WO1999006536A1

WO1999006536A1 - Catalytic monoclonal antibodies for the in vivo transformation of corticosteroid prodrugs

Info

Publication number: WO1999006536A1
Application number: PCT/EP1998/004682
Authority: WO
Inventors: Adriano Podesta'; Silvia Trasciatti; Sergio Rosini
Original assignee: Abiogen Pharma S.P.A.
Priority date: 1997-07-30
Filing date: 1998-07-27
Publication date: 1999-02-11
Also published as: CN1268174A; NZ502526A; NO20000395D0; EP1003842A1; JP2001512006A; AU736187B2; IT1293510B1; CA2299623A1; ITMI971825A1; AU8732898A; NO20000395L

Abstract

Catalytic monoclonal antibodies (abzymes) for the in vivo transformation of corticosteroid prodrugs ae herein disclosed. These abzymes are advantageously used in emergency therapy of acute adrenal insufficiency allowing a prompt pharmacological action of the corticosteroid. Haptens against which the abzymes are generated are also disclosed.

Description

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, hypovole ic conditions and burns, yocardial 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 hand, can not be attributed to methodological errors nor to a lack of pharmacological activity fcorn 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 cytoplas atic 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 g 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 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 glomerulonephritiε , kidney alterations, Systemic Lupus Erithematosus , arthritis, rheumatoid carditis, bronchial asthma, allergic diseases, intestinal and hepatic disorders, dermis alterations, lymphomas, Hodgkin'ε 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 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, prostaglandinε , 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 membrane and bind to their cytoplasmatic receptor in order to exploit their pharmacological acti-vity. 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 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 haptenε 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 tendε to aεεume the final arrangement of the reaction aε 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 abzy e is finding out the chemical structure of the suitable hapten. WO8910754 discloεeε catalytic monoclonal antibodieε 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 biomoleculeε , and a series of peptidic haptenε iε provided. 09302703 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, linkε to εaid εecond reactant, releaεing thereafter the active moiety. The reactantε can have antibodieε aε functional groupε 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, releaεing the active part iε alεo 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 i any case there is a leading effect of the active drug to the action site. The specific teaching iε directed to leading effective moleculeε in the diagnosis or treatment of tumors.

JP94220072 discloses abzymes against antibiotics.

It is desirable to dispoεe of a unique criterion in the design of a εuitable homologous of the transition state valid for a whole class of drugs, and not for a εingle compound. Moreover, it iε also desirable to dispose of such a homologouε 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 the organism a specific esteraεe, capable of releaεing the drug in queεtion, doeε 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 phoεphonic function is indicated aε mimicking the tranεition εtate, the coupling group with the carrier protein iε alεo described. However, there is no indication to the person skilled in the art about the criticality of how positioning the coupling group on εteroidal molecule and in which relation with the mimicking group of the transition εtate.

.Therefore, the type of hapten to be use iε highly critical, and the reciprocal poεition of the coupling and mimesis groupε of the tranεition state is still more critical.

WO8910754, 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 gpl20 envelope protein. O9302703, 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 εolved 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. It has now been found that the hapten suitable for the production of abzymes against hydroεoluble 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 acromolecule at position C-3 of the εteroidal εkeleton, the fartheεt poεεible from said phosphate group. Accordingly, it is an object of the present invention a compound of formula (I)

. R-[A]- OR - PO3X2 (I) wherein: A representε the steroidal nucleus of a corticosteroid ' hormone;

R is a chemical group linked to poεition C-3 of the pregnane ring A and iε εuitable for the conjugation with an immunogenic macromolecule;

PO3X2 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 (I), 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 antibodieε (abzymeε) εpecific against the different hydr.osoluble prodrugs of corticosteroids will be obtained.

Said abzyme or a fragment thereof or an engineerized fragment thereof are another object of the present invention. Another object of the preεent invention is the use of the abzymes for the preparation of medicaments uεeful in the treatment of acute adrenal insufficiency.

The esεential advantage obtained by the preεent invention, by consequence of the higher bioavailability of the active ingredient, conεiεts in the posεibility of obtaining an immediate therapeutic effect, which, in emergency situations, will allow to achieve a higher number of succeεseε in εurvival terms of the treated subjects.

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

The poεεibilitieε of uεing lower doses and the opportunity to diminish therapy timeε, due to the higher bioavailability of the active ingredient, could allow effective treatmentε of the above listed chronical diseases, avoiding unwanted effects.

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

In the compounds of formula (I), (A) is the εteroidal nucleus of a natural or synthetic corticosteroid.

Examples of adrenocortical hormones are: cortisol, fluorocortiεone , triamcinolone , dexamethaεone, predniεone, cortiεone, and betamethaεone .

Exampleε of R chemical groupε εuitable .for the conjugation with an immunogenic macromolecule are azido, p-azophenyl-β-D-lactoεide (Lac), p-azophenyl-β-D-glucoεide (Glu), p-azophenyl- arεonate (Arε), sodium trinitrobenzene sulfonate ( TNBS ) , εodium dinitrobenzene εulfonate (DNBS), dinitrophenyl (DNP) and S-acetyl-mercaptoεuccinic anhydride. Among theεe, the azido group* iε preferred.

Examples of hydroεoluble corticosteroid prodrugs are those used in injectable formulations. Preferred examples are: betamethasone sodium phosphate, betamethasone εodium phosphate and acetate, cortisone acetate, cortisol sodium phosphate, cortisol sodium succinate, cortisone acetate, dexamethasone acetate, dexamethaεone εodium phoεphate, prednisolone acetate, methylprednisolone εodium εuccinate, predniεolone acetate, predniεolone εodium phosphate, prednisolone tebutate, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide . Cortisol 21-hemisuccinate , cortiεol 21-acetate, cortisol 21-phosphate, cortisol 17-hemisuccinate , cortisol 17- ace^'tate, cortisol 17-phosphate are more preferred exampleε .

Compoundε of formula (I) are prepared εtarting from the reεpective active ingredientε, namely from the pharmacologically active εteroidε or their commercially available derivativeε. For example, the active ingredientε 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 (I) (hapten) is obtained, it iε conjugated with the immunogenic macromolecule. Exampleε of εuch macromolecules are KLH, BSA, ovoalbumine, thyreoglobuline , chicken IG, polysaccharides.

The conjugate hapten-carrier is then used for 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 iε 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 preεent invention are absolutely conventional and within the normal activity of the skilled person. A firεt preferred embodiment of the present invention relates to the hapten of cortisol substituted at position

21. It is the compound 21ι3,17α-21-trihydroxy-4-pregnene-

20-one-3-hydrazono-4 ' -azidobenzamide-21-phosphate , namely the compound of formula (I) wherein [A] is the steroidal moiety of cortisol, R is the 3-hydrazono-4 ' -azidobenzamide group, X is hydrogen.

H O

I il

H—C-O— P-o

αzidθ This compound constituteε 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

5 cortisol. Instead of synthesizing the analogous of the tranεition state for each of the - exiεting 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

-H) 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 hemiεuccinate,

15 phosphate or other well known carriers, is leεε 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

20 formulations only when linked to a hemisuccinic residue (methylprednisolone hemisuccinate , URBASON(R) SOLUBILE, 8, 20, 40, 250 g), can be used as therapeutic agent.

In the hapten molecule, a phosphate group is present in position -21 mimicking the tranεition state of the

25 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

30 position 21 or 17, on which the hydrophilic substituent is present . 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 macro olecular carrier is attached to the azide in poεition C3, leaving available the moiety of the molecule for the immunological recognition where the reaction muεt actually occur. Then the evoked antibody recognizes both the binding εite in C21 and the major part of the εteroidal molecule. The hapten haε been obtained by condenεing commercial dihydrocortiεone-21-phoεphate with the hydrazide of the 4- azidobenzoic acid; the latter compound haε 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 4- azidobenzoic acid; the latter was esterified with hydrochloric methanol; the reaction waε followed by hydrazinolysiε in methanol of the intermediate methyl ester. The condensation of the hapten with dihydrocortisone-21-phoεphate was carried out by heating equimolar amounts (2 m ol) 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 εolvent evaporation. The preεence of the hapten in the reaction mixture waε 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 macromolecular carrierε. In the uεual experimental conditions, more frequently used molecular carriers are proteins. It has been neceεεary 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 modeε of conjugation to a macromolecular carrier depend on the type of functional group available on the hapten molecule. Different ethodε of chemical modification of proteinε with haptenε are available, and can be uεed indifferently, then εelecting the moεt effective. The εubstitution molar ratio is estimated by absorption spectrophotometry .

