CZ145593A3 - Polymeric conjugate of cyclosporin a, process for preparing and use thereof in in vitro diagnostic tests - Google Patents

Abstract

Polymeric conjugates of cyclosporine A consisting of a polymeric carrier containing 40 to 1,000 polymerised monomeric units, of which 20 to 99 molar per cent are N-(2-hydroxypropyl)methacrylamide, N-ethylmethacrylamide, N-ethylacrylamide, acryl amide, N-vinyl pyrrolidone or acrylic acid and methacrylic acid as sodium salts, or the copolymers of the above-mentioned monomers, but preferably N-(2-hydroxypropyl)methacrylamide, 0.2 to 20 molar per cent of the units are methacrylated amino acids or methacrylated oligopeptides with terminally covalently bound cyclosporine, and 0.2 to 15 molar per cent of the units are methacrylated amino acids or methacrylated oligopeptides, where the units of methacrylated amino acids, selected from the group which includes glycine, alpha-alanine, beta-alanine, phenylalanine, leucine and aminocaproic acid, or methacrylated oligopeptides GlyGly, GlyPheLeuGly, GlyLeuPheGly, GlyLeuGly, GlyPheGly and GlyPheGly, is covalently bound with an enzyme selected from the group which includes horse-radish peroxidase, alkaline phosphatase and β-galactosidase, the method of preparing and using them for the in-vitro determination of free cyclosporine A and its biologically active metabolites in human body fluids.

Description

Technical field

The subject invention is a polymeric conjugate of cyclosporin ^{A: 3} * ^ Joshah) j ^ ^- způšó'b jehd * * * přlprávy''á use in'viťř6 ^^ * duTgnoštickycR test ', in particular qualitative and quantitative analysis of the levels of free cyclosporin A (CsA ) and its metabolites present in human body fluids.

State of the art

Monitoring of cyclosporin A (CsA) levels in the blood of patients immunosuppressed by Sandimun (Sandoz) or Consupren ® (Galena, Opava) is a prerequisite to ensure optimal dosing of the drug. The narrow concentration range of effective CsA levels, the high risk of CsA side effects, the individual pharmacokinetics of CsA in the individual patients to be treated require a sustained control of the CsA levels in the blood. At present, the determination of CsA levels is performed by high-performance chromatography (Hold, D., Marsden, J., Johnston, A., Transplant. Proc., 22, 1234, 1990), and in clinical practice primarily by saturation radioimmunoassay (RIA). using 3A labeled CsA (Quesmiaux, V., Tees, R., Schreier, et al., Clin. Chem. 44, 43, 1987) or 1251 (Knepil, J., McPhilips, M. Clin. Chem., 1989, 35, 181). Another routinely used method of polarized immunofluorescence (FPIA) is based on measuring the fluorescence changes of fluorescein isothiocyanate (FITC) labeled CsA (Wang, O., Morrison, M., Wang, N., Clin. Chem. 32, 1061, 1986).

Furthermore, an immunoassay known as ACMIA (Af-Column-Mediated-Immunoassay) has been developed which uses an enzyme (β-galactosidase) coupled to a specific antibody (Prince, C., Davey, C., Medculp, E., et al., Clin. Chem., 36, 1932 (1990). Another non-isotopic iraunochemical method, the chemiluminescence immunoassay of CsA (CLIA), was developed using N- (4-aminobutyl) -N-ethylisolaminol-labeled hemisuccinate-CsC (Stabler, T., Lieger, A., Clin. Chem. 36, 906, 1990). In 1990, kits for the determination of CsA levels in the EMIT system (Enzyme-Multiplica-Immunoassay Technique) with CsA labeled glucose-6-phosphate dehydrogenase (Beresini, M., Totan-: Quinn, R., Barnett, B., et al., Clin Chem 36, 1034 (1990). In addition to traditional immunochemical techniques, a receptor analysis method using the target drug molecule as an analytical reagent was published in 1990-1992 to determine CsA levels. The advantage of this methodological approach over the traditional reagent antibody is the selectivity of the interaction that reflects the biological activity of the drug and its metabolites (Paul, K. ', Harding, WH, Marks, MW, Handschuniacfter, RG, Lorber, MS, Transplant. 974, 191, Haskova, V., Rozprimova, L., Svobodova, J., Folia

Biol. 37, 134 (1991); Kullinger, B., Wolfram, J., Hamilton, G. , Hrozemsky, M., Kovarik, K., Woluszuk, W., Steiner, G., Transplant. Why. 22, 1702 (19909, 10). The technique proposed by us uses the above approach, the drug molecule itself being covalently linked to an enzyme-modified polymer carrier molecule allowing easy detection and evaluation of the assay by methods commonly available in any biochemical laboratory.

