Method of localizing and quantifying regional energy metabolism in a warm-blooded living being and composition therefor

The invention relates to a method of localizing and quantifying regional energy metabolism, in particular regional ischemia, in a warm-blooded living being, comprising administering to said being a diagonstically effective quantity of a radiolabelled L-homocysteine or a radiolabelled functional derivative thereof. The invention further relates to a pharmaceutical composition to be used for performing the above method.

The invention relates to a method of localizing and quantifying regional 
energy metabolism, in particular regional ischemia, in a warm-blooded 
living being. The invention further relates to a pharmaceutical 
composition to be used for said method. 
The diagnosis and localisation of ischemia in various organs in present 
clinical practice relies mainly on angiographic methods and the study of 
tissue perfusion using thallium scintigraphy. While angiography permits 
the localisation of diseased arteries, it does not allow to draw 
conclusions as to the functional consequence and extent of the ischemic 
process. Furthermore, it is often difficult to relate changes in tissue 
perfusion to abnormalities observed with angiographic techniques. In order 
to gain information into the dynamics of tissue metabolism during ischemia 
use was made of various positron emitting tracer substances. Previous 
studies have largely relied upon only a few tracers for example N-13 
ammonia for blood flow, C-11 palmitate for fatty acid metabolism and F-18 
2--fluoro-2-desoxyglucose (FDG) for glucose utilisation. None of these 
tracers however, permitted a direct insight into the dynamics of the 
energy metabolism, mainly because the metabolism of substances applied 
differs greatly from organ to organ and is dependent on the dietary state 
of the organism. 
Thallium scintigraphy is only a measure of tissue perfusion but not 
suitable for assessing the dynamics of energy metabolism. Therefore this 
method has a serious restriction in evaluating and quantifying ischemia. 
Radiolabelled fatty acids have the drawback, that their kinetic behaviour 
is seriously influenced by other substrates which play a role in the 
metabolism of the organs to be investigated.

It is the object of the invention to enable the localization and 
quantification of regional energy metabolism, in particular of regional 
ischemia, without invasive methods and avoiding the above disadvantages. 
It has been found that this object can be achieved according to the 
present invention by administering to a warm-blooded living being a 
diagnostically effective quantity of a radiolabelled L-homocysteine or a 
radiolabelled functional derivative thereof. 
The quantity of radioactive material effective for diagnosing depends on 
various factors such as the diagnostic method, e.g. planar scintigraphy or 
emission tomography, the radiolabel used and the organ to be examined. In 
case the method of the invention is used to localize and quantify regional 
ischemia, such ischemia does not only encompass cardiac ischemia but also 
includes ischemia of other organs like the brains and the 
gastro-intestinal tract. It will be obvious from the above, that the 
quantity of radioactive material which is effective for diagnosing 
purposes may vary within broad ranges. Generally the radioactive material 
is administered to the living being in a quantity of 1 to 1000 MBq per 70 
kg of body weight. The radiolabel may be chosen from radionuclides 
selected from the group consisting of positron emitting nuclides and gamma 
radiation emitting nuclides. Functional derivatives of L-homocysteine are 
to be understood to include derivatives of L-homocysteine wherein the S-H 
function is intact, or, in case the radiolabel is selenium-73, wherein the 
.sup.73 Se-H function is intact. 
A preferred radiolabelled compound to be used for the method of the 
invention is L-homocysteine or its functional derivative, labelled with a 
radioisotope selected from the group consisting of carbon-11, selenium-73, 
fluoride 18, bromine-76, bromine-77 and iodine-123. Typical positron 
emitting nuclides like carbon-11, selenium-73 and fluorine-18 enable the 
in vivo application of the labelled compounds by using the so-called 
"positron-emission tomography" (PET) technique. By using this technique a 
computer tomogram can be obtained of the organ to be investigated, e.g. 
the heart or the brains, enabling the localization and quantification of 
regional energy metabolism. In the PET technique very short living 
radioisotopes are used which emit positrons, for example carbon-11 and 
fluorine-18 with half-lives of 20 and 110 minutes respectively. Gamma 
radiation emitting isotopes like bromine-76, bromine-77 and iodine-123 can 
be used for the labelling of compounds to be detected by conventional 
scanning techniques or in the so-called "single photon emission computer 
tomography" (SPECT) technique. By using conventional scanning techniques 
the emitted gamma radiation can be detected by suitable apparatuses, e.g. 
a gamma camera, to produce images of the organ to be investigated. The 
more advanced SPECT technique is also based upon the detection of gamma 
radiation by sensible detectors. 
The invention further relates to a pharmaceutical composition to be used 
for the method defined before, comprising in addition to a 
pharmaceutically acceptable carrier and, if desired, at least one 
pharmaceutical acceptable adjuvant, as the active substance a 
radiolabelled L-homocysteine or a radiolabelled functional derivative 
thereof, in a diagnostically effective quantity. If desired, said 
composition may be brought into a form more suitable for intravenous or 
subcutaneous administration, for example by the addition of a 
pharmaceutically acceptable liquid vehicle, preferably a physiological 
saline solution. It will be evident, that the composition should be 
sterile for intravenous or subcutaneous administration. If desired, one or 
more adjuvants may be present in the composition, for example suitable 
stabilizers like ascorbic acid, gentisic acid or salts of these acids, 
and/or fillers like glucose, lactose mannitol etc. Dependent on the 
investigation to be performed and the results desired by performing these 
experiments, the composition may be administered to the living being, 
preferably a human being, at once, as a so-called bolus injection, or 
gradually by a continuous infusion. 
The radiolabelled compounds can be prepared by methods which are known per 
se for related compounds by using readily available radiolabelled synthons 
like [C-11]CO.sub.2, [C-11]CH.sub.3 I, [C-11]HCN, [C-11]CO, 
[F-18]F.sub.2,[F-18]CH.sub.3 CO.sub.2 F and [I-123]NaI. The use of 
[C-11]CO.sub.2 for the preparation of 1-[C- 11]-homocysteine thiolactone: 
a precursor for carbon-11 labelled homocysteine, is described in the 
examples. The position of carbon-11 as the radiolabel in the homocysteine 
molecule is not relevant and can be chosen according to the ease of 
synthesizing the [C-11]homocysteine. Selenium-73 can be introduced as a 
radioactive label into the homocysteine molecule by substituting the 
mercapto group by a .sup.73 Se-H group. Radioactive halogen can be 
substituted for one of the hydrogen atoms at choice in the homocysteine 
molecule. The same holds, mutatis mutandis, for introducing a radioactive 
label in a functional derivative of homocysteine. 
The invention will now be described in greater detail with reference to the 
ensuing specific examples. 
EXAMPLE I 
Preparation of (C11)-homocysteine thiolactone (11) 
The preparation is carried out according to the reaction scheme illustrated 
in the FIGURE. The precursor, viz. S-(tetrahydropyranyl-(2))-3-thiopropyl 
isonitrile (8) is prepared starting with 3-chloropropylamine (1) via the 
intermediates 2 and 3. The oily formamide (2) is purified by distillation; 
intermediate (3) is purified by column chromatography. The alternative 
synthetic way (1.fwdarw.4.fwdarw.5) gives reasonably good yields with 
column chromatography of 5 only without purification of 4. The formamide 
of 3-mercaptopropylamine (6) is known e.g. from U.S. Pat. No. 3,278,526. 
This mercapto compound is converted into compound 7 with an equimolar 
quantity of 3,4-dihydro-2H-pyran in diethylether at room temperature; TSOH 
as a catalyst. Formamide 7 is dehydrated in the presence of excess 
diisopropylamine in dimethoxyethane as a solvent by slowly adding 
POCl.sub.3 while cooling. The overall chemical yield of the precursor (8) 
synthesis is approx. 10%. 
The labelling procedure is based on the carboxylation of the corresponding 
.alpha.-lithioisocyanide (9) with [C-11]CO.sub.2 and subsequent 
hydrolysation and lactonisation. The labelling procedure is carried out in 
THF as a solvent. An equimolar quantity of n-butyllithium in hexane is 
added to a solution of the isonitrile precursor (8) in THF. The labelling 
is performed by flushing this solution while cooling with [C-11]CO2 in 
helium, followed by flushing with CO.sub.2. Subsequent deprotection, 
hydrolysation and lactonisation of 10 with aqueous acetic acid and 6M 
hydrochloric acid successively yields the desired thiolactone (11). The 
thiolactone (11) obtained is purified by reverse phase HPLC with 0.1M 
sodium dihydrogenphosphate as an eluent. The radiochemical yield obtained 
is approx. 15%. 
EXAMPLE II 
Use of (C-11) homocysteine 
Animal experiments were carried out in anaesthetized, thoracotomized dogs. 
(C-11)Homocysteine thiolactone (370 MBq) was given i.v. at a dose of 20 
mg/kg either as bolus or over 3-10 min. The thiolactone administered is 
converted within the test animals to (C-11) homocysteine, the active 
species. Thereafter a side branch of the left anterior descending coronary 
artery was occluded. Imaging was performed with a positron emission 
tomograph (Scanditronix.RTM. PC-4096) and data were collected for 60 min. 
after the start of homocysteine infusion. Tracer concentration in plasma 
decreased with a T.sub.1/2 of 40 min Topograms revealed that accumulation 
of tracer in the heart was strictly confirmed to the ischemic tissue.