FR2575160A1 - Monovalent carboxylic ionophoric compounds - Google Patents

Abstract

Carboxylic ionophoric compounds containing an optionally acetylated thiol group artificially introduced by the action of S-acetylmercaptosuccinic anhydride on the said ionophore and optional removal of the acetyl group with hydroxylamine. Application: preparation of immunotoxin potentiating agents.

Description

Monovalent carboxylic iontophoric compounds.

 The present invention relates to monovalent carboxylic ionophoric compounds useful for obtaining new conjugates combining by covalent bonding on monovalent carboxylic ionophore and a macromolecule, said conjugates being suitable as potentiators of immunotoxins.

 In its French patent application No. 82-02 091, the Applicant mentioned the ability of carboxylic ionophores to potentiate immunotoxins.

 The term “immunotoxins” denotes anticancer substances obtained by coupling, by covalent bond, of a cytotoxic protein (for example the A chain of ricin) with antibodies or fragments of antibodies directed against an antigen carried 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. 81121836. The potentiating properties of ionophores could be used advantageously in certain types of situations. In particular, they give favorable results whenever an immunotoxin is used as a selective cytotoxic agent in vitro to destroy the target cells. This is particularly the case when the immunotoxin is used as a cytotoxic agent in the treatment of bone marrow in leukemia patients to whom the bone marrow thus treated is subsequently transplanted. This is also the case when the immunotoxin is used to reduce the T cell population of a transplant in view applications of allogeneic bone marrow transplantation in order to prevent disorders linked to the graft reaction against hSte.

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

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

 According to a first aspect, the subject of the present invention is therefore, as new products, conjugates (mixed artificial molecules) constructed by covalent bonding of a monovalent carboxylic ionophore with macromolecules (such as antibodies, proteins, peptide ligands ). In the remainder of this request, such conjugates will be designated under the name of "ionophoric conjugates.

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

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

 The macromolecules used can be antibodies (or fragments of antibodies), 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 may be either of polyclonal character if it results from a conventional immunization carried out in animals, or of monoclonal character if it is produced by a ~ clone of hybrid cells obtained by fusion between lymphocytes and myeloma cells. This antibody can be used either in the form of whole immunoglobulin molecules comprising the capacity to recognize the chosen antigen, or in the form of any fragment of these immunoglobulin molecules which have retained the capacity to recognize the chosen 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 carried out by various methods provided that the method chosen - preserves the respective biological activities of the
components of the conjugate; - ensures satisfactory reproducibility and good
coupling efficiency;

- allows to control the value of the report
ionophore / macromolecule in the conjugate obtained; - leads to a stable and soluble product in batten.

 Among all the chemical coupling methods meeting these characteristics, one can choose those which use one or more thiol functions for the establishment of the bond. 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 the thiol functions, and this in an aqueous medium of pE between 5 and 9, at a temperature not exceeding 300C, to provide a stable, covalent and defined bond.

 For example, one can artificially introduce a thiol on the ionophores by the action of acetyl-mercapto-succinic anhydride S which will be able to acylate one of the alcohol functions of the ionophore. This thiol function can then be released 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). coupling will then be carried out with the macromolecule on which one or more functions capable of establishing a covalent bond with the thiol will 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 diagram
ISH + PRSSX --t ISSRP + XSH in which
I represents Itionophore to be coupled,
P the macromolecule to be modified, -SSX denotes an activated mixed disulfide group
of which X is the activating radical.

The macromolecule substituted by an activated sulfur atom is obtained from the macromolecule itself, by substitution using a reagent itself carrying an activated sulfur atom according to the scheme.
P + YRSSX 'r PRSSX in which
P is the macromolecule to be modified,
Y represents a function allowing the fixing co
valent of the reagent on the protein,
R denotes a group which can carry simultaneously
the substituents Y and -SSX,
X denotes the activating radical.

 The functional group Y is a function capable of binding covalently with any of the functions carried by the side chains of the amino acids constituting the protein to be substituted.

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

Among these, the terminal amino functions of the lysyl radicals contained in the protein are particularly indicated. In this case, Y can in particular represent - a carboxylic group which can bond to the func
amino acid protein in the presence of an agent
coupling such as a carbodiimide and in particular a
water-soluble derivative such as ethyl-1 (diethyl
3-amino-propyl) -3-carbodiimide, - a carboxylic acid chloride which is susceptible
to react directly with amino functions to
acylate them, - an ester called "activated" such as an ester of ortho- or
para-, nitro- or dinitro-phenyl, or an ester
of N-hydroxysuccinimide which reacts directly with
the amino functions for acylating them, - an internal anhydride of a dicarboxylic acid such,
for example, succinic anhydride which reacts spon
temporarily with the amine functions to create
amide bonds, - an imidoester group

or R1 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 mixed disulfide, X may denote a pyridyl-2 or pyridyl-4 group optionally substituted by one or more alkyl, halogen or carboxylic radicals. X can also denote a phenyl group, preferably substituted by one or more nitro- or carboxylic groups. X can also represent an alkoxycarbonyl group, such as the methoxycarbonyl group.

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

ou
R5 désigne un groupe alkyle droit ou ramifié ayant de 1 à 5 atomes de carbone et notamment le groupe tertiobutyle.The radical R denotes any radical capable of carrying the substituents Y and SSX simultaneously. It should be chosen so as not to contain functions which are likely to interfere during 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 even a group

in which R4 denotes hydrogen or an alkyl group having from 1 to 8 carbon atoms, and R3 denotes a substituent inert towards the reactants used subsequently, such as a carbamate group

or
R5 denotes a straight or branched alkyl group having from 1 to 5 carbon atoms and in particular the tert-butyl group.

 The reaction of compound Y-R-S-S-X with 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 room temperature for a time varying from a few minutes to 24 hours. After which, dialysis makes it possible to remove the products of low molecular mass, and in particular the excess of reagent. This process makes it possible to introduce a number of substituents per mole of macromolecule usually between 1 and 50.

 By using such compounds, the coupling of the macromolecule and the ionophore is carried out by bringing the two compounds into aqueous solution at a temperature not exceeding 30 ° C. for a time varying from a few minutes to 1 day. the aqueous solution obtained 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 your thioether bond
The preparation of the conjugate then consists in reacting I-SH with the protein P, on which a maleimide radical will have previously been introduced. The reaction is then represented by the diagram

in which
Z represents a spacing structure, aliphatic
or aromatic 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 carbo
diimide et notamment un dérivé soluble dans liteau,
tel que l'ethyl-1 (diéthylamino-3 propyl)-3-carbo
diimide, - soit un ester dit "activé" tel qu'un ester d'ortho- ou
para-, nitro- ou dinitrophényle,ou encore un ester de
N-hydroxysuccinimi-de qui réagit directement avec les
fonctions aminées pour les acyler.Protein P substituted by maleimide is obtained from protein P itself, by substitution of amino functions of the protein using a reagent itself carrying the maleimide group, according to the scheme.

in which Y1 represents: - either a carboxylic group, the reaction then being
performed after activation of the carboxylic function
in the presence of a coupling agent, such as a carbo
diimide and in particular a derivative soluble in batten,
such as ethyl-1 (diethylamino-3 propyl) -3-carbo
diimide, - either a so-called "activated" ester such as an ortho- or
para-, nitro- or dinitrophenyl, or an ester of
N-hydroxysuccinimi-de which reacts directly with
amino functions to acylate them.

 The preparation of such reagents is described in particular in Helvetica Chimica Acta, 58, 531-541 (1975).

Other reagents of the same class are commercially available.

The reaction of the compound

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.

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

 The reaction is carried out at room temperature for a time varying from a few hours to 24 hours.

After which, dialysis makes it possible to remove the products of low molecular mass and, in particular, the excess of reagents. This process makes it possible to introduce a number of substituent groups per mole of protein usually between 1 and 50.

 By using such compounds, the coupling of the macromolecule with the ionophore is carried out by bringing the two compounds into aqueous solution at a temperature not exceeding 300C for a time varying from a few hours to a day. The solution obtained is dialyzed to remove the low molecular weight products, then 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 carried out on ionophore conjugates have shown that the potentiating effect of ionophores is preserved
after coupling with the macromolecule.

The binding of the ionophore to the macromolecule provides
to this one a great solubility in aqueous medium
favorable for in vivo administration of the product; - the binding of the ionophore to the elongated macromolecule
significantly the plasma half-life time 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 effector substances essential to the result will be directed to the target cells - on the one hand, a cytotoxic protein (such as by
example ricin chain A) which will be the effector
cytotoxic included in the conjugate called immunotoxin; - on the other hand, the ionophore (such as for example the
monensin) which is the constituent of the poten system
tialisateur included in the conjugate called conjugate
ionophore.

 In the case where the ionophore is coupled to a macromolecule having a receptor on the surface of target cells (for example when the macromolecule will be an antibody or an antibody fragment recognizing a specific antigen of 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 cells to be destroyed, the probability that the two chosen targets are present together on the surface of non-target cells becomes very weak and an extremely powerful means is thus available to further increase the specific character of the cytotoxicity. of the immunotoxins.

 The present invention therefore also relates to the drug combination of at least one immunotoxin and at least one ionophore conjugate according to the invention.

 The following examples illustrate the invention without limiting its scope.

EXAMPLE 1
Ionophoric conjugate obtained by reaction between the antibody T 29-33 substituted by an activated disulfide group and the monensin on which a thiol has been introduced.

a) Antibody T 29-33
This antibody is a monoclonal antibody directed against the T 200 antigen of human leukocytes. This antibody is described in J. Exp. Met., 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 solution of T 29-33 antibody at 10 mg / ml (i.e. 0.133 micromole of antibody) is added an aqueous solution containing 3 mg of (pyridyl2-disulfanyl) -3-propionic acid previously dissolved in tertiary butanol and 1.8 mg ethyl-1-dimethylamino-3propyl-3-carbodiimide. The mixture is stirred for 15 minutes at 300C and then dialyzed continuously against 125 mM phosphate buffer pH 7 (40 hours at 500 ml / h). After dialysis, the protein solution is centrifuged and 2.5 ml of a solution containing 6 * 6 mg of modified antibody per ml are obtained. By spectrophotometric assay at 343 nm of pyridine thione-2 released by exchange with mercapto-2 ethanol, it is found that an antibody was obtained carrying 7.2 activating groups per mol-e 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-acetyl mercaptosuccinic anhydride (SAMSA). The reaction is allowed to proceed for half an hour at room temperature. The reaction medium is then evaporated to dryness under vacuum then taken up in ethyl acetate and washed extensively with water. The organic phase is then dried and drawn to dryness under vacuum. The product is obtained crystallized after drying under vacuum.

It is identified by its NMR spectrum and its mass spectrum.

d) Preparation of the ionophore conjugate
8 mg- (i.e. 9 micromoles) of activated monensin are dissolved in the minimum amount of terbutanol and added to 2.5 ml of the solution of activated antibody at 6.6 mg / ml (i.e. O, 11 micromoles) in phosphate buffer 125 mM pH 7.

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

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

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 particularly its capacity to potentiate the activity of the anti-T65 immunotoxin in an appropriate cell model.

 This model is made up of cells of the CEM human lymphoblastoid line which naturally carry the T65 and T2OO antigens. The T65- antigen against which the immunotoxin used is directed constitutes the first target antigen of the model. This immunotoxin is that which has been described in a previous patent application of the Applicant filed in France under the number 81/21836. The T200 antigen against which the ionophore conjugate is directed will be the second target antigen in the model.

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

 From these determinations, one can draw the effects / dose curves presenting on the abscissa the molar concentration in chain A 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 of 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 various tests were carried out as follows; the corresponding experimental results are shown in Figure nO I.

a) CEM cells are incubated for 18 hours in the presence
known concentrations of anti-T65 immunotoxin
used for reference and then the cells
are subjected to the step of incorporating the tracer
radioactive. 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 hours at 37 "C in
presence of a mixture of known concentrations
anti-T65 immunotoxin and respectively
1. either free monensin at the concentration of
50 nu (curve 2);
2. either of the anti-T200 ionophore conjugate previously described, at a concentration of 10 NM (i.e. 70 nM
monensin) (curve 3).

 It has been verified beforehand that the mone-nsine and the ionophore conjugate are not cytotoxic for the cells used at the indicated concentrations.

The CI 50 obtained are respectively:
2,4.10-14 M for 50 nM monensin
1.4.10 M for the ionophore conjugate 10-8 M.

 These results show remarkable potentiating effects, by a factor of 1000 times for 50 nM monensin and 2000 times for the ionophore conjugate at a concentration of 10 H M (ie 70 nM in monensin).

EXAMPLE 3
Ionophoric conjugate obtained by reaction between the antibody 3E10 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-human melanoma SK MEL 28 lymphocyte fusion. The hybridoma 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 1. 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 activated monensin (i.e. 104 micromoles) are dissolved in the minimum of terbutanol and added to 50 ml of the solution of activated antibody at 3.9 mg / ml (i.e. 1.3 micromoles) in the 125 mM ph phosphate buffer 7.

 The incubation lasts 2 hours at 25 ° C., then the reaction medium is purified of the excess reagent 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 IgG solution were obtained, carrying on average 8 monensins per IgG.

EXAMPLE 4
Toxicity of the 3E10-monensin conjugate.

 The 3E10-monensin conjugate has been studied for its biological properties in vivo. More special, its toxicity and pharmacokinetics have been compared to that of free monensin.

 The toxicity of free monensin has been determined dear mice. The 50% lethal dose after IV injection is 4 mg / kg9, i.e. 80 micrograms / mouse.

 The toxicity of the conjugate was tested on mice in a single administration also 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 m2. of conjugate is respectively the equivalent of 163 g, 81.5 pg, 40.75 g, 20.4 iig of aonensine. No mortality was observed in any of the batches of mice.

EXAMPLE 5
Pharmacokinetics of the conjugate -3E10-monensine.

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

The mice are injected - either 1 ml of ionophore conjugate at 3.7 mg / ml (or
163 g of coupled monensin), - i.e. 163 μg of free monensin (control batch).

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

 The assay is carried out on CEM cells in the presence of the anti-T65 immunotoxin.

 The results are presented in FIG. 2, on which the time in hours has been plotted on the abscissa and the monensin concentration in g / ml on the ordinate.

These results show 1) that free monensin cannot be evi
dence in plasmas (except at time zero where we
finds a plasma concentration of 5.10 7 M,
either about 0.5 llg / ml, or in the blood mass
total mouse an amount equal to 0.007 Z of
the injected dose).

2) In the case of the conjugate, we observe that the conjugate
can be highlighted for at least 8 hours
at a concentration of the order of 10 g / ml, or approximately 1 x 10 5 M. After 24 hours, the concentration
tration found is no more than close to 1.5 g / ml.

This concentration is however 100 times higher
to that required for maximum activation in
conditions of in vitro tests.

EXAMPLE 6
Ionophoric conjugate obtained by reaction between Human serum albumin (SA) substituted peacock activated disulfide group and monensine on which a thioL has been introduced.

a) Activated human serum albumin
The SA was obtained from the above SA according to a technique analogous 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 of activated monensin, i.e. 268 micromoles, are dissolved in the minimum of terbutanol and added to 23 ml of the activated SA solution at 9.4 mg / ml (i.e. 52.7 micromoles) in the 125 mM phosphate buffer pH 7 .

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

 After dialysis and centrifugation, 28 ml of a modified SA solution at 8.7 mg / ml were obtained, carrying on average 16 monensins per mole of albumin.

EXAMPLE 7
Pharmacokinetics of the SA-Monensine conjugate.

 The pharmacokinetics was studied in male 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 on which the time in hours has been plotted on the abscissa and the monensin concentration in pg / ml on the ordinate.

They show that 1) As in Example 5, free la-monensin
cannot be highlighted in plasmas.

2) The conjugate can be highlighted during
minus 4 hours at a concentration of around
30 g / ml. After 8 hours, the concentration
found is only about 6 pg / ml.

This concentration is however 400 times higher
to that required for maximum activation in
in-glass test conditions.

Claims (5)

1. As new products, carboxylic ionophoric compounds containing a thiol group, optionally acetylated, introduced artificially by the action of the anhydride S-acetylmercaptosuccinic on said ionophore and possible elimination of the acetyl group by hydroxylamine. 2. Ionophoric compound according to claim 1, characterized in that it comes from monensin. 3. Ionophoric compound obtained by reaction between monensin and S-acetylmercapto succinic anhydride in an organic solvent and at room temperature. 4. Method for obtaining an ionophore compound according to claim 1, characterized in that an ionophore compound is treated with S-acetyl-mercaptosuccinic anhydride in an organic solvent and at room temperature and then released the thiol function by the action of hydroxylamine. 5. Method according to claim 4, characterized in that the ionophore compound is monensin.