Patent Application
20090246127
The present invention pertains to targeting contrast agents and targeting therapeutic agents, methods for their production and use thereof.
Known imaging techniques with tremendous importance in medical diagnostics are positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), single photon computed tomography (SPECT) and ultrasound (US). Although today's imaging technologies are well developed they rely mostly on non-specific, macroscopic, physical, physiological, or metabolic changes that differentiate pathological from normal tissue.
Targeting molecular imaging (MI) has the potential to reach a new dimension in medical diagnostics. The term “targeting” is related to the selective and highly specific binding of a natural or synthetic ligand (binder) to a molecule of interest (molecular target) in vitro or in vivo.
MI is a rapidly emerging biomedical research discipline that may be defined as the visual representation, characterization and quantification of biological processes at the cellular and sub-cellular levels within intact living organisms. It is a novel multidisciplinary field, in which the images produced reflect cellular and molecular pathways and in vivo mechanism of disease present within the context of physiologically authentic environments rather than identify molecular events responsible for disease.
Several different contrast-enhancing agents are known today and their unspecific or nontargeting forms are already in clinical routine. Some examples listed below are reported in literature.
For example, Gd-complexes could be used as contrast agents for MRI according to “Contrast Agents I” by W. Krause (Springer Verlag 2002, page one and following pages). Furthermore, superparamagnetic particles are another example of contrast-enhancing units, which could also be used as contrast agents for MRI (Textbook of Contrast Media, Superparamagnetic Oxides, Dawson, Cosgrove and Grainger Isis Medical Media Ltd, 1999, page 373 and following pages). As described in Contrast Agent II by W. Krause (Springer Verlag 2002, page 73 and following pages), gas-filled microbubbles could be used in a similar way as contrast agents for ultrasound. Moreover “Contrast Agents II” by W. Krause (Springer Verlag, 2002, page 151 and following pages) reports the use of iodinated liposomes or fatty acids as contrast agents for X-Ray imaging.
Contrast-enhancing agents that can be used in functional imaging are mainly developed for PET and SPECT.
One example of these contrast agents are 18F-labelled molecules such as desoxyglucose (Beuthien-Baumann B, et al., (2000), Carbohydr. Res., 327, 107). The use of these labeled molecules as contrast agents for PET is described in “Contrast Agents II” by W. Krause (Springer Verlag, 2002, page 201 and following pages). But they only accumulate in tumor tissue without any prior specific cell interaction. Further on, 99Tc-labelled molecules like antibodies or peptides could be used as targeting contrast agents for SPECT (Verbruggen A. M., Nosco D. L., Van Nerom C. G. et al., 99mTc-L,L-ethylenedicysteine: a renal imaging agent, Nucl. Med. 1992, 33, 551-557), but the labeling of such complex molecules is very difficult and costly.
The same can be said about several other ligands already existing for use in PET/SPECT, e.g. L-DOPA (dopamine receptor, Parkinson) (Luxen A., Guillaume M, Melega W P, Pike V W, Solin O, Wagner R, (1992) Int. J. Rad. Appl. Instrm B 19, 149); Serotonin analogue (serotonin receptor) (Dyck C H, et al., 2000, J. Nucl. Med., 41, 234); Somatostatin analogue (somatostatin, oncology) (Maecke, H. R. et al., Eur. J. Nucl. Med. Mol. Imaging, 2004, Mar. 17), Peptide for integrin receptors (angiogenesis) (Wicklinde, S. A. et al., Cancer Res., 2003 Sep. 15, 63(18), 5838-43; Wicklinde, S. A. et al., Circulation 2003 Nov. 4, 108, (18), 2270-4).
WO 01/09193 A1 and U.S. Pat. No. 6,437,095 B1 claim chimeric polypeptides, a method for production by directed association of peptides and disulfide bond formation and uses thereof for multimeric pharmaceutical agents (therapeutics). Electrostatic interaction promotes the directed association of two synthetic peptides and subsequent disulfide bond formation as described in S. A. Richter et al., Protein Engineering, 2001, vol. 14, no. 10, pp 775-783.
However, the identification of specific molecular events responsible for disease is of increasing importance in medicine. Targeting agents which are equipped with molecular recognition mechanisms to enrich contrast-enhancing materials specifically in certain tissues in vivo or in vitro and allow insights into molecular pathology are therefore essential to diagnosis and future therapy as well.
Thus, it is an object of the present invention to provide a new generation of improved contrast agents which allow an early diagnosis with high sensitivity and specificity as well as differential diagnosis and to supply methods for producing said improved contrast agents which are less costly and time consuming. Here is would also be advantageous to provide production processes and targeting agents produced thereby, which can be easily adapted to actually occurring problems which have to be solved in short time and with low effort concerning cost and man power. Apart from their potential for imaging diagnostics, targeting contrast agents will also play a crucial role in the development of new therapeutics. Such targeting contrast agents are still not available at the moment.
