WO2012067670A1 - Immunodosage d'irinotécan - Google Patents

Immunodosage d'irinotécan Download PDF

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Publication number
WO2012067670A1
WO2012067670A1 PCT/US2011/028441 US2011028441W WO2012067670A1 WO 2012067670 A1 WO2012067670 A1 WO 2012067670A1 US 2011028441 W US2011028441 W US 2011028441W WO 2012067670 A1 WO2012067670 A1 WO 2012067670A1
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WIPO (PCT)
Prior art keywords
compound
formula
conjugate
antibody
carrier
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PCT/US2011/028441
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English (en)
Inventor
Salvatore J. Salamone
Jodi Blake Courtney
Alexander Volkov
Hongxia Zhang
Howard Sard
Vishnumurthy Hegde
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Saladax Biomedical Inc.
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Priority claimed from PCT/US2010/057264 external-priority patent/WO2011071672A1/fr
Priority claimed from US13/039,868 external-priority patent/US20110165699A1/en
Application filed by Saladax Biomedical Inc. filed Critical Saladax Biomedical Inc.
Publication of WO2012067670A1 publication Critical patent/WO2012067670A1/fr

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    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline

Definitions

  • This invention relates to the field of immunoassays and reagents for determining the presence and/or quantifying the amount of the
  • Cancer is a term used to describe a group of malignancies that all share the common trait of developing when cells in a part of the body begin to grow out of control. Most cancers form as tumors, but can also manifest in the blood and circulate through other tissues where they grow. Cancer malignancies are most commonly treated with a combination of surgery, chemotherapy, and/or radiation therapy. The type of treatment used to treat a specific cancer depends upon several factors including the type of cancer malignancy and the stage during which it was diagnosed.
  • the chemotherapeutic agent whose common chemical name is irinotecan has the formula:
  • hydrochloride is a pro-drug for administering the active SN-38 and its pharmaceutically acceptable salts.
  • SN-38 has the formula:
  • Irinotecan and its pharmaceutically acceptable salts, particularly the hydrochloride salt is one of the more commonly used chemotherapeutic agents for treatment of first and second stage metastatic colorectal cancer in combination with 5-fluorourac.il and leucovorin (Camptostar package insert, Pfizer, July 2008).
  • Irinotecan and the active metabolite have been shown to have high inter-patient variability with CV of 25.97% and 38,91% in AUCs respectively (Sasaki, Jpn. J. Cancer, 86, 101-110, 1995) and greater than a 40 fold variation in AUCs (Canal, JCO, 14(10): 2688-2695 1996)
  • Routine therapeutic drug management of the active metabolite of irinotecan would require the availability of simple automated tests adaptable to general laboratory equipment. Routine therapeutic drug management of irinotecan and its active metabolite SN-38 and its pharmaceutically acceptable salts would require the availability of simple automated tests adaptable to general laboratory equipment.
  • X is a terminal functional group capable of binding to a carrier, or its pharmaceutically acceptable salts
  • the compound of formula II can be used both to produce an immunogen for monoclonal antibodies which can be used in an immunoassay for accurately monitoring the levels of the compound of formula I or its pharmaceutically acceptable salts.
  • the compound of formula II where X is a terminal functional group capable of binding to an amino group in a polyamine containing carrier not only can be used to provide a binding partner but can also be used to provide the immunogen for an antibody for use in an immunoassay for determining the presence and/or quantifying the amount of the pharmacologically active metabolites of irinotecan(the compound of Formula I and its pharmaceutically acceptable salts.
  • the antibody and binding partner so produced provide a simple immunoassay but can be easily carried out without extensive washing and incubating steps.
  • the immunogen for producing monoclonal antibodies unlike the immunogen in Gaita, Biol. Pham bull. 23(8) 911-916 (2000), and the binding partner of this invention contain the whole active structure of irinotecan and the compound of formula I, this immunogen produces monoclonal antibodies specifically reactive with the compounds of formula I and its pharmaceutically acceptable salts without any cross reactivity to therapeutically or
  • the binding partner and the antibodies for an immunoassay including reagents and kits for such immunoassay for detecting and/ or quantifying the compound of formula and its pharmaceutically acceptable salts
  • the pharmaceutically active metabolite of irinotecan in blood, plasma or other body fluid samples.
  • this immunoassay the presence and amount of the compound of formula I and its pharmaceutically acceptable salts in body fluid samples, preferably a blood or plasma sample, can be detected and/or quantified.
  • a patient being treated with irinotecan and/or its pharmaceutically acceptable salts can be monitored during therapy and treatment adjusted in accordance with said monitoring.
  • this invention one achieves the therapeutic drug management of irinotecan or its pharmaceutically acceptable salts in cancer patients being treated with irinotecan and/or its pharmaceutically acceptable salts as a chemotherapeutic agent.
  • the reagents utilized in the assay of this invention are conjugates of a carrier, preferably containing polyamine functional groups, with the compounds of formula II. These conjugates are competitive binding partners with the compound of formula I and it's pharmaceutically acceptable salts present in the sample for the binding with the antibodies of this invention. Therefore, the amount of conjugate reagent which binds to the antibody will be inversely proportional to the amount of compound of formula I and it's
  • the assay utilizes any conventional measuring means for detecting and measuring the amount of said conjugate which is bound or unbound to the antibody. Through the use of said means, the amount of the bound or unbound conjugate can be determined.
  • the amount of compound of formula I and it's pharmaceutically acceptable salts in a sample is determined by correlating the measured amount of the bound or unbound conjugate produced by the compound of formula I and it's pharmaceutically acceptable salts in the sample with values of the bound or unbound conjugate determined from standard or calibration curve samples containing known amounts of compound of formula I, and it's pharmaceutically acceptable salts which known amounts are in the range expected for the sample to be tested.
  • the conjugates, as well as the immunogens, are prepared from compounds of the formula II.
  • the carrier and the polyamine polymer are linked to ligand portions of the compounds of formula II.
  • the ligand portions have the formula:
  • X * is -CH 2 - or a functional linking group
  • This ligand portion may be linked to one or more active sites on the carrier of the conjugate or the immunogen.
  • these carriers contain polymers, most preferably polyamine polymers having a reactive amino group.
  • X is preferably a functional group which can react with an amino group.
  • X can be any functional group which can react with any conventional carrier.
  • X in the compound of formula II is preferably any functional group capable of binding or linking to a polyamine polymer.
  • alkylene designates a divalent saturated straight or branch chain hydrocarbon substituent containing from one to ten carbon atoms
  • immunogen and “immunogenic” refer to substances capable of eliciting, producing, or generating an immune response in an organism.
  • conjugate refers to any substance formed from the joining together of two parts.
  • Representative conjugates in accordance with the present invention include those formed by the joining together of a small molecule, such as the compound of formula II and a large molecule, such as a carrier, preferably carriers which comprise a polyamine polymer, particularly a protein.
  • the small molecule maybe joined or linked at one or more active sites on the large molecule.
  • conjugate includes the term immunogen.
  • the carrier can be any carrier and X can be any functional group which can be linked to a carrier.
  • the carrier is a polyamine polymer and X is any functional group capable of linking to a polyamine polymer.
  • Haptens are partial or incomplete antigens. They are protein-free substances, mostly low molecular weight substances, which are not capable of stimulating antibody formation, but which do react with antibodies. The latter are formed by coupling a hapten to a high molecular weight
  • a spacing group refers to a portion of a chemical structure which connects two or more substructures such as haptens, carriers, immunogens, labels, or tracer through a CH 2 or functional linking group. These spacer groups will be enumerated hereinafter in this application.
  • the atoms of a spacing group and the atoms of a chain within the spacing group are themselves connected by chemical bonds. Among the preferred spacers are straight or branched, saturated or unsaturated, carbon chains. Theses carbon chains may also include one or more heteroatoms within the chain or at termini of the chains.
  • heteroatoms is meant atoms other than carbon which are chosen from the group consisting of oxygen, nitrogen and sulfur. Spacing groups may also include cyclic or aromatic groups as part of the chain or as a substitution on one of the atoms in the chain.
  • the immunogenic carrier can also be a poly(nucleic acid) either alone or conjugated to one of the above mentioned poly(amino acids) or
  • the immunogenic carrier can also include solid particles.
  • the particles are generally at least about 0.02 microns ( ⁇ ) and not more than about 100 ⁇ , and usually about 0.05 ⁇ to 10 ⁇ in diameter.
  • the particle can be organic or inorganic, swellable or non-swellable, porous or non-porous, optimally of a density approximating water, generally from about 0.7 to 1.5 g/ mL, and composed of material that can be transparent, partially transparent, or opaque.
  • the particles can be biological materials such as cells and
  • Poly(amino acid) or “polypeptide” is a polyamide formed from amino acids.
  • Poly(amino acids) will generally range from about 2,000 molecular weight, having no upper molecular weight limit, normally being less than 10,000,000 and usually not more than about 600,000 daltons. There will usually be different ranges, depending on whether an immunogenic carrier or an enzyme is involved.
  • a “peptide” is any compound formed by the linkage of two or more amino acids by amide (peptide) bonds, usually a polymer of a-amino acids in which the a-amino group of each amino acid residue (except the NH2 terminus) is linked to the a-carboxyl group of the next residue in a linear chain.
  • the terms peptide, polypeptide and poly(amino acid) are used synonymously herein to refer to this class of compounds without restriction as to size. The largest members of this class are referred to as proteins.
  • a “label,” “detector molecule,” or “tracer” is any molecule which produces, or can be induced to produce, a detectable signal.
  • the label can be conjugated to an analyte, immunogen, antibody, or to another molecule such as a receptor or a molecule that can bind to a receptor such as a ligand, particularly a hapten.
  • Non-limiting examples of labels include radioactive isotopes, enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes, chemiluminescers, luminescers, or sensitizers; a non-magnetic or magnetic particle, a solid support, a liposome, a ligand, or a receptor.
  • derivative refers to a chemical compound or molecule made from a parent compound by one or more chemical reactions.
  • carrier refers to solid particles and/or polymeric polymers such as immunogenic polymers such as those mentioned above. Where the carrier is a solid particle, the solid particle may be bound, coated with or otherwise attached to the polymeric material which preferably is a polyamine polymer to provide one or more reactive sites for bonding to the terminal functional group X in the compounds of the formula II.
  • reagent kit refers to an assembly of materials that are used in performing an assay.
  • the reagents can be provided in packaged combination in the same or in separate containers, depending on their cross- reactivities and stabilities, and in liquid or in lyophilized form.
  • the amounts and proportions of reagents provided in the kit can be selected so as to provide optimum results for a particular application.
  • a reagent kit embodying features of the present invention comprises antibodies specific for the compound of formula I.
  • the kit may further comprise ligands of the analyte and calibration and control materials.
  • the reagents may remain in liquid form or may be lyophilized.
  • calibration and control materials refers to any standard or reference material containing a known amount of a drug to be measured.
  • concentration of drug is calculated by comparing the results obtained for the unknown specimen with the results obtained for the standard. This is commonly done by constructing a calibration curve.
  • biological sample includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing.
  • living things include, but are not limited to, humans, mice, monkeys, rats, rabbits, horses, and other animals.
  • substances include, but are not limited to, blood, serum, plasma, urine, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovial macrophages, endothelial cells, and skin.
  • a conjugate of the compound of formula I is constructed to compete with the compound of formula I in the sample for binding sites on the antibodies.
  • the reagents are conjugates of a carrier with the compound of formula II.
  • the linker spacer constitutes the "-B-(Y)p -X'-" portion of this molecule .
  • the linker X' and the spacer "-B-(Y)p-" - are conventional in preparing conjugates and immunogens. Any of the conventional spacer-linking groups utilized to prepare conjugates and immunogens for immunoassays can be utilized in the compound of formula III.
  • Such conventional linkers and spacers are disclosed in U.S. Patent 5,501,987 and U.S. Patent 5,101,015.
  • n and o are integers from o to 6, and n is an integer from 1 to 6 with alkylene being the especially preferred spacing group
  • m is o
  • n is preferably an integer of from 1-6, most preferably l or 2 and o is preferably o or ⁇ .
  • X' is -CH2- or a functional group linking the spacer to the carrier, preferably to an amine group on a polymeric carrier.
  • the group X' is the result of the terminal functional group X in the compound of Formula II which is capable of binding to a carrier, preferably to an amino group in the polyamine polymer present in the carrier or used as the immunogen.
  • Any terminal functional group capable of binding to a carrier, preferably capable of reacting with an amine can be utilized as the functional group X in the compound of formula II.
  • These terminal functional groups preferably included within X are:
  • R 3 is hydrogen or taken together with its attached oxygen atom forms a reactive ester and R4 is oxygen or sulfur.
  • the active esters formed by OR3 include imidoester, such as N-hydroxysuccinamide, l-hydroxy benzotriazole and p-nitrophenyl ester. However any active ester which can react with an amine group can be used.
  • any conventional means of condensing a reactive hydroxy group with the amine group in the compound of formula VI can be used in carrying out this condensation reaction.
  • the reactive groups on the compound of formula VI can be protected as described hereinabove with a conventional protecting group. These protecting groups can be removed after this amine condensation by conventional means. Also the 10-protected hydroxy group can be removed after the amine condensation.
  • the compound of formula I when B is in 0 is produced by treating the free hydroxy group at the 20- position of the 10-hydroxy protected compound of formula I with a carboxylic an acid compound of the formula.
  • the amines are protected by forming the corresponding N-trifluoroacetamide, N-tertbutyloxycarbonyl urethane (N-t-BOC urethane), N-carbobenzyloxy urethane or similar structure.
  • N-t-BOC urethane N-tertbutyloxycarbonyl urethane
  • N-carbobenzyloxy urethane or similar structure.
  • Such reagents and methods include weak or strong aqueous or anhydrous acids, weak or strong aqueous or anhydrous bases, hydride-containing reagents such as sodium borohydride or sodium cyanoborohydride and catalytic hydrogenation.
  • the present invention also relates to novel monoclonal antibodies to the compound of formula I produced by utilizing the aforementioned immunogens.
  • these antibodies produced in accordance with this invention are reactive with the compound of formula I and do not substantially react with irinotecan, its pharmaceutically acceptable salts or with pharmacologically inactive metabolites of irinotecan which would interfere with immunoassays for the compound of formula I.
  • B lymphocytes obtained from the spleen, peripheral blood, lymph nodes or other tissue of the host may be used as the monoclonal antibody producing cell. Most preferred are B lymphocytes obtained from the spleen.
  • Hybridomas capable of generating the desired monoclonal antibodies of the invention are obtained by fusing such B lymphocytes with an immortal cell line, which is a cell line that which imparts long term tissue culture stability on the hybrid cell.
  • the immortal cell may be a lymphoblastoid cell or a plasmacytoma cell such as a myeloma cell.
  • Murine hybridomas which produce SN-38 monoclonal antibodies are formed by the fusion of mouse myeloma cells and spleen cells from mice immunized with the aforementioned immunogenic conjugates.
  • Polypeptide fragments comprising only a portion of the primary antibody structure may be produced, which fragments possess one or more
  • immunoglobulin activities These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in expression vectors containing the antibody genes using site-directed mutageneses to produce Fab fragments or (Fab')2 fragments.
  • Single chain antibodies may be produced by joining VL and VH regions with a DNA linker (see Huston et al., Proe. Natl. Acad. Sci. U.SA., 85:5879-5883 (1988) and Bird et al, Science, 242:423-426 (1988))
  • the antibodies of this invention are selectively reactive with the compound of Formula I and it's pharmaceutically acceptable salts and not irinotecan.
  • the preferred antibodies do not have any substantial cross-reactivity with pharmaceutically inactive metabolites of irinotecan or its
  • any immunoassay in which the reagent conjugates formed from the compound of formula II compete with the compound of formula I in the sample for binding sites on the antibodies generated in accordance with this invention can be utilized to determine the presence of the compound of formula I in a patient sample.
  • the manner for conducting such an assay for the compound of formula I in a sample suspected of containing SN-38 comprises combining an (a) aqueous medium sample, (b) an antibody to the compound of formula I generated in accordance with this invention and (c) the conjugates formed from the compound of formula II.
  • the compound of formula I in the sample can be determined by measuring the inhibition of the binding to the specific antibody of a known amount of the conjugate added to the mixture of the sample and antibody.
  • Various means can be utilized to measure the amount of conjugate formed from the compound of formula II bound to the antibody.
  • One method is where binding of the conjugates to the antibody causes a decrease in the rate of rotation of a fluorophore conjugate.
  • the amount of decrease in the rate of rotation of a fluorophore conjugate in the liquid mixture can be detected by the fluorescent polarization technique such as disclosed in U.S. Patent 4,269,511 and U.S. Patent 4,420,568.
  • the antibody can be coated or absorbed on nanoparticles so that when these particles react with the compound of formula I and conjugates formed from the compounds of formula II these nanoparticles form an aggregate.
  • the antibody coated or absorbed on nanoparticles react with the SN-38 in the sample, the SN-38 from the sample bound to these nanoparticles does not cause aggregation of the antibody nanoparticles.
  • the amount of aggregation or agglutination can be measured in the assay mixture by absorbance.
  • these assays can be carried out by having either the antibody or the compounds of formula II attached to a solid support such as a microtiter plate or any other conventional solid support including solid particles. Attaching antibodies and proteins to such solid particles is well known in the art. Any conventional method can be utilized for carrying out such attachments.
  • labels may be placed upon the antibodies, conjugates or solid particles , such as radioactive labels or enzyme labels, as aids in detecting the amount of the conjugates formed from the compound of formula II which is bound or unbound with the antibody.
  • suitable labels include chromophores, fluorophores, etc.
  • assay components of the present invention can be provided in a kit, a packaged combination with predetermined amounts of new reagents employed in assaying for SN-38.
  • reagents include the antibody of this invention, as well as, the conjugate formed from the compounds of formula II.
  • the radicals p, X, Y and B in the reagent and the immunogen which forms the antibody used in a given immunoassay can be the same or be a different substituent within the groups defined for each of theses radicals.
  • additives such as ancillary reagents may be included, for example, stabilizers, buffers and the like.
  • ancillary reagents may be included, for example, stabilizers, buffers and the like.
  • the relative amounts of the various reagents may vary widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • Reagents can be provided in solution or as a dry powder, usually lyophilized, including excipients which on dissolution will provide for a reagent solution having the appropriate concentrations for performing the assay.
  • the phosphate buffer composition has an aqueous solution containing
  • SN-38 acid derivatives were activated with EDC and NHS to produce the NHS activated ester of SN-38 for eventual conjugation to proteins (examples 6, 7a and 7b).
  • Example 5a
  • SN-38 derivative [6] example 1, scheme l, (91.8 mg) was dissolved in 10 mL of DMF to which was added NHS (56.1 mg) and EDC (92.8 mg). The reaction mixture was stirred for 20 hours at room temperature under a nitrogen atmosphere to produce the NHS activated ester of SN-38 [7]. The reaction mixture was used directly in examples 6 and 7a.
  • a protein solution of KLH was prepared by dissolving 300 mg of KLH in 19.6 mL of phosphate buffer (50 mM, pH 7.5) and then slowly adding 39.2 mL of DMSO while stirring the protein solution of KLH on ice, followed by addition of 5.1 mL of NHS activated SN-38 derivative [7] prepared in Example 5a.
  • the reaction mixture of KLH and activated SN-38 derivative [7] was allowed to stir for 20 hours at room temperature to produce an SN-38-KLH conjugate [8].
  • the SN-38-KLH conjugate was then purified by dialysis against 50% DMSO in phosphate buffer (50 mM, pH 7.5) at room temperature.
  • a protein solution of BSA was prepared by dissolving 990 mg BSA in phosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/n L. 19.8 mL of DMSO was slowly added to the protein solution of BSA while stirring on ice, followed by addition of 0.9 mL of NHS activated SN-38 derivative [7] prepared in Example 5a. The amount of NHS activated SN-38 derivative [7] added to the protein solution of BSA was calculated for a 1:1 molar ratio between the derivative of SN-38 [7] and BSA. The mixture of BSA and activated SN-38 derivative [7] was allowed to stir for 18 hours at room temperature to produce the conjugate of the activated SN-38 ester [7] and BSA.
  • This conjugate was then purified by dialysis against 50% DMSO in phosphate buffer (50 mM, pH 7.5) at room temperature. Thereafter the DMSO proportion was reduced stepwise: 40%, 30%, 20%, 10% and 0%. The last dialysis was performed against phosphate buffer at 4°C. The purified SN-38- BSA conjugate was characterized by UV/VIS spectroscopy.
  • a protein solution of BSA was prepared by dissolving 990 mg BSA in phosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL. 19.8 mL of DMSO was slowly added to the protein solution of BSA while stirring on ice, followed by addition of 1 mL of NHS activated SN-38 derivative [12] prepared in Example 5b. The amount of NHS activated SN-38 derivative [12] added to the protein solution of BSA was calculated for a 1:1 molar ratio between the derivative of SN-38 [12] and BSA. The mixture of BSA and activated SN-38 derivative [12] was allowed to stir for 18 hours at room temperature to produce the conjugate of the activated SN-38 ester [12] and BSA.
  • mice from Example 8a that were immunized with SN-38-KLH conjugate [8] prepared in Example 6 were used to produce monoclonal antibodies.
  • the mice were injected i.p. with 400 ⁇ g (3 days before fusion), 200 ⁇ g (2 days before fusion), and 200 ⁇ g (1 day before fusion) of SN-38-KLH conjugate [8] in PBS prepared in Example 6.
  • Spleen cells were isolated from the selected mice and fused with 2 x 107 SP2/0 cells with 50% polyethylene glycol 1500 according to the method of Coligan, J.E.
  • Hybridoma culture supernatants containing monoclonal antibody from selected subclones were confirmed for SN-38 binding by a competitive ELISA (Example 11). These monoclonal antibodies were tested for SN-38 binding and cross-reactivity to irinotecan, the SN-38 prodrug, by indirect competitive microtiter plate assay as described in example 11.
  • hybridoma supernatants of Example 8b which were found to be positive for the presence of antibody by the procedure of Example 10b were diluted 1:10, 1:100, 1:1000, etc. (volume/volume) in phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal.
  • phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal 50 ⁇ , phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal and 50 ⁇ . of diluted antibody were added and incubated for 10 minutes at room temperature with shaking.
  • the ELISA method for screening SN-38 monoclonal antibodies was performed with the microtiter plates that were sensitized with SN-38-BSA conjugate as described in examples 9a and 9b.
  • SN-38-BSA sensitized wells prepared in examples 9a and 9b
  • 50 ⁇ phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal and then 50 ⁇
  • monoclonal culture supernatant were added and incubated for 10 minutes at room temperature with shaking. During this incubation antibody binds to the SN-38-conjugate in the well.
  • the ELISA method for determining IC50 values and cross- reactivity was performed with the mierotiter plates that were sensitized with SN-38-BSA conjugates as described in Examples 9a and 9b.
  • the analytes - SN-38 and irinotecan were diluted in diH 2 0 - SN-38 over a concentration range of 1 to 10,000 ng/mL for SN-38 [6]-BSA mierotiter plate and 0.1 to 1,000 ng/mL for SN-38 [ii]-BSA mierotiter plate.
  • Irinotecan was diluted in diH 2 0 over a concentration range of 2000 to 10,000 ng/mL for SN-38 [6]-BSA mierotiter plates and a concentration range of 1 to 100,000 ng/mL for SN-38 [n]-BSA mierotiter plates.
  • Each of the assays were performed by incubating 50 ⁇ L of the analyte solution with 50 ⁇ , of one of the antibodies selected from the polyclonal antibodies produced in Example 8a with the immunogen of Example 6 and the monoclonal antibody produced in Example 8b.
  • the assays were all performed by diluting the concentration of the antibodies in each of the wells to the titer determined in Example 10a.
  • Immunoresearch diluted to a predetermined specific activity (approximately 1/3000) in PBS with 0.1% BSA, 0.05% ANS, 0.01% thimerosal, capable of binding specifically with murine immunoglobulins and producing a colored product when incubated with a substrate, in this example TMB, were added to each well. After an incubation of 10 minutes at room temperature with shaking, during which the goat anti- mouse antibody - HRP enzyme conjugate binds to SN-38 antibodies in the wells, the plates were again washed three times to remove unbound secondary conjugate.
  • TMB TMB Substrate, BioFx
  • stop solution (1.5% sodium fluoride in diH 2 0
  • the cross-reactivity was calculated as the ratio of the IC 50 for SN-38 to the IC 50 for irinotecan and expressed as a percent. When measured with this pool of antibodies, the percent cross-reactivities relative to SN-38 for irinotecan were less than or equal to 0.12%. Results for polyclonal antibodies to SN-38 are in table I below. When measured with selected monoclonal antibodies the percent cross- reactivities relative to SN-38 for irinotecan were less than 0.13%. Results for monoclonal antibodies to SN-38 are in table II.
  • the antibodies of this invention are substantially selectively reactive with the active form of SN-38 and are not substantially cross-reactive with the inactive prodrug irinotecan.

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Abstract

L'invention concerne de nouveaux conjugués du métabolite pharmaceutiquement actif d'irinotécan, de nouveaux immunogènes dérivés dudit métabolite et des anticorps monoclonaux générés par lesdits immunogènes qui sont utilisés dans des immunodosages pour quantifier et surveiller le métabolite pharmaceutiquement actif d'irinotécan dans des fluides biologiques.
PCT/US2011/028441 2010-11-18 2011-03-15 Immunodosage d'irinotécan WO2012067670A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
PCT/US2010/057264 WO2011071672A1 (fr) 2009-12-08 2010-11-18 Immunoessai d'irinotécan
USPCT/US2010/057264 2010-11-18
US13/039,868 US20110165699A1 (en) 2009-12-08 2011-03-03 Irinotecan immunoassay
US13/039,868 2011-03-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021005583A1 (fr) 2019-07-11 2021-01-14 Sun Pharma Advanced Research Company Ltd. Dérivés de camptothécine ayant une fraction disulfure et une fraction pipérazine

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