WO1994025072A1 - Conjugues de rapamycine et anticorps contre la rapamycine - Google Patents

Conjugues de rapamycine et anticorps contre la rapamycine Download PDF

Info

Publication number
WO1994025072A1
WO1994025072A1 PCT/US1994/004463 US9404463W WO9425072A1 WO 1994025072 A1 WO1994025072 A1 WO 1994025072A1 US 9404463 W US9404463 W US 9404463W WO 9425072 A1 WO9425072 A1 WO 9425072A1
Authority
WO
WIPO (PCT)
Prior art keywords
rapamycin
carbon atoms
conjugate
derivative
hydrogen
Prior art date
Application number
PCT/US1994/004463
Other languages
English (en)
Inventor
Katherine Lu Molnar-Kimber
Timothy Donald Ocain
Craig Eugene Caufield
Thomas Joseph Caggiano
Amedeo Arturo Failli
Original Assignee
American Home Products Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by American Home Products Corporation filed Critical American Home Products Corporation
Priority to AU67119/94A priority Critical patent/AU6711994A/en
Publication of WO1994025072A1 publication Critical patent/WO1994025072A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • This invention relates to derivatives of rapamycin which are useful as immunogenic molecules for the generation of antibodies specific for rapamycin or ring opened derivatives thereof, for measuring levels of rapamycin or derivatives thereof; for isolating rapamycin binding proteins; and detecting antibodies specific for rapamycin or derivatives thereof.
  • Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus. which was found to have antifungal activity, particularly against Candida albicans. both in vitro and in vivo [C. Vezina et al., J. Antibiot. 28, 721
  • Rapamycin alone (U.S. Patent 4,885,171) or in combination with picibanil (U.S. Patent 4,401,653) has been shown to have antitumor activity.
  • R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in the experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the formation of IgE-like antibodies.
  • Rapamycin has also been shown to be useful in preventing or treating systemic lupus erythematosus [U.S. Patent 5,078,999], pulmonary inflammation [U.S. Patent 5,080,899], insulin dependent diabetes mellitus [Fifth Int. Conf. Inflamm. Res. Assoc. 121 (Abstract), (1990)], adult T-cell leukemia/lymphoma [European Patent Application 525,960 Al], and smooth muscle cell proliferation and intimal thickening following vascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197 (1992)].
  • Patent 5,130, 307 discloses aminoesters of rapamycin.
  • U.S. Patent 5,177,203 discloses sulfonates and sulfamates of rapamycin.
  • U.S. Patent 5,194,447 discloses sulfonylcarbamates of rapamycin.
  • PCT Publication WO 92/05179 discloses carboxylic acid esters of rapamycin.
  • This invention provides a rapamycin conjugate of formula I, having the structure
  • R 1 and R 2 are each, independently, hydrogen or -(R 3 -L-R ) a - ;
  • L is a linking group
  • R 3 is selected from the group consisting of carbonyl, -S(O)- , -S(O)2 , -P(O)2- ,
  • the linking group, L is any moiety that contains the group R 3 on one end and
  • linking group on other end, therefore enabling the linking group to be connected to the 42- and/or 31-hydroxyl groups of rapamycin on one end and connected to another linking group or the immunogenic carrier material, detector material, or matrix on the other end.
  • each R 3 , R 4 or L group can be the same or different.
  • the first L group is designated as L 1
  • the second L group designated as L 2 and so on.
  • the rapamycin conjugates of the present invention may be prepared in such ways as to encompass a wide range of linking groups (L) and terminal functional groups R 4 .
  • L may be linear or branched alkylenes comprising from 1 to as many as 15, more usually 10 or less, and normally less than 6 carbon atoms (i.e., methylene, ethylene, n-propylene, iso-propylene, n-butylene, and so forth).
  • alkylenes can contain other substituent groups such as cyano, amino (including substituted amino, e.g., mono- or di- alkylamino), acylamino (e.g., alkanoylamino or aroylamino), halogen, thiol, hydroxyl, carbonyl groups, carboxyl (including substituted carboxyls such as esters (e.g., alkyl or aralkyl esters), amides, and substituted amides (e.g., mono- or di- alkylamides).
  • substituent groups such as cyano, amino (including substituted amino, e.g., mono- or di- alkylamino), acylamino (e.g., alkanoylamino or aroylamino), halogen, thiol, hydroxyl, carbonyl groups, carboxyl (including substituted carboxyls such as esters (e.g., alkyl or aralkyl est
  • the linking group L can also contain or consist of substituted or unsubstituted aryl (including heteroaryl, e.g., where the heteroatom(s) is (are) selected from one (or more) of oxygen, nitrogen and sulphur) or aralkyl (e.g., phenylene, phenethylene, and so forth). Additionally, such linkages can contain one or more heteroatoms selected from nitrogen, sulfur and oxygen in the form of ether, ester, amido, amino, thio ether, amidino, sulfone, or sulfoxide. Also, such linkages can include unsaturated groupings such as olefinic or acetylenic bonds, disulfide, imino, or oximino groups.
  • L will be a chain, usually aliphatic comprising between 1 and about 20 atoms, more usually between 1 and 10, excluding hydrogen, of which between 0 and 5 are heteroatoms preferrably selected from nitrogen, oxygen, and sulfur. Therefore, the choice of linking group L is not critical to the present invention and may be selected by one of ordinary skill taking normal precautions to assure that stable compounds are produced.
