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WO1997029114A1 - Composes contenant de la biotine, reactifs et procedes de biotinylation - Google Patents

Composes contenant de la biotine, reactifs et procedes de biotinylation Download PDF

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Publication number
WO1997029114A1
WO1997029114A1 PCT/US1997/002560 US9702560W WO9729114A1 WO 1997029114 A1 WO1997029114 A1 WO 1997029114A1 US 9702560 W US9702560 W US 9702560W WO 9729114 A1 WO9729114 A1 WO 9729114A1
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Prior art keywords
biotin
moiety
moieties
water soluble
containing compound
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PCT/US1997/002560
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English (en)
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Scott D. Wilbur
Pradip M. Pathare
S. Ananda Weerawarna
Donald K. Hamlin
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Board Of Regents Of The University Of Washington
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Priority to AU20524/97A priority Critical patent/AU2052497A/en
Publication of WO1997029114A1 publication Critical patent/WO1997029114A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/009—Neutron capture therapy, e.g. using uranium or non-boron material
    • A61K41/0095—Boron neutron capture therapy, i.e. BNCT, e.g. using boronated porphyrins
    • 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
    • A61K47/54—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 organic compound
    • A61K47/555—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 organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/557—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 organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
    • 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
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6893—Pre-targeting systems involving an antibody for targeting specific cells clearing therapy or enhanced clearance, i.e. using an antibody clearing agents in addition to T-A and D-M
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates generally to biotin-containing compounds, biotinylation reagents, and methods for synthesizing such compounds and reagents.
  • the present invention relates, more specifically, to biotin-containing compounds and biotinylation reagents suitable for in vitro and in vivo applications that are soluble in aqueous solution.
  • the biotin-containing compounds and biotinylatin reagents may additionally comprise constituents that confer other properties or functionalities, such as biotinidase-stabilizing properties, cleavable moieties, reactive moieties, and functional moieties, including diagnostic or therapeutic moieties such as dyes, radionuclides and drugs, targeting moieties, and the like.
  • the present invention also relates to methods for synthesizing and using such biotin-containing compounds and biotinylation reagents.
  • biotin illustrated below as chemical structure 1, with the proteins avidin and streptavidin renders biotin compounds useful for numerous applications. For example, many diagnostic tests use biotinylated derivatives.
  • ELISA enzyme-linked immunosorbent assay
  • Other biotinylated compounds have been used as probes.
  • Biotinylated nucleic acids have also been widely used. Purification techniques such as affinity chromatography frequently employ biotinylated materials.
  • biotin derivatives have been used in diagnosis and therapy of human disease.
  • investigators have shown that use of a combination of monoclonal antibodies, streptavidin and/or avidin. and radiolabeled biotin derivatives improves the diagnostic and therapeutic characteristics of the radiolabeled monoclonal antibody tumor targeting system.
  • One important application under investigation by a number of research groups is the use of "pretargeted" monoclonal antibody conjugates using the biotin/(strept)avidin ligand/anti-ligand pair for imaging and therapy of cancer.
  • biotin derivatives for many of these applications are their generally low solubility in aqueous media.
  • Biotin derivatives and biotinylation reagents generally need to be solubilized in organic solvents, or media comprising a substantial level of organic constituents. Insolubility of biotin derivatives and biotinylation reagents in aqueous solutions is particularly problematic for in vivo applications where organic solvents cannot be used.
  • biotin derivatives for binding with avidin- and streptavidin-containing compounds may be greater when a spacer molecule is used between the biotin moiety and the other moieties to which it is attached.
  • the spacer molecules used have generally been molecules having a low solubility in aqueous media, such as aliphatic molecules.
  • Such linkers generally reduce the at.ueous solubility of the biotin-containing compound.
  • the lipophilic nature of the biotin derivatives with linkers of low water solubility may cause them to associate with blood components, rendering their biological half-life in in vivo applications longer than desired.
  • the linker is referred to as a long, flexible hydrophilic spacer arm. Levenson et al.,
  • U.S. Patent 9,550,249 discloses water soluble biotin salts for oral, parenteral or topical administration. Salts of biotin with an aminoalcohol are disclosed, and biotinate of ethanolammonium is preferred.
  • U.S. Patent 4,478,915 discloses a process for applying layers of a protein and a ligand extender to a surface and to a multiple layer system.
  • Biotin derivatives used as extenders include caproylamidobiotin and biocytin. Extenders such as fibrogen, albumin, succinylated polylysine and ribonuclease modified with biotin or biotin derivatives are also disclosed.
  • biotin-containing compounds Little attention has been devoted to the water solubility of biotin-containing compounds, or the synthesis of biotinylation reagents having improved solubility in aqueous solutions. Because biotin-containing compounds are increasingly being used in biological systems for diagnostic and therapeutic applications, where organic solvents cannot be used, there is a need for biotin-containing compounds exhibiting enhanced water solubility and improved linker systems. There is also a need for biotin-containing compounds that are resistant to cleavage by the serum enzyme biotinidase lor use in in vivo applications.
  • Biotin compounds are conjugated with water soluble linker moieties to form biotin-linker adducts that are water soluble.
  • Such biotin-linker adducts may additionally comprise one or more functional group that confers resistance to cleavage by biotinidase.
  • the biotin-linker adducts or conjugated biotin compounds may also comprise one or more constituents that are cleavable in vitro or in vivo.
  • Biotinylation reagents of the present invention comprise water soluble biotin- linker adducts linked to a reactive moiety that provides a site for reaction with yet another moiety, such as a targeting, diagnostic or therapeutic lunctional moiety.
  • Conjugates including functional moieties are disclosed.
  • Biotin Jiuiers. trimers and multimers comprising water soluble linker moieties are also disclosed and demonstrate enhanced water solubility. Methods for synthesizing the biolin-containing compounds and biotinylation reagents, and methods for using such compounds are described.
  • Fig. 1 illustrates the results of the experiment described in Example 14 demonstrating the successful cross-linking of streptavidin with biotin trimers.
  • Naturally occurring biotin illustrated below as structure 1. is useful for many different applications as a result of its strong binding affinity for avidin or streptavidin.
  • Many modified forms of biotin have been synthesized for various applications. Modifications of biotin at positions other than the carboxylate group, for example, provide molecules that have weaker interactions with avidin or streptavidin.
  • modified biotin molecules such as desthiobiotin, shown below as structure 2, and biotin sulfone, shown below as structure 3, are useful for some applications because they bind tightly enough to provide a strong association, yet they bind in a reversible fashion such that they can be displaced by tighter binding biotin derivatives.
  • These modified biotin molecules (2 and 3) have low water solubility.
  • biotins of varying binding strengths.
  • conversion of the ureido functionality to an guanidinium functionalih e.g. iminobiotin, shown below as structure 4
  • conversion of the amide-NH by alkylation (e.g. methylation) or by acylation (e.g. acelyl) produce biotins of varying binding strengths.
  • biotin or biotin derivatives by leaction with amines of sterically bulky groups such as proteins (e.g. insulin) or steric small molecules such as branched chain moieties including 2-dimethyl-amino compounds or amino-aryl moieties are also described above, and any other modifications to biotin or constituents thereof that bind to avidin or streptavidin or modifications to or constituents thereof.
  • proteins e.g. insulin
  • steric small molecules such as branched chain moieties including 2-dimethyl-amino compounds or amino-aryl moieties
  • biotin The solubility of naturally-occurring biotin in water is approximately 0.2 mg/mL at neutral pH and ambient temperature. Modifications to biotin such as those described above generally reduce its water solubility, and biotin compounds having more than one biotin moiety per molecule are generally substantially insoluble in aqueous media.
  • a biotin compound comprising two biotin moieties joined by an aliphatic 1 , 12 diaminododecane linker moiety, for example, has such low water solubility that routine HPLC analysis does not detect any dimers in aqueous solution.
  • trimeric biotin molecules comprising aliphatic linkers exhibit water solubilities below normal detection levels.
  • the solubility of biotin moieties in aqueous media is enhanced according to the present invention by attaching a water soluble linker moiety, preferably through the carboxylate functionality of the biotin moiety.
  • Water soluble linker moieties compromise any linker moiety that, when coupled to a biotin moiety, increases the water solubility of the biotin moiety.
  • water soluble used in connection with a linker moiety, biotin-containing compound or a biotinylation reagent indicates that the compound or reagent has a water solubility greater than that of naturally-occurring biotin: that is.
  • Biotin-containing compounds and biotiny latin reagents of the present invention preferably exhibit water solubility of at least about 1 mg/mL at neutral pH and ambient temperature, and most preferably exhibit a water solubility of at least about 5 mg/mL at neutral pH and ambient temperature. Solubility may be ascertained by dissolving the compound in water, stirring the solution, and allowing the solution to stand at room temperature for about 24 hours. The solution is then centrifuged and the resultant aqueous layer analyzed using high-pressure liquid chromatography ("HPLC"). The HPLC analysis is conducted with a radiant using acetonitrile or methanol and water as the solvent mixture using a reverse-phase column.
  • HPLC high-pressure liquid chromatography
  • a carboxylic acid or amine modifier is used in the solvent mixture.
  • Solubility of a compound may alternatively be determined using any of the techniques described in the Handbook of Solubility Parameters and Other Cohesion Parameters by A.F.M. Benton, CRC Press, 1983.
  • Water-soluble linker moieties preferably comprise hydrophilic moieties (e.g., polar functional groups) including electronically neutral and charged (i.e., ionic) moieties.
  • Suitable hydrophilic moieties include electronically neutral moieties containing polar functional groups (i.e., groups that contain atoms of differing electronegativities such as organic compounds containing nitrogen, oxygen, and sulfur) that increase their hydrophilicity.
  • these neutral hydrophilic moieties contain functional groups that hydrogen bond with water.
  • Such hydrogen bonding groups include ether (-O-), hydroxy (-OH), amino (-NR 2 , -NHR, -NH 2 ), and to a lesser extent thioether (-S-), and thiol (-SH) groups.
  • Moieties that comprise multiple polar functional groups are more hydrophilic than those moieties that comprise a single polar functional group.
  • Suitable moieties comprising multiple polar groups include, for example, polyhydroxy, polyamino, polyether, polyphosphoric acid, polyalcohol and polyamine moieties.
  • Polyhydroxy moieties include, for example, glycols, glycerols, and polysaccharides including glucose, fructose, galactose, idose, inositol, mannose, tagatose, and N-methylglucamine.
  • Polyalcohol moieties include, for example, N-methylglucamine and glucose derivatives.
  • Polyether moieties include, for example, polyethelyne glycol, ethoxy ethanol. and ethoxy ethoxy ethanol.
  • Polyamine moieties include, for example, spermine or spermidine.
  • Suitable charged hydrophilic moieties become cither formally negatively or positively charged in water.
  • Suitable negatively charged moieties include acid anions resulting from the dissociation of acids in water.
  • carboxylic acids (-CO 2 H) dissociate to form negatively charged carboxylate ions (-CO 2 -) at pH greater than about 5.
  • Other stronger acids such as phosphoric (-PO 3 H 2 ) and sulfonic (-SO 3 H) acids ionize to form phosphonate (-PO 3 2 -) and sulfonate (-SOX anions, respectively, at pH greater than about 2.
  • Suitable charged solubilizing moieties may also dissociate to form their corresponding anionic derivatives that are also water solubilizing.
  • abasic moieties may become formally positively charged moieties in water. These moieties become highly water soluble through protonation in aqueous solution. For example, at pH about 5, amines (-NR 2 , -NHR, -NH 2 ) become ammonium ions (-NHR 2 +, -NH 2 R+, -NH 3 +), which are water soluble moieties. Quaternary ammonium moieties (-NR 3 +) are water soluble at all pHs. Suitable charged solubilizing moieties also include polylysine groups.
  • Water soluble linkers are preferably relatively linear molecules greater than 4 atoms in length, preferably between 6 and 50 atoms in length, and most preferably about 8 to 20 atoms in length.
  • the linker is a linear molecule of 12-15 atoms in length.
  • the term "atom" refers to a chemical element such as C, N, O, S. or the like. The ranges provided herein are based on the relatively linear accounting of the linker.
  • a linker may be linear, branched, and may contain ring or cone structures.
  • Suitable water soluble linkers preferably comprise at least two coupling or reactive groups allowing the linker to bind lo both a biotin moiety and another moiety, such as another biotin moiety, a reactive moiety, or a functional moiety.
  • Suitable linkers may be homobifunctional, heterobifunctional, homotrifunctional, or heterotrifunctional. Homobifunctional agents may facilitate cross-linking, or dimerization of biotin moieties in a single step.
  • Suitable water soluble homobifunctional. heterobifunctional, homotrifunctional, and heterotrifunctional linkers are available, and additional water soluble linkers may be prepared using commercially available linkers.
  • Empirical factors such as the size (e.g., molecular weight and molecular conformation) and the nature (e.g., charge and constituency) of the biotin moiety or moieties, the reactive moiety or moieties, and the functional moiety it binds affect the choice of linker.
  • Homo- and heterotrifunctional linkers may be coupled to a functional moiety and a biotin moiety as described above, with the additional advantage of a third coupling site on the linker.
  • markers such as radiolabeled and flourescent molecules
  • proteins and peptides such as antibodies: and conjugating molecules.
  • Suitable trifunctinal linkers include, but are not limited to 5-N-Boc amino isophthoyl ditetrafluorophenyl ester; 3,5-diamino methyl benzoate; 5-(p-iodobenzoyl) amino- 1 ,3-isophthaloyl ditetrafluorophenyl ester; 5-(p-tri-N- butylisoamylbenzoyl)-amino-1,3-isophthaloyl ditetrafluorophenyl ester; 4-(4-iodo)3- amidoethyl)-4'-2-(1 ,3-bistosyl)propyl phthalate.
  • Two principle types of water soluble linker moieties are preferred for use in the biotin-containing compounds and biotinylation reagents of the present invention.
  • One type is a non-ionized linker which is made more soluble by functional groups such as ethers or hydroxyl groups.
  • Non-ionized linker moieties render the biotin moiety more water soluble, while retaining the neutral character of the biotin moiety.
  • Non-ionized linker moieties comprise a diamino-ether moiety, such as the commercially available molecule 4,7,10-trioxa-1 ,13-tridecanediamine, shown below as structure 5, or 2,2'-(ethylenedioxy)diethylamine, shown below as structure 6, and tetraethylene glycol, shown below as structure 7.
  • diamino-ether molecules are also advantageous in this application.
  • water soluble linkers may be coupled to a biotin moiety to form a biotin moiety-linker adduct using any one of several means, including, for example, an amide forming reaction, employing an amine group on the linker and the carboxylate coupling site on the biotin moiety.
  • a water soluble linker may be coupled to a biotin moiety through an amide forming reaction employing a carboxylate group on the linker and an amino group on a biotin moiety.
  • the amide forming reaction may include the use of a coupling agent.
  • Suitable coupling agents include carbodiimide coupling agents, such as 1-thyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), 1-benzyl-3-(3- dimethylaminopropyl) carbodiimide (BDC), 1-cyclohexyl-3-(2-morpholinyl-4- ethyl)carbodiimide (CMC), and 1 ,3-dicyclohexylcarbodiimide (DCC).
  • EDC 1-thyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride
  • BDC 1-benzyl-3-(3- dimethylaminopropyl) carbodiimide
  • CMC 1-cyclohexyl-3-(2-morpholinyl-4- ethyl)carbodiimide
  • DCC 1- ,3-dicyclohexylcarbodiimide
  • the amide forming reaction coupling the linker to a water soluble biotin moiety may employ a reactive carboxylic acid group and an amine.
  • Suitable reactive carboxylic acid groups include carboxylic acid derivatives that yield an amide upon reaction with an amine.
  • Such reactive groups include, for example, any reactive carboxylic acid derivative, such as carboxylic acid halides, including acid chlorides and bromides; carboxylic acid anhydrides including mixed acetic anhydrides and triflouroacetic anhydrides; esters including, p-nitrophenyl esters, N-hydroxysuccinimide esters, and tetrafluorophenyl esters. Suitable techniques are described in detail in Bodanszky, Principles of Peptide Synthesis. Springer Verlag, Berlin, 1984.
  • the first reaction scheme depicts conjugation with the carboxylate (or activated carboxylate) of a biotin moiety.
  • the second reaction scheme depicts conjugation wherein the biotin carboxylate functionality has been reduced and activated towards reactions with nucleophiles.
  • the syntheses of representative biotin-containing compounds are set forth below in the examples.
  • R good leaving group (e.g. tetrafluorophen ⁇ l. p-nitrophenol, N- hydroxysuccinimide, etc.)
  • R' good leaving group (e.g. Cl, Br, I, OTs, OMs, etc.)
  • X NH 2 , OH, SH
  • biotin moiety-linker adducts comprising preferred water soluble linkers are shown below as structures 8 - 12.
  • the water soluble linker moieties may also contain other functional groups attached to or within, the chain (e.g. amides) as illustrated in structures 13 and 14, and may be synthesized in a sequential step of reactions.
  • a second type of water soluble linker moiety comprises an ionized or ionizable moiety within the linker.
  • the ionized functionality is preferably at least 3 atoms away from the point of conjugation with the biotin moiety.
  • Functional groups containing a sulfonate or ammonium ion are advantageous.
  • Linker moieties comprising anionic borane and carborane cage molecules are especially preferred, since these moieties provide biotin compounds having enhanced water solubility and a site for radiolabeling..
  • R good leaving group (e.g. tetrafluorophenol, p-nitrophenol, N-hydroxysuccinimide. etc.)
  • R' good leaving group (e.g. Cl, Br, I, OTs, OMs, etc.)
  • A moiety containing an ionic or ionizable functional group
  • Structures 15 - 17 illustrate preferred biotin-linker adducts comprising an ionic or ionizable functional group.
  • Other combinations of ionic or ionizable functionalities and linker moieties may be used (e.g. charged functionalities as part of aromatic ring substitution - aryl sulfonates, aryl ammonium ions) in combination with differing lengths of chains containing the ionic moiety branched from the linker moiety.
  • Another preferred ionic water soluble linker moiety contains anionic boron cage moieties.
  • Structures 18 and 19, shown below, comprise a dodecaborane (icosahedral) cage moiety which has a 2- charge
  • structure 20 comprises a nido-dicarbon carborane cage moiety which has a 1- charge.
  • Other anionic boranes or carboranes may be used as water soluble linkers as well.
  • the borane cage water solubilized linkers are preferably used when it is desirable to radiohalogenate the biotin compound.
  • Yet another water soluble linker moiety comprises polyhydroxyl groups.
  • Biotin-containing compounds illustrated below in structures 21 - 23 are exemplary of biotin-containing compounds comprising water soluble polyhydroxyl linking moieties.
  • Linker moieties comprising from about 2-20 hydroxyl moieties may also be used, wherein the hydroxyl groups are bonded to the linking chain itself (as shown below in structures 21 and 22), or are bonded to a branch point of the linker (as exemplified in structure 23).
  • a cleavable linker is desired between the biotin moiety and the functional molecule, or, preferably, between the water soluble linker moiety bound to the biotin moiety and a reactive or functional group.
  • the cleavable functional group is prepared by the combination of a reactive moiety on the biotin compound with a functional group on a drug or reporter moiety.
  • Compounds 37 and 38, illustrated below, are exemplary of this type of compound.
  • Suitable cleavable linkers include disulfides, esters, imines and specific peptide sequences. Diol-ether containing linker moieties, such as triethylene glycol or tetraetheylene glycol, are used as cleavable linkers for many applications.
  • linker moieties can be used to will form an ester with the biotin carboxylate, rendering it cleavable, either in vitro or in vivo.
  • Linker moieties comprising ether or hydroxyl functionalities, which also contain cleavable disulfide functionalities, are also preferred for some applications.
  • the cleavable linker release the drug in its bioactive form.
  • Modification of the linker moiety attached to the biotin moiety is desirable under certain circumstances to prevent the serum enzyme biotinidase from cleaving the water soluble linker from biotin.
  • Introduction of a steric group alpha to the amine (or another functionality) of the linker which is attached to the biotin carboxylate provides resistance to cleavage by biotindase.
  • Suitable steric moieties include carboxylates. larger alkyl groups, aryl groups, heteroaryl groups and other groups in the same manner.
  • some reduction in binding affinity for biotin-binding proteins may result.
  • biotin compound determines how much steric bulk is desired or can be tolerated in the branched group.
  • Modifications of biotin by conjugation with water soluble linkers possessing a branched chain alpha methyl (or other steric) group are desirable to produce conjugates that are more resistant to in vivo degradation by the enzyme biotinidase.
  • Preferred alpha methyl group containing linkers include 3-aminobutyric acid, 1,2-diaminopropane, and 1 ,5-diaminohexane (Dytek A).
  • biotin moieties By combining a variety of biotin moieties with carboxylate coupled steric moieties and a water soluble linker moiety, water soluble biotin compounds having varying binding affinities with biotin-binding proteins and enhanced resistance to in vivo degradation are provided.
  • biotinylate various compounds such as small molecules, peptides, proteins, oligonucleotides, targeting moieties, diagnostic or therapeutic moieties, and the like.
  • the biotinylation of these compounds is preferably accomplished by conjugation with an activated biotinylation reagent of the present invention comprising a water soluble linker with a reactive functionality.
  • Suitable reactive moieties include amino reactive moieties such as carboxylate active esters including hydroxysuccinimidyl, hydroxybenztriazole, N-hydroxypyrrolidone, phenyl, 2- and 4-nitrophenyl, 2-chloro-4-nitrophenyl, 2-nitro-4-sulfophenyl, cyanomethyl, 2-mecaptopyridyl, 4-fluorophenyl, 2, 4-dichlorophenyl, trichlorophenyl, tetrafluorophenyl, tetrafluorothiophenyl, pentafluorophenyl, and the like; imidate esters such as methyl imidate esters and methyl benzimidates; isocyanates or isothiocyanates; alpha- haloacetamides such as alpha-iodoacetamides and alpha-bromoacetamides; aldehydes such as alkylaldehydes or benzaldehyde,
  • Suitable reactive moieties may alternatively or additionally comprise sulfhydryl reactive moieties such as maleimides and alpha-haloacetamides; oxidized carbohydrate reactive moieties such as amines, hydrazines, acyl hydrazines. and hydroxylamines; and pH-, photo- or heat-activated reactive moieties such as arylazides, diazonium salts, and diazirines.
  • sulfhydryl reactive moieties such as maleimides and alpha-haloacetamides
  • oxidized carbohydrate reactive moieties such as amines, hydrazines, acyl hydrazines. and hydroxylamines
  • pH-, photo- or heat-activated reactive moieties such as arylazides, diazonium salts, and diazirines.
  • biotiny latin reagents of the present invention comprising an activated ester (tetrafluorophenyl), 24; a maleimide group, 25; an iodoacetamide group, 26; an acyl hydrazine group, 27; a hydroxyl amine group, 28; and a nitrophenylazide moiety, 29 are illustrated below.
  • activated ester tetrafluorophenyl
  • maleimide group 25
  • an iodoacetamide group 26
  • an acyl hydrazine group 27
  • These compounds can be conjugated through amines (e.g. 24, 25), sulfhydrvl groups (e.g. 25,26).
  • oxidized carbohydrate, ketone or aldehyde functionalities oxidized carbohydrate, ketone or aldehyde functionalities
  • water soluble biotin-containing compounds may also be prepared by reaction of a biotin-water soluble linker adduct with a variety of cross-linking reagents, such as those described in the Pierce Catalog and Handbook. Homobifunctional, heterobifunctional. homotrifunctional and heterotrifunctional linkers are commercially available. For example, sulfhydryl and disulfide containing water solubilized biotin derivatives are readily available by reaction with various sulfide or disulfide containing cross-linking reagents (e.g. DTSSP, SMPT, SPDP and 2-iminothiolane).
  • cross-linking reagents e.g. DTSSP, SMPT, SPDP and 2-iminothiolane.
  • Biotinylation reagents and biotin-containing compounds of the present invention may be linked to targeting moieties such as monoclonal antibodies, or fragments or constituents thereof.
  • targeting moieties such as monoclonal antibodies, or fragments or constituents thereof.
  • biotin-containing compound be water soluble, since binding proteins are most stable in aqueous media.
  • the monoclonal antibody may be conjugated, through chemical or molecular biology techniques, with the biotin binding protein prior to its binding with the target material, or alternatively, it may be bound with the target material, then the biotin binding protein can be introduced in a second step. In this latter case, the antibody must be biotinylated using one of many different biotinylation reagents prior to targeting of the biotin-binding protein.
  • Water-soluble biotin-containing compounds and biotinylation reagents linked to imageable or therapeutic functional moieties can be used in in vivo applications for imaging and/or therapy of human disorders (e.g. cancer, blood clotting, myocardial infarcts).
  • Example 18 describes a method for preparing a water soluble biotin compound stabilized from biotinidase cleavage and having a radio-iodine reporter moiety.
  • BNCT Boron Neutron Capture Therapy
  • radionuclides can be, and have been, used with targeting moieties such as monoclonal antibodies. See, for example Srivastava, Ed., Radiolabeled Monoclonal Antibodies for Imaging and Therapy, 1988.
  • Biotinylation reagents and biotin-containing compounds comprising a water soluble linker moiety may improve the properties of radionuclides that are used in combination with monoclonal antibody targeting.
  • the p-(radio)halobenzoyl adduct of structure 8, illustrated below as structure 30, can be prepared by radiohalogenati ⁇ n of p-tri-n- butylstannylbenzoyl derivative shown below as structure 31.
  • X 18 F, 75 Br, 76 Br, 77 Br, 80m Br, 122 I, 123 I, 124 I, 125 I, 131 I, 211 At.
  • diagnostic tests are routinely conducted with 125 I. Diagnostic in vivo applications may be accomplished for example, using 77 Br, 123 I or 131 I and employing gamma cameras or SPECT instruments as detection devices. Alternatively, PET scanning may be achieved with the positron emitting radionuclides 18 F, 75 Br, 76 Br, and 124 I.
  • Therapeutic applications may employ beta or positron emitting radionuclides, or the alpha emitting radionuclide
  • Radiohalogen compounds comprising 123 I (shown in structure 30a), 211 At (shown in structure 30b). 123 I (shown in structure 32b), and 21 1 At (shown in structure 32c), below, are particularly beneficial for in vivo diagnostic imaging and therapy applications. Exemplary syntheses of these compounds are illustrated below. Other linking moieties with aryl or vinyl radiohalogen derivatives can also be prepared as described in Wilbur, Bioconjugate Chem., 1992.
  • Biotinylation reagents and biotin-containing compounds that are chelatable to radionuclides other than radiohalogens also benefit from having a water soluble linker moiety between the chelate and the biotin moiety.
  • the water soluble linker moiety provides better interaction with biotin-binding proteins.
  • Water solubilizing biotin-chelate conjugates may be synthesized, for example, by conjugating the trioxobiotin-glycolate TFP ester illustrated in structure 24, with ⁇ -terminal amino derivatives of cheiates known as EDTA and DTPA (described in Subramanian, Cancer Imaging and Radiolabeled Antibodies, pp 183-199, 1990) and cyclic cheiates known as NOTA, DOTA.
  • biotin-containing compounds may provide cheiates for many different radionuclides, including In-1 1 1, Y-90, Ga-67, Ga-68, Cu-64, Cu-67, Sm-153, and Bi-212.
  • the amino-trioxo-biotin compound illustrated as structure 8 may be conjugated with cheiates known as N 2 S 2 (described in Fritzberg, Proc. Natl. Acad. Sci. USA, 85, 4025-4029, 1988) or N 3 S (described in Gerretsen, Cancer Res. 53, 3524-3529, 1993) to chelate Tc-99, Tc-99m, Re-186, Re-188.
  • Another important aspect of the invention is the preparation of radionuclide carrying biotin-containing compounds that exhibit reduced binding affinity for the biotin-binding proteins avidin and streptavidin.
  • Such reduced binding affinity biotin compounds are suitable for targeting a specific sites such as a tumor with a diagnostic radionuclide having reduced binding affinity to obtain information on location, size, vascularity, etc.
  • a higher affinity biotin-containing compound conjugated to a therapeutic radionuclide can then be administered to displace the diagnostic radiolabeled biotin moiety from its target site. In this manner, similar localized antibody-biotin binding protein complexes can be used for both diagnosis and therapy.
  • Biotin-containing compounds comprising UV, visible, or infrared active compounds may be prepared for a variety of applications.
  • the dansyl derivative, 33, or the fluorescein derivative, 34 are useful. These are but two examples of a large number of imaging agents that have various adsorption and photochemical properties. Numerous other imaging agents, such as those described in Haugland, Molecular Probes catalog and references therein, may be conjugated to biotinylation reagents of the present invention and utilized for in vitro or in vivo applications.
  • Biotin-dye conjugates can be used for in vivo detection of disorders when combined with targeting moieties such as monoclonal antibodies.
  • diagnostic dyes preferably have absorption maximum in the range of 550 - 1200 run.
  • Preferred diagnostic dyes are cyanine dyes, and a biotin conjugate of a cyanine dye is illustrated below as compound 35.
  • Many different cyanine dye-biotin moiety conjugates can be prepared by reaction of a terminal amine containing a water soluble biotin moiety, with a cyanine dye containing a carboxylate group, or activated carboxylate group.
  • Suitable conjugation techniques and dyes are described, for example, in Mujumdar, Cytometry 10, 1 1-19, 1989; Southwick, Cytomelry 1 1 , 418-430, 1990; and Mujumdar, Bioconjugate Chem. 4, 105-1 1 1, 1993.
  • Water soluble biotin-containing compounds of the present invention which contain a terminal sulfhydryl group can be reacted with cyanine dyes which are appropriately derivatized, as described in Ernst, Cytometry 10, 3-10, 1989.
  • Photodynamic dyes that are useful for therapeutic applications, such as photodynamic therapy ("PDT”) are described in Rosenthal, Ann Med. 26, 405-409, 1994.
  • Biotinylated PDT-active dye conjugates used in combination with targeting functions provided by monoclonal antibodies/biotin binding proteins, can improve PDT.
  • An exemplary benzoporphyrin derivative which has a desthiobiotin attached is illustrated as compound 36.
  • Compound 36 has an ester linkage to the desthiobiotin so that it can be cleaved from the porphyrin prior to cellular localization.
  • photosensitizer dyes such as those described in Diwu, Pharm. Ther. 63, 1-35, 1994, can be also conjugated with biotin moieties to prepare
  • biotin-containing compounds and biotinylation reagents Another application for water soluble biotin-containing compounds and biotinylation reagents is to provide a targeting system that can be used with therapeutic drugs. Targeting of any number of therapeutic drugs to sites such as tumors can be accomplished with this system, and biotinylated therapeutic drugs can be released at a selected site. However, it is also important in many instances to release the biotin from the drug such that its most active form is in the cell. This may be accomplished by introducing one or more cleavable functional groups at the point of attachment of the drug. For example, a morpholino-doxorubicin (See. Mueller, Antibody,
  • adduct of desthiobiotin is illustrated as compound 37.
  • compound 38 an example of a vinca alkaloid conjugate (Laguzza, J. Med. Chem. 32, 548-555, 1989) of desthiobiotin is illustrated as compound 38. Both of these examples are designed to be released from the antibody-biotin binding protein complex at a preselected site, such as a tumor. Release may be brought about by biotin in the diet (slow release), or it may be brought about by introduction of biotin by other means such as osmotic pump, injection, or the like.
  • Compound 37 has a hydroiyzable hydrazide linkage to the desthiobiotin for its release in the cell (or in acid tumor environment), and
  • biotin-boron-10-containing polymer is prepared from a starburst and cascade dendrimer (See, Tomalia, In Topics in Current Chemistry, 165, 193-313, 1993), where 1 -20 biotins (depending on the size of polymer) are first conjugated with the dendrimer, then 10-200 boron- 10 containing cage molecules (e.g.
  • biotinylated boron-10 containing polymer may be used with prelocalized monoclonal antibody/biotin binding proteins to localize boron-10 to selected sites, such as tumors for subsequent neutron irradiation in a therapeutic protocol.
  • Dimeric, trimeric, and other multimeric biotin compounds can be prepared for cross-linking of the biotin-binding proteins.
  • Biotin dimers, trimers and multimers made using hydrophobic linker moieties are highly insoluble in aqueous solutions, which renders them unusable for in vivo applications.
  • One application for polymerization of biotin-binding proteins is clearance of the antibody-biotin binding protein, or non-bound biotin-binding protein, from the blood of patients which are involved in procedures where targeting of tumor sites in the body is achieved with monoclonal antibodies conjugated with biotin-binding proteins.
  • a few examples of the many possible dimeric biotin-containing compounds comprising water solubilizing linkers are illustrated below as compounds 39 - 47.
  • the compounds illustrated above are considerably more water soluble than their aliphatic counterparts. While biotin dimers linked by aliphatic linker moieties are substantially insoluble, the biotin dimer illustrated as compound 39 has a solubility of about 9 mg/mL in water at ambient temperature. Importantly, the distances between the two biotin moieties are long enough (> 15 ⁇ ) to bind two proteins, but short enough ( ⁇ 20 ⁇ ) such that the two biotins will not bind to the same avidin or streptavidin molecule.
  • Pre-incubation of any of the dimeric biotin derivatives to saturate the biotin binding sites without polymerization may be used to increase the biological half-life of the biotins, and may be used to prepare mixed polymers, wherein, the saturated biotin binding protein can polymerize in a 1 : 1 ratio with another biotin binding protein (as a tetramer) (e.g. ...avidin- streptavidin-avidin-streptavidin-avidin).
  • another biotin binding protein as a tetramer
  • Trimeric biotin compounds may also be prepared for cross-linking of biotin- binding proteins.
  • Compounds 48-51, illustrated below, are exemplary of trimeric biotin compounds comprising water soluble linkers.
  • the biotin moieties are at a distance from one another that permits two of the biotin moieties to bind with one tetrameric biotin binding molecule, while the third biotin moiety will be free because it does not have a linker of sufficient length to bind at a third site on the same protein molecule.
  • the distance between each biotin moiety in a biotin trimer is preferably from about 20 to about 50 ⁇ .
  • the free biotin moiety is the weaker binding desthiobiotin, and this can result in preparing polymers which are unstable. However, unstable polymers can also be cleared from the blood, which is desirable under some circumstances.
  • Biotin trimer 51 has two desthiobiotin moieties. In this example, four of the trimeric biotin molecules can combine with one (tetrameric) avidin or streptavidin molecule. This permits more (total of 8) desthiobiotin moieties on the molecule, and can lead to branching in the polymers formed.
  • Biotin multimers having more than three biotin moieties are useful for blood clearance of biotin binding proteins, and are also useful for other applications such as amplification of signals in diagnostic or therapeutic systems. Multimers in which the biotin moieties are joined by water soluble linker moieties demonstrate enhanced water solubility.
  • a preferred series of compounds which are biotinylated with multiple biotins are known as starburst and cascade dendrimers (See Tomalia, In Topics in Current Chemistry, 165, 193-313, 1993). For example, reaction of a terminal amine containing starburst dendrimer (e.g. generation 2, available from commercial sources) with the activated carboxyl-biotin derivative 24 produces a compound which has up to 16 biotin molecules attached. Attachment of multiple water solubilized biotin derivatives to proteins is achieved through the use of biotinylation reagents such as those shown in compounds 24-29.
  • Dimeric, trimeric and multimeric biotin compounds comprising other functional moieties such as radionuciides, photoactive groups, or drugs preferably incorporate water soluble linker moieties.
  • Such dimeric, trimeric and multimeric compounds are of interest in polymerizing biotin-binding proteins.
  • An important application of these compounds is to increase the amount of radioactivity, photoactive moiety, or drug at a preselected site, such as a tumor, by introducing new biotin sites for biotin-binding proteins to bind to.
  • methods for amplifying the number of sites for binding biotin-binding proteins at a preselected target would involve multiple alternating administrations of a biotin trimer or the like, and a binding protein, such as avidin or streptavidin.
  • a functional reporter moiety such as a targeting, diagnostic, or therapeutic agent, or the like may be linked to the biotin compound or binding protein compound.
  • biotin dimer which can cross-link two streptavidin or avidin molecules while having a radiohalogen (X as previously defined) attached is illustrated as compound 52.
  • the corresponding arylstannane is illustrated below as compound 53.
  • the water solubilizing linkers cannot be attached to the biotin moieties in these compounds as that would lead to the two biotins being of a distance apart that would result in both biotins binding to the same biotin-binding protein, which would not lead to cross-linking or amplification. Therefore, two water solubilizing moieties are combined in the molecule in a location that is unimportant in the binding process.
  • a water soluble (ionic) biotin dimer which does not have the long ether chain is shown as compound 46. Radiohalogenation of 46 provides 54.
  • Water soluble biotinylation reagents can be prepared comprising a biotin moiety, a functionality for conjugation (e.g. amine, carboxylate ester, maleimide, acyl hydrazine, alkoxylamine) and another functionality.
  • a functionality for conjugation e.g. amine, carboxylate ester, maleimide, acyl hydrazine, alkoxylamine
  • These trifunctional reagents have many applications, which are made possible by the water solubility conferred by the water soluble linker moiety.
  • the linker moieties also provide for a distance of separation of the molecule such that they can be conjugated more efficiently.
  • An exemplary reagent is illustrated as compound 55, wherein the biotin-containing trifunctional reagent also has two maleimide groups. This compound may be used to cross-link two other compounds.
  • F(ab') 2 molecules have application in the methods of tumor targeting already presented herein.
  • Reactive groups other than maleimides may also be used for cross-linking.
  • Another trifunctional biotin-containing reagent is shown in compound 56. In that compound there is a biotin, a conjugation group (maleimide) and a radiohalogenated moiety (X as previously defined).
  • Water soluble linker moieties that provide a desired distance between an avidin or streptavidin binding biotin moiety and another moiety that binds to a specific carrier or receptor protein improve such compounds. Water solubility is necessary for the protein interaction, but the water soluble linker moiety also permits the biotin and other molecule to extend from one another in aqueous media such that finding with two different proteins can be achieved. This is particularly important to have an availability of the compound to bind a second protein once the first protein has been bound, and where the protein binding molecule is sensitive to steric encumbrance to its binding pocket.
  • An example of such a protein binding molecule is the cyanocobalamin conjugate of biotin, illustrated below as structure 57.
  • Compound 57 was found to be much more water soluble than the corresponding 1,12-diaminododecane linked biotin and cyanocobalamin.
  • the improved water solubility of biotin compound 57 over the alkyl counterpart made this compound much more useful.
  • the combination of other specific protein binding molecules such as hormones (e.g. steroids), neurologic binding molecules, such as those that bind to dopamine or seratonin receptors, peptides that bind to receptors (e.g. somatastatin), and the like with biotin with the water solubilizing linkers improve their properties as well.
  • Biotin (10 g, 40.9 mmol) was dissolved in 200 mL warm (70°C) DMF under argon atmosphere. The solution was allowed to cool to ambient temperature, and 10 mL (82 mmol) triethylamine was added, followed by the addition of 16 g (61 mmol) 2,3,5,6-tetrafluorophenyl trifluoroacetate. The reaction was stirred at room temperature for 30 min and solvent was removed under vacuum. The product was triturated in 100 mL ether and was filtered.
  • This example sets forth a general methodology for preparing a biotin moiety linked to a soluble linker moiety.
  • a diamino-ether linker is used.
  • Ether linkers containing terminal functionalities such as: an amine and a carboxylate; an amine and an alcohol; an alcohol and a carboxylate; and two alcohols, can also be prepared using the method described.
  • the general method can also be used when the linker contains polyhydroxyl groups if the terminal functionalities are two amines, or are an amine and a carboxylate.
  • This example sets forth a general methodology for preparing a biotin moiety linker adduct comprising a diamino-linker molecule, in which the linker has one amine group protected.
  • a diamino-ether linker is protected as a t-BOC derivative prior to addition to the biotin moiety active ester molecule.
  • Any number of amine protecting groups can be utilized in this reaction.
  • the primary purpose of the reaction method (versus example 2) is to be able to react one equivalent of diamino linker molecule with one equivalent of the biotin active ester.
  • Reaction Step 1 Preparation of N-BOC-4,7,10-trioxa-tridecane-13-amine: To a solution of 151.40 g (687.25 mmol) of 4,7,10-Trioxa-1,13-tridecaneamine in 700 mL CHCl 3 was added 6.00 g (27.5 mmol) of di-tert-butyl dicarbonate in 100 mL CHCl 3 with stirring at ambient temperature over 30 min.
  • Reaction Step 2 Preparation of N-(13-N-BOC-4,7,10-trioxatridecanyl)biotinamide: To a solution containing 1.00 (2.54 mmol) 2,3,5,6-tetrafluorophenol ester of biotin in 100 mL CH 3 CN at 55 °C was added 0.80 g (2.49 mmol) N-BOC-4,7,10-trioxa-13-tridecaneamine in 25 mL CH 3 CN. The mixture was allowed to cool to ambient temperature and was stirred for 10 h. The solvent was evaporated under reduced pressure and the residue was redissolved in 200 mL EtOAc.
  • Reaction Step 3 Preparation of 13-N-Biotinamidