Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2001-01-09
2003-10-21
Ceperley, Mary E. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S007930, C435S007940, C435S007950, C530S403000, C530S404000, C530S388900
Reexamination Certificate
active
06635745
ABSTRACT:
This invention relates to monoclonal antibodies to rapamycin and rapamycin derivatives, which are useful, e.g., in assay kits for monitoring blood levels of drug.
Rapamycin is a macrolide antibiotic produced by
Streptomyces hygroscopicus
, which has been found to be pharmaceutically useful in a variety of applications, particularly as an immunosuppressant, e.g., for use in the treatment and prevention of organ transplant rejection and autoimmune diseases. Rapamycin, however, does exhibit side effects at higher dosages, and it has a somewhat variable bioavailability. Monitoring blood levels of rapamycin in patients being treated with rapamycin is thus very desirable in order to be able to regulate the dosage so as to maintain the minimum level sufficient for pharmacologic activity and to avoid any undue risk of side effects. The lack of a sensitive and reliable assay which can be performed quickly and easily in a clinical setting has been a major obstacle to the development of rapamycin as a pharmaceutical.
Previous efforts to develop assay kits for clinical monitoring of rapamycin have not been particularly successful. EP 041795, for example, describes a microbiological assay in which rapamycin concentration is measured as a function of antifungal activity. WO 92/02946 provides an assay system which measures rapamycin levels indirectly by measuring competition for binding to macrophilin. Both of these assays are cumbersome and not particularly sensitive. Even more importantly, both of these assays may have considerable variation under slightly different test conditions, making comparisons of test results from different hospitals difficult.
There have been no previous reports of monoclonal antibodies which recognize rapamycin. There are inherent difficulties in making monoclonal antibodies to rapamycin because rapamycin is not immunogenic and is itself extremely immunosuppressive. Moreover, as the metabolites of rapamycin have not been well characterized in the literature, it is difficult to identify a monoclonal antibody capable of differentiating between rapamycin and its metabolites.
The present invention provides monoclonal antibodies which are highly sensitive to rapamycin. The antibodies of the invention are produced in response to inoculation with a novel immunogenic conjugate comprising a novel derivative of rapamycin linked to an immunogenic protein. Assay kits using these antibodies are well suited for use in a clinical setting and provide far more accurate and reproducible results than was previously possible. The antibodies are also useful in the purification and isolation of rapamycin.
Providing assay systems for immunosuppressive derivatives of rapamycin present similar challenges. Of particular interest are 40-O-derivatives of rapamycin, i.e., rapamycins which are O-substituted at the hydroxy on the cyclohexyl ring (position 40), e.g., as described in U.S. Pat. No. 5,258,389 and PCT/EP 93/02604 (O-aryl and O-alkyl rapamycins) (both incorporated herein by reference); especially 40-O-alkylated rapamycins where the 40-O-substituent is alkyl or substituted alkyl; e.g., hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, wherein “alk-” or “alkyl” refers to C
1-6
alkyl, branched or linear, preferably C
1-3
alkyl, in which the carbon chain may be optionally interrupted by an ether (—O—) linkage; most especially 40-O-(2-hydroxyethyl)-rapamycin, 40-O-(3-hydroxypropyl)-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and 40-O-(2-acetaminoethyl)-rapamycin). Thus a further object of the invention is to provide monoclonal antibodies to such 40-O-derivatives. Such antibodies are useful in diagnostic assays and also in the purification and production of the derivatives.
The novel activated derivatives of rapamycin used to make the novel immunogenic conjugates of the invention are rapamycins which are linked through one of the hydroxy groups on the rapamycin, preferably the hydroxy group located on the cyclohexyl portion of the rapamycin (position 40) or the hydroxy at position 28, to an activated coupling group, i.e., a group capable of direct reaction with a protein to form a covalent linkage without the requirement for the use of a coupling agent (e.g., carbodiimide reagents) to enable, effect, or promote the reaction with the protein. Preferably, the activated coupling group has an activated ester or carboxy group, i.e., of formula —CO—O—X where X is a carboxy activating group such as o- or p-nitrophenyl, 1-benztriazole, pentafluorophenyl, or (especially) N-succinimido. Other suitable activated coupling groups are, for example, i) activated dithio groups, e.g., of formula —S—S—Z wherein Z is a dithio activating group such as 2-pyridyl, which may be linked to the rapamycin; or ii) epoxy groups, e.g., epoxy methyl. The activated coupling group may be linked to the rapamycin by means of an ester, ether, amide, thio or other suitable linkage, but ester linkage is preferred. Most preferably, the activated coupling group contains a bis-ester moiety, e.g., succinyl, having an ester linkage to the rapamycin at one end and the activated ester or activated carboxy group at the other.
The preferred rapamycin derivatives of the invention are those of formula III below which are produced according to Reaction I:
wherein formula I is rapamycin, which is a) reacted with an acylating agent, e.g., a cyclic anhydride or a dicarboxylic acid (optionally in hemi-O-protected form), under suitable conditions and deprotection if necessary to yield the rapamycin of formula II, wherein Y is a spacer moiety, preferably a lower alkylene, e.g., C
2-6
alkylene, most preferably ethylene. This rapamycin of formula II is then b) activated by reaction with a carboxy activating group, e.g. of formula HO—X where X is as defined above, to yield the activated rapamycin of formula III.
A preferred activated derivative of rapamycin is the succinimido derivative of formula III below, prepared, e.g., according to Reaction II:
wherein formula I is rapamycin, which is a) 0-acylated using succinic anhydride in the presence of DMAP and pyridine to form the rapamycin hemisuccinate of formula II′ (40-O-(3-Carboxy)propanoyl-rapamycin); which is then b) activated with N-Hydroxy succinimide in the presence of EDC, Et
3
N, and CH
2
Cl
2
to form the 40-O-succinimidooxysuccinyl rapamycin of formula III′, e.g., as described more fully in example 1 below. Monoclonal antibodies produced using a hapten such as this which is linked through the 40-position will ordinarily be cross reactive between rapamycin and a 40-O-derivative of rapamycin, such as described above. Such monoclonal antibodies can be selected as described below for compounds which recognize a particular region of the rapamycin or 40-O-derivative of the rapamycin, e.g., in the binder domain or effector domain, as described below.
It is in some cases desirable to have monoclonal antibodies capable of fine sensitivity to modifications in the cyclohexyl region, e.g., for distinguishing between rapamycin and the 40-O rapamycin derivatives, or for identifying metabolites in the cyclohexyl region. In such a case, the hapten is preferably linked through the 28-O position rather then the 40-O position. For example, the rapamycin derivative of formula A:
wherein R is an O-protecting group, or a substituent as described above, e.g., hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, optionally in protected form, is reacted according to Reaction I, deprotecting if necessary, to give the analogous 28-O activated hapten, for example a compound of formula B:
wherein R1 is H, or an O-substituent as described above, e.g., hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, Y is a linker moiety as defined above, and X is a carboxy activating group as defined above. In preparing this hapten, where R is an O-protecting group or an O-protected substituent, the acylating agent may optionally be, e.g., a dicarboxylic acid in hemi-O-protected form, so that following acylation, both O-protecting groups may be remove
Quesniaux Ryffel Valerie
Sedrani Richard
Ceperley Mary E.
Furman Diane E.
Lopez Gabriel
Novartis AG
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