  • aryl as a group or part of a group such as arylalkyl includes any carbocyclic aromatic group of 6 to 10 carbon atoms or heteroaromatic group of 5 to 10 ring atoms of which up to 3 are heteroatoms selected from the group consisting of oxygen, nitrogen and sulphur.
  • substituents are one or more, the same or different, of the following: alkyl of 1-6 carbon atoms, arylalkyl in which the alkyl portion contains 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, mono- or di- alkylamino of 1-6 carbon atoms per alkyl group, aminocarbonyl, alkylthio of 1-6 carbon atoms, -SO3H and -CO2H.
  • the aryl group may be mono- or bi-cyclic.
  • alkyl as a group or part of a group, e.g., arylalkyl, alkoxy or alkanoyl (alkylcarbonyl), are straight or branched chains of 1-6 carbon atoms, preferably 1-4 carbon atoms, e.g., methyl, ethyl, propyl, isopropyl and n-butyl.
  • a preferred embodiment of this invention provides a conjugate of formula II, having the structure
  • R 1 and R 2 are each, independently, hydrogen or -R 3 -L-R 4 - ;
  • A is -CH2- or -NR 9 - ;
  • B is -O- , -NR 9 - , -S- , -S(O)- , or -S(O) 2 - ;
  • R 3 is selected from the group consisting of carbonyl, -S(O)- , -S(O)2 , -P(O)2- ,
  • R 5 , R 6 , R 7 , and R 8 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, halo, hydroxy, trifluoromethyl, arylalkyl in which the alkyl portion contains 1-6 carbon atoms, aminoalkyl of 1-6 carbon atoms, hydroxyalkyl of 1-4 carbon atoms, alkoxy of 1-6 carbon atoms, carbalkoxy of 2-7 carbon atoms, cyano, amino, -CO2H, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or -CO 2 H; R 9 is hydrogen, alkyl of
  • a second preferred embodiment of this invention provides a conjugate of formula III, having the structure
  • R 1 and R 2 are each, independently, hydrogen or -(R 3 -L 1 -R 4 ) f -(R 10 -L 2 -R 11 ) g -Carrier,
  • L 1 is -(CH 2 )h-CHR 12 -(CH 2 )j- ;
  • L 2 is -(CH 2 ) k -D-(CH 2 ) m -E- ;
  • E is -CH 2 - or ⁇ C-
  • R 3 and R 10 are each, independently, selected from the group consisting of carbonyl
  • R 4 and R 11 are each, independently, selected from the group consisting of carbonyl,
  • R 12 is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl in which the alkyl portion contains 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -(CH2) n C02R 13 , -(CH2) p NR 14 R 1 - 5 , carbamylalkyl of 2-3 carbon atoms, aminoalkyl of 1-4 carbon atoms, hydroxyalkyl of 1-4 carbon atoms, guanylalkyl of 2-4 carbon atoms, mercaptoalkyl of 1-4 carbon atoms, alkylthioalkyl of 2-6 carbon atoms, indolylmethyl, hydroxyphenylmethyl, imidazoylmethyl, halo, trifluoromethyl, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of
  • R 14 , and R 15 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, or arylalkyl of 7-10 carbon atoms;
  • R 13 is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl in which the alkyl portion has 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or -CO2H;
  • f 0-3;
  • This invention also provides a conjugate of formula IV, having the structure
  • the immunogenic carrier material can be selected from any of those conventionally known.
  • the carrier will be a protein or polypeptide, although other materials such as carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids and the like of sufficient size and immunogenicity can likewise be used.
  • immunogenic proteins and polypeptides will have molecular weights between 5,000 and 10,000,000, preferably greater than 15,000 and more usually greater than 40,000. Generally, proteins taken from one animal species will be immunogenic when introduced into the blood stream of another species.
  • Particularly useful proteins are those such as albumins, globulins, enzymes, hemocyanins, glutelins or proteins having significant non-proteinaceous constituents, e.g., glycoproteins, and the like.
  • Conventional immunogenic carrier materials and techniques for coupling haptens thereto may be had to the following: Parker, Radioimmunoassay of Biologically Active Compounds, Prentice-Hall (Englewood Cliffs, N.J., USA, 1976), Butler, J. Immunol. Meth. 7:1- 24 (1975) and Pharmacol. Rev. 29(2):103-163 (1978); Weinryb and Shroff, Drug Metab. Rev.
  • Preferred immunogenic carrier materials for use in the present invention are ovalbumin and keyhole limpet hemocyanin. Particularly preferred for use in the present invention is ovalbumin.
  • the detector carrier material can be a rapamycin-linking moiety conjugated to an enzyme such as horseradish peroxidase, alkaline phosphatase, luciferase, a fluorescent moiety such as fluorescein, Texas Red, or rhodamine, a chemiluminescent moiety, and the like.
  • the solid matrix carrier material can be resin beads, an ELIS A plate, glass beads as commonly used in a radioimmunoassay, plastic beads, solid matrix material typically used in a dipstick-type assay.
  • rapamycin is conjugated to a solid matrix
  • the resulting conjugate can be used in a dipstick assay, as described in this disclosure, for the affinity purification of antibodies, or for isolating rapamycin binding proteins.
  • z represents the number of rapamycin conjugated to the carrier material.
  • the value z is sometimes referred to as the epitopic density of the immunogen, detector, or solid matrix and in the usual situation will be on the average from about 1 to about 120 and more typically from 1 to 50. The densities, however, may vary greatly depending on the particular carrier material used.
  • the aryl portion is a phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, quinoxalyl, thienyl, thionaphthyl, furyl, benzofuryl, benzodioxyl, benzoxazolyl, benzoisoxazolyl, or benzodioxolyl group that may be optionally mono-, di-, or tri- substituted with a group selected from alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, nitro, carbalkoxy of 2- 7 carbon atoms, trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms per alkyl group, alkylthio of 1-6 carbon atoms, -SO3H and
  • the aryl moiety is a phenyl group that is optionally mono-, di-, or tri- substituted with a group selected from alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms per alkyl group, alkylthio of 1-6 carbon atoms, -SO3H and -CO2H.
  • the salts are those derived from such inorganic cations such as sodium, potassium, and the like; organic bases such as: mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkyl group and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms per alkyl group, and the like; and organic and inorganic acids as: acetic, lactic, citric, tartaric, succinic, maleic, malonic, gluconic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, and similarly known acceptable acids.
  • organic bases such as: mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkyl group and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms per alkyl group, and the like
  • organic and inorganic acids as: acetic, lactic, citric, tartaric, succinic
  • This invention also provides a process for preparing the rapamycin conjugates and antibodies of this invention.
  • this invention provides a process for preparing rapamycin conjugates including those of formula I and IV as defined above which comprises: reacting a compound of formula (V) or (VI):
  • R 21 and R 22 are each selected from hydrogen or -(R 3 -L-R ) a -T 1 providing that
  • R 21 and R 22 are not both hydrogen (i.e. when both R 21 and R 22 are hydrogen the compound of formula (V) is rapamycin); R 23 is -O(CH 2 )(CH 2 ) q R 4 -T l ;
  • T 1 is an electrophilic or nucleophilic group depending on the value for R 4 ; and q and R 4 are as hereinbefore defined; with a carrier material or matrix having available at least z nucleophilic or electrophilic groups; said carrier being represented by the formula
  • T 2 is a nucleophilic or electrophilic group of the carrier
  • the compounds of this invention can be prepared by reacting the 42- and/or 31-hydroxyl groups of rapamycin with a suitable electrophilic reagent that will serve as the linker moiety.
  • a suitable electrophilic reagent that will serve as the linker moiety.
  • the following patents exemplify the preparation of the 42- and/or 31 -derivatives of rapamycin that can be used as linking groups for the preparation of the compounds of this invention.
  • the preparation of fluorinated esters of rapamycin is described in U.S. Patent 5,100,883.
  • the preparation of amide esters is disclosed in 5,118,677.
  • carbamates of rapamycin is disclosed in U.S. Patent 5,118,678.
  • the preparation of aminoesters of rapamycin is described in U.S. Patent 5,130,307.
  • rapamycin-linking group moieties are provided as examples below.
  • the preparation of ether derivatives of rapamycin can be accomplished using the methodology disclosed in Example 18.
  • the electrophile (or activated electrophile) is reacted with rapamycin to typically provide a mixture of the 42- and 31,42-derivatized rapamycin that can be separated by chromatography.
  • the 42-hydroxyl group must be protected with a suitable protecting group, such as with a tert- butyldimethyl silyl group.
  • the 31 -hydroxyl can then be reacted with a suitable electrophile to provide the derivatized rapamycin, followed by deprotection of the 42- hydroxyl group.
  • the linker group attached to rapamycin can be coupled to a second linker group using standard methodology described in the peptide literature; typically by activating the electrophilic moiety, with DCC type coupling reagent, or with N- hydroxysuccinimide, or as an activated ester or anhydride.
  • the activated electrophilic end of one linking moiety can then be reacted with the nucleophilic end of the other linker moiety.
  • the coupling of the rapamycin linking group moiety to the immunogenic carrier can be accomplished under standard literature conditions.
  • an electrophilic moiety such as a carboxylic acid
  • a suitable activating agent such as N-hydroxysuccinimide
  • Examples 2 and 3 specifically exemplify this technique.
  • Similar methodology is employed for the coupling of a nucleophilic moiety on the linking group to an electrophilic moiety on the immunogenic carrier material.
  • the electrophilic moiety on the immunogenic carrier material is activated as described above, and then reacted with the nucleophilic end of the linking group.
  • the linking group can be attached to the carrier and then reacted with rapamycin at the 42- and/or 31 -hydroxy function.
  • the reagents used to prepare the compounds of the invention are commercially available or can be prepared by methods that are disclosed in the literature.
  • This invention also covers analogous conjugates of other rapamycins such as, but not limited to, 29-demethoxyrapamycin, [U.S. Patent 4,375,464, 32- demethoxyrapamycin under CA. nomenclature]; rapamycin derivatives in which the double bonds in the 1-, 3-, and/or 5-positions have been reduced [U.S. Patent 5,023,262]; 42-oxorapamycin [U.S. Patent 5,023,262]; 27-oximes of rapamycin [U.S. Patent 5,023,264]; 27-hydrazones of rapamycin [U.S. Patent 5,120,726]; 29-desmethylrapamycin [U.S.
  • the disclosures in the above cited U.S. Patents are hereby incorporated by reference.
