Radiotracer Compositions.
Field of the Invention.
The present invention relates to improved radiotracer imaging agent compositions,
which comprises 1 F-labelled biological targeting moieties, wherein impurities which
affect imaging in vivo are identified and suppressed. Also provided are
radiopharmaceuticals comprising said improved compositions, together with
radiofluorinated aldehyde compositions useful in preparing said radiotracer
compositions. The invention also includes methods of imaging and/or diagnosis using
the radiopharmaceutical compositions described.
Background to the Invention.
WO 2004/080492 discloses a method of radiofluorination of a vector which comprises
reaction of a compound of formula (I) with a compound of formula (II):
R — I vector
(I)
F-(Linker)-R2 (II)
or a compound of formula (III) with a compound of formula (IV)
R3- vector
(III)
F-(Linker)-R4 (IV)
wherein:
Rl is an aldehyde moiety, a ketone moiety, a protected aldehyde such as an
acetal, a protected ketone, such as a ketal, or a functionality, such as diol or N-
terminal serine residue, which can be rapidly and efficiently oxidised to an
aldehyde or ketone using an oxidising agent;
R2 is a group selected from primary amine, secondary amine, hydroxylamine,
hydrazine, hydrazide, aminoxy, phenylhydrazine, semicarbazide, and
thiosemicarbazide and is preferably a hydrazine, hydrazide or aminoxy group;
R3 is a group selected from primary amine, secondary amine, hydroxylamine,
hydrazine, hydrazide, aminoxy, phenylhydrazine, semicarbazide, or
thiosemicarbazide, and is preferably a hydrazine, hydrazide or aminoxy group;R4 is an aldehyde moiety, a ketone moiety, a protected aldehyde such as an
acetal, a protected ketone, such as a ketal, or a functionality, such as diol or
terminal serine residue, which can be rapidly and efficiently oxidised to an
aldehyde or ketone using an oxidising agent;
a conjugate of formula (V) or (VI) respectively:
(Linker) X— = — vector
Y
vector -X - N= (Linker)— F
(VI)
wherein X is -CO-NH- , -NH- , -0-, - HCO H-, or - HCS H-, and is
preferably -CO-NH- , -NH- or -O- ; Y is H, alkyl or aryl substituents; and
the Linker group in the formulae (II), (IV), V) and (VI) is selected from:
- - C
o
wherein n is an integer of 0 to 20; m is an integer of 1 to 10; p is an integer of 0 or 1;
Z is O or S.WO 2006/030291 discloses a method for radiofluonnation comprising reaction of a
compound of formula (I):
vector 1 O-NH.
(I)
wherein the vector com rises the fragment:
with a compound of formula (II):
wherein:
n is an integer of 0 to 20;
m is an integer of 0 to 10;
Y is hydrogen, Ci-
6alkyl, or phenyl
to give a compound of formula (III):
ri
wherein m, n, and Y are defined as for the compound of formula (II) and the vector is
as defined for the compound of formula (I).
Glaser et al [Bioconj.Chem., 19(4), 951-957 (2008)], describe the synthesis of 1 F-
labelled aldehydes, including 1 F-fluorobenzaldehyde, and their conjugation to amino-
oxy functional! sed cyclic RGD peptides.Speranza et al [Appl.Rad.Isotop., 67, 1664-1669 (2009)] describe an automated
synthesis of [1 F]-fluorobenzaldehyde ([1 F]-FBA) using a TRACERlab™ apparatus.
A hand-made purification device is used to purify the [1 F]-FBA. Speranza et al
describe the fact that cartridge purification is preferred over FIPLC purification for
automated synthesizer apparatus syntheses. Their cartridge methodology, however,
suggests that dichloromethane or chloroform are the best solvents for [1 F]-FBA
purification. Both solvents have unsuitable toxicological properties for in vivo use,
and are immiscible with water. The method is therefore unsuitable for
radiopharmaceutical preparations.
Battle et al [J.Nucl.Med., 52(3), 424-430 (201 1)] disclose monitoring anti-angiogenic
thera with [1 F]-fluciclatide:
[ F]-fluciclatide
Battle et al mention that the [ FJ-FBA used was purified by diluting with water, and
trapping on a solid-phase extraction (SPE) cartridge. Impurities such as precursor,
DMSO, Kryptofix-222 and hydrophilic by-products were said to be eluted to waste,
and the [1 F]-FBA subsequently eluted with ethanol. The present inventors have,
however, found that using a CI8 SPE cartridge only some of the precursor is eluted to
waste, and the remainder co-elutes when the [1 F]-FBA is eluted with ethanol.
There is therefore still a need for alternative methods of labelling biological targeting
moieties with 1 F.The Present Invention.
The present invention provides improved 1 F-radiolabelled biological targeting moiety
(BTM) compositions, derived from the conjugation of 1 F-labelled aldehydes. To
aminooxy- or hydrazine- functionalised BTMs. The invention is based on detailed
analyses of the impurities present in such aldehydes, and an understanding of how
they may be carried through into the radiolabelled BTM product - plus how best to
suppress all undesired impurity species. Many of these impurities were not
recognised in the prior art, and hence such prior art agents contained undesirable
species which would adversely affect the imaging characteristics.
In addition, the improved compositions of the present inventions can be achieved in
shorter preparation times, which minimises any loss of 1 F (half-life 109 minutes)
radioactive content during the preparation and purification steps prior to use. The
compositions of the present invention can be obtained using methodology which is
amenable to automation on a commercial automated synthesizer apparatus - an
advantage over prior art HPLC methods (which cannot be automated in this way).
Automation confers improved reproducibility, as well as reduced operator radiation
dose.
In addition, the higher radiochemical yield and purity of the product means that less
functionalised BTM need be used in obtain the same amount of radioactive product.
Since the unlabelled BTM will compete for the same biological site in vivo, lowering
the amount of functionalised BTM present helps preserve the efficacy of the
radiolabelled product. In addition, since the BTM may be e.g. a complex polypeptide
or protein which is expensive and time-consuming to obtain, that is an important
efficiency of time/materials.
The present invention provides compositions wherein the concentration of the desired
1 F-radiofluorinated BTM is enhanced by a factor of about 40, while the chemical
impurities are reduced by about 99% (ie. by a factor of about 100).Detailed Description of the Invention.
In a first aspect, the present invention provides a composition which comprises an
imaging agent of Formula (I) together with one or more non-radioactive aryl
derivatives of Formula (II):
wherein:
BTM is the biological targeting molecule;
L1 is a synthetic linker group of formula -(A)
m- wherein each A is
independently -CR2- , -CR=CR- , -C≡C- , -CR2C0 2- , -C0 2CR2- ,
- RCO- , -CO R- , -CR=N-0-, - R(C=0 ) R-, - R(C=S) R-, -
S0 2 R- , - RS0 2- , -CR2OCR2- , -CR2SCR2- , -CR2 RCR2- , a C4-8
cycloheteroalkylene group, a C4-8
cycloalkylene group, -Ar-, -NR-Ar-,
-O-Ar-, -Ar-(CO)-, an amino acid, a sugar or a monodisperse
polyethyleneglycol (PEG) building block, wherein each Ar is
independently a C5-12
arylene group, or a C3-12
heteroarylene group;
each R is independently chosen from H, C1-
alkyl, C2-4
alkenyl, C2-4
alkynyl, C1-
alkoxyalkyl or C1-
hydroxyalkyl;
m is an integer of value 1 to 20;
Y is -O- or - H-;
X1 is 1 F, -0(CH 2) 1 F or -OCH2-CH(OH)-CH2
1 F,
wherein q is 2, 3 or 4;
X2 is -N+(CH3)
3, -N(CH3)
2, -OCH3, H, -CH3, -OH, -SCH3,
-OC6H4CHO or 1 F;
wherein BTM, L1 and Y are the same in Formula (I) and (II);
and wherein the total concentration of derivatives of Formula (II) present in
the composition is less than 150 g/mL.The term "composition" has its conventional meaning and refers to a mixture of the
radiolabelled BTM of Formula (I), with one or more non-radioactive aryl derivatives
of Formula (II). Multiple derivatives of Formula (II) may be present in the
composition - but in Formulae (I) and (II) BTM, L1 and Y are the same for all
components of the composition, and X1 and X2 are located at the same ortho, meta or
para- position on the phenyl ring relative to the -C=N group. The components of the
composition of the first aspect therefore differ only in the identity of X1 and X2.
The term "concentration of derivatives of Formula (II) present" refers to the total
concentration of all compounds of Formula (II) present, even though X2 may differ.
As an illustration, present Example 2 uses 3.3 mg of trimethylammonium
benzaldehyde precursor in 1.1 mL volume (3.0 mg/mL or 3000 g/mL). 77 GBq of
[1 F]-fluorobenzaldehyde equates to approximately 0.072 g . Hence, in chemical
terms, the [1 F]-fluoride consumes a relatively small proportion of the precursor, and
without the methods of the present invention, the level of Formula (II) impurities
would be significantly greater. The level of less than 150 g/mL requires that at least
90%, preferably at least 95% of such impurities present have been removed.
Preferably the concentration of derivatives of Formula (II) present is less than 100
g/mL, more preferably less than 45 µg/mL. Quantification is by HPLC-MS, by
reference to a calibration curve based on authentic samples of known impurities. The
extinction coefficients of authentic samples were also determined to aid
quantification. When X2 is 1 F, that corresponds to any 1 F carrier present in the 1 F
radioisotope used. Such species contribute to the non-radioactive impurities of
Formula (II).
The terms "comprising" or "comprises" have their conventional meaning throughout
this application and imply that the composition must have the components listed, but
that other, unspecified compounds or species may be present in addition. The terms
therefore include as a preferred subset "consisting essentially of which means that
the composition has the components listed without other compounds or species being
present.
The imaging agent of Formula (I) comprises a radiofluorinated biological targeting
moiety (BTM). By the term "imaging agent" is meant a compound suitable forimaging the mammalian body. Preferably, the mammal is an intact mammalian body
in vivo, and is more preferably a human subject. Preferably, the imaging agent can be
administered to the mammalian body in a minimally invasive manner, i.e. without a
substantial health risk to the mammalian subject when carried out under professional
medical expertise. Such minimally invasive administration is preferably intravenous
administration into a peripheral vein of said subject, without the need for local or
general anaesthetic.
The term "in vivo imaging" as used herein refers to those techniques that non-
invasively produce images of all or part of an internal aspect of a mammalian subject.
A preferred imaging technique of the present invention is positron emission
tomography (PET).
By the term "biological targeting moiety" (BTM) is meant a compound which, after
administration, is taken up selectively or localises at a particular site of the
mammalian body in vivo. Such sites may be implicated in a particular disease state or
be indicative of how an organ or metabolic process is functioning.
By the term "amino acid" is meant an L- or J -amino acid, amino acid analogue (eg.
naphthylalanine) which may be naturally occurring or of purely synthetic origin, and
may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of
enantiomers. Conventional 3 -letter or single letter abbreviations for amino acids are
used herein. Preferably the amino acids of the present invention are optically pure.
By the term "sugar" is meant a mono-, di- or tri- saccharide. Suitable sugars include:
glucose, galactose, maltose, mannose, and lactose. Optionally, the sugar may be
functionalised to permit facile coupling to amino acids. Thus, eg. a glucosamine
derivative of an amino acid can be conjugated to other amino acids via peptide bonds.
The glucosamine derivative of asparagine (commercially available from
NovaBiochem) is one example of this:The term "polyethyleneglycol polymer" or "PEG" has its conventional meaning, as
described e.g. in "The Merck Index", 14th Edition entry 7568, i.e. a liquid or solid
polymer of general formula H(OCH2CH2)
nOH where n is an integer greater than or
equal to 4 . The polyethyleneglycol polymers of the present invention may be linear or
branched, but are preferably linear. The polymers are also preferably non-
dendrimeric. Preferred PEG-containing linker groups comprise units derived from
oligomerisation of the monodisperse PEG-like structures of Formulae Biol or Bio2:
(Biol)
17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula Biol
wherein p is an integer from 1 to 10. Alternatively, a PEG-like structure based on a
propionic acid derivative of Formula Bio2 can be used:
(Bio2)
where p is as defined for Formula Biol and q is an integer from 3 to 15.
In Formula Bio2, p is preferably 1 or 2, and q is preferably 5 to 12.
In Formulae (I) and (II), isomerism at the C=N bond means that E- or Z-
diastereomers may occur. Although drawn as only a single isomer, Formulae (I) and
(II) are intended to encompass mixtures of such isomers, as well as mixtures enriched
in one such diastereomer, as well as pure diastereomers.Preferred embodiments.
In Formulae (I) of the first aspect, X1 is preferably 1 F, or -0(CH 2)
q
1 F where q is 2 or
3; more preferably 1 F or -0(CH 2 ) 3
1 F, most preferably 1 F.
In Formulae (I) and (II) of the first aspect, X1 and X2 are preferably located at either
the ortho or para- position, more preferably at the para- position.
In Formulae (I) and (II), Y is preferably -0-. More preferably, Y is -O- and X1 and X2
are preferably located at the para- position. For each such combination, preferred
BTM groups are as described below.
The BTM may be of synthetic or natural origin, but is preferably synthetic. The term
"synthetic" has its conventional meaning, i.e. man-made as opposed to being isolated
from natural sources eg. from the mammalian body. Such compounds have the
advantage that their manufacture and impurity profile can be fully controlled.
Monoclonal antibodies and fragments thereof of natural origin are therefore outside
the scope of the term 'synthetic' as used herein. The BTM is preferably non-
proteinaceous, i.e. does not comprise a protein.
The molecular weight of the BTM is preferably up to 10,000 Daltons. More
preferably, the molecular weight is in the range 200 to 9,000 Daltons, most preferably
300 to 8,000 Daltons, with 400 to 6,000 Daltons being especially preferred. When the
BTM is a non-peptide, the molecular weight of the BTM is preferably up to 3,000
Daltons, more preferably 200 to 2,500 Daltons, most preferably 300 to 2,000 Daltons,
with 400 to 1,500 Daltons being especially preferred.
The biological targeting moiety preferably comprises: a 3-80 mer peptide, peptide
analogue, peptoid or peptide mimetic which may be a linear or cyclic peptide or
combination thereof; a single amino acid; an enzyme substrate, enzyme antagonist
enzyme agonist (including partial agonist) or enzyme inhibitor; receptor-binding
compound (including a receptor substrate, antagonist, agonist or substrate);
oligonucleotides, or oligo-DNA or oligo-RNA fragments.By the term "peptide" is meant a compound comprising two or more amino acids, as
defined below, linked by a peptide bond (i.e. an amide bond linking the amine of one
amino acid to the carboxyl of another). The term "peptide mimetic" or "mimetic"
refers to biologically active compounds that mimic the biological activity of a peptide
or a protein but are no longer peptidic in chemical nature, that is, they no longer
contain any peptide bonds (that is, amide bonds between amino acids). Here, the term
peptide mimetic is used in a broader sense to include molecules that are no longer
completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.
The term "peptide analogue" refers to peptides comprising one or more amino acid
analogues, as described below. See also Synthesis o f Peptides and Peptidomimetics,
M. Goodman et al, Houben-Weyl Vol E22c ofMethods in Organic Chemistry,
Thieme (2004).
When the BTM is an enzyme substrate, enzyme antagonist, enzyme agonist, enzyme
inhibitor or receptor-binding compound it is preferably a non-peptide, and more
preferably is synthetic. By the term "non-peptide" is meant a compound which does
not comprise any peptide bonds, i.e. an amide bond between two amino acid residues.
Suitable enzyme substrates, antagonists, agonists or inhibitors include glucose and
glucose analogues such as fluorodeoxyglucose; fatty acids, or elastase, Angiotensin II
or metalloproteinase inhibitors. A preferred non-peptide Angiotensin II antagonist is
Losartan. Suitable synthetic receptor-binding compounds include estradiol, estrogen,
progestin, progesterone and other steroid hormones; ligands for the dopamine D - l or
D -2 receptor, or dopamine transporter such as tropanes; and ligands for the serotonin
receptor.
The BTM is most preferably a 3-100 mer peptide or peptide analogue. When the
BTM is a peptide, it is preferably a 4-30 mer peptide, and most preferably a 5 to 28-
mer peptide. When the BTM is a peptide, preferred such peptides include:
somatostatin, octreotide and analogues,
- peptides which bind to the ST receptor, where ST refers to the heat-stable
toxin produced by E.coli and other micro-organisms;
- bombesin;
- vasoactive intestinal peptide;
neurotensin;- laminin fragments eg. YIGSR, PDSGR, IKVAV, LRE and
KCQAGTFALRGDPQG,
- N-formyl chemotactic peptides for targeting sites of leucocyte accumulation,
- Platelet factor 4 (PF4) and fragments thereof,
- RGD (Arg-Gly-Asp)-containing peptides, which may eg. target angiogenesis
[R.Pasqualini et al, Nat Biotechnol. 1997 Jun;15(6):542-6]; [E. Ruoslahti,
Kidney Int. 1997 May;5 1(5): 1413-7].
peptide fragments of a.2-antiplasmin, fibronectin or beta-casein, fibrinogen or
thrombospondin. The amino acid sequences of a.2-antiplasmin, fibronectin,
beta-casein, fibrinogen and thrombospondin can be found in the following
references: a.2-antiplasmin precursor [M.Tone et al, J.Biochem, 102, 1033,
(1987)]; beta-casein [L.Hansson et al, Gene, 139, 193, (1994)]; fibronectin
[A.Gutman et al, FEBS Lett., 207, 145, (1996)]; thrombospondin- 1 precursor
[V.Dixit et al, Proc. Natl. Acad. Sci., USA, 83, 5449, (1986)]; R.F.Doolittle,
Ann. Rev. Biochem., 53, 195, (1984);
peptides which are substrates or inhibitors of angiotensin, such as:
angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (E. C. Jorgensen et al, J.
Med. Chem., 1979, Vol 22, 9, 1038-1044)
[Sar, e] Angiotensin II: Sar-Arg-Val-Tyr-Ile-His-Pro-Ile (R.K. Turker et
al, Science, 1972, 177, 1203).
Angiotensin I : Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu.
When the BTM is a peptide, one or both termini of the peptide, preferably both, have
conjugated thereto a metabolism inhibiting group (M ) . Having both peptide termini
protected in this way is important for in vivo imaging applications, since otherwise
rapid metabolism would be expected with consequent loss of selective binding affinity
for the BTM peptide. By the term "metabolism inhibiting group" (M ) is meant a
biocompatible group which inhibits or suppresses enzyme, especially peptidase such
as carboxypeptidase, metabolism of the BTM peptide at either the amino terminus or
carboxy terminus. Such groups are particularly important for in vivo applications, and
are well known to those skilled in the art and are suitably chosen from, for the peptide
amine terminus:N-acylated groups -NH(C=0)R where the acyl group -(C=0)R has R chosen
from: Ci_6 alkyl, C 3-10 aryl groups or comprises a polyethyleneglycol (PEG) building
block. Suitable PEG groups are described for the linker group (L1), above. Preferred
such PEG groups are the biomodifiers of Formulae Biol or Bio2 (above). Preferred
such amino terminus M groups are acetyl, benzyl oxycarbonyl or trifluoroacetyl,
most preferably acetyl.
Suitable metabolism inhibiting groups for the peptide carboxyl terminus include:
carboxamide, t r t-butyl ester, benzyl ester, cyclohexyl ester, amino alcohol or a
polyethyleneglycol (PEG) building block. A suitable M group for the carboxy
terminal amino acid residue of the BTM peptide is where the terminal amine of the
amino acid residue is N-alkylated with a C1-4 alkyl group, preferably a methyl group.
Preferred such M groups are carboxamide or PEG, most preferred such groups are
carboxamide.
Preferred BTM peptides are RGD peptides. A more preferred such RGD peptide
comprises the fragment:
A most preferred such RGD peptide is when the BTM is a peptide of Formula
(BTM1):PEG1
wherein a is an integer of from 1to 10.
In Formula BTM1, X is preferably PEG1 with 'a' preferably equal to 1.
A referred imaging agent of Formula (I) is [1 F]-fluciclatide of Formula (IA):
In the composition of the first aspect, when X1 is 1 F, X2 is preferably -N+(CH3)
3,
-N(CH3)
2 or -OH, more preferably multiple derivatives of Formula (II) are present
such that X2 is equal to all 3 of these groups. That is described more fully in the third
aspect (below).Preferably, the imaging agent composition is provided in sterile form, i.e. in a form
suitable for mammalian administration as is described in the second aspect (below).
The imaging agent compositions of the first aspect can be obtained as described in the
fourth aspect (below).
In a second aspect, the present invention provides a radiopharmaceutical composition
which comprises the imaging agent composition of the first aspect, together with a
biocompatible carrier, in a form suitable for mammalian administration.