The hapten-protein carrier conjugate waε uεed 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 schemeε.

At the end of the treatment period, serum of the animalε 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 antibodieε waε carried out according to the uεual methodε (fuεion 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 strategies : to modify the analogous of the transition state; to carry out the selection of antibodies fro combinatory libraries expresεed on phageε (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 hydroεoluble 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 abzymeε against hydroεoluble 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 interventionε , hypovolemic εtateε and burns, myocardial infarct, pulmonary embolism, severe edematous states (Quincke's edema, glottis edema, pulmonary edema), acute pancreatitis, anaphylactic reactionε following sera injections, transfusions accidents, drug hypersenεitivity , εevere allergic reactionε, apoplexy, childhood acute toxicosis and accidental poisonings, hepatitis and hepatic coma, thyrotoxic and addisonian criεes, thromboagiitis obliterans . Still another object of the preεent invention iε the use of abzymeε 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 glo erulonephritis and other renal alterations, Systemic Lupus Erithematosus , arthritis, rheumatoid carditis, bronchial asthma, allergic diseases, disorders of the intestinal tract and of liver, dermatological alterations, lymphomaε, Hodgkin'ε 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 aεsociated.

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 phyεician. Conveniently, the abzyme according to the preεent 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 Scienceε Handbook, Mack. Pub., N.Y. , U.S.A. 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 illustrateε the invention. EXAMPLE a) Preparation of the hapten 21β,17α-21-trihydroxy-4- pregnene-20-one-3-hydrazono-4 ' -azidobenzamide-21- phosphate The compound of the title iε obtained by evaporating to dryness" 50 mg of 3-azidobenzoylhydrazine and 150 mg of not purified cortiεol 21-phoεphate . b) Conjugation with KLH

20 mg of KLH (hexamer, MW 450000 g/mol) were diεεolved into 10 M potaεsium bicarbonate, pH 7,4, 5 ml for a final concentration of 4 mg/ml.

The above products, without purification, were disεolved in the same solution, calculating a 200 molar excess of cortisol 21-phosphate derivative. The amount of product to be uεed is calculated arbitrarily presuming that all the steroid is in the active subεtituted azido form:

20 x 10^-3 g KLH _x 200 x 645 g/mol x 4/3 = 7.64 x 10^~3 g 450000 g/mol

(4/3 correctε the maεs amount taking into account the fact that in the starting reaction mixture there are 1 part of azide and 3 parts of cortisol 23,-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 Δ 2 cm x 5 cm equilibrated with the same buffer of the reaction.

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

A28O = 0.981.

Since the extinction coefficient of the test is A28O

= 0.240/0.01 μmoles 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

10 x 10^{" 3} g KLH 1 x = 5.55 x 10^~6 moles/1

450000 g/moleε 4 x 10^"3* 1 of KLH, from which the substitution molar ratio of: fP] 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 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 schemeε . At the end of thiε treatment period the animal εeru was withdrawn in order to verify that the animalε developed an immune reεponεe againεt 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]-llβ, 17 - dihydroxy-4-pregnene-3 , 20-dione-21 hemisuccinate labeled with. tritium ( H-C21-H) waε synthesized, starting from 37

Mbq of [1,2,6,7-³H] Cortisol (Amersham) .

In order to obtain an analytic signal of 50.000 cpm, with an counting efficiency b^a 60%, the working concentration from a volume of 100 μl iε 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 10⁶ bq ₌ 2.664 1 2.11 x 10¹⁵ bq/mol x 6.6 x 10^~9 moles/1

Since working at real concentrations of subεtrate equal to 10 μM ( aε obtainable from literature) is appropriate, it iε 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^ bq/2.11 x 10^•**•*■ bq/mol = 1.7 x 10^-8 moles, its contribution to the total masε 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 ol 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) 30°C.

The cortisol was dissolved into 200 μl 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 waε left 15 hours at r.t. under constant stirring. ^' At the end of incubation, the mixture was transferred into a separating funnel, waεhing the React. Vial, with further 200 μl of pyridine.

