AU6463100A

AU6463100A - Radiopharmaceutical products and their preparation procedure

Info

Publication number: AU6463100A
Application number: AU64631/00A
Authority: AU
Inventors: Emmanuel Bellande; Benoit Denizot; Pierre Jallet
Original assignee: Schering AG
Current assignee: Bayer Pharma AG
Priority date: 1999-09-01
Filing date: 2000-08-23
Publication date: 2001-03-26
Also published as: EE200200105A; YU14202A; JP2003508455A; FR2797769A1; CN1371292A; BG106438A; BR0013729A; HK1044893A1; SK2772002A3; MXPA02001923A; IL148076A0; WO2001015746A1; NO20021001L; ZA200201057B; EA200200307A1; HUP0202714A2; NZ517377A; PL353803A1; CZ2002782A3; NO20021001D0

Description

WO01/15746 PCT/IB00/01161 1 RADIOPHARMACEUTICAL PRODUCTS AND THEIR PREPARATION PROCEDURE Technical field The present invention relates to radiopharmaceutical products which can be used for 5 diagnosis or therapy and to their preparation procedure. In particular, it relates to radiopharmaceutical products formed for example from a suspension of particles labelled by a radioactive isotope utilisable 10 in particular for pulmonary scintigraphy, for example in order to establish a diagnosis when a pulmonary embolism is suspected. In this application, the products are used under the form of particles which are preferably spherical in 15 shape and of a size ranging from 10 to 100im. In fact, since the pulmonary capillaries have a diameter of about 7pm, the particles remain blocked in the capillaries after their intravenous injection, which makes it possible to visualise anomalies of pulmonary 20 blood perfusion. Evidently these products must fulfil a certain number of pharmaceutical restrictions. In particular they must have a suitable degradation rate in vivo, that is sufficiently slow to allow imagery to be 25 carried out, for example by a gamma-ray camera, a minimum of about one hour, but also sufficiently rapid so as not to provoke permanent obstruction of the pulmonary capillaries, which could give rise to small thromboses. In addition, these products must not be WO01/15746 PCT/IB00/01161 2 toxic for the organism, they must be able to be sterilised for example by autoclaving or by irradiation, they must be able to be labelled easily with a radioactive metal and be able to be packaged 5 under the form of a stable labelling kit. Prior Art For example, the application for French brevet FR A-2 273 516, deposited in 1975 by the PHARMACIA AKTIEBOLAG Company, resident in Sweden, describes the 10 use of microspheres of amyl pectin reticulated by epichlorhydrin and labelled by a simple mixture with 99 mTc for pulmonary perfusion scintigraphy. These particles present several inconveniences. In fact, only the hydroxyl groupings of amyl pectin used can allow 15 this mixture labelling, and unfortunately they only form weak bonds with technetium and do not make stable labelling possible. In addition, the preparation procedure described uses many solvents and emulsifiers which are difficult to eliminate from the particles 20 prepared. Furthermore, the exact rate of reticulation cannot be measured accurately nor controlled on this particle type. Moreover, this document does not describe the kit compatible with routine utilisation in nuclear 25 medicine. In fact, for an injectable preparation for humans, several manipulations such as adjunction of tin to the sterile flask, a centrifuging, a restoration of suspension, etc. are necessary, which is not compatible with sterility requirements.

WO01/15746 PCT/IB00/01161 3 Finally, the solutions obtained are not stable and the epichlorhydrin used for reticulation is recognised as being very toxic and mutagenic. The inventors demonstrated other defects of these 5 microparticles in the comparative examples 1 and 2 below. The application for French brevet FR-A-2 285 857 deposited in 1975 by the PHARMAGIA FINE CHEMICALS AB Company, resident in Sweden, describes the utilisation 10 of polysaccharide particles linked to different sequestering agents and labelled with the aid of radioactive isotopes. The particles comprise chelating groups linked by covalent bonds to which the radioactive nucleus is linked under the form of chelate 15 type complexes which are principally composed of at least four, and preferably at least five to eight cyclic nuclei with 5 to 6 groups, enclosing the metal, and two metal-coordinating atoms. The polysaccharide is a polysaccharide reticulated chemically, for example by 20 means of epichlorhydrin or epibromhydrin. Leaving the labelling aside, these particles present the same problems as those mentioned previously for the particles described in FR-A-2 273 516. Moreover, this document does not give any examples of labelling with 25 technetium. Further, the labelling procedure comprises heating to 100 0 C in the presence of the radioactive element, a washing and a drying after labelling, which is not at all compatible with the idea of the above mentioned labelling kit and the restrictions of 30 sterility of usage.

WO01/15746 PCT/IB00/01161 4 Even though the labelling method described allows the particles to be labelled in a relatively stable manner, it does not make it possible to prepare a labelling kit which is pharmaceutically acceptable, in 5 particular because it contains epichlorhydrin, and easily usable in a nuclear medicine service. The microspheres described in these two brevet applications are thus not adapted to the pharmaceutical restrictions and they cannot be exploited. Moreover 10 they have never been used for pulmonary scintigraphy. This type of product has been abandoned since. The many researches carried out since 1975 for perfecting new radiopharmaceutical products have concentrated on products based on albumin-serum and its 15 derivatives. These blood products do in fact correspond to pharmaceutical restrictions and can be used in particular for pulmonary scintigraphy. These are the products used at present in nuclear medicine. For example, in 1975, M.A. Davis, in the document 20 "Radiopharmaceuticals N.Y.", 1975, pages 267 to 281, described the radioactive particles intended for the study of pulmonary perfusion. The particles described in this document are macro-aggregates of radio iodinated serum albumin (1 31 I-MAA) or microspheres of 25 denatured human serum albumin labelled with technetium

(

99 mTc-HAM) . The microspheres of 99 mTc-HAM are preferable, because of their uniformity of particle size ranging essentially between 40 and 50 am. Moreover this document describes the general characteristics 30 required for such radiopharmaceutical particles.

WO01/15746 PCT/IB00/01161 5 The document of R. Guiraud "Macro-aggregates and radioactive microspheres", Radiopharmaceuticals, 1997, 519, describes macro-aggregates of albumin (MAA) and microspheres of human serum albumin. It describes the 5 labelling of such micro-aggregates and microparticles with technetium 99m by a solution of stannous chloride. It also notes that the optimum size for the microparticles is 15±5 upm. It mentions organic microspheres of starch. 10 At present, these macro-aggregates and microspheres of human serum albumin labelled with 99 mTc are by far the most utilised in nuclear medicine. However, they present several inconveniences. For example, the variability and quality of batches of 15 human albumin sometimes make preparation of diagnosis kits difficult, containing particles which can vary in size and number. But one of the major inconveniences is their human origin, which can pose serious problems of potential vital contamination of the type HIV, 20 hepatitis, or Creutzfeld-Jacob disease. It would therefore be very interesting to be able to have microspheres labelled with 99 mTc which are not of human origin in order to ensure perfect safety. With this in view, the very recent document of 25 A.C. Perkins, Nuclear Medicine Communications, 1999, 20, 1-3 describes ways of replacing radiopharmaceutical products obtained from blood. In particular it mentions the utilisation of recombinant materials, synthetic polymers and polypeptides. But, this document does not 30 mention polysaccharides.

WO01/15746 PCT/IBOO/011 61 6 Description of the invention The precise aim of the invention is to overcome the inconveniences mentioned above for prior art products, by providing a radiopharmaceutical product 5 being able to be easily labelled, for example with 99 mTc, presenting a very good pulmonary captation which has been demonstrated by inventors for rats, non-toxic, easily biodegradable, easily sterilisable and able to be packaged as a kit ready for labelling, stable and 10 fulfilling the pharmaceutical restrictions for this type of product. These advantages and others will be evident from the following description. The radiopharmaceutical product of the present invention is characterised in that it comprises a 15 polysaccharide provided with sequestering agents linked to the polysaccharide by covalent bonds and chosen among the groups of formulae R-NH-, R-N=, and R-N -N I R' in which R is a hydrocarbonic or aromatic group 20 comprising at least one atom of sulphur, and R' is a hydrogen atom or an alkyl grouping, for example methyl, said sequestering groups forming a chelate type complex with a radioactive metal chosen from among technetium, rhenium, copper, yttrium, erbium, gallium and samarium. 25 The utilisable alkyl groups for R' can be linear or branched, and preferably they have 1 to 5 carbon atoms. According to the invention, the polysaccharide can be soluble, or in the form of microparticles. According WO 01/15746 PCT/IB00/01161 7 to the invention, the polysaccharide can be chosen, for example, from among natural starch, cellulose or reticulated amyl pectin. The natural starch can, for example, be maize 5 starch. The polysaccharide can be in the form of microparticles, for example in the form of microspheres. The present inventors have also demonstrated that 10 modified cellulose according to the present invention offers very good pulmonary captation and an elimination speed slower than with starch. The modified cellulose of the present invention can therefore also be used for radiotherapy, for example with labelling with rhenium, 15 copper, or with one of the above-mentioned metals, since it corresponds to the radiotherapy necessity of using microparticles with a longer half-life. According to the invention, the sequestering groups can be chosen for example from the groups with 20 formulae: s SH -N- N-C -N-N-C

/

R 2

SR

2 N R1

R

3 R R SHI SN C SH and - NH C SH

R

5 n n WO 01/15746 PCT/IB00/01161 8 in which R', R 2 , R 3 , R 4 and R 5 are independently hydrogen atoms, saturated or unsaturated hydrocarbonic groups, carboxylic groups or aromatic groups, Sis an aromatic nucleus possibly containing one 5 or several hetero-atoms, and n is a whole number between 1 and 5. For example, they can be chosen from among the formula groups: -N-NH-C , N-N -C SCHI SCH3