Preferred aspects of the imaging agent composition in the second aspect are as defined
in the first aspect (above).
By the phrase "in a form suitable for mammalian administration" is meant a
composition which is sterile, pyrogen-free, lacks compounds which produce toxic or
adverse effects, and is formulated at a biocompatible pH (approximately pH 4.0 to
10.5). Such compositions lack particulates which could risk causing emboli in vivo,
and are formulated so that precipitation does not occur on contact with biological
fluids (e.g. blood). Such compositions also contain only biologically compatible
excipients, and are preferably isotonic.
The "biocompatible carrier" is a fluid, especially a liquid, in which the imaging agent
can be suspended or preferably 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; an aqueous solution such as saline (which
may advantageously be balanced so that the final product for injection is isotonic); an
aqueous buffer solution comprising a biocompatible buffering agent (e.g. phosphate
buffer); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of
plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose),
sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic
polyol materials (e.g. polyethyleneglycols, propylene glycols and the like). Preferablythe biocompatible carrier is pyrogen-free water for injection, isotonic saline or
phosphate buffer.
The imaging agents and biocompatible carrier are each supplied in suitable vials or
vessels which comprise a sealed container which permits maintenance of sterile
integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen
or argon), whilst permitting addition and withdrawal of solutions by syringe or
cannula. A preferred such container is a septum-sealed vial, wherein the gas-tight
closure is crimped on with an overseal (typically of aluminium). The closure is
suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on
septum seal closure) whilst maintaining sterile integrity. Such containers have the
additional advantage that the closure can withstand vacuum if desired (eg. to change
the headspace gas or degas solutions), and withstand pressure changes such as
reductions in pressure without permitting ingress of external atmospheric gases, such
as oxygen or water vapour.
Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 50 cm3
volume) which contains multiple patient doses, whereby single patient doses can thus
be withdrawn into clinical grade syringes at various time intervals during the viable
lifetime of the preparation to suit the clinical situation. Pre-filled syringes are
designed to contain a single human dose, or "unit dose" and are therefore preferably a
disposable or other syringe suitable for clinical use. The pharmaceutical compositions
of the present invention preferably have a dosage suitable for a single patient and are
provided in a suitable syringe or container, as described above.
The pharmaceutical composition may contain additional optional excipients such as:
an antimicrobial preservative, pH-adjusting agent, filler, radioprotectant, solubiliser or
osmolality adjusting agent. By the term "radioprotectant" is meant a compound
which inhibits degradation reactions, such as redox processes, by trapping highly-
reactive free radicals, such as oxygen-containing free radicals arising from the
radiolysis of water. The radioprotectants of the present invention are suitably chosen
from: ascorbic acid, ^ara-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid
(i.e. 2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cation. By the
term "biocompatible cation" (B ) is meant a positively charged counterion whichforms a salt with an ionised, negatively charged group, where said positively charged
counterion is also non-toxic and hence suitable for administration to the mammalian
body, especially the human body. Examples of suitable biocompatible cations
include: the alkali metals sodium or potassium; the alkaline earth metals calcium and
magnesium; and the ammonium ion. Preferred biocompatible cations are sodium and
potassium, most preferably sodium.
When the radiopharmaceutical composition comprises fluciclatide of Formula (IA),
the composition preferably comprises a radioprotectant. Preferably, the
radioprotectant is sodium 4-aminobenzoate (Na-pAB A). A preferred concentration of
Na-pABA to use is 1 to 3 mg/mL, preferably 1.5 to 2.5 mg/mL, most preferably about
2.0 mg/mL.
By the term "solubiliser" is meant an additive present in the composition which
increases the solubility of the imaging agent in the solvent. A preferred such solvent is
aqueous media, and hence the solubiliser preferably improves solubility in water.
Suitable such solubilisers include: C1-4 alcohols; glycerine; polyethylene glycol
(PEG); propylene glycol; polyoxyethylene sorbitan monooleate; sorbitan
monooloeate; polysorbates; poly(oxyethylene)poly(oxypropylene)poly(oxyethylene)
block copolymers (Pluronics™); cyclodextrins (e.g. alpha, beta or gamma
cyclodextrin, hydroxypropyl -P-cyclodextrin or hydroxypropyl-y-cyclodextrin) and
lecithin.
By the term "antimicrobial preservative" is meant an agent which inhibits the growth
of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The
antimicrobial preservative may also exhibit some bactericidal properties, depending on
the dosage employed. The main role of the antimicrobial preservative(s) of the present
invention is to inhibit the growth of any such micro-organism in the pharmaceutical
composition. The antimicrobial preservative may, however, also optionally be used to
inhibit the growth of potentially harmful micro-organisms in one or more components
of kits used to prepare said composition prior to administration. Suitable antimicrobial
preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben ormixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred
antimicrobial preservative(s) are the parabens.
The term "pH-adjusting agent" means a compound or mixture of compounds useful to
ensure that the pH of the composition is within acceptable limits (approximately pH
4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting
agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS
[i.e. tm(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such
as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition
is employed in kit form, the pH adjusting agent may optionally be provided in a
separate vial or container, so that the user of the kit can adjust the pH as part of a
multi-step procedure.
By the term "filler" is meant a pharmaceutically acceptable bulking agent which may
facilitate material handling during production and lyophilisation. Suitable fillers
include inorganic salts such as sodium chloride, and water soluble sugars or sugar
alcohols such as sucrose, maltose, mannitol or trehalose.
The radiopharmaceutical compositions of the fourth aspect may be prepared under
aseptic manufacture (i.e. clean room) conditions to give the desired sterile, non-
pyrogenic product. It is preferred that the key components, especially the associated
reagents plus those parts of the apparatus which come into contact with the imaging
agent (eg. vials) are sterile. The components and reagents can be sterilised by
methods known in the art, including: sterile filtration, terminal sterilisation using e.g.
gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene
oxide). It is preferred to sterilise some components in advance, so that the minimum
number of manipulations needs to be carried out. As a precaution, however, it is
preferred to include at least a sterile filtration step as the final step in the preparation
of the pharmaceutical composition.
The radiopharmaceutical compositions of the present invention may be prepared as
described in the fourth aspect (below).In a third aspect, the present invention provides a composition which comprises a
radioactive aldehyde of Formula (A) together with one or more non-radioactive
aldehydes of Formula (B):
wherein X1 and X2 are as defined in the first aspect;
and wherein the total concentration of derivatives of Formula (B) present in
the composition is less than 150 µ π ΐ .
Preferred aspects of X1 and X2 in the third aspect are as defined in the first aspect
(above). Preferably the concentration of derivatives of Formula (B) present is less
than 100 µ more preferably less than 45 µ .
In the composition of the third aspect, X1 is preferably 1 F and X2 is -N+(CH3)
3, -
N(CH3)
2 or -OH or combinations thereof.
Thus, the present inventors have found that, when [1 F]-fluorobenzaldehyde ([1 F]-
FBA) is prepared by conventional radiosynthesis from TMAB, over 95% of the
chemical impurities present are derived from TMAB, DMAB and HBA:TMAB DMAB
In addition, impurities of Formula (II) can arise where X2 = -SCH3 or -OC6H4CHO.
The X2 = -SCH3 species can arise when DMSO is used as the solvent. These
impurities have been identified by LC-MS studies.
Since these impurities are all aldehydic in nature, they compete with [1 F]-FBA with
the functionalised BTM of interest. That has three important effects:
(i) the radioactive yield is reduced;
(ii) the overall chemical purity is reduced;
(iii) the imaging agent composition arising therefrom may contain multiple
BTM-functionalised impurities [of Formula (II)] which may compete with the
1 F-labelled imaging agent for the desired biological site in vivo.
Issue (iii) may therefore impact on the effectiveness of the imaging agent in vivo. In
addition, since the BTM conjugates of Formulae (I) and (II) are of similar structure,
they can be difficult to separate once formed and present in the composition. The
radioisotope 1 F has a half-life of 109 minutes, consequently time spent in purifying
the composition also has an impact on issue (i). Hence, the improved composition of
the third aspect is an important contributor to achieving the imaging agent
composition of the first aspect.
The composition of the third aspect is preferably provided as a solution. Suitable
solvents for such solution are: ethanol, aqueous ethanol, acetonitrile or aqueous
acetonitrile. Preferred solvents are ethanol or aqueous ethanol, more preferably
ethanol.
The composition of the third aspect may be obtained as follows. The 1 F-aldehyde is
diluted with ammonium hydroxide solution and then purified on an MCX mixedmode solid-phase extraction (SPE) cartridge (commercially available from Waters,
part #186003516). The mixed mode cartridge has both cation exchange and reversed
phase (CI 8) chromatography characteristics. The alkaline conditions of the crude
mixture ensures that HBA, Kryptofix 222 and potassium carbonate plus any unreacted
[1 F]-fluoride ion, are in ionized form. Consequently, they do not bind to the cartridge
and are thus washed to waste. The [1 F]-aldehyde is subsequently eluted from the
MCX cartridge with ethanol. Cationic species such as TMAB are retained by the
cartridge - and not eluted when the FBA is eluted with organic solvent.
In a fourth aspect, the present invention provides a method of radiolabelling a
biological targeting molecule which comprises:
(i) provision of a biological targeting moiety functionalized with an aminooxy
or hydrazine group;
(ii) reaction of the functionalized-biological targeting moiety from step (i)
with the radioactive aldehyde composition of the third aspect, such that the
radioactive aldehyde of Formula (A) condenses with said aminooxy or
hydrazine group, to give the radiolabelled biological targeting molecule.
The biological targeting moiety (BTM) of the fourth aspect, and preferred
embodiments thereof are as described in the first aspect (above).
The term "amino-oxy group" is meant the BTM having covalently conjugated thereto
an amino-oxy functional group. Such groups are of formula - 0 -NH2, preferably
-CH 2O- H2 and have the advantage that the amine of the amino-oxy group is more
reactive than a Lys amine group in condensation reactions with aldehydes to form
oxime ethers.
The "hydrazine group" is a functional group of formula - H- H2.
In the method of the fourth aspect, the functionalized biological targeting moiety is
preferably of Formula (IV):
[BTM] -L1-Y- H2 (IV)and the radiolabeled product is of Formula (I):
wherein:
BTM, L1, Y and X1 and preferred aspects thereof are as described in the first
aspect (above).
The method of the fourth aspect is preferably carried out using an automated
synthesizer apparatus. By the term "automated synthesizer" is meant an automated
module based on the principle of unit operations as described by Satyamurthy et al
[Clin.Positr.Imag., 2(5), 233-253 (1999)]. 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. Such automated synthesizers are preferred for the
method of the present invention especially when a radiopharmaceutical composition is
desired. They are commercially available from a range of suppliers [Satyamurthy et
al, above], including: GE Healthcare; CTI Inc; Ion Beam Applications S.A. (Chemin
du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and
Bioscan (USA).
Commercial automated synthesizers also provide suitable containers for the liquid
radioactive waste generated as a result of the radiopharmaceutical preparation.
Automated synthesizers 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 provides suitable radiation shielding to protect the operator from
potential radiation dose, as well as ventilation to remove chemical and/or radioactive
vapours. The automated synthesizer preferably comprises a cassette. By the term
"cassette" is meant a piece of apparatus designed to fit removably and
interchangeably onto an automated synthesizer apparatus (as defined above), in such a
way that mechanical movement of moving parts of the synthesizer controls the
operation of the cassette from outside the cassette, i.e. externally. Suitable cassettes
comprise a linear array of valves, each linked to a port where reagents or vials can be
attached, by either needle puncture of an inverted septum-sealed vial, or by gas-tight,
marrying joints. Each valve has a male-female joint which interfaces with acorresponding moving arm of the automated synthesizer. External rotation of the arm
thus controls the opening or closing of the valve when the cassette is attached to the
automated synthesizer. Additional moving parts of the automated synthesizer are
designed to clip onto syringe plunger tips, and thus raise or depress syringe barrels.
The cassette is versatile, typically having several positions where reagents can be
attached, and several suitable for attachment of syringe vials of reagents or
chromatography cartridges (e.g. solid phase extraction or SPE). The cassette always
comprises a reaction vessel. Such reaction vessels are preferably 0.5 to 10 mL, more
preferably 0.5 to 5 mL and most preferably 0.5 to 4 mL in volume and are configured
such that 3 or more ports of the cassette are connected thereto, to permit transfer of
reagents or solvents from various ports on the cassette. Preferably the cassette has 15
to 40 valves in a linear array, most preferably 20 to 30, with 25 being especially
preferred. The valves of the cassette are preferably each identical, and most
preferably are 3-way valves. The cassettes are designed to be suitable for
radiopharmaceutical manufacture and are therefore manufactured from materials
which are of pharmaceutical grade and ideally also are resistant to radiolysis.
Preferred automated synthesizers of the present invention comprise a disposable or
single use cassette which comprises all the reagents, reaction vessels and apparatus
necessary to carry out the preparation of a given batch of radiofluorinated
radiopharmaceutical. The cassette means that the automated synthesizer has the
flexibility to be capable of making a variety of different radiopharmaceuticals with
minimal risk of cross-contamination, by simply changing the cassette. The cassette
approach also has the advantages of: simplified set-up 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.
Included in this aspect of the invention, is the use of an automated synthesizer
apparatus to prepare the radiopharmaceutical composition of the second aspect.The method of the fourth aspect is preferably carried out in a sterile manner, such that
the pharmaceutical composition of the second aspect is obtained. The
radiopharmaceutical compositions of the present invention may be prepared by
various methods:
(i) aseptic manufacture techniques in which the 1 F-radiolabelling step is
carried out in a clean room environment;
(ii) terminal sterilisation, in which the 1 F-radiolabelling is carried out without
using aseptic manufacture and then sterilised at the last step [eg. by gamma
irradiation, autoclaving dry heat or chemical treatment (e.g. with ethylene
oxide)];
(iii) kit methodology in which a sterile, non-radioactive kit formulation
comprising a suitable precursor and optional excipients is reacted with a
suitable supply of 1 F;
(iv) aseptic manufacture techniques in which the 1 F-radiolabelling step is
carried out using an automated synthesizer apparatus.
Method (iv) is preferred.
Amino-oxy functionalised peptides can be prepared by the methods of Poethko et al
[J.Nucl.Med., 45, 892-902 (2004)], Schirrmacher et al [Bioconj.Chem., 18, 2085-
2089 (2007)], Solbakken et al [Bioorg.Med.Chem.Lett, 16, 6190-6193 (2006)] or
Glaser et al [Bioconj. Chem., 19, 951-957 (2008)]. The amino-oxy group may
optionally be conjugated in two steps. First, the corresponding N-protected amino-
oxy carboxylic acid or N-protected amino-oxy activated ester is conjugated to the
peptide. Second, the intermediate N-protected amino-oxy functionalised peptide is
deprotected to give the desired product (see Solbakken and Glaser papers cited
above). N-protected amino-oxy carboxylic acids such as Boc-NH-0-CH 2(C=0)OH
and Eei-N-0-CH 2(C=0)OH are commercially available, e.g. from Novabiochem and
IRIS.
Methods of conjugating hydrazine functional groups to polypeptides and subsequent
condensations with radiolabeled aldehydes to form hydrazine-linked conjugates are
described by Y.Wang et al [Nucl.Med.Biol., (201 1) "Synthesis and evaluation of
[1 F]exendin (9-39). . ." epublished before print], as well asMeszaros et al
[Inorg.Chim.Acta, 363(6). 1059-1069 (2010)].The term "protected" refers to the use of a protecting group. By the term "protecting
group" is meant 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 under mild enough conditions that do not modify the rest
of the molecule. After deprotection the desired product is obtained. Amine protecting
groups are well known to those skilled in the art and are suitably chosen from: Boc
(where Boc is t r t-butyloxycarbonyl); Eei (where Eei is ethoxyethylidene); Fmoc
(where Fmoc is fluorenylmethoxycarbonyl); trifluoroacetyl; allyloxycarbonyl; Dde
[i.e. l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl] orNpys (i.e. 3-nitro-2-pyridine
sulfenyl). The use of further protecting groups are described in Protective Groups in
Organic Synthesis, 4th Edition, Theorodora W. Greene and Peter G.M.Wuts, [Wiley
Blackwell, (2006)]. Preferred amine protecting groups are Boc and Eei, most
preferably Eei.
1 F-labelled aliphatic aldehydes of formula 1 F(CH2)
20[CH 2CH20 ] CH2CHO, where
q is 3, can be obtained by the method of Glaser et al [Bioconj.Chem., 19(4), 951-957
(2008)]. [1 F]-fluorobenzaldehyde can be obtained by the method of Glaser et al
[J.Lab.Comp.Radiopharm., 52, 327-330 (2009)]. The precursor to [1 F]-
fluorobenzaldehyde, i.e. Me3N+-C6H 4-CHO. CF3S0 3 is obtained by the method of
Haka et al [J.Lab.Comp.Radiopharm., 27, 823-833 (1989)].
Other peptides can be obtained by solid phase peptide synthesis as described in P.
Lloyd-Williams, F. Albericio and E. Girald; ChemicalApproaches to the Synthesis o f
Peptides and Proteins, CRC Press, 1997.
In a fifth aspect, the present invention provides a method of imaging the human or
animal body which comprises generating a PET image of at least a part of said body
to which the radiopharmaceutical composition of the second aspect has distributed.
Preferred aspects of the radiopharmaceutical composition and the imaging agent
therein in the fifth aspect are as described in the second and first aspects of the present
invention respectively (see above).The method of the fifth aspect is preferably carried out where the part of the body is
disease state where abnormal expression of the integrin α β3 receptor is involved, in
particular where angiogenesis is involved. Such disease states include rheumatoid
arthritis, psoriasis, restenosis, retinopathy and tumour growth. A preferred such
diseases state of the fifth aspect is tumour growth. Positron Emission Tomography
(PET) imaging of integrin α β3 expression is described by Beer et al [Theranostics, I ,
48-57 (201 1)].
The imaging method of the fifth aspect may optionally be carried out repeatedly to
monitor the effect of treatment of a human or animal body with a drug, said imaging
being effected before and after treatment with said drug, and optionally also during
treatment with said drug. Of particular interest is early monitoring of the efficacy of
anti-angiogenic cancer therapy to ensure that malignant growth is controlled before
the condition becomes terminal. Such therapy monitoring imaging is described by
Battle et al [J.Nucl.Med., 52(3), 424-430 (201 1)] and Morrison et al [J.Nucl.Med.,
50(1), 116-122 (2009) and Theranostics, 1, 149-153 (201 1)].
The method of the fifth aspect is preferably carried out whereby the
radiopharmaceutical composition has been previously administered to the mammalian
body. By "previously administered" is meant that the step involving the clinician,
wherein the imaging agent is given to the patient e.g. as an intravenous injection, has
already been carried out prior to imaging.
In a sixth aspect, the present invention provides a method of diagnosis of the human
or animal body which comprises the imaging method of the fifth aspect.
Preferred aspects of the imaging agent, composition and imaging method in the sixth
aspect are as described in the first, second and fifth aspects (above).
The invention is illustrated by the non-limiting Examples detailed below. Example 1
provides the synthesis of Precursor 1 of the invention. Example 2 provides the
synthesis of [1 F-FBA, and Example 3 the purification of [1 F]-FBA to obtaincompositions of the invention. Example 4 provides the synthesis of Compound 1 of
the invention using the purified [1 F]-FBA composition of the invention. Example 5
provides an impurity analysis demonstrating how impurity species are removed using
the methods of the invention.
Abbreviations.
Conventional single letter or 3-letter amino acid abbreviations are used.
Ac: Acetyl.
ACN: Acetonitrile.
Boc: t r t-Butyloxycarbonyl .
DIPEA: Ν,Ν D-diisopropylethylamine.
DMAB: 4-(dimethylamino)benzaldehyde.
DMSO: Dimethylsulfoxide.
EOS: End of synthesis.
FBA: 4-Fluorobenzaldehyde.
Fmoc: 9-Fluorenylmethoxycarbonyl .
HATU: 0-(7-Azabenzotriazol- 1-yl)-N,N V,N -tetramethyluronium hexafluorophosphate
HBA: 4-hydroxybenzaldehyde.
HPLC: High performance liquid chromatography.
MCX Mixed mode cation exchange cartridge
MM: N-methymorpholine.
MP: 1-Methyl-2-pyrrolidinone.
PBS: Phosphate-buffered saline.
PyBOP: Benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.
RAC: radioactive concentration.
RCP: Radiochemical purity.
RT: room temperature.
SPE: solid-phase extraction.
tBu: t r t-Butyl.
TFA: Trifluoroacetic acid.
TFP: Tetrafluorophenyl .
TMAB: 4-(trimethylammonium)benzaldehyde.
TR: retention time.able 1: Compounds of the Invention.Example 1: Synthesis of Precursor 1.
Peptide 1was synthesised using standard peptide synthesis.
(a) 1,17-Diazido-3 ,6,9,12,15 -pentaoxaheptadecane .
A solution of dry hexaethylene glycol (25 g, 88 mmol) and methanesulfonyl chloride
(22.3 g, 195 mmol) in dry THF (125 mL) was kept under argon and cooled to 0 °C in
an ice/water bath. A solution of triethylamine (19.7 g, 195 mmol) in dry THF (25
mL) was added dropwise over 45 min. After 1 hr the cooling bath was removed and
the reaction was stirred for another for 4 hrs. Water (55 mL) was then added to the
mixture, followed by sodium hydrogencarbonate (5.3 g, to pH 8) and sodium azide
(12.7 g, 195 mmol). THF was removed by distillation and the aqueous solution was
refluxed for 24 h (two layers were formed). The mixture was cooled, ether (100 mL)
was added and the aqueous phase was saturated with sodium chloride. The phases
were separated and the aqueous phase was extracted with ether (4 x 50 mL). The
combined organic phases were washed with brine (2 x 50 mL) and dried (MgSC^).
Filtration and evaporation of the solvent gave a yellow oil 26 g (89 %). The product
was used in the next step without further purification.
(b) 17-Azido-3,6,9,12,15-pentaoxaheptadecanamine.
To a vigorously stirred suspension of l,17-diazido-3,6,9,12,15-pentaoxaheptadecane
(25 g, 75 mmol) in 5 % HC1 (200 mL) was added a solution of triphenylphosphine
(19.2 g, 73 mmol) in ether (150 mL) over 3 hrs at room temperature. The reaction
mixture was stirred for additional 24 hrs. The phases were separated and the aqueous
phase was extracted with dichloromethane (3 x 40 mL). The aqueous phase was
cooled in an ice/water bath and the pH was adjusted to 12 by addition of solid
potassium hydroxide. The aqueous phase was concentrated and the product was taken
up in dichloromethane (150 mL). The organic phase was dried (Na2S0 4) and
concentrated giving a yellow oil 22 g (95 %). The product was identified by
electrospray mass spectrometry (ESI-MS) (MH+ calculated: 307.19; found 307.4).
The crude oil was used in the next step without further purification.
(c) 23-Azido-5-oxo-6-aza-3,9, 12, 15,1 8,2 1-hexaoxatricosanoic acid.
To a solution of 17-azido-3,6,9,12,15-pentaoxaheptadecanamine (15 g, 50 mmol) in
dichloromethane (100 mL) was added diglycolic anhydride (Acros, 6.4 g, 55 mmol).The reaction mixture was stirred overnight. The reaction was monitored by ESI-MS
analysis, and more reagents were added to drive the reaction to completion. The
solution was concentrated to give a yellow residue which was dissolved in water (250
mL). The product was isolated from the aqueous phase by continuous extraction with
dichloromethane overnight. Drying and evaporation of the solvent gave a yield of 18
g (85 %). The product was characterized by ESI-MS analysis (MH+ calculated:
423.20; found 423.4). The product was used in the next step without further
purification.
(d) 23-Amino-5-oxo-6-aza-3,9, 12, 15,1 8,2 1-hexaoxatricosanoic acid.
23-Azido-5-oxo-6-aza-3, 9,12,15, 18,21-hexaoxatricosanoic acid (9.0 g, 2 1 mmol) was
dissolved in water (50 mL) and reduced using H2(g)-Pd/C (10 %). The reaction was
run until ESI-MS analysis showed complete conversion to the desired product (MH+
calculated: 397.2; found 397.6). The crude product was used in the next step without
further purification.
(e) (Boc-aminooxy)acetyl-PEG(6)-diglycolic acid.
A solution of dicyclohexycarbodiimide (515 mg, 2.50 mmol) in dioxan (2.5 mL) was
added dropwise to a solution of (Boc-aminooxy)acetic acid (477 mg, 2.50 mmol) and
N-hydroxysuccinimide (287 mg, 2.50 mmol) in dioxan (2.5 mL). The reaction was
stirred at RT for l h and filtered. The filtrate was transferred to a reaction vessel
containing a solution of 23-amino-5-oxo-6-aza-3, 9,12,15, 18,21-hexaoxatricosanoic
acid (1.0 g, 2.5 mmol) and NMM (278 µΐ , 2.50 mmol) in water (5 mL). The mixture
was stirred at RT for 30 min. ESI-MS analysis showed complete conversion to the
desired product (MH+ calculated: 570.28; found 570.6). The crude product was
purified by preparative HPLC (column: Phenomenex Luna 5µ C18 (2) 250 x 21.20
mm, detection: 214 nm, gradient: 0-50 % B over 60 min where A = H2O/0.1 % TFA
and B = acetonitrile/0.1 % TFA, flow rate: 10 mL/min) affording 500 mg (38 %) of
pure product. The product was analyzed by HPLC (column: Phenomenex Luna 3
C18 (2), 50 x 2.00 mm, detection: 214 nm, gradient: 0-50 % B over 10 min where A =
H2O/0.1 % TFA and B = acetonitrile/0.1 % TFA, flow rate: 0.75 mL/min, Rt = 5.52
min). Further confirmation was carried out by MR analysis.(f) Conjugation of (Boc-aminooxy)acetyl-PEG(6)-diglycolic acid to Peptide 1.
(Boc-aminooxy)acetyl-PEG(6)-diglycolic acid (0.15 mmol, 85 mg) and PyAOP (0.13
mmol, 68 mg) were dissolved in DMF (2 mL). NMM (0.20 mmol, 20 µ ) was added
and the mixture was stirred for 10 min. A solution of Peptide 1 (0.100 mmol, 126
mg) and NMM (0.20 mmol, 20 µ ) in DMF (4 mL) was added and the reaction
mixture was stirred for 25 min. Additional NMM (0.20 mmol, 20 µ ) was added and
the mixture was stirred for another 15 min. DMF was evaporated in vacuo and the
product was taken up in 10 % acetonitrile-water and purified by preparative FIPLC
(column: Phenomenex Luna 5µ C18 (2) 250 x 21.20 mm, detection: UV 214 nm,
gradient: 5-50 % B over 40 min where A = H2O/0. 1% TFA and B = acetonitrile/0. 1
% TFA, flow rate: 10 mL/min,) affording 100 mg semi-pure product. A second
purification step where TFA was replaced by HCOOH (gradient: 0-30 % B, otherwise
same conditions as above) afforded 89 mg (50 %). The product was analysed by
HPLC (column: Phenomenex Luna 3µ C18 (2) 50 x 2 mm, detection: UV 214 nm,
gradient: 0-30 % B over 10 min where A = H2O/0. 1% HCOOH and B =
acetonitrile/0.1 % HCOOH, flow rate: 0.3 mL/min, Rt: 10.21 min). Further product
characterisation was carried out using ESI-MS (MH22+ calculated: 905.4, found:
906.0).
(g) Deprotection.
Deprotection was carried out by addition of TFA containing 5% water to 10 mg of
peptide.
Example 2 : Radiosynthesis of F-benzaldehyde F-FBA).
[1 F]-fluoride was produced using a GEMS PETtrace cyclotron with a silver target via
the [1 0](p,n) [1 F] nuclear reaction. Total target volumes of 1.5 - 3.5 mL were used.
The radiofluoride was trapped on a Waters QMA cartridge (pre-conditioned with
carbonate), and the fluoride is eluted with a solution of Kryptofix 2 2 2.
(4 mg, 10.7 µΜ)
and potassium carbonate (0.56 mg, 4 .1 µΜ) in water (80 ) and acetonitrile (320
) . Nitrogen was used to drive the solution off the QMA cartridge to the reaction
vessel. The [1 F]-fluoride was dried for 9 minutes at 120°C under a steady stream of
nitrogen and vacuum. Trimethylammonium benzaldehyde triflate, [Haka et al,
J.Lab.Comp.Radiopharm., 27, 823-833 (1989)] (3.3 mg, 10.5 µΜ), in DMSO (1.1mL) was added to the dried [1 F]-fluoride, and the mixture heated at 105°C for 7
minutes to produce 4-[ 1 F]-fluorobenzaldehyde.
Example 3 : Purification of F-Fluorobenzaldehyde F-FBA).
The crude labelling mixture from Example 2 was diluted with ammonium hydroxide
solution and loaded onto an MCX+ SPE cartridge (pre-conditioned with water as part
of the FASTlab sequence). The cartridge was washed with water, dried with nitrogen
gas before elution of 4-[ 1 F]-fluorobenzaldehyde back to the reaction vessel in ethanol
(1.8 mL). A total volume of ethanol of 2.2 mL was used for the elution but the initial
portion (0.4 mL) was discarded as this did not contain [1 F]-FBA. 4-7% (decay
corrected) of the [1 F] radioactivity remained trapped on the cartridge.
Example 4: Preparation of [ Fl-fluciclatide (Compound 1).
The conjugation of [1 F]-FBA with Precursor 1 (5 mg) was performed in a solution of
ethanol (1.8 mL) and water (1.8 mL) in the presence of aniline hydrochloride. The
reaction mixture was maintained at at 60°C for 5 minutes.
Example 5: Impurity Analysis.
The levels of benzaldehyde type impurities before and after SPE purification were
determined as shown in Table 1 (based on 5250 g of TMAB triflate salt; mol. wt
313):
Table 1
The SPE method thus achieved removal of 91% of the impurity benzaldehydes.CLAIMS.
1. A composition which comprises an imaging agent of Formula (I) together with
one or more non-radioactive aryl derivatives of Formula (II):
wherein:
BTM is a biological targeting moiety;
L1 is a synthetic linker group of formula -(A)
m- wherein each A is
independently -CR2- , -CR=CR- , -C≡C- , -CR2C0 2- , -C0 2CR2- ,
- RCO- , -CO R- , -CR=N-0-, - R(C=0 ) R-, - R(C=S) R-, -
S0 2 R- , - RS0 2- , -CR2OCR2- , -CR2SCR2- , -CR2 RCR2- , a C4-8
cycloheteroalkylene group, a C4-8
cycloalkylene group, -Ar-, -NR-Ar-,
-O-Ar-, -Ar-(CO)-, an amino acid, a sugar or a monodisperse
polyethyleneglycol (PEG) building block, wherein each Ar is
independently a C5-12
arylene group, or a C3-12
heteroarylene group;
each R is independently chosen from H, C1-
alkyl, C2-4
alkenyl, C2-4
alkynyl, C1-
alkoxyalkyl or C1-
hydroxyalkyl;
m is an integer of value 1 to 20;
Y is -O- or - H-;
X1 is 1 F, -0(CH 2) 1 F or -OCH2-CH(OH)-CH2
1 F,
wherein q is 2, 3 or 4;
X2 is -N+(CH3)
3, -N(CH3)
2, -OCH3, H, -CH3, -OH, -SCH3,
-OC6H4CHO or 1 F;
wherein BTM, L1 and Y are the same in Formula (I) and (II);
and wherein the total concentration of derivatives of Formula (II) present in
the composition is less than 150 g/mL.2 . The composition of claim 1, where Y is -O-
3 . The composition of claim 1 or claim 2, where the BTM comprises a single
amino acid, a 3-100 mer peptide, an enzyme substrate, an enzyme antagonist an
enzyme agonist, an enzyme inhibitor or a receptor-binding compound.
4 . The composition of any one of claims 1 to 3, where the BTM comprises an
RGD peptide.
5 . The composition of any one of claims 1 to 4, wherein BTM is a peptide comprising
the fragment:
6 . The composition of any one of claims 1 to 5, where the imaging agent is of
Formula IA) :
( A )
7 . The composition of any one of claims 1 to 6, where X1 is 1 F and X2 is
-N+(CH3)
3, -N(CH3)
2 or -OH.8 . A radiopharmaceutical composition which comprises the composition of any
one of claims 1 to 7, together with a biocompatible carrier, in a form suitable for
mammalian administration.
9 . A composition which comprises a radioactive aldehyde of Formula (A)
together with one or more non-radioactive aldehydes of Formula (B):
wherein X1 and X2 are as defined in claim 1;
and wherein the total concentration of derivatives of Formula (B) present in
the composition is less than 150 g/mL.
10. The composition of claim 9, where X1 is 1 F and X2 is -N+(CH3)
3, -N(CH3)
2 or
-OH.
11. The composition of claim 9 or claim 10, which is provided as a solution.
A method of radiolabelling a biological targeting moiety which comprises:
(i) provision of a biological targeting moiety functionalized with an
aminooxy or hydrazine group;
(ii) reaction of the functionalized- biological targeting moiety from
step (i) with the radioactive aldehyde composition of any one of claims
9 to 11, such that the radioactive aldehyde of Formula (A) condenses
with said aminooxy or hydrazine group, to give the radiolabelled
biological targeting moiety.13. The method of claim 12, where the functionalized biological targeting moiety
is of Formula (IV):
[BTM] -L1-Y- H2 (IV)
and the radiolabeled product is of Formula (I):
wherein:
BTM, L1, Y and X1 are as defined in any one of claims 1 to 7 .
14. The method of claim 12 or claim 13, which is carried out using an automated
synthesizer apparatus.
15. The method of claim 14, which is carried out in a sterile manner, such that the
radiopharmaceutical composition of claim 8 is obtained.
16. A method of imaging the human or animal body which comprises generating a
PET image of at least a part of said body to which the radiopharmaceutical
composition of claim 8 has distributed.
17. The method of claim 16, which is carried out repeatedly to monitor the effect
of treatment of a human or animal body with a drug, said imaging being effected
before and after treatment with said drug, and optionally also during treatment with
said drug.
18. The method of claim 16 or claim 17, wherein said composition has been
previously administered to said body
19. A method of diagnosis of the human or animal body which comprises the
imaging method of any one of claims 16 to 18.