EP2046392A2 - Conjugue radiopaque - Google Patents
Conjugue radiopaqueInfo
- Publication number
- EP2046392A2 EP2046392A2 EP07786390A EP07786390A EP2046392A2 EP 2046392 A2 EP2046392 A2 EP 2046392A2 EP 07786390 A EP07786390 A EP 07786390A EP 07786390 A EP07786390 A EP 07786390A EP 2046392 A2 EP2046392 A2 EP 2046392A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptide
- conjugate
- conjugate according
- cells
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- 238000003325 tomography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
- A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
- A61K49/0043—Fluorescein, used in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/04—X-ray contrast preparations
- A61K49/0433—X-ray contrast preparations containing an organic halogenated X-ray contrast-enhancing agent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Definitions
- the present invention relates to a radiopaque conjugate, the use of the conjugate for the preparation of a diagnostic and therapeutic composition, a pharmaceutical and / or diagnostic composition comprising the conjugate, a method for the diagnostic and / or analytical treatment of biological material or a living being, and a method for the therapeutic treatment of a living being.
- Computed tomography is a very widespread imaging method in emergency and routine examinations, which enables a particularly high resolution, for example of the lung parenchyma, in comparison to magnetic resonance tomography.
- Contrast agents are used to increase contrast in CT.
- iodine-containing substances such as iopromide (Ultravist®) or ipitridol (Xenetix®).
- These contrast agents have as a backbone, due to its radiopaque iodine atoms, 2,3,5-triiodobenzoic acid (TIBA), which was originally mainly used to induce a premature flowering / maturation period Plants was used; see.
- TIBA 2,3,5-triiodobenzoic acid
- the currently used iodine-containing contrast agents have the disadvantage that they can not penetrate the membrane of the biological cells and therefore only in the intercellular space, i. in the interstitium of tumors (see Krause W., Delivery of diagnostic agents in computed tomography, Adv. Drug Deliv. Rev., 159-173 (1999)).
- a contrast agent which has an amphiphilic character and forms in physiological fluids micelles.
- the contrast agent contains three molecules of TIBA per total molecule bound to a carboxylic acid backbone. Both components form a hydrophobic block.
- This hydrophobic block is in turn bound to a hydrophilic polymer component consisting of monomethoxypolyethylene glycol (MPEG).
- MPEG monomethoxypolyethylene glycol
- the contrast agent because of its retention in the bloodstream, be used exclusively for the presentation of bloodstreams, as a so-called "blood pool” contrast agent, for example in the context of angiography, which is eliminated again after a short time from the body.
- a comparable very high molecular weight "blood pool” contrast agent is described in the publication by Fu et al., "Dendritic ionidated contrast agents with PEG cores for CT imaging: synthesis and preliminary characterization", Bioconjugate Chem. 17, 1043-1056 (2006 ) having triiodophtalamide groups coupled via beta-alanine to polylysine in the form of "lysine trees".
- US 2005/0119470 A1 discloses a composition consisting of an iodine-containing compound, namely 2,3,5-triiodobenzoic acid (TIBA), and two peptides or nucleic acids which can at least partially hybridize with one another.
- TIBA 2,3,5-triiodobenzoic acid
- RNA silencer two peptides or nucleic acids which can at least partially hybridize with one another.
- the known extracellular contrast agents are not able to penetrate into biological cells, tumor tissue, for example, can not be exactly distinguished from healthy tissue by computer tomography. Only a washed out representation of the tumor borders occurs. If the said extracellular contrast agents are administered during or directly after an operation, then the contrast agent runs along the intercellular space opened by the surgeon to beyond the tumor borders.
- WO 2006/069677 A2 describes a composition which comprises a derivative of 2,3,5-triiodobenzoic acid (TIBA) and a "targeting group", for example a nuclear localization sequence (NLS), in combination with a material for producing an implantable medical Device and a therapeutically active agent.
- TIBA 2,3,5-triiodobenzoic acid
- NLS nuclear localization sequence
- the metal clip Since the biopsied tumor does not remain constantly large but changes in size, the metal clip does not remain in its original position and shifts. In biopsies of the mammary gland tissue, one year later, the metal clips could be found far away from the original biopsy site even in another breast quadrant; Philpotts L.E. & Lee C.H., Clip migration after 11-gauge vacuum-assisted stereotactic biopsy: case report. Radiology 222, 794-796 (2002). If bleeding occurs during a biopsy, the metal clip can also be flushed out of the biopsy area via the puncture channel to below the skin; see. Parikh J., Ultrasound Demonstration of Skin Migration within 6 weeks of 11-gauge vacuum-assisted stereotactic breast biopsy. Breast J. 10, 539-542 (2004).
- radioactive substances may be used, such as radioactive iodine.
- radioactive iodine Currently, this radioiodine therapy in humans only succeeds in thyroid carcinoma, since this special type of tumor cell expresses the sodium iodine symporter on the cell surface.
- radioactive iodine is administered to a patient, it is taken up by the thyroid carcinoma cells together with sodium, ie in the sodium iodine symport, into the cell interior, where it can develop its radiochemotherapeutic effect.
- the sodium-iodine-symporter is present only in thyroid carcinoma, other aggressive cancers, such as brain, prostate or intestinal tumors, in humans so far can not be treated with radioactively labeled iodine ( 131 I), since these tumors can not absorb the iodine .
- radioactively labeled iodine 131 I
- these tumors can not absorb the iodine
- to access human colon carcinoma cells for radioactively labeled iodine they must first be transfected with the gene of the sodium iodine symporter; see. Scholz IV et al., Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer. Gene Ther. 12, 272-80 (2005).
- These human colon carcinoma cells then express, just like the thyroid carcinoma cells, the cell surface sodium iodine symporter and can now also absorb the radioactively labeled iodine.
- radioiodine therapy of thyroid carcinoma healthy tissues such as, for example, salivary glands, gastric mucosa and the tactile breast, also receive radioactively labeled iodine, since the sodium iodine symporter is also expressed here; see. Spitzweg C. et al., Analysis of human sodium iodide expression gene in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa. J. Clin. Endocrinol. Metab. 83, 1746-51 (1998). Therefore, with radioiodine therapy currently used, e.g. Salivary inflammation and changes in taste occur; see. Alexander C.
- an object of the invention to provide a radiopaque substance which is suitable as a contrast agent and with which the disadvantages of the currently used iodine contrast agents are avoided.
- it is intended to provide such a contrast agent with which tomographic sharply richer tumor margins can be displayed than is the case with the current contrast agents.
- Another object of the present invention is to provide a substance which enables improved biopsy site marking than is the case with metal clips currently used.
- a further object of the invention is to provide a radiopaque substance which can be used therapeutically, in particular for the treatment of tumors, and with which the disadvantages of current radioiodine therapy can be avoided.
- a substance is to be provided which shows a rapidly occurring effect after administration and is preferably non-radioactive.
- the first compound may have 1, 2, 3, 4 or 5 halogens or 1, 2, 3, 4 or 5 hydrogen atoms.
- the halogens or hydrogens can be arranged at any of the indicated positions R 1 to R s .
- the first compound may have different or identical halogens.
- R 6 is a carboxyl group
- 5-iodobenzoic acid, 4-iodobenzoic acid 5-IBA / MIBA, 4-IBA / MIBA; Position R 1 or R 5 or R 4 ), 2,3- or 3,5-diiodobenzoic acid (2,3-DIBA, 3,5-DIBA, two iodine atoms at position R 1 and R 2 or R 4 and R 3 5 or R 3 and R s ), 3,5-diiodobenzoic acid (3,5-DIBA, two iodine atoms at position R3 and R 5 or Ri and Rs), 2,4,6-, 2,3,6- , or 2,3,5-trichlorobenzoric acid (TCBA, three chlorine atoms at the positions R 2 , R 4 , R s , or R 2 , R 3 , R s , and R 2 , R 3 , R s
- a conjugate is understood to be the linking product of a plurality of substances.
- the substances linked to one another comprise the first compound and optionally further, for example, second and third compounds and also the first peptide and optionally further, for example second and third peptides.
- the linkage can be carried out in any desired manner, for example by covalent or ionic bonding.
- the inventors have found that a particularly good signal in imaging processes can be achieved by the radiopaque halogens with the conjugate according to the invention.
- the inventors have further surprisingly found that the first compound in complex with a first peptide can penetrate the membrane of biological cells and, moreover, can penetrate into the cell nucleus.
- the cell membrane and nuclear membrane permeability of the conjugate according to the invention has the great advantage that now the boundaries of a particular tissue or a tumor in the context of an imaging method, such as a computed tomography, can be displayed sharply.
- the conjugate when administered during or immediately after surgery, can no longer extend beyond the tumor borders along the opened intercellular space.
