CA1080432A - Stannous-pyrophosphate complex - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A stannous phosphate complex is provided which can be manufactured as a kit for forming a bone seeking complex with technetium-99m, comprising the stannous-phosphate complex sealed in a sterile, non-pyrogenic container, the phosphate moiety of the complex comprising pyrophosphate, the weight ratio of stannous to phosphate moiety ranging from 10-3 to 0.5, the kit can be employed as needed to provide a 99mTc-stannous-phosphate complex which is characterized by good uptake into the bone marrow for radioactively imaging the bone marrow in diagnosis.

Description

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This invention relates to a stannous-phosphate complex and its preparation as well as its use in a kit for ~;
forming a bone seeking complex with technetium-99m.
This application is a divisional application of Canadian Patent Application Ser. ~o. 179,088, filed August 17, 1973.
It has been known for some time that phosphates, including long chain linear polyphosphates, when introduced into the blood stream of mammals will selectively seek out and collect in the bone or skeletal structure. Pro. Soc. Exp, Biol. Med. Volume 100, pages 53-55 (1959), Journal of Labelled Compounds, April-June 1970, Vol. VI, No. 2, pages 166-173, ~ournal of Nuclear Medicine, Vol. 11, No. 6, pages 380-381, 1970, Journal of Nuclear Medicine, Vol. 1, No. 1, January 1960, pages 1-13. In these cases a phosphorous atom or atoms of the phosphate are radioactive, i.e. 3 P. ;~
It has also been known for some time that technetium-99m (99mTcj is a preferred radionuclide for radioactively scan-ning organs because of its short half life and because it 20 radiates gamma rays which can be easily measured, compared, ~or example, to beta rays. See Radiology, Vol, 99, April 1971, pages 192-196.
It has also been known for some time to use divalent stannous tin (Sn++) in the form of stannous chloride, or di-valent iron (Fe+ ) or reduced zirconium to bind radioactive 99mTc to carriers, such as chelating agents, red blood cells, albumin and other proteins, which selectively seek out certain ~ organs of the body, in order to carry the 99mTc with them to i~ such organs of the body where it is concentrated, whereby such organ can be radioactively scanned or imaged for diagnostic or other purposes, e.gO radioactive treatment of a pathological - ..... , :..... . . : ,. . .
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condition. See Journal of Nuclear Medicine, Vol. 11, ~o. 12, 1970, Page 761, Journal of Nuclear Medicine, Vol. 12, No. 1, 1971, pages 22-24, Journal of Nuclear Medicine, Vol. 13, No~ 2, 1972, pages 180-181, Journal of Nuclear Medicine, Vol. 12, No.
5, May 1971, pages 204-211, Radiology, Vol. 102, January 1972, pages 185-196, Journal of Nuclear Medicine, Vol. 13, No. 1, 1972, pages 58-65.
Also, it has been sugges-ted to label a stannous com-pound with 99mTc for radioactively imaging bone marrow, Journal of Nuclear Medicine, Vol. 11, 1970, pages 365-366.
It has also been known for some time that the stannous ion Sn~+ forms soluble complexes with long chàin polyphosphates, Journal Inorganic ~uc. Chem., Vol 28, 1966, pages 493-502.
It has been suggested to employ the aforesaid 9mTc for radioactively scanning the skeletal bone structure of mammals by complexing or binding it to tripolyphosphate carrier by use of the aforesaid stannous ion as a binding agent in order for such phosphate to selectively carry the 99mTc to, and con-centrate it in, the skeletal bone structure upon in vivo intravenous administration for subsequent radioactive scanning or imaging the skeletal structure. Radiology, Vol. 99, April 1971, pages 192-196. The use of 9 mTc in this manner is alleged to have certain advantages over the use of strontium, e.g. 85Sr, as the radioactive label which has been used for radioactive bone scanning in the past. These advantages are those which are inherent in mTc, i.e. short half life and pure, near optimal energy gamma rays. However, the bone uptake (the percent of the total dosage which becomes concentrated in the skeletal structure within a certain time after in vivo intravenous administration) of such 99mTc-containing complex and the ratios of such bone uptake to uptake of the mTc by the other organs .
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~08~432 of the body (the higher these ratios the better), i.e. radio-active contrast, are not nearly as high as with radioactive strontium.
It has been discovered that if the phosphate moiety of the 99mTc-stannous-phosphate complex, which has been suggested in the aforesaid Radiology publication, comprises pyrophosphate (P207 ) (which is a linear polyphosphate moiety of molecular weight less than 300), the bone uptake, bone/blood ratio, bone/
liver ratio, bone/G.I. ratio and bone/kidneys ratio are sub- ;
stantially increased.
It has also been discovered that optimum results are achieved if such phosphate moiety contains no more than about 15 to 20 or 25%~ preferably no more than 5 to 10~/o and more preferably no more than 5% (less than 5% is the most preferred), by weight of linear or branched chain polyphosphate (formula PnO3n+l (n~2)) of molecular weight greater than that of pyro- ~
phosphate. ~ -Maximum bone/liver ratios are achieved when the 99mTc-Sn +-pyrophosphate complex is administered to the mammal in relatively small dosages of substantially less than 20 or 25 preferably substantially less than 8 or 10 and still more pre-ferably less than 5 or 6 (between 0.01 or 0.10 and 3 and even ;~
less provide excellent results), milligrams pyrophosphate moiety per kilogram of body weight of the mammal.
The term "phosphate moiety" as used herein refers tothe phosphorus and oxygen atoms only of the phosphate.
The presence of polyphosphates of formula PnO3n+l (n~2) and molecular weight greater than that of pyrophosphate seems to reduce bone take-up and the aforesaid ratios, as compared to complexes without such higher molecular weight polyphosphates.

