Extended-linear polymeric contrast agents, and synthesizing methods, for medical imaging

#This invention relates to nuclear magnetic resonance imaging (MRI) and, more particularly, to extended-linear polymeric contrast agents for magnetic resonance imaging of tumors and methods of synthesizing such agents.

#Tumor angiogenesis is the recruitment of new blood vessels by a growing tumor from existing neighboring vessels. This recruitment of new microvasculature is a central process in tumor growth and in the potential for aggressive spreading of the tumor through metastasis. All solid tumors require angiogenesis for growth and metastasis. Thus, the level of angiogenesis is thought to be an important parameter for the staging of tumors. Furthermore, new therapies are being developed which attack the process of angiogenesis for the purpose of attempting to control tumor growth and tumor spread by restricting or eliminating the tumor blood supply. It is therefore of clinical importance to be able to monitor angiogenesis in tumors in a noninvasive manner.

#To assess angiogenic activity of tumors, two parameters are of primary importance: vascular volume and vascular permeability. Invasive techniques utilizing tissue staining can be used to assess microvascular development, but the sensitivity of existing staining methods is not high enough and the prognostic value of such methods is not yet well established (N. Weidner, et al., New Eng. J. Med. 324:1-8, 1991). At present there is no single imaging method capable of providing quantitative characterization of tumor angiogenesis.

#FIG. 1 is a block diagram of an MRI system which employs contrast agents of the present invention;

#FIG. 2 is a graphic representation of a pulse sequence performed by the MRI system of FIG. 1 to assess tumor angiogenosis; and

#FIG. 3 is a graphic representation of the relationship between proton relaxivity and lysine content for linear extended polymeric paramagnetic chelates usable as MRI contrast agents.

#In characterizing tumor angiogenesis, a contrast agent comprising a reptating polymer is intravenously injected and a series of timed medical images is obtained. A signal enhancement (in T1 weighted images) above a certain threshold, preferably 10%, constitutes an indicator of angiogenic activity. Signals beyond the threshold level will indicate increased angiogenic activity in the form of increased microvascular density, usually at the periphery edges of the tumor, and increased vascular permeability at the periphery and throughout the interior of the tumor.

#One contrast agent for characterizing tumor angiogenesis is a reptating polymer, preferably as described in Uzgiris U.S. Pat. No. 5,762,909, issued Jun. 9, 1998 and assigned to the instant assignee. U.S. Pat. No. 5,762,909, incorporated by reference herein, describes the creation of elongated, worm-like macromolecules. A particularly preferred polymer is a homopolymer of lysine where the lysine residues are substituted with Gd-DTPA, or gadolinuim- diethylentriaminepentaaceticacid. The degree of substitution must be very high, in excess of 90%, for the polymer to assume an elongated worm-chain conformation. The polymers described in U.S. Pat. No. 5,762,909 have such a conformation as determined by their measured persistence length (in the range of 100 to 600 Å) which is similar to the persistence length of double-stranded DNA. Double-stranded DNA is a classic reptating polymer and is separated according to length in gel electrophoresis by the mechanism of reptation (R. H. Austin, et al., Physics Today, pp. 32-37, 1997). The polymers of U.S. Pat. No. 5,762,909 remain in the vasculature as a blood pool agent and leak out of the endothelium only in tumors which have a hyperpermeable endothelium. The hyperpermeability is a result of angiogenesis signals emanating from tumor cells under nutrient and oxygen stress. The polymers are shown to be ideal agents for MR imaging methods to measure tumor blood volume and tumor endothelium permeability. The polymers for use in characterizing tumor angiogenesis are made by substituting the lysine residues of polylysine with DTPA in a mixed anhydride reaction (Sieving, et al. Bioconjugate Chem. 1:65-71, 1990). However, in order to attain the reptating conformation, the anhydride reaction and the coupling reaction are modified: the synthesis of the anhydride of DTPA is as previously described by Sieving, but the reaction is preferably run between −25° C. and −28° C. for 30 minutes under dry nitrogen atmosphere. The coupling of the anhydride to the lysines is modified in that a much higher molar ratio of anhydride to lysines residues is used in the coupling (from 7 to 10). After the coupling reaction, the reaction solution is subject to roto-vaporation at 50° C. to release all the volatile organic molecules and then the product is purified through extensive dia-filtration (Amicon, 10 kD molecular weight cutoff filters). To achieve the final MR active agent, the paramagnetic ion gadolinium is incorporated into the product polymer chelating DTPA groups by dropwise addition of GdCl3 in 0.1 M HCl (50 mM in Gd) into the polymer solution (15 mM NaHCO3). The dropwise addition of Gd continues until a slight indication of free Gd (not chelated by available DTPA groups) is noted (small aliquots of polymer solution added to 10 μM of arzenzo III in acetate buffer-free Gd turns the dye solution blue). The reptating polymer is then introduced into a blood vessel of the subject.

#Other paramagnetic ions besides Gd may be used. However, Gd is the most paramagnetic (i.e., has the most unpaired electrons) and thus is the most effective as contrast agent. A chelator such as DTPA must be used because free Gd is toxic. The chelator folds around the Gd and tightly binds it, but the water protons can come into one Gd coordination site and be relaxed.