- the cell membrane and nuclear membrane permeability of the conjugate according to the invention without coupling of additional large transmembrane transport units, such as, for example, penetratin or transportan. This avoids an unnecessary increase in molecular weight, which could affect the signaling in computed tomography.
- the conjugate according to the invention is relatively small, ie it is in the range from about 1400 Da to about 3300 Da, preferably from 1600 Da to 1800 Da.
- the conjugate according to the invention is free of a hydrophilic polymer component, such as monomethoxypolyethylene glycol (MPEG), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) etc., which are already due to its size would prevent penetration of a compound coupled thereto into the interior of biological cells.
- a hydrophilic polymer component such as monomethoxypolyethylene glycol (MPEG), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) etc.
- MPEG monomethoxypolyethylene glycol
- PEG polyethylene glycol
- PVP polyvinylpyrrolidone
- the conjugate according to the invention is substantially smaller and preferably has a molecular weight of about 0.5 to about 5 kDa, more preferably from about 1 to about 3 kDa, most preferably about 2 kDa.
- the conjugate according to the invention comprises an amino acid consisting of peptide bonds linked together by peptide bonds, which preferably contains at least two different amino acids. that the conjugate according to the invention can penetrate into biological cells, whereas the known contrast agent remains exclusively in the blood and does not even get into the interstitium.
- the conjugate according to the invention also has a very large halogen content, up to about 40%, in contrast to the known contrast agent on the whole construct, is not toxic to the whole organism and does not form micelles.
- the operability of the conjugate according to the invention does not require such a peptide which is hybridizable with an oligomeric compound, such as another peptide or a nucleic acid.
- the conjugate according to the invention is particularly well suited for labeling a biopsy site since it is biopsied after administration to the biopsy site Tissue remains fixed in place. A later finding of the biopsy site is thus possible without problems.
- the conjugate of the invention after its uptake into the cells in the latter within a short time the programmed cell death, the so-called apoptosis, triggers.
- the first compound alone in the form of triiodobenzoic acid (TIBA)
- TIBA triiodobenzoic acid
- the initiation of apoptosis by the conjugate according to the invention in the ingested cells takes place already at low concentrations, e.g. in the range of ⁇ 300 ⁇ g conjugate / ml.
- contrast agents such as diatrizoates, ioxaglates, iopromides, iotroan, do not trigger apoptosis until very high concentrations, e.g. at 250 mg iodine per milliliter); see. Zhang et al. (2000), Effects of radiographic contrast media on proliferation and apoptosis of human vascular endothelial cells, The British Journal of Radiology 73, pp. 1034-1041.
- the therapeutically usable apoptosis-initiating property of the conjugate according to the invention already shows itself without the presence of a further therapeutically active agent or / and a material for producing an implantable medical device.
- the conjugate according to the invention has a significant therapeutic potential, which can be used for the treatment of tumor diseases.
- the uptake into the cells is independent of the sodium-iodine-symporter, so that now not only thyroid carcinomas can be treated, but also other tumors, such as prostate carcinomas, brain tumors and breast cancer.
- Radiolabeling of the halogens can be dispensed with, since, surprisingly, the apoptosis of the tumor cells is already triggered by the compound that accumulates in the nucleus and is bound to the peptide. Thereby remains a radioactive strain on the body, in particular the gastric mucosa, the salivary glands and the lactating breast from. Patients also no longer need to spend therapy in screened rooms.
- the conjugate according to the invention can be given into a tumor cavity during an operation, for example for 20 minutes. Following this, the resection cavity is rinsed with conjugate-free buffer solution to remove excess conjugate that was not taken up by the cells.
- the tumor cells lining the resection cavity are thereby stained intracellularly or intranuclearly and the tumor borders can be sharply visualized in comparison to the previous interstitial iodine contrast agents.
- the computed tomography devices currently available on the market for use in the operating room and intraoperative resection control can be used, e.g. the mobile CT scanner Philips Tomoscan M, Philips Medical Systems, Eindhoven, The Netherlands.
- the uptake into healthy tissue is prevented on account of the blood brain barrier intact in the healthy brain parenchyma, whereas in the brain tumor the blood-brain barrier is permeable, so that the conjugate can penetrate and specifically trigger apoptosis.
- the higher signal density of the tumor cells in computed tomography can be regarded as a sign of tumor apoptosis.
- a reservoir for example an omaya reservoir, can be applied intratumorally during a brain operation.
- the solution with the conjugates according to the invention may optionally be infused over a number of cycles via this access.
- the first peptide may be attached to the first compound in a variety of ways by methods known to those skilled in the art, for example by taking advantage of Compound-side carboxyl group (R 6 ) and formation of a peptidic bond with a peptide-side free NH 2 group.
- halogen of the first compound is one selected from the group consisting of: iodine, bromine, fluorine, chlorine, and astatine.
- the first compound is one selected from the group consisting of: triiodobenzoic acid (TIBA), 5-iodobenzoic acid (5-IBA, 5-MIBA), 4-iodobenzoic acid (4- IBA, 4-MIBA), 2,3-diiodobenzoic acid (2,3-DIBA), 3,5-diiodobenzoic acid (3,5-DIBA), 2,5-diiodobenzoic acid (2,5-DIBA), trichlorobenzoic acid ( TCBA), trifluorobenzoic acid (TFBA), tribromobenzoic acid (TBBA), tribromophenyl isothiocyanate (TBPI).
- TIBA triiodobenzoic acid
- 5-IBA 5-iodobenzoic acid
- 5-MIBA 5-MIBA
- 4-iodobenzoic acid (4- IBA, 4-MIBA
- TIBA has the molecular formula I3C6H2CO2H and a molecular weight of 499.81 Da.
- TIBA is listed under CAS number 88-82-4.
- the benzene ring of the compound is iodine-iodinated 3 times, preferably at positions 2, 3 and 5 (R 1 , R 2 and R 4 ) or 2, 4 and 6 (R 1 , R 3 and R 5 ). The iodination is also possible at other positions of the benzene ring.
- IBA / MIBA has the molecular formula IC6H4CO2H and a molecular weight of 248.02 Da.
- IBA / MIBA is listed under CAS number 88-67-5.
- 2,3-DIBA, 3,5-DIBA, 2,5-DIBA have the molecular formulas C7H4I2O2 and the molecular weights of 373,914 Da on.
- 2,3-DIBA, 3,5-DIBA, 2,5-DIBA are listed under CAS numbers 19094-48-5 (3.5) and 14192-12-2 (2.5), respectively.
- TCBA has the molecular formula C 7 H 3 Cl 3 O 2 and a molecular weight of 225.45862 Da.
- TCBA is listed under CAS number 50-73-7.
- TFBA has the molecular formula C 7 H 3 F 3 O 2 and a molecular weight of 176.09 Da.
- TFBA is listed under CAS number 121602-93-5.
- TBPI has the molecular formula C 7 H 2 Br 3 NS and a molecular weight of 371.87158.
- TBPI is listed under CAS number 22134-11-8.
- TBBA has the molecular formula C? H 3 Br 3 O 2 and a molecular weight of 358.81 Da.
- TBBA is listed under CAS number 633-12-5.
- the first peptide of the conjugate according to the invention has a net positive charge.
- Net charge is understood as meaning the charge of the first peptide which, under physiological conditions (pH 7), results from the charge contribution of the individual positively or negatively charged amino acid residues of the peptide.
- the inventors have surprisingly found that the cell membrane and nuclear permeability of the conjugate of the invention is particularly well achieved when the first peptide has a net positive charge.
- the first peptide preferably has such amino acids that are positively charged under physiological conditions.
- the first peptide according to the invention therefore preferably has one or more molecules of the "basic" amino acids arginine (R), lysine (K) or histidine (H).
- the first peptide in the conjugate according to the invention prefferably has 2-20, more preferably 5-10, and most preferably seven amino acids.
- the inventors have surprisingly found that such short peptides are sufficient to mediate the transport of the conjugate through both the cell membrane and the nuclear membrane.
- large transport peptides such as penetratin or transportan, are therefor not mandatory. Due to the small size of the first peptide, the ratio between the TIBA signaling in the imaging method and the non-signaling first peptide is kept as low as possible, and a good signal is generated even with a small amount of conjugate.
- the first peptide is derived from a nuclear localization sequence (NLS).
- NLS nuclear localization sequence
- NLS Nuclear localization sequences
- the nuclear membrane permeability of the conjugate of the invention was also achieved using a mutant NLS. Already a 20% or 40%, preferably 60%, more preferably 80%, most preferably 90% sequence identity with a natural NLS is sufficient. It is crucial that at least one or more of the "basic" amino acids contained in the natural NLS remain present.
- the first compound is bound to the peptide via its carboxyl group.
- This measure has the advantage that a group which is particularly suitable for coupling the peptide to the benzene ring of the first compound is used without as a result, the halogen substituents are involved in the bond or, if appropriate, sterically hindered or shielded, but rather are available for realizing the invention.
- the first peptide has a free amino function in the side chain via which it is bonded to the first compound.
- This measure has the advantage that even on the first peptide a particularly suitable reactive group is exploited, via which a binding to the first compound, for example.
- a particularly suitable reactive group is exploited, via which a binding to the first compound, for example.
- free amino functions are found, for example, in the side chain of asparagine (N), glutamine (Q), or lysine (K) and at the N-terminus of the peptide.
- the peptide has an ⁇ -amino function of a C-terminal lysine residue, via which it is bound to the compound.
- the conjugate according to the invention assumes a particularly advantageous conformation which ensures the imaging and apoptosis-inducing properties.
- the terminal lysine residue may either be part of the first peptide or may be attached to a terminal end of the first compound as a kind of "hanger".
- the first peptide has the amino acid sequence PKKKRKV (SEQ ID NO: 1) or PKKTRKV (SEQ ID NO: 2) or PLGLA (SEQ ID NO: 13).
- the C-terminus is on the right and the N-terminus on the left.
- the usual one-letter code was used for Amino acids.
- This measure has the advantage of providing the design prerequisites for a first peptide which has a net positive charge and is derived from the NLS sequence of the SV40 T antigen.
- the sequence PKKKRKV was identically taken from the NLS of the SV40-T antigen, while in the sequence PKKTRKV at the fourth position opposite to the NLS from the SV40-T antigen a lysine (K) was replaced by a threonine (T).
- first peptides with sequences having homology of 80%, 85%, 90%, 95%, 98% to SEQ ID NO: 1 or NO 2 are also suitable.
- a lysine residue can readily be provided, for example, at the C-terminus, so that, for example, the following sequences are obtained: PKKKRKVX or PKKTRKVK, wherein the lysine "hanger" is shown in italics.
- the conjugate according to the invention has a detectable marker.
- a detectable marker means any compound which can be identified by means of imaging methods. These include color indicators such as dyes having fluorescent, phosphorescent or chemiluminescent properties, dansyl or coumarin dyes, AMPPD, CSPD, non-radioactive indicators such as biotin or digoxigenin, alkaline phosphatase, peroxidase, etc.
- radioactive indicators such as 32 P, 3 S, 132 I, 31 I, 14 C or 3 H can be used, in which case the disadvantages described above in connection with the radioiodine therapy in Purchase must be taken.
- microscopy, blotting, hybridization techniques or autoradiography may be considered as imaging techniques.
- the detectable marker is a fluorescent dye, preferably fluorescein isothiocyanate (FITC).
- FITC fluorescein isothiocyanate
- Fluorescence microscopes are available in most operating theaters. Thus, after administration of the conjugates according to the invention into the tumor cell, the tumor borders can already be displayed by fluorescence microscopy during the operation. This allows an even better localization of the tumor and possibly better therapeutic or surgical measures.
- the detectable marker has a free amino function of an amino acid, preferably an ⁇ -amino function of a lysine residue, via which it is bound to the peptide.
- the lysine residue can either be part of the first peptide or can be attached to a terminal, preferably C-terminal, end of the first peptide as a kind of "hanger" for the detectable marker.
- a Aminoklarespacer is arranged, which preferably has 2 amino acids, more preferably the amino acid sequence GG (SEQ ID NO: 3).
- This measure has the advantage that interfering steric interactions between the detectable marker and the first compound are largely avoided and thus the conjugate according to the invention remains functional despite the bound further detectable marker.
- the amino acid spacer can have any amino acid sequence or even a length of 1 or 3 amino acids, since this essentially serves to space the first peptide or the detectable marker from the first compound, although the sequence GG has proved to be particularly suitable.
- the spacer can furthermore have C and / or N-terminal lysine residues as "hangers" for the detectable marker or the first compound
- the amino acid spacer can also be replaced by a nonpeptidic spacer as long as the function described above is ensured.
- the conjugate according to the invention further comprises at least one component which confers tumor cell specificity or specificity for virus-infected cells.
- Such a component which may be coupled to or incorporated into the conjugate in a manner known to those skilled in the art, may be realized, for example, in the form of an antibody and / or an aptamer having a specificity or affinity for such Cell, for example, binds to a tumor marker or an infection marker that is specifically expressed on the surface of a tumor or infected cell.
- Such a component may also be realized, for example, in the form of a synthetic ligand having specificity for such cells, or by viruses or components thereof, which are coupled to the conjugate of the invention and confer tropism to tumor cells or virus-infected cells to the latter.
- the component has a third peptide which (i) has a charge which at least neutralizes the net positive charge of the first peptide, and (ii) is linked to the conjugate via a second peptide which has an amino acid recognition sequence for a tumor cell or virus-specific enzyme having.
- tumor cell- or virus-specific enzyme are preferably matrix metalloproteinases (MMP), cathepsins, prostate-specific antigen (PSA), herpes simplex virus protease, human immunodeficiency virus protease, cytomegalovirus protease, interleukin-lß-converting enzyme in question.
- This measure has the advantage that a tumor cell specificity or specificity for virus-infected cells is imparted to the conjugate according to the invention in a particularly effective manner.
- various tumor or carcinoma cells express characteristic enzymes and secrete them into their cellular environment, for example in order to digest the surrounding connective tissue in order to invade previously healthy tissues or organs.
- Glioblastomas for example, predominantly express and secrete matrix metalloproteinase 2 (MMP2).
- MMP2 matrix metalloproteinase 2
- Breast cancers express and secrete predominantly cathepsins which recognize and cut a specific amino acid sequence.
- Prostate cancers express and secrete mainly prostate specific antigen (PSA). It is also known that virus-infected cells express and secrete virus-specific proteases.
- PSA prostate specific antigen
- Herpes simplex virus (HSV) infected cells secrete herpes simplex virus protease.
- Cells infected with the HIV virus express and secrete HIV proteases.
- Cells infected with cytomegalovirus express and secrete a protease specific for this type of virus.
- MMP2 recognizes the sequence PLGVR (SEQ ID NO: 4), PLGVA (SEQ ID NO: 5) or PLGLA (SEQ ID NO: 13), while cathepsin B recognizes the specific sequences KK (SEQ ID NO: 6) and / or RR (SEQ ID NO. SEQ ID NO. 7).
- Cathepsin D recognizes the sequence PIC (Et) FF, where "Et” denotes an ester branch, Cathepsin K recognizes the specific sequence GGPRGLPG (SEQ ID No. 8), whereas PSA recognizes the amino acid sequence HSSKLQ (SEQ ID No.
- Enzymes and their specific recognition and cleavage sites are well described in the art, an overview of which is given in Hahn, WC and Weinberg, RA, Rules for making human tumor cells, N. Engl. J. Med. 347, pages 1593-1603 (2002), the content of which is incorporated herein by reference.
- the HSV protease recognizes the amino acid sequence AEAGALVNASSAAHVDV (SEQ ID NO: 10), the HIV protease recognizes the sequence SQNYPIVQ (SEQ ID NO: 11), the cytomegalovirus protease recognizes the sequence GWNASCRLA (SEQ ID NO: 12).
- Second peptides with sequences having homology of 80%, 85%, 90%, 95%, 98% to the above sequences are also suitable.
- the third peptide of the component having a charge that at least neutralizes the net positive charge of the first peptide is of particular importance for tumor cell specificity of the conjugate or specificity for virus-infected cells.
- the third peptide has a net negative charge that cancels the positive charge of the first peptide.
- the negative net charge of the third peptide can be realized, for example, by negatively charged amino acids, such as glutamic acid (E) or aspartic acid (D).
- At least neutralizing in this context means that the third peptide can also result in a net negative charge of the modified construct of the invention As a result of this neutralization or negativity of the charge of the conjugate of the present invention so modified, the latter accumulates in the interstitium and is no longer in However, the situation is different in the environment of tumor cells, where the tumor cell-specific extracellular proteases mentioned above cause the cleavage of the second peptide, the corresponding one This results in the environment of tumor cells or of virus-infected cells at a loss of the neutralizing or negative charge of the third peptide.
- the positively charged first peptide re-affords a net positive charge whereby the conjugate of the present invention can penetrate both the cell membrane and the nuclear membrane of the tumor cells and virus-infected cells, respectively. This process is thus dependent on the environment of the tumor cells or virus-infected cells, so that the modified conjugate according to the invention can only penetrate into such cells and there develop the apoptotic effect.
- the component has the following, namely a second compound with the following formula:
- R 1 , R 2 , R 3 , R 4 and R s each independently correspond to a halogen or a hydrogen
- a second one peptide linked to the second compound which has an amino acid recognition sequence for a tumor cell- or virus-specific enzyme, the component being linked to the conjugate via the second peptide.
- the inventors thus provide such a conjugate which itself blocks in its ability to penetrate into the cytoplasm or cell nuclei of healthy cells. This self-lock is ensured by the presence of the second connection. Due to the associated size and configuration of the conjugate, an uptake into the cytoplasm or the Nucleus of healthy cells prevented. Rather, the conjugate remains in the interior and is excreted from the organism after some time. By contrast, in the vicinity of tumor cells or virus-infected cells, the second, specificity-mediating peptide is recognized and cut by the tumor- or virus-specific proteases.
- the second compound is thereby cleaved from the conjugate and the latter can penetrate due to the first peptide in the cytoplasm and the cell nucleus of the tumor cell.
- the separated second compound remains after cleavage by the tumor or virus-specific protease for some time in the interstitium and acts in a configuration as a contrast agent advantageously significantly in the signaling in the tumor or infected area with them until they are removed from the interstitium and the organism is excreted. It is particularly advantageous that the separated second compound has no neurotoxic properties, as described, for example, for peptides with a negative net charge; see. Garattini et al. (2000), Glutamic Acid, Twenty Years later, J. Nutr. 130 (4S Suppl.): 901S-9S.
- the conjugate according to the invention thus modified becomes, as it were, "activated” in the vicinity of tumor or virus-infected cells and cell membrane and nuclear membrane permeable, whereas such activation does not occur in the presence of healthy cells modified conjugate unfolds its properties exclusively in tumor cells or virus-infected cells.
- the halogen of the second compound is selected from a group consisting of: iodine, bromine, fluorine, chlorine, and astatine.
- halogens are characterized by their high radiopacity and therefore provide particularly good signals in imaging processes.
- the second compound is selected from the group consisting of: triiodobenzoic acid (TIBA), 5-iodobenzoic acid (5-IBA, 5- MIBA), 4-iodobenzoic acid (4-IBA, 4-MIBA), 2,3-diiodobenzoic acid (2,3-DIBA), 3,5-diiodobenzoic acid (3,5-DIBA), 2,5-diiodobenzoic acid (2, 5-DIBA), trichlorobenzoic acid (TCBA), trifluorobenzoic acid (TFBA), tribromobenzoic acid (TBBA), tribromophenyl isocyanate (TBPI).
- TIBA triiodobenzoic acid
- 5-IBA 5-iodobenzoic acid
- 4-MIBA 4-iodobenzoic acid
- the second peptide has the amino acid sequence PLGLR (SEQ ID NO: 4) and / or PLGVA (SEQ ID NO: 5) and / or PLGLA (SEQ ID NO: 13).
- MMP-2 matrix metalloproteinase 2
- Second peptides with sequences having homology of 80%, 85%, 90%, 95%, 98% to SEQ ID NO: 4 or NO. 5 are also suitable.
- MMP-2 inhibitors such as prinomastate
- the second peptide is bound to the second compound via the ⁇ -amino function of a C-terminal lysine residue.
- the lysine residue may be part of the second peptide, but also connect to it C- or N-terminal, so that, for example, the following sequences for the second peptide are obtained: PLGLRiC or PLGVAiC, wherein the lysine "hanger" is shown in italics .
- the Lysine residue may preferably be covalently bound to the second peptide via its ⁇ -amino group or its ⁇ -carboxyl group.
- the conjugate according to the invention has a third peptide, which preferably has a net positive charge, more preferably 2 to 20, preferably 5 to 10, more preferably 7 amino acids. Further, it is preferred if the third peptide is derived from a nuclear localization sequence (NLS). The third peptide preferably has a free amino function of a side chain via which it is bonded to the second compound, preferably via an ⁇ -amino function of an N-terminal lysine residue. It is preferred if the third peptide has the amino acid sequence PKKKRKV (SEQ ID NO: 1) or the amino acid sequence PKKTRKV (SEQ ID NO: 2).
- the third peptide thus has the same properties as the first peptide.
- the comments made on the first peptide therefore apply correspondingly to the third peptide.
- a detectable marker such as FITC
- another dye such as rhodamine
- rhodamine may also be coupled to the third peptide in the appropriate manner so that the markers differ from one another. This measure creates a more or less symmetrical tumor-specific conjugate, wherein after cleavage of the second peptide by a tumor cell-specific protease, such as MMP-2, both cleavage products can penetrate specifically into the tumor cell.
- the tumor cell specificity-mediating component comprises a nucleic acid molecule which hybridizes under stringent conditions to tumor cell-specific molecules, preferably oncogenes.
- nucleic acid molecule can be bound in a variety of ways to the conjugate according to the invention by methods known to those skilled in the art. There is a coupling both to the first and / or second peptide and to TIBA conceivable.
- the nucleic acid sequence is chosen so that it is largely complementary to the nucleic acid sequence of tumor cell-specific molecules, such as, for example, the coding sequence of an oncogene.
- nucleic acid molecule can thereby act in the manner of an "anchor” and accumulate the conjugate according to the invention in the tumor cells, so that only there can its activity be unfolded For this purpose, in contrast, there is no accumulation of the construct according to the invention in non-transformed, healthy cells
- the nucleic acid molecule can be designed to hybridize either to the mRNA or to the DNA coding for the tumor cell-specific molecules: an overview of known oncogenes and genes, the are involved in the growth of tumors, and from which the sequences for the realization of the nucleic acid molecule can be derived, can be found in Vogelstein, B. and Kinzler KW, Cancer genes and pathways day control, Nat. Med. 10, pages 789- 799 (2004). The content of this publication is incorporated herein by reference logon application.
- Another object of the present invention relates to the use of the conjugate described above for the preparation of a diagnostic composition, which is preferably a contrast agent for computed tomography (CT) and / or radioiodine therapy.
- a diagnostic composition which is preferably a contrast agent for computed tomography (CT) and / or radioiodine therapy.
- Another object relates to the use of the conjugate according to the invention for the preparation of a therapeutic composition, which is preferably an apoptosis-inducing composition.
- a further subject relates to a pharmaceutical and / or diagnostic composition
- a pharmaceutical and / or diagnostic composition comprising the conjugate according to the invention and optionally a pharmaceutically and / or diagnostically acceptable carrier.
- Diagnostically and pharmaceutically acceptable carriers with optionally further additives are well known in the art and are described, for example, in the paper by Kibbe A., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association and Pharmaceutical Press 2000. These include, for example, binders, disintegrants, lubricants, salts and other substances used in the formulation of drugs.
- Another object of the present invention relates to a method for the diagnostic and / or analytical treatment of biological material or a living being, comprising the following steps: (a) incubation or administration of the conjugate according to the invention with biological material or into a living being, (b) carrying out a imaging process.
- Imaging techniques are generally nuclear medicine / radiology techniques, including angiography, positron emission tomography, scintigraphy, and near-infrared imaging and conventional fluorescence microscopy, respectively, with computed tomography (CT) being preferred.
- CT computed tomography
- Another object of the present invention relates to a method for the therapeutic treatment of a living being, in which the conjugate according to the invention is administered into the animal.
- Fig. 1 shows by way of example the ESI mass spectra for the conjugates 1-4 with molecular masses of 2123.4 Da (Kl) (A), 2096.3 Da (K2) (B), 1641.8 Da (K3) (C ) and 1614.6 Da (K4) (D).
- Fig. 2 shows fluorescence and transmission light microscopic images of human malignant U373 glioma cells after incubation with PBS alone for 20 minutes (native, column 1), TIBA-containing conjugate 2 (mutant NLS) (126 ⁇ M, 260 ⁇ M, 2.6 mM, columns 2-4) and non-TIBA-containing conjugate 4 (mutant NLS) (26 ⁇ M, 260 ⁇ M, 2.6 mM, columns 5-7).
- TIBA-containing conjugate 2 126 ⁇ M, 260 ⁇ M, 2.6 mM, columns 2-4
- non-TIBA-containing conjugate 4 mutant NLS
- FITC channel Localization of the FITC-labeled conjugate. Untreated cells show no autofluorescence.
- Second column (propidium iodide (P ⁇ ) channel): result of the PI vitality test. The nuclei of dead cells are stained. The living cells remain dark.
- FIG. 3A FACS (fluorescence activated cell sorting) analysis demonstrating a low percentage of highly labeled cells after incubation with non-TIBA-containing conjugates (conjugate 3: 26 ⁇ M / 6%, 260 ⁇ M / 7%, and 2.6 mM / 10%) (conjugate 4: 26 ⁇ M / 4%, 260 ⁇ M / 11% and 2.6 mM / 13%).
- FIG. 3B After incubation with the TIBA-containing conjugates (conjugate 1: 26 uM / 22 ° / 0/260 uM / 82% and 2.6 mM / 93%) (conjugate 2: 26 uM / 0%, 260 uM / 78% and 2.6 mM / 86%) a marked increase of strongly stained cells to more than 90% could be observed.
- FIG. 3C After incubation with the TIBA-containing conjugates at 260 ⁇ M and 2.6 mM, two cell populations could be distinguished on a morphological basis (side scatter versus forward scatter FACS analysis).
- Figure 4A CLSM images of human malignant U373 glioma cells.
- Annexin V Alexa TM 568 reagent was used to detect phosphatidyl serine in the outer membrane of necrotic or apoptotic cells. Incubation with TIBA alone or non-tiba-containing conjugates 3 and 4 did not result in the binding of annexin V-Alexa TM 568 reagent to the surface of the glioma cells. The co-incubation of TIBA with conjugates 3 and 4 also did not result in the binding of Annexin V Alexa TM 568 reagent to the surface of the glioma cells and did not result in a higher cell staining rate.
- Glioma cells A very large number of cell nuclei were stained with the FITC-labeled TIBA-containing conjugates 1 (row 1) and 2 (row 2) (260 ⁇ M) (left column). Most of these cells show the expression of phosphatidylserine in the outer membrane and were therefore stained with the Annexin V Alexa TM 568 reagent (right column).
- FIG. 4C Fluorescence microscopy of semithin sections (approximately 0.4 ⁇ m) of human malignant U373 glioma cells after incubation with the TIBA conjugates 1 (correct NLS) and 2 (mutated NLS). The nucleoli of the cells are clearly colored.
- Fig. 5B Corresponding MTT test results of cell pellets from computed tomography after incubation with conjugate 2 (top) and conjugate 1 (bottom) both: 26 ⁇ M, 260 ⁇ M and 2.6 mM). Only vital cells oxidize the yellow methyl thiaoctyl tetrazolium (MTT) salt in blue formazan (incubation either with PBS alone or with both conjugates at 26 ⁇ M).
- MTT yellow methyl thiaoctyl tetrazolium
- Figure 5C CT uptake (InSpace) of U373 human malignant glioma cells after incubation (20 minutes) with PBS alone (left), TIBA-containing conjugate 1 (correct NLS) (middle), and TIBA-containing conjugate 2 (mutant NLS) (right) (both 2.6 mM). There were no significant differences between conjugates 1 and 2.
- FIG. 5D Corresponding signal density for the cell pellets in the tubes 1-9 (partial illustration A).
- Fig. 6A Top: CT image of human prostate cancer cells (PC3) after
- PC3 FACS (fluorescence activated cell sorting) analysis similar to that of human malignant U373 cells.
- the upper cloud represents the population of strongly stained cells (high, right histogram peak, upper left image).
- the lower cloud represents the population of the weakly colored cells (flat, left histogram peak, lower left image)
- Fig. 7 Confocal laser microscopy of human malignant LN18 glioma cells.
- Annexin-V-Alexa TM 568 reagent was used to detect phosphate! - Dylserin in the outer membrane leaf necrotic or apoptotic cells ver used.
- Coincubation of either MIBA (4-monoiodo benzoic acid) or DIBA (2,5-diiodobenzoic acid) with Conjugate 3 did not bind Annexin-V-Alexa TM 568 to the glioma cell surface and was compared to incubation with Conjugate 3 alone is not associated with a higher staining rate.
- Fig. 8 Confocal laser microscopy of human malignant LNI 8 glioma cells.
- U373 glioma cells after incubation with conjugate 12 Many nuclear stained nuclei; top left: FITC channel: determination of the localization of the conjugate; top right: Annexin channel for staining with annexin-Alexa as a sign of strong expression of phosphatidylserine in cell death; bottom left: transmitted light microscopy; bottom right: superposition of FITC- u. Annexin channel.
- LN18 and U373 glioma cells with conjugate 12 (260 ⁇ M and 2.6 mM). At the higher concentration significantly more cells are stained in both cell lines. This staining is more pronounced in U373 glioma cells.
- the cloud diagram shows two morphologically different populations at the higher concentration.
- FIG. 12 CT image (InSpace mode) of the LN18 and U373 glioma cells according to FIG.
- Fig. 13 Confocal laser microscopy of LN18 glioma cells.
- TFBA free, uncoupled trifluorobenzoic acid
- conjugate 13 trifluorobenzoic acid is tightly coupled to K3
- PI propidium iodide
- Figure 14 CLSM of LN18 glioma cells. After incubation with conjugate 3 together with free trichlorobenzoic acid, only a few nuclear-colored cells are present. These cells remain vital (lack of staining with Anne). xin-Alexa, column 2). Only after incubation with conjugate 14 (TCBA tightly coupled to K3) do almost all nuclei stain. The cells are dead (color, with Alexa-annexin).
- Fig. 15 Confocal laser microscopy of LN18 glioma cells. Incubation with free tribromophenyl isocyanate (TBPI) alone does not lead to cell death (no Annexin-Alexa staining, 2nd column). The TBPI-free conjugate 3 does not nuclearly nucleate a small number of cells, which remain vital (no staining with annexin). The co-incubation of free TBPI with conjugate 3 also leads to only a few nuclear-stained cells without impairment of vitality (lack of staining with annexin). Only after incubation of Kl 5 (TBPI fixed to K3) does a massive nuclear staining of almost all cells appear (column 1). Alexa-annexin binds to the surface of cells (2nd column, signs of cell death).
- TBPI tribromophenyl isocyanate
- Fig. 16 A Confocal laser microscopy (superimposition of the FITC and Alexa
- the nuclei of the LN18 glioma cells accumulate the FITC-labeled tribromophenyl isocyanate-NLS conjugate (K 15).
- the red Alexa annexin binds to phosphatidylserine, which is strongly expressed on the cell surface in cell death.
- B Fluorescence activated cell sorting (FACS) analysis of LN 18 and U373 glioma cells after incubation with either conjugate 3 alone, conjugate 3 plus free tribromophenyl isocyanate (TBPI) or the TBPI-NLS conjugate 15.
- a marked increase in strong cells stained with FITC shift of the histogram peak to the right
- C Semi-thin section of an LN18 glioma cell. The nucleolus (in the center) is strongly stained by the tribromophenyl isocyanate-NLS conjugate 15.
- FIG. 18 Active principle of the tumor-specific conjugate 16.
- Fig. 19 Confocal laser microscopy of LN18 glioma cells after incubation with conjugate 16 in the presence of inactive (top row) or active matrix metalloproteinase 2 (MMP-2).
- MMP-2 active matrix metalloproteinase 2
- the nuclei first stain through the cleaved conjugate after exposure to the active MMP-2 (left column). Only the cleaved conjugate triggers cell death (uptake of propidium iodide, second column).
- FACS fluorescence activated cell sorting
- Fig. 21 CT image (in space mode).
- Left tube LN18 glioma cells (native,
- Conjugates 1-8 (Table 1) were synthesized on an Eppendorf ECOSYN P solid phase synthesizer with Fmoc-Rink amide Tentagel SRAM (0.25 mM / g) (Rapp Polymere, Tübingen, Germany). All amino acids (0.1 mM per 0.4 g resin), except for the N-terminal proline, were incorporated with amino functions protected by the 9-fluorenylmethyloxycarbonyl (Fmoc) group: The functions of the side chains were protected as tert-butyl ether (threonine), 2.2.4.6.7.
- Fmoc-Lys N * -2,3,5-triiodobenzoyl was prepared by the coupling of KP-Fmoc-Lys-OH with 2,3,5-triiodobenzoic acid (TIBA) by activation with isobutylchloroformate (1 eq.). and N-methylmorpholine (1 eq.) (mixed anhydride coupling). The substance was recrystallized from the DMF / diethyl ether.
- the FITC portions were introduced into the lysine 8 residue with fluorescein-5 (6) -isothiocanate in DMSO-N-methylmorpholine (1 eq.) After the 4-methyltrityl group of lysine-8 with TFA in dichloromethane. romethan (1%) + triisopropylsilane (1%) for 1 hour at room temperature.
- the N-terminal proline was introduced as a Boc derivative.
- a simultaneous cleavage of the protecting groups of the amino acid side chains was carried out by reacting the resin in a mixture of 12 ml trifluoroacetic acid, 0.3 ml ethanedithiol, 0.3 ml anisole, 0.3 ml water and 0.1 ml triisopropylsilane for 2 hours was incubated. The mixture was filtered and washed with TFA, and the combined filtrates were precipitated with anhydrous diethyl ether.
- the crude products were further purified by HPLC on a Nucleosil column 100 C18 (7 ⁇ m) 250 ⁇ 10, monitoring elution at 214 nm (buffer A: 0.07% TFA / H 2 O, buffer B: 80% CH 3 CN / 0.058 % TFA / H 2 O, 4 ml / min).
- the peptides were analyzed for purity by analytical high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI / MS) (see 1.2). The purity of the substance was at least 98%.
- Fmoc Lys (N-benzoyl), Fmoc Lys (N -4-monoiodobenzoyl) and Fmoc Lys (N-2,5-diiodobenzoyl) were prepared by coupling N-Fmoc Lys-OH with benzoic acid (BA), 4-monoiodobenzoic acid (MIBA ) and 2,5-diiodobenzoic acid (DIBA) (Sigma-Aldrich, Taufkirchen, Germany) [activation with isobutylchloroformate (iBuOCOCI) (1 eq.) (Merck) and N-methylmorpholine (NMM) (1 eq.) (Fluka, Buchs, Switzerland) (mixed anhydride coupling)].
- BA benzoic acid
- MIBA 4-monoiodobenzoic acid
- DIBA 2,5-diiodobenzoic acid
- IBA 2,5-diio
- conjugates 9, 10 and 11 were otherwise described in the same way as in 1.1.1.
- the purity of the conjugates (at least 98%., was checked by analytical HPLC (high performance liquid chromatography).
- the mass was determined by electrospray ionization mass spectrometry (ESI / MS) (see 1.2).
- Fmoc Pro N-triiodobenzoyl was prepared by coupling N-Fmoc-Pro-OH with triiodobenzoic acid.
- the remaining synthesis of conjugate 12 was carried out on an Eppendorf ECOSYN P solid-phase peptide synthesizer (Eppendorf-Biotronik, Hamburg, Germany) as described under 1.1.1 [purity of the conjugate at least 98%, analytical HPLC (high-performance liquid chromatography)] , The mass was determined by electrospray ionization mass spectrometry (ESI / MS) as in Sch. 1 (see 1.2).
- Fmoc Lys (N-benzoyl), Fmoc Lys (N-trichlorobenzoyl), Fmoc Lys (N -2,5-trifluorobenzoyl), and Fmoc Lys (N-2,4,6-tribromo-phenyl-ureido) -OH were prepared by coupling N -Fmoc Lys-OH with either benzoic acid (BA), trichlorobenzoic acid (TCBA), trifluorobenzoic acid (TFBA) (Sigma-Aldrich, Taufkirchen, Germany) or 2,4,6-tribromophenyl isocyanate (TBPI) (Sigma-Aldrich) Activation with isobutyl chloroformate (iBuOCOCI) (1 eq.) (Merck) and N-methylmorpholine (NMM) (1 eq.) (Fluka, Buchs, Switzerland) (mixed anhydride coupling).
- BA benzoic acid
- Lysine derivatives with 4-methoxytrityl (Mmt) protection were used for the side chains to be provided with the triiodobenzoyl groups.
- the N-terminal amino acid proline is introduced into the peptide as Boc-proline.
- the Mmt side-chain protecting group is cleaved by one-hour addition of 1% TFA / dichloromethane (DCM) solution containing 1% triisopropylsilane.
- DCM dichloromethane
- the exposed side chain becomes available for coupling with 2.3.5% triiodobenzoic acid Available (3 eq in the presence of 3 eq of TBTU and 6 eq of diisopropylethylamine within 1.5 h at room temperature).
- the Dde-protecting group is cleaved by multiple additions of the resin with a 2.5% hydrazine hydrate solution in DMF within one hour.
- the fluorescein urea derivative is prepared by coupling 0.5 mM fluorescein 5 (6) isothiocyanate in the presence of eq. Amount of diisopropylethylamine in DMSO prepared overnight at room temperature.
- the remaining protective groups and the peptide are split off from the resin after drying. This is done by stirring the dried resin for three hours in a mixture of 12 ml of TFA, 0.3 ml of ethanedithiol (EDT), 0.3 ml of anisole, 0.3 ml of water and 0.1 ml of triisopropylsilane at room temperature.
- the crude peptides thus obtained are purified by semipreparative HPLC using a Nucleosil 100 7 mm C18 column (10 ⁇ 250 mm) (buffer A: 0.07% TFA / H 2 O, buffer B: 80% CH 3 CN in 0.058% TFA / H 2 O) (4 ml / min 90 bar, 214 nm); 10® 90% B in 13 min.
- the resulting conjugate is homogeneous in analytical HPLC (purity at least 98%) and consistent with its structure (ESI-MS).
- the conjugates were analyzed by ESI-MS on an Esquire 3000+ spectrometer ion trap mass spectrometer (Bruker-Daltonics, Bremen, Germany).
- the peptides were dissolved in 40% ACN, 0.1% formic acid in water (v / v / v) (20 pmol / ⁇ l) was dissolved and uniformly infused using a syringe pump (5 ⁇ l / min flow rate).
- the mass spectra were generated in the positive ion mode. Dry gas (6 l / min) was brought to a temperature of 325 0 C, the nebulizer to 20.0 psi and the electrospray voltage to - 3700 V.
- the conjugate 16 was dissolved in HEPES buffer containing once the active MMP-2 (Calbiochem, Bad Soden, Germany) and once the inactive MMP-2 proform (Calbiochem). Incubation in HEPES with active MMP-2 took 2 hours.
- APMA 4-aminophenyl mercuric acetate
- conjugate 16 was incubated with HEPES buffer only. The experiments were also performed with an MMP-2 inhibitor. The cleavage products were evaluated by HPLC:
- Buffer B 0.058% CF 3 COOH / 80% CH 3 CN
- U373 human malignant glioma cells were exposed to up to 70% confluence in RPMI 1640 ready mix medium containing L-glutamine and 10% FBS-GoId (PAA). bora tories, Pasching, Austria) at 37 0 C, 5% CO 2 (vol / vol) in 4-well PIatten (NUNC, Wiesbaden, Germany) with approximately 300,000 cells per well grown.
- the cells were washed with Dulbecco's PBS (D-PBS; GIBCO, Invitrogen, Germany) alone (negative control) and 26 micromolar to 260 micromolar and 2.6 millimolar solutions of the conjugates 1-4 in D-PBS for 20 minutes at 37 0 C.
- D-PBS Dulbecco's PBS
- GIBCO Invitrogen, Germany
- a reversal microscope (Axiovert 135 M, Carl Zeiss, Jena, Germany), Long-distance (LD) Objective (Carl Zeiss, Jena, Germany), an illuminator N HBO103 (Carl Zeiss, Jena , Germany) and standard fluorescence filters for excitation and emission of FITC and PI.
- the images were taken with a 3-CCD color video camera (MC3254P, Sony, Japan) and the Axiovision software (Carl Zeiss, Jena, Germany). The intensity of cell fluorescence was recorded as the exposure time required to produce fluorescence images.
- the inner ear spiral CT protocol is as follows: tube voltage 120 KV, effective mAs 550, acquisition time TI: 1.5, SL 0.75 / 0.75 / 4.5, FOV: 50 0/52, core : U70, window: invertebral disc.
- 3D images were obtained using the InSpace software (Syngo CT 2006G) (Siemens AG, Er Weg, Germany).
- the FACS analysis was carried out as described under 1.4.
- the cells in the other ten tubes were each treated with benzoic acid (BA), monoiodobenzoic acid (MIBA) or diiodobenzoic acid (DIBA) alone (260 ⁇ M), conjugate 3 alone (260 ⁇ M), conjugate 3 plus either BA, MIBA or DIBA (260 ⁇ M ), as well as conjugates 9, 10 and 11 alone (260 ⁇ M).
- BA benzoic acid
- MIBA monoiodobenzoic acid
- DIBA diiodobenzoic acid
- human LN-18 and U373 glioma cells were cultured in 75 cm 2 culture bottles (Corning Costar) (70% confluence) (conditions as in 1.4).
- Accutase TM (PAA Laboratories) was added to allow the cells to detach from the bottom of the culture flask. The cells were collected and then divided into Eppendorf tubes (6 x 10 6 cells per tube). The cells in the first two tubes served as control (only PBS, LN18 and U373 glioma cells native).
- the cells in the other tubes were incubated with a 260 ⁇ M or 2.6 mM solution of conjugate 12 for 20 minutes at 37 ° C and 5% CO 2 and then 3 times with PBS buffer washed out and centrifuged at 800 rpm (rounds per minute) for 5 min. In a small part of the cells, the MMT test (see 1.4) was used to check how many of the cells were still alive. Computed tomography of Cell centrifugation was performed with a Somatom Sensation 16 (Siemens).
- An orbital CT coil was used: tube voltage 120KV, effective mAs 550, time of imaging TI: 1.5, SL 0.75 / 0.75 / 4.5, FOV: 50 0/52, GT: 0.0, kernel: U70, window: intervertebral disc.
- 3D images were taken using the InSpace software (Syngo CT 2006G) (Siemens AG, Er Weg, Germany).
- human LN18 and U373 glioma cells were cultured in 75 cm 2 culture flasks (70% confluency) (conditions as described in section 1). Accutase TM (PAA Laboratories) was added to allow the cells to detach from the culture soil. The cells were collected and then divided into Eppendorf tubes (6 x 10 6 cells per tube). The first 4 tubes were incubated for 20 minutes with each of the conjugates 3, 13, 14 and 15 dissolved in PBS buffer at a concentration of 260 ⁇ M.
- TCBA trichlorobenzoic acid
- TFBA trifluorobenzoic acid
- TBPI tribromophenyl isocyanate
- NLS-FITC conjugate 3 260 ⁇ M
- Cells served as controls were incubated with either CIBA, FIBA, TBPI (each 260 ⁇ M in PBS) or with PBS buffer (native control) alone. After incubation, the cells were washed three times with PBS buffer and centrifuged at 800 rpm (rounds per minute). Computed tomography and FACS analysis of cell centrifugates were performed three times as in 1.5.1 and 1.4, respectively.
- APMA 4-aminophenyl mercuric acetate
- the cells in the other four tubes were incubated with 65 and 130 ⁇ M of jugate 10 for 1 and 2 hours both with and without MMP-2 inhibitor I (37 ° C and 5% CO2).
- the MMP-2 inhibitor I was as previously described by Yin et al. 2006 described applied. After incubation for 1 or 2 hours, it was washed out 3 times with PBS buffer and centrifuge at 800 rpm for 5 min. Cell vitality was then checked using methyl thiazoyl tetrazolium (MTT) salt (Sigma Aldrich, Germany) (15 mg / ml). The formation of formazan was examined after 20 minutes in a transmitted light microscope.
- MTT methyl thiazoyl tetrazolium
- PI propidium iodide
- a temporal bone CT coil was used: tube voltage 120KV, effective mAs 550, time of imaging TI: 1.5, SL 0.75 / 0.75 / 4.5, FOV: 50 0/52, GT: 0.0, kernel: U70, window: intervertebral disc.
- the signal density of each cell pellet was measured.
- the FACS analysis was performed on a Becton Dickinson FACSCalibur [100 ⁇ l of the cell suspension (IxIO 6 cells) plus 300 ⁇ l FACS buffer (D-PBS buffer with 1% paraformaldehyde)]. Approximately 25000-35000 cells were measured per sample [fluorescence excitation: argon ion laser (488nm), fluorescence detection: 540-565nm bandpass filter]. The experiments were each carried out three times.
- Human malignant glioma cells (U373) were grown in 4-well plates under the same conditions as described for fluorescence microscopy in 1.5. The cells were incubated for 20 minutes with each of the conjugates dissolved in 0.1% DMSO / PBS at 260 ⁇ m. The cells were further co-incubated with TIBA and the two non-TIBA-containing conjugates (3 and 4) in 0.1% DMSO / PBS at 260 ⁇ m. Due to the insolubility of TIBA (but not the conjugates) in pure PBS, both TIBA and the conjugates were dissolved in 0.1% DMSO / PBS at 260 ⁇ m for incubation. As controls, the cells were incubated with PBS alone, 0.1% DMSO / PBS alone and TIBA alone (260 ⁇ m in 0.1% DMSO / PBS).
- phosphatidylserine in the outer membrane of apoptotic cells was carried out with Annexin V Alexa TM 568 reagent according to the manufacturer's protocol (Roche Molecular Biochemicals, Indianapolis, USA). Confocal laser scanning microscopy was performed using a LSM510 reversal laser scanning microscope (Carl Zeiss, Jena, Germany) (objectives: LD Achroplan 40x0.6, Plan Neofluar 20x0.50, 40x0.75). For fluorescence excitation, the 488 nm line of an argon-ion laser and the 534 nm line of a helium-neon laser were used with suitable beam splitters and barrier filters for FITC and Alexa, respectively. Superimposed images of FITC and Alexa stained samples were generated by overlaying overlapping views. All measurements were made on live, unfixed cells.
- Human malignant LN18 and U373 glioma cells were grown in four-well plates (NUNC, Wiesbaden, Germany) (approximately 300,000 cells per well) as under 1.5. Cells were incubated at 37 ° C / 5% CO 2 for 20 minutes with each of conjugates 3, 9, 10 and 11 at a concentration of 260 ⁇ M (dissolved in PBS) (GIBCO, Invitrogen, Germany). Cells were also co-incubated with either CIBA, FIBA or TBPI, as well as NLS-FITC conjugate 3 (260 ⁇ M each). Control cells were incubated with only CIBA, FIBA or TBPI (260 ⁇ M in PBS) alone.
- NUNC Wiesbaden, Germany
- the cells were washed three times with PBS and subsequently Then incubate again in Ready Mix medium.
- the FITC-labeled conjugates as well as the Alexa annexin for the detection of phosphatidylserine as a sign of cell death were localized with confocal laser microscopy (CLSM) as described in 1.6.1. All measurements were made on living cells.
- Human malignant LN18 and U373 glioma cells were grown in four-well plates (NUNC, Wiesbaden, Germany) (approximately 300,000 cells per well) as in 1.5. The cells were incubated at 37 ° C / 5% CO 2 for 20 minutes with the conjugate 12 at concentrations of 260 ⁇ M and 2.6 mM (dissolved in PBS buffer) (GIBCO, Invitrogen, Germany). As a control, cells were incubated with PBS buffer only. After the incubations, the cells were washed three times with PBS and then incubated again in Ready Mix medium. The detection of phosphatidylserine and CLSM was as in 1.6.1. All measurements were made on living cells three times.
- Human malignant LN18 and U373 glioma cells were grown in four-well plates (NUNC, Wiesbaden, Germany) (approximately 300,000 cells per well) as described under Sh. 1 bred. The cells were incubated at 37 0 C / 5% CO 2 for 20 minutes with each of the conjugates 3, 13, 14 and 15 at a concentration of 260 uM (dissolved in PBS) (GIBCO, Invitrogen, Germany) incubated. Cells were also co-incubated with either CIBA, FIBA or TBPI, as well as NLS-FITC conjugate 3 (260 ⁇ M each). Control cells were incubated with only CIBA, FIBA or TBPI (260 ⁇ M in PBS) alone.
- NUNC Wiesbaden, Germany
- Human malignant glioma cells (U373 and LN18) were seeded in 25 cm 2 culture flasks containing 3 ml RPMI-1640 Ready Mix medium with L-glutamine and 10% fetal bovine serum (FBS) (PAA laboratories, Pasching, Austria) [ 37 ° C, 5% CO2 (vol / vol)]. The medium was left for one day so that enough MMP-2 secreted by the glioma cells could accumulate. To activate the MMP-2 present in the medium in the inactive proform, the medium was incubated with APMA on the day of the study (final APMA concentration in the medium: ImM, with 1% DMSO) (confluency of the cells: 70%).
- FBS fetal bovine serum
- the BIS-TIBA conjugate 16 was in each case dissolved without MMP-2 inhibitor in the APMA-containing media of 4 bottles (26 and 130 ⁇ M) (both cell lines).
- the BIS-TIBA conjugate 16 was again dissolved in the APMA-containing media of 4 bottles (26 and 130 ⁇ M) but now with MMP-2 inhibitor I.
- both glioma cell lines were incubated in a one day old medium both with and without APMA and inhibitor.
- MTT-SaIz and propidium iodide (PI) were used as under 1.4.
- phosphatidylserine in the outer membrane leaf of apoptotic cells was detected with Annexin-V-Alexa TM 568 reagent as per the recommendation of the manufacturer (Roche Molecular Biochemicals, Indianapolis, USA).
- a portion of the cells stained for FACS analysis were fixed in paraformaldehyde with 2% agar, dehydrated in ethanol, embedded in Lowricyl K4M (Polysciences, Eppelheim, Germany) and at room temperature according to the manufacturer's instructions UV polymerized. Semi-thin sections (about 0.4 ⁇ m) were cut and evaluated by fluorescence microscopy.
- FITC-labeled conjugates were synthesized: the correct NLS of the SV-40 T antigen with TIBA (conjugate 1 Kl), a mutant NLS of the SV-40 T antigen with TIBA (conjugate 2, K2) and both conjugates without TIBA (Conjugates 3 and 4, K3 and K4).
- the same conjugates again without FITC are designated as conjugates 5 to 8, K5 to K8; Table 1, on the left side for all conjugates as usual, the C-terminus and shown on the right side of the N-terminus, Fig. 1.
- the conjugate Kl has a molecular weight of 2123.4 Da, the conjugate K2 of 2096.3 Da, the conjugate K3 of 1641.8 Da, the conjugate K4 of 1614.6 Since the K5 conjugate is 1735.4 Da, the K6 conjugate is 1708.3 Da, the K7 conjugate is 1493.1 Da, the K8 conjugate is 1466.0 Da.
- FITC-labeled conjugates were synthesized: the NLS of the SV40 T antigen with the non-iodinated benzoic acid (BA) (conjugate 9), the 4-monoiodobenzoic acid (MIBA) (conjugate 10) or the 2,5-diiodobenzoic acid (DIBA) (Conjugate 11); Table 1
- the conjugate 9 has a molecular weight of 1745.95 Da, the conjugate 10 of 1871.83 Da and the conjugate 11 of 1997.72 Da.
- FITC-labeled conjugate was synthesized in which triiodobenzoic acid (TIBA) was coupled to the proline; Table 1.
- TIBA triiodobenzoic acid
- the conjugate 12 has a molecular weight of 2123.4 Da.
- FITC-labeled conjugates were synthesized: the NLS of the SV40 T antigen with the trifluorobenzoic acid (TFBA) (Conjugate 13), trichlorobenzoic acid (TCBA) (Conjugate 14), and tribromobenzoic acid (TBPI) (Conjugate 15); Table 1.
- TFBA trifluorobenzoic acid
- TCBA trichlorobenzoic acid
- TBPI tribromobenzoic acid
- the conjugate 13 has a molecular weight has a molecular weight of 1799.90 Da, the conjugate 14 of 1847.81 Da and the conjugate 15 of 1997.75 Da.
- 2.1.5 Conjugate 16 :
- the conjugate 16 has a molecular weight of 3255.76 Da.
- Intracellular staining was confirmed by examination of semithin sections (about 0.4 ⁇ m) of the incubated cells ( Figure 4C).
- the human malignant LN18 and U373 glioma cells showed no autofluorescence in confocal laser microscopy after incubation with PBS buffer alone.
- conjugate 9 (conjugate 3 coupled to BA) stained only a small number of U373 glioma cells (8%) and LN18 glioma cells (9%) (comparable to conjugate 3) ( Figure 8). These few stained cells showed no signs of cell death (lack of binding of Annexin-V-Alexa TM 568 reagent to phosphatidylserine in the outer membrane leaf) ( Figure 8).
- the human malignant LNl 8 and U373 glioma cells showed no autofluorescence in confocal laser microscopy after incubation with PBS buffer.
- the human malignant LN 18 and U373 glioma cells showed no autofluorescence in confocal laser microscopy after incubation in pure PBS buffer (FIG. 13).
- the sole incubation with trifluorobenzoic acid, with trichlorobenzoic acid or the tribromophenyl isocyanate (in each case 260 ⁇ M) led to no impairment of cell vitality (FIG. 15).
- the principle of action of the conjugate 16 is schematically illustrated by a particular embodiment.
- the first peptide is located in the form of a nuclear localization sequence (NLS).
- NLS nuclear localization sequence
- the first compound in the form of triiodobenzoic acid (TIBA) is covalently bound to the NLS.
- the second peptide which has an interface which is recognized and cleaved by the tumor-specific protease MMP-2, is bound to the carboxyl group of the lysine residue by means of peptide binding.
- the second peptide is shown as a thin bar.
- the second compound connects. This is likewise bound to the second peptide via a lysine residue in the form of triodoiodobenzoic acid (TIBA), which is not shown in the second peptide in the second peptide.
- TIBA triodoiodobenzoic acid
- This conjugate 16 of the present invention is unable to invade healthy untransformed cells due to its size and lack of MMP-2 (left). Only in the presence of transformed tumor cells that secrete MMP-2 into the environment, the second peptide is cleaved. The liberated residual conjugate 16, due to the presence of NLS and its reduced size in the cytoplasm and the cell nucleus of the tumor cell can be taken (right). After the induction of apoptosis in the tumor cells by the residual conjugate 9, the cleavage products are then "disposed of" via macrophages and possibly excreted from the organism.
- the human malignant LN18 and U373 glioma cells showed no autofluorescence in confocal laser microscopy (CLSM) after incubation alone with APMI (4-aminophenyl mercuric acetate) -containing RPMI medium both with and without inhibitor. Both the inhibitor and APMA in the medium did not affect cell viability.
- CLSM confocal laser microscopy
- the cells showed only a slight increase in signal density after incubation with inhibitor-containing medium compared to the untreated control (13 and 130 ⁇ M) (FIG. 21).
- a significant increase in the signal density occurred after incubation of the cells with the BIS-TIBA conjugate (Kl 6) (130 ⁇ M) in inhibitor-free medium (FIG. 21).
- HPLC High Performance Liquid Chromatography
- the BIS-TIBA conjugate (Kl 6) is cleaved both in the presence of the active MMP-2 and the APMA-activated MMP-2 proform, and that this cleavage can be prevented by the presence of the inhibitor (FIG. 22).
- the inventors thus provide a conjugate which is both an improved contrast agent and an apoptosis-inducing therapeutic.
- the conjugate is tumor-specific and thus enables targeted diagnosis and / or therapy of a tumor disease.
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Abstract
La présente invention concerne un conjugué radiopaque, l'utilisation de ce conjugué pour préparer une composition à usage diagnostique et thérapeutique, une composition pharmaceutique et/ou diagnostique contenant le conjugué, un procédé de traitement diagnostique et/ou analytique d'un matériel biologique ou d'un être vivant, ainsi qu'un procédé de traitement thérapeutique d'un être vivant.
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DE102006035577A DE102006035577A1 (de) | 2006-07-27 | 2006-07-27 | Röntgendichtes Konjugat |
PCT/EP2007/006678 WO2008012102A2 (fr) | 2006-07-27 | 2007-07-27 | Conjugué radiopaque |
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EP3416692B1 (fr) | 2016-02-18 | 2020-12-16 | Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. | Sonde basée sur l'activité et ses utilisations pour l'imagerie |
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WO2005054279A1 (fr) * | 2003-11-21 | 2005-06-16 | Orthogen Ag | Peptides d'administration, leurs structures avec agents actifs et leur utilisation |
WO2006056227A1 (fr) * | 2004-11-27 | 2006-06-01 | Eberhard-Karls- Universität Tübingen Universitätsklinikum | Conjugues renfermant un agent actif et des peptides a chaine ramifiee ou flanquant ledit agent |
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US5567410A (en) * | 1994-06-24 | 1996-10-22 | The General Hospital Corporation | Composotions and methods for radiographic imaging |
US6310243B1 (en) * | 1994-09-23 | 2001-10-30 | Nycomed Imaging As | Iodinated x-ray contrast media |
US20050119470A1 (en) * | 1996-06-06 | 2005-06-02 | Muthiah Manoharan | Conjugated oligomeric compounds and their use in gene modulation |
AU2002246744B2 (en) * | 2000-10-19 | 2008-04-10 | Target Discovery, Inc. | Methods for determining protein and peptide terminal sequences |
EP1424343A1 (fr) * | 2002-11-29 | 2004-06-02 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Conjugué peptidique pour l'imagerie moléculaire des noyeux cellulaires et la thérapie des tumeurs |
WO2006071754A2 (fr) * | 2004-12-23 | 2006-07-06 | Purdue Research Foundation | Procede d'imagerie par tomographie par emission de positrons |
CA2590386A1 (fr) * | 2004-12-30 | 2006-07-06 | Cinvention Ag | Ensemble comprenant un agent produisant un signal, un implant et un medicament |
US20060292077A1 (en) * | 2005-03-18 | 2006-12-28 | Zhao Jonathon Z | Dendritic and star-shaped contrast agents for medical devices and bioabsorbable radiopaque bulk material and method for producing same |
US7795380B2 (en) * | 2006-06-02 | 2010-09-14 | University Of Iowa Research Foundation | Compositions and methods for nucleic acid delivery |
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WO2005054279A1 (fr) * | 2003-11-21 | 2005-06-16 | Orthogen Ag | Peptides d'administration, leurs structures avec agents actifs et leur utilisation |
WO2006056227A1 (fr) * | 2004-11-27 | 2006-06-01 | Eberhard-Karls- Universität Tübingen Universitätsklinikum | Conjugues renfermant un agent actif et des peptides a chaine ramifiee ou flanquant ledit agent |
Non-Patent Citations (2)
Title |
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JIANG T ET AL: "Tumor imaging by means of proteolytic activation of cell-penetrating peptides", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC; US, vol. 101, no. 51, 21 December 2004 (2004-12-21), pages 17867 - 17872, XP002369426, ISSN: 0027-8424, DOI: 10.1073/PNAS.0408191101 * |
PENG C T ED - WOLFF M E: "BURGER'S MEDICINAL CHEMISTRY, CHAPTER SIXTY-ONE. ÖRADIOPAQUES", 1 January 1979, BURGER'S MEDICINAL CHEMISTRY. BASIS OF MEDICINAL CHEMISTRY; [BURGER'S MEDICINAL CHEMISTRY], NEW YORK, WILEY & SONS, US, PAGE(S) 1139 - 1203, XP002005069 * |
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