EP0750640A1 - Delipidated purified polysaccharide compound d25 preparation, and injectable composition comprising same - Google Patents

Abstract

A preparation of a delipidated purified polysaccharide compound D25 with a molecular weight of around 34 KD, extracted from the membrane proteoglycans of Klebsiella pneumoniae, wherein said polysaccharide compound is delipidated and purified so that an aqueous solution containing the compound no longer comprises any C16 fatty acids and peptides whether free or combined with said compound. The use of said preparation in imaging, given the targeting properties of said compound, is also disclosed.

Description

 PREPARATION OF A DELIPID AND PURIFIED POLYSACCHARIDE COMPOUND D25 AND INJECTION COMPOSITION COMPRISING SAME

The present invention relates to a new compound derived from D25. The present invention also relates to a process for the production of this new compound.

The present invention also relates to preparations of said compound in particular in the form of aqueous solutions.

The present invention also relates to compositions comprising said compound intended for imaging, diagnosis or therapy, in particular of infectious, inflammatory and tumor foci. These are injectable compositions, in particular administered intravenously.

The present invention finally relates to diagnostic kits comprising a colyophilisate of said compound, of a labeling reagent and of a stabilization excipient making it possible to reconstitute an aqueous solution which, by simple mixing with a labeling element, provides compositions according to the invention. invention in which the delipidated and purified derivative of D25 is labeled with a detectable element. The product called D25 is a polysaccharide compound extracted from bacterial membrane proteoglycan. The polysaccharide compound was originally described in patent applications No. 84 13844 and No. 86 06765.

This polysaccharide compound D25 is preferably isolated from a wild, non-pathogenic encapsulated strain of Klebsiella pneumoniae biotype A, deposited in the National Collection of the Pasteur Institute under the number 145-I-IP.

This molecule was initially exploited for its immunostimulatory properties described in patent applications in France No. 84 13844 and No. 86 06765.

In patent application FR 89 03034, the compound D25 has been described as having an affinity for macrophages and constituting a vectoring agent for recognizing and fixing on inflammatory and tumor foci mobilizing macrophages in their immediate environment. Patent application in France No. 89,03034 described the use of D25 in aerosol compositions for scintigraphic imaging by inhalation after labeling with an appropriate radioactive tracer, in particular 99mTc. The D25 molecule had a lipid part which was an element essential to allow it to cross the pulmonary barrier spontaneously.

In these previous patent applications, D25 is described as comprising on the one hand a linear polysaccharide chain constituted by repetition of a monomeric unit of 10 sugars, and on the other hand a single, more complex linking sequence to which are linked two short peptide chains. The repeating monomeric unit of the linear polysaccharide chain is described as containing only galactose in pyran and furan form in the following proportions: 3 β Gai p, 3 α Gai p, 2 β Gai f, and 2 α Gai f. The unique binding structure is attached to the end of the linear polysaccharide chain. It contains glucose, galactose, glucosamine, heptose and mannodeoxyoctulosonic acid residues. The two short peptide chains are linked to this structure.

As described in these previous patent applications, the process for manufacturing D25 actually leads to a product onto which are grafted by N-glycosidic bonds respectively on two glucosamines located in the terminal position of the single binding structure, two β-hydroxymyristic acids representing approximately 1.5% of the mass of D25. These β-hydroxymyristic acids are associated in esterified form or in free form of traces of fatty acid in CI _Ô (palmitic) representing approximately 0.5% of the mass of D25.

It is the presence of these fatty acids which gives the molecule the amphiphilic nature which allows it, by aerosol, to cross the pulmonary barrier by fusing with the alveolar surfactant without requiring the presence of liposomes or without requiring the use of other dosage forms. The free fatty acids in C i ₆ fulfill the role of lipophilic excipient. The presence of a hydrophobic pole on the molecule also plays an essential role in the interaction with the membrane of monocytes and macrophages to allow access to the D25 receptor and its attachment to these cells. The aerosol route, although it allows the detection of pathological foci of millimeter size, has the disadvantage of making it impossible to explore certain anatomical regions which are contaminated either directly by the administration of radio-aerosol ( facial mass) or by the inevitable presence of the product in the digestive tract as a result of swallowing or pulmonary mucocilliary clearance. This drawback of the aerosol form linked to the inhalation technique does not allow for "whole body" imaging.

In addition, the administration of a radio-aerosol poses problems for the large-scale use of such a diagnostic form, taking into account the precautions which must be taken to avoid the risk of false positive results linked to skin contaminations. by the patients themselves.

It is for these reasons that the development of an injectable form was then sought.

However, the highly lipophilic nature of one end of the D25 molecule leads, through the hydrophobic interactions to which it is subjected in aqueous medium, the formation of micelles (or "polymers") with an apparent molecular weight greater than 150 KD. These "polymeric" forms are in equilibrium in D25 solutions with the monomeric form of 34 KD and can represent up to 30% by weight. By aerosol, these micellar aggregates are dissociated in the alveolar surfactant "and it is the free form which then crosses the pulmonary barrier. The systemic distribution allows an adequate imaging of inflammatory, infectious or tumor pathological foci mobilizing macrophages. intravenously, these micelles are very quickly taken up by the reticuloendothelial system of the liver and the spleen where they remain fixed.

Thus, French patent n ° 90 02957 presents modifications carried out on D25 to reduce the presence of micellar forms. The modifications presented in this patent No. 90 02957 mainly concern the selective elimination of esterified fatty acids (C16), not involved in the specific recognition of target cells, in order to reduce the lipophilic nature of the molecule and therefore the formation of micelles. In fact, the D25 derivative described in patent application FR 90 02957 still contains approximately 2% of "polymers" residues so that the injectable compositions can only be by lymphatic route. The existence of a population of macrophages present in normal ganglia and greatly increased during inflammation or tumor proliferation, led in fact to use as targeting agent for this cell type D25 delipidated by locoregional administration, by lymphatic route, thereby allowing maximum detection sensitivity. The defatted D25 is injected by subcutaneous or intra-lymphatic route and is taken in charge by the lymphatic flow then concentrated in the pathological nodes which it allows the visualization. The residual presence of polymers in the lymphatic route does not pose a problem because the filtering effect of the ganglia retains the "polymers" in the first ganglion and only the monomeric form progresses beyond towards the other ganglionic relays then towards the systemic circulation.

The use of D25 in this lymphatic application overcomes the disadvantages of macromolecular compounds such as colloid or nanoparticulate agents used previously, which led to blockage at the level of the first pathological nodes and the impossibility of accessing the entire chain. lymphatic.

Furthermore, in the two patents no. 89 03 094 and no. 90 02957, the labeling of the active principle with a radioactive tracer such as 99mχ _c was obtained extemporaneously via stannous tin (Sn ²⁺ ) either in the form chloride (Sn C12), or fluoride (SnF2). The conditions of labeling and the lack of stability of this labeling of D25 are other reasons which limited its use intravenously. D25 is a stronger 99mJc acceptor than SnF2, however the formation of tin colloids linked to an excess of SnF2 is responsible for the presence of labeled tin colloids. This is not a problem for an aerosol form because the lung functions as a filter for colloids which never reach the systemic circulation. On the other hand, as has been recalled, in the case of intravenous administration, the colloids directly poured into the blood stream are immediately trapped by the liver and the spleen where they persist for a very long time. In addition, the lack of stability of the labeling results in an exchange of Technetium with serum proteins or in a concentration by the thyroid gland of free Technetium. This is completely incompatible with the "whole body imaging objective" for which a homogeneous and rapid diffusion of the product in the body must be obtained while avoiding passive fixation in organs such as the liver and the thyroid.

The aim of the present invention is to provide an intravenous injection composition intended for imaging, diagnosis or therapy, in particular of infectious, inflammatory and tumor foci via the targeting of macrophages. The object of the present invention is therefore more precisely to obtain a new preparation of a compound D25 which improves the biodistribution of said compound by avoiding the hepatosplenic trapping of the micellar forms and which allows its labeling in a stable manner in vivo, in particular by 99m χ.

To do this, the present invention consisted not only in separating the "polymeric" form from the partially defatted D25 described in patent FR 90 02957 in order to keep only the "monomeric" form, but also in modifying the molecule and its environment to obtain a preparation in which the "monomeric" form is stabilized to avoid spontaneous micelle formation, while retaining the targeting properties of compound D25.

The present invention firstly relates to a preparation of a polysaccharide compound D25, defatted and purified, of molecular weight of approximately 34 KD, obtainable by extraction from the membrane proteoglycans of Klebsiella pneumoniae, characterized in that said polysaccharide compound is defatted and purified so that said preparation does not contain micellar forms of said compound. According to another characteristic, the subject of the present invention is a preparation of a polysaccharide compound D25, defatted and purified, with a molecular weight of approximately 34 KD, which can be obtained by extraction from membrane proteoglycans of Klebsiella pneumoniae, characterized in that that said compound is defatted and purified so that said preparation does not contain palmitic fatty acids and peptides free or associated with said compound. The term "preparation" is understood here to mean an aqueous solution of said compound, in particular a pyrogenic injectable solution or a lyophilisate thereof.

The present invention also provides a new derivative of D25 which is chemically modified by defatting and purification, which makes it possible to avoid the "polymerization" of D25 in aqueous solution by reducing its hydrophobic character without however eliminating it completely , in order not to destroy the properties of the product, in particular as regards its specific fixation on macrophages.

The present invention therefore also relates to a defatted and purified polysaccharide compound D25, of about 34 KD, which can be obtained by extraction from the membrane proteoglycans of Klebsiella pneumoniae, characterized in that it can be represented by the formula (I ) next :

[PS]

in which [PS] represents a polysaccharide chain. Analysis of the sequence of the polysaccharide chain made it possible to specify the sequence [PS] as being a linear polysaccharide chain composed by the regular alternation of the two following disaccharide sequences linked by sequences (1 -> 3) repeated approximately 30 times :

- [-> 3) αD Gal _p (1 -> 3) β D Gal _f (1-] ->, - [-> 3) aD Gal _p (1 -> 3) β D Gal _p (1 -] - > at the end of which is a unique binding structure consisting of 3 glucose residues, 1 galactose residue, 2 heptose residues and 1 mannodeoxyoctulosonic acid residue. This unique binding structure is attached to the two terminal glucosamines represented in the formula (I ) to each of which is linked a β-hydroxymyristic acid in the N-glycosidic bond.

The hypothetical formula for this unique binding structure is as follows:

It appears that these two β-hydroxymyristic fatty acids, linked to the terminal diglucosamine by amide bonds, are essential but sufficient to confer the targeting property of macrophages and that the absence of the two associated peptides and of palmitic fatty acids is not not necessary in this regard.

In addition, the quantitative elimination of the fatty acids esterified in Cl 6 and of the peptides made it possible to remove the very weak capacity for activation of the residual complement in the previous forms of delipidated D25. These undesirable residual immunostimulatory properties could be troublesome in patients with severe deficiencies of the complement system. Finally, the elimination of the peptides associated with the compound D25 made it possible to unmask a terminal pyrophosphate group which is responsible for the stability of the labeling with technetium observed with the compound according to the present invention compared to the previous forms of D25. It has been demonstrated by selective elimination of this pyrophosphate group that it was not involved in the specific recognition of cellular receptors, but responsible for the high affinity 99m χ binding during labeling of the compound.

Even after defatting, the compound according to the present invention retains two β-hydroxymyristic fatty acids. Therefore, it retains an amphiphilic nature which can potentially induce the formation of micelles in solution. It appears, however, that the formation of these micelles depends on the ionic environment and that the addition of salts at low concentration makes it possible to definitively oppose the formation of these micelles in aqueous solution.

This is why, advantageously, the preparation of the compound according to the present invention comprises a physiologically acceptable salt, at low molar concentration, so that, in the form of an aqueous solution, this has a low ionic strength. Other salts than NaCl can be used but NaCl appears to be quite suitable because it is the same salt which is used in the operations of labeling with technetium before administration both to ensure isoosmolarity and to elute the technetium from the generator.

The NaCl range in terms of molarity can be quite wide since at 0.15 M (isoosmolarity), there is still no micellization.

In a particular embodiment, the aqueous solution according to the present invention comprises an NaCl salt at a concentration of ImM at 0.15 M.

The process for the preparation of this defatted D25 derivative, as described in the prior art, is characterized in that: a) from bacterial lysates of a gram negative bacterial strain, the crude membrane proteoglycan is extracted, b) the crude proteoglycan from step a) is solubilized by alkaline hydrolysis, and the soluble proteoglycan which is found in the aqueous solution is recovered, c) a polysaccharide compound is isolated and, if necessary, the proteins present in the isolated fraction, d) extracting the free fatty acids associated with said polysaccharide compound using an appropriate organic solvent. Among the gram negative bacteria capable of being used, mention is made of Klebsiella pneumoniae, Serratia marcescens and Escherichia coli; in particular and preferably, as we have seen, Klebsiella pneumoniae which is the subject of a deposit at the National Collection of Cultures of Microorganisms (CNCM) under the n ^β 145-I-IP.

In a particular embodiment of the method described in the prior art, the following steps are carried out: a) from a gram negative bacterial strain, the crude membrane proteoglycan is extracted. This crude proteoglycan is collected in the surganant obtained after centrifugation of the bacterial lysates; b) the crude proteoglycan obtained in step a) is solubilized by alkaline hydrolysis, in particular with an alkaline hydroxyl, preferably sodium hydroxide with a molarity of between 0.3 and 1 M, preferably from 0.5 to 0.75 M at a temperature below 90 ° C, preferably between 50 and 60 ° C, for example 56 ° C, preferably with stirring, the treatment being continued for at least 1 hour; c) after cooling, the suspension is neutralized with an acid, for example hydrochloric acid and, if necessary, an enzymatic hydrolysis of the proteins present in the solution is carried out, in particular by the action of proteinase; d) the polysaccharide compound D25 is then purified by alcoholic precipitation and collected, for example by centrifugation; e) the free fatty acids are extracted using an appropriate organic solvent, for example chloroform or a mixture of chloroform and methanol, for example the precipitate of the polysaccharide compound D25 is dispersed in the organic solvent with stirring and then the solvent is removed, then finally the precipitate is rinsed with the same solvent; f) the precipitate is taken up in water, and the defatted D25 solution according to the invention is clarified by centrifugation, then the supernatant is collected and, finally, g) a new alkaline hydrolysis is carried out, then after cooling, it is neutralized the suspension with an acid, for example hydrochloric acid, and to the solution is added deoxycholate of sodium in a proportion not exceeding 0.5%, then the polysaccharide compound is precipitated, and the precipitate is taken up in water, then the defatted compound D25 obtained is isolated by fractionation or ultrafiltration. The compound according to the present invention was obtained by purification of the defatted compound D25 obtained by the above method, described in patent application FR 90 02957, under the following specific conditions. The deoxycholate treatment in step g), which was intended to prevent or reduce the micellar fraction of the solution obtained after the last alkaline hydrolysis, was replaced by a step of HPLC chromatography of the solution obtained after the second alkaline hydrolysis in weakly saline conditions and with an appropriate chromatographic support, which allow on the one hand to exclude a fraction of apparent mass greater than 100 KD, corresponding to the formation of micelles or aggregates, but above all, on the other hand, avoid hydrophobic interactions with the chromatographic support in order to be able to elute at its theoretical mass of approximately 34 KD a polysaccharide compound according to the present invention.

Other theoretically possible approaches had been attempted to eliminate the polymer such as ultracentrifugation, salting out by divalent cation salts, or ultrafiltration. Only the chromatography under the specific conditions used has made it possible to obtain a solution of D25 in stable "monomeric" form with the defatted and purified derivative according to the present invention, with which no fatty acid in Cl 6 and peptides are no longer associated. .

This method also has other advantages. First of all, HPLC chromatography is more easily transposable on an industrial scale. Furthermore, one of the major critical points in the production of intravenous injectable substances in humans is to rule out any contamination by endotoxins in the finished product. However, D25 comes from a gram negative bacterium rich in LPS, it is itself an analog of LPS detoxified and the reagents (enzymes) and environment of the manufacturing process (premises, materials ...) are likely to introduce this type of contaminant. In this regard, chromatography as it is used in the process according to the present invention, in particular at the end of the process makes it possible to remove these possible contaminants, at the same time as the "polymeric" form. Another advantage of a preparation of the compound according to the invention of low ionic strength is to avoid interactions with the chromatographic support. The increase in the salt concentration generates interactions with the chromatographic support which prevent the elution of the product.

A preparation according to the present invention comprising 20 mM NaCl is particularly advantageous for several reasons:

First of all, it is the concentration which makes it possible to obtain the best resolving power in the chromatography step and therefore, in industrial application, to treat samples of maximum size;

Then, this concentration as a function of the industrial yield of compound according to the invention and of the following stage of manufacture of the corresponding radiopharmaceutical makes it possible to directly obtain the good final concentration of NaCl in the finished product. According to another original aspect of the present invention, the injectable product ready to be labeled can be presented in the form of a single colyophilisate of a compound according to the present invention, a labeling reagent and a stabilizing excipient. This colyophilisate makes it possible to reconstitute an aqueous solution which is ready to be mixed just before use directly with the labeling element proper to provide an aqueous solution of said compound marked with a detectable element. The product in lyophilized form is more stable than in solution. Another essential and original aspect of the present invention is the development of colyophilization. SnF2 is a labeling reagent known to label molecules at 99m Te by its ability to reduce the pertechnetate ion. Its stability in neutral medium makes it compatible with the compound according to the present invention, which is not the case of SnCl ₂ stable only in acid medium where the compound according to the present invention is labile. It is therefore SnF2 which is advantageously selected to mark the compound with

99m Te, but under limiting conditions to prevent or limit the formation of tin colloids.

The presence of ascorbic acid as stabilizing agent increases the stability of the complex between 99m Te at the degree of oxidation + V and the weak acceptor sites of the compound according to the present invention by simultaneously reducing the amount of exchangeable technetium. The weak acceptor sites for D25 are the functional groups exposed on the polysaccharide such as hydroxyl groups.

According to another characteristic, the kit according to the present invention is a single-bottle kit for preparing the product ready for marking, which has many advantages compared to the previous presentation in two bottles:

- ease of use with a simplified marking procedure;

- greater security with less risk of error;

- better product quality for intravenous imaging.

Indeed, the objective of the intravenous route is to carry out a "whole body" imagery by avoiding the useless irradiation of target organs such as the thyroid or the stomach (by 99m Te free) and a troublesome fixation, in particularly from the liver, by capturing labeled tin colloids.

However, the excess of tin spontaneously forms colloids which will become fixed in the liver. It is therefore necessary to use the quantity of SnF2 strictly necessary for labeling. This is unfortunately incompatible with good stability of the radiopharmaceutical and, with the fact that not only the absolute quantity of tin intervenes, but also the instantaneous concentration at the time of labeling. There is therefore a lower limit below which the quality of the labeling is deeply degraded, leading to free technetium which will then become fixed in the thyroid and introducing background noise by its fraction linked to albumin which reduces the specificity and the contrast of the scintigraphic image. A presentation in two bottles, to respect these parameters, requires the use of a tin concentration higher than that which is strictly necessary (two solutions to be mixed); in addition, it increases the number of manipulations at the time of marking and the risk of oxidation of Sn2 + in contact with air which facilitates the formation of colloids.

The single-bottle presentation therefore makes it possible to minimize all of these risks and to reduce the quantity of SnF2 to be used to the strict minimum. All marking operations are thus carried out in the absence of air in the same bottle without transferring solutions. In addition, experience also shows that the stability of the finished product is much better in colyophisate and therefore makes it possible to claim a longer shelf life.

The increase in labeling stability in vivo can be linked to the unmasking of pyrophosphate groups. This stability of in vivo labeling of the compound according to the invention is further improved by the formulation in the presence of ascorbic acid, which increases the stability of the complex between the 99m Te reduced to the degree of oxidation + V, and the acceptor sites of the compound according to the present invention (such as the pyrophosphate group, but also the other acceptor groups).

The present invention therefore relates to compositions intended for imaging, diagnosis or therapy, characterized in that they comprise the compound D25 partially defatted according to the invention.

In a preferred embodiment of the invention, the compositions are intended for imaging and for the diagnosis or therapy of inflammatory and tumor infectious foci by targeting macrophages.

The compositions according to the invention can be injectable by the intravenous route, but also by the lymphatic route, in particular subcutaneous or intrapleural, or administered by aerosol route.

When the compositions are intended for in vivo or ex vivo imaging and diagnosis, the polysaccharide compound according to the invention can be labeled with a detectable element such as a radioactive, paramagnetic or fluorescent element. Said polysaccharide compound can also be detectable indirectly, that is to say it can be detected via a substance itself labeled with a detectable element as mentioned above, said substance recognizing and specifically binding to said polysaccharide compound. As a substance recognizing and specifically binding to said purified polysaccharide compound according to the invention, there may be mentioned an antibody raised against said compound.

The in vivo or ex vivo imaging or diagnostic kits according to the invention will therefore comprise either the polysaccharide compound D25 defatted and purified according to the present invention and means for marking it, or the polysaccharide compound D25 defatted and purified according to the present invention and a substance recognizing said compound and means for labeling said substance. A field of application of the defatted and purified derivative D25 according to the present invention is scintigraphic imaging or intraoperative detection when this agent is coupled to a radioactive isotope. The radionuclides can indeed be detectable by visualization or by intraoperative counting by a manually guided probe. It is then a diagnosis by counting and not imaging. Mention may be made, as suitable radioactive element according to the invention, of a radionuclite detectable by scintigraphy such as technetium Te 99m or alternatively, iodine 123 and indium m. The derivative D25 defatted and purified according to the present invention can also be associated with a paramagnetic agent such as gadolinium, for example to constitute a contrast agent in magnetic resonance imaging (MRI).

More specifically, the injectable compositions comprising defatted and purified D25 according to the present invention are particularly suitable for imaging, diagnosis and therapy of infectious, inflammatory and tumor foci of the lymphatic pathways via the targeting of macrophages.

A lymphoscintigraphic application of the compound according to the invention relates to the imaging of the mediastinal nodes.

The diagnostic applications of the compound according to the invention via the targeting of macrophages relate in particular to the following inflammatory pathologies: primary pulmonary fibrosis, pneumoconiosis, pulmonary fibrosis during connectivities, sarcoidosis, drug-induced pneumopathy, allergic pneumopathy, pneumocystosis, asthma, rheumatic fever, deep infectious foci, system diseases, grafts and implants (biomaterials).

They also relate to the following tumor pathologies, such as the early detection of metastases by targeting recruited macrophages, and the specific early detection of melanoma metastases by targeting specific receptors of the compound according to the invention expressed on these cells such as galactose MAC-2 receptor, an expression which seems to be correlated with a gravity factor. We have indeed identified new specific receptors expressed on melanoma cells, implying another site of the molecule in the binding (poly galactose chain), the hydrophobic pole remaining for its part necessary for the interaction of the compound with the cell membrane . This application, important for specific indications in oncology, had not been highlighted previously.

In targeting melanoma, the compound according to the invention is particularly advantageous since there are no competing products specifically targeting metastases from melanomas. The other imaging techniques give anatomical information (NMR, X-ray CT) but not specific and their sensitivity is currently insufficient to detect metastases of melanoma at a sufficiently early stage to modify the dramatic prognosis of this cancer.

The present invention therefore in particular relates to compositions intended for the imaging and diagnosis of melanoma and other tumors expressing the same receptor, in particular colon cancer.

Finally, given its characteristics allowing the targeting of macrophages and melanoma cells, the compound according to the invention can of course be used for therapeutic purposes as a vectoring agent for targeted radiotherapy, for vectoring cytotoxic substances or else for the vectorization of immunogen and / or immunomodulatory or anti-inflammatory agents.

Finally, the defatted and purified D25 according to the invention may also be used directly as a medicament, in particular taking into account its endotoxin anti-shock properties.

Other therapeutic applications, linked to the targeting properties of the compound according to the invention are appropriate. In some applications, the compound will be labeled with a radioactive element. These are, for example, applications in radiotherapy for localized irradiation with an appropriate emitter of tumor pathological foci fixing the labeled compound. The excessive hepatic fixation of D25 intravenously (the only possible for this type of application) did not allow to consider it.

The present invention also relates to derivatives of the compound D25 defatted and purified according to the present invention and the use of these derivatives in the same applications as those mentioned above. These are oxidized derivatives, the oxidized derivatives of compound D25 defatted and purified according to the present invention being understood to be compounds in which the galactofuranose residues (gai f) of the linear polysaccharide chain have been transformed into arabinose; and amide, ester or ether derivatives, as well as their quaternary ammonium salts and derivatives. The amide, ester or ether derivatives, as well as the quaternary ammonium salts and derivatives which are derivatives of hemisynthesis of D25, have been described in patent applications No. 86 06765 and No. 87 05690. Derivatives oxidized D25 in which the galactofuranose residue (Gai f) of the linear polysaccharide chain of D25 has been transformed into arabinose, have also been described in patent application No. 87 05690.

In the detailed description which will follow in the light of which other characteristics and advantages of the present invention will appear, the compound D25 partially defatted, which has undergone the additional purification and the chemical modification according to the present invention in order to make it compatible with intravenous injection to completely eliminate its pharmacological properties and increase its affinity by a radioisotope, in particular Te, is called "JOO1".

Figure 1 shows the results of binding J001 FITC to human leukocytes.

FIG. 2 represents the results at different doses of the binding of JOOl-biotinylated on the BAL macrophages and the blood monocytes of 23 different individuals (mean values).

Figure 3 represents the cellular distribution of JOOl-biotinylated by binding on BAL macrophages and leukocytes from blood of healthy volunteers (average values). Figures 4 to 6 show the links of J00-1-biolinylated to different melanoma lines. Example 1: process for manufacturing the active ingredient T001

1) Obtaining the clarified bacterial lysate

The biomass of Klebsiella pneumoniae is obtained by culture in a fermenter in a liquid medium, under conventional conditions. The only specific constraint being the blocking of growth at the end of the exponential phase by abrupt cooling to 4 ° C to preserve the biological structures. The biomass is separated from the culture medium by continuous centrifugation at low temperature and washed by centrifugation. The cell concentrate is stored in the freezer while waiting to be processed.

The concentrate is thawed in a reactor and suspended in tris-HCl buffer (10 mM) pH 7.0 containing Mg C12 (10 mM) and NaCl (15 mM) at 4 ° C to have a final concentration equivalent to 50 g of dry cells per liter of suspension. The production of industrial batches uses the equivalent of 1 to 3 kg of dry cells per operation. 5 mg of ase DN are then added per liter of suspension.

The microbial cells are then disintegrated by continuous passage on industrial grinders of the APV - Manton Gaulin type. The bacterial lysate thus obtained is subjected to a first continuous clarification at 15,000 x g on a Sharples separator at 4 ° C to remove the unmilled cells and the grinding residues. The centrifugation pellet is eliminated and the supernatant collected, it constitutes the clarified lysate.

2) Isolation of the membrane fraction

The clarified and acidified bacterial lysate to pH 4.2 +. 0.2 with acetic acid is left to stand for 30 minutes at + 4 ° C. The impurity precipitate is removed by continuous centrifugation at 15,000 xg on a Sharples separator. The opalescent surganant containing the membrane fraction is neutralized by NaOH then dialysis by ultrafiltration at constant volume against distilled water on a membrane cutting at 10,000 Daltons. When the resistivity reaches 1000 Ω.cπr ¹ , the volume is concentrated to reach 6 1 per kg of equivalent in dry starting cells. After assay by the reaction of biuret, the protein titer is adjusted to 10 mg / ml by dilution with distilled water. The membrane suspension of Klebsiella pneumoniae thus obtained will then be used for the extraction and purification of the active ingredient J001.

3) Process for preparing the active ingredient J001 (to obtain an injectable monomeric form)

a) First alkaline hydrolysis This step is intended to dissolve the membranes to allow the isolation of the crude D25 and to hydrolyze most of the esterified fatty acids.

To the membrane suspension obtained above, concentrated sodium hydroxide is added slowly with stirring until a molarity of 0.5 M is reached. The solution is then brought to

56 ° C and maintained for 1 hour with stirring at this temperature.

After rapid cooling to room temperature, the solution is neutralized with hydrochloric acid.

b) Enzymatic hydrolysis

This step is intended to remove protein impurities and to digest the peptidoglycan residues remaining after the first alkaline hydrolysis.

To the neutralized solution, 10 mM tris buffer and EDTA q.s.p. 1 mM. The pH is adjusted to 7.5. An enzymatic digestion is then carried out for two hours at 37 ° C. in the presence of 400 mg / 1 of proteinase K and 100 mg / 1 of lysozyme.

c) Isolation of the crude D25 by alcoholic precipitation The crude J001 is separated by precipitation with 2 volumes of ethanol previously cooled to -20 ° C. After standing for 30 min at + 4 ° C., the precipitate is collected by centrifugation on Sharples. d) Delipidation

This step is intended to extract from the raw D25, the fatty acids previously de-esterified by the first alkaline hydrolysis.

The alcoholic residue is immediately dispersed at room temperature using a homogenizer (of the Turrax type) in a chloroform: methanol mixture (3: 1) at the rate of 1 1 per 1 kg of dry starting cells. (All the volumes indicated are given for 1 kg of dry biomass cells).

The precipitate is collected on sintered glass No. 3 where it is rinsed with 500 ml of the same chloroform: methanol mixture and then dried under a stream of nitrogen. The dry residue is dispersed in 1500 ml of distilled water and then kept stirring overnight at 4 ° C.

The solution for taking up the defatted D25 is clarified by centrifugation at 30,000 x g for 60 minutes and the supernatant dialyzed up to 5000 Ω cm-i on a membrane cutting at 10,000 daltons. The volume is adjusted to 1500 ml with distilled water.

e) Second alkaline hydrolysis

It has multiple objectives at this stage: (1) quantitatively de-esterify the fatty acids in Cl 6 which may remain at this stage of the purification of defatted D25, (2) eliminate the associated peptides, (3) eliminate the micellar forms which may have been forming after defatting, (4) destroying the contaminating endotoxins that may have been introduced during the process, in particular during enzymatic digestions, (5) minimizing the level of residual polymers.

To the previous dialysate, concentrated sodium hydroxide is added to have a final molarity of 0.5M. The solution is then brought to 56 ° C. and kept stirring for 1 hour at this temperature.

After rapid cooling to room temperature, the solution is neutralized with hydrochloric acid.

f) Molecular sieving chromatography

It is carried out under conditions which reduce molecular interactions to prevent the formation of micelles and on a chromatographic support of appropriate porosity, this purification step makes it possible to eliminate in the exclusion peak, all of the residual polymeric forms in the preparation. from J001. The solution neutralized after the second alkaline hydrolysis is balanced into 20 mM NaCl by dialysis on a membrane cutting at 10,000 daltons. When the ion balance is reached, the volume is concentrated to have a final concentration in J001 of 20 to 30 mg / ml. this solution constitutes the sample intended to be deposited on the chromatography column.

