Patent Application
20070020177
The present invention relates to stabilised technetium and rhenium metal complex compositions comprising a radioprotectant and a radiometal complex of a tropane-tetradentate chelating agent conjugate. Radiopharmaceuticals comprising the stabilised metal complex compositions, and kits for the preparation of the radiopharmaceuticals are also described.
Tropanes labelled with 123I, 18F or 99mTc are known as diagnostic imaging radiopharmaceuticals for brain imaging [Morgan and Nowotnik, Drug News Perspect. 12(3), 137-145 (1999)]. Tropanes are known to target the dopamine transporter in the brain, and the dopamine transporter has been implicated in several diseases including Parkinson's Disease, Parkinsonian Syndrome and attention-deficit hyperactivity disorder.
Tropanes labelled with 99mTc are known. The development of 99mTc-TRODAT-1 has been described by Kung [Nucl. Med. Biol., 28, p. 505-508 (2001)]:
TRODAT-1 is also described in U.S. Pat. No. 5,980,860 and equivalents.
Technepine has also been described by Meltzer et al [J. Med. Chem., 40, 1835-1844 (1997)]:
Technepine is described in U.S. Pat. No. 6,171,576 and equivalents.
WO 03/055879 describes chelator-tropane conjugates wherein the 6- or 7- positions of the tropane are functionalised. Kits are briefly described, but the use of radioprotectants is not disclosed.
A range of 99mTc complexes of N2S2 diaminedithiol chelator conjugates of tropanes (including TRODAT-1) have been reported to show good in vitro stability at 4 and 24 hours post preparation, with little change in radiochemical purity [Meegalla et al, J. Med. Chem., 40, p. 9-17 (1997)]. Fan et al [Chin. J. Nucl. Med., 19(3) 146-148 (1999)] report that 99mTc-TRODAT-1 is stable for 24 hours at room temperature.
An improved kit formulation for the preparation of 99mTc-TRODAT-1 has been described [Choi et al, Nucl. Med. Biol., X, p. 461-466 (1999)]. Choi et al report that, as long as a minimum of 10 μg (microgrammes) of the tropane conjugate is present in the kit, the radiochemnical purity consistently reaches greater than 90%. Heating is necessary to achieve a satisfactory radiochemical purity (RCP), and Choi et al use autoclave heating for 30 minutes.
Technetium-99m (99mTc) is a radioisotope which decays with a half-life of 6.02 hours to technetium-99 (99Tc). 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, 99Tc, is also radioactive and decays by emission with a half-life of 2.1×105 years (to the stable isotope 99Ru), but the radioactive emissions from 99Tc are insufficient for medical imaging. Conventional 99mTc “generators” comprise the radioisotope 99Mo, which decays with a half-life of 66.2 hours. About 86% of 99Mo decays result in the production of 99mTc, however ca. 14% of 99Mo decays result in the direct production of 99Tc. Therefore, if a 99mTc generator is eluted a very short time after the previous elution, the 99mTc content will be low but will be about 86% of the total technetium content. As time passes since the previous elution of the generator, 99Tc is being produced both from 99Mo and from the decay of 99mTc to 99Tc. Consequently, as the time interval between generator elutions increases, the 99Tc/99mTc ratio increases. The 99Tc and 99mTc technetium isotopes are chemically identical, and consequently any radiopharmaceutical preparation must be able to cope with a wide range of 99Tc 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 99mTc 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 99mTc radiopharmaceutical preparation thus needs to be able to provide satisfactory RCP performance even when such reactive free radicals are present. These characteristics of the 99mTc generator are illustrated in most radiochemistry or nuclear chemistry textbooks, and the problems that different eluate properties can give to the performance of 99mTc kits have been described by Saha, G. B. “Radiopharmaceuticals and Methods of Radiolabeling”; Chapter 6 (pages 80-108) in Fundamentals of Nuclear Pharmacy (3rd Edn.), and Hung et al for Cardiolite™ [Nucl. Med. Biol., 23, 599-603 (1996)].
The present invention provides improved radiometal complex compositions comprising a technetium or rhenium metal complex of a tropane-tetradentate chelating agent conjugate and a radioprotectant. The improved compositions 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 radiometal tropane conjugates 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 99mTc generator. The present invention provides compositions comprising a radioprotectant which solve this previously unrecognised problem.
In a first embodiment, the present invention provides a stabilised composition which comprises:
The term “tropane” has its conventional meaning, i.e. a bicyclic amine of formula (with numbering of the ring positions shown):
where the amine nitrogen at the 8-position may be secondary or tertiary.
By the term “metal complex” is meant a coordination complex of the technetium or rhenium metal ion with a ligand, here the tetradentate chelating agent. The chelated metal complex is “resistant to transchelation”, i.e. does not readily undergo ligand exchange with other potentially competing ligands for the radiometal coordination sites. Potentially competing ligands include the tropane moiety itself, the radioprotectant or other excipients in the preparation in vitro (e.g. antimicrobial preservatives), or endogenous compounds in vivo (e.g. glutathione, transferrin or plasma proteins).
Suitable radioactive isotopes of technetium or rhenium include: 94mTc, 99mTc, 186Re and 188Re. A preferred such radioisotope is 99mTc.
The term “tetradentate” has its conventional meaning, i.e. the chelating agent has four donor atoms, each of which coordinate to the metal giving chelate rings on formation of the metal complex. The tetradentate chelating agent is preferably attached at the 2-, 6-, 7-or 8-positions of the tropane, and is most preferably attached at either the 2- or the 8-position of the tropane, ideally at the 2-position.
By the term “radioprotectant” is meant a compound which inhibits degradation reactions induced by radioactive emissions (e.g. 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, para-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e. 2,5-dihydroxybenzoic acid), gentisyl alcohol and salicyclic acid, including salts thereof with a biocompatible cation. Preferred radioprotectants are ascorbic acid and para-aminobenzoic acid, or salts thereof with a biocompatible cation. Especially preferred radioprotectants are ascorbic acid and salts thereof with a biocompatible cation. A preferred such salt is sodium ascorbate. The radioprotectants of the present invention are commercially available to a pharmaceutical grade specification.
By the term “biocompatible cation” 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 technetium and rhenium metal complexes of the present invention are “neutral”, i.e. any positive charge on the central metal core is balanced by the sum of the negative charge on the four metal donor atoms of the tetradentate chelating agent, to give an overall electrically neutral metal complex. Examples of likely technetium cores are O═Tc+═O and Tc3+═O, which both represent technetium in the Tc(V) oxidation state. Similar cores O═Re+═O and Re3+═O are known for rhenium.
The neutral radioactive technetium or rhenium complexes of the present invention are suitably of Formula I:
[{tropane}-(A)n]m-[metal complex] (I)
The “linker group” (A), is as defined below for Formula Ia. The metal complexes of Formula I are derived from tropane “conjugates”. The tropane tetradentate chelating agent “conjugates” of the present invention are as defined in Formula Ia:
[{tropane}-(A)n]m-[tetradentate chelating agent] (Ia)
where:
In Formulae I and Ia, m is preferably 1 or 2, and is most preferably 1; and (A), is preferably (CR2)n, where n is chosen to be 1 to 3.
Examples of suitable tetradentate chelating agents for technetium and rhenium which form neutral metal complexes include, but are not limited to:
(i) N2S2 ligands having a diaminedithiol donor set such as BAT, an amideaminedithiol donor set such as MAMA, a phenylenediaminethioetherthiol donor set such as PhAT, or a dithiosemicarbazone donor set;
(ii) diaminedioximes;
(iii) N3S ligands having a diamidepyridinethiol donor set such as Pica;
(iv) open chain or macrocyclic ligands having an amidetriamine donor set, such as monoxocyclam.
(v) N2O2 ligands having a diaminediphenol donor set
The above described ligands are particularly suitable for complexing technetium e.g. 94mTc or 99mTc, and are described more fully by Jurisson et al [Chem. Rev., 99, 2205-2218 (1999)]. N2S2 dithiosemicarbazone chelators are described by Arano et al [Chem. Pharm. Bull., 39, p. 104-107 (1991)]. N2S2 phenylenediaminethioetherthiol chelators are described by McBride et al [J. Med. Chem., 3, p. 81-6 (1993)]. Macrocyclic amidetriamine ligands and their tropane conjugates are described by Turpin et al [J. Lab. Comp. Radiopharm., 45, 379-393 (2002)). Diaminedioximes are described by Nanjappan et al [Tetrahedron, 50, 8617-8632 (1994)]. N3S ligands having a diamidepyridinethiol donor set such as Pica are described by Bryson et al [Inorg. Chem., 29, 2948-2951 (1990)]. N2O2 ligands having a diaminediphenol donor set are described by Pillai et al [Appl. Rad. Isot., 41, 557-561 (1990)].
Preferred 99mTc metal complexes of the present invention are those suitable for crossing the blood-brain barrier (BBB) as described by Volkert et al [Radiochim. Acta, 63, p. 205-208 (1993)]. Especially preferred 99mTc metal complexes of the present invention are 99mTc-TRODAT-1 and Technepine.
Preferred tetradentate chelating agents are those having an N2S2 diaminedithiol or amideaminedithiol donor set of Formula II:
where: E1-E5 are each independently an R′ group;
By the term “protecting group” is meant a group which inhibits or suppresses undesirable chemical reactions (e.g. oxidation of the free thiol to the corresponding disulphide), but which is designed to be sufficiently reactive that it may be cleaved from the thiol under mild enough conditions that do not modify the rest of the molecule during radiolabelling of the conjugate. Thiol protecting groups are well known to those skilled in the art and include but are not limited to: trityl, 4-methoxybenzyl, benzyl, tetrahydropyranyl, methyltetrahydrofuranyl (MTHF), acetamidomethyl and ethoxyethyl. The use of further thiol protecting groups are described in ‘Protective Groups in Organic Synthesis’, Theorodora W. Greene and Peter G. M. Wuts, (John Wiley & Sons, 1991). In Formula II, P1 and P2 are preferably both H.
Preferred Q groups are as follows: —CH2CH2—, —CH2CH2CH2— or —(C═O)CH2—, most preferably N2S2 diaminedithiol chelators where Q is —CH2CH2— or —CH2CH2CH2—, with —CH2CH2— (i.e. BAT type chelators) being especially preferred.
E1 to E5 are preferably chosen from: H, C1-3 alkyl, C1-3 alkoxyalkyl, C1-3 hydroxyalkyl or C1-3 fluoroalkyl. Most preferably, each E1 to E4 group is H, and E5 is C1-3 alkyl.
A most particularly preferred chelator of Formula II is the N2S2 diaminedithiol chelator of TRODAT-1, i.e. the chelator of Formula II where: Q is —CH2CH2—, E1 to E5 are all H and P1=P2=H.
The tetradentate chelating agents of Formula II are preferably attached to the tropane via either the bridging group Q or the E5 group. Most preferably, the tropane is attached via the E5 group.
Preferably, the tropane of the present invention is a phenyl tropane of Formula III:
where:
R1 is preferably C1-3 alkyl or C1-3 fluoroalkyl. R2 is preferably CO2CH3 or C1-2 alkyl. R3 is preferably 4-chloro, 4-fluoro or 4-methyl, and R4 is preferably H or CH3. R1 is most preferably CH3.
It is envisaged that the role of the linker group -(A)n- of Formula I is to distance the relatively bulky metal complex from the tropane, so that binding of the tropane to biological target sites (e.g. the dopamine transporter in the mammalian brain) is not impaired. This can be achieved by a combination of flexibility (e.g. simple alkyl chains), so that the bulky group has the freedom to position itself away from the active site and/or rigidity such as a cycloalkyl or aryl spacer which orientates the metal complex away from the active site.
The nature of the linker group can also be used to modify the biodistribution of the resulting metal complex of the conjugate. Thus, e.g. the introduction of ether groups in the linker will help to minimise plasma protein binding. Preferred linker groups -(A)n-have a backbone chain of linked atoms which make up the -(A)n- moiety of 2 to 10 atoms, most preferably 2 to 5 atoms, with 2 or 3 atoms being especially preferred. A minimum linker group backbone chain of 2 atoms confers the advantage that the chelator is well-separated from the tropane, so that any interaction is minimised.
Non-peptide linker groups such as alkylene groups or arylene groups have the advantage that there are no significant hydrogen bonding interactions with the conjugated tropane, so that the linker does not wrap round onto the tropane. Preferred alkylene spacer groups are —(CH2)q— where q is 2 to 5. Preferred arylene spacers are of formula:
It is strongly preferred that the tropane is bound to the metal complex in such a way that the linkage does not undergo facile metabolism in blood, since that would result in the metal complex being cleaved off before the labelled tropane inhibitor reached the desired in vivo target site. The tropane is therefore preferably covalently bound to the metal complexes of the present invention via linkages which are not readily metabolised.
The stabilised composition of the present invention may be prepared by reaction of a solution of the radiometal in the appropriate oxidation state with the chelator conjugate at the appropriate pH in the presence of the radioprotectant, in solution in a suitable solvent The radioprotectant may be supplied either together with the conjugate or the solution of the radiometal. Preferably, the radioprotectant is pre-mixed with the conjugate, and that precursor composition is subsequently reacted with the radiometal, (as described in the second embodiment below). The conjugate solution may preferably contain a ligand which complexes weakly but rapidly to the radiometal, such as gluconate or citrate i.e. the radiometal complex is prepared by ligand exchange or transchelation. Such conditions are useful to suppress undesirable side reactions such as hydrolysis of the metal ion. When the radiometal is rhenium, the usual radioactive starting material is perrhenate, i.e. ReO4−. When the radiometal is 99mTc, the usual radioactive starting material is sodium pertechnetate from a 99Mo generator. In both perrhenate and pertechnetate the metal (M) is present in the M(VII) oxidation state, which is relatively unreactive. The preparation of technetium or rhenium complexes of lower oxidation state M(I) to M(V) therefore usually requires the addition of a suitable biocompatible reductant. The “biocompatible reductant” is a pharmaceutically acceptable reducing agent such as sodium dithionite, sodium bisulphite, formamidine sulphinic acid, stannous ion, Fe(II) or Cu(I), to facilitate complexation. Ascorbic acid can function both as a radioprotectant and a biocompatible reductant, and hence it is possible to use quantities of ascorbic acid greater than that necessary for radioprotection alone to facilitate reduction also. The biocompatible reductant preferably comprises stannous i.e. Sn(II), preferably as a stannous ion or salt. Preferred stannous salts are stannous chloride, stannous fluoride and stannous tartrate. The stannous salt may be employed in either anhydrous or hydrated form.
Alternatively, the stabilised composition of the present invention may be prepared in a stepwise manner by first forming the radiometal complex in a suitable solvent, and subsequently adding the radioprotectant. In such an approach, the radioprotectant should be added as soon as possible after formation of the radiometal complex, so that the stabilising effect of the radioprotectant is brought-into effect to minimise radiolysis and possible degradation. Methods of preparation wherein the radioprotectant is present prior to formation of the radiometal complex are preferred.
The concentration of radioprotectant for use in the present invention is suitably 0.3 to 5.0 millimolar, preferably 0.4 to 4.0 millimolar, most preferably 1.0 to 3.5 millimolar. For ascorbic acid, this corresponds to a suitable concentration of 50 to 900 μg/cm3, preferably 70 to 800 μg/cm3, most preferably 90 to 700 μg/cm3. For the 99mTc radiopharmaceutical 99mTc-TRODAT-1, the preferred concentration of an ascorbic acid or ascorbate radioprotectant is in the range 0.5 to 3.8 millimolar.
When the radiometal complexes of the present invention are to be used in radiopharmaceutical compositions, a preferred method of preparation is the use of a sterile, non-radioactive kit as described in the third and fourth embodiments below. The kit provides a convenient supply of the necessary reactants at the right concentration, which needs only be reconstituted with perrhenate or pertechnetate in saline or another suitable solvent.
In a second embodiment, the present invention provides a precursor composition useful in the preparation of the above stabilised composition, which comprises:
Preferably, the tropane of the precursor composition is a phenyl tropane of Formula III (above). Preferred and most preferred phenyl tropanes for the precursor composition are as described above for the first embodiment. Most preferably, the conjugate of the precursor composition is of Formula IV:
The term “protecting group” is as defined for Formula II above. Preferred conjugates of Formula IV are where P1 and P2 are both H.
The conjugates used in the precursor compositions of the present invention may be prepared via the bifunctional chelate approach. Thus, it is well known to prepare chelating agents which have attached thereto a functional group (“bifunctional chelates”). Functional groups that have been attached include: amine, thiocyanate, maleimide and active esters such as N-hydroxysuccinimide or pentafluorophenol. Such bifunctional chelates can be reacted with suitable functional groups on the tropane to form the desired conjugate. Such suitable functional groups on the tropane include:
carboxyls (for amide bond formation with an amine-functionalised bifunctional chelator);
amines (for amide bond formation with an carboxyl- or active ester-functionalised bifunctional chelator);
halogens, mesylates and tosylates (for N-alkylation of an amine-functionalised bifunctional chelator) and
thiols (for reaction with a maleimide-functionalised bifunctional chelator). Further details of the bifunctional chelate approach are described by Arano [Adv. Drug Deliv. Rev., 37, 103-120 (1999)]. Further details specific to the conjugation of tropanes with the tetradentate chelating agents indicated are described in: the methods of Meegalla et al [J. Med. Chem., 40, 9-17 (1997)] for N2S2 diaminedithiol chelators; Meltzer et al for N2S2 amideaminedithiol (MAMA) chelators [ibid, 40, 1835-1844 (1997)] and Turpin et al [J. Lab. Comp. Radiopharm., 4, 379-393 (2002)] for monoxocyclam chelators.
In a third embodiment, the present invention provides a radiopharmaceutical which comprises the stabilised composition of the first embodiment together with a biocompatible carrier, in a form suitable for mammalian administration. The “biocompatible carrier” is a fluid, especially a liquid, in which the imaging agent can be suspended or dissolved, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); 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).
The radiopharmaceuticals of the present invention may optionally further comprise an antimicrobial preservative. 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 concentration. The main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the radiopharmaceutical composition post-reconstitution, i.e. in the radioactive diagnostic product itself. Suitable antimicrobial preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservative(s) are the parabens.
Such radiopharmaceuticals are suitably supplied in either a container which is provided with a seal which 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 may contain single or multiple patient doses. 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. Pre-filled syringes are designed to contain a single human dose, and are therefore preferably a disposable or other syringe suitable for clinical use. The pre-filled syringe may optionally be provided with a syringe shield to protect the operator from radioactive dose. Suitable such radiopharmaceutical syringe shields are known in the art and preferably comprise either lead or tungsten.
When the radioactive isotope is 99mTc, a radioactivity content suitable for a diagnostic imaging radiopharmaceutical is in the range 180 to 1500 MBq of 99mTc, depending on the site to be imaged in vivo, the uptake and the target to background ratio. 99mTc is suitable for SPECT imaging and 94mTc for PET imaging.
The radiopharmaceuticals of the present invention comprise the improved radiometal compositions of the first embodiment. This has the advantage that radioactive impurities are suppressed. Such radioactive impurities may either contribute to unnecessary radiation dose for the patient, or may in some cases have an adverse effect on imaging by reducing the signal to background ratio.
The radiopharmaceuticals of the present invention may be prepared from kits, as is described in the fourth embodiment below. Alternatively, the radiometal complexes of the present invention in a biocompatible carrier may be prepared under aseptic manufacture conditions to give the desired sterile product. The radiopharmaceuticals may also be prepared under non-sterile conditions, followed by terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). Preferably, the radiopharmaceuticals of the present invention are prepared from kits.
In a fourth embodiment, the present invention provides a kit for the preparation of the radiopharmaceuticals of the present invention, which comprises:
The “radioprotectant” of the kit is as defined above. Preferred radioprotectants correspond to those described for the stabilised composition of the first embodiment.
The conjugate of Formula (Ia) comprises the amine which forms the 8-position of the tropane ring, plus possibly further amine donor atoms of the tetradentate chelating agent. Hence, the conjugate may optionally be used in the kit as “a salt thereof with a biocompatible counterion”, i.e. an acid salt of the conjugate. Suitable such salts include but are not limited to: hydrochlorides, trifluoroacetates, sulphonates, tartrates, oxalates and sulphosalicyclates. When the conjugate is of Formula IV, preferred salts are the trifluoroacetate or hydrochloride salts, especially the trifluoroacetate salt.
The non-radioactive kits may optionally further comprise additional components such as one or more transchelator(s), antimicrobial preservative(s), pH-adjusting agent(s) or filler(s). The “transchelator” comprise one or more compounds which react rapidly to form a weak complex(es) with technetium, then are displaced by the ligand. This minimises the risk of formation of reduced hydrolysed technetium (RHT) due to rapid reduction of pertechnetate competing with technetium complexation. Suitable such transchelators are salts of a weak organic acid, i.e. an organic acid having a pKa in the range 3 to 7, with a biocompatible cation. Suitable such weak organic acids are acetic acid, citric acid, tartaric acid, gluconic acid, glucoheptonic acid, benzoic acid, phenols or phosphonic acids, or aminocarboxylic acids, such as ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA). Hence, suitable salts are acetates, citrates, tartrates, gluconates, glucoheptonates, benzoates, phenolates, phosphonates or edetates. Preferred such salts are edetates, gluconates, glucoheptonates, benzoates, or phosphonates, most preferably edetates, gluconates, glucoheptonates or phosphonates, most especially gluconates, glucoheptonates or edetates. Preferred edetate salts are disodium edetate and calcium edetate. A preferred transchelator is a gluconate or glucoheptonate salt of a biocompatible cation.
When the kit comprises a diaminedithiol N2S2 tetradentate chelator, the transchelator preferably comprises a combination of a gluconate or glucoheptonate salt, together with an edetate salt.
The “antimicrobial preservative” is as defined for the radiopharmaceutical (i.e. third) embodiment (above). For the kit, the inclusion of an antimicrobial preservative means that, once reconstituted, the growth of potentially harmful micro-organisms in the preparation is inhibited.
The term “pH-adjusting agent” means a compound or mixture of compounds useful to ensure that the pH of the reconstituted kit 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. tris(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the conjugate is employed in acid salt 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. A preferred filler is mannitol.
For 99mTc, the kit is preferably lyophilised and is designed to be reconstituted with a sterile solution of 99mTc-pertechnetate (TcO4) from a 99mTc radioisotope generator to give a solution suitable for human or mammalian administration with the minimum of further manipulation. In ideal circumstances, the desired radiopharmaceutical product is formed at room temperature in a few minutes directly from 99mTc generator eluate, i.e. a one-step preparation. An alternative possibility is a multi-step process in which it is necessary to add two or more solutions (e.g. eluate and buffer solution) to the kit. In some instances, the reaction time at room temperature may be found to be unduly long. This can readily be determined by RCP measurements at time intervals as is known in the art. Heating may therefore need to be applied to drive the radiolabelling reaction to completion in a shorter timeframe. When heating is necessary, the heating process may employ any suitable methodology such as: hot baths of fluid, such as water or a high-boiling oil (e.g. silicone); heating blocks; hot plates or microwave radiation; as long as the desired temperature control can be achieved. After the heating is complete, the reaction mixture is either allowed to cool to room temperature, or may be actively cooled (e.g. in a stream of a cooling fluid such as a gas or water) or via a heating block with integral inductive cooling.
Preferred kits of the present invention comprise:
When the conjugate of Formula (Ia) comprises an N2S2 diaminedithiol chelator, preferred kits of the present invention further comprise:
When the N2S2 diaminedithiol chelator is that which forms part of TRODAT-1, the transchelator preferably comprises a combination of ethylenediaminetetraacetic acid (EDTA), and biocompatible salts thereof together with a salt of gluconic acid or glucoheptonic acid. When the tropane-tetradentate chelator conjugate is TRODAT-1, the kit of the present invention preferably comprises:
Preferred TRODAT-1 kits comprise ascorbic acid or sodium ascorbate as the radioprotectant and a combination of sodium gluconate and disodium edetate as the transchelator. A most preferred TRODAT-1 kit formulation is that given as Formulation P in Example 1.
In a fifth embodiment, the present invention provides a method of preparation of the radiopharmaceutical of the present invention, which comprises formation of the metal complex of the tropane chelator conjugate in a biocompatible carrier in a form suitable for mammalian administration by either:
In a sixth embodiment, the present invention provides the use of the radiopharmaceutical of the third embodiment in a method of diagnostic imaging of the mammalian brain.
In a further embodiment, the present invention provides a method of diagnostic imaging of the mammalian brain which comprises imaging a mammal which had previously been administered with the radiopharmaceutical of the third embodiment. In this embodiment the radiopharmaceutical is used in a method of imaging, or image processing wherein the term “previously been administered” means that any step requiring a medically-qualified person to administer the agent to the patient has already been carried out.
The invention is illustrated by the following non-limiting Examples. Example 1 describes the materials and methods used in the comparative studies described in later Examples. Thus a formulation of the present invention (Formulation P) is compared with a prior art formulation (Formulation Q). Example 2 describes the radiolabelling protocol and quality control methodology employed.
Example 3 studies the effect of different 99mTc generator elution characteristics on the performance of the kit formulations P and Q. 99mTc generators are designed to be used over a period of several days, and depending on the age of the generator, and the time since the last elution, a range of characteristics of the eluate resulting from elution are obtained. A commercial kit must give satisfactory RCP preparations under the full range of storage and elution conditions in use by customers. Example 3 studies a range of four sets of elution conditions (“Elution Conditions 1 to 4). The results show that both formulations give acceptable initial RCPs (92 and 88% for P and Q respectively) under ‘best case’ generator elution conditions (“elution conditions 1”). Formulation P gave an RCP of 90% at 3.5 hours post-preparation, whereas for Formulation Q (prior art), the RCP fell off sharply to 77% over the same period of time. These results show that Formulation P exhibits both an improved initial RCP and an improved post-preparation stability.
Under generator elution conditions 2, Formulation P had an RCP of 92% immediately after preparation and 90% at 4 hours. The initial RCP for Formulation Q was 88%, but again it fell off significantly to 77% at 3.5 hours. These results demonstrate that even with generator eluate at the end of its usable shelf-life, Formulation P displays an improved initial RCP, and greater post-preparation stability than prior art Formulation Q.
Under generator elution conditions 3, in spite of the long interval between generator elutions, both formulations give acceptable initial RCPs (91 and 88% for P and Q respectively). At 4 hours post-preparation the RCP of Formulation P was still as high as 88%, whereas the RCP of Formulation Q was down to 73% after only 2 hours.
Under generator elution conditions 4, Formulation P exhibits an RCP of 90% after 1.5 hours and 89% at 3.5 hours post-preparation. When subjected to these most challenging eluate conditions, the RCP of prior art Formulation Q is only 76% at 1.5 hours, falling to 71% at 3.5 hours.
Example 4 shows that p-ABA is also effective as a radioprotectant for 99mTc-TRODAT-1 preparations. Addition of p-ABA led to a significant improvement in RCP. Increasing the level of p-ABA from 200 to 500 μg increased further the stability at both initial and 4 hours.
Example 5 studies the effect of the volume of 99mTc-pertechnetate used to reconstitute the kit vial (“reconstitution volume”) on the RCP, at the same eluate radioactive concentration (0.75 GBq/ml). At the standard 2 ml reconstitution volume the Formulation P kits perform better than Formulation Q (prior art). The Formulation P kits continue to radiolabel well even when reconstituted with 3 GBq in 4 ml, but the RCP drops markedly when kits are reconstituted with 4.5 GBq in 6 ml. These results show that the RCP of both formulations are affected by reconstitution volume. This effect may be attributable to an increased path length effect for radiolysis of the solvent. The inclusion of the radioprotectant ascorbate in Formulation P suppresses the volume effect compared with the prior art Formulation (O), but does not eliminate it completely.
Example 6 studies the effect of use of an autoclave heating cycle (121° C., 30 minutes) as part of the radiolabelling procedure, since Choi et al [Nucl. Med. Biol., 26, p. 461-466 (1999)] employ that vial heating methodology. The present experiments were typically conducted using heating via a boiling water bath, since the use of an autoclave as part of the preparation procedure is not an attractive option for a commercial product. Hence, a comparative study was carried out to determine if the different heating procedure might contribute to the RCP differences observed for Formulations P and Q. Example 6 indicates that the use of an autoclave heating cycle has a detrimental effect on the RCP of both formulations. Low RCPs were observed for both formulations at both analysis time points and high levels of hydrophilic impurities were seen in the radioactive HPLC chromatograms. Hence, the reported stability of the prior art Choi et al 99mTc-TRODAT-1 preparations cannot be ascribed to the use of autoclaving.
Example 7 shows that the useful regional brain biodistribution properties of 99mTc-TRODAT-1 are maintained in the presence of the radioprotectant sodium ascorbate.
Example 8 shows that a 99mTc kit formulation of the present invention gives satisfactory RCP over a range of eluate conditions with three different commercial 99mTc generators.
Example 9 shows that a Formulation P kit of the present invention can be reconstituted successfully at room temperature, using a two-step protocol. The radioactivity is added first, followed by a buffer solution at pH 7.4. The buffer raises the pH of the reaction mixture and drives the radiolabelling to completion.
Experimental.
A series of comparative experiments has been carried out to generate radiolabelling data for a radioprotectant formulation of the present invention vs the optimised one for 99mTc-TRODAT-1 described in the prior art. The present formulation demonstrates significant advantages over the prior art published TRODAT-1 formulation [Choi et al, Nucl. Med. Biol., 26, p. 461-466 (1999)].
All studies were carried out using lyophilised kit formulations. The kit vials were prepared under the same conditions but to different formulations—that of the present invention (Formulation P), and that of the prior art Choi et al formulation (Formulation Q). All vials were stored upright, in the dark at −20° C. until required for use. The 99mTc-pertechnetate eluate was obtained from Amertec II™ generators (for Examples 3 to 7), Drytec™ generators (for Examples 8 and 9), and Ultra Technekow™ and Elutec™ generators (for Example 8). The kit formulations are given in Table 1:
*Formulated as the trifluoroacetic acid salt
The most significant difference is that Formulation P contains a radioprotectant (sodium ascorbate), whereas Formulation Q does not.
Unless otherwise stated, all test items were radiolabelled and analysed in the same way. Thus, once equilibrated to ambient temperature, each kit was reconstituted with 2 ml of sodium 99mTc-pertechnetate solution containing 1.5 GBq (±10%) of radioactivity (1.5 GBq corresponds to 2 patient doses of 740 MBq), heated in a boiling water bath for 20 minutes and then cooled for 10 minutes before RCP analysis by HPLC and ITLC. Time of analysis is reported as ‘post-preparation’.
RCP Determination
HPLC:
Column: Xterra RP18 3.5 μm 3.0×50 mm.
Loop size: 50 μL,
Mobile Phase: 60% 50 mM Ammonium Acetate pH 7: 40% Acetonitrile
Flow rate: 0.5 ml/min.
ITLC:
Pall ITLC-SG sheet (part number 61886) cut into strips 20 mm×200 mm and eluted with 50% 1M Ammonium Acetate: 50% Acetone
RCP calculation:
RCP=(A+B)*((100-RHT)/100)
A=species A from HPLC, B=species B from HPLC, RHT=reduced hydrolysed technetium, species at origin from ITLC.
Species A and Species B are the diastereomers of 99mTc-TRODAT-1 as described by Meegalla et al (J. Med. Chem., 41, 428-436 (1998)].
Kits of formulations P and Q (as described in Example 1) were reconstituted, heated and analysed in exactly the same way, as per Example 2. Four generator elution conditions were investigated:
RCP determinations were carried out at two post-preparation time points. The results are shown in Table 2:
A freshly prepared, nitrogen purged solution of sodium p-ABA (sodium para-aminobenzoate was added to a kit of Formulation Q. Radiolabelling of the kits was performed by first adding the radioprotectant (0.2 ml), followed by the immediate addition of pertechnetate solution (1 GBq in 1.8 ml). The kits were then heated as described in Example 2. The results are given in Table 3:
A comparison of the effects of reconstitution volume on the radiolabelling performance of the P and Q Formulations was carried out. Kits of both formulations were reconstituted, heated and analysed as per Examples 1 and 2. The radioactive concentration of eluate used to reconstitute the kits was kept constant at 1.5 GBq/ml for each test item and eluate reconstitution volumes of 2, 4 and 6 ml were investigated.
Two vials each of Formulations P and Q were reconstituted in the standard manner with 2 ml of sodium pertechnetate solution containing 1.5 GBq of radioactivity. The vials were subjected to an autoclave cycle of 121° C. for 25 minutes. The total duration of the cycle (heating and cooling) was about 120 minutes. As a result the earliest RCP analysis time point acquired was 2 h 20 min post-reconstitution. Analysis times are reported in Table 5 as post-reconstitution (as opposed to post-preparation) time points:
Kit formulation P was reconstituted to give 99mTc-TRODAT-1 as described in Examples 1 and 2, which was the Test Item. The radiochemical purity (RCP) of the Test Item was 92% pre-injection, falling to 91% by the post-injection analysis time point. At the pre- and post-injection analysis time points, there were a low percentage of lipophilic (approximately 2%) and hydrophilic (approximately 6%) radiolabelled species present. The ratio of the A and diastereomers (46:54) remained constant at the pre- and post-injection analysis time points. Experiments were performed at 6 predetermined time points (2 and 20 minutes, 1, 2, 4 and 7 hours) post injection (p.i.) of the Test Item in normal male Wistar rats (180 to 220 g). Animals were anaesthetised with Halothane (6% in oxygen), injected with 0.1 ml (500 MBq/ml) Test Item, sacrificed, dissected and the samples assayed for radioactivity. A comparative study was carried out using a 99mTc-TRODAT-1 kit preparation corresponding to Formulation P, but lacking the ascorbate radioprotectant.
The percentage of the injected dose present in the blood was approximately three-fold lower for Formulation P at all the time points post-injection. The uptake and retention of radioactivity into the brain was similar at all except the 20 minute pi time point for both formulations. By 20 minutes pi, approximately 0.45% of the injected dose (id) was retained within the brain after administration of the radioprotectant formulation, relative to 0.29% id after administration for the unstabilised formulation. This difference in brain uptake was reflected in the elevated percentage injected dose present in the brain regions at 20 minutes pi when expressed per gram of brain region.
The main difference observed was the elevated selective retention in the striatum after administration of the radioprotectant formulation, which peaked at 2.31±0.31 after 2 hours pi and stayed at this peak level out to 4 hours pi (2.42±0.80). In comparison, after administration of the unstabilised formulation the selective retention in the striatum was 1.74±0.96 by 2 hours pi and 0.76±0.30 by 4 hours pi.
Kits of Formulation P of the present invention were reconstituted with 2 GBq of 99mTc in 2.5 ml of eluate from 3 different European 99mTc generators, heated and cooled as per Example 2 and then stored at either 5° C. or 25° C. and analysed at 0, 4 and 6 hours post-preparation. Tests were carried out on kits reconstituted with both fresh and aged eluate from 99mTc-generators. The results are shown in Table 7 (overleaf):
The kit of Formulation P was reconstituted in two steps. First 1.5 ml of 99mTc sodium pertechnetate solution containing 2 GBq of radioactivity was added to the kit vial. Phosphate buffer solution of pH 7.4 (1 ml) was then added immediately, and the RCP determined 30 minutes after the addition of the pertechnetate solution. The results are given in Table 6:
| Number | Date | Country | Kind |
|---|---|---|---|
| 03002809.6 | Feb 2003 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB04/00443 | 2/9/2004 | WO | 10/12/2006 |
