DD281659A5 - METHOD FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINES - Google Patents

METHOD FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINES Download PDF

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DD281659A5
DD281659A5 DD88319577A DD31957788A DD281659A5 DD 281659 A5 DD281659 A5 DD 281659A5 DD 88319577 A DD88319577 A DD 88319577A DD 31957788 A DD31957788 A DD 31957788A DD 281659 A5 DD281659 A5 DD 281659A5
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concentration
cyclosporins
cyclosporin
enzyme
inhibition
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DD88319577A
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German (de)
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Gunter Fischer
Gerhard Kuellertz
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Univ Halle Wittenberg
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Priority to DD88319577A priority Critical patent/DD281659A5/en
Priority to EP89115759A priority patent/EP0360029B1/en
Priority to ES89115759T priority patent/ES2058425T3/en
Priority to AT89115759T priority patent/ATE92536T1/en
Priority to DE8989115759T priority patent/DE58905125D1/en
Priority to SU894742104A priority patent/RU2074392C1/en
Priority to HU894650A priority patent/HU204132B/en
Priority to JP1232660A priority patent/JP2860118B2/en
Publication of DD281659A5 publication Critical patent/DD281659A5/en
Priority to US08/004,643 priority patent/US5480779A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
    • C12Q2337/10—Anilides
    • C12Q2337/12—Para-Nitroanilides p-NA

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

The invention relates to a method and an assay for determining the concentration of cyclosporin and its biologically active derivatives. The aim of the invention is to provide a straightforward method for determining the concentration of cyclosporins in biological materials. The measurement of the concentration of cyclosporins is important for monitoring a therapy in patients treated with medicaments which have cyclosporins as active drugs. The amount of cyclosporins in blood serum or plasma can be measured on the basis of the inhibition of peptidyl-prolyl cis-trans-isomerase by cyclosporins. The activity of this enzyme in the plasma can be determined by methods known per se. The method according to the invention quantifies with high accuracy the concentration of biologically active cyclosporins in the blood and is particularly suitable for monitoring therapy with these medicaments. Cyclosporin Derivatives biologically active Concentration determination Enzyme inhibition Substrates Proline-containing Peptide linkage Isomerising enzymes

Description

Hierzu 2 Seiten ZeichnungenFor this 2 pages drawings

Anwendungsgebiet der ErfindungField of application of the invention

Die Erfindung betrifft ein Verfahren zur Konzentrationsbestimmung von Cyclosporin und seinen biologisch wirksamen Derivaten. Es kann in medizinischen und biologischen Laboratorien genutzt werden. Die Bestimmung hat bei Patienten mit Organtransplantaten und Autoimmunerkrankungen eine diagnostische Bedeutung.The invention relates to a method for determining the concentration of cyclosporin and its biologically active derivatives. It can be used in medical and biological laboratories. The determination has a diagnostic significance in patients with organ transplants and autoimmune diseases.

Der Nachweis von Cyclosporinkonzentrationon ist wichtig zur Thenpieüberwachung bei Patienten, die mit Medikamenten therapiert werden, die die Substanzklasse der Cyclosporine als therapeutisch wichtiges Pharmakon besitzen.The detection of cyclosporin concentration is important for monitoring the aftermath in patients treated with drugs that possess the class of cyclosporine as a therapeutically important drug.

Die Konzentration von Cyclosporin und seinen biologisch wirksamen Derivaten ist in wäßrigen Systemen, vor allem in biologischen Flüssigkeiten, insbesondere im Blutserum oder Plasma, aber au.-.h in Liquor, Synovialflüssigkeit, Urin oder Suspensionen von Blutzellen bzw. Gewebehomogenaten zu bestimmen.The concentration of cyclosporin and its biologically active derivatives is to be determined in aqueous systems, especially in biological fluids, in particular in blood serum or plasma, but excluding cerebrospinal fluid, synovial fluid, urine or suspensions of blood cells or tissue homogenates.

