Radiopharmaceutical Compositions.
Field of the Invention.
The present invention relates to mTc-maraciclatide radiopharmaceutical
compositions, which are stabilised with a radioprotectant. Also described are kits for
the preparation of the radiopharmaceutical compositions, as well as methods of
preparing such compositions from the kit. The invention also includes methods of
imaging the mammalian body using the radiopharmaceutical compositions.
Background to the Invention.
mTc-maraciclatide is the recommended INN (USA Approved Name) for mTc-
NC100692. mTc-NC 100692 has been described in both patents and publications, as
a radiopharmaceutical which targets integrin receptors in vivo.
WO 03/006491 discloses compounds of Formula (I):
(= 0 ) - X1 - X2 - X3 - G- D-X 4 - X - X - X7
S (CH2 ) h
( I )
or pharmaceutically acceptable salt thereof
wherein:
G represents glycine
D represents aspartic acid
R i represents -(CH2) - or -(CH2)n-C6H4- wherein
n represents a positive integer 1 to 10,
h represents a positive integer 1 or 2,
Xi represents an amino acid residue wherein said amino acid possesses a
functional side-chain such as an acid or amine,
X2 and X4 represent independently an amino acid residue capable of forming a
disulfide bond,
X3 represents arginine, N-methylarginine or an arginine mimetic,
X5 represents a hydrophobic amino acid or derivatives thereof,
X6 represents a thiol-containing amino acid residue,
X7 is absent or represents a biomodifier moiety,
Zi represents an anti-neoplastic agent, a chelating agent or a reporter moiety
and
Wi is absent or represents a spacer moiety.
WO 03/006491 discloses that a preferred chelating moiety has the formula shown, and
includes mTc complexes of said helator conjugate of Formula I therein:
WO 03/006491 does not disclose kits.
Edwards et al [Nucl.Med.Biol, 35, 365-375 (2008)] disclose that mTc-maraciclatide
( mTc-NC 100692) can be prepared from a kit. Edwards et al state that each
lyophilised kit contains approximately 44 nmol maraciclatide (NCI 00692), plus a
number of excipients including buffer, stannous reducing agent and methylene
diphosphonic acid (present as a Sn2+ solubiliser). Edwards et al report that the
radiochemical purity (RCP) of each reconstituted kit was determined at 20 minutes
post reconstitution, and was found to be at least 90%. The RCP was found to be
stable over the period that the reconstituted kits were used.
The Present Invention.
Technetium-99m ( mTc) is a radioisotope which decays with a half-life of 6.02 hours
to technetium-99 ( Tc). The radioactive decay is accompanied by the emission of a
gamma ray with an energy that is near ideal for medical imaging with a modern
gamma-camera. The decay product, Tc, is also radioactive and decays by b-
emission with a half-life of 2.1xl0 5 years (to the stable isotope Ru), but the
radioactive emissions from Tc are insufficient for medical imaging.
Conventional mTc "generators" comprise the radioisotope Mo, which decays with
a half-life of 66.2 hours. The chemical form of the technetium eluted from such a
generator is mTc-pertechnetate. About 86% of Mo decays result in the production
of mTc, however ca. 14% of Mo decays result in the direct production of Tc.
Therefore, if a mTc generator is eluted a very short time after the previous elution,
the mTc content will be low but will be about 86% of the total technetium content.
As time passes since the previous elution of the generator, Tc is being produced both
from Mo and from the decay of mTc to Tc. Consequently, as the time interval
between generator elutions increases, the Tc/ mTc ratio increases. The Tc and
mTc technetium isotopes are chemically identical, and consequently any
radiopharmaceutical preparation must be able to cope with a wide range of Tc
chemical content in the eluate in order to be able to function effectively over the
usable lifetime of the generator. It is also the case that elutions made with a fresh
mTc generator are likely to have a higher radioactive concentration, and thus have a
higher concentration of reactive free radicals arising from radiolysis of the solvent
(water). A viable mTc radiopharmaceutical preparation thus needs to be able to
provide satisfactory RCP performance even when such reactive free radicals are
present. These characteristics of the mTc generator are illustrated in most
radiochemistry or nuclear chemistry textbooks, and the problems that different eluate
properties can give to the performance of mTc kits have been described by Saha, G.
B. "Radiopharmaceuticals and Methods o Radiolabeling' '; Chapter 6 (pages 80-108)
in Fundamentals of Nuclear Pharmacy (3 Edn.).
The present inventors have found that the Maraciclatide kit reported by Edwards et al
(above) suffers from various problems not previously recognised in the prior art:
(i) relatively low initial RCP post-reconstitution of the kit with mTcpertechnetate;
(ii) insufficient post-reconstitution stability;
(iii) the need to store and ship the kit at -15 to -20°C to maintain kit stability
and performance;
(iv) only a single patient dose being obtainable from the kit.
The present invention provides improved mTc-maraciclatide radiopharmaceutical
compositions which exhibit more reproducible initial radiochemical purity (RCP) and
improved stability post-reconstitution, so that an RCP of 85 to 90% is maintained at 6
hours post-reconstitution. The problem of unsatisfactory RCP for mTc-maraciclatide
preparations under certain conditions of radioactivity levels, radioactive
concentrations or reconstitution volumes was not recognised in the prior art. Such
conditions are those that could arise under normal conditions of use of a commercial
radionuclide generator, such as a mTc generator.
Berger [Int.J.Appl.Rad.Isotop., 33, 1341-1344 (1982)] discloses that a wide range of
antioxidants can be used to stabilise mTc- radiopharmaceuticals. Methylene Blue
and ascorbic acid have since been highlighted as particularly suitable stabilisers
[Weisner et al, Eur.J.Nucl.Med., 20, 661-666 (1993) and Liu et al, Bioconj.Chem.,
14(4), 1052-1056 (2003)].
The present inventors have also established that the well-known radioprotectant
ascorbic acid/ascorbate actually has a deleterious effect on the RCP of mTcmaraciclatide.
A further known radioprotectant gentisic acid caused discolouration
problems which negated its use in the present composition. The present invention
provides compositions comprising a radioprotectant which solve this previously
unrecognised problem. The kits of the present invention have the advantages of:
higher initial RCP; more robust RCP over longer time periods post-reconstitution;
compatibility with various commercial mTc radiopharmaceutical generators and
under a range of elution conditions; the facility to obtain two patient doses per kit (i.e.
to reconstitute successfully with higher levels of radioactivity); adequate stability to
be stored and shipped at fridge (+5 ±3 °C) rather than freezer temperature (-10 to -
20 °C) and kit shelf-life stability of at least 3 years.
Detailed Description of the Invention.
In a first aspect, the present invention provides a radiopharmaceutical composition
which comprises:
(i) mTc-maraciclatide;
(ii) a radioprotectant chosen from /?ara-aminobenzoic acid or a salt
thereof with a biocompatible cation;
in a biocompatible carrier in a form suitable for mammalian administration.
The term "maraciclatide" refers to the compound known in the scientific literature as
NC100692 [D.Edwards et al, Nucl.Med.BioL, 35, 365-375 (2008)]. The chemical
name is: 1,5-pentanedioic acid-(5-[2-hydroxyimino-l,l-dimethyl-propylamino]-3-(2-
[2-hydroxyimido- 1,1-dimethyl-propylamino]-ethyl)-pentyl)-amide 5-[ 13-benzyl- 19-
carboxymethyl-25-(3 -guanidino-propyl)- 10-(4,7, 10,1 6-tetraoxa- 14,1 8-dioxo- 1,13,19-
triazanonadecyl)-carbamoyl-3,6, 12,15,18,2 1,24,27-octaoxo-8,29,30-trithia-
2,5,1 l,14,17,20,23,26-octaaza-bicyclo[14.1 1.4]hentriacont-4-yl] pentyl-amide.
The chemical structure of maraciclatide is as follows:
Maraciclatide
Maraciclatide can be used in the free base form, or in the salt form (eg. the
trifluoroacetate).
The term "radiopharmaceutical" has its conventional meaning, and refers to a
radioactive compound suitable for in vivo mammalian administration for use in
diagnosis or therapy.
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
radioprotectant of the present invention is suitably chosen from /?ara-aminobenzoic
acid (i.e. 4-aminobenzoic acid) and salts thereof with a biocompatible cation. These
radioprotectants are commercially available, including in pharmaceutical grade purity.
Preferably, pharmaceutical grade material is used.
By the term "biocompatible cation" (B ) is meant a positively charged counterion
which forms 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.
The "biocompatible carrier" is a fluid, especially a liquid, in which the
radiopharmaceutical 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 (eg. salts of plasma cations with biocompatible
counterions), sugars (e.g. glucose or sucrose), sugar alcohols (eg. sorbitol or
mannitol), glycols (eg. glycerol), or other non-ionic polyol materials (eg.
polyethyleneglycols, propylene glycols and the like). Preferably the biocompatible
carrier is pyrogen-free water for injection, isotonic saline or phosphate buffer.
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, preferably 6.5 to 9.5 for the agents of the present invention) and physiologically
compatible osmolality. 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.
Preferably, the mammal is an intact mammalian body in vivo, and is more preferably a
human subject. Preferably, the radiopharmaceutical can be administered to the
mammalian body in a minimally invasive manner, i.e. without a substantial health risk
to the mammalian subject even 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 "comprising" has its conventional meaning throughout this application and
implies that the composition must have the components listed, but that other,
unspecified compounds or species may be present in addition. The term 'comprising'
includes as a preferred subset "consisting essentially of which means that the
composition has the components listed without other compounds or species being
present.
The radiopharmaceutical composition may contain additional optional excipients such
as: an antimicrobial preservative, pH-adjusting agent, filler, solubiliser or osmolality
adjusting agent.
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 or
mixtures thereof; benzyl alcohol; ethanol, phenol; cresol; cetrimide and thiomersal.
Preferred antimicrobial preservative(s) are the parabens or ethanol.
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, preferably 6.5 to 9.5 for the agents of the present invention) for human or
mammalian administration. Suitable such pH-adjusting agents include
pharmaceutically acceptable buffers, such as tricine, phosphate, acetate 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.
By the term "solubiliser" is meant an additive present in the composition which
increases the solubility of the radiopharmaceutical 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 (e.g. Tween™);
poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers
(Pluronics™); cyclodextrins (e.g. alpha, beta or gamma cyclodextrin, hydroxypropyl-
b-cyclodextrin or hydroxypropyl-y-cyclodextrin) and lecithin.
Preferred solubilisers are cyclodextrins, C1-4 alcohols, polysorbates and Pluronics™,
more preferably cyclodextrins and C2 -4 alcohols. When the solubiliser is an alcohol, it
is preferably ethanol or propanol, more preferably ethanol. Ethanol has potentially
several roles, since it can also function as a biocompatible carrier, radioprotectant or
antimicrobial preservative. When the solubiliser is a cyclodextrin, it is preferably a
gamma cyclodextrin, more preferably hydroxypropyl -P-cyclodextrin (HPCD). The
concentration of cyclodextrin can be from about 0.1 to about 40 mg/ml, preferably
between about 5 and about 35 mg/ml, more preferably 20 to 30 mg/ml, most
preferably around 25 mg/ml.
Preferred features.
The radioprotectant of the present invention preferably comprises sodium paraaminobenzoate.
An additional radioprotectant may also be present. If such an
additional radioprotectant is used, it preferably does not comprise ascorbic acid or
gentisic acid or salts thereof. More preferably, the radioprotectant of the present
invention consists essentially of /?ara-aminobenzoic acid or a salt thereof with a
biocompatible cation. Most preferably, the radioprotectant of the present invention
consists essentially of sodium/?ara-aminobenzoate.
The radiopharmaceutical composition of the first aspect is suitably provided in a
pharmaceutical grade container. 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 30 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.
The radiopharmaceutical composition of the first aspect may also be provided in a
syringe. Pre-filled syringes are designed to contain a single human dose, or "unit
dose" and are therefore preferably a single-use or other syringe suitable for clinical
use. The radiopharmaceutical syringe is preferably provided with a syringe shield to
minimise radiation dose to the operator.
In a second aspect, the present invention provides a kit for the preparation of the
radiopharmaceutical composition of the first aspect, wherein said kit comprises:
Maraciclatide;
a radioprotectant chosen from /?ara-aminobenzoic acid
thereof with a biocompatible cation;
a stannous reductant;
methylene diphosphonic acid or a salt thereof with a
biocompatible cation.
By the term "kit" is meant one or more pharmaceutical grade containers, comprising
the necessary non-radioactive chemicals to prepare the desired radiopharmaceutical
composition, together with operating instructions. The kit is designed to be
reconstituted with mTc to give a solution suitable for human administration with the
minimum of manipulation. The kit of the present invention preferably comprises a
lyophilised composition containing all the kit components in a single lyophilised
formulation in a single container.
The kit of the second aspect is non-radioactive. Preferred aspects of the
radioprotectant in the kit are as described in the first aspect (above).
The term "stannous reductant" has its conventional meaning in the field of mTc
radiopharmaceutical kits, and refers to a salt of Sn2+, ie. tin in the Sn(II) oxidation
state. Suitable such salts may be in the hydrated or anhydrous form, and include:
stannous chloride, stannous fluoride and stannous tartrate. A preferred such stannous
reductant is stannous chloride.
The term "methylene diphosphonic acid" has its conventional chemical meaning, and
has the chemical structure shown:
methylene diphosphonic acid (MDP)
The kit is preferably lyophilised and is designed to be reconstituted with sterile mTcpertechnetate
(Tc(V) from a mTc radioisotope generator to give a solution suitable
for human administration without further manipulation. The non-radioactive kits may
optionally further comprise additional components such as a transchelator,
antimicrobial preservative, pH-adjusting agent or filler - as defined for the first aspect
above.
The kit of the second aspect preferably further comprises a buffer which comprises a
mixture of sodium hydrogen carbonate and anhydrous sodium carbonate.
A most preferred kit formulation is as follows (Formulation C of Example 8):
In a third aspect, the present invention provides a method of preparation of the
radiopharmaceutical composition of the first aspect, which comprises either:
(i) reconstitution of the kit of the second aspect with a supply of a
biocompatible carrier, followed by addition of a supply of mTc in a
biocompatible carrier to the reconstituted kit; or
(ii) reconstitution of the kit of the second aspect with a supply of mTc in a
biocompatible carrier.
Preferred aspects of the kit in the third aspect are as described in the second aspect
(above).
The supply of mTc is suitably in sterile form, and is preferably mTc-pertechnetate
(Tc(V) from a mTc radioisotope generator. Such generators are commercially
available.
When option (i) is used, that means first reconstituting the kit with a non-radioactive
biocompatible carrier in sterile form (as defined above; e.g. saline), followed by the
addition of the mTc. When option (ii) is used, that means reconstituting the kit by
addition of mTc in a biocompatible carrier directly to the kit. Option (ii) is preferred,
especially in conjunction with a single, lyophilised kit container, since that gives a
solution suitable for human administration without further manipulation.
The method of the third aspect is preferably carried out at room temperature, i.e. no
heating is required.
The method of the third aspect is preferably used to prepare unit patient doses, by
withdrawing the radiopharmaceutical composition into a clinical grade syringe, as
described in the first aspect (above).
In a fourth aspect, the present invention provides the use of the kit of the second
aspect in the preparation of the radiopharmaceutical composition of the first aspect.
Preferred aspects of the kit and radiopharmaceutical composition in the fourth aspect
are as described in the second and first aspects respectively (above).
In a fifth aspect, the present invention provides the use of /?ara-aminobenzoic acid or
a salt thereof with a biocompatible cation, as a radioprotectant to stabilise either:
(i) mTc-maraciclatide radiopharmaceutical compositions;
(ii) kits for the preparation of mTc-maraciclatide radiopharmaceutical
compositions.
The radiopharmaceutical compositions and kits of the fifth aspect, and preferred
embodiments thereof, are preferably as described in the first and second aspects
respectively (above). Preferred embodiments of the radioprotectant in the fifth aspect
are as described in the first aspect (above).
In a sixth aspect, the present invention provides the use of the radiopharmaceutical
composition of the first aspect in a method of imaging of the mammalian body.
Preferred aspects of the radiopharmaceutical composition in the sixth aspect are as
described in the first aspect (above). Preferably, the mammal is an intact mammalian
subject in vivo, and is more preferably a human subject. The method of imaging is
preferably used to assist in a method of diagnosis of said mammalian subject.
In a seventh aspect, the present invention provides a method of imaging of the
mammalian body which comprises imaging a mammal which had previously been
administered with the radiopharmaceutical composition of the first aspect. Preferred
aspects of the radiopharmaceutical composition in the seventh aspect are as described
in the first aspect (above). Preferably, the mammal is an intact mammalian body in
vivo, and is more preferably a human subject.
The imaging of the sixth and seventh aspects is preferably to image a mammalian
subject suffering from a disease in which in which integrins are expressed, such as
angiogenesis, fibrosis or inflammation.
The invention is illustrated by the non-limiting Examples detailed below. Examples 1
to 3 provide the synthesis of Chelator 1 (also sometimes called carba-Pn216) of the
invention. Example 4 provides the synthesis of Chelator 1A of the invention - an
active ester-functionalised version of Chelator 1. Example 5 provides the synthesis of
cyclic peptides of the invention and chelator conjugation. Example 6 provides the
synthesis of maraciclatide. Example 7 provides a study on the choice of
radioprotectant. Example 8 provides data on optimising the amount of radioprotectant
used. Example 9 describes comparative kit formulations vs a prior art kit formulation.
Example 10 provides a mTc generator compatibility study for kits of the invention,
showing that they are compatible with a range of commercial generators under a range
of conditions. Example 11 provides data on the shelf-life stability of kits of the
invention.
Abbreviations.
Conventional single letter or 3-letter amino acid abbreviations are used.
Ac: Acetyl
Boc: t r t-Butyloxycarbonyl
tBu: tertiary-butyl
DMF: Dimethylformamide
DMSO: Dimethyl sulfoxide
Fmoc: 9-Fluorenylmethoxycarbonyl
HATU: 0-(7-Azabenzotriazol- 1-yl)-N,N V,N -tetramethyluronium hexafluorophosphate
HBTU: 0-Benzotriazol- 1-yl-N,N,N,N4etramethyluronium hexafluorophosphate
HPLC: High performance liquid chromatography
MM: N-Methylmorpholine
pABA: /?ara-amino-benzoic acid sodium salt
PBS: Phosphate-buffered saline
PEG: polyethyleneglycol, repeat units of (OCH2CH2)n, where n is an integer,
tBu: t r t-butyl
RCP: radiochemical purity.
RP-HPLC: reversed-phase HPLC.
TFA: Trifluoroacetic acid
THF: Tetrahydrofuran
TIS: Triisopropyl silane
TLC: thin layer chromatography
Trt: Trityl.
Compounds of the Invention .
Compound Structure
Chelator 1
Chelator 1A
I I
Example 1: Synthesis of l,l,l -rmf2-aminoethyl)methane.
Step 1(a): 3(methoxycarbonylmethylene)glutaric acid dimethylester.
Carbomethoxymethylenetriphenylphosphorane (167g, 0.5mol) in toluene (600ml) was
treated with dimethyl 3-oxoglutarate (87g, 0.5mol) and the reaction heated to 100°C
on an oil bath at 120 °C under an atmosphere of nitrogen for 36h. The reaction was
then concentrated in vacuo and the oily residue triturated with 40/60 petrol
ether/diethylether (1:1, 600 ml). Triphenylphosphine oxide precipitated out and the
supernatant liquid was decanted/filtered off. The residue on evaporation in vacuo was
Kugelrohr distilled under high vacuum Bpt (oven temperature 180-200°C at 0.2 torr)
to give 3-(methoxycarbonylmethylene)glutaric acid dimethylester (89.08g, 53%).
NMR ^(CDCl,): d 3.31 (2H, s, CH2), 3.7(9H, s, 3xOCH3), 3.87 (2H, s, CH2), 5.79 (1H, s,
=CH, ) ppm.
NMR 1 C(CDC13), d 36.56,CH3, 48.7, 2xCH3, 52.09 and 52.5 (2xCH2); 122.3 and 146.16
C=CH; 165.9, 170.0 and 170.5 3xCOO ppm.
Step Kb): Hydrogenation of 3-(methoxycarbonylmethylene)glutaric acid
dimethylester.
3-(Methoxycarbonylmethylene)glutaric acid dimethylester (89g, 267mmol) in
methanol (200ml) was shaken with (10% palladium on charcoal: 50% water) (9 g)
under an atmosphere of hydrogen gas (3.5 bar) for 3Oh. The solution was filtered
through kieselguhr and concentrated in vacuo to give 3-
(methoxycarbonylmethyl)glutaric acid dimethylester as an oil, yield (84. 9g, 94 %).
NMR ^(CDCy, d 2.48 (6H, d, J=8Hz, 3xCH2), 2.78 (1H, hextet, J=8Hz CH, ) 3.7 (9H, s,
3xCH3) .
NMR 1 C(CDC1 ), d 28.6, CH; 37.50, 3xCH ; 51.6, 3xCH2; 172.28,3 x COO.
Step 1(c): Reduction and esterification of trimethyl ester to the triacetate.
Under an atmosphere of nitrogen in a 3 necked 2L round bottomed flask lithium
aluminium hydride (20g, 588 mmol) in THF (400ml) was treated cautiously with
tm(methyloxycarbonylmethyl)methane (40g, 212 mmol) in THF (200ml) over lh. A
strongly exothermic reaction occurred, causing the solvent to reflux strongly. The
reaction was heated on an oil bath at 90°C at reflux for 3 days. The reaction was
quenched by the cautious dropwise addition of acetic acid (100ml) until the evolution
of hydrogen ceased. The stirred reaction mixture was cautiously treated with acetic
anhydride solution (500ml) at such a rate as to cause gentle reflux. The flask was
equipped for distillation and stirred and then heating at 90°C (oil bath temperature) to
distil out the THF. A further portion of acetic anhydride (300ml) was added, the
reaction returned to reflux configuration and stirred and heated in an oil bath at 140°C
for 5h. The reaction was allowed to cool and filtered. The aluminium oxide
precipitate was washed with ethyl acetate and the combined filtrates concentrated on a
rotary evaporator at a water bath temperature of 50°C in vacuo (5 mmHg) to afford an
oil. The oil was taken up in ethyl acetate (500ml) and washed with saturated aqueous
potassium carbonate solution. The ethyl acetate solution was separated, dried over
sodium sulfate, and concentrated in vacuo to afford an oil. The oil was Kugelrohr
distilled in high vacuum to give tm(2-acetoxyethyl)methane (45. 3g, 95.9%) as an oil.
Bp. 220 °C at 0.1 mmHg.
NMR ^(CDCy, d 1.66(7H, m, 3xCH2, CH), 2.08(1H, s, 3xCH3); 4.1(6H, t, 3xCH20).
NMR 1 C(CDC13), d 20.9, CH3; 29.34, CH; 32.17, CH2; 62.15, CH20 ; 171, CO.
Step 1(d): Removal of Acetate groups from the triacetate.
Jm(2-acetoxyethyl)methane (45. 3g, 165mM) in methanol (200ml) and 880 ammonia
(100ml) was heated on an oil bath at 80°C for 2 days. The reaction was treated with a
further portion of 880 ammonia (50ml) and heated at 80°C in an oil bath for 24h. A
further portion of 880 ammonia (50ml) was added and the reaction heated at 80°C for
24h. The reaction was then concentrated in vacuo to remove all solvents to give an
oil. This was taken up into 880 ammonia (150ml) and heated at 80°C for 24h. The
reaction was then concentrated in vacuo to remove all solvents to give an oil.
Kugelrohr distillation gave acetamide bp 170-180 0.2mm. The bulbs containing the
acetamide were washed clean and the distillation continued. Tris{2-
hydroxyethyl)methane (22.53g, 92%) distilled at bp 220 °C 0.2mm.
NMR ^(CDCy, d 1.45(6H, q, 3xCH2), 2.2(1H, quintet, CH); 3.7(6H, t 3xCH2OH); 5.5(3H,
brs, 3xOH).
NMR 1 C(CDC13), d 22.13, CH; 33.95, 3xCH2; 57.8, 3xCH2OH.
Step 1(e): Conversion of the triol to the tm(methanesulfonate).
To an stirred ice-cooled solution of tm(2-hydroxyethyl)methane (lOg, 0.0676mol) in
dichloromethane (50ml) was slowly dripped a solution of methanesulfonyl chloride
(40g, 0.349mol) in dichloromethane (50ml) under nitrogen at such a rate that the
temperature did not rise above 15°C. Pyridine (21.4g, 0.27mol, 4eq) dissolved in
dichlorom ethane (50ml) was then added drop-wise at such a rate that the temperature
did not rise above 15°C, exothermic reaction. The reaction was left to stir at room
temperature for 24h and then treated with 5N hydrochloric acid solution (80ml) and
the layers separated. The aqueous layer was extracted with further dichloromethane
(50ml) and the organic extracts combined, dried over sodium sulfate, filtered and
concentrated in vacuo to give tm[2-(methylsulfonyloxy)ethyl]methane contaminated
with excess methanesulfonyl chloride. The theoretical yield was 25. 8g.
NMR ^(CDCy, d 4.3 (6H, t, 2xCH 2), 3.0 (9H, s, 3xCH 3), 2 (1H, hextet, CH), 1.85 (6H, q,
3xCH 2) .
Step 1(f): Preparation of 1,1,1- tm(2-azidoethyl)methane.
A stirred solution of tra[2-(methylsulfonyloxy)ethyl]methane [from Step 1(e),
contaminated with excess methylsulfonyl chloride] (25. 8g, 67mmol, theoretical) in
dry DMF (250ml) under nitrogen was treated with sodium azide (30. 7g, 0.47mol)
portion-wise over 15 minutes. An exotherm was observed and the reaction was
cooled on an ice bath. After 30 minutes, the reaction mixture was heated on an oil
bath at 50°C for 24h. The reaction became brown in colour. The reaction was
allowed to cool, treated with dilute potassium carbonate solution (200ml) and
extracted three times with 40/60 petrol ether/di ethyl ether 10: 1 (3x150ml). The
organic extracts were washed with water (2x1 50ml), dried over sodium sulfate and
filtered. Ethanol (200ml) was added to the petrol/ether solution to keep the triazide in
solution and the volume reduced in vacuo to no less than 200ml. Ethanol (200ml)
was added and reconcentrated in vacuo to remove the last traces of petrol leaving no
less than 200ml of ethanolic solution. The ethanol solution of triazide was used
directly in Step 1(g).
CARE: DO NOT REMOVE ALL THE SOLVENT AS THE AZIDE IS
POTENTIALLY EXPLOSIVE AND SHOULD BE KEPT IN DILUTE SOLUTION
AT ALL TIMES.
Less than 0.2ml of the solution was evaporated in vacuo to remove the ethanol and an
NMR run on this small sample: NMR 1H(CDC13), d 3.35 (6H, t, 3xCH2), 1.8 (1H,
septet, CH, ), 1.6 (6H, q, 3xCH2) .
Step Kg): Preparation of l,l,l-tris(2-aminoethyl)m ethane.
rm(2-azidoethyl)methane (15.06g, 0.0676 mol), (assuming 100% yield from
previous reaction) in ethanol (200ml) was treated with 10% palladium on charcoal
(2g, 50% water) and hydrogenated for 12h. The reaction vessel was evacuated every
2 hours to remove nitrogen evolved from the reaction and refilled with hydrogen. A
sample was taken for NMR analysis to confirm complete conversion of the triazide to
the triamine.
Caution: unreduced azide could explode on distillation. The reaction was filtered
through a celite pad to remove the catalyst and concentrated in vacuo to give tris{2-
aminoethyl)methane as an oil. This was further purified by Kugelrohr distillation
bp.l80-200°C at 0.4mm/Hg to give a colourless oil (8.1g, 82.7% overall yield).
NMR ^(CDCy, d 2.72 (6H, t, 3xCH2N), 1.41 (H, septet, CH), 1.39 (6H, q, 3xCH2) .
NMR 1 C(CDC13), d 39.8 (CH2NH2), 38.2 (CH2), 31.0 (CH).
Example 2 : Preparation of 3-chloro-3-methyl-2-nitrosobutane.
A mixture of 2-methylbut-2-ene (147ml, 1.4mol) and isoamyl nitrite (156ml,
1.16mol) was cooled to -30 °C in a bath of cardice and methanol and vigorously
stirred with an overhead air stirrer and treated dropwise with concentrated
hydrochloric acid (140ml, 1.68mol ) at such a rate that the temperature was
maintained below -20°C. This requires about l h as there is a significant exotherm
and care must be taken to prevent overheating. Ethanol (100ml) was added to reduce
the viscosity of the slurry that had formed at the end of the addition and the reaction
stirred at -20 to -10°C for a further 2h to complete the reaction. The precipitate was
collected by filtration under vacuum and washed with 4x3 0ml of cold (-20°C) ethanol
and 100ml of ice cold water, and dried in vacuo to give 3-chloro-3-methyl-2-
nitrosobutane as a white solid. The ethanol filtrate and washings were combined and
diluted with water (200ml) and cooled and allowed to stand for l h at -10°C when a
further crop of 3-chloro-3-methyl-2-nitrosobutane crystallised out. The precipitate
was collected by filtration and washed with the minimum of water and dried in vacuo
to give a total yield of 3-chloro-3-methyl-2-nitrosobutane ( 115g 0.85mol, 73%) >98%
pure by NMR.
NMR ^(CDCy, As a mixture ofisomers (isomer1, 90%) 1.5 d, (2H, CH3), 1.65 d, (4H, 2
xCH3), 5.85,q, and 5.95,q, together 1H. (isomer2, 10%), 1.76 s, (6H, 2x CH3), 2.07(3H,
CH ) .
Example 3 : Synthesis of [N- l -dimethyl-2-N-hvdrox imine propyl)2-
aminoethyll-f2-aminoethyl)methane (Chelator 1).
To a solution of tm(2-aminoethyl)methane (Example 1; 4.047g, 27.9mmol) in dry
ethanol (30ml) was added potassium carbonate anhydrous (7.7g, 55.8mmol, 2eq) at
room temperature with vigorous stirring under a nitrogen atmosphere. A solution of
3-chloro-3-methyl-2-nitrosobutane (Example 2; 7.56g, 55.8mol, 2eq) was dissolved in
dry ethanol (100ml) and 75ml of this solution was dripped slowly into the reaction
mixture. The reaction was followed by TLC on silica [plates run in dichloromethane,
methanol, concentrated (0.88sg) ammonia; 100/30/5 and the TLC plate developed by
spraying with ninhydrin and heating]. The mono-, di- and tri-alkylated products were
seen with RF's increasing in that order. Analytical HPLC was run using PRP reverse
phase column in a gradient of 7.5-75% acetonitrile in 3% aqueous ammonia. The
reaction was concentrated in vacuo to remove the ethanol and re-suspended in water
( 110ml). The aqueous slurry was extracted with ether (100ml) to remove some of the
trialkylated compound and lipophilic impurities leaving the mono and desired
dialkylated product in the water layer. The aqueous solution was buffered with
ammonium acetate (2eq, 4.3g, 55.8mmol) to ensure good chromatography. The
aqueous solution was stored at 4°C overnight before purifying by automated
preparative HPLC.
Yield (22%, 6.4mmol, 23%).
Mass spec; Positive ion 10 V cone voltage. Found: 344; calculated M+H= 344.
NMR ^(CDCy, d 1.24(6H, s, 2xCH3), 1.3(6H, s, 2xCH3), 1.25-1.75(7H, m, 3xCH2,CH),
(3H, s, 2xCH2), 2.58 (4H, m, CH2N), 2.88(2H, t CH2N), 5.0 (6H, s, NH2, 2xNH, 2xOH).
NMR 1H ((CD3)2SO) d 1.1 4xCH; 1.29, 3xCH2; 2.1 (4H, t, 2xCH 2);
NMR 1 C((CD3)2SO), d 9.0 (4xCH3), 25.8 (2xCH ), 31.0 2xCH2, 34.6 CH2, 56.8 2xCH2N;
160.3, C=N.
HPLC conditions: flow rate 8ml/min using a 25mm PRP column [A=3% ammonia
solution (sp.gr = 0.88) /water; B = Acetonitrile].
Load 3ml of aqueous solution per run, and collect in a time window of 12.5-13.5 min.
Example 4: Synthesis of Tetrafluorothiophenyl ester of Chelator 1-glutaric acid
(Chelator 1A .
(Step 4a) Synthesis of [Chelator l]-glutaric acid intermediate.
Chelator 1 (100 mg, 0.29 mmol) was dissolved in DMF (10 ml) and glutaric
anhydride (33 mg, 0.29 mmol) added by portions with stirring. The reaction was
stirred for 23 hours to afford complete conversion to the desired product. The pure
acid was obtained following RP-HPLC in good yield.
(Step 4b) Synthesis of Chelator 1A.
Chelator 1A
To [Chelator l]-glutaric acid (from Step 4a; 300 mg, 0.66 mmol) in DMF (2 ml) was
added HATU (249 mg, 0.66 mmol) and MM (132 mΐ , 1.32 mmol). The mixture
was stirred for 5 minutes then tetrafluorothiophenol (0.66 mmol, 119 mg) was added.
The solution was stirred for 10 minutes then the reaction mixture was diluted with 20
% acetonitrile/water (8 ml) and the product purified by RP- PLC yielding 110 mg of
the desired product following freeze-drying.
Example 5: Synthesis of disulfide [Cys l thioether [CHiCO-Lysf Chelator
l-glutarylVCvs -Arg-Glv-Asp-Cvs -Phe-Cvsl-N¾.
(Step 5a) Synthesis of ClCH CO-Lvs-Cvs(tBu)-Arg-Glv-Asp-Cvs(tBu)-Phe-Cvs-
Molecular Weight = 8.844
Exact Mass = 1117.464
Molecular Formula = C46H76CIN1301 1S3
The peptide was synthesised on an ABI 433A automatic peptide synthesiser starting
with Rink Amide AM resin on a 0.25 mmol scale using 1 mmol amino acid cartridges.
The amino acids were pre-activated using HBTU before coupling. N-terminal amine
groups were chloroacetylated using a solution of chloroacetic anhydride in DMF for
30 min. The simultaneous removal of peptide and side-chain protecting groups
(except tBu) from the resin was carried out in TFA containing TIS (5 %), H20 (5 %)
and phenol (2.5 %) for two hours. After work-up 295 mg of crude peptide was
obtained (Analytical HPLC: Gradient, 5-50 % B over 10 min where A = H2O/0. 1%
TFA and B = CH3CN/0. 1% TFA; column, Phenomenex Luna 3m 18 (2) 50 c 4.6
mm; flow, 2 ml/min; detection, UV 214 nm; product retention time, 6.42 min).
Further product characterisation was carried out using mass spectrometry: Expected,
M+H at 1118.5, found, at 1118.6).
(Step 5b) Synthesis of thioether v / [CH CO-Lvs-Cvs(tBu)-Arg-Gly-Asp-Cvs(tBu)-
Phe-Cvs - ¾.
Molecular Weight = 1082.383
Exact Mass = 1081 .487
Molecular Formula = C46H75N1301 1S3
295 mg of ClCH2CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-NH 2 was
dissolved in water/acetonitrile. The mixture was adjusted to pH 8 with ammonia
solution and stirred for 16 hours. After work-up 217 mg of crude peptide was
obtained (Analytical HPLC: Gradient, 5-50 % B over 10 min where A = H2O/0. 1%
TFA and B = CH3CN/0. 1% TFA; column, Phenomenex Luna 3m 18 (2) 50 c 4.6
mm; flow, 2 ml/min; detection, UV 214 nm; product retention time, 6.18 min).
Further product characterisation was carried out using mass spectrometry: Expected,
M+H at 1882.5, found, at 1882.6).
(Step 5c) Synthesis of disulfide [Cvs2 6 thioether cvc/o[CFLCO-Lys-Cvs -Arg-Glv-
A -Cvs -Phe-Cvs - .
Molecular Weight = 968.150
Exact Mass = 967.346
Molecular Formula = C38H57N1301 1S3
217 mg of thioether / [CH2CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-
H2 was treated with a solution of anisole (500 m , DMSO (2 ml) and TFA (100 ml)
for 60 min following which the TFA was removed in vacuo and the peptide
precipitated by the addition of diethyl ether. Purification by preparative FIPLC
(Phenomenex Luna 10m CI8 (2) 250 x 50 mm column) of the crude material (202 mg)
was carried out using 0-30 % B, where A = H2O/0. 1% TFA and B = CH3CN/0. 1%
TFA, over 60 min at a flow rate of 50 ml/min. After lyophilisation 112 mg of pure
material was obtained (Analytical FIPLC: Gradient, 5-50 % B over 10 min where A =
H2O/O.I % TFA and B = CH3CN/O.I % TFA; column, Phenomenex Luna 3 CI 8 (2)
50 x 4.6 mm; flow, 2 ml/min; detection, UV 214 nm; product retention time, 5.50
min). Further product_characterisation was carried out using mass spectrometry:
Expected, M+H at 968, found, at 971).
(Step 5d) Synthesis of disulfide [Cvs2 6 thioether v / [CH CO-Lvs(Chelator 1-
glutaryl)-Cvs -Arg-Glv-Asp-Cvs -Phe-Cvsl-NH .
9.7 mg of disulfide[Cys 2 6] thioether / [CH2CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-
Cys]-NH2, 9.1 mg of Chelator 1A (Example 5) and 6 of MM was dissolved in
DMF (0.5 ml). The mixture was stirred for 3 hours. Purification by preparative
HPLC (Phenomenex Luna 5m C18 (2) 250 x 21.20 mm column) of the reaction
mixture was carried out using 0-30 % B, where A = H2O/0. 1% TFA and B =
CH3CN/0. 1% TFA, over 40 min at a flow rate of 10 ml/min. After lyophilisation 5.7
mg of pure material was obtained (Analytical FIPLC: Gradient, 0-30 % B over 10 min
where A = H2O/0.1 % TFA and B = CH3CN/O.I % TFA; column, Phenomenex Luna
3 m C18 (2) 50 x 4.6 mm; flow, 2 ml/min; detection, UV 214 nm; product retention
time, 7.32 min). Further product characterisation was carried out using mass
spectrometry: Expected, M+H at 1407.7, found, at 1407.6).
Example 6: Synthesis of disulfide [Cys l thioether [CHiCO-Lysf Chelator
l-glutaryn-Cys -Arg-Gly-Asp-Cys -Phe-Cys1-fPEG)s-NHWMaraciclatide).
(Step 6a) Synthesis of 17-(Fmoc-amino)-5-oxo-6-aza-3, 9,12,15-
tetraoxaheptadecanoic acid.
This building block is coupled to the solid-phase using Fmoc chemistry.
l,l l-Diazido-3,6,9-trioxaundecane.
A solution of dry tetraethyleneglycol (19.4 g, 0.100 mol) and methanesulfonyl
chloride (25.2 g, 0.220 mol) in dry THF (100 ml) was kept under argon and cooled to
0 °C in an ice/water bath. To the flask was added a solution of triethylamine (22.6 g,
0.220 mol) in dry THF (25 ml) dropwise over 45 min. After 1 hr the cooling bath was
removed and stirring was continued for 4 hrs. Water (60 ml) was added. To the
mixture was added sodium hydrogen carbonate (6 g, to pH 8) and sodium azide (14.3
g, 0.220 mmol), in that order. THF was removed by distillation and the aqueous
solution was refluxed for 24 h (two layers formed). The mixture was cooled and ether
(100 ml) was added. The aqueous phase was saturated with sodium chloride. The
phases were separated and the aqueous phase was extracted with ether (4 x 50 ml).
Combined organic phases were washed with brine (2 x 50 ml) and dried (MgSC^).
Filtration and concentration gave 22.1 g (91%) of yellow oil. The product was used in
the next step without further purification.
1l-Azido-3,6,9-trioxaundecanamine.
To a mechanically, vigorously stirred suspension of 1,1 l-diazido-3,6,9-
trioxaundecane (20.8 g, 0.085 mol) in 5% hydrochloric acid (200 ml) was added a
solution of triphenylphosphine (19.9 g, 0.073 mol) 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 pH was adjusted to ca 12
by addition of KOH. The product was extracted into dichloromethane (5 x 50 ml).
Combined organic phases were dried (MgSC^). Filtration and evaporation gave 14.0
g (88%) of yellow oil. Analysis by MALDI-TOF mass spectroscopy (matrix: acyano-
4-hydroxycinnamic acid) gave a M+H peak at 219 as expected. Further
characterisation using 1H (500 MHz) and 1 C (125 MHz) MR spectroscopy verified
the structure.
17-Azido-5-oxo-6-aza-3,9J2J5-tetraoxaheptadecanoic acid.
To a solution of 1l-azido-3,6,9-trioxaundecanamine (10.9 g, 50.0 mmol) in
dichloromethane (100 ml) was added diglycolic anhydride (6.38 g, 55.0 mmol). The
reaction mixture was stirred overnight. HPLC analysis (column Vydac 218TP54;
solvents: A = water/0. 1% TFA and B = acetonitrile/0. 1% TFA; gradient 4-16% B
over 20 min; flow 1.0 ml/min; UV detection at 214 and 284 nm), showed complete
conversion of starting material to a product with retention time 18.3 min. The
solution was concentrated to give quantitative yield of a yellow syrup. The product
was analysed by LC-MS (ES ionisation) giving [MH]+ at 335 as expected. 1H (500
MHz) and 1 C (125 MHz) NMR spectroscopy was in agreement with structure
The product was used in the next step without further purification.
17-Amino-5-oxo-6-aza-3,9J2J5-tetraoxaheptadecanoic acid.
A solution of 17-azido-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid (8.36 g,
25.0 mmol) in water (100 ml) was reduced using H2(g)-Pd/C (10%). The reaction
was run until LC-MS analysis showed complete conversion of starting material
(column Vydac 218TP54; solvents: A = water/0.1% TFA and B = acetonitrile/0. 1%
TFA; gradient 4-16% B over 20 min; flow 1.0 ml/min; UV detection at 214 and 284
nm, ES ionisation giving M+H at 335 for starting material and 309 for the product).
The solution was filtered and used directly in the next step.
17-(Fmoc-amino)-5-oxo-6-aza-3,9J2J5-tetraoxaheptadecanoic acid.
To the aqueous solution of 17-amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic
acid from above (corresponding to 25.0 mmol amino acid) was added sodium
bicarbonate (5.04 g, 60.0 mmol) and dioxan (40 ml). A solution of Fmoc-chloride
(7.1 1 g, 0.275 mol) in dioxan (40 ml) was added dropwise. The reaction mixture was
stirred overnight. Dioxan was evaporated off (rotavapor) and the aqueous phase was
extracted with ethyl acetate. The aqueous phase was acidified by addition of
hydrochloric acid and precipitated material was extracted into chloroform. The
organic phase was dried (MgSC^), filtered and concentrated to give 11.3 g (85%) of a
yellow syrup. The structure was confirmed by LC-MS analysis (column Vydac
218TP54; solvents: A = water/0.1% TFA and B = acetonitrile/0.1% TFA; gradient 40-
60% B over 20 min; flow 1.0 ml/min; UV detection at 214 and 254 nm, ES ionisation
giving M+H at 53 1 as expected for the product peak at 5,8 minutes). The analysis
showed very low content of side products and the material was used without further
purification.
(Step 6b) Synthesis of ClCH CO-Lvs-Cvs(tBu)-Arg-Glv-Asp-Cvs(tBu)-Phe-Cvs-
PEG H .
The PEG unit was coupled manually to Rink Amide AM resin, starting on a 0.25
mmol scale, mediated by HATU activation. The remaining peptide was assembled on
an ABI 433A automatic peptide synthesiser using 1 mmol amino acid cartridges. The
amino acids were pre-activated using HBTU before coupling. N-terminal amine
groups were chloroacetylated using a solution of chloroacetic anhydride in DMF for
30 min.
The simultaneous removal of peptide and side-chain protecting groups (except tBu)
from the resin was carried out in TFA containing TIS (5 %), H20 (5 %) and phenol
(2.5 %) for two hours. After work-up 322 mg of crude peptide was obtained
(Analytical HPLC: Gradient, 5-50 % B over 10 min where A = H2O/0. 1% TFA and B
= CH3CN/O.I % TFA; column, Phenomenex Luna 3m C18 (2) 50 x 4.6 mm; flow, 2
ml/min; detection, UV 214 nm; product retention time, 6.37 min). Further product
characterisation was carried out using mass spectrometry: Expected, M+H at 1409,
found, at 1415).
(Step 6c) Synthesis of thioether v / [CH CO-Lvs-Cvs(tBu)-Arg-Glv-Asp-Cvs(tBu)-
Phe-Cvsl-(PEGVNH .
322 mg ofClCH 2CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys-(PEG ) 3- H2
was dissolved in water/acetonitrile. The mixture was adjusted to pH 8 with ammonia
solution and stirred for 16 hours.
After work-up crude peptide was obtained (Analytical HPLC: Gradient, 5-50 % B
over 10 min where A = H2O/0. 1% TFA and B = CH3CN/0. 1% TFA; column,
Phenomenex Luna 3m C18 (2) 50 x 4.6 mm; flow, 2 ml/min; detection, UV 214 nm;
product retention time, 6.22 min). Further product characterisation was carried out
using mass spectrometry: Expected, M+H at 1373, found, at 1378).
(Step 6d) Synthesis of disulfide [Cvs2 6 thioether v / [CH CO-Lvs-Cvs -Arg-Gly-
Asp-Cvs -Phe-Cvsl-(PEGVNH .
Thioether / [CH2CO-Lys-Cys(tBu)-Arg-Gly-Asp-Cys(tBu)-Phe-Cys]-(PEG) 3- H2
was treated with a solution of anisole (200 mί , DMSO (2 ml) and TFA (100 ml) for
60 min following which the TFA was removed in vacuo and the peptide precipitated
by the addition of diethyl ether. Purification by preparative FIPLC (Phenomenex
Luna 5 CI8 (2) 250 x 21.20 mm column) of 70 mg crude material was carried out
using 0-30 % B, where A = H2O/0. 1% TFA and B = CH3CN/0. 1% TFA, over 40 min
at a flow rate of 10 ml/min. After lyophilisation 46 mg of pure material was obtained
(Analytical HPLC: Gradient, 0-30 % B over 10 min where A = H2O/0.1 % TFA and B
= CH3CN/O.I % TFA; column, Phenomenex Luna 3m C18 (2) 50 x 4.6 mm; flow, 2
ml/min; detection, UV 214 nm; product retention time, 6.80 min). Further product
characterisation was carried out using mass spectrometry: Expected, M+H at 1258.5,
found, at 1258.8).
(Step 6e) Synthesis of disulfide [Cvs2 6 thioether / [CH CO-Lys(Chelator 1-
glutaryl)-Cvs -Arg-Glv-Asp-Cvs -Phe-Cvsl-(PEGVNH .
13 mg of [Cys2 6] / [CH2CO-Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG)3-NH 2,
9.6 mg of Chelator 1A and 8 of MM was dissolved in DMF (0.5 ml). The
mixture was stirred for 2 hours and 30 minutes. Purification by preparative HPLC
(Phenomenex Luna 5 CI8 (2) 250 x 21.20 mm column) of the reaction mixture was
carried out using 0-30 % B, where A = H2O/0. 1% TFA and B = CH3CN/0. 1% TFA,
over 40 min at a flow rate of 10 ml/min. After lyophilisation 14.2 mg of pure material
was obtained (Analytical HPLC: Gradient, 0-30 % B over 10 min where A = H2O/0. 1
% TFA and B = CH3CN/0. 1% TFA; column, Phenomenex Luna 3m 18 (2) 50 c 4.6
mm; flow, 2 ml/min; detection, UV 214 nm; product retention time, 7.87 min).
Further product characterisation was carried out using mass spectrometry: Expected,
M+H at 1697.8, found, at 1697.9).
Example 7: Choice of Radioprotectant.
The effect, on radiolabelling efficiency and radiostabilisation, of three
radioprotectants, pABA, gentisic acid and ascorbic acid, was evaluated for freezedried
kits. The formulations were identical to Formulation A (see Example 9) except
for the addition of the radioprotectant and an increased amount of sodium carbonate to
maintain the pH close to 9.3, see Table 2 :
Table 2: radioprotectant formulations
Formulation A had an RCP at 20 minutes post-reconstitution of ca. 90-91%, falling to
82-85% at 4-hours, and 75-82% at 8-hours post-reconstitution.
The freeze-dried kits containing ascorbic acid had a poor radiolabelling efficiency -
the RCP was 80%> at both 20 minutes and 4 hours post-reconstitution.
The freeze-dried kits with gentisic acid showed a good radiolabelling efficiency, 94%
at 20 minutes post reconstitution and a good radiostabilising effect, the RCP was 90%
at 4 hours post reconstitution. However, the reconstituted kit solution discoloured
(turned pink) after some time. Similar discolouration was observed in solution
chemistry experiments.
The freeze-dried kits with pABA showed a good radiolabelling efficiency and the
RCP was stabilised. The RCP values were approximately 90% at both 20 minutes
and 4 hours post reconstitution, with stability maintained even at 8-hours post
reconstitution (see Example 8). No new radioactive impurities were observed
compared to Formulation A.
Example 8: Optimisation of Amount of Radioprotectant.
To optimise the amount of pABA in the formulation, a factorial design with 3 levels
of pABA [100 to 600 g per vial] and 2 levels of pH [8.7 to 9.3] was prepared. The
levels of other kit components were as for Formulation A. The sodium carbonate
levels were used to adjust the pH.
Table 3 RCP results from pABA optimisation study
pr = post-reconstitution
Analysing the results from the factorial design, shown in Table 3, showed that the
optimal pABA amount with respect to initial RCP and radiostabilising capacity is
between 200 and 350 g/vial.
Two further batches with respectively 200 g (Batch #9; 630 g Na2C0 3 pH 9.3) and
300 g (Batch #10; 800 mg Na2C0 3 pH 9.3) pABA, gave very similar RCP values at
an initial time point and during stability. The results showed that there was no
significant difference between freeze dried kits containing 200 and 300 mg pABA
with respect to radiolabelling efficiency or radiostabilising capacity.
Example 9: Comparative Lyophilised Kit Formulations.
Lyophilised kits were prepared as follows, to compare the prior art kit of Edwards et
al [Nucl.Med.Biol, 35, 365-375 (2008)] and the stabilised kit of the present
invention:
Table 1 : kit formulations
Example 10: Generator Compatibility Study.
Two generator compatibility studies have been performed to investigate the
compatibility of Formulation C. The generators studied in the first study were:
Technelite Technetium-99m, Sterile generator [Bristol Myers Squibb Medical
Imaging], Drytec Technetium-99m, Sterile generator [GE Heathcare, UK], Ultra-
Technekow® DTE generator [Tyco Healthcare Mallinckrodt, USA] and ISOTEC Mo-
99-Tc-99m, Sterile generator [Amersham Health, Norway]. The test samples were
Formulation C (Batch #9 of Example 8). All samples were reconstituted with
generator eluate of 3.1 GBq/6 ml. The variables studied for the generators were
generator age (time between elutions) and eluate age (time after elution).
All four generators tested in this first study were compatible with Formulation C.
There was only a small difference in the RCP values, 1.6%. The eluate age had a
negative effect on both RCP and post reconstitution stability for all four generators.
Example 11: Stability of Freeze-Dried kits.
Shelf-life stability testing was performed on several batches of kit stored at various
temperatures [-20°C, 5°C and 25°C], and for some temperatures up to 12 months.
The RCP was determined after storage under different temperature conditions.
Formulation A kit.
5° storage 12 months: RCP at 4 hours post-reconstitution was 87%
25°storage 3 months: RCP at 4 hours post-reconstitution was 87%
Formulation C kit
5° storage 12 months: RCP at 4 hours post-reconstitution was 91.7%
25°storage 3 months: RCP at 4 hours post-reconstitution was 91.5%
25°storage 6 months: RCP at 4 hours post-reconstitution was >90%
Formulation C has a shelf-life of at least 50 months when stored at 5°C, whereas
Formulation A would have to be stored at -20°C to ensure adequate kit RCP
performance.
CLAIMS.
1. A radiopharmaceutical composition which comprises
(i) mTc-maraciclatide;
(ii) a radioprotectant chosen from /?ara-aminobenzoic acid or a salt
thereof with a biocompatible cation;
in a biocompatible carrier in a form suitable for mammalian
administration.
2 . The radiopharmaceutical composition of claim 1, where the radioprotectant is
sodium /?ara-aminobenzoate.
3 . The radiopharmaceutical composition of claim 1 or claim 2, where the
radiopharmaceutical is provided in a syringe.
4 . The radiopharmaceutical composition of claim 1 or claim 2, where the
radiopharmaceutical is provided in a vial fitted with a closure.
5 . A kit for the preparation of the radiopharmaceutical composition of any one of
claims 1 to 4, which comprises:
(i) Maraciclatide;
(ii) a radioprotectant chosen from /?ara-aminobenzoic acid or a salt
thereof with a biocompatible cation;
(iii) a stannous reductant;
(iv) methylene diphosphonic acid or a salt thereof with a
biocompatible cation.
6 . The kit of claim 5, where all the kit components are lyophilised together.
7 . The kit of claim 5 or claim 6, which further comprises a buffer.
8 . The kit of any one of claims 5 to 7, where the stannous reductant is stannous
chloride.
9 . A method of preparation of the radiopharmaceutical composition of any one of
claims 1 to 4, which comprises either:
(i) reconstitution of the kit of any one of claims 5 to 8 with a supply of a
biocompatible carrier, followed by addition of a supply of mTc in a
biocompatible carrier to the reconstituted kit; or
(ii) reconstitution of the kit of any one of claims 5 to 8 with a supply of mTc
in a biocompatible carrier.
10. Use of the kit of any one of claims 5 to 8 in the preparation of the
radiopharmaceutical composition of any one of claims 1 to 4 .
11. Use of /?ara-aminobenzoic acid or a salt thereof with a biocompatible cation,
as a radioprotectant to stabilise either:
(i) mTc-maraciclatide radiopharmaceutical compositions;
(ii) kits for the preparation of mTc-maraciclatide radiopharmaceutical
compositions.
12. Use of the radiopharmaceutical composition of any one of claims 1 to 4 in a
method of imaging of the mammalian body.
13. A method of imaging of the mammalian body which comprises imaging a
mammal which had previously been administered with the radiopharmaceutical
composition of any one of claims 1 to 4 .
14. The method of claim 13, where the mammal is suffering from a disease in
which suffering from a disease in which in which integrins are expressed.