The present invention relates to a drug product composition and in particular to a
composition comprising a positron emission tomography (PET) tracer. The
composition of the present invention and its method of synthesis have certain
advantages over the prior art.
Description of Related Art
The non-natural amino acid 1 F-1 -amino-3-fluorocyclo butane- 1-carboxylic acid ( FFACBC,
also known as 1 F-Fluciclovine) is taken up specifically by amino acid
transporters and shows promise for positron emission tomography (PET) imaging of
prostate cancer (Nanni et al 2014 Clinical Genitourinary Cancer; 12(2): 106-1 10).
Production of 1 F-FACBC comprises labelling of a triflate precursor compound with
1 F-fluoride:
I II
before removal of the two protecting groups:
II III
Following the deprotection steps purification is carried out to remove impurities. In the
currently-practiced methods a combination of solid phases is used: ion retardation to
remove excess Na+ and excess Cl~ left over from the deprotection steps, alumina to
remove 1 F-fluoride and a reversed phase to remove FACBC-related impurities such as
l-amino-3-hydroxyl-cyclo butane- 1-carboxylic acid (hydroxy1-ACBC) and l-amino-3-
chloro-cyclo butane- 1-carboxylic acid (chloro-ACBC).
The synthesis is currently typically carried out by means of an automated radiosynthesis
procedure employing a so-called "cassette" or "cartridge" designed to fit removably and
interchangeably onto an automated synthesis apparatus such as those that are
commercially available from GE Healthcare, CTI Inc, Ion Beam Applications S.A.
(Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and
Bioscan (USA). The cassette comprises all the reagents, reaction vessels and apparatus
necessary to carry out the preparation of 1 F-FACBC following introduction of suitably
prepared 1 F-fluoride by methods well-known in the field of PET tracer production.
A known cassette for the synthesis of 1 F-FACBC is a FASTlab ™ cassette from GE
Healthcare. Each cassette is built around a one-piece-moulded manifold with 25 threeway
stopcocks, all made of polypropylene. The cassette includes a quaternary
methylammonium (QMA) solid phase extraction (SPE) cartridge, a 5 ml cyclic olefin
copolymer reactor, one 1ml syringe and two 5 ml syringes, spikes for connection with
five prefilled reagent vials A-E, one water bag (100 ml), three SPE cartridges (tC18,
HLB and alumina) and filters. The five FASTlab™ cassette reagent vials are filled as
follows: vial A contains eluent solution comprising Kryptofix 2.2.2. and K2CO3 in
acetonitrile (MeCN), vial B contains HC1, vial C contains MeCN, vial D contains dry
precursor compound of Formula I from the above-illustrated reaction scheme and vial E
contains NaOH. A known method for production of 1 F-FACBC drug product using this
FASTlab™ cassette is described in Example 1 of WO 2013/093025. The
radiosynthesis is started by trapping aqueous 1 F-fluoride onto the QMA followed by
elution into the reactor using eluent from vial A, and then concentrated to dryness by
azeotropic distillation with acetonitrile from vial C. Approximately MeCN is mixed
with precursor compound from vial D and the dissolved precursor is added to the
reactor and heated for 3 min at 85°C. The reaction mixture is then diluted with water
and sent through the tC18 cartridge. The reactor is washed with water and sent through
the tC18 cartridge. The labelled intermediate, fixed on the tC18 cartridge is washed
with water, and then incubated with NaOH for 5 min to remove the ester group. The
deesterified intermediate is eluted off the tC18 cartridge and back into the reactor using
water. The BOC group is hydro lysed in the reactor by adding HC1 and heating for 5
min at 60°C. The crude 18F-FACBC is then sent through the HLB (HLB = hydrophilic
lipophilic balanced) cartridge for removal of FACBC-related impurities, the alumina
cartridge for removal of 1 F-fluoride, and thereafter into a 30 ml product vial containing
citrate buffer. The HLB and alumina cartridges are then washed with water, which is
sent to the product vial. Finally, NaOH and water are added to the product vial to
provide the final purified formulation of 1 F-FACBC. Prior to intravenous
administration, this formulation is passed through a sterile filter.
The present inventors have found that the quality of the final 1 F-FACBC drug product
obtained using the above-described known FASTlab™ cassette and process can be
somewhat variable. Residual acetonitrile levels have been found to range from about
100 mg/ml to about 600 mg/ml. While acceptable in terms of permitted daily exposure
and in the context of the acceptance criteria for 1 F-FACBC drug product, the amount
and observed variability is less than ideal. Furthermore, residual aluminium have been
found to range from about 7 mg/ml to nearly 20 mg/ml, which would mean a potential
amount of 100 mg in a 5 ml 1 F-FACBC injection. Where the 1 F-FACBC drug product
also comprises citrate buffer, complexes of aluminium and citrate are likely to be
present, which is problematic as it is known that such complexes cross the blood-brain
barrier (Rengel 2004 Biometals; 17: 669-689).
There is therefore scope to for an improved 1 F-FACBC drug product formulation.
Summary of the Invention
The present invention provides a drug product composition comprising 1 F-FACBC that
overcomes the problems seen with known such compositions. In particular, the
composition of the present invention has an improved impurity profile, making it safer
and more effective for imaging as compared with the prior art. Low and predictable
levels of acetonitrile and/or aluminium in the final drug product mean that the
composition of the invention more easily meets worldwide pharmacopeia requirements.
In addition to a significant reduction in the concentration of aluminium in the final drug
product, removal of the alumina cartridge has the allied advantages that a shorter and
simplified process is permitted and that no particles arising from this cartridge are
present, which the present inventors have noted can block the sterile filter used prior to
injection of the drug product. Furthermore, the advantages of the present invention are
achieved with only minor changes to the known process and without impairing the
desirable qualities of known 1 F-FACBC compositions.
Detailed Description of the Preferred Embodiments
To more clearly and concisely describe and point out the subject matter of the claimed
invention, definitions are provided in the detailed description hereinbelow for specific
terms used throughout the present specification and claims. Any exemplification of
specific terms herein should be considered as non-limiting examples.
In one aspect the present invention relates to a positron emission tomography (PET)
tracer composition comprising t -l-amino-3- 1 F-fluorocyclobutyl-l-carboxylic acid
(1 F-FACBC) characterised in that said composition comprises no more than 5.0 mg/mL
dissolved aluminium (Al).
In one aspect the present invention relates to a positron emission tomography (PET)
tracer composition comprising t -l-amino-3- 1 F-fluorocyclobutyl-l-carboxylic acid
(1 F-FACBC) characterised in that said composition comprises no more than 5.0 mg/mL
dissolved aluminium (Al) and no more than 50 mg/mL acetonitrile (MeCN).
In the context of the present invention a "PET tracer composition" refers to a
composition comprising a PET tracer together with a biocompatible carrier in a form
suitable for mammalian administration. The PET tracer composition of the invention is
referred to hereunder also as the composition of the invention. A "PET tracer" is
defined herein as a biologically active molecule comprising an atom which is a positron
emitter suitable for intravenous administration to a mammalian subject followed by
PET imaging to obtain one or more clinically-useful images of the location and/or
distribution of the PET tracer. A "biocompatible carrier" as defined herein is a fluid,
especially a liquid, in which a pharmaceutical is suspended or dissolved, such that the
composition is physiologically tolerable, i.e. can be administered to the mammalian
body without toxicity or undue discomfort. The biocompatible carrier is suitably an
injectable carrier liquid such as sterile, pyrogen-free water for injection or an aqueous
solution such as saline.
The compound "1 F-FACBC" is represented by the following chemical structure:
The term "not more than" as used herein should be understood to mean any amount less
than and including the quoted quantity. In an idealized embodiment of the composition
of the present invention there would be zero mg/mL of each impurity present. However,
in reality, zero m /h of an impurity is unlikely and at least a trace amount of each
impurity remains in the composition. The term "not more than" acknowledges that a
trace amount of one or more impurities is present in a PET tracer composition, and
defines a concentration limit above which the composition would not be deemed
acceptable for use.
In one embodiment, the composition of the invention comprises not more than not more
than 3.0 mg/mL dissolved Al, and in another embodiment not more than 1.5 mg/mL
dissolved Al.
In one embodiment the composition of the present invention comprises MeCN at a
concentration not more than 20 mg/mL.
The composition of the invention in one embodiment has an end of synthesis (EOS)
radiochemical purity (RCP) of at least 95%, in another embodiment at least 98%, and in
yet another embodiment at least 99%.
The term "end of synthesis" refers to the point in time when the labelled compound is
collected in the product collection vial.
EP 2 119458 (Al) teaches that a more stable formulation of 1 F-FACBC is achieved
when the pH is maintained within the range 2.0-5.9. As discussed in WO 2013/093025,
use of citrate buffer allows the pH to be maintained within an even narrower range,
provides resistance to degradation and enables the formulation to be autoclaved. In one
embodiment, the composition of the present invention therefore comprises around 50-
100 mM citrate buffer, in another embodiment around 60-90 mM citrate buffer and in
yet another embodiment around 75-85 mM citrate buffer. The term "around" in this
context incorporates the exact values of the ranges as well as a small variation around
these values that would be expected by the skilled person to achieve the same
stabilisation effect.
In another aspect, the present invention provides a method to prepare a PET tracer
composition of the invention wherein said method comprises:
(a) reacting in a reaction vessel a source of 1 F-fluoride with a precursor
compound of Formula I :
wherein:
LG is a leaving group;
PG1 is a carboxy protecting group; and,
PG2 is an amine protecting group;
to obtain a reaction mixture comprising a compound of Formula II:
wherein PG and PG are as defined for Formula I;
(b) carrying out removal of PG1 and PG2 to obtain a reaction mixture
comprising 1 F-FACBC; and
(c) purifying said reaction mixture comprising 1 F-FACBC by passing it
through a hydrophilic lipophilic balanced (HLB) solid phase, characterised in
that said purifying does not comprise passing the reaction mixture comprising
1 F-FACBC through an alumina solid phase.
In another aspect, the present invention provides a method to prepare a PET tracer
composition of the invention wherein said method comprises:
(a) reacting in a reaction vessel a source of 1 F-fluoride with a precursor
compound of Formula I :
wherein:
LG is a leaving group;
PG1 is a carboxy protecting group; and,
PG2 is an amine protecting group;
wherein said reacting step is carried out in acetonitrile;
to obtain a reaction mixture comprising a compound of Formula II:
wherein PG and PG are as defined for Formula I;
(b) transferring said reaction mixture comprising said compound of Formula
II out of said reaction vessel and carrying out removal of PG1 to obtain a
reaction mixture comprising a compound of Formula III:
wherein PG2 is as defined for Formula I;
(c) applying heat to said reaction vessel at the same time as carrying out
removal of PG1;
(d) transferring said reaction mixture comprising said compound of Formula
III back into said reaction vessel and carrying out removal of PG2 to obtain a
reaction mixture comprising 18F-FACBC;
(e) purifying said reaction mixture comprising 1 F-FACBC by passing it
through a hydrophilic lipophilic balanced (HLB) solid phase, characterised in
that said purifying does not comprise passing the reaction mixture comprising
1 F-FACBC through an alumina solid phase.
The "source of 1 F-fluoride" suitable for use in step (a) of the method of the invention is
normally obtained as an aqueous solution from the nuclear reaction 1 0(p,n) 1 F. In
order to increase the reactivity of fluoride and to reduce or minimise hydroxy lated by
products resulting from the presence of water, water is typically removed from 1 Ffluoride
prior to the reaction, and fluorination reactions are carried out using anhydrous
reaction solvents (Aigbirhio et al 1995 J Fluor Chem; 70: 279-87). A further step that
is used to improve the reactivity of 1 F-fluoride for radiofluorination reactions is to add
a cationic counterion prior to the removal of water. Suitably, the counterion should
possess sufficient solubility within the anhydrous reaction solvent to maintain the
solubility of the 1 F-fluoride. Therefore, counterions that are typically used include
large but soft metal ions such as rubidium or caesium, potassium complexed with a
cryptand such as Kryptofix™, or tetraalkylammonium salts, wherein potassium
complexed with a cryptand such as Kryptofix™, or tetraalkylammonium salts are
preferred.
The "precursor compound " for step (a) of the method of the invention comprises a non
radioactive derivative of a radiolabelled compound, designed so that chemical reaction
with a convenient chemical form of the detectable label occurs site-specifically, can be
conducted in the minimum number of steps (ideally a single step), and without the need
for significant purification (ideally no further purification), to give the desired
radiolabelled compound. Such precursor compounds are synthetic and can
conveniently be obtained in good chemical purity.
A suitable "leaving group " in the context of the compound of Formula I in step (a) of
the method of the present invention is a chemical group that can be displaced by
nucleophilic displacement reaction with fluoride ion. These are well-known in the art
of synthetic chemistry. In some embodiments the leaving group of the present
invention is a linear or branched C1-10 haloalkyl sulfonic acid substituent, a linear or
branched C1-10 alkyl sulfonic acid substituent, a fluorosulfonic acid substituent, or an
aromatic sulfonic acid substituent. In other embodiments of the invention the leaving
group is selected from methanesulfonic acid, toluenesulfonic acid, nitrobenzenesulfonic
acid, benzenesulfonic acid, trifluoromethanesulfonic acid, fluorosulfonic acid, and
perfluoroalkylsulfonic acid. In some embodiments the leaving group is either
methanesulfonic acid, trifluoromethanesulfonic acid or toluenesulfonic acid and in
another embodiment the leaving group is trifluoromethanesulfonic acid.
The term "protecting group " as used in connection with the substituents PG1 and PG2
refers to a group which inhibits or suppresses undesirable chemical reactions, but which
is designed to be sufficiently reactive that it may be cleaved from the functional group
in question to obtain the desired product under mild enough conditions that do not
modify the rest of the molecule. Protecting groups are well known to those skilled in
the art and are described in 'Protective Groups in Organic Synthesis', Theorodora W.
Greene and Peter G. M. Wuts, (Fourth Edition, John Wiley & Sons, 2007).
The PG1 "carboxy protecting group" herein is preferably linear or branched C1-10 alkyl
chain or an aryl substituent. The term "alkyl" used either alone or as part of another
group is defined as any straight, branched or cyclic, saturated or unsaturated C H2n+i
group. The term "aryl" refers to any C6-14 molecular fragment or group which is
derived from a monocyclic or polycyclic aromatic hydrocarbon, or a monocyclic or
polycyclic heteroaromatic hydrocarbon. In one embodiment of the method of the
invention PG1 is selected from methyl, ethyl, t-butyl and phenyl. In another
embodiment of the invention PG1 is methyl or ethyl and in yet another embodiment PG1
is ethyl.
The PG2 "amine protecting group" herein refers to a chemical group that suitably
prevents reaction between 1 F and the amino group in the process of providing the
compound of Formula II. Examples of suitable amine protecting groups include
various carbamate substituents, various amide substituents, various imide substituents,
and various amine substituents. Preferably, the amine protecting group is selected from
the group consisting of linear or branched C2_ alkyloxycarbonyl substituents, linear or
branched C _7 alkenyloxycarbonyl substituents, C7_i 2 benzyloxycarbonyl substituents
that may have a modifying group, C2_7 alkyldithiooxycarbonyl substituents, linear or
branched Ci_ alkylamide substituents, linear or branched C2_ alkenylamide
substituents, C6-11 benzamide substituents that may have a modifying group, C4-10 cyclic
imide substituents, C6-11 aromatic imine substituents that may have a substituent, linear
or branched Ci_ alkylamine substituents, linear or branched C2_ alkenylamine
substituents, and C6-11 benzylamine substituents that may have a modifying group. In
some embodiments of the invention PG2 is selected from t-butoxycarbonyl,
allyloxycarbonyl, phthalimide, and N-benzylideneamine. In other embodiments PG2 is
selected from t-butoxycarbonyl or phthalimide. In one embodiment of the invention
PG2 is t-butoxycarbonyl.
The term "reacting " in step (a) of the method of the invention as is well known to those
of skill in the art refers to bringing two or more chemical substances (typically referred
to in the art as "reactants" or "reagents") together to result in a chemical change in one
or both/all of the chemical substances.
The "removal of PG1" in step (b) of the method of the invention is suitably carried out
by contacting the compound of Formula II, comprised within the reaction mixture
obtained in step (a), with a carboxy deprotecting agent. A suitable carboxy
deprotecting agent may be either an acid or an alkaline solution, as is well-known to the
skilled person (see Greene and Wuts, supra). The concentration of the carboxy
deprotecting agent is suitably just sufficient to remove the carboxy protecting group.
Preferably the carboxy deprotecting agent is an alkaline solution. In certain
embodiments the carboxy deprotecting agent is a sodium hydroxide or a potassium
hydroxide solution and in a preferred embodiment is a sodium hydroxide solution, for
example of 0.5-2.0M. The temperature and the duration used for deprotection may in
some embodiments be tailored to permit removal of PG1. For example, in certain
embodiments the reacting step is carried out at room temperature and for a duration of
around 1-5 minutes. In one embodiment, removal of PG1 is carried out by passing the
reaction mixture comprising the compound of Formula II through a solid phase
extraction (SPE) column where the compound of Formula II binds to the solid phase.
Once the compound of Formula II is bound, the outlet of the SPE column is closed so
that the carboxy deprotecting agent is retained therein for a defined amount of time. A
suitable solid phase for use in this manner is a reversed phase solid phase, e.g. tC18.
Step (c) comprises applying heat to the reaction vessel using methods well-known to
those of skill in the art, e.g. using a dedicated heater into which the reaction vessel is
placed for the duration of the radiosynthesis. The application of heat must be so that
the reaction vessel can be used for the subsequent step (d), i.e. so that the reaction
vessel is intact and undamaged, and also so that residual solvent is effectively removed.
This step (c) is carried out at the same time as removal of PG1, i.e. after the reaction
mixture comprising the compound of Formula II has been transferred out of said
reaction vessel. A suitable temperature for this heating step should be no greater than
the tolerance of the reaction vessel, e.g. for a reaction vessel made from cyclic olefin
copolymer (COC) a temperature of no greater than about 130°C and for a reaction
vessel made from polyetheretherketone (PEEK) a temperature of no greater than about
200°C. For convenience, the temperature used to heat the reaction vessel in step (c)
may be selected to be as close as possible to the temperature used during the labelling
step (a). Suitable temperatures for radiolabelling step (a) are in the range of about 80-
140°C, in other embodiments 85-130°C.
The "removal of PG2" in step (d) of the method of the invention is carried out by
contacting the compound of Formula III with an amine deprotecting agent. A suitable
amine deprotecting agent may be either an acid or an alkaline solution, as is wellknown
to the skilled person (see Greene and Wuts, supra). The concentration of the
amine deprotecting agent is suitably just sufficient to remove PG2. Preferably the
amine deprotecting agent is an acid solution. A suitable acid is an acid selected from
inorganic acids such as hydrochloric acid (HCl), sulfuric acid (H2SO4) and nitric acid
(HNO 3), and organic acids such as perfluoroalkyl carboxylic acids, e.g. trifluoroacetic
acid (CF 3CO2H). In certain embodiments, the amine deprotecting agent is HCl, e.g. at a
concentration of 1.0-4.0M. Removal of PG2 is in one embodiment carried out with heat
to allow the deprotection to proceed more rapidly. The time depends on the reaction
temperature or other conditions. For example, in one embodiment removal of PG2 is
carried out at 60°C, with a reaction time of 5 minutes.
The aim of the "purifying" step (e) is to obtain substantially pure 1 F-FACBC. The
term "substantially" refers to the complete or nearly complete extent or degree of an
action, characteristic, property, state, structure, item, or result. The term "substantially
pure" as used herein in the context of 1 F-FACBC encompasses completely pure 1 FFACBC
or 1 F-FACBC that is sufficiently pure to be suitable for use as a PET tracer.
The term "suitable for use as a PET tracer " means that the purified 1 F-FACBC product
is suitable for intravenous administration to a mammalian subject followed by PET
imaging to obtain one or more clinically-useful images of the location and/or
distribution of 1 F-FACBC.
A "HLB solid phase" is a reversed phase solid phase having hydrophilic and lipophilic
components suitable for a range of purposes. HLB solid phase is commerciallyavailable
as SPE cartridges suitable for use in the method of the present invention, e.g.
the Oasis HLB SPE cartridge.
An "alumina solid phase" is an aluminium oxide normal phase solid phase routinely
used in 1 F labelling methods as a means to remove free 1 F-fluoride and optimise the
radiochemical purity of the final product. Alumina solid phase is commerciallyavailable
as SPE cartridges suitable for use in the method of the present invention, e.g.
the Waters Alumina N Light.
In the method of the invention, steps (a)-(c) or (a)-(e) are carried out in sequence.
In one embodiment of the method of the present invention, the substituent LG in the
compound of Formula I is a linear or branched C1-10 haloalkyl sulfonic acid substituent,
a linear or branched C1-10 alkyl sulfonic acid substituent, a fluorosulfonic acid
substituent, or an aromatic sulfonic acid substituent. Examples of LG include
methanesulfonic acid, toluenesulfonic acid, nitrobenzenesulfonic acid, benzenesulfonic
acid, trifluoromethanesulfonic acid, fluorosulfonic acid, and perfluoroalkylsulfonic
acid. In one embodiment LG is trifluoromethanesulfonic acid.
In one embodiment of the method of the present invention the substituent PG1 in the
compounds of Formula I and II is a linear or branched C1-10 alkyl chain or an aryl
substituent. For example, PG1 can be methyl, ethyl, t-butyl or phenyl. In one
embodiment PG1 is methyl or ethyl. In another embodiment, PG1 is ethyl.
In one embodiment of the method of the present invention the substituent PG2 in the
compounds of Formulas I-III is a carbamate substituent, an amide substituent, an imide
substituent or an amine substituent. Examples include t-butoxycarbonyl,
allyloxycarbonyl, phthalimide, and N-benzylideneamine. In one embodiment, PG2 is tbutoxycarbonyl.
The method of the present invention may further comprise the step of formulating the
purified reaction mixture obtained in step (e) with citrate buffer. In one embodiment,
this formulating step results in a concentration of 50-100 mM citrate buffer, in another
embodiment 60-90 mM citrate buffer and in yet another embodiment 75-85 mM citrate
buffer.
In one embodiment, the method of the invention is automated, e.g. carried out on an
automated synthesis apparatus. 1 F-labelled PET tracers are often conveniently
prepared on automated radiosynthesis apparatus. By the term "automated
radiosynthesis apparatus" is meant an automated module based on the principle of unit
operations as described by Satyamurthy et al (1999 Clin Positr Imag; 2(5): 233-253).
The term "unit operations " means that complex processes are reduced to a series of
simple operations or reactions, which can be applied to a range of materials. Suitable
automated synthesiser apparatus are commercially available from a range of suppliers
including: GE Healthcare Ltd (Chalfont St Giles, UK); CTI Inc. (Knoxville, USA); Ion
Beam Applications S.A. (Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve,
Belgium); Raytest (Straubenhardt, Germany) and Bioscan (Washington DC, USA).
Commercial automated radiosynthesis apparatus also provide suitable containers for the
liquid radioactive waste generated as a result of the radiopharmaceutical preparation.
Automated radiosynthesis apparatus are not typically provided with radiation shielding,
since they are designed to be employed in a suitably configured radioactive work cell.
The radioactive work cell, also termed a hot cell, provides suitable radiation shielding
to protect the operator from potential radiation dose, as well as ventilation to remove
chemical and/or radioactive vapours.
Preferred automated radiosynthesis apparatus of the present invention are those which
interact with a disposable or single use "cassette" (also commonly referred to as a
"cartridge") which comprises all the reagents, reaction vessels and apparatus necessary
to carry out the preparation of a given batch of radiopharmaceutical. By use of such
cassettes the automated radiosynthesis apparatus has the flexibility to be capable of
making a variety of different radiopharmaceuticals with minimal risk of crosscontamination,
by simply changing the cassette. The cassette approach also has the
advantages of: simplified set-up and hence reduced risk of operator error; improved
GMP (Good Manufacturing Practice) compliance; multi-tracer capability; rapid change
between production runs; pre-run automated diagnostic checking of the cassette and
reagents; automated barcode cross-check of chemical reagents vs the synthesis to be
carried out; reagent traceability; single-use and hence no risk of cross-contamination,
tamper and abuse resistance.
The cassette has been simplified by removal of the alumina cartridge. The alumina
cartridge was present in prior cassette configurations to remove residues of free 1 Ffluoride
from insufficient purification and/or from radio lysis. However, the present
inventors have found that the rest activity on the alumina cartridge is very low (0.1-
0.3%) indicating both a robust purification process and a low degree of radiolysis.
These data suggest that the alumina cartridge is superfluous and can be removed. This
has the additional benefit of there being no risk of any particles from the alumina
cartridge being present in drug product, which pose a risk of blocking the sterile filter.
The process has been improved by the addition of a concurrent step of removal of
residual acetonitrile from the reactor while the deesterification step proceeds on the
tC18 cartridge. This results in a final drug product having a lower and more predictable
concentration of residual acetonitrile than that obtained using prior art methods.
Brief Description of the Figures
Figure 1 illustrates an exemplary cassette to carry out the method of the invention.
Brief Description of the Examples
The following non-limiting examples serve to illustrate particular embodiments of the
subject matter of the present invention.
List of Abbreviations used in the Examples
BOC tert-Butyloxycarbony1
DP drug product
HLB hydrophobic-lipophilic balance
K222 Kryptofix 222
MeCN acetonitrile
QMA quaternary methyl ammonium
RAC radioactive concentration
RCP: radiochemical purity
Examples
Comparative Example 1: Prior Art Synthesis of F-FACBC
Ifi) FASTlab CassetteAll radiochemistry was performed on a commerciallyavailable
GE FASTlab™ with single-use cassettes. Each cassette is built around a onepiece-
moulded manifold with 25 three-way stopcocks, all made of polypropylene.
Briefly, the cassette includes a 5 ml reactor (cyclic olefin copolymer), one 1ml syringe
and two 5 ml syringes, spikes for connection with five prefilled vials, one water bag
(100 ml) as well as various SPE cartridges and filters. Fluid paths are controlled with
nitrogen purging, vacuum and the three syringes. The fully automated system is
designed for single-step fluorinations with cyclotron-produced 1 F-fluoride. The
FASTlab was programmed by the software package in a step-by-step time-dependent
sequence of events such as moving the syringes, nitrogen purging, vacuum, and
temperature regulation. Vial A contained K222 (58.8 mg, 156 mihoΐ), K2C0 3 (8.1 mg,
60.8 m hoΐ) in 79.5% (v/v) MeCN(aq) ( 1105 mΐ) . Vial B contained 4M HC1 (2.0 ml). Vial
C contained MeCN (4.1ml). Vial D contained the precursor (48.4 mg, 123.5 mihoΐ) in
its dry form (stored at -20°C until cassette assembly). Vial E contained 2 M NaOH (4.1
ml). The 30 ml product collection glass vial was filled with 200 mM trisodium citrate
(10 ml).
1(H) Production of F-Fluoride
No-carrier-added 1 F-fluoride was produced via the 1 0(p,n) 1 F nuclear reaction on a
GE PETtrace 6 cyclotron (Norwegian Cyclotron Centre, Oslo). Irradiations were
performed using a dual-beam, 30mA current on two equal Ag targets with HAVAR foils
using 16.5 MeV protons. Each target contained 1.6 ml of > 96% [1 0]water (Marshall
Isotopes). Subsequent to irradiation and delivery to a hotcell, each target was washed
with [1 0]water (Merck, water for GR analysis). Aqueous 1 F-fluoride was passed
through the QMA and into the 1 0-H 20 recovery vial. The QMA was then flushed with
MeCN and sent to waste.
1(Hi) F-Fluoride Labelling
The trapped 1 F-fluoride was eluted into the reactor using eluent from vial A and then
concentrated to dryness by azeotropic distillation with acetonitrile (vial C). MeCN was
mixed with precursor in vial D from which the dissolved precursor was added to the
reactor and heated to 85°C.
1(iv) Removal of Ester Protecting Group
The reaction mixture was diluted with water and sent through the tC18 cartridge.
Reactor was washed with water and sent through the tC18 cartridge. The labelled
intermediate, fixed on the tC18 cartridge was washed with water, and then incubated
with 2M NaOH after which the 2M NaOH was sent to waste.
l(v) Removal of BOC Protecting Group
The labelled intermediate (without the ester group) was then eluted off the tC18
cartridge into the reactor using water. The BOC group was hydrolysed by adding 4M
HC1 and heating the reactor.
l(vi) Purification
The reactor content with the crude 1 F-FACBC was sent through the HLB and Alumina
cartridges and into the 30 ml product vial. The HLB and Alumina cartridges were
washed with water and collected in the product vial.
vii) Formulation
2M NaOH and water was added to the product vial, giving a purified drug product (DP)
with a total volume of 26 ml.
(lviii) Acetonitrile Concentration
Acetonitrile (MeCN) concentration was determined using a gas chromatographic
system with FID, an automated liquid injector, a fused silica capillary column with USP
stationary phase G43 (6% cyanopropylphenyl-94% dimethyl polysiloxane) and a
reporting integrator or data system with reintegration capacity. 1000 mg/ml of MeCN
was used as a standard. Blank was prepared by transferring 1ml of purified water to a
2 ml GC crimp cap vial, which was capped immediately. 1ml of the standard was
transferred to a 2 ml GC crimp cap vials and capped immediately. 0.20 ml of the
sample was transferred to a 2 ml GC crimp cap vial with low volume insert (0.25 ml)
and capped immediately. The experimental conditions of the GC instrument were as
follows:
Carrier gas flow, Helium : 2.0 ml/min
Oven temperature program : 40°C for 6 minutes then 20°C/min to 240°C for 4
minutes
Injector temperature : 225°C
Split ratio : 10:1
Detector : FID
Detector temperature : 250°C
Hydrogen flow rate : 30 ml/min
Air flow rate : 400 ml/min
Make up gas flow rate (He) : 25 ml/min
The experimental conditions of the automatic liquid injector were as follows:
Solvent pre washes : 3
Sample pumps : 3
Solvent post washes : 3
Injection volume : 1ml
The column was conditioned at 250°C for at least one hour prior to use.
One injection of each standard and two replicate injections of the sample solution were
performed in addition to blank injections in the following order:
1. Blank
2. Calibration standard
3. Calibration standard
4. Blank
5. Sample, replicate 1
6. Sample, replicate 2
7. Blank
The concentration of each analyte, C s ampie was calculated in mg/ml using the following
formula:
A x
_ sample std
' sample
st
where:
Asampie- Peak area of the analyte in sample
std . Concentration of the analyte in calibration standard ^g/ml)
A st . Peak area of the analyte in calibration standard, average of 2 injections
(lix) Aluminium Concentration
Aluminium concentration was determined by inductively coupled plasma atomic
emission spectroscopy (ICP-AES).
fix) Radiochemical Parameters
Radiochemical purity (RCP) and radioactive concentration (RAC) of 1 F-FACBC were
measured.
RCP was determined by thin layer chromatography (TLC). The TLC strip was eluted
using a mobile phase consisting of acetonitrile:methanol: water:acetic acid, 20:5:5:1 v/v.
The RCP and any radiochemical impurities including 1 F-fluoride were reported as
percentages of the net sum of all peaks.
(lxi) Results
The following results were obtained:
Example 2: Synthesis of F-FACBC using Inventive Method
2( ) Modified Sequence
A modified FASTlab™ cassette was used, as illustrated in Figure 1. The sequence
described in Example 1 was used except that the sequence included the extra
heating/purging of the reaction vessel. The hydrolysis step was replaced with two
steps, the first step of which included hydrolysis and in parallel heating of the reactor at
85°C, nitrogen purging (600 mbar HF) of the reaction vessel and vacuum (-600 mbar).
The second step also included hydrolysis but heating of the reaction vessel was stopped.
Nitrogen purging (600 mbar HF) and vacuum (-600 mbar) were used for cooling of the
reaction vessel. Furthermore, the alumina SPE was removed and the sequence was
changed to transfer the product directly to the formulation buffer vial after the HLB
cartridge step.
2(H) Analysis
The analysis methods as described in Example 1were
Claims

(1) A positron emission tomography (PET) tracer composition comprising anti-lamino-
3-1 F-fluorocyclobutyl-l-carboxylic acid (1 F-FACBC) characterised in that said
composition comprises no more than 5.0 mg/mL dissolved aluminium (Al).
(2) The PET tracer composition as defined in Claim 1 wherein said composition
comprises no more than 50 mg/mL acetonitrile (MeCN).
(3) The PET tracer composition as defined in Claim 1 or Claim 2 wherein said
composition comprises no more than 3.0 mg/mL dissolved Al.
(4) The PET tracer composition as defined in Claim 3 wherein said composition
comprises no more than 1.5 mg/mL dissolved Al.
(5) The PET tracer composition as defined in any one of Claims 1-4 having an end
of synthesis (EOS) radiochemical purity (RCP) of at least 95%.
(6) The PET tracer composition as defined in Claim 5 having an EOS RCP of at
least 98%.
(7) The PET tracer composition as defined in Claim 6 having an EOS RCP of at
least 99%.
(8) The PET tracer composition as defined in any one of Claims 1-7 comprising 50-
100 mM citrate buffer.
(9) In another aspect, the present invention provides a method to prepare a PET
tracer composition of the invention wherein said method comprises:
(a) reacting in a reaction vessel a source of 1 F-fluoride with a precursor
compound of Formula I :
wherein:
LG is a leaving group;
PG is a carboxy protecting group; and,
PG2 is an amine protecting group;
to obtain a reaction mixture comprising a compound of Formula II:
wherein PG1 and PG2 are as defined for Formula I;
(b) carrying out removal of PG1 and PG2 to obtain a reaction mixture
comprising 1 F-FACBC; and
(c) purifying said reaction mixture comprising 1 F-FACBC by passing it
through a hydrophilic lipophilic balanced (HLB) solid phase, characterised in
that said purifying does not comprise passing the reaction mixture comprising
1 F-FACBC through an alumina solid phase.
(10) A method according to claim 9 wherein said method comprises:
(a) reacting in a reaction vessel a source of 1 F-fluoride with a precursor
compound of Formula I :
wherein:
LG is a leaving group;
PG1 is a carboxy protecting group; and,
PG2 is an amine protecting group;
wherein said reacting step is carried out in acetonitrile;
to obtain a reaction mixture comprising a compound of Formula II:
wherein PG1 and PG2 are as defined for Formula I;
(b) transferring said reaction mixture comprising said compound of Formula
II out of said reaction vessel and carrying out removal of PG1 to obtain a
reaction mixture comprising a compound of Formula III:
wherein PG2 is as defined for Formula I;
(c) applying heat to said reaction vessel at the same time as carrying out
removal of PG1;
(d) transferring said reaction mixture comprising said compound of Formula
III back into said reaction vessel and carrying out removal of PG2 to
obtain a reaction mixture comprising 1 F-FACBC;
(e) purifying said reaction mixture comprising 1 F-FACBC by passing it
through a hydrophilic lipophilic balanced (HLB) solid phase,
characterised in that said purifying does not comprise passing the
reaction mixture comprising 1 F-FACBC through an alumina solid
phase.
( 11) The method as defined in Claim 9 or Claim 10 wherein LG is a linear or
branched C1-10 haloalkyl sulfonic acid substituent, a linear or branched C1-10 alkyl
sulfonic acid substituent, a fluorosulfomc acid substituent, or an aromatic sulfonic acid
substituent.
(12) The method as defined in Claim 11 wherein LG is methanesulfonic acid,
toluenesulfonic acid, nitrobenzenesulfonic acid, benzenesulfonic acid,
trifluoromethanesulfonic acid, fluorosulfomc acid, and perfluoroalkylsulfonic acid.
(13) The method as defined in Claim 9 or Claim 10 wherein LG is
trifluoromethanesulfonic acid.
(14) The method as defined in any one of Claims 9-13 wherein PG1 is a linear or
branched C1-10 alkyl chain or an aryl substituent.
(15) The method as defined in Claim 14 wherein PG1 is methyl, ethyl, t-butyl and
phenyl.
(16) The method as defined in Claim 15 wherein PG1 is methyl or ethyl.
(17) The method as defined in Claim 16 wherein PG1 is ethyl.
(18) The method as defined in any one of Claims 9-17 wherein PG2 is a carbamate
substituent, an amide substituent, an imide substituent or an amine substituent.
(19) The method as defined in Claim 18 wherein PG2 is t-butoxycarbonyl,
allyloxycarbonyl, phthalimide, or N-benzylideneamine.
(20) The method as defined in Claim 19 wherein PG2 is t-butoxycarbonyl.
(21) The method as defined in any one of Claims 9-20 further comprising
formulating said purified reaction mixture obtained in steps (c) or (e) with citrate buffer.
(22) The method as defined in any one of Claims 9-21 which is automated.
(23) A positron emission tomography (PET) tracer composition comprising anti-lamino-
3- 1 F-fluorocyclobutyl-l-carboxylic acid (1 F-FACBC) wherein the PET tracer
is made by a method according to any one of Claims 9-22 characterised in that said
composition comprises no more than 5.0 mg/mL dissolved aluminium (Al).
(24) A positron emission tomography (PET) tracer composition as defined in claim
23 characterised in that said composition comprises no more than 5.0 mg/mL dissolved
aluminium (Al) and no more than 50 mg/mL acetonitrile (MeCN).