However, as aforesaid, some of such higher molecular weight - 3 ~
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polyphosphates can be tolerated, preferably not more than about 15% to 2~/o or 25%~ more preferably no more than 5% to l~/o and still more preferably not more than 5% (less than 5% is the most preferred), by weight of the total phosphate moiety.
Where the pyrophosphate does not constitute 10~/o of the phosphate moiety of the 99mTc-Sn++-phosphate complex, the rest of the phosphate moiety is preferably a ring phosphate of formula Pn03n n (with n preferably being 3 which is trimeta-phosphate) and/or ortho phosphate, and preferably a ring phosphate only, although the a-foresaid limited amounts of higher molecular weight linear polyphosphates can be tolerated.
The complex is made from a water soluble alkali metal ~`
(preferably sodium) or ammonium salt or acid salt of the pyro-phosphate, e.g. sodium pyrophosphate.
Preferably, the sodium pyrophosphate is admixed with a stannous salt, e.g. SnC12 (the stannous salts of other acids which are pharmaceutically acceptable, i.e. safely intravenously administered, can be used) to form the stannous-pyrophosphate complex, the pH of which is adjusted to 3-8, preferably 5-8, by 20 a pharmaceutically acceptable acid, such as HCl, or base, such as NaOH or Na2CO3 or NaHCO3, followed by admixing with the stannous-pyrophosphate complex, an aqueous saline solution of radioactive sodium pertechnetate ( mTc) to form the mTc-stannous-pyrophosphate complex at the time it is desired to intravenously administer the 9 Tc complex. The stannous-pyro-phosphate complex may be sealed in a sterile, non-pyrogenic container or vial as a solution or a lyophilized solid and shipped as a ]~it with the freshly generated sterile and non-pyrogenic 99mTc being added aseptically at the situs just prior to use.

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According to the invention there is provided a method o~ making stannous-pyrophosphate complex comprising admixing a solution of a water soluble phosphate salt or acid salt having a pharmaceutically acceptable cation, the phosphate moiety of -~
which comprises pyrophosphate with stannous compound to form the complex, and adjusting the pH of the complex to between 3 and 8 with a pharmaceutically acceptable pH adjusting agent, the weight ratio of 3tannous to phosphate moiety being from 10 to 0.5~ `' In another aspect of the invention there is provided a stannous-pyrophosphate complex produced in accordance with the invention The stannous compound is conveniently employed in solid form, for example, as a lyophilizate.
According to another aspect of the invention there is provided a kit for forming a bone seeking complex with technetium-99m, comprising a stannou~-phosphate complex sealed in a sterile, non-pyrogenic container, the phosphate moiety of the complex comprising pyrophosphate, the weight ratio of stannous to phosphate moiety ranging from 10 3 to 0.5.
According to yet another aspect of the invention there is provided a method of making the kit comprising admixing with a stannou~ compound, a solution of a water soluble phosphate salt or acid salt, the phosphate moiety of which comprises pyrophosphate to form a ~tannous-pyrophosphate complex, adjusting the pH of the complex to between 3 and 8 with a pharmaceutically acceptable pH adjusting agent, sterilizing the complex and sealing it in a sterile non- :
pyrogenic container, the weight ratio of stannous to phosphate moiety ranging from 10 3 to 0.5.
: The following compositions were prepared:

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TABLE
Sample No. De~cr~ ion 1 A commercial sodium polyphosphate sold by FMC
Corporation under the trade name FMC Glass H
(average chain length of 21 and average M.W.
about 2100).
1-1 A first high molecular weight fraction of the FMC Glass H of Sample 1 obtained by fractionating an aqueou~ solution of Sample 1 with acetone according to the technique described in Van Wazer, Phosphorous And Its Compounds, Inter- :
science Publi~hers, Inc. 1961 (pages 744-747) to '' ':
:
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_ 5a-1C)8~3~32 Sample No . De scr~
precipitate out of the aqueous solution of -the FMC Glass H, as an oil, the highest molecular weight fraction of polyphosphates (composition given in TABL~ 2).

1-2 A second acetone fraction of FMC Glass H
achieved by adding more acetone to precipitate out of the remaining supernatant of 1-1, as an oil, the next higher molecular weight poly~
10 ` phosphates (composition given in TABLE 2). The acetone decreases the solubility of the poly-phosphates in the water: the higher the mole-cular weight of the polyphosphate the less soluble it is so that the highest molecular weights are forced out of solution first.

1-3 A third acetone frac-tion of FMC Glass H contain-ing the next higher molecular weight polyphos-phates is precipitated out of the remaining super-natant solution of 1-2, as an oil, upon addition of further amounts of acetone (composition given !
in TABLE 2).

1-4 A fourth acetone fraction of FMC Glass H contain-ing the next higher molecular weight polyphos-phates (composition given in TABLE 2) is pre-cipitated out of the supernatant solution of 1-3, as an oil, by adding more acetone.

1-5 A fifth acetone fraction of the FMC Glass X (con-, taining the next higher molecular weight poly-- phosphates) (composition given in TABLE 2) is precipitated out of the remaining supernatant : ,.;
.. . . . . . .. . . . ...
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, ~ . .: ;, Sample No. Description solution of l-4, as an oil, by adding more ace-tone.

1-6 A sixth ace-tone fraction of FMC Glass H
(composition given in TABLE 2) is precipitated out of the remaining supernatant solution of .
l-5, as a solid precipitate of the next higher molecular weight polyphosphates by adding more acetone. .. .

1-7 A seventh acetone fraction of FMC Glass H
(composition given in TABLE 2) is precipitated out of the remaining supernatant solution of 1-6, as a solid precipitate of the next higher molecular weight polyphosphates by adding more acetone.

1-8 The residue fraction in the supernatant liquid left after removal of the 1 7 fraction (com-position given in TABLE 2) is recovered by evaporating off the supernatant liquid. .

2 An end acetone fraction of Sample l after 90~/O
by weight had been previously fractionated off :
and leaving after removal of such end fraction

3% by weight in the supernatant (composition given in TABLE 2).

4 A mixture o-f 86% sodium trimetaphosphate (Na3P309), 3% sodium orthophosphate (Na3P04) ; (molecular weight of phosphate moiety - 95) and 10% sodium pyrophosphate (~a4P207) (linear polyphosphate - molecular weight of phosphate moiety - 174) obtained by acetone fractionation 8~32 Sample No. D~scription of sodium trimetaphosphate obtained from Monsanto. Sodium trimetaphosphate, as aforesaid, is one of a plurality of cyclic phosphates having the general formula PnO3 n. Sodium orthophosphate is a phosphate monomer. Sodium pyrophosphate is a dipolyphosphate.

An acetone end fraction of a food grade poly-phosphate sold by FMC under the name FMC FG ~
(composition given in TABLE 2). ~-6 A commercial cyclic trimetaphosphate sold by Stauffer Chemical. (Composition given in TABLE 2).

7 Sodium orthophosphate~ ;
, 8 Sodium pyrophosphate.

9 Sodium tripolyphosphate.
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Sodium tetrapolyphosphate - Na6P4013 - a poly-phosphate - phosphate moiety havin~ a M.~. of 348.
It together with the pyrophosphate and tripoly-phosphate, fall in the class of linear chain polyphosphates having the general formula -(n-~2) n 3n+1 An aqueous solu-tion of each of the phosphate com-position samples 1 through 10 ~40 mg. phosphate/l ml. solution) were made with distilled water in which the dissolved oxygen content was reduced in a conventional manner by bubbling through such water gaseous nitrogen for a period of two hours.
The water and phosphates were mixed to form the solutions in a nitrogen atmosphere and in a nitrogen flushed container. The :

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reason for this is to reduce oxidation o~ the divalent Sn++ to be subsequently admixed with each solution sample. However, it is not essential (but highly preferred) to use nitrogen-treated water or a nitrogen atmosphere or a nitrogen-flushed container. Other known pharmaceutically acceptable conditions, which will inhibit oxidation of the Sn+~ upon subsequent mixing thereof with the phosphate solution, can be used, including the use of conventional pharmaceutically acceptable reducing agents and anti-oxidants in the products used.
Each of these solutions, samples 1 through 10, in an amount equal to 100 ml, was mixed with 0.16g of solid S~ 12 2H20 under a nitrogen atmosphere. The SnC12 2H20 was made by adding to 84.5 mg. of metallic tin, sufficient concentrated HCl with mixing until all the tin has dissolved followed by removing excess acid and water by lyophilization (this operation also being carried out in a vacuum or in a nitrogen atmosphere and in a nitrogen flushed container to prevent oxidation of stannous to stannic). Antioxidants, which can be administered intravenously, may also be used. A stannous (Sn~ phosphate complex or mixture of some kind ~as formed in each case, the phosphate moiety of each sample corresponding to the phosphate moieties of the phosphates set forth in TABLE 2.
Sufficient aqueous solution 3N sodium hydroxide (sodium carbonate or bicarbonate can also be used), in the case of samples 1 through 7 and 9 and 10, and 3N HCl, in the case of sample 8, is then added to each sample to give a pH of 5.0 to achieve a pH suitable for subsequent intravenous in vivo administration into the body of a mammal, in this case adult mice. The pH adjustment is preferably done under a nitrogen atmosphere also.
After thorough mixing, the solutions are sterilized by passing them through a Millipore*biological filter of 0.22 micron pore size under a nitrogen atmosphere. Thereafter milli-* trademark _ 9 _ : - ~ ,: , .:

108~432 liter portions of each of the sterile solutions are poured into individual sterile and non-pyrogenic storage glass vials under a nitrogen atmosphere.
In the case of each sample, vials are lyophilized by conventional freeze drying equipment under aseptic conditions to remove water. This provides a solid stannous-phosphate complex which aids in shipping and storage and which is more stable than the ~omplex in solution.
Each vial contains 1.35 mg. SnC12 and ~0 mg. of the phosphate.
The vials can be sealed and stored until needed sub-sequently to form the technetium-99m-stannous-phosphate complex at the use situs.
To prepare the technetium-99m complex, 3 to 7 (5) ml.
of fresh sodium pertechnetate, removed as a sterile non-pyro- ``
genic eluate from a sterile NEN 99mTc Generator (any other ~`
source of pharmaceutically acceptable 99mTc can be used, including mTc generators manufactured by other than NEN), in a 0.9% saline solution is aseptically added to each vial con-taining the sterile and non-pyrogenic stannous-phosphate com-plex and the vial is swirled until a solution is obtained. In each`case a technetium-99m-stannous-phosphate complex or mixture of some kind is formed in aqueous solution (8 mg.
phosphate per ml solutibn when 5 ml of pertechnetate is used), the phosphate moiety of which corresponds to the phosphate moieties of the phosphate compounds of each sample set forth in TABLE 2.
Aseptic techniques and sterile, non-pyrogenic ingred-ients and containers were used at all steps, such procedures being standard to those skilled in the art.
An eleventh sample was prepared in the manner set forth above by diluting sample 8 to a concentration of 1 mg of .

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phosphate per ml of solution. This was labeled Sample 11.
Each o the technetium-99m-stannous-phosphate complex-containing solutions is aseptically intravenously injected in vivo into a vein in the tail of adult mice (average weight 0.040 kgs) in an amount equal to between 1 and 3 mCi and a volume of 0.12 ml (8 mg. of phosphate per ml solution in samples 1 through 10 and 1 mg. phosphate per ml. solution in sample 11). Also sample 8 was injected in the same manner except that a volume of 0.015 ml was injected instead of 0.12 ml to reduce the dosage of the complex by a factor of ~. This was labeled Sample 12.
Three hours after intravenous administration, some of the mice to which each sample was administered were sacrificed and the various organs of their bodies (skeletal, liver, G.I., blood, kidneys) were counted by conventional gamma ray counting techniques to determine uptake of 99mTc by each organ and thereby determine contrast of bone uptake as compared to uptake by the other organs. As aforesaid, it is not only important to have a high bone uptake (based on total technetium-99m-dosage) but it is also important that the ratio of uptake by the bone to uptake by the other organs be high.
The results are set forth in TABLE 2 below, in which the uptakes (the bone uptake figures represent the average bone uptake for the skeletal system) are in terms of percent of the total technetium-99m activity injected (corrected for radio-active decay) which has collected in the various organs indicated three hours after in vivo intravenous injection, in which the ratio amounts are computed from the uptake amounts, in which "Percent Having Phosphate Moiety M. W. Less Than 300" refers to weight percent of the phosphate moiety based on the total phos-phate moiety of the sample identified in the first horizontal column, in which the percents referred to under Phosphate 3~:

Composition are weight percents of the whole phosphate moiety :
of the sample (as aforesaid, phosphate moiety as used herein is limited to that part of -the compound or complex made up of phosphate phosphorus and oxygen atoms), in which Ortho Pl refers to the phosphate moiety of sodium orthophosphate, Pyro P2 refers to the phosphate moiety of sodium pyrophosphate, Tripoly P3 refers to the phosphate moiety o-f sodium tripoly-phosphate, Tetrapoly P4 refers to the phosphate moiety of sodium tetrapolyphosphate, Trimeta R3 refers to the phosphate moiety of sodium trimetaphosphate, Tetrameta R4 refers to the phosphate moiety of sodium tetrametaphosphate, both trimeta and tetrametaphosphates falling within the class of cyclic or ring phosphates having the formula P303n n, in which "Pentapoly and Longer Linear Chains" refers to the phosphate moiety of sodium pentapolyphosphate and longer linear (linear as used herein includes straight and branched linear phosphate chains) poly-phosphates of formula Pn03rl+l (n+2), in which "Average M.W."
refers to the average molecular weight of the phosphate moiety of the sample and in which "Fraction In Raw Stock" with reference to samples 1-1, 1-2, 1-3, 1-4, 1-6, 1-7 and 1-8 refer :
to the normalized percent by weight of each of these samples in sample 1, which is the raw stock which is fractionated.

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Conventional gamma counting techniques for measuring techne~ium 99m take-up in ~he organs are conventional gamma ray-excitable scintillation counters for radioassaying multiple samples of the organs of the sacrificed mice.
Also, conventional scanning by radioac-tive imaging using a gamma ray-excited scintillation or gamma camera and a dual crystal rectilinear scanner was used in vivo. In vivo scintiphotos of the total body using the Anger camera were obtained as well as rectilinear total body scans.
The figures given in TAsLE 2 are average figures achieved by the aforesaid conventional counting techniques, each sample having been intravenously administered to mice followed by radioactive counting.
Following intravenous administration, the Tc-Sn++-pyrophosphate complexes of the present invention are rapidly cleared from the blood by deposition in bone and excretion into urine. Thus, the technetium-99m-stannous-pyrophosphate complexes are metabolizable. The deposition of the 99mTc-stannous-pyrophosphate complexes of the invention appears to be ; 20 primarily a function of the bone blood flow as well as being related to the efficiency of the bone in extracting the complex from the blood which perfuses the bones.
It was observed that the deposition of the 99mTc in the skeleton is bilaterally symmetrical with increased accumulations being present in the axial skeleton as compared to the appendicular skeleton. There is also increased deposition in the distal aspect of long bones.
Localized areas of abnormal accumulation of the radio-pharmaceutical may be seen in primary malignancies of the bone, metastatic malignancies of the bone, acute or chronic osteo-myelitis, arthritides, recent fractures, areas of ectopic calcification, Paget's disease, regional migra-tory osteoporosis, -.~

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areas of aseptic necrosis and in general any pathological situation involving bone in which there is increased osteogenic activity or localized increased osseous blood perfusion.
The acute toxicity level in mice (LD50/30) for Sample No. 2 has been determined to be lS0 mg/Kg body weight and for Sample 6 it is 800 mg/Kg and for Sample 8 it is 70 mg/Kg.
Subacute toxicity studies in mice of Sample 2 have shown no ` signs of toxicity after 15 daily injections at dose levels as ; high as 63 mg/Kg body weight/day. A similar subacute study in dogs indicates no signs of toxicity at a dose level of 3.6 mg/Kg body weight/day.
It was found that samples 4 and 6 were only one fourth as toxic to mice as sample 2 and one-eighth as toxic to mice as sample 1.
The complexes of the invention have been used success-fully as a skeletal imaging or scanning agent to visualize areas of altered blood flow to the bone and altered osteogenic activity, including suspected bone lesions not shown on X-ray, bone survey performed as part of a work-up in patients with known or suspected malignancy, to follow the response of meta-static or primary bone lesions to radiation therapy, metabolic bone disease, to diagnose arthritis and osteomyelitis, and to diagnose and determine healing rate of bone fractures.
Seven clinical studies by seven doctors on humans using sample 2 and involving 91 patients showed no adverse reactions and were deemed to be highly successful and clinically useful for skeletal diagnostic purposes in the case of 90 patients.
The technetium-99m (99mTc) labeling reactions involved in preparing the 99mTc-stannous-phosphate complexes of the invention~depend on maintaining the tin in the reduced or stan-nous (Sn+2) sta-te. Oxidants present in the pertechnetate supply may adversely affect quality.
.
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The radioactive dosage of the 99mTc complex of the invention may vary from 1 to 25 mCi (millicuries) but preferably is from 10 to 15 mCi. The dosage should preferably be sub-stantially less than 20 or 25, preferably less than 8 or 10 and more preferably less than 5 or 6 mg. of pyrophosphate moiety per kilogram of body weight of the mammal since greater pyro- ;
phosphate dosages than this reduces the bone-liver ratio too much. ~ote for example the low bone-liver ratio in sample 8 ' where the dosage was 25 mg. pyrophosphate per Kg body weight compared to the bone-liver ratio of sample 11 where the dosage was 3.1 mg/Kg body weight.
Only trace amounts of pyrophosphate moiety in the dosage, e.g. as low as 0.001, more preferably 0.01, mg/Kg body weight, gives good bone take-up and bone-to-other organ take-up ratios.
The dosage of pyrophosphate can be kept small either by use of more dilute dosage solutions of the pure pyrophosphate, as in sample 11 or by administering smaller doses of a more con-centrated complex solution, the phosphate moiety of which con-tains a high concentration of pyrophosphate or by more con-centrated solutions of phosphate containing, in addition to -the pyrophosphate, ring phosphate and/or orthophosphate which effectively dilute the pyrophosphate concentration of the dose.
It is preferred to use a 9mTc--Sn ~-phosphate ` solution containing between 0.1 and 40, more preferably between 0.5 and 4 or 5 mgs of pyrophosphate moiety per ml of solution.
,~ An advantage of a complex containing a relatively ~' large amount of ring phosphate and a smaller amount of pyro-, phosphate is that the ring phosphate in addition to providing ~ 30 excellent~,;bone up-take and bone-to-other-organ ratios is less -~ toxic than pyrophosphate, although pyrophosphate, alone, is , still not unduly toxic.

Scanning may be commenced as early as one hour after intravenous administration and may be as long after injection ;~
as clinically useful amounts of Tc remain in the organ. -Another manner of making the complex of the invention ~ :
- is to weigh 4 mg t of SnC12 2~I20 and 100 mg. of sodium pyro-phosphate into a flask (the flasX is sterile and non-pyrogenic - -~
.` and is flushed with nitrogen before weighing and is kept under . .
nitrogen during this step and for the next step). Add, under aseptic conditions, 12 ml of sterile, non-pyrogenic sodium `~ 10 pertechnetate in 0.9% saline solution. Shake the mixture until a solution is obtained followed by intravenous injection (pre- ~:
ferably the pH of the mi~ture is aseptically adjusted to pH `~
`~` 3-8 before intravenous injection). ~ .
, Also, the sterile stannous chloride can first be ~ ~
:~ aseptically mixed with the sterile 99mTc saline solution to : ;
form a mTc-stannous complex, followed by adding the sterile sodium pyrophosphate under aseptic conditions to form the 99mTc-stannous-pyrophosphate, adjusting the pH to 3-8, followed by intravenous in]ection.
It can be seen from TABLE 2 that a 9mTc-stannous-phosphate complex in which the phosphate moiety comprises pyro-phosphate and in which such rnoiety contains no more than 25% by weight of linear polyphosphate of molecular weight greater than .
that of pyrophosphate (sarnples 1-7, 1-8, 2, 4, 5 and 6, 8, 11 and 12) gives surprising higher bone uptake and ratio of bone uptake to other organs, as compared to orthophosphate and other polyphosphates, e.g. tripolyphosphate, tetrapolyphosphate and longer chain polyphosphates (see samples 1, 1-2 to 1-6, 7, 9 ~.
and 10).
I-t can also be seen by comparing samples 8, 11 and 12 in TABLE 2 that the bone to liver ratio is substantially increased by reducing the amount of pyrophosphate in the dosage admlnistered - ~80~3Z
to the mammal.
The pyrophosphate moiety of the 99mTc-stannous-phosphate complex may be from l or 2% or even less up to 100%
by weight of the total phosphate moiety. Preferably, the pyro-phosphate moiety consists of 5 or 10% or more of the total phosphate moiety, more preferably 5~/O or 60% or more and most :: .
preferably between 90 and 10~/o.
Although the stannous (Sn +) ion is by far preferred, ~ the divalent ferrous (Fe++) ion in the form of ferrous ascor-; lO bate, and reduced zirconium can also be used but without as : good results. All these metals can exist in a plurality of redox states.
The phosphate may be added to the solid SnC12 as an aqueous solution, or it may be added to a solution of the SnCl2 to form the Sn -phosphate complex followed by adding the 99mTc solution.
Very little Sn~+ need be used to form the complex of ; the invention, e.g. less than 7 or 10~/o of the phosphate based on ' molecular weights, The weight ratio of Sn + ion to the pyrophosphate moiety may vary over a wide range, i.e. from lO 3 to 0.50, pre- `~
ferably 0.01 to 0.4. It is preferred that the molecular ratio of Sn + to pyrophosphate moiety not exceed 2/l. The maximum ratio is dictated by the amount beyond which the precipitation of Sn occurs. The minimum amount required is that amount necessary to bind a sufficient amount of 99mTc to the pyrophos-phate to achieve good bone uptake and contrast. This can be determined by routine experiment.
The pH of the stannous-phosphate complex may be between 3 and 8.

The water used-for making the complexes of the invention is distilled and is at an elevated temperature of 3'~

200~F during removal of dissolved oxygen and reduction of oxidants by bubbling the nitrogen gas therethrough.
The maximum amount of99mTc is that beyond the ` capacity of the Sn~-pyrophosphate complex to bind the 9mTc.
This can be determined by routine thin layer radiochromatography to determine the percent of free or unbound mTc in the complex. The minimum amount is dictated by that amount below which there is an insufficient amount to give good scanning of bone uptake and contrast, which also can be determined by routine experiment. Generally, the amount of 9 ~ c added to the Sn++ pyrophosphate complex should be sufficient to achieve the counting rate desired by the doctor or laboratory personnel ~ , .
~;~ for the volume to be injected, ordinarily, as aforesaid, the activity dosage varies from 5 to 25 millicuries.
Although sodium pyrophosphates are preferred, any `~ alkali metal, such as potassium and lithium, or ammonium can be used as the cation so long as it is pharmaceutically accept-able so that it can be safely administered intravenously. Also ~`~ the acid pyrophosphates of such cations can be used.
; 20 Although in the examples given above saline water was used as the vehicle, any other vehicle which is pharmaceutically acceptable for intravenous administration can be used.
It is not intended that the invention be limited to any theory which may have been given above or to the specific examples set forth above but only by the claims appended hereto and their equivalents.

:, .

Claims (37)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A method of making stannous-phosphate complex com-prising admixing a solution of a water soluble phosphate salt or acid salt having a pharmaceutically acceptable cation, the phosphate moiety of which comprises pyrophosphate with solid stannous compound to form a complex, and adjusting the pH of the complex to between 3 and 8 with a pharmaceutically accept-able pH adjusting agent, the weight ratio of stannous to phos-phate moiety being from 10-3 to 0.5.

2. A method according to claim 1, said pyrophosphate being admixed with said stannous compound under non-oxidizing conditions.

3. A method according to claim 1, wherein the cation is an alkali metal or ammonium ion.

4. A method according to claim 1, wherein the cation is sodium ion.

5. A method according to claim 1, said phosphate moiety containing no more than 25% by weight of linear polyphosphates of formula PnO3n+1-(n+2) having a molecular weight greater than pyrophosphate.

6. A method according to claim 5, at least 5% by weight of said phosphate moiety being said pyrophosphate and at least the major portion of any phosphate in said phosphate moiety other than pyrophosphate being selected from the group consist-ing of a ring phosphate of formula PnO3n-n orthophosphate and combinations thereof.

7. A method according to claim 6, said phosphate moiety consisting of said pyrophosphate, a ring phosphate of formula PnO3n-n and orthophosphate.

8, A method according to claim 5 or 6, at least 90%
of said phosphate moiety being pyrophosphate.

9. A complex made according to claim 1, 2 or 3.

10. A complex made according to claim 4, 5 or 6.

11. A method of making a kit comprising admixing with a stannous compound, a solution of a water soluble phosphate salt or acid salt having a pharmaceutically acceptable cation, the phosphate moiety of which comprises pyrophosphate to form a stannous-pyrophosphate complex, adjusting the pH of the complex to between 3 and 8 by a pharmaceutically acceptable pH adjust-ing agent, sterilizing the complex and sealing it in a sterile non-pyrogenic container, the weight ratio of stannous to phos-phate moiety ranging from 10-3 to 0.5.

12. A method according to claim 11, said phosphate moiety containing no more than 25% by weight of linear polyphosphates of formula PnO3n+l-(n+2) having a molecular weight greater than pyrophosphate.

13. A method according to claim 12, at least 5% by weight of said phosphate moiety being said pyrophosphate and at least the major portion of any phosphate in said phosphate moiety other than pyrophosphate being selected from the group consisting of a ring phosphate of formula PnO3n-n ortho-phosphate and combinations thereof.

14. A method according to claim 13, said phosphate moiety consisting of said pyrophosphate, a ring phosphate of formula PnO3n-n and orthophosphate.

15. A method according to claim 12 or 13, at least 90%
of said phosphate moiety being pyrophosphate.

16. A method according to claim 11, 12 or 13, in which the sterilized complex is lyophilized and sealed in said container in a lyophilized state.

17. A method according to claim 11, wherein said phosphate is admixed with solid stannous compound.

18. A method according to claim 17, wherein said phosphate is admixed with said stannous compound in the form of a solution and said complex is sterilized and sealed in a non-oxidizing atmosphere.

19. A method according to claim 11, wherein at least 5%
by weight of said phosphate comprises pyrophosphate, said complex being sealed in said container in a non-oxidizing atmosphere.

20. A method according to claim 11, said phosphate moiety being substantially free from linear polyphosphates,

21. A method according to claim 19, substantially 100%
by weight of said phosphate moiety being said pyrophosphate.

22. A method according to claim 11, wherein at least a major portion of any phosphate in said phosphate moiety other than pyrophosphate being selected from the group consisting of a ring phosphate of formula PnO3n-n, orthophosphate and combinations thereof.

23. A method according to claim 22, where n is equal to 3.

24. A method according to claim 23, at least 5% by weight of said phosphate moiety being said pyrophosphate and said phosphate moiety containing no more than 10% by weight of said linear polyphosphates of molecular weight greater than pyrophosphate.

25. A method according to claim 11, at least 1% of said phosphate moiety being pyrophosphate and any remaining phos-phate moiety comprising phosphate of the group consisting of a ring phosphate of formula PnO3n-n where n is 3 and ortho-phosphate and combinations thereof.

26. A method according to claim 11, any phosphate in said phosphate moiety other than pyrophosphate being selected from the group consisting of ring phosphate of formula PnO3n-n, one or more phosphates of formula PnO(3n+1)-(n+2) of which not more than 15% by weight has a n value greater than 2, and combinations thereof.

27. A method according to claim 11, wherein said complex is a freeze dried solid.

28. A method according to claim 11, wherein said complex is a freeze dried solid, said complex containing a pH adjust-ing agent to provide said complex with said pH of between 3 and 8, said complex being packaged in a nitrogen atmosphere in said container.

29. A kit for forming a bone seeking complex with technetium-99m, comprising a sterilized stannous-phosphate complex sealed in a sterile, non-pyrogenic container, the phosphate moiety of said complex comprising pyrophosphate, said complex having a pH of between 3 and 8, the weight ratio of stannous to phosphate moiety ranging from 10-3 to 0.5, pre-pared by the method of claim 11.

30. A kit prepared by the method of claim 12.

31. A kit prepared by the method of claim 13 or 14.

32. A kit prepared by the method of claim 17 or 18.

33. A kit prepared by the method of claim 19 or 21.

34. A kit prepared by the method of claim 20.

35. A kit prepared by the method of claim 22, 23 or 24.

36. A kit prepared by the method of claim 25 or 26.

37. A kit prepared by the method of claim 27 or 28.