Cathepsin-binding compounds bound to a carrier and their diagnostic use

Abstract

The invention relates to Cathepsin-binding compounds bound to a carrier comprising a diagnostic moiety, for use in the diagnosis of inflammatory diseases, and/or for use in the diagnosis of neoplastic diseases, wherein the Cathepsin-binding compound binds to inflammatory cells of the tumour stroma. The invention also relates to Cathepsin B-targeting compounds and Cathepsin B-binding and liposome-binding compounds.

Claims

1. A Cathepsin binding and liposome-binding compound having the structure of formula (XIII): ##STR00006## wherein n=38 to 53.

2. A Cathepsin targeting compound, comprising the Cathepsin binding and liposome-binding compound of claim 1, bound to a liposome.

3. The Cathepsin targeting compound according to claim 2, comprising a diagnostic moiety.

Description

FIGURES

(1) FIG. 1: Preferred embodiment of the present invention. A, cathepsins are normally localized in the intracellular organelles: endosomes or lysosmes. However, in several pathological conditions cathepsins could be translocated to the extracelluar milieu. B, An example of the cathepsin-binding compounds bound to a carrier, represented as LNP—NS-629. NS-629 represents the preferred Cathepsin-binding and liposome-binding compound according to formula (III), wherein n=38 to 53. In exemplary embodiments, the cathepsin-binding compounds bound to a carrier are 90-110 nm. C, Targeting system based on cathepsin-binding compounds binding to extracellular cathepsins enabling active targeting of encapsulated drug.

(2) FIG. 2: Chemical structure of the lipidated inhibitor NS-629 (length distribution of the PEG segment: n=38-53).

(3) FIG. 3: Titration of Cathepsin B with free NS-629 (a) and liposome-coupled lipidated NS-629 (b) at pH 6.0 and 25° C. The solid lines were generated by linear regression analysis.

(4) FIG. 4: Size distribution of liposomes functionalized with lipidated inhibitor as determined by dynamic light scattering (DLS). Average size is 85.66 nm.

(5) FIG. 5: Validation of targeting of liposomes with lipidated Cathepsin inhibitor to the immune cells. Liposomes functionalized (NS-Lip-Alx) and not functionalized (Lip-Alx) with NS-629 were loaded with fluorescence marker (Alexa Fluor 546™; (Invitrogen)) and incubated with mouse bone marrow-derived macrophages for 15 minutes at 4° C. Fluorescence of accumulated marker was measured with TECAN plate reader. As a control liposomes without labeling were used.

(6) FIG. 6: Representation of liposomes with lipidated Cathepsin inhibitor targeting efficiency in primary mouse immune cells. Non-functionalized liposomes (Lip-Alx) and liposomes functionalized with NS-629 (NS-Lip-Alx) were loaded with fluorescence marker (Alexa Fluor 555™ (Invitrogen)) and incubated with mouse bone marrow-derived macrophages for 15 minutes at 4° C. Fluorescence of accumulated marker was examined with an Olympus fluorescence microscope (Olympus IX 81) with Imaging Software for Life Science Microscopy Cell.sup.f.

(7) FIG. 7: Targeted delivery of liposomes labeled by lipidated inhibitor carrying D-luciferin into transgenic mouse expressing luciferase (FVB.luc.sup.tg/+). The high-intensity luciferase signal associated with the induced paw edema demonstrates selective accumulation of labelled liposomes in the inflammation area. The scale is in photons/sec/sm.sup.2/sr.

(8) FIG. 8: T.sub.1-weighted MR images (TE=8.5 ms, TR=400 ms) of an orthotopic transplanted breast cancer mouse before, 1 and 24 hours after (T.sub.1) intraperitoneal injection of 200 μl NS-629 labeled liposomes containing Magnevist® (Bayer HealthCare Pharmaceuticals). The tumour tissue possess negative MR signal on T.sub.1-weighted images. The bright signal at 1 and 24 hours after injection in T.sub.1-weighted MR image indicates successful targeting of Magnevist® (Bayer HealthCare Pharmaceuticals) loaded NS-629 labelled liposomes.

EXAMPLES

Example 1

Synthesis of the Lipidated Inhibitor NS-629

(9) DSPE-PEG(2000) refers to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (ammonium salt) (CAS Reg. 474922-20-8). DSPE-PEG(2000) carboxylic acid with PEG unit length of n=45 (PEG lengthes of n=38 to 53 are present) has the following formula I:

(10) ##STR00005##

(11) DSPE-PEG(2000) carboxylic acid (Avanti Polar Lipids, Inc.) (30.0 mg, 10.5 μmol) was suspended in MeCN (1 mL) and CHCl.sub.3 was added dropwise until a clear solution was obtained. Then at room temperature under stirring a solution of DSC (Fluka, Buchs) in MeCN (c=0.0105 mol/L, 1 mL) and a solution of DIPEA in MeCN (c=0.105 mol/L, 0.1 mL) were added. In parallel, H.sub.2N—(CH.sub.2).sub.6—NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH×TFA (16.5 mg, 21 μmol; prepared by treating H.sub.2N—(CH.sub.2).sub.6—NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OtBu (Schaschke et al. (1), 2000) with TFA/H.sub.2O (95:5, v/v)) was suspended in MeCN (1 mL) and H.sub.2O was added dropwise until a clear solution was obtained. H.sub.2N—(CH.sub.2).sub.6—NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH×TFA and its synthesis are described in Schaschke et al. (2), 2000, and in Schaschke et al., 1998. Then to this solution a solution of DIPEA in MeCN (c=0.105 mol/L, 0.4 mL) was added. After 60 min, this solution was added to the formed active ester of DSPE-PEG(2000) carboxylic acid and stirring was continued for 48 h. The solvent was evaporated under reduced pressure and the resulting material was dissolved in CHCl.sub.3 (50 mL). The organic phase was washed with 5% aq KHSO.sub.4 (3×20 mL), brine (1×20 mL), dried (Na.sub.2SO.sub.4), and the solvent evaporated. The obtained crude product was dissolved in MeCN/H.sub.2O (1:3, v/v; 10 mL), lyophilized, and purified by thick-layer chromatography using glass plates from Merck, Darmstadt (type: PLC Silica gel 60 F.sub.254, 1 mm). Prior to use the plates were developed with MeOH twice. Upon development with CHCl.sub.3/MeOH (2:1, v/v containing 1% AcOH) as solvent system, the area containing homogeneous product was scraped from the plate, the product extracted from the collected silica gel with MeOH, and the solvent evaporated. The lipidated inhibitor (NS-629) was obtained upon lyophilization as colorless powder; yield: 2.8 mg (8%); TLC (CHCl.sub.3/MeOH/AcOH 13:5:0.18, v/v/v) R.sub.f 0.62; ESI-MS: m/z=1762.0 [M−2H].sup.2−; calcd for C.sub.167H.sub.319N.sub.8O.sub.66P: 1761.6 (most abundant signal for n=46). The chemical structure of NS-629 for is shown in FIG. 2.

Example 2

Titration of Cathepsin B Active Site with Lipidated Inhibitor NS-629

(12) Powdered lipidated inhibitor of Cathepsin B, NS-629, was dissolved in 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA and 0.1% (v/v) PEG for final concentration of 0.05 μM. The kinetic reaction between Cathepsin B and its lipidated inhibitor was analyzed by continuous measurements of the loss of enzymatic activity at different concentration of inhibitor in the presence of fluorogenic substrate Z-Arg-Arg-AMC (AMC=7-amido-4-methylcoumarin) (Bachem). Inhibitor NS-629 in increasing concentrations (0.01-0.06 mM concentration), recombinant Cathepsin B (0.05 mM) and the dithiothreitole (DTT) (0.5 mM) were mixed in a plate with 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA and 0.1% (v/v) PEG. After 15 minutes incubation at 37° C. the inhibition kinetics of Cathepsin B and NS-629 were determined. The reaction was started by the addition of 150 μl of Cathepsin substrate Z-Arg-Arg-AMC solution and the kinetics of substrate hydrolysis was monitored continuously during 10 min by a TECAN plate reader at excitation and emission wavelengths of 370 and 460 nm, respectively. As can be seen in FIG. 3a, the inhibitor bound to Cathepsin B with an apparent 1:1.5 stoichiometry, suggesting that Cathepsin B efficiently binds NS-629.

Example 3

Lipid Vesicles Linked with Lipidated Inhibitor Preparation by Extrusion

(13) Aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of the 0.1 mM phosphate buffer pH 6.0. Dispersions are homogenized with vortex mixing and extruded under pressure through polycarbonate filters of decreasing pore diameter 0.1 μm using extruder. FIG. 4 shows the size distribution of liposomes functionalized with lipidated inhibitor as determined by dynamic light scattering (DLS). The average size was shown to be 85.66 nm.

Example 4

Lipid Vesicles Linked with Lipidated Inhibitor Preparation by Sonification

(14) Aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of the 0.1 mM phosphate buffer pH 6.0. Dispersions are homogenized with vortex mixing and then emulsified by sonication in bath sonicator during 20 min.

Example 5

Titration of Cathepsin B Active Site with Liposomes Labelled by Lipidated Inhibitor NS-629

(15) The liposomes labelled by lipidated inhibitor were prepared as described above in Example 3. The kinetic reaction between Cathepsin B and liposomes labelled by lipidated Cathepsin inhibitor NS-629 was analyzed by continuous measurements of the loss of enzymatic activity at different concentration of inhibitor in the presence of fluorogenic substrate Z-Arg-Arg-AMC. Liposomes labelled by Cathepsin inhibitor NS-629 in increasing inhibitor concentrations (0.01-0.09 mM concentration) in increasing concentrations (0.01-0.06 mM concentration), recombinant Cathepsin B (0.05 mM) and the DTT (0.5 mM) were mixed in a plate with 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA and 0.1% (v/v) PEG. After 15 minutes incubation at 37° C. the inhibition kinetics of Cathepsin B and NS-629 were determined. The reaction was started by the addition of 150 μl of Cathepsin substrate Z-Arg-Arg-AMC solution and the kinetics of substrate hydrolysis was monitored continuously during 10 min by a TECAN plate reader at excitation and emission wavelengths of 370 and 460 nm, respectively. As can be seen in FIG. 3a, the liposomes labelled by Cathepsin inhibitor NS-629 bound to Cathepsin B with an apparent 1:2.5 stoichiometry, suggesting that Cathepsin B could efficiently bind liposomes labelled by Cathepsin inhibitor NS-629.

Example 6

Ex Vivo Binding of Alexa Fluor 555™ (Invitrogen) Loaded Liposomes Labeled by Lipidated Inhibitor to Mouse Bone Marrow-Derived Macrophages

(16) Liposomes were prepared as: aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of 0.1 mg Alexa Fluor 555™ (Invitrogen) containing 0.01 M phosphate buffer, pH 7.4. Active endocytosis of macrophages was stopped by incubation at 4° C. during 15 minutes. Next, 200 μl of liposomes were placed on the cells and incubated for 15 minutes at 4° C. After incubation cells were washed by PBS and fluorescence intensity was examined with TECAN plate reader. FIG. 4 shows that this experiment proves targeting of liposomes with lipidated Cathepsin inhibitor to the immune cells. Liposomes functionalized (NS-Lip-Alx) and not functionalized (Lip-Alx) with NS-629 were loaded with fluorescence marker (Alexa Fluor 555™ (Invitrogen)) and incubated with mouse bone marrow-derived macrophages for 15 minutes at 4° C. Fluorescence of accumulated marker was measured with TECAN plate reader. As a control liposomes without labeling were used.

Example 7

Ex Vivo Binding of Alexa Fluor 555™ (Invitrogen) Loaded Liposomes Labeled by Lipidated Inhibitor to Mouse Bone Marrow-Derived Macrophages

(17) Liposomes were prepared as: aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of 0.1 mg Alexa Fluor 555™ (Invitrogen) containing 0.01 M phosphate buffer, pH 7.4. Active endocytosis of macrophages was stopped by incubation at 4° C. during 15 minutes. Next, 200 μl of liposomes were placed on the cells and incubated for 15 minutes at 4° C. After incubation cells were washed by PBS and examined with an Olympus fluorescent microscope (Olympus IX 81, Olympus) with Imaging Software for Life Science Microscopy Cell. Non-functionalized liposomes (Lip-Alx) and liposomes functionalized with NS-629 (NS-Lip-Alx) were examined with an Olympus fluorescence microscope (Olympus IX 81, Olympus) with Imaging Software for Life Science Microscopy Cell. FIG. 6 shows representation of liposomes with lipidated Cathepsin inhibitor targeting efficiency in primary mouse immune cells.

Example 8

Encapsulation of Magnevist® (Bayer HealthCare Pharmaceuticals) (Dimeglumine Salt of Gd-DTPA; Bayer AG) into the Liposome with Following Extrusion

(18) Aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of Magnevist® (Bayer HealthCare Pharmaceuticals). Dispersions are homogenized with vortex mixing and extruded under pressure through polycarbonate filters of decreasing pore diameter 0.1 μm using extruder. T.sub.1-weighted MR images (TE=8.5 ms, TR=400 ms) of an orthotopic transplanted breast cancer mouse before, 1 and 24 hours after (T.sub.1) intraperitoneal injection of 200 μl NS-629 labeled liposomes containing Magnevist® (Bayer HealthCare Pharmaceuticals) are shown in FIG. 8. The tumour tissue possess negative MR signal on T.sub.1-weighted images. The bright signal at 1 and 24 hours after injection in T.sub.1-weighted MR image shows successful targeting of Magnevist® (Bayer HealthCare Pharmaceuticals) loaded NS-629 labelled liposomes.

Example 9

Encapsulation of D-luciferine into the Liposome with Following Extrusion

(19) Aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of D-luciferine in PBS (15 mg/ml). Dispersions are homogenized with vortex mixing and extruded under pressure through polycarbonate filters of decreasing pore diameter 0.1 μm using extruder. Targeted delivery of liposomes labeled by lipidated inhibitor carrying D-luciferin into transgenic mouse expressing luciferase (FVB.luc.sup.tg/+) is shown in FIG. 7. The high-intensity luciferase signal associated with the induced paw edema demonstrates selective accumulation of labelled liposomes in the inflammation area.

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