METHOD FOR PREDICTING THE BIOAVAILABILITY OF A RADIOELEMENT FOLLOWING CONTAMINATION, AND USES THEREOF

Abstract

Method for predicting the bioavailability of a radioelement in an animal living organism following the contamination of the organism by the radioelement, the method comprising the steps of: (a) producing a gel imitating 5 the area of contamination in the animal living organism and in which the radioelement is uniformly distributed; (b) bringing the gel produced in the step (a) into contact with a solution imitating a biological fluid associated with the area of contamination in the animal living organism, then stirring the mixture; and (c) measuring the quantity of the radioelement in the solution at a given moment t, the measurement allowing the 10 prediction of the bioavailability of the radioelement in the animal living organism. The invention also relates to the use of such a method for identifying a molecule exhibiting chelating properties in relation to a given radioelement and/or for characterising the chelating properties of a molecule.

Claims

1) Method for predicting the bioavailability of a radioelement in a living animal body further to contamination of this body by said radioelement, comprising the steps of: a) preparing a gel in which said radioelement is uniformly distributed, said gel mimicking the contamination site in said living animal body; b) placing the gel prepared at said step (a) in contact with a solution mimicking a biological fluid associated with the contamination site in said living animal body, then placing the whole under agitation; and c) measuring, at a time t, the amount of said radioelement in said solution, said measurement allowing prediction of the bioavailability of said radioelement in said living animal body.

2) The method according to claim 1, wherein said radioelement is an actinide.

3) The method according to claim 1, wherein the gel is prepared at step said (a) from at least one compound having colloidal properties, a solution called gel solution and at least one radioelement.

4) The method according to claim 3, wherein said compound having colloidal properties is a polysaccharide selected in particular from the group consisting of agarose, sucrose, sepharose, chitosan, xanthan, carrageenan, dextran, agar, alginate or mixtures thereof.

5) The method according to claim 3, wherein the ratio between compound(s) having colloidal properties (weight expressed in g)/gel solution (volume expressed in ml) is between 1 and 5%, typically between 2 and 4%.

6) The method according to claim 3, wherein said gel solution is an aqueous solution e.g. physiological saline solution optionally completed with one or more organic or inorganic salts selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), sodium bicarbonate (NaHCO.sub.3), sodium phosphate (Na.sub.3PO.sub.4), sodium hydrogen phosphate (Na.sub.2HPO.sub.4), sodium dihydrogen phosphate (NaH.sub.2PO.sub.4), potassium hydrogen phosphate (K.sub.2HPO.sub.4), potassium dihydrogen phosphate (KH.sub.2PO.sub.4), hydrochloric acid (HCl), magnesium chloride (MgCl.sub.2), calcium chloride (CaCl.sub.2), sodium sulfate (Na.sub.2SO.sub.4), sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium acetate (CH.sub.3COONa), sodium tartrate (Na.sub.2C.sub.4H.sub.4O.sub.6), sodium lactate (C(OH)(CH.sub.3)COONa), sodium pyruvate (CH.sub.3C(O)COONa), sodium citrate (Na.sub.3C.sub.6H.sub.5O.sub.7) and citric acid (C.sub.6H.sub.8O.sub.7).

7) The method according to claim 3, wherein the gel comprises one or more compounds selected from the group consisting of pepsin, lecithin, glycine, glucose, lysozyme, albumin, sodium taurocholate, maleic acid, transferrin, ferritin, fibrin, hyaluronic acid, glucosamine, fibronectin, laminin, mucin, keratins, collagens, chondroitin, osteopontin, hydroxyapatite and glutamic acid.

8) The method according to claim 3 wherein, at step (a), the radioelement is added in the form of a suspension or of a solution to the gel solution containing at least one compound having colloidal properties.

9) The method according to claim 3, characterized in that the solution of said step (b) is an aqueous solution comprising one or more organic or inorganic salts selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), sodium bicarbonate (NaHCO.sub.3), sodium phosphate (Na.sub.3PO.sub.4), sodium hydrogen phosphate (Na.sub.2HPO.sub.4), sodium dihydrogen phosphate (NaH.sub.2PO.sub.4), potassium hydrogen phosphate (K.sub.2HPO.sub.4), potassium dihydrogen phosphate (KH.sub.2PO.sub.4), hydrochloric acid (HCl), magnesium chloride (MgCl.sub.2), calcium chloride (CaCl.sub.2), sodium sulfate (Na.sub.2SO.sub.4), sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium acetate (CH.sub.3COONa), sodium tartrate (Na.sub.2C.sub.4H.sub.4O.sub.6), sodium lactate (C(OH)(CH.sub.3)COONa), sodium pyruvate (CH.sub.3C(O)COONa), sodium citrate (Na.sub.3C.sub.6H.sub.5O.sub.7) and citric acid (C.sub.6H.sub.8O.sub.7).

10) The method according to claim 3, characterized in that said gel solution and said solution used at step (b) comprise one or more salts, the same or different.

11) The method according to claim 3, characterized in that the solution of said step (b) further comprises one or more compounds selected from the group consisting of pepsin, lecithin, glycine, glucose, lysozyme, albumin, sodium taurocholate, maleic acid, transferrin, ferritin and fibrin.

12) The method according to claim 3, characterized in that the solution of said step (b) is prepared in an incubator with agitation having a temperature of approximately 37 C., the controlled composition of the atmosphere being humid air comprising 5% CO.sub.2.

13) Use of a method according to claim 1, to identify a molecule having chelating properties towards a given radioelement and/or to characterize the chelating properties of a molecule.

14) Method for identifying a molecule having chelating properties towards a radioelement, comprising the steps of: i) measuring the bioavailability of said radioelement with the method according to claim 1, wherein the solution mimicking the biological fluid comprises a molecule likely to exhibit chelating properties; ii) measuring the bioavailability of said radioelement with the method according to claim 1 under the same conditions as at step (i), the solution mimicking the biological fluid being free of said molecule likely to exhibit chelating properties; and iii) comparing the bioavailability obtained at step (i) with that obtained at step (ii) whereby, if the bioavailability obtained at step (i) is higher than obtained at step (ii), it is an indication that the molecule tested at step (i) has chelating properties towards said radioelement.

15) The method according to claim 14, characterized in that at said steps (i) and (ii), the same conditions are used in terms of composition of the gel mimicking a contamination site, type and amount of radioelement initially contained in this gel, composition of the solution mimicking the biological fluid, technique for measuring the radioelement contained in the solution, and time t at which this measurement is performed, the only difference being the presence or absence of the molecule to be tested in the solution mimicking the biological fluid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 illustrates the quantity of Evans Blue recovered when implementing a method of invention, as obtained after different experiments performed by two different operators.

[0075] FIG. 2 gives total -activity of MOX, of Pu in nitrate salts and of Am in nitrate salts, recovered by implementing a method of the invention.

[0076] FIG. 3 gives total -activity of Pu recovered from gels mimicking different contamination sites after injury, when implementing a method of the invention.

[0077] FIG. 4 gives total -activity of Pu recovered when implementing a method of the invention on gels in contact with different media containing or not containing chelating molecules (DTPA and 3,4-LIHOPO).

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

I. Repeatability and Reproducibility of the Method of the Invention

[0078] I.1. Operating Protocol.

[0079] Two hundred and fifty mg of agarose (Type IIA; Medium EEO) are added to 10 ml of physiological saline solution (8.8 g/L NaCl, 0.37 g/L KCl) in a beaker. This mixture is weighed and heated in a microwave oven (170 W30 s2; 170 W20 s1). After cooling for 2 min, the beaker is again weighed to adjust the volume to the value of the initial volume with physiological saline.

[0080] One hundred l of Evans Blue (0.2% in water) are added to the agarose under gentle mixing. After cooling for about 5-10 minutes, 700 l of the agarose solution containing the Evans Blue are distributed in the wells of a culture plate (12-well plate) using a Gilson P1000 pipette, the conical tip being cut to facilitate pipetting.

[0081] Three ml of physiological saline solution are added to the gels. The plates are incubated at 37 C. in a humid atmosphere in the presence of 5% CO.sub.2 for 2 h under slow agitation (5 rpm). Two hours after the start of incubation, the medium is taken from each well and the percentage of Evans Blue is measured by spectrophotometry at 610 nm. The gels are dissolved in formamide and optical density is measured. The concentrations of Evans Blue in the samples are calculated from a range of increasing concentrations of the colouring agent.

[0082] I.2. Results.

