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Evaluation of N,N′-bis-dimethyldiatrizoic acid analogs as liver imaging agents
Evaluation of N,N'-bis-dimethyldiatrizoic Acid Analogs as Liver Imaging Agents' R. S. Ranganathan, T. Arunachalam, B. Song, S. Mantha M. Ogan, P. Wedeking, F. Yost, E. Jagoda, M. Tweedle
The detection of focal hepatic lesions by computed tomography using water-soluble ionic (ICM) or nonionic (NICM) contrast agents (CA) is a widely used radiological procedure (1). Limitations stemming from nonspecific tissue distribution and short residence time, which render small lesions undetectable, make this procedure less than optimal for hepatic imaging. Liver specificity and increased residence time provide agents of higher sensitivity (2), in addition to enabling the requisite longer imaging window. Examples of such agents that target the nonparenchymal (Kupffer) system are ethiodized-oil emulsions (3), iodinated starch particles (4), liposomat CA formulations (5), water-insoluble esters of ICM (6), and triiodinated PEG-based micelles (7). However, this approach is accompanied by unacceptable side effects (1) and hence has been abandoned. An alternative approach targeting the parenchymal (hepatocytes) system using polyiodinated triglycerides is currently under active investigation (8). In this paper we present another approach for the development of liver-specific agents by the hydrophobic modification of diatrizoic acid (la). Hydrophobic modification is known (9,10) to favor liver uptake by promoting plasma protein binding and thereby reducing kidney excretion. Amphophils, mimicking bile acids (11), are also taken up by hepatocytes by specific transport pathways. Our approach for the synthesis of hydrophobic and amphophilic diatrizoate derivatives consisted in N,N' bis-methylation to obtain N,N'-bis-
Acad Radio11998; 5(suppl 1):$23-$27 1From Bracco Research USA, 305 College Rd E, Princeton, NJ 08540.
Address reprint requests to R.S.R, © AUR, 1998
dimethyldiatrizoic acid (MDTA) (lb) or by N,N'-bis-methylation, followed by acylation with c0-aminophenylalkanoic acids, to obtain PMDTA derivatives 2 and 3. These modifications furnished low-molecular-weight (<1,000 Da) triiodobenzenoids with differing hydrophobicity and amphophilicity characteristics. Our results on the synthesis, physical properties, and tissue distribution of the compounds 1, 2, and 3 are described.
Synthesis The chemical structures of the CM candidates synthesized are given in Figure 1. MDTA (lb) was prepared from diatrizoic acid (la) as described (12). The fatty acid conjugates 2 were synthesized (Ranganathan RS, unpublished material) by amidation of MDTA acid chloride with the respective w-aminophenylalkanoic acid t-butyl esters. Compound 3 was prepared (Ranganathan RS, unpublished material) by the amidation of MDTA acid chloride with 9(10)- [(p-aminobenzoyl)amino]stearic acid methyl ester. All new compounds were unambiguously characterized by all applicable analytical and spectroscopic methods. The sodium and meglumine salts of MDTA (lb) were made by neutralizing the free acid with either sodium hydroxide or meglumine in aqueous solution.
Physical Studies Radiolabeling was achieved by the exchange-labeling (13) of the cold iodinated compounds with Na125Iin the presence of CuSO 4 and Na2S205 in glacial acetic acid, followed by HPLC purification. HPLC capacity factor logK' was determined on a Nucleosil C18 column (4.6 x 25 cm) using CH3CN/H:O (9:1) elution at 1 mL/min and detection at 254 nm. Hydrophobicity was evaluated by the parameter clogP, which was computed using the clogP for Win-
$23
o
.o.
H3C*~1~~ R I
0,~ ~L~(C CH 3
R
I"13 CH3 I
la:R=H; lb:R=CHa
I CH3
H2)nc OOH
CH 3
2a:n=l; 2b:n=3; 2c:n=6; 2d:n=10; 2e:n=14; 2f:n=18
. _ ~ ~ "(C Hz)n'COOH ]
0
I~_~1
0
L "(CH2 n-CH3 j
,r. CH3
I
CH3 3:11=7
Figure 1. Chemical structures of the diatrizoate analogs. dows program (BioByte, Claremont, Calif). Binding to bovine serum albumin (BSA) was determined by equilibrium dialysis in HEPES buffer using six different concentrations and Scatchard analysis.
Biological Studies Tissue distribution studies on all the compounds in Figure 1 were performed in male CD-1 mice using four animals per time period. The animals were injected intravenously with tracer levels (-0.3-0.6 nmol/kg; 0.1 mL) of the'radiolabeled CM candidate. They were sacrificed at 1', 5' and 60' post injection and the residual radioactivity measured in the various organs of interest. Higher dose tissue distribution studies (gmol/kg to mmol&g) were performed on selected candidates. In the case of the fatty acid analogs with poor water solubility, an aqueous vehicle consisting of 0.9% saline and 10% BSA was used to solubilize them. Acute tolerance studies on MDTA-NMG were conducted in male CD-1 mice at dose levels of 7.5, 10, and 15 mmol/kg using three mice for each dose. An aqueous solution (1.3 M) of the compound was injected intravenously into a tail vein at a rate of 0.0l mL/sec. The animals were examined for symptoms of adverse effects for a period of 7 days postinjection. Tissue distribution studies were also performed on MDTA (lb) in rats using four different doses and two'rats at each dose. The rats were anesthetized with sodium pentobarbital for the injections. The test compounds were administered into an exposed jugular
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Table 1 C A Candidates and Physical Properties
Compound
clogP
Diatrizoic acid (la) MDTA (1 b) PMDTA02 (2a) PMDTA04 (2b) PMDTA07 (2c) PMDTA11 (2d) PMDTA15 (2e) PMDTA19 (2f) 9(10)PMDTA (3)
-1.8 1.9 1.4 2.2 3.8 5.9 8,1 10,2 7.7
HPLC log K'
~],21 ~].07 0.46
vein at a dose volume of 1.0 mL/kg. The animals were sacrificed at 5' and 15' postinjection and selected tissues were assayed for residual radioactivity.
A total of eight CA candidates were made. These are listed along with their clogP and HPLC log K" values in Table 1. Physical Studies The solubility of the long chain fatty acid conjugates 2 a - f and 3 in water was poor, requiring solubilization by 10% BSA at concentrations in the 30 HM to 0.75 mM
Vol 5, Suppl I, April 1998
EVALUATION OF N,N'-BIS-DIMETHYLDIATRIZOIC A C I D A N A L O G S
Table 2 Distribution of Radiolabeled Analogs in Mouse Tissuesat 5' (%lD/Organ)* Compound Diatrizoic a c i d ( l a ) * MDTA ( l b ) PMDTA02 (2a) PMDTA04 (2b) PMDTA07 (2c) PMDTA11 (2d) PMDTA15 (2e) PMDTA19 (2f) 9(10)PMDTA (3)
Liver 8.6 + 0.6 30.9 + 2.6 3t.7+2.0 67.2 + 1.0 68.7+3.2 82.4+2.3 71.9 + 1.4 35.8+ 11.2 80.1 + 1.2
Blood Poop 9.9 _+0.9 3,4 + 0.4 3,2+0.4 1.8 + 0.2 1,3+0.2 1.3+0.6 6.6 + 0.4 29.1 + 8 . 9 1.6 + 0.1
Kidney
Intestine t
4.5 + 0.6 12.7 + 1.4 4.4+0.1 3.8 + 0.2 1.4+0.1 1.1 + 0 . 2 1.7 + 0.3 2.5+0.8 1.7 + 0
3.6 + 1.2 46.0 + 2.6 83.1 + 2 . 6 80.6 + 2.3 80.7+4.1 91.6+ 1.3 82.6 + 6.6 43.8+ 11.9 85.3 + 2.2
* P e r c e n t a g e of t h e injected label present in t h e o r g a n a t t i m e of sacrifice. t Blood p o o l v o l u m e f a c t o r used was 78 mL/kg b o d y weight. t Values a t 60'.
range. Above this range only emulsions resulted with or without BSA. MDTA (lb), on the other hand, was highly water soluble, the sodium and meglumine salts exhibiting solubilities of 0.35 and 1.30 M, respectively, at room temperature. The corresponding values for diatrizoic acid (la) are reported (14) to be 0.94 and 1.10 M, respectively. In order to evaluate the validity of the hydrophobic parameter clogP, the calculated values were correlated with the experimentally determined hydrophobicity parameter, HPLC log K', for three of the analogs, 2d, 2e, and 2f. A good linear correlation (r 2 = .9) was observed, indicating that the clogP values could be assumed to be a reliable measure of the hydrophobicity for all the analogs synthesized. The clogP values as expected increased with hydrophobic substitution. The seemingly anomalous lower clogP values obtained for 2a and 3, in comparison to 1 and 2f, respectively, are due to the presence of the highly hydrophilic amide group linker that is not present in the two latter compounds. BSA Binding BSA binding of the compounds 2d and 2e was studied. While the method failed in the case 2e due to nonspecific binding, a K value of 2.9 x 106 M -1 was found for 2d. This compares well with the values of 0.2 x 106 and 1.3 x 106 reported for dodecanoic acid (15) and c0-dansylaminododecanoic acid (16), respectively. Biological Studies Acute tolerance study of MDTA (lb) in mice.--At 7.5 mmol/kg, no symptoms were observed. At 10 mmol/kg,
ataxia, slight tremors, increased respiratory rate, and peripheral dilation of blood vessels were observed. These disappeared within 30" postinjection. All mice maintained normal body weights during the 7-day observation period. At the highest dose of 15 mmol/kg, one of three mice died, while the surviving mice behaved analogous to the mice administered 10 mmol/kg. It is concluded that the LDs0 of MDTA (lb) is -15 mmol/kg, comparable to the reported (14) value of 14.2 mmol/kg for diatrizoic acid (la). Tissue distribution studies at tracer dose.--Results of the tissue distribution studies in mice at tracer level at 5' are given in Table 2 for liver, blood, and kidney. Distributions in the intestine at 60' are also listed. The corresponding values for diatrizoic acid (la) are also listed for comparison. It is seen from the data in Table 2 that, upon N,N'-bismethylation of diatrizoic acid, liver uptake increased by a factor of 3.6 and that upon further amidation with o)-aminophenylalkanoic acids it further increased substantially. The increase in liver uptake with chain length reaches a maximum of 82% at C-11 for 2d. The decreased uptake of the lower analogs is attributed to competing renal excretion, while that of the higher analogs is ascribable to longer blood retention. Amphophils 2a and 3 mimicked the behavior of the lower homologues lb and 2d, respectively, indicating that introduction of hydrophilic moieties such as an amide linker in the fatty acid chain has the effect of abolishing the hydrophobic effect of the rest of the lipophilic chain. The clogP values actually predict such an outcome. In order to evaluate the fate of the material taken up by the liver, the liver value at 5" was plotted against the
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intestine value at 60'. A good linear correlation (r: = .98) was obtained, allowing the conclusion that the material taken up by the liver is quantitatively excreted via the hepatobiliary system. Tissue distribution studies at higher d o s e . - - W e next carried out higher dose studies on MDTA (lb) and the fatty acid conjugate 2e to evaluate whether at or below saturation levels there would be enough iodine concentration to enable x-ray CT imaging of the liver. The approximate minimum concentration of iodine needed to attain barely visible x-ray opacification is - 1 0 mM based on published work (17). Figure 2 depicts the results of these studies in mice. It is seen from Figure 2 that in the case of the fatty acid-conjugate 2e the minimum imaging concentration is not reached at the BSA solubilized doses of 0.1, 1.0, or 2.5 mol I/kg or at the suspension dose of 100 gmol I/kg. The study with suspensions was also extended to analog 2c. The suspensions behaved differently from the solubilized preparations. Liver distributions were lower (52.5% for 2e at 5" postinjection) though the absolute concentrations were higher. The residence times in the liver and blood were prolonged, suggesting that the in vivo treatment of these emulsions may be more like that of chylomicrons than that of free fatty acids. The highly water-soluble MDTA (lb), on the other hand, approached the threshold iodine concentration of 10 mM at dose levels close to 1 mmol I/kg at 15' postinjection. Additional studies with higher dose levels indicated that the minimum imaging concentration is reached at doses of 1-3 mmol I/kg and that liver saturation levels are reached at doses of 15-22.5 mmol I/kg yielding tissue iodine concentrations of -30 mM at 15' postinjection. A similar study in which rats were injected at 1.5, 3.0, 15.0, and 30.0 mmol I/kg revealed that the liver distributions at 15' were lower than in mice, the minimum imaging concentration being reached only at dose levels >15 mmol I/ kg. Unlike the mice, there was no indication of liver saturation in the dose range studied. CONCLUSION N,N'-bis-methylation of diatrizoic acid (la) gave MDTA (lb) which exhibited 3.6-fold higher uptake (30.6%) in liver than diatrizoic acid (8.6%) at 5' postinjection, while maintaining similar water solubility and acute tolerance profiles. Further conjugation of MDTA (lb) with (0-aminophenylalkanoic acids led to significantly higher liver distribution, which peaks (82.4% at 5' post-
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15" 14" 13" 12" 11" Minimum Imaging Concentration 10" 9" 8" [I] (mM) 7" 6" 5" MDTA |b) 4" Suspension---~ , . 3" ........... PMDTA15~[e) in10% BSA 2" 1" ................... O - - ' O ' " ' 0" r -1 0.01 0.1 1 10 ID (pmollKg)
! _~_~
/ / . L T , - ~
L /
i
Figure 2.
*
i
100
100~
E f f e c t o f d o s e o n liver distribution in m i c e a t 5'.
injection) at the chain length of 11 in the case of compound 2d. Interruption of amphophilicity by hydrophilic substitution at the middle of the fatty acid chain, as in compound 3, renders the analog to behave as a lower homologue in tissue distribution studies as predicted by the hydrophilicity parameter clogP. The fatty acid conjugates suffer from low water solubility, and the minimum imaging concentration for iodine is not reached even with the use of suspension formulations. The suspensions behave differently from the soluble formulations in having lower liver distribution (52.5% for 2e at 5" postinjection) and longer residence times. MDTA (lb), on the other hand, affords the minimum imaging concentration for iodine in the dose range - 1 - 3 mmol I/kg in mice. The bimodal clearance profile of MDTA (lb) could be an advantage in subjects with impaired hepatic function. It is concluded that MDTA (lb) has the potential to serve as a liver-specific xray CT imaging agent and that further evaluation of this molecule is warranted. EFERENCE.¢ 1, Leander P. Liver specific contrast media for MRI and CT experimental studies, A c t a Radio11995; 396(suppl):1-36, 2. Ivancev K, Lunderquist A, Isaksson A, Hochbergs P, Wretlind A. Clinical trials with a new iodinated lipid emulsion for c o m p u t e d t o m o g r a p h y of the liver, A c t a Radio11989; 30:449-457, 3. Bhalffacharya S, Dhillon AP, Winslet MC, et al. Human liver cancer cells and endothelial cells incorporate iodised oil. Br J Cancer 1996; 73:877-881. 4. Cohen Z, Seltzer SE, Davis MA, Hanson RN. Iodinated starch particles: new contrast material for c o m p u t e d t o m o g r a p h y of the liver, J C o m p u t Assist Tomogr 1981; 5:843-836. 5. Krause W, Leike J, Schuhmann-Giampieri G, Sachse A, Schmiedl U, Strunk H. Iopremide carrying liposomes as a contrast a g e n t for the liver, A c a d Radio11996; 3:S235-$237. 6. Violante MR, Mare K, Fischer HW. Biodistribution of a particulate h e p a t o g r a p h i c CT contrast agent: a study of iodipamide ethyl ester in the rat, Invest Radio11981; 16:40-45, 7, TorchiIin VP, Trubetskoy VS. Amphiphilic polyethylene glycol de-
8.
9.
10.
11.
12.
rivatives: long-circulating micellar carriers for therapeutic and diagnostic agents. Polym Preprints 1997; 38:545-546, Weichert JP, Lee FT Jr, Longino MA, et al. Computed tomography scanning of hepatic tumors with polyiodinated triglycerides. Acad Radio11996; 3:$229-$231. Urich VG, Speck U. Biliary excretion of contrast media. In: Siegers CP, Watkings JB Ill, eds. Progr. Pharm. Clin. Pharm.: Biliary excretion of drugs and other chemicals. New York, NY: Gustav Fischer, 1991; 8(4):307-322. Schuhmann-GiampieriG. Liver contrast media for magnetic resonance imaging: interrelations between pharmacokinetics and imaging. Invest Radio11993; 28:753-761. Anelli PL, Calabi L, de Haen C, Laffuada L, Lorusso V. Hepatocyte-directed MRI agents: can we take a d v a n t a g e of bile acids? Acta Radiol (in press). Holtermann H, Haugen, LG, Nordal V, Haavaldsen JL. Process for the N-alkylation of acyl anilides halogen substituted in the nucleus. US patent 3,178,473 (Apr. 13, 1965).
13. Neves M, Paulo A, Patricio L. A kit formulation of [~3~l)metaiodobenzylguanidine (MIBG) using Cu(I) generated "in situ" by sodium disulphite. Appl Radiat Isot 1992; 43:737-740. 14. Hoey GB, Smith KR. Chemistry of X-ray contrast media. In: Sovak M, ed. Radiocontrast agents. New York, NY: Springer-Verlag, 1984; 23-125. 15. Koh SM, Means GE. Characterization of a small apolar binding site of human serum albumin. Arch Biochem Biophys 1979; 192:73-79. 16. Wilton DC. The fatty acid analogue 11-(dansylamino)undecanoic acid is a fluorescent probe for the bilirubin binding sites of albumin and not for the high affinity fatty acid-binding sites. Biochem J 1990; 270:163-166. 17. Janower ML, Lundstrom P. A JR 1981; 136:515-516.
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The detection of focal hepatic lesions by computed tomography using water-soluble ionic (ICM) or nonionic (NICM) contrast agents (CA) is a widely used radiological procedure (1). Limitations stemming from nonspecific tissue distribution and short residence time, which render small lesions undetectable, make this procedure less than optimal for hepatic imaging. Liver specificity and increased residence time provide agents of higher sensitivity (2), in addition to enabling the requisite longer imaging window. Examples of such agents that target the nonparenchymal (Kupffer) system are ethiodized-oil emulsions (3), iodinated starch particles (4), liposomat CA formulations (5), water-insoluble esters of ICM (6), and triiodinated PEG-based micelles (7). However, this approach is accompanied by unacceptable side effects (1) and hence has been abandoned. An alternative approach targeting the parenchymal (hepatocytes) system using polyiodinated triglycerides is currently under active investigation (8). In this paper we present another approach for the development of liver-specific agents by the hydrophobic modification of diatrizoic acid (la). Hydrophobic modification is known (9,10) to favor liver uptake by promoting plasma protein binding and thereby reducing kidney excretion. Amphophils, mimicking bile acids (11), are also taken up by hepatocytes by specific transport pathways. Our approach for the synthesis of hydrophobic and amphophilic diatrizoate derivatives consisted in N,N' bis-methylation to obtain N,N'-bis-
Acad Radio11998; 5(suppl 1):$23-$27 1From Bracco Research USA, 305 College Rd E, Princeton, NJ 08540.
Address reprint requests to R.S.R, © AUR, 1998
dimethyldiatrizoic acid (MDTA) (lb) or by N,N'-bis-methylation, followed by acylation with c0-aminophenylalkanoic acids, to obtain PMDTA derivatives 2 and 3. These modifications furnished low-molecular-weight (<1,000 Da) triiodobenzenoids with differing hydrophobicity and amphophilicity characteristics. Our results on the synthesis, physical properties, and tissue distribution of the compounds 1, 2, and 3 are described.
Synthesis The chemical structures of the CM candidates synthesized are given in Figure 1. MDTA (lb) was prepared from diatrizoic acid (la) as described (12). The fatty acid conjugates 2 were synthesized (Ranganathan RS, unpublished material) by amidation of MDTA acid chloride with the respective w-aminophenylalkanoic acid t-butyl esters. Compound 3 was prepared (Ranganathan RS, unpublished material) by the amidation of MDTA acid chloride with 9(10)- [(p-aminobenzoyl)amino]stearic acid methyl ester. All new compounds were unambiguously characterized by all applicable analytical and spectroscopic methods. The sodium and meglumine salts of MDTA (lb) were made by neutralizing the free acid with either sodium hydroxide or meglumine in aqueous solution.
Physical Studies Radiolabeling was achieved by the exchange-labeling (13) of the cold iodinated compounds with Na125Iin the presence of CuSO 4 and Na2S205 in glacial acetic acid, followed by HPLC purification. HPLC capacity factor logK' was determined on a Nucleosil C18 column (4.6 x 25 cm) using CH3CN/H:O (9:1) elution at 1 mL/min and detection at 254 nm. Hydrophobicity was evaluated by the parameter clogP, which was computed using the clogP for Win-
$23
o
.o.
H3C*~1~~ R I
0,~ ~L~(C CH 3
R
I"13 CH3 I
la:R=H; lb:R=CHa
I CH3
H2)nc OOH
CH 3
2a:n=l; 2b:n=3; 2c:n=6; 2d:n=10; 2e:n=14; 2f:n=18
. _ ~ ~ "(C Hz)n'COOH ]
0
I~_~1
0
L "(CH2 n-CH3 j
,r. CH3
I
CH3 3:11=7
Figure 1. Chemical structures of the diatrizoate analogs. dows program (BioByte, Claremont, Calif). Binding to bovine serum albumin (BSA) was determined by equilibrium dialysis in HEPES buffer using six different concentrations and Scatchard analysis.
Biological Studies Tissue distribution studies on all the compounds in Figure 1 were performed in male CD-1 mice using four animals per time period. The animals were injected intravenously with tracer levels (-0.3-0.6 nmol/kg; 0.1 mL) of the'radiolabeled CM candidate. They were sacrificed at 1', 5' and 60' post injection and the residual radioactivity measured in the various organs of interest. Higher dose tissue distribution studies (gmol/kg to mmol&g) were performed on selected candidates. In the case of the fatty acid analogs with poor water solubility, an aqueous vehicle consisting of 0.9% saline and 10% BSA was used to solubilize them. Acute tolerance studies on MDTA-NMG were conducted in male CD-1 mice at dose levels of 7.5, 10, and 15 mmol/kg using three mice for each dose. An aqueous solution (1.3 M) of the compound was injected intravenously into a tail vein at a rate of 0.0l mL/sec. The animals were examined for symptoms of adverse effects for a period of 7 days postinjection. Tissue distribution studies were also performed on MDTA (lb) in rats using four different doses and two'rats at each dose. The rats were anesthetized with sodium pentobarbital for the injections. The test compounds were administered into an exposed jugular
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Table 1 C A Candidates and Physical Properties
Compound
clogP
Diatrizoic acid (la) MDTA (1 b) PMDTA02 (2a) PMDTA04 (2b) PMDTA07 (2c) PMDTA11 (2d) PMDTA15 (2e) PMDTA19 (2f) 9(10)PMDTA (3)
-1.8 1.9 1.4 2.2 3.8 5.9 8,1 10,2 7.7
HPLC log K'
~],21 ~].07 0.46
vein at a dose volume of 1.0 mL/kg. The animals were sacrificed at 5' and 15' postinjection and selected tissues were assayed for residual radioactivity.
A total of eight CA candidates were made. These are listed along with their clogP and HPLC log K" values in Table 1. Physical Studies The solubility of the long chain fatty acid conjugates 2 a - f and 3 in water was poor, requiring solubilization by 10% BSA at concentrations in the 30 HM to 0.75 mM
Vol 5, Suppl I, April 1998
EVALUATION OF N,N'-BIS-DIMETHYLDIATRIZOIC A C I D A N A L O G S
Table 2 Distribution of Radiolabeled Analogs in Mouse Tissuesat 5' (%lD/Organ)* Compound Diatrizoic a c i d ( l a ) * MDTA ( l b ) PMDTA02 (2a) PMDTA04 (2b) PMDTA07 (2c) PMDTA11 (2d) PMDTA15 (2e) PMDTA19 (2f) 9(10)PMDTA (3)
Liver 8.6 + 0.6 30.9 + 2.6 3t.7+2.0 67.2 + 1.0 68.7+3.2 82.4+2.3 71.9 + 1.4 35.8+ 11.2 80.1 + 1.2
Blood Poop 9.9 _+0.9 3,4 + 0.4 3,2+0.4 1.8 + 0.2 1,3+0.2 1.3+0.6 6.6 + 0.4 29.1 + 8 . 9 1.6 + 0.1
Kidney
Intestine t
4.5 + 0.6 12.7 + 1.4 4.4+0.1 3.8 + 0.2 1.4+0.1 1.1 + 0 . 2 1.7 + 0.3 2.5+0.8 1.7 + 0
3.6 + 1.2 46.0 + 2.6 83.1 + 2 . 6 80.6 + 2.3 80.7+4.1 91.6+ 1.3 82.6 + 6.6 43.8+ 11.9 85.3 + 2.2
* P e r c e n t a g e of t h e injected label present in t h e o r g a n a t t i m e of sacrifice. t Blood p o o l v o l u m e f a c t o r used was 78 mL/kg b o d y weight. t Values a t 60'.
range. Above this range only emulsions resulted with or without BSA. MDTA (lb), on the other hand, was highly water soluble, the sodium and meglumine salts exhibiting solubilities of 0.35 and 1.30 M, respectively, at room temperature. The corresponding values for diatrizoic acid (la) are reported (14) to be 0.94 and 1.10 M, respectively. In order to evaluate the validity of the hydrophobic parameter clogP, the calculated values were correlated with the experimentally determined hydrophobicity parameter, HPLC log K', for three of the analogs, 2d, 2e, and 2f. A good linear correlation (r 2 = .9) was observed, indicating that the clogP values could be assumed to be a reliable measure of the hydrophobicity for all the analogs synthesized. The clogP values as expected increased with hydrophobic substitution. The seemingly anomalous lower clogP values obtained for 2a and 3, in comparison to 1 and 2f, respectively, are due to the presence of the highly hydrophilic amide group linker that is not present in the two latter compounds. BSA Binding BSA binding of the compounds 2d and 2e was studied. While the method failed in the case 2e due to nonspecific binding, a K value of 2.9 x 106 M -1 was found for 2d. This compares well with the values of 0.2 x 106 and 1.3 x 106 reported for dodecanoic acid (15) and c0-dansylaminododecanoic acid (16), respectively. Biological Studies Acute tolerance study of MDTA (lb) in mice.--At 7.5 mmol/kg, no symptoms were observed. At 10 mmol/kg,
ataxia, slight tremors, increased respiratory rate, and peripheral dilation of blood vessels were observed. These disappeared within 30" postinjection. All mice maintained normal body weights during the 7-day observation period. At the highest dose of 15 mmol/kg, one of three mice died, while the surviving mice behaved analogous to the mice administered 10 mmol/kg. It is concluded that the LDs0 of MDTA (lb) is -15 mmol/kg, comparable to the reported (14) value of 14.2 mmol/kg for diatrizoic acid (la). Tissue distribution studies at tracer dose.--Results of the tissue distribution studies in mice at tracer level at 5' are given in Table 2 for liver, blood, and kidney. Distributions in the intestine at 60' are also listed. The corresponding values for diatrizoic acid (la) are also listed for comparison. It is seen from the data in Table 2 that, upon N,N'-bismethylation of diatrizoic acid, liver uptake increased by a factor of 3.6 and that upon further amidation with o)-aminophenylalkanoic acids it further increased substantially. The increase in liver uptake with chain length reaches a maximum of 82% at C-11 for 2d. The decreased uptake of the lower analogs is attributed to competing renal excretion, while that of the higher analogs is ascribable to longer blood retention. Amphophils 2a and 3 mimicked the behavior of the lower homologues lb and 2d, respectively, indicating that introduction of hydrophilic moieties such as an amide linker in the fatty acid chain has the effect of abolishing the hydrophobic effect of the rest of the lipophilic chain. The clogP values actually predict such an outcome. In order to evaluate the fate of the material taken up by the liver, the liver value at 5" was plotted against the
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intestine value at 60'. A good linear correlation (r: = .98) was obtained, allowing the conclusion that the material taken up by the liver is quantitatively excreted via the hepatobiliary system. Tissue distribution studies at higher d o s e . - - W e next carried out higher dose studies on MDTA (lb) and the fatty acid conjugate 2e to evaluate whether at or below saturation levels there would be enough iodine concentration to enable x-ray CT imaging of the liver. The approximate minimum concentration of iodine needed to attain barely visible x-ray opacification is - 1 0 mM based on published work (17). Figure 2 depicts the results of these studies in mice. It is seen from Figure 2 that in the case of the fatty acid-conjugate 2e the minimum imaging concentration is not reached at the BSA solubilized doses of 0.1, 1.0, or 2.5 mol I/kg or at the suspension dose of 100 gmol I/kg. The study with suspensions was also extended to analog 2c. The suspensions behaved differently from the solubilized preparations. Liver distributions were lower (52.5% for 2e at 5" postinjection) though the absolute concentrations were higher. The residence times in the liver and blood were prolonged, suggesting that the in vivo treatment of these emulsions may be more like that of chylomicrons than that of free fatty acids. The highly water-soluble MDTA (lb), on the other hand, approached the threshold iodine concentration of 10 mM at dose levels close to 1 mmol I/kg at 15' postinjection. Additional studies with higher dose levels indicated that the minimum imaging concentration is reached at doses of 1-3 mmol I/kg and that liver saturation levels are reached at doses of 15-22.5 mmol I/kg yielding tissue iodine concentrations of -30 mM at 15' postinjection. A similar study in which rats were injected at 1.5, 3.0, 15.0, and 30.0 mmol I/kg revealed that the liver distributions at 15' were lower than in mice, the minimum imaging concentration being reached only at dose levels >15 mmol I/ kg. Unlike the mice, there was no indication of liver saturation in the dose range studied. CONCLUSION N,N'-bis-methylation of diatrizoic acid (la) gave MDTA (lb) which exhibited 3.6-fold higher uptake (30.6%) in liver than diatrizoic acid (8.6%) at 5' postinjection, while maintaining similar water solubility and acute tolerance profiles. Further conjugation of MDTA (lb) with (0-aminophenylalkanoic acids led to significantly higher liver distribution, which peaks (82.4% at 5' post-
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15" 14" 13" 12" 11" Minimum Imaging Concentration 10" 9" 8" [I] (mM) 7" 6" 5" MDTA |b) 4" Suspension---~ , . 3" ........... PMDTA15~[e) in10% BSA 2" 1" ................... O - - ' O ' " ' 0" r -1 0.01 0.1 1 10 ID (pmollKg)
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injection) at the chain length of 11 in the case of compound 2d. Interruption of amphophilicity by hydrophilic substitution at the middle of the fatty acid chain, as in compound 3, renders the analog to behave as a lower homologue in tissue distribution studies as predicted by the hydrophilicity parameter clogP. The fatty acid conjugates suffer from low water solubility, and the minimum imaging concentration for iodine is not reached even with the use of suspension formulations. The suspensions behave differently from the soluble formulations in having lower liver distribution (52.5% for 2e at 5" postinjection) and longer residence times. MDTA (lb), on the other hand, affords the minimum imaging concentration for iodine in the dose range - 1 - 3 mmol I/kg in mice. The bimodal clearance profile of MDTA (lb) could be an advantage in subjects with impaired hepatic function. It is concluded that MDTA (lb) has the potential to serve as a liver-specific xray CT imaging agent and that further evaluation of this molecule is warranted. EFERENCE.¢ 1, Leander P. Liver specific contrast media for MRI and CT experimental studies, A c t a Radio11995; 396(suppl):1-36, 2. Ivancev K, Lunderquist A, Isaksson A, Hochbergs P, Wretlind A. Clinical trials with a new iodinated lipid emulsion for c o m p u t e d t o m o g r a p h y of the liver, A c t a Radio11989; 30:449-457, 3. Bhalffacharya S, Dhillon AP, Winslet MC, et al. Human liver cancer cells and endothelial cells incorporate iodised oil. Br J Cancer 1996; 73:877-881. 4. Cohen Z, Seltzer SE, Davis MA, Hanson RN. Iodinated starch particles: new contrast material for c o m p u t e d t o m o g r a p h y of the liver, J C o m p u t Assist Tomogr 1981; 5:843-836. 5. Krause W, Leike J, Schuhmann-Giampieri G, Sachse A, Schmiedl U, Strunk H. Iopremide carrying liposomes as a contrast a g e n t for the liver, A c a d Radio11996; 3:S235-$237. 6. Violante MR, Mare K, Fischer HW. Biodistribution of a particulate h e p a t o g r a p h i c CT contrast agent: a study of iodipamide ethyl ester in the rat, Invest Radio11981; 16:40-45, 7, TorchiIin VP, Trubetskoy VS. Amphiphilic polyethylene glycol de-
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rivatives: long-circulating micellar carriers for therapeutic and diagnostic agents. Polym Preprints 1997; 38:545-546, Weichert JP, Lee FT Jr, Longino MA, et al. Computed tomography scanning of hepatic tumors with polyiodinated triglycerides. Acad Radio11996; 3:$229-$231. Urich VG, Speck U. Biliary excretion of contrast media. In: Siegers CP, Watkings JB Ill, eds. Progr. Pharm. Clin. Pharm.: Biliary excretion of drugs and other chemicals. New York, NY: Gustav Fischer, 1991; 8(4):307-322. Schuhmann-GiampieriG. Liver contrast media for magnetic resonance imaging: interrelations between pharmacokinetics and imaging. Invest Radio11993; 28:753-761. Anelli PL, Calabi L, de Haen C, Laffuada L, Lorusso V. Hepatocyte-directed MRI agents: can we take a d v a n t a g e of bile acids? Acta Radiol (in press). Holtermann H, Haugen, LG, Nordal V, Haavaldsen JL. Process for the N-alkylation of acyl anilides halogen substituted in the nucleus. US patent 3,178,473 (Apr. 13, 1965).
13. Neves M, Paulo A, Patricio L. A kit formulation of [~3~l)metaiodobenzylguanidine (MIBG) using Cu(I) generated "in situ" by sodium disulphite. Appl Radiat Isot 1992; 43:737-740. 14. Hoey GB, Smith KR. Chemistry of X-ray contrast media. In: Sovak M, ed. Radiocontrast agents. New York, NY: Springer-Verlag, 1984; 23-125. 15. Koh SM, Means GE. Characterization of a small apolar binding site of human serum albumin. Arch Biochem Biophys 1979; 192:73-79. 16. Wilton DC. The fatty acid analogue 11-(dansylamino)undecanoic acid is a fluorescent probe for the bilirubin binding sites of albumin and not for the high affinity fatty acid-binding sites. Biochem J 1990; 270:163-166. 17. Janower ML, Lundstrom P. A JR 1981; 136:515-516.
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