AU736187B2 - Catalytic monoclonal antibodies for the in vivo transformation of corticosteroid prodrugs - Google Patents

Description

WO 99/06536 PCT/EP98/04682 CATALYTIC MONOCLONAL ANTIBODIES FOR THE IN-VIVO TRANSFORMATION OF CORTICOSTEROID PRODRUGS The present invention relates to catalytic monoclonal antibodies, in the following named "abzymes", which are useful for the in-vivo transformation of corticosteroid prodrugs and their use in the preparation of medicaments useful for the treatment of acute adrenal insufficiency.

The present invention also relates to processes for the preparation of said abzymes.

Background of the invention.

.Acute adrenal insufficiency is an extremely dangerous condition and has often a fatal outcome. It can arise in case of sepsis, acute asthma crisis, shock and collapse states following traumas, surgery, hypovolemic conditions and burns, myocardial infarct, pulmonary embolia, severe edemas states (Quincke's edema, glottis edema, pulmonary edema), acute pancreatitis, anaphylactic reactions following sera injections, transfusions accidents, drug hypersensitivity, severe allergic reactions, apoplexy, childhood acute toxicosis and accidental poisonings, hepatitis and hepatic coma, thyrotoxic and addisonian crises, thromboagiitis obliterans.

Presently used therapy consists in the intravenous treatment with high doses of corticosteroids. However, the clinical use of corticosteroids in such therapeutic application has not been proved by suitable clinical studies, on the contrary, the major part of clinical trials did not give clear results of efficacy. The pharmacokinetic characteristics of the used products can give account of this uncertainty, which, on the other WO 99/06536 PCT/EP98/04682 2 hand, can not be attributed to methodological errors nor to a lack of pharmacological activity from the steroids in said indications. Since corticosteroids are water insoluble, they must be conjugated with hydrophilic carriers, so that they can be injected. However, their biological activity can be carried on only after the separation of the hydrophilic portion, thus releasing the corticosteroid, which in this way can cross the cell membrane and bind to the cytoplasmatic receptor. In-vivo cleavage of the bond between the cortisonic molecule and the hydrophilic portion is very slow, since the molecule is moderately stable and specific esterase enzymes are not present in blood flow. Accordingly, bioavailability of the active ingredient is not immediate and therapeutic response can be observed only after several hours, with consequent danger for patient survival.

Corticosteroids are still the drug of choice in the treatment of acute syndromes requiring emergency intervention, notwithstanding their mechanism of action is not yet completely clarified and that controlled studies are not yet available for an objective ascertainment of their efficacy. Situations like acute adrenal insufficiency, anaphylactic shock, hypovolemic shock and cardiogenic shock, larynx edema and other pathologic states are generally treated with high dosages of cortisonic drugs (from 500 mg up to 10 g) by intravenous route and with administrations repeated every 1-2 hours in the course of 24 hours, until the patient gives a clinical response. The therapy can further be lengthened, with lower doses, the days following the crisis, until the control of symptoms and recovery of vital parameters are WO 99/06536 PCT/EP98/04682 3 reached. Subsequently, it is usually necessary a maintenance therapy with low doses, even by oral route.

Moreover, it is well known that corticosteroids are widely used also in the therapy of subacute and chronic disorders of the degenerative, inflammatory and neoplastic type, such as glomerulonephritis, kidney alterations, Systemic Lupus Erithematosus, arthritis, rheumatoid carditis, bronchial asthma, allergic diseases, intestinal and hepatic disorders, dermis alterations, lymphomas, Hodgkin's disease, pericarditis, pemphigus, erythrodermia and others. Also in these cases prolonged treatments and high. dosages are necessary to achieve suitable concentrations of the active ingredient at the action site, with resulting onset of side effects, typical of corticosteroids.

Adverse effects occurring after prolonged therapies consist mainly in suppression of the pituitary and suprarenal functions, disturbances of hydric and electrolytic homeostasis, hypertension, hyperglycemia, glycosuria, enhanced sensitivity to infections, osteoporosis and neuropathies. It is evident that such adverse reactions are due to the drug intrinsic activity, but also the kinetic characteristics of corticosteroids are strongly responsible of the side reaction entity.

These characteristics make mandatory such treatment schemes, as explained later.