[0083] Eight experiments were performed under the same conditions over several days, and by two operators using 3 to 12 samples per experiment (FIG. 1). The data obtained show repeatability by one same operator with a coefficient of variation of about 8.6%, and reproducibility between two operators with a coefficient of variation of about 8.5%.

II. Dissolution of Actinides

[0084] II.1. Operating Protocol.

[0085] The solutions and suspensions of actinides are prepared as follows:

[0086] MOX: 25 mg of MOX (Mixed OXide i.e. mixture of uranium and plutonium oxides) are placed in 5 ml of absolute ethanol and vigorously vortexed. Five hundred l of this suspension are diluted 1:4 in 0.9% NaCl. An aliquot is taken to measure total -activity via liquid scintillation.

[0087] Pu: An aliquot of plutonium solution (Pu) in 2N nitric acid (isotopic composition as % of total activity: 12.4% .sup.239Pu, 85.6% .sup.238Pu and 1.8% .sup.241Am) of about 4-5 kBq is placed in a glass liquid scintillation vial placed over a hot plate (100 C.) until complete evaporation. The Pu is then taken up in 100 l of water or NaCl/KCl buffer. Total activity is measured by liquid scintillation.

[0088] Am: An aliquot of americium (Am) solution in 2N nitric acid of about 4-5 kBq is placed in a glass liquid scintillation vial over a hot plate (100 C.) until complete evaporation. The Am is then taken up with 100 l of water. Total activity is measured by liquid scintillation.

[0089] A 2.5% agarose solution is prepared in physiological saline solution (8.8 g/L NaCl, 0.37 g/L KCl, pH 5.6) through the addition of 0.375 g agarose (low melting point, A11-EE0 Sigma Aldrich) to 15 ml of physiological saline solution in a beaker. The solution is heated for 30 s in a microwave oven at 250 W power, homogenized then heated 30 s, again homogenized and heated 20 s. After verifying the translucency of the solution, the volume is readjusted to 15 ml if needed.

[0090] After cooling to ambient temperature for 5 to 10 min, approximately 100 kBq of MOX, 4-5 kBq of Pu or Am in 100-150 l are added to the 15 ml of agarose solution. The mixture is carefully homogenized. 700 l of the mixture are placed in 3.9 cm.sup.2 wells of a 12-well culture plate using an automatic P1000 pipette of which the tip of the end part has been cut away. The culture plate is placed under very slight agitation to distribute the agarose solution homogeneously over the bottom of the well. The plate is then left 10-15 min at ambient temperature, without lid to prevent condensation, to reach complete solidification of the gels.

[0091] Each condition is performed at least three times (3 identical wells).

[0092] At this step, the plate can be sealed with vinyl film and stored at +4 C. for later use (1-3 days) or used immediately.

[0093] Three ml of physiological saline solution are added to each well. The culture plate is then placed in a cell culture incubator at 37 C., in a humid atmosphere with 5% CO.sub.2, under slow agitation (5 rpm).

[0094] Two hours after the start of incubation, the buffer of each well is taken and placed in a liquid scintillation vial. Fifteen ml of scintillator (Ultimagold) are added and total -activity is measured by liquid scintillation (Packard counter). Three ml of buffer are added to each well and the plate is placed back in the incubator. The supernatants are collected in similar manner at 24 h and 48 h after the start of incubation.

[0095] At 48 h, the gels are very carefully recovered from the culture plates using a metallic spatula and can be placed in a Petri dish wrapped in film of Parafilm M type to prevent drying and for direct counting by a detector. As a variant, the carefully collected gels are directly placed in liquid scintillation vials. 2 ml of 2N nitric acid are added to the vials. These are placed over a hot plate (100 C.) until complete evaporation. One ml of H.sub.2O.sub.2 (30%) is then added. After complete evaporation 1 ml of 2N nitric acid is added followed by 15 ml of scintillating liquid. The activity of each sample is measured i.e. the supernatants already collected and the gel.

[0096] II.2. Results.

[0097] The activities of each well, measured in the supernatants at 2 h, 24 h and 48 h are added to that of the gels. This value forms 100% of each well i.e. the initial activity deposited in the wells. The results are then expressed as activity measured in the supernatants/initial activity and are given in FIG. 2. It is to be noted that the activity at 24 h is equal to the sum of activity of the supernatant at 2 h and of the supernatant at 24 h, and that the activity at 48 h is the sum of the activity of the supernatant at 2 h, of the supernatant at 24 h and of the supernatant at 48 h.

