NZ517377A

NZ517377A - Radiopharmaceutical products and their preparation procedure

Info

Publication number: NZ517377A
Application number: NZ517377A
Authority: NZ
Inventors: Emmanuel Bellande; Pierre Jallet; Benoit Denizot
Original assignee: Schering Ag
Priority date: 1999-09-01
Filing date: 2000-08-23
Publication date: 2003-08-29
Also published as: IL148076A0; SK2772002A3; FR2797769A1; AU6463100A; CN1371292A; CA2383517A1; EE200200105A; BR0013729A; FR2797769B1; MXPA02001923A; KR20020040799A; CZ2002782A3; NO20021001L; JP2003508455A; NO20021001D0; HK1044893A1; WO2001015746A1; HUP0202714A2; BG106438A; PL353803A1

Abstract

A radiopharmaceutical product comprising a polysaccharide provided with sequestering groups linked to the polysaccharide by covalent bonds and chosen from among the groups of formulae R-NH-, R-N=, and the formula as illustrated where R is a hydrocarbonic or aromatic group comprising at least one atom of sulphur, and R1 is an atom of hydrogen or an alkyl. The sequestering groups form with a radioactive metal a complex of the chelate type, where the polysaccharide is in the form of microparticles. The radioactive metal is chosen from technetium, rhenium, copper, yttrium, erbium, gallium and samarium.

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 517377 51737 WO 01/15746 ■ / W ' * PCT/IB00/01161 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 100pm. In fact, since the pulmonary capillaries have a diameter of about 7vim, 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 23- vQ.r.pJ..: 'y .2., ; BREVATOME :P?irited:26-10-^001 :r_r> «=» h /f m u n I c h ; + 33 «■ + -< — ^51784-]B0001,1 TNTEUECTUALPRO^RtY-f OFFICE OF N.Z. 2 2 MAR 2002 received 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 (equivalent to GB 1 518 813), deposited in 1975 by the PHARMACIA AKTIEBOLAG Company, resident in Sweden, describes 10 the use of microspheres of amylopectin reticulated by epichlorhydrin and labelled by a simple mixture with 99n,Tc for pulmonary perfusion, scintigraphy. These particl'es present several inconveniences. In fact, only the hydroxyl groupings of <amyT.—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 paxticles 2 0 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 v. 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. i WO 01/15746 3 INTEoFEncEAoFPR^z-R^AB00/01161 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. 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 (equivalent to GB 1 518 813). Moreover, this document does not give any examples 25 of labelling with technetium. Further, the labelling procedure comprises heating to 100°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. * available on request The application for French brevet FR-A-2 285 857 WO 01/15746 4 PCT/IB00/01161 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 (131I-MAA) or microspheres of 25 denatured human serum albumin labelled with technetium (99mTc-HAM) . The microspheres of "mTc-HAM are preferable, because of their uniformity of particle size ranging essentially between 40 and 50 pm. Moreover this document describes the general characteristics 30 required for such radiopharmaceutical particles. WO 01/15746 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 microparticies with technetium 99m by a solution of stannous chloride. It also notes that the optimum size for the microparticies is 15±5 pm. It mentions organic microspheres of starch. 10 At present, these macro-aggregates and microspheres of human serum albumin labelled with 99inTc 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. — i It would therefore be very interesting to be able to have microspheres labelled with "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 3 0 mention polysaccharides. WO 01/15746 6 PCTVTBOO/01161 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 99mTc, 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 = | . - - ■ R' --i 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 microparticies. According -«-/ i , • s : '< £ ; omc.V^ i oeo/municn /PrTntf^ ■DESc;;.-r *" "*" ** 5 / '^95OT#IBGi|lf 7 to the invention, the polysaccharide can be chosen, for example, from among natural starch, cellulose or reticulated amyl pectin-. QA(\jilopedx/f\ The natural starch can, for example, be maize 5 starch. The polysaccharide can be in the form of microparticies, 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 microparticies with a longer half-life. According to the invention, the sequestering groups can be chosen for example from the groups with 20 formulae: ' c S SH N N C /K f A A N- N- \ SR2 ^ R2 \X xn ^ R3 •SH gl N /r\ -SH and ■ NH \ R;/n / | \ ■ c - h •SH IPONZ 2 JUL 2003 '2 ■9W 0-?0 WO 01/15746 PCT/IB00/01161 8 in which R1, R2, R3, R4 and R5 are independently hydrogen atoms, saturated or unsaturated hydrocarbonic groups, carboxylic groups or aromatic groups, is 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: ^ S c ^ sch3 N NH C N N SCH-, CH, ^ S = N NH C / N NH C ^ NH c H ^ NHCH3 10 6 5 tPOl\}£ " 2 JUL 2003 WO 01/15746 9 PCT/IB00/01161 :N NH c N(CH3)2 :N NH c i \ NH. :N NH c •NH i \ HS nhch2ch=ch2 -(2) N nh /r\ \R5 L (ch2)2 sh /f\ sh "NH sh \RsL N •nh SH /and = N C N \/ nh nh According to the invention, the microparticies, for example in the shape of microspheres, can be of a dimension from 0.01 to 100 jam, preferably from 10 to 5 50 pm for diagnosis by pulmonary scintigraphy and from 0.1 to 5 um for therapy. According to the invention, the levels of sequestering groups can be from 0.1 to 50% compared to the saccharide units of the polysaccharide, preferably 10 from 2 to 15%. According to the invention, in the radiopharmaceutical product, particularly when it is IPONZ - 2 JUL 2003 N SH WO 01/15746 PCT/IBOO/OII6I 10 used for diagnosis, the radioactive metal can be "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-NH2 or r n nh2 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- WO 01/15746 PCT/IB00/01161 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 NH2 - (CH2) 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 12 pct/iboo/ou61 ^ S NH2 NH c , NH2 N C< | \ CH sch3 i SCH3 ^ S NH2 NH c , NH2 NH c ^ nhc6h5 nhch3 NH2 NH c , NH2 NH C N-N(CH3)2 ^ NH— ch2CH=CH2 NH2 NH C , NH0 and ^ nh2 N C SH II II NH, 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 "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 13 PCT/IB00/01161 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: 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 RNH2 or RNHNH? 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 14 PCT/IBOO/OH61 \ c + R NH2 H aldehyde grouping amine or hydrazin R C = N R I H functionalised aldehyde grouping with: 1. R=NRi (C=S)SR2 (Schiff bases issued from 5 dithiocarbazate -2. R=NRi (C=S)NR2R3 (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 99niTc04~ in the presence of a reducing agent, for example stannous chloride. According to the invention, microparticies, for 20 example microspheres, for example maize starch or ; its: i z ibHtVAir oeo/mun » cn iiiaiwii* 15 starch with a base of reticulated amyl- poo tin can thus p/m^lLoptfbu. 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, "mTc. The present invention thus provides in particular microparticies 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 microparticies of the present invention also have the advantage of being able to be sterilised 15 easily, for example by irradiation, and to be processed tinder 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 microparticies 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 3 0 without final purification which makes it possible to adapt to the requirements of sterility and simplicity ?3I t R mds FmpfanRs7fii+ 93 flirt 1 C • 00 23:10-2001 WO 01/15746 16 PCT7IB00/01161 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' 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 microparticies,-for example in the shape of .microspheres, ; the microparticies 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 microparticies, in the first flask, said first flask can besides contain lyophilised stannous chloride. WO 01/15746 17 PCT/IB00/01161 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 yim, 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) (NaX04),. that is 3.6 g, dissolved in 100 ml" of water. _The suspension is then stirred for 18 hours" at room temperature. The —a. 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 3 0% 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) SCH3) , 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 WO 01/15746 18 PCT/IB0O/O1161 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 "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 ug of SnCL2, 2H20 (20 ul of a solution at 0.5 mg/ml in HC1 0.1 N) . Then 1 ml of a solution of 99mTc04~ (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 ytm then the —i. 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)xlOO It is 98.9%. WO 01/15746 19 PCT/IB00/01161 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 "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 99mTc —i 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 3 0% 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 (NH2N(CH3) (C=S)SCH3) , 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 WO 01/15746 20 PCT/IB00/01161 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 "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 (NH2NHj (C=S)NH(C6H5) , 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 9SmTc WO 01/15746 PCT/IB00/01161 21 One proceeds as for example 1. The RCP is 98%. Example € 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 (NH2NH) (C=S)NH (CH3) , 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 "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 3 0%. 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 (NH2NH) (C=S)N(CH3) 2, M=119, that is 1.3 0 g dissolved in 10 ml of ethanol. The suspension is WO 01/15746 22 PCT/IBOO/OII6I 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 99mTc 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 3 0%. 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 (NH2NH) (C=S)NH(CH2CH=CH2) , M=131, that —j. 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%. WO 01/15746 23 PCT/IBOO/OII6I Labelling reaction with "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 (NH2NH) (C=S)NH2, 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 °f- ' 7.3% - (7.3 units thiosemicarbazide for 100 theoretical aldehyde 20 functions, that is 14.6 thiosemicarbazone units for 100 ~"V ~ ~' units glucose). The coupling yield is thus 73%. Labelling reaction with "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 3 0% in 60 ml of a mixture of water/ethanol (2/1 by volume). Next one adds 0.1 eq 3 0 (0.1x0.055x2), that is 0.011 mole of 2 - amino thiophenol (C6H4) (NH2) (SH) , M=125, that is 1.37 g dissolved in WO 01/15746 PCT/IBOO/OH61 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 "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) (NH2CH2CH2SH) , 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 (NaBH4) 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 2 0 ml ethanol and then vacuum dried. 3 0 One thus obtains about 10 g of modified starch. The assay of the powder by elementary analysis gives a WO 01/15746 25 PCT/IB00/01161 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 99mTc 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 3 0%. A suspension is prepared of 10 g of starch oxidised at 3 0% 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 (C4N2H) (SH) (NH2) , 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 "mTc One proceeds as for example 1. The RCP is 85%. Comparative example 1 3 0 One uses 10 g of sieved pharmacopoeia maize which has not undergone chemical transformation and one wo 01/15746 caries out the same procedu: 2 intellectual property pct/ib00/01 161 OFFICE OF N.Z. 2 2 MAR MM , received n example 1 to label it with 99tnTc. The RCP is 19%. This example is a good demonstration of the fact sequestering groups) carried out according to the invention is certainly necessary to allow labelling with 99mTc. In addition, one cannot obtain durable pulmonary captation if the microparticies 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 (equivalent to GB 1 518 813). 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 20 Labelling reaction to 99mTc 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 v. Q0 labelled with 99mTc 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 3 0 images are shot over a period of 3 hours. One thus obtains successive images after acquiring 15,000 shots 5 that the chemical modification (fixation of 7%. 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 I.V. Example 1 Example 2 Example 3 Example 4 Example 5 % pulmon. activity 90% 35% 80% 80% 85% \ % hepat activity <5% <5% ■ <5% <10% <10% pufmon. half-life 2 hours 1 hour 30 mins 2 hours 2 hours f % activity 15 min. after I.V. Example 6 Example 7 Example 8 Example 9 • Example 10 Exorovfk 43 % pulmon. activity 85% 85% 85% 85% 90% % hepat. activity <5% <5% <5% <5% <5% pulmon. haif-life 2 hours 2 hours 2 hours 2 hours > 4 hours 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 4. B 13345 EE r.ni . . - . ! A A A /Mi 23-10-200 ~ •viAp.-. ; BBEVATOME O n 1 CD fPriritecfc26-,1G _00V ;D£-S.G|' ■■&>■ ■ ;4*vf-i*r l.i INTELLECTUAL PROPERTY OFFICE OF N.Z. * 2 2 MAR 2002 RECEIVED 00951784-IB00011 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 anyIs pectin reticulated by XT' 2.1 epichlorhydrin as in the patent FR-A-2 273 516 (equivalent to GB 1 518 813). 