REDUCED METASTABLE COMPLEX MACROCYCLIC CONTRAST AGENTS

Abstract

Gadolinium based contrast agents (GCA) incorporating linear ligand chelation are fundamentally different from GCAs incorporating macrocyclic ligands. The macrocyclic GCAs are synthesized by pathways characterized by the formation of a sequence of metastable complexes before obtaining the final stable complex. The synthesis of linear GCAs do not form metastable complexes. Commercial macrocyclic GCAs contain unstable metastable complexes. These metastable species are not regulated and quickly release free Gd3+ ions upon administration into the body. Gadolinium based contrast agents with near zero metastable species content and methods of synthesizing the same are disclosed. Gadolinium based contrast agents with long dissociation time in the body, and low free Gd3+ ion formation are obtained using a synthesis method which departs in novel ways from the traditional free Gd3+-based synthesis methods.

Claims

1. A contrast agent for enhancing a biologic image obtained by magnetic resonance imaging (MRI), the contrast agent being in an aqueous solution for injection, the contrast agent comprising: a plurality of Gd-DOTA complexes and meglumine; and at most 100 ppm of free Gd.sup.3+ ion, wherein greater than 90% of the Gd-DOTA complexes are maximally stable complexes.

2. The contrast agent of claim 1, wherein the maximally stable complexes consist of one water molecule in the coordination sphere.

3. The contrast agent of claim 1, wherein the contrast agent is metastable free such that the contrast agent has less than 1 metastable complex for every 10.sup.6 maximally stable complexes.

4. The contrast agent of claim 3, wherein the metastable complexes comprise four carboxylates coordinated to the Gd.sup.3+ ion and 4 or 5 water molecules in a coordination sphere of the complex.

5. The contrast agent of claim 1, comprising: 3.7 ppm free Gd3+ ion or less after 30 minutes in vitro.

6. The contrast agent of claim 5, comprising: 2.9 ppm free Gd3+ ion or less after 30 minutes in vitro.

7. The contrast agent of claim 6, comprising: 2.1 ppm free Gd3+ ion or less after 30 minutes in vitro.

8. The contrast agent of claim 1, comprising: 35.5 ppm free Gd3.sup.+ ion or less after 30 minutes in vivo.

9. The contrast agent of claim 8, comprising: 34.8 ppm free Gd3+ ion or less after 30 minutes in vivo.

10. The contrast agent of claim 9, comprising: 34.1 ppm free Gd3+ ion or less after 30 minutes in vivo.

11. The contrast agent of claim 1, comprising: 3.7 ppm free Gd3+ ion or less after 30 minutes in vitro and 35.5 ppm free Gd3+ ion or less after 30 minutes in vivo.

12. A method for synthesizing a gadolinium-based contrast agent, the method comprising: performing a complexation reaction between Gd3+ and DOTA; deprotonation of the [Gd—H(dota)] or [Gd—H.sub.2(dota)].sup.+ metastables; and adding meglumine after verification of full complexification.

13. The method of claim 12, wherein the deprotonation of metastables is by hydroxide insertion.

14. The method of claim 12, wherein the deprotonation of metastables is by nitrogen inversion.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0081] FIG. 1 provides a schematic of the dynamical proton spin during the MRI process.

[0082] FIG. 2 depicts the gadoterate meglumine (Gd-dota) coordination complex.

[0083] FIG. 3 depicts the gadolinium coordination sphere.

[0084] FIG. 4 depicts the hydrated ligand H2DOTA2−.

[0085] FIG. 5 depicts the metastable complex [Gd—H.sub.2(dota)]+.

[0086] FIG. 6 illustrates tetraethyl-substituted ligand H4Et4dota.

[0087] FIG. 7 illustrates tetramethyl substituted ligand H4Me4dota.

[0088] FIG. 8 illustrates phenyl-substituted ligand H4Phdota.

[0089] FIG. 9 illustrates nitrobenzyl-substituted ligand H4nb-dota.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0090] The present disclosure is a novel class of macrocyclic GCAs, and associated synthesis methods, where the metal ion is chosen from the lanthanide series and the coordination ligand has macrocyclic structure. The lanthanide series of chemical elements comprises the 15 metallic chemical elements with atomic numbers 57-71, from lanthanum through lutetium.

[0091] Here, the chemical symbol Ln is used in general to indicate lanthanide chemistry. All but one of the lanthanides are f-block elements, corresponding to the filling of the 4f electron shell. All lanthanide elements form trivalent cations, Ln3+, whose chemistry is largely determined by the ionic radius, which decreases steadily from lanthanum to lutetium

[0092] Methods to Quantify Metastable States

[0093] Laser excitation spectra of the transition of F D electrons of Gd3+ in the presence of ligands can be used to quantify complexation metastables and final product. It is known laser excitation at 577-581 nm, emission at 614 nm, is useful in detecting the .sup.5D.sub.0.fwdarw..sup.7F.sub.2 transition.

[0094] During the process of complexation, the initial association of Gd3+ with DOTA and the final formation of Gd(dota) result in different ligand coordination fields and therefore different excitation spectra. The complexation reaction between Gd3+ and DOTA involves the following three steps. The first step is a fast equilibrium; the second and/or the third deprotonation reaction is considered rate-determining. Good excitation spectra can be obtained from 0.2 mM Gd3+(dota) solution at pH 6.10. To verify stability, the excitation spectra of a 0.2 mM Gd3+(dota) solution (pH 6.10) prepared and stored for 200 h and a solution freshly prepared by titrating a an acidic 0.2 mM Gd3+(dota) solution with (CH.sub.3).sub.4NOH to pH 9.90 were compared. If the two spectra are the same, the reaction product is stable.

[0095] A simple inline test to evaluate how quickly Gd3+ is released from any chelated form is simply to add the complex to strong acid (0.1 M HCl) and measure the appearance of Gd3+ with time. The method can be modified by the addition of calcium or zinc ion, or the use of a synthetic blood serum preparation.

[0096] The relative kinetic stability of multiple runs of GCA batches is to expose the GCA to a solution containing phosphate anions at physiological pH. Even chelates with very high thermodynamic stability constants will equilibrate to form some free chelate and non-chelated gadolinium. Since gadolinium phosphate is insoluble, any non-chelated gadolinium will precipitate as insoluble gadolinium phosphate.

[0097] The detection system is to react according to Le Chatelier's Principle to establish a new equilibrium to replenish the “free gadolinium” until the phosphate anions are almost all precipitated from the solution. The rate at which this happens is determined by the rate of dissociation of Gd3+ from each metastable complex. metastable complexes will have relatively high thermodynamic instability and a relatively fast rate of dissociation.

[0098] Removing Metastables

[0099] The phosphate precipitation can be used in the synthesis process, since the metastables can be completely dissociated and the free gadolinium removed by filtration.

Synthesis Methods

[0100] Up date previous process with metastable detection, meglumine titration, and pH control

Examples of Macrocyclic GCAs with Reduced Metastables

Example 1: Metastable-Free Gd-Dota Synthesis

[0101] Preparation of DOTA solution [0102] 1. Heat reactor to 25-30° C. [0103] 2. Charge water with DOTA [0104] 3. Begin stirring (stir unless otherwise indicated, nominal rate 300 rpm) [0105] 4. Charge 10% of the DOTA [0106] 5. Stir until uniformly distributed in the water [0107] 6. If all the DOTA is charged, then go to step 8 [0108] 7. Go to step 4 [0109] 8. Stir 10 min

[0110] Preparation of Gadolinium: DOTA complex [0111] 9. Charge 25% by weight of the Gadolinium oxide [0112] 10. Stir until uniformly distributed [0113] 11. If all the Gadolinium oxide is charged then go to step 13 [0114] 12. Go to step 9 [0115] 13. Stir 10 min [0116] 14. Raise temperature to 95+/−2° C. [0117] 15. Stir 3 hrs. [0118] 16. Check clarity [0119] 17. If not clear continue for 1 hr, go to step 16 (this step took about 12 hours) [0120] 18. If clear, continue 1 hr and then cool to 40-45° C. [0121] 19. If precipitate forms, heat to 95+/−2° C. and stir for 1 hr, go to step 16

[0122] Verify Gd-Dota is Metastable-Free

[0123] A standard metastable-free Gd-dota reference solution is obtained by taking 5 ml of reactant obtained from steps 1-19 and diluting to a 0.2 mM Gd-dota solution. One measures the metastable content after synthesis for 5, 10, 15, 20, 25, 30, and 35 hours, or until the concentration of the metastable asymptotes as close to zero as possible.

[0124] The metastable content is measured by placing a 5 ml Gd-dota solution titrated with meglumine to pH 6.10 in a square 10 ml quartz vessel. The output from a copper vapor laser tuned to 578.2 nm (Oxford Lasers, Didcot, United Kingdom) is directed through the reactant and the excitation spectrum obtained at 578.2 nm. Baseline is established by directing the laser through the quartz vessel filled with distilled water. A graph of excitation intensity (measured in millivolts) vs reaction time is plotted, and the reference solution obtained after the slope of this plot is less than 0.01 or the absorption intensity does not change by more than 1% between 5 hour reaction intervals.

[0125] The stability of the reference solution can be verified by checking that the excitation spectra of a 0.2 mM Gd-dota solution (pH 6.10) does not change after storing for 200 h at 20° C.

[0126] Once the reference solution is prepared, then product runs can be indexed against this standard.

[0127] If inline monitoring of metastable presence is desired, the reactant can be titrated with a strong acid (0.1 M HCl) and the free Gd3.sup.+ ion quantified by xylenol orange titration. The metastable will release free Gd3+ more quickly than fully complexed Gd-dota. The presence of multiple metastable forms can be detected by dynamic titration over the span of an hour or more, and calculating the Gd3.sup.+ ion as a function of time.

[0128] In addition, the relative kinetic stability of a Gd-dota complex is accessed by exposing the solution to phosphate anions at physiological pH. Even chelates with very high thermodynamic stability constants will equilibrate to form some free chelate and non-chelated gadolinium. Since gadolinium phosphate is insoluble, any non-chelated gadolinium will precipitate as insoluble gadolinium phosphate, and the free Gd3.sup.+ ion can be quantified by weighing the precipitate.

[0129] Verify Complex Formation [0130] 20. Verify absence of free gadolinium using Xylenol orange [0131] 21. If free gadolinium detected, add X DOTA, raise temperature to 95+1-2° C., stir for 1 hr and proceed to step 16 [0132] 22. If not, proceed to step 23

[0133] Preparation of Gadoteric Acid Meglumine Solution [0134] 23. Add 90% of the meglumine at 40-45° C. [0135] 24. Sir 10 minutes [0136] 25. Measure pH—inline probe calibrated to 25° C. (USP) [0137] 26. If pH is >7.5, discard [0138] 27. If pH is between 7.0 and 7.5, then go to step 29 [0139] 28. If pH<than 7.0, add 2% of the Meglumine, go to step 24 [0140] 29. Stir for 1 hr at 40-45° C. [0141] 30. Check solution is clear, if yes proceed to 31, if not repeat 29

[0142] Gadoteric Acid Meglumine Solution Filtration [0143] 31. Cool the solution to 20-25° C. [0144] 32. Filter the solution using the carbon filter [0145] 33. Rinse the reactor with 20-25° C. water using ¼ V [0146] 34. Pass rinse through the filter [0147] 35. Repeat rinse steps 33 & 34 for a total of 2 rinses [0148] 36. Place filtrate and rinses back in reactor [0149] 37. Stir at 25-30° C. for 10 min [0150] 38. Measure Free DOTA by HPLC [0151] 39. If Free DOTA is 0.01-0.06% ww proceed to 42 [0152] 40. If Free DOTA <0.01% ww, add 0.03% ww equivalent of DOTA [0153] 41. Stir for ½ hr and go to step 38 [0154] 42. Measure pH—inline probe calibrated to 25° C. (USP) [0155] 43. If pH is between 7.0 and 7.5, then go to step 45 [0156] 44. If pH<7.0, add meglumine. Stir 10 min. Go to step 42. [0157] 45. Stir ½ hr. [0158] 46. Check solution is clear, if yes proceed to 47, if not repeat 45

[0159] Verify Purity [0160] 47. Measure Purity by HPLC [0161] 48. If individual impurity >0.05%, go to step 32

[0162] Final API Adjustments [0163] 49. Measure Free DOTA by HPLC [0164] 50. If Free DOTA >0.06% ww, repeat steps 32-42 [0165] 51. If Free DOTA is 0.01-0.06% ww proceed to 55 [0166] 52. If Free DOTA <0.01% ww, add 0.03% ww equivalent of DOTA [0167] 53. Stir ½ hr [0168] 54. Go to Step 49 [0169] 55. Measure pH [0170] 56. If pH is 7.0-7.5, then go to step 53 [0171] 57. If pH<7.0, add Meglumine. Stir 10 min. Go to step 55

[0172] Final API Testing [0173] 58. Perform full API testing: Gadolinium content; Meglumine Content; Assay; Water

[0174] Content; Heavy Metals

Example 2: Metastable-Free Gd-Dota Synthesis

[0175] If it is desired to minimize the synthesis time, then the metastables can be removed after step 20 or after step 59 of Example 1 using the phosphate precipitation method described in Example 1. The complete complexation of Gd-dota is a statistical process, and eliminating all the metastable complexes by synthesis, the intent of Example 1, can more than double the synthesis time. Therefore, it may be cost effective to terminate the synthesis in the incompletely complexed state, wherein greater than 90% of the Gd-dota complexes are fully complexed, then removal of the metastable forms by phosphate precipitation may be economically advantageous.

Example 3: Establishing Minimum Detection Levels

[0176] The ability to measure a minimum concentration of 1 ppm Gd3.sup.+ ion depends on being able to prepare a 10.sup.−6 molar solution of Gd3+ and a 1 molar solution of Xylenol orange to an accuracy of 10.sup.−6.

[0177] The Xylenol orange titration relies on a solution color change. Since the photon source used to assess color change will not be isomorphic in wavelength, one needs to calibrate the source and detector for each of the two wavelengths quantified in the xylenol orange method (see USP Memorandum on Gadolinium Contrast Agents). The calibration is only as good as the thermal stability of the source and detector. Therefore, it will be important to put source and detector into thermal equilibrium before measurement, therefore a 1 hour baseline must be established where the calibration varies by less than 10 ppm signal.

[0178] The entire measurement setup is to be placed in a thermally controlled environmental chamber at 20° C.

[0179] For these reasons, 10 separate 1 ppm Gd(III) solutions were prepared and equilibrated to 20° C. It is not sufficient to make one solution and take 10 measurements. Each of the 10 standard solutions are to be measured a minimum of 10 times, by performing a time integration until the variability reaches the 1 ppm threshold. This is possible by the programmable algorithm interface of commercial spectrometers.

[0180] The above procedure is to be repeated for 10 ppm solution and 100 ppm. Based on naive thermodynamic considerations, it is expected the robust (standard deviation less than 10%) detection range will start at between 10 ppm and 100 ppm.

[0181] Once the robust detection range is established, then the variability in decades is to be quantified: 1000 ppm, 10,000 ppm (1%), and 100,000 ppm (10%).

Calibration Results

[0182] The concentration of a solution of Gd(III) ion is determined stoichiometrically where the concentration is given by:

[00001]$\frac{\left[\mathrm{Gd}\left(\mathrm{III}\right)\right]}{\left[XO\right]}X{10}^6=\mathrm{ppmGd}\left(\mathrm{III}\right)$

[0183] [ . . . ] is the molar amount of Gd(III) or Xylenol Orange (XO). In terms of spectral measurement

[00002]$\frac{{\mathrm{Abs}}^{573}}{{\mathrm{Abs}}^{573}+{\mathrm{Abs}}^{433}}X{10}^6=\mathrm{ppmGd}\left(\mathrm{III}\right)$

[0184] Ten 1 ppm Gd(III) ion solutions were titrated to 7.0+/−0.1 pH with NaOH and the ammonia removed by vacuum.

TABLE-US-00001 1 ppm 10 ppm 100 ppm 1000 ppm 10,000 ppm 100,000 ppm Solution 1  5.6 +/− 0.6 14.8 +/− 0.6  93.7 +/− 0.7 1009.3 +/− 0.5 10,006.6 +/− 0.9 100,005.7 +/− 0.5 Solution 2  3.1 +/− 0.7 17.0 +/− 0.4  95.5 +/− 0.6  992.8 +/− 0.5 10,009.3 +/− 0.8  99,993.1 +/− 0.3 Solution 3  2.6 +/− 0.6 11.5 +/− 0.9 102.6 +/− 0.3 1005.6 +/− 0.7  9,996.8 +/− 0.2  99,998.9 +/− 0.0 Solution 4  0.1 +/− 0.2  8.9 +/− 0.2 109.2 +/− 0.0 1009.7 +/− 0.5  9,991.6 +/− 0.3  99,991.8 +/− 0.3 Solution 5  1.5 +/− 0.0  8.5 +/− 0.9  97.3 +/− 0.3 1006.5 +/− 0.8 10,006.3 +/− 0.4  99,993.1 +/− 0.5 Solution 6  2.3 +/− 0.4  5.9 +/− 0.3  92.8 +/− 0.1 1001.9 +/− 0.6 10,002.4 +/− 0.0  99,995.8 +/− 0.7 Solution 7  6.9 +/− 0.1 12.7 +/− 0.2  97.1 +/− 0.4  991.4 +/− 0.3  9,992.2 +/− 0.0  99,992.9 +/− 0.6 Solution 8  4.9 +/− 0.0  4.9 +/− 0.0  92.5 +/− 0.9 1003.9 +/− 0.5 10,009.5 +/− 0.6 100,000.6 +/− 0.5 Solution 9  0.7 +/− 0.1 10.6 +/− 0.7 103.6 +/− 0.0  994.0 +/− 0.1 10,002.8 +/− 0.9 100,002.1 +/− 0.5 Solution 10 1.8 +/− 0.3 12.9 +/− 0.3  94.6 +/− 0.9 1007.9 +/− 0.4 10,003.7 +/− 0.4  99,995.3 +/− 0.5 MEAN 3.0 +/− 2.1 10.8 +/− 3.6  97.9 +/− 5.2 1002.3 +/− 6.7 10,002.1 +/− 6.2  99,996.9 +/− 4.4 (sdev result of integration of 10 spectrometer runs)

Example 4. Identification of Metastables in Incompletely Synthesized Complex Solution

[0185] Using Example 1, metastable forms and free Gd3+ was quantified. 40.5 g of DOTA was suspended in 150 ml of water at a temperature of 75° C. 17.8 g of gadolinium oxide was added, and the batch was stirred at 75° C. for 2 hours. The solution that was produced was mixed with 19.5 g of meglumine and stirred at 75° C. for one hour. Then, the content of free DOTA, free gadolinium, and complex was determined, and the final content of excess free DOTA was set.

[0186] Five reactions were run, using 4.05 g of DOTA, 15 ml water, and 1.78 g of gadolinium oxide. Ten spectroscopic measurements using Xylenol orange were conducted. Here the calculation of ppm is different, where the amount of xylenol orange equals the number of theoretical drug molecules, that is for each gadolinium oxide molecule one will generate two gadolinium ions which can either complex with DOTA or remain free, accordingly [XO]=[GdO X 2]. This is the molar amount of xylenol orange added to each reaction product. We one uses

[00003]$\frac{{\mathrm{Abs}}^{573}}{{\mathrm{Abs}}^{573}+{\mathrm{Abs}}^{433}}X{10}^6=\mathrm{pprnGd}\left(\mathrm{III}\right)$

[0187] Understanding that the denominator is a constant and Abs.sup.573 is the number of free Gd(III) ions that react with the xylenol orange. This is a conservative measure of ppm, given by the ratio of free gadolinium and total gadolinium in the reactant (X 10.sup.6). Another possible definition, which would give a higher number (less conservative) is simply comparing free gadolinium to complexed gadolinium, which would mean ppm of free gadolinium for every million complexed gadolinium. The two definitions depart significantly at free gadolinium concentrations above 1%.

TABLE-US-00002 Reaction # Free Gd (III) Reaction 1 30,415.3 +/− 4.4 Reaction 2 19,567.6 +/− 3.5 Reaction 3 22,989.0 +/− 0.6 Reaction 4 36,080.7 +/− 3.8 Reaction 5 45,890.4 +/− 7.6 MEAN 30,989 +/− 9410 ppm

Example 5. Metastables as a Function of Reaction Time

[0188] Stoichiometric ratios of gadolinium oxide were reacted with DOTA according to the procedure outlined in Example 1. Measurements were taken (N=1) to conserve volume.

[0189] The final measurement was performed 5 times.

TABLE-US-00003 Hours of reaction metastables [ppm]  3 hours 28,094 +/− 9.7   N = 1   8 hours 3,853 +/− 12.4  N = 1  24 hours  72 +/− 5.0 N = 1  32 hours 0.8 +/− 1.3 N = 10

[0190] Now titrate with meglumine to pH 7.0

TABLE-US-00004 1.2 +/− 3.8 N = 2

Example 5: Optimizing Contrast and Stability

[0191] Due to the low sensitivity of MRI as an imaging technique, large quantities of a contrast agent, often on the gram scale, must be injected into the patient to obtain useful images. The ability to reduce the quantity of GBCAs required is highly desirable, especially when considering the toxicity problems discussed above. One way in which the amount of contrast agent required can be reduced is to enhance its relaxivity.

[0192] Relaxivity is a measure of how water relaxation rate changes with concentration of a contrast agent, and high relaxivities are indicative of more effective agents. The greater the number of coordinated inner sphere water molecules the higher the complex relaxivity.

[0193] All existing clinical GBCAs are based on octadentate polyaminocarboxylate ligands. As trivalent gadolinium prefers a coordination number of 9, this leaves one available coordination site free for an inner sphere water molecule.

[0194] The hydration state of the complex can be increased by stopping the coordination process early so that metastables are formed by reducing the number of coordination sites devoted to the Gd3.sup.+ ion. Importantly though, this lowers the thermodynamic stability of the complex and renders the metal ion more accessible to endogenous anions.

[0195] This accessibility leads to demetallation of gadolinium(III) complexes in vivo, causing further toxicity issues. Many in the field have tried to master the subtle interplay between maximizing relaxivity through accessing higher hydration states and forfeiting thermodynamic stability and kinetic inertness.

[0196] This strategy is flawed, and one objective of this application is to achieve greater relaxivity without sacrificing complex inertness. The inventors have surprisingly found that chemical stability is clinically less important than complex inertness. Kinetic inertness indicates the rate of Gd3+ release, while thermodynamic stability describes how much Gd3+ is released at equilibrium under certain conditions. Since the rate at which equilibrium is reached for macrocyclic gadolinium(III) complexes in vivo is very slow and normally cannot be reached during their residence time, thermodynamic stability cannot accurately predict Gd3+ release for macrocyclic GBCAs. These inventors conclude that thermodynamic stability alone is insufficient to predict the in vivo dissociation of macrocyclic chelates.

[0197] This conclusion is based on the following observation. The complex [Gd(OH2)(dota)]− is somewhat thermodynamically stable but is more kinetically inert and results in low in vivo deposition; while [Gd(OH2)(dtpa)]2− is highly thermodynamically stable but has a low kinetic inertness and has had its use restricted.

[0198] The same complex with Gd3+ replaced by Eu3+ is much more kinetically inert, despite a much lower thermodynamic stability than (fully complexed) [Gd(OH2)(dota)]−. This combined information suggests that it is kinetic inertness rather than thermodynamic stability that appears to be the useful predictor of in vivo Gd3+ release from GBCAs. Maximizing kinetic inertness should be a critical concern for those seeking to develop future GBCAs.

[0199] In this embodiment, chemical inertness is enhanced so that the presence of metastables (higher hydration states) does not contribute to Gd3+ deposition in the body.

[0200] To understand the logic behind the present embodiment it is necessary to consider the solution behavior of lanthanide(III) complexes of dota. There are known to be four different stereoisomers of [Ln(dota)]− in solution, which arise from the orientation of the five-membered coordination metallacycles formed by ethylene bridges in the macrocycles λλλλ and δδδδ and the corresponding positions of the pendant arms Λ and Δ.

[0201] In geometric terms, the shapes adopted by these stereoisomers are described as square antiprismatic (SAP) or twisted SAP (TSAP). Each of these isomers may be characterized by the twist angle between the nitrogen and oxygen donor atom planes. This angle varies according to which Ln3+ center the ligand is complexed to, but is typically around 40° for SAP structures and between −20° and −30° for TSAP geometries. The two SAP and two TSAP isomers are enantiomeric pairs, and interconversion between them is possible on the nuclear magnetic resonance (NMR) timescale, leading to the broadening often seen in proton NMR spectra of lanthanide(III) complexes of dota.

[0202] Interconversion occurs through two mechanisms: rotation of the acetate arms or inversion of the macrocyclic ring. In order to maximize complex inertness, it is imperative that interconversion is minimized. Chirality can be exploited to maximize complex inertness. Introduction of chirality to the macrocycle itself and the pendant arms can render both interconversion mechanisms unfavorable and cause the complex to favor a particular geometry, thus reducing kinetic lability of the complex.

[0203] The introduction of chirality locks ring conformation and facilitates ligand pre-organization during synthesis, resulting in greater complex inertness. chirality has the added advantage of making the metastable forms far less likely to persist in the reaction, if at all.

[0204] The gadolinium(III) complexes comprising the present embodiment possess increased steric bulk resulting in a greater propensity for TSAP isomer formation, potentially due to steric clashes with the acetate arms rendering SAP geometry unfavorable. SAP geometry is associated with the more hydrate metastables.

[0205] For example, SAP and TSAP isomers of a Gd3+ complex of a tetraethyl-substituted ligand [H4Et4dota], see FIG. 6. Analysis of rate equations of both isomers of the gadolinium(III) complex of H4Et4dota have greater than 1 day persistence in the presence of 1000 equivalents of a strong competing chelate (H5dtpa) and are expected to exhibit no demetallation after 100 h at 50° C. in the presence of a 100-fold excess of ZnCl.sub.2. This theoretically remarkable kinetic inertness can be attributed to the influence of the chiral substituents which enhance complex rigidity, leading to the restricted and non-interconvertible SAP and TSAP structures. Based on calculation, it is the interconversion between SAP and SAP structures and not the structures themselves which reduces kinetic inertness.

[0206] Chirality can be introduced by the examples given in FIGS. 6-9. FIG. 6 illustrates tetraethyl-substituted ligand H4Et4dota. FIG. 7 illustrates tetramethyl substituted ligand H4Me4dota. FIG. 8 illustrates phenyl-substituted ligand H4Phdota. FIG. 9 illustrates nitrobenzyl-substituted ligand H4nb-dota.

[0207] These chiral modifications to dota should have no effect on toxicity of the ligand and greatly reduce the Gd3+ toxicity of GBCA based on the chiral forms of dota.

Example 6: Free Gd3+ Results—Comparative Data

[0208] The contrast agents of the present disclosure were compared to a commercially available gadolinium-based contrast agents both in vitro and in vivo. After 30 minutes at standard dosing the contrast agents of the present disclosure released significantly less free GAD as summarized in the table below.

TABLE-US-00005 GBCA In Vitro PPM In Vivo PPM Contrast agents of 2.9 +/− 0.8 34.8 +/− 17   the present disclosure DOTAREM ® 1,204 +/− 3.9  2.9 +/− 24  (gadoterate meglumine) 415 times 256 times less free GAD less free GAD