State of the art.

In order to overcome the disadvantages of emergency therapy with corticosteroids, several therapeutic strategies have been tried, for example against septic shock. In order to modulate acute systemic inflammatory WO 99/06536 PCT/EP98/04682 4 event, monoclonal antibodies blocking inflammation mediators of the cytokine type or inactivating endotoxins have been used. However, being the molecules implied in the acute process particularly numerous and redundant, the selective inhibition of only one or some of them resulted not therapeutically sufficient, therefore said approaches have been abandoned. Very recent pharmacological studies, devoted to clarify the action mechanism of glucocorticoids, showed that these drugs, by interacting with their specific cytoplasmatic receptor, are able to control the inflammation by inhibiting the different aspects of phlogistic process (production of cytokines, nitric oxide, prostaglandins, leucotrienes, expression of adhesion molecules on cell surface, etc.) through the stimulus or depression of DNA transcription. Following these results, a revaluation of the use of corticosteroids in emergency therapy is being observed. On the other hand, some characteristics, in particular structural ones, of this class of drugs does not allow to fully exploit their actual therapeutic capacities, and for this reason doubts on their clinical efficacy still exist.

It is well known that, in order to produce its characteristic effects, a drug must reach a suitable concentration at its action site, this concentration depending on both the amount of administered product and the absorption degree of the substance and other kinetic parameters. Absorption process, in particular, is affected by factors such as molecular form and dimensions, ionisation degree, solubility in lipophilic solvents.

As far as corticosteroids are concerned, they are lipophilic molecules therefore they must cross cell WO 99/06536 PCT/EP98/04682 membrane and bind to their cytoplasmatic receptor in order to exploit their pharmacological activity. This aspect constitutes the major problem for their therapeutic use.

Lipophilicity of said compounds evidently hinders solubilization in aqueous solvents, as well as their diffusion in blood stream, thus compromising their availability at the action site. In order to obviate to this drawback, molecules of this class have been conjugated, through covalent bonds, to particular hydrophilic molecules (carriers), allowing their solubilization. For example, in the case of cortisol, positions 11, 17 or 21 have been esterified with phosphate, sulphate, succinate, hemisuccinate or acetate, tebutate, acetonide, diacetate, hexaacetone groups.

Unfortunately, if on one side this ester bond makes the substance soluble and allows its administration and diffusion in blood circle, at the same time it hinders its passage through cell membrane. Therefore the product will be able to exploit its pharmacological activity only after the cleavage of the ester bond of the prodrug and the release of the lipophilic active ingredient. The organism, on the other hand, has not esterase enzymes capable of cleaving a bond of this type, thus, hydrolytic process is extremely slow and bioavailability of the active ingredient is equally scarce. It is evident that, such a pharmacokinetic characteristic often produces negative results in case of pathological events wherein emergency therapeutic actions are required.

Accordingly, there is the need to have a form of the active ingredient that is soluble in aqueous pharmaceutical formulations, for example the injectable WO 99/06536 PCT/EP98/04682 6 ones, and, once in the blood stream, promptly available in the lipophilic form in order to rapidly cross cell membrane and exert its own pharmacological action.

Monoclonal catalytic antibodies and their use in the activation of prodrugs are well known, such as for example disclosed in Chemical Abstract (114) 610w, (119) 210716q, (121) 195943g and (122)28553d. The general strategy is based on the development of catalytic monoclonal antibodies (abzymes), which can be generated against haptens mimicking the molecular structure of the transition state of the compound taking part in a chemical reaction. An intermediate configuration, in which potential energy of the constituting atoms and of all molecules reaches a maximum value, exists among the starting and final arrangement of the molecules taking part in a chemical reaction. This configuration corresponds to the transition state and tends to assume the final arrangement of the reaction as to bring potential energy back to lower levels.

Abzymes against corticosteroids or their derivatives are not known.

A problem encountered in the preparation of the abzyme is finding out the chemical structure of the suitable hapten.

W08910754 discloses catalytic monoclonal antibodies capable of cleaving an ester bond, and in any case to activate a prodrug in-vivo. The enabling teaching provided in this piece of prior art is directed to solve the problem of activating or inactivating peptidic biomolecules, and a series of peptidic haptens is Sprovided.

WO 99/06536 PCT/EP98/04682 7 W09302703 specifically relates to antitumor therapy, wherein corticosteroids are not foreseen.

W09416734 relates to a method of treatment which foresees addressing effector molecules, among which drugs, on predetermined cellular sites by means of a complex construction that provides a first reactant linking to the cellular site, a second reactant linking to said first reactant in an amplified mode, the effector molecule, in turn, links to said second reactant, releasing thereafter the active moiety. The reactants can have antibodies as functional groups for the formation of the different bonds, but can also be assimilated to antibodies. The effector molecule can be a drug and can be linked to a carrier molecule, such as a protein. The drug can also be a prodrug, since an enzymatic agent, releasing the active part is also provided; or the effector molecule can be an enzyme catalyzing the conversion of a prodrug.

The. optional release of the active moiety occurs at or near the receptor site and in any case there is a leading effect of the active drug to the action site. The specific teaching is directed to leading effective molecules in the diagnosis or treatment of tumors.

JP94220072 discloses abzymes against antibiotics.

It is desirable to dispose of a unique criterion in the design of a suitable homologous of the transition state valid for a whole class of drugs, and not for a single compound. Moreover, it is also desirable to dispose of such a homologous independently from the hydrophilic part conjugating the steroid molecule.

The specific problem consists in the quick hydrolysis of the ester function in the cortisonic prodrug, since in WO 99/06536 PCT/EP98/04682 8 the organism a specific esterase, capable of releasing the drug in question, does not exist. The general terms of the problem relating to the obtainment of an abzyme against a prodrug in the form of ester are all comprised in the state of the art: the phosphonic function is indicated as mimicking the transition state, the coupling group with the carrier protein is also described. However, there is no indication to the person skilled in the art about the criticality of how positioning the coupling group on steroidal molecule and in which relation with the mimicking group of the transition state.

.Therefore, the type of hapten to be use is highly critical, and the reciprocal position of the coupling and mimesis groups of the transition state is still more critical.

W08910754, other than a reference to generic computer modeling techniques, in particular dwells upon cleaving selected peptide bonds of certain proteins, such as human rennin, HIV gp120 envelope protein.

W09302703, dealing with the specific problem of prodrugs of immunosuppressive, antiviral, antitumor and cytotoxic agents, selects the parts esterifying the drug in order to protect a possible prodrug from endogenous esterases, and only subsequently, once the suitable therapeutic level has been reached, releases the active ingredient. In a certain aspect, the technical problem is the opposite of the one solved by the present invention, wherein hydrolysis must be achieved in the shortest possible time, since an action must be performed in emergency therapy, not in chronic therapy.

Abstract of the invention.

9 It has now been found that the hapten suitable for the production of abzymes against hydrosoluble prodrugs of corticosteroids is a compound of steroidal structure bearing a phosphate group on the hydroxyl group which in the corresponding corticosteroid is conjugated with the hydrophilic carrier, and a chemical group suitable for the conjugation with the macromolecule at position C-3 of the steroidal skeleton, the farthest possible from said phosphate group.

Accordingly, it is an object of the present invention a compound of formula (I) P03X2 (I) wherein: A represents the steroidal nucleus of a corticosteroid hormone; R is a chemical group linked to position C-3 of the pregnane ring A and is suitable for the conjugation with an immunogenic macromolecule; P03X2 is a phosphate group linked to the hydroxyl group at 21- or 17- position (which on the corresponding corticosteroid hormone bears a hydrophilic molecule); X is hydrogen or a pharmaceutically acceptable cation.

The compounds of formula appropriately conjugated through R with a molecule suitable for giving rise to an antibody response in an animal, constitute the haptens from which the catalytic monoclonal antibodies (abzymes) specific against the different hydrosoluble prodrugs of corticosteroids will be obtained.

Said abzyme or a fragment thereof or an engineerized fragment thereof are another object of the present Sinventi6n.

IN

AMENDED SHEET WO 99/06536 PCT/EP98/04682 Another object of the present invention is the use of the abzymes for the preparation of medicaments useful in the treatment of acute adrenal insufficiency.

The essential advantage obtained by the present invention, by consequence of the higher bioavailability of the active ingredient, consists in the possibility of obtaining an immediate therapeutic effect, which, in emergency situations, will allow to achieve a higher number of successes in survival terms of the treated subjects.

