IMAGING TUBERCULOSIS WITH PYRAZINAMIDE CONTRAST AGENTS
Technical Field of the Invention
The present invention relates to in vivo imaging and more particularly to in
vivo imaging to detect the presence of tuberculosis. A novel in vivo imaging
agent is provided by the present invention which has properties that make it
advantageous compared with similar known in vivo imaging agents.
Description of Related Art
Pulmonary tuberculosis (TB) is an airborne infection caused by
Mycobacterium tuberculosis (MTB) that causes high mortality and morbidity,
particularly in developing countries (Dye et al JAMA 1999; 282(7): 677-686). A
recent factsheet produced by the World Health Organisation reported that the
number of new cases of TB continues to increase each year in South-East Asia,
the Eastern Mediterranean, and in Africa
(http://www.who.int/mediacentre/factsheets/fs104/en/print.html ). The
emergence of drug-resistant strains of MTB has resulted in efforts to identify
new agents to treat an otherwise incurable disease.
Accurate and prompt diagnosis is important in order to control the infection
and also to ensure the appropriate therapy for infected patients. Currently, a
definitive diagnosis of TB requires culture of MTB from a sample taken from a
patient. Patients with clear signs and symptoms of pulmonary disease with a
sputum smear-positive result present no problems to diagnose. However, there
can be difficulty culturing the slow-growing MTB organism in the laboratory.
Furthermore the emergence of HIV has resulted in a decreased likelihood of
sputum smear positivity and an increase in non-respiratory disease, such that
ease of diagnosis is more difficult in these cases (see reviews by Jeong &Lee Am
J Roent 2008; 191: 834-844; Davies &Pai Int J Tuberc Lung Dis 2008; 12(11):
1226-1234; and, Lange &Mori Respirology 2010; 15: 220-240).
In vivo imaging methods can be useful in the diagnosis of TB. Chest x-ray is a
widely-used in vivo imaging method for screening, diagnosis and treatment
monitoring in patients with known or suspected TB. Chest computed
tomography (CT) is more sensitive than conventional x-ray and may be applied
to identify early parenchymal lesions or mediastinal lymph node enlargements
and to determine disease activity in tuberculosis (Lee &Im A R 1995; 164(6):
1361-1367). Nuclear imaging methods have also been reported for diagnosis
and treatment monitoring of TB. The positron-emission tomography (PET)
tracer F-fluorodeoxyglucose ([ F]FDG) has been proposed as useful in the
diagnosis of disease activity and therapy monitoring in patients with TB
(Demura et al Eur J Nuc Med Mol Imag 2009; 36: 632-639).
Pyrazinamide (PZA) is a well-known and important first-line drug used in the
treatment of TB. The mechanism of action of PZA is poorly understood,
although experimental evidence suggests that PZA diffuses into MTB and is
converted into pyrazinoic acid by pyrazinamidase causing pH imbalance in the
bacteria followed by disruption of the bacterial cell wall (Zhang &Mitchison
2003 Int J Tuberc Lung Dis; 7(1): 6-21). There are some known teachings
relating to labelled versions of PZA.
US 2008/0107598 teaches a metal chelate-targeting ligand conjugate wherein
the targeting ligand can be selected from a broad variety of different
pharmaceutical agents, including antimicrobial agents. Amongst a wide
selection of other antimicrobial agents, PZA is disclosed as a suitable targeting
ligand. However, there is no specific teaching in US 2008/ 0107598 as to how
any specific metal chelate-PZA conjugate may be obtained.
Chohan et al (Metal-based Drugs 1998; 5(6): 347-354) teach a PZA derivative
complexed with a metal selected from cobalt(II), copper(II), nickel(II) and
zinc(II). The PZA derivative is disclosed as having the following structure:
wherein X can be O, S or NH. The metal complexes are disclosed as being
biologically active against one or more bacterial species, with the metal
complexes being more active than the uncomplexed ligands. The ligands were
not tested against MTB, and there is no disclosure in Chohan et al relating to
diagnosis or in vivo imaging of TB.
Liu et al (J Med Chem 2010; 53: 2882-2891) disclose C-labelled tuberculosis
therapeutics and their use in investigating the biodistribution of these
therapeutics using positron-emission tomography (PET) imaging. C-labelled
PZA is disclosed (wherein the asterisk denotes the position of the Clabel):
The half life of Cis relatively short (20.4 minutes) and its production is based
on the nuclear reaction N , ) on a nitrogen target that requires use of a
cyclotron. The production of C-labelled PET tracers therefore requires
proximity to a cyclotron facility, which limits the geographical availability of
these tracers. This is particularly true in some countries having a high
incidence of TB such as India, China and African countries, where much of the
population lives in remote locations. In any case, a cyclotron facility is
expensive to set up and therefore not widely available. For these reasons, C
PET tracers are not ideal in the context of diagnosing TB.
KR 2007/0092536 discloses radiolabeled PZA for imaging tuberculosis,
teaching that the radiolabel may be selected from T C n 131^ n
2 31.
However, a specific structure is provided only in respect of radioiodinated PZA:
123I has a longer half-life than C of 13.22 hours, but like Crequires a cyclotron
for its production and therefore needs to be transported from a cyclotron
facility to its intended site of use. This can be logistically difficult in the case of
remote locations and as for this reason 2 l is not an ideal tracer for an in vivo
TB imaging agent.
US 5955053 teaches chelators which comprise a PZA moiety, e.g. L-CEPZ,
which is of the following structure:
When a metal ion suitable for in vivo imaging such as T c S coordinated by
the above structure, one of the nitrogens of the PZA moiety is involved in the
coordination, as is set out in the general formula provided in claim 1 of US
5955053. As such, the desired biological activity of the PZA moiety towards
MTB is likely to be negatively impacted. Indeed there is no mention in US
5955053 that these compounds are of use for use in the diagnosis of TB.
There is therefore scope for improved in vivo imaging agents useful in the
diagnosis of TB.
Summary of the Invention
The present invention provides novel in vivo imaging agents useful for
detecting the presence of mycobacteria using in vivo imaging methods. The in
vivo imaging agents of the invention are radiolabeled derivatives of unlabelled
small molecules that are known to have good properties as treatment agents for
mycobacterial infections. The in vivo imaging agents of the present invention
have similar or improved biological properties to the related unlabelled
compounds. Also provided by the present invention is a precursor compound
useful in the synthesis of the in vivo imaging agents of the invention, and a
method to obtain the in vivo imaging agent of the invention using said
precursor compound. Methods of in vivo imaging and diagnosis in which the
in vivo imaging agent of the invention finds use are also provided.
Detailed Description of the Invention
In Vivo Imaging Agent
In one aspect, the present invention provides an in vivo imaging agent of
Formula I :
wherein:
X1represents a direct bond or a linker -(L)n- wherein each Lis independently -
C(=0)-, -CRV, -CR'=CR'-, -CºC-, -CR' C0 -, -C0 CRV, -NR'-, -NR'C(=0)-, -
C(=0)NR'-, -NR'(C=0)NR'-, -NR'(C=S)NR'-, -S0 2NR'-, -NR'S0 -, -CR'2-0-
CR'2-, -CR'2-S-CR' -, -CR'2-NR'-CR'2-, wherein eachR' group is independently
H or Ci-6 alkyl;
Ch -M1 is a metal ion complex wherein Ch1 is a chelating agent and M1 is a
metal ion suitable for in vivo imaging.
An "in vivo imaging agent " in the context of the present invention is a labelled
compound that facilitates the generation of an image in an in vivo imaging
procedure. The term "in vivo imaging" as used herein refers to those
techniques that noninvasively produce images of all or part of the internal
aspect of a subject.
The term "linker " as used herein refers to a bivalent chain of between 10 and 100
atoms, preferably between 10 and 50 atoms. Specifically excluded are linkers
wherein 2 or more carbonyl groups are linked together, or wherein 2 or more
heteroatoms are linked together. The skilled person would understand that these
are either not chemically feasible, or are too reactive or unstable to be suitable for
use in the field of the present invention. Where X1 of Formula I represents the
linker -(L)n-, preferred L groups are selected from: -C(=0)-; -CH2-; -NH-; -
NHC(=0)-; -C(=0)NH-; and, -CH2-0-CH 2-. Aparticularly preferred linker group
is of the formula -(CH2)m- wherein m is 1-6, preferably 2-4.
The term "metal complex" is taken to mean a coordination complex wherein a
metal ion is bonded to a surrounding array of molecules or anions, which in the
present invention are comprised in the chelating agent Ch . It is strongly preferred
that the metal complex of the present invention is "resistant to transchelation " i.e.
does not readily undergo ligand exchange with other potentially competing ligands
for the metal coordination sites. Potentially competing ligands include the in vivo
imaging agent itself plus other excipients in the preparation in vitro (e.g.
radioprotectants or antimicrobial preservatives used in the preparation), or
endogenous compounds in vivo (e.g. glutathione, transferrin or plasma proteins).
A "chelating agent" is an organic compound capable of forming coordinate bonds
with a metal ion through two or more donor atoms. In a typical chelating agent
suitable for the present invention 2-6, and preferably 2-4, metal donor atoms are
arranged such that 5- or 6-membered chelate rings result (by having a noncoordinating
backbone of either carbon atoms or non-coordinating heteroatoms
linking the metal donor atoms). Examples of donor atom types which bind well to
metal ions as part of chelating agents are: amines, thiols, amides, oximes, and
phosphines. Other arrangements are also envisaged, such as by means of
c(CO)3 radiochemistry when the metal suitable for in vivo imaging is .
Examples of suitable chelating agents for technetium which form metal complexes
resistant to transchelation include, but are not limited to:
(a) diaminedioximes;
(b)N3S ligands having a thioltriamide donor set;
(c) N2S2 ligands having a diaminedithiol donor set;
(d)N4 ligands which are open chain or macrocyclic ligands having a
tetramine, amidetriamine or diamidediamine donor set; or,
(e) N2O2 ligands having a diaminediphenol donor set.
The above described chelating agents are particularly suitable wherein the metal
ion suitable for in vivo imaging is technetium e.g. 4m c o r T C a n a e described
more fully by Jurisson et al (Chem Rev 1999; 99: 2205-2218). These chelating
agents are also useful for other metals, such as copper ( C or Cu), vanadium
(e.g. V), iron (eg. 52Fe), or cobalt (e.g. Co). Other suitable chelating agents are
described in Sandoz WO 91/01144, which are particularly suitable for indium,
yttrium and gadolinium. Examples of such suitable chelating agents include
i,4,7,io-tetraazacyclododecane-i,4,7,io-tetraacetic acid (DOTA) and
diethylenetriaminepentaacetic acid (DTPA). Non-ionic (i.e. neutral) metal
complexes of gadolinium are known and are described in US 4885363.
Furthermore as highlighted above, where the radioactive metal ion is , the
radioactive metal complex may also be formed by means of mTc(CO)3
radiochemistry, as described more fullyby Schibli in Chapter 2.2 of "Technetium-
99m Pharmaceuticals: Preparation and Quality Control in Nuclear Medicine (2007
Springer; Zolle, Ed.). Abenefit of this chemistry is that it can further reduce the
likelihood of non-specific binding of the 9¥ to the pyrazinamide active moiety.
The radioactive metal complex using this chemistry would therefore take the form
of a tridentate chelate linked to mTc(CO)3.
It is envisaged that the role of the linker group is to distance the relatively bulky
metal complex, which results upon metal coordination, from the active site of the
pyrazinamide so that e.g. substrate binding 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 technetium complex of the conjugate. Thus, e.g. the introduction
of ether groups in the linker will help to minimise plasma protein binding, or the
use of polymeric linker groups such as polyalkyleneglycol, especially
polyethyleneglycol (PEG) can help to prolong the lifetime of the in vivo imaging
agent in the blood in vivo. It is strongly preferred that the pyrazinamide is bound
to the chelator in such a way that the linkage does not undergo facile metabolism
in blood. That is because such metabolism would result in the imaging metal
complex being cleaved offbefore the in vivo imaging agent of the invention reaches
the desired in vivo target site.
A "metal ion suitable for in vivo imaging " means a metal ion that may be
detected externally by an in vivo imaging technique following administration to
a subject. Preferably, said metal ion suitable for in vivo imaging of Formula I is
either a radioactive metal ion or a paramagnetic metal ion. When the metal ion
is a radioactive metal ion it may be a gamma-emitting radioactive metal ion or a
positron-emitting radioactive metal ion. Apreferred gamma-emitting
radioactive metal ion is selected from 9 c , in, 3 In, and Ga, with 99mT c
being most preferred. A preferred positron-emitting radioactive metal ion is
selected from u, , 2Fe, o, 94mT c and Ga. When the metal ion is a
paramagnetic metal ion it is preferably is selected from Gd(III), Mn(II), Cu(II),
Cr(III), Fe(III), Co(II), Er(II), Ni(II), Eu(III) and Dy(III), with Gd(III) being
most preferred. A most preferred metal ion suitable for in vivo imaging of the
present invention is a gamma-emitting radioactive metal ion, an in particular
A preliminary in vitro assessment was carried out on a rhenium derivative of an
imaging agent of the invention (see Example 5). The data obtained suggests
that the rhenium complex is at least as active as PZA, and probably has more
favourable activity than PZA.
Pharmaceutical Composition
Preferably, the in vivo imaging agent of the invention is provided as a
pharmaceutical composition together with a pharmaceutically acceptable
carrier. The "pharmaceutically acceptable carrier " is a fluid, especially a liquid,
in which the in vivo imaging agent is suspended or dissolved, such that the
pharmaceutical composition is physiologically tolerable, i.e. can be
administered to the mammalian body without toxicity or undue discomfort.
The pharmaceutically acceptable 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
pharmaceutically acceptable carrier may also comprise biocompatible organic
solvents such as ethanol. Such organic solvents are useful to solubilise more
lipophilic compounds or formulations. Preferably the biocompatible carrier
medium is pyrogen-free water for injection, isotonic saline or an aqueous
ethanol solution. The pH of the biocompatible carrier medium for intravenous
injection is suitably in the range 4.0 to 10.5.
The pharmaceutical composition of the invention is suitably supplied in 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 cm volume) which contains multiple patient doses,
whereby single patient doses can thus be withdrawn into clinical grade syringes
at various time intervals during the viable lifetime of the preparation to suit the
clinical situation. Pre-filled syringes are designed to contain a single human
dose, or "unit dose" and are therefore preferably a disposable or other syringe
suitable for clinical use. Where the pharmaceutical composition is a
radiopharmaceutical composition (i.e. when the metal ion is a gamma- or a
positron-emitter), 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.
The pharmaceutical composition of the present invention may be prepared
from a kit, as is described below in an additional aspect of the invention.
Alternatively, it may be prepared under aseptic manufacture conditions to give
the desired sterile product. The pharmaceutical composition 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 pharmaceutical composition of the present
invention is prepared from a kit.
Precursor Compound
In another aspect, the present invention provides a precursor compound of
Formula II:
wherein X2 is as defined herein for X1, and Ch2 is as defined herein for Ch1.
The suitable and preferred embodiments of X1and Ch1are equally applicable to
X2 and Ch2.
A"precursor compound " comprises a derivative of the in vivo imaging agent of
Formula I wherein the metal ion is not complexed by the chelating agent. Such
precursor compounds are typically designed so that chemical reaction with a
convenient chemical form of the metal ion occurs site-specifically; can be
conducted in the minimum number of steps (ideally a single step); and without
the need for significant purification (ideally no further purification), to give the
desired in vivo imaging agent. Such precursor compounds are synthetic and
can conveniently be obtained in good chemical purity. In order to facilitate sitespecific
reaction, the precursor compound of the invention may optionally
comprise a suitable protecting group.
By the term "protecting group" is meant a group which inhibits or suppresses
undesirable chemical reactions, but which is designed to be sufficiently reactive
that it maybe cleaved from the functional group in question to obtain the
desired product under mild enough conditions that do not modify the rest of
the molecule. Protecting groups are well known to those skilled in the art and
are described in 'Protective Groups in Organic Synthesis', Theorodora W.
Greene and Peter G. M. Wuts, (Fourth Edition, John Wiley&Sons, 2007).
Precursor compounds of the present invention may be obtained in a
straightforward manner starting by coupling a commercially-available pyrazine
derivative with a suitable derivative of the desired chelate e.g. as illustrated
below in Scheme 1:
wherein and Ch are as suitably and preferably defined herein for X1and Ch1,
respectively. Reaction conditions suitable for the coupling reaction are wellknown
to the person skilled in the field of organic chemistry (see March's
Advanced Organic Chemistry 6th Edition; Wiley: Smith &March, Eds.).
The precursor compound of the invention is ideally provided in sterile,
apyrogenic form. The precursor compound can accordingly be used for the
preparation of the above-described pharmaceutical composition of the
invention, and for inclusion as a component in a kit for the preparation of such
a pharmaceutical composition, as described in greater detail below.
Method of Preparation
In a further aspect, the present invention provides a method for the preparation
of the in vivo imaging agent as defined herein wherein said method comprises:
(i) providing a precursor compound of Formula II as defined herein;
(ii) reacting said precursor compound with a source of a metal ion suitable for
in vivo imaging wherein said metal ion is as defined herein.
The step of "reacting " the precursor compound with the source of a metal ion
suitable for in vivo imaging involves bringing the two reactants together under
reaction conditions suitable for formation of the desired in vivo imaging agent
in as high a radiochemical yield (RCY) as possible. Synthetic routes for
obtaining particular in vivo imaging agents of the present invention are
presented in the experimental section below.
The term "source of a metal ion " refers to the metal ion in a chemical form that
will react in a single step with the precursor compound of the invention to form
the metal chelate -Ch -M1of Formula I. For example, when the metal ion is
technetium, the usual source of technetium for labelling is pertechnetate, i.e.
Tc0 4 , which is technetium in the Tc(VII) oxidation state. Pertechnetate itself
does not readily form complexes, hence the preparation of technetium
complexes usually requires the addition of a suitable reducing agent such as
stannous ion to facilitate complexation by reducing the oxidation state of the
technetium to the lower oxidation states, usually Tc(I) to Tc(V). The solvent
may be organic or aqueous, or mixtures thereof. When the solvent comprises
an organic solvent, the organic solvent is preferably a biocompatible solvent,
such as ethanol or dimethylsulfoxide (DMSO). Preferably the solvent is
aqueous, and is most preferably isotonic saline. Where it is desired to label
with Gd(III), Gd20 3 can be reacted with the precursor compound of the
invention. The person skilled in the art of in vivo imaging agents will be
familiar with other sources of metal ion that are suitable for application in the
present invention. For more detail, the reader is referred to the "Handbook of
Radiopharmaceuticals" (2003; Wiley: Welch and Redvanly, Eds), and to
"Contrast Agents: Magnetic Resonance Imaging" (2002; Springer-Verlag:
Krause, Ed).
The suitable and preferred embodiments of the in vivo imaging agent of the
invention and precursor compound of the invention as applied to this aspect of the
invention are as defined above.
Kit
In a yet further aspect, the present invention provides a kit for carrying out the
above-described method for the preparation of the in vivo imaging agent of the
invention, and preferably for preparing the pharmaceutical composition of the
invention, wherein said kit comprises a vial containing the precursor compound of
the invention as defined herein.
The precursor compound of the invention is preferably provided in the kit of the
invention in sterile non-pyrogenic form, so that reaction with a sterile source of a
suitable metal ion gives the desired pharmaceutical with the minimum number of
manipulations. Such considerations are particularly important in the case of
radiopharmaceuticals, in particular for radiopharmaceuticals where the
radioisotope has a relatively short half-life, for ease of handling and hence reduced
radiation dose for the radiopharmacist. Hence, the reaction medium for
reconstitution of such kits is preferably a "pharmaceutically acceptable carrier"as
defined above, and is most preferably aqueous.
Suitable kit containers comprise a sealed container which permits maintenance of
sterile integrity and/ or radioactive safety, plus optionally an inert headspace gas
(e.g. nitrogen or argon), whilst permitting addition and withdrawal of solutions by
syringe. Apreferred such container is a septum-sealed vial, wherein the gas-tight
closure is crimped on with an overseal (typically of aluminium). Such containers
have the additional advantage that the closure can withstand vacuum if desired
e.g. to change the headspace gas or degas solutions.
Preferred aspects of the precursor compound of the invention when employed in
the kit are as herein described. The precursor compound for use in the kit maybe
employed under aseptic manufacture conditions to give the desired sterile, nonpyrogenic
material. The precursor compound may also be employed under nonsterile
conditions, followedby terminal sterilisation using e.g. gamma-irradiation,
autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). Preferably,
the precursor compound is employed in sterile, non-pyrogenic form. Most
preferably the sterile, non-pyrogenic precursor compound is employed in the
sealed container as described above.
For T C e kit is preferably lyophilised and is designed tobe reconstituted with
sterile 99 e e netate (TcO4-) from a 9 radioisotope generator to give a
solution suitable for human administration without further manipulation.
Suitable kits comprise a container (e.g. a septum-sealed vial) containing the
uncomplexed chelating agent, together with a pharmaceutically acceptable
reducing agent such as sodium dithionite, sodium bisulphite, ascorbic acid,
formamidine sulphinic acid, stannous ion, Fe(II) or Cu(I); together with at least
one salt of a weak organic acid with a pharmaceutically acceptable cation. By the
term "pharmaceutically acceptable 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 pharmaceutically acceptable cations include: the alkali metals sodium or
potassium; the alkaline earth metals calcium and magnesium; and the ammonium
ion. Preferred pharmaceutically acceptable cations are sodium and potassium,
most preferably sodium.
The kits for preparation of 99 T c imaging agents may optionally further comprise a
second, weak organic acid or salt thereof with a biocompatible cation, which
functions as a transchelator. The transchelator is a compound which reacts
rapidly to form a weak complex with technetium, then is displaced by the chelator
of the kit. 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 the weak organic acids and salts
thereof described above, preferably tartrates, gluconates, glucoheptonates,
benzoates, or phosphonates, preferably phosphonates, most especially
diphosphonates. A preferred such transchelator is MDP, ie.
methylenediphosphonic acid, or a salt thereof with a biocompatible cation.
Also in relation to 9¥ kits, the kit may optionally contain a non-radioactive
metal complex of the chelator which, upon addition of the technetium, undergoes
transmetallation (i.e. ligand exchange) giving the desired product. Suitable such
complexes for transmetallation are copper or zinc complexes.
The pharmaceutically acceptable reducing agent used in the T c imaging agent
kit is preferably a stannous salt such as stannous chloride, stannous fluoride or
stannous tartrate, and may be in either anhydrous or hydrated form. The stannous
salt is preferably stannous chloride or stannous fluoride.
The kits may optionally further comprise additional components such as a
radioprotectant, antimicrobial preservative, pH-adjusting agent or filler.
By the term "radioprotectant " is meant a compound which inhibits degradation
reactions, such as redox processes, by trapping highly-reactive free radicals, such
as oxygen-containing free radicals arising from the radiolysis of water. The
radioprotectants of the present invention are suitably chosen from: ascorbic acid,
para-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e. 2 ,5-
dihydroxybenzoic acid) and salts thereof with a pharmaceutically acceptable
cation. The "pharmaceutically acceptable cation " and preferred embodiments
thereof are as described above.
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 dose. 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 post-reconstitution. The antimicrobial preservative
may, however, also optionally be used to inhibit the growth of potentially harmful
micro-organisms in one or more components of the kit prior to reconstitution.
Suitable antimicrobial preservative(s) include: the parabens, i.e. methyl, ethyl,
- 5 -
propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol;
cetrimide and thiomersal. Preferred antimicrobial preservative(s) are the
parabens.
The term "pH-adjusting agent " means a compound or mixture of compounds
useful to ensure that the pH of the 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 precursor compound 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.
Clinical Use
The in vivo imaging agent of the invention finds use in the diagnosis and
monitoring of infection caused by Mycobacteria tuberculosis (MTB).
Accordingly, the present invention provides a method to determine the location
and/or amount of MTB present in a subject, wherein said method comprises:
(a) administering the in vivo imaging agent as defined herein to said subject in
an amount suitable for in vivo imaging;
(b) allowing the administered in vivo imaging agent to bind to any MTB
present in said subject;
(c) detecting by a suitable in vivo imaging procedure signals emitted by said
metal ion suitable for in vivo imaging comprised in said in vivo imaging
agent;
(d) generating an image representative of the location and/ or amount of said
signals; and,
(e) attributing the location and/ or amount of said signals to the location
and/or amount of MTB present in said subject.
"Administering " the in vivo imaging agent is preferably carried out parenterally,
and most preferably intravenously. The intravenous route represents the most
efficient way to deliver the in vivo imaging agent throughout the body of the
subject, and also does not represent a substantial physical intervention on the
body of the subject. By the term "substantial " is meant an intervention which
requires professional medical expertise to be carried out, or which entails a
substantial health risk even when carried out with the required professional care
and expertise. The in vivo imaging agent of the invention is preferably
administered as the pharmaceutical composition of the invention, as defined
herein. The in vivo imaging method of the invention can also be understood as
comprising the above-defmed steps (b)-(e) carried out on a subject towhom the in
vivo imaging agent of the invention has been pre-administered.
Following the administering step and preceding the detecting step, the in vivo
imaging agent is allowed to bind to any MTB present within said subject. For
example, when the subject is an intact mammal, the in vivo imaging agent will
dynamically move through the mammal's body, coming into contact with various
tissues therein. Once the in vivo imaging agent comes into contact with MTB, the
two entities bind such that clearance of the in vivo imaging agent from tissue in
which MTB is present takes longer than from tissue without any, or less, MTB
present. A certain point in time will be reached when detection of in vivo imaging
agent specifically bound to MTB is enabled as a result of the ratio between in vivo
imaging agent bound to tissue with MTB versus that bound in tissue without any
MTB. This is the optimal time for the detecting step to be carried out.
The "detecting " step of the method of the invention involves detection of signals
emitted by the radioisotope by means of a detector sensitive to said signals. This
detection step can also be understood as the acquisition of signal data. For
gamma-emitting metal ions, single-photon emission tomography (SPECT) is used;
for positron-emitting metal ions, positron-emission tomography (PET) is used;
and, for paramagnetic metal ions, magnetic resonance imaging (MRI) is used.
The "generating " step of the method of the invention is carried out by a computer
which applies a reconstruction algorithm to the acquired signal data to yield a
dataset. This dataset is then manipulated to generate images showing the location
and/or amount of signals emitted by the metal ion which is comprised in the in
vivo imaging agent. The signals emitted directly correlate with the presence of
MTB such that the "determining " step can be made by evaluating the generated
image.
The "subject " of the invention can be any human or animal subject. Preferably the
subject of the invention is a mammal. Most preferably, said subject is an intact
mammalian body in vivo. In an especially preferred embodiment, the subject of
the invention is a human. The in vivo imaging method may be used in subjects
known or suspected to have TB caused by MTB. The in vivo imaging method of
the invention may be carried out repeatedly during the course of a treatment
regimen for said subject, said regimen comprising administration of a drug to
combat TB caused by MTB.
In another aspect, the present invention provides the pharmaceutical composition
of the invention for use in a method to determine the location and/ or amount of
MTB present in a subject, wherein said method is as previously defined herein.
Furthermore, the present invention provides for use of the in vivo imaging agent of
the invention in the manufacture of the pharmaceutical composition of the
invention for use in a method to determine the location and/ or amount of MTB
present in a subject, wherein said method is as previously defined herein.
Brief Description of the Examples
Example i describes the synthesis of a starting material for the preparation of a
precursor of the invention.
-i8-
Example 2 describes how a particular precursor compound of the invention was
obtained.
Example 3 describes how the precursor compound from Example 1was labelled
with rhenium.
Example 4 describes how the precursor compound of Example 1can be
radiolabelled with T c 0 obtain an in vivo imaging agent of the present
invention.
Example 5 describes the in vitro assessment of a rhenium derivative of a
compound of the invention.
List of Abbreviations used in the Examples
DCM dichloromethane
EDTA ethylenediaminetetraacetic acid
HPLC high-performance liquid chromatography
MeOH methanol
mg milligram(s)
mL millilitre(s)
mmol millimole(s)
MS mass spectrometry
MSA methanesulfonic acid
NMR nuclear magnetic resonance
RT room temperature
THF tetrahydrofuran
Examples
Example 1: N'-[2-C4-Methoxy-benzylsulfariyl)-ethyl]-N,-{2-[4-
rnethoxy-benzylsulfanyD-ethylarnino ]-ethyl}-propan-i,^-diamine
ill
i(a) Synthesis of 2-(4-Methoxy-benzylsulfanyl)-ethylamine
Sodium (1.50 g, 65.2 mmol) was added in small portions (about 100 mgs) under a
blanket of nitrogen to vigorously stirred methanol (50 ml HPLC grade). When the
effervescence had ceased 2-aminoethanethiol hydrochloride (3.6 g 31.6 mmol) was
added in one portion precipitating sodium chloride from solution pmethoxybenzyl
chloride (5.0 g, 32.0 mmol) was added in one portion and the
mixture heated under reflux at 75-8o°C for 30 minutes. After cooling the solid was
removed by filtration and the filter cake washed with methanol (15 ml). The
organic extracts were combined and volatiles removed under reduced pressure (10
mm Hg, 40°C) to leave a colourless oil which contained sodium chloride crystals.
This residue was redissolved in DCM (25 ml), extracted with water (25 ml x 3),
dried (MgS04), filtered and solvent removed to leave a colourless oil. This material
was about (95 %) of desired compound and not purified any further. Yield 5.4 g
(87 %).
Ή -NMR (CDCI3) d 1.25 (2H, bs, NH ), 2.42 (2H, t, J = 7 Hz , SCH ), 2.73 (2H, t, J
= 7 Hz, CH N), 2.73 (2H, s, SCH Ar), 3 -74 (3H, s, OCH3), 6.77 (2H, d, J = 7 Hz, CH
x 2), 7.15 (2H, d, J = 7 Hz, CH x 2).
1 b ) Synthesis ofN- {(4-Methoxy-benzylsulfanyl)-ethyl}-2-chloroacetamide
Chloroacetyl chloride (630 mg, 5.6 mmol) in dry DCM (5 ml) was added dropwise
over 5 minutes with stirring to an ice bath cooled (o-5°C) solution of 2-(4-
Methoxy-benzylsulfanyl)-ethylamine (1.0 g, 5 mmol) and triethylamine (600 mg,
5.9 mmol) in dry DCM (20 ml). After addition the cooling bath was removed and
stirring continued for 30 minutes. The solution was extracted with water (50 ml x
2), dried (MgS04), filtered and solvent evaporated under reduced pressure toleave
a fawn coloured solid. Yield 1.30 g (95 %). This material required no further
purification.
Ή -NMR (CDCI3) : d 2.54 (2H, t, J = 7 Hz , SCH ), 3.39 (2H, t, J = 7 Hz, NCH ),
3-66 (2H, s, SCH Ar), 3.99 (3H, s, OCH3), 6.82(2H, d, J = 8 Hz, CH x 2), 6.90 (lH
,bs, NH), 7.21 (2H, d, J = 8 Hz, CH x 2)
i(c) Methyl s-[f4-Methoxy-benzylsufanyl)-ethylamino]propanoate
Methyl acrylate (440 mg 5.1 mmol) in methanol (1ml) was added in one portion to
a stirred solution of 2-(4-Methoxy-benzylsulfanyl)-ethylamine (1.0 g, 5 mmol) in
methanol (5 ml). The colourless solution was allowed to stir at RT for 2 hours.
Volatiles were removed by rotary evaporation to leave the product as colourless
viscous oil in about 95 %purity. Yield 1.35 g (96 %). An analytical sample was
obtained by chromatography over silica eluting with DCM/MeOH 98:2 . The
product was isolated as a colourless viscous oil r = 0.2.
Ή -NMR (CDCI3) d 1.67 (lH, bs, NH), 2.45 (2H, t, J = 7 Hz, CH C=0), 2.53 (2H, t, J
= 7 Hz, SCH2), 2.72 (2H, t, J = 7 Hz, NCH2), 2.81 (2H, t, J = 7 Hz, NCH2), 3.64
(2H, s, SCH2-C-N), 3.66 (3H, s, OCH3 ester), 3.75 (3H, s, OCH3 methoxy), 6.80
(2H, d, J = 8 Hz, CH x 2), 7.20 (2H, d, J = 8 Hz, CH x 2).
i(d) s-[2 -(4-Methoxybenzylsulfanyl)-ethylamino] propanamide
Methyl 3-[(4-Methoxy-benzylsufanyl)-ethylamino] propanoate (1.35 g4.8 mmol),
methanol (15 ml) and ammonia solution (25 ml) were stirred at RT for 16 hours.
Volatiles were removed under reduced pressure (10 mmHg, 50°C) to leave a
viscous oil which solidifies on standing. Yield 1.28 g (99 %). This material is about
95 %pure and can be used in the next step. An analytical sample was obtained
after purification by chromatography over silica eluting with DCM/Methanol 80 :
20. The product had a r =0.15 and was isolated as a white solid.
Ή -NMR (CDCI3 ) d 2.54 (2H, t, J = 7 Hz, CH C=0), 2.65 (2H, t, J = 7 Hz, SCH2-CN),
2.91 (2H, t, J = 7H z, NCH ), 3.00 (2H, t, J = 7 Hz, NCH ), 3.84 (2H, s,
SCHPh), 3.95 (3H, s, OCH3), 7.00 (2H, d, J = 8 Hz, CH x 2), 7.48 (2H, d, J = 8 Hz,
CH x 2)
lie) 3-f [2 -f4-Methoxy-benzylsulfanyl)-ethyl]-{[2 -f4-methoxy-benzylsulfanyl)-
ethylcarbamoylj-methyl }-amino )propionamide
3-[2-(4-Methoxybenzylsulfanyl)-ethylamino] propanamide (670 mgs, 2.5 mmol),
N-{(4-Methoxy-benzylsulfanyl)-ethyl}-2-chloroacetamide (680 mgs, 2.5 mmol),
trimethylamine ( 300 mgs 3mmol) and acetonitrile (smls) were heated under
reflux at 70°C for 16 hours. The solvent was removed under reduced pressure to
leave an orange/brown residue which was redissolved in DCM (25 ml), extracted
with water (25 ml x 2), dried (MgS04), filtered and solvent evaporated by rotary
evaporation. The residue was purified over silica eluting with DCM/Methanol 95:5
(rf =0.15) to give a colourless viscous oil (Yield 450 mg 36%).
Ή -NMR (CDCI3) d 2.27 (2H, t, J = Hz, CH C0), 2.51 (2H, t, J = 7 Hz, SCH ), 2.55
(2H, t, J = 7 Hz, SCH ), 2.62 (2H, t, J = 7 Hz, NCH ), 2.76 (2H, t, J = 7 Hz, NCH2),
3·04( 2H, s, NCH2C0), 3-39 (2H, q, J = 6 Hz, H2CNH), 3.62 (2H, s, SCH2Ph), 3.65
((2H, s, SCH2Ph), 3.76 (6H, s, OMe x 2), 5.52 (lH bs, NH), 5.97 (lH, bs, NH), 6.81
(4H, bd, J = 8 Hz, CH x 4), 7.18 (2H. d, J = 8 Hz, CH x 2), 7.20 (2H, d, J = 8 Hz,
CH x 2), 7.70 (lH, bt, J = 6 Hz, HNCO)
i(f) N'-[2-(4-Methoxy-benzylmlfanyl)-ethyl]-N'-{2-[4-metto
ben2Mlsulfanyl)-ethylaminol-ethyl}-OroOan-i,3-diamine (1)
1.0M Borane in THF (12 mis, 12 mmol) was added via a syringe under a nitrogen
atmosphere to 3-([2-(4-Methoxy-benzylsulfanyl)-ethyl]-{ [2-(4-methoxybenzylsulfanyl)-
ethylcarbamoyl]-methyl} -amino) propionamide (450 mgs, 0.89
mmol). The resulting colourless solution was heated under reflux at 70°C. Awhite
gum precipitated after about 40 minutes but heating was continued for a further
16 hours. After cooling to RT water (2 mis) was added dropwise allowing for the
vigorous effervescence to cease. The solvent was removed under reduced pressure
to leave a waxy solid to which dilute HCl (2%, 20 mis) was added and the mixture
heated under reflux at ioo°C for 3 hours. After cooling, sodium hydroxide was
added until a pH 10 -11 was obtained. This mixture was extracted with DCM (25
mis x 3) and the fractions combined, dried (MgS0 4), filtered and solvent
evaporated to leave a waxy solid. This material was analysed by 13Cnmr and found
to be a mixture of mono and di reduced products. This material was subjected to
silica gel chromatography eluting with DCM/MeOH/NH 4OH 90:10:1. The
product eluted with an r =0.35 and was isolated as a colourless oil. Yield 180 mg
(42 %).
Ή -NMR (CDCI3 ) d 1.48 (2H, quintet, J = 7 Hz, -CH -), 2.29 - 2.52 (15H, SCH x 2 +
4xCH N + NH + NH, m), 2.64 (2H, t, J = 7 Hz, NCH2), 2.67 (2H, t, J = 7 Hz,
NCH2), 3.59 (4H, s, SCH2Ph x 2), 3.70 (6H, s, OCH3 x 2), 6.76 (4H, d, J = 9 Hz,
CH x 4), 7.15 (4H, d, J = 9 Hz, CH x 4).
Example 2: Precursor Compound 1 4- y drzinecarbonly -N- - 2-
(4-methoxylbenzylthio)ethyl)(2-(2-(4-
In to a mixture of pyrazinoic acid (2 I5 m , o.i25mmol), i-ethyl-343-
dimethylaminopropyl]carbodiimide hydrochloride (30mg, o.i56mmol),
hydroxybenzotriazole (2.8mg, o.02mmol) and triethylamine (2img, o.2o8mmol)
in dimethylformamide was added 1 (50mg, o.i04mmol) slowly and allowed to stir
at RT overnight. The reaction mixture was concentrated, dissolved in ethylacetate
and washed with water. The organic layer was dried with sodium sulphate,
concentrated and purified by combiflash chromatography. Yield, 2img (29%). Ή
NMR (CDCI3): d. 9-37 (s, iH), 8.73-8.77 (d, lH), 8.5 (s, lH), 7.17-2.27 (dd, 4 H),
6.77-6.87 (dd, 4H), 3.78 (s, 6H), 3.72 (s, 2H), 3.66 (s, 2H), 3-52-3-6 (m, 2H), 2.48-
3.02 (m, 14H) and 1.72-1.82 (m, 2H). MS (m/z): 584.6 (M + H+).
Example 3: Re-labelling of Precursor Compound 1
The methods described by Zhen et al (J Med Chem 1999; 42: 2805-2815) were
adapted to label precursor compound 1with rhenium. Precursor compound 1,
70mg (0.117 mmol) was dissolved in lmL of trifluoroacetic acid (TFA), added
one drop of anisole and 0.2 mL of MSA. The reaction mixture was stirred at
6o°C for 90 minutes under nitrogen atmosphere. Later it was concentrated
under vacuum for 2 hours to remove TFA/anisole/MSA. Resulting mass was
taken in 14 mL of 7:1 CH3OH:THF and heated to reflux. Tin(II) chloride (49 mg,
0.25 mmol, in 300 uL of 0.1 M HC1) was added, followed immediately by a
solution of sodium perrhenate (70 mg, 0.25 mmol, in 300 uL of distilled water).
Refluxing was continued for 16 hours, after which the solution was filtered and
concentrated. The product was purified by preparative HPLC to a purity of 99%.
MS (m/z): 558.3 (M + H÷).
Example 4: mTc-labelling of Precursor Compound 1
The c a e conditions described by Meegalla et al (J Med Chem 1998;
41: 428-436) can be used to label precursor compound 1with T c n
summary, precursor compound 1 (0.2-0.4 mihoΐ ) is treated with
TFA/anisole/MSA as per the procedure given in Example 3 above. After the
removal of the volatiles, the residue is dissolved in 100 ΐ of EtOH and 100 ΐ
of HC1 (l N). HC1 (500 mL, l N), 1mL of Sn-glucoheptonate solution (containing
136 g of SnCl2 and 200 g of Naglucoheptonate, pH 6.67), and 50 mL of EDTA
solution (0.1 N) are successively added. [99mTc]Pertechnetate (100-200 mΐ ;
from 1-20 mCi) in saline solution is then added. The reaction mixture is heated
for 30 min at ioo°C (or i2i °Cin an autoclave for 30 min), cooled to RT, and
neutralized with a saturated NaHCO3 solution. After the complex is extracted
from the aqueous reaction medium with ethyl acetate (1 x3, 2 xi.5 mL) and
passed through a small column of Na2SO4, the ethyl acetate extracts are
condensed under a flow of N2. The residue is dissolved in 200 mL of EtOH and
purified by HPLC.
Example 5: In VitroAssessment
A series of in vitro tests were carried out to determine the properties of known
compounds alongside a rhenium derivative of an in vivo imaging agent of the
present invention. These tests were the Microplate Alamar Blue Assay
(MABA)(Franzblau S et al., J Clin Microbiol 1998, 36, 362-366), the Low
Oxygen Recovery Assay (LORA)(Cho et al., Antimicro Agents Chemother 2007,
51, 1380-1385), and the VERO cell cytotoxicity assay to determine IC 50(Cory
AH et al., Cancer Commun 1991, 3, 207-12).
(a) Microplate Alamar Blue Assay (MABA)
The initial screen was conducted against Mycobacterium tuberculosis H37RV
(ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue
Assay (MABA). Compounds were tested in ten 2-fold dilutions, typically from
100 g/mL to 0.19 g/mL at pH 6.8. The MIC90 is defined as the
concentration effecting a reduction in fluorescence of 90% relative to controls.
This value was determined from the dose-response curve using a curve-fitting
program.
5(b ) Low Oxygen Recovery Assay fLORA)
This assay was done with single concentration, typically at >io uMor ug/ml vs.
hypoxia-adapted M. tuberculosis H37RV carrying luciferase gene; exposure for
10 days to test compound under anaerobic conditions; luminescent %
inhibition readout.
c) Methodfor Determining IC50
The VERO cell cytotoxicity assay was done in parallel with the TB Dose
Response assay. After 72 hours exposure, viability was assessed using
Promega's Cell Titer Glo Luminescent Cell Viability Assay, a homogeneous
method of determining the number of viable cells in culture based on
quantitation of the ATPpresent. Cytotoxicity was determined from the doseresponse
curve as the IC50 using a curve-fitting program.
Table 1below sets out the data obtained for the parent PZA compound, the
chelator obtained by the method of Example i(f) above, PZA conjugated to said
chelate obtained by the method of Example 2, and a rhenium complex of PZA
conjugated to said chelate obtained by the method of Example 3.

Claims
i ) An in vivo imaging agent of Formula I :
wherein:
X1represents a direct bond or a linker -(L)n- wherein each Lis independently -
C(=0)-, -CRV, -CR'=CR'-, -CºC-, -CR' C0 -, -C0 CR' -, -NR'-, -NR'CO-, -
CONR'-, -NR' (C=0)NR'-, -NR'(C=S)NR'-, -S0 NR'-, -NR'S0 -, -CR' 0CR' -, -
CR' SCR' -, -CR' NR'CR' -, wherein each R' group is independently H or C1-6
alkyl;
Ch -M1 is a metal ion complex wherein Ch1 is a chelating agent and M1 is a
metal ion suitable for in vivo imaging.
2) The in vivo imaging agent as defined in Claim i wherein said metal ion suitable
for in vivo imaging is either a radioactive metal ion or a paramagnetic metal
ion.
3) The in vivo imaging agent as defined in Claim 2 wherein said radioactive metal
ion is a gamma-emitting radioactive metal ion selected from , In, mIn,
and Ga.
4) The in vivo imaging agent as defined in Claim 3wherein said radioactive metal
5) The in vivo imaging agent as defined in Claim 2 wherein said radioactive metal
ion is a positron-emitting radioactive metal ion selected from 4Cu, V, 2Fe,
6) The in vivo imaging agent as defined in Claim 2 wherein said paramagnetic
metal ion is selected from Gd(III), Mn(II), Cu(II), Cr(III), Fe(III), Co(II), Er(II),
Ni(II), Eu(III) and Dy(III).
7) The in vivo imaging agent as defmed in Claim 1wherein Ch1is selected from:
(a) diaminedioximes;
(b) N3S ligands having a thioltriamide donor set;
(c) N S ligands having a diaminedithiol donor set;
(d) N4 ligands which are open chain or macrocyclic ligands having a
tetramine, amidetriamine or diamidediamine donor set; or,
(e) N O ligands having a diaminediphenol donor set.
8) Apharmaceutical composition comprising the in vivo imaging agent as defmed
in any one of Claims 1-7 together with a pharmaceutically acceptable carrier.
9) A precursor compound of Formula II:
wherein X2 is as defmed in Claim 1for X1, and Ch2 is as defmed for Ch1in either
Claim 1 or Claim 7.
10)Amethod for the preparation of the in vivo imaging agent as defmed in any one
of Claims 1-7 wherein said method comprises:
(i) providing a precursor compound of Formula II as defmed in Claim 9;
(ii) reacting said precursor compound with a source of a metal ion suitable for
in vivo imaging wherein said metal ion is as defmed in any one of
Claims 1-6.
11) The method as defmed in Claim 10 wherein said source of metal ion suitable
for in vivo imaging is a source of .
12) The method as defined in Claim 11 wherein said source of 9¥ is
pertechnetate.
13)Akit for carrying out the method as defined in any one of Claims 10-12 wherein
said kit comprises a vial containing the precursor compound as defined in
Claim 9.
14)A method to determine the location and/or amount of Mycobacteria
tuberculosis (MTB) present in a subject, wherein said method comprises:
(a) administering the in vivo imaging agent as defined in any one of Claims 1-7
to said subject in an amount suitable for in vivo imaging;
(b) allowing the administered in vivo imaging agent to bind to any MTB
present in said subject;
(c) detecting by a suitable in vivo imaging procedure signals emitted by said
metal ion suitable for in vivo imaging comprised in said in vivo imaging
agent;
(d) generating an image representative of the location and/ or amount of said
signals; and,
(e) attributing the location and/ or amount of said signals to the location
and/or amount of MTB present in said subject.
15) The method as defined in Claim 14 wherein said administration step is carried
out by intravenous injection.
16) The method as defined in either Claim 14 or Claim 15 wherein said in vivo
imaging agent is administered as the pharmaceutical composition as defined in
Claim 8.
17) The method as defined in any one of Claims 14-16 which is carried out
repeatedly during the course of a treatment regimen for said subject, said
regimen comprising administration of a drug to combat TB caused by MTB.
18) The pharmaceutical composition as defined in Claim 8 for use in a method to
determine the location and/or amount of MTB present in a subject, wherein
said method is as defined in any one of Claims 14-17.
19) Use of the in vivo imaging agent as defined in any one of Claims 1-7 in the
manufacture of the pharmaceutical composition as defined in Claim 8 for use
in the method as defined in any one of Claims 14-17.