Complex of gadolinium and a chelating ligand derived from a diastereoisomerically enriched PCTA and preparation and purification process

Abstract

The present invention relates to a complex of formula (II) constituted of at least 90% of a diastereoisomeric excess comprising a mixture of isomers II-RRR and II-SSS of formulae: ##STR00001##
The present invention also relates to a process for preparing and purifying said complex of formula (II), and also to a composition comprising said complex.

Claims

1. A composition comprising: 1) A complex of formula (II) below: ##STR00022## having a diastereoisomeric excess of at least 80% of a mixture of isomers II-RRR and II-SSS of formulae: ##STR00023## and ##STR00024## and 2) A free macrocyclic ligand, wherein the composition has a concentration of free gadolinium of less than 1 ppm (m/v), and wherein the composition comprises from 0.002 to 0.4 mol/mol % of free macrocyclic ligand relative to the complex of formula (II).

2. The composition of claim 1, wherein the composition comprises from 0.01 to 0.3 mol/mol % of free macrocyclic ligand relative to the complex of formula (II).

3. The composition of claim 1, wherein the complex of formula (II) has a diastereoisomeric excess of at least 90%.

4. The composition of claim 1, wherein the degree of purity of the complex of formula (II) is greater than 95% evaluated by chromatography.

5. The composition of claim 1, wherein the degree of purity of the complex of formula (II) is greater than 97% evaluated by chromatography.

6. The composition of claim 1, wherein the complex of formula (II) has a diastereoisomeric excess of at least 92%.

7. The composition of claim 1, wherein the complex of formula (II) has a diastereoisomeric excess of at least 94%.

8. The composition of claim 1, wherein the isomers II-RRR and II-SSS are present in the mixture in a ratio of between 60/40 and 40/60.

9. The composition of claim 1, wherein the composition has a concentration of complex of formula (II) of between 0.01 and 1.5 mol.Math.L.sup.−1.

10. The composition of claim 1, wherein the composition has a concentration of complex of formula (II) of between 0.2 and 0.7 mol.Math.L.sup.−1.

11. The composition of claim 1, wherein the composition has a concentration of complex of formula (II) of between 0.3 and 0.6 mol.Math.L.sup.−1.

12. The composition of claim 1, wherein the free macrocyclic ligand is selected from the group constituted of DOTA, NOTA, DO3A, BT-DO3A, HP-DO3A, PCTA, DOTA-GA and derivatives thereof.

13. The composition of claim 1, wherein the free macrocyclic ligand is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

14. The composition of claim 1, wherein the pH of the composition is between 4.5 and 8.5.

15. The composition of claim 1, wherein the pH of the composition is between 6.5 and 8.

16. The composition of claim 1, wherein the composition further comprises a buffer selected from the group consisting of lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, Tris (Tris(hydroxymethyl)aminomethane), HEPES (2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid), MES (2-morpholinoethanesulfonic acid) buffers and mixtures thereof.

17. The composition of claim 1, wherein the composition further comprises a buffer being Tris (Tris(hydroxymethyl)aminomethane).

18. A composition comprising: 1) A complex of formula (II) below: ##STR00025## having a diastereoisomeric excess of at least 90% of a mixture of isomers II-RRR and II-SSS of formulae: ##STR00026## and ##STR00027## and 2) A free macrocyclic ligand being DOTA, wherein the composition has a concentration of free gadolinium of less than 1 ppm (m/v), and wherein the composition comprises from 0.002 to 0.4 mol/mol % of DOTA relative to the complex of formula (II).

19. The composition of claim 18, wherein the composition has a concentration of complex of formula (II) of between 0.3 and 0.6 mol.Math.L.sup.−1.

20. The composition of claim 18, wherein the composition further comprises a buffer being Tris (Tris(hydroxymethyl)aminomethane) and has a pH between 6.5 and 8.

Description

EXAMPLES

(1) The examples given below are presented as non-limiting illustrations of the invention.

(2) Separation of the Groups of Isomers Iso1, Iso2, Iso3 and Iso4 of the Complex of Formula (II) by UHPLC

(3) A UHPLC machine constituted of a pumping system, an injector, a chromatography column, a UV detector and a data station is used. The chromatography column used is a UHPLC 150×2.1 mm−1.6 μm column (Waters Cortecs® UPLC T3 column).

(4) Mobile Phase: Route A: 100% acetonitrile and Route B: aqueous solution of H.sub.2SO.sub.4 (96%) at 0.0005% v/v

(5) Preparation of the Test Solutions: Solution of the complex of formula (II) at 2 mg/mL in purified water

(6) Analytical Conditions:

(7) TABLE-US-00005 Column temperature 40° C. Sample temperature Room temperature (20-25° C.) Flow rate 0.3 ml/min Injection volume 1 μl UV detection 200 nm Analysis time 20 min

(8) Gradient:

(9) TABLE-US-00006 Time % Acn % H.sub.2SO.sub.4 0.0005% 0 1 99 3 5 95 12 10 90 15 25 75 16 1 99 20 1 99

(10) Four main peaks are obtained. Peak 4 of the UHPLC plot, namely iso4, corresponds to a retention time of 6.3 minutes.

(11) Preparation of the Butyl Hexaester of Formula (VI)

(12) 184 kg (570 mol) of dibutyl 2-bromoglutarate and 89 kg (644 mol) of potassium carbonate are mixed in a reactor and heated to 55-60° C. An aqueous solution of 29.4 kg (143 mol) of pyclene in 24 kg of water is added to the preceding preparation. The reaction mixture is maintained at 55-60° C. and then refluxed for about 10 hours. After reaction, the medium is cooled, diluted with 155 kg of toluene and then washed with 300 litres of water. The butyl hexaester is extracted into the aqueous phase with 175 kg (1340 mol) of phosphoric acid (75%). It is then washed three times with 150 kg of toluene. The butyl hexaester is re-extracted into a toluene phase by dilution with 145 kg of toluene and 165 kg of water, followed by basification with 30% sodium hydroxide (m/m) to reach a pH of 5-5.5. The lower aqueous phase is removed. The butyl hexaester is obtained by concentrating to dryness under vacuum at 60° C., in a yield of about 85%.

(13) Preparation of the Hexaacid of Formula (III)

(14) 113 kg (121 mol) of butyl hexaester are placed in a reactor along with 8 kg of ethanol. The medium is brought to 55±5° C. and 161 kg (1207.5 mol) of 30% sodium hydroxide (m/m) are then added over 3 hours. The reaction mixture is maintained at this temperature for about 20 hours. The butanol is then removed by decantation of the reaction medium. The hexaacid of formula (III) obtained in sodium salt form is diluted with water to obtain an aqueous solution of about 10% (m/m). This solution is treated on an acidic cationic resin. The hexaacid of formula (III) in aqueous solution is obtained in a yield of about 90% and a purity of 95%.

(15) Preparation of the Hexaacid Gadolinium Complex of Formula (I)

(16) Experimental Protocol

(17) Complexation and Isomerization

(18) Without Acetic Acid

(19) 418 kg (117 kg of pure hexaacid of formula (III)/196 mol) of an aqueous solution of hexaacid of formula (III) at 28% by weight are placed in a reactor. The pH of the solution is adjusted to 2.7 by adding hydrochloric acid, and 37 kg (103.2 mol) of gadolinium oxide are then added. The reaction medium is heated at 100-102° C. for 48 hours to achieve the expected isomeric distribution of the hexaacid of formula (III).

(20) With Acetic Acid

(21) Gadolinium oxide (0.525 molar eq.) is suspended in a solution of hexaacid of formula (III) at 28.1% by mass.

(22) 99-100% acetic acid (50% by mass/pure hexaacid of formula (III)) is poured into the medium at room temperature.

(23) The medium is heated to reflux followed by distillation up to 113° C. by mass by refilling the medium with acetic acid gradually as the water is removed. Once the temperature of 113° C. is reached, a sufficient amount of acetic acid to arrive at the starting volume is added.

(24) The medium is maintained at 113° C. overnight.

(25) Crystallization, Recrystallization

(26) Crystallization

(27) The hexaacid gadolinium complex of formula (I) in solution is cooled to 40° C., the primer is added and the agents are left in contact for at least 2 hours. The product is then isolated by filtration at 40° C. and washed with osmosed water.

(28) Recrystallization

(29) 180 kg of the hexaacid gadolinium complex of formula (I) obtained previously (solids content of about 72%) are suspended in 390 kg of water. The medium is heated to 100° C. to dissolve the product, and then cooled to 80° C. to be primed by adding a small amount of primer. After cooling to room temperature, the hexaacid gadolinium complex of formula (I) is isolated by filtration and drying.

(30) Selective Decomplexation

(31) The dry product is placed in the reactor with osmosed water at 20° C. The mass of water added is equal to twice the theoretical mass of hexaacid gadolinium complex of formula (I). 30.5% sodium hydroxide (m/m) (6.5 eq.) is poured into the medium at 20° C. At the end of the addition of NaOH, the medium is left in contact at 50° C. for 16 hours. The medium is cooled to 25° C. and the product is filtered off on a bed of Clarcel.

(32) Content of the Mixture of Diastereoisomers I-RRR and I-SSS

(33) The ratio in which the various isomers of the complex of formula (I) are present in the mixture of diastereoisomers depends on the conditions under which the complexation and isomerization steps are performed, as is seen in Table 3 below.

(34) TABLE-US-00007 TABLE 3 content of the mixture I-RRR and I-SSS as a function of the complexation/isomerization conditions Diastereoisomeric Content of excess in the hexaacid of mixture pH Temperature formula (III) Time I-RRR and I-SSS 5.7  80° C. 40%  3 hours   19% 3.5  90° C. 50% 10 hours   49% 3.0 101° C. 40% 10 hours   68% 2.7 101° C. 28% 48 hours 98.04%

(35) The additional steps of recrystallization and selective decomplexation make it possible to increase the diastereoisomeric excess of the mixture I-RRR and I-SSS (see Table 4).

(36) TABLE-US-00008 TABLE 4 content of the mixture I-RRR and I-SSS after crystallization/recrystallization/selective decomplexation After the first After After selective crystallization recrystallization decomplexation Diastereoisomeric 98.04% 99.12% 99.75% excess in the mixture I-RRR and I-SSS
Preparation of the Complex of Formula (II)

(37) 90 kg (119 mol) of the hexaacid complex of formula (I) and 650 kg of methanol are placed in a reactor. The mixture is cooled to about 0° C. and 111 kg (252 mol) of a methanolic solution of hydrochloric acid (8.25% of HCl in methanol) are then poured in while maintaining the temperature at 0° C. The reaction medium is brought to room temperature and stirring is then continued for 16 hours. After cooling to 0-5° C., 120 kg (1319 mol) of 3-amino-1,2-propanediol are added. The reaction medium is then heated while distilling off the methanol under vacuum until a temperature of 60-65° C. is reached. The concentrate is maintained for 16 hours at this temperature under vacuum. At the end of contact, the medium is diluted with 607 kg of water while cooling to room temperature. The solution of the crude complex of formula (II) is neutralized with 20% hydrochloric acid (m/m). 978.6 kg of solution are thus obtained, with a concentration of 10.3%, representing 101 kg of material. The yield obtained is 86.5%, the purity of the complex of formula (II) is 92.3% (HPLC s/s). The amount of dicoupled impurities is 6.4% (HPLC s/s).

(38) Purification of the Complex of Formula (II)

(39) Nanofiltration

(40) The nanofiltration membrane used has a cut-off threshold of 200 daltons (Koch Membran System SR3D). This treatment is performed in the following manner:

(41) The solution of crude complex of formula (II) is heated to 30° C. The nanofilter is filled with said solution. The pump is switched on first at a low rate to purge the system, then the rate of the nanofilter pump is gradually increased to the desired recirculation rate (1.0 m.sup.3/h for a membrane of 2.5×40 inches). The system is then placed in total recirculation at 30° C. for at least 2 hours to establish a polarization layer. The medium is then passed to diafiltration at 30° C. under 2.5 bar while keeping the volume constant by adding pure water until a conductivity of the retentate of less than 1000 μS is obtained. At the end of diafiltration, the medium is concentrated to obtain a concentration of about 40% (m/m).

(42) Treatment on Resins

(43) The solution of complex of formula (II) obtained from the nanofiltration is diluted with purified water with stirring to obtain a 15% solution (m/m). This solution is eluted in series on 50 litres of strong anionic resins (FPA900) in OH.sup.− form and then on 50 litres of weak cationic resins (HP336) in H.sup.+ form at a mean elution flow rate of 2V/V/H (2 volumes of solution per volume of resin per hour). The resins are then rinsed with about 450 litres of purified water until a refractive index of less than 1.3335 is obtained.

(44) The solution of complex of formula (II) is then concentrated by heating to 50-60° C. under a vacuum of 20 mbar to reach a concentration of 35% (m/m).

(45) Ultrafiltration

(46) The ultrafiltration membrane is a UF 10KD Koch Spiral membrane.

(47) The ultrafilter is fed with the preceding solution of complex of formula (II) at 35% heated to 40° C. The ultrafiltration is applied at a flow rate of 3 m.sup.3/h with a transmembrane pressure of 2.5-3 bar. The system is rinsed several times with 13 litres of apyrogenic purified water until a final dilution of the complex of formula (II) of 25% (m/m) is reached.

(48) Atomization

(49) The complex of formula (II) is obtained in powder form by atomization of the preceding solution of complex of formula (II) concentrated to 25%.

(50) The atomization is performed in the following manner:

(51) The atomizer is equilibrated with apyrogenic pure water by setting the inlet temperature to 165° C.-170° C. and adapting the feed rate such that the outlet temperature is between 105 and 110° C.

(52) The concentrated solution of complex of formula (II) is then added and the flow rate is adjusted so as to conserve the above parameters.

(53) These operating conditions are maintained throughout the atomization, while ensuring good behaviour of the powder in the atomization chamber and at the atomizer outlet. It should notably be ensured that there is no adhesion of the product.

(54) At the end of feeding the atomizer with the solution, the container of this complex of formula (II) and the atomizer are rinsed with apyrogenic pure water until maximum recovery of the powder is obtained.

(55) A 99.6% pure complex of formula (II) is obtained.

(56) This degree of purity was determined by reverse-phase liquid chromatography.

(57) Composition According to the Invention and Results of Studies Thereon

(58) Example of a Manufacturing Process in Accordance with the Invention

(59) The process for manufacturing a composition according to the invention is performed according to the following steps:

(60) a) 485.1 g (i.e. 0.5 M) of complex of formula (II) are dissolved in water (qs 1 litre), heating the tank to a temperature of between 39 and 48° C. and stirring the solution vigorously until this complex has fully dissolved in the water. The solution is then cooled to about 30° C.
b) 0.404 g (i.e. 0.2 mol/mol % relative to the proportion of complex added in step a)) of DOTA (Simafex, France) is added with stirring to the solution obtained in step a) via a solution of DOTA at 10% m/v.
c) Trometamol (Tris) is added to the solution obtained in step b) with stirring. The pH is then adjusted to a value of between 7.2 and 7.7 by addition of hydrochloric acid solution with stirring.
d) The target concentration (0.5 mol/L) is obtained by adding water for injection in two steps until a density value of between 1.198 and 1.219 g/mL is obtained.

(61) The liquid composition is then filtered through a polyethersulfone membrane and placed in its final container, which is finally sterilized at 121° C. for 15 minutes.

(62) Example of a Composition in Accordance with the Invention.

(63) The following formulation is obtained by means of the process described above:

(64) TABLE-US-00009 Ingredients Proportions in the composition Complex of formula (II) 485.1 g (0.5 M) DOTA** 0.404 g (1 mM, i.e. 0.2 mol/mol % versus complex) NaOH or HCl qs pH 7.2 to 7.7 Trometamol 1.211 g Free gadolinium* <1 ppm m/v Water for injection (injection-grade) qs 1 L *Measurement performed by the colorimetric method with xylenol orange **expressed on an anhydrous and pure basis

(65) Formulation Tests Performed

(66) Various concentrations of trometamol from 0 to 100 mM were tested. The results of these tests showed that a content of 10 mM (0.12% w/v) was sufficient to ensure the pH stability of the formulation while limiting the formation of degradation impurities.

(67) Various concentrations of DOTA from 0 to 2.5 mM were tested. The results of these tests showed that a content of 1 mM, which corresponds to 0.04% m/v or 0.2 mol/mol %, makes it possible to ensure the absence of release of free Gd during the process and during the lifetime of the product.

(68) Stability Studies Under Accelerated Conditions of a Composition According to the Invention

(69) The formulation of the preceding example is analysed just after its manufacture (T.sub.0) and after storage at 40° C. for 6 months after its manufacture (T+6 months).

(70) At T.sub.0: Purity evaluated by chromatography*: 99.6% Concentration of Gd-DOTA: 0.007% (m/V) Concentration of Gd: below 0.0001% (m/V) pH: 7.5

(71) At T+6 months: Purity evaluated by chromatography*: 97.2% *reverse-phase liquid chromatography Concentration of Gd-DOTA: 0.014% (m/V)−0.25 mM Concentration of Gd: below 0.0001% (m/V) pH: 7.5

(72) These results demonstrate that this formulation has good stability over time.

(73) Comparative Stability Studies

(74) The stability of the compositions below was evaluated over time. The term “non-optimized AP” denotes the active principle, namely the complex of formula (II), obtained according to the process described in EP 1 931 673. The term “optimized AP” denotes the diastereoisomerically enriched and purified complex of formula (II) obtained via the process according to the invention.

(75) TABLE-US-00010 AP [DOTA] Trometamol (0.5 M) mol/mol % mM pH.sub.adjustment C1 Not 0.3 — 5.0 optimized C2 Optimized 0.2 — 7.5 C3 Optimized 0.1 — 7.5 C4 Optimized 0.2 10 7.5 C5 Optimized 0.1 10 7.5 C6 Optimized 0.2 — 5.0 C7 Optimized 0.1 — 5.0 Free Gd in ppm m/v DOTA-Gd in mol/mol% (xylenol) (LC formate*) T 6 months T 6 months T 0 40° C. T 0 40° C. C1 <DL 0.18 0.27 0.3 C2 <DL <DL 0.02 0.05 C3 <DL <DL 0.02 0.05 C4 <DL <DL 0.02 0.05 C5 <DL <DL 0.02 0.08 C6 <DL <DL 0.03 0.03 C7 <DL <DL 0.02 0.07 *LC formate: chromatographic method involving fluorimetric detection. The separation is performed on a reverse-phase C18 grafted chromatography column with elution in gradient mode.

(76) The results reported above indicate that formulation of the non-optimized AP with free DOTA is not possible. The reason for this is that the chelation excipient is entirely consumed by the trans-ligation reaction between the complex of formula (II) and DOTA and consequently can no longer play its role of trapping the leached Gd.sup.3+.

(77) On the other hand, the diastereoisomerically enriched and purified complex of formula (II) obtained via the process according to the invention may be formulated with free DOTA. Specifically, the absence of free Gd in the composition at 6 months, 40° C., is observed, this being the case irrespective of the pH of the formulation and whether or not buffering species are present. In addition, the consumption of chelation excipient is very low, since it does not exceed 0.08 mol/mol %.