AU7776591A - Contrast medium, process for its preparation and application to imagery - Google Patents

Description

WO 91/16080 - 1 - PCT/FR91/00301 CONTRAST MEDIUM, PROCESS FOR ITS PREPARATION AND APPLICATION TO IMAGERY The invention relates to a contrast-modifying medium for X-ray imagery, nuclear magnetic resonance 5 imagery (NMRI) or ultrasound imagery. It also relates to a process for preparing this medium and to its application in angiography, in the imagery of the volume and blood flow rate of organisms, and in the monitoring, for example, of ischemias and 10 infarctions without reinjection. X-ray imagery is based on varying attenuations of X-rays from one tissue to another. Anatomical structures have different radiological opacities which are reflected in radiological contrasts. This contrast is due to dif 15 ferences in average density and anatomical composition. It can be produced artificially. Thus, a hollow organ can be made visible by filling it with a contrast product or by replacing its liquid content by a contrast product, for example air for cerebral ventriculography and iodi 20 nated solutions for radiography of vessels. Some organisms can be visualized by introducing into the circulation a contrast product which becomes concentrated in these regions. This is in particular the case for urography or cholecystography. 25 To visualize the vascular system using X-rays, water-soluble iodinated contrast products are used which, after injection via the vascular route, are distributed in the vascular system and the interstitial space. They are not metabolized and are rapidly eliminated via the 30 urinary route. These products are characterized by a high extravascular diffusion which requires the product to be injected as a bolus and where appropriate reinjections at regular intervals in order to maintain the contrast at a sufficient level. 35 In magnetic resonance imagery, a homogeneous principal magnetic field and a high frequency electromagnetic field, perpendicular to the first, are applied to the tissue examined. The images found are Wo 91/16080 - 2 - PCT/FR91/00301 reconstructed from signals emitted by atoms in the organism. When the second field is interrupted, the energy absorbed by the material during the excitation process is restored by the nuclei whose magnetic moment 5 will again coincide with the direction of the principal magnetic field. The weak energy emitted, called energy of precession, decreases by means of a relaxation phenomenon. The relaxation can be separated into two processes: 10 - a spin-spin relaxation, characterized by a time constant

T

2 , - a spin-lattice relaxation characterized by a time constant T1. In MRI, the T1- or T2-weighted signal acquisition 15 sequences make it possible to obtain information on the morphology and the physiology of the organs studied but also on their composition. However, for the NMR phenomenon to be observable, the sample should contain a large number of nucleae 20 possessing a magnetic moment, especially the hydrogen ('H), phosphorus ( 31 P) and carbon (1 3 C) atom. MRI is therefore confronted with a problem of sensitivity and contrast. Paramagnetic and superparamagnetic contrast agents are particularly suitable for locally modifying 25 the relaxation times Ti and T2. They permit diagnosis depending on their localization. Visualization of the circulatory system and the perfusion rate of organs can prove particularly useful especially in hypoperfusion and ischemia phenomena or in 30 hyperperfusions of some tumor processes. Because of substantial renal or hepatic removal and an extravascular diffusion, conventional paramagnetic and superparamagnetic contrast agents appear to be unsuitable. 35 Of the conventional agents, metal chelates such as DTPA gadolinium (Patent U.S. 4,647,447) or iron oxide dextran type superparamagnetic colloids can be mentioned. These agents possess a molecular weight (590 for Gd DTPA) WO 91/16080 - 3 - PCT/FR91/00301 which results in their passage into the interstitial space and a rapid renal removal. The colloids are rapidly entrapped by the reticulo endothelial system and fixed in the hepatic and 5 splenic region. Another route for improving the parameters for relaxation of the vascular system is the use of chromium labeled red blood cells (Invest. Radiol., 1989, 24, 742 753). The labeling of red blood cells with 51 Cr is 10 conventionally used in nuclear medicine. Red blood cells are incubated with varying concentrations of Na 2 CrO 4 ; after penetrating across the membrane, CrO 4 2 is reduced to Cr 3 which has paramagnetic properties and binds to the hemoglobin molecule. The relaxation times for the 15 globular pellets are substantially reduced. However, chromium is cytotoxic at doses which are effective in NMR and hemolysis of chromium-labeled red blood cells is greater. The half-life of these red blood cells is reduced by a factor of 4 compared with normal canine red 20 blood cells whose half-life is 19 days. Ultrasound imagery is based on the fact that ultrasound waves are provided in a given medium at a speed which is dependent on the nature of the latter. At the interface between two media of different natures, 25 part of the wave is reflected and the other is transmitted. By analyzing the reflected or transmitted signals it is thus possible to obtain either transmission images or reflection images which make it possible to visualize the interfaces between the various media, this 30 latter technique being used under the name of ultrasound echography. It may therefore be advantageous to use a contrast product capable of modifying the ultrasound wave transmission and reflection parameters so as to obtain better visualization, for example, of an organ or of the 35 vascular system. The present invention therefore relates to a contrast medium, characterized in that it comprises a hydrophilic or water-soluble or colloidal contrast Wo 91/16080 - 4 - PCT/FR91/00301 product, f or example a superparamagnetic colloid or a gas in bubble form, encapsulated by a technique for lysing/resealing inside erythrocytes, and in that it comprises, in addition, an acceptable carrier for enteral 5 or parenteral administration. Contrast product is understood to mean any product capable of improving contrast in imagery, especially in medical imagery, in particular in X-ray imagery, in magnetic resonance imagery and in ultrasound 10 imagery. For X-ray imagery, the product is opaque to X-rays. It can be in particular a polyiodinated acid salt. Among the preferred molecules which can be 15 encapsulated into erythrocytes, the following polyiodinated acid salts can be mentioned: - acetrizoic acid, - iothalamic acid, - ioxithalamic acid, 20 - ioxaglic acid, - and especially their sodium, lysine or N-methyl glucamine salts or nonionic polyiodinated compounds: - Iopamidol, N-N'-bis[2-hydroxy-1-(hydroxymethyl)ethyl] 25 5-[(2-hydroxy-1-oxopropyl)amino]-2,4,6-triiodo-1, 3 -ben zenedicarboxamide - 5-[acetyl(2,3-dihydroxypropyl)amino]-N,N'-bis( 2

,

3 -di hydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide - Ioxilan, 5-[acetyl(2,3-dihydroxypropyl) amino[-N- (2,3 30 dihydroxypropyl)-N'-(2-hydroxyethyl)-2, 4

,

6 -triiodo 1,3-benzenedicarboxamide -Iopromide, N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo 5-(methoxyacetyl)amino-N-methyl-1,3-benzenedicarboxamide - Iopentol, 5-[acetyl(2-hydroxy-3-methoxypropyl)amino 35 N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1, 3 -benzene dicarboxamide - Iotrolan, 5,5'-((1,3-dioxo-1,3-propanediyl)bis(methyl imino)]bis[N,NI-bis[2,3-dihydroxy-1-(hydroxymethyl)- WO 91/16080 - 5 - PCT/FR91/00301 propyl]-2,4,6-triiodo-1,3-benzenedicarboxamide - Iodixanol, 5,5'-( (2-hydroxy-1,3-propanediyl)bis(acetyl imino)]bis[N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo 1,3-benzenedicarboxamide 5 - Ioversol, N,N '-bis(2,3-dihydroxypropyl)-5-[(hydroxy acetyl)(2-hydroxyethyl)amino]-2,4,6-triiodo-1,3-benzene dicarboxamide. For MRI, the contrast-modifying product is a sub stance which can locally-influence the applied magnetic 10 field, preferably a biodegradable paramagnetic complex or a superparamagnetic colloid such as dextran-coated iron oxide particles. The effect on the acceleration of proton relaxation is due: - either to a conventional dipolar interaction between 15 the nuclear spin of the protons of water molecules and the unpaired electrons of the metal (in this case, manganese Mn 2 + and gadolinium Gd 3 complexes are preferred) - or to a difference in magnetic susceptibility linked to the presence of an intraerythrocytic paramagnetic complex 20 (in this case, dysprosium complexes are preferred). This complex may be chosen from the complexes formed from a cyclic or acyclic ligand possessing coordi nation atoms chosen from oxygen, nitrogen, phosphorus, sulfur and a metal chosen from the lanthanides or the 25 transition metals, especially gadolinium, dysprosium, manganese and iron. Among the preferred complexes which can be encapsulated in the human erythrocytes, there may be mentioned: 30 - the N-methylglucamine or sodium salt of the gadolinium complex of diethylenetriaminepentaacetic acid (Gd-DTPA), - the N-methylglucamine or sodium salt of the gadolinium complex of 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' tetraacetic acid (Gd-DOTA), 35 - the N-methylglucamine or sodium salt of the gadolinium complex of 2-methyl-1,4,7,10-tetraazacyclododecane N,N',N'',N'''-tetraacetic acid (Gd-MCTA) WO 91/16080 - 6 - PCT/FR91/00301 - the gadolinium complex of 1,4,7,10 tetraazacyclododecane-N,N',N''-triacetic acid (Gd-DO3A), - the gadolinium complex of 1,4,7,10-tetraazacyclo dodecane-N-2-hydroxypropyl-N',N'',N'''-triacetic acid 5 (Gd-HPDO3A), - the gadolinium complex of N,N''-bis(methylcarbamoyl methyl)diethylenetriamine-N,N',N''-triacetic acid (Gd-DTPA-BMA). These complexes can be neutral or ionic depending 10 on the respective charges on the ligand and the complexed metal. This list is not limited to neutral complexes or the complexes salified with N-methylglucamine and can be extended to the salts obtained with other inorganic bases 15 (sodium, calcium) or organic bases, especially amino acids (lysine, arginine and the like). The erythrocytes containing superparamagnetic -agents (dextran-coated particles of various metal oxides, especially iron in the form of Fe 3

C

4 and Fe 2

(

3 ) can also be used as contrast 20 agents. Contrast products of the triiodinated acid salt or paramagnetic complex type can also be used in ultra sound imagery. The product encapsulated into erythrocytes can 25 also be a gas in bubble form. Preferably, this gas can be air, nitrogen or carbon dioxide. Hydrophilic or water-soluble products denote both the contrast products exhibiting these characteristics and the products having undergone this treatment which 30 renders them hydrophilic and/or water-soluble. This product is encapsulated in erythrocytes by a lysing/resealing technique. Erythrocytic encapsulation techniques are of three types: - using osmotic shocks, 35 - using electric shocks, - using isotonic hemolysis. Indeed, erythrocytes, like all cells, can be lysed under certain conditions, especially of osmotic WO 91/16080 - 7 - PCT/FR91/00301 pressure. But the erythrocytic membrane can be reconsti tuted after the lysis, this is known as resealing. It is therefore possible to introduce certain exogenous components into the erythrocytes. 5 The technique that has been described below is that of encapsulation using "osmotic shocks". The various stages of the hemolysis .and the resealing have been described by Schwoch G. and Passow H. (1973). 10 Red blood cells subjected to a decrease in ionic strength swell until a critical volume (150 to 175% of their initial value) is reached where the membrane becomes permeable to macromolecules and to ions. By restoring the isotonicity of the medium, the 15 pores are closed again and the impermeability of the membrane to macromolecules is restored. However, the membrane remains permeable to mainly alkali metal ions. An incubation stage at 37*C is required for the red blood cells to recover the permeability characteristics identi 20 cal to the initial red blood cells. Three different hypotonic hemolysis techniques have been proposed: Dilution technique The red blood cells are directly diluted in a 25 hypotonic medium. The pores are immediately opened and equilibrium between the extra- and intracellular medium is obtained in less than a minute. However, in this technique, 60 to 70% of the cytoplasmic content of the red blood cells is- lost, considerably reducing the 30 viability of the stromata obtained. Preswelling technique In this technique, proposed by Rechsteiner M. (1975), the red blood cells are diluted in a hypotonic buffer at 220 mos/kg and centrifuged. The lysis is then 35 obtained by adding a small volume of water. This pro cedure is rapid and makes it possible to retain more small molecules such as ATP and cytoplasmic enzymes.

WO 91/16080 - 8 - PCT/FR91/00301 Dialysis technique In this technique, proposed independently by Dale G. et al. (1977) and Deloach J.P. et al. (1977), the erythrocytic suspension with a high hematocrit value (70 5 to 80%) is kept in a dialysis bag and dialyzed against a hypotonic buffer. The hemolysis is thus gradual and can be easily controlled by modifying the dialysis time. This procedure offers several advantages. By comparing the yields of the various encapsulation 10 techniques, it is noted that dialysis leads to a better result in most cases. This can be explained by the smaller dilution of the product to be encapsulated. Furthermore, losses of the cytoplasmic content of red blood cells during encapsulation are considerably reduced 15 in this technique. It is the one which is used in the present invention. In particular, a dialysis technique providing excellent yields is that developed in Patent EP 101 341. This application describes a process for encapsulating into human or animal erythrocytes a 20 substance exhibiting biological activity, by a suitable continuous flow dialysis technique. Accordingly, the present invention relates to a process for preparing a medium, characterized in that: -a) The erythrocytes are placed in a dialysis 25 compartment, the other compartment containing an aqueous solution hypotonic relative to the erythrocyte suspension so as to lyse the erythrocytes. -b) The erythrocytes brought into contact with the 30 contrast product are resealed by increasing the osmotic pressure on encapsulating the said contrast product. -c) Where appropriate, the erythrocytes are separated from the reaction medium. 35 -d) The erythrocytes are placed in a carrier suitable for enteral or parenteral administration in order to obtain a contrast medium.

WO 91/16080 - 9 - PCT/FR91/00301 For the simplest technique permitting encapsu lation of the contrast product into human or animal erythrocytes, in the case of small volumes, the dialysis is carried out in a dialysis bag against a hypotonic 5 solution. The lysate is exposed to the active product and the resealing is carried out by adding a hypertonic solution. The preferred encapsulation method is the continuous flow dialysis process. For that, the 10 erythrocytes are separated from the plasma and washed and placed in aqueous suspension, and then continuously supply the primary compartment of a dialyzer. In the secondary compartment circulates countercurrentwise a dialysis buffer which is hypotonic relative to the 15 erythrocyte suspension. The erythrocyte lysate is brought into contact with the product to be encapsulated, this substance being introduced before, during or after the lysis. The product penetrates inside the erythrocyte and the cell is resealed by increasing the osmotic pressure 20 by adding a hypertonic solution. After resealing, the erythrocytic suspension is centrifuged so as to remove nonencapsulated molecules and hemoglobin from the intercellular liquid. The erythrocytes are then washed and resuspended in plasma at a physiological hematocrit 25 value. For the iodinated products encapsulated by this method, the intraerythrocytic iodine concentration of the medium may be between 1 and 300 g of iodine per liter of globular pellet. 30 For the media in which a paramagnetic complex is encapsulated by this method, the intraerythrocytic complexed paramagnetic metal concentration is between 0.02 mM and 0.5 M in the globular pellet. The use of encapsulated contrast products in red 35 blood cells makes it possible to target the space where these products are distributed by strictly confining them to the vascular space.

WO 91/16080 - 10 - PCT/FR91/00301 Accordingly, the present invention relates to the application of the above-defined contrast medium to the imagery of the circulatory system, of vascularized organs and of small cavities, characterized in that the medium 5 is injected enterally or parenterally, and in that these elements are visualized by an appropriate means, chosen from X-ray, nuclear magnetic resonance or ultrasound imagery. Administration of the medium can be performed 10 enterally. The medium can also be introduced into a small cavity of the organism, whose contrast it is desired to modify during imagery. Preferably, the medium according to the invention can be injected parenterally, in particular intravenously 15 or intraarterially. The pharmacokinetics of the product will be that for erythrocytes. This application will therefore permit the imagery of all vascularized organs and of situations of vascular pathology or of vascular rupture or modifications. 20 A very distinct improvement will be observed in the anatomical imagery of vessels by virtue of the reduction, on the one hand, of the leak into the inter stitial space and, on the other hand, of the urinary removal which are encountered with current products, in 25 particular with iodinated products and gadolinium chelates. An improvement will also be obtained in the imagery of the local blood volume and flow rate, and therefore of organ perfusion abnormalities. On the one 30 hand, hypofusion phenomena or ischemias can be detected and, on the other hand, hyperperfusion phenomena occur ring during certain tumor processes. The encapsulation of contrast products by this lysis/resealing method does not modify the half-life of 35 erythrocytes. -After parenteral administration of the contrast medium, stability will be observed for the contrast product in circulation, it being possible for its half-life to be equal to 60 days in the case of human WO 91/16080 - 11 - PCT/FR91/00301 erythrocytes. It will therefore be possible to perform the visualization at various times ranging up to 60 days. It will thus be possible to monitor infarction or ischemia 5 processes without reinjection. The medium will be eliminated by destruction of the erythrocytes in the spleen. The medium can be injected at doses ranging from 1 to 500 cm 3 of treated globular pellet (with a hematocrit 10 value of between 30 and 80%). One application of this invention is charac terized in that the erythrocytes are collected from a patient, in that they are treated so as to encapsulate a contrast product and in that they are reinjected into the 15 patient. In one embodiment of the process, the human erythrocytes are collected over an anticoagulant solution of citrate phosphate dextrose. The plasma is decanted and the leucocytes, platelets and plasma are removed by 20 prewashing and centrifugations of the erythrocytes. The globular pellet is adjusted to the desired concentration and incubated for 30 min at 4C. The erythrocyte lysis/resealing step is then carried out. For that, an erythrocytic solution and a 25 hypotonic lysis solution is circulated countercurrent wise in a plate dialyzer. These steps are carried out at 4*C. Swelling of the erythrocytes and opening of the pores occur. At the outlet of the dialyzer, DOTA Gd is added to the erythrocytic lysate continuously. A contact 30 time permitting penetration of the active ingredient into the cells should be provided for. The properties of the erythrocyte are restored by reheating the solution at 37*C and adding a hypertonic resealing solution. The resealing is continued by 35 incubating for 30 min at 37 0 C. The erythrocytic suspension is then centrifuged to remove' the nonencapsulated molecules and hemoglobin.

WO 91/16080 - 12 - PCT/FR91/00301 After three successive washes with 150 mM NaCl, the erythrocytes are resuspended in a physiological medium. Figure 1 : Stability of encapsulated gadolinium at 4*C. 5 Figure 2 : Stability of encapsulated gadolinium at 37*C. EXAMPLE 1 Encapsulation of DOTA Gd into human erythrocytes by continuous flow dialysis. 10 a) PreEarationof the human-erythrocytes The blood units used during this study are: - either units collected over citrate phosphate dextrose (CPD) solution at a concentration of 63 ml per 450 ml of blood collected, which are preserved 15 at 4*C for one week or more, - or globular concentrates preserved in SAG.M at 4*C for one week or more. CPD : Citric acid 1H 2 0 ...... 0..*............. 3.27 g 20 Sodium citrate 2H 2 0 ...................... 26.30 g Monosodium phosphate H 2 0 ........... *.. 2.22 g Glucose ............................-. 23.20 g Water for injection qs ................... 100 ml Decantation of the plasma and prewashing of the 25 red blood cells are then carried out so as to remove the leucocytes, platelets and plasma. These steps are carried out by means of 300 and 600-ml transfer pouches (Terumo). The washes of the globular pellets are carried out in 150 mM NaCl using 30 equal volumes, with 15 min centrifugations at 1,000 g. The globular pellet is adjusted to the desired cell concentration and the hematological parameters are measured on a sample. Finally, the pellet is incubated for 30 mm [sic] 35 at 4*C. b) Lysf the erythrgcytes The lysis is performed in a Gambro plate dialyser (Lundia 1C) whose membrane (thickness 13.5 pm and effective surface area 0.41 mn) is an acryla-butadiene WO 91/16080 - 13 - PCT/FR91/00301 styrene, high and low density polyethylene, cuprophan and cellulose polymer. The erythrocytic solution and lysis buffer, which is prepared as follows, are circulated countercurrentwise: 5 Stock solution: Na 2

HPO

4 ; NaH 2 PO4, pH 7.4 ................... 0.2 M NaHCO 3 -......----. *.-*** * -********** 0.2 M Glucose ................... ........ ...... 0.04 M The lysis buffer consists of this solution 10 diluted 1/20 in distilled water, a buffer at 40 mos/kg and pH 7.4 is obtained. The flow of dialysis buffer is steady: 160 ml/min. In contrast, the dialysis conditions vary as a function of the blood flow and the hematocrit value. 15 The hematocrit value of the globular pellet should be sufficiently high to avoid substantial loss of the said cytoplasmic content of the red blood cells; but it should remain sufficiently low to allow substantial swelling of the red blood cell during dialysis without 20 inducing too high a pressure in the dialyzer. c) Introduction of the active ingredient In the case of dialyzable molecules, it is preferable continuously to add the active ingredient directly into the lysate at the outlet of the dialyser. 25 d) Resealing and regeneration of the red blood cells Restoration of the isotonicity of the lysed globular suspension is carried out after resealing at 37*C by addition of a hypertonic resealing solution in an amount of one volume per ten volumes of lysate. The 30 resealing is continued for 30 minutes by incubation of the red blood cells in a water bath at 37*C. The resealing solution consists of a 2 M NaCl and PIGPA mixture. PIGPA C (BRUNEAU Laboratory) 35 Adenine .-.................................... 5 mM Inosine ............................. 100 mM Na pyruvate ......................... 100 mM Na phosphate ........................ 100 mM WO 91/16080 - 14 - PCT/FR91/00301 Glucose ............................. 100 mM e) Post-resealing washes of the red blood cells After resealing, the erythrocytic suspension is centrifuged in order to remove the nonencapsulated 5 molecules and hemoglobin from the intercellular liquid. The red blood cells are then washed according to the same procedure as that described for the prewashes. After these three washes in 150 mM NaCl, one wash is carried out in plasma in order to resuspend the red blood 10 cells in physiological medium. EXAMPLE 2: Encapsulation of DOTA Gd in mouse erythrocytes by batch dialysis a) Preparation of the mouse red blood cells 15 The mice used in this study are Swiss mice obtained from the Janvier breeding or from laboratory breeding. Whole blood is collected over heparin after anesthetizing the animals with ether, by incision in the lateral thoracic region, section of the brachial artery 20 and collection with a pasteur pipette. The washing operations are carried out in a tube. b) Lysis The dialysis is carried out in a glycerinated cellulose dialysis bag (Poly labo). The flat diameter is 25 2.54 cm whereas once swollen, the diameter is 1.59 cm. The membrane of these 20-ym thick bags has a permeability limit of 12,000 to 14,000. It is performed using 5 ml of 70% globular pellet in a dialysis bag placed in a 250 ml flask containing 30 lysis buffer. The flask is fixed upon a rotary stirrer at 12 rpm kept at 4*C. c) Introduction of DOTA Gd and resealing These steps are carried out in a tube. 35 Table -1 gives the hematological parameters and the parameters for encapsulating DOTA.Gd into human red blood cells as a function of the dialysis time. The encapsulation yields are calculated relative to the WO 91/16080 - 15 - PCT/FR91/00301 initial red blood cell suspension (encap. yield/pool) or relative to the concentration obtained in the lysate (encap. yield/lys.). Table 2 represents the hematological parameters 5 and the parameters for encapsulating DOTA-Gd into human and murine red blood cells for a dialysis time of 45 min (mean ± SD with N = number of experiments). Table 1 Encapsulation with DOTA.Gd 10 Human red blood cells Time Controls 30 min 45 min 60 min N=3 N= 8 N= 1 Parameters 15 Cell yield (%) 82.8 ± 4.2 73.2 ± 3.5 70.3 Encap. yield/pool (%) 28.4 ± 6.7 36.4 ± 4.1 40.1 Encap. yield/lysis (%) 40.9 ± 4.7 65.0 ± 4.6 76.2 mg/ml* 0.47 ± 0.11 0.70 ± 0.09 0.73 VGM (fl) 95.9 ± 1.7 91.3 ± 0.7 86.8 ± 2.5 84.8 20 TGMH (pg) 30.1 ± 0.2 27.0 ± 0.1 25.7 ± 1.0 24.6 CCMH (g/d) 31.3 ± 0.3 29.5 ± 0.1 29.2 ± 0.7 29.1 * mg of DOTA Gd/ml at 100% standardized hematocrit value for 1 mg of DOTA Gd/ml in the initial erythrocytic suspension. 25 VGM: mean globular volume (mean volume of a red blood cell) TGMH: mean globular quantity of hemoglobin CCMH: mean globular concentration of hemoglobin.

WO 91/16080 - 16 - PCT/FR91/00301 Table 2 Murine erythrocytes Human erythrocytes Parameters Before After Before After 5 Cell yield (%) 65.0 t 2.6 73.2 t 3.5 Encap. yield/pool (%) 31.6 t 4.0 36.4 t 4.1 Encap. 10 yield/lysis (%) 59.1 t 5.7 65.0 t 4.6 mg/ml* 0.67 t 0.09 0.70 t 0.09 VGM (fl) 50.9 t 0.3 48.3 t 0.9 96.5 ± 2.0 86.8 t 2.5 TGMH (pg) 17.5 t 0.9 16.2 ± 0.7 30.2 t 0.2 25.7 t 1.0 CCMH (g/dl) 34.4 t 1.7 33.5 t 1.5 31.2 t 0.4 29.2 t 0.7 15 * mg of DOTA Gd/ml at 100% standardized hematocrit value for 1 mg of DOTA Gd/ml in the initial erythrocytic suspension. EXAMPLE 3: Stability of DOTA Gd after erythrocytic encapsulation 20 After erythrocytic encapsulation, the suspension of red blood cells at 40% plasma hematocrit value is kept at 4C or 37*C. For varying incubation times, an aliquot is collected and centrifuged at 1,000 g for 10 minutes at 4*C. Gadolinium is then assayed on the globular pellet 25 and on the supernatant. The hematocrit value for the pellet is measured so as to standardize all the assays at the same hematocrit value. Figures 1 and 2 show the variation, as a function of time, of the intraerythrocytic concentration of 30 gadolinium after encapsulation of DOTA-gadolinium for two intraerythrocytic concentrations. GR1 and GR2 are the intraerythrocytic DOTA Gd concentrations, SP1 and SP2 are the plasma DOTA Gd concentrations corresponding to GR1 and GR2 respectively. 35 The temperature has little impact on the release of DOTA Gd.

WO 91/16080 - 17 - PCT/FR91/00301 This study, carried out using human red blood cells, shows a slight gadolinium leakage with a half-life of 9.1 days at 4*C and 7.5 days at 37*C. On average, the intraerythrocytic gadolinium concentration after 48 hours 5 is equal to 81% of the initial concentration. EXAMPLE 4: Study of the relaxivity The relaxivity of DOTA Gd was measured in washed globular pellets (suspension of red blood cells at high 10 hematocrit value 80/90%) after incorporation of DOTA Gd. Gadolinium was assayed by atomic emission spectrophotometry (DCP). The relaxation time Ti was determined on a Minispec BRUKER PC 20 at 20 MHz 0.5 T by inversion-recovery sequence at 37*C. 15 Table 3 Mouse red blood cells CONCENTRATION RELAXIVITY MEDIUM (mmol/l) 20 Gd DOTA (mM~1 s~1) Mouse globular pellet 2.9 8.1* Mouse globular pellet 0.2 12.3 Mouse plasma 0.5 3.9 25 Water 0.5 3.9 * This result was confirmed at various dilutions of the pellet. An increase in the relaxivity of DOTA Gd is 30 therefore observed in the globular pellet.

WO 91/16080 - 18 - PCT/FR91/00301 Table 4 Human red blood cells CONCENTRATION RELAXIVITY 5 MEDIUM Gd (mmol/l) (mM 1 s~1) Human globular pellet 0.5 4.7 Water 0.5 3.9 10 EXAMPLE 5: Efficacy during imagery The imagery of tubes containing the human globu lar pellets after incorporation of DOTA Gd was performed by comparison with aqueous solutions of DOTA Gd of 15 equivalent concentration. Imagery . MR MAX 0.5 Tesla, . SE sequences 300/20/1, . section thickness: 7 mm, 20 - generation of intensity profiles as a function of the concentration of gadolinium permitting a relative quantification of the samples relative to each other. Results: an efficacy of the same order of magnitude is 25 observed in vitro between DOTA Gd and DOTA Gd encapsu lated into erythrocytes.

WO 91/16080 - 19 - PCT/FR91/00301 Table 5 MEDIUM CONCENTRATION PEAK INTENSITY HEIGH. Gd (mmol/l) (cm) 5 9.48 2 4.75 4.3 0.93 4.6 Globular pellet 0.37 3.6 10 0.10 2 0.013 1.4 0 1 8.87 4.7 15 4.47 6.9 Water 0.85 6.6 0.41 3.6 0.08 1.6 0 0.8 20 1 The DOTA Gd incorporated into red blood cells results in a substantial modification of intensity during imagery (multiplied by 4.6 for a concentration of 0.93 mM) for concentrations of between 2 x 10~ and 10~ 4 M 25 as with DOTA Gd. EXAMPLE 6: Lifetime of the red blood cells and pharmacokinetics of gadolinium The lifetime of mouse red blood cells containing 30 Dota Gd (9 mM) was monitored by 5 Cr-labeling. Gadolinium was measured by AES (atomic emission spectrophotometry) in parallel with the gamma counting of 5 Cr in mice, and alone in the case of pharmacokinetics in beagle dogs. 35 Results: in the first 24 hours, the disappearance of 18% of 51 Cr and 22.5% of Gd is observed in mice and 25.5% of Gd in beagle dogs.

WO 91/16080 - 20 - PCT/FR91/00301 Table 6 Product incorporated Species Half-life of the into the red blood cells red blood cells 5 (days) Dota Gd 9 mM mice 10.6 None mice 10.4 10 Cr 10 mM(d) dogs 4.3 Dota Gd 7.5 mM dogs 19.0* None dogs 18.9 * half-life of gadolinium measured by AES 15 (d) technique of Eisenberg et al. (1989) It can be observed that there is neither release of Dota Gd nor substantial hemolysis due to the incorporation. Example 7 20 Preparation of the composition DTPA-Gd methylglucamine salt-erythrocytes This composition was prepared according to the process described in Example 1. Table 7 gives the results for the encapsulation of DTPA-Gd for human red blood 25 cells and for an initial concentration of 10 to 15 mg/ml.

WO 91/16080 - 21 - PCT/FR91/00301 Table 7 Encapsulation of DTPA-Gd methylglutamine salts into human red blood cells 5 Controls DTPA Gd N - 3 Parameters Cell yield (%) 85.4 ± 4.9 Encap. yield/pool (%) 26.2 ± 4.3 10 Encap. yield/lysis (%) 44.8 ± 4.8 mg/ml 0.45 ± 0.08 VGM (fl) 97.2 ± 2.8 87.9 ± 3.6 TGMH (pg) 30.3 ± 1.1 26.7 ± 1.4 CCMH (%) 31.3 ± 0.9 30.2 ± 0.6 15 Example 8 Encapsulation of iopamidol into human and murine erythrocytes The preparations are carried out according to the 20 process described in Example 1 and 2. Table 8 gives the hematological parameters and the parameters for the encapsulation of iopamidol into human red blood cells as a function of the dialysis time for an initial concentration of 10 mg/ml of erythrocytic 25 suspension. It is possible to increase the final intra erythrocytic concentration by increasing the initial concentration of the erythrocytic suspension, thus for 60 minutes of dialysis and an initial concentration of 50 mg/ml, a concentration of 41.4 mg/ml at 100% hemato 30 crit value can be obtained.