The chromatography conditions are as follows: Industrial column in pyrex of 20 cm in diameter molded with Sephacryl S300 gel over a height of 60 cm and balanced in 20 mM NaCl under sterile and pyrogen-free conditions. The deposited sample represents from 3 to 5% of the volume of the gel. Elution is carried out in 20 mM NaCl with continuous UV detection at 206 nm and refractive index. An automatic pilot system makes it possible to directly collect the monomeric form of defatted D25 after elution of the peak of polymers, excluding. The yield of this chromatography step is close to 90% in monomeric forms of J001.

The support used in this step (Sephacryl S300) was not chosen solely for its separation characteristics. It also has the advantage of being able to be decontaminated by high concentration alkaline treatments which are validated for the destruction of endotoxins and also with regard to BSE risks. This is important for use in humans at the level of good manufacturing practices.

g) Sterilization and storage of the active ingredient J001

The fraction collected during the chromatography is kept in

20 mM NaCl to prevent subsequent formation of micelles. For the same reason, it does not undergo an intermediate stage of lyophilization which could promote molecular aggregations. After sterilization by filtration on a 0.22 μm membrane and sampling for quality controls, the solution is kept frozen.

The frozen, sterile and pyrogen-free solution as obtained is a solution of active ingredient J001 which will be used for the production of a colyophilisate containing all the ingredients making it possible to reconstitute an aqueous solution ready for labeling by a radioisotope hereinafter called "J001C". Example 2: Preparation of the T001C

1) J001C formulation strategy (injectable route)

1.1) Basic composition of 1001C

- J001 1.0 mg (active ingredient)

- SnF2 0.080 m g (labeling reagent)

- NaCl 1.0 mg (monomer stabilizer J001Y)

- Ascorbic acid 0.50 m g (stabilizer of labeling with 99m Te)

Limits of variation of the components of the formula:

- J001 0.100 - 2.0 mg

- SnF2 0.040 - 0.1 mg

- NaCl 0.50 - 2.0 mg - Ascorbic acid 0.20 - 0.80 mg

0.040 mg is a critical threshold for labeling because SnF2 does not ensure the stability of the radiopharmaceutical below this dose.

At more than 0.80 mg of ascorbic acid, a fixation of 99m Te is observed on the ascorbic acid.

1.1.1) SnF2 labeling reagent

The technetium obtained from marketed generators is in stable chemical form corresponding to sodium pertechnetate. It must be subjected to an extemporaneous reduction to allow the formation of a complex with the J001 acceptor sites. The strategy for reduction of the pertechnetate anion by stannous tin according to [UN, M., S. (1975) - Labeling of proteins with 99m Te - Radiopharm. Int. Nice. 1974 (eds. Subramanian, Rhodes, Cooper and Saad p.p. 36-48). The Society of Nuclear Medicine, New York)] was selected. At this stage, two important points must be taken into account.

The choice of a tin salt compatible with the stability conditions of J001. Boundary conditions to cause minimal formation of radiolabelled tin colloids. SnC12 is most classically used for the reduction of TC04-, unfortunately to avoid the formation of hydroxide, it must be stabilized in hydrochloric medium at pH <3.5, which is completely incompatible with the stability of J001. In weak acid medium, the lipophilic pole of J001 responsible for the specific binding to macrophagic receptors is hydrolysed, SnF2 has the advantage of being stable at neutral pH. That is why it was selected. The boundary conditions are fixed by physical and biological measurements of the quality of the marking. Were used as methods of investigation and control: the measurement of radiochemical purity by thin layer radiochromatography on paper and HPLC radio which must remain above 97% at least 6 hours after labeling. Diffusion exclusion HPLC chromatography which makes it possible to control the content of colloids. Laser granulometry and multiangular laser scattering which analyze the distributions of particles present. The in vivo study of biodistribution in rats and rabbits which makes it possible to measure the percentage of hepatosplenic fixation (critical thresholds) and the distribution of the product in the organism.

The margins of variation deduced from systematic studies show that whatever the quantity of J001, the minimum, critical quantity of SnF2 to be able to ensure the quantitative reduction of Tc04- is around 20 μg of SnF2 per vial at the time of marking. In the presence of 1 mg of J001, at least 40 μg of SnF2 must be added to ensure radiochemical purity stable at more than 97% for 6 hours and 60 μg of SnF2 for stability of J001C for 2 to 3 years.

The limit margins for variation of SnF2 are therefore: minimum: 40 μg - maximum: 100 μg. The optimum for 1 mg of J001Y is 80 μg of SnF2.

1.1.2) NaCl

NaCl has been introduced into the formulation to stabilize J001 in its monomeric form by reducing molecular interactions and the formation of micelles. Other components such as glycine and phosphate buffer have been studied, glycine is effective in reducing the formation of micelles of J001, but it causes labeling of ascorbic acid with 99m Te which results in non-binding specific to the brain and adrenal capsules. The phosphate buffer cannot be used "in vivo" because it results in bone fixation of the non-specific 99m Te JOO 1 in the metaphyses and diaphyses. NaCl at a concentration of 20 mM is already introduced in the final purification phase of J001 to prevent micellization (ie 1.16 mg NaCl / ml).

The labeling is carried out in physiological serum at 9 V "of NaCl. An amount of 1 mg of NaCl per bottle of J001C is optimal.

1.1.3) Ascorbic acid

Different reducing agents have been tested to stabilize the labeling of J001 Y in vitro and then in vivo in order to limit the exchange of 99m Te between the weak acceptor sites of J001 and the blood proteins. In particular, gentisic acid and ascorbic acid. HPLC radios, performed 30 minutes after injection of the labeled product, on the sera of rats and rabbits, have shown that only ascorbic acid is capable of reducing the exchange of 99m Te by approximately 50%. The dose of ascorbic acid must however remain below 0.80 mg to avoid marking the excess with 99m Te and non-specific fixation in the brain and adrenal capsules. The lower limit in ascorbic acid for a bottle of J001C is around 0.20 mg, a threshold below which the stability of the "in vivo" labeling is degraded.

1.2) Quantity of Tc04 added to 1001 C

It is normally 740 MBq (20 mCi) for a human examination but can be adjusted according to the objectives of the examination. It is a routine operation, according to standard operating procedures, which is performed in all nuclear medicine centers at the time of the examination (1/2 life of 99m Te = 6 hours).

1.3) Examples of studies carried out to evaluate the 1001C formulation Various studies have been carried out to validate the development of the J001C formulation. The techniques used make it possible to evaluate the stability of labeling with 99m Te in vitro and in vivo, the formulation of colloids or aggregates and the distribution of the product in the healthy organism. a) Determination of radiochemical purity 1 hour and 6 hours after labeling by paper radiochromatography.

Determined by radiochromatography on whatman paper in methanol / water 80/20 v), the radiochemical purity always remains greater than 97% for the formulation selected.

b) HPLC radio exclusion-diffusion on TSK 2000 column according to the formulation excipients to control the radiochemical purity, the presence of 99m Te free or fixed with ascorbic acid.

The chromatograms obtained clearly demonstrate that in the presence of glycine phosphate, the radiochemical purity is very insufficient for the peak corresponding to J001 (36.3%) with a transfer of 99m Te on ascorbic acid (54.8%) and the presence of 8.9% of 99m free Te.

c) HPLC radio exclusion-diffusion on TSK 2000 column of rat sera 30 min. after intravenous injection of J001C with or without ascorbic acid. Radiochromatograms of rat sera taken 30 minutes after the intravenous injection of the labeled product without or with ascorbic acid have made it possible to demonstrate the beneficial role of ascorbic acid on the "in vivo" stabilization of labeling with 99m Te. In the presence of ascorbic acid, the fraction of 99m Te free or exchangeable with serum proteins is considerably reduced.

% of total radioactivity

Acid Exchange on ascorbic J001 albumin Tc O4 ^" with 68 26.7 5.3 without 44.4 44.8 10.7 d) Study of biodistribution in rats.