Hauptsächlich wird die Bestimmung der Konzentration von Cyclosporin und seinen Derivaten bei der Einstellung und Überwachung des therapeutisch sinnvollen Konzentra*ionsbereiches in der Humanmedizin bei Patienten mit transplantierten Organen angewendet, die zur Vermeidung von Abstoßungsreaktionen Verbindungen wie Cyclosporin als Medikament erhalten.The determination of the concentration of cyclosporin and its derivatives is mainly used in the setting and monitoring of the therapeutically meaningful range of concentrations in human medicine in patients with transplanted organs who receive compounds such as cyclosporin as medicament in order to avoid rejection reactions.

Auf Grund der relativ geringen therapeutisch sinnvollen Breite der Medikamentation ist es zur Vermeidung vo.i immunologischen Reaktionen gegenüber dem Transplantat bei zu geringer Dosierung bzw. zur Vermeidung von toxischen Nebenwirkungen bei zu hoher Dosierung notwendig, die Konzentration des Pharmakons in Körperflüssigkeiten von Patienten zu messen. Die Notwendigkeit dieser Konzentrationsbestimmung ergibt S'ch weiterhin aus den interindiv;duellen Unterschieden der natürlichen Elimination dieser Pharmaka und aus der sich bei längerei Medikamentation ändernden intraindividuellen Eliminationsrate, die für eine differenzierte Dosierung des Medikamentes Voraussetzung ist.Due to the relatively small therapeutically meaningful width of the medication, it is necessary to avoid vo.i immunological reactions to the graft at too low a dose or to avoid toxic side effects at too high a dose to measure the concentration of the drug in body fluids of patients. The necessity of this determination of concentration continues to give S'ch the interindividual ; differences in the natural elimination of these drugs, and in the intra-individual elimination rate, which varies with prolonged medication, which is a prerequisite for a differentiated dosage of the drug.

Üblicherweise erfolgt die Konzentrationsbestimmung von Cyclosporin und seinen Derivaten mittels immunologischer (1) oder chromatografischer (2) Verfahren.Usually, the determination of the concentration of cyclosporin and its derivatives by means of immunological (1) or chromatographic (2) method.

Immunologische Verfahren stützen sich auf die Herstellung geeigneter Antikörper gegen das interessierende Cyclosporin. Mit Hilfe dieses Antikörpers gelingt es dann mitielj der aufwendigen Methode des Immunoassays die Konzentration des Antigens zu ermitteln.Immunological methods rely on the production of suitable antibodies against the cyclosporin of interest. With the help of this antibody, it then succeed mitielj the complex method of immunoassay to determine the concentration of the antigen.

Die Nachteile dieser Methode sind insbesondere die manuell teilweise sehr aufwendige Methodik und die mögliche Kreuzreaktion mit therapeutisch nicht wirksamen Verbindungen.The disadvantages of this method are in particular the manual, sometimes very complicated methodology and the possible cross-reaction with therapeutically inactive compounds.

Chromatografische Methoden nutzen die Trennung der nachzuweisenden Verbindung von die Detektion störenden Substanzen mittels physikalischer Effekte. Die Empfindlichkeit des Nachweises wird dadurch stark durch die Güte des Detektors als auch durch die Qualität der Trennung beeinflußt. Besondere Nachteile dieses Verfahrens sind die aufwendige Gerätetechnik und die relativ große Materialmenge, um beispielsweise den Cyclosporinspiegel im Blutplasma bestimmen zu können.Chromatographic methods use the separation of the compound to be detected from the detection of interfering substances by means of physical effects. The sensitivity of the detection is thereby strongly influenced by the quality of the detector as well as by the quality of the separation. Particular disadvantages of this method are the complex device technology and the relatively large amount of material in order to be able to determine, for example, the cyclosporin level in the blood plasma.

