Radioactive diagnostic agent for bone scanning and non-radioactive carrier therefor

A radioactive diagnostic agent for bone scanning, which comprises .sup.99m Tc and a non-radioactive carrier comprising (A) at least one chosen from methanehydroxydiphosphonic acid and their salts and (B) at least one reducing agent for pertechnetates in a weight ratio of about 1:1 to 7:1 and prevents the accumulation of radioactivity in liver so that definite diagnosis can be assured.

The present invention relates to a radioactive diagnostic agent for bone 
scanning and a non-radioactive carrier therefor. More particularly, it 
relates to a .sup.99m Tc-labeled radioactive diagnostic agent for bone 
scanning, which prevents the accumulation of radioactivity in liver and 
makes definite diagnosing possible, and a non-radioactive carrier 
therefor. 
The diagnostic detection of bone diseases accompanied with abnormality of 
calcium hydroxyapatite such as metastasis of cancer to bone, especially at 
the initial stage, is highly desired. For this purpose, however, the 
conventional diagnostic system using X-ray perspective inspection 
technique is unsatisfactory. 
Attempts to use radioactive diagnostic agents for said purpose were made 
with compositions comprising radioactive isotopes such as fluorine-18 and 
strontium-85. These isotopes have a tendency to be selectively accumulated 
at the physiologically active sites such as a joint site or a tumor site 
and are recognized to be useful for detecting skeletal metastasis of 
cancer. However, fluorine-18 is limited in the use because of its too 
short half life (i.e. 110 minutes). Further, its radioactive gamma ray 
energy is so high (i.e. 511 KeV) as being unsuited for imaging with a 
usual scintillation camera. On the other hand, strontium-85 has a too long 
half life (i.e. 65 days) and, when administered within an allowable 
exposure limit, requires a very long time to obtain a scintigram 
permitting diagnosis. As above, fluorine-18 and strontium-85 are not 
suitable for radionuclear diagnosis, and the focus of study in this field 
of the nuclear medicine has been directed to technetium-99m (.sup.99m Tc). 
.sup.99m Tc has an adequate half life of six hours. Further, the energy of 
the gamma ray emitted by .sup.99m Tc (140 KeV) is most suitable to get a 
scintigram. Moreover, it has a merit of being usable at any optional time 
due to the popularization of a technetium-99m generator. Commercialized 
.sup.99m Tc as well as .sup.99m Tc eluted from a technetium-99m generator 
are considered to be in a heptavalent state and as such can not combine 
with a carrier for bone scanning. But, by reducing to a lower valency 
state with a suitable reducing agent, .sup.99m Tc comes to be readily 
combined with such carrier. .sup.99m Tc per se has no property to be 
selectively accumulated in bone but, when combined with a suitable 
bone-seeking carrier, can be accumulated on a skeleton site so as to make 
bone imaging possible. 
In this respect, U.S. patent 4,016,249 discloses the use of .sup.99m Tc in 
combination with a certain water-soluble phosphate and a reducing agent 
for accumulation of the radio-activity of .sup.99m Tc on a skeleton site. 
Also, U.S. Pat. No. 3,983,227 discloses the use of a radioactive 
pertechnetate solution incorporated with a reductive salt and an organic 
phosphonate for bone scanning. Further, Japanese Patent Publication 
(unexamined) No. 1040/1977 discloses that various organic phosphonates are 
usable in radioactive diagnostic agents for bone scanning. 
According to Japanese Patent Publication (unexamined) No. 54439/1980, the 
use of a methanehydroxydiphosphonate in a radioactive diagnostic agent for 
bone scanning comprising a pertechnetate and a reducing agent therefor can 
provide a sharp bone mineral image and an excellent lesion detection, when 
the weight ratio of the methanehydroxydiphosphonate and the reducing agent 
is in a range of about 10:1 to 15:1, particularly of about 8:1 to 13:1. 
Up to the present time, various radioactive diagnostic agents comprising 
.sup.99m Tc and organic phosphonates or inorganic phosphates have appeared 
in the market and are subjected to the practical use. The greatest problem 
common to these radioactive diagnostic agents is the retention of the 
radioactive component in various organs. Particularly when the 
accumulation of radioactivity in liver exceeds a certain level, the large 
portion of the skeleton scintigram is made obscure. Because of this 
reason, a great effort has been made to provide a radioactive diagnostic 
agent which suppresses the uptake of the radioactivity in liver. 
As a result of the extensive study, it has now been found that a 
radioactive diagnostic agent comprising a pertechnetate, a reducing agent 
therefor and a methanehydroxydiphosphonate wherein the weight ratio of the 
methanehydroxydiphosphonate and the reducing agent is kept in a certain 
specific range is quite effective in detection of any lesion in the 
skeleton site and can provide a sharp and clear bone scintigram within a 
short time after the administration without any serious uptake of 
radioactivity in liver. 
When, for instance, aqueous solutions comprising .sup.99m Tc in the form of 
pertechnetate with methanehydroxydiphosphonic acid and stannous chloride 
as the reducing agent for the pertechnetate in various weight proportions 
were administered to rats by injecting into the tail vein, the proportion 
of the radioactivity concentrations in femur and in liver was varied with 
the said weight proportion as shown in the following table: 
______________________________________ 
Weight ratio of methane- 
Ratio of radio- 
hydroxydiphosphonic acid 
activity concen- 
Experiment 
and anhydrous stannous 
trations (%/g) in 
No.*.sup.1 
chloride femur and in liver 
______________________________________ 
1 0.7:1 18.9 
2 1:1 56 
3 1.5:1 38 
4 1.9:1 15 
5 5.0:1 2.1 
6 7.0:1 1.5 
7 10.0:1 0.8 
8 12.4:1 0.6 
______________________________________ 
Note: 
*.sup.1 Using anhydrous stannous chloride (1 mM concentration) as the 
reducing agent and Lascorbic acid (0.167 mM concentration) as the 
stabilizer, an injection solution containing .sup.99m Tc in a 
concentration of 1.25 mCi/ml was prepared. The injection solution was 
administered to groups of SD strain female rats, each group consisting of 
three rats, in an amount of 0.25 mCi per rat. After 2 hours, the rats wer 
sacrificed, and the desired organs were taken out and subjected to 
measurement of radioactivity. The values are the average in each group. 
With respect to the radioactive diagnostic agent comprising a 
pertechnetate, an organic phosphonate and a reducing agent for the 
pertechnetate, the suppression of the uptake of radioactivity in liver has 
heretofore been attempted by limiting the amount of the reducing agent to 
the minimum. For instance, a typical example of radioactive diagnostic 
agents as commercially available contains the organic phosphonate and the 
reducing agent in a weight ratio of about 10:1 to 40:1. Further, for 
instance, Japanese Patent Publication (unexamined) No. 54439/1980 relating 
to a .sup.99m Tc-labeled radioactive diagnostic agent using 
methanehydroxydiphosphonic acid teaches the weight ratio of the 
methanehydroxydiphosphonic acid and the reducing agent being from about 
8:1 to 13:1 as the preferred range. 
Contrary to the past attempt as above, the results in the said table show 
that the lower weight ratio of methanehydroxydiphosphonic acid and 
stannous chloride as the reducing agent produces remarkable suppression. 
More specifically, the larger proportion of methanehydroxydiphosphonic 
acid to stannous chloride increases the uptake of radioactivity into 
liver, whereby the ratio of the radioactivity concentrations in bone and 
in liver is lowered, and the clarity and detectability of the scintigram 
or scanning image are deteriorated. 
According to the present invention, there is provided a non-radioactive 
carrier, which comprises (A) at least one chosen from 
methanehydroxydiphosphonic acid and their salts and (B) at least one 
reducing agent for pertechnetate in a weight ratio of about 1:1 to 7:1. 
There is also provided a radioactive diagnostic agent for bone scanning, 
which comprises .sup.99m Tc in the form of pertechnetate and the said 
non-radioactive carrier. 
Methanehydroxydiphosphonic acid is representable by the formula: 
##STR1## 
and its salt may be any pharmaceutically acceptable, water-soluble one. 
Specific examples are alkali metal salts (e.g. monosodium salt, disodium 
salt, trisodium salt, tetrasodium salt, dipotassium salt), ammonium salts 
(e.g. diammonium salt), etc. The most preferred is a mixture of 
methanehydroxydiphosphonic acid and its sodium salt(s). 
As the reducing agent for pertechnetate, there is usually employed a 
stannous salt, i.e. a salt of divalent tin ion (Sn.sup.++). Specific 
examples are stannous halides (e.g. stannous chloride, stannous fluoride), 
stannous sulfate, stannous nitrate, stannous acetate, stannous citrate, 
etc. Sn.sup.++ ion-bearing resins such as ion-exchange resins charged with 
Sn.sup.++ ion are also usable. 
In addition to the components (A) and (B), the carrier of the invention may 
preferaly comprise (C) a reductive stabilizer for preventing the oxidation 
of a stannous salt and/or inhibiting the re-oxidation of .sup.99m Tc as 
once reduced. The reductive stabilizer may also be effective in 
suppressing the production of any unfavorable impurity in the carrier or 
the diagnostic agent prepared by its use. The stabilizer may be any 
pharmaceutically acceptable one, particularly suitable for intravenous 
injection. Specific examples are ascorbic acid, erythorbic acid, gentisic 
acid, etc. These may be used in a free form or in the form of salt or 
ester. Further, any conventional additive(s) such as an isotonizing agent 
(e.g. sodium chloride) or a preservative (e.g. benzyl alcohol) may be 
incorporated in the carrier. In general, the components (B) and (C) are 
preferred to be water-soluble, but this is not essential. 
On preparation of the non-radioactive carrier of the invention, the 
essential components (A) and (B) may be mixed in an optional order, if 
necessary, with any optional component(s) such as the component (C). The 
carrier may be formulated in the form of powdery preparation, particularly 
of lyophilized powder, or in the form of liquid preparation, particularly 
of aqueous solution. In the thus prepared carrier, the components (A) and 
(B) should be included in a weight ratio of about 1:1 to 7:1, preferably 
of about 1:1 to 3:1. In case of the component (B) being used in a larger 
proportion than the said upper limit, the ratio of the radioactivity 
concentrations in bone and in liver is still relatively high but that in 
bone and in blood is considerably lowered. Thus, the disappearance of 
radioactivity from blood becomes very late, and a sharp image on the 
scintigram is hardly obtainable. In case of the component (B) being used 
in a smaller proportion than the said lower limit, the ratio of the 
radioactivity concentrations in bone and in liver becomes less than 1, 
which is the lowest limit for obtaining a sharp skeletal image. When the 
component (C) is incorporated therein, its amount is usually from about 1 
to 1/20 part by weight, preferably from about 1/2 to 1/5 part by weight, 
to 1 part by weight of the component (A), although any particular 
limitation is not present. 
For preparation of the radioactive diagnostic agent of the invention, 
.sup.99m Tc in the form of pertechnetate may be contacted with the 
non-radioactive carrier. .sup.99m Tc in the form of pertechnetate is used 
normally in its aqueous solution, which may include additionally any 
conventional additive(s) such as an isotonizing agent (e.g. sodium 
chloride) or a preservative (e.g. benzyl alcohol). While the concentration 
of .sup.99m Tc in the aqueous solution as the radioactive diagnostic agent 
is not particularly limited, it should have such a concentration as can 
afford a sufficient radioactivity concentration for bone scanning, 
preferably from about 0.1 to 50 mCi in about 0.5 to 5.0 ml at the time of 
administration. The amount of the carrier to be combined with .sup.99m Tc 
in the form of pertechnetate may be such that the reducing agent in the 
carrier is sufficient to reduce said .sup.99m Tc. 
As the result of the combination of the non-radioactive carrier in a 
powdery form or in a liquid form with an aqueous solution comprising 
.sup.99m Tc in the form of pertechnetate, there is prepared the .sup.99m 
Tc-labeled radioactive diagnostic agent in situ. Naturally, the weight 
ratio of the components (A) and (B) in the diagnostic agent is from about 
1:1 to 7:1. Sn.sup.++ ions act on .sup.99m Tc in a heptavalent state to 
reduce it to be in a tetravalent state. 
As stated above, the .sup.99m Tc-labeled radioactive diagnostic agent of 
the present invention suppresses the accumulation of radioactivity in 
liver so that a sharp and clear scintigram or scanning image is 
obtainable. Thus, definite diagnosis is made possible by the use of the 
radioactive diagnostic agent of the invention.

The following examples will illustrate the present invention in more 
detail. In these examples, methanehydroxydiphosphonic acid is referred to 
as "HMDP," and ethanehydroxydiphosphonic acid is referred to as "EHDP." 
EXAMPLE 1 
Preparation of the non-radioactive carrier using the disodium salt of HMDP 
and stannous chloride with L-ascorbic acid as the stabilizer: 
Into sterilized water containing no pyrogen substance, nitrogen gas freed 
from bacteria through a filter of 0.2 .mu.m in pore size was introduced to 
remove the dissolved oxygen. In 1 liter of this water, there were 
dissolved aseptically the disodium salt of HMDP (142 mg, 0.6 mmol), 
anhydrous stannous chloride (66 mg, 0.35 mmol) and L-ascorbic acid (35 mg, 
0.2 mmol) under nitrogen stream, and the resulting solution was adjusted 
to a pH of 4 to 6 with sodium hydrogen carbonate. The resultant 
composition (referred to as "H.Sn.A.") was passed through a filter of 0.22 
.mu.m in pore size under nitrogen stream, and each 2.2 ml was filled in 
each ampoule, followed by sealing. The H.Sn.A. composition was colorless 
and transparent. 
EXAMPLE 2 
Preparation of the .sup.99m Tc-labeled radioactive diagnostic agent using 
the H.Sn.A. composition: 
The H.Sn.A. composition (1.5 ml) obtained in Example 1 was mixed with 
physiological saline water (1.5 ml) containing technetium-99m (15 mCi) in 
the form of sodium pertechnetate and stirred well. Thereafter, the mixture 
was allowed to stand for 15 minutes to give a diagnostic composition 
(referred to as "Tc-H.Sn.A."). 
In order to check the labeling efficiency, the Tc-H.Sn.A. composition was 
developed on a cellulose acetate thin layer plate with a solvent mixture 
of a 2 M ammonium chloride solution and a 10 M urea solution in a volume 
ratio of 1:1 and scanned with a radiochromato-scanner. The radioactivity 
was detected as a single peak at Rf=0.97, and no other radioactive peak 
was observed. In the same thin layer chromatography system as above, 
technetium-99m in the form of pertechnetate was developed at Rf=0.7, and 
tin colloid labeled with technetium-99m remained at the original point. 
Thus, the labeling efficiency of the Tc-H.Sn.A. composition may be 
considered as 100%. 
EXAMPLE 3 
Distribution of the .sup.99m Tc-labeled radioactive diagnostic agent in 
rat: 
The Tc-H.Sn.A. composition (0.2 ml) obtained in Example 2 was intravenously 
administered to each of SD strain female rats, which were dissected 1 
hour, 2 hours or 3 hours after the administration. The organs were taken 
out to measure the radioactivity in each organ and the weight of each 
organ. The radioactivity concentration in each organ as determined is 
shown in Table 1. The radioactivity concentration ratios of bone/blood, 
bone/muscle and bone/liver were calculated and are also shown in Table 1. 
TABLE 1 
______________________________________ 
Distribution of Tc--H.Sn.A. in rat 
(% to administered radioactivity per gram) 
1 hr. after 2 hrs. after 
3 hrs. after 
Organ administration 
administration 
administration 
______________________________________ 
Bone (femur) 
3.71 4.30 3.88 
Liver 0.07 0.05 0.04 
Kidney 0.52 0.87 0.32 
Muscle 0.038 0.019 0.013 
Blood (1 ml) 
0.101 0.022 0.012 
Bladder*.sup.1 
47.6 46.8 54.4 
Bone/Muscle 
97.6 226.3 298.5 
Bone/Blood 
36.7 195.5 323.3 
Bone/Liver 
53.0 86.0 97.0 
______________________________________ 
Note: 
*.sup.1 % to administered radioactivity. 
For comparison, the distribution of .sup.99m Tc-EHDP injection as a 
commercially available bone scanning radioactive diagnostic agent in rat 
was examined in the same manner as above, and the results are shown in 
Table 2. 
TABLE 2 
______________________________________ 
Ratio of radioactivity concentration 
of .sup.99m Tc--EHDP in bone/muscle, bone/blood 
and bone/liver in rat 
1 hr. after 2 hrs. after 
3 hrs. after 
Organ administration 
administration 
administration 
______________________________________ 
Bone/Muscle 
67.3 105.3 172.5 
Bone/Blood 
38.4 78.6 109.9 
Bone/Liver 
41.2 55.6 76.3 
______________________________________ 
It is well known to those skilled in the art that, in order to obtain a 
clear skeleton image within a short time after administration of a 
radioactive diagnostic agent for bone scanning, the ratios of bone/muscle, 
bone/blood and bone/liver should give higher values. The results in Tables 
1 and 2 show that the Tc-H.Sn.A. composition is more excellent as a bone 
scanning radioactive diagnostic agent than the commercially available 
Tc-EHDP injection. 
EXAMPLE 4 
Stability of the non-radioactive carrier (i.e. the H.Sn.A. composition): 
The H.Sn.A. composition obtained in Example 1 was stored at 4.degree. to 
10.degree. C. for 2 months. Using the resultant composition, there was 
prepared a .sup.99m Tc-labeled radioactive diagnostic agent in the same 
manner as in Example 2. As to this diagnostic agent, the labeling rate and 
the distribution in rat were examined in the same manner as in Examples 2 
and 3. The obtained results were substantially the same as those with the 
non-radioactive carrier immediately after its production. Thus, no 
material difference was recognized between the H.Sn.A compositions 
immediately after the production and after the storage over a period of 2 
months. 
EXAMPLE 5 
Stability of the .sup.99m Tc-labeled radioactive diagnostic agent (i.e. the 
Tc-H.Sn.A. composition): 
The Tc-H.Sn.A. composition obtained in Example 2 was stored at room 
temperature for 24 hours. As to this diagnostic agent, the labeling rate 
and the distribution in rat were examined in the same manner as in 
Examples 2 and 3. The obtained results were substantially the same as 
those with the diagnostic agent immediately after its production. Thus, no 
material difference was recognized between the Tc-H.Sn.A. composition 
immediately after the production and after the storage over 24 hours. 
Since the half life of .sup.99m Tc is 6 hours, the assurance of the 
stability for 24 hours is sufficient for the practical use. 
EXAMPLE 6 
Toxicity of the .sup.99m Tc-labeled radioactive diagnostic agent (i.e. the 
Tc-H.Sn.A. composition): 
The H.Sn.A. composition obtained in Example 1 was admixed with a 
physiological saline water containing .sup.99m Tc radioactively attenuated 
in the form of sodium pertechnetate in a weight proportion of 1:1, and the 
resultant mixture was intravenously administered to groups of SD strain 
male and female rats, each group consisting of 10 animals, in an amount of 
1 ml per 100 g of body weight (which corresponds to 300 times of the 
amount to be administered to human beings) and also to groups of ICR 
strain male and female mice, each group consisting of 10 animals, in an 
amount of 0.5 ml per 10 g of body weight (which corresponds to 1500 times 
of the amount to be administered to human beings). In the control groups 
having the same number of animals as above, the same volume of 
physiological saline water as above was administered intravenously. All 
the groups were bred for 10 days, and the variation of the body weight was 
recorded everyday. No significant difference was observed between the 
groups which received the Tc-H.Sn.A. composition and the control groups. 
After the observation over 10 days, all the animals were sacrificed, and 
any abnormality was not observed on any organ taken out from them. Thus, 
it is understood that the toxicity of the Tc-H.Sn.A. composition is 
extremely low.