CN1223146A - Tris(isontrile) cooper (1) sulfates for preparing radionuclide complexes - Google Patents

Abstract

Tris(isonitrile) copper(I) sulfate complexes and their use in synthetic methods for making radionuclide isonitrile coordination complexes such as (1-isocyano-2-methoxy-2-methylpropane)6. The coordination complexes are useful as radiopharmaceutical imaging agents.

Description

Copper triisonitrile sulfate for preparing radionuclide

The present application is a divisional application of the invention patent application having the title "cuprous triisocarbonitrile sulfate for preparing radionuclide complexes" with the application number of 94193524.8, the filing date of which is 29/7/1994.

The present invention relates to methods, compounds and formulations for the preparation of radiopharmaceutical developers, particularly Tc-99m isonitrile complexes.

Nitrile complexes with some radionuclides are very useful radiopharmaceuticals. Jones et al, U.S. Pat. No.4,452,774 have reported the use of this complex in the visualization of cardiac tissue, the detection of blood clotting in the lungs and other types of congestion defects, lung function studies, renal excretion studies, and the visualization of the bone marrow and hepatobiliary system. However, in practice, these complexes containing simple hydrocarbon isonitrile ligands have a relatively high uptake in human lungs and liver, see e.g. Holman et al j.nuclear.med.25, 1380 (1984). Such ingestion can interfere with visualization of myocardial tissue.

The problem of pulmonary liver uptake can be partially overcome by using isonitrile complexes described by Jones et al in U.S. Pat. nos.4,735,793 and 4,872,561, and these ester or amine isonitrile complexes generally have better pulmonary liver clearance and can give earlier or higher contrast in development. A series of better ether substituted isonitrile complexes have been reported by Bergstein and Subramanyan in U.S. patent No.4,988,827, and these ether substituted isonitrile complexes have been extensively evaluated in vivo experiments. Clinical evaluation of ether-substituted isonitrile complexes of technetium-99 m (Tc-99m) has been reported, see Kahn et al Circulation 791282-1293 (1989); iskandriam et al, Amer.J.Cardiol.64,270-275 (1989); and Christian et al Circulation 83.1615-1620 (1991).

The development of a commercial manufacturing process for preparing a Tc-99m isonitrile complex lyophilized kit is complicated by the volatility of the isonitrile ligand. Carpenter, Jr et al in U.S. Pat. No.4,894,445 propose a solution to this problem for the synthesis of isonitrile adducts of non-radioactive metals such as Cu, Mo, Pd, Co, Ni, Cr, Ag and Rh. When the selected metal isonitrile adducts and a radioactive metal are mixed in a suitable medium, the metal is displaced by the radioactive metal to produce the desired radiopharmaceutical. Where the copper complex is a complex of a diisonitrile phenanthroline and a tetraisonitrile, many of these adducts react with the desired metal radionuclide (e.g., Tc-99m) at elevated temperatures to produce the radiopharmaceutical relatively rapidly. However, in hospitals, the need for such heating becomes cumbersome and inconvenient.

Iqbalet al, in U.S. Pat. No.4,885,100, report cuprous triisonitrile with BF selected from₄、PF₆、ClO₄I, Br, Cl and CF₃COO, an anion. These adducts react with radionuclides such as Tc-99m to produce radiopharmaceuticals more rapidly at room temperature than the complexes described by Carpenter, Jr et al. However, the technique described by Iqbal et al does not allow a sufficiently high yield of Tc-99m isonitrile complex to be obtained in a short period of time and is therefore not suitable for use in a busy hospital.

Therefore, there is a need for a convenient, efficient, and cost-effective reagent and method for preparing radionuclide complexes.

One aspect of the present invention is a copper (III) sulfate triisonitrile complex which is useful in the rapid synthesis of radionuclide isonitrile complexes and gives high yields at room temperature.

Another aspect of the present invention is a method for preparing cuprous triisocyanide sulfate, comprising:

(a) reacting one equivalent of cuprous tetraethyl sulfate with six equivalents of isonitrile ligand; and

(b) and (3) separating triisonitrile sulfate cuprous complex solid.

In a third aspect, the invention is a process for preparing a coordination complex of an isonitrile ligand and a radionuclide comprising reacting a cuprous sulfate isonitrile ligand complex with the radionuclide in a solvent to displace the copper from the radionuclide and form the corresponding coordination complex.

In a fourth aspect, the invention is a sterile, non-pyrogenic kit for preparing a complex of a radionuclide and an isonitrile ligand, comprising the above-described copper triisonitrile sulphate complex, a transfer agentand a reducing agent capable of reducing a correspondingly sufficient amount of the radionuclide to form the complex of the radionuclide and the isonitrile ligand.

In one aspect, the invention is a copper (I) triisocarbonitrile sulfate complex for use in the preparation of a radiopharmaceutical diagnostic imaging agent. Generally, copper triisonitril sulfate complexes are more convenient and efficient to use and have higher yields than previous complexes when used to prepare developers.

The copper (I) triisonitrile sulfate complex of the present invention can be prepared using any isonitrile ligand. Typical isonitrile ligands include those of the formula CNR, wherein R is an aliphatic or aromatic organic group containing 1 to 30 carbon atoms which may be substituted with a variety of charged or uncharged groups. The aromatic R groups may include phenyl, tolyl, xylyl, naphthyl, and diphenyl groups, each of which may be optionally substituted with halogen, hydroxy, nitro, alkyl of 1-15 carbon atoms, alkyl ether of 1-15 carbon atoms, and alkyl ester of 1-15 carbon atoms. The aliphatic R groups may include alkyl groups, preferably containing 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, n-hexyl, 2-ethylhexyl, dodecyl and stearyl, alkenyl, alkynyl or cycloalkyl groups, each of which may be optionally substituted by halogen, hydroxy, nitro, alkyl of 1 to 10 carbon atoms, alkyl ether of 1 to 10 carbon atoms and alkyl ester of 1 to 10 carbon atoms. Examples of suitable isonitrile ligands are described in U.S. Pat. nos.4,452,774,4,872,561 and 4,988,827, which are incorporated herein by reference.

A preferred triisonitrile cuprous sulfate of the present invention can be represented by the following formula (I):

[Cu(CNR)₃]₂[SO₄](Ⅰ)

where R is an alkyl group of 1 to 20 carbon atoms, or of formula (II) or (IIA):

wherein A is a linear or branched alkyl group, R¹And R²Each independently of the other, a linear or branched alkyl group or together a linear or branched alkenyl group, provided that:

(1) in the formula (II), A and R¹The sum of the carbon atoms above is 4 to 6, with the further proviso that if the total number of carbon atoms is 6, then the β carbon atom on the isonitrile group is a quaternary carbon atom, and

(2) a and R in formula (IIA)¹、R²The sum of the carbon atoms in (a) is 4 to 9.

The most preferred sulfate salt is one in which the isonitrile ligand is Methoxyisobutylisonitrile (MIBI), i.e., R is a methoxyisobutyl group. This compound, cuprous tris (methoxyisobutylisonitrile) sulfate, was named as cuprous tris (1-isocyano-2-methoxy-2-methylpropane) sulfate according to the IUPAC rules, hereinafter with [ Cu (MIBI]₃]₂[SO₄]And (4) showing.

The copper (I) triisonitrile sulfate complex of the present invention is more water soluble than the copper (II) triisonitrile adduct disclosed in U.S. patent No.4,885,100 to Igbal et al. Igbal et al give adducts containing one compound selected from BF₄、PF₆、ClO₄I, Br, Cl andCF₃the anion of COO, in the form of a cationic or neutral complex, has a maximum solubility in water of only 2-3 mg/ml, due to limited water solubility of the anion or lack of charge in the complex. In contrast, the water solubility of the sulfuric acid complexes of the present invention is in excess of 2-3 mg/ml, especially in [ Cu (MIBI)]₃]₂[SO₄]In this case, it is preferably more than 100 mg/ml.

Another aspect of the invention is the above-described method of making a copper triisonitrile sulfate complex. The sulfuric acid complex can be prepared by reacting tetraacetonitrile cuprous sulfate, namely [ Cu (CH)₃CN)₄]₂[SO₄]In (b) is synthesized by exchanging the acetonitrile molecule of formula (c) with an isonitrile ligand of formula (CNR), wherein R is as defined above.

[Cu(CH₃CN)₄]₂[SO₄]Can be prepared in situ by heating a mixture of copper sulfate, an excess of one equivalent of copper powder and an excess of eight equivalents of acetonitrile. In a suitable organic solvent such as acetone, acetonitrile, dichloromethane or chloroform at about 0 deg.C to one equivalent of [ Cu (CH)₃CN)₄]₂[SO₄]Adding six equivalents of isonitrile ligand to quantitatively produce [ Cu (CNR)₃]₂[SO₄]. Equations 1 and 2 summarize these reaction steps.

(1)

(2)

The crude product of copper (III) sulfate complex is isolated by filtering the reacted solution, distilling off the volatiles, and precipitating by adding ether to acetone. The product is then recrystallized from hot acetone.

Another aspect of the invention is a method of preparing a radionuclide isonitrile coordination compound. The radionuclide is a radioisotope of Tc, Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb or Ta. The preferred radionuclide is Tc-99 m. The radiolabeled isonitrile complex is prepared by mixing a copper isonitrile complex and a radionuclide in a solvent to replace the copper with the radionuclide. Typical solvents include water, dimethyl sulfoxide, dimethylformamide, methanol, ethanol, 1-or 2-propanol, acetone or acetonitrile. The preferred solvent is water or brine. The reaction temperature may be from room temperature to reflux temperature or even higher, and it is preferable to carry out the reaction at around room temperature. The radiolabelled isonitrile complex is co-labelled (isolable) and gives higher yields in shorter reaction times.

When preparing the isonitrile complex of technetium (Tc-99m), it is preferable to mix an appropriate amount of triisonitrile cuprous sulfate, an appropriate amount of a transfer agent and an appropriate amount of a reducing agent (which can reduce pertechnetate in an aqueous medium: (Tc-99 m))^99mTcO₄ ^-) In sufficient quantity for each to form a radiolabeled isonitrile complex. The order of addition of these compounds is in any case applicable. In some cases, a sufficient amount of cyclodextrin may be added prior to the addition of pertechnetate to facilitate the formation of the radiolabeled isonitrile complex. Also, sometimes a pharmaceutically acceptable buffer such as citrate, phosphate or a lyophilization aid such as maltol, maltose or both may be added. Preferably, the formulation is such that the amount of copper trisacetonitrile sulfate is from about 0.1 mg to about 100 mg, the amount of transfer agent is from about 0.05 mg to about 5 mg, the amount of reducing agent is from about 5 micrograms to about 5 mg, the amount of optional dextrin is from about 1 mg to about 100 mg, the amount of optional buffer is from about 0.1 mg to about 25 mg, and the amount of any one of the freezing aids is from one percent to ten percent by weight.

Preferred transfer agents are the hydrochlorides of homocysteine or salts thereof, with alkyl esters of cysteine such as Cysteine Methyl Ester (CME) and Cysteine Ethyl Ester (CEE) also preferred,with CME being most preferred.

Some of the isonitrile ligands used in the present invention can be used as reducing agents, eliminating the need for additional reducing agents. When needed or when the reaction rate is to be increased, a reducing agent is additionally added. Typical reducing agents are stannous salts such as stannous chloride dihydrate, formamidine sulfite, sodium hydrosulfite, sodium bisulfite, hydroxylamine, ascorbic acid, and the like.

A typical cyclodextrin that can be used in the labeling reaction is gamma cyclodextrin. Cyclodextrins are believed to act by pre-organizing the reactants into their hydrophobic cavities or pockets, thereby increasing the reaction rate.

Depending on the particular reagents and reaction conditions used, the reaction is generally complete in about 1 minute to about 2 hours. The yield of the radionuclide isonitrile coordination compound prepared by the method of the present invention is about 71 to 85% in a reaction time of 15 minutes at 26 ℃ and about 87 to 95% in a reaction time of 35 minutes. The yield obtained in 15 minutes of reaction exceeded the best yield obtained in 30 minutes of reaction with the technology of Iqbal et al (U.S. Pat. No.4,885,100).

For example, when in the greenhouse, appropriate amounts of [ Cu (MIBI)]₃]₂[SO₄]Cysteine hydrochloride (as a transfer agent), stannous chloride dihydrate as a reducing agent and^99mTcO₄ ^-when the reaction is carried out, the reaction is carried out for 15 minutes,^99mTc(MIBI)₆ ⁺the yield of (A) was 71 to 76%, and at 35 minutes of reaction, 87%.

When a cysteine ester is used as the transfer agent, a higher yield of Tc-99m isonitrile complex is obtained. For example, at room temperature, a suitable amount of [ Cu (MIBI)]₃]₂[SO₄]Cysteine ethyl ester hydrochloride, stannous chloride dihydrate and^99mTcO₄ ^-when the reaction is carried out, the reaction is carried out for 15 minutes,^99mTc(MIBI)₆ ⁺the yield of (A) was about 74%, and the reaction time was 35 minutes, about 90%. At room temperature, proper amount of [ Cu (MIBI)₃]₂[SO₄]Cysteine methyl ester hydrochloride, stannous chloride dihydrate and^99mTcO₄ ^-when the reaction is carried out, the reaction is carried out for 15 minutes,^99mTc(MIBI)₆ ⁺the yield of (A) was about 85%, and the reaction time was 35 minutes, about 91%. When gamma cyclodextrin is introduced into proper amount of [ Cu (MIBI)]at room temperature₃]₂[SO₄]Cysteine methyl ester hydrochloride, stannous chloride dihydrate, and^99mTcO₄ ^-when the reaction is carried out, the reaction is carried out for 15 minutes,^99mTc(MIBI)₆ ⁺the yield of (A) was about 78%, and the reaction time was 35 minutes, about 95%.

The kit for preparing the radionuclide and isonitrile ligand complex of the invention is sterile and non-pyrolytic and comprises a copper triisocyanide sulfate complex, a transfer agent and a reducing agent. The reducing agent is used to reduce a substantial amount of the radionuclide to form a complex of the radionuclide and the isonitrile ligand. In some cases, the kit may contain a cyclodextrin, a buffer, a lyophilization aid, or combinations thereof. Preferred kits contain from about 0.1 mg to about 100 mg of triisonitrile cuprous sulfate complex, from about 0.05 mg to about 5 mg of a transfer agent, from about 0.005 mg to about 5000 mg of a reducing agent, and optionally from about 1 mg to 100 mg of a cyclodextrin, from 0.1 mg to 25 mg of a buffer, or from 1% to 10% by weight of alyophilization aid. Preferably, if possible, the contents of the kit are lyophilized, which facilitates storage; if lyophilized unconditionally, these kits can be stored under freezing conditions. The components of the kit are preferably contained in sealed, non-pyrolyzed sterile containers.

The invention is described in detail below by way of a few individual, non-limiting examples.

Analytical method

The radiochemical purity (RCP) of the Tc-99m labelled product was determined by High Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). The radiochemical purity reflects the percent yield of the radionuclide-isonitrile complex.

The labeling reaction mixture portion described below was used with Whatman C₁₈Reverse phase thin layer chromatographyDeveloped with a solvent system of 40% acetonitrile, 30% methanol, 20% 0.5M ammonium acetate and 10% tetrahydrofuran. In which the system is produced from pertechnetate and radionuclide isonitrile complexes^99mThe Tc species is separated from the colloidal material that is the by-product of the labeling reaction. Radio-analysis high-pressure liquid chromatography on μ Bondapak C₁₈(4.6 mm. times.250 mm) column (hydrate). The column was eluted at a flow rate of 1.5 ml/min in a linear gradient from 100% solvent A (700: 300: 1 water: acetonitrile: trifluoroacetic acid) to 100% solvent B (100: 900: 1, water: acetonitrile: trifluoroacetic acid) and continued for 1 min at 100% solvent BAfter a while, the reaction is returned to 100% of solvent A. The radiochemical purity, amine body and corrected radiochemical purity data in the examples below are reported in percentages. The corrected radiochemical purity data are determined by averaging two HPLC values, each corrected by averaging three colloid values obtained by thin-layerchromatography, i.e.the corrected radiochemical purity percentage is equal to [ (100-% colloid (by thin-layer chromatography))/100%](% radiochemical purity (by high pressure liquid chromatography)).

Example 1

Multivariate parametric analysis of the technique described in U.S. Pat. No.4,885,100

Experience has shown that in a busy hospital practice, the technique described in U.S. patent No.4,885,100 by Igbal et al does not provide sufficiently high yields of Tc-99m isonitrile complexes in a short period of time. Based on a broad multivariate parametric analysis, it has been determined that the technique of Igbal et al produces Tc-99m isonitrile complex [ 2]^99mTc(MIB)₆]⁺The maximum yield was 30% and 68% at reaction times of 10 minutes and 30 minutes.

This study was conducted using a commercially available software package RSDiscover (Bolt Beranek)&Newman, Cambridge, MA) was statistically designed. A 5-factor, 32 experiment face-centered cubic design was applied. Table 1 lists various factors such as [ Cu (MIBI)]₃][BF₄]Levels, stannous chloride dihydrate levels, cysteine hydrochloride hydrate levels, mannitol levels, and PH. IIThe composition of the hydrated sodium citrate buffer is fixed. The three levels selected for each factor are: [ Cu (MIBI)₃][BF₄]0.5,1.25 and 2.0 mg/vial; stannous chloride 10,105 and 200 micrograms per vial; cysteine 3,7.5 and 12 mg/vial; mannitol 5,15 and 25 mg/vial, PH 3,4.5 and 6. The ingredients listed in Table 1, mannitol, cysteine hydrochloride hydrate, [ Cu (MIBI)]₃][BF₄]Stannous chloride dihydrate and a fixed amount of sodium citrate dihydrate were dissolved in a 10.0 ml volumetric flask with deionized water sparged with argon, PH adjusted and diluted to scale. Adding 1.0 ml of the above solution into three medicine tubes placed in a constant temperature water bath at 26 ℃, and adding 1.0 ml of the solution into each medicine tubeSalt 1.8% (wt.) Na^99mTcO₄ ^-Solutions (50 mJ/ml from⁹⁹Mo/^99mObtained in a Tc radionuclide generator). The product [ 2]after 10 minutes and 30 minutes was measured by using the thin layer chromatography and the high pressure liquid chromatography described above^99mTc(MIBI)₆]⁺The yield of (1). Two cartridges were analyzed by thin layer chromatography and high pressure liquid chromatography simultaneously, and the other cartridge was analyzed by thin layer chromatography only. The data are shown in Table 2.

Table 1 levels of components in response surface studies

Serial number	Mannitol (mg)	Cysteine ester (mg)	[Cu(MIBI)3] (BF4)(mg)	Stannous salt (ug)	pH
						1	25.77	11.5	1.95	200	6.17
2	15.05	7.7	1.26	105	4.37
						3	25.60	2.94	0.51	10	3.22
4	4.96	3.07	0.50	200	3.20
						5	14.95	2.85	1.28	105	4.33
6	5.07	7.90	1.30	105	4.37
						7	14.82	7.56	1.29	200	4.41
8	24.93	12.16	0.47	200	3.01

9	4.88	12.07	1.99	200	2.82
						10	4.96	11.96	0.49	200	6.25
11	14.82	7.56	1.93	105	4.44
						12	25.00	7.69	1.30	105	4.50
13	24.90	12.00	0.51	10	6.10
						14	15.00	7.40	1.30	105	4.57
15	4.91	2.98	1.95	200	6.12
						16	5.19	3.01	1.97	10	2.98
17	5.11	12.06	1.95	10	5.90
						18	14.98	7.57	1.28	10	4.62
19	14.98	7.43	1.31	105	3.16
						20	25.03	12.12	2.01	10	3.17
21	15.14	7.47	1.27	105	4.46
						22	25.31	3.06	2.01	200	3.01
23	15.06	7.49	1.26	105	4.44
						24	25.14	2.95	0.55	200	5.97
25	15.00	7.50	1.30	105	4.43
						26	15.30	7.40	0.60	105	4.43
27	24.90	3.00	2.00	10	5.97
						28	4.93	2.9	0.50	10	5.96
29	15.03	12.08	1.24	105	4.63
						30	14.91	7.41	1.35	105	4.61

31	5.00	12.03	0.53	10	3.12
						32	14.89	7.41	1.31	105	6.07

Table 2 response surface study data

Serial number	Colloid (average)		Purity of radiochemistry (average)			Corrected for Purity of radiochemistry
									10 minutes	30 minutes	10 minutes		30 minutes	10 minutes	30 minutesClock (CN)
1	2.29	1.92	18.16		51.08	17.74	50.10
								2	14.14	17.12	25.74		45.07	22.10	37.35
3	16.29	10.24	7.66		17.67	6.41	15.86
								4	30.53	42.31	7.80		12.62	5.42	7.28
	11.73	21.08	29.62		50.74	26.15	40.04
								6	10.88	18.52	25.62		47.49	22.83	38.69
7	14.58	22.76	25.60		51.32	21.87	39.64
								8	29.49	41.81	17.44		34.93	12.30	20.33
9	22.46	33.65	31.42		60.12	24.36	39.89
								10	12.22	21.48	9.03		37.15	7.93	29.17
11	8.13	10.61	28.90		48.04	26.55	42.94
								12	9.37	13.27	25.16		48.55	22.80	42.11
13	0.0	0.0	0.0		0.0	0.0	0.0
								14	10.61	14.53	26.06		51.41	23.30	43.94
15	4.88	4.65	32.14		70.32	30.57	67.05
								16	6.86	9.85	28.70		46.96	26.73	42.33

17	0.0	0.0	0.0	0.0	0.0		0.0
								18	3.13	2.21	28.42	55.04	27.53		53.82
19	19.16	34.81	23.87	42.58	19.30		27.76
								20	6.28	4.84	31.00	58.15	29.05		55.34
21	13.07	11.93	42.01	57.37	36.52		50.53
								22	36.65	45.23	38.02	55.22	24.09		30.24
23	15.23	18.46	26.57	46.80	22.52		38.16
								24	13.24	15.01	25.71	62.94	22.31		53.49
25	9.56	15.16	24.80	47.81	22.43		40.56
								26	15.38	25.54	20.48	39.17	17.33		29.17
27	0.0	0.0	0.37	0.31	0.37		0.31
								28	0.35	1.37	23.49	55.48	23.41		54.72
29	8.78	13.29	24.71	55.19	22.54		47.86
								30	9.57	14.24	31.29	53.52	28.30		45.90
31	7.20	7.04	17.95	37.29	16.66		34.66
								32	2.55	2.20	25.71	55.02	25.05		53.81

Corrected [ 2]^99mTc(MIBI)₆]⁺Radiochemical purity (%) data was used in the experimental design as a response. The data was then modeled with RSDiscover. Tables 3 and 4 show that the time is 10 minutes^99mTc(MIBI)₆]⁺Yield model and 30 minutes^99mTc(MIBI)₆]⁺And (5) analyzing the deviation of the yield model. In tables 3 and 4, M represents mannitol, CY represents cysteine, and MI represents [ Cu (MIBI)₃][BF₄]T represents SnCl₂·2H₂O and P represent pH values.

TABLE 3 reaction least squares component of 10 min radiochemical purity analysis of variance

Sources of deviation	Degree of freedom	General prescription	Mean square	F ratio	Significance of
						Constant number	1	12579.756		-	-
M	1	11.203	11.203	0.53	0.4754
						CY	1	61.130	61.130	2.89	0.1053
MI	1	276.770	276.770	13.10	0.0018
						T	1	71.992	71.992	3.41	0.0806
P	1	96.000	96.000	4.54	0.0463
						M*CY	1	144.581	144.581	6.84	0.0170
M*T	1	88.552	88.552	4.19	0.0547
						CY*P	1	351.481	351.481	16.63	0.0006
MI**2	1	563.046	563.046	26.65	0.0001
						MI*T	1	86.777	86.777	4.11	0.0570
MI*P	1	312.008	312.008	14.77	0.0011
						T*P	1	291.250	291.250	13.78	0.0015
Residual error	19	401.465	21.130	-	-

Root mean square error =0.8491

Corrected root mean square error =0.7538

TABLE 4 reaction least squarescomponent of 30 min radiochemical purity analysis of variance

Sources of deviation	Degree of freedom	General prescription	Mean square	F ratio	Significance of
						Constant number1t	1	43001.447	-	-	-
M	1	49.237	49.237	0.80	0.3827
						CY	1	56.214	56.214	0.91	0.3517
MI	1	432.923	432.923	7.04	0.0162
						T	1	352.968	352.968	5.74	0.0277
p	1	66.529	66.529	1.08	0.3121
						M*CY	1	605.900	605.900	9.85	0.0057
M*T	1	324.563	324.563	5.28	0.0338
						CY*MI	1	142.077	142.077	2.31	0.1459
CY*P	1	1640.296	1640.296	26.67	0.0001
						MI**2	1	993.176	993.176	16.15	0.0008
MI*T	1	424.118	424.118	6.90	0.0171
						MI*P	1	846.989	846.989	13.77	0.0016
T*P	1	2380.371	2380.371	38.71	0.0000
						Residual error	18	1106.913	61.495	-	-

Root mean square error =0.8780

Corrected root mean square error =0.7898

The deviation analysis table shows that the time can be 10 minutes^99mTc(MIBI)₆]⁺The yield data was modeled to account for 75% of the data variation. Can also be 30 minutes time^99mTc(MIBI)₆]⁺Yield data a similar model was established to account for 79% of the data variation.

According to these models, the method and the reagent disclosed in U.S. Pat. No.4,885,100 to Iqbal et al are used, at 10 minutes and 30 minutes, respectively^99mTc(MIBI)₆]⁺The maximum expected values for the yield were 31% and 75%, respectively. Upon reaching the maximum expected value, the values of the 5 factors are, [ Cu (MIBI)]₃][BF₄]1.9 mg, SnCl₂·2H₂O192 μ g, PH6, anhydro sugar alcohol 5 mg, cysteine 3 mg. The above formula was verified experimentally to yield 30% and 68% yield values at 10 and 30 minutes respectively. The observed yield value is slightly lower than the expected yield value but within the standard deviation of the expected value.

Example 2

Synthesis of [ Cu (MIBI)]₃]₂[BF₄]·0.5CH₃COCH₃

In a 500 ml Schlenk flask, CuSO was placed under a nitrogen atmosphere₄·5H₂O (24.5 g, 98.1 mmol) and metallic copper (12.6 g, 198 mmol) 200 ml acetone and 75 ml acetonitrile flushed with nitrogen were added. The reaction mixture was refluxed for 1.5 hours under nitrogen and then cooled in an ice bath. A large amount of white crystalline solid was formed. 66.6 g, i.e. 588 mmol of 2-methoxy-isobutyl isonitrile (MIBI) are added dropwise over a period of 2 hours. The reaction mixture was warmed to room temperature and stirred for 12 hours. Excess copper metal was filtered off by Schlenk technique and volatile components were distilled off from the green filtrate. With a minimum amount (about 200 ml) of acetone (from B)₂O₃Evaporated, degassed) dissolved the yellow-green syrup residue, 400 ml of anhydrous ether were added dropwise with vigorous stirring, and the precipitated off-white oily solid was separated on a medium Schlenk filter and dried under vacuum. The crude product was recrystallized 3 times with a minimum amount of hot acetone in an argon-filled glove box to give a white crystalline solid (15.0 g, 16.1 mmol).¹H NMR(CDCl₃,270MH₂) The spectral data are as follows: 3.58(s,12H, CH)₂),3.20(s,18H,OCH₃),2.12(s,3H,CH₃COCH₃),1.24(s,36H,CH₃)。C_37.5H₆₉N₆O_10.5SCu₂Calculation results of elemental analysis: % C, 48.37; % H, 7.47; % N, 9.03; % Cu, 13.65.; the measurement result is as follows: % C, 48.56; % H, 7.43; % N, 8.79; % Cu, 13.4.

Examples 3 to 5

Copper triisonitrile sulfate and the para [ transpotant]^99mTc(MIBI)₆]⁺Influence of the yield

In a 10.0 ml volumetric flask, the amount shown in Table 5 was added [ Cu (MIBI)]₃]₂[SO₄]·0.5CH₃COCH₃And cysteine hydrochloride hydrate, then 0.27 mmole of dry mannitol, 0.17 mmole of sodium citrate dihydrate and 0.009 mmole of stannous chloride dihydrate, dissolved in deionized water stirred with argon, adjusted in pH and dilutedTo scale. In each of three cartridges, 1.0 ml of the above solution was added and placed in a thermostatic water bath at 26 ℃. 1.0 ml of Na prepared as in example 1 was added to each tube^99mTcO₄(50 mS/ml) and the corrected values for radiochemical purity at 15, 35 or 40 minutes of reaction were determined and the data are presented in Table 5.

TABLE 5[ Cu (MIBI)]₃]₂[SO₄]Level and cysteine level pairs

[^99mTc(MIBI)₆]⁺Influence of the yield

Example No. 2	Cysteine Amino acid (mmol)	MIBI* (mmol)	pH	Colloid 15 minutes	Colloid 35 minutes	Corrected for RCP 15 minutes	Corrected for RCP 35 minutes
								3	0.008	0.067	5.8	3.3	n.d.	71	n.d.
4	0.008	0.200	5.8	2.2	n.d.	74	n.d.
								5	0.016	0.067	5.2	4.6	4.0	76	87

MIBI means [ Cu (MIBI)]₃]₂[SO₄]Molar concentration of MIBI in the form of [ Cu salt]]And 6, calculating.

The results show that more soluble sulfate [ Cu (MIBI)]is used₃]₂[SO₄]Higher concentrations of [ Cu (MIBI)]can be obtained₃]⁺. Reaction time of 15 minutes^99mTc(MIBI)₆]⁺Is significantly higher than that obtained with the Iqbal et al (u.s.pat. No.4,885,100) technique. In fact, the yield of 15 minutes of reaction exceeded the yield of 30 minutes of reaction with the prior art. The cysteine level is improved, and the effect on yield improvement is also good. Therefore, under the conditions of example 5, the reaction was carried out for 35 minutes to obtain a product of 87%^99mTc(MIBI)₆]⁺。

Examples 6 to 7

Cysteine alkyl ester pair [ alpha], [ beta]-cysteine^99mTc(MIBI)₆]⁺Influence of the yield

In a 10.0 ml volumetric flask, the amount shown in Table 6 was added [ Cu (MIBI)₃]₂[SO₄]·0.5CH₃COCH₃And cysteine methyl ester hydrochloride (CME) or cysteine ethyl ester hydrochloride (CEE), then 0.27 mmol mannitol, 0.17 mmol sodium citrate dihydrate and 0.009 mmol stannous chloride dihydrate were added, dissolved in deionized water with argon stirring, PH adjusted and diluted to the mark. In each of three cartridges, 1.0 ml of the above solution was added and placed in a thermostatic water bath at 26 ℃. 1.0 ml of Na prepared as in example 1 was added to each vial^99mTcO₄(50 mCur/ml) solution, and the reaction was monitored for 15 minutes and 35 minutes. The data are presented in table 6.

TABLE 6 cysteine alkyl esters as transfer agents

Para 2^99mTc(MIBI)₆]⁺Influence of the yield

Example No. 2	Transfer agent	Transfer agent mmol	MIBI* mmol	pH	Colloid d 15 minutes	Colloid 35 minutes	Corrected for RCP 15 minutes	Corrected for RCP 35 minutes
									6	CME	0.016	0.067	5.6	0	0	85	91
7	CEE	0.016	0.067	5.6	0.8	0.6	74	90

MIBI means [ Cu (MIBI)]₃]₂[SO₄]Molar concentration of MIBI in the form of [ Cu salt]]And 6, calculating.

The results show that the use of cysteine alkyl esters instead of cysteine as transfer agents works better. The yield is improved mainly due to great reduction in^99mFormation of Tc colloid by-product. Using the preferred cysteine methyl ester of FIG. 7 as transfer agent, up to 85% yield was obtained in 15 minutes. In examples 6 and 7, the reaction was carried out for 35 minutes, and the yield was not less than 90%.

Example 8

Cyclodextrin pair [ alpha], [ beta]-cyclodextrin^99mTc(MIBI)₆ ⁺]Influence of the yield

In a 10.0 ml volumetric flask, 0.011 mmol [ Cu (MIBI)]₃]₂[SO₄]0.5CH₃COCH₃0.022 mmole cysteine methyl ester hydrochloride, 0.38 mmole gamma cyclodextrin, 0.008 mmole sodium citrate dihydrate, 0.008 mmole anhydrous chromium dichloride and 0.009 mmole stannous chloride dihydrate, dissolved with argon stirred deionized water, adjusted PH and diluted to the mark. In three cartridges, 1.0 ml of the above solution was added and the mixture was left at 26 ℃ constantlyIn a warm water bath. Na prepared as in example 1 was added to each tube^99mTcO₄ ^-(50 mCur/ml) solution, and the reaction was monitored for 15 minutes and 35 minutes. The data are presented in table 7.

TABLE 7 [ gamma]cyclodextrin pair [ gamma], [ beta]^99mTc(MIBI)₆ ⁺]Influence of the yield

Example No. 2	Gamma-cyclodextrin mmol	CME mmol	MIBI* mmol	pH	Colloid 15 minutes	Colloid 35 minutes	Corrected for RCP. 15 minutes	Corrected for RCP. 35 minutes
									8	0.036	0.002	0.006	6.4	0.7	0.8	78	95

MIBI means [ Cu (MIBI)]₃]₂[SO₄]Molar concentration of MIBI in the form of [ Cu salt]]And 6, calculating.

These data indicate that the addition of gamma cyclodextrin to the reaction mixture works better. Under these reaction conditions, much smaller amounts of [ Cu (MIBI)]are used₃]₂[SO₄](0.001 vs. 0.01 mmol) and much smaller amounts of cysteine methyl ester (0.002 vs. 0.016 mmol), yields of 78% and 95% were obtained at 15 and 35 minutes of reaction, respectively. This effect is due to the effect of the pre-organization of the reactants on the reaction rate.

The present invention may be embodied in other specific forms without departing from its spirit or essential attributes. Reference must therefore be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Claims (11)

1. A sterile, non-pyrogenic kit for preparing a radionuclide and an isonitrile ligand complex, comprising a copper triisonitrile sulphate complex, a transfer agent and a reducing agent capable of reducing a correspondingly sufficient amount of a radionuclide to form the complex of the radionuclide and the isonitrile ligand.

2. The kit of claim 1 wherein the amount of triisonitrile cuprous sulfate complex is about 0.1 to 100 mg, the amount of transfer agent is about 0.05 to 5 mg, and the amount of reducing agent is about 0.005 to 5000 mg.

3. The kit of claim 1 wherein the components are lyophilized or frozen and the radionuclide is Tc-99 m.

4. The kit of claim 1 wherein the transfer agent is cysteine or a salt thereof.

5. The kit of claim 4 wherein the transfer agent is a cysteine alkyl ester.

6. The kit of claim 5, wherein the cysteine alkyl ester is cysteine methyl ester.

7. The kit of claim 5, wherein the cysteine alkyl ester is cysteine ethyl ester.

8. The kit of claim 1 wherein the reducing agent is stannous chloride dihydrate.

9. The kit of claim 1, further comprising a cyclodextrin in an amount sufficient to promote the formation of a complex between the radionuclide and the isonitrile ligand.

10. The kit of claim 9, wherein the cyclodextrin is gamma cyclodextrin.

11. The kit of claim 10, wherein the amount of gamma cyclodextrin is about 1 to 100 mg.