[0098] Compared with initial activity, there is less activity derived from the MOX contained in the supernatant than from Pu or Am in nitrate salts. The latter are therefore less retained in the agarose gel than MOX: Pu and Am are therefore more bioavailable when in the form of nitrate salts than when contained in MOX.

III. Identification of Plutonium-Retaining Compartments

[0099] III.1. Operating Protocol.

[0100] The Pu solution is prepared in the same manner as described under item II.1 above, then added to the agarose solution prepared in buffers of varying composition, as a function of the mimicked physiological compartment. A solution of agarose prepared in NaCl/KCl is used as reference.

[0101] To mimic synovial fluid (SYN): a solution of 8.8 g/L NaCl, 0.37 g/L KCl, 1.44 g/L Na.sub.2HPO.sub.4, 0.24 g/L KH.sub.2PO.sub.4, 3 g/L hyaluronic acid is used to prepare the agarose gel.

[0102] To mimic the extracellular matrix (ECM-1): a solution of 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na.sub.2HPO.sub.4, 0.24 g/L KH.sub.2PO.sub.4, is used to prepare the agarose gel as described under item II.1 above. After cooling at ambient temperature for 5 to 10 min, collagen (type I, from rat tail) is added to obtain a final concentration of 0.5 g/L. The pH is adjusted to 7 with acetic acid.

[0103] To mimic the extracellular matrix (ECM-2): a solution of 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na.sub.2HPO.sub.4, 0.24 g/L KH.sub.2PO.sub.4, 3 g/L hyaluronic acid, 1.1 g/L glucosamine is used to prepare the agarose gel. After cooling at ambient temperature for 5 to 10 min, collagen (type I, from rat tail) is added to obtain a final concentration of 0.5 g/L. The pH is adjusted to 7 with acetic acid.

[0104] To mimic cartilage (CHON): a solution of 8 g/L NaCl, 0.2 g/L KCl, 1 g/L chondroitin is used to prepare the agarose gel.

[0105] After homogenization, 4-5 kBq of Pu in 100-150 l are added to each of the different agarose solutions. The remainder of the experiment is similar to that described under item II.1 above.

[0106] III.2. Results.

[0107] As set forth in the protocol under item II above, the activities of each well, measured in the supernatants at 2 h, 24 h and 48 h, are added to that of the gels. This value forms 100% of each well i.e. the initial activity deposited in the wells. The results are therefore expressed as activity measured in the supernatants/initial activity and are given in FIG. 3.

[0108] It follows from the mimicked compartments that there are two types of behaviour with (1) the compartments in which Pu is retained namely the synovial fluid and extracellular matrix, the two curves corresponding to ECM-1 and ECM-2 being juxtaposed, and (2) those which do not retain either Pu or the reference agarose, namely the cartilage. Therefore, Pu appears to be less bioavailable in synovial fluid and the extracellular matrix than in cartilage.

IV. Evaluation of the Efficacy of Plutonium-Chelating Molecules

[0109] IV.1. Operating Protocol.

[0110] The Pu solution and agarose solution are prepared in the same manner as described under item II.1 above. The preparation of the Pu-containing gels is carried out as described under item II.1 above.

[0111] Incubation of the agarose gels takes place in buffers of different compositions as a function of the chelating molecule to be tested. Incubation in NaCl/KCl buffer is used as reference. A solution of diethylenetriamine pentaacetic acid (Ca-DTPA, Pharmacie centrale des armes) is diluted in NaCl/KCl to obtain a 10 M concentration. A solution of 3,4-LIHOPO is prepared with a final concentration of 10 M. The remainder of the experiment is similar to the description under item II.1 above.

[0112] IV.2. Results.

[0113] As set forth in the protocol under item II above, the activities of each well, measured in the supernatants at 2 h, 24 h and 48 h, are added to that of the gels. This value forms 100% of each well i.e. the initial activity deposited in the wells. The results are therefore expressed as activity measured in the supernatants/initial activity and are given in FIG. 4.

[0114] As expected, given the nature of Ca-DTPA and 3,4-LIHOPO, there is less retention of Pu in the agarose gel, Pu therefore being more bioavailable when the medium surrounding this gel comprises a chelating molecule, compared with a medium devoid thereof.

REFERENCES

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