Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• 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.
• compositions comprising said compound intended for imaging, diagnosis or therapy, in particular of infectious, inflammatory and tumor foci.
• 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.
• patent application FR 89 030344 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.
• 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 ⁇ Gai p, 3 ⁇ Gai p, 2 ⁇ Gai f, and 2 ⁇ 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.
• 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 ⁇ -hydroxymyristic acids representing approximately 1.5% of the mass of D25.
• These ⁇ -hydroxymyristic acids are associated in esterified form or in free form of traces of fatty acid in CI ⁇ (palmitic) representing approximately 0.5% of the mass of D25.
• 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.
• 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.
• 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 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.
• 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.
• 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 ⁇ .
• 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.
• 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.
• 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] 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 :
• 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.
• 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 ⁇ binding during labeling of the compound.
• the compound according to the present invention retains two ⁇ -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.
• 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.
• a physiologically acceptable salt at low molar concentration
• 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.
• 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 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.
• Klebsiella pneumoniae which is the subject of a deposit at the National Collection of Cultures of Microorganisms (CNCM) under the n ⁇ 145-I-IP.
• CNCM National Collection of Cultures of Microorganisms
• 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
• 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.
• 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.
• 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.
• 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:
• 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.
• 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
• 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.
• 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:
• 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.
• 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.
• compositions are intended for imaging and for the diagnosis or therapy of inflammatory and tumor infectious foci by targeting macrophages.
• 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.
• 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.
• 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.
• radioactive element 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).
• MRI magnetic resonance imaging
• 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).
• 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.
• galactose MAC-2 receptor an expression which seems to be correlated with a gravity factor.
• 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.
• 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.
• 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.
• the compound will be labeled with a radioactive element.
• radioactive element 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 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.
• 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.
• 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
• 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.
• 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.
• 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 ⁇ .c ⁇ r 1 , the volume is concentrated to reach 6 1 per kg of equivalent in dry starting cells.
• 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.
• This step is intended to remove protein impurities and to digest the peptidoglycan residues remaining after the first alkaline hydrolysis.
• 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 ⁇ cm-i on a membrane cutting at 10,000 daltons. The volume is adjusted to 1500 ml with distilled water.
• 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.
• 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 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.
• 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".
• J001C a radioisotope
• 0.040 mg is a critical threshold for labeling because SnF2 does not ensure the stability of the radiopharmaceutical below this dose.
• 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 limit margins for variation of SnF2 are therefore: minimum: 40 ⁇ g - maximum: 100 ⁇ g.
• the optimum for 1 mg of J001Y is 80 ⁇ g of SnF2.
• 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.
• 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.
• 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.
• 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.
• the solution obtained above is immediately sterilized by filtration on a 0.22 ⁇ m membrane under nitrogen. The integrity of the filter must be checked after filtration.
• 3rd stage distribution in vials (in sterile block)
• 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.
• the lyophilization is then carried out under usual conditions taking into account the eutectic points of the mixture.
• 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.
• 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.
• 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.
• the bound JOOl is very quickly internalized in cells via its receptors.
• 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 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.
• 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 ⁇ -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.
• cytokines such as IL-1, TNF- ⁇ , IFN- ⁇ mindfulIL-6 ...
• cytokines are markers characteristic of the activation of macrophages and translate the pro inflammatory and immunostimulatory drugs of D25.
• 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 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 ⁇ g / ml.
• the scintigraphys 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.
• 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.
• JOOl inhibits the production of TNF induced by the 5 different types of LPS tested.
• 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.

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• 1994
  • 1994-03-16 FR FR9403072A patent/FR2717483B1/fr not_active Expired - Fee Related
• 1995
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