JP2009541221A - Biodegradable polymeric MRI contrast agents and their preparation and use - Google Patents

Abstract

Disclosed is a novel method for preparing degradable polymeric magnetic resonance imaging contrast agents. Disclosed are degradable polymeric magnetic resonance imaging contrast agents of defined or controlled molecular weight distribution for use in various diagnostic methods and methods for synthesizing, using, and degrading these agents. The
The macromolecular contrast agents disclosed in various aspects of the present invention are degradable paramagnetic polydisulfide compounds that have prolonged plasma retention and increased in various tissues, organs, or tumors. Shows permeability and retention time, but can still be quickly removed from the body.

Description

Priority Claim This application is a US provisional patent filed on June 16, 2006 for "Biodegradable Macromolecular MRI Contrast Agent and its Preparation and Use (BIODEGRABLE MACROMOLECULAR MRI CONTRAST AGENTS OF METHODS OF PREPARATION AND USE THEOROF)". Claim the benefit of the filing date of application 60 / 814,449.

US Government Assisted Research and Development Statement The work described herein was supported by the National Institute of Health Grants CA 0958873. The United States government may have certain rights in the invention.

  The present invention relates generally to biodegradable polymeric contrast agents used in diagnostic imaging and methods for synthesizing, purifying, using and degrading such compounds.

Magnetic resonance imaging (MRI) is a non-invasive method for medical diagnosis. Paramagnetic metal complexes are often used as contrast agents to enhance image contrast between normal and diseased tissue. Paramagnetic metal ions commonly used in diagnostic methods include manganese (Mn ²⁺ ), iron (Fe ³⁺ ), and gadolinium (Gd ³⁺ ). Gd ³⁺ chelates are often used as MRI contrast agents because of their long electron relaxation times and high magnetic moments. Low molecular gadolinium complexes such as Gd (III) (DTPA) [diethylenetriaminepentaacetate], Gd (III) (DOTA) [1,4,7,10-tetraazadodecanetetraacetic acid], Gd (DTPA-BMA) (diethylenetriamine) Contrast agents based on gadolinium, including pentaacetic acid bismethylamide) and derivatives thereof, are routinely used as contrast agents for MRI in clinical practice. These agents are extracellular contrast agents that quickly overflow from the blood vessel into the extracellular fluid gap, and because of their small size, have a short tissue retention time, a short imaging time window and a low signal-to-noise ratio. Several inconveniences occur. In order to prolong the circulation and retention of the contrast agent, chemical conjugation of the Gd (III) chelate to a medical polymer (Non-patent Document 1) or incorporation into the polymer backbone (Non-patent Document 2) A considerable amount of effort has been made to increase the size.

Polymeric MRI contrast agents, sometimes referred to as long-circulating or blood pool agents, are particularly useful because of their long retention time in the blood pool. For example, the half-life of albumin- (Gd-DTPA) conjugate is about 3 hours in blood (Non-patent Document 3). Gd-DTPA-labeled dextran (molecular weight ˜75 kDa) has a longer half-life of 6.1 hours in rats compared to 13 minutes for Gd-DTPA (Non-Patent Document 4). Macromolecular contrast agents also have an increased proton T ₁ relaxation that results from longer rotation times compared to smaller sized molecules (5). Because of increased permeability and retention, these contrast agents accumulate effectively in solid tumors and have potential for contrast enhancement in MR cancer imaging.

  However, polymeric contrast agents have potential toxicity associated with slow excretion and long-term tissue Gd accumulation, thus hindering further development. Due to the low clearance rate, the use of Gd polymer agents can result in accumulation of Gd in bone and other tissues, resulting in toxicity and adverse side effects. A prototype of a polymeric MRI contrast agent, (Gd-DTPA) -albumin conjugate, shows high Gd accumulation in bone and liver as a result of its slow excretion (Non-Patent Document 3), which indicates endocytosis. Increased the possibility of cell uptake of the drug by the lysosome and dissociation of the Gd-DTPA complex due to low pH and enzymatic degradation in the lysosome (Non-patent Documents 6 and 7). Gd high and long term tissue accumulation was also observed for other macromolecular Gd (III) complexes. It was reported that conjugation of Gd-DO3A to carboxymethylhydroxyethyl starch became a micromolecular agent (72 kDa) in which about 47% of the injected dose was detected in the body of rats 7 days after injection. (Non-Patent Document 8). The Gd-DTPA polypropyleneimine dendrimer (second generation) conjugate (7 kDa) resulted in a 45% retention of the injected dose in rats 14 days after injection (Non-patent Document 9). The long in vivo high accumulation of contrast agent significantly increases the potential for metabolic release of toxic Gd (III) from the chelate.

  The present inventors recently designed and developed a biodegradable polymeric MRI contrast agent based on polydisulfide Gd (III) complex to promote Gd chelate excretion by in vivo degradation of the polymeric agent ( Non-patent literature 10-12). Disulfide bonds in the polymer backbone can be rapidly reduced by free plasma thiols, such as glutathione and cysteine, or by enzymatic degradation. The first polydisulfide MRI contrast agent, (Gd-DTPA) -cystamine copolymer (GDCC), has a more significant blood pool in rats than the clinically available MRI contrast agent, Gd- (DTPA-BMA). It has been shown to have occurred and then quickly removed from the blood pool. GDCC showed minimal long-term Gd tissue accumulation comparable to clinically used Gd- (DTPA-BMA).