- Biodistribution in healthy animals is carried out in male Wistar rats weighing 250 g (3 animals per batch). The measurements are carried out 30 minutes after the intravenous injection of the labeled product with or without ascorbic acid. The target organs are: the liver and the spleen for fixing colloids, the stomach and the thyroid to detect the free fraction of 99m Te dissociated in vivo, and the blood to evaluate the bioavailable fraction for targeting macrophages.

An example of the results obtained is presented in Table 1 below: Table

% of total radioactivity injected by JOO 1C without J001C with organ ascorbic acid ascorbic acid

Stomach <Q50 <0.50 Thyroid <0.05 0.09 Liver 18.33 10.11 Spleen 2.13 0.49 Whole blood 22.25 47.53

In all cases, the fixation in the liver and thyroid is insignificant and reflects the absence of an appreciable amount of 99m free Te.

The addition of ascorbic acid has a very clear beneficial effect on the reduction of hepato-splenic fixation and the enhancement of the bioavailable fraction in whole blood.

2) Industrial manufacturing process for J001C

This example is given for the manufacture of an industrial batch of the basic formulation (values indicated for 1000 bottles of J001C). The quality of the environment in the work areas must be adapted to the manufacture of sterile and pyrogen-free lyophilized products, in accordance with good manufacturing practices.

All adequate precautions must be taken to protect the stannous tin from oxidation during operations, in particular by sweeping the working areas with inert gases and degassing the solutions beforehand.

1st stage: dissolution

1000 ml of water for injections (ppi) are introduced into a 5 l pyrex reactor, followed by bubbling with nitrogen gas sterilized by filtration in order to degas the medium with permanent stirring.

The bubbling and the stirring are successively dissolved: 420 mg of NaCl then 500 mg of ascorbic acid and finally 80 mg of SnF2.

The bubbling is maintained for a few minutes and then the gas inlet is placed above the surface in order to have a sweep and no longer a bubbling. The amphiphatic properties of J001 when it was introduced under bubbling would result in an abundant production of foam which would disturb subsequent operations.

500 ml of a solution of J001 containing 2 mg / ml of J001 are then introduced with 20 mM NaCl previously degassed under vacuum. Continue gentle agitation to homogenize while maintaining the nitrogen sweep.

2nd stage: sterilizing filtration

The solution obtained above is immediately sterilized by filtration on a 0.22 μm membrane under nitrogen. The integrity of the filter must be checked after filtration.

3rd stage: distribution in vials (in sterile block)

15 ml glass vials, previously sterilized, are placed in stainless steel freeze-drying trays. Liquid nitrogen is introduced into the trays to allow instant freezing and create an inert atmosphere. The bottles are purged by injecting nitrogen or preferably argon (more dense) before the introduction of the solution. 2 ml of solution are then introduced into each bottle and the fluted caps placed on the bottles. The trays are then immediately transferred to the pre-cooled tank of the freeze dryer.

4th step: freeze-drying

A critical point of the process relates particularly to the lyophilization stage due to the nature of the components and in particular of the ascorbic acid. The brutal freezing in liquid nitrogen guarantees the later stability of the product but it does not allow to properly control the forms of crystallization and leads to poor lyophilization of the product.

A specific optimization had to be carried out showing the importance of respecting a preliminary stage before putting under vacuum. Maintaining the vials at a constant temperature close to -45 ° C for two hours ensures that the crystal form is balanced and allows correct lyophilization in the presence of ascorbic acid.

The lyophilization is then carried out under usual conditions taking into account the eutectic points of the mixture.

At the end of lyophilization, the bottles are capped under vacuum in the enclosure of the lyophilizer before the latter is opened. They are then crimped and stored away from light.

Example 3: Targeting Properties of Macrophages

J001 has the ability to selectively bind to monocytes and macrophages via its lipophilic end. Indeed, the selective elimination of this part of the molecule leads to a total loss of the binding capacities.

The binding capacity of J001 to leukocytes in human blood in vitro has been widely studied by cytofluorometry, using fluorescent derivatives of J001 and specific antibodies. The results are shown in Figure 1.

At concentrations below 5 μg / ml, J001 specifically binds to human monocytes. The link of J001 increases with maturation in macrophages and then with activation induced by phorbol esters or gamma interferon. The binding specificity was confirmed by competitive tests with the unlabeled JOOl. The murine inflammatory macrophages obtained after thioglycolate injection have a higher JOOl binding capacity than resident macrophages. At 37 ° C, the bound JOOl is very quickly internalized in cells via its receptors.

The role of membrane antigens as JOOl receptors on monocytes was explored using biotinylated JOOl in the absence of serum. To characterize these receptors, competitive binding studies with different monoclonal antibodies specific for the monocyte membrane antigens were carried out.

Preincubation of cells with Class I or Class II anti-HLA, anti-gamma III FRC (C16), anti-CDl la, anti-CDl lc, or anti CD I l / CD 18 β chain integrins does not cause specific inhibition of JOOl-biotinylated binding. On the other hand, a significant inhibition is observed when the cells are pre-incubated with anti CD14 or antiCDl lb (53% and 41% respectively) with a precise epitopic restriction because all the monoclonal antibodies against CD1 lb or CD14 cannot not inhibit JOOl fixation. The combination of the CD1b and CD14 antibodies results in an increase in inhibition to 70%. The results are presented in Table 2 below, which represents the inhibition of the specific binding of JOOl - biotinylated to monocytes of human blood by monoclonal antibodies. In Taleau 2, "ΔMFI" corresponds to the measurement of the mean fluorescence intensity corrected for "background noise"("Mean Fluorescence Intensity").

Table 2

Percentage Link

Antibody Specific antigen for JOOl-Biot membrane monoclonal inhibition binding

Δ specific MFI

Middle witness - 41.6 -

IOT 16 CDlla 42.8 0

Anti MAC-1 CDllb 24.4 41

OKM1 u 38.3 8

Anti-Leu 15 u 41.0 1

Anti-Leu M5 CDllc 38.7 7

IOMc u 41.7 0

BL5 CD18 38.1 8

GQ3 α 42.5 0

IOM2 CD14 19.4 53

BA.8 α 41.4 0

MAC-1 + IOM2 CDllb CD14 11.8 72

3G8 CD16 42.9 0 (RFcgamma III

W6.32 42.9 0

HLA - Class I

BL2 41.2 1

HLA - Class II The results clearly indicate that the specific binding of JOOl to monocytes does not take place via the β chain CD11 / CD18 common in the family of adhesion molecules nor by the α chain of LFA-1 since the binding does not is not inhibited by anti CD1 la, anti CD 1 le and anti CD18. However, the α chain of CR3 (CDl lb) and the CD14 molecule participate in the binding of JOOl to monocytes.

In order to confirm these results, clinical studies have made it possible to evaluate the binding of JOOl to cells derived from brochoalveolar lavage and human blood originating from patients suffering from various pulmonary inflammatory pathologies.

The binding to human leukocytes was studied by direct cytofluorometry using biotinylated JOOl at three different concentrations, in the presence of an excess (x 10-fold) of unlabeled JOOl.

The results confirm that the binding is concentration dependent and totally inhibited in the presence of an excess of unlabeled JOOl. It is more intense on bronchoalveolar lavage cells than on less differentiated monocytes.

Binding to macrophage monocytes is always much higher than that observed with other cell types. The results are presented in Figures 2 and 3.

EXAMPLE 4 Targeting Properties of Melanoma Cells

1) Cells and culture conditions

Eight lines or clones of human melanomas were used. Three cell lines were isolated from a metastasized lymph node in 3 patients (1, 2 and 3) as described previously (Jacubovich and Dore, 1979). 2 and 3 have a high potential to develop tumors while 1 has a very low potential in nude mice (Berthier-Vergnes et al., 1985). Clones 4, 5, 6, 7 and 8 were all derived from the 3 cell lines. Cell line 8 has a low metastatic potential while that of the other 5 clones is high in the immunocompromised newborn rat. (Bailly and Dore, 1991). These cells were cultured in Mac Coy's modified medium (SIGMA, St. Qμentin Fallavier, France), containing 10% fetal calf serum and eliminated with a buffer of Trypsin PBS-EDTA- (1/5000).