10 Preparation of reticulated microspheres of starch One dissolves 8 g of maize amy^Qpcgtln in 40 ml of a solution containing 4 g of NaOH and 0.15 g of sodium borohydride. The aiuy 10pectin is left for 24 hours to Arri\»*vptcfc/iv 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 amy\Epoetin- and then 3.2 ml of epichlorhydrin. The emulsion is heated to 55 °C for 4 hours and then left to be stirred 2 0 overnight. The microspheres obtained of a size around 50 pm are washed by 3 times 250 ml of acetone, dried and then lyophilised. Label lino with 99n)Tc One proceeds as for example 1 but using 1 mg of 25 Sn£l2, 2H20. The RCP is 90%. Starch modification One proceeds as for example 1 but using 10 g of microspheres of amyio pectin reticulated by the epichlorhydrin previously prepared. One thus obtains 10 g of microspheres of amylopectin oxidised at 30% and 3 0 coupled to the DTCZ at 7%. ,s v.rT.. ; 0REVATOME Printed 26-10 J01 un I cn DESC • 29 i-33 +++++*++++ Qjs , 00951784-IBOOo 111 INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 2 MAR 2002 received Labelling reaction with 99;nTc One proceeds as for example 1. The RCP is 99%. Example 15 One follows the same operational mode as in example 14 to test the microspheres of reticulated amyM-pectin labelled with "Tc of the comparative example 2. The results obtained are given in table II below. Table II % activity 15 min. after I.V. Gompamtr/3 example 2 * Example 17 **" % pulmonary activity • <10% 85% •% hepatic activity 70% <5% - pulmonary half-life • 2 hours 10 15 20 25 One notes that contrary to the description in FR-A-2 273 516 (equivalent to GB 1 518 813) the microspheres of reticulated amylopectin not modified chemically are labelled by 99mTc but do not present any pulmonary captation, doubtless due to the weak link between "raTc and the microspheres. On the other hand, these microspheres transformed chemically by the procedure of the invention demonstrate good pulmonary captation. Example 16 Starch microspheres prepared as in example 1 (Starch oxidised at 3 0%, coupled with DTCZ at 7%) are used to produce sterile labelling kits and are ready for labelling with 99rnTc. Sterilisation of the microspheres 10 g of microspheres are "introduced into a flask crimped and then irradiated by a source of cobalt-60. * cj&rt. ZXaJLujJt X «/XOA^ I PajJt i WO 01/15746 30 PCT/IB00/01161 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 NaCl 9/1000. The solution is de-aerated by nitrogen bubbling and then one adds 400 pi of a sterile solution of SnCl2, 2H2O 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 SnCl2, 2H20 9 mg of NaCl Labelling reaction with 99mTc 15 5 ml of Tc04" (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%. Ki't stability trial" Labelling kits prepared as above are stored at --L different temperatures and then the labelling reaction 20 to "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 2-8° C 98.5% 98.4% 25°C 97% 96% 45° C 94% 90% 25 One notes the very high stability of the kit stored at a temperature between 2 and 8°C. WO 01/15746 31 PCT/IBOO/OII6I 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 99niTc 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. WO 01/15746 32 PCT/IB00/01161 Example 18 One uses cellulose oxidised at 3 0% 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 SnCl2, 2H20 (100 pi 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 Re04_ solution corresponding to an activity of 2 mc. The flask is heated in a water bath at 100°C 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 —i. 20 stability test in vitro is carried out. The mixture is incubated with HSA (human serum albumin) at 20 mg/ml at 37°C. The following results are obtained: Incubation time 0 2 hours 6 hours 24 hours 48 hours 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. WO 01/15746 33 PCT/IB00/01161 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. activity 80% 85% 85% 85% 90% % hepatic activity <5% <5% <5% <5% <5% 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 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO 01/15746 34 PCT/IB00/01161 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 among the groups of formulae R-NH-, R-N=, and R N N-rzr 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, said 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 microparticies. 2. A radiopharmaceutical product according to claim 1 wherein R' is methyl. 3. A radiopharmaceutical product according to claim 1 in which the sequestering groups are chosen from among the groups of formulae: lpONz 2 JUL 2053 WO 01/15746 36 PCT/IB00/01161 in which R1, R2, R3, R4 and R5 are independently atoms of hydrogen atoms, saturated or unsaturated hydrocarbonic groups, carboxylic groups or aromatic groups, or several heteroatoms, and n is a whole number between 1 and 5. 4. A radiopharmaceutical product according to 10 Claim 3 in which the sequestering groups are chosen from among the groups of formulae: 5 an aromatic nucleus possibly containing one = n nh — c n n c sch 3 ch. '3 s s nh c- n nh c 15 nhch IPONZ 2 JUL 2003 37 :N ——NH c "N NH c N(CH3)2 :N NH C 'NIL *NH' ■ nhch2ch=ch2 N (CH2)2 SH / R4' •NH ■SH R 5 /n SH and SH N \ NH NH 5. A radiopharmaceutical product according to any one o£ Claims 1 to 4, in which the polysaccharide is chosen from among natural starch, cellulose and reticulated amylopectin. 10 6. A radiopharmaceutical product according to any one of Claims 1 to 5, in which the microparticies have a diameter between 0.01 and 100 um. 7. A radiopharmaceutical product according to any one of Claims 1 to 6, in which the level of IPONZ 2 JUL 2003 38 sequestering groups is from o.l to 50% relative to the saccharide units of polysaccharide. 8. A radiopharmaceutical product according to any one of claims 1 to 7, in the form of a suspension of microspheres in a physiologically acceptable liquid or in lyophilised form. 9. Utilisation of a radiopharmaceutical product according to any one of Claims 1 to 7, in which the radioactive metal is "mTc or S7Ga to prepare a product intended for diagnosis. 10. utilisation of a radiopharmaceutical product according to any one of Claims 1 to 7, in which the radioactive metal is rheniuiti-186 or 188, copper-64 or 67, yttrium 90, erbium 169 or samarium 153, to prepare a drug. 11. Utilisation of a radiopharmaceutical product according to any one of Claims 1 to 1, in which the radioactive metal is 99mTc- to prepare a product intended for pulmonary scintigraphy. 12. A procedure for preparation of a radiopharmaceutical product according to any one o£ Claims 1 to 7, which comprises the following stages: (a) submit a- polysaccharide to an oxidation carried out by means of a periodate, IPONZ " 2 JUL 2003 WO Oi, 15746 39 PCT/IB00/01161 (b) make the oxidated polysaccharide react with a compound containing a primary amine function or hydrazin of formula R-NH2 or R N NH2 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 metal sequestering groups of formulae R-NH-, R-N= or R-NH-10 N=, and R' is a hydrogen atom or an alkyl, (c) make the polysaccharide comprising the sequestering groups react with a salt of a radioactive metal chosen from among technetium, rhenium, copper, yttrium, erbium and samarium. 15 13. A procedure according to claim 11 wherein R' is methyl. 14. A procedure according to claim 12, in which the compound containing a primary amine function corresponds to the formula NH2- (CH2)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) . 15. a procedure according to Claim 12, in which 25 the compound bonded to the oxidised polysaccharide corresponds to one of the following formulae: IPONZ ~2 JUL iw/3 WO

01/15746 40 PCT/IBOO/OH61 ^ S NH, NH C , NH. N C \sch3 Ihj \sch3 ^3 NH, NH C , NHr—NH C 2 \ 2 \ NHCgHg XNHCH3 ^ S NH2 NH C , NH- NH c ^ N (CH3) 2 . ^NH CH2CH=CH2 NH2 NH C , (M) NH2 and N c SH NH2 C N \ / NH 16. A procedure according to any one of Claims 12 5 to 15, 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). 17- A procedure according to Claim 16, in which 10 the oxidation level of the polysaccharide is from 10 to 50%. 18. a procedure according to Claim 16, in which the level of sequestering groups is from 2 to 15%. IPONZ "2 JUL 2003 WO Ox/15746 41 PCT/IB00/01161 19- a procedure according to any one of Claims 12 to is, in which the stage (c) consists of putting into contact microspheres of polysaccharide comprising 5 sequestering groups with a solution of pertechnetate 99mTcC>4~, in the presence of a reducing agent. 20. 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 = 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, in which the polysaccharide is in the form of lyophilised 20 microparticies or in suspension in a pharmaceutically acceptable liquid. 21. A diagnosis kit according to claim 20 wherein R' is methyl. 25 22. A kit according to claim 20 comprising also a second flask containing stannous chloride in lyophilised form. 23. A kit according to claim 20, in which the polysaccharide being in lyophilised - 2 JUL 2003 WOOl/15746 PCT/IBOO/OH61 42 microparticies in the first flask, said first flask also contains lyophilised stannous chloride. IPONZ -2 JUL 2003 43 24. A radiopharmaceutical product according to claim 1 substantially as herein described or exemplified. 25. A utilisation according to claim 9 substantially as herein described or exemplified. 26. A utilisation according to claim 10 substantially as herein described or exemplified. 27. A utilisation according to claim 11 substantially as herein described or exemplified. 28. A procedure according to claim 12 substantially as herein described or exemplified. 29. A diagnosis kit according to claim 20 substantially as herein described or exemplified. IPONZ 2 M 2003 </div>