WO1994012523A1 - Steroid-21-phosphate complex of technetium-99m - Google Patents

Steroid-21-phosphate complex of technetium-99m Download PDF

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Publication number
WO1994012523A1
WO1994012523A1 PCT/US1992/010312 US9210312W WO9412523A1 WO 1994012523 A1 WO1994012523 A1 WO 1994012523A1 US 9210312 W US9210312 W US 9210312W WO 9412523 A1 WO9412523 A1 WO 9412523A1
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Prior art keywords
phosphate
steroid
complex
dianion
technetium
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PCT/US1992/010312
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French (fr)
Inventor
M. T. Ercan
Original Assignee
Mallinckrodt Medical, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mallinckrodt Medical, Inc. filed Critical Mallinckrodt Medical, Inc.
Priority to AU32299/93A priority Critical patent/AU3229993A/en
Priority to PCT/US1992/010312 priority patent/WO1994012523A1/en
Publication of WO1994012523A1 publication Critical patent/WO1994012523A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/0026—Oxygen-containing hetero ring cyclic ketals
    • C07J71/0031—Oxygen-containing hetero ring cyclic ketals at positions 16, 17
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00

Definitions

  • the present invention relates to a complex of technetium-99m, to a method of preparing this complex, and to a method of detecting and locating inflammatory lesions and/or processes by using a composition comprising said complex.
  • the invention also relates to a kit for preparing said composition.
  • the detection and location of the inflammation s ⁇ te(s) in an early stage is of the utmost importance.
  • a good diagnostic agent is also indispensable for supporting the therapy used.
  • Various requirements have to be imposed on such a diagnostic agent, for example, non-toxic, no adverse influence on the host resistance and/or therapeutic treatment, well detectable and selective.
  • the required high selectivity means that the diagnostic agent, after having been introduced into the body, must accumulate selectively at the site of the inflammation to be detected.
  • the diagnostic agent should be labelled, preferably with a radionuclide. In that case the radioactive radiation can be detected by using a suitable detector (scanning).
  • the selectivity i.e. the specific accumulation of the labelled agents in the inflamed sites
  • Selectivity is to be understood to mean the target -to-non- target ratio. This means that a comparatively large radioactivity is found in the non-target tissues, e.g. in the blood, as a result of which the image of the inflammation to be examined is obscured.
  • the locating of inflammations is disturbed in case these are present in certain sites of the body.
  • a good imaging in particular in an early stage of the inflammation, is disturbed so that a correct diagnosis is impeded. Therefore there exists a need for agents for diagnosing inflammations with a better selectivity than the above- described labelled immunoglobulins known for this purpose.
  • the present invention relates to a complex of cationic technetium-99m with a stero ⁇ d-21-phosphate dianion of the general formula
  • a and B are both hydrogen atoms or together constitute a C-C bond;
  • R is a hydroxy group or an esterified hydroxy group;
  • R 1 is a hydrogen atom, a hydroxy group or a methyl group
  • R 2 is a hydrogen atom, a methyl group or a halogen atom selected from fluorine and chlorine;
  • R 3 is a hydrogen atom or a halogen atom selected from fluorine and chlorine;
  • R 4 is a hydrogen atom or a methyl group
  • R 5 is a hydrogen atom or a methyl group
  • R 6 is a hydrogen atom or a halogen atom selected from fluorine and chlorine;
  • R and R 1 together constitute a group of the general formula
  • R 7 is a hydrogen atom or a methyl group
  • R 8 is a (C 1 -C 6 )alkyl group
  • R 7 and R 8 together constitute a (C 2 -C 5 )alkylidene group.
  • Disodium salts of the above steroid-21-phosphate esters are known to be effective as anti-inflammatory drugs.
  • the drastic modification of the molecule viz. the complexation with a technetium cation, does not affect the receptor binding affinity.
  • the complexes of the present invention remain stable in the organism to which they have been administered. It is well-known in the art, that phosphate esters are readily hydrolized in the body of a living being. Such an unexpected resistance to hydrolysis permits the desired in vivo application of the complexes of the invention.
  • the high selectivity i.e. the high target-to -non-target ratio
  • the favourable metabolic fate viz. the fast blood clearance and the excretion mainly through the kidneys
  • the technet ⁇ um-99m complex of the invention may exhibit a variety of stoichiometries, depending on the oxidation state of the metal ion, with respect to the dianion ligands.
  • the above complex of cationic technet ⁇ um-99m with a stero ⁇ d-21-phosphate dianion should be interpreted broadly and also includes complexes of technet ⁇ um-99m with mixed ligands, viz. with ligands of formula I mixed with well-known ligands for technetium complexes. Suitable examples of such well-known ligands are: citrate, tartrate, phosphonate, pyrophosphate and glucoheptonate.
  • R means an esterified hydroxy group
  • said hydroxy group may be esterified to a benzoate ester, to a benzoate ester substituted with one or more substituents selected from halogen, methyl and trfluoromethyl, or to a furoate ester.
  • Examples of derivatives of hydrocort ⁇ sone-21-phosphate dianions are dianions of:
  • prednisolone-21-phosphate dianions are dianions of:
  • Preferred complexes of the present invention wherein prednisolone-21- phosphate dianions and derivatives thereof are used as ligands, have the
  • R, R 1 , R 2 and R 3 have the meanings given hereinbefore .
  • the new complexes of the invention can be prepared in a manner known per se for the preparation of related compounds. So the new complexes can be prepared in such a manner, that an at least substantially aqueous solution of a stero ⁇ d-21-phosphate of the general formula
  • M is ammonium or an alka l i metal selected from sodium and potassium, and the other symbols have the meanings given above;
  • the pH of said steroid-phosphate solution is adjusted to approximately 5 by adding a mineral acid or an acidifying buffer, e.g. hydrochloric acid, citrate buffer, acetate buffer or phosphate buffer, after which the reaction is carried out in the presence of a reducing agent, preferably a metallic reducing agent, e.g. Sn(II), Fe(II), Cu (I),) Ti(III) or Sb(III), at a temperature of approx. 0°C to approx. 35 °C, preferably at ambient temperature. So the desired complexes can be prepared in a simple manner and under moderate reaction conditions.
  • a mineral acid or an acidifying buffer e.g. hydrochloric acid, citrate buffer, acetate buffer or phosphate buffer
  • a metallic reducing agent e.g. Sn(II), Fe(II), Cu (I),) Ti(III) or Sb(III
  • compositions suitable for diagnostic purposes are usually processed to compositions suitable for diagnostic purposes.
  • a composition preferably in a sterile form to allow its intravenous administration, comprises, in addition to a pharmaceutically acceptable liquid carrier material and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a complex of cationic technet ⁇ um-99m with a stero ⁇ d-21-phosphate dianion, as defined above.
  • the composition for performing a diagnostic examination the composition, as described above, if desired after dilution with a pharmaceutically acceptable liquid, preferably a physiological saline solution, can be administered to a warm-blooded living being in a quantity sufficient for externally imaging said being to detect and locate an inflammation, in particular inflammatory lesions and/or processes, in the body of said being.
  • the radioactive material is generally administered to the living being in a quantity from 1 to 2000 MBq, preferably from 100 to 1200 Mbq, per 70 kg of body weight.
  • the being is subjected to external imaging to detect accumulated radioactivity and thus to determine the targeted sites in the body of said being in relation to the background activity. Both sterile and asterile inflammations can be detected by using the above compositions.
  • kit' It will be obvious tnat the manipulations necessary to perform the desired reaction should be as sirrpie as possible to enable the user to prepare from the kit the radioactive labelled composition by using the facilities that are at his disposal. Technet ⁇ um-99m in the form of a pertechnetate solution is readily available to the user by eluton of a Mo-Tc generator.
  • the present invention also relates to a kit for preparing a radiopharmaceutical composition, said kit comprising (i) as the substance to be labelled a stero ⁇ d-21-phosphate, as defined hereinbefore, in an optionally dry condition, (ii) a reducing agent, and an aqueous mineral acid or an acidifying buffer, in an optionally combined condition, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with technet ⁇ um-99m in the form of a pertechnetate solution.
  • kit may in addition comprise an inert, pharmaceutically acceptable carrier, e.g. a physiological saline solution, and/or auxiliary substances, such as stabilisers, antioxidants and filling agents.
  • a suitable pertechnetate reducing agent e.g.
  • a dithionite or, preferably, a metallic reducing agent, such as Sn(II), Fe(II), Cu(I), T ⁇ (III) or Sb(III), preferably Sn(II), or a complex-stabilizing reducing agent, preferably a Sn(II) compound, such as Sn(II)-tartrate, Sn(II)-phosphonate or -pyrophosphate, or Sn(II)- glucoheptonate, so that labelling is achieved by ligand exchange.
  • certain ingredients of the kit may be combined, provided they are compatible.
  • the substance to be labelled may be supplied as a solution, e.g.
  • a physiological saline solution in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry state, for example, in the lyophilized condition.
  • a component for an injection liquid it should be sterile, in which, when the constituent is in the dry state, the user should preferably use a sterile physiological saline solution as a solvent.
  • the above-mentioned constituent may be stabilized in a conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitcol , and the like.
  • the pH is adjusted to 5 with 1N HCl, 0.2 ml SnCl 2 .2H 2 O aq. solution (1 mg/ml) is added and mixed well.
  • the mixture is passed through 0.22 ⁇ m Millipore ® membrane filter into a sterile vial.
  • 1-2 ml generator eluate cotaining 370-555 MBq of wm Tc as pertechnetate is added and left to react at room temperature (R.T.) for 10 min.
  • ITLC Chromatography
  • the stability of the prepared complex viz. wm Tc-DMP, is determined after storage at R.T. for 0.5, 1, 3 and 24 h by using the ITLC method above.
  • the labelling efficiency found is very high, viz.>98%, and remains at this level for up to 24 h at R.T.
  • Table A The results are presented in Table A below.
  • Turpentine-induced abscesses are produced in mice according to the method of Thakur et al. (Nucl Med. Biol. 1991, 18, 605-612).
  • 40 Swiss albino mice weighting 20-25 g are injected with 50 ⁇ l turpentine into the right thigh muscle.
  • the biodistribution studies are carried out when the abscess age is 6 days instead of 2 days proposed by Thakur et al., because it is easier to dissect and isolate the abscesses intact.
  • 18 mice are injected with 3.7 MBq wm Tc-DMP, prepared as described in Example I, in 0.2 ml containing 0.2 mg DMP through the tail vein. They are sacrificed in groups of 3 at 1, 3, 6 and 24 h.
  • Static images of all mice are obtained by a gamma camera.
  • the organs and tissues are weighed and counted at the photopeak of wm Tc (140 keV) in the gamma counter against a standard prepared from 1/100 dilution of the injected solution.
  • the percentage uptake of each organ or tissue and % injected dose/g tissue are calculated.
  • the average results are calculated.
  • Bacterial abscesses are induced by the injection of Staphylococcus aureus. 5x10 9 bacteria in 0.1 ml are injected into the right thigh muscle in 20 mice. After 6 days biodistribution studies with wm Tc-DMP are repeated following the same above procedure.
  • the biodistributions of wm Tc-DMP in mice with turpentine- and Staphylococcus-induced abscesses are given in Tables B and C, respectively.
  • the % uptake/g tissue in all the organs are low except for the kidneys, the only organs with appreciable amounts of radioactivity. High levels of radioactivity in urine indicate excretion by the kidneys.
  • the distribution of wm Tc-DMP in the organs is simular in both types of abscesses. Abscess/muscle (A/M) ratios are calculated for both abscess types. The max.
  • A/M ratios for wm Tc-DMP are obtained at 1 h post- injection (4.00 + 1.39 and 4.47 + 1.76 for turpentine- and Staphylococcus-induced abscesses).
  • the A/M ratios remain at about the same level' up to 24 h.
  • the max. abscess/blood (A/B) ratios are 2.11 + 1.10 at 1 h and 4.19 + 2.05 at 6 h for turpentine- and Staphylococcus- induced abscesses, respectively.
  • the scintigrams can be taken any time between 1-6 h post-injection of wm Tc-DMP.
  • the abscesses are well delineated on scintigrams up to 24 h. The best images are obtained at 3 h post-injection. The A/C and A/N ratios increase as a function of age of abscess. The max. values are obtained between 6-10 days for turpentine-induced abscesses.
  • wm Tc-DMP is stable in vivo, has an excretion mainly through the kidneys and has a favourable accumulation in experimentally induced abscesses, allowing its scintigraphic visualization.
  • Experimental arthritis is produced in 10 New Zealand white rabbits (2.5- 3 kg) by intra-articular injection of 1 ml ovalbumin in 0.9% saline (20 mg/ml), emulsified with an equal volume of Freund's incomplete adjuvant as an antigen, into the right front knee according to well-known methods.
  • the contralateral knees are used as control joints.
  • the animals are kept under supervision up to 21 days.
  • scintigrams are obtained 3 h after the injection of 37 MBq wm Tc-DMP in 1 ml containing 1 mg DMP through the ear vein.
  • ROI 's over the arthritic and contralateral knee joints are drawn and the counts are compared.
  • DMP in 1 ml ( 1 mg DMP) is injected through the ear vein.
  • Blood samples (1-2 ml) are obtained from a vein of the other ear at 5 mm, 30 mm, 1, 3, 6 and 24 h. They are counted in the gamma counter against a standard prepared from 1/100 dilution of the injected solution.
  • the blood clearance of wm Tc-DMP found in normal rabbits is biexponential.
  • the clearance half-times are 51 mm and 22 h for the slow and fast components, respectively.
  • Electrophoretic analysis of urine samples shows only two peaks at 0.0 and 20 cm, indicating that wm Tc-DMP is excreted intact without protein- binding or conjugation to another molecule.
  • the amount of wm TcO (20 cm peak) is negligible (3.60 + 3.50 %).
  • Plasma and urine samples obtained from mice at 1 and 3 h, are analyzed by ITLC ( ITLC-strips; acetone or saline) and by electrophoresis (500 V, 2 h) with phosphate buffer (pH 7.5)
  • Freshly drawn human blood is used in the following experiments. 4 ml samples (5) are mixed with 3.7 MBq wm Tc-DMP and incubated at 37°C for 2 h. After centrifugation, the separated plasma and the red blood cells (RBC's) are counted in the gamma counter. The % wm Tc-DMP bound to RBC's is calculated. To check the stability of the erythrocyte binding of wm Tc- DMP, the labelled cells are repeatedly washed with physiological saline. 4 ml saline is added to the RBC's, mixed well, incubated at 37°C for 10 min and centrifuged. The wash solution is removed and counted together with the RBC's. This procedure is repeated six times. The results show that only approximately 10% of radioactivity is bound to RBC's.
  • In vitro protein binding of wm Tc-DMP is determined in plasma samples by protein precipitation with trichloroacetic acid (TCA).
  • TCA trichloroacetic acid

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Abstract

The invention relates to a complex of cationic technetium-99m with a steroid-21-phosphate dianion. The invention also relates to a method of detecting and locating inflammatory lesions and/or processes in the body of a warm-blooded living being by administering to said being a composition comprising, in a quantity sufficient for external imaging, as the active substance said complex of cationic technetium-99m with a steroid-21-phosphate dianion. The invention further relates to a kit for preparing a radiopharmaceutical composition, comprising as the substance to be labelled a steroid-21-phosphate.

Description

Steroid-21-phosphate Complex of Technetium-99M
The present invention relates to a complex of technetium-99m, to a method of preparing this complex, and to a method of detecting and locating inflammatory lesions and/or processes by using a composition comprising said complex. The invention also relates to a kit for preparing said composition.
Inflammations in the body of a warm-blooded living being cause many diseases and disorders, and may even turn out to be life- threatening. In order to be able to achieve a specific therapy, the detection and location of the inflammation sιte(s) in an early stage is of the utmost importance. A good diagnostic agent is also indispensable for supporting the therapy used. Various requirements have to be imposed on such a diagnostic agent, for example, non-toxic, no adverse influence on the host resistance and/or therapeutic treatment, well detectable and selective. The required high selectivity means that the diagnostic agent, after having been introduced into the body, must accumulate selectively at the site of the inflammation to be detected. In order to be detectable from outside the body, the diagnostic agent should be labelled, preferably with a radionuclide. In that case the radioactive radiation can be detected by using a suitable detector (scanning).
Modern techniques in this field use emission tomography; when gamma radiating isotopes are used, the so-called single photon emission computerized tomography (SPECT) may be applied. A number of radiopharmaceuticals has been introduced in nuclear medicine for the scintigraphic visualization of focal inflammatory lesions, such as indium-111 and technetium-99m labelled leukocytes: see e.g. J.G. McAfee et al., Sem. Nucl. Med. 1984, 14, 83-104. It will be evident, that the time-consuming steps involved in cell harvesting and labelling make these agents less attractive for practical application. Therefore, direct intravenous administration of wmTc- or '''In-labelled agents which localize at inflammatory sites, such as anti-granulocyte antibodies and polyclonal humar immunoglobulin, are more recently proposed for the imaging of focal infections: see in this connection the publications of J.G. McAfee in J. Nucl. Med. 1990, 31, 413-416 and of M.C. Pike in J. Nucl Med. 1991, 32, 2034-2036.
However, the selectivity, i.e. the specific accumulation of the labelled agents in the inflamed sites, is still unsatisfactory in practice. Selectivity is to be understood to mean the target -to-non- target ratio. This means that a comparatively large radioactivity is found in the non-target tissues, e.g. in the blood, as a result of which the image of the inflammation to be examined is obscured. Moreover, when a considerable quantity of radioactivity remains circulating, the locating of inflammations is disturbed in case these are present in certain sites of the body. As a result of this a good imaging, in particular in an early stage of the inflammation, is disturbed so that a correct diagnosis is impeded. Therefore there exists a need for agents for diagnosing inflammations with a better selectivity than the above- described labelled immunoglobulins known for this purpose.
Surprisingly it has now been found, that said need can be met by using a new complex of technetium-99m for the above-defined purpose, viz. a complex with a steroid-21-phosphate dianion.
Consequently, the present invention relates to a complex of cationic technetium-99m with a steroιd-21-phosphate dianion of the general formula
Figure imgf000004_0001
wherein: A and B are both hydrogen atoms or together constitute a C-C bond; R is a hydroxy group or an esterified hydroxy group;
R1 is a hydrogen atom, a hydroxy group or a methyl group;
R2 is a hydrogen atom, a methyl group or a halogen atom selected from fluorine and chlorine;
R3 is a hydrogen atom or a halogen atom selected from fluorine and chlorine;
R4 is a hydrogen atom or a methyl group;
R5 is a hydrogen atom or a methyl group; and
R6 is a hydrogen atom or a halogen atom selected from fluorine and chlorine;
or wherein:
R and R1 together constitute a group of the general formula
wherein:
Figure imgf000005_0001
R7 is a hydrogen atom or a methyl group, and
R8 is a (C1-C6)alkyl group; or
wherein:
R7 and R8 together constitute a (C2-C5)alkylidene group.
Disodium salts of the above steroid-21-phosphate esters are known to be effective as anti-inflammatory drugs. However, it is a surprise that the drastic modification of the molecule, viz. the complexation with a technetium cation, does not affect the receptor binding affinity.
Further, it is beyond all expectation, that the complexes of the present invention remain stable in the organism to which they have been administered. It is well-known in the art, that phosphate esters are readily hydrolized in the body of a living being. Such an unexpected resistance to hydrolysis permits the desired in vivo application of the complexes of the invention. In addition, as will also be evident from the accompanying Examples, the high selectivity, i.e. the high target-to -non-target ratio, in combination with the favourable metabolic fate, viz. the fast blood clearance and the excretion mainly through the kidneys, make the complexes of the invention promising tools in the diagnosis of inflammatory lesions.
It is presumed that two of the dianions of the formula I above constitute the ligands for technetιum-99m, thus producing a stable and uncharged complex as defined above. In such a complex, technetium is present in a lower oxidation state than the pertechnetate condition, e.g. in the Tc(V) or Tc(IV) oxidation state, probably forming a technetium-oxo core in the centre of the complex. However, the technetιum-99m complex of the invention may exhibit a variety of stoichiometries, depending on the oxidation state of the metal ion, with respect to the dianion ligands. The above complex of cationic technetιum-99m with a steroιd-21-phosphate dianion, according to the invention, should be interpreted broadly and also includes complexes of technetιum-99m with mixed ligands, viz. with ligands of formula I mixed with well-known ligands for technetium complexes. Suitable examples of such well-known ligands are: citrate, tartrate, phosphonate, pyrophosphate and glucoheptonate.
If the above symbol R means an esterified hydroxy group, said hydroxy group may be esterified to a benzoate ester, to a benzoate ester substituted with one or more substituents selected from halogen, methyl and trfluoromethyl, or to a furoate ester.
Suitable dianions which can be used as ligands for technetιum-99m in the complex of the invention are hydrocortιsone-21-phosphate dianions and derivatives thereof ( A = B = H ) and prednιsolone-21-phosphate dianions and derivatives thereof ( A + B = C-C bond ).
Examples of derivatives of hydrocortιsone-21-phosphate dianions are dianions of:
9α-fluorohydrocortιsone-21-phosphate,
16α-methylhydrocortιsone-21-phosphate, and
16α-methyl-9α-fluorohydrocortisone-21-phosphate,
as well as the corresponding 10-nor- and 19-nor-hydrocortisone-21- phosphate dianions. It has been found, however, that technetium-99m complexes with prednisolone-21-phosphate dianions and derivatives thereof are even more promising than the above hydrocortisone-compounds. Examples of suitable derivatives of prednisolone-21-phosphate dianions are dianions of:
6α-methylprednisolone-21-phosphate,
9α-fluoroprednisolone-21-phosphate,
16α-hydroxy-9α-fluoroprednisolone-21-phosphate,
16α-methylprednisolone-21-phosphate,
16α-methyl-9α-fluoroprednisolone-21-phosphate (dexamethasone-21- phosphate),
16β-methyl-9α-fluoroprednisolone-21-phosphate (betamethasone-21- phosphate),
16,17-(but-1-ylidenedioxy)prednisolone-21-phosphate and corresp. 9a- fluoro compound (budesonide derivatives),
16α-methyl-17α-furoyloxyprednisolone-21-phosphate and corresp.9α-fluoro compound (mometasone derivatives),
16,17-cyclopentylidenedioxyprednisolone-21-phosphate and corresp. 9a- fluoro compound (amcinonide derivatives),
16 ,17-(prop-2-ylidenedioxy)prednisolone-21-phosphate and corresp. 9a- fluoro compound (benetonide derivatives),
6-fluoro-16α-methylprednisolone-21-phosphate and corresp. 9α-fluoro compound (fluocortolone derivatives), and
7α-chloro-16α-methyl-17α-hydroxyprednisolone-21-phosphate and corresp.
9α-fluoro compound (alclometasone derivatives),
as well as the corresponding 10-nor- and 19-nor-prednisolone-21- phosphate dianions.
Preferred complexes of the present invention, wherein prednisolone-21- phosphate dianions and derivatives thereof are used as ligands, have the
Figure imgf000007_0001
wherein R, R1 , R2 and R3 have the meanings given hereinbefore .
From the last-mentioned complexes the most promising are the complexes of cationic technetιum-99m with prednιsolone-21-phosphate dianion , with betamethasone-21-phosphate dianion and with dexamethasone-21-phosphate dianion, as well as with mixed l igands involving citrate , tartrate , phosphonate, pyrophosphate or glucoheptonate ligands .
The new complexes of the invention can be prepared in a manner known per se for the preparation of related compounds. So the new complexes can be prepared in such a manner, that an at least substantially aqueous solution of a steroιd-21-phosphate of the general formula
Figure imgf000008_0001
wherein
M is ammonium or an alka l i metal selected from sodium and potassium, and the other symbols have the meanings given above;
is acidified to a pH between approx. 3 and approx. 7, after which the acidified solution so obtained is reacted, in the presence of a reducing agent, with technetιum-99m in the form of a pertechnetate solution. The preferred complexes of the invention, as defined above, are prepared in a corresponding manner.
Preferably the pH of said steroid-phosphate solution is adjusted to approximately 5 by adding a mineral acid or an acidifying buffer, e.g. hydrochloric acid, citrate buffer, acetate buffer or phosphate buffer, after which the reaction is carried out in the presence of a reducing agent, preferably a metallic reducing agent, e.g. Sn(II), Fe(II), Cu (I),) Ti(III) or Sb(III), at a temperature of approx. 0°C to approx. 35 °C, preferably at ambient temperature. So the desired complexes can be prepared in a simple manner and under moderate reaction conditions.
The new complexes according to the invention are usually processed to compositions suitable for diagnostic purposes. Such a composition, preferably in a sterile form to allow its intravenous administration, comprises, in addition to a pharmaceutically acceptable liquid carrier material and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a complex of cationic technetιum-99m with a steroιd-21-phosphate dianion, as defined above.
For performing a diagnostic examination the composition, as described above, if desired after dilution with a pharmaceutically acceptable liquid, preferably a physiological saline solution, can be administered to a warm-blooded living being in a quantity sufficient for externally imaging said being to detect and locate an inflammation, in particular inflammatory lesions and/or processes, in the body of said being. The radioactive material is generally administered to the living being in a quantity from 1 to 2000 MBq, preferably from 100 to 1200 Mbq, per 70 kg of body weight. Thereupon the being is subjected to external imaging to detect accumulated radioactivity and thus to determine the targeted sites in the body of said being in relation to the background activity. Both sterile and asterile inflammations can be detected by using the above compositions. An example of the former is rheumatoscintigraphy; bacterial inflammations, abscesses and arthritis are examples of asterile inflammations. It is frequently impossible or undesirable to put the ready-for-use composition at the disposal of the user, in connection with the often poor shelf life of the radiolabelled compound. In such cases the user will carry out the labelling reaction with the radionuclide in the clinical hospital or laboratory. For this purpose the various reaction ingredients are then offered to the user in the form of a so-called
"kit' . It will be obvious tnat the manipulations necessary to perform the desired reaction should be as sirrpie as possible to enable the user to prepare from the kit the radioactive labelled composition by using the facilities that are at his disposal. Technetιum-99m in the form of a pertechnetate solution is readily available to the user by eluton of a Mo-Tc generator.
On account of the simple manner in which the complex of the invention can be prepared, such a kit for preparing the complex of the invention is extremely attractive. Therefore, the present invention also relates to a kit for preparing a radiopharmaceutical composition, said kit comprising (i) as the substance to be labelled a steroιd-21-phosphate, as defined hereinbefore, in an optionally dry condition, (ii) a reducing agent, and an aqueous mineral acid or an acidifying buffer, in an optionally combined condition, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with technetιum-99m in the form of a pertechnetate solution.
Such a kit may in addition comprise an inert, pharmaceutically acceptable carrier, e.g. a physiological saline solution, and/or auxiliary substances, such as stabilisers, antioxidants and filling agents. The kit should contain a suitable pertechnetate reducing agent, e.g. a dithionite, or, preferably, a metallic reducing agent, such as Sn(II), Fe(II), Cu(I), Tι(III) or Sb(III), preferably Sn(II), or a complex-stabilizing reducing agent, preferably a Sn(II) compound, such as Sn(II)-tartrate, Sn(II)-phosphonate or -pyrophosphate, or Sn(II)- glucoheptonate, so that labelling is achieved by ligand exchange. If desired, certain ingredients of the kit may be combined, provided they are compatible. The substance to be labelled may be supplied as a solution, e.g. in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry state, for example, in the lyophilized condition. When used as a component for an injection liquid it should be sterile, in which, when the constituent is in the dry state, the user should preferably use a sterile physiological saline solution as a solvent. If desired, the above-mentioned constituent may be stabilized in a conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitcol , and the like. The invention will now be described in greater detail with reference to the ensuing specific examples.
Example I
Preparation of the complex
The disodium salt of dexamethasone-21-phosphate (DMP), in a quantity of 4 mg, is dissolved in 2 ml water, placed in a glass vial. The pH is adjusted to 5 with 1N HCl, 0.2 ml SnCl2.2H2O aq. solution (1 mg/ml) is added and mixed well. The mixture is passed through 0.22 μm Millipore® membrane filter into a sterile vial. 1-2 ml generator eluate cotaining 370-555 MBq of wmTc as pertechnetate is added and left to react at room temperature (R.T.) for 10 min.
The labelling efficiency is determined by impregnated-Thin-Layer-
Chromatography (ITLC), using ready plates of ITLC-SG® and solvents such as saline and methyl ethyl ketone, and by electrophoresis, using 3 buffer systems, viz. veronal, phosphate and acetate buffer (samples of radioactive material in saline). The inability to show any mobility, as opposed to pertechnetate on electrophoresis, indicates that a neutral complex of wmTc-DMP is formed. The complex obtained is highly water soluble.
The stability of the prepared complex, viz. wmTc-DMP, is determined after storage at R.T. for 0.5, 1, 3 and 24 h by using the ITLC method above. The labelling efficiency found is very high, viz.>98%, and remains at this level for up to 24 h at R.T. The results are presented in Table A below.
In a corresponding manner a complex with betamethasone-21-phosphate (BMP), viz. wmTc-BMP, is prepared, also with a high labelling efficiency. Table A
Stability of wmTc-DMP at R.T.
Figure imgf000012_0001
N.B. The above results are average values.
Example II
Animal studies in mice
Turpentine-induced abscesses are produced in mice according to the method of Thakur et al. (Nucl Med. Biol. 1991, 18, 605-612). 40 Swiss albino mice weighting 20-25 g are injected with 50 μ l turpentine into the right thigh muscle. The biodistribution studies are carried out when the abscess age is 6 days instead of 2 days proposed by Thakur et al., because it is easier to dissect and isolate the abscesses intact. 18 mice are injected with 3.7 MBq wmTc-DMP, prepared as described in Example I, in 0.2 ml containing 0.2 mg DMP through the tail vein. They are sacrificed in groups of 3 at 1, 3, 6 and 24 h. Static images of all mice are obtained by a gamma camera. The organs and tissues are weighed and counted at the photopeak of wmTc (140 keV) in the gamma counter against a standard prepared from 1/100 dilution of the injected solution. The percentage uptake of each organ or tissue and % injected dose/g tissue are calculated. The average results are calculated.
Bacterial abscesses are induced by the injection of Staphylococcus aureus. 5x109 bacteria in 0.1 ml are injected into the right thigh muscle in 20 mice. After 6 days biodistribution studies with wmTc-DMP are repeated following the same above procedure.
In order to find the effect of abscess age on the abscess/contralateral (A/C) tissue ratios of wmTc-DMP, 12 mice with turpentine-induced abscesses are i.v. injected with 18.5 MBq wmTc-DMP on 2, 6, 10 and 20 days following turpentine injection and are sacrificed 3 h later. The scintigrams are obtained and the regions of interest (ROI's) over abscess and contralateral tissues are compared.
The biodistributions of wmTc-DMP in mice with turpentine- and Staphylococcus-induced abscesses are given in Tables B and C, respectively. The % uptake/g tissue in all the organs are low except for the kidneys, the only organs with appreciable amounts of radioactivity. High levels of radioactivity in urine indicate excretion by the kidneys. The distribution of wmTc-DMP in the organs is simular in both types of abscesses. Abscess/muscle (A/M) ratios are calculated for both abscess types. The max. A/M ratios for wmTc-DMP are obtained at 1 h post- injection (4.00 + 1.39 and 4.47 + 1.76 for turpentine- and Staphylococcus-induced abscesses). The A/M ratios remain at about the same level' up to 24 h. The max. abscess/blood (A/B) ratios are 2.11 + 1.10 at 1 h and 4.19 + 2.05 at 6 h for turpentine- and Staphylococcus- induced abscesses, respectively. According to biodistribution studies in mice the scintigrams can be taken any time between 1-6 h post-injection of wmTc-DMP. The abscesses are well delineated on scintigrams up to 24 h. The best images are obtained at 3 h post-injection. The A/C and A/N ratios increase as a function of age of abscess. The max. values are obtained between 6-10 days for turpentine-induced abscesses.
From the results it can be concluded, that wmTc-DMP is stable in vivo, has an excretion mainly through the kidneys and has a favourable accumulation in experimentally induced abscesses, allowing its scintigraphic visualization.
Figure imgf000014_0001
Figure imgf000015_0001
Example IV
Animal studies in rabbits
Experimental arthritis is produced in 10 New Zealand white rabbits (2.5- 3 kg) by intra-articular injection of 1 ml ovalbumin in 0.9% saline (20 mg/ml), emulsified with an equal volume of Freund's incomplete adjuvant as an antigen, into the right front knee according to well-known methods. The contralateral knees are used as control joints. The animals are kept under supervision up to 21 days. On 4, 7, 13 and 21 days following the induction of arthritis, scintigrams are obtained 3 h after the injection of 37 MBq wmTc-DMP in 1 ml containing 1 mg DMP through the ear vein. ROI 's over the arthritic and contralateral knee joints are drawn and the counts are compared. On day 4, 4 rabbits are i.v. injected with 37 MBq wmTc-DMP. Scintigrams are obtained at 1 , 3, 6 and 24 h with wmTc-DMP. Again the ROI's over arthritic and contralateral normal knees are compared.
Scintigraphic images of the rabbits demonstrate the arthritic knees very well; wmTc-DMP clearly demonstrates the synovial structures. The arthritic/ normal knee (A/N) ratios, obtained by ROI 's over respective areas on scintigrams, are taken at 96 h following the induction of arthritis. The max. A/N ratio is obtained at 24 h, viz. 1.95 + 0.55. The A/N ratios increase as a function of age of arthritis. The max. values are obtained after 21 days for the arthritic knee joints. Blood clearance of wmTc-DMP is studied in 5 normal rabbits. 10 MBq wmTc-
DMP in 1 ml ( 1 mg DMP) is injected through the ear vein. Blood samples (1-2 ml) are obtained from a vein of the other ear at 5 mm, 30 mm, 1, 3, 6 and 24 h. They are counted in the gamma counter against a standard prepared from 1/100 dilution of the injected solution. The blood clearance of wmTc-DMP found in normal rabbits is biexponential. The clearance half-times are 51 mm and 22 h for the slow and fast components, respectively.
Electrophoretic analysis of urine samples shows only two peaks at 0.0 and 20 cm, indicating that wmTc-DMP is excreted intact without protein- binding or conjugation to another molecule. The amount of wmTcO (20 cm peak) is negligible (3.60 + 3.50 %).
It can be concluded from the above experimental results, that the accumulation of wmTc-DMP in experimentally induced arthritis is favourable for scintigraphic visualization, and that the complex has a fast blood clearance. Example V
In vitro studies
Plasma and urine samples, obtained from mice at 1 and 3 h, are analyzed by ITLC ( ITLC-strips; acetone or saline) and by electrophoresis (500 V, 2 h) with phosphate buffer (pH 7.5)
Freshly drawn human blood is used in the following experiments. 4 ml samples (5) are mixed with 3.7 MBq wmTc-DMP and incubated at 37°C for 2 h. After centrifugation, the separated plasma and the red blood cells (RBC's) are counted in the gamma counter. The % wmTc-DMP bound to RBC's is calculated. To check the stability of the erythrocyte binding of wmTc- DMP, the labelled cells are repeatedly washed with physiological saline. 4 ml saline is added to the RBC's, mixed well, incubated at 37°C for 10 min and centrifuged. The wash solution is removed and counted together with the RBC's. This procedure is repeated six times. The results show that only approximately 10% of radioactivity is bound to RBC's.
In vitro protein binding of wmTc-DMP is determined in plasma samples by protein precipitation with trichloroacetic acid (TCA). To 0.5 ml plasma containing a few kBq wmTc-DMP, 5 ml of a 5% TCA solution are added. 10 min later the mixture is centrifuged. The supernatant is decanted into another tube. 5 ml of TCA solution are added to the precipitate and the mixture is again centrifuged. The washing is repeated 5 times. Both the precipitate and the wash solutions are counted. The fraction of radioactivity precipitated with the proteins is calculated. The results show that approximately 18% of radioactivity in plasma is protein-bound. It appears from the above results, that wmTc-DMP has only a low erythrocyte and protein binding, and is stable in vitro at R.T.

Claims

Claims
1. A complex of cationic technetium-99m with a steroid-21-phosphate dianion of the formula
Figure imgf000018_0001
wherein:
A and B are both hydrogen atoms or together constitute a C-C bond;
R is a hydroxy group or an esterified hydroxy group;
R1 is a hydrogen atom, a hydroxy group or a methyl group;
R2 is a hydrogen atom, a methyl group or a halogen atom selected from fluorine and chlorine;
R3 is a hydrogen atom or a halogen atom selected from fluorine and chlorine;
R4 is a hydrogen atom or a methyl group;
R5 is a hydrogen atom or a methyl group; and
R6 is a hydrogen atom or a halogen atom selected from fluorine and chlorine;
or wherein:
R and R1 together constitute a group of the general formula
wherein:
Figure imgf000018_0002
R7 is a hydrogen atom or a methyl group, and
R8 is a ( C1-C6 ) alky l group ; or
where in : R7 and R8 together constitute a (C2-C5)alkylidene group.
2. A complex as claimed in claim 1, containing mixed ligands of a steroid-21-phosphate dianion, as defined in claim 1, with a ligand selected from the group consisting of citrate, tartrate, phosphonate, pyrophosphate and glucoheptonate.
3. A complex as claimed in claim 1, wherein the steroid-21-phosphate dianion has the formula
Figure imgf000019_0001
wherein R, R1, R2 and R3 have the meanings given in claim 1.
4. A complex as claimed in claim 2, wherein the steroid-21-phosphate dianion has the formula II, shown in claim 2, wherein the symbols have the meanings given in claim 1.
5. A complex as claimed in claim 1, wherein the steroid-21-phosphate dianion is selected from the group consisting of prednisolone-21- phosphate dianion, betamethasone-21-phosphate dianion and dexamethasone- 21-phosphate dianion.
6. A complex as claimed in claim 2, wherein the steroid-21-phosphate dianion is selected from the group consisting of prednisolone-21- phosphate dianion, betamethasone-21-phosphate dianion and dexamethasone-
21-phosphate dianion.
7. A method of preparing a complex as claimed in claim 1,
characterized in that an at least substantially aqueous solution of a steroid-21-phosphate of the formula
Figure imgf000020_0001
wherein
M is ammonium or an alkali metal selected from sodium and potassium, and the other symbols have the meanings given in claim 1;
is acidified to a pH between approx. 3 and approx. 7, after which the acidified solution so obtained is reacted, in the presence of a reducing agent, with technetium-99m in the form of a pertechnetate solution.
8. A method of preparing a complex as claimed in claim 2,
characterized in that an at least substantially aqueous solution of a steroid-21-phosphate of the formula III, wherein M has the meaning given in claim 3, and the other symbols have the meanings given in claim 1, is acidified to a pH between approx. 3 and approx. 7, after which the acidified solution so obtained is reacted, in the presence of a ligand, selected from citrate, tartrate, phosphonate, pyrophosphate and glucoheptonate, and in the presence of a reducing agent, with technetium-99m in the form of a pertechnetate solution.
9. A method as claimed in claim 7 or 8, wherein said steroid-21- phosphate has the formula
Figure imgf000021_0001
wherein :
M has the meaning given in claim 7, and
the other symbols have the meanings given in claim 1.
10. A method as claimed in claim 7 or 8, wherein said steroid-21- phosphate is selected from an ammonium salt, a sodium salt and a potassium salt of a phosphate ester, said phosphate ester being selected from prednisolone-21-phosphate ester, betamethasone-21-phosphate ester and dexamethasone-21-phosphate ester.
11. A method as claimed in claim 7 or 8, wherein the pH of said steroid- phosphate solution is adjusted to approximately 5 by adding a mineral acid or an acidifying buffer, and wherein the reaction is performed in the presence of Sn(II) as the reducing agent at a temperature of approx. 0°C to approx. 35°C.
12. A radiopharmaceutical composition comprising, in addition to a pharmaceutically acceptable liquid carrier material and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a complex of cationic technetium-99m with a steroid-21-phosphate dianion, as claimed in claim 1.
13. A composition as claimed in claim 12, comprising as the active substance a complex of cationic technetium-99m with mixed ligands, as defined in claim 2.
14. A method of detecting and locating inflammatory lesions and/or processes in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising, in a quantity sufficient for external imaging, as the active substance a complex of cationic technetium-99m with a steroid-21-phosphate dianion, as claimed in claim 1, and thereupon (ii) subjecting said being to external imaging to determine the targeted sites in the body of said being in relation to the background activity.
15. A method as claimed in claim 14, wherein a composition is administered comprising as the active substance a complex of cationic technetium-99m with mixed ligands, as defined in claim 2.
16. A kit for preparing a radiopharmaceutical composition, said kit comprising (i) as the substance to be labelled a steroid-21-phosphate of the formula III, as defined in claim 7, in an optionally dry condition, (ii) a reducing agent, and an aqueous mineral acid or an acidifying buffer, in an optionally combined condition, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with technetium-99m in the form of a pertechnetate solution.
17. A kit as claimed in claim 16, comprising in addition to the ingredients defined in claim 16 a ligand, selected from citrate, tartrate, phosphonate, pyrophosphate and glucoheptonate.
18. A kit as claimed in claim 16 or 17, wherein the substance to be labelled is a steroid-21-phosphate of the formula IV, as defined in claim 9.
19. A kit as claimed in claim 16 or 17, wherein the substance to be labelled is a steroid-21-phosphate as defined in claim 10.
PCT/US1992/010312 1992-11-30 1992-11-30 Steroid-21-phosphate complex of technetium-99m WO1994012523A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3989730A (en) * 1972-06-15 1976-11-02 Research Corporation Bone-seeking technetium-99m complex
US3998701A (en) * 1974-01-04 1976-12-21 Lark S.P.A. Process for the preparation of 17-acyl esters of 17α, 21-dihydroxysteroids of the pregnane series and novel products
US4229427A (en) * 1978-06-28 1980-10-21 The Procter & Gamble Company Radioactive scanning agents with hydroquinone stabilizer
US4263290A (en) * 1978-12-13 1981-04-21 Roussel Uclaf Novel 19-nor-steroids

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3989730A (en) * 1972-06-15 1976-11-02 Research Corporation Bone-seeking technetium-99m complex
US3998701A (en) * 1974-01-04 1976-12-21 Lark S.P.A. Process for the preparation of 17-acyl esters of 17α, 21-dihydroxysteroids of the pregnane series and novel products
US4229427A (en) * 1978-06-28 1980-10-21 The Procter & Gamble Company Radioactive scanning agents with hydroquinone stabilizer
US4263290A (en) * 1978-12-13 1981-04-21 Roussel Uclaf Novel 19-nor-steroids

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