  • Patent 5,252,579 which is hereby incorporated by reference), in which one or more of the hydroxyl groups has been esterified into a carboxylic ester, a carbamate, a sulfonate ester, an amide, or the like, or one or more of the ketones has been reduced to a hydroxyl group, or one or more of the double bonds has been reduced, or one ketones has been converted to an oxime or a hydrazone.
  • Other rapamycin derivatives for which the compounds of this invention can be used for measuring levels of or generating antibodies to will be apparent to one skilled in the art based on this disclosure.
  • Antibodies specific for rapamycin or a derivative thereof using the rapamycin immunogen conjugates of this invention may be generated by standard techniques that are known in the art. Typically, a host animal is inoculated at one or more sites with the immunogen conjugate, either alone or in combination with an adjuvant The typical host mammals include, but are not limited to, mice, goats, rabbits, guinea pigs, sheep, or horses. Subsequent injections can be made until a sufficient titer of antibodies are produced.
  • the antibodies generated from the rapamycin immunogen conjugates of this invention can be used in numerous immunoassays, for determining rapamycin levels, in ELISAs, radioimmunoassays, in chemiluminesence immunoassays, and in fluorescent immunoassays.
  • immunoassays for determining rapamycin levels
  • ELISAs ELISAs
  • radioimmunoassays chemiluminesence immunoassays
  • fluorescent immunoassays fluorescent immunoassays.
  • a basic competitive inhibition immunoassay can be performed as follows: Antibody specific for the ligand is usually bound to a matrix. A solution is applied to decrease nonspecific binding of the ligand to the matrix. After rinsing the excess away, the antibody coupled matrix may be treated in some cases so it can be stored.
  • the ligand standard curve is made and added with the rapamycin detector conjugate to compete for binding to the rapamycin-specific antibody. If necessary, the excess is removed.
  • the detector molecule is detected by the standard methods used by one skilled in the art. Different formats can be used, which include but are not limited to, dipstick assays, FPIA, EMIT, ELISA, VISTA, RIA, and MEIA.
  • Detector conjugates of the present invention can be prepared to use in the above assays.
  • the detector conjugates can be Carrier material with labeled fluorescent, chemiluminescent, or enzymatic moieties.
  • This invention also provides for the use of the rapamycin immunogen conjugates or antibodies specific for rapamycin or a derivative thereof in a test kit that can be commercially marketed.
  • the test kit may be used for measuring levels of rapamycin in biological or laboratory fluids.
  • Test kit components may include antibodies to rapamycin or a derivative thereof, antisera, or rapamycin carrier conjugates.
  • the conjugates or antibodies may be bound to a solid matrix, and rapamycin derivatives or antibodies may be radiolabeled if the assay so requires.
  • Standard concentrations of rapamycin can be included so that a standard concentration curve can be generated.
  • Suitable containers, microtiter plates, solid supports, test tubes, trays, can also be included in any such kit. Many variations of reagents can be included in the kit depending on the type of assay used.
  • rapamycin immunogen conjugate of this invention to generate antibodies specific for rapamycin or a derivative thereof and detect them using an ELISA format immunoassay.
  • Five mice were immunized with 50 ⁇ g rapamycin 31,42-diester with glutaric acid conjugate with keyhole limpet hemocyanin in Complete Freund's Adjuvant) intrasplenically and after about one month were boosted with 50 ⁇ g of rapamycin 31,42-diester with glutaric acid conjugate with keyhole limpet hemocyanin in incomplete Freund's Adjuvant) into the footpads.
  • Microtiter plates (Immunolon I) were coated overnight with 100 ⁇ l of goat anti-mouse antibody (10 ⁇ g ml in 10 mM potassium phosphate buffer, pH 7.2) at 4° C The plates were flicked and blocked with 100 ⁇ l of 1% bovine sera albumin in phosphate buffered saline overnight at 4° C After flicking and washing the plates thrice with 10 mM phosphate buffer, pH 7.05, 30 mM NaCl, 0.02% Triton X-100, and 0.004% thimerosal wash buffer, 100 ⁇ l of each mouse sera diluted with phosphate buffer solution was added to a well and incubated at room temperature for overnight.
  • rapamycin 31,42-diester with glutaric acid conjugate with horseradish peroxidase (100 ⁇ l, 0.5 ng/ml) was added and incubated for 1 hour at room temperature in the dark.
  • tetramethyl benzidine (TMB) substrate with H2O2 was added and the plates were incubated covered for 30 min. at room temperature in the dark.
  • the optical density was read on a spectrophotometer at 450 nm. As shown in Table I, five of the five mice had antibodies reactive for rapamycin 31,42-diester with glutaric acid conjugate with horseradish peroxidase (compound of Example 10).
  • mice 6904 produced the most antibodies to the compound of Example 10.
  • Hybridomas were generated using standard methodology. Following a splenectomy of a mouse immunized and boosted 3 times with the compound of Example 4, spleen cells were fused to SP20 cells to produce hybridomas. The hybridomas were evaluated for the production of antibodies specific for rapamycin or a derivative thereof using an ELISA assay as briefly described below.
  • Microtiter plates (Immunolon I) were coated overnight with 100 ⁇ l of goat anti- mouse antibody (10 ⁇ g/ml in lOmM potassium phosphate buffer, pH 7.2) at 4° C The plates were flicked and blocked with 100 ⁇ l of 1% bovine sera albumin in phosphate buffered saline (PBS) overnight at 4° C After flicking and washing the plates thrice with 0.2x PBS containing 0.02% Triton X-100 and 0.004% thimerosal, 100 ⁇ l of each hybridoma supernatant was added to a well and incubated at room temperature for overnight.