The object of the present invention is advantageously solved through the present invention as described below and additionally through the claims and examples. Preferred variants are described in the Figures and used for explanation of the invention but are not limiting.
The present invention pertains to one advantageous variant of a method for the production of a targeting contrast agent or targeting therapeutic agent, which method comprises the steps of:
a) providing a core;
b) adding a shell to the core;
c) modifying the shell through attaching at least a first polypeptide comprising at least one cysteine;
d) providing a ligand bearing a second complementary polypeptide comprising at least one cysteine; and
e) linking at least one ligand to the shell via a linking unit which is formed through electrostatic association between said first polypeptide and said second complementary polypeptide followed by at least one disulfide bond formation between said cysteines.
In a further embodiment of said method more than one shell can be added to the core in step b). Or in other words, the outer shell can be separated from the core by one to several inner shells. In preferred embodiments of the present invention the core can be separated from the outer shell by 1 to 100 inner shell(s), more preferred by 1 to 50 inner shell(s). Thereby the shell(s) can comprise a monolayer or a polylayer. Each of these shells (which can comprise a monolayer or a polylayer of an appropriated material in preferred embodiments of the present invention) has a thickness of about 0.5 nm to 100 nm. In a preferred embodiment of the present invention, each shell has a thickness of about 05 nm to 500 nm. Furthermore, each shell or even several shells can comprise the same material or different materials.
In a further variant of the present invention the shell(s) can cover the core at least partially. This is preferably the case when e.g. an organic polymer (e.g. polyethylenglycol/PEG, polyvinylalcohol/PVA, polyamide, polyacrylat, polyurea), an organic polymer with functional end groups (e.g. 1.2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(polyethylene glycol)2000] ammonium salt), a biopolymer (e.g. polysaccharide such as dextran, xylan, glycogen, pectin, cellulose or polypeptide such as collagen, globulin), cysteine or a peptide with high cysteine content or a phospholipid is used as shell(s). In the sense of the present invention adding a shell to the core means completely surrounding the core, covering only some distinct areas and preferably all ranges between these situations.
For a special application it might be suitable not to add one or more shell(s) to the core. Thus the present invention also pertains to a variant method for the production of a targeting contrast agent, the method comprising the steps of:
a) providing a core;
b) modifying the core through attaching at least a first polypeptide comprising at least one cysteine;
c) providing a ligand bearing a second complementary polypeptide comprising at least one cysteine; and
d) linking at least one ligand to the core via a linking unit which is formed through electrostatic association between said first polypeptide and said second complementary polypeptide followed by a disulfide bond formation between said cysteines.
Advantageous variations of present methods for the production of a targeting contrast agent or targeting therapeutic agents are described through the depending claims as attached.
In detail the present invention has several particular advantageous variations described as set out below:
The “core”, means material suitable as a contrast-enhancing part and/or the therapeutic part of the present targeting contrast agent. Said core is covalently and ionically bonded to the ligand, because of the particular structure of the polypeptides used as a linking unit.
In the sense of the present invention the expression “ligand” can be used as a synonym for binder or preferably for biologically active ligand.
The expression “linking unit” means the association of at least two polypeptides during the production process.
Advantageously, the “first polypeptide” comprises 1 to 3 cysteines and 4 to 12 basic amino acids selected from the group consisting of arginine, lysine and ornithine or 4 to 12 acidic amino acids selected from the group consisting of glutamate and aspartate and the “second complementary polypeptide” comprises 1 to 3 cysteines and 4 to 12 acidic amino acids selected from the group consisting of glutamate and aspartate or 4 to 12 basic amino acids selected from the group consisting of arginine, lysine and ornithine, wherein the group of basic amino acids selected for the first and second polypeptides are different.
Consequently, “complementary sequences” means either basic or acidic amino acids or any other amino acids which interact contrary to each other with respect to their electrostatic charging.
The “shell(s)” means material that can allow a good dispersion of the targeting contrast agent, can decrease its toxicity or can prevent adverse effects, depending on which material is used as a shell. If nanoparticles are used as the core, the use of an appropriated shell (e.g. a shell of ZnS) can reduce the number of surface defects of the nanoparticles. These defects considerably reduce the contrast generated by the nanoparticles. Therefore, reducing the number of defects leads to better targeting contrast-enhancing agents.
The ionic and covalent bond between said polypeptides can be generated under mild reaction condition in aqueous media, so that preferably the ligand keeps its full biological activity. This is possible because the electrostatic interactions between the two complementary polypeptides allow the formation of the disulfide bond under particularly mild conditions and because both the bonding unit and the contrast-enhancing cores (or eventually the core/shell(s) assemblies) are water-soluble.
“Mild conditions” preferably means art-known conditions under which the ligand will retain its activity and specificity, respectively, e.g. condition in aqueous solution or blood- or serum-such as solutions, physiological pH values at room temperature.
The “linking” is performed site-specifically at the cysteines of said polypeptides attached to the core and/or shell(s) or ligand, respectively. Because fixing said polypeptides to the ligand (or core/shell(s)) is possible in a directly controlled and highly selective way the catalytic or regulatory center or the recognition sites for specific binding of the ligand will retain its natural activity or avoids the deactivation of the ligand.
The expression “modifying the core/shell(s) through attaching” has to be understood in the same way as the fixing of said polypeptides to the ligand.
The shell(s) can comprise further components”, e.g. proteins which enable the passage of the complete targeting agents through e.g. cell membranes (e.g. the HIV-tat peptide, etc) or to increase the biocompatibility or decrease the toxicity.
The methods disclosed in this invention are potentially applicable to any ligand and any contrast-enhancing material or therapeutic material, providing a very versatile and easily adaptable system for the preparation of any kind of targeting contrast agent or targeting therapeutic agent.
The present invention further pertains to targeting contrast agents and targeting therapeutic agents and the use thereof.
The present targeting contrast agent has the following characteristics which describe the invention but are not limiting:
Depending on the contrast-enhancing material the targeting contrast agent can be applied in different imaging procedures such as MU, US, SPECT, CT, PET, optical imaging or multimodalit approaches like PET/CT.
The targeting contrast agent comprising a contrast-enhancing core (e.g. magnetic nanoparticle) or therapeutic core that can be covered by one ore more shells to improve stability and /or biocompatibility and/or to reduce toxicity in vivo (e.g. PEG shell).
If nanoparticles are used as the core, the size of these particles may vary from about 1 nm to 200 nm. In preferred embodiments of the present invention, the size of particles may vary from 1 nm to 100 nm.
If polymers are used as shell(s), the molecular weight of these polymers may vary from 200 g/mol to 200000 g/mol. In preferred embodiments of the present invention, the molecular weight of these polymers may vary from 200 g/mol to 100000 g/mol.
The targeting contrast agent comprises a targeting ligand.
A most preferred variant of a present targeting contrast agent comprises a core, at least one linking unit and at least one ligand.
Moreover, the present invention pertains to advantageous specifications of targeting contrast agents or targeting therapeutic agents comprising a core, at least one shell, at least one linking unit and at least one ligand.
Object of the present invention are further targeting contrast agents or targeting therapeutic agents produced by any one of the described or claimed methods.
The present targeting contrast agents or targeting therapeutic agents are suitable for use in diagnosis or therapy, preferably in targeting molecular imaging.
Targeting contrast agents for use in CT, MRI, PET, SPECT or US are also included in the present invention.
Moreover, the use of the present targeting contrast agents or targeting therapeutic agents for the production of compounds suitable in diagnosis or therapy are object of the present invention as well as the use of the targeting contrast agents or targeting therapeutic agents for the production of compounds suitable for targeting molecular imaging. Additionally, the present invention pertains to the use of the present targeting contrast agents for the production of compounds suitable in CT, MRI, PET, SPECT or US.
A most preferred variant of the present targeting contrast agent is described schematically in
Core (1): e.g. (not limited to this) contrast-enhancing material; or therapeutical material for:
Paramagnetic ion (e.g. lanthanide, manganese, iron, copper) based contrast-enhancing units e.g. Gadolinium chelates like Gd(DTPA), Gd(BMA-DTPA), Gd(DOTA), Gd(DO3A); oligomeric structures; macromolecular structures such as Albumin Gd(DTPA)20-35, Dextran Gd(DTPA), Gd(DTPA)-24-cascade polymer, polylysine-Gd(DTPA), MPEG polylysine-Gd(DTPA); dendrimeric structures of lanthanide based contrast-enhancing units; Manganese-based contrast-enhancing units such as Mn(DPDP), Mn(EDTA-MEA), poly-Mn(EED-EEA), and polymeric structures; liposomes as carriers of paramagnetic ions e.g. liposomal Gd(DTPA); non-proton imaging agents;
Shell(s) (2): e.g. (not limited to these) may comprise carboxylic acids, acid halides, amines, acid anhydrides, activated esters, maleimides, isothiocyanates, amines, gold, SiO2, a polyphosphate (e.g. calcium polyphosphate), an amino acid (e.g. cysteine), an organic polymer (e.g. polyethylenglycol/PEG, polyvinylalcohol/PVA, polyamide, polyacrylat, polyurea), an organic functional polymer (e.g. 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(polyethylene glycol)2000] ammonium salt), a biopolymer (e.g. polysaccharide such as dextran, xylan, glycogen, pectin, cellulose or polypeptide such as collagen, globulin), cysteine or a peptide with high cysteine content or a phospholipid.
The core can be separated from the outer shell (3) by 1 to 100 inner shell(s) (2). In preferred embodiments of the present invention, the core can be separated from the outer shell by 1 to 50 inner shell(s). Each of these shells (which can consist of a monolayer or a polylayer of an appropriated material in preferred embodiments of the present invention) has a thickness of about 0.5 nm to 100 nm. In a preferred embodiment of the present invention, each shell has a thickness of about 0.5 nm to 500 nm and can be made of different materials or of the same material. Furthermore, the shell can cover the core at least partially.
Linking Unit (4):
Ligand (5):
Reaction scheme for the surface modification of a contrast-enhancing unit with a Cys-Glu-Glu-Glu-Glu-Glu-Glu-Glu-Glu (=Cys-Glu8) functionality by a one pot reaction of carboxylic acids, linked to the contrast enhancing unit, with 1-Ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) to form a o-Acylisourea intermediate (room temperature, pH≈5). This intermediate reacts with sulfo-NHS to give a sulfo-NHS-ester intermediate. The excess of EDC is quenched by the addition of 2-mercaptoethanol. Finally the reaction with a primary amine containing Cys-Glu8 leads to the desired amide bond (room temperature, pH≈7).
Reaction scheme for the coupling of Cys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (=Cys-Lys8) to contrast-enhancing material bearing a Cys-Glu8 functionality by a one pot reaction of Cys-Lys8 and a contrast-enhancing material bearing a Cys-Glu8 functionality, followed by the disulfide bond formation, due to the addition of an oxidation agent, GSSG (oxidized Glutathione).
CdSe/ZnS Quantum Dots (contrast-enhancing units) are surface modified with a carboxylic acid functionality by an acid by a water soluble polymer bearing a carboxylic acid function at one end and a 1.2-Distearoyl-sn-Glycero-3-Phosphoethanolamine function at the other end.
The COOH coated Quantum Dots are obtained by mixing (4 h at 50° C.):
And finally removing the Chloroform by vacuum and dispersing the COOH coated Quantum Dots in water by an ultrasonic treatment
The 1.2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(polyethylene glycol)2000] ammonium salt binds to the surface of the nanoparticles by hydrophobic interactions (or adsorption)by the 1.2-Distearoyl-sn-Glycero-3-Phosphoethanolamine end group. Furthermore the 1.2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(polyethylene glycol)2000] ammonium salt provides a carboxy function, which is protonated, at an acid pH, to obtain a carboxylic acid.
DPPC is used as a dummy (or spacer) to leave spaces between the COOH functions fixed on the nanoparticles. Actually the covering of the whole nanoparticle surface only by COOH functions could have adverse effects by creating interactions, and therefore contrast, in undesired tissues or undesired areas of the body.
The contrast-enhancing unit is surface modified with Cys-Glu8 functionality by a coupling via an acid.
Other examples would be e.g. coupling via an activated ester, via maleimide or via isothiocyanate.
This can be done by:
1) Modifying water soluble CdSe/ZnS Quantum Dots (
55 μl water
40 l 10× PBS solution (PBS=phosphate buffer saline: 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4)
100 μl 0.1 M EDC solution (EDC=1-Ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride)
5 μl 20 mM sulfo-NHS solution (N-Hydroxysulfosuccimide sodium salt)
200 μl 2 82 M CdSe/ZnS Quantum Dots (COOH terminated) solution
Incubation at room temperature (30 min)
10 μl 2-mercaptoethanol
mixing for 15 mins
50 μl 29 mM Cys-Glu8
mixing at r.t. (2 h)
separation of QDs by centrifugation
2) Coupling of Cys-Lys8 to contrast-enhancing unit QD-Cys-Glu8 unit (
50 μM Cys-Lys8
5 μM QD-Cys-Glu8
2 mM EDTA
2.5 mM oxidized Glutathione (GSSG)
Incubation at room temperature (5 h)
separation of QD-conjugates by ultracentrifugation
| Number | Date | Country | Kind |
|---|---|---|---|
| 04106696.0 | Dec 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB05/54187 | 12/12/2005 | WO | 00 | 6/11/2007 |