SUMMARY OF THE INVENTION

An object of the present invention is a polymeric cyclosporin conjugate comprising 40-1000 polymerized monomer units, of which 20 to 99 mol%. N- (2-hydroxypropyl) methacrylamide, N-ethylmethakrylamid, N-ethyl acrylamide, acrylamide, N- _lt vinylpyrrolidone, methacrylic acid and acrylic acid in the form of their sodium salts or copolymers of the aforementioned monomers, but preferably N- (2- hydroxypropyl) methacrylamide, 0.2 to 20 mol%; units of methacryloylated amino acids or methacryloylated oligopeptides with terminally bound cyclosporin and 0.2 to 15 mol%; units of methacryloylated amino acids or methacryloylated oligopeptides, said methacryloylated. For example, amino acids-or-methacryloyl-bound oligopeptides are covalently bound by an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, and β-galactosidase.

The process for preparing the polymeric cyclosporin conjugates of the present invention is characterized in that cyclosporin A bearing an aldehyde, hydrazine or primary amino function is first bound to the prepared polymeric carrier and then coupled with an amide bond by the amine bond formed by reaction of the primary amino groups present in the enzyme molecule with the polymeric carrier. or by linkages resulting from the reaction of the polymeric carrier with the aldehyde groups introduced into the enzyme molecule by slight oxidation or linkages; resulting from their careful reduction. ''

Another aspect of the invention is the use of the cyclosporin A polymer conjugates of the present invention for the non-isotopic determination of free cyclosporin A and its biologically active metabolites in vitro in human body fluids.

The invention also provides a method of using polymeric cyclosporin A conjugates for non-isotopic determination of free cyclosporin A and its metabolites, wherein the determination of the level of cyclosporin and its metabolites in solution is based on the binding competition of the determined cyclosporin or its metabolites and polymeric cyclosporin conjugate. with an enzyme bound according to the present invention, to cyclophilin as a. free and polymer-bound cyclosporin receptor, wherein the concentration of cyclophilin-bound polymer conjugate (enzyme-bound cyclosporin) is determined by the enzymatic reaction to the polymer-bound enzyme with a chromogen-containing substrate solution. enzyme with a solution ^- substrate containing chromogen to determine the concentration of this cyclophilin bound polymer conjugate of cyclosporin with bound enzyme.

The method of using the cyclosporin A polymer conjugates of the invention can also be accomplished by using an anti-cyclosporin antibody selected from the group consisting of a monoclonal murine anti-CsA antibody, a polyclonal antibody, instead of cyclophilin as a receptor for binding free cyclosporin and a polymeric cyclosporin conjugate. a sheep anti-CsA antibody, a polyclonal rabbit anti-CsA antibody, or a polyclonal chicken anti-CsA antibody.

The enzyme-linked cyclosporin polymer conjugates of the present invention (CsA-β-Enz) starting from N-substituted acrylic and methacrylamides can be described by the general formula:

; .....------ Rq. —W —-_ GH5 i,. · »,». »,» ». ·, .-» '··' · Τ

III - (CH ₂ -C-) _x - (CH-C) (CH ₂ -C-)

I -4 I. C = O C = O C = o

I I f ^R 2 R 3 R where R is -H, (amido), or -CH ₃ (methacrylamides), R ₂ is NHCH2CH3, -NHCH 2 CH (OH) CH 3 or -NH ₂ group, R3 is an amino acid or oligopeptide with terminally bound cyclosporin and R 4 is an amino acid or oligopeptide with a terminally bound enzyme, x is from 20 to 99 mol%, y is from 0.2 to 20 mol%, and z is from 0.2 to 15 mol%.

In the case of carriers prepared on the basis of N-vinylpyrrolidone copolymers, the expression for x equals 20 to 99 mol%. corresponds to the molar proportion of N-vinylpyrrolidone in the copolymers.

In preparing the polymeric cyclosporin conjugates of the present invention, the cyclosporin A carrying an aldehyde, hydrazine or primary amino function is first coupled to the prepared polymeric carrier and then the enzyme is coupled. The enzyme is bound by an amide bond formed by the reaction of primary amino groups. present in the enzyme molecule with the polymeric carrier, or by linkages resulting from the reaction of the polymeric carrier with 5 aldehyde groups introduced into the enzyme molecule by slight oxidation or by bonds resulting from their careful reduction.

Semisynthetic derivatives of natural cyclosporins and described in the patents (PV 6498-89 and PV 6499-89) containing an aldehyde group or derivatives thereof modified with ethylenediamine, hexamethylenediamine or hydrazinera were used for coupling to the carrier. The cyclosporin A derivative in the conjugate is bound to the amino acid or oligopeptide in the side chain of the polymer by an amidic (-C0NH-) bond, a hydrazidic bond (-NHNHCO-), or by bonds, (-C = N-NH-), (-CH-NH- ) and (-CH-NH-NH-).

The units of N-methacryloylated amino acid monomers and oligopeptides involved in the polymeric carrier chain are based on biogenic amino acids such as glycine, alanine, phenylalarsine, aminocaproic acid, or oligopeptides thereof. The enzymes used to label polymer conjugates are horseradish peroxidase, alkaline phosphatase, and β-galactosidase.

These enzymes are attached to the polymer via an amino acid or oligopeptide spacer of the amino acid composition set forth above via the -COOH end of the amino acid or oligopeptide via an amide -CONH- bond (formed by acylating the amino groups of the enzyme with a carboxyl-activated enzyme in the polymer side chain), or -CONHfCHzJnNHCH- -CONHN = CH- and CONHNHCH2 - formed by the reaction of a polymer whose amino acid or oligopeptide side chains are terminated with an amino or hydrazo group, with an enzyme slightly oxidized by sodium periodate and optionally subsequent reduction with borohydride or sodium cyanoborohydride.

The method of determining free cyclosporin A of the invention utilizes the principle of saturation receptor analysis. As a receptor for free cyclosporin (CsA) or polymer-enzyme conjugated cyclosporin (CsA-β-Enz), cyclophilin (Cph) or anti CsA antibodies are used as monoclonal mouse, polyclonal sheep, rabbit, hen. The evaluation of the assay, ie the determination of free CsA from the sample and the enzyme-labeled polymeric carrier (CsA-P-Enz), both bound to the receptor, is based on the competition of binding both CsA and the CsA-P-Enz concentration using enzyme enzymatic reactions. carrier-bound with a chromogen-containing substrate substrate solution.

Analytical determination of free cyclosporin (CsA) based on competitive binding of free and bound CsA to substrate can be performed in triplicate.

a) Both types of cyclosporin (CsA from the test solution and CsA bound in the Reagent - polymer conjugate) are first bound to cyclophilin in solution and then anchored on microtiter plates with immobilized cyclophilin-specific immunoglobulin. The plate-anchored conjugated CsA concentration is determined from the absorbance of the solution stained due to the color enzymatic reaction.

b) Incubate with reagent (CsA-P-Enz) and test sample solutions. The test is carried out directly on a microtiter plate with pre - fixed cyclophilin, the evaluation is similar to that of.

a), i.e., based on the color enzymatic reaction of the polymer conjugate with the substrate.

c) Incubate the solution of reagent and test substance with anti-cyclosporin antibody (anti-CsA-Ab) in solution, the cyclosporin-modified antibody is anchored in a microtiter plate with anti-CsA-Ab-modified surface and the concentration of bound conjugate (CsA-P). (Ennz) is determined by measuring the absorbance of a solution stained with the same enzymatic reaction as in both previous cases.

A method for determining the CsA-β-Enz conjugate binding activity in a saturation / recipe recipe system is shown using an example of a cyclosporin A horseradish peroxidase (CsA-P-HRP) polymer conjugate in Example 3. .

The principle of the ciclosporin assay is based on the use of a water-soluble polymeric carrier, to which both the ciclosporin drug and the enzyme giving the color reaction substrates are coupled by oligopeptide sequences to allow quantitative evaluation of the conjugate concentration in solution.

. ---------------- V-da-1-s-m-is-exemplified-without-being exemplified without exemplifying it. ----- limits.

List of abbreviations used

CsA cyclosporin A # MA methacryloyl. Gly, Phe, Leu, Gly, Val L-amino acids

P - polymer carrier. -.-. HRkřenoyá _in peroxidase. _mtTr -r- ..... _rr . _r . _,, -. _r .,. _r ....

-ONp p-nitrophenoxy group

TRIS.HCl buffer prepared from tris (hydroxymethyl) aminomethane, pH adjusted with HCl

Cph cyclophilin

GPC gel permeation chromatography

DMF dimethylformamide

Enz enzyme

Examples

Examples of synthesis of CsA-P-Enz polymer conjugate

Example 1: Synthesis of polymeric conjugate CsA-P-HRP, aminolytically coupled enzyme

The synthesis consists of three phases: a) synthesis of the polymeric carrier, b) binding of cyclosporin to the carrier, c) binding of the enzyme to the carrier, purification and characterization of the conjugate.

a) Polymeric carrier (R 1 methyl, R 2 -NHCH 2 CH (OH) CH 3, R 3 oligopeptide GlyPheLeuGlyONp, x = 92 mol%, y = 8 mol% az = 0) was prepared by radical precipitation polymerization of 1.5 g of N- (2) hydroxypropyl) methacrylamide with methacryloylated 0.58 g of p-nitrophenyl ester glycylfenylalanylleucylglycinu (MAGlyPheLeuGlyONp) in 18 ml of acetone under initiation with 90 mg of azo-bis-isobutyronitrile at 50 ^e C. the polymerization mixture was purged incandescent ampoule with nitrogen, the ampoule sealed and placed to the thermostat for 24 hours. The molecular weight of the prepared polymer was 25,000 g / mol.

b) 0.1 g of the hydra20-modified cyclosporin A was dissolved in 1.8 ml of dimethylsulfoxide (DMSO) and a solution of 0.30 g of polymeric carrier (see preparation a) in 2 ml of DMSO was added with stirring. After 6 hours of reaction at 25 ° C, the polymer was isolated by precipitation into 1: 1 acetone-diethyl ether. The bound CsA content was determined by amino acid analysis of the polymer hydrolyzate (18 wt% content), the UV content of the remaining p-nitrophenyl ester groups being 3.7 mol%.

c) 90 mg of the polymer prepared in case b) was dissolved in ml of water and refrigeration (4 C ^c) .přidán solution of 30 mg of horseradish peroxidase in phosphate buffer pH-seventh The pH of the solution was raised to 9.2 by addition of sodium tetraborate solution over 4 hours. After an additional 3 hours of reaction, the product was purified by GPC (Sephadex G-15) and lyophilized. The polymer conjugate was characterized by GPC gel permeation chromatography (Sepharosa 4B and 6B, 1: 1) and electrophoresis.

Example 2: Synthesis of CsA-P-HRP polymer conjugate, an enzyme bound via aldehyde groups introduced into the enzyme by slight oxidation.

For the preparation of the conjugate, a polymeric carrier was used, the preparation of which was described in Example 1 a). A 10% solution of this polymer in dimethylformamide (DMF) was added to a 100-fold molar excess of ethylenediamine (calculated on the content of reactive -ONp groups) with vigorous stirring. (R - GlyPheLeuGlyNH (CH _{2) 2} NH _2). In the same way, however, the reaction with s-hydradine hydrate also prepared polymeric precursor 2 bearing a hydrazo group in the side chains (R ₃ -GlyPheLeuGlyNHNH ₂ )

Both polymer precursors were used to synthesize the final conjugate, which was a two step process.

..__. In a first reaction step of 100 µg-polymeric precursors (1 or 2)

2) was reacted in methanol with 33 mg of cyclosporin A (CsA) modified with aldehyde group (polymer intermediate isolated by precipitation into acetone-diethyl ether), in the next step 33 mg of HRP enzyme (oxidized in o.iM acetate buffer pH 4.0 by periodate) Sodium) onto a polymeric carrier in a borate buffer at pH 9 (precursor i) or pH 8 (precursor 2). Polymer conjugates were purified by GPC (Sephadex G 15), binding between carrier and bound components stabilized by reduction with sodium cyanoborohydride and final, purified, lyophilized and characterized analogously to Example x.