  • PBS phosphate buffered saline
  • Example 22 After flicking and washing the plates thrice with 0.2x PBS containing 0.02% Triton X-100 and 0.004% thimerosal, the compound of Example 22 (100 ⁇ l, 0.17 ⁇ M) was added and incubated for 1 hour at 4° C After flicking and washing the plates thrice with 0.2x PBS containing 0.02% Triton X-100 and 0.004% thimerosal, strepavidin or avidin conjugated to horseradish peroxidase (100 ⁇ l, 0.2 ⁇ g/ml) was added and incubated at room temperature for 1 hour in the dark.
  • hybridoma cell line in P4G1 was cloned by limiting dilution and is designated as hybridoma cell line, RAP-42-OVAF2#lhc-.
  • hybridoma cell line, RAP-42-OVAF2#lhc was deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 12301 Parklawn Drive, Rockville, Maryland, 20852, USA, on March 10, 1994, and was granted accession number HB 11568.
  • FPIA Fluorescent Polarization Immunoassay
  • the ring opened non-enzymatically transformed product of the above tracer (secorapamycin 42-ester with succinic acid conjugate with 5-glycinylfluoresceinamine) was isolated on TLC plate (50:chloroform:4 methanol:0.5 acetic acid; migrated slowest of three components).
  • the slowest migrating material termed A3, had a background reading of 75 mP and a reading of 178 mP in presence of RAP-42-OVAF2#lMoAb. Background levels were observed in the presence of FKBP12 (79 mP).
  • Competition of the binding between the antibody and A3 tracer with rapamycin or secorapamycin at 3 min was 155 mP and 105 mP, respectively and after 38 min. gives 121 mP and 89 mP, respectively.
  • the ring opened rapamycin-specific antibody designated as RAP-42-OVAF2#lMoAb, was isolated and purified using conventional methodology.
  • the compounds of Examples 12 and 13 can be used in an assay for the detection of polyclonal antibodies and monoclonal antibodies specific for rapamycin or a derivative thereof as described below.
  • Microtiter plates (Immunolon I) were coated overnight with 100 ⁇ l of goat anti- mouse antibody (10 ⁇ g/ml in lOmM potassium phosphate buffer, pH 7.2) at 4° C The plates were flicked and blocked with 100 ⁇ l of 1% bovine sera albumin in phosphate buffered saline overnight at 4° C After flicking and washing the plates thrice with wash buffer, 100 ⁇ l of rabbit sera diluted 1:5 in phosphate buffered saline was added to a well and incubated at room temperature for overnight After flicking and washing the plates thrice with wash buffer, rapamycin 42-ester with 3-[3-(4-imino- butylthio)succinimidyl]phenacylglycine conjugate with horseradish peroxidase (compound of Example 12) (100 ⁇ l, 0.5 ng/ml) or rapamycin 42 ester with (N-(3- carboxyphenyl)-3-thi
  • TMB substrate with H2O2 was added and the plates were incubated covered for 30 min. at room temperature in the dark.
  • the optical density was read on a spectrophotometer at 450 nm. The results are shown in Table HI.
  • Table 3 show that the compounds of Examples 12 and 13 can be used to detect antibodies specific for rapamycin or a derivative thereof in a mammal, as seen in rabbit number 81.
  • the following is an example of the measurement of rapamycin concentrations using a competitive inhibition assay for rapamycin with an ELISA format using an antibody specific for rapamycin.
  • Microtiter plates (Immunolon I) were coated overnight with 100 ⁇ l of goat anti-mouse antibody (10 ⁇ g/ml in 10 mM potassium phosphate buffer, pH 7.2) at 4' C The plates were flicked and blocked with 100 ⁇ l of 1% bovine sera albumin in phosphate buffered saline overnight at 4° C After flicking and washing the plates thrice with wash buffer, the rapamycin specific antibody described above (100 ⁇ l of l ⁇ g ml) was added to each well and incubated at room temperature for 1-4 hour.
  • rapamycin 31,42-bis(hemiglutarate) conjugate with horseradish peroxidase 100 ⁇ l, 0.5 ng/ml was added and incubated for 1 hour at room temperature in the dark.
  • TMB substrate was added and the plates were incubated covered for 5 min at room temperature in the dark. The optical density was read on a spectrophotometer at 450 nm. Results of the competition between rapamycin and rapamycin 31,42-diester with glutaric acid conjugate with horseradish peroxidase binding to mouse sera are shown in Table 4. From these results, a standard curve can be constructed and the concentration of rapamycin in a sample can be determined.
  • the compound of Example 11 (rapamycin 42-ester with N-[9H-fluoren-9- ylmethoxy)carbonyl]glycine) can be deprotected by the procedure used in Example 12 (to give rapamycin 42-ester with glycine) and conjugated to a solid matrix. It can bind antibodies specific for rapamycin or a derivative thereof as used in some dipstick immunoassay methods or to isolate rapamycin binding proteins.
  • the following example illustrates that 803 resonance units (RU) of the compound of Example 11 can be immobilized on a solid matrix using the BIAcore's standard protocol based on EDC and NHS used in a BIAcore. This matrix bound 1401 RU units of rapamycin specific antibody.
  • Deprotected rapamycin 42-ester with N- [9H-fluoren-9-ylmethoxy)carbonyl]glycine can be used to isolate binding proteins of rapamycin-FKBP complex by one of the following methods.
  • tissue or cell lysates containing the appropriate protease inhibitors are incubated with FKBP which has been incubated with a deprotected-rapamycin 42-ester with N-[9H-fluoren- 9-ylmethoxy)carbonyl]glycine conjugated matrix for a sufficient time to allow binding.
