Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-09
2002-01-15
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007800, C435S007920, C514S011400, C514S015800, C530S402000
Reexamination Certificate
active
06338946
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of accurately assaying calcineurin-inhibiting immunosuppressants, such as FK506 and cyclosporin A, which method can be used in the field of medicine.
BACKGROUND ART
It is well known that a compound represented by the structural formula and chemical name shown below and also designated as FK506 or FR-900506 has potent immunosuppressive activity and can be used as a prophylactic or therapeutic agent for organ transplant rejection or autoimmune diseases (for example, EP-0184162-A2).
Chemical name: 17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0
4,9
]octacos-18-ene-2,3,10,16-tetraone
As a result of investigations of the mechanism of immunosuppressive activity of FK506, said activity is supposed to be displayed as follows. FK506 binds to FKBP-12, which is a cytoplasmic FK506-binding protein, followed by association with calcineurin, together with calmodulin and calcium ion, to form a complex (FK506:FKBP-12: calcineurin=1:1:1), whereby the phosphatase activity of calcineurin is inhibited. This phosphatase inhibition leads to suppression of the activation of nuclear factor of activated T cells (NFAT) and to inhibition of IL2 production, whereby immunosuppression is caused.
Cyclosporin A, which has similar immunosuppressive activity, is also supposed to show its immunosuppressive activity by forming a similar complex [cf. for example, Biochemical and Biophysical Research Communications, 192 (3), 1388-1394 (1993) and Angew. Chem., Int. Ed. Engl., 31 (1992) 384-400].
The utility of FK506 as an immunosuppressant has been amply studied and, in Japan, FK506 is already on the market as a rejection reaction suppressant particularly in liver transplantation.
However, since its activity is very potent, the determination of an optimum dose is an important issue. It is very important to administer it at a dose with displaying its effective immunosuppressive activity and without producing adverse effects or the like.
For that purpose, several assay methods have been proposed, including an enzyme immunoassay method with an antibody recognizing the antigenic determinant of FKS506 (e.g. EP-0293892-A2), a method in which the above-mentioned enzyme immunoassay is combined with HPLC, and a radioreceptor method utilizing an FK506-binding protein (FKBP-12) [cf. for example, Clin. Chem., 38/7, 1307-1310 (1992)].
On the other hand, studies on the mechanism of its metabolism have revealed that while FK506 undergoes metabolism in the living body, some of its metabolites still retain immunosuppressive activity, and others are capable of binding to a monoclonal antibody to FK506 with only weak immunosuppressive activity [e.g. Drug Metabolism and Disposition, 21 (6), 971-977 (1993)].
Furthermore, the existence of a substance (e.g. 506BD) capable of binding to FK506 binding proteins (FKBPs) but having no immunosuppressive activity has been revealed [e.g. Angew. Chem., Int. Ed. Engl., 31 (1992) 384-400].
Therefore, the previous assay methods using, as an index, either the binding of FK506 to an antibody recognizing the antigenic determinant of FK506, or the binding of an FKBP to FK506, can hardly be said to be capable of accurately measuring the actual state of immunosuppression. The development of an assay method capable of accurately measuring the total concentration of active drug substances including metabolites actually having immunosuppressive activity has been awaited.
DISCLOSURE OF THE INVENTION
Giving their attention to the fact that an immunosuppressant, such as FK506 or cyclosporin A, binds to an immunophilin (protein capable of binding to an immunosuppressant; e.g. FKBP-12 or cyclophilin) and then form a complex with calcineurin, calmodulin and calcium ion and thereby inhibits the activity of calcineurin, the inventors of the present invention succeeded in establishing a method of assaying immunosuppressants making use of the complex forming ability of said substances.
The present invention thus provides a method of assaying immunosuppressants having calcineurin-inhibiting activity, which comprises assaying a complex comprising (1) an immunophilin, (2) calcineurin, (3) calmodulin, (4) calcium ion and (5) an immunosuppressant having calcineurin-inhibiting activity.
In the following, the particular terms used herein within the scope of the present invention are defined and explained in detail and preferred examples are given.
The “immunophilin” means a cytoplasmic receptor protein to which an immunosuppressant binds and includes, for example, FKBP-12 which is an FK506 binding protein having a molecular weight of about 12K and peptidyl prolyl cis-trans isomerase (PPIase) activity, and cyclophilin which is an intracellular receptor for cyclosporin A and has similar PPIase activity and a molecular weight of about 17K. Preferred are FKBP-12 and cyclophilin produced by mammals such as cattle or humans.
They are already known and can be obtained in the same manner as described in J. Am. Chem. Soc., 113, 1409-1411 (1991), Proc. Natl. Acad. Sci. USA, 88, 6229-6233 (1991), Nature, 346, 671-674 (1991), WO 92/01052, WO 91/17439, Nature, 337, 473-475, 476-478 (1989), or Japanese Kokai Tokkyo Koho Hei 02-209897, for instance.
“Calcineurin” is known as a calcium ion- and calmodulin-dependent serine-threonine phosphatase, and calcineurin obtained from mammals such as rats, cattle or humans can be used. Rat or bovine calcineurin, for example, is known to be a heterodimer composed of A and B subunits and it is further known that the A subunit includes two isoforms, A&agr; and A&bgr;. Rat calcineurin can be isolated and purified, for example, from the rat brain [cf. for example, J. Neurochem., 58, 1643-1651 (1992)]. Bovine calcineurin can be obtained in the same manner as described in Adv. Enzymol., 61, 149-200 (1989) or Methods Enzymol., 102, 244-256 (1983). It is also commercially available from Upstate Biotechnology Co. Ltd or Sigma Co. Ltd under the product name “protein phosphatase 2B”, for instance, and such product may also be used.
“Calmodulin” is a substance known as a calcium binding protein and is known to activate various enzymes including the above-mentioned calcineurin in the presence of the calcium ion. Calmodulin derived from mammals such as cattle or humans can be used. Bovine calmodulin, for instance, can be prepared and obtained as described in J. Biochem., 87, 1313-1320 (1980), and a commercial product available from Upstate Biotechnology Co. Ltd or Sigma Co. Ltd can also be used.
The “immunosuppressant having calcineurin-inhibiting activity” to be assayed in accordance with the present invention means a compound which inhibits the phosphatase activity of calcineurin by forming a complex with an immunophilin, calcineurin, calmodulin and calcium ion, and has immunosuppressive activity. Preferred examples are compounds of the following formula:
(wherein R
1
is hydroxy or protected hydroxy, R
2
is hydrogen, hydroxy or protected hydroxy, R
3
is methyl, ethyl, propyl or allyl, R
4
is hydroxy or methoxy, R
5
is hydrogen, or R
4
and R
5
together form oxo, n is an integer of 1 or 2, and the symbol comprising a solid line and a dotted line means a single bond or a double bond, provided that when R
4
is hydroxy and R
5
is hydrogen or when R
4
and R
5
together form oxo, R
2
is not protected hydroxy.)
The term “lower” as used in defining the symbols used in the above general formula [I], and in the subsequent description means, unless otherwise indicated, that the number of carbon atoms is 1 to 6.
Suitable protective groups for use in the “protected hydroxy” include the following: 1-(lower alkylthio) (lower) alkyl groups such as lower alkylthiomethyl (e.g. methylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, hexylthiomethyl, etc.), and the like, more preferably C
1
-C
4
alkylthiomethyl, most pref
Kobayashi Masakazu
Tamura Kouichi
Fujisawa Pharmaceutical Co. Ltd.
Riley Jezia
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