The pyridine waε εolidified with 400 μl of HC1 37%. After dilution with water, it was extracted with ethyl acetate (3 times).

In this way, aε known by earlier experienceε with coldε, it is possible to obtain the labeled cortisol, evaporating cryεtalε of the desired product.

After the preparation of the labeled substrate of the reaction, the radioenzymatic aεεay waε εet up.

The method is baεed on solubility variation following the detachment of the cold εuccinic moiety from the nucleuε 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 evaporation of the solvent and measurement in the extract of the radioactive signal, emitted by ³H Cortisol. Incubation is terminated at the deεired 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 2^ timeε the reaction speed (remembering that every 10°C of T reduction, with a AE = 20 hJ/moles, speed halves), and can be therefore considered satisfying.

.The extraction phase with ether iε 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^'aqueouε phaεe during the extraction itself.

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

The spontaneous esterase activity of the cortisol-21- he iεuccinate 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 animalε that developed a positive immune response, thanks to the production of antibodieε with esterase activity against the cortisol-21-hemiεuccinate . The reεultε 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 esteraεe activity higher than the one present in the serum of non treated animals and blank controls was found.

Claims

1. Abzyme or a fragment thereof or an engineerized fragment thereof against a hydrosoluble prodrug of a corticosteroid hormone.

2. Abzyme against a hydrosoluble prodrug of a cortico╬╡teroid hormone obtainable by a proce╬╡╬╡ comprising the following steps: a) conjugation of a hapten analogous of the transition ╬╡tate of ╬╡aid prodrug, ╬╡aid hapten having formula (I):

R-^[A^]-OR-P03X2 ⁽D wherein

A is the ╬╡teroidal nucleu╬╡ 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;

PO3X2 is a phosphate group linked to the hydroxyl in position 21- or 17- (which on the corresponding cortico╬╡teroid hormone carrie╬╡ 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 conjugate.

3. Abzyme according to claim 1 or 2, wherein said prodrug i╬╡ a hydro╬╡oluble derivative of cortisol, fluorocorti╬╡one, triamcinolone, dexamethasone , prednisone, cortisone, betamethasone.

4. Abzyme according to claim 3, wherein said prodrug i╬╡ a hydrosoluble derivative of cortisol.

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

6. Abzyme according to claim 4, wherein ╬╡aid prodrug i╬╡ selected from the group consi╬╡ting of corti╬╡ol 17- hemisuccinate , cortisol 17-acetate, cortisol 17- phosphate .

7. Abzyme according to a anyone of claims 1 or 2-6, wherein said hapten is the compound 11-beta, 17-alpha- 21-trihydroxy-4-pregnene-20-dione-3-hydrazono-3 ' - azidobenzamide-21-phosphate .

8. Abzyme according to anyone of claims 2-7, wherein said macromolecule is a protein.

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

10. Abzyme according to anyone of claims 2-8, wherein said monoclonal antibody is obtained by immunization of an animal with said conjugate obtained in the ╬╡tep a).

11. Abzyme according to anyone of claim╬╡ 2-8, wherein said monoclonal antibody is obtained by in vitro immunization.

12. The use of the abzyme of claims 1 or 2-11 as medicament .

13. The use of the abzyme of claims 1 or 2-10 for the preparation of a medicament u╬╡eful in the therapies curable with cortisonic drugs.

14. The use of the abzyme of claims 1 or 2-10 for the preparation of a medicament useful in the therapy of the acute adrenal in╬╡uf f iciency .

15. Pharmaceutical compo╬╡ition╬╡ containing an effective amount of the abzyme of claim╬╡ 1 or 2-10, in admixture with pharmaceutically acceptable carrier╬╡ and excipients.

16. Pharmaceutical composition╬╡ according to claim 15, in injectable form.

17. Kit comprising at least a pharmaceutical composition containing an effective amount of the abzyme of claims 1 or 2-10, 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.

18. Compounds of formula (I)

R-[A]-0-P0 X₂ (I) wherein

A is the steroidal nucleus of a corticosteroi*d hormone; 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;

PO3X2 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.

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

20. Use of the compounds of claims 18 or 19 as haptens in the preparation of abzymes of claims 1 or 2-10.