AU2007261421A2 - Biodegradable macromolecular MRI contrast agents and methods of preparation and use thereof - Google Patents

Description

WO 2007/149408 PCT/US2007/014223 -1- BIODEGRADABLE MACROMOLECULAR MRI CONTRAST AGENTS AND METHODS OF PREPARATION AND USE THEREOF PRIORITY CLAIM This application claims the benefit of the filing date of United States Provisional Patent Application Serial Number 60/814,449, filed June 16, 2006, for "BIODEGRADABLE MACROMOLECULAR MRI CONTRAST AGENTS AND METHODS OF PREPARATION AND USE THEREOF." STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

Work described herein was supported by National Institutes of Health Grants CA095873. The United States government may have certain rights in the invention.

151 TECHNICAL FIELD This invention relates generally to biodegradable macromolecular contrast agents used in diagnostic imaging, and methods of synthesizing, purifying, using and degrading such compounds.

201

BACKGROUND

Magnetic resonance imaging (MRI) is a non-invasive method for medical diagnosis. Paramagnetic metal complexes are often used as contrast agents to enhance the image contrast between normal tissue and diseased tissue. Paramagnetic metal ions that 251 are typically used in diagnostic procedures include manganese (Mn 2 iron (Fe 3 and gadolinium (Gd3). Chelates of Gd 3 are frequently used as MRI contrast agents because of their long electronic relaxation time and high magnetic moment. Gadolinium-based contrast agents including small molecular gadolinium complexes such as Gd(III)(DTPA) [diethylenetriaminepentaacetate], Gd(III)(DOTA) [1,4,7,10-tetraazadodecanetetraacetic 301 acid], Gd(DTPA-BMA) (diethylenetriaminepentaacetic bismethyl amide), and their derivatives are routinely used as contrast agents for MRI in clinical practice. These agents are extracellular contrast agents, extravasate rapidly into extracellular fluid space and have short tissue retention time due to their small sizes, leading to some disadvantages including short imaging time window and low signal to noise ratio. In order to prolong 351 the circulation and retention of contrast agents, significant amounts of efforts have been WO 2007/149408 PCT/US2007/014223 -2made on the increase in the sizes of the contrast agents by either chemical conjugation of Gd(III) chelates to biomedical polymers or incorporation into the backbone of polymers Macromolecular MRI contrast agents, sometimes referred to as long-circulating or S blood pool agents, are particularly useful because of their prolonged retention time in the blood pool. For example, the half-life of albumin-(Gd-DTPA) conjugate is approximately 3 hours in the blood Gd-DTPA labeled dextran (MW 75 kDa) has a long half-life of 6.1 hours as compared to 13 minutes of Gd-DTPA in rats Polymeric contrast agents also possess increased proton Ti relaxivity resulting from long rotational time compared to small size molecules Because of the enhanced permeability and retention, these contrast agents accumulate effectively in solid tumors and have a potential in contrast enhancement in MR cancer imaging.

However, macromolecular contrast agents have potential toxicities related to slow excretion and long-term tissue Gd accumulation, thus hinders their further development.

As a result of poor clearance rates, use of Gd macromolecular agents may result in Gd accumulation in the bone and other tissues, resulting in toxicity and adverse side-effects.

(Gd-DTPA)-albumin conjugate, a prototype of macromolecular MRI contrast- agent, showed high accumulation of Gd in the bone and liver consequential of its slow excretion which increased possibility of cellular uptake of the agent through endocytosis and dissociation of Gd-DTPA complexes in the lysosome due to low pH and enzymatic degradation High long-term Gd tissue accumulation was also observed for other macromolecular Gd(III) complexes. It has been reported that the conjugation of Gd- DO3A to carboxylmethyl hydroxylethyl starch resulted in a macromolecular agent (72 kDa) that had about 47% of injected dose detected in rat body seven days after the injection A Gd-DTPA polypropyleneimine dendrimer (generation 2) conjugate (7 kDa) resulted in the retention of 45% of injected dose in rats 14 days after injection High long-term in vivo accumulation of the contrast agents significantly increases the possibility of metabolic release of toxic Gd(III) ions from the chelates.

The inventors recently designed and developed biodegradable macromolecular MRI contrast agents based on polydisulfide Gd() complexes to facilitate excretion of Gd chelates via in vivo degradation of the macromolecular agents [10-12]. Disulfide bonds in the polymer backbone can be rapidly reduced by free plasma thiols, e.g., glutathione and cysteine, or by enzymatic degradation. It has been shown that (Gd- DTPA)-cystamine copolymers (GDCC), the first polydisulfide MRI contrast agent, WO 2007/149408 PCT/US2007/014223 -3produced more significant blood pool contrast enhancement in rats than a clinically available MRI contrast agent, Gd-(DTPA-BMA), and then cleared rapidly from the blood pool. GDCC exhibited minimal long-term tissue accumulation of Gd comparable to the clinically used Gd-(DTPA-BMA).

Functional groups around the disulfide bonds have been introduced to tune the degradation rate of the paramagnetic polydisulfide agents and to prepare biodegradable macromolecular agents with various pharmacokinetic properties. Two modified polydisulfide MRI contrast agents, (Gd-DTPA)-cystine copolymers (GDCP) and (Gd- DTPA)-cystine diethyl ester copolymers (GDCEP), and preliminary results of these agents in contrast enhanced tumor MR imaging have been reported in Ref. [11].

DISCLOSURE OF INVENTION The present invention provides, among other things, degradable macromolecular contrast agents with a defined or controlled molecular weight and/or molecular weight distribution. In a preferred embodiment, the contrast agents comprise polydisulfide. In another preferred embodiment, these contrast agents are complexes with metals, e.g., Mn 2 Fe 3 and Gd 3 Examples of various contrast agents are disclosed in US Patent 6,982,324, Ref. Ref. or Ref. These contrast agents can be used in various medical procedures, diagnostic and treatment procedures. In one embodiment, these contrast agents are used in magnetic resonance imaging. In an alternative embodiment, these contrast agents are used in X-ray computed tomography. In other embodiments, the degradable polymer can form chelates with radioactive metal ions for scintigraphy, positron emission tomography and radiotherapy. In another aspect of the invention, the contrast agent complexes include targeting molecules. The incorporation of targeting molecules, including, but are not limited to, antibodies, antibody fragments, peptides, other proteins and other chemical entities results in macromolecular contrast agents with targeting ability.

The invention provides a method of preparing degradable macromolecular contrast agents in an aqueous medium. In a preferred embodiment, the reaction medium is a basic aqueous solution. Use of aqueous solution as a medium for polymerization avoids polluting solvents, and also facilitates easy scale-up for manufacture.

The invention provides a method of purifying degradable macromolecular ligands and contrast agents. In one embodiment, the ligands and contrast agents are purified by chromatography methods. In another embodiment, the contrast agents are purified by WO 2007/149408 PCT/US2007/014223 -4ultrafiltration. Biodegradable macromolecular MRI contrast agents comprising Gd can also be purified by raising pH of the contrast agent solutions to remove any residual free Gd(III) ions as Gd 2 0 3 precipitates.

The invention also provides a method of fractionating degradable macromolecular contrast agents to provide contrast agents with narrower or desired molecular weight distributions. In one embodiment of the invention, contrast agents are fractionated by chromatography methods such as size exclusion chromatography.

The invention also provides a method of controlling the molecular weight and/or molecular weight distribution of degradable macromolecular contrasting agents. In one embodiment, the molecular weights of the contrasting agents are controlled by varying polymerization conditions, such as reaction temperature and/or feed ratios of polymerization reactants.

The invention provides a method for obtaining a magnetic resonance image of a tissue or organ of a mammal by administering one or more degradable macromolecular contrast agents and obtaining a magnetic resonance image. In several embodiments, the macromolecular contrast agents are capable of being degraded by both endogenous and exogenous compounds. In one embodiment, the macromolecular contrast agents are degraded by endogenous mercaptans and/or enzymes into small stable chelates.

The invention also provides a method to degrade or stimulate degradation of the macromolecular contrast agents by administering one or more disulfide bond reducing compounds or other compounds that stimulate the degradation of the macromolecular contrast agents. In one embodiment, exogenous mercaptans are delivered to the mammal.

Another object of several embodiments of the current invention is to administer macromolecular contrast agents in conjunction with other agents. In various aspects, physiologically acceptable agents, such as diluents and carriers, are also administered.

One aspect of the invention includes a method for obtaining a magnetic resonance image of a tissue or organ of a mammal by administering an effective amount of one or more macromolecular contrast agents to the mammal and obtaining a magnetic resonance image. In a preferred embodiment, one or more of the macromolecular 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 said macromolecular contrast agent is also administered. In another aspect, one or more disulfide bond reducing compounds is also administered. The disulfide bond reducing compound is selected from the group consisting of one or more of WO 2007/149408 PCT/US2007/014223 the following: mercaptans, NADH, NADPH, hydrazines, phosphines, zinc, tin(II), sodium sulfide, performic acid, hydrogen peroxide.

Mercaptans used in various aspects of the present invention are selected from the group consisting of one or more of the following: cysteine and its derivatives, glutathione and its derivatives, cysteinylglycine and its derivatives, 2,3-dimercaptosuccinic acid and its derivatives, 2,3-dimercapto-l-propanesulfonic acid and its derivatives, 2mercaptoethanol, penicillamine and its derivatives, mercaptoacetic acid and its derivatives, mercaptoanisole, 2-mercaptobenzoic acid and its derivatives, 4mercaptobenzoic acid and its derivatives, 2-mercapto-5-benzimidazolesulfonic acid and its derivatives, 2-mercaptobenzothiazole, 3-mercapto-iso-butyric acid, mercaptocyclohexane, 2-mercaptoethanesulfonic acid, 2-mercaptoethylamine, 2mercaptoethylamine hydrochloride, 3-mercapto-1,2-propanediol, 3-mercapto-lpropanesulfonic acid, 3-mercapto-l-propanol, 2-mercaptopropionic acid, 3mercaptopropionic acid, diethyldithiocarbamate, dithioerythritol, and dithioglycol.

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

In another embodiment, at least one of the macromolecular contrast agents is administered in conjunction with one or more physiologically acceptable agents selected from the group consisting of: diluents, carriers, antibodies, antibody Fab' fragments, antibody F(ab') 2 fragments, and delivery systems.

In a preferred embodiment, at least one of the macromolecular contrast agents is administered in conjunction with one or more contrast agents 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 an object of several embodiments of the current invention to obtain a magnetic resonance image of healthy or tumorous tissues or organs, including but not limited to, liver, spleen, lung, heart, kidney, tumors, ovary, pancreas, biliary system, peritoneum, muscles, head, neck, esophagus, bone marrow, lymph node, lymph vessels, nervous system, brain, spinal cord, blood capillaries, stomach, small intestine, and large intestine.

It is an object of several embodiments of the present invention to provide a method to obtain a magnetic resonance image of magnetic resonance image of healthy or WO 2007/149408 PCT/US2007/014223 -6tumorous tissues or organs, the method comprising selecting a suitable molecular weight range for one or more contrasting agents. In one embodiment, contrast agents of high (preferably larger than 40 kDa) molecular weight provide more prolonged and significant contrast enhancement for cardiac and vasculature imaging. In another embodiment, contrast agents of low (preferably smaller than 40 kDa) molecular weight result in more significant enhancement in tumor tissues than high molecular weight agents.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 Effects of reaction temperatures on molecular weight distributions of polymer ligand DTPA-cystine copolymers, as also illustrated in the Table 1.

FIG. 2 Effect of feeding ratio of DTPA dianhydride to cystine on molecular weight distributions of copolymers, as also illustrated in Table 2.

FIG. 3 Comparison of purification of poly(GdDTPA-co-L-cystine) (GDCP) by columns vs. ultrafiltration.

FIG. 4 Time signal intensity curve of major fractions of Gd-DTPA cystamine copolymers (GDCC) using AKTA P-920 FPLC, Superose 6 10/200 GL column and a Knauer RI detector.

FIG. 5 Three-dimensional maximum intensity projection MR images of mice before injection and 2 5 10 15 30 and 60 min after injection of GDCP 23 kDa, B: 43 kDa, C: 109 kDa) and GDGP 21 kDa, E: 43 kDa, F: 108 kDa) at a dose of 0.1 mmol Gd/kg via a tail vein.

FIG. 6 Two-dimensional axial MR images of mice with human breast cancer (MB-231) (see arrows) before injection and 2 5 10 15 30 and 60 (g) min after injection of GDCP 23 kDa, B: 43 kDa, C: 109 kDa) and GDGP 21 kDa, E: 43 kDa, F: 108 kDa).

MODES FOR CARRYING OUT THE INVENTION Traditionally, polydisulfide ligands are prepared in organic solvents, for example, polydisulfide ligands are obtained by condensation polymerization of DTPA dianhydride and disulfide-containing diamines in DMSO. Organic solvents are, in general, not environmentally friendly, especially when they are used in large scale manufacturing processes. In addition, it takes extra steps to remove the organic solvents prior to application of the polydisulfide agents.

WO 2007/149408 PCT/US2007/014223 -7- The present invention provides a method for preparing degradable macromolecular contrasting agents in an aqueous medium, thus offering advantages over traditional methods in less pollution and ease of scaling up. Preferably, the aqueous medium is a basic aqueous solution. Using this novel method, polydisulfide MRI contrast agents, with different chemical structures and molecular weights, have been made successfully. For example, in vivo MR imaging in mice showed that polydisulfide MRI contrast agents had long retention in blood pool compared to a clinically-used agent with small size. These polydisulfide contrast agents show great promises to diagnose tumor and other cardiovascular diseases.

The invention provides a method of purifying degradable macromolecular contrast agents. The contrast agents can be purified by various chromatography methods, such as HPLC (high performance liquid chromatography), GPC (gel permeation chromatograph), and SEC (size exclusion chromatography). In a particular embodiment, the contrast agents are purified by SEC equipped with a column packed with G-25 medium. The contrast agents can also be purified by various filtration methods, such as ultrafiltration using a filter with a desired molecular weight cut-off.

The present invention also provides a method to prepare biodegradable macromolecular MRI contrast agents with different molecular weights, and/or narrow molecular weight distribution for different clinical applications. Biodegradable macromolecular MRI contrast agents with various molecular weights can be readily controlled by varying the reaction conditions, for example, by varying the reaction temperature, or by varying the feed ratio of polymerization reactants. Methods to adjust reaction conditions are exemplified in the Examples herein. It is to be noted that other reaction conditions, such as pH of the reaction medium, reaction time, mixing speed of reactants, and other factors, can also affect the molecular weight and/or molecular weight distribution.

It has been demonstrated that high molecular weight biodegradable agents, for example, of 108 kDa, provide more prolonged and significant contrast enhancement for cardiac and vasculature imaging than the corresponding agents of low molecular weights.

The polymer agents with low molecular weight, for example, of 23 kDa, resulted in more significant enhancement in the tumor tissues than high molecular weight agents.

Therefore, the invention also provides a method to obtain a magnetic resonance image of healthy or tumorous tissues or organs, the method comprising selecting one or more contrast agents with a suitable molecular weight for a specific application.

WO 2007/149408 PCT/US2007/014223 -8- A macromolecular contrast agent may have a broader molecular weight distribution than desired. For example, in order to accurately assess the size effects of a paramagnetic polydisulfide on its pharmacokinetics and in vivo contrast enhancement, the paramagnetic polydisulfide with a narrower molecular weight distribution are prepared by fractionation with size exclusion chromatography. It is to be noted other methods such as ultrafiltration or dialysis can also offer 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(II) complexes, (Gd-DTPA)-cystine copolymers (GDCP), (Gd-DTPA)-glutathione (oxidized form) copolymers (GDGP) and (Gd-DTPA)-cystine diethyl ester copolymers (GDCEP) behaved differently in mice. The data and analysis with respect to degradation, pharmacokinetics and in vivo contrast enhancement for these polydisulfide contrasting agents are shown in FIGs. 5 and 6, and detailed in Ref. the entire contents of which are incorporated herein by reference.

The effects of structure, size and/or charges of a polydisulfide contrast agent suggests that optimal polydisulfide Gd(III) complexes can be designed and prepared by structural optimization as safe and effective biodegradable macromolecular MRI contrast agents for various clinical applications. Further, contrast agents with different pharmacokinetic properties may be suitable for different applications. In general, agents with an acceptably long blood circulation are more effective in contrast enhanced cardiovascular imaging and cancer imaging. Therefore, the invention also provides a method to obtain magnetic resonance images of healthy or tumorous tissues or organs, the method comprising selecting one or more contrasting agents with suitable properties of pharmacokinetic, degradation, and/or in vivo contrast enhancement for a specific application.

In another aspect, the present invention provides polydisulfide MRI contrast agents of defined or controlled molecular weight and/or molecular weight distribution.

Polydisulfide MRI contrast agents of the present invention include, but are not limited to various contrast agents as disclosed in US Patent 6,982,324, Ref. 10, Ref. 11 or Ref. 12.

In one embodiment of various contrast agents, the contrast agents are prepared in aqueous medium. In another embodiment, the contrast agents of defined molecular weight and/or molecular weight distribution are prepared by varying reaction conditions. In another embodiment, the contrast agents of a narrow distribution range or a desired distribution range are prepared by purification or fractionation methods such as ultrafiltration, size exclusion, and dialysis.

WO 2007/149408 PCT/US2007/014223 -9- In another aspect, the invention provides a method for obtaining magnetic resonance images of a tissue or organ of a mammal by administering an effective amount of one or more macromolecular contrast agents to the mammal and obtaining magnetic resonance images. One skilled in the art will understand that many known methods of obtaining magnetic resonance images exist in the scientific and medical field. In a preferred embodiment, an MRI procedure is performed on a human subject. One skilled in the art will appreciate that a variety of tissues and organs may be examined using different aspects of this invention, including, but not limited to, liver, spleen, lung, esophagus, bone marrow, lymph node, lymph vessels, nervous system, brain, spinal cord, blood capillaries, stomach, small intestine, large intestine. One skilled in the art will appreciate that both normal tissues and abnormal tissues, such as tumors, can be examined.

Another aspect of this invention relates to a method of clearing metal complexes.

Preferably, the clearance procedure is performed after the MRI procedure has been completed or substantially completed. In one embodiment, mercaptans, or other similar agents, are administered after the MRI procedure. In various embodiments, these agents facilitate the excretion process by cleaving the macromolecular backbone. Alternatively, or in addition, clearing occurs by removal of the paramagnetic metal complexes from the polymer carriers by cleavage of the disulfide bond. Several embodiments are particularly advantageous because the paramagnetic metal complexes released from the macromolecules can be cleared at a rate comparable to that of the small molecular contrast agents used clinically today.

In a preferred embodiment, the macromolecular compounds have a prolonged retention time in the blood pool, favorable accumulation in the solid tumor tissues, and are cleared rapidly after MRI. These macromolecular agents, and the methods described thereof, will be indispensable tools in a variety of medical procedures, including, but not limited to, angiography, plethysmography, lymphography, mammography, cancer diagnosis, and functional and dynamic MRI.

The present invention is further described in the following non-limiting examples, which are offered by way of illustration and are not intended to limit the invention in any manner.

WO 2007/149408 PCT/US2007/014223

EXAMPLES

Examples of preparation of DTPA-cystine copolymers, DTPA-cystine diethyl ester copolymers, and paramagnetic complexes GDCP and GDCEP, degradation of GDCP and GDCEP in rat plasma, MR imaging and Data Analysis, have been presented in Ref. [12] Zong, T. Ke, A.M. Mohs, J. Guo, D.L. Parker, Lu Effect of size and charge on in vivo MRI contrast enhancement of polydisulfide Gd(III) complexes. J.

Controlled Rel. 2006, 112, 350-356), the entire contents of which are incorporated herein by this reference.

EXAMPLE 1 Preparation of polydisulfide copolymers in aqueous phase Effects of reaction temperature on molecular weight distribution: Cystine (5 mmol, 1.200 g, was dissolved in 2 ml aqueous solution and the pH of the solution was adjusted with NaOH to 11 at room temperature. The reactions were then carried out at three different temperatures, -10 0 C (ice-NaC1 bath), 0 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 in portions within 1 h with fast stirring. The pH of the reaction mixture was maintained at 11 with saturated NaOH aqueous solution. 30% more DTPA dianhydride was added in portions in 30 minutes at constant pH 11. Five minutes after adding the last portion of DTPA-DA, 10 (by weight) of HC1 was added to adjust pH of the reaction mixture to 7. The molecular weight distribution of the copolymers was analyzed by size exclusion chromatography (SEC) using AKTATM FPLC system (Amersham Bioscience Corp., Piscataway, NJ) with a Superose 12 T M column (Table FIG. 1 shows the molecular weight distribution of DTPA cystine copolymers prepared at different temperature. It shows that the reaction temperature has significant impact on the molecular weight distribution. High reaction temperature increases the molecular weights of copolymers.

Table 1. The weight-average (Mw) and number-average (Mn) molecular weights of the biodegradable macromolecular ligands, DTPA-cystine copolymers prepared at different reaction temperature.

Reaction temperature Mw (kDa) Mn (kDa) oC 50 39 0 oC 40 33 room temperature (25 OC) 38 22 WO 2007/149408 PCT/US2007/014223 -11- Effects of feed ratio of DTPA-DA to diamine on molecular weight distribution: Cystine (5 mmol, 1.200 g) was dissolved in 2 ml water and the pH was adjusted with NaOH to 11 at room temperature. DTPA-DA of different molar ratios to cystine 1.0, 1.1, 1.2 and 1.3) was added in portions within 1.5 h with fast stirring to the cystine solutions. The pH of the reaction mixtures was maintained at pH 11 with saturated NaOH aqueous solution. Five minutes after addition of the last portion of DTPA-DA, (by weight) of HC1 was added to adjust pH of the reaction mixture to 7. The molecular weight of the DTPA-cystine copolymers prepared at different molar ratios was analyzed by size exclusion chromatography with a Superose 12 column. See Table 2 and FIG. 2. High molar ratio (1.2 and 1.3) of DTPA dianhydride to cystine gives high molecular weight. The molecular weight of the copolymers was then decreased with decreasing molar ratios.

Table 2. The weight-average (Mw) and number-average (Mn) molecular weights of the biodegradable macromolecular ligands, DTPA-cystine copolymers prepared at different DTPA to cystine ratios.

[DTPA- Mw (kDa) Mn (kDa) dianhydride]/[cystine] 0.9 28 22 40 32 1.1 49 38 1.2 51 1.3 50 39 Copolymerization of DTPA-Cystine: Cystine (10 mmol, 2.403 g) was dissolved in 5 ml of aqueous NaOH at pH 11 at room temperature and then the mixture was cooled in an ice water bath. DTPA dianhydride (10 mmol, 3.573 g) was then added in portions within 1 h, maintaining at pH 11 with NaOH aqueous solution. Five minutes after addition of the last portion of DTPA-DA, 10% (by weight) of HCI was added to adjust pH to 7 and the solution was dialyzed against deionized water using membrane with molecular weight cutoff of 6-8000 Da for 24 h. The copolymer solution was lyophilized giving 3.2 g colorless solid product The number (Mn) and weight (Mw) average molecular weights of the copolymers were 18 and 33 kDa as determined by SEC using AKTA FPLC system with a Superose T M 12 column. The system was calibrated with standard poly[N-(2hydroxypropyl)methacrylanide] (PHPMA).

WO 2007/149408 PCT/US2007/014223 -12- Copolymerization of DTPA-Glutathione (oxidized form): Glutathione (8 mmol, 4.901 g, oxidized form) was dissolved in 5 ml of aqueous NaOH at pH 11 at room temperature and then the mixture was cooled in an ice water bath.

DTPA dianhydride (8 mmol, 2.850 g) was then added in portions within 1 h, maintaining at pH 11 with NaOH aqueous solution. Five minutes after addition of the last portion of DTPA-DA, 10 (by weight) of HC1 was added to adjust pH to 7 and the solution was dialyzed against deionized water using membrane with molecular weight cutoff of 6-8000 Da for 24 h. The copolymer solution was lyophilized giving 4.00 g colorless solid product The number (Mn) and weight (Mw) average molecular weights of the copolymers were 37 and 61 kDa as determined by SEC using AKTA FPLC system with a SuperoserM 6 column.

Copolymerization of DTPA and Cystamine: Cystamine hydrochloride (5 mmol, 1.126 g) was dissolved in 2 ml of de-ionized water at room temperature, and the pH was adjusted to 11 using saturated NaOH aqueous solution. Under fast stirring, DTPA dianhydride (6 mmol, 2.144 g) was then added in portions within 1 hour at room temperature, maintaining at pH 11 with NaOH aqueous solution. Five minutes after the addition of the last portion of DTPA-DA, 10% (by weight) of HC1 was added to adjust pH to 7 and the solution was dialyzed against de-ionized water using membrane with molecular weight cutoff of 6-8000 Da for 24 h.

The copolymer solution was lyophilized giving 1.46 g colorless solid product The number (Mn) and weight (Mw) average molecular weights of the copolymers were 35 and 42 Da as determined by size exclusion chromatography (SEC) using AKTA FPLC system with a Superose 6 column.

EXAMPLE 2 Preparation of paramagnetic polydisulfide copolymers complexes Complexation of DTPA disulfide copolymers with Gde: The paramagnetic complexes, (Gd-DTPA)-cystine copolymers (GDCP), (Gd- DTPA)-glutathione copolymers (GDGP) and (Gd-DTPA)-Cystamine (GDCC), were prepared by the complexation of DTPA-cystine copolymers (DCP), DTPA-glutathione copolymers (DGP) and DTPA-cystamine copolymers (DCC) with Gd 3 respectively.

Briefly, 0.5 g of DCP or DGP or DCC was dissolved in de-ionized water. Xylenol orange will be added as the indicators of free Gd 3 Then a slight excess of Gd(OAc) 3 was added to the solution at pH 5.5-6.0 until the color was changed to red from orange. The pH was WO 2007/149408 PCT/US2007/014223 -13then adjusted to 10 using 5 M of NaOH to precipitate the excess of Gd 3 as GdO0 3 After centrifugation, the supematant will be collected and dried to give GDCP or GDGP or

GDCC.

EXAMPLE 3 Purification of paramagnetic polydisulfide copolymers complexes Purification of paramagnetic polydisulfide copolymers complexes was further carried out by chromatographic methods or by ultrafiltration methods in the presence of citric acid to remove residual Gd 3 In the first purification method, free Gd3+ was removed by size exclusion chromatography with a column packed with G-25 medium. The column was eluted with pure water and polymer factions were collected.

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

EXAMPLE 4 Fractionation of paramagnetic polydisulfide copolymers complexes The fractionation of polydisulfide Gd-DTPA complexes were carried out by SEC (Size exclusion chromatography) using AKTA M FPLC system equipped with Hiload T M 16/60 Superdex

T

200 or Superose T M 6 XK50/100 prep grade column (Amersham Bioscience). Briefly, 50 mg of GDCP or GDGP was dissolved in water and loaded to the aforementioned columns. The flow phase is 0.02 M of Tris buffer at pH 7.4, and flow rate was 60 ml/hour for the SuperdexTM 200 column and 5.0 mn/min for the Superose T 6 column. The fractions were collected and their molecular weights were determined with SEC equipped with Superose T 6 column (Table Gd contents of polydisulfide contrast agents were determined using inductive coupling plasma-optical emission spectrum (ICP-

OES).

Table 3. GDCP and GDGP fractions with different molecular weight GDCP GDGP Mw (kDa) 109 43 23 108 43 21 Polydispersity Index 1.07 1.08 1.05 1.10 1.08 1.09 mmol Gd/g of polymer 0.90 0.82 0.88 0.63 0.63 0.62 WO 2007/149408 PCT/US2007/014223 -14- Procedure for fractionation of Gd-DTPA cystamine copolymers (GDCC) using AKTAprime m plus FPLC and Superose T 6 prep grade XK 50/100 column is set forth as follows: Before fractionation, wash pump of FPLC using Tris buffer (pH then wash the column using same buffer for total 18 hr at 4.5 ml/min. Prepare 2.7 ml Gd- DTPA cystamine copolymers solution at a concentration of 250 mg lyophilized polymer ml DI water, then inject into the FPLC via a 0.2 pL filter. Flow rate is set to 5.0 ml/min.

Discard first 700 ml eluent and collect fractions in the following 1200 ml. Each fraction contains 20 ml eluent.

The characterization of the fractions of Gd-DTPA cystamine copolymers is done with AKTA P-920 FPLC, Superose 6 10/200 GL column and a Knauer RI detector using PHPMA as standard. The flow rate is set to 0.5 ml/min. The characterization of major fractions is shown in FIG. 4. The number average, weight average molecular weight and polydispersion index (PDI) of some combined fractions and fractions are listed in Table 4.

Table 4. The number average, weight average molecular weight and polydispersion index (PDI) for major fractions.

Fraction(s) 6and 7 8and9 10 11 12 13 14 15 17 Mn, KDa 225 192 163 152 136 127 116 102 84 Mw, KDa 234 198 169 158 141 131 120 106 89 Mw/Mn (PDI) 1.04 1.03 1.04 1.04 1.04 1.03 1.04 1.04 1.06 32 and 34 and Fraction(s) 20 21 22 24 26 28 3031 33 Mn, KDa 66 60 55 47 38 34 28 22 19 Mw, KDa 69 63 58 49 41 36 30 24 Mw/Mn (PDI) 1.06 1.06 1.05 1.05 1.06 1.06 1.06 1.05 1.06 EXAMPLE 5 Application of Gd-containing polydisulfide MRI contrast agents in MR imaging The in vivo contrast enhancement by GDCP and GDGP was investigated in female athymic nude mice (Charles River Lab) with human breast cancer (MB-231) using a Siemens Trio 3T scanner with a human wrist coil. The mice were anesthetized by the intramuscular administration of a mixture of ketamine (80 mg/kg) and xylazine (12 mg/kg). Contrast enhanced MR images of the mice were acquired using spin echo sequence before and at 2, 5, 10, 15, 30 and 60 minutes after injection of the contrast agents at a dose of 0.1 mmol-Gd/kg via a tail vein. The imaging parameters were 2.7 ms TE, 7.8 ms TR, 25 OC RF tip angle, 0.5 mm axial slice thickness.

WO 2007/149408 PCT/US2007/014223 Three-dimensional maximum intensity project (MIP) MR images of mice before and at various time points after injection of GDCP and GDGP with different molecular weights are shown in FIG. 5. Strong contrast enhancement was observed in the liver, kidneys and blood in the heart and vasculature with all agents 2 min post-injection and the signal intensity gradually decreased thereafter. Both agents with highest MW can strongly enhanced blood vessel up to 30 minutes. Both the signal intensity and enhancement duration for GDCP and GDGP were increased with molecular weight. Gradual enhancement in the urinary bladder was observed for all compounds, indicating urinary clearance of the agents. High molecular biodegradable agents (108 kDa) provide more prolonged and significant contrast enhancement for cardiac and vasculature imaging than the corresponding agents of low molecular weights.

Tl weighted axial images of tumors were also acquired with human wrist coil using spin echo sequence with 10 ms TE, 400 ms TR, 900 RF tip angle, 2 mm axial slice thickness. T1 weighted tumor images showed strong enhancement by both GDCP and GDGP in the tumor periphery 2 minutes pot-injection and up to 60 minutes (FIG. The contrast enhancement in tumor center is weaker than in periphery. The polymer agents with low molecular weight (23 kDa) resulted in more significant enhancement in the tumor tissues than high molecular weight agents.

While this invention has been described in certain 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, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.

All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The references 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.

WO 2007/149408 PCT/US2007/014223 -16-

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Claims (12)

1. A method for preparing a polydisulfide, the method comprising reacting a thiol-containing compound with a dianhydride compound in an aqueous medium in 00 O the presence of a base, wherein the base is a hydroxide base.

2. The method of claim 1, wherein the thiol-containing compound is cysteine, glutathione or cystamine hydrochloride and the dianhydride compound is diethylenetriamine-N, N, N' N"-pentaacetic acid dianhydride.

3. The method in claims 1 or 2, wherein the base is an alkali metal 0 hydroxide.

4. The method of claim 3, wherein the alkali metal hydroxide is NaOH. The method in any of claims 1-4, wherein the amount of hydroxide base is present so that pH of the aqueous medium is maintained at a pH of 11.

6. The method in any of claims 1-5, wherein the reaction is conducted at room temperature or less than room temperature.

7. The method in any of claims 1-6, wherein there is a molar excess of dianhydride compound relative to the thiol-containing compound.

8. The method of claim 1, wherein the thiol-containing compound is cystamine hydrochloride, the dianhydride compound is diethylenetriamine-N, N, N' N"-pentaacetic acid dianhydride, and the hydroxide base is NaOH, wherein the reaction is conducted at room temperature at a pH of 11.

9. A method for preparing a polydisulfide, the method comprising reacting a thiol-containing compound with a dianhydride compound in an aqueous medium in the presence of a base, wherein the amount of base is present so that pH of the aqueous medium is maintained at a pH of 11.

10. A polydisulfide made by the method in any of claims 1-9.

11. The polydisulfide of claim 10, wherein the molecular weight of the polydisulfide is 40 to 200 kDa. N:\Melboume\CaoeB\Patent\79000-79999\P9932 AU\Specio\P79932.AU GH spect fir doc 8/01/09 -19-

12. A method for preparing a biodegradable polydisulfide macromolecular Scontrast agent, the method comprising complexing Gd 3 with a polydisulfide made by the method in any of claims 1-9. 00 S13. The method of claim 12, wherein the Gd 3 is Gd(OAc) 3

14. The method of claims 12 or 13, further comprising precipitating excess N of Gd 3 at pH greater than or equal to A biodegradable polydisulfide macromolecular contrast agent produced by the method in any of claims 12-14. C 16. The use of a biodegradable polydisulfide macromolecular contrast agent of claim 15 for obtaining a magnetic resonance image of a tissue or organ of a mammal. NA\Melborne\C~aoo\PatenL\790D0-9999\P79932.AU\Sec1.\P9932.AU GM *peci flrotdOC 8/01109