CH

3 S S -N-NH-C , = N- NH-C 10 NH -C 6H 5

NHCH

3 10 WO 01/15 746 PCT/IBOO/01161 9 S /S N -NH -C N -NH -C N (CH) 2

NHCH

2 CH=CH HS SK N -NH -C - NH 0

NH

2 HS - N N - (CH 2 ) 2 -SH

R

4 R4 - NH C SH N C SH

R

5 n

R

5 n N C - SH N- C SH 11 11 11 11 -NH -C and N - C NH NH According to the invention, the microparticles, for example in the shape of microspheres, can be of a dimension from 0.01 to 100 jm, preferably from 10 to 5 50 pimn for diagnosis by pulmonary scintigraphy and from 0.1 to 5 pimn for therapy. According to the invention, the levels of sequestering groups can be from 0.1 to 50% compared to the saccharide patterns of the polysaccharide, 10 preferably from 2 to 15%. According to the invention, in the radiopharmaceutical product, particularly when it is WO01/15746 PCT/IB00/01161 10 used for diagnosis, the radioactive metal can be 99 mTc or gallium-67. This can be the case, for example, when the radiopharmaceutical product is used for pulmonary 5 scintigraphy. According to the invention, in the radiopharmaceutical product, in particular when it is used for therapy, the radioactive metal can be rhenium 186 or 188, copper-64 or 67, yttrium-90, erbium-169 or 10 samarium-153. According to the invention, said radiopharmaceutical product can be in the form of a suspension of microspheres in a physiologically acceptable liquid or in lyophilised form. 15 The present invention also provides a preparation procedure for the radiopharmaceutical product of the invention comprising the following stages: (a) submit a polysaccharide, for example such as those mentioned above, to oxidation controlled by means of 20 a periodate, (b) react the oxidised polysaccharide with a compound containing a primary amine function or hydrazin of formula R-NH 2 or R -N NH R' 25 (c) in which R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, in order to bond in a covalent manner to the polysaccharide with sequestering groups the metals of formulae R-NH-, R- WO01/15746 PCT/IBOO/01161 11 N= or R-NH-N=, and R' is a hydrogen atom or an alkyl grouping, for example methyl, (d) react the polysaccharide comprising the sequestering groups with a salt of a radioactive 5 metal chosen from among technetium, rhenium, copper, yttrium, erbium and samarium. The oxidation controlled by means of a periodate can be that described, for example, in C.L. Mehltretter, "Methods in Carbohydrate Chemistry", vol. 10 IV, 1964, applied in particular to the oxidation of starch, dextrane or cellulose. It is used in the examples given below. According to the invention, the compound containing a primary amine function can correspond to 15 the formula NH 2

-(CH

2 )n-SH with n being a whole number between 1 and 5, and can include a supplementary reducing stage of this compound with sodium borohydride between stages (b) and (c). According to the invention, the compound bonded to 20 the polysaccharide can correspond, for example, to one of the following formulae: WO 01/15746 PCT/IB00/01161 12 S

NH

2 - NH- C NH 2 - N -C

SCH

3 SCH,

CH

3 NH -NH-C , NH -NH -C SNHC6H5 NHCH 3 NH - NH- C NH 2 - NH-C N (CH3) 2 NH - CH2CH=CH 2 S

NH

2 -NH- C NH 2 and

NH

2 SH N- C -SH II II

NH

2 - C N NH According to the invention, the level of sequestering groups fixed on the polysaccharide can be 5 regulated by controlling the level of oxidation of the polysaccharide in stage (a) mentioned above. This oxidation level of polysaccharide can, for example, be between 10 to 50%. The level of the sequestering group can, for example, be between 2 and 15%. 10 In order to allow labelling of the polysaccharide according to the invention, for example with 99 mTc, the inventors have therefore used a two-stage transformation method. This method can be presented as consisting of 15 carrying out a controlled oxidation of the WO 01/15746 PCT/IB00/01161 13 polysaccharide by the periodate in the first stage. Each unit of oxidised glucose thus generates two aldehyde groups in neighbouring positions following the chemical reaction diagram given below:

CH

2 OH CH20 H S10 - O0 0-* OH 0 S OH n --- 0 -n 5 natural glucoside monomer oxidised glucoside monomer The level of oxidation of the polysaccharide can be variable and easily adjusted. In fact, the yield of this oxidation reaction is close to 100% and the level 10 of oxidaton can be calculated from the quantities of periodate added. In general, oxidation levels lower than 50% are used so as to modify the structure of the macromolecule only slightly. The real oxidation level, ranging from 1 to 100%, can easily be determined by a 15 colorometric method. In the second stage, the oxidised polysaccharide is made to react with a molecule containing an amine or hydrazin grouping with the general formula RNH 2 or

RNHNH

2 to form a chelating grouping able to sequester 20 technetium. Thus one obtains a Schiff base type ligand or thiosemicarbazone. This second stage can be summarised as follows: WO 01/15746 PCT/IBOO/011 61 14 R \ #0 C R - NH 2 H aldehyde grouping amine or hydrazin R C -N - R I H functionalised aldehyde grouping with: 1. R=NRi(C=S)SR 2 (Schiff bases issued from 5 dithiocarbazate 2. R=NRi (C=S)NR 2

R

3 (thiosemicarbazones) 3. R= aromatic grouping (aromatic Schiff bases) 4. R= alkyl grouping (alkylic Schiff bases); in this case the Schiff bases are not stable and one carries 10 out a second reduction stage of the C=N bond with borohydride so as to stabilise it and then an amine C-NHR bond is obtained. According to the invention, stage (c) can for example consist of putting into contact the 15 microspheres of polysaccharide comprising the sequestering groups for example with a solution of pertechnetate 99mTcO4- in the presence of a reducing agent, for example stannous chloride. According to the invention, microparticles, for 20 example microspheres, for example maize starch or WO01/15746 PCT/IBOO/01161 15 starch with a base of reticulated amyl pectin can thus be oxidised, then coupled to a molecule containing an amine or hydrazin function, for example S-methyl dithiocarbazate. These particles modified in this way 5 can easily be labelled with, for example, 99 mTc. The present invention thus provides in particular microparticles prepared for example from a base of starch particles, which therefore do not present the inconveniences of the albumin mentioned above. In 10 addition, the starch is described as an excipient in the pharmacopoeia. It is therefore easily available and at low cost. The microparticles of the present invention also have the advantage of being able to be sterilised 15 easily, for example by irradiation, and to be processed under the form of a kit ready for labelling. Moreover, the present inventors have demonstrated according to the present invention that the speed of pulmonary clearance can be modified according to the 20 level of oxidation of the microparticles used in the present invention, which is not possible, for example, with human albumin microspheres. Another advantage of the present invention lies in the simplicity of operation of the procedure: the 25 reaction conditions being very gentle: reactions at ambient temperature, in an aqueous medium, quasi quantitative yields. In addition, the sequestering reactions, for example with technetium, are quantitative; they take place at room temperature and 30 without final purification which makes it possible to adapt to the requirements of sterility and simplicity WO01/15746 PCTIB00/01161 16 of preparation necessary for the utilisation of technetised kits in the hospital environment. The present invention also provides a diagnosis kit which can be used, for example, for pulmonary 5 scintigraphy. This kit comprises: a first flask containing a polysaccharide according to the invention, that is to say provided with sequestering groups linked to the polysaccharide by covalent bonds and chosen among 10 the groups of formulae R-NH-, R-N= and R-N -N I R' in which R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, and in which R' is a hydrogen atom or an alkyl grouping 15 such as methyl. According to the invention, the polysaccharide can, for example, be in the form of microparticles, for example in the shape of microspheres, the microparticles being able to be in lyophilised form or 20 in suspension in a pharmaceutically acceptable liquid. The kit of the present invention can furthermore comprise a second flask containing stannous chloride preferably uin lyophilised form, or also when the polysaccharide is in lyophilised form, for example in 25 the form of microparticles, in the first flask, said first flask can besides contain lyophilised stannous chloride.

WO01/15746 PCT/IBOO/01161 17 The kits of the present invention are stable for at least twelve months as demonstrated in the examples given below. The radiopharmaceutical product of the present 5 invention therefore presents all the qualities required for a use such as radiopharmaceutical usage, for example for scintigraphy of pulmonary perfusion or for radiotherapy. Other advantages will also appear when reading the 10 following examples related to the present invention. EXAMPLES Example 1 A suspension of 10 g of maize starch from the pharmacopoeia is prepared, previously sieved between 10 15 and 40 pm, containing about 10% water, that is 0.055 mole of glucose in 100 ml of water. One adds 0.0168 mole of sodium periodate (0.3 eq) (NaI0 4 ), that is 3.6 g, dissolved in 100 ml of water. The suspension is then stirred for 18 hours at room temperature. The 20 solution is filtered and the oxidised starch is rinsed by 5 times 20 ml of water and then 2 times 50 ml of acetone. The starch is vacuum dried and one obtains 10 g of starch oxidised at 30% (yield = 100%). A suspension of 10 g of starch oxidised at 30% is 25 prepared in 60 ml of a water/ethanol mixture 2/1 by volume. Next one adds 0.1 eq (0.1x0.055x2) that is 0.011 mole of S-methyl dithiocarbazate (NH2NH(C=S)SCH 3 ), M=122, that is 1.34 g, dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room 30 temperature. The solution is next filtered and the WO01/15746 PCT/IBOO/011 61 18 modified starch is washed by 3 times 20 ml of ethanol and then vacuum dried. One thus obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a sulphur content of 5.4%, which 5 corresponds to a coupling level of S-methyl dithiocarbazate (DTCZ) of 7% (7 units dithiocarbazate for 100 theoretical aldehyde functions that is 14 dithiocarbazate units for 100 glucose units). The coupling yield is therefore 70%. One thus obtains 10 g 10 of starch oxidised at 30% and coupled to the DTCZ at 7%. Labelling reaction with 99 mTc 10 mg of starch thus modified are introduced into a flask of the penicillin type. One then adds 4 ml of 15 physiological serum then 10 pg of SnCL 2 , 2H 2 0 (20 pl of a solution at 0.5 mg/ml in HC1 0.1 N). Then 1 ml of a solution of 99 mTcO4- (5mc) is added. The solution is stirred for 15 minutes and the radiochemical purity control (RCP) is carried out by filtration of 1 ml of 20 the solution on a Millipore filter of 0.22 pmn then the filter is rinsed by 2ml of physiological serum. The labelled microspheres are retained on the filter contrary to the radioactive impurities not linked to the microspheres which are found in the filtrate. The 25 radiochemical purity corresponds to: RCP= (activity on the filter/total activity)x100 It is 98.9%.

WO01/15746 PCT/IB00/01161 19 Example 2 Starch modification One proceeds as for example 1 but one uses 0.2 eq of periodate during the oxidation reaction. One obtains a starch oxidised at 20%. 5 One proceeds in the same way as for example 1 for the coupling reaction and one obtains a starch oxidised at 20% and coupled to the DTCZ at 7%. Labelling reaction with 99 mTc One proceeds as for example 1. The radiochemical 10 purity (RCP) is 99%. Example 3 Starch modification One proceeds as for example 1 but one uses 0.1 eq of periodate during the oxidation reaction. One obtains a starch oxidised at 10%. 15 One proceeds in the same way as for example 1 for the coupling reaction and one obtains a starch oxidised at 10% and coupled to the DTCZ at 7%. Labelling reaction with 99 mTc One proceeds as for example 1. The radiochemical 20 purity (RCP) is 98.8%. Example 4 One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of 25 water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of N-methyl-S-methyl dithiocarbazate (NH 2

N(CH

3 ) (C=S)SCH 3 ), M=136, that is 1.50 g dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. The solution WO01/15746 PCT/IB00/01161 20 is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a sulphur content 5 of 5%, which corresponds to a coupling level of the N methyl S-methyl dithiocarbazate of 6.5% (6.5 units dithiocarbazate for 100 theoretical aldehyde functions, that is 13 dithiocarbazate units for 100 units glucose). The coupling yield is thus 65%. 10 Labelling reaction to 99 mTc One proceeds as for example 1. The RCP is 95%. Example 5 Starch modification One proceeds as for example 1 to obtain 10 g of 15 starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of 4-phenyl 3 thiosemicarbazide (NH 2 NH) (C=S)NH(C 6

H

5 ), M=167, that is 20 1.83 g dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus obtains about 10 g of modified starch. The assay of the 25 powder by elementary analysis gives a sulphur content of 3.16%, which corresponds to a coupling level of the 4-phenyl 3-thiosemicarbazone of 8% (8 units thiosemicarbazide for 100 theoretical aldehyde functions, that is 16 thiosemicarbazone units for 100 30 units glucose). The coupling yield is thus 80%. Labelling reaction with 99 mTc WO 01/15746 PCT/IBOO/011 61 21 One proceeds as for example 1. The RCP is 98%. Example 6 Starch modification One proceeds as for example 1 to obtain 10 g of 5 starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of 4-methyl 3 thiosemicarbazide (NH 2 NH) (C=S)NH(CH 3 ), M=105, that is 10 1.15 g dissolved in 10 ml of ethanol. The suspension is shaken for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus obtains about 10 g of modified starch. The assay of the 15 powder by elementary analysis gives a sulphur content of 2.9%, which corresponds to a coupling level of the 4-methyl 3-thiosemicarbazone of 7.3% (7.3 units thiosemicarbazide for 100 theoretical aldehyde functions, that is 14.6 thiosemicarbazone units for 100 20 units glucose). The coupling yield is thus 73%. Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 97%. Example 7 Starch modification 25 One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of 4,4-dimethyl 3 30 thiosemicarbazide (NH 2 NH) (C=S)N(CH 3

)

2 , M=119, that is 1.30 g dissolved in 10 ml of ethanol. The suspension is WO01/15746 PCTIB00/01161 22 stirred for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus obtains about 10 g of modified starch. The assay of the 5 powder by elementary analysis gives a sulphur content of 3%, which corresponds to a coupling level of the 4,4-dimethyl 3-thiosemicarbazone of 7.5% (7.5 units thiosemicarbazide for 100 theoretical aldehyde functions, that is 15 thiosemicarbazone units for 100 10 units glucose). The coupling yield is thus 75%. Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 96%. Example 8 Starch modification 15 One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of 4-allyl 3 20 thiosemicarbazide (NH 2 NH) (C=S)NH(CH 2

CH=CH

2 ), M=131, that is 1.44 g dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. 25 One thus obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a sulphur content of 3%, which corresponds to a coupling level of the 4-allyl 3-thiosemicarbazone of 7.5% (7.5 units thiosemicarbazide for 100 theoretical aldehyde 30 functions, that is 15 thiosemicarbazone units for 100 units glucose). The coupling yield is thus 75%.

WO01/15746 PCT/IB00/01161 23 Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 98%. Example 9 Starch modification 5 One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of 3 10 thiosemicarbazide (NH 2 NH) (C=S)NH 2 , M=91, that is 1 g dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus 15 obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a sulphur content of 2.9%, which corresponds to a coupling level of the 3-thiosemicarbazone of 7.3% (7.3 units thiosemicarbazide for 100 theoretical aldehyde 20 functions, that is 14.6 thiosemicarbazone units for 100 units glucose). The coupling yield is thus 73%. Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 95%. Example 10 25 Starch modification One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq 30 (0.1x0.055x2), that is 0.011 mole of 2-aminothiophenol

(C

6

H

4 ) (NH 2 ) (SH), M=125, that is 1.37 g dissolved in WO01/15746 PCT/IB00/01161 24 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus obtains 5 about 10 g of modified starch. The assay of the powder by elementary analysis gives a sulphur content of 3%, which corresponds to a coupling level of the 2 aminothiophenol of 7.5% (7.5 units aminothiophenol for 100 theoretical aldehyde functions, that is 15 10 aminothiophenol units for 100 units glucose). The coupling yield is thus 75%. Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 94%. Example 11 15 Starch modification One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq 20 (0.1x0.055x2), that is 0.011 mole of 2 mercaptoethylamine (or 2-aminoethanethiol)

(NH

2

CH

2

CH

2 SH), M=91, that is 1 g dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. Next one adds 0.015 mole of sodium 25 borohydride (NaBH 4 ) so as to reduce the Schiff base formed to stabilise it (non-aromatic Schiff bases not being stable) and leaves it to react for 1 hour. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. 30 One thus obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a WO 01/15746 PCT/IB00/01161 25 sulphur content of 3.4%, which corresponds to a coupling level of the 2-mercaptoethylamine of 8.5% (8.5 units 2-mercaptoethylamine for 100 theoretical aldehyde functions, that is 17 2-mercaptoethylamine units for 5 100 units glucose). The coupling yield is thus 85%. Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 95%. Example 12 Starch modification 10 One proceeds as for example 1 to obtain 10 g of starch oxidised at 30%. A suspension is prepared of 10 g of starch oxidised at 30% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq (0.1x0.055x2), that is 0.011 mole of 2-amino 4 15 mercaptotriazole (C 4