Examples of the use of conjugate to determine CsA concentration

Example 3: Determination of CsA-β-HRP binding activity in a system, saturation receptor analysis

Cyclophilin (Cph) isolated from bovine thymus is used as the ligand-receptor binding system by ultrafiltration of tissue homogenate through Diaflo membranes XM-300-XM-3G on an AMICON. For use in analytics, a protein-containing filtrate of mw less than 30 kD is thickened by filtration through a YM-10 membrane. Cyclosporin A (CsA) receptor binding activity is determined using tritium-labeled CsA ( ³ H-CsA). Cyclophilin concentration (Cph) corresponding to 50% of the maximal binding activity was used for saturation radioreceptor analysis (RRA).

Essential solutions (20 mg / L) of the test conjugates - (CsA-P-HRP reagents) were prepared by dissolving the preparation in 10 mM. TRIS.HCl buffer pH 7.3, containing 50 mM mercaptoethanol> 2 mM dithiotreitol, 2 mM calcium lactate and 1% (w / v) bovine serum albumin (dilution buffer). Working solutions of 10-1 mg conjugate / 11 buffer were prepared from the stock solutions. To -200 µL of test sample was added 50 µL of 3H-CsA and after addition of 50 µL of optimally diluted Cph, the mixture was incubated for 60 minutes at 3 ° C. After the incubation was completed, 500 µL of activated carbon suspension (10 mg / mL) was added to the reaction mixture and incubated for 8 minutes in an ice water bath. After pre-incubation, the suspension was centrifuged a

500 ^_ / ur ^'_ supernatant ^- be - transferred -do- 10 mL · scintillant SLD 31 and the measured activity of β - rays. The measured values were graphically processed after subtracting the background and converting to B _o .

The results of the inhibition of the Cph x 3H-CsA interaction by the prepared CsA-P-HRP conjugates show identity between the two types of CsA derivatives (3H-CsA and CsA-P-HRP) in relation to the binding ligand, i.e. cyclophilin. This means that CsA-β-HRP conjugates can be used as detection compounds in both qualitative and quantitative analysis.

'** Výsledkysaturační »^ proká2aly analysis rúčinnost _{j <k} jugátu.v. ^ receptor analysis system in a concentration range of CsA necessary for monitoring therapy imunosupr.esivní SANDIMUN Consuprenem ^{R R} respectively. ''. .

Examples of experimental determination of cyclosporin A (CsA)

Example 4

100 µl of optimally diluted Cs-A-HRP conjugate in dilution buffer is added to 100 µl of the test sample (extract) of body fluid. After thorough mixing of both components, an optimal amount of Cph (corresponding to 50¾ Β ^ _χ ) is added in 50 µl dilution buffer. The mixture is again thoroughly mixed and incubated at 8 ° C for 60 minutes,

After incubation, 200 µl of the reaction mixture is transferred to a well of a microtiter plate (MaxiSorp F8 NUNC), on which the immunoglobulin fraction of rabbit immune serum against bovine cyclophilin is mobilized. After 60 minutes incubation at 18 ° C, the wells are aspirated, the wells are washed 3 times with bovine albumin-free dilution buffer and the presence of CsA-P-HRP conjugate is demonstrated by enzyme activity by adding 150 µl of chromogen-containing substrate solution. After the end of the enzyme reaction, the absorbances of the individual samples are measured and the results graphically processed. The enzyme activity value of a given sample is inversely proportional to the CsA concentration. in the test material, i.e. body fluid and tissue extract, respectively.

Example 5

The optimum amount of cyclophilin (Cph) is immobilized on the surface of the wells of the microtiter plates (MaxiSorp F8 NUNC). To prevent non-specific adsorption of the analyte, blocking of the surface of the wells with 1% gelatin solution is performed after immobilization of Cph. Until use, microtiter plates with immobilized Cph are stored at 4 ° C under a 1% gelatin solution containing 0.01% sodium azide. Before use, the wells after aspiration of gelatin are washed 3 times with a wash buffer (0.8% (w / v) NaCl, pH 7.3, 0.002% (v / v) Tween 20). After aspiration of the wash solution residues, 100 µl of the test sample (extract) of body fluid is dispensed into the wells, immediately thereafter 100 µl of optimally diluted CsA-P-HRP conjugate is added to each well. After a 60 minute incubation at 8 ° C, the reaction mixture was aspirated and the wells were washed as in Example 1.

Example 6

100 µl of optimally diluted CsA-HRP conjugate in dilution buffer without mercaptoethanol and dithiotreitol is mixed with 100 µl of test sample (extract) of body fluid. After mixing, 200 µl of optimally diluted antibody (monoclonal mouse, polyclonal sheep, rabbit, chicken) is added to the mixture and incubated at 8 ° C for 60 minutes. After completion of the incubation period, 200 µl of the mixture is transferred to the wells of a microtiter plate, on whose surface a fraction of antibodies against antibodies of the respective animal species ie anti-mouse and anti-sheep, anti-rabbit, anti-chicken and the mixture in the wells are incubated at 8 ° C for 60 minutes. After the incubation was complete, the well contents were aspirated wells 3 times with wash solution and then proceeded as in Example 1.

Utility of the invention

The polymer conjugate of the invention and the method of its use for the in vitro diagnostic tests of the invention is a non-isotopic technique for determining free cyclosporin A that can be used in all biochemical laboratories of the "human" 7 where cyclosporin levels in body fluids need to be monitored. in patients receiving this medicine. The dosage of the drug can be individually adjusted based on the test results. The advantage of the method according to the invention is its simplicity and possibility to carry out in any biochemical laboratory, because the method does not require permission to work with radioisotopes.

Claims (5)

Patent claims Polymeric cyclosporin A conjugates containing 40 to 1000 polymerized monomer units, of which 20 to 99 mol%. is N- ( 2-hydroxypropyl) methacrylamide, N-ethylmethakrylamid, Ν'ethyl acrylamide, acrylamide, N-vinylpyrrolidone, methacrylic acid and acrylic acid in the form of their sodium salts or copolymers _ITI .. ,, of the aforementioned monomers, but preferably N- (2 -hydroxypropyl) methacrylamide *** 0 ' ^, 72' to '20 ^, 20% moles of methacrylated amino acids or methacrylated oligopeptides with terrain covalently bound cyclosporin, and 0.2 to 15 mol% of methacryloylated amino acid or methacryloylated oligopeptide units characterized in that an enzyme selected from the group consisting of horseradish peroxidase 2U, alkaline phosphatase and B-galactosidase is covalently bound to the methacryloylated amino acid or methacryloylated oligopeptide units. I. Process for the preparation of polymeric cyclosporin conjugates according to claim 1, characterized in that the prepared polymeric carrier is first coupled with a cyclosporin A bearing an aldehyde, hydrazine or primary amino functional group and then bound by the enzyme by an amide bond formed by the reaction of the primary araino groups present in the molecule. an enzyme with a polymeric carrier or linkages; formed by the reaction of the polymeric carrier with the aldehyde groups introduced into the enzyme molecule by slight oxidation or by the bonds resulting from their careful reduction. Use of the polymeric cyclosporin A conjugates according to claim 1 for the determination of free cyclosporin A and its biologically active -metabolites in vitro in human body fluids. 4. A method according to claim 1 for the determination of free cyclosporin A and its metabolites, characterized in that the determination of the level of cyclosporin and its metabolites in solution is based on correcting the binding of the determined free cyclosporin or its metabolites and the polymeric cyclosporin S-linked polymer conjugate. to cyclophilin as a receptor for both free and polymer bound cyclosporin, wherein the concentration on The cyclophilin-bound cyclosporin polymer-bound enzyme conjugate is determined by the enzymatic reaction to the polymer-bound enzyme with a substrate solution containing a chromogen. 5. The method of claim 4, wherein the cyclosporin A polymer conjugate is an anti-cyclosporin antibody selected from monoclonal mouse anti-cyclosporin antibody instead of cyclophilin. CsA antibody, polyclonal sheep anti-CsA antibody, polyclonal rabbit anti-CsA antibody, or polyclonal chicken anti-CsA antibody.