1 Keown. F.A.1 Stiller, C. R., Sinclair, N. R., et al. The clinical relevance of cyclosporine blood levels as measured by radioimunoassay. Transplant Proc. (1933), 15. 2438-2441.1 Keown. F.A.1 Stiller, C.R., Sinclair, N.R., et al. The clinical relevance of cyclosporine blood levels as measured by radioimunoassay. Transplant Proc. (1933), 152438-2441.

2 Kabra, P K., Wall. J.H., Dimson, P. Automated solid-phase extraction and liquid chromatography for assay of cyclosporine in whole blood. Clinical chemistry (1987), 33, 2272-2274.2 Kabra, P K., Wall. J.H., Dimson, P. Automated solid-phase extraction and liquid chromatography for the assay of cyclosporine in whole blood. Clinical chemistry (1987), 33, 2272-2274.

3 Fischer, G., Bang, H., Mech, C. Biomed. Biochem. Acta 43 1101-1111, (1984).3 Fischer, G., Bang, H., Mech, C. Biomed. Biochem. Acta 43 1101-1111, (1984).

Ziel der ErfindungObject of the invention

Ziel der Erfindung ist die Bereitstellung eines einfachen Verfahrens zur quant:tativen Bestimmung von biologisch wirksamen Cyclosporinen in humanen Materialien von Patienten, die mit dieser Verbindungsklasse therapiert werden.The aim of the invention is to provide a simple process for quant: tative determination of biologically active materials cyclosporins in human patients who are treated with this class of compounds.

-2- 281 Darlegung des W esens der Erfindung-2- 281 Presentation of the essence of the invention

Aufgabe der Erfindung ist ein einfaches Verfahren, welches auf Grund seiner hohen Empfindlichkeit mit geringen Probenmengen zur Bestimmung von Cyclosporinen, nach Zusatz einer bestimmten Mv,ge eines die Peptidbindung N-terminal zur Prolin itomerisierenden Enzyms, die Hemmung der enzymkatalysierten !comb '!sierung eines prolinhaltigen Substrates gemessen und daraus über eine Eichkurve die Konzentration an Cyclosporin ,n ermitteltThe object of the invention is a simple process which, owing to its high sensitivity with small sample quantities for the determination of cyclosporins, after addition of a certain Mv, a peptide binding N-terminal to the proline itomerizing enzyme, the inhibition of the enzyme-catalyzed! Proline-containing substrate measured and determined from a calibration curve, the concentration of cyclosporin, n

Als Cyclosporine gelten auch die Verbindungen, die sich chemisch von der Substanzklasse der Cyclosporine im Sinne von Derivaten ableiten und biologisch wie Cyclosporin A wirksam sind. Zur Ausführung des erfindungsgemäßen Verfahrens wird katalytisch aktive und für Cyclosporine empfindliche Peptidyl-Prolpyl cis-trans Isomerase (PPIase) eingesetzt.Cyclosporins are also the compounds which are chemically derived from the substance class of cyclosporins in the sense of derivatives and are biologically active such as cyclosporin A. Catalytically active and cyclosporin sensitive peptidyl-prolyl cis-trans isomerase (PPIase) is used to carry out the process according to the invention.

Geeignete Substrate sind Verbindungen des Typs Xaa-Pro-Yaa, mit deren Hilfe die Reaktion der PPIase nachgewiesen werden kann. Vorzugsweise werden dabei solche Substrate genutzt, in denen Yaa für eine chromogene Gruppierung wie zum Beispiel Phenylalanyl-4-nitroanilid und in denen Xaa für Aminoacylreste oder Peptidylreste, wie zum Beispiel Suc-Ala-Ala steht. Ein weiteres Kennzeichen geeigneter Substrate ist die Substrateigenschaft für das zum Nachweis der PPIase notwendige Indikatorsystem mittels einer geeigneten Protease, wie z. B. Chymotrypsin, Thrombin oder humane Leukozyten Elastase unter Millieubedingungen (pH-Wert, PufferzusammensetzLng, Temperatur und lonenstärke) die für diese proteolytischen Systeme bekannt sind.Suitable substrates are compounds of the type Xaa-Pro-Yaa, with the aid of which the reaction of the PPIase can be detected. Preferably, such substrates are used in which Yaa is a chromogenic moiety such as phenylalanyl-4-nitroanilide and in which Xaa is aminoacyl or peptidyl, such as Suc-Ala-Ala. Another characteristic of suitable substrates is the substrate property for the necessary for the detection of PPIase indicator system by means of a suitable protease, such as. Chymotrypsin, thrombin or human leukocyte elastase under millie conditions (pH, buffer composition, temperature and ionic strength) known for these proteolytic systems.

Eine für Cyclosporine empfindliche PPIase mit einem Molekulargewicht von ~ 17,OkD kann beispielsweise aus Schwdinenieren (3) nach bekannten Vorschriften isoliert werden. Die Cyclosporinkonzentration kann aus dem Vergleich der Aktivitätsmossung von Messung ohne Cyclosporin (Vergleichswert) und Messung der PPIase-Aktivität mit Cyclosporinzusatz ermittelt werden.A cyclosporin-sensitive PPIase with a molecular weight of ~ 17, OkD, for example, can be isolated from Schwdinenieren (3) according to known procedures. The cyclosporin concentration can be determined from the comparison of the activity kill of measurement without cyclosporin (reference value) and measurement of PPIase activity with cyclosporin addition.

Die Durchführung der Aktivitätsbestimmung der PPIase einschließlich der Konzentrationsbestimmung von Cyclosporinen soll im folgenden an zwei Ausführungsbeispielen näher erläutert werden.The implementation of the activity determination of the PPIase including the determination of the concentration of cyclosporins will be explained in more detail below with reference to two exemplary embodiments.

Ausführungsbeispieleembodiments

Berechnung: üblicherweise wird die Konzentration eines Hemmstoffes aus der prozentualen Inhibierung einer bestimmten zugesetzten Enzymmenge anhand einer Eichkurve gemessen. Eine solche typische Eichkurve ist in Fig. 1 für Ausführungsbeispiel 1 und in Fig. 2 für Ausführungsbeispiel 2 dargestellt.Calculation: Usually, the concentration of an inhibitor is measured from the percentage inhibition of a certain amount of enzyme added using a calibration curve. Such a typical calibration curve is shown in FIG. 1 for exemplary embodiment 1 and in FIG. 2 for exemplary embodiment 2.

Beispiel 1:Example 1:

Puffer: 0,035m HEPES pH 7,8Buffer: 0.035m HEPES pH 7.8

Chymotrypsin: alpha-Chymotrypsin aus Rindarpankreas (3fach kristallisiert, Boerhringer/BRD) 24mg in 4,2ml Puffer Substrat: Glt-Ala-Ala-Pro-Phe-NHNp 14mg in 10m! DimethylsuifoxidChymotrypsin: alpha-chymotrypsin from bovine pancreas (crystallized 3 times, Boerhringer / FRG) 24mg in 4.2ml buffer Substrate: Glt-Ala-Ala-Pro-Phe-NHNp 14mg in 10m! Dimethylsuifoxid

PPIase: aus Schweinenieren isoliert spez. Aktivität = 500pMol/min x mgPPIase: isolated from pork kidneys spec. Activity = 500 pmol / min x mg

Meßgerät: registrierendes Spektrallinienphotome'.erMeasuring instrument: recording spectral line photometer

Meßwellenlänge: 410nmMeasuring wavelength: 410nm

Reaktionstemperatur: H0CReaction temperature: H 0 C

Probe: humaner Plasri.apool, dem 5,8,18, 30, 36,40 ng/ml Cydosporin-A zugesetzt warenSample: human Plasri.apool to which 5, 8, 18, 30, 36.40 ng / ml of Cydosporin-A were added

