pharmaceutically acceptable metal chelating compounds, wherein the metal chelating compound is one or more cyclic or acyclic organic chelating agents that complex with one or more metal ions. Consists of. Preferred paramagnetic metal ions for MRI include metal ions having atomic numbers 21-29, 42, 44, or 57-83.

The paramagnetic metal ion should dissociate from the chelating ligand to any significant extent during the passage of the imaging reagent through the body, including the passage of tissue through which the contrast agent may undergo biological modification. Absent. The significant release of free metal ions can result in large MRI alterations and can also be associated with toxicity. This release is only tolerated in diseased tissues. Preferably, the physiological activity does not significantly impair the stability of the metal chelate complex,
This metal is excreted intact.

[0027]   In general, the degree of toxicity of a metal chelate depends on its degree of dissociation in vivo prior to excretion.
is connected with. Toxicity generally increases with the amount of free metal ions. That is,
High production constants are preferred to prevent toxic concentrations of free metal ions. Generation constant
But at least 10¹⁵M^-1, Or at least 10¹⁶M^-1, Or few
At least 10¹⁷M^-1, Or at least 10¹⁸M^-1, Or at least 1
0¹⁹M^-1, Or at least 10²⁰M^-1, Or at least 10²²M ^-1 , Or at least 10²⁴M^-1Or even more preferred
Good If the metal ion dissociation rate is very slow, the lower formation constant
(Ie at least 10¹⁰M^-1A complex with) may be sufficient.

Preferred paramagnetic metals are selected from the group consisting of: Gd ³⁺ , Fe ³⁺ , Mn ²⁺ , Mn ³⁺ , Cr ³⁺ , Cu ²⁺ , Dy ³⁺ , Tb ³⁺ , Ho ³⁺ ,
Er ³⁺ and Eu ³⁺ . The most preferred metal is Gd ³⁺ . Many suitable chelating ligands for MRI reagents are known in the art. They can also be used as metal chelates for other forms of biological imaging. MRI
For imaging purposes, preferred chelating ligands include, but are not limited to, the following derivatives:

[0029]

[Chemical 2]

[0030] .

Although all of these chelating ligands are shown with chelated Gd ³⁺ , it is known in the art that other metals can substitute for Gd ^{3+ in} certain applications.

The present application describes novel compounds containing gadolinium complexes with poor HSA binding and low relaxivity. This compound can be converted via enzymatic cleavage to a species with improved HSA binding and enhanced relaxivity. The compounds of the present invention may be modified so that cleavage is accomplished by specific proteases that have been identified as useful targets in diagnostics and treatment of diseases.

One property of the invention is the linking group containing the amino acid side chain. Such linking groups allow for a simplified synthesis of the contrast agent, as the contrast agent can be synthesized using standard peptide synthesis techniques.

Another property of the invention is a cleavable group containing an amino acid. This also allows for a simplified synthesis of this contrast agent, as standard peptide synthesis techniques are used. Amino acids can be selected for their ability to interfere with the binding of this contrast agent to the target. In a particularly preferred embodiment, the cleavable polypeptide comprises positively charged amino acids. The amino acid of the cleavable group can also be selected based on the specificity of the protease that cleaves the amino acid. Other factors can influence the choice of amino acids containing the linking group and the cleavable polypeptide.

[0035]   Another particular property of the invention is the structure of the amino acid targeting group. Multiple targeting groups
Can bind to a particular target molecule. This targeting group can be either a direct or short phosphorus group.
Is an amino acid that is covalently bound to a chelating ligand, either through the Kerr
. A preferred embodiment of the invention comprises a linking group that binds to the protein target. One
Amino acids containing the above aromatic groups, preferably phenyl groups, are particularly preferred. Or less
Amino containing a phenyl group substituted with 1 to 5 groups selected from the group consisting of
More preferred are acids: halogen, CN, NO._Two, CF_Three, OCF_Three, OH, S (
C₁~ C_Four) -Alkyl, SO (C₁~ C_Four) -Alkyl, SO_Two(C₁~ C_Four ) -Alkyl, NH_Two, NH (C₁~ C_Four) -Alkyl, N ((C₁~ C_Four) −
Alkyl)_Two, COOH, C (O) O (C₁~ C_Four) -Alkyl, O (C₁~ C _Four ) -Alkyl ,; (C₁~ C₆) -Alkyl, (C_Two~ C₆) -Alkenyl,
(C_Two~ C₆) -Alkynyl, and (C_Three~ C₇) -Cycloalkyl).

An optional linker can connect the chelating ligand and the targeting amino acid. Preferred linkers are relatively short, especially when bound to HSA, limiting chelate mobility. Most preferred are linkers that are 6 atoms or less in length and that link to the atom containing the amino acid targeting group and chelate. Carbonyl, glycine,
Or both are preferred linkers.

Preferred embodiments of the invention include cleavable polypeptide groups that are cleaved in vivo. A preferred embodiment comprises a cleavable group that is cleaved by an enzyme selected from: Thrombin activatable fibrinolytic inhibitor (Throm).
bin Activatable Fibrinolysis Inhibit
or) (TAFI), a member of the carboxypeptidase B family, trypsin, factor Xa, 7B2 protein, proprotein convertase 2, subtilisin, kexin endoproteinase, pancreatic carboxypeptidase, endoproteinase Lys-C, myxobacterial ( Myxobacter
) Protease, elastase, matrix metalloproteinase (MMP),
Clostripain, and Armillaria
Protease. The invention further contemplates the use of other enzymes known as site-specific cleavage peptides (eg, chymotrypsin), particularly where the shielding polypeptide comprises positively charged terminal amino acids. The most preferred embodiment comprises a cleavable group that is cleaved by the proteolytic enzyme TAFI, a member of the carboxypeptidase B class of proteolytic enzymes. TAFI acts in vivo by cleaving the exposed C-terminal lysine on fibrin. After fibrin is cleaved in vivo, tissue plasminogen activator and plasminogen inhibit clot degradation. After cleavage of the contrast agent of the present invention by the TAFI enzyme, the contrast agent binds more closely to the target protein, resulting in increased relaxivity and improved image contrast.

Screening a number of candidate contrast agents revealed that the incorporated aryl groups in the structure of conventional gadolinium polyaminocarboxylate ligands (eg DOTA or DTPA) resulted in increased binding of the contrast agent to HSA,
Already shown. To maximize relaxivity, the linking group should be about 20 from the metal center.
It should not be arranged with more than one carbon-carbon bond length. Because the intervening atoms provide additional flexibility to the molecular structure. This may result in increased unwanted tumbling of the molecule or increased movement of the chelating paramagnetic metal ion at the chelating site. Any reduction in tumbling of molecules or movement of chelates results in increased relaxivity. Therefore,
The linker between the chelating ligand and the targeting amino acid should be relatively short.

Positively charged amino acids (eg lysine, arginine, ornithine, 2,4-
Contrast agents comprising a shielding polypeptide comprising diaminobutanoic acid, 2,3-diaminopropionic acid, or other residues) are more likely to be associated with HSA than contrast agents lacking positively charged amino acids in an aqueous medium containing HSA. It binds less tightly and exhibits a lower relaxivity. The positive charge significantly diminishes the molecule's affinity for HSA. Therefore, cleavage of the charged amino acid by a suitable enzyme (eg, TAFI, which cleaves polylysine) causes the contrast agent to bind more tightly to HSA. The close binding of this contrast agent to HSA results in increased relaxivity. The peptide is preferably covalently attached to the linker / chelate via its N-terminus. This leaves the negatively charged C-terminus exposed, allowing the peptide to be cleaved by carboxypeptidase. The negatively charged carboxylate groups remaining after such cleavage and removal of the positively charged amino acids can facilitate the binding of "unshielded" factors to HSA.

III. Examples In certain embodiments, a modified DTPA chelating ligand that comprises Gd ³⁺ complexed at the chelation site binds an amino acid, where the side chain is diphenylalanine. Or 3,5-diiodotyrosine, both of which bind well to HSA. The Gd ³⁺ chelate acts as a signaling domain and binds to the HSA binding moiety masked by an HSA-blocking polypeptide (preferably polylysine) that inhibits binding to HSA. Enzymatic cleavage activates the contrast agent by releasing this masking polypeptide group and promoting HSA binding. FIG. 1 shows diphenylalanine (M11-01) and 3,5.
-Shows the structure of the diiodotyrosine (M11-02) compound. Addition of multiple charged groups (eg, lysine residues) to amino acid targeting groups inhibits binding to HSA.
This is because it is known that charged groups do not bind well to HSA.
The lysine residues of M11-01 and M11-02 can be cleaved by TAFI to yield compounds M11-03 and M11-04, respectively. Cleavage of this lysine residue results in activation.

The synthesis of the preferred embodiments (M11-01 and M11-02) is shown in FIG. The left side of this scheme shows glycine-bonded diethylenetriaminepentaacetic acid (DT
3 shows the synthesis of PA) derivatives. The starting material is the t-butyl ester of carboxylated DTPA, the synthesis of which is described in US Pat. No. 5,637,759 (“Hears”).
t "patent). The "Hearst" patent also has a related EDT
The synthesis of the A analog is described. Carboxylation of DTPA to a glycine conjugate t
Conversion of the -butyl ester is accomplished by reaction of glycine with the benzyl ester under standard peptide coupling conditions followed by hydrogenation to remove the benzyl protecting group.

The right side of the scheme shows a parallel synthesis of the shielding peptide and the targeting amino acid on a solid support, for example as used by an automated peptide synthesizer. The amino acid bound to the solid support is bound to the carboxylated glycine conjugate of DTPA t-butyl ester. The carboxylate groups are deprotected and the contrast agent precursor is removed from the solid support as shown. The active contrast agent is then D
It is synthesized by the addition of a metal that is chelated by the TPA derivative.

Peptide synthesis can be either N to C or C to N and the linker / chelate can be attached to either end of the peptide. Preferably, the peptide is attached to the N-terminus during synthesis, and the resin preferably produces a free carbonate at the C-terminus.

The linker, if present, can be attached to the growing polypeptide chain, which is attached to the resin using conventional solid phase peptide synthesis. The amine-containing linker is preferably attached to the C-terminus of the peptide and the carbonyl-containing linker is preferably attached to the N-terminus of the peptide. This is because the peptide bond occurs in each case. US Pat. No. 5,637
, 759 ("Hearst" patent) DTPA chelate and E
DTA chelates are particularly preferred. Because these compounds can be attached to peptides using conventional solid phase peptide synthesis. For syntheses using these compounds, higher yields are achieved when the glycine linker is inserted between the target amino acid of the chelate. In this embodiment, the linker is -C (O) -gly-, where the carbonyl is derived from the compound in the Hurst patent above and a glycine residue is added to the peptide attached to the solid phase. It

Synthetic routes to other compounds are also illustrated in FIG. For example, a linker attached to the peptide can be attached to the acetate group of the chelating ligand. The bond to the acetate group can be formed directly to the carbonyl of the acetate (eg, acetate carbonyl can form a peptide bond with an amino group linker-conjugated peptide) or to the carbonyl via the α carbon atom. It can either be formed indirectly.

Preferred linkers are relatively short, especially when attached to HSA, and limit the flexibility of the chelate. Most preferred is a linker that is no more than 6 atoms in length and connects the atoms containing the amino acid targeting group and the chelate.

The linker, when present, binds the target amino acid to the metal chelate complex, and its main purpose is to facilitate the synthesis of the compound. Examples of linkers include: linear alkyl groups, branched or cyclic alkyl groups, aryl groups and heterosubstituted.
ituted) analogs (eg ether, amine, amide). In general, the smallest linker and greatest relaxivity (that allows proper expression of the bond of interest).
relaxivity) is preferred. In the case of a linear alkyl linker,
Less than 10 atoms are preferred, less than 6 atoms are more preferred and less than 4 atoms are most preferred. In some cases, hetero-substituted linkers are preferred for convenient synthesis or desired physical properties. For example, a short amino acid sequence (eg Al
a, Gly, Gly-Gly, or Ala-Ala) may provide the optimal space between the metal complex and the amino acid of interest. Some examples of linkers are shown in Figure 3.

In addition to the linker described in FIG. 3, other preferred linkers contain 1 to 6 atoms and are linear and unbranched. The linker preferably connects the chelating group (eg, via the carbon of the ethylene backbone or the carbon of the acetate group) and the α-nitrogen or carbonyl carbon of the amino acid targeting group. The linker portion of the molecule is preferably derived, either wholly or partially, from synthetic chemical intermediates terminating with amines or carboxylates, including both their protected and activated forms. It These are typically compatible with the synthetic chemistry conditions used for solid phase peptide synthesis. Finally, the carbon atoms of the linker can be optionally replaced with hydroxyl groups or halogens.

The solid phase resin used for the synthesis of the DTPA-peptide conjugate is [acetic acid (3
-(4-hydroxymethylphenoxy))] (PAC) handle (handle)
) And fluorenyl methyloxycarbonyl (fmo
c) Polyethylene glycol polystyrene having a protected amino acid (PEG-PS
) Resin. This method involves DT on a support structure according to known standard procedures.
Due to the stepwise attachment of individual amino acids, including PA-peptide molecules, the coupling agent O- (7- in the presence of diisopropylethylamine (DIPEA).
Azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) is utilized. The resulting molecule is cleaved from the resin and the t-butyl ester is converted to the free carboxylic acid moiety by mixing with trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / H ₂ O for 2 hours. It

The product is 0.1% TFA and 0.1% TFA in acetonitrile.
Purified by reverse phase high performance liquid chromatography (HPLC) on a C ₁₈ column using a linear gradient of aqueous solution. The Gd ⁺³ complex has a pH of 3.0-7.5, preferably p, in acetate buffer by mixing the contrast agent precursor with GdCl _3.
Obtained in aqueous solution between H4.5 and 5.5. Final Gd ⁺³ concentration is assessed by inductively coupled plasma (ICP) atomic spectroscopy. The identification and purification of both the ligand and the complex are confirmed by electrospray mass spectrometry (ES-MS) and liquid chromatography mass spectrometry (LC-MS).

FIG. 4 shows the time course for cleavage of polylysine residues from the preferred compound M11-01. FIG. 4A shows that the time course of TAFI induction increases 1 / T ₁ . TAFI induction involves the cleavage of polylysine residues from the M11-01 compound to form M11-03. The 1 / T ₁ increase was measured at a magnetic field strength of 0.5 T and a reaction temperature of 24 ° C. The initial concentration of M11-01 is 0.2 mM,
The reaction was then carried out in the presence of 4.5% (w / v) HSA and 75 nM TAFI enzyme.

FIG. 4B shows the time course for conversion of M11-01 to M11-03 by cleavage of lysine residues. The percentage of species present is plotted on the vertical axis against time in minutes. Cleavage is not 100% efficient; therefore some partially cleaved M11-01 compound remains after 60 minutes. M11-01 represents a compound with three lysine residues, M11-01-1Lys represents a partial cleavage product that still contains two lysine residues (one lysine is cleaved), while so,
M11-01-2Lys is a partial cleavage product containing one lysine residue (two lysines are cleaved). M11-03 is a fully cleaved and fully active contrast agent.

The increase in 1 / T ₁ most closely matches the disappearance of M11-01 (FIG. 4B). 0
During minutes of 30 minutes, 1 / _{T 1} is increased from 3.6 s ^-1 to 4.8 s ^-1, whereas in, M11-01 of about 85% is converted into M11-03 or intermediate It was Three
Between 0 and 60 minutes, 1 / T ₁ changes only slightly (4.8s ⁻¹ to 5.0.
s ⁻¹ ), while the concentration of M11-03 increases from 100 μM to 160 μM.
Increased by 5%. Removal of the two C-terminal lysine residues resulted in a large 1 / T ₁ increase, while removal of the third lysine appeared to be less critical to achieve a significant effect.

After proceeding with enzymatic turnover by TAFI at 24 ° C., HPLC was performed and quantified by peak integration of the ligand form of the substrate and reaction product. HPLC analysis of the reaction mixture, quenched at various time points, confirmed the formation of the dilysine and monolysine intermediates and M11-03. TAF
I prior to addition of substrate under the following conditions: 250 μM TAFI in 10 mM HEPES, 10 nM a-thrombin, 25 nM thrombomodulin, 150.
Activated in mM NaCl and 5 mM CaCl ₂ for 10 minutes at room temperature.
TFA was used to quench the reaction. The effect of HSA on enzyme kinetics was not clear. The HSA binding percentage was determined by ultrafiltration.

Enzyme kinetics was studied in more detail for one compound, M11-01. FIG. 5 shows four compounds (M11-01, M11-01-1Lys, M11-01-
2Lys and M11-03 each show HPLC and mass spectrometry (LC-MS) profiles for sequential less than one lysine). The left side of the figure shows the relative concentrations of the four compounds determined by HPLC during the enzymatic cleavage between 0 and 60 minutes. This part of the figure shows that M11-01 is present almost exclusively at the beginning of the experiment and M11-03 (“-3Lys label”) is present almost exclusively at the end of the experiment. The right part of the figure shows the LC-MS profiles for the four compounds. Mass versus conversion (m / z) is plotted on the horizontal axis. This figure shows that the mass of the compound decreases in proportion to the disappearance of the lysine residue.

Relaxability and HSA of preferred uncleaved compounds (M11-01 and M11-02) and final products of enzymatic reactions (M11-03 and M11-04).
Binding data is shown in Figure 6 and illustrated in Figure 1. This table is 24 ℃, 37 ℃
Shown is the relaxivity in and the percentage of compounds bound to HSA at 37 ° C.

The increase in relaxivity at 24 ° C. is due to M11-03 and M11-0, respectively.
Observed for both M11-01 and M11-02 cleavage to form 4. Cleavage of M11-01 resulted in a 26% increase in relaxivity, while M11-0.
Cleavage of 2 results in a greater than 100% increase in relaxivity. A 120% increase in relaxivity at 37 ° C. was caused by cleavage of M11-01, while
The 0% increase results from cleavage of M11-02. No increase in relaxivity is observed for cleavage of this compound in phosphate buffered saline (PBS) (lacking HSA) at any temperature.

The second compound, M11-02, showed an improved relaxivity increase after cleavage of polylysine residues compared to M11-01. At 24 ° C., relaxivity M11-02 and non lysine compound M11-04 in the presence of 4.5% HSA, respectively, ^{12.5 mM} -1 ^{s -1} and ^{25.2 mm} -1 ^{s -1} Met.
Complete conversion of M11-02 to M11-04 by TAFI was achieved at micromolar enzyme concentrations, and expected 100% relaxivity due to the observed 18-fold increase in HSA binding activity. Occurred. Nanomolar levels (200 μM 2,75 nM TA monitored by 1 / T ₁ change
The time course of the TAFI reaction with FI, 4.5% w / v HSA) produced a smaller effect due to a slower cleavage of the third lysine residue that competed with TAFI self-inactivation. . At 30 minutes, the monolysine intermediate represented 83% of all species, while M11-04 accounted for 5% of the total. In contrast to M11-01, the removal of the third lysine was found to be essential to achieve maximal 1 / T ₁ gain, but full realization of maximal relaxivity was found in clinical applications. May not be necessary for. At the end of turnover by TAFI, the monolysine intermediate and M11-04 represented 44% and 51% of the reaction mixture, respectively.
However, 1 / T ₁ only increased to 26% over 3.83s ⁻¹ . This indicates that M11-02 must be exposed to TAFI for a longer period of time to achieve a beneficial signal increase. However, an almost 3-fold higher relaxivity increase can finally be achieved at 37 ° C. (FIG. 6).

The relaxivity (r ₁ ) was calculated from the slope of the plot of 1 / T ₁ vs. sample concentration according to the following formula:

[0060]

[Equation 1]

The relaxivity unit is mM ⁻¹ s ⁻¹ as indicated. The T ₁ value is 20 according to the standard recovery recovery methods.
Measured in MHz.

The percentage of each compound bound to HSA is shown in the right column of FIG. H
The percentage of SA binding increases after cleavage for both compounds. Cleavage of M11-01 resulted in a 220% increase in the percentage bound to HSA, while cleavage of M11-02 resulted in a 18% increase in the percentage bound to HSA.
It produces an increase of more than zero.

At physiological concentrations of TAFI (75 nM), MM11-01 is rapidly converted to M11-03. The kinetic parameters for the disappearance of MM11-01 (K _m = 340 μM; K _cat = 5.3S ⁻¹ ) were found to be hippuryl arginine (K _m = 140 μM; K _cat = 21).
It is comparable to other TAFI substrates such as S- ¹ ). The time course for turnover of M11-01 (230 μM) by TAFI in the presence of 4.5% HSA was completed within 1 h.

[Brief description of drawings]

FIG. 1 shows the structures of diphenylalanine (M11-01) and 3,5-diiodotyrosine (M11-02) compounds.

FIG. 2 shows the synthesis of the preferred embodiments (M11-01 and M11-02).

[Figure 3]   Figure 3 shows some examples of linkers.

FIG. 4 shows the time course for cleavage of polylysine residues from the preferred compound M11-01. FIG. 4A shows that the time course of TAFI induction increases 1 / T ₁ . FIG. 4B shows the time course for conversion of M11-01 to M11-03 by cleavage of lysine residues.

FIG. 5 shows four compounds (M11-01, M11-01-1Lys, M11-0).
1-2 Lys and M11-03 each show HPLC and mass spectrometry (LC-MS) profiles for sequentially containing less than one lysine).

FIG. 6 shows relaxivity and HSA binding data for preferred uncleaved compounds (M11-01 and M11-02) and final products of enzymatic reactions (M11-03 and M11-04). .

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, J P, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, R O, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor McMurray, Thomas Jay.             United States Massachusetts             01890, Winchester, Bonad               Road 4 (72) Inventor Corojay, Andrew             United States Massachusetts             01890, Winchester, S.             Border Road 390 F-term (reference) 4C085 HH07 JJ01 KA09 KB45 KB82                       KB99

Claims (16)

[Claims] 1. A magnetic resonance imaging contrast agent having the following structure: [chelating ligand and metal ion] | [linker] _n | [targeting amino acid] | [masking poly Peptide] where: the chelating ligand has atomic numbers 13, 21-34, 39-42, 44-5.
0, and forms a complex with one or more paramagnetic metal ions selected from the group consisting of metal ions from 57 to 83; wherein the linker, if present, contains from 1 to 20 carbon atoms, And the carbon atom forms a linear, branched, or cyclic alkyl group, aryl group, or hetero-substituted analog thereof, wherein 1 to 6 carbon atoms are a nitrogen atom, an oxygen atom, or a sulfur atom. Substituted by an atom; n is 0 or 1; the targeted amino acid comprises 1 to 3 phenyl rings linked as side chains at the α-carbon of the amino acid, wherein: each phenyl group is Optionally substituted with up to 5 substituents selected from the Z group; wherein Z is halogen, CN, NO ₂ , CF ₃ , OCF ₃ , OH, S (C ₁ -C ₄ ) -alkyl, SO (C ₁ -C ₄₎ - A Alkyl, SO ₂ (C ₁ -C ₄ ) -alkyl, NH ₂ , NH (C ₁ -C ₄ ) -alkyl, N ((C ₁ -C ₄ -alkyl) ₂ ,
COOH, C (O) O (C ₁ -C ₄ ) -alkyl, O (C ₁ -C ₄ ) -alkyl; (C ₁ -C ₆ ) -alkyl, (C ₂ -C ₆ ) -alkenyl, (C ₂ -C ₆₎ -
Alkynyl, and (C 3 _-C 7) _- consists cycloalkyl; said masking polypeptide comprises 1 to 10 amino acid residues; and wherein the cleavage site, the target amino acids and said masking polypeptide Exists between
A contrast agent, wherein the cleavage site is a peptide bond. 2. The compound according to claim 1, wherein the chelating ligand is tetraamine 1,4,7,10-tetraazacyclododecane-N, N.
', N ", N'"-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), or selected from one of the following structures Done: [Chemical 1a] [Chemical 1b] Wherein: R _is selected from -CX 2 C (O) O- or _{-CX 2 C (O) NX 2} ; X _{is, H, (C 1 -C 6} ) linear or branched alkyl, ( C 2 _{-C 6)} straight or branched alkenyl, or (C 2 _{-C 6)} straight or branched alkynyl; single bond using a wavy line intersecting is a point where the linker is attached; And C (O) shows a carbon-oxygen double bond. 3. Z is halogen, CN, NO ₂ , CF ₃ , OCF ₃ or O
The compound of claim 2, which is H. 4. The compound of claim 3, wherein the targeted amino acid is 3,5 diiodotyrosine. 5. The compound according to claim 3, wherein the targeted amino acid is a diphenylalanine group. 6. The paramagnetic metal ion has an atomic number of 21 to 29, 42, 44.
Or the compound of claim 3, selected from the group of metals having 57-83. 7. The compound according to claim 6, wherein the paramagnetic metal ion is Gd ³⁺ . 8. The compound of claim 7, wherein the chelating ligand and paramagnetic metal ion are gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). 9. The compound of claim 3, wherein the metal chelate has a formation constant greater than about 10 ²⁰ M ⁻¹ . 10. The compound of any of claims 1-9, wherein the covalently cleavable masking polypeptide group comprises the group consisting of: thrombin activated fibrinolysis inhibitor (TAFI). ), A member of the carboxypeptidase B family, trypsin, factor Xa, 7B2 protein,
Removed by an enzyme selected from proprotein convertase 2, subtilisin, kexin endoproteinase, pancreatic carboxypeptidase, endoproteinase Lys-C, myxobacterial protease, elastase, matrix metalloproteinase (MMP), clostrypan, and Armillaria protease ,Compound. 11. The covalently cleavable masking polypeptide group comprises:
11. The compound of claim 10, which is removed by TAFI or another member of the carboxypeptidase B family. 12. A pharmaceutical composition comprising the contrast agent according to any one of claims 1 to 9 and a carrier, adjuvant or vehicle. 13. A method of MRI imaging, the method comprising the step of administering a diagnostically effective amount of any one of claims 1-9. 14. A method of MRI imaging, the method comprising the step of administering a diagnostically effective amount of the composition of claim 12. 15. A method for physiologically activating a contrast agent, the method comprising administering a compound according to any one of claims 1 to 9 and cleaving the masking polypeptide with a protease. And activating the contrast agent. 16. A method of physiologically activating a contrast agent, the method comprising administering the pharmaceutical composition according to claim 12 and cleaving the masking polypeptide with a protease. And activating the method.