  • Various buffers are used to rinse the proteins which are nonspecifically bound. Proteins are released by the addition of additional buffers which disrupt the bond between the rapamycin nucleus-FKBP and the binding proteins.
  • Example 12 Ranamvcin 42-ester with 3-r3-(4-iminohutvlthio succinimidvnphenacvl- glvcine conjugate with horseradish peroxidase
  • rapamycin 42-ester with N-[9H-fluoren-9-ylmethoxy)- carbonyl]glycine 10 mg, 8.4 ⁇ mol
  • acetonitrile 84 ⁇ L
  • diethylamine 10 ⁇ L (in acetonitrile at 0.84 M) of diethylamine.
  • the reaction mixture was stirred at room temperature for 60 minutes and d e solvent was removed with a stream of nitrogen.
  • rapamycin 42-ester with glycine was taken up in a solution of m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) (2 mg) in DMF (200 ⁇ L) and allowed to incubate for two hours at 4 ⁇ C, followed by the addition of 50 nM ethanolamine (20 ⁇ L) in 50 mM Tris HC1, pH 8.0.
  • MFS m-maleimidobenzoyl-N-hydroxysuccinimide
  • Horseradish peroxidase (5 mg) and Rabbit IgG (10 mg) were treated with 2-iminothiolane and purified with Sephadex G- 25, followed by the addition of the MBS-rapamycin glycine ester adduct The mixture was incubated overnight at 4"C and purified by gel filtration on Sephadex G-25 to provide the title compound.
  • rapamycin 42-ester with glycine was taken up in a solution of N-succinimidyl S-acetylthioacetate (2 mg) in DMF (200 ⁇ L). The reaction mixture was stirred at room temperature for 15 minutes and then at 4°C overnight. A solution of hydroxylamine HC1 (7 mg in 50 ⁇ L DMF) was added to die solution of rapamycin reaction mixture, incubated for one hour, followed by the addition of MBS-horseradish peroxidase adduct and MBS-Rabbit IgG to give the title compound which was purified by Sephadex G-25 gel filtration.
  • Example 14 Ranamvcin 1.3. Diels Alder adduct with diethvl a/idodicarhoxvlate
  • Rapamycin (0.66g, 721 mmol) was dissolved in dichloromethane (10 ml) and cooled to 0 ⁇ C To this was added, dropwise, a solution of phenyltriazolinedione (0.133 g, 758 mmol) in dichloromethane (10 ml). The solution was stirred overnight, TLC showed the reaction was not complete. Additional phenyltriazenedione (0.025g, 27 mmol) was added. The reaction was purified using HPLC (4.1x31cm, Si ⁇ 2) with ethyl acetate as eluant to provide the title compound as a solid.
  • rapamycin derivatives that can be conjugated via a linker at the 31 -position of rapamycin.
  • Rapamycin (459 mg, 0.5 mmol) and pyrenebutyric acid (216 mg, 0.75 mmol) were dissolved in THF/CH2CI2 (10 ml, 1:1).
  • l-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (146 mg, 0.67 mmol) and 4-dimethylaminopyridine (15 mg) were added to the solution.
  • the reaction was allowed to warm to room temperature over 15 hours.
  • the reaction was diluted with CH2CI2 and washed with 5% HCl, then brine.
  • the solution was dried over MgSO4, filtered and evaporated to a solid.
  • rapamycin derivatives that can be conjugated to immunogenic carriers by the procedures described above or can be connected to another linker and then conjugated.
  • Rapamycin (2.0 g, 2.187 mmol) and rhodium (II) acetate (0.37 g, 0.08 mmol) were heated to reflux in benzene and treated with a solution ethyl diazoacetate (500 ml) in benzene (10 ml) over 10 minutes. The solution was cooled to room temperature and was stirred overnight. TLC showed that the reaction was incomplete. Two additional portions of ethyldiazoacetate (3 ml) were added at 24 hour intervals. The mixture was concentrated and purified by flash chromatography over silica using ethyl acetate. This provided the 42-monoether (1 g) and the 31,42 diether (0.850 g) as oils.
  • the 42- monoether was triturated in a mixture of hexane, ethyl acetate and dichloromethane over the weekend to give the product as a powder.
  • the diether was purified on HPLC on a silica gel column with ethyl acetate as eluant. This provided the product as a solid.
  • Analytical data for the monoether :
  • Rapamycin (0.450 g, 0.49 mmol) was dissolved in dry dichloromethane (10 ml) and cooled to 0°C To this solution was added pyridine (0.4 ml, 5.7 mmol) and a crystal of 4-dimethyl aminopyridine.
  • pyridine 0.4 ml, 5.7 mmol
  • Rapamycin (1.030 g, 1.12 mmol) was dissolved in dry dichloromethane (100 ml) and was cooled to 0°C To this solution was added pyridine (0.27 ml, 3.33 mmol) and a crystal of 4-dimethyl aminopyridine. A solution of thiophenyl chloroformate (0.47 ml 1.49 mmol) in dichloromethane (5 ml) was added to the reaction mixture. The solution was allowed to warm to room temperature overnight and was stirred at room temperature for 24 hours. The reaction was quenched into 0.1N HCl (5 ml) and the aqueous layer was washed with dichloromethane.
  • Example 21 Ranamvcin- -carhoxvmethvl-27-oxime To a solution of 600 mg (650 ⁇ M) of rapamycin in 6 mL of methanol was added at room temperature, 100 mg (1.2 mmol) of anhydrous sodium acetate and 140 mg (660 ⁇ M) of carboxymethoxylamine hemihydrochloride. After stirring overnight at room temperature, the reaction was complete. The reaction mixture was concentrated in vacuo and the residue was triturated with water. The solids were filtered and washed thoroughly with water. The product was dried under high vacuum to give 575 mg (89.7%) of a white solid. 13 C and *H NMR indicated a mixture of E and Z isomers for the oxime derivative at position 27.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Abstract

