U.S. patent application number 17/701021 was filed with the patent office on 2022-07-07 for solvent free gadolinium contrast agents.
This patent application is currently assigned to INVENTURE, LLC. The applicant listed for this patent is INVENTURE, LLC. Invention is credited to Jonathan BALFOUR, Richard J. DESLAURIERS, Michael MILBOCKER.
Application Number | 20220211879 17/701021 |
Document ID | / |
Family ID | 1000006211266 |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220211879 |
Kind Code |
A1 |
DESLAURIERS; Richard J. ; et
al. |
July 7, 2022 |
SOLVENT FREE GADOLINIUM CONTRAST AGENTS
Abstract
Disclosed herein are complexes of gadolinium metal, ligand and
meglumine that are substantially free of non-aqueous solvents. In
particular, solvent-free complexes of 1) gadopentetate dimeglumine
and 2) gadoterate meglumine are disclosed and methods of their
preparation are disclosed. In addition, methods are disclosed for
purifying reactants, monitoring and controlling pH, quantifying the
free gadolinium content, quantifying the concentration of
gadolinium-ligand complex in aqueous solution, and procedures for
producing a drug product in one step. The one step process
eliminates the need to dry the gadolinium-ligand complex, which is
typically highly hygroscopic. The one step process includes
purification steps that do not require the use of non-aqueous
solvents.
Inventors: |
DESLAURIERS; Richard J.;
(Woodbury, CT) ; BALFOUR; Jonathan; (Toronto,
CA) ; MILBOCKER; Michael; (Holliston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INVENTURE, LLC |
Southbury |
CT |
US |
|
|
Assignee: |
INVENTURE, LLC
Southbury
CT
|
Family ID: |
1000006211266 |
Appl. No.: |
17/701021 |
Filed: |
March 22, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16420046 |
May 22, 2019 |
|
|
|
17701021 |
|
|
|
|
15855570 |
Dec 27, 2017 |
|
|
|
16420046 |
|
|
|
|
62439893 |
Dec 29, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/105 20130101;
A61K 49/103 20130101; A61K 2123/00 20130101; A61K 49/18 20130101;
A61K 49/101 20130101; A61K 49/108 20130101; A61K 49/106 20130101;
A61K 49/1806 20130101 |
International
Class: |
A61K 49/10 20060101
A61K049/10; A61K 49/18 20060101 A61K049/18 |
Claims
1. An API for a gadolinium contrast agent that is suitable for
injection in a mammal, the API comprising: a complex of Gd(III)
ion, ligand and meglumine in an aqueous formulation; wherein the
aqueous formulation is produced by a solvent-free method so that
the aqueous formulation comprises not more than 0.025% by weight of
free ligand, the aqueous formulation comprises less than 1 part per
million of free Gd(III) ion, and the aqueous formulation is
substantially free of non-gadolinium and ligand complexes; and
wherein API has a concentration that is in a range from 5 to 10
mg/ml of the aqueous formulation and a pH greater than 7.5.
2. The API of claim 1, wherein the formulation comprises less than
0.5 part per million of free Gd(III) ion.
3. The API of claim 1, wherein the formulation comprises less than
0.1 part per million of free Gd(III) ion.
4. The API of claim 1, wherein the formulation comprises less than
1 part per million of non-aqueous solvent.
5. The API of claim 1, wherein the formulation comprises less than
0.020% by weight of free ligand.
6. The API of claim 1, wherein the formulation comprises less than
50 parts per million of non-aqueous solvent.
7. The API of claim 1, wherein the ligand is DOTA.
8. The API of claim 1, wherein the complex is gadopentetate
dimeglumine.
9. The API of claim 1, wherein the complex is gadoterate
meglumine.
10. The API of claim 1, wherein the complex has a conditional
thermodynamic stability constant, at pH 7.4, in the range of from
about 18.1 to about 18.6.
11. The API of claim 1, wherein the gadolinium contrast agent has a
pH in the range of from about 7.2 to about 7.5.
12. The API of claim 1, wherein the solvent free method comprises
reacting and stirring the Gd(III) ion and ligand at a reaction
temperature for at least about 12 hours and until there is no free
Gd(III) ion to yield a Gd(III) ion and ligand complex of the
formulation.
13. The API of claim 12, wherein the solvent free method further
comprises: cooling the Gd(III) ion and ligand complex to
40-45.degree. C., adding meglumine, and stirring at 40-45.degree.
C. for at least 1 hour to yield the formulation.
14. The API of claim 12, wherein the Gd(III) ion and ligand are
reacted and stirred at a reaction temperature for at least about 24
hours.
15. The API of claim 12, wherein the Gd(III) ion and ligand are
reacted and stirred at a reaction temperature for at least about 32
hours.
16. A gadolinium contrast agent comprising a complex of Gd(III)
ion, ligand and meglumine in an aqueous, wherein: the gadolinium
contrast agent is produced by a solvent-free method in which: i)
the Gd(III) ion and ligand are reacted and stirred at a reaction
temperature for at least about 12 hours and until there is no free
Gd(III) ion to yield a Gd(III) ion and ligand complex, and ii) the
Gd(III) ion and ligand complex are cooled to 40-45.degree. C.,
meglumine is added, and stirring is performed at 40-45.degree. C.
for at least 1 hour to yield a concentrated API of the complex that
has a pH >7.5; and iii) the concentrated API is diluted with
water for injection to yield a formulation suitable for injection
in a mammal; wherein the formulation comprises not more than 0.025%
by weight of free ligand; wherein the formulation comprises less
than 1 part per million of free Gd(III) ion; and wherein the
formulation is substantially free of non-gadolinium and ligand
complexes.
17. The gadolinium contrast agent of claim 22, wherein the
formulation comprises less than 0.5 part per million of free
Gd(III) ion.
18. The gadolinium contrast agent of claim 22, wherein the
formulation comprises less than 0.1 part per million of free
Gd(III) ion.
19. The gadolinium contrast agent of claim 22, wherein the
formulation comprises less than 1 part per million of non-aqueous
solvent.
20. The contrast agent of claim 22, wherein the complex is
gadopentetate dimeglumine or gadoterate meglumine.
21. The contrast agent of claim 22, wherein the formulation has a
pH in the range of from about 7.2 to about 7.5.
22. The contrast agent of claim 22, wherein the complex has a
conditional thermodynamic stability constant, at pH 7.4, in the
range of from about 18.1 to about 18.6.
23. The contrast agent of claim 22, wherein the Gd(III) ion and
ligand are reacted and stirred at a reaction temperature for at
least about 24 hours.
24. The contrast agent of claim 22, wherein the ligand is DOTA.
25. The contrast agent of claim 22, wherein the formulation
comprises less than 50 parts per million of non-aqueous solvent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/420,046, filed May 22, 2019, which is a continuation-in-part
of U.S. application Ser. No. 15/855,570, filed Dec. 27, 2017, which
claims benefit of U.S. Provisional Application No. 62/439,893,
filed Dec. 29, 2016, all of which are hereby incorporated by
reference in their entireties.
TECHNICAL FIELD
[0002] The present disclosure relates generally to metal chelates,
particularly those of lanthanide metals, and in one specific
embodiment, those of Gd(III), which are useful as contrast agents
in magnetic resonance imaging for therapeutic and diagnostic
applications, as well as clinical and biomedical research
applications.
BACKGROUND OF THE INVENTION
[0003] Magnetic resonance imaging (MRI) is a powerful diagnostic
method that yields three-dimensional images of body tissues in
vivo. The tissue features obtained are the result of variations in
the distribution of water in these tissues. MM contrast agents
administered prior to imaging alter the relaxation times of protons
in their vicinity enhancing specific features of an image. MM
contrast agents improve the sensitivity and utility of MM
diagnostics.
[0004] The use of contrast agents for MRI in the clinical setting
has become a routine standard of practice for the enhancement in
resolution and tissue specificity of medical MM images.
Paramagnetic metal chelates, such as
Gd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA)
(Magnevist),
Gd(III)--N,N',N',N'',N'''-tetracarboxymethyl-1,4,7,10-tetraaza
cyclododecane (Gd(III)-DOTA), and their analogs have proven to
increase the relaxation rate of surrounding protons and have been
widely used as MM contrast agents.
[0005] The thermodynamic stability of gadolinium complexes are
strongly pH dependent, and while the pH in vivo is not highly
variable, the current manufacturing methods yield compositions of
gadolinium complexes that vary considerably in pH. Reduced
thermodynamic stability can result in the release of toxic Gd(III)
ion from the ligand, and may be linked to nephrogenic systemic
fibrosis. While the formation of the Gd(III) ion occurs due to
manufacturing variations in product pH, and this product pH
eventually equilibrates to in vivo pH when injected, the dilution
due to injection is sufficiently rapid compared to pH equilibration
to separate Gd(III) ion and the ligand (for example, pentetic
acid), such that when favorable pH is reached, the metal ion and
ligand are sufficiently separated that they do not recombine as a
conjugate of ligand and gadolinium.
[0006] Consequently, medical contrast agents with a large pH range
in the product specification present a safety concern regarding
product stability and the potential for formation the release toxic
Gd(III) from the complex. The large pH range in the drug product is
linked to the use of solvents in the drug purification process,
which tends to remove ligand in an unpredictable fashion.
[0007] The synthetic methods attempted in the past to prepare
paramagnetic metal chelates have one or more drawbacks such as the
use of large excess of ligand to reduce free Gd(III) ion; or the
need to carry out extensive solvent purification of product due to
impurities in the original reagents. Ironically, the biocompatible
solvents used to purify the drug product can complex with the
impurities they are meant to remove. If pure ingredients are used
initially, the need for solvent purification is removed.
Nevertheless, solvents are still needed because the gadolinium
complex must be precipitated in an anhydrous state in order to
formulate the drug product at the therapeutic potency.
[0008] The sequence of complexing Gd(III) and the ligand in water,
drying, and then reformulating in water is a multi-step process
that results in dramatic shifts in the delicate balance between the
gadolinium ion and the ligand. Ultimately, this multi-step process
is responsible for solvent complexed impurities, shifts in pH and
gadolinium-ligand balance. As a consequence, it has become standard
in the industry to allow large ranges of pH and meglumine
content.
[0009] In particular, a significant excess of ligand, for example
pentetic acid, is intentionally formulated in the current MM
contrast agent Magnevist.RTM.. In Magnevist, the formation of
Gd(III) ion is reduced in the presence of excess pentetic acid. The
formation of the Gd(III) ion is largely the result of variation in
the thermodynamic stability of the macromolecular conjugate of
pentetic acid ligand and gadolinium in the presence of solvent.
[0010] The shortening of proton relaxation times by gadolinium is
mediated by dipole-dipole interactions between the unpaired valence
electrons of gadolinium and adjacent water protons. The magnitude
of gadolinium magnetic dipole interaction drops off very rapidly as
a function of its distance from these protons (as the sixth power
of the radius). Consequently, the only protons which are relaxed
efficiently are those able to enter the gadolinium metal.
[0011] The protons can enter the first or second coordination
spheres of the gadolinium metal and metal complex. In coordination
chemistry, metal ions are described as consisting of two concentric
coordination spheres. The first coordination sphere refers to a
central atom or ion (in this case gadolinium). The second
coordination sphere can consist of ions (especially in charged
complexes), molecules (especially those that hydrogen bond to
ligands in the first coordination sphere) and portions of a ligand
backbone. Compared to the first coordination sphere, the second
coordination sphere has a less direct influence on the reactivity
and chemical properties of the metal complex. Nonetheless, the
second coordination sphere is relevant to understanding reactions
of the metal complex, including the mechanisms of ligand exchange
and catalysis.
[0012] The protons enter the first or second coordination spheres
of the gadolinium metal complex during the interval between an rf
pulse and a signal detection. This interval ranges in duration from
105 to 106 protons/second (Brown (1985) Mag. Res. Imag. V 3, p
3).
[0013] Gadolinium has seven unpaired valence electrons in its 4f
orbital and consequently has the largest paramagnetic dipole (7.9
Bohr magnetons) and exhibits the greatest paramagnetic relaxivity
of any element (Runge et al. (1983) Am. J. Radiol V 141, p 1209 and
Weinman et al. (1984) Am. J. Radiol V 142, p 619). Consequently,
gadolinium has the highest potential of any element for enhancing
magnetic resonance images.
[0014] In order to take advantage of the large paramagnetic dipole
of gadolinium one must recognize the toxicity of free gadolinium
metal ion (Gd(III)) in vivo. Thus, the use of gadolinium metal in
vivo, for example gadolinium chloride or gadolinium oxide, is not
safe and a water-soluble chelate of gadolinium must be used. While
a water soluble chelated gadolinium-based contrast agent is safer
to inject in patients, the toxicity issues are not entirely solved.
Latent toxicity is in part the result of precipitation of the
gadolinium that can occur at body pH as gadolinium hydroxide.
[0015] However, Gd(III) ion, even if it does not form a
water-insoluble compound, can still be toxic, since the reactivity
of Gd(III) is very similar to Ca(II), and Ca(II) is ubiquitous in
chemical pathways in the mammalian body.
[0016] In order to increase solubility and decrease toxicity,
gadolinium has been chemically chelated by small organic molecules.
To date, the chelator most satisfactory from the standpoints of
general utility, activity, and toxicity is diethylenetriamine
pentaacetic acid (DTPA) (Runge et al. (1983) Am. J. Radiol V 141, p
1209 and Weinman et al. (1984) Am. J. Radiol V 142, p 619). The
first formulation of this chelate to undergo extensive clinical
testing was developed by Schering-Berlex AG according to a patent
application filed in West Germany by Gries, Rosenberg and Weinmann
(DE-OS 3129906 A 1 (1981)). The chelate consists of Gd-DTPA which
is pH-neutralized and stabilized with an organic base,
N-methyl-D-glucamine (meglumine or methyl meglumine).
[0017] A direct relationship exists between the concentration of an
X-ray attenuator and its efficacy in contrast enhancement. The
relationship between concentration and contrast effect is not
linear with respect to MRI contrast agents, where a threshold
concentration of the paramagnetic entity is required to affect the
proton relaxation rates in a physiologic region that is being
imaged. Beyond this threshold concentration, any further increase
in concentration results in little improvement in contrast
enhancement. Thus, MRI contrast agents are formulated as close as
practicable to the threshold concentration to help reduce toxic
effects not mitigated by chelation. However, if the gadolinium
complex is unstable, then the formulation must be hedged and the
chelate concentration made greater than the threshold value.
[0018] The ionic radii of the trivalent lanthanide cations range
from 1.1 A for La(III) to 0.85 .ANG. for Lu(III) while Gd(III),
sitting exactly in the center of the lanthanide series, has an
ionic radius of 0.99 .ANG., very nearly equal to that of divalent
Ca(II). Gd(III) can compete with Ca(II) in the chemical pathways of
biological systems, and this substitution potential results in
gadolinium toxicity to organisms. In fact, the trivalent ion of
gadolinium binds with much higher affinity than the divalent ion of
calcium. When bound to a Ca(II)-binding enzyme, lanthanide ion
replacement often alters the kinetics of the biological process
catalyzed by that enzyme.
[0019] The toxicity of gadolinium has placed emphasis on the
stability of the gadolinium-ligand (GdL) complex, since the complex
form is significantly less toxic than the metal ion form. The
thermodynamic stability of a complex simply describes the
concentrations of all species present in solution at equilibrium as
given by the following equations:
Gd(H2O)+L.revreaction.GdL(H2O)+7H2O
K st=[GdL][Gd][L]
[0020] where Gd is gadolinium ion, L is the ligand, K is the
stability constant, GdL is the gadolinium-ligand complex, [L] is
the ligand protonation constant, [GdL] is the thermodynamic
stability constant of the complex, and [Gd] is the Gd(III) ion
formation constant. When solvent is introduced into the equilibrium
equation, the thermodynamic stability constant of the complex is
reduced and the Gd(III) ion formation constant increased.
[0021] Free gadolinium metal ion has 8 inner-sphere sites for
water, and the complex form has only 1 inner-sphere for water. The
Gibbs free energy of the equilibrium process between complex and
free metal ion will have large favorable entropy toward the complex
form due to the release of seven of the eight inner-spheres for
water. This entropy contribution is referred to as the "chelate
effect". This chelate effect can be compromised by the presence of
solvent, which can form binding spheres with solvent rather than
water.
[0022] In addition, the gadolinium ion-ligand interaction possesses
a large electrostatic component that contributes a favorable
enthalpy term. The result is that the overall free energy change
becomes quite favorable toward the complex form. For these reasons,
the solvated Gd(III) ion forms very stable complexes with ligands
having more basic donor atoms. That stability is enhanced by the
absence of solvent.
[0023] The desirability of maximally basic groups in ligands
results in the universal selection of ligands comprised of amines.
This consideration also explains why amine groups with
amide-containing side-chains are considerably less basic than amine
groups with acetate side-chains, for example diethylene triamine
pentaacetic acid (DTPA) or pentetic acid.
[0024] Higher thermodynamic stability of a complex is expressed by
a larger thermodynamic stability constant Kst. It should be
appreciated that small differences in the ligand protonation
constants can have a significant impact on the thermodynamic
stabilities of the resulting GdL complex. Unlike the relatively
small variations in the log [L] values for the ligands, the log Kst
values for a complex can vary by over 10 orders of magnitude. The
stability constant is widely used to compare contrast agents
because it reduces comparisons to a single convenient number.
[0025] The thermodynamic stability constant describes the
equilibrium under conditions where the ligand is entirely
deprotonated. At physiological pH values, the ligand will be
partially protonated so one can argue that a better way to compare
GdL stabilities is to use what are called conditional stability
constants, set forth in Table 1.
TABLE-US-00001 TABLE 1 Thermodynamic and Conditional Stability
Constants for Common Gd Complexes Ligand protonation constants DTPA
DTPA-BMA DOTA DOTA-(gly).sub.4 Thermodynamic stability constants
22.46 16.85 24.7 14.54 pH 14 (log K.sub.GdL) Conditional stability
constant at 18.4 14.8 17.2 12.7 pH 7.4 (log K.sub.eff)
(Schmitt-Willich H, Brehm M, Ewers CL, Michl G, Mueller-Fahrnow A,
Petrov O, et al. Inorg Chem 1999;38:1134-44.)
[0026] Table 1 compares the stability constant of complexes formed
between gadolinium and various ligands at pH 14 (deprotonated, and
standard "thermodynamic stability constant") and the conditional
stability constant at pH 7.4. Stronger acid conditions clearly
results in lower complex stability.
[0027] There are additional ionic competitors besides protons that
can affect complex stability. For instance, ions like zinc, copper,
and iron form very stable complexes with these ligands, and can at
the right activation energy force gadolinium out of the less toxic
complex state. At the same time, gadolinium has a high affinity for
some contaminants and will leave the complex for phosphate,
citrate, and carbonate ions which may be present in solution. The
magnitude of the effect of these contaminants is generally
determined by the thermodynamic stability constant at product
pH.
Transmetallation of Gadolinium Complexes
[0028] Magnevist.RTM. list on its label a pH range of 6.5-8 pH.
Reported impurities of gadolinium oxide, used in the preparation of
Magnevist, are usually 99.9% pure based on the presence of rare
earth metals only. Thus the presence of iron, which may be the
source of the yellow color, is not assessed. Iron-DTPA complex is
yellow in color.
[0029] Much attention has been paid to the potential of Zn(II) to
react with a gadolinium contrast agent and displace the gadolinium.
Such exchange of one metal for another is termed transmetallation.
Of the commonly encountered metal ion contaminants in chemical
compounds, Na(I), K(I), Mg(II), and Ca(II), all form very weak
complexes with the chelators used in contrast agents and are
thermodynamically disfavored from such transmetallation reactions.
The order of affinity of contrast agent chelators for other
endogenous ions is Fe(III)>Cu(II)>Zn(II).
[0030] The fact that transmetallation of gadolinium complexes
results in the formation of a more unstable metal complex implies
that the synthesis methodology can impact the magnitude of
transmetallation. In particular, if reaction temperatures are kept
as low as possible during the complexation process can
significantly reduce the incidence of non-gadolinium metal complex
formation.
[0031] Accordingly, there is a need to improve the safety profile
of MRI contrast agents. The present disclosure addresses this need
by providing a gadolinium complex formulation for injection, where
solvent is absent, and the competitive interaction with ligand
eliminated.
BRIEF SUMMARY OF THE INVENTION
[0032] Generally, it is an object of the present invention to
provide drug products and methods for synthesizing contrast agents
capable of enhancing magnetic resonance images of body organs and
tissues, and in particular, a ligand-gadolinium complex possessing
an improved safety profile, reduced variability of product and
absence of solvent contaminants. Other objects and features will be
in part apparent and in part pointed out hereinafter.
[0033] Among the several objects of the invention may be noted the
synthesis of organic solvent-free complexes of ligand and
gadolinium balanced by a counterion, for example meglumine, with
only one central metal ion of gadolinium for use in enhancing
magnetic resonance images of body organs and tissues.
[0034] Another object of the invention is the provision of methods
for forming complexes of ligand and gadolinium in a one step
process which begins with complex formation and ends with drug
product formulation, and beneficially eliminates the use of
solvents.
[0035] Another object of the invention is to provide a gadolinium
contrast agent, such as gadopentetate dimeglumine or gadoterate
meglumine, having a pH range that is smaller than currently
available formulations.
[0036] Another object of the invention is to provide a method of
preparation of gadolinium contrast agent formulations in which the
formation of non-gadolinium and ligand complexes is reduced
significantly or eliminated. In particular, the formation of
solvent-ligand complexes is excluded by the present methods.
[0037] Another object of the invention is to provide a method of
preparation of gadolinium contrast agent formulations with enhanced
weights and measures such that the color of the formulation is
substantially reproducible and preferably colorless, whereas
currently marketed gadopentetate dimeglumine ranges in color from
colorless to yellow.
[0038] Another object of the invention is the combination of the
provisions cited above such that the result is a ligand complex of
gadolinium, for example gadopentetate dimeglumine, with a
thermodynamic stability of low variability and enhanced
stability.
[0039] Another object of the invention to provide a solvent-free
ligand-gadolinium complex with reduced variability of thermodynamic
stability constant, and having a reduced propensity for causing or
contributing to a etiology of nephrogenic systemic fibrosis.
[0040] Accordingly, the present disclosure provides, a gadolinium
contrast agent comprising a complex of Gd(III) ion, ligand and
meglumine in a formulation suitable for injection in a mammal,
wherein the complex comprises less than 50 parts per million of
non-aqueous solvent. In some embodiments, the complex less than 1
part per million of non-aqueous solvent. In some embodiments, the
non-aqueous solvent is selected from the group consisting of
acetone, methanol, ethanol, heptane, hexane, acetonitrile, toluene
or a combination thereof. In more particular embodiments, the
solvent is methanol, ethanol, or a combination thereof
[0041] The gadolinium contrast is in some embodiments gadopentetate
dimeglumine or gadoterate meglumine.
[0042] In certain embodiments, the formulation comprises less than
0.025% by weight of free ligand, and more particularly, less than
0.020%, or 0.010%.
[0043] In further embodiments, the formulation has a pH ranging
from about 7.2 to about 7.5. In other embodiments, the complex has
a thermodynamic stability constant ranging from about 18.1 to about
18.6.
[0044] The gadolinium contrast agents of the present disclosure
advantageously have reduced impurities. In some embodiments, the
contrast agent comprises less than 1 part per million of free
Gd(III) ion. In some embodiments, the contrast agent comprises less
than 10 parts per million of non-Gd pentetic acid complexes.
[0045] The present disclosure further provides a method of
synthesizing a gadolinium contrast agent comprising a complex of
Gd(III) ion, ligand and meglumine in a formulation suitable for
injection in a mammal, wherein the method uses no non-aqueous
solvent. The present method advantageously maintains a hydrated
state during all of the process steps, meaning that removal of
water is not necessary or desired. In some embodiments, the complex
is in a hydrated state of at least 1% by weight water during each
method step.
[0046] In a particular embodiment, the method comprises the steps
of: i) preparing an aqueous solution of DOTA, ii) preparing a
gadolinium:DOTA complex in water, iii) verifying free gadolinium
content in the complex, iv) verifying gadolinium:DOTA complex
formation, v) preparing gadoteric acid meglumine solution, and vi)
filtering the gadoteric acid meglumine solution. In other
embodiments, the method comprises the steps of: i) preparing an
aqueous solution of pentetic acid, ii) preparing a
gadolinium:pentetate complex in water, iii) verifying free
gadolinium content in the complex, iv) verifying
gadolinium:pentetate complex formation, v) preparing a
gadopentetate dimeglumine solution, and vi) filtering the
gadopentetate dimeglumine solution.
[0047] The present disclosure further provides a method of reducing
the risk of nephrogenic systemic fibrosis in a patient receiving a
gadolinium contrast agent comprising administering to the patient a
gadolinium contrast agent of claim 1.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0048] FIG. 1 illustrates the reaction between Gd(III), DTPA, and
meglumine to form gadopentetate dimeglumine.
[0049] FIG. 2 illustrates the molecular structure of
diethylenetriamine pentaacetic acid.
[0050] FIG. 3 illustrates the molecular structure of
diethylenetriamine pentaacetic dianhydride.
[0051] FIG. 4 is a graph comparing the amount of methanol and
ethanol in a commercially available gadopentate dimeglumine
formulation and a gadopenteate dimeglumine formulation prepared
according to the present disclosure.
[0052] FIG. 5 is a graph comparing the impurity content of a
commercially available gadopentate dimeglumine formulation to a
gadopenteate dimeglumine formulation prepared according to the
present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0053] Disclosed herein is a method of synthesizing solvent-free
gadolinium complexes which significantly reduce or eliminate the
occurrence of sub-optimal product features that may be linked to
adverse clinical outcomes. These sub-optimal product features are:
1) presence of solvent impurities, 2) variation of product pH, 3)
variation of product color, 4) variation of product thermodynamic
stability constant, 5) formation of free Gd(III) ion and 6)
formation of non-gadolinium complexes with pentetic acid.
[0054] The structure of an example of an enhanced MRI contrast
agent of the present invention is provided in FIG. 1. FIG. 1 shows
the chemical reactions between one atom of Gd(III), two molecules
of DTPA (diethylene triamine pentaacetic acid) and two molecules of
meglumine (N-methylglucamine). In steps 1-20 of the present
methods, the complex of Gd and DTPA is formed. Concurrently during
the complex formation process, the co-ligand (meglumine) is
conjugated with the complex for greater stability. The result is
gadopentetate dimeglumine (Gd-DTPA-meglumine).
[0055] Commercially sold gadopentetate dimeglumine contains
0.027-0.04% non-complexed (excess) pentetic acid contaminant
(Sources of Contamination in Medicinal Products and Medical
Devices, p. 157, Denise Bohrer). There is no theoretical
requirement that this magnitude of excess pentetic acid should be
present in commercial formulations of gadopentetate dimeglumine.
The common explanation of the excess pentetic acid is that it is
provided as a safety feature against the formation of Gd(III) ion.
In reality, the excess is there, in part, due to shifts in pH when
the dry gadolinium complex is rehydrated.
[0056] The thermodynamic stability constants describe the
equilibrium between concentrations of the Gd-complex (GdL) on one
hand and disassociated concentrations of free Gd(III) and free
ligand (L) on the other hand. Since free ligand is far safer than
free Gd(III), increasing the concentration of free ligand can
inhibit the formation of free Gd(III). In this sense, excess free
ligand can be viewed as a safety measure.
[0057] In practice it is found that in a closed environment, an
increase in the concentration of the free ligand in the formulation
of a gadolinium-based contrast agents results in a reduction of the
concentration of free Gd(III). This protective effect is more
pronounced in the group of linear ligands, in which gadopentetate
dimeglumine is a member, and less effective among cyclic ligands.
The equilibrium between the concentrations of the GdL complex and
the concentrations of the individual complex partners is shifted to
the side of the GdL complex through the use of excess ligand to
maintain the equilibrium described by the thermodynamic stability
constant.
[0058] This safety feature is somewhat dubious in that one
extremely toxic component is displaced by the existence of a less
toxic component, but the highest degree of safety is achieved when
the Gd(III) and ligand are perfectly balanced and exist only in the
form GdL.
[0059] It is natural to consider variations in product pH to be
dependent upon how and to what degree the pentetic acid forms the
complex with gadolinium. However, in practice variations in final
pH are primarily the result of removal of meglumine during the
solvent precipitation step. Secondarily, the association of
meglumine with the chelate is of interest. And that association can
also be affected by the presence of residual solvents. In order to
achieve low variance in the product pH, than control of the
environment in which the gadolinium-ligand complex is formed is
important, and in particular keeping the gadolinium complex in a
hydrated state, i.e., in an aqueous solution, can serve to reduce
the evolution of anhydride states of the ligand.
[0060] Pentetic acid is not an inert chemical compound. Review of
the Material Safety Data Sheet reveals that pentetic acid
(diethylenetriaminepentaacetic acid) carries several potential
health effects:
[0061] Potential Health Effects [0062] Inhalation Toxic if inhaled.
Causes respiratory tract irritation. [0063] Skin May be harmful if
absorbed through skin. Causes skin irritation. [0064] Eyes Causes
eye irritation. [0065] Ingestion May be harmful if swallowed
[0066] The present invention reduces the amount of intentional
excess pentetic acid without increasing the concentration of
Gd(III) ion in the product. The general approach to achieving this
end is to reduce the variability in parameters that can shift the
equilibrium toward Gd(III). The shift toward Gd(III) can result
from one or more of the following: 1) metallic contaminants
resulting in transmetallation, 2) ionic contaminants resulting in
pulling the ligand away from the Gd(III), and 3) a change in the
thermodynamic stability constant itself.
[0067] Metallic contaminants can be reduced or eliminated in the
product by precluding them energetically from forming chelates. In
this instance one can take advantage of the preference of amine
containing ligands to form complexes with gadolinium before other
metals. Thus, precision in controlling the temperature of the
reaction, and in particular taking care to avoid activation
energies characteristic for common transmetallation reactions, the
effect of contaminant induced transmetallation can be significantly
reduced.
[0068] Ionic contaminants present in the ligand that tend to
complex with the ligand by excluding Gd(III) can be reduced by
introducing scavenger species, for example carbon filtration) that
can bind to the contaminants and which are more easily removed from
the reaction than the contaminant moiety.
[0069] The thermodynamic stability constant increases (greater
stability) as pH rises. A particular ratio of Gd(III) and DTPA is
optimal for a given value of thermodynamic stability constant. The
practice of intentionally adding excess ligand arises out of the
necessity for compensating changes in the thermodynamic stability
caused by manufacturing variability of product pH and ligand loss
during the usual solvent drying process. Ligand is lost when excess
solvent is poured off the crystallized drug product.
[0070] The clinical rationale for using excess ligand in
gadolinium-based contrast agents is in part based on an increased
incidence of NSF(nephrogenic systemic fibrosis) in patients
receiving contrast agent. NSF is a very rare disease that, thus
far, has predominantly been observed in patients with severe renal
impairment. The etiology of NSF is still unknown but is thought to
be multifactorial. The particular combination and severity of
co-factors necessary to trigger the development of NSF has not, as
yet, been elucidated.
[0071] Exposure to Gd-based contrast agents (GBCAs) has been
identified as a potential risk factor for acquiring this serious
and disabling disease. This theory was first proposed in 2006. A
number of other mechanisms and potential risk factors have also
previously been proposed, including surgery and/or the occurrence
of thrombosis or other vascular injury, proinflammatory state, and
the administration of high doses of erythropoietin.
[0072] Published studies in the medical literature suggest the
incidence rates of NSF following the administration of Magnevist to
be lower than that of non-ionic linear GBCAs, ie, Omniscan. Since
ionic linear GBCAs are more stable than non-ionic, this suggests
NSF may be linked to the thermodynamic stability of metal-ligand
complexes contained in GBCAs.
[0073] The importance of water in all steps of the drug preparation
process can be understood by considering the anhydride state of
diethylenetriaminepentaacetic acid. FIG. 2 illustrates the
diethylenetriamine pentaacetic acid molecule. The sites A can bond
to sites B under anhydrous conditions to form two hexacyclic
structures and the release of two molecules of water. The resulting
molecule is either diethylenetriamine pentaacetic anhydride, or in
saturation diethylenetriaminepentaacetic dianhydride (illustrated
in FIG. 3).
[0074] The anhydride of diethylenetriaminepentaacetic is less
acidic than diethylenetriaminepentaacetic. This can be a source of
the observed pH variation of current product pH, and ultimately the
cause of variation of the thermodynamic stability constant.
[0075] For example, looking at commercial specifications for
diethylenetriaminepentaacetic dianhydride (Sigma-Aldrich, St.
Louis, Mo.), it can be seen that, depending on the degree of
dehydration, diethylenetriaminepentaacetic dianhydride varies in
color from colorless to very dark yellow. This observation is
consistent with labeling of Magnevist, and suggests some
diethylenetriaminepentaacetic dianhydride is present in Magnevist.
Further, the source of this contaminant can be associated with the
anhydrous precipitation of the gadolinium complex in solvent.
[0076] The effect of the presence of diethylenetriaminepentaacetic
dianhydride can be seen by considering its acid number: Titration
with NaOH 97.5-102.5%.
[0077] In a manufacturing process that yields a product that
contains diethylenetriaminepentaacetic dianhydride might be
titrated down to a pH specification by excess addition of
diethylenetriamine pentaacetic after the conjugation step. When the
commercial product is formulated, it is formulated in an aqueous
state. Consequently, the relatively basic anhydride is converted to
the more acidic diethylenetriamine pentaacetic, which reduces the
product pH, reduces the thermodynamic stability constant, and
results in the formation of Gd(III) ion.
[0078] If acid groups are still present in the resulting complex
salt, it is often advantageous to convert the acidic complex salt
into a neutral complex-salt by reaction with inorganic and/or
organic bases or amino acids, which form physiologically
biocompatible cations, and isolate them. In many cases, the
procedure is unavoidable since the dissociation of the complex salt
is moved toward neutrality to such an extent by a shift in the pH
value during the preparation that only in this way is the isolation
of homogeneous products or at least their purification made
possible. Production is advantageously performed with organic bases
or basic amino acids. It can also be advantageous, however, to
perform the neutralization by means of inorganic bases (hydroxides,
carbonates or bicarbonates) of sodium, potassium or lithium.
[0079] The present novel method for synthesis of gadopentetate
dimeglumine encompasses all the considerations described above to
provide a product that: 1) contains no solvent residues, 2)
possesses a product pH variation in the range of 7.2 to 7.5, 3) is
colorless, 4) possesses a product thermodynamic stability constant
variation in the range of 18.2-18.6, 5) less than 1 ppm free
Gd(III) ion and 6) less than 10 ppm non-gadolinium complexes with
pentetic acid.
[0080] The present methods advantageously take aqueous solution to
a drug product concentration and purity, without the need to obtain
a dry powder of gadolinium complex is advantageous. The absence of
organic solvent in the drug product is a distinguishing
characteristic of the present novel drug compounds. In practice, it
is impossible to remove all solvent from a gadolinium contrast
agent when a solvent other than water is used during synthesis. All
present gadolinium contrast agents have measureable solvent
contaminations; and in most commercial products one or more
solvents represent the most impurities.
[0081] Generally, contrast agents of the invention may be
pre-formed, or may alternatively be prepared directly before
administration, by mixing in aqueous solution the chelating agent
and a soluble compound containing the paramagnetic metal with a
physiologically acceptable counter ion. Generally, the chelating
entity is itself in salt form. The counterion should also be
physiologically acceptable and may, for example, be meglumine.
[0082] Furthermore, solvents such as acetone, various alcohols,
heptane and the like form complexes with naturally occurring
impurities present in the ligand. The solvents intended to create a
crystalline pure drug compound ironically bind impurities in the
drug product. Ultimately, the solvents primarily serve as
azeotropic agents for removing water and not as purifying agents.
Solvents are not used in the present invention.
[0083] The product gadopentetate dimeglumine is hygroscopic and
difficult to separate from the water used in the synthesis. The
final product form is formulated as an aqueous solution.
Consequently, it is important to determine the final mass or mass
proportion of gadopentetate dimeglumine in a batch output. This can
be done using the HPLC analysis techniques disclosed here. It is a
simple matter to determine how much additional water is to be added
to a particular hydrated batch product to obtain a desired product
formulation specification.
Examples
[0084] In the following, absolute weights of ingredients and
volumes of the equipment used are illustrative only, and it should
be understood that it is the mass ratios between ingredients that
is the important aspect of this invention. Those ingredients are
given in the Table below.
TABLE-US-00002 Ingredient Mol. Wt. Amount Equivalents Amount in
Example DTPA 393.35 1 mole 393.35 g/mol 393.35 kg Gd(III) oxide
362.50 1/2 mole 181.25 g/mol Gd 181.25 kg n-methylglucamine 195.21
2 moles 390.42 g/mmol 390.42 kg
[0085] The following procedure is the result of complicated
equilibria calculations involving the calculation of protonation
constants, thermodynamic stability constants, and equilibrium
speciation diagrams.
[0086] Purification of Ligand
[0087] As an example, a procedure for purification of DTPA is
provided. It should be understood the procedure is applicable to
all ligands used in forming complexes of gadolinium useful as
contrast agents in medical imaging.
[0088] In a 5.0 litre four neck round bottom flask equipped with a
mechanical stirrer, condenser and heating jacket was charged 3000
ml of distilled water (pH 7) and 500 g of DTPA at 25 to 30.degree.
C.
[0089] After stirring for 10 minutes, raise the temperature of the
reaction mixture to 95-100.degree. C., stir until the mixture forms
a clear solution and maintain at this temperature for 0.5 hrs after
forming clear solution.
[0090] Cool the reaction mixture to 35-40.degree. C. and stir for 1
h. The DTPA should precipitate out of solution as a
crystallite.
[0091] Filter the slurry through a Buchner funnel and wash with
distilled water. Perform HPLC and quantify impurities, total
impurities should be less than 0.5% NN. If not, return to step
1.
[0092] Dry the purified DTPA under reduced pressure at
55-60.degree. C. for 1 h.
[0093] Determination of % Free Ligand
[0094] It is important to determine the amount of free ligand in
the complexation synthesis between gadolinium and ligand. In this
example, an HPLC procedure is provided for determining % w/w of
free DTPA compared to the weight of the complex in a solution of
gadolinium-DTPA complex.
[0095] Chromatographic System: [0096] Instrument: Agilent 1200
Series (or) equivalent [0097] Column: Hypersil MOS-1, 150.times.4.6
mm, 5.mu. [0098] Wavelength: 195 nm [0099] Mobile Phase: [0100]
Pump `A`: 10 mM potassium phosphate monobasic in water, pH 3.0 with
Orthophosphoric acid (0.1 M) [0101] Pump `B`: 1.5 mM
Tetra-n-butylammonium perchlorate in Acetonitrile: Water (20:80)
[0102] Isocratic: A:B (30:70) [0103] Flow Rate: 1.5 ml/min [0104]
Column oven: 25.degree. C. [0105] Injection Volume: 20 .mu.l [0106]
Run Time: 20 minutes [0107] Diluent: Water
Definitions
[0108] Purity: The mass amount of gadopentetate dimeglumine
relative to other HPLC peaks. The purity is not specific to
dilution, the amount of water in the sampled API does not change
the purity.
[0109] Wet API sample Potency: The mass amount of gadopentetate
dimeglumine API in a mass amount of diluent, usually water. Applies
to in-process/wet API.
General Notes:
[0110] The following flask sizes and standard weights may be
adjusted as long as the final concentration of each solution is
maintained. Sonication or other appropriate means may be used to
aid dissolution. Mix each solution until all solids are
dissolved.
DTPA Standard Preparation:
[0111] Prepare a solution containing 0.0033 mg/ml of purified DTPA
in diluent. Weigh about 0.33 mg of DTPA in a 100 ml volumetric
flask, add 10 ml of diluent, gently warm up to 60 degree in water
bath and allow to cool. Swirl to dissolve. Bring up to volume with
water.
API Sample Preparation (Wet or Dry):
[0112] Prepare a solution that yields a product value=(API
concentration (mg/ml).times.Wet API Potency) in the range of 5-10
mg/ml of API in water. In other words, when the solution is made it
should satisfy:
5<(API concentration (mg/ml).times.Wet API Potency)<10 mg/ml
of API in water
[0113] Weigh about 500.0.times. (1/Potency) mg of API in a 100 ml
volumetric flask, add 10 ml of diluent, mix thoroughly. Bring up to
volume with diluent. Assess by HPLC that the concentration of
Gadopentetate dimeglumine meets target specification.
Evaluation of System Suitability:
[0114] DTPA retention time should be about 4.5 minutes. The
chromatographic procedure is set forth in the following table:
TABLE-US-00003 Description # Injection Blank 3 DTPA Standard 5 API
solution 2
[0115] The gadopentetate dimeglumine peak will be quite large in
the API sample, the mV scaling of the HPLC should be adjusted so
that the DTPA peak is discernible. It should be verified visually
that the DTPA peak and the Gadopentetate Dimeglumine peak in the
API sample do not overlap. Overlap will give an erroneous
integrated area.
Reporting Criteria:
[0116] Integrate only the DTPA peaks in the DTPA standard and API.
Calculate % Free DTPA relative the API (w/w) using one the
following equations:
% .times. .times. Free .times. .times. DTPA /Wet .times. .times. A
.times. .times. P .times. .times. I .times. .times. sample .times.
.times. ( w / w ) = Area .times. .times. A .times. .times. P
.times. .times. I .times. .times. D .times. .times. T .times.
.times. P .times. .times. A .times. .times. peak .times. Std
.times. .times. D .times. .times. T .times. .times. P .times.
.times. A .times. .times. Concentration .times. .times. ( mg/ml)
.times. Purity .times. .times. D .times. .times. T .times. .times.
P .times. .times. A .times. 100 .times. % / Mean .times. .times.
peak .times. .times. area .times. .times. D .times. .times. T
.times. .times. P .times. .times. A .times. .times. standard
.times. .times. ( n = 5 ) .times. A .times. .times. P .times.
.times. I .times. .times. concentration .times. .times. ( mg/ml) (
1 ) % .times. .times. Free .times. .times. DTPA / API .times.
.times. ( w / w ) = ( 1 ) .times. ( 1 / Wet .times. .times. A
.times. .times. P .times. .times. I .times. .times. Potency ) ( 2 )
##EQU00001##
[0117] Potency is obtained from the API HPLC Potency Procedure.
Determination of Potency of Gadolinium-Ligand Complex.
[0118] In this example, an HPLC procedure is provided for
determining % w/w of gadolinium-ligand complex compared to the
weight of the solution of gadolinium-DTPA complex. This method
determines the chromatographic potency of gadopentetate dimeglumine
drug substance by HPLC with UV detector: [0119] Chromatographic
System: [0120] Instrument: Agilent 1200 Series (or) equivalent
[0121] Column: Hypersil MOS-1, 150.times.4.6 mm, 5.mu. [0122]
Wavelength: 195 nm [0123] Mobile Phase: [0124] Pump `A`: 10 mM
potassium phosphate monobasic in water, pH 3.0 with dilute
orthophosphoric acid (0.1 M) [0125] Pump `B` [0126] Isocratic: A:B
(30:70) [0127] Flow Rate: 1.5 ml/min [0128] Column oven: 25.degree.
C. [0129] Injection Volume: 20 .mu.l [0130] Run Time: 20 minutes
[0131] 1.5 mM Tetra-n-butylammonium perchlorate in Acetonitrile:
Water (20:80)
[0132] The following flask sizes and standard weights may be
adjusted as long as the final concentration of each solution is
maintained. Sonication or other appropriate means may be used to
aid dissolution. Mix each solution until no solid remains.
Stock Standard Preparation:
[0133] Prepare a solution containing 0.5 mg/ml of Gadopentetate
Dimeglumine standard in diluent. For example, weigh about 50.0 mg
of Gadopentetate Dimeglumine standard in a 100 ml volumetric flask,
add 30 ml of diluent, and gently swirl to dissolve and dilute to
volume with diluent.
Working Standard Preparation:
[0134] Prepare a solution containing 0.005 mg/ml of Gadopentetate
Dimeglumine standard in diluent. Pipette 2.0 ml into a 200 ml
volumetric flask, add diluent to mark and mix well.
Sample Preparation (API):
[0135] Prepare a solution containing 5.0 mg/ml of sample in
diluent. Weigh about 100.0 mg of sample in a 20 ml volumetric
flask, add 10 ml of diluent and gently swirl to dissolve. Bring up
to volume with diluent.
Evaluation of System Suitability:
[0136] % RSD for retention time for the first 5 injections of
standard solution is not more than 2.0%. % RSD for peak area for
the first 5 injections and throughout the run of standard solutions
is not more than 15%. Diluent peak should not show any interfering
peaks at retention time of Gadopentetate peak greater than 5% of
the peak area response of Gadopentetate peak from the analysis of
the working standard. Tailing factor for the working standard
solution should not be more than 2, assess based on the 1st
Injection of working standard solution.
[0137] Integrate only the gadopentetate dimeglumine peaks in the
sample and in the working standard.
[0138] Calculate the Potency (% w/w) of gadopentetate dimeglumine
API in the Wet API sample using the following equation:
Potency API/Wet API sample (% w/w)=Area Gad. Dimeg. peak in
Sample.times.Standard Concentration (mg/ml).times.100%/Mean peak
area of working standard (n=5).times.sample concentration
(mg/ml)
Determination of Purity of Gadolinium-Ligand Complex
[0139] In this example, an HPLC procedure is provided for
determining % w/w of non-complex moieties (impurities) compared to
the weight of the complex in a solution of gadolinium-DTPA complex.
This method determines the chromatographic purity of Gadopentetate
Dimeglumine drug substance by HPLC with UV detector: [0140] Column:
Hypersil MOS-1, 150.times.4.6 mm, 5 .mu.m, Part #30205-154630
[0141] Purified Water or HPLC Grade, Fisher Scientific Cat #W5-4 or
equivalent [0142] Acetonitrile, HPLC Grade, Fisher Scientific Cat
#A996-4 or equivalent [0143] Tetra-n-butylammonium Perchlorate,
Alfa-Aesar Cat #30801 or equivalent [0144] Potassium Phosphate
monobasic, ACS grade, BDH Cat #BDH9268 or equivalent [0145]
Phosphoric Acid, HPLC grade, EMD Cat #PX0996-6 or equivalent [0146]
Meglumine (N-Methylglucamine), Acros Organics Cat #126841000 or
equivalent [0147] Gadopentetate Dimeglumine Reference Standard
[0148] Analytical Balance capable of reading 0.01 mg [0149]
Calibrated pH meter [0150] Class "A" volumetric glassware
Solution Preparations
[0151] Dilute Phosphoric Acid: Pipette 5.0 mL of Phosphoric acid
into a 50-mL volumetric flask and dilute to volume with purified
water, mix well.
[0152] Mobile Phase A: (10 mM Potassium Phosphate monobasic in
water) Weigh accurately 1.36 g of Potassium phosphate monobasic
(KH2P04) and transfer into a 1 L HPLC bottle already containing
1000 mL of water and mix well. Scale up the volume as required.
Adjust to pH 3.0 using dilute phosphoric acid.
[0153] Mobile Phase B: (1.5 mM Tetra-n-butylammonium perchlorate in
Acetonitrile: Water/20:80) Weigh and transfer accurately 0.51 g of
tetra-n-butylammonium perchlorate into a 1 L HPLC bottle. Add 200
mL acetonitrile and dissolve the solids. Add 800 mL of purified
water and mix it well. Scale up the volume as required.
[0154] Diluent: Purified or HPLC grade water.
[0155] Meglumine 41.5% Solution: weigh about 41.5 mg of Meglumine
in to a 20-mL volumetric flask. Dissolve and dilute to mark with
diluent and mix well.
[0156] Note: Use Anti-static gun to remove charge from spatula,
hand gloves, sample standard bottle and weighing pan or volumetric
flasks while weighing gadopentetate dimeglumine standard and
sample.
Stock Standard Preparation (0.5 mg/mL)
[0157] Weigh accurately 50 mg.+-.1 mg of Gadopentetate Dimeglumine
Reference Standard into a 100-mL volumetric flask. Add
approximately 30 mL of diluent to the flask and gently swirl or
vortex to dissolve. Bring up to volume with diluent. Sonicate for
about 5 minutes, if necessary, to ensure complete dissolution of
the material. Allow to cool to room temperature and Mix the
solution well. Stock Standard Concentration (mg/mL)=Standard Weight
(mg).times.Decimal Purity/100 mL.
Working Standard Preparation (0.005 mg/mL)
[0158] Accurately pipette 2.0 mL of Gadopentetate Dimeglumine stock
Standard into a 200 mL volumetric flask. Add diluent to the mark
and mix well. Transfer the standard solution into HPLC vial and
seal for analysis. Working Standard Concentration (mg/mL)=Stock
standard concentration (mg/ml).times.2.0 mL/200 mL.
Sample Preparations
[0159] Weigh 100 mg.+-.1 mg of Gadopentetate Dimeglumine sample
into a 20-rnL volumetric flask. Add approximately 10 mL of diluent
to the flask and gently swirl or vortex to dissolve. Bring up to
volume with diluent. Sonicate for about 5 minutes, if necessary, to
ensure complete dissolution of the material. Allow to cool to room
temperature and mix the solution well. Transfer the sample solution
into a HPLC vial and seal for analysis. Sample Concentration
(mg/mL)=Sample Weight (mg)/20 mL.
Instrument Operating Conditions
[0160] Typical starting column pressure is approximately 96
bar.
TABLE-US-00004 A. 10 mM Potassium Phosphate monobasic in Water
Mobile Phase: B: 1.5 mM TBAP in Acetonitrile: Water/20:80 Mobile
Phase ration (Isocratic): Time Solvent A (%) Solvent B (%) 0 30 70
15 30 70 Flow Rate: 1.5 mL/min Total Run Time: 20 min Column
Temperature: 25.degree. C. Detection Wavelength: 195 mm Injection
Volume: 20 .mu.l
Operating Procedure
[0161] Injection Sequence:
[0162] (Inject Standard Solution after every 5 sample preparation
injections.) [0163] 1. Blank (3.times., at least to ensure a clean
baseline) [0164] 2. Working Standard Solution (5.times.) [0165] 3.
Meglumine 41.5% Solution (1.times.) [0166] 4. Blank (1.times.)
[0167] 5. Sample Solution (2.times., for each sample) [0168] 6.
Blank (1.times.) [0169] 7. Standard Solution (1.times.)
System Suitability
[0169] [0170] 1. Diluent blank does not show any interfering peaks
at retention time of Gadopentetate peak greater than 5% of the peak
area response of Gadopentetate peak from the analysis of the
working standard solutions. [0171] 2. Tailing factor for the
working standard solution is NMT 2; assess based on the 1st
injection of working standard solution. [0172] 3. % RSD for
Retention time for the first 5 injections of Standard Solutions is
NMT 15%. [0173] 4. % RSD for Peak area for the first injections and
throughout the run of Standard Solutions is NMT 15.
[0174] Calculations [0175] 1. Integrate all peaks excluding peaks
present in blank and Meglumine 41.5% solution injection (Meglumine
and related peaks). [0176] 2. Calculate % Weight of Gadopentetate
Dimeglumine related substances using the following equation:
[0176] % Wt/Wt=Peak area of related substances.times.Standard
Concentration (mg/mL).times.100%/Mean Peak area of working standard
(n=6).times.Sample Concentration (mg/mL)
Example 1: Solvent-Free Gadopentetate Dimeglumine
[0177] Raw Materials
TABLE-US-00005 Raw Material Mole Ratio 1 DTPA 1 2 Gd.sub.2O.sub.3
0.496 3 Water 2.2 V.sup.1 (DTPA) 4 Meglumine 0.992
Preparation of DTPA Solution
[0178] 1. Heat reactor to 25-30.degree. C. [0179] 2. Charge water
[0180] 3. Begin stirring (stir unless otherwise indicated, nominal
rate 300 rpm) [0181] 4. Charge 10% of the DTPA [0182] 5. Stir until
uniformly distributed in the water [0183] 6. If all the DTPA is
charged, then go to step 8 [0184] 7. Go to step 4 [0185] 8. Stir 10
min
Preparation of Gadolinium: DTPA Complex
[0185] [0186] 9. Charge 25% by weight of the Gadolinium oxide
[0187] 10. Stir until uniformly distributed [0188] 11. If all the
Gadolinium oxide is charged then go to step 13 [0189] 12. Go to
step 9 [0190] 13. Stir 10 min [0191] 14. Raise temperature to
95+/-2.degree. C. [0192] 15. Stir 3 hrs. [0193] 16. Check clarity
[0194] 17. If not clear continue for 1 hr, go to step 16 [0195] 18.
If clear, continue 1 hr and then cool to 40-45.degree. C. [0196]
19. If precipitate forms, heat to 95+/-2.degree. C. and stir for 1
hr, go to step 16
Verify Complex Formation
[0196] [0197] 20. Verify absence of free gadolinium using Xylenol
orange [0198] 21. If free gadolinium detected, add 0.05% additional
DTPA, raise temperature to 95+/-2.degree. C., stir for 1 hr and
proceed to step 16 [0199] 22. If not, proceed to step 23
Preparation of Gadopentetate Dimeglumine Solution
[0199] [0200] 23. Add 90% of the Meglumine at 40-45.degree. C.
[0201] 24. Stir until in solution .about.1 hr [0202] 25. Measure
pH--inline probe calibrated to 25.degree. C. (USP) [0203] 26. If pH
is >7.5, discard [0204] 27. If pH is between 7.0 and 7.5, then
go to step 29 [0205] 28. If pH<than 7.0, add 1% of the
Meglumine, stir for 10 min, go to step 25 [0206] 29. Stir for 1 hr
at 40-45.degree. C. [0207] 30. Check solution is clear, if yes
proceed to 31, if not repeat 29
Gadopentetate Dimeglumine Solution Filtration
[0207] [0208] 31. Measure pH--inline probe calibrated to 25.degree.
C. (USP) [0209] 32. If pH is between 7.0 and 7.5, then go to step
34 [0210] 33. If pH<than 7.0, add Meglumine, stir for 10 min, go
to step 31 [0211] 34. Filter the solution using the carbon filter
[0212] 35. Rinse the reactor with 20-25.degree. C. water using 1/4
V [0213] 36. Pass rinse through the filter [0214] 37. Repeat rinse
steps 35 & 36 for a total of 2 rinses [0215] 38. Place filtrate
and rinses back in reactor [0216] 39. Stir at 40-45.degree. C. for
10 min [0217] 40. Verify absence of free gadolinium using Xylenol
orange [0218] 41. If free gadolinium detected, add 0.05% additional
DTPA, stir for 1 hr, and go to step 40 [0219] 42. If not, proceed
to step 43 [0220] 43. Measure pH--inline probe calibrated to
25.degree. C. (USP) [0221] 44. If pH is between 6.0 and 6.6, then
go to step 46 [0222] 45. If pH<6.0, add Meglumine. Stir 10 min.
Go to step 43. [0223] 46. Stir 1/2 hr. [0224] 47. Check solution is
clear, if yes proceed to 48, if not go to step 46
Verify Purity
[0224] [0225] 48. Measure Purity by HPLC [0226] 49. If individual
impurity >0.05%, go to step 31
Final API Adjustments
[0226] [0227] 50. Measure Free DTPA by HPLC [0228] 51. If Free DTPA
>0.06% ww, go to step 31 [0229] 52. If Free DTPA is 0.01-0.06%
ww proceed to 56 [0230] 53. If Free DTPA <0.01% ww, add 0.05% of
DTPA [0231] 54. Stir 1 hr [0232] 55. Go to Step 50 [0233] 56.
Measure pH [0234] 57. If pH is 6.0-6.6, then go to step 59 [0235]
58. If pH<6.0, add Meglumine. Stir 10 min. Go to step 56
Final API Testing
[0235] [0236] 59. Perform full API testing: Gadolinium content;
Meglumine Content; Assay; Water Content; Heavy Metals
Comparison: Present Invention and Magnevist
[0237] The brand name for Gadopentetate Dimeglumine is Magnevist.
Using HPLC, direct comparison of solvent content in Magnevist can
be made to the present invention. FIG. 4 depicts a representative
sample of the parts per million of solvent in commercially
available Magnevist. By not using solvents in the manufacturing
process the amount of all impurities (including non-solvent
impurities) is improved.
[0238] In FIG. 5, the impurity content of Magnevist (Gd reference
std) is compared to the complex of the present invention "purified"
with solvents (Gd solvent sample) and using the solvent-free
procedure (Gd solventless sample). These data illustrate an overall
improvement in impurity levels when solvents are removed from the
drug product process.
Example 2: Solvent-Free Gadoterate Meglumine
[0239] Preparation of DOTA Solution [0240] 1. Heat reactor to
25-30.degree. C. [0241] 2. Charge water [0242] 3. Begin stirring
(stir unless otherwise indicated, nominal rate 300 rpm) [0243] 4.
Charge 10% of the DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) [0244]
5. Stir until uniformly distributed in the water [0245] 6. If all
the DOTA is charged, then go to step 8 [0246] 7. Go to step 4
[0247] 8. Stir 10 min
[0248] Preparation of Gadolinium: DOTA Complex [0249] 9. Charge 25%
by weight of the Gadolinium oxide [0250] 10. Stir until uniformly
distributed [0251] 11. If all the Gadolinium oxide is charged then
go to step 13 [0252] 12. Go to step 9 [0253] 13. Stir 10 min [0254]
14. Raise temperature to 95+/-2.degree. C. [0255] 15. Stir 3 hrs.
[0256] 16. Check clarity [0257] 17. If not clear continue for 1 hr,
go to step 16 (this step took about 12 hours, slower than Magnevist
synthesis) [0258] 18. If clear, continue 1 hr and then cool to
40-45.degree. C. [0259] 19. If precipitate forms, heat to
95+/-2.degree. C. and stir for 1 hr, go to step 16
[0260] Verify Complex Formation [0261] 20. Verify absence of free
gadolinium using Xylenol orange [0262] 21. If free gadolinium
detected, add X DOTA, raise temperature to 95+/-2.degree. C., stir
for [0263] 1 hr and proceed to step 16 [0264] 22. If not, proceed
to step 23
[0265] Preparation of Gadolinium: DOTA Complex [0266] 23. Add 90%
of the Meglumine at 40-45.degree. C. [0267] 24. Sir 10 minutes
[0268] 25. Measure pH--inline probe calibrated to 25.degree. C.
(USP) [0269] 26. If pH is >7.5, discard [0270] 27. If pH is
between 7.0 and 7.5, then go to step 29 [0271] 28. If pH<than
7.0, add 2% of the Meglumine, go to step 24 [0272] 29. Stir for 1
hr at 40-45.degree. C. [0273] 30. Check solution is clear, if yes
proceed to 31, if not repeat 29
[0274] Gadoteric Acid Meglumine Solution Filtration [0275] 31. Cool
the solution to 20-25.degree. C. [0276] 32. Filter the solution
using the carbon filter [0277] 33. Rinse the reactor with
20-25.degree. C. water using 1/4 V [0278] 34. Pass rinse through
the filter [0279] 35. Repeat rinse steps 33 & 34 for a total of
2 rinses [0280] 36. Place filtrate and rinses back in reactor
[0281] 37. Stir at 25-30.degree. C. for 10 min [0282] 38. Measure
Free DOTA by HPLC [0283] 39. If Free DOTA is 0.01-0.06% ww proceed
to 42 [0284] 40. If Free DOTA <0.01% ww, add 0.03% ww equivalent
of DOTA [0285] 41. Stir for 1/2 hr and go to step 38 [0286] 42.
Measure pH--inline probe calibrated to 25.degree. C. (USP) [0287]
43. If pH is between 7.0 and 7.5, then go to step 45 [0288] 44. If
pH<7.0, add Meglumine. Stir 10 min. Go to step 42. [0289] 45.
Stir 1/2 hr. [0290] 46. Check solution is clear, if yes proceed to
47, if not repeat 45
[0291] Verify Purity [0292] 47. Measure Purity by HPLC [0293] 48.
If individual impurity >0.05%, go to step 32
[0294] Final API Adjustments [0295] 49. Measure Free DOTA by HPLC
[0296] 50. If Free DOTA >0.06% ww, repeat steps 32-42 [0297] 51.
If Free DOTA is 0.01-0.06% ww proceed to 55 [0298] 52. If Free DOTA
<0.01% ww, add 0.03% ww equivalent of DOTA [0299] 53. Stir 1/2
hr [0300] 54. Go to Step 49 [0301] 55. Measure pH [0302] 56. If pH
is 7.0-7.5, then go to step 53 [0303] 57. If pH<7.0, add
Meglumine. Stir 10 min. Go to step 55
[0304] Final API Adjustments [0305] 58. Perform full API testing:
Gadolinium content; Meglumine Content; Assay; Water Content; Heavy
Metals
[0306] The effect of reaction time on free Gd (III) ion content was
evaluated by carrying out reactions as described above for various
periods of time. The free Gd (III) ion content was determined using
the xylenol orange method. See Jeong, Y. and Na, K., "Synthesis of
a gadolinium based-macrocyclic MRI contrast agent for effective
cancer diagnosis," Biomaterials Research (2018) 22:17. The
resulting aqueous formulations, comprising a complex of Gd(III)
ion, ligand and meglumine, contained not more than 0.025% by weight
of free ligand, less than 50 parts per million of non-aqueous
solvent, and various amounts of free Gd (III) ion. The results
tabulated below show that reactions as described above can be used
to obtain such formulations having a broad range of levels of free
Gd (III) ion, such as less than 10,000 ppm, less than 5,000 ppm,
less than 1,000 ppm, less than 500 ppm, less than 100 ppm, less
than 25 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm,
less than 0.5 ppm, or less than 0.1 ppm, by controlling the
reaction time.
TABLE-US-00006 Reaction Time, hr Free Gd(III), ppm 3 28,094 .+-.
9.7 8 3,853 .+-. 12.4 24 72 .+-. 5.0 32 0.8 .+-. 1.3
[0307] Production of a medical contrast media according to the
invention can be clinically formulated in a way known in the art.
For example, the gadopentetate dimeglumine solution is diluted in
an aqueous medium and then the solution or suspension is
sterilized. Suitable additives include, for example,
physiologically biocompatible buffers (as, for example,
tromethamine hydrochloride), slight additions of complexing agents
(as, for example, DTPA) or, if necessary, electrolytes (for
example, sodium chloride).
[0308] Preferred embodiments of this invention are described
herein. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *