FR2567521A1 - New activated ionophores - Google Patents

Abstract

New activated ionophores of formula: in which I DEG represents the residue of an ionophore which has lost one of its hydroxyls and W represents hydrogen or an acetyl group, which are useful as intermediates in the preparation of modified antibodies.

Description

New activated ionophores.

 The present invention relates to activated ionophores and their use as synthesis intermediates for immunotoxins.

 In its earlier patent application, filed in France under number 82/02 091, the Applicant mentioned the ability of carboxylic ionophores to potentiate immunotoxins.

 By immunotoxins is meant anticancer substances obtained by covalently coupling a cytotoxic protein (e.g. ricin chain A) with antibodies or antibody fragments directed against an antigen posted by a cell to be destroyed.

 Such immunotoxins have in particular been described by the Applicant in French Patent No. 78/27838 and its addition No. 79/24655 and in French Applications No. 81/07596 and No. 81/21836.

The potentiating properties of ionophores have been advantageously used in certain types of situations. In particular, they give favorable results whenever an immunotoxin is used as a selective cytotoxic agent in vitro to kill the target cells. This is particularly the case when the immunotoxin is used as a cytotoxic agent in the treatment of the bone marrow of leukemic patients to whom the bone marrow thus treated will subsequently be transplanted.

This is also the case when the immunotoxin is used to reduce the T-cell population of a graft for allogeneic bone marrow transplant applications in order to prevent disorders related to the graft-versus-graft reaction. host.

In contrast, when the immunotoxin is used in vivo as a therapeutic agent in humans, the in vivo use of an ionophore to benefit from the potentiating effect has certain inherent limitations.
- the toxicity of ionophores
their low solubility in solvents suitable for parenteral administration
- their very rapid elimination of blood plasma.

 According to the present invention, it has been found that, surprisingly, these limitations can be eliminated by replacing the carboxylic ionophores with conjugates covalently associating a monovalent carboxylic ionophore and a macromolecule, such as an antibody, a fragment or a of antibodies, a protein, a peptide, etc.

 According to a first aspect, the subject of the present invention is therefore, as novel products, conjugates (mixed artificial molecules) constructed by covalent binding of a monovalent carboxylic ionophore with macromolecules (such as antibodies, proteins, peptide ligands). ).

 In the following description, such conjugates will be referred to as "ionophore conjugates".

 The ionophores used are known molecules: they are in particular natural substances isolated from strains of Streptomycetes fungi which comprise a hydrocarbon backbone in which are included oxygenated erythrocytes. At one end of the chain there is always a carboxylic acid function while at the other end are one or more alcohol functional groups (B.C. Pressman, Annual Review of Biochemistry, 45, 501-530, 1976).

 These natural substances, as well as certain semisynthetic compounds derived therefrom, have in common an ionospheric activity, that is to say the ability to allow the transfer of metal ions (and in particular of monovalent ions) from a hydrophilic phase. to an immiscible lipophilic phase.

 The macromolecules used may be antibodies (or antibody fragments), proteins (such as peptide hormones or human proteins, for example human serum albumin), peptide ligands (such as natural or synthetic polypeptides and in particular polylysine).

 In the case where an antibody is used, it can be either of polyclonal character if it results from a conventional immunization carried out in the animal, or of monoclonal character if it is produced by a hybrid cell clone. obtained by fusion between lymphocytes and myeloma cells. This antibody can be used either in the form of whole immunoglobulin molecules having the ability to recognize the selected antigen, or in the form of any fragment of these immunoglobulin molecules having retained the ability to recognize the selected antigen, and in particular the fragments known under the names of F (ab ') 2, Fab and Fab'.

The chemical coupling between the macromolecules and the ionophore can be achieved by various methods provided that the chosen method
preserves the respective biological activities of the components of the conjugate
ensures satisfactory reproducibility and a good coupling efficiency;
- makes it possible to control the value of the ionophore / macromolecule ratio in the conjugate obtained
- lead to a stable product that is soluble in water.

 Among all the chemical coupling methods corresponding to these characteristics, one can choose those which implement one or more thiol functions for the establishment of the link.

In this case, it is possible to use a thiol group artificially introduced onto one of the compounds to be coupled, and to introduce onto the other compound one or more functions capable of reacting with thiol functions, and this in an aqueous medium of pH between 5 and 9, at a temperature not exceeding 300C, to provide a stable, covalent and defined bond.

For example, a thiol can be artificially introduced onto the ionophores by the action of S-acetyl-mercaptosuccinic anhydride which will be able to acylate one of the alcohol functions of the ionophore. This thiol function can then be liberated by elimination of the protective acetyl radical by the action of hydroxylamine, as has already been described (Archives of Biochemistry and
Biophysics, 119, 41-49, 1967).

Suitable ionophores are monensin, nigericin, grisonixin and lasalocid; monensin and niger

<tb> ricin, <SEP> having <SEP> the <SEP> formulas
<tb><SEP> ifOH3 <SEP> ICH
<tb><SEP> HÇ / -Ft - CH20H
<tb><SEP> CH, O <SEP> Ln. <SEP> jLH <SEP> v <SEP> fl <SEP> 1 <SEP> n <SEP> cr --- n3VC
<tb><SEP> CH (CH3) CoeH <SEP> monensin
<Tb>

<tb><SEP> 3
<tb> H <SEP> H <SEP>><SEP> HO
<tb><SEP> CCOH
<tb><SEP> nigericine
<tb> are particularly preferred.

The introduction of the artificial thiol is carried out by reaction of the ionophore with S-acetyl-mercaptosuccinic anhydride

<tb> of <SEP> formula <SEP> O
<tb><SEP> Lo <SEP> S-CO-CH3
<tb> in an inert organic solvent at about room temperature.

où I" représente le résidu du ionophore privé d'un de ses hydroxyles, l'hydroxyle primaire dans le cas de la monensine et de la nigéricine.By this reaction, an ester bond is established between one of the hydroxyls of the ionophore, the primary hydroxide in the case of monensin and nigericin, and acetylmercaptosuccinic acid and obtaining a compound of formula

where I "represents the residue of the ionophore deprived of one of its hydroxyls, the primary hydroxyl in the case of monensin and nigericin.

où IO est tel que défini ci-dessus, peut être utilisé in situ pour l'opération de couplage.The thiol group of the above compound can be released by the action of hydroxylamine and the product thus obtained, of formula

where IO is as defined above, can be used in situ for the coupling operation.

dans laquelle W est l'hydrogène ou un groupe acétyle, sont nouveaux et reprsentent un autre aspect de la présente invention.Said activated ionophores, which are represented by the formula

wherein W is hydrogen or an acetyl group, are novel and represent another aspect of the present invention.

est indiqué ci-après I et désigné "ionophore à coupler".The group

is indicated hereinafter and designated "ionophore to be coupled".

 The coupling on the macromolecule can be carried out with one or other of the compounds I and II, but said coupling is faster with the compound II which is therefore preferably used.

 The coupling will then be carried out with the macromolecule onto which one or more functions capable of establishing a covalent bond with the thiol have been introduced. This covalent bond may be either a disulfide bond or a thioether bond.

Disulfide bond case
In this case, the preparation of the conjugate can be represented by the scheme
ISH + PRSSX> ISSRP + XSH in which
I represents the ionophore to be coupled,
P the macromolecule to be modified, -SSX denotes an activated mixed disulfide group of which X is the activator radical.

The macromolecule substituted with an activated sulfur atom is obtained from the macromolecule itself, by substitution with a reagent itself carrying an activated sulfur atom according to the scheme.
P + YRSSX> PRSSX in which
P is the macromolecule to be modified,
Y represents a function allowing the covalent attachment of the reagent to the protein,
R denotes a group which can simultaneously carry the substituents
Y and -SSX,
X denotes the activator radical.

où R1 est un groupe alkyle réagissant avec les groupes aminés de la macromolécule selon la réaction

The functional group Y is a function capable of binding covalently with any of the functions carried by the side chains of the constituent amino acids of the protein E substitute. Among these, the terminal amine functions of the lysyl radicals contained in the protein are particularly indicated. In this case, Y may in particular represent
a carboxylic group which can bind to the amino functions of the protein in the presence of a coupling agent such as a carbodiimide and especially a water-soluble derivative such as ethyl-1 (3-diethylamino-propyl) -3 -carbodiimide,
a carboxylic acid chloride which is capable of reacting directly with the amine functions to acylate them,
an "activated" ester such as an ortho- or para-nitro- or dinitro-phenyl ester, or an N-hydroxysuccinimide ester which reacts directly with the amine functions to acylate them,
an internal anhydride of a dicarboxylic acid such as, for example, succinic anhydride, which reacts spontaneously with the amine functions to create amide bonds, an imidoester group;

where R 1 is an alkyl group reacting with the amino groups of the macromolecule according to the reaction

 The radical -S-S-X denotes an activated mixed disulfide capable of reacting with a free thiol radical. In particular, in the mixed disulfide, X may denote a pyridyl-2 or pyridyl-4 group optionally substituted with one or more alkyl, halogen, carboxylic radicals. X may also denote a phenyl group preferably substituted by one or more nitro or carboxylic groups.

X may still be an alkoxycarbonyl group, such as methoxycarbonyl.

dans lequel R4 désigne l'hydrogène ou un groupe alkyle ayant de 1 à 8 atomes de carbone, et R3 désigne un substituant inerte vis-à-vis des réactifs utilisés ultérieurement, tel qu'un groupe carbamate -NH-C-OR5 où R5 désigne un groupe alkyle droit ou ramifié.ayant de
o 1 à 5 atomes de carbone et notamment le groupe tertiobutyle.The radical R denotes any radical capable of simultaneously carrying the substituents Y and -SSX. It should be chosen in such a way that it does not include any functions that may interfere with subsequent reactions with the reagents used and the products synthesized. In particular, the group R can be a group
(CH2) n with n between 1 and 10, or a group

wherein R4 denotes hydrogen or an alkyl group having 1 to 8 carbon atoms, and R3 denotes a substituent inert to the reactants used later, such as a carbamate group -NH-C-OR5 where R5 means a straight or branched alkyl group having
1 to 5 carbon atoms and in particular the tert-butyl group.

 The reaction of the compound Y-R-S-S-X with the macromolecule P is carried out in a homogeneous liquid phase, most often in water or a buffer solution. When the solubility of the reagents requires it, it is possible to add to the reaction medium up to 30% by volume of an organic solvent miscible with water, such as an alcohol and in particular tertiary butanol. The reaction is carried out at ambient temperature for a time varying from a few minutes to 24 hours. After that, dialysis eliminates low molecular weight products, particularly excess reagents.

This method makes it possible to introduce a number of substituents per mole of macromolecule usually between 1 and 50.

 Using such compounds, the coupling of the macromolecule and the ionophore is carried out by placing the two compounds in an aqueous solution at a temperature of not more than 30 ° C. for a time varying from a few minutes to 1 i. The resulting aqueous solution is dialyzed to remove the low molecular weight products.

dans lequel :
Z représente une structure d'espacement, aliphatique ou aromatique comportant de 1 à 10 atomes de carbone.Case of the thioether bond
The preparation of the conjugate then consists in reacting
I-SH with the P protein, on which a maleimide radical has previously been introduced. The reaction is then represented by the diagram

in which :
Z represents an aliphatic or aromatic spacer structure having from 1 to 10 carbon atoms.

dans lequel Y1 représente
- soit un groupe carboxylique, la réaction étant alors effectuée après activation de la fonction carboxylique en présence d'un agent de couplage, tel qu'un carbodiimide et notamment un dérivé soluble dans l'eau, tel que l'éthyl-1 (diéthylamino-3 propyl)-3carbodiimide,
- soit un ester dit "activé" tel qu'un ester d'orthoou para-, nitro- ou dinitrophényle, ou encore un ester de N-hydroxysuccinimide qui réagit directement avec les fonctions aminées pour les acyler. The P protein substituted with the maleimide is obtained from the protein P itself, by substitution of amino functions of the protein with a reagent which carries the maleimide group, according to the scheme.

in which Y1 represents
or a carboxylic group, the reaction then being carried out after activation of the carboxylic function in the presence of a coupling agent, such as a carbodiimide and especially a water-soluble derivative, such as ethyl-1 (diethylamino); 3-propyl) -3 carbodiimide,
or an "activated" ester, such as an ortho or para-nitro- or dinitrophenyl ester, or an N-hydroxysuccinimide ester which reacts directly with the amine functions to acylate them.

 The preparation of such reagents is especially described in Helvetica Chimica Acta, 58, 531-541 (1975). Other reagents of the same class are commercially available.

avec l'ionophore est effectuée en phase liquide homogène, le plus souvent dans l'eau ou une solution tampon. Lorsque la solubilité des réactifs l'exige, il est possible d'ajouter au milieu réactionnel jusqu'à 20 % en volume d'un solvant organique miscible à l'eau tel qu'un alcool et notamment le butanol tertiaire.The reaction of the compound

with the ionophore is carried out in homogeneous liquid phase, usually in water or buffer solution. When the solubility of the reagents requires it, it is possible to add to the reaction medium up to 20% by volume of a water-miscible organic solvent such as an alcohol and in particular tertiary butanol.

 The reaction is carried out at ambient temperature for a time varying from a few hours to 24 hours. After that, dialysis eliminates low molecular weight products and, in particular, excess reagents. This method makes it possible to introduce a number of substituent groups per mol of protein usually between 1 and 50.

 Using such compounds, the coupling of the macromolecule with the isophoron is carried out by placing the two compounds in an aqueous solution at a temperature not exceeding 300 ° C. for a time varying from a few hours to a day. The resulting solution is dialyzed to remove the low molecular weight products, and the conjugate can be purified by various known methods.

 According to a second aspect, the invention relates to the use of ionophore conjugates as potentiators of immunotoxins.

Studies on ionophore conjugates have shown that
the potentiating effect of the ionophores is preserved after coupling with the macromolecule.

 The binding of the ionophore to the macromolecule gives it a high solubility in an aqueous medium favorable to the in vivo administration of the product.

the binding of the ionophore to the macromolecule considerably lengthens the plasma half-life of the ionophore, which is essential for maintaining the potentiating effect in vivo
the toxicity of the conjugate is lower than that of the ionophore itself.

Thus, for each patient, at least two essential effector substances will be directed to the target cells.
on the one hand, a cytotoxic protein (such as, for example, ricin A chain) which will be the cytotoxic suppressor included in the immunotoxin conjugate
- On the other hand, the ionophore (such as for example monensin) which is the constituent of the potentiating system included in the conjugate called "ionophore conjugate".

 In the case where the ionophore is coupled to a macromolecule having a receptor on the surface of the target cells (for example when the macromolecule will be an antibody or an antibody fragment recognizing an antigen specific to the cell population to be destroyed different from that recognized by the immunotoxin) or if the ionophore is coupled to a peptide hormone having a receptor on the surface of the cells to be destroyed, the probability of the two chosen targets being present together on the surface of the non-target cells becomes very low and thus provides an extremely powerful means for further enhancing the specificity of the cytotoxicity of immunotoxins.

 The following examples illustrate the invention without limiting its scope.

Example 1
An ionophore conjugate obtained by reaction between the activated disulfide group T 29-33 and monensin on which a thiol has been introduced.

a) T 29-33 Antibody
This antibody is a monoclonal antibody directed against the T 200 antigen of human leukocytes. This antibody is described in J. Exp. Med., 1980, 152, 842. It is commercially available from Hybritech Inc., San Diego - California - USA.

b) T 29-33 Antibody Activated
To 2 ml of a T 29-33 antibody solution at 10 mg / ml (ie 0.133 pmol of antibody) is added an aqueous solution containing 3 mg of (pyridyl-2-disulfanyl) -3-propionic acid dissolved in tertiary butanol and 1.8 mg of ethyl-1-dimethylamino-3-propyl-3-carbodiimide. The mixture is stirred for 15 min at 300 ° C. and then dialyzed continuously against 125 mM phosphate buffer pH 7 (40 h at 500 ml / h). After dialysis, the protein solution is centrifuged and 2.5 ml of a solution of 6.6 mg modified antibody per ml is obtained. By spectrophotometric determination at 343 nm of the pyridine thione-2 released by exchange with the mercapto-2-ethanol, it is found that an antibody was obtained carrying 7.2 activating groups per mole of antibody.

c) Activated monensin
The monensin used is a commercial product. It has been modified as follows: 693 mg of monensin are dissolved in chloroform and then reacted with 350 mg of S-acetylmercaptosuccinic anhydride (SAMSA). The reaction is allowed to evolve for 1/2 hour at room temperature. The reaction medium is then evaporated to dryness under vacuum and then taken up in ethyl acetate and washed extensively with water. The organic phase is then dried and dried under vacuum. The product is obtained crystallized after drying under vacuum. It is identified by its NMR spectrum and its mass spectrum.

 The product thus obtained (S-acetyl activated monensin) has formula III above, wherein 10 is the residue of monensin deprived of its primary hydroxyl and W is an acetyl group.

 The acetyl radical can be released by the action of hydroxylamine at a concentration of 50 mS final and the product thus obtained, corresponding to formula III above where IO is the monensin residue deprived of its primary hydroxide and W is the hydrogen, can be directly used for coupling.

However, for the next step it is preferable to release the thiol group in situ in the presence of activated monensin
S-acetyl and activated antibody.

d) Preparation of the ionophore conjugate
8 mg (ie 9, mol) of S-acetyl activated monensin are dissolved in the minimum of terbutanol and added to 2.5 ml of the activated antibody solution at 6.6 mg / ml (ie 0.11 μmol) in the 125 mM phosphate buffer pH 7. 125 μl of a 1N hydroxylamine solution are added.

 The incubation lasts for 2 h at 250 ° C. and then the reaction medium is purified by dialysis of the excess of reagents against PBS buffer (10 mM phosphate, 140 mM sodium chloride pH 7.4).

 After dialysis and centrifugation, 2.8 ml of a solution of IgG T 29-33 5.6 mg / ml carrying an average of 7 monensins per mol of antibody was obtained.

Example 2
Potentiation of the anti-T65 immunotoxin
The conjugate according to the invention, obtained as indicated previously, has been studied with regard to its biological properties and more especially its capacity to potentiate the activity of the anti-T65 immunotoxin in a suitable cellular model.

 This model consists of cells of the human lymphoblastoid line CEM which naturally carry the T65 and T200 antigens. The T65 antigen against which the immunotoxin used is directed is the first target antigen in the model. This immunotoxin is that which has been described in a previous patent application of the applicant filed in France under No. 81/21836.

The T200 antigen against which the ionophore conjugate is directed will be the second target antigen in the model.

The fundamental property of immunotoxins being to inhibit the protein synthesis of the target cells, the test used consists in measuring the effect of the substances studied on the incorporation of 14C-leucine into the cancerous cells in culture. This measurement is carried out according to a technique adapted from that described in
Journal of Biological Chemistry, 1974, 249 (11), 3557-3562, using the 14C-leucine tracer for the determination of the protein synthesis rate. The determination of the incorporated radioactivity is carried out here on the whole cells isolated by filtration.

 On the basis of these determinations, we can draw the effects / dose curves showing, on the abscissa, the molar concentration in the A chain of the substances studied and, on the ordinate, the incorporation of 14C-leucine expressed as a percentage of the incorporation of the control cells in the absence. any substance affecting protein synthesis.

 It is thus possible to determine for each substance studied the concentration which inhibits 50% of the incorporation of 14C-leucine or "inhibitory concentration 50" (IC 50).

 The different tests were conducted as follows. The corresponding experimental results are shown in FIG.

a) EMC cells are incubated for 18 h in the presence of known concentrations of the anti-T65 immunotoxin used as reference, and then the cells are subjected to the incorporation step.
-11 radioactive tracer. The IC 50 obtained is 2.8 × 10 11 M (curve 1), which shows that the cells are normally sensitive to the effect of the immunotoxin.

b) The CEM cells are incubated for 18 h at 370 ° C. in the presence of a mixture of known anti-T65 concentrations and, respectively,
1 - free monensin at a concentration of 50 nM (curve 2)
Or 2 - anti-T200 ionophore conjugate previously described, at the concentration of 10 M (or 70 nM monensin) (curve 3).

 It has been previously verified that monensin and ionophore conjugate are not cytotoxic for the cells employed at the indicated concentrations.

The IC 50 values are respectively
-14 2.4.10 14 M for monensin 50 nM
1.4 × 10 M for the 10 M ionophore conjugate
These results show remarkable potentiating effects, by a factor of 1000-fold for 50 nM monensin and 2000-fold for the ionophore conjugate at a concentration of 10 -8 M (ie 70 μM monensin).

Example 3
Ionophoric conjugate obtained by reaction between the 3E10 antibody substituted by an activated disulfide group and the monensin on which a thiol has been introduced.

a) 3E10 Antibody
This antibody is a monoclonal antibody directed against a human membrane antigen. It was obtained during an anti-cell lymphocyte fusion of the following species: SK MEL 28. Hybrid corne was cloned and multiplied; the antibody was produced in ascites and then purified on protein A Sepharose.

b) Anti-3E10 antibody activated
This antibody is obtained from the above antibody according to a technique described in Example i. There was thus obtained 195 mg of 3E10 antibody having 8 activating groups per mole of antibody.

c) Activated monensin
Monensin is activated according to the method described in Example 1.

d) Preparation of the ionophore conjugate
91 mg of S-acetyl activated monensin (ie 104 pmol) are dissolved in the minimum of terbutanol and added to 50 ml of the activated antibody solution at 3.9 mg / ml (ie 1.3 μmol) in phosphate buffer 125 mM pH 7. 2.5 ml of 1 N hydroxylamine solution are added.

 The incubation lasts 2 hours at 250 ° C., and then the reaction medium is purified from the excess of reagents by dialysis against PBS buffer (10 mM phosphate, 140 mM sodium chloride, pH 7.4). After dialysis and centrifugation, 2.8 ml of a 3.7 mg / ml IgC solution carrying an average of 8 monensins per IgC was obtained.

Example 4
3E10-monensin conjugate toxicity
The 3E10-monensin conjugate has been studied with respect to its biological properties in vivo. More specifically, its toxicity and pharmacokinetics were compared to those of free monensin.

 The toxicity of free monensin was determined in mice. The lethal dose 50% after I.V. injection is 4 mg / kg, ie 80 μg / mouse.

 The toxicity of the conjugate was tested on a single mouse also administered by the IV route., For the mice of each batch, the product was injected intravenously at the following doses: 3.7 mg, 1.85 mg, 0.952 mg, 0.496 mg of conjugate, being respectively the equivalent of 163 μg, 81.5 μg, 40.75 μg, 20.4 μg of monensin. No mortality was observed in any of the lots of mice.

Example 5
Pharmacokinetics of the conjugate 3E10-monensin

 Pharmacokinetics was studied in female nude mice using the ionophore conjugate described in Example 3.

The mice are injected
or 1 ml of ionophore conjugate at 3.7 mg / ml (ie 163 μg of coupled monensin),
- 163 ug of free monensin (control group).

 For each time, the plasmas of two mice are taken. The active monensin is assayed in the plasma by dilution thereof and comparison with a free monensin standard curve in the protein synthesis inhibition test described in Example 1.

 The assay is performed on CEM cells in the presence of the anti-T65 immunotoxime.

The results are shown in FIG. 2, in which the time in hours is plotted on the abscissa and the concentration of monensin in μg / ml is ordered. These results show
1) that free monensin can not be demonstrated in plasmas (except at zero time where a plasma concentration of 5.10 * -7 M is found, ie approximately 0.5 μg / ml, or in the total blood volume of the mouse an amount equal to 0.007% of the injected dose).

 2) In the case of the conjugate, it is observed that the conjugate can be detected for at least 8 h at a concentration of the order of 10 μg / ml, ie approximately 1 × 10 -5 M.

 After 24 hours, the concentration found is now close to 1.5 μg / ml. This concentration is, however, 100 times greater than that required for maximal activation under the conditions of the in vitro tests.

Example 6
Ionophoric conjugate obtained by reaction between the activated human serum albumin (SA) substituted with an activated disulfide group and the monensin on which a thiol has been introduced
a) Activated human serum albumin
SA was obtained from SA above using a technique similar to that described for the antibodies in the previous examples. There was thus obtained 220 mg of SA having 16 activating groups per mole of albumin.

b) Activated monensin
Monensin is activated according to the method described in Example 1.

c) Preparation of the ionophore conjugate
228 mg activated S-acetyl monensin, ie 268 μmol, are dissolved in the minimum of terbutanol and added to 23 ml of the activated SA solution at 9.4 mg / ml (ie 52.7 μmol) in 125 mM phosphate buffer. pH 7. 1.15 ml of 1 N hydroxylamine solution is added.

 The incubation lasts 1 h at 250C, then the reaction medium is dialyzed against PBS buffer (10 mM phosphate, 140 mM sodium chloride, pH 7.4).

 After dialysis and centrifugation, 28 μl of a solution of SA modified at 8.7 mg / ml carrying an average of 16 monensins per mol of albumin was obtained.

Example 7
Pharmacokinetics of SA-monensin conjugate
Pharmacokinetics was studied in nude mice using the ionophore conjugate described in Example 6.

 The experimental protocol used is the same as that described in Example 5.

The results are presented in FIG. 3, in which the time in h and the ordinate the concentration of monensin in pg / ml are plotted on the abscissa. They show that
1) As in the case of Example 5, the free monensin can not be demonstrated in the plasmas.

 2) It is observed that the conjugate can be detected for at least 4 h at a concentration of about 30 Clg / ml.

After 8 hours, the concentration found is only about 6 ug / ml. This concentration is, however, 400 times greater than that required for maximal activation under the conditions of the in vitro tests.

Claims (3)

1. Compound of formula I -O-CO-CH-S-W  CH2COOH wherein IO represents the residue of an ionophore deprived of one of its hydroxyls and W represents hydrogen or an acetyl group. 2. Compound according to claim 1 having the formula I -O-CO-CH-S-W  CH2COOH wherein IO represents the residue of monensin deprived of its primary hydroxyl and W is as defined in claim 1. 3. A compound according to claim 2 having the formula I -O-CO-CH-S-CO-CH3  CH2COOH wherein IO is as defined in claim 2.