BONE-SEEKING COMPLEXES OF TECHNETIUM-99m

Abstract: A composition for use in the preparation of a bone-scanning agent comprises a mixture of a bis-phosphonic acid of formula (I), wherein R<1> is H and R<2> is -COOH or formula (II), or R<1> is -NMe2 and R<2> is -CH2OH, or a non-toxic salt thereof, with a reducing agent for pertechnetate. Technetium-99m, as an aqueous solution of pertechnetate ions, is added to the composition to form a complexe of Tc-99m and the bis-phosphonate. The complex is useful as a bone-scanning agent and is rapidly taken up in bone to give scans of high definition.

Description: Bone-Seeking Complexes of Technetium-99m In April 1971, G. Subramanian and J.G. McAfee described (Radiology, 99, 192-6) a bone scanning agent prepared by reducing pertechnetate TcO-, " with stannous chloride in the presence of tripolyphosphate. The resulting labelled complex gave good skeletal uptake but suffered from several disadvantages, the most important of which were a 24-hour delay between injection and scanning (so that high levels of radioactivity were required in order to obtain adequate instrument response), and the instability of the tripolyphosphate with respect to hydrolysis. An intensive search in the 1970's for better phosphate- and phosphonate-based bone scanning agents has resulted in a large number of publications and several commercial products. The most widely used compound is methylene diphosphonate (MDP), the complex of which with tin and Technetium-99m is the subject of U.S. Patent 4032625. Recent introductions to the market have included hydroxymethylene diphosphonate (HDP), which is the subject of European Patent Application 7676; and 1 , 1-diphosphonopropane- ,3- dicarboxylic acid (DPD), which is described in German O.S. 2755874. A successful bone scanning agent requires inter alia high and rapid uptake of the agent in bone with rapid clearance from the blood and soft tissues such as muscle of that part of the agent not taken up in the bone. In order to achieve scans of high definition, current bone agents normally require an interval of two hours or even more between injection of the agent into the patient and performance of the scan. (The word "scan" is here taken to include gamma-camera imaging techniques). Even small reductions of the interval between injection and scanning are highly desirable and could lead to worthwhile increases in convenience to patient and physician and in the efficiency of the running of nuclear medicine units. The present invention arises from our discovery of some new bis-phosphonic acid technetium-99m complexes which show high and rapid uptake in bone without any apparent concomitant disadvantages. In one aspect the invention provides a composition for the preparation of a bone-scanning agent comprising a mixture of a bis-phosphonic acid selected from the compounds having the following formulae:- H 2 0 3 P UMe- I 2 C-CH 2 0H (ID H 2 0 3 P non-toxic salts of these, together with a reducing agent for pertechnetate. On addition, to this composition, of an aqueous solution of pertechnetate, for example the isotonic saline eluate from a technetium-99m generator, the technetium is reduced from the 7+ to a lower •*' valency in which it forms a complex with the bis- phosphonic acid compound. This bis-phosphonate- technetium-99m- complex forms another aspect of the invention. Accordingly, the present invention provides a bone-seeking composition f0 which comprises an aqueous solution of a complex of technetium-99m and a bis-phosphonic acid selected from compounds having the formula I, II or III and non-toxic salts thereof. The exact nature of the complexes formed are 15 not known; the reducing agent may also possibly form part of these complexes. After shaking or standing Tor a short period of time to ensure complete reduction and complex formation, the liquid is ready for injection. N-(Aminomethylene)aminomethane-diphosphonic 0 acid (I) is a known compound and is described in DE 2439355 as being useful as a chelating agent for divalent metal ions. It may be prepared by the reaction of formamide with phosphorus trichloride either in the presence of phosphorous acid or by 5 subsequent treatment with water. Diphosphonoacetic acid (hydroxycarbonylmethane diphosphonic acid - III) is mentioned as a component in electropolishing baths for Cr-Co alloys in East German Patent 205198 although no indication of 0 the method of preparation of this compound has been reported. However, we have found that this compound may be prepared from tetramethyl methanediphosphonate according to the scheme below. 0 0 II tl (Me 0)- P (Me 0)~ P— CHCOOCH,Ph CH 2 1)NaH 2 / (MeO). P 2)ClC00CH ? Ph (Me 0). P tι 3) H " 0 0 CHCOOCH 2 Ph H 2 Pd/C 0 II < H0 > 2 P CHCOOH •(HO)- P / ** - II The compound l-dimethylamino-2-hydroxyethane-l ,1- diphosphonic acid (II) may be prepared from benzyloxy- acetyl chloride and dimethylamine according to the following reaction scheme:- 2 0CH 2 CONMe 2 + PC1-, + H 3 P0 3 NMe-, In the present invention, instead of using one of the bisphosphonic acids, described above, one may use a non-toxic salt thereof, for instance the sodium salt. Known reducing agents for pertechnetate include tin (2+), iron (2+), and chromium (2+) salts and tin metal. Tin, as the metal or more particularly as a stannous salt, especially stannous chloride, or stannous fluoride, is the most widely used reducing agent for this purpose, and is preferred for use in this inventio When the reducing agent is in the form of a salt, the use of the bisphosphonic acid in a molar excess over the salt helps to stabilise the composition.. The composition may be prepared simply by mixing an aqueous solution, typically a saline 5 solution, of the bis-phosphonic acid or salt with a solution of the reducing agent. If the reducing agent is stannous chloride , it may be used in solution in ethanol to minimise problems of hydrolysis. The pH of the mixture may be adjusted, preferably to a 10. " value in the range 3 - 9, especially 4 - 8. If desired, the mixture may be dispensed into sealed vials , freeze-dried and finally sterilized , if not already sterile. An inert bulking agent may if desired be added 15 to assist in dispensing small amounts into individual vials. Other known additives may also be included, particularly stabilising agents such as nitrate or nitrite, or para-aminobenzoic acid. A preferred formulation includes from 0.1 to ' 0. 1.Omg of stannous chloride or other reducing agent with from 1 to 50 mg of the bis-phosphonic acid I, II αr III, or its sodium salt, in a vial for activation by from 1 to 15 ml of saline eluate from a technetium-99m generator . EXAMPLE I A. Preparation of N-(aminomethylene)aminomethane diphosphonic acid (I) Preparation 1 To a stirred solution of phosphorus trichloride (412 g) and phosphorous acid (82 g) was slowly added formamide (45 g). The mixture was heated to 70°C, kept at that temperature for 1.5 h, and then cooled in an ice bath. Water (200 cm J ) was then slowly added. The resultant solution was then reduced to one third of its original volume by removing the water under reduced pressure. The white precipitate which formed was filtered off and the filtrate concentrated to give a viscous oil. This oil was then taken up in a small volume of water and the desired * compound precipitated by the addition of acetone. Re-precipitation of this material from an aqueous solution gave the N-(aminomethylene)aminomethane-diphosphonic acid as a white solid (10 g). Found: C, 11.20; H, 3.42; N 12.97; C 2 HgN 2 0 6 P 2 requires C, 11.01; H, 3.70; N, 12.84%. g( 31 P) (D 2 0) 9.7 p.p.m. J pH 20 Hz S( 13 C) (D 2 0,pH 8) 156.3 (t,6 Hz), 153.0 (t, 5 Hz), 58.6 (t, 123 Hz) 54.2 (t, 121 Hz) Preparation 2 Formamide (45 g) was added dropwise over a period of about 1 h to a vigorously stirred quantity of phosphorus trichloride (138 g) at 6θ°C. The mixture was then heated for a further 2 h at 70°C during which time the viscosity of the mixture increased until it could no longer be stirred. The mixture was cooled and then slowly hydrolysed by the dropwise addition of water (250 cm ). The resulting solution was warmed at 5 35°C under reduced pressure to remove most of the excess hydrogen chloride. This solution was then poured into acetone (1 1) and left until precipitation was complete. The precipitate (48 g) was filtered off and dried. P n.m.r. spectroscopy indicated that the 10- material was almost entirely the desired diphosphonic acid. Purification of this material was achieved by reprecipitating the product from an aqueous solution by the addition of acetone. N.B. In 50% hydrochloric acid, this diphosphonic acid 15. can be seen as a mixture of the two protonated forms Major component 11.01 p.p.m. Minor component 11.63 p.p.m. B. Labelling 2.0 mg of the ligand was dissolved in 1.8 ml of 0 saline solution (9 mg NaCl/ml) in a 10 ml vial. 0.1 ml of an ethanolic solution of stannous chloride (3 mg SnCl p .2H 0/ml of ethanol) was added and the pH was adjusted to 4.5 with 0.2 M sodium hydrogen carbonate solution. After nitrogen flushing the vial was capped. 5 i mi of technetium generator eluate (29 mCi Tc) was added. After mixing and standing for 5 minutes the preparation was checked for completeness of reduction and freedom from colloid by chromatography on silica gel ITLC strips run in saline solution (9 mg NaCl/ml) 0 and butan-2-one, and on a Whatman 541 paper strip run in saline solution. The biodistribution of this preparation in rats was determined according to Example IV. EXAMPLE II A. Preparation of 1~dimethylamino-2- hydroxyethane-1 , 1-diphosphonic acid (II) Benzyloxyacetyl chloride (30 g) in dry diethyl ether (120 cm J ) was added dropwise to a stirred solution of N,N-dimethylaraine (40 g) in dry diethyl ether at 0°C. The mixture was filtered and the solvent removed to give N,N-dimethyl benzyloxyacetamide in essentially quantitative yield. This amide (32 g) was added slowly to a mixture of phosphorus trichloride (76 g) and phosphorous acid (15.2 g) and the mixture was then heated at 70°C for 2 h. The excess phosphorus trichloride was decanted off and the residue hydrolysed - with water (150 cm J ). This hydrolysed material was evaporated to dryness under reduced pressure and then dissolved in the minimum volume of water. The addition of a large quantity of acetone to this solution caused the crude product to precipitate. P n.m.r. spectroscopy showed this material to be a mixture of 2- benzyloxy-1-dimethylaminoethane-1.1-diphosphonic acid and 1-dimethylaminoethane-1 , 1-diphosphonic acid together with some phosphorous acid. The mixture was separated by chromatography on cellulose using methanol as eluant. Hydrogenolysis of 2-benzyloxy-1- dimethylaminoethane-1 , 1-diphosphonic acid was carried out in aqueous solution using palladium on charcoal as the catalyst. After removing the catalyst by filtration the water was removed under reduced pressure to give a mixture of the desired hydroxy-compound together with some 1-dimethylaminoethane-1 , 1- diphosphonic acid. A pure sample of l-dimethylamino-2- hydroxyethane-1 ,1-diphosphonic acid (2 g) was obtained as a white crystalline solid. Found C, 19.02; H, 5.34; N, 5.62; C 4 H 13 N0 ? P 2 requires C, 19.28; H, 5.27; N τ 5.6 " 2%. Mp 158-160°C g( 31 P) (D 2 0) 8.4 p.p.m. J pH 8 Hz, triplet. ζ κ (D 2 0, ext TMS) 3.05 (6H), 4.10 (t,J pR 12) (2H) S(13 c ) (D 2 0) 74.5 (t, J pc 245) 60.3 (t, J CH 151) 42.1 (q, J CH 145) B. Labelling 2.0 mg of the ligand was dissolved in 0.9 ml of saline solution (9 mg NaCl/ml) in a 10 ml vial. 0.1 ml of an ethanolic solution of stannous chloride (3 mg SnCl .2H 2 0/ml of ethanol)added. pH adjusted to 4.5 with 0.2M sodium hydrogen carbonate solution. After nitrogen flushing the vial was capped, 0.92 ml of QQm technetium generator eluate (20 mCi TC) added. After mixing and standing for 5 minutes purity was determined as in Example I. The biodistribution of this preparation in rats was determined according to Example IV. EXAMPLE III A. Preparation of hydroxycarbonylmethane -r diphosphonic acid (III) i. Tetramethyl benzyloxycarbonylmethane- diphosphonate Tetramethyl methanediphosphonate (9.28 g) was ~ dissolved in dry dimethoxyethane (50 cm ) and sodium hydride (1g, 50% dispersion in oil) was then added. When the anion formation was complete (about 2 h) benzyl chloroformate (6.62 g) was added. A precipitate formed immediately. When the reaction was complete dry diethyl ether (50 cm ) was added and the suspension filtered. The solid was taken up in water (100 cm ) and acidified (pH 2) with concentrated hydrochloric acid. This * solution was then extracted with chloroform (3 x 30 cm ) and the chloroform then removed under ■ z> reduced pressure. Benzene (50 cm ) was added and then removed under reduced pressure to dry the crude material (4 g). The mixture was shown to be largely (85%) tetramethyl benzyloxycarbonylmethanediphosphonate * 3 Λ by P n.m.r. spectroscopy. £( 31 P) (CDC1 3 ) 15.7 p.p.m. $( 1 H) (CDC1 3 ) 7.34 (s), 5.23 (s), 3.80 (d, 11 Hz), 3.68 (t, 22 Hz). g( 13 C) (CDC1 3 ) 162.3 (t, 6Hz), 134.2, 127.6-127.9 (5C), 67.5, 53.0 (d, 24 Hz),46.4 (t,128 Hz) ii. Tetra(trimethylsilyl)benzyloxycarbonylmethane- diphosphonate. The crude product from the previous reaction was. dissolved in chloroform (20 cm ) and trimethylsilyl 5 bromide (7 g) added until the reaction was complete (as indicated by 31P n.m.r. spectroscopy) . The solution was evaporated under reduced pressure to give the crude tetra(trimethylsilyl) benzyloxycarbonylmethane- diphosphonate (6 g) ( g - 5.8 p.p.m.). 10. iii. Benzyloxycarbonylmethane-diphosphonic acid. The crude trimethylsilyl ester was stirred with water (25 cm J ) until the hydrolysis was complete. This resulting solution was then evaporated under reduced pressure to give the crude benzyloxycarbonylmethane 15 -diphosphonic acid ( 3 g). The pure diphosphonic- acid was obtained by chromatrography on cellulose using a 50:50 mixture of ethylacetate and iso-propanol as the eluant. SC 3T ) (D 2 0) 10.6 p.p.m. J pH 22 Hz. 0 S 1 H) (H 2 0) 7.29 (5H, br s), 5.14 (2H, s), 3.69 (1H, t, 122 Hz) S( 13 C) (H 2 0) 166.7 (t, 6Hz), 135.1, 127.7-128.8 (5C), 67.9, 49.4 (t, 119 Hz). iv.Hydroxycarbonylmethane-diphosphonic acid. 5 Benzyloxycarbonylmethane-diphosphonic acid (540 mg) was dissolved in water (25 cm ) and a 10% palladium on charcoal catalyst (90 mg) added. This material was hydrogenated at room temperature and at atmospheric pressure until the hydrogenolysis was complete (16 h). The catalyst was removed by filtration and the filtrate evaporated to dryness under reduced pressure at a temperature below 45°C. This yielded the desired hydroxycarbonylmethane-diphosphonic acid as the monohydrate (400 mg). Found: C, 10.30; H, 3.64; C 2 H 6 P 2 0g.H 2 0 requires C, 10.08; H, 3-36%. $( 31 P) (H 2 0/HC1) 10.7 p.p.m. J pH 22 Hz. S( 1 H) (H 2 0) 3.44 (t, 21.5 Hz) S( 13 C) (H 2 0) 169.2 (t, 5 Hz),50.0 (t, 119 Hz) B. Labelling 3.3 mg of the ligand (syrup) was dissolved in 1 ml of saline solution (9 mg NaCl/ml) in a 10 ml vial. 0.1 ml of an ethanolic solution of stannous chloride (3 mg SnCl p .2H 2 0/ml of ethanol) added. pH adjusted to 4.5 with 0.2M sodium hydrogen carbonate solution. The vial was nitrogen flushed and capped. 1 ml of technetium generator eluate (23.7 mCi 99mTc) was added. After mixing, the preparation was allowed to stand for 5 minutes after which its purity was checked as in Example I. The biodistribution of this preparation in rats was determined according to Example IV. EXAMPLE IV This example shows the unexpected good results achieved using the preparations containing the bis- phosphonates according to this invention. The biodistributions in rats of the preparations made according to Examples I to III were determined by the following procedure. A preparation was injected into the rat followed by screening on a gamma camera for 2 hours. At the end of screening the rat was sacrificed and dissected to measure the activity uptake in the bone, blood, muscle, liver and spleen of the rat. Results are presented in the Table. Data on the performances of bis-phosphonates I and II at a range of different pH values and the performances of bisphosphonate III are presented in the Table together with typical data for MDP (methylene diphosphonate). Results are also given in the Table for the activity uptake in rats disected 15 minutes from injection. In the Table, the abbreviations B:M, B:B1 and B:L + S represent the activity ratios for bone: muscle, bone: blood and bone: liver + spleen. LIGAND TIME pH % BONE B:M B:B1 B:L+S COMPOUND I 2 hrs 5.0 42.3 299 153 182 48.7 607 149 171 15 mins 4.0 37.2 56.6 13.1 60.6 35.0 50.5 12.1 49.5 36.9 46.6 10.0 46.4 It 4.5 36.7 48.1 18.7 85.1 41.6 62.2 14.9 54.3 34.4 44.4 10.6 47.7 It 5.0 42.1 57.2 14.2 65.8 42.9 62.7 14.1 68.3 36.5 50.7 11.8 57.2 tt 6.0 37.6 41.8 12.4 45.8 38.5 46.6 12.1 56.4 35.9 40.8 11.3 45.2 II 7.0 41.2 87.7 25.2 64.4 38.5 55.4 15.3 68.5 38.4 63.8 16.8 71.7 COMPOUND II 2 hrs 4 . 0 35.5 474 120 165 36.5 371 104 151 1 5 mins 4 36.7 48.2 13-3 49.0 29.7 26.2 9.8 39.3 31.5 38.5 9.6 32.0 4 .5 32.8 46.4 10.9 42.1 33.1 43.2 9.5 43.6 32.4 37.7 9.2 42.5 LIGAND TIME pH % BONE B:M B:B1 B:L+S COMPOUND II 15 min 5.0 33.8 33.8 7.9 38.7 30.7 26.4 6.8 31.3 27.7 23.1 6.0 29.7 tl 6.0 28.8 26.6 6.2 30.0 33.3 28.2 7.5 33.4 33.0 25.0 6.1 27.3 It 7.0 37.9 45.1 9.2 45.7 35.9 24.5 6.0 33.6 33.7 27.9 6.8 34.0 COMPOUND III 2 hr 4.5 44.5 252 75 125 58.1 177 64 94 15 min 4.5 34.4 41.5 10.5 17.3 35.9 15.5 9.1 16.2 31.9 29.9 7.7 11.1 MDP 2 hr 4.0 46.9 351 115 93 50.8 341 120 126 15 min 4.0 36.1 38.7 10.9 15.3 37.5 30.1 10.1 17.2 39.2 39.3 11.6 27.0 SUESTITUT - 1 6a - EXAMPLE V Ampoules were prepared so that each contained the following reagent mixture, in sterile freeze dried form, under nitrogen: N-(aminomethylene)aminomethane diphosphonic acid 2 mg Stannous fluoride 0.4 mg p-aminobenzoic acid sodium salt 5 mg Ampoules were reconstituted by the addition of 5 ml of technetiura generator eluate containing 10 to 15 mCi of Tc-99m. 2 mCi amounts of the resulting solution were injected into three healthy human volunteers, and gamma camera scans were performed at 30, 60, 90 and 120 minut.es after injection. Good skeletal images were obtained, and were generally comparable with those obtained using commercially available MDP bone scanning agent.

Claims: - 1 7 - CLAIMS 1 -. A composition for use in the preparation of a bone-scanning agent which comprises a mixture of a bis¬ phosphonic acid of the formula (A) H„0.-P. '2 W 3 J _-R H 2 0 3 P wherein R 1 is H and R2 is -COOH or the group -N = C ^NH 2 or R 1 is• -N•Me 2 and R2 is -CH 2 0H; or a non-toxic salt thereof, together with a reducing agent for pertechnetate ions. A composition as claimed in claim 1 which comprises a mixture of the compound having the following formula:- or a non-toxic salt thereof together with a reducing agent for pertechnetate ions. 3. A composition as claimed in either claim 1 or claim 2, wherein the reducing agent is a non-toxic salt of tin (2+), iron (2+) or chromium (2+). - 18 - 4. A composition as claimed in claim 3, wherein the reducing agent is selected from stannous chloride and stannous fluoride. 5. A method of preparing a Technetium-99m labelled bone-scanning agent which comprises adding Tc-99m, as an aqueous solution of pertechnetate ions to a composition as claimed in any one of claims 1 to 4. 6. A method as claimed in claim 5, wherein the pertechnetate ions are added as a solution in isotonic saline. 7. A bone-seeking composition comprising an aqueous solution of a complex of Tc-99m and a bis¬ phosphonic acid of the formula (A):- wherein R 1 is H and R2 is -COOH or the group or R 1 is is -CH 2 0H. A bone-seeking composition comprising an aqueous solution of a Tc-99m-tin-N-(aminomethylene)amino- methane diphosphonate complex.

Inventors: GRIFFITHS DAVID VAUGHAN, KELLY JAMES DUNCAN, GRIFFITHS, DAVID, VAUGHAN, KELLY, JAMES, DUNCAN
Applicants: AMERSHAM INT PLC, AMERSHAM INTERNATIONAL PLC
Application year: 1986
Main classification: A61K49
IPC codes: A61K51, A61L26, C07F9