Meßansatzthe mixture for measurement Leeransatzempty approach Puffer:Buffer: 500 μΙ500 μΙ 540 μΙ540 μΙ Chymotrypsinlösung:chymotrypsin: 100 μΙ100 μΙ 100 μΙ100 μΙ PPIase-Lösung:PPIase solution: 20 μΙ20 μΙ -- Probe:Sample: 20 μΙ20 μΙ -- Substratlösung:Substrate Solution: 50 μΙ .50 μΙ. 50 μΙ50 μΙ

Aktivitätsbestimmung: a) Berechnung der Geschwindigkeitskonstante von Meßansatz (k-Meßanr.atz) undDetermination of activity: a) Calculation of the rate constant of the measuring batch (k-gauge) and

Leeransatz (k-Leeransatz) der Kinetik nach einem Zeitgesetz erster Ordnung d) Berechnung der gemessenen Aktivität der Probe nach:Empty approach (k-empty approach) of the kinetics according to a first order time law d) Calculation of the measured activity of the sample according to:

k-Meßansatzk-mixture for measurement

Aktivität -· 1Activity - · 1

k-Leeransatzk-Space Approach

Cyclosporinkonzentration: Ernvttelung mit Hilfe einer Eichkurve wie in Abbildung 1 angegebenCyclosporin concentration: Recovery by a calibration curve as shown in Figure 1

Beispiel 2:Example 2:

Puffer: 0,035m HEPES pH 7,8Buffer: 0.035m HEPES pH 7.8

Chymotrypsin: alpha-Chymotrypsin aus Rinderpankreas (3fach kristallisiert, Boerhringer (BRD)) 35mg in 4,2ml Puffer Substrat. Glt-Ala-Pro-Phe-NHNp 14mg in 10ml DimethylsuifoxidChymotrypsin: alpha-chymotrypsin from bovine pancreas (3x crystallized, Boerhringer (FRG)) 35mg in 4.2ml buffer substrate. Glt-Ala-Pro-Phe-NHNp 14mg in 10ml dimethylsulfoxide

PPiase: aus Schweinenieren isoliert spez Aktivität = 500μΜοΙ/πιίη χ mgPPiase: isolated from pig kidney spec activity = 500μΜοΙ / πιίη χ mg

Meßgerät: registrierendes SpektrallinienphotometerMeasuring instrument: registering spectral line photometer

Meßwelleniänge: 410nmMeasuring wave length: 410nm

Reaktionstemperatur: 5CCReaction temperature : 5 C C

Probe: humaner Plasmapool, dem 5. 8,15, 30, 36,40ng/m! Cyclosporin-A zugesetzt waren Meßansatz, Leeransatz und Aktivitätsbestimmung wie in Beispiel 1Sample: human plasma pool, the 5. 8,15, 30, 36,40ng / m! Cyclosporin-A added were measurement batch, blank batch and activity determination as in Example 1

Cyclosporinkonzen'ration: Ermittelung mit Hilfe einer Eichkuive entsprechend Abb.2Cyclosporin concentration: Determination with the aid of a calibration cuvette according to Fig.2

Claims (4)

1. Verfahren zu * Konzentrationsbestimmung von Cyclosporinen, dadurch gekennzeichnet, daß nach Zusatz einer bestimmten Menge eines die Peptidbildung N-terminal zu Prolin isomerisierenden Enzyms die Hemmung der enzymkatalysierten Isomerisierung eines prolinhaltigen Substrates gemessen und daraus über eine Eichkurve die Konzentration an Cyclosporinen ermittelt wird.1. A method for * concentration determination of cyclosporins, characterized in that after addition of a certain amount of peptide formation N-terminal to proline isomerizing enzyme, the inhibition of the enzyme-catalyzed isomerization of a proline-containing substrate measured and determined from a calibration curve, the concentration of cyclosporins. 2. Vorfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß als isomerisierendes Enzym eine Peptidyl-Propyl cis-trans Isomerase eingesetzt wird.2. An ancestor according to claim 1, characterized in that a peptidyl-propyl cis-trans isomerase is used as the isomerizing enzyme. 3. Verfahren gemäß Anspruch 1 und 2, dadurch gekennzeichnet, daß als Substrate Verbindungen des Typs Xaa-Pro-Yaa eingesetzt werden, in denen Yaa für eine chromogene Gruppierung, ζ. Β. Phenylalanyl-4-nitroanilid oder Alanyl-4-nitroanilid und Xaa für Aminoacyl- oder Peptidylreste, z. B. Suc-Ala-Ala, steht.3. The method according to claim 1 and 2, characterized in that are used as substrates compounds of the type Xaa-Pro-Yaa, in which Yaa for a chromogenic grouping, ζ. Β. Phenylalanyl-4-nitroanilide or alanyl-4-nitroanilide and Xaa for aminoacyl or peptidyl radicals, e.g. B. Suc-Ala-Ala. 4. Verfahren gemäß Anspruch 1 bis 3, dadurch gekennzeichnet, daß die Isomerasesubstrate auch Substrateigenschaft für das zum Nachweis der Hemmung der Isomerase notwendige Indikatorsystem mit einer Protease, ζ. Β. Chymotrypsin-, Thrombin oder humane Leukozyten-Elastase, aufweist.4. The method according to claim 1 to 3, characterized in that the Isomerasesubstrate also substrate property for the necessary for the detection of inhibition of the isomerase indicator system with a protease, ζ. Β. Chymotrypsin, thrombin or human leukocyte elastase.
DD88319577A 1988-09-07 1988-09-07 METHOD FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINES DD281659A5 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
DD88319577A DD281659A5 (en) 1988-09-07 1988-09-07 METHOD FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINES
EP89115759A EP0360029B1 (en) 1988-09-07 1989-08-26 Method and assay to determine the concentration of cyclosporins
ES89115759T ES2058425T3 (en) 1988-09-07 1989-08-26 PROCEDURE AND TEST FOR THE DETERMINATION OF CONCENTRATION OF CYCLOSPORINS.
AT89115759T ATE92536T1 (en) 1988-09-07 1989-08-26 METHOD AND ASSAY FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINS.
DE8989115759T DE58905125D1 (en) 1988-09-07 1989-08-26 METHOD AND ASSAY FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINES.
SU894742104A RU2074392C1 (en) 1988-09-07 1989-09-06 Method of blood cyclosporine a assay
HU894650A HU204132B (en) 1988-09-07 1989-09-06 Method and reagent set for detecting concentration of the cyclosporines
JP1232660A JP2860118B2 (en) 1988-09-07 1989-09-07 Methods and kits for measuring cyclosporin concentration
US08/004,643 US5480779A (en) 1988-09-07 1993-01-12 Cyclosporine assay

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
DD88319577A DD281659A5 (en) 1988-09-07 1988-09-07 METHOD FOR DETERMINING THE CONCENTRATION OF CYCLOSPORINES

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JP (1) JP2860118B2 (en)
AT (1) ATE92536T1 (en)
DD (1) DD281659A5 (en)
DE (1) DE58905125D1 (en)
ES (1) ES2058425T3 (en)
HU (1) HU204132B (en)
RU (1) RU2074392C1 (en)

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DE58905125D1 (en) 1993-09-09
ES2058425T3 (en) 1994-11-01
RU2074392C1 (en) 1997-02-27
HU204132B (en) 1991-11-28
JP2860118B2 (en) 1999-02-24
ATE92536T1 (en) 1993-08-15
HUT54809A (en) 1991-03-28
EP0360029A1 (en) 1990-03-28
EP0360029B1 (en) 1993-08-04

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