Being the corticosteroid immediately available for the .pharmacological action, it will be probably possible to use it at lower doses.

The possibilities of using lower doses and the opportunity to diminish therapy times, due to the higher bioavailability of the active ingredient, could allow effective treatments of the above listed chronical diseases, avoiding unwanted effects.

These and other objects of the present invention will be disclosed in higher detail also by means of examples.

Detailed description of the invention In the compounds of formula is the steroidal nucleus of a natural or synthetic corticosteroid.

Examples of adrenocortical hormones are: cortisol, fluorocortisone, triamcinolone, dexamethasone, prednisone, cortisone, and betamethasone.

Examples of R chemical groups suitable .for the conjugation with an immunogenic macromolecule are azido, p-azophenyl-P-D-lactoside (Lac), p-azophenyl-P-D-glucoside (Glu), p-azophenyl- arsonate (Ars), sodium trinitrobenzene Ssulfonate (TNBS), sodium dinitrobenzene sulfonate (DNBS), WO 99/06536 PCT/EP98/04682 11 dinitrophenyl (DNP) and S-acetyl-mercaptosuccinic anhydride. Among these, the azido groupis preferred.

Examples of hydrosoluble corticosteroid prodrugs are those used in injectable formulations. Preferred examples are: betamethasone sodium phosphate, betamethasone sodium phosphate and acetate, cortisone acetate, cortisol sodium phosphate, cortisol sodium succinate, cortisone acetate, dexamethasone acetate, dexamethasone sodium phosphate, prednisolone acetate, methylprednisolone sodium succinate, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide.

Cortisol 21-hemisuccinate, cortisol 21-acetate, cortisol 21-phosphate, cortisol 17-hemisuccinate, cortisol 17acetate, cortisol 17-phosphate are more preferred examples.

Compounds of formula are prepared starting from the respective active ingredients, namely from the pharmacologically active steroids or their commercially available derivatives. For example, the active ingredients already bearing the phosphate ester group in the desired position are commercially available or can be prepared with well known methods. Then, the selected R group is inserted according to conventional methods described in literature.

Once the compound of formula (hapten) is obtained, it is conjugated with the immunogenic macromolecule. Examples of such macromolecules are KLH, BSA, ovoalbumine, thyreoglobuline, chicken IG, polysaccharides.

The conjugate hapten-carrier is then used for WO 99/06536 PCT/EP98/04682 12 immunizing the animal from which, with conventional methods, the monoclonal catalytic antibody (abzyme) is obtained.

It is an object of the present invention an abzyme or a fragment thereof or an engineerized fragment thereof against a hydrosoluble prodrug of a corticosteroid hormone.

Typically, splenocytes of the immunized animal are fused with a myeloma line, and after the selection of the hybridomas and the positive clones, the purification of the abzyme is carried out.

.The procedure for obtaining the abzyme is not critical and there are no particular limitations in the selecting the animal, the immunisation protocol, the particular myeloma cell line (or other immortalized cells).

Purification methods of the abzyme of the present invention are absolutely conventional and within the normal activity of the skilled person.

A first preferred embodiment of the present invention relates to the hapten of cortisol substituted at position 21. It is the compound 2 18,17a-21-trihydroxy-4-pregnene- 20-one-3-hydrazono-4'-azidobenzamide-21-phosphate, namely the compound of formula wherein is the steroidal moiety of cortisol, R is the 3-hydrazono-4'-azidobenzamide group, X is hydrogen.

H O I ii H- C P O I I c:=o 0 azide WO 99/06536 PCT/EP98/04682 13 This compound constitutes the hapten.

It can be observed that the molecular structure of the compound used as hapten does not have any side chain corresponding to the hydrophilic part of any prodrug of cortisol. Instead of synthesizing the analogous of the transition state for each of the -existing cortisol prodrug, the present invention allows, for each determined active ingredient, to provide the analogous of the transition state of the cleaving reaction of every TM prodrug, independently from the hydrophilic residue.

Therefore, the antibody will recognize that part of the molecule represented by the cleaving point, other than the steroidal moiety, while the difference of the hydrophilic moiety, which can be one of hemisuccinate, phosphate or other well known carriers, is less important.

According to the same principle, also other cortisol derivatives, such as for example methylprednisolone that differs from cortisol for the presence of a methyl group at position .6-alpha, which is available in injectable formulations only when linked to a hemisuccinic residue (methylprednisolone hemisuccinate, URBASON(R) SOLUBILE, 8, 40, 250 mg), can be used as therapeutic agent.

In the hapten molecule, a phosphate group is present in position -21 mimicking the transition state of the hydrolysis of the ester group, being sterically analogous to a tetrahedric carbon.

The group intended for establishing the bond with the carrier protein is linked at position 3, which in the molecule represents the carbon atom most distant from position 21 or 17, on which the hydrophilic substituent is present.

WO 99/06536 PCT/EP98/04682 14 According to the present invention, the catalytic antibody is capable of recognizing a significant part of the steroidal molecule near the reaction center. In fact, the macromolecular carrier is attached to the azide in position C3, leaving available the moiety of the molecule for the immunological recognition where the reaction must actually occur. Then the evoked antibody recognizes both the binding site in C21 and the major part of the steroidal molecule.

The hapten has been obtained by condensing commercial dihydrocortisone-21-phosphate with the hydrazide of the 4azidobenzoic acid; the latter compound has been prepared starting from 4-amminobenzoic acid through the following reaction steps: diazotation with nitrous acid in water followed by a reaction with sodium azotide to obtain 4azidobenzoic acid; the latter was esterified with hydrochloric methanol; the reaction was followed by hydrazinolysis in methanol of the intermediate methyl ester. The condensation of the hapten with dihydrocortisone-21-phosphate was carried out by heating equimolar amounts (2 mmol) of the reactants in methanol (3 ml) in a sealed vial heated to 90*C for 3 h, until a clear solution was obtained, from which the hapten was isolated as a solid by solvent evaporation. The presence of the hapten in the reaction mixture was inferred from the shifting of the vinyl signal linked to carbon-4 in the RMN spectrum.

Due to their low molecular weight, the synthesized molecules as analogues lack in immunogenic capacity, which is instead acquired when they are conjugated to I macromolecular carriers. In the usual experimental WO 99/06536 PCTIEP98/04682 conditions, more frequently used molecular carriers are proteins. It has been necessary then, to conjugate the modified steroid (hapten) to a protein, in order to guarantee the maximum exposition of the molecular domain of interest (epitope). In a preferred embodiment, KLH was the selected protein.

The modes of conjugation to a macromolecular carrier depend on the type of functional group available on the hapten molecule. Different methods of chemical modification of proteins with haptens are available, and can be used indifferently, then selecting the most effective. The substitution molar ratio is estimated by absorption spectrophotometry.

The hapten-protein carrier conjugate was used to immunize 8-10 weeks Balb/c mice, both by subcutaneous route (with complete and incomplete Freund adjuvant) and by intraperitoneal route, according to different treatment schemes.

At the end of the treatment period, serum of the animals was withdrawn to verify the development of an immune response against the antigen and then produce the desired catalytic activity.

Once the animals that developed the higher antibody response were selected, the preparation of monoclonal antibodies was carried out according to the usual methods (fusion of splenocytes with myeloma cells, screening of the families of hybridomas by the radiometric assay disclosed below, selection of the positive clones, purification of the antibody).

In order to maximize the efficiency of the catalytic antibody it is possible to make resort to different WO 99/06536 PCT/EP98/04682 16 strategies: to modify the analogous of the transition state; to carry out the selection of antibodies from combinatory libraries expressed on phages (phage display); to modify the catalytic monoclonal antibody, previously obtained with standard methods, by means of site directed mutagenesis.

The abzymes according to the present invention have catalytic activity against hydrosoluble prodrugs of corticosteroid hormones, therefore they are useful in the therapy of adrenal insufficiency, in particular the emergency therapy, where the fastest action of the drug is necessary.

Accordingly, it is an object of the present invention the use of abzymes against hydrosoluble prodrugs of corticosteroid hormones for the preparation of a medicament useful in the treatment of pathologies curable with cortisonic drugs. In particular, the use according to the present invention finds application in the treatment of the acute adrenal insufficiency, sepsis, acute asthma crisis, shock and collapse states following traumas, surgical interventions, hypovolemic states and burns, myocardial infarct, pulmonary embolism, severe edematous states (Quincke's edema, glottis edema, pulmonary edema), acute pancreatitis, anaphylactic reactions following sera injections, transfusions accidents, drug hypersensitivity, severe allergic reactions, apoplexy, childhood acute toxicosis and accidental poisonings, hepatitis and hepatic coma, thyrotoxic and addisonian crises, thromboagiitis obliterans.

WO 99/06536 PCT/EP98/04682 17 Still another object of the present invention is the use of abzymes against hydrosoluble prodrugs of corticosteroid hormones for the preparation of a medicament useful in the treatment of subacute and chronical disorders of degenerative, inflammatory and neoplastic type, such as glomerulonephritis and other renal alterations, Systemic Lupus Erithematosus, arthritis, rheumatoid carditis, bronchial asthma, allergic diseases, disorders of the intestinal tract and of liver, dermatological alterations, lymphomas, Hodgkin's disease, pericarditis, pemphigo, erythrodermatitis and others.

The administration of the abzyme will be determined by the attending physician, as to the times and doses, according to the type of pathologies, the state of the patient and type of prodrug associated.

The abzyme according to the present invention can be administered in concomitance with the corresponding prodrug or in a certain sequence, established by the expert physician.

Conveniently, the abzyme according to the present invention is formulated into pharmaceutical.compositions, which are also comprised in the present invention. The compositions contain an effective amount of abzyme, which can be determined in relation to the specific activity with respect to the prodrug.

The compositions according to the present invention contain the abzyme in admixture with pharmaceutically acceptable carriers and excipients and can be prepared with well known methods for example as described in Remington's Pharmaceutical Sciences Handbook, Mack. Pub., S U.S.A.

WO 99/06536 PCT/EP98/04682 Injectable compositions are preferred.

The present invention also comprises a kit containing, in separated form, a pharmaceutical composition comprising a suitable amount of an abzyme according to the present invention and a pharmaceutical composition comprising a therapeutically effective amount (with respect to the abzyme) of a hydrosoluble prodrug of a corticosteroid.

The following example further illustrates the invention.

EXAMPLE

a) Preparation of the hapten 213,17a-21-trihydroxy-4pregnene-20-one-3-hydrazono-4'-azidobenzamide-21phosphate The compound of the title is obtained by evaporating to dryness 50 mg of 3-azidobenzoylhydrazine and 150 mg of not purified cortisol 21-phosphate.

b) Conjugation with KLH mg of KLH (hexamer, MW 450000 g/mol) were dissolved into 10 mM potassium bicarbonate, pH 7.4, ml for a final concentration of 4 mg/ml.

The above products, without purification, were dissolved in the same solution, calculating a 200 molar excess of cortisol 21-phosphate derivative. The amount of product to be used is calculated arbitrarily presuming that all the steroid is in the active substituted azido form: x 10 3 g KLH x 200 x 645 g/mol x 4/3 7.64 x 10 3 g 450000 g/mol Z (4/3 corrects the mass amount taking into account the WO 99/06536 PCT/EP98/04682 19 fact that in the starting reaction mixture there are 1 part of azide and 3 parts of cortisol 21-phosphate).

The solution is irradiated for 20 minutes with an unshielded 200 W-mercury vapor lamp, with cooled light, under stirring.

The product is passed through Sephadex G 50 Zim A 2 cm x 5 cm equilibrated with the same buffer of the reaction.

The fraction of the (A280) peak is collected, by measuring the volume. In order to predict the content of phosphate per volume unit, it is assumed that the maximum molar substitution ratio (200:1) is obtained, and the amount of phosphates is evaluated on suitable volumes and dilutions by means of the Ames test. The Ames test was applied to 10 pl of the reaction KLH and gave a value of A280 0.981.

Since the extinction coefficient of the test is A280 0.240/0.01 ptmoles of phosphates, it comes out: (0.981 0.193) x 1 x 10- 8 moles 3.28 x 10 3 moles/l of organic P 0.240 x 10 x 10 6 1 x 10 3 g KLH 1 x 5.55 x 10- 6 moles/1 450000 g/moles 4 x 10 1 of KLH, from which the substitution molar ratio of: P] 3.28 x 10 3

M

591 [KLH] 5.55 x 10 6

M

c) Immunization of mice with the conjugate and control of the antibody activity The conjugate hapten-protein carrier was used to WO 99/06536 PCT/EP98/04682 immunize 8-10 weeks Balb/c mice, both by subcutaneous route (with complete and incomplete Freund's adjuvant) and by intraperitoneal route, according to different treatment schemes.

At the end of this treatment period the animal serum was withdrawn in order to verify that the animals developed an immune response against the antigen and then produced the desired catalytic activity.

In order to carry out this control a specific radiometric assay was put in order: [1,2,6,7-3H]-llP,17adihydroxy-4-pregnene-3,20-dione-21 hemisuccinate labeled with.tritium 3 H-C21-H) was synthesized, starting from 37 Mbq of [1,2,6,7- 3 H] Cortisol (Amersham).

In order to obtain an analytic signal of 50.000 cpm, with an counting efficiency ba 60%, the working concentration from a volume of 100 p1 is then the following: 100 x 50.000 6.6 x 10- 9

M

60 x 60 su x 2.11 x 1015 bq/mol x 10 4 1 The mass amount of available labeled would allow, in theory, to prepare a volume of working solution equal to: 37 x 10 6 bq 2.664 1 2.11 x 1015 bq/mol x 6.6 x 10 9 moles/1 Since working at real concentrations of substrate equal to 10 PM (as obtainable from literature) is appropriate, it is then necessary to prepare the following total amount of substrate: 2.664 x 10- 6 moles/1 2.664 x 10 5 moles Being the amount of marker equal to 37 x 10 6 bq/2.11 WO 99/06536 PCT/EP98/04682 21 x 1015 bq/mol 1.7 x 10- 8 moles, its contribution to the total mass is negligible.

The amount of cold carrier, for a M.W. of Cortisol 62.5 g/mol. is obtained from: 2.664 x 10 5 mol x 362 g/mol 9.6 x 10 3 g This mass of cortisol is distributed in React. Vial Pierce in which the solvent of the labeled has been evaporated under nitrogen stream at T° (max) The cortisol was dissolved into 200 pi of dry pyridine, for the reaction with a basic catalysis with three molar fold excess of succinic anhydride .2.664 x 10 5 moles x 3 x 100.1 g/mol 8.0 x 10- 3 g The reaction mixture was left 15 hours at r.t. under constant stirring.

At the end of incubation, the mixture was transferred into a separating funnel, washing the React. Vial. with further 200 p~ of pyridine.

The pyridine was solidified with 400 )l of HC1 37%.

After dilution with water, it was extracted with ethyl acetate (3 times).

In this way, as known by earlier experiences with colds, it is possible to obtain the labeled cortisol, evaporating crystals of the desired product.

After the preparation of the labeled substrate of the reaction, the radioenzymatic assay was set up.

The method is based on solubility variation following the detachment of the cold succinic moiety from the nucleus of tritiated cortisol. After incubation of the substrate in contact with a reaction matrix (buffer solution, supernatant of clones or whole human serum), the aqueous phase is extracted with ether, followed by WO 99/06536 PCT/EP98/04682 22 evaporation of the solvent and measurement in the extract of the radioactive signal, emitted by 3 H Cortisol.

Incubation is terminated at the desired time by a jumping dilution (16 x) with distilled water at its melting point.

Given the dependence of catalysis speed from the catalyst concentration and from temperature, and considering that incubation occurs at 38.5*C (corresponding to internal human temperature), this procedure allows to reduce by 16 x 24 times the reaction speed (remembering that every of T reduction, with a AE 20 hJ/moles, speed halves), and can be therefore considered satisfying.

.The extraction phase with ether is carried out by vortexing for 10 seconds. A so limited time has the scope of permitting the reaction to remain out of equilibrium in the'aqueous phase during the extraction itself.

The definition of the procedure was carried out by using five different molar concentrations of substrate incubated in human serum at different times.

The spontaneous esterase activity of the cortisol-21hemisuccinate in human serum, in comparison with control samples, was detected only at concentrations of substrate 100 mM and at incubation times higher than 1 hour.

The method has been used therefore for identifying the immunized animals that developed a positive immune response, thanks to the production of antibodies with esterase activity against the cortisol-21-hemisuccinate.

The results of this assay, carried out at a substrate concentration equal to 10 mM and after one hour incubation, gave the following indications: in the serum of immunized mice with the hapten-carrier conjugate an esterase activity higher than the one present in the serum WO 99/06536 PCT/EP98/04682 of non treated animals and blank controls was found.

Claims (19)

1. Abzyme against a hydrosoluble prodrug of a corticosteroid hormone obtained by a process comprising the following steps: a) conjugation of a hapten analogous of the transition state of said prodrug, said hapten having formula R-[A]-O-P0 3 X 2 .20 wherein A is the steroidal nucleus of a corticosteroid hormone; R is a chemical group linked to the position C-3 of ring A of pregnane and suitable for the conjugation with an immunogenic macromolecule; PO 3 X 2 is a phosphate group linked to the hydroxyl in position 21- or 17- (which on the corresponding corticosteroid hormone carries a hydrophilic molecule); X is hydrogen or a pharmaceutically acceptable cation with an immunogenic macromolecule to give a conjugate; b) obtaining the catalytic monoclonal antibody against said conjuctate.

2. Abzyme according to claim 1, wherein said prodrug is a hydrosoluble dreivative of cortisol, fluorocortisone, triamcinolone, dexamethasone, prednisone, cortisone, betamethasone.

3. Abzyme according to claim 2, wherein said prodrug is a hydrosoluble derivative of cortisol.

4. Abzyme according to claim 3, wherein said prodrug is selected from the group consisting of cortisol 21- hemisuccinate, cortisol 21-acetate, cortisol 21- phosphate.

5. Abzyme according to claim 3, wherein said prodrug is selected from the group consisting of cortisol 17- hemisuccinate, cortisol 17-acetate, cortisol 17- phosphate.

6. Abzyme according to a anyone of claims 1-5, wherein said hapten is the compound 11-beta,17-alpha-21- trihydroxy-4-pregnene-20-dione-3-hydrazono-3'- azidobenzamide-21-phosphate.

7. Abzyme according to anyone of claims 1-6, wherein said macromolecule is a protein.

8. Abzyme according to claim 7, wherein said protein is KLH.

9. Abzyme according to anyone of claims 1-7, wherein said monoclonal antibody is obtained by immunization of an animal with said conjugate obtained in the step a).

10. Abzyme according to anyone of claims 1-7, wherein said monoclonal antibody is obtained by in vitro immunization.

11. The use of the abzyme of claims 1-10 as medicament.

12. The use of the abzyme of claims 1-9 for the preparation of a medicament useful in the therapies curable with cortisonic drugs.

13. The use of the abzyme of claims 1-9 for the preparation of a medicament useful in the therapy of the acute adrenal insufficiency. AMENOED SHEET 26

14. Pharmaceutical compositions containing an effective amount of the abzyme of claims 1-9, in admixture with pharmaceutically acceptable carriers and excipients.

Pharmaceutical compositions according to claim 14, in injectable form.

16. Kit comprising at least a pharmaceutical composition containing an effective amount of the abzyme of claims 1 or 1-9, in admixture with pharmaceutically acceptable carriers and excipients and a pharmaceutical composition containing an effective amount with respect to the amount of the abzyme of a hydrosoluble prodrug of a corticosteroid hormone.

17. Compounds of formula (I) R-[A]-O-P0 3 X 2 (I) wherein *9 S* A is the steroidal nucleus of a corticosteroid hormone; S R is a chemical group linked to the position C-3 of the A ring of pregnane and suitable for the conjugation with an immunogenic macromolecule, wherein R is not a C=O group; P0 3 X 2 is a phosphate linked to the hydroxyl in position 21- or 17- (which on the corresponding corticosteroid hormone carries a hydrophilic molecule); X is hydrogen or a pharmaceutically acceptable cation. **o

18. Compound according to claim 17 which is 11-beta, 17-alpha-21-trihydroxy-4- pregnene-20-dione-3-hydrazono-3'-azidobenzamide-21-phosphate.

19. Use of the compounds of claims 17 or 18 as haptens in the preparation of abzymes of claims 1-9.