N

2 H)(SH) (NH 2 ), M=116, that is 1.27 g dissolved in 10 ml of ethanol. The suspension is stirred for 18 hours at room temperature. The solution is then filtered and the modified starch washed by 3 times 20 ml ethanol and then vacuum dried. One thus 20 obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a sulphur content of 2.8%, which corresponds to a coupling level of the 2-amino 4-mercaptotriazole of 7% (7 units mercaptotriazole for 100 theoretical aldehyde 25 functions, that is 14 mercaptotriazole units for 100 units glucose). The coupling yield is thus 75%. Labelling reaction with 99mTc One proceeds as for example 1. The RCP is 85%. Comparative example 1 30 One uses 10 g of sieved pharmacopoeia maize which has not undergone chemical transformation and one WO01/15746 PCT/IB00/01161 26 caries out the same procedure as in example 1 to label it with 99 mTc. The RCP is 19%. This example is a good demonstration of the fact 5 that the chemical modification (fixation of sequestering groups) carried out according to the invention is certainly necessary to allow labelling with 99 mTc. In addition, one cannot obtain durable pulmonary captation if the microparticles are labelled 10 without prior chemical transformation contrary to the product relating to the present invention. These results are therefore in contradiction with that which is described in FR-A-2 273 516. Example 13 15 Cellulose modification One proceeds as in example 1 but using sieved cellulose between 10 and 40 pm. Thus one obtains 10 g of cellulose oxidised at 30% and coupled to the DTCZ at 7%. 20 Labelling reaction to 99 mTc One proceeds as in example 1. The RCP is 99.1%. Example 14 Sprague Dawley rats weighing about 200 g are anaesthetised with sodium thiopental, and are injected 25 intravenously with different solutions of microspheres labelled with 99 mTC according to examples 1 to 9 and 13. Each animal receives 0.2 ml of solution in the penis vein, that is 0.2 mc per animal. The animals are then placed under a gamma-ray camera and successive static 30 images are shot over a period of 3 hours. One thus obtains successive images after acquiring 15,000 shots WO 01/15746 PCT/IB00/01161 27 per image. Then, manually, one defines the zones of interest in order to estimate the activity present in the different organs 15 minutes after the injection. The results are given in tables I below. 5 Tables I: Results % activity 15 min. after Example 1 Example 2 Example 3 Example 4 Example 5 I.V. % pulmon. 90% 85% 80% 80% 85% activity % hepat. <5% <5% <5% <10% <10% activity pulmon. 2 hours 1 hour 30 mins 2 hours 2 hours half-life % activity 15 min. after Example 6 Example 7 Example 8 Example 9 Example 10 I.V. % pulmon. 85% 85% 85% 85% 90% activity % hepat. <5% <5% <5% <5% <5% activity pulmon. 2 hours 2 hours 2 hours 2 hours > 4 hours half-life 10 One thus notes that the modified microspheres show very good pulmonary captation. In addition, one can modulate the speed of pulmonary elimination by varying WO01/15746 PCT/IB00/01161 28 the oxidation level as shown in examples 1, 2 and 3 (oxidation levels 30, 20 and 10%). The usage of cellulose makes it possible to lengthen the speed of elimination considerably (example 5 10, half-life > 4 hours). Comparative example 2 In this example, natural starch is not used, but microspheres prepared from amyl pectin reticulated by epichlorhydrin as in the patent FR-A-2 273 516. 10 Preparation of reticulated microspheres of starch One dissolves 8 g of maize amyl pectin in 40 ml of a solution containing 4 g of NaOH and 0.15 g of sodium borohydride. The amyl pectin is left for 24 hours to dissolve. Next one prepares an emulsion by stirring 15 60 ml of fluid paraffin and 1.6 g of soy lecithin dissolved in 4 ml of hexane at 800 revs/min. Then one adds the aqueous phase containing the amyl pectin and then 3.2 ml of epichlorhydrin. The emulsion is heated to 55 0 C for 4 hours and then left to be stirred 20 overnight. The microspheres obtained of a size around 50 um are washed by 3 times 250 ml of acetone, dried and then lyophilised. Labelling with 99 mTc One proceeds as for example 1 but using 1 mg of 25 SnC12, 2H 2 0. The RCP is 90%. Starch modification One proceeds as for example 1 but using 10 g of microspheres of amyl pectin reticulated by the epichlorhydrin previously prepared. One thus obtains 10 g of microspheres of amyl pectin oxidised at 30% and 30 coupled to the DTCZ at 7%.

WO 01/15746 PCT/IB00/01161 29 Labelling reaction with 99 mTc One proceeds as for example 1. The RCP is 99%. Example 15 One follows the same operational mode as in 5 example 14 to test the microspheres of reticulated amyl pectin labelled with 99 mTc of the comparative example 2. The results obtained are given in table II below. Table II % activity 15 min. after I.V. Comparative example 2 Example 17 % pulmonary <10% 85% activity % hepatic 70% <5% activity pulmonary - 2 hours half-life 10 One notes that contrary to the description in FR A-2 273 516 the microspheres of reticulated amyl pectin not modified chemically are labelled by 99 mTc but do not present any pulmonary captation, doubtless due to the 15 weak link between 99 mTc and the microspheres. On the other hand, these microspheres transformed chemically by the procedure of the invention demonstrate good pulmonary captation. Example 16 20 Starch microspheres prepared as in example 1 (starch oxidised at 30%, coupled with DTCZ at 7%) are used to produce sterile labelling kits and are ready for labelling with 99 mTc. Sterilisation of the microspheres 10 g of microspheres are introduced into a flask 25 crimped and then irradiated by a source of cobalt-60.

WO01/15746 PCT/TB00/01161 30 The microspheres receive a total dose of gamma radiation of 25kGy over 20 hours. Preparation of the kits 200 mg of sterilised microspheres are introduced in a sterile way into a reactor containing 20 ml of 5 NaCI 9/1000. The solution is de-aerated by nitrogen bubbling and then one adds 400 pl of a sterile solution of SnC1 2 , 2H20 at 0.5 mg/ml in HC1 0.1N. One separates 1 ml of solution for each of 20 flasks, which are then lyophilised and placed in a nitrogen atmosphere. 10 Each flask thus contains: 10 mg of modified microspheres 10 pg of SnC1 2 , 2H20 9 mg of NaCl Labelling reaction with 99 mTc 15 5 ml of Tc0 4 - (5mc) solution is added to a flask in lyophilised form and left to react for 15 minutes. One proceeds as in example 1. The RCP is 98.7%. Kit stability trial Labelling kits prepared as above are stored at different temperatures and then the labelling reaction 20 to 99 mTc is tested so as to evaluate their stability. The results obtained are given in table III below: Table III : RCP (%) Storage temperature 6 months 12 months 2-80 C 98.5% 98.4% 25 0 C 97% 96% 45 0 C 94% 90% 25 One notes the very high stability of the kit stored at a temperature between 2 and 8 0

C.

WO01/15746 PCT/IB00/01161 31 Example 17 Sprague Dawley rats weighing about 200 g are anaesthetised with sodium thiopental, and are then injected intravenously with different solutions of 5 microspheres labelled with 99 mTc as in example 14. Each animal receives 0.2 ml of solution in the penis vein, that is 0.2 mc per animal. The animals are then killed 15 minutes after the injection. Next, the different organs are retrieved, the measurement of radioactivity 10 present in each organ is counted and thus the percentage of activity present in each organ is determined. The results are given in table IV below: Table IV - Results 15 % of the dose injected 15 minutes after injection Organs Example 1 Example 13 Blood (1 ml) 0.1% 0.2% Liver 2.2% 5.6% Kidneys 0.4% 0.4% Lungs 91% 82% Spleen 0.1% 0.1% Intestines 1.5% 0.7% Bladder 0.1% 1.3% One thus notes a very high pulmonary captation whereas as there is weak captation in the other organs for natural starch microspheres as well as reticulated 20 starch base microspheres. The sterile product prepared under kit form thus seems completely adapted to usage as a radiopharmaceutical product for pulmonary perfusion substituting for albumin, and for radiotherapy.

WO01/15746 PCT/IB00/01161 32 Example 18 One uses cellulose oxidised at 30% and coupled with the DTCZ at 7% as in example 13. The cellulose thus modified is labelled with rhenium 186 in order to 5 illustrate the utilisation of the support according to the present invention for therapy. Labelling reaction with Re 186 10 mg of modified cellulose are introduced into a penicillin type flask. One then adds 2 ml of physiological serum and then 20 mg of citric acid and 10 finally 1 mg of SnC1 2 , 2H 2 0 (100 pl of a solution at 10 mg/ml in HC1 0.1 N). Next one adds to the contents of the flask 0.1 ml of ReO4- solution corresponding to an activity of 2 mc. The flask is heated in a water bath at 1000C for 30 15 minutes. The radiochemical purity (RCP) is determined by filtration on a Millipore as in example 1. RCP = 92% In order to prove the stability of the link between rhenium 186 and the cellulose microspheres, a 20 stability test in vitro is carried out. The mixture is incubated with HSA (human serum albumin) at 20 mg/ml at 370C. The following results are obtained: incubation 0 2 hours 6 hours 24 hours 48 hours time RCP 92% 92% 91% 89% 90% 25 These results therefore prove the very high stability of labelling of cellulose microspheres with rhenium 186 and the high stability of the microspheres themselves vis-a-vis HSA.

WO01/15746 PCT/IB00/01161 33 The expert will easily understand that these results can be extrapolated to the utilisation of rhenium 188. Example 19 5 Sprague Dawley rats weighing about 200 g are anaesthetised and then injected with 0.2 ml (0.1 mc) of cellulose microsphere solution labelled with Re 186 as in example 18. The animals are then placed under a gamma-ray 10 camera and images are registered over 48 hours. The activity present in the zones of interest is then calculated as in example 14. Time after injection 1 hour 2 hours 6 hours 24 hours 48 hours % pulmon. 80% 85% 85% 85% 90% activity % hepatic <5% <5% <5% <5% <5% activity 15 These results therefore prove that the activity remains blocked at the pulmonary level for at least 48 hours. This type of microsphere can thus be used for therapy. The most important clinical application can be the 20 treatment of liver cancers after injection, not intravenously but directly into the hepatic artery (metabolic radiotherapy). Another possible application is to inject this type of particle subcutaneously in breast cancer. The 25 particles migrating through the lymphatic system may WO01/15746 PCT/IB00/01161 34 make it possible to treat the sentinel nodes invaded by cancerous cells.

Claims

1. A radiopharmaceutical product comprising a polysaccharide provided with sequestering groups linked to the polysaccharide by covalent bonds and chosen from 5 among the groups of formulae R-NH-, R-N=, and R-N -N R' in which R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, and R' is an atom of hydrogen or an alkyl or methyl grouping, said 10 sequestering groups forming, together with a radioactive metal chosen from among technetium, rhenium, copper, yttrium, erbium, gallium and samarium, a complex of the chelate type, in which the polysaccharide is in the form of microparticles. 15

2. A radiopharmaceutical product according to Claim 1 in which the sequestering groups are chosen from among the groups of formulae: s SH /5 S S R2 S =-NH -N-C =N--N--C / SR 2 NN R I R I R 3 R4 R SH CH N C SH and - NH C SH n n 20 WO01/15746 PCT/IB00/01161 36 in which R', R 2 , R 3 , R 4 and. R 5 are independently atoms of hydrogen atoms, saturated or unsaturated hydrocarbonic groups, carboxylic groups or aromatic groups, 5 is an aromatic nucleus possibly containing one or several heteroatoms, and n is a whole number between 1 and 5.