L'invention se rapporte à des conjugués de rapamycine aptes à être utilisés comme molécules immunogènes pour la génération d'anticorps spécifiques contre la rapamycine, afin de mesurer des niveaux de rapamycine ou de ses dérivés; d'isoler des protéines de liaison de rapamycine et de détecter des anticorps spécifiques contre la rapamycine ou ses dérivés.
PCT/US1994/004463 1993-04-23 1994-04-22 Conjugues de rapamycine et anticorps contre la rapamycine WO1994025072A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU67119/94A AU6711994A (en) 1993-04-23 1994-04-22 Rapamycin conjugates and antibodies

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5303093A 1993-04-23 1993-04-23
US22420794A 1994-04-14 1994-04-14
US08/053,030 1994-04-14
US08/224,207 1994-04-14

Publications (1)

Publication Number Publication Date
WO1994025072A1 true WO1994025072A1 (fr) 1994-11-10

Family

ID=26731365

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/004463 WO1994025072A1 (fr) 1993-04-23 1994-04-22 Conjugues de rapamycine et anticorps contre la rapamycine

Country Status (2)

Country Link
AU (1) AU6711994A (fr)
WO (1) WO1994025072A1 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045333A1 (fr) * 1997-04-09 1998-10-15 Isotechnika, Inc. Procede permettant la production d'anticorps a des sites specifiques de la rapamycine
US6432973B1 (en) * 2000-09-19 2002-08-13 Wyeth Water soluble rapamycin esters
US6635745B2 (en) 1993-04-08 2003-10-21 Novartis Ag Rapamycin assay
US6709873B1 (en) 1997-04-09 2004-03-23 Isodiagnostika Inc. Method for production of antibodies to specific sites of rapamycin
US7189582B2 (en) 2005-04-27 2007-03-13 Dade Behring Inc. Compositions and methods for detection of sirolimus
US7445916B2 (en) 2004-04-14 2008-11-04 Wyeth Process for preparing rapamycin 42-esters and FK-506 32-esters with dicarboxylic acid, precursors for rapamycin conjugates and antibodies
USRE40596E1 (en) * 1993-04-08 2008-12-02 Novartis Ag Rapamycin assay
US7883855B2 (en) 2006-07-21 2011-02-08 Abbott Laboratories Immunosuppressant drug extraction reagent for immunoassays
US7914999B2 (en) 2006-12-29 2011-03-29 Abbott Laboratories Non-denaturing lysis reagent
US7993851B2 (en) 2006-12-29 2011-08-09 Abbott Laboratories Lysis reagent for use with capture-in-solution immunoassay
US8021849B2 (en) 2004-11-05 2011-09-20 Siemens Healthcare Diagnostics Inc. Methods and kits for the determination of sirolimus in a sample
US8129127B2 (en) 2006-12-29 2012-03-06 Abbott Laboratories Assay for immunosuppressant drugs
US8221986B2 (en) 2006-12-29 2012-07-17 Abbott Laboratories Diagnostic test for the detection of a molecule or drug in whole blood
WO2019212990A1 (fr) * 2018-05-01 2019-11-07 Revolution Medicines, Inc. Analogues de rapamycine liés à c40, c28 et c32 en tant qu'inhibiteurs de mtor
US11685749B2 (en) 2018-05-01 2023-06-27 Revolution Medicines, Inc. C26-linked rapamycin analogs as mTOR inhibitors
RU2805211C2 (ru) * 2018-05-01 2023-10-12 Революшн Медсинз, Инк. С40-, с28- и с-32-связанные аналоги рапамицина в качестве ингибиторов mtor
US11819476B2 (en) 2019-12-05 2023-11-21 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof
US11944605B2 (en) 2018-06-15 2024-04-02 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005179A1 (fr) * 1990-09-19 1992-04-02 American Home Products Corporation Esters d'acide carboxylique de rapamycine
US5164495A (en) * 1991-09-18 1992-11-17 Abbott Laboratories Method for preparing a dicarboxylic acid half-acid ester of FK506
US5177203A (en) * 1992-03-05 1993-01-05 American Home Products Corporation Rapamycin 42-sulfonates and 42-(N-carboalkoxy) sulfamates useful as immunosuppressive agents
US5233036A (en) * 1990-10-16 1993-08-03 American Home Products Corporation Rapamycin alkoxyesters
WO1993019752A1 (fr) * 1992-04-07 1993-10-14 Dana-Farber Cancer Institute, Inc. Inhibition de kinase s6 p70
WO1993025533A1 (fr) * 1992-06-05 1993-12-23 Abbott Laboratories Procedes et reactifs pour le dosage d'immnosuppresseurs

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005179A1 (fr) * 1990-09-19 1992-04-02 American Home Products Corporation Esters d'acide carboxylique de rapamycine
US5233036A (en) * 1990-10-16 1993-08-03 American Home Products Corporation Rapamycin alkoxyesters
US5164495A (en) * 1991-09-18 1992-11-17 Abbott Laboratories Method for preparing a dicarboxylic acid half-acid ester of FK506
US5177203A (en) * 1992-03-05 1993-01-05 American Home Products Corporation Rapamycin 42-sulfonates and 42-(N-carboalkoxy) sulfamates useful as immunosuppressive agents
WO1993019752A1 (fr) * 1992-04-07 1993-10-14 Dana-Farber Cancer Institute, Inc. Inhibition de kinase s6 p70
WO1993025533A1 (fr) * 1992-06-05 1993-12-23 Abbott Laboratories Procedes et reactifs pour le dosage d'immnosuppresseurs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
THOMAS HULTSCH ET AL.: "IMMUNOPHILIN LIGANDS DEMONSTRATE COMMON FEATURES OF SIGNAL TRANSDUCTION LEADING TO EXOCYTOSIS OR TRANSCRIPTION.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, July 1991 (1991-07-01), WASHINGTON US, pages 6229 - 6233 *
YANQIU CHEN ET AL.: "CONFORMATIONAL CHANGES OF RAPAMYCIN AND ANALOGS UPON COMPLEXING WITH FKBP ASSOCIATED WITH ACTIVITY: AN APPLICATION OF SECOND DERIVATIVE CD SPECTROSCOPY.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 116, March 1994 (1994-03-01), WASHINGTON, DC US, pages 2683 - 2684 *

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE40596E1 (en) * 1993-04-08 2008-12-02 Novartis Ag Rapamycin assay
US8039599B1 (en) 1993-04-08 2011-10-18 Novartis Ag Rapamycin assay
US6635745B2 (en) 1993-04-08 2003-10-21 Novartis Ag Rapamycin assay
US8039600B2 (en) 1993-04-08 2011-10-18 Novartis Ag Rapamycin assay
US6709873B1 (en) 1997-04-09 2004-03-23 Isodiagnostika Inc. Method for production of antibodies to specific sites of rapamycin
WO1998045333A1 (fr) * 1997-04-09 1998-10-15 Isotechnika, Inc. Procede permettant la production d'anticorps a des sites specifiques de la rapamycine
US6432973B1 (en) * 2000-09-19 2002-08-13 Wyeth Water soluble rapamycin esters
US7445916B2 (en) 2004-04-14 2008-11-04 Wyeth Process for preparing rapamycin 42-esters and FK-506 32-esters with dicarboxylic acid, precursors for rapamycin conjugates and antibodies
US7625726B2 (en) 2004-04-14 2009-12-01 Wyeth Process for preparing rapamycin 42-esters and FK-506 32-esters with dicarboxylic acid, precursors for rapamycin conjugates and antibodies
US8021849B2 (en) 2004-11-05 2011-09-20 Siemens Healthcare Diagnostics Inc. Methods and kits for the determination of sirolimus in a sample
US7189582B2 (en) 2005-04-27 2007-03-13 Dade Behring Inc. Compositions and methods for detection of sirolimus
US7883855B2 (en) 2006-07-21 2011-02-08 Abbott Laboratories Immunosuppressant drug extraction reagent for immunoassays
US8541554B2 (en) 2006-07-21 2013-09-24 Abbott Laboratories Immunosuppressant drug extraction reagent for immunoassays
US8129127B2 (en) 2006-12-29 2012-03-06 Abbott Laboratories Assay for immunosuppressant drugs
US7993851B2 (en) 2006-12-29 2011-08-09 Abbott Laboratories Lysis reagent for use with capture-in-solution immunoassay
US8221986B2 (en) 2006-12-29 2012-07-17 Abbott Laboratories Diagnostic test for the detection of a molecule or drug in whole blood
US8329415B2 (en) 2006-12-29 2012-12-11 Abbott Laboratories Lysis reagent for use with capture-in-solution immunoassay
US8404452B2 (en) 2006-12-29 2013-03-26 Abbott Laboratories Assay for immunosuppressant drugs
US8440416B2 (en) 2006-12-29 2013-05-14 Abbott Laboratories Diagnostic test for the detection of a molecule or drug in whole blood
US7914999B2 (en) 2006-12-29 2011-03-29 Abbott Laboratories Non-denaturing lysis reagent
US8697365B2 (en) 2006-12-29 2014-04-15 Abbott Laboratories Non-denaturing lysis reagent
US10980889B1 (en) 2018-05-01 2021-04-20 Revolution Medicines, Inc. C40-, C28-, and C-32-linked rapamycin analogs as mTOR inhibitors
WO2019212990A1 (fr) * 2018-05-01 2019-11-07 Revolution Medicines, Inc. Analogues de rapamycine liés à c40, c28 et c32 en tant qu'inhibiteurs de mtor
CN112771054A (zh) * 2018-05-01 2021-05-07 锐新医药公司 作为mTOR抑制剂的C40-、C28-及C-32连接的雷帕霉素类似物
US11364300B2 (en) 2018-05-01 2022-06-21 Revolution Medicines, Inc. C40-, C28-, and C-32-linked rapamycin analogs as mTOR inhibitors
US11685749B2 (en) 2018-05-01 2023-06-27 Revolution Medicines, Inc. C26-linked rapamycin analogs as mTOR inhibitors
AU2019262978B2 (en) * 2018-05-01 2023-07-13 Revolution Medicines, Inc. C40-, C28-, and C-32-linked rapamycin analogs as mTOR inhibitors
RU2805211C2 (ru) * 2018-05-01 2023-10-12 Революшн Медсинз, Инк. С40-, с28- и с-32-связанные аналоги рапамицина в качестве ингибиторов mtor
EP4234031A3 (fr) * 2018-05-01 2024-02-28 Revolution Medicines, Inc. Analogues de rapamycine liés à c40, c28 et c32 en tant qu'inhibiteurs de mtor
US11944605B2 (en) 2018-06-15 2024-04-02 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof
US11819476B2 (en) 2019-12-05 2023-11-21 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof

Also Published As

Publication number Publication date
AU6711994A (en) 1994-11-21

Similar Documents

Publication Publication Date Title
US7897733B2 (en) Rapamycin conjugates and antibodies
US6328970B1 (en) Rapamycin position 27 conjugates
US5504091A (en) Biotin esters of rapamycin
WO1994025072A1 (fr) Conjugues de rapamycine et anticorps contre la rapamycine
JP4576429B2 (ja) タキソールの免疫学的検定
US8030458B2 (en) Monoclonal antibodies to tacrolimus and immunoassays methods for tacrolimus
JP4896959B2 (ja) ドキソルビシン免疫測定法
JP4540704B2 (ja) エベロリムスの測定方法および測定キット
CZ258395A3 (en) Method of determining rapamycin concentration
US7195882B2 (en) Monoclonal antibodies specific for buprenorphine and metabolites thereof
CA2559998C (fr) Conjugues de rapamycine et anticorps
US20110136253A1 (en) Irinotecan Immunoassay
US20110165699A1 (en) Irinotecan immunoassay

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR BY CA CN CZ FI GE HU JP KG KP KR KZ LK LV MD MG MN MW NO NZ PL RO RU SD SI SK TJ TT UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA