Process for producing a complex of a lanthanide with a macrocyclic ligand.
Description
Technical Field
[0001] The present invention relates to a process for producing a complex of a
lanthanide or a similar compound with a ligand and which can be used as
contrast agent for magnetic resonance imaging.
Background Art
[0002] Magnetic resonance imaging (MRI) is a powerful, non-invasive technique
used to produce detailed two or three-dimensional anatomical images of
tissues in the body. Conventional MRI uses the proton H as its signal
source which is highly abundant in tissues and it has the highest sensitivity
of all the biologically relevant nuclei.
[0003] The contrast, which makes the differentiation of internal structures
possible in the image, arises from how the signal decays and is the
difference between the resulting signals from two tissue regions. The route
by which the protons release the energy they absorbed from the radiofrequency
pulse, thus reducing the transverse magnetisation and causing
signal decay, is known as relaxation. In MRI two independent relaxation
processes occur simultaneously: spin-lattice or longitudinal relaxation
characterised by the time constant 7 , and spin-spin or transverse
relaxation, characterised by the time constant T .
[0004] Often, when suitable 7 - or ^-weighting sequences are used, the natural
contrast between two tissues is enough to produce a diagnostically-useful
image. However, some conditions do not lead to specific enough changes
in the relaxation times of the affected tissue though and then a contrast
agent is used to locally change the relaxation times of the diseased tissue,
improving the image contrast.
[0005] Most contrast agents work by shortening the relaxation times of the water
protons in the targeted tissue. 7Ϊ contrast agents are based on
paramagnetic metal ion chelates which make the tissue appear brighter on
the 7i-weighted image (positive contrast). T contrast agents are usually
superparamagnetic iron oxide nanoparticles which create dark spots on
the ^-weighted image (negative contrast). 7 agents are the most widely
used and the majority of these are based on chelates of the gadolinium ion
Gd(lll).
[0006] To be an effective 7 agent the lanthanide chelate must significantly
increase the proton relaxation rates in water. Lanthanide elements are
most commonly found in the +3 oxidation state (Ln+3) , corresponding to
the electronic configuration [Xe]6s24fn. Gadolinium (Gd) is the seventh
element in the lanthanide series and has an electronic configuration
[Cb]4 . This means that Gd(lll) has seven unpaired electrons, making it
highly paramagnetic i.e. Gd(lll) ions have large permanent magnetic
moments (due to electron spin angular momentum), but in the absence of
an external magnetic field these are randomly oriented. Due to its large
size the Gd(lll) and other lanthanides ions typically have a coordination
number of nine in its complexes.
[0007] Free ions of lanthanides, and in particular gadolinium, are very toxic for the
tissues. Indeed, a pathology known as NSF (nephrologic systemic fibrosis,
or fibrogenic dermopathy, with very severe effects on human skin), may be
at least partly correlated to the existence of free gadolinium ions, i.e. noncomplexed
gadolinium, in the body. This disease has led to health
authorities being alerted with respect to marketed gadolinium-based
contrast agents. Briefly, NSF could be associated to the transmetallation of
some lanthanide from the complex [lanthanide-chelate] by endogenic ions
such as zinc and resulting in unwanted release of free lanthanide ions.
[0008] The level of toxicity depends on the strength of the chelating agent, also
known as ligand, chelator or sequestering agent to form a complex with
the lanthanide ions. Usually these ligands are organic compounds which
form two or more separate coordinate bonds with a single central metal
ion, in this case, the lanthanide ion, inactivating it and thus reducing or
eliminating its toxic effect in the tissues.
[0009] Polyaminopolycarboxylic acid compounds are the ligand type of choice
because they form exceptionally stable complexes with the Gd(lll) ion,
which can be explained by a number of reasons. These compounds can
be linear (such as pentetic acid or diethylene triamine pentaacetic acid
also named as DTPA) or macrocyclic (such as 1,4,7,10-
tetraazacyclododecane-1 ,4,7,1 0-tetraacetic acid, DOTA). Complexes of
macrocyclic ligands are much more kinetically inert and thus, present an
exceptionally high solution stability.
[0010] Several contrast agents comprising gadolinium as the lanthanide are
marketed, Magnevist® (Bayer Healthcare), Multihance® (Bracco),
Omniscan® (GE Healthcare), Optimark® (Mallinckrodt Inc.), Dotarem®
(Guerbet), Prohance® (Bracco) and Gadovist® (Bayer Healthcare).
[001 1] EP0270483 discloses contrast agents based on gadolinium with addition
of one or more free ligands, such DOTA ( 1 ,4,7,1 0-tetraazacyclododecane-
1,4,7,1 0-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid) or
DTPA (diethylene triamine pentaacetic acid) and/or one or more weak
metal complexes (presenting relatively low stability constant, such as
calcium, magnesium, zinc and iron). The metal ions of the weak metal
complex must however be removed via an extra purification step e.g.
ultrafiltration to obtain a pharmaceutical formulation which can be directly
administrated to the patient.
[0012] US4647447 discloses complex salts from the anion of a complexing acid
and one or more central ions of an element with an atomic number of 2 1 to
29, 42, 44 or 57 to 83 and, optionally, also formed from one or more
physiologically biocompatible cations of an inorganic and/or organic base
or amino acid. This document also discloses the production of contrast
agents without isolating the respective complex salts. In this case, to avoid
the presence of free toxically active metal ions, such as Gd(lll), the
chelating step is done by using colour indicators such as xylenol orange by
control titrations. If the chelates take up water very quickly, it is not
possible to assure that the correct balance between chelate and
lanthanide is present in the body, in particular when other ions are present,
such Ca2+, Zn+2 and Cu2+ which are able to replace the lanthanide ion in
some extent, in the complex and thus releasing these to the body.
Moreover, no step is disclosed in the method too guarantee that, if an
excess of chelate is obtained, the concentration of the free chelate is less
than the upper limit of the specifications of the liquid pharmaceutical
formulation.
[0013] WO201 0130814 discloses a process for preparing contrast agents in
powder form based on a lanthanide chelate, said powder including an
excess of free chelate of 0.002% to 0.4% mol/mol to address the problem
of in vivo intolerance of lanthanide chelates related to the presence of free
lanthanide ions in the formulation to be administered. Said process
includes a step a) of mixing the chelate and the lanthanide wherein the
free chelate is in excess in relation to the amount of the lanthanide; b)
measuring the amount of free chelate and adjusting said amount to an
excess of 0.002 to 0.4% mol/mol in relation to the amount of the
lanthanide; and c) precipitating the solution containing the complex
obtained with or without the adjustment step in an organic solvent, thus
obtaining a powder of chelate-lanthanide, wherein said powder contains
an amount of free chelate in excess in relation to the amount of the
lanthanide. Besides the fact that it is not possible to assure that the correct
balance between chelate and lanthanide is present in the final formulation
to be administered, a redissolution of the powder to obtain an injectable
pharmaceutical formulation is required.
[0014] The undesired release of lanthanide ion is prevented by an amount of
excess of ligand. In US 5 876 695 the ligands (ligands L) added in excess
are described under the form of an excipient having the formula X[X',L],
where X and X' are metal ions (especially calcium, sodium, zinc or
magnesium) and L is the ligand in excess. These excipients are designed
to scavenge free lanthanide. Table 1 of US 7 385 041 illustrates with LD50
values that free macrocyclic ligands (HP-D03A, D03A, DOTA) are about
at least 10 times more toxic than these macrocycles under the form X[X'
,L]. In particular for DOTA, the LD50 is at least about 40 times better for
Na2[Ca-DOTA] than for free DOTA. The formulation of a macrocyclic
ligand administered to the patient should contain, besides the macrocyclic
ligand complexed by the lanthanide, an excess of free macrocyclic ligand
but in a low range. The free ligand L is not complexed with any metal ions
and in particular not under the form of an excipient X[X',L]
[0015] In WO20091 03744 it was demonstrated, that a very satisfying tolerance
can be obtained when using an amount of excess free macrocyclic chelate
at a particular low dose range, and which is not under the form of an
excipient X[X',L]. It was shown that with macrocyclic chelates, and in
particular DOTA, results are very advantageous, using a very low excess
of free chelate L, so that the pharmaceutical composition administered to
the patient contains more specifically between 0.02% and 0.4% and in
particular between 0.025% and 0.25 mol % of the free macrocyclic chelate
L in relation to the complexed chelate.
[0016] As a result, the target values of the free ligand are in a very narrow range.
As an injectable product for diagnostic, it is of very high importance that
the final pharmaceutical formulation should be manufactured with
extremely precise and delicate industrial scale control of the
concentrations of free macrocyclic ligands. If this is achieved, no additional
purification or isolation steps are required anymore.
[0017] By respecting the stoichiometric proportions and by adding an excess of
ligand intended not to be complexed with the lanthanide, it is not possible
at the industrial scale to achieve sufficient reproducibility in the final
pharmaceutical solution of an excess of free DOTA in the target range.
This is because of two reasons:
1) the uncertainty of weighing on an industrial scale, which does not make
it possible to correctly ensure the ratio (of the order of 1000 to 1) between
the chelate and the excess chelate, given the small amount of excess
chelate allowed;
2) the rapid uptake from the atmosphere of water by the chelate.
[0018] This problem has been solved in WO20091 03744 by means of measuring
in the liquid pharmaceutical formulation after the complex of the lanthanide
with the ligand has been formed, the concentrations of free macrocyclic
chelate or of free lanthanide and by adjusting these concentrations by
adding additional chelate or lanthanide so as to obtain the desired
concentration of an excess of free ligand. This process, however, requires
the presence of two measuring methods, one for the lanthanide and one
for the ligand in a production installation. Consequently two solutions, one
for adjusting the concentration of the lanthanide and another one for the
ligand depending on which compound is present in excess, have to be
present in the production installation. This increases the complexity of the
production process and increase the risk of errors and of introducing
microbiological contaminants. Furthermore, it also makes the process
more expensive and time consuming. Moreover, adjusting the
concentration of the ligand or lanthanide in the final pharmaceutical
formulation with a solution of ligand can not be done very accurately since
the ligand in powder form takes up water very quickly which can give
considerable weighing errors. To avoid this, the ligand such as DOTA
should be kept in very dry conditions or the adjustment of the
pharmaceutical formulation should be done in more than one step with
intermediate measuring the concentration of the macrocyclic ligand.
[0019] It is thus desirable to obtain an optimized process for producing a
pharmaceutical liquid formulation on an industrial scale comprising a
complex of macrocyclic ligand with a lanthanide, which requires no or only
one adjustment of the concentration of the macrocyclic ligand and is fast,
accurate and straightforward without the need of a further purification or
isolation step(s).
Summary of invention
[0020] The above stated problem is solved by the object of the process as
defined in claim 1.The process is based on the determination of the
moisture content of the macrocyclic ligand which guarantees an excess of
macrocyclic ligand after the complex formation of a lanthanide with a
macrocyclic ligand on an industrial scale.
[0021] Preferred embodiments are described by the dependent claims 2 to 13.
[0022] Further advantages and embodiments of the present invention will become
apparent from the following description and the dependent claims.
Description of embodiments
[0023] According to the present invention the production of the complex of a
lanthanide with a macrocyclic ligand encompasses the following steps:
A. Measurement of the moisture content of the material comprising a
macrocyclic ligand.
[0024] In the scope of the present invention the macrocyclic ligand is preferably a
derivative of tetraaza macrocycles such as 1,4,7,10-
tetraazacyclododecane (cyclen), 1,4,7,10-tetrazacyclotridecan
(homocyclen) and ,4,8,1 1-tetraazacyclotetradecane (cyclam), preferably
DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid), NOTA
( 1H-1 ,4,7-Triazonine-1 ,4,7-triacetic acid, hexahydro), DOTAGA ( 1 ,4,7,10-
Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid, a-(2-carboxyethyl)),
D03A ( 1 ,4,7,10-Tetraazacyclododecane-1 ,4,7-triacetic acid), D03A-butrol
( 1 ,4,7,1 0-Tetraazacyclododecane-1 ,4,7-triacetic acid, 10-[2,3-dihydroxy-1 -
(hydroxymethyl)propyl]), HP-D03A ( 1 ,4,7,1 0-Tetraazacyclododecane-
1,4,7-triacetic acid, 10-(2-hydroxypropyl)) and PCTA (3,6,9,1 5-
Tetraazabicyclo[9.3.1]pentadeca-1 (15),1 1,13-triene-3,6,9-triacetic acid),
more preferably DOTA, D03A, HP-D03A and even more preferably DOTA.
Most of these macrocyclic ligands take up water from the air very rapidly.
The moisture content of macrocyclic ligands can be in the order of 1 to 10
(wt.)% or even higher and lead to a considerable error in weighing the
macrocyclic ligand on an industrial scale. Indeed, large batches of
macrocyclic ligands can not be stored in complete moisture free
circumstances to avoid water uptake as can be in a lab environment. If no
moisture content is taken into account, the amount of macrocyclic ligand in
a weighed batch may be too low to complex all the lanthanide and hence
the risk is high that free lanthanide in the liquid pharmaceutical formulation
will be present or an additional step of adding macrocyclic ligand is
required. The macrocyclic ligand including moisture is denoted hereafter
as the material comprising the macrocyclic ligand.
[0025] In the first step of the method according to the present invention, at least
one sample of the material comprising the macrocyclic ligand to be used in
production is taken to determine the moisture content. The material
comprising the macrocyclic ligand may be homogenised prior to the taking
of the sample. Another preferred embodiment is to homogenise the
different samples taken prior to the measurement of the moisture. Any
known method for the measurement of the moisture content can be used.
A preferable method according to the invention is the Karl Fisher method.
Another preferable method is measuring the content of the macrocyclic
ligand by a suitable analytical technique, e.g. HPLC and calculate the
moisture content of the material comprising the macrocyclic ligand. With
HPLC, other volatile substances than water such as alcohols are
determined. The amount of alcohols can be taken into account based on
the information of the Certificate of Analysis of the macrocyclic ligand from
the supplier. In order to compensate for the additional amount of moisture
which will be taken up by the macrocyclic ligand after the measurement of
the moisture content and before the formation of the complex, it is
preferred to use an extrapolated moisture content. The extrapolated
moisture content can be obtained using a linear or non-linear
extrapolation. The moisture content is hereafter presented by 'M and
expressed in % by weight.
[0026] The total amount of macrocyclic ligand can be produced by any known
method in the art and is preferably homogenised in a container. Said
container may be the container where the reaction for producing the ligand
occurred, a blender, a homogeniser or any container suitable for
homogenising the obtained macrocyclic ligand in the desired amount for a
certain batch of formulation.
B. Mixing the lanthanide with the macrocyclic ligand.
B.1 Lanthanide.
[0027] In the scope of the present invention, "lanthanides" comprise the fifteen
metallic chemical elements with atomic numbers 57 through 7 1, from
lanthanum through lutetium. "Similar compounds" comprise scandium and
yttrium. Together with scandium and yttrium, the trivial name "rare earths"
is sometimes used to describe all the lanthanides and similar compounds.
[0028] Preferred lanthanides and similar compound are Gadolinium (Gd), Yttrium
(Y) and Terbium (Tb) and most preferred is Gadolinium.
B.2 Determination of the amount of the material comprising the
macrocyclic ligand, X3.
[0029] The determination of X3 is done in two steps:
B.2.1 . Calculating the amount X 1 of macrocyclic ligand necessary for
obtaining an excess of ligand after complexation.
[0030] The total amount of macrocyclic ligand (X1) for a specific batch of complex
of lanthanide with a macrocyclic ligand, must be calculated in a way that it
is present in the final formulation in an excess (Lf) in relation of the total
amount of lanthanide or a similar compound (G), being said amount (Lf) in
the range from 0.002% to 0.4% mol in relation of the amount of lanthanide
or similar compound (in mol), preferably in the range from 0.02% to 0.3%
mol/mol, more preferably in the range from 0.025% to 0.25%, meaning
that a small amount of excess ligand should be present after the complex
formation. This may ensure that metal traces originated during its
production and/or sterilization can be trapped, thus avoiding any possibility
of replacement of the lanthanide or similar compound ions in the complex
that may result in their release in free form in the contrast agent
formulation.
[0031] This means that in the final formulation there is an amount LG of
macrocyclic ligand forming a complex with lanthanide or similar
compound. Hence LG is the amount of macrocyclic ligand necessary for
complexing the amount G of lanthanide and an amount in free form Lf, not
complexing the lanthanide or similar compound and is thus represented by
formula 1:
X 1 = LG + Lf ( 1)
Wherein:
- Lf is the amount of macrocyclic ligand in its free form after complex
formation, which according to the present invention is in the range set out
above in relation of the total amount of the lanthanide or similar compound
(G).
[0032] The size of the batch of the complex can be small, suitable for laboratory
scale i.e. for example of 250g, 500g 1000g, etc. but the advantage of the
method of the invention is that the method is particularly suited for bigger
sizes such as 20kg, 50kg, 200kg, 500kg, etc., i.e. for pilot and industrial
scale.
B.2.2. Calculation of the total amount X3 of material comprising a
macrocyclic iigand for the production of a complex of a macrocyclic ligand
and a lanthanide.
[0033] Based on the result of the measurement of the moisture content (MC) in
the macrocyclic ligand as described in section A, it is now possible to
calculate or determine the total amount of material comprising the
macrocyclic ligand, hereafter denoted as X3 which is required for the
production of a batch of the complex. X3 being the sum of X 1 and M
according to formula 2:
X3 = X 1 + M (2)
Wherein M is the amount of moisture present in the amount of material
comprising the macrocyclic ligand (X3) which will be used in the
production of the complex of the lanthanide and the macrocyclic ligand. M
must be expressed in the same units as the units of X 1. Since MC is the
moisture content of X3, MC = M* 100 / (X1 + M).
[0034] The total amount of moisture M can be calculated based on X 1 and the
measured moisture content MC according to formula 3:
M= MC * X 1 / (100 - MC) (3)
Wherein MC is expressed in (wt.) %.
[0035] In another embodiment, when a certain amount (Y) of material comprising
a macrocyclic ligand is taken as a sample for controlling purposes, ex.
quality control, then said amount (Y) may be added to the calculated
amount of material in order to compensate the amount that was taken for
testing.
[0036] If the amount of material comprising the macrocyclic ligand + moisture is
introduced into the formulation, the amount of ligand available to form the
complex with a lanthanide or similar compound may be not enough when
the amount of moisture (M) is not taken into account. Therefore, by
calculating X3 it is possible to know the exact amount of material
comprising the macrocyclic ligand necessary to add to obtain a certain
batch of the complex of a lanthanide with the macrocyclic ligand and
avoiding the presence of free lanthanide after complexation and in the final
formulation.
[0037] Alternatively it is possible to further calculate the amount of material
comprising the macrocyclic ligand necessary to fill each unit package
when the batch is optionally divided into portions. Thus according formula
4:
X2 = X3 / N 1 (4)
Wherein,
- X2 is the amount in weight of material comprising the macrocyclic ligand
with moisture (M) in each unit package; and
- N 1 is the number of unit packages for one final formulation batch. In
another embodiment, the batches of material comprising the macrocyclic
ligand may be divided into portions witch are not equal to each other.
In the embodiment where the material comprising a macrocyclic ligand is
divided into portions, each unit package is then filled with a homogenised
and specifically measured amount (X2) of material under controlled
conditions of humidity so that the powder ligand does not further absorb or
desorb water during this phase. In consequence, the real content of
macrocyclic ligand able to form a complex with a lanthanide or similar
compound and of moisture in each unit package is known and thus no
further measurements or adjustments are needed; when it is desired to
use a certain amount of ligand to produce a batch of formulation it is only
necessary to calculate the number of unit packages to achieve the total
amount of necessary ligand.
[0039] The total amount (X2) of the material of the batch can be divided into
portions and then packed. In such case, the amount (N1) of unit packages
necessary to pack the material comprising the macrocyclic ligand can be
also calculated in function of the total volume of the batch and the size of
each unit package.
B.3. Complex formation.
[0040] The complex formation according to the invention is now described. In one
embodiment, the lanthanide or similar compound is added in the form of
oxides or salts thereof to the material comprising the macrocyclic ligand to
form the complex. In another embodiment, the material comprising the
macrocyclic ligand is added as a solution, preferably an aqueous solution,
to the lanthanide. Both compounds may also be added simultaneously to
the solvent, preferably water. Preferably the macrocyclic ligand is
dissolved in a solvent, most preferably water before the lanthanide is
added as an oxide or salt. The total amount G of lanthanide or similar
compound necessary to form the said complex depends on the amount LG
of ligand desired to form a complex, i.e. G should correspond to LG. '
Corresponding' means that the amounts are calculated taken the
stoichiometry of the complexation reaction into account. The preferred
lanthanide or similar compound is gadolinium, terbium or yttrium, most
preferable compound is gadolinium. The preferred form of providing
gadolinium is in the form of the oxide, i.e. Gd203.
[0041] Another preferred embodiment is to fix the amount G of the lanthanide
depending on the batch size of the pharmaceutical formulation to be
obtained. Depending on G, the amount of ligand LG, necessary for forming
a complex, is then calculated.
[0042] Preferably the macrocyclic ligand is dissolved in a solvent, most preferably
water before the lanthanide is added. The precise experimental conditions
of the mixing are detailed in the examples. Advantageously, the
temperature for the complex formation is between 60 and 100°C, and is
advantageously about 80°C. The mixture of the lanthanide and the ligand
is kept at the elevated temperature for 1 to 4 hours, preferably at least 3
hours. After the complex formation has taken place the obtained solution
can be cooled down to room temperature. Moreover, it is understood that
the complex formation may be performed in several sub-steps which
would be equivalent to an overall complexation step. The sub-steps
comprise the addition of the lanthanide or the macrocyclic ligand via
multiple steps instead of adding them in one step.
C. Adjustment of the amount or concentration of macrocyclic ligand in a
free form.
[0043] According to the present invention, an excess of macrocyclic ligand in
solution is obtained. This excess, also called free macrocyclic ligand may
be, due to small variations in the process, too high. This means that the
concentration of free macrocyclic ligand in solution is then higher than a
target level Ct. This target level is for example determined by the upper
limit of the product specifications related to the concentration of the free
macrocyclic ligand of the liquid pharmaceutical formulation. In a preferred
embodiment of the present invention an additional adjustment step is
therefore provided. According to that embodiment of the invention, the
concentration of the free macrocyclic ligand in the solution after formation
of the complex is measured. Preferably, a sample from the solution
obtained as described in section B.3. is taken and eventually cooled down
to room temperature. The measurement can be performed by any suitable
analytical method. Suitable methods are detailed in the examples; also
HPLC can be used to measure the concentration of the macrocyclic ligand
in free form. The concentration of the macrocyclic ligand is then compared
with the target value Ct. If the concentration is higher than the target value,
an amount of lanthanide can be added to the solution. Preferably the
solution is therefore kept at an elevated temperature between 60 and 00°
C.The amount of required lanthanide, preferably added as a salt or oxide
can be added in one step, based on a calculation or in multiple steps.
Since the oxide or salts of lanthanides take up much less water than the
macrocyclic ligands, the adjustment of the concentration of macrocyclic
ligand in the solution by means of adding lanthanide is much more
accurate than adjusting an excess of free lanthanide with a solution of
macrocyclic ligand. If the lanthanide is added in multiple steps a
measurement of the concentration of the macrocyclic ligand in free form
can be performed between the multiple steps. Additionally the presence of
lanthanide in its free from in the solution can be checked for safety
reasons. The solution obtained after the optional adaptation comprises the
complex of the lanthanide with the macrocyclic ligand and an excess of
free macrocyclic ligand which concentration is within the product
specifications of the final pharmaceutical formulation. This solution is now
ready to be used in the process of producing a liquid pharmaceutical
formulation without the need for additional isolation or purification steps.
The analysis of the free lanthanide can be performed by using, for
example, a solution of EDTA in the presence of xylenol orange or
Arsenazo as indicator. Free gadolinium can be analysed advantageously
with a colorimetric method using 0.01 M edetate disodium titration solution
in the presence of xylenol orange as an indicator. Titration is then
preferably carried out at pH=5 in a sodium acetate / acetic acid buffered
solution on a sample of the solution comprising the complex until the
colour of the indicator turns from red to yellow.
[0044] The intended amount of free ligand (Lf) in the liquid formulation is in the
range from 0.002 mol/mol % to 0.4 mol/mol % in relation of the total
amount of lanthanide or similar compound (G), preferably is in the range
from 0.02 mol/mol % to 0.3 mol/mol %, more preferably in the range from
0.025 mol/mol % to 0.25 mol/mol % in relation to the total amount of
lanthanide or similar compound (G).
D. Process for producing a liquid pharmaceutical formulation
[0045] The remaining components necessary to obtain a liquid pharmaceutical
formulation of the invention and comprising the complex of the lanthanide
with the macrocyclic ligand, prepared as described above, are added to
the material prepared as described above or optionally to a certain number
of unit packages in the desired amounts in function of the different contrast
agent formulations. This step may include the addition of the necessary
excipients to obtain a formulation with the desired pharmaceutical
properties. Examples of such excipients are water, meglumine,
hydrochloric acid and/or sodium hydroxide for a pH adjustment.
[0046] Examples of excipients added to the complex of a macrocyclic ligand with
a lanthanide or similar compound to obtain a liquid pharmaceutical
formulation as described above are water for injection, an organic base,
hydrochloric acid and/or sodium hydroxide for pH adjustment. The pH is
preferably in a range from 4.0 to 8.5 and more preferably in a range from
6.5 to 7.9. The organic base is preferably meglumine.
[0047] These excipients may be added to the reaction vessel containing the
complex of a macrocyclic ligand and the selected lanthanide or similar
compound produced as described above. The macrocyclic ligand is
preferably present in the final liquid formulation in a concentration in the
range between 0.3 to 0.7 M, preferably in the range between 0.4 to 0.6 M
and most preferably at 0.5 M.
[0048] Specific embodiments will now be described in detail. The examples are
intended to be illustrative and the claims are not limited to the materials,
conditions or parameters set forth in the examples.
EXAMPLES
Measurements
Moisture content in DOTA.
[0049] The measurement of the moisture content in DOTA is performed as
described in the semi-micro method of Karl Fischer (Fischer, Karl - Pharm.
Eur. Section 2.5.12).
Analysis of DOTA in free form (free DOTA) in solution.
[0050] Solution A: 50 g of sodium acetate was dissolved in 10 ml of glacial acetic
acid and the volume was adjusted to 1000.0 ml with water free from
carbon dioxide. The obtained solution was adjusted to pH (5 ± 0.05) with
0.1 M sodium hydroxide solution or glacial acetic acid.
[0051] Solution B: 50.8 mg of xylene orange was dissolved in water free from
carbon dioxide, the volume was adjusted the volume to 100.0 ml with the
same solvent. Freshly prepared solutions were used.
[0052] Solution C: 3 ml of solution B was added to 30 ml of solution A, the volume
of the solution was added to 200.0 ml with water free from carbon dioxide.
[0053] 0.005 M gadolinium sulphate solution was prepared as follows: 3.735 g of
gadolinium sulphate octahydrate was dissolved in water free from carbon
dioxide. The volume of solution was adjusted to 1000 ml with the same
solvent.
[0054] A test solution was prepared as follows: 4.88 ± 0.5 g of meglumine and 50
± 1 g of hot (70 - 90 °C) sample of the solution comprising the gadolinium-
DOTA complex, were transferred into a 100 ml conical flask, mixed for 5 to
10 minutes at 70° C to 90° C and cooled to room temperature. To 2 ml of
this solution, 20 ml of water free from carbon dioxide and 10 ml of solution
C were added and mixed. The resultant solution was adjusted to pH 5 ±
0.05 with 0.1 M sodium hydroxide solution or glacial acetic acid. The
yellow coloration indicates the presence of free DOTA. The solution was
titrated with the 0.005 M gadolinium sulphate solution until colour alters to
reddish-pink.1 ml of 0.005 M gadolinium sulphate solution corresponds to
4.044 g of DOTA.
Check for presence of free gadolinium ions in solution
[0055] 4.88 ± 0.5 g meglumine and 50 ± 1 g of hot (70 - 90 °C) solution
comprising the gadolinium-DOTA complex were transferred into a 100-ml
conical flask, mixed for 5 to 10 minutes at 70° C to 90° C and cooled to
room temperature. 2 ml of this solution was added to 20 ml of water free
from carbon dioxide and 10 ml of solution C. The resultant solution was
adjusted to pH (5 ± 0.05) with 0.1 M sodium hydroxide solution or glacial
acetic acid. A red-violet coloration indicates the presence of free
gadolinium ions.
Materials
[0056] All reagents used in the following examples were readily available from
commercial sources unless otherwise specified:
Gadolinium oxide : Gd2O3 from Rhodia
Meglumine: N-methyl-D-glucamine from Merck KGaA, Darmstadt
DOTA: was obtained as described in the unpublished patent
application EP1 3 152873.9. The obtained DOTA was stored in drums
with a PE bag inside.
Calculation of X3.
Preparation of a batch of macrocyclic ligand DOTA for the production of a
200 L batch of the Gd-DOTA complex.
[0057] In this section it is explained how the amount X3 of DOTA was calculated
for the production of a batch of 200 L of a Gd-DOTA complex at a
concentration of 0.5 M (mol/L) taking into account a measured moisture
content of the DOTA and a certain Lf value.
[0058] In a first step, X 1 was calculated. A batch of 200 L of Gd-DOTA complex
at a concentration of 0.5 M corresponds with an amount LG of DOTA
required for the complex formation of 100 moles or 40.442 kg. For a
chosen excess of free DOTA of 0.2 % mol /mol Gd, Lf is equal to 0.2
moles or 80.88 g. The amount X 1 was calculated according to formula 1
defined above and hence the obtained value of X 1 was 40.523 kg.
[0059] In a second step, the moisture content MC, expressed as weight
percentage, in the sample taken from a DOTA batch was measured by the
method described above. The result was 5.0 (wt.)%., which allows to
calculate the total amount M of moisture present in the batch of DOTA
required for the production of the 200L batch of Gd-DOTA complex, as
follows (see formula 3 defined above) :
M = MC * X 1 / ( 00 - MC) = 5.0*40.523kg / ( 100-5.0) = 2.133 kg
[0060] X3 was then calculated as X1+M (formula 2 defined above), resulting in a
value of 42.656 kg.
Measuring the moisture content of the DOTA.
[0061] This section and the next section illustrates how the moisture content in a
batch of a macrocyclic ligand changed when the product was exposed to
different conditions of relative humidity. The moisture content in the
samples was measured using methods known to those skilled in the art. In
this example, the measurement of the content of moisture in DOTA was
performed as described above.
[0062] Two samples of DOTA were kept in conditions of relative humidity (RH) =
30+5% at a temperature of Temp = 20+2°C. From these samples, small
samples were taken at different times (To = beginning of the experiment;
T30 = To + 30 min; T90 = TO + 90 min; and T300 = To + 300 min) and the
moisture content was measured as described above. Each experiment
(Exp.) was performed twice and the average of each (Ind.) was
determined (Avg.). The results in percentage of the initial weight of DOTA
are shown in Table 1.
Table 1
These results show that at 30% RH the moisture content in the selected
DOTA batch increased in time. These experimental values could be used
for extrapolating the moisture content to obtain an extrapolated moisture
content.
Measuring the moisture content of the DOTA ligand in conditions of higher
RH.
This measurement, as the previous one, illustrated how the moisture
content in a batch of a macrocyclic ligand was affected by the
environmental relative humidity conditions. Therefore, the previous
experiments were repeated in the same conditions but at a RH = 75+5%.
Again, each experiment (Exp.) was performed twice and the average of
each (Ind.) was determined (Avg.). The results in percentage of the initial
weight of DOTA are shown in Table 2.
Table 2
4.929 6.694 7.200 5.813
Exp.4 5.346 6.449 6.962 4.1 11
5.25 6.65 6.88 4.36
5.1 50 6.851 6.789 4.609
[0065] These results showed that the moisture content in DOTA increased with
time and with relative humidity of the atmosphere at which the DOTA was
stored.
Preparation of a complex of DOTA with gadolinium and adjusting the
concentration of the free DOTA in solution.
[0066] Two batches, INV01 and INV02 of DOTA were prepared for the production
of gadolinium-DOTA complex batches of respectively 50 L and 5 L. The
amount X 1 of DOTA was calculated in order to obtain an Lf value of
respectively 12.97 g for INV01 and 1.30 g for INV02. This corresponds to a
free DOTA ratio to gadolinium of 0.124 % mol / mol. A target value for the
concentration of the free DOTA after the complex formation, Ct, was set to
0.045 g/100mi or 0.128 % mol / mol gadolinium, as being the upper limit of
the product specification of the liquid pharmaceutical formulation which
had to be obtained.
[0067] The moisture content of the samples taken from batch INV01 and batch
INV02 was determined according to the method as described above.
The results of the measurements, the determined amounts M and the
calculated amounts X3 according to formula 3 are reported in Table 3.
Table 3
INV01 (50 L) INV02 (5L)
Amount X 1 of DOTA (kg) 10.123 1.012
Moisture content of DOTA batch (wt. %) 6.71 6.00
Total amount of moisture M (kg) 0.727 0.065
Amount X3 of DOTA (kg) 10.85 1.077
Amount G of gadolinium oxide (kg) 4.59 0.454
[0068] INV01 was further prepared by weighing DOTA in an amount X3 = 10.85
kg of DOTA including moisture (see Table 3) . This amount of DOTA was
stepwise added via a pipe into a tank already containing 35.00 kg of water.
Hereafter 4.59 kg of gadolinium oxide was stepwise added via a pipe into
the tank containing the water and the DOTA. The moisture content of the
gadolinium oxide was 0.2 (wt.) %. The pipes were rinsed with 3.96 kg of
water. The reaction mixture was heated to 97°C and kept at this
temperature for 180 minutes. From the solution, samples were taken to
check for the presence of free gadolinium and for determining the
concentration of free DOTA. The results are reported in Table 4. The
solution after using the batch DOTA of INV01 for the complex formation
showed no free gadolinium ions.
[0069] INV02 was further prepared by weighing DOTA in an amount X3 as
reported in Table 3 and added through a funnel into a 6.0 L glass reactor
equipped with a stirrer, a thermometer, and a backflow condenser. An
amount G of gadolinium oxide (see Table 3) corresponding with LG was
added through the same funnel. The moisture content of the gadolinium
oxide was 0.2 (wt.) %. 3.9 L of water for injection was added through the
same funnel and was washing down the raw material residing from the
funnel. The mixture was heated during stirring to a temperature of 95 - 99°
C. The mixture was stirred for 3 hours at this temperature. The stirrer was
turned off and the presence of free gadolinium was checked and the
concentration of free DOTA in the reaction mixture was determined as
described above. The results are reported in Table 4. The reaction mixture
after using the batch DOTA of INV02 did not contain free gadolinium ions.
[0070] As the concentration of free DOTA in both the solutions after gadolinium-
DOTA complex formation using the DOTA batches INV01 and INV02 were
exceeding the target value Ct of 0.045 g / 00 ml of DOTA, additional
amounts A of gadolinium oxide were further added to adjust the
concentration of free DOTA. The solution is therefore kept at a
temperature of 95 - 99°C for 95 minutes. The amounts A added were
calculated on the basis of formula 5 and are reported in Table 4.
V * (Cd - 0.025) * 181.25
A = (5)
404.42 *100
wherein:
- V= volume of the solution comprising the gadolinium-DOTA complex in
ml.
- Cd= concentration in solution of free DOTA in g/100ml.
[0071] The resulting concentrations of free DOTA obtained in the final
composition related to INV01 and INV02, showed an excess which was
still sufficient to avoid free gadolinium in the final solution and which was
lower than the target value Ct of 0.045 g/ 100ml. Only one adjustment with
gadolinium oxide was required to obtain a final composition with the
concentration of free DOTA within the specifications. This final
composition could now be used for obtaining a liquid pharmaceutical
formulation without further isolation or purification steps as described
below.
Table 4
INV01 (50L) INV02 (5L)
Free gadolinium Below detection limit Below detection limit
Measured concentration of free 0.425 (g/100ml) 0.259 (g/ 00ml)
DOTA after complex formation 2.102 (% mol/mol Gd) 1.281 (% mol/mol Gd)
Additional amount A of 89.63 5.24
gadolinium oxide (g)
Free gadolinium after addition of Below detection limit Below detection limit
gadolinium oxide
Free DOTA concentration in 0.018 g/1 00ml 0.014 g/100ml
final composition after addition 0.089 (% mol/mol Gd) 0.069 (% mol/mol Gd)
of gadolinium oxide
Preparation of the liquid pharmaceutical formulation
The final composition prepared with the DOTA batch INV01 was cooled to
a temperature between 40°C and 50°C and 4.88 kg meglumine was added
under agitation. After 40 minutes, the agitation was stopped. The pH of the
solution was then measured. 3 g of meglumine was further added to the
solution to obtain a pH value of the solution between 7.1 and 7.8. The
obtained final liquid pharmaceutical formulation was now ready to be put
into vials.

we claim
A process for producing a complex of a lanthanide or similar compound with a
macrocyclic ligand, wherein the ratio of macrocyclic ligand in free form in
relation to the lanthanide or similar compound is equal or more than 0.002%
mol/mol , comprising the following steps:
a) measuring the moisture content in a sample of the macrocyclic ligand; and
b) mixing an amount G of the lanthanide with an amount X3 of the macrocyclic
ligand with the proviso that X3= LG+Lf + M, wherein LG is the amount of
macrocyclic ligand necessary for complexing the amount G of lanthanide or
similar compound; Lf is an excess amount of the macrocyclic ligand; and M is
the amount of moisture present in the amount X3 of the macrocyclic ligand.
The process according to claim 1 wherein the ratio of macrocyclic ligand in free
form in relation to the lanthanide or similar compound is in the range from
0.002% to 0.4% mol/mol.
The process according to the claims 1 or 2 further comprising the step c)
wherein the concentration of macrocyclic ligand in free form is measured, and
if this concentration is higher than a target value Ct, extra lanthanide or similar
compound is further added so as to obtain a ratio of macrocyclic ligand in free
form in relation to the lanthanide or similar compound of more than 0.002%
mol/mol.
The process according to claim 3 wherein the ratio of macrocyclic ligand in a
free form in relation to the lanthanide or similar compound after adding the
extra lanthanide is in the range from 0.002% to 0.4% mol/mol.
The process according to any of the preceding claims wherein the macrocyclic
ligand is DOTA, D03A or HP-D03A.
The process according to any of the preceding claims wherein the lanthanide
or similar compound is gadolinium, terbium or yttrium.
The process according to any of the preceding claims wherein the lanthanide
or similar compound used for mixing in step b) is gadolinium oxide.
The process according to the claims 3 to 7 wherein the macrocyclic ligand is
DOTA and the lanthanide used in step b) and step c) is gadolinium oxide.
The process according to any of the preceding claims wherein the macrocyclic
ligand is homogenised prior to step a).
0. The process according to any of the preceding claims wherein M is an
extrapolated value based on the moisture content measured in step a).
11. A process of producing a liquid pharmaceutical formulation comprising the
following steps:
- producing a complex as defined in any of the preceding claims; and
- adding an organic base; and
- optionally adjusting the pH of the solution to a range from 6.5 to 7.9 with
hydrochloric acid and / or sodium hydroxide.
12. The process according to claim 11 wherein the organic base is meglumine or a
salt thereof and the macrocyclic ligand is DOTA and the lanthanide added in
step b) and step c) is gadolinium oxide.
13. Use of a liquid pharmaceutical formulation as obtained by the method of claim
11 or 12 as a contrast agent for magnetic resonance imaging.