3. A radiopharmaceutical product according to 10 Claim 2 in which the sequestering groups are chosen from among the groups of formulae: //s / =N-NH-C ,= N-N -C SCHl 3 1SCH 3 SCHCH 3 3 S 1 N-NH-C ,= N - NH -C 15 WO 01/15746 PCT/IB00/01161 37 S /S -N -NH -C , -N -NH-C , N (CH 3 ) 2 NHCH 2 CH=CH 2 HS N -NH -C N -NH O NH 2 2 HS - N N - (CH 2 ) 2 -- SH R4 R4 I- N I S - NH C SH -N C SH R 5 n \R 5 n N C -SH N- C -SH -NH - C and - N - C NH NH

4. A radiopharmaceutical product according to any one of Claims 1 to 3, in which the polysaccharide is chosen from among natural starch, cellulose and 5 reticulated amyl pectin.

5. A radiopharmaceutical product according to any one of Claims 1 to 5, in which the microparticles have a dimension between 0.01 and 100 pm 10

6. A radiopharmaceutical product according to any one of Claims 1 to 5, in which the level of WO01/15746 PCT/IB00/01161 38 sequestering groups is from 0.1 to 50% relative to the saccharide patterns of polysaccharide.

7. Utilisation of a radiopharmaceutical product 5 according to any one of Claims 1 to 6, in which the radioactive metal is 99 mTc or 67Ga to prepare a product intended for diagnosis.

8. Utilisation of a radiopharmaceutical product 10 according to any one of Claims 1 to 6, in which the radioactive metal is rhenium-186 or 188, copper-64 or 67, yttrium 90, erbium 169 or samarium 153, to prepare a drug. 15

9. Utilisation of a radiopharmaceutical product according to any one of Claims 1 to 7, in which the radioactive metal is 9 9mTc to prepare a product intended for pulmonary scintigraphy. 20

10. A radiopharmaceutical product according to any one of Claims 1 to 6, under the form of a suspension of microspheres in a physiologically acceptable liquid or in lyophilised form. 25

11. A procedure for preparation of a radiopharmaceutical product according to any one of Claims 1 to 6, which comprises the following stages: (a) submit a polysaccharide to an oxidation carried out by means of a periodate, WO01/15746 PCT/IBOO/011 61 39 (b) make the oxidated polysaccharide react with a compound containing a primary amine function or hydrazin of formula R-NH 2 or R- N - NH 2 RI R' 5 in which R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, in order to bond in a covalent manner to the polysaccharide with sequestering groups the metals of formulae R-NH-, R-N= 10 or R-NH-N=, and R' is a hydrogen atom or an alkyl or methyl grouping. (c) make the polysaccharide comprising the sequestering groups react with a salt of a radioactive metal chosen from among technetium, rhenium, copper, 15 yttrium, erbium and samarium.

12. A procedure according to Claim 11, in which the compound containing a primary amine function corresponds to the formula NH2-(CH 2 )n-SH with n being a 20 whole number from 1 to 5, and comprising a supplementary stage of reduction of this compound by sodium borohydride between stages (b) and (c).

13. A procedure according to Claim 11, in which 25 the compound bonded to the oxidised polysaccharide corresponds to one of the following formulae: WO 01/15746 PCT/IB00/01161 40 NH 2 - NH- C NH 2 N -C SCH 3 CH 3 SCH 3 NH -NH-C NH- NH -C NHCN H NHCH S S NH 2-NH- C 2 NH--NH -C N (CH )2 NH- CH 2 CH=CH 2 N2 -NH-IC NH)2 an >NH2 SH NH 2-NH- O -N 2 a NH2 R SH N - C-SH II II NH2-- C N NH

14. A procedure according to any one of Claims 11 5 to 13, in which the level of sequestering groups fixed on the polysaccharide is regulated by controlling the level of oxidation of the polysaccharide in stage (a).

15. A procedure according to Claim 14, in which 10 the oxidation level of the polysaccharide is from 10 to 50%.

16. A procedure according to Claim 14, in which the level of sequestering groups is from 2 to 15%. WO 01/15746 PCT/IB00/01161 41

17. A procedure according to any one of Claims 10 to 16, in which the stage (c) consists of putting into contact microspheres of polysaccharide comprising 5 sequestering groups with a solution of pertechnetate 99 mTcO4-, in the presence of a reducing agent.

18. A diagnosis kit which can be used for pulmonary scintigraphy which comprises: 10 a first flask containing a polysaccharide provided with sequestering groups linked to said polysaccharide by covalent bonds and chosen among the formulae groups R-NH-, R-N= and R-N -N 15 15 R' in which R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, and in which R' is an atom of hydrogen or an alkyl or methyl grouping, in which the polysaccharide is in the form of 20 lyophilised microparticles or in suspension in a pharmaceutically acceptable liquid.

19. A kit according to Claim 18 comprising also a second flask containing stannous chloride in 25 lyophilised form.

20. A kit according to Claim 18, in which the polysaccharide being in the form of lyophilised WO01/15746 PCT/IB00/01161 42 microparticles in the first flask, said first flask also contains lyophilised stannous chloride.