WO 91/16080 - 22 - PCT/FR91/00301 Table 8 Encapsulation of iopamidol into human red blood cells Time Controls 30 min 45 min 60 min 5 N= 1 N= 4 N= 2 Parameters Cell yield (%) 82.4 71.8 t 1.6 71.8 t 1.8 Encap. yield/pool (%) 42.5 46.9 t 5.6 50.7 t 2.0 10 Encap. yield/lysis (%) 59.3 69.0 t 5.6 71.2 t 0.8 mg/ml* 0.74 0.9 t 0.1 1.07 ± 0.04 VGM (fl) 94.5 ±2.3 88.4 78.5 t 1.5 72.4 t 6.7 TGMH (pg) 29.5 t 1.7 27.1 26.5 t 0.9 23.3 t 1.8 15 CCMH (%) 32.0 t 0.9 30.1 33.3 ± 0.6 32.2 t 0.5 * mg/ml at 100% standardized hematocrit value for 1 mg/ml in the initial suspension. Table 9 gives the encapsulation parameters and 20 the hematological parameters f or the encapsulation of iopamidol into murine red blood cells. Table 9 Encapsulation of iopamidol into murine red blood cells 25 Controls Iopamidol N = 4 Parameters Cell yield (%) 70.8 t 1.1 30 Encap. yield/pool (%) 32.6 t 2.0 Encap. yield/lysis (%) 50.7 & 2.6 mg/ml 16.5 t 0.6 VGM (fl) 53.5 t 2.2 48.8 t 1.1 TGMH (pg) 17.4 t 0.3 14.7 ± 1.4 35 CCMH (%) 32.7 1 2.0 29.7 t 2.2 WO 91/16080 - 23 - PCT/FR91/00301 Example 9 Lifetime of red blood cells after internalization of iopamidol The lifetime of mouse red blood cells containing 5 iopamidol was generated as in Example 6. Iodine is measured by spectrophotometry in parallel with the gamma counting of 51 Cr. Results: Blood disparance after 24 hours is of the 10 monoexponential type with a half-life of 12.9 to 7.9 days for 5 Cr and iodine respectively. Table 10 Product incorporated Species Half-life of the 15 into the red blood cells red blood cells (days) iopamidol 16.3 mg/ml mice 12.9 ± 1.3 none mice 10.4 ± 0.7 20 REFERENCES 1. DALE G., VILLACORTE D. and BEUTLER E.: High-yield entrapment of proteins into erythrocytes. Biochem. Med 18. 220-225 (1977). 25 2. DELOACH J. and IHLER G.: A dialysis procedure for loading erythrocytes with enzymes and lipids. Biochem. Biophys. Acta 496. 136-145 (1977). 3. EISENBERG A.D., CONTURO T.E., PRICE R., HOLBURN G.E., PARTAIN C.L. -et JAMES A.E.: MR1 and Enhancement of 30 perfused tissues using chromium labeled red blood cells as an intravascular contrast agent. Invest. Radiol., 24. 742-753 (1989).

WO 91/16080 - 24 - PCT/FR91/00301 4. SCHWOCK G. et PASSOW H.: Preparation and properties of human erythrocyte ghosts. Mol. Cell. Biochem. 2. 197-218 (1973).

Claims (9)

1. Contrast modifying medium for X-ray, nuclear magnetic resonance and ultrasound imagery, characterized in that it comprises a hydrophilic or water-soluble or 5 colloidal contrast product, for example a superparamagnetic colloid or a gas in bubble form, encapsulated by a technique for lysing/resealing inside erythrocytes, and in that it comprises, in addition, an acceptable carrier for enteral or parenteral 10 administration.

2. Medium according to claim 1, characterized in that the encapsulated contrast product is a polyiodinated contrast agent.

3. Medium according to claims 1 and 2, characterized 15 in that the intraerythrocytic concentration of iodine is between 1 and 300 g of iodine per liter of globular pellet.

4. Medium according to claim 1, characterized in that the encapsulated contrast product is a gas in bubble 20 form.

5. Medium according to claim 4, characterized in that the encapsulated gas is chosen from air, nitrogen and carbon dioxide.

6. Medium according to claim 1, characterized in 25 that the encapsulated contrast product is a paramagnetic complex.

7. Medium according to claim 6, characterized in' that the complex is chosen from the complexes formed from a cyclic or acyclic ligand possessing coordination atoms 30 chosen from oxygen, nitrogen, phosphorus, sulfur and a metal chosen from the lanthanides or the transition metals, especially gadolinium, dysprosium, manganese and iron.

8. Medium according to one of claims 6 or 7, 35 characterized in that the intraerythrocytic concentration of complexed paramagnetic metal is between 0.02 mM and 0.5 M in the globular pellet. REPLACEMENT SHEET WO 91/16080 - 25a - PCT/FR91/00301

9. Medium according to claims 1 and 6 to 8, characterized in that the encapsulated contrast product is an N-methylglucamine or sodium salt of the gadolinium complex of diethylene-triaminepentaacetic acid (Gd-DTPA). REPLACEMENT SHEET