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

• 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 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 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 aqueous solution according to the present invention comprises an NaCl salt at a concentration of ImM at 0.15 M.
• 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 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 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 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.
• 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.
• 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
• 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
  • 1995-03-15 EP EP95913199A patent/EP0750640A1/de not_active Withdrawn
  • 1995-03-15 AU AU20755/95A patent/AU2075595A/en not_active Abandoned
  • 1995-03-15 JP JP7523887A patent/JPH09512842A/ja active Pending
  • 1995-03-15 WO PCT/FR1995/000311 patent/WO1995025128A1/fr not_active Application Discontinuation
  • 1995-03-15 CA CA002185663A patent/CA2185663A1/fr not_active Abandoned

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