2) Bibliography

- Jacubovich R. and Doré J.F. - Tumor associated antigens in culture médium of malignant melanoma cell strains - 1979 - Cancer Immunol. Immunother. 7, 59-64.

- Berthier-Vergnes O. Portoukalian and Doré J.F. - Expression of cell surface glycoproteins in human melanoma cell lines with different tumorigenic properties - 1985 - Int. J. Cancer.

- Bailly M. and Doré J.F. - Human tumor spontaneous metastasis in immunosuppressed newborn rats. Multiple selections of human melanoma metastatic clones and variants - 1991 - Int. J. Cancer 49, 750-

757.

3) Bonds of biotinylated JOOl to melanoma cells

Origin from ^a lineage: Laboratory of Immunology and Cancer

Experimental, Dr. Doré, INSERM

Reagents: biotinylated JOOl according to the procedure described in

Immunobiol., Vol. 186, p.183-198 (1992), Z. Hmama et al. JOOl not biotinylated batch P242.

The operating conditions are those described in reference Z. Hmama et al. referred to above. 128

32

Results in ΔIMF: (net MFI i.e. deducted background noise).

Total: Biotinylated JOOl binding in the presence of an excess of

^• JOOl not biotinylated 1.

Specific: total bond - specific bond.

The results are presented in the Table of Figures 4 and 5 and in Figure 6.

The results presented in the Tables (Figures 4 and 5) and summarized graphically (Figure 6) clearly indicate that the JOOl binds specifically to the human melanoma lines tested and this in proportion to the dose. It is interesting to note that line 8, which has a low metastatic power, fixes JOOl in a much less intense way in vitro.

EXAMPLE 5 Elimination of Immunostimulatory Properties

The immunostimulatory properties of D25 were undesirable for a scintigraphic diagnostic product. Conventionally, for this type of molecule, the immunostimulatory activities observed are consecutive to the binding to receptors and generally inseparable from this binding.

The originality of the optimization of the JOOl molecule lies precisely in the fact that the pharmacological properties of D25 have been completely eliminated, while preserving intact the binding capacity to the target cells. This in particular at the level of the manufacturing process by strict control of the elimination of the esterified fatty acids which are responsible for the immunostimulating properties, and of the polymeric forms which, at high concentrations, can cause activation of the complement.

Important safety immunopharmacology studies have been carried out, comparing JOOl to the different fractions isolated from the membrane of K. pneumoniae. Particularly studied were the properties of the membrane proteoglycan (PGM) which serves as a raw material for the extraction of D25 (partially defatted) with those of D25 itself, then those of JOOl. The JOOl polysaccharide chain (GC-APG) obtained after selective hydrolysis of the lipophilic end responsible for binding to the target cell receptors served as a negative control in these studies, its elimination resulting in a total loss of the binding and cellular activation.

The complement activation properties observed at high concentration with D25 are due to the presence of residues of polymeric forms with a mass greater than 100 kDa. Their elimination in the. JOOl purification process by a selective chromatography step completely eliminates these properties.

The immunostimulatory properties are themselves strictly dependent on the composition of the lipophilic end of the molecule and, in particular, on the presence of esterified fatty acids. The manufacturing conditions optimized for JOOl allow quantitative elimination of fatty acids while fully preserving the 2 β-OH C14 fatty acids linked by amide bonds and strictly necessary for the recognition of target cell receptors, as well as phosphate and especially pyrophosphate allowing stable labeling at 99m Te. Among the characteristic properties of the membrane proteoglycan, preserved in D25, it should be noted: a polyclonal activation of B lymphocytes, an absence of activation of T lymphocytes, a very strong activation of macrophages which results in an increase in their oxidative metabolism and the release of cytokines, such as IL-1, TNF-α, IFN-α „IL-6 ... These cytokines are markers characteristic of the activation of macrophages and translate the pro inflammatory and immunostimulatory drugs of D25. The strictly controlled emination of the esterified fatty acids in the preparation of JOOl makes it possible to completely eliminate these immunostimulatory properties that the only β-OH-C14 acids linked by amide bonds to the terminal diglucosamine are incapable of inducing. The originality of JOOl obtained under these conditions is that despite the loss of all its activities, it retains all of its binding capacity to macrophages, which is generally not the case for this type of molecules of bacterial origin. .

1) Measurement of the secretion of cytokines by adherent human monocytes in culture

The adherent human monocytes are cultured for 24 hours in the presence of the products to be tested. The concentration of cytokines in the supernatants is then measured by ELISA technique.

The products tested are: Standard medium: background noise

- GC-APG (J001Y polysaccharide chain) negative control

- J001Y

- D25

- PGM-p (raw material of D25 and JOOl) Sm-Re LPS - positive control (LPS of S-typhimurium Sigma)

Table 3

Secretion of cytokines (ng / ml)

Concen¬

IL-lβ IL-6 TNFα tration product tested final

- Medium 0 ... 1.1 .. <0.3 ... <0.1

- GC-APG ... 100 ... 4.2 .... 2.5 ... <0.1

- J001Y ... 100 .. <0.6 .... 5.4 ... <0.1

- D25 ... 100 .. 32.6 ... 40.2 .... 1.9

- PGM-Kp ... 100 .. 47.0 ... 38.5 4.6

- Sm-Re-

LPS .... 10 .. 47.0 ... 40.4 .... 6.8

These results demonstrate the loss of activity of JOOl with respect to the induction of cytokines by activation of human monocytes compared to PGM-Kp and to D25. 2) Measurement of the polyclonal activation of BALB / c mouse splenocytes

The splenocytes are cultured at 10 ⁶ C / ml, in the presence of the products to be tested. Cell proliferation is determined by measuring the incorporation of [3 H] TdR during the last 24 hours of a 72 hour culture. Immunoglobin secretions are measured by ELISA after 6 days of culture. All products are tested at the final concentration of 100 μg / ml.

Results

Table 4

Ig secretion (μg / ml)

Incorporation of

Test product [3H] TdR -cpm x l0-3) IgM IgG IgA

- Middle 1.65 ± 0.23 1.4 0.46 0.54

- GC-APG Not done 1.1 0.45 0.61

- J001Y 1.35 ± 0.29 1.0 0.40 0.51

- D25 7.67 ± 0.11 15.4 1.54 1.76

- PGM-Kp 29.30 ± 0.65 46.1 5.61 6,%

These results confirm the loss of activity of JOOl with respect to PGM-Kp (raw material) and to D25 with respect to the polyclonal activation of mouse B lymphocytes. EXAMPLE 6 Application in imaging of the radiopharmaceutical J001C

1) Imaging on experimental models of inflammatory pathology

1.1) Study of the scintigraphic potentials of J001C in the model of immunological arthritis in the New Zealand rabbit and the monkey. cynomolgus

This model of experimental pathology is induced according to Consden R. et al. "Production of chronic arthritis with ovalbumin" - Ann. Rheum. Say. 1971, 30, 307-315 "by an intra-articular injection of ovalbumin in animals previously sensitized with respect to this antigen injected by sc route, in the presence of complete Freund's adjuvant. It makes it possible to obtain a chronic localized arthritis, without significant associated edema, and recruiting immunocompetent cells including many macrophages as soon as the inflammation is in chronic phase, ie 2 to 3 weeks after induction. This model offers the possibility of having a pathological focus well delimited at the knee with a healthy territory in a contralateral situation constituting a control zone for each animal which allows a precise determination of the scintigraphic report.

The scintigraphies were carried out on two male monkeys of 4 and 6 kg and two male rabbits of 3-4 kg from the third to the sixth week following the induction of arthritis. Three hours after the intravenous injection of 2 mCi (37 MBq) of 99mT Tc - J001C, corresponding to a dose of 0.050 to 0.200 mg of JOOl, the animals anesthetized with sodium pentobarbital are subjected to a scintigraphic acquisition at previous incidence. The Siemens Orbiter 75 gamma camera, equipped with a high-resolution parallel collimator, records images of 2 min in a matrix of 128 x 128 pixels. The processing of scintigraphic data and the quantification of the scintigraphic ratio R according to the classical technique of the regions of interest are carried out on a SIEMENS microdelta computer system. The images obtained in monkeys show a very intense focal focus on the pathological right knee (Scintigraphic reports of 2.2) with positive imagery of the drainage lymph node. The healthy contralateral knee shows no significant activity at the joint level.

Three hours after intravenous injection of J001C and one month after induction of arthritis in the right knee, there is a high scintigraphic contrast (R = 2.2) at the level of the articular lesion and a very significant fixation in the drainage nodes. inguinal area.

Similar results are obtained in arthritic rabbits for preparations of J001C marked with specific activities ranging from 370 to 2590 MBq / mg (10 to 70 mCi / mg) of JOOl, or for 74 MBq (2 mCi) injected for doses of JOOl from 200 to 290 μg per animal. Three hours after intravenous injection of J001C and one month after induction of arthritis in the right knee, there is a high scintigraphic contrast (R = 2.8) at the level of the joint lesion.

2) Melanoma imaging

2.1) Experimental model of human melanoma grafted in nude mice

Three weeks after subcutaneous injection in nude mice of 5 x 105 cells of the human melanoma line TW12 (Dr. JF Doré - Center Léon Bérard - Lyon), in the left inguinal area, the animal develops a tumor of 6-8 mm in diameter. At this stage, it is subjected to a scintigraphy by J001 C administered intravenously in the retro-orbital sinus. Three hours after injection, high-resolution imaging shows a significant fixation of J001C in the tumor area, with a very significant scintigraphic contrast (R = 1.6).

2.2) Illustration of a clinical case of melanoma

- Patient: FOR ... TH (obs N ° 401), male, 36 years old. - Right posterior nodular thoracic melanoma with a Breslow index = 2.1 and a grade = IV (Clark).

- Treatment: surgical excesses, then two cures of MUPHORAN

- Recurrence: diagnosis of a right axillary recurrence.

- J001C scintigraphy by injection. Operating protocol:

. 0.100 mg of JOOl Y labeled with 74 MBq (2 mCi) of 99m Te are injected; . Acquisitions of 2 minutes are performed in anterior (AF) and posterior (FP) incidences at 1 hour after injection using a gamma camera equipped with a high resolution parallel collimator with a matrix of 128 x 128 Pixels.

Results:

Hyperfixation is observed in the right axillary hollow corresponding to the clinically detected lymphadenopathy, with a fixation index at 1 hour = 1.7, and a fixation index at 2 hours = 2.5. Lymph node dissection performed on 10.12.91 effectively reveals a metastatic lymph node of (3.5 x 2.5 x 1.5 cm).

Example 7: Anti-LPS Properties of 1001

The fact that JOOl retains the binding properties of D25 and binds to macrophages on receptors partly common with those of LPS (CDl lb - CD14) without immunostimulatory activity has prompted the search for possible anti-LPS therapeutic properties in protection against endotoxin shock. In view of the results obtained "in vitro", it seems that simple competition at the level of common receptors does not explain all the activity observed. Even if it does not completely block the binding of LPS to macrophages, JOOl appears to have a "post-binding signaling" effect which results in a prolonged blocking of the production of TNFα, a marker characteristic of LPS activity. Antagonistic properties vis-à-vis LPS

1. Human monocytes

On isolated mononuclear cell cultures and in the absence of serum, JOOl inhibits the production of TNF induced by the 5 different types of LPS tested. ^•

2. Mouse spleen lymphocytes

JOOl induces an inhibition of proliferation induced by LPS. This effect is observed by adding JOOl up to 24 hours after LPS to cell culture. The same effect could be observed, but less systematically, on an anti-IgM antibody.

Claims

1. Preparation of a defatted and purified polysaccharide compound D25, of molecular weight of approximately 34 KD, obtainable by extraction from the membrane proteoglycans of Klebsiella pneumoniae, characterized in that said polysaccharide compound is defatted and purified so that said preparation does not include micellar forms of said compound.

2. Preparation of a delipidized and purified polysaccharide compound D25, of molecular weight of approximately 34 KD, obtainable by extraction from membrane proteoglycans of Klebsiella pneumoniae, characterized in that said compound is defatted and purified so that said preparation does not contain palmitic fatty acid and peptides free or associated with said compound.

3. Preparation according to claim 1 or 2, characterized in that it comprises a physiologically acceptable salt, at low molar concentration, so that in the form of an aqueous solution, it has a low ionic strength.

4. Preparation according to claim 1 or 2, characterized in that it comprises an NaCl salt at a concentration of ImM at 0.15 M.

5. Preparation according to claim 1 or 2, characterized in that it comprises an NaCl salt at a concentration of approximately 20 mM.

6. Polysaccharide compound D25 defatted and purified, of approximately 34 KD, obtainable by extraction from the membrane proteoglycans of Klebsiella pneumoniae, characterized in that it can be represented by the following formula (I): [PS]

in which [PS] represents a polysaccharide chain,

7. A polysaccharide compound D25 with a molecular weight of approximately 34 KD capable of being extracted from the membrane proteoglycans of Klebsiella pneumoniae, according to claim 6, characterized in that the linear polysaccharide chain [PS] is composed by the repetition of the alternation -of two following disaccharide sequences linked by sequences (1 -> 3): - [-> 3) αD Gal _p (1 -> 3) β D Gal _f (1-] ->, - [-> 3) αD Gal _p (1 -> 3) β D Gal _p (l -] -> and at the end of which is a single binding structure consisting of 3 glucose residues, 1 galactose residue, 2 heptose residues and 1 mannodeoxyoctulosonic acid residue, said unique binding structure being linked to the two terminal glucosamines represented in formula (I), on each of which is linked a β-hydroxymyristic acid by an N-glycosidic amide bond.

8. Composition intended for imaging, diagnosis or therapy, characterized in that it comprises a preparation of the defatted and purified compound according to one of the preceding claims.

9. The composition as claimed in claim 8, intended for imaging and diagnosis of infectious, inflammatory and tumor foci by targeting macrophages.

10. The composition as claimed in claim 8, intended for the imaging and diagnosis of melanoma and other tumors expressing the same receptor, in particular colon cancer.

11. Composition according to one of claims 8 to 10 characterized in that it is injectable intravenously.

12. Composition according to one of claims 8 to 10, characterized in that it is injectable by lymphatic route, in particular by subcutaneous route or intrapleural route.

13. Composition according to one of claims 8 to 10, characterized in that it can be administered by aerosol.

14. Imaging or diagnostic composition according to one of claims 8 to 13, characterized in that the polysaccharide compound is labeled with a detectable element such as a radioactive, paramagnetic or fluorescent element.

15. Composition according to one of claims 13 and 14, characterized in that the radioactive element is a radionuclide detectable by scintigraphy.

16. Composition according to claim 15, characterized in that the radionuclide is technetium 99m, iodine 123 ₍ or indium m.

17. Use of the compound according to one of claims 1 to 7 as a medicament.

18. Use of the compound according to claim 17 as a medicament in the protection against endotoxin shock.

19. Use of the compound according to one of claims 1 to 7 for therapeutic purposes as a vectoring agent for targeted radiotherapy, for vectoring cytotoxic substances or also for vectoring immunogenic or anti-inflammatory substances.

20. Monoflacon kit intended for labeling the compound in aqueous solution according to one of claims 1 to 7 comprising a colyophilisate of said compound, a labeling reagent and a stabilizing excipient.

21. Kit according to claim 1 intended for labeling with technetium, characterized in that it comprises tin fluoride (SnF ₂ ) as labeling reagent and ascorbic acid as stabilization excipient.