United States Patent 1191 [11] Patent Number: 5,686,061 Li Et Al

United States Patent 1191 [11] Patent Number: 5,686,061 Li et al. [45] Date of Patent: Nov. 11, 1997

[54] PARTICULATE CONTRAST MEDIA Sands et al., “Computed Tomographic Enhancement of DERIVED FROM NON-IONIC WATER Liver and Spleen in the Dog with Iodipamide Ethyl Ester SOLUBLE CONTRAST AGENTS FOR CT Particulate Suspensions,” pp. 408-416 (Jun. 1987). _ ENHANCEMENT OF HEPATIC TUMORS Violante et al., “Maximizing Hepatic Contrast Enhancement With A Particulate Contrast Agent in Computed Tomogra [75] Inventors: Chun Li; Sidney Wallace; Zuxing phy,” pp. 69-75 (Mar. 1981). Kan, all of Houston; David J. Yang; Masazumi Ishikawa, et al., Fundamental Study of Positive Li-Ren Kuang, both of Sugar Land, all Contrast Media of Hepatic CT by Micro-Barium Sulphate of Tex. Particles, 1478-1488 (Nov. 1987). MR. Violante, et al., Biodistribution of a Particulate Hepa [73] Assignee: The Board of Regents of the tolienographic CI‘ Contrast Agent; A Study of Iodipamide University of Texas System, Austin, Ethyl Ester in the Rat, Investigative Radiology, vol. 16, Jan. Tex. 1981, 40-45. Michael R. Violante, et al., Particulate Contrast Media for [21] Appl. No.: 225,665 Computed Tomographic Scanning of the Liver, Investigative [22] Filed: Apr. 11, 1994 Radiology, vol. 15, Nov. 1980, 8171-8175. M. Sovak, et al., Current Contrast Media and Ioxilan Com [51] Im. c1.6 ...... A61K 9/16 parative Evaluation of Vascular Pain by Aversion Condi [52] US. Cl...... 424/9454; 424/945; 424/9451; tioning, Investigative Radiology, vol. 23, Sep. 1988, 424/9452; 424/9453; 549/229 $84-$87. [58] Field of Search ...... 424/9, 9.45, 9.451, Steven E. Seltzer, et al., Liposomes Carrying Diatrizoate 424/9452, 9.453, 9.454, 9.455; 549/229 Characterization of Biophysical Properties and Imaging Applications, Investigative Radiology, Mar. 1984, 142-151. [56] ‘ References Cited David J. Yang, et al., Evaluation of P0ly(dl—latide) Encap U.S. PATENT DOCUMENTS sulated Rad/‘opaque Microcapsules, American Chemical Society, 371-381 (Apr. 1993). 4,716,225 12/1987 Ledley et al...... 536/122 4,873,075 10/1989 Counsel] et a1...... 424/11 Peter R. Mueller, et al., Medical Progress: Interventional 4,957,729 9/1990 Counsell et al...... 424/5 Radiology in the Chest and Abdomen, The New England 5,093,042 3/1992 Counsell et al...... 260/408 Journal of Medicine, May 1990, 1364-1374. L Ko? Adzarnli, et al., Production and Charaterization of FOREIGN PATENT DOCUMENTS Improved Liposomes Containing Radiographic Contrast 0 083 964 7/1983 European Pat. Off. . Media, Investigative Radiology, Nov. 1990, 1217-1223. 0 133 248 2/1985 European Pat. Off. . Paul H. Sugarbaker, et al., Improved Detection of Focal 0 431 338 Al 6/1991 European Pat. O?. . Lesions with Computerized Tomography Examination of the 5208921 8/1993 Japan . Liver Using Ethiodized Oil Emulsion (EOE-13) Liver Con WO 90/07491 7/1990 WIPO . WO 94/14478 7/1994 WIPO . trast, Cancer, Oct. 1984, 1489-1495. Errol Lewis, et al., CI‘ Detection of Hepatic Metastases with OTHER PUBLICATIONS Ethiodized Oil Emulsion 13, Journal of Computer Assisted Newton, “Structure-Toxicity Relationships of Iodinated Tomography, Dec. 1982, 1108-1114. Aromatic Carbonates and Related Compounds,” Jnl of Phar Primary Examiner-Brian M. Burn maceutical Sciences, vol. 67, No. 8 (Aug. 1978). Attorney, Agent, or Finn-Rosenblatt & Redano, RC. Newton, “Iodine-Containing Organic Carbonates as Inves tigative Radiopaque Compounds,” J n1 of Medicinal Chem, [57] ABSTRACT vol. 19, No. 12 (Dec. 1976). Pillai, et al., “Heterocyclic Nonionic X-ray ContrastAgents. The present invention provides a method for chemically 3. The Synthesis of modifying non-ionic, water soluble particulate contrast 5-[4-(HydroxymethyD-2-oxo-3—oxazolidinyl]—2,4,6-tri agents so that they degrade in vivo to their non-ionic parent iodo-l,3-benzene-dicarboxamide Derivatives,” Jnl. Org. contrast material and carbon dioxide. According to the Chem 59, pp. 1344-1350 (Mar. 1994). present invention, known particulate, non-ionic contrast Ranganathan, et al., ‘The Chemical, Biological, and Physi agents are chemically modi?ed to form a precursor or cal Properties of S-Heterocycle Substituted 2,4,6-Tri “prodrug” comprising cyclic carbonates and carbamates of iodo-1,3-Benzenedicarboxarnide Derivatives,” Investiga the parent compound. The resulting cyclic carbonates and tive Radiology, vol. 26, Supp. No. 1 (Nov. 1991). carbamates are lipid soluble, biodegradable, and can be Morris, “Inotropic E?’ects of Sodium Citrate in a Nonionic prepared in large quantities using well-established methods. Contrast Medium,” Jnl of Clinical and Laboratory Investi These cyclic carbonates and carbamates can be converted to gative Radiology, vol. 25, Supp. (Sep. 1990). particulate contrast media using simple, well known Li, et al., “Preparation, Characterization, and Evaluation of techniques, such as solvent-extraction or solvent evapora Ioxilan Carbonate Particles for Computed Tomography Con tion. trast Enhancement of Liver,” Jnl of Clinical and Laboratory Investigative Radiology, vol. 29. No. 11 (Nov. 1994). 16 Claims, 7 Drawing Sheets