  Functional groups around disulfide bonds have been introduced to adjust the degradation rate of paramagnetic polydisulfide agents and to prepare biodegradable polymer agents with various pharmacokinetic properties. Two modified disulfide MRI contrast agents, (Gd-DTPA) -cystine copolymer (GDCP) and (Gd-DTPA) -cystine diethyl ester copolymer (GDPEP), and preliminary use of these agents in contrast-enhanced tumor MR imaging The results are reported in Non-Patent Document 11.

US Pat. No. 6,982,324

R. B. Lauffer, T. J. Brady, "Preparation and water relaxation properties of paramagnetic metal, paramagnetic metal chelates and water relaxation properties." Magnetic Resonance Imaging (Magn. Reson. Imaging), 1985, Vol. 3, No. 1, p. 11-16. Desser (T.S. Desser), Rubin (DL, Rubin), Mueller (H. H. Muller), King (F. Qing), Coder (S. Khodor), Zanaji (G. Zanazzi), Young ( S. W. Young, D. L. Ladd, JA Wellons, K. K. Keller, "kinetics of tumor imaging with Gd-DTPA-polyethylene glycol polymer: Dependence on molecular weight (Dynamics of tumor imaging with Gd-DTPA-polyethylene polymer polymers: Journal of Magnetic Resonance M.) gn.Reson.Imaging) ", 1994, Vol. 4, No. 3, p. 467-472. U. Schmiedl, O. M. Ogan, H. Paajanen, M. Marotti, L. E. Crooks, Burito (AC Brito), B. R. C. Brusch), “Gd-DTPA labeled albumin as an intravascular blood pool enhancer for MR imaging: Biodistribution with Gd-DTPA as an intravasular, blood pool-enhancing. agent for MR imaging: biodistribution and imaging studies), Radiology ", 1987, 162. , P. 205-210. W (S. C. Wang), Wigström (MG Wikstrom), White (DL White), Clavenes (J. Klavenes), Holz (E. Holtz), Long Bed (P. Longved), M.E. Mosley, RC Brasch, "Evaluation of gadolinium-DTPA-labeled dextran as an intravascular MR contrast agent: Evaluation of gadolinium-DTPA- labeled astran intra MR MR contrast agent: imaging charactaristics in normal rat tumors), radiology ", 990 years, 175 Volume, No. 2, p. 483-488. By KE Keller, PM Henrichs, R. Hollister, SH Koening, J. Eck, and D. Wei. High relaxivity linear Gd (DTPA) -polymer conjugates: role of hydrophobic interactions (DTPA) -polymer conjugations: the role of hydrophobic interactions, Magnetic Resonance M. Reson. Med.), 1997, Vol. 38, No. 5, p. 712-716. "Evidence of gadolinium dissociation from protein-DTPA-dadolinium complex" by Duncan, F.N.Franano, Edward WB, MJ Welch. (Evidence of gadolinium dissemination from protein-DTPA-gadolinium complexes), Investigative Radiology ", 1994, Vol. 29, Appendix 2, s58-s61. F. N. Francano, F. Nicholas, Edwards (WB Edwards), MJ Welch, MW Brechbiel, OA Gansow ), Duncan (JR Duncan), “Biodistribution and metabolism of targeted and non-targeted protein-chelate-gadolinium complexes: in vitro and in vivo gadolinium dissociation (Biodistribution and metabodism of targeted and non-targeted protein-). chelate-Gadolinium complexs: Evidence for gadolinium dissociation in vitro and in vivo), Magnetic Resonance Imaging ", 1995, Vol. 13, No. 2, p. 201-214. T.H. Helbich, A. Gossman, P. A. Mareski, B. Raduchel, T.P. Roberts, Shermes (D. M. Shames) ”, M. Muhler, K. Turetschek, RC Brasch,“ New polysaccharide polymer contrast agent for MR imaging: A new polysaccharide macromolecular contrast ”. agent for MR imaging: Biodistribution and imaging characturistics), Journal of Magnetic Resonance Imaging ", 2000, Vol. 11, No. 6, p. 694-701. Characteristic of a novel MRI contrast agent prepared from SJ Wang, MW Brechbiel, E. C. Wiener, “Polypropylenimine dendrimer, 2nd generation” (Characteristics of a New MRI Contrast Agent Prepared From Polypropyleneimine Dendrimers, Generation 2), Investigative Radiology ", 2003, Vol. 38, No. 10, p. 662-668. Wang (X. Wang), Fang (Y. Feng), Key (T. Ke), Shovel (M. Schabel), Lou (ZR Lu), "Biodegradable polymer magnetic resonance imaging contrast agent, Pharmacokinetics and tissue retention of (Gd-DTPA) -Cystamine copolymer oligomers, a biodegradable macromolecular pharmacology and tissue retention of (Gd-DTPA) -cystamine copolymer moleculars, pharmacokinetics and tissue retention of (Gd-DTPA) -Cystamine copolymer ligands, 2005, Vol. 22, No. 4, p. 596-602. Zong, X.Wang, Goodrich (K.C. Goodrich), Mohs (AM Mohs), Parker (DL Parker), Lou (ZR Lu) MRI (Contrast-enhanced MRI with new biodegradable macromolecular Gd (III) complex in tumor-bearing mice, tumor-bearing mice), contrast enhanced with a novel biodegradable polymer Gd (III) complex In Medicine ", 2005, Vol. 53, p. 835-842. By Zong, T. Ke, AM Mohs, J. Guo, Parker Parker, Z.R. Lu, “ Effect of size and charge on in vivo MRI contrast enhancement of polydisulphide Gd (III) complex, Journal of Controls, Journal of Controls Controlled Rel.), 2006, 112, p. 350-356).

The present invention provides, inter alia, degradable polymeric contrast agents having a defined or controlled molecular weight and / or molecular weight distribution. In a preferred embodiment, the contrast agent comprises polydisulfide. In another preferred implementation variety, these contrast agents are complexes with metals such as Mn ²⁺ , Fe ³⁺ , and Gd ³⁺ . Examples of various contrast agents are disclosed in Patent Literature 1, Non-Patent Literature 10, Non-Patent Literature 11, or Non-Patent Literature 12. These contrast agents can be used in various medical methods, such as diagnostic and therapeutic methods. In one embodiment, these contrast agents are used in magnetic resonance imaging. In another embodiment, these contrast agents are used in X-ray computed tomography. In another embodiment, the degradable polymer may form a chelate with a radioactive metal ion for scintigraphy, positron emission tomography, and radiation therapy. In another aspect of the invention, the contrast agent complex includes a targeting molecule. Incorporation of targeting molecules including, without limitation, antibodies, antibody fragments, peptides, other proteins, and other chemical entities will result in macromolecular contrast agents with targeting capabilities.

  The present invention provides a method for preparing degradable polymeric contrast agents in an aqueous medium. In a preferred embodiment, the reaction medium is a basic aqueous solution. Using an aqueous solution as a polymerization medium avoids solution contamination and also facilitates easy scale-up for manufacturing.

The present invention provides a method for purifying degradable polymeric ligands and contrast agents. In one embodiment, the ligand and contrast agent are purified by chromatographic methods. In another embodiment, the contrast agent is purified by ultrafiltration. Biodegradable polymeric MRI contrast agents comprising Gd can also be purified by raising the pH of the contrast solution and removing any remaining free Gd (III) ions as Gd ₂ O ₃ precipitates. It is.

  The present invention also provides a method for fractionating a degradable polymeric contrast agent to provide a contrast agent having a narrower and desired molecular weight distribution. In one embodiment of the invention, the contrast agent is fractionated by chromatographic methods such as size exclusion chromatography.

  The present invention also provides a method for controlling the molecular weight and / or molecular weight distribution of a degradable polymeric contrast agent. In one embodiment, the molecular weight of the contrast agent is controlled by changing the polymerization conditions, such as the reaction temperature and / or the feed ratio of the polymerization reactants.

  The present invention provides a method for obtaining a magnetic resonance image of a mammalian tissue or organ by administering one or more degradable polymeric contrast agents and obtaining a magnetic resonance image. In some embodiments, the polymeric contrast agent can be degraded by both endogenous and exogenous compounds. In one embodiment, the polymeric contrast agent is degraded to small stable chelates by endogenous mercaptans and / or enzymes.

  The present invention also provides a method of degrading or stimulating degradation of a polymeric contrast agent by administering one or more disulfide bond reducing compounds or other compounds that stimulate degradation of the polymeric contrast agent. To do. In one embodiment, exogenous mercaptan is delivered to the mammal. Another object of some embodiments of the present invention is to administer a polymeric contrast agent in combination with other agents. In various aspects, pharmaceutically acceptable agents such as diluents and carriers are also administered.

  One aspect of the present invention is a method for obtaining a magnetic resonance image of a mammalian tissue or organ, administering an effective amount of one or more polymeric contrast agents to the mammal, and obtaining the magnetic resonance image. Including the method. In a preferred embodiment, the one or more polymeric contrast agents are degraded by endogenous mercaptans and / or enzymes.

  According to one aspect of the invention, one or more compounds that stimulate the degradation of the polymeric contrast agent are also administered. In another aspect, one or more disulfide bond reducing compounds are also administered. The disulfide bond reducing compound is selected from the group consisting of one or more of the following: mercaptan, NADH, NADPH, hydrazine, phosphine, zinc, tin (II), sodium sulfide, performic acid, hydrogen peroxide.

  The mercaptan used in the various aspects of the present invention is selected from the group consisting of one or more of the following: cysteine and its derivatives, glutathione and its derivatives, cysteinyl glycine and its derivatives, 2,3-dimercaptosuccin Acid derivatives thereof, 2,3-dimercapto-1-propanesulfonic acid and derivatives thereof, 2-mercaptoethanol, penicillamine and derivatives thereof, mercaptoacetic acid and derivatives thereof, mercaptoanisole, 2-mercaptobenzoic acid and derivatives thereof, 4-mercapto Benzoic acid and its derivatives, 2-mercapto-5-benzimidazolesulfonic acid and its derivatives, 2-mercaptobenzothiazole, 3-mercaptoisobutyric acid, mercaptocyclohexane, 2-mercaptoethanesulfonic acid, 2-mercaptoethylamino 2-mercaptoethylamine hydrochloride, 3-mercapto-1,2-propanediol, 3-mercapto-1-propanesulfonic acid, 3-mercapto-1-propanol, 2-mercaptopropionic acid, 3-mercaptopropionic acid, diethyl Dithiocarbamate, dithioerythritol, and dithioglycol.

  According to one aspect of the invention, two or more polymeric contrast agents are administered simultaneously. In one embodiment, the polymer contrast formulation comprises a first polymer contrast agent and a second polymer contrast agent, wherein the second polymer contrast agent comprises the first polymer contrast agent. Administered after administration of the polymeric contrast agent.

In another embodiment, the at least one macromolecular contrast agent is selected from the group consisting of a diluent, a carrier, an antibody, an antibody Fab ′ fragment, an antibody F (ab ′) ₂ fragment, and a delivery system. It is administered together with the above physiologically acceptable drugs.

  In a preferred embodiment, the at least one polymeric contrast agent is selected from the group consisting of paramagnetic metal complexes, radioactive metal complexes, therapeutic agents, proteins, DNA, RNA, drug delivery systems, and gene delivery systems. It is administered together with the above contrast agents.

  The purpose of some embodiments of the present invention is, without limitation, liver, spleen, lung, heart, kidney, tumor, ovary, pancreas, bile duct, peritoneum, muscle, head, neck, esophagus, bone marrow, lymph node Obtaining magnetic resonance images of healthy or neoplastic tissues or organs, including lymphatic vessels, nervous system, brain, spinal cord, capillaries, stomach, small intestine, and large intestine.

  An object of some embodiments of the present invention is to provide a method for obtaining magnetic resonance images of healthy or neoplastic tissues or organs, the method comprising an appropriate molecular weight for one or more contrast agents. Selecting a range. In one embodiment, high (preferably greater than 40 kDa) molecular weight contrast agents provide longer and significant contrast enhancement for cardiac and vascular imaging. In another embodiment, a low (preferably less than 40 kDa) molecular weight contrast agent results in a significant enhancement in tumor tissue over a high molecular weight agent.

FIG. 2 is a diagram illustrating the influence of reaction temperature on the molecular weight distribution of the polymer ligand DTPA-cystine copolymer, which is also exemplified in Table 1. FIG. 3 illustrates the effect of DTPA dianhydride to cystine feed ratio on the molecular weight distribution of the copolymer, also exemplified in Table 2. FIG. 6 shows a comparison of the purification of poly (GdDTPA-co-L-lysine) (GDCP) by PD-10 column versus ultrafiltration. A time signal intensity curve of the main fraction of Gd-DTPA cystamine copolymer (GDCC) using an AKTA P-920 FPLC, a Superose 610/200 GL column, and a Knauer RI detector. FIG. Pre-injection (a) of GDCP (A: 23 kDa, B: 43 kDa, C: 109 kDa) and GDGP (D: 21 kDa, E: 43 kDa, F: 108 kDa) at a dose of 0.1 mmol Gd / kg from the tail vein. And three-dimensional maximum intensity projection MR images of mice at 2 minutes (b), 5 minutes (c), 10 minutes (d), 15 minutes (e), 30 minutes (f), and 60 minutes (g) after injection FIG. GDCP (A: 23 kDa, B: 43 kDa, C: 109 kDa) and GDGP (D: 21 kDa, E: 43 kDa, F: 108 kDa) before injection (a) and 2 minutes after injection (b), 5 minutes (c) ) Shows 2D axial MR images of mice with human breast cancer (MB-231) (see arrow) at 10 min (d), 15 min (e), 30 min (f), and 60 min (g). FIG.

  Conventionally, polydisulfide ligands have been prepared in organic solvents, for example, polydisulfide ligands are obtained by condensation polymerization of DTPA dianhydride and disulfide-containing diamines in DMSO. In general, organic solvents are not environmentally friendly, especially when they are used in large-scale manufacturing processes. In addition, it requires an extra step to remove the organic solvent prior to application of the polydisulfide agent.

  The present invention is a method for preparing degradable polymeric contrast agents in an aqueous medium and thus provides a method that has advantages over conventional methods in terms of less contamination and ease of scale-up. Preferably, the aqueous medium is a basic aqueous solution. Using this novel method, polydisulfide MRI contrast agents with various chemical structures and molecular weights have been successfully made. For example, in vivo MR imaging in mice showed that polydisulfide MRI contrast agents have a long retention time in the blood pool compared to smaller size clinically used agents. These polydisulfide contrast agents are highly promising for the diagnosis of tumors and other cardiovascular diseases.

  The present invention provides a method for preparing degradable polymeric contrast agents. The contrast agent can be purified by various chromatographic methods such as HPLC (high performance liquid chromatography), GPC (gel permeation chromatography), and SEC (size exclusion chromatography). In one particular embodiment, the contrast agent is purified by SEC with a column packed with G-25 media. The contrast agent can also be purified by various filtration methods, such as ultrafiltration using a filter with the desired molecular weight cutoff.

  The present invention also provides methods for preparing biodegradable polymeric MRI contrast agents with various molecular weights and / or narrow molecular weight distributions for various clinical applications. Biodegradable polymeric MRI contrast agents of various molecular weights can be easily controlled by changing the reaction conditions, for example, by changing the reaction temperature or by changing the feed ratio of the polymerization reactants . Methods for adjusting the reaction conditions are illustrated in the examples herein. It should be noted that other reaction conditions such as reaction medium pH, reaction time, reactant mixing rate, and other factors may also affect molecular weight and / or molecular weight distribution.

  High molecular weight biodegradable drugs, such as those of 108 kDa, have been shown to provide longer and significant contrast enhancement for cardiac and vascular imaging than the corresponding low molecular weight drugs. Low molecular weight polymer drugs, such as those of 23 kDa, resulted in a more significant enhancement in tumor tissue than high molecular weight drugs. Thus, the present invention also provides a method for obtaining a magnetic resonance image of a healthy or neoplastic tissue or organ, comprising selecting one or more contrast agents having a molecular weight suitable for a particular application. This method is also provided.

  The polymeric contrast agent may have a broader molecular weight distribution than desired. For example, to accurately assess the size effect of paramagnetic polydisulfides on their pharmacokinetics and in vivo contrast enhancement, paramagnetic polydisulfides with narrower molecular weight distributions can be obtained by fractionation using size exclusion chromatography. Prepared. It should be noted that other methods, such as ultrafiltration or dialysis, can also provide partial fractionation.

  It will be appreciated that the structure, charge, and / or size of a polydisulfide contrast agent can affect its degradation, pharmacokinetics, and in vivo contrast enhancement. For example, polydisulfide Gd (III) complex, (Gd-DTPA) -cystine copolymer (GDCP), (Gd-DTPA) -glutathione (oxidized) copolymer (GDGP), and (Gd-DTPA) -cystine diethyl ester copolymer ( GDCEP) behaves differently in mice. Data and analysis on the degradation, pharmacokinetics, and in vivo contrast enhancement of these polydisulfide contrast agents are shown in FIGS. 5 and 6 and are detailed in [12], the entire contents of which are hereby incorporated by reference. Included in the description. The structure, size, and / or charge effects of polydisulfide contrast agents indicate that the optimal polydisulfide Gd (III) complex can be safely and effectively biodegradable for various clinical applications by structural optimization. It suggests that it can be designed and prepared as a polymeric MRI contrast agent. In addition, contrast agents with different pharmacokinetics may be appropriate for different applications. In general, agents with an acceptable long blood circulation are more effective in contrast-enhanced cardiovascular imaging and cancer imaging. Thus, the present invention is also a method for obtaining magnetic resonance images of healthy and neoplastic tissues or organs, with appropriate pharmacokinetic, degradation, and / or in vivo enhancement properties for special applications There is also provided the method comprising selecting one or more contrast agents.

  In another aspect, the present invention provides a polydisulfide MRI contrast agent having a defined or controlled molecular weight and / or molecular weight distribution. The polydisulfide MRI contrast agent of the present invention includes, without limitation, various contrast agents disclosed in US Pat. No. 6,982,324, Non-Patent Document 10, Non-Patent Document 11, or Reference 12. In one embodiment of the various contrast agents, the contrast agent is prepared in an aqueous medium. In another embodiment, contrast agents of defined molecular weight and / or molecular weight distribution are prepared by changing reaction conditions. In another embodiment, a narrow or desired distribution range of contrast agent is prepared by purification or fractionation methods such as ultrafiltration, size exclusion, and dialysis.

  In another embodiment, the invention provides a method for obtaining a magnetic resonance image of a mammalian tissue or organ comprising administering an effective amount of one or more polymeric contrast agents to the mammal, and magnetic A method is provided by obtaining a resonance image. One skilled in the art will appreciate that many known methods for obtaining magnetic resonance images exist in the scientific and medical fields. In one preferred embodiment, MRI treatment is performed on a human patient. Those skilled in the art will recognize that various tissues or organs including, but not limited to, liver, spleen, lung, esophagus, bone marrow, lymph node, lymphatic vessel, nervous system, brain, spinal cord, capillary, stomach, small intestine, and large intestine are used in the present invention. It will be appreciated that various aspects of the inspection may be used. One skilled in the art will recognize that both normal tissues and abnormal tissues such as tumors can be examined.

  Another aspect of the invention relates to a method for removing a metal complex. Preferably, the clearance process is performed after the MRI procedure is completed or substantially completed. In one embodiment, the mercaptan or other similar agent is administered after the MRI treatment. In various embodiments, these agents facilitate the excretion process by cleaving the polymer backbone. Alternatively or additionally, removal occurs by removing the paramagnetic metal complex from the polymer support by cleavage of disulfide bonds. Some embodiments are particularly advantageous because paramagnetic metal complexes released from macromolecules can be removed at a rate comparable to the low molecular contrast agents used clinically today.

  In one preferred embodiment, the polymeric compound has an extended retention time in the blood pool that favors accumulation in solid tumor tissue and is rapidly removed after MRI. These polymeric agents and the methods described there are numerous medical procedures including, without limitation, angiography, plethysmography, lymphangiography, mammography, cancer diagnosis, and functional and dynamic MRI. Would be an indispensable tool.

  The invention is further described in the following non-limiting examples, which are given by way of example only and are not intended to limit the invention in any way.

  Examples of preparation of DTPA-cystine copolymer, DTPA-cystine diethyl ester copolymer, and paramagnetic complexes GDCP and GDCEP, degradation of GDCP and GDEP in rat plasma, MR imaging, and data analysis are described in Non-Patent Document 12 (Zong ( Y. Zong, T. Ke, AM Mohs, J. Guo, Parker, Z.-R. Lu, “Poly Effect of size and charge on in vivo MRI contrast enhancement of polydisulphide Gd (III) complex, Journal of disulfide Gd (III) complex in vivo MRI contrast enhancement Of Controlled Releases, 2006, Vol. 112, p. 350-356), the entire contents of which are hereby incorporated by reference. .

Example 1 Preparation of polydisulfide copolymer in aqueous phase. Effect of reaction temperature on molecular weight distribution:
Cysteine (5 mmol, 1.200 g,> 99%) was dissolved in 2 ml of aqueous solution and the pH of this solution was adjusted to 11 with NaOH at room temperature. The reaction was then performed at three different temperatures, -10 ° C (ice-NaCl bath), 0 ° C (ice water bath), and room temperature, respectively. Diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid dianhydride (DTPA-DA) (5 mmol, 1.787 g) was added portionwise within 1 hour with rapid stirring. The pH of the reaction mixture was maintained at 11 with saturated aqueous NaOH. More than 30% DTPA dianhydride was added in portions over 30 minutes at a constant pH of 11. Five minutes after the addition of the last portion of DTPA-DA, 10% (by weight) HCl was added to adjust the pH of the reaction mixture to 7. The molecular weight distribution of the copolymer was analyzed by size exclusion chromatography (SEC) using an AKTA ^™ FPLC system equipped with Superose 12 ^™ (Amersham Bioscience Corp., Piscataway, NJ). FIG. 1 shows the molecular weight distribution of DTPA cystine copolymers prepared at different temperatures. This indicates that the reaction temperature has a significant impact on the molecular weight distribution. A high reaction temperature increases the molecular weight of the copolymer.

Effect of DTPA-DA to diamine feed ratio on molecular weight distribution Cystine (5 mmol, 1.200 g) was dissolved in 2 ml of water and the pH was adjusted to 11 with NaOH at room temperature. For this cystine solution, various molar ratios of cystine to DTPA-DA (0.9, 1.0, 1.1, 1.2, and 1.3) were divided within 1.5 hours with rapid stirring. Added. The pH of the reaction mixture was maintained at pH 11 with saturated aqueous NaOH. Five minutes after the addition of the last portion of DTPA-DA, 10% (by weight) HCl was added to adjust the pH of the reaction mixture to 7. The molecular weight of DTPA-cystine copolymers prepared at various molar ratios was analyzed by size exclusion chromatography using a Superose 12 column. See Table 2 and FIG. High molar ratios (1.2 and 1.3) DTPA dianhydride to cystine yield high molecular weights. The molecular weight of the copolymer therefore decreased with decreasing molar ratio.

DTPA-cystine copolymerization:
Cystine (10 mmol, 2.403 g) was dissolved in 5 ml NaOH aqueous solution at room temperature at pH 11 and then the mixture was cooled in an ice-water bath. Next, DTPA dianhydride (10 mmol, 3.573 g) was added portionwise within 1 hour while maintaining pH 11 using aqueous NaOH. Five minutes after the addition of the last portion of DTPA-DA, 10% (by weight) HCl was added to adjust the pH to 7, and this solution was used with a membrane having a molecular weight cut-off of 6-8000 Da. Dialyzed against deionized water for 24 hours. The copolymer solution was lyophilized to give 3.2 g of a colorless solid product (54%). The number of copolymers (Mn) and weight (Mw) average molecular weights as determined by SEC using an AKTA ^™ FPLC system equipped with a Superose ^™ 12 column were 18 and 33 kDa. The system was calibrated using standard poly [N- (2-hydroxypropyl) methylacrylamide] (PHPMA).

DTPA-glutathione (oxidized) copolymerization:
Glutathione (8 mmol, 4.901 g oxidized form) was dissolved in 5 ml NaOH aqueous solution at room temperature at pH 11 and then the mixture was cooled in an ice-water bath. Next, DTPA dianhydride (8 mmol, 2.850 g) was added portionwise within 1 hour while maintaining pH 11 using aqueous NaOH. Five minutes after the addition of the last portion of DTPA-DA, 10% (by weight) HCl was added to adjust the pH to 7, and this solution was used with a membrane having a molecular weight cut-off of 6-8000 Da. Dialyzed against deionized water for 24 hours. The copolymer solution was lyophilized to give 4.00 g of a colorless solid product (52%). The number of copolymers (Mn) and weight (Mw) average molecular weights, determined by SEC using an AKTA ^™ FPLC system equipped with a Superose ^™ 6 column, were 37 and 61 kDa.

Copolymerization of DTPA and cystamine:
Cystamine hydrochloride (5 mmol, 1.126 g) was dissolved in 2 ml of deionized water at room temperature and the pH was maintained at 11 using saturated aqueous NaOH. DTPA dianhydride (6 mmol, 2.144 g) was then added portionwise within 1 hour at room temperature under rapid stirring while maintaining pH 11 with aqueous NaOH. Five minutes after the addition of the last portion of DTPA-DA, 10% (by weight) HCl was added to adjust the pH to 7, and this solution was used with a membrane having a molecular weight cut-off of 6-8000 Da. Dialyzed against deionized water for 24 hours. The copolymer solution was lyophilized to give 1.46 g of a colorless solid product (50%). The number of copolymers (Mn) and weight (Mw) average molecular weights as measured by size exclusion chromatography (SEC) using an AKTA ^™ FPLC system equipped with a Superose 6 column were 35 and 42 Da.

Example 2 Preparation of paramagnetic polydisulfide copolymer complex. Complexation of DTPA disulfide copolymer with Gd ³⁺ :
Paramagnetic complexes, (Gd-DTPA) -cystine copolymer (GDCP), (Gd-DTPA) -glutathione copolymer (GDGP), and (Gd-DTPA) -cystamine (GDCC) are DTPA-cystine copolymer (DCP), DTPA. -Glutathione copolymer (DGP) and DTPA-cystamine copolymer (DCC) were each prepared by complexation with Gd3 ⁺ . Briefly, 0.5 g DCP or DGP or DCC was dissolved in deionized water. Xylenol orange can be added as an indicator of free Gd ³⁺ . Then, at pH 5.5-6.0, a slight excess of Gd (OAc) ₃ was added to the solution until its color changed from orange to red. The pH was then adjusted to 10 with 5M NaOH to precipitate excess Gd ³⁺ as Gd ₂ O ₃ . After centrifugation, the supernatant was collected and dried to obtain GDCP or GDGP or GDCC.

Example 3 Purification of Paramagnetic Polydisulfide Copolymer Complex The purification of the paramagnetic polydisulfide copolymer complex can be further purified by chromatographic methods or by ultrafiltration in the presence of citric acid to remove residual Gd ^3+. went.

In the first purification method, free Gd ³⁺ was removed by size exclusion chromatography on a column packed with G-25 medium. The column was eluted with pure water and the polymer fraction was collected.

  In the second method, the polymer solution was placed in an ultrafiltration chamber equipped with a semipermeable membrane with MWCO = 10000 Da (protein). This solution was stirred under pressure and diluted several times in the chamber. The concentrated polymer solution was evaporated to dryness. The SEC profile from the polymer purified by ultrafiltration was similar to that purified by SEC (Figure 3).

Example 4 Fractionation of Paramagnetic Polydisulfide Copolymer Complex Fractionation of polydisulfide Gd-DTPA complex was determined by SEC (size exclusion chromatography) using the Hiload ^TM 16/60 Superdex ^TM 200, or This was performed using an AKTA ^™ FPLC system equipped with a Superose ^™ 6 XK50 / 100 preparation grade column (Amersham Biosciences). Briefly, 50 mg of GDCP or GDGP was dissolved in water and loaded onto the column. The flow phase was 0.02 M Tris buffer pH 7.4 and the flow rate was 60 ml / hour for the Superdex ™ 200 column and 5.0 ml / min for the Superose ^™ 6 column. Fractions were collected and their molecular weight was measured by SEC equipped with a Superose ^™ 6 column (Table 3). The Gd content of the polydisulfide contrast agent was measured using inductively coupled plasma optical emission spectrometry (ICP-OES).

The method for fractionation of Gd-DTPA cystamine complex (GDCC) using AKTAprime ^™ plus FPLC and Superose ^™ 6 preparation grade XK50 / 100 column is shown as follows: (1) Prior to fractionation The FPLC pump is washed with Tris buffer (pH 7.5) and the column is then washed with 4.5 ml / min for the entire 18 hours with the same buffer. (2) 2.7 ml of Gd-DTPA cystamine copolymer solution is prepared to a concentration of 250 mg lyophilized polymer / ml deionized water and then injected into the FPLC through a 0.2 μL filter. Set the flow rate to 5.0 ml / min. (3) Discard the first 700 ml of eluate and collect the subsequent fractions in 1200 ml. Each fraction contains 20 ml of eluate.

The characterization of the fraction of Gd-DTPA cystamine complex is AKTA P-920 FPLC, Superose 6 10/200 GL column, and Knauer RI detector using PHPMA as standard. The flow rate is set at 5.0 ml / min. The characterization of the main fraction is shown in FIG. The number average, weight average molecular weight, and polydispersity index (PDI) of some combined fractions and fractions are listed in Table 4.

Example 5 Application of Gd-containing polydisulfide MRI contrast agent in MR imaging In vivo contrast enhancement by GDCP and GDGP was performed in female athymic nude mice with human breast cancer (MB-231) (Charles River Labs). Lab)) using a Siemens Trio 3T scanner equipped with a human wrist coil. Mice were anesthetized by intramuscular administration of a mixture of ketamine (80 mg / kg) and xylazine (12 mg / kg). Contrast-enhanced MR images of the mice were obtained from the tail vein at a dose of 0.1 mmol-Gd / kg of contrast agent before injection and at 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 60 minutes after injection. In minutes, a spin echo sequence was used. Imaging parameters were 2.7 ms TE, 7.8 ms TR, 25 ° C. RF chip angle, and axial slice thickness 0.5 mm.

  Three-dimensional maximum projection (MIP) MR imaging of mice at various time points before and after injection of various molecular weights of GDCP and GDGP is shown in FIG. A strong contrast enhancement was observed in the liver, kidney, and blood in the heart and vasculature 2 minutes after administration for all drugs, and the signal intensity then gradually decreased. Both drugs with maximum MW can strongly enhance blood vessels for up to 30 minutes. Both signal intensity and enhancement duration of GDCP and GDGP increased with molecular weight. A gradual enhancement in the bladder has been observed for all compounds, suggesting urinary clearance of this drug. The high molecular weight biodegradable drug (108 kDa) provides further extended and significant contrast enhancement for cardiac and vascular imaging than the corresponding low molecular weight drug.

A T1-weighted axial image of the tumor was also obtained with a human wrist coil using a spin echo sequence at 10 ms TE, 400 ms TR, 90 ° RF tip angle, axial slice thickness 2 mm. T1-weighted tumor images showed strong enhancement by both GDCP and GDGP in the tumor periphery up to 2 and 60 minutes after injection (FIG. 6).
The contrast enhancement at the center of the tumor is weaker than at the periphery. The low molecular weight (23 kDa) polymer drug resulted in a more significant enhancement in the tumor tissue than the high molecular weight drug.

  While this invention has been described in several embodiments, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, and adaptations using its general principles of the invention. Furthermore, this application is intended to cover such deviations from this disclosure as being within the scope of known or routine practice in the art to which this invention pertains, which is incorporated in the appended claims. Enter within the limits.

  All non-patent literature, including publications, patents, and patent applications cited herein are indicated as if each non-patent literature should be included individually and specifically as a reference, and the entire description. Are included herein by reference to the same extent as indicated in the specification. The non-patent documents discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

Claims (23)

  A method for preparing a biodegradable polymeric contrast agent comprising at least one step of polymerization in an aqueous medium, wherein said polymerization results in polydisulfide.   The method of claim 1, wherein the aqueous medium is a basic aqueous solution. The process of claim 1 or 4 comprising complexing the polydisulfide with Gd ³⁺ . A method for preparing a polydisulfide contrast agent comprising:
Selecting the reaction conditions, wherein the reaction conditions determine the molecular weight and / or molecular weight distribution of the contrast agent.   The method of claim 4, wherein the reaction condition is a reaction temperature.   The method of claim 4, wherein the reaction condition is a feed ratio of a polymerization reactant. The method of claim 1, 3 or 4, wherein:
The method comprising purifying the contrast agent by size exclusion chromatography, ultrafiltration, and / or dialysis. The method of claim 1, 3 or 4, wherein:
The method comprising fractionating the contrast agent by size exclusion chromatography. The method of claim 1, 3 or 4, wherein:
Dissolving at least one reactant in a reaction medium;
Adjusting the pH of the reaction medium as necessary,
Adding at least one other reactant to the reaction medium;
Stirring the reactants and the reaction medium for a period of time, and adjusting the pH of the reaction medium as needed;
The method comprising. 4. The method of claim 3, further comprising:
The method comprising precipitating excess Gd ³⁺ at pH = 10 or> 10.   The method according to claim 1, 3 or 4, wherein the molecular weight of the contrast agent is 40 to 200 kDa.   The method of claim 1, 3 or 4, wherein the contrast agent has a molecular weight of 5 to 40 kDa. 10. The method of claim 9, further comprising:
The process comprising lyophilizing the product resulting from the reactants. 10. The method of claim 9, further comprising:
The method comprising purifying the product resulting from the reactants by dialysis, chromatography, and / or ultrafiltration. 10. The method of claim 9, further comprising:
A method comprising fractionating a contrast agent using size exclusion chromatography.   A polymeric contrast agent prepared by the method of claim 1 or claim 3 or claim 4, wherein the contrast agent is a structural formula of GDCC, GDCP, GDEP, GDGP, US Pat. No. 6,982,324. Comprising a compound selected from the group consisting of compounds I to VII and compounds 1 to 11 of US Pat. No. 6,982,324, wherein the contrast agent is controlled or defined The contrast agent having a molecular weight and / or molecular weight distribution.   17. The polymeric contrast agent of claim 16, comprising at least one targeting molecule.   17. The polymeric contrast agent of claim 16, wherein the contrast agent can provide extended and effective contrast enhancement for cardiac and vascular imaging, and the contrast agent has a molecular weight of 40-200 kDa. The contrast agent.   17. The polymeric contrast agent of claim 16, wherein the contrast agent can provide effective contrast enhancement for tumor tissue imaging, and the contrast agent has a molecular weight of 5 to 40 kDa. A method of applying the contrast agent of claim 16 in a medical method comprising:
17. The contrast agent of claim 16, or a pharmaceutically acceptable salt thereof, or the contrast agent of claim 16, comprising administering to a mammal together with one or more pharmaceutically acceptable agents. Method.   21. The method of claim 20, wherein the medical method comprises magnetic resonance imaging, x-ray computed tomography, scintigraphy, positron emission tomography, and radiation therapy.   21. The method of claim 20, wherein the contrast agent is degraded by an endogenous agent.   21. The method of claim 20, wherein the mammal is administered one or more disulfide reducing agents simultaneously or sequentially with the contrast agent.

Images