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C: D gm N (DH) uolwmuanv Patent Nov. 11, 1997 Sheet 7 of 7 5,686,061 5,686,061 1 2 PARTICULATE CONTRAST MEDIA ' high osmolarity resulting when it is present. A by-product of DERIVED FROM NON-IONIC WATER the degradation of other similar radiopaque esteri?ed par SOLUBLE CONTRAST AGENTS FOR CT ticles also should be the original ionic form of the radio ENHANCEMENT OF HEPATIC TUMORS paque compound. The osmolarity of body ?uids and cell contents must be FIELD OF THE INVENTION maintained within a narrow physiological range. The par The present invention relates to contrast media used to ticles present in particulate contrast media are targeted to, detect hepatic lesions, and more particularly to non-ionic, and should be degraded by Kupffer cells. Therefore, the particulate contrast media having particles that are‘not water osmotic in?uence of the degradation lay-products of these soluble, but which will degrade to their parent, water contrast media, particularly if the by-products are ionic, is of soluble, non-ionic contrast agent and carbon dioxide. major concern. A particulate contrast agent which was biodegradable and produces non-ionic, non-toxic BACKGROUND OF THE INVENTION by-products would be highly desirable. Assessment of the liver for metastases is important in staging a wide variety of malignancies. Surgical removal of SUMMARY OF THE INVENTION hepatic lesions requires precise diagnosis of the number, The present invention provides a method for chemically size, and location of the tumor(s). Typically, hepatic lesions modifying non-ionic, water soluble particulate contrast are diagnosed using computed tomography scanning (CT), agents so that they degrade in vivo to their non-ionic, water or computed tomographic portography (CT 20 soluble parent compound and carbon dioxide. According to angioportography). CT angioportography is performed after the present invention, known particulate, non-ionic contrast the superior mesenteric or splenic artery has been selectively agents are chemically modi?ed to form a precursor or catheterized and injected with contrast media (CM) to '“prodrug” comprising cyclic carbonates and carbamates of opacify the hepatic parenchyma. During CI‘ angioportogra the parent compound. The resulting cyclic carbonates and phy lesions in the liver appear as defects in the enhanced 25 carbarnates are lipid soluble, biodegradable, and can be parenchyrna. Unfortunately, benign and malignant masses prepared in large quantities using well-established methods. can’t be differentiated, and the time available for optimal These cyclic carbonates and carbamates can be converted imaging is limited. into particulate contrast media using simple, well known CT detection of hepatic lesions generally can be improved techniques, such as solvent-extraction or solvent evapora using rapid intravenous injection of water-soluble contrast 30 tion. agents combined with fast, incremental CI‘ scanning (bolus dynamic CI‘). Bolus dynamic CI‘ has an accuracy between DETAILED DESCRIPTION OF THE DRAWINGS about 73%—75% in identifying patients with hepatic metastases (Freeny PC, Marks WM, Ryan JA, Bolen JW. FIG. 1 is a diagrammatic representation of the scheme and the structure of the cyclic carbonate of IOXILAN obtained Colorectal carcinoma evaluation with CI‘. Preoperative stag 35 ing and detection of postoperative recurrence. Radiology by reacting IOXILAN with CD1 (Carbonyldiimidazole). 1986;158:1347-353). Furthermore, currently available water This synthesis can be used to prepare other non-ionic soluble contrast agents produce contrast enhancement for a particulate contrast agents, as well. duration of only minutes before CT densities return to FIG..2 represents the lX-C microparticles’ (1-2 pm) size baseline levels. distribution when prepared according to the solvent Particulate contrast agents are a promising avenue for extraction/evaporation process of the present invention. selectively opacifying the liver and for prolonging the FIG. 3 is a scanning electron micrograph of the particles radiocontrast e?’ect of the contrast media. Phagocytic cells from FIG. 2. of the reticulo-endothelial system (RES), the Kup?ier cells FIG. 4 illustrates the particles of FIG. 2 suspended in (KC) of the liver, very e?iciently remove foreign particles saline in the presence of rat plasma. from the blood. A large proportion of small particles (<5 pm FIG. 5 is a scanning electron micrograph of the particles in size) will be removed by the RES within a few minutes. of FIG. 2 after incubation in saline at 37° C. for two weeks. Furthermore, most neoplastic lesions do not contain mac FIG. 6 is a plot of liver attenuation enhancement as a rophages. Therefore, targeting the liver with particulate function of time for three doses of lX-C particles. contrast media enhances the liver parenchyma, causing 50 tumors to appear as defects. FIG. 7 is a plot of the CT pharrnaookinetics of aorta, Several experimental particulate contrast agents have kidney (cortex and medulla), and bladder following injec been developed during the last few decades. These roughly tion of 200 mg Ilkg body weight of IX-C particles (1-2 pm) can be divided into three categories: (1) iodinated lipid presented as a histogram. (EOE-13); (2) radiopaque liposomes; and, (3) particles FIG. 8 represents CI‘ imaging of a rabbit liver six days derived from water-soluble ionic contrast media (iodipamide after tumor inoculation: “a” is the CI‘ image before contrast ethyl ester, or “IDE”). Unfortunately, each of these particu injection; “b” after contrast injection; “c” and “d” after about late media has drawbacks. 2 hours. EOE-l3 is a lipid soluble contrast agent which can be DETAILED DESCRIPTION OF THE formulated as an oil emulsion for intravenous injection. The INVENTION drawback of EOE-13 is that EOE-l3 is not biodegradable. Liposome-based contrast agents have the drawback that they The invention is described with reference to a third usually have a short shelf-life and the efficiency of encap generation non-ionic, water soluble contrast agent called sulation is low. Although iodipamide ethylester (IDE) par IOXILAN. Toxicological and pharmacological studies of ticles appeared promising, one of the degradation products IOXILAN indicate that the body has a high overall biologi of DE particles in vivo is iodipamide. Iodipamide is ionic cal tolerance for IOXILAN. However, other water soluble and therefore is toxic to endothelial cells, perhaps due to the non-ionic contrast agents, including but not limited to 5,686,061 3 4 IOHEXOL, IOPROMIDE, IOTROLAN, IOPAMIDOL, tn'chloromethyl chloroforrnate, or other activating/coupling MEI‘RIZAMIDE, IOGLUNIDE, IOGULAMIDE, and simi agents known in the art, in (b) a polar aprotic solvent, such lar agents, also are suitable for use according to the present as dry dimethyl sulfoxide (DMSO), dirnethylforrnamide, invention. 1-methy1-2-pyrrolidinone, or other polar aprotic solvents Generally, water soluble non-ionic contrast agents suit known in the art, in the presence of (c) a catalyst capable of able for use in the present invention are aromatic compounds catalyzing the formation of cyclic carbonates and carbam substituted with an amount of a radiopaque element su?i ates from said water soluble non-ionic CM. Suitable cata cient to render the compound detectable by standard diag lysts include salts of alkyl oxides, such as sodium nostic tools, such as computed tomography. Even a single methoxide, sodium ethoxide, potassium methoxide or simi radiopaque substituent may be su?icient for purposes of the 10 lar salts. The mixing process typically requires about 30 present invention; however, the presence of two or more minutes. After mixing, the solution should be stirred for a radiopaque substituents renders the material more detect time and at a temperature suf?cient to permit the formation able. Therefore, it is preferred to have as many radiopaque of cyclic carbonates and carbamates. Typically, the solution substituents on the aromatic ring as possible, preferably should be stirred between about 2-20 hours, preferably at three such agents on alternating carbons of the aromatic ring. 15 least about 10 hours, at a temperature between about The radiopaque element can be any suitable non-toxic 40°~90° C., preferably at about 70° C. element; however, the preferred radiopaque element is The reaction then may be terminated by adding an organic iodine. solvent, such as methylene chloride, and washing with cold The aromatic compound also has at least one amide water. The solution should separate into an organic and a substituent with an aliphatic vicinal diol and/or 1,3-diol water phase, the cyclic carbonates and carbamates remain substituent bound to either the carbon or nitrogen of the ing in the organic phase, and the DMSO remaining in the amide moeity. This hydrophilic aliphatic polyol substituent water phase. Once the organic phase has been separated, the renders the contrast agent water soluble. In other words, a organic solvent is dried over dehydrating agents, such as preferred embodiment of the invention comprises an aro MgSO4, Na2SO4, or a similar agent. The solvent then is matic ring alternately substituted at the ring carbons with a 25 ?ltered and evaporated to dryness so that the product may be radiopaque element, preferably iodine, and an aliphatic collected for further use. FIG. 1 is a diagrammatic repre amide group. Each of the aliphatic amide groups preferably sentation illustrating the reaction of IOXlLAN to form contains at least one hydroxyl group, and at least one of the IOXHJAN Carbonate according to the present invention. amide groups must contain a vicinal diol or a 1,3-diol. The following is a general formula which, without lim The following is an illustration of the general structure of 30 iting the present invention, is believed to represent biode suitable water soluble non-ionic contrast agents: gradable contrast prodrug made from water-soluble non ionic CM prepared according to the method of the present R1 invention:

35 R1

R 40

R is a radiopaque element; R1 is an amide group bonded to said aromatic ring at either the nitrogen or the carbon of the amide, the unbonded nitrogen or carbon having a substituent In the foregoing structure, R is a radiopaque element. selected from the group consisting of an aliphatic vicinal 45 Preferably all three R groups are radiopaque elements. diol and an aliphatic 1,3-diol; and R2 is selected from the preferably iodine. R1 is an amide group bonded to the group consisting of a radiopaque element, a hydrogen, an aromatic ring at either the nitrogen or the carbon of the alkyl group having between about 1-4 carbon atoms, and an amide moiety, and the unbonded nitrogen or carbon of the amide group bonded to the aromatic carbon at either the amide moiety is substituted by an aliphatic group which nitrogen or the carbon of the amide, the unbonded nitrogen 50 includes a cyclic carbonate and/or a carbamate. R2 prefer or carbon having a substituent selected from the group ably is another amide group which contains another cyclic consisting of hydrogen, an alkyl group having between carbonate and/or a carbamate; however, R2 also may be a about 1-3 carbon atoms, and a hydroxylated aliphatic side radiopaque element, hydrogen, an alkyl group having chain having between about 1-8 carbon atoms. Preferably, between about 1-4 carbon atoms. or any other substituent the aromatic carbon is substituted with at least two amide 55 which will not interfere with the function of the contrast groups having a vicinal diol or 1,3-dio1 substituent and at agent-—that is, to opacify the liver parenchyma while bio least two radiopaque elements, preferably iodine. degrading to water soluble, non-ionic by-products. The foregoing non-ionic contrast agents may be chemi The prodrugs or precursors of the invention are formu cally modi?ed to form cyclic carbonates and carbamates. lated as injectable microparticles (mean diameter about l-2 One such suitable method is described in Kutney, J. P., and micron) in the following manner. The cyclic carbonate and Ratcliffe A. H. “A novel and mild procedure for preparation carbamate derived from the water soluble non-ionic contrast of cyclic carbonates. An excellent protecting group for agent(s) is dissolved in organic solvent or solvent mixture, vicinal diols.” Synth. Commun. 1975;5;47-52 (incorporated which may include but is not limited to acetone. chlorinated herein by reference). carbon, tetrahydrofuran, dimethylforrnamide, etc., prefer Generally the radiopaque contrast agent is thoroughly 65 ably a mixture of acetone and methylene chloride. The mixed with (a) an activating and/or coupling agent, such as organic solution containing the prodrug is added to an carbonyldiimidazole (CDI), a phosgene, a triphosgene, aqueous solution containing an emulsi?er, such as polyvinyl 5,686,061 5 6 alcohol, Tween 80, cellulose, polyvinylpyrrolidone. A pre Nicomp 370 Submicron Particle Sizer (Nicomp Instruments ferred emulsi?er is polyvinyl alcohol. The mixture then is Corp., Goleta, Calif). emulsi?ed mechanically with or without sonication for up to Suspension Stability about 10 minutes, and stirred for about another 4 hrs to Suspension stability of radiopaque particles were ensure complete removal of organic solvent. The resulting observed under light microscopy 20><2.5, Zeiss, Germany) microparticles are collected following repeated centrifuga and recorded with a video camera. lX-C particle suspensions tion and washing steps. in saline and in saline solution of New 80 (0.1%, w/w) The invention will be more clearly understood with were studied. After 5 minutes of observation, fresh rat reference to the following examples. plasma was added onto the suspensions, and the mixtures 10 were observed for an additional 5 minutes to record any EXAMPLES changes. Materials Hydrolyric and Enzymatic Degradation of lX-C Particles For purposes of the following examples, IOXILAN was Degradation of IX-C particles was investigated by incu supplied by Cook Imaging Corp. (Bloomington, Ind.). Car bating a suspension of the particles (25 mg) in the following bonyldiimidazole (CDI), dimethyl sulfoxide (DMSO), mag solutions (each 1 ml) at 37° C.:0.l N HCl, 0.1 N NaOH, nesium sulfate, methylene chloride, and sodium methoxide saline, and rabbit plasma. The disappearance of the particles were obtained from Aldrich Chemical Co. (Milwaukee, in the suspensions was noted by visual observations and the Wis.). Poly(vinyl alcohol) (PVA, MW 30 to 70K) was integrity of the particles was examined by scanning electron purchased from Sigma Chemicals Co. (St. Louis. Mo.). microscopy. To identify the degradation products, the Synthesis of IOELAN Carbonate 20 residual solutions were subjected to analysis by HPLC. The The cyclic carbonate of IOX]LAN (IX-C) was prepared HPLC system consisted of a RP-l8 column, a Perkin-Elmer using the method of Kutney and Ratcliffe, Synth. Commun. isocratic LC pump (Model 250), a PE Nelson 900 series 1975; 5; 47-52, incorporated herein by reference. A solution interface, a Spectra-Physics UV/Vis detector (Model SP of CD1 (4 g, 24 mM) in dry DMSO (15 mL) was dropped 8540) and a data station. The eluant (10% methanol in into a solution of IOXILAN (4 g, 2.5 mM) in DMSO (10 double distilled watm') was run at 0.8 ml/min. with UV mL) over a period of 30 minutes and stirred at 70° C. detection at 254 nm. Samples in HCl or NaOH were overnight. A catalytic amount of sodium methoxide was neutralized before injection. Plasma samples were ?rst added to facilitate the formation of cyclic carbonates. To treated with PCA (0.4 N) and centrifuged to remove pre terminate the reaction, the DMSO solution was diluted with cipitate proteins. The supematants were then injected for methylene chloride and washed with cold water. The meth 30 HPLC analysis. ylene chloride layer was dried over MgSO4 and evaporated Acute Toxicity to dryness to yield 2.2 g product (yield 50%). TLC (Silica, LD50 of IX-C particles was determined by injecting chloroformrmethanol, 10:1) indicated only one spot (Rf: diiferent volumes of particulate suspension (80 mg Ilml 0.75). IR (ICBr, cyclic carbonate): 1780 cm“. Mass spec 35 saline) into the tail veins of mice. Swiss Webster mice trum was determined by Fast Atom Bombardment (Kratos (Harlan Sprague Dawley Inc., Indianapolis, Ind.) weighing MS50, England) using nitrobenzyl alcohol as matrix mate between 25 and 30 g were given doses ranging from 0.2 to rial: MH+=870. Elemental analysis, calculated C:29.0%, 1 mllmouse. Five animals were used for each dose. No H:2.07%, N:4.83%, I:43.8%; found C:30.6%, H:2.47%, anesthesia was used for injection. Following the injection, N:4.63%, I:42.0%. Carbon-l3 NMR (DMSO-d6) revealed animals were monitored daily for 7 days. The percentage the presence of acetyl methyl carbon (22.3 ppm), aliphatic survival vs. dose curve was constructed to estimate LDSO. carbons (40.7 to 74.9 ppm, 8C), aromatic carbons (91.0, Computed Tomography Studies in Normal Rabbits 99.5, 100.4, 146.7, 151.0, and 151.9 ppm), and amide New Zealand white rabbits (male, 3.0 to 3.5 kg) were carbonyl carbons (167.3 to 170.2 ppm, 3C), representing the anesthetized by an intramuscular injection of a solution basic structure of IOXILAN. Cyclic carbonate carbons and containing xylazine (8.6 mgml), ketamine (42.9 mg/rnl), carbarnate carbon (148.6, 154.2, 154.5) were also present. and acepromazine (1.4 mg/ml) at a dose of 0.4 ml/kg for a Preparation of IX-C Particles long-lasting effect. Intravenous catheters (22-gauge) were IX-C particles were prepared by a solvent extraction/ placed in a marginal ear vein for the introduction of particle evaporation method. A solution of IX-C (3.0 g) in acetone suspension. Rabbits were positioned supine in a GE model (20 mL) and methylene chloride (60 mL) Was added to an 50 9800 Quick scanner (Milwaukee, Wis). The particulate aqueous solution of PVA (400 ml, 1%, w/v). The mixture contrast agent of proper volume (8% I, w/v in saline) was was emulsi?ed with an emulsi?er (Tekman, Germany) for 1 injected through the catheterized ear vein over a period of 10 minute and then stirred at 400 rpm for 4 hours to ensure to 15 minutes. complete removal of organic solvent. The resulting emulsion CI‘ imaging of radiopaque particles (80 mg Ilml) was was centrifuged at 3000 rpm, resuspended in distilled water, 55 carried out at doses of 100, 200, and 270 mg l/kg body ?ltered through a nylon ?lter (S-pm pore size), and centri weight respectively. Three rabbits were used for each dose fuged again. The process was repeated three times. Finally, level. CI‘ imaging was done with a scan speed of 1.0 the centrifuged product was resuspended in saline and seconds, 120 kV, 280 mAs, and a 25-cm ?eld of view. adjusted to proper volume for in vitro and in vivo testing. Sequential, contiguous 3- -thick slices through the abdo Physical Characterization of IX-C Particles men and S-mm-thick-slices through the pelvis were obtained Surface characteristics of the particles were evaluated before contrast injection, immediately after injection and at with a scanning electron microscope (Hitachi Model S520). various times (15 and 30 minutes, 1, 2, and 6 hours, and l, For sample preparation, microspheres were placed onto a 2, and 7 days after injection). The rabbits were killed with 0.1-um Nuclepore membrane, mounted onto stubs and an overdose of pentobarbital sodium (50 mglkg) adminis sputter-coated with 200A gold-palladium (80:20) in a Hum 65 tered via the catheterized ear vein. _ mer VI (Technics, Spring?eld, Va). The size distribution of Densitometric analysis of the liver, kidney, aorta, and IX-C particles was measured by light scattering with a bladder were performed. The density attenuation (HU) was 5,686,061 7 8 obtained from 10 areas of interest from at least three slices. but is labile towards basic solution. To test their hydrolytic To minimize the partial volume effect, care was taken to stability, lX-C particles were suspended in HCl. NaOH, ensure that no visible blood vessels were included in the area saline, and plasma solutions at 37° C. As expected, when of interest. Organ enhancement vs. time curves for each dose placed in NaOH solution, the lX-C particles were com administered were constructed to determine the pharmaco pletely dissolved within 1 horn‘. The degradation of lX-C lrinetic pro?les. particles in both HCl and saline solutions was much slower. Computed Tomography Studies of Rabbit Liver Bearing No gross changes in suspension appearance was observed VX2 Tumor ' during a 2-week period. However, the degradation did occur in both solutions as UV absorbance of the supernatants from Four New Zealand white rabbits (3.0 to 3.5 kg) were the lX-C suspension increased steadily over the incubation inoculated at a single site in the liver with a 0.5 cc suspen 10 period. As con?rmed by scanning electron microscopy, IX-C sion of minced VX2 tumor fragments (~10'5 cells). The VX2 started to crumble and disintegrate after being incubated in tumors were maintained through serial animal passage and saline for 2 weeks (FIG. 5). In plasma suspension, where pH were available from'l‘he University of Texas MD. Anderson is slightly acidic, lX-C particles were completely dissolved Cancer Center. in 6 days, indicating that an enzymatic e?’ect played a CI‘ scans were performed 5 days after inoculation. After 15 signi?cant role in the degradation of IX-C particles. preinjection scanning, IX-C particles (80 mg I/ml) of dose In order to determine the identity of lX-C degradation 200 mg I/kg body weight were injected intravenously and products, the supernatants of all samples were subjected to abdominal scans were performed immediately after injec reverse-phase HPLC analysis. All samples had a distinct tion and at 15, 30, 60, and 120 minutes after injection. The peak at 6.88 minutes. Standard IOXILAN had the same animals were killed after scanning. The livers were cut 20 retention time under the same analytical conditions. Thus, it transversely into slices of 2-3 mm to con?rm the size and appeared that the degradation of lX-C yielded IOXILAN location of the hepatic tumors. The attenuation of tumor and and carbon dioxide. To ascertain that the observed peak was the surrounding liver parenchyma were measured directly not an artifact from plasma component, the plasma samples from CI‘ scans. were also analyzed by FAB Mass spectroscopy. The pres Statistics 25 ence of IOXILAN was con?rmed by the molecular peak A P value less than 0.05 was considered to be signi?cant. (MH+) of IOXILAN at 792. An unpaired two-tailed Student’s t~test was used to compare Acute Toxicity liver attenuations between pre- and postcontrast groups. The LDso of IX-C particles determined with Swiss Web RESULTS 30 ster mice was 1.4 g I/kg body weight for males and 1.2 g I/kg body weight for females. The doses correspond to 3.1 and lX-C Synthesis 2.6 g/kg bodyweight lX-C respectively. The reaction scheme and the structure of the cyclic carbonate of IOXILAN obtained by reacting IOXILAN with Computed Tomography CD1 is shown in FIG. 1. The structure was con?rmed by Liver attenuation enhancement (AHU) is plotted as a Infrared spectroscopy (IR), mass spectroscopy, and elemen 35 function of time for three doses of lX-C particles (FIG. 6). tal analysis. Carbon-l3 NlVIR indicated the presence of Signi?cant attenuation enhancement of the liver was cyclic carbonate carbons. The spectrum was complicated by achieved over a period of 6 hours in a dose-dependent the existence of optical isomers conferred by the chiral manner. Following intravenous administration of 100, 200. carbons of the secondary alcohol and rotational isomers and 270 mg I/kg body weight of IX-C particles, maximum resulted from N-acetylated anilide nitrogens. liver CI‘ attenuation increases were 23, 38, and 110 respec Particle Preparation and Characterization tively. Liver attenuation reached maximum at approximately 30 minutes postinjection. At 270 mg I/kg body weight, the Di-C particles could be easily prepared by a solvent attenuation enhancement was much greater compared with extraction/evaporation process. Because IX~C has limited those of lower doses and reached maximum earlier. The solubility in methylene chloride, a cosolvent (acetone) is 45 attenuation enhancement persisted for 1 hour and started to necessary to facilitate lX-C solubilization. The presence of decrease at 2 hours postinjection. Liver attenuation water-soluble acetone in the organic phase resulted in rapid decreased to the preinjection value by 48 hours (FIG. 7). The phase separation because acetone was quickly extracted by increase in attenuation of the spleen was even more striking. the aqueous phase upon emulsi?cation. When acetone was Immediately after injection of 200 mg I/kg body weight of used alone, irregular particles were produced 50 radiopaque particles, the Houns?eld units increased from a lX-C particles thus prepared had an average diameter of precontrast level of 20 to 265 HU. The attenuation of the 1.1 pm. with 95% of them ranging between 0.6 and 2.0 pm spleen had reduced to 63 EU by 2 days postinjection. (number average) as determined by a submicron particle Gallbladder and bowel activity were observed at 6 hours analyzer (FIG. 2). The iodine content of the particles was postinjection (data not shown). 45%. Scanning electron microscopy revealed that the par 55 The CI‘ pharmacokinetics of aorta, kidney (cortex and ticles were spherical in shape and had smooth surfaces (FIG. medulla), and bladder following the injection of 200 mg I/kg 3). body weight of lX-C particles are presented as a histogram Suspension Stability in FIG. 7. ‘The attenuation of the aorta reached a maximum All lX-C particle formulations were stable. No particle immediately after injection (AHU 43) and decreased rapidly aggregation was observed either in saline or in 0.1% Tween to the preinjection level 1 hour after injection. IX-C or 80 solution. The lX-C particle suspensions were also stable metabolites of lX-C could be visualized in the kidney when mixed with rat plasma (FIG. 4), indicating that the immediately after injection. The kidney cortex attenuation interactions between the lX-C particles and blood compo reached maximum values of 94 HU at 2 hours postinjection, nents (e.g., ?brinogen) were which was 50 HU higher than that of preinjection value. The Hydrolytic and Enzymatic Degradability 65 kidney activity fell back to the preinjection level by 2 days Cyclic carbonate of 1,2-diol has been prepared as a means (FIG. 7). For all doses studied, attenuation changes of the to protect hydroxyl groups. It is stable in acidic condition, lungs were found to be negligible. 5,686,061 9 10 Computed Tomography of Rabbits Bearing VX2 'Ilumors of IX-C in saline and other lX-C formulations was investi The CI‘ imaging of a rabbit liver 6 days after tumor gated. IX-C particles were stable in saline with no tendency inoculation is shown in FIG. 8. The tumor was barely to ?occulate upon the addition of rat plasma (FIG. 4). detectable at any level before contrast injection (FIG. 8a). Administration of lX-C suspension in saline at concentration Immediately after the injection of 200 mg I/kg body weight 5 as high as 8% I (w/v) did not cause lung embolization in of lX-C particles, a tumor measuring 6-8 mm was clearly rabbits, con?rming the nonaggregation nature of lX-C par visible at the anterior-lateral portion of the right lobe (FIG. ticles. 8b). The visibility of the tumor persisted up to 2 hours The ability of IX-C particles to opacify the liver in rabbits (FIGS. 80 and 8d). The presence of the tumor was veri?ed was demonstrated in FIG. 6. The fact that the spleen was also by necropsy in exactly the same location. For all four highly opaci?ed con?rmed that the selective enhancement of rabbits, the average increases in liver and tumor attenuation the liver was due to macrophage uptake of the radiopaque were 39 and 4 HU respectively at 30 minutes after injection. particles. At a dose of only 100 mg I/kg body weight, IX-C These values re?ect an increase in the attenuation di?erence particles enhanced attenuation to a satisfactory level of 35 HU between the liver and the tumor. (AHU>20). Moreover, the attenuation enhancement per sisted for a period of 2 hours, allowing adequate time to DISCUSSION conduct CI‘ examination. Thus, radiopaque particles such as The goal of the foregoing experiments is to develop a lX-C overcome one of the disadvantages of water-soluble novel contrast agent that can be selectively delivered to the CM, namely, fast distribution to the interstitial space. RES and improve the detectability of liver lesions on CI‘ Pharmacokinetic data were obtained by measuring scans. The feasibility of using particulate CM as a hepatic 20 changes in the attenuation of various organs in the rabbits. macrophage imaging agent has been demonstrated. IX-C particles were rapidly cleared from the blood. Signi? However, adverse reactions often have been associated with cant enhancement of gallbladder attenuation and enhanced the administration of particulate CM, which has impeded its bowel activity at 6 hours postinjection, indicating that lX-C further development. One possible solution is to develop particles were cleared via the hepatobiliary system. This particulate CM that can be quickly degraded and cleared 25 observation is consistent with other particulate CM that also from the Kupffer cells and the liver. In this way, the impact produced increased gallbladder opacity. The relatively short of foreign particles on the function of the RES and the time (2 days) for the elimination of IX-C particles from the subsequent side reactions can be reduced to a minimum. liver was clearly demonstrated. Thus, the degradability of Among the methods used to develop particulate CM, the IX-C particles was con?rmed in vivo. 30 prodrug approach has the advantage of being easier to Surprisingly, lX-C particles were found to cause signi? prepare, less expensive, and having a higher iodine content cant kidney attenuation enhancement immediately after con on a weight basis. Since the degradation product is the trast injection (FIG. 7). This observation may be attributed original water-soluble CM, it is conceivable that radiopaque to the following. First. IX-C particles were quickly degraded particles made of a non-ionic contrast agent would cause 35 to water-soluble products. The observed kidney activity was less osmotic toxicity than ionic CM. due to the excretion of the resulting water-soluble CM. Based on the above considerations, a new iodinated Second, lX-C particles were caught in the tubule of the compound using IOXlLAN as the substrate was designed kidney. Although the exact cause of lX-C uptake in the Treatment of IOXILAN with CD1 in DMSO yielded cyclic kidney is not clear at present, metabolism and eventual carbonate and carbamate derivatives of IOXILAN, IX-C excretion of lX-C particles by the kidney pathway was (FIG. 1). This compound is soluble in acetone, is slightly clearly demonstrated. The bladder CT attenuation at 6 hours soluble in methylene chloride, and is insoluble in water. The after contrast injection was 240 HU higher than the precon lipid ‘soluble property of the IX-C compound allowed the trast level. I-IPLC analysis of urine samples taken at 2 hours easy preparation of IX-C particles by a solvent extraction! and 6 hours postinjection revealed the presence of the evaporation procedure. 45 degradation production IOXILAN. It was noted that the Because phagocytosis of foreign particles by the Kupffer liver attenuation increased at a much faster pace when the cells generally results in Kupifer cell activation and distur injected dose reached a certain level (270 mg I/kg body bance in the microcirculation of the liver (Li et al., unpub weight) (FIG. 6). This observation can also be explained by lished data). it is desirable that particulate CM designed for the saturation of the kidney elimination pathway, which macrophage imaging will quickly be cleared from the liver 50 resulted in more particles being redirected to the liver. after their functions are over. As shown in in vitro degra Therefore, unlike other previously reported radiopaque dation studies, lX-C particles were extremely unstable in particles, IX-C particles were eliminated via both the hepatic basic solutions. IX-C particle suspensions in saline at neutral and the urinary pathways. pH underwent a slow, yet de?nite degradation. Of interest is Toxicity of particulate CM has been a major concern. The the ability of IX-C particles to dissolve completely in rabbit 55 determined LD50 of lX-C of 1.4 and 1.2 g I/kg body weight plasma. This observation implies that various enzymes play corresponded to 3.1 and 2.6 g of IX-C/kg body weight for a signi?cant role in the dissolution of IX-C particles and will male and female mice respectively. These values are slightly be an important factor in the in vivo fate of IX-C particles. higher than those reported for other particulate CM. Since The degradation of lX-C particles produced IOIGLAN and the suspension used in this study was very concentrated (800 carbon dioxide, both of which are not expected to impose a mg Ilml), it is possible that the LDSO value would be higher signi?cant toxicity problem. if this suspension was diluted and injection was made in For the- particles to be ef?ciently taken up by the RES and several portions (to reduce the volume etfect). Using data able to pass through capillaries without causing from the CI‘ imaging study, one can predict that the diag embolization, they must have proper shape, size, and size nostic dose for Di-C is 100 mg I/kg body weight. This would distribution. Furthermore, interactions of plasma compo 65 give a safety margin of more than ten-fold. nents with small particles have to be minimized since they At a dose of 200 mg I/kg body weight, a tumor (6 mm in usually lead to particle aggregation. The suspension stability the smallest dimension) could be clearly detected in the 5,686,061 11 12 postcontrast images (FIG. 8). The tumor was not visible in emulsifying said mixture; and the precontrast image because it was either too small or removing said organic solvent to form said particulate isodense to liver parenchyma. Studies with rabbits bearing contrast agent. VX2 tumors demonstrated that IX-C particles could opacify 5. The method of claim 1 wherein said catalyst is a salt of the liver for about 2 hours without signi?cant reduction of 5 an alkyl oxide. . contrast enhancement, which allowed suf?cient time for CI‘ 6. The method of claim 3 wherein said catalyst is a salt of examinations. an alkyl oxide. The results showed that IX-C particles were 7. The method of claim 3 wherein said organic solvent biodegradable, with IOXILAN and carbon dioxide as the comprises acetone and methylene chloride. degradation products. The particles had an average size of 10 8. The method of claim 4 wherein said organic solvent 1-2 pm, and were stable in saline suspension. The LD5,0 determined for lX-C particles was 2.6 and 3.1 g/kg body comprises acetone and methylene chloride. weight for females and males respectively. A dose of 200 mg 9. The method of claim 3 wherein said emulsifying step Ilkg body weight caused an increase of 38 HU in liver comprises adding an emulsi?er selected from the group attenuation. In rabbit, hepatic clearance of the contrast consisting of polyvinyl alcohol, TWEEN 80, cellulose, and medium in 2 days was demonstrated. A tumor barely visible polyvinylpyrrolidone. in precontrast scans could be detected after contrast injec 10. The method of claim 4 wherein said emulsi?er com tion. prises polyvinyl alcohol. 11. The method of claim 1 wherein said radiopaque CONCLUSION 20 particulate contrast agent has the following structure: wherein Biodegradable IX-C particles have suitable physico chemical characteristics as a particulate CI‘ contrast agent, R comprises a radiopaque element; and are e?‘ective as a macrophage imaging agent. R1 is an amide group bonded to said aromatic ring at either the nitrogen or the carbon of the amide, ?ue The foregoing invention was explained with reference to 25 a particular embodiment. One skilled in the art will recog unbonded nitrogen or carbon having a substituent nize that many modi?cations may be made to the present selected from the group consisting of an aliphatic invention without departing from the spirit and scope of the vicinal diol and an aliphatic 1.3-diol; and invention. The embodiment described herein is meant to be R2 is selected from the group consisting of a radiopaque illustrative only and should not be taken as limiting the element, a hydrogen, an alkyl group having between invention. which is de?ned in the following claims. about 1-4 carbon atoms, and an amide group bonded to We claim: an aromatic carbon at either the nitrogen or the carbon 1. A method for producing a precursor for a radiopaque of the amide moiety, the unbonded nitrogen or carbon particulate contrast agent for selectively detecting lesions in of said amide moeity having a substituent selected from the liver and spleen. said contrast agent being biodegradable 35 the group consisting of hydrogen, an alkyl group hav into non-ionic by-products. said method comprising the ing between about l-3 carbon atoms, and a hydroxy steps of: lated aliphatic side chain having between about 1-8 providing a non-ionic, water soluble, radiopaque contrast carbon atoms. agent comprising an aromatic ring bonded to at least 12. The method of claim 11 wherein said radiopaque one radiopaque substituent and to at least one amide substituents comprise iodine. group at the carbon or the nitrogen of the amide moiety, 13. The method of claim 12 wherein at least two of said said amide group having a substituent selected from the R groups comprise iodine and at least one of said R2 groups group consisting of an aliphatic 1,3-diol and a vicinal comprises said amide group having a substituent selected diol; from the group consisting of an aliphatic vicinal diol and an 45 aliphatic 1,3-diol. thoroughly mixing said contrast agent with an activating 14. The method of claim 1 wherein said Water-soluble agent and a polar aprotic solvent in the presence of a catalyst; non-ionic contrast agent is selected from the group consist ing of IOHEXOL, IOPROMIDE, IOTROLAN, reacting said mixture for a time and at a temperature IOPAMIDOL, METRIZAMIDE, IOGLUNIDE, su?icient to form said precursor by changing said diol 50 IOGULAMJDE, and combinations thereof. substituent on said amide into a compound selected 15. The method of claim 3 wherein said water-soluble from the group consisting of a cyclic carbonate, a non-ionic contrast agent is selected from the group consist carbamate. and mixtures thereof; and ing of IOHEXOL, IOPROMIDE, IOTROLAN, separating said precursor from said polar aprotic solvent. IOPAMIDOL, METRIZAMIDE, IOGLUNIDE, 2. The method of claim 1 further comprising the step of 55 IOGULAMIDE, and combinations thereof. isolating a puri?ed form of said precursor. 16. The method of claim 4 wherein said water-soluble 3. The method of claim 1 further comprising the steps of non-ionic contrast agent is selected from the group consist mixing said precursor with an organic solvent; emulsifying ing of IOHEXOL, IOPROMIDE, IOTROLAN, said mixture; and removing said organic solvent to form said IOPAMIDOL, METRIZAMIDE, IOGLUNIDE, particulate contrast agent. IOGULAMIDE, and combinations thereof. 4. The method of claim 2 further comprising the steps of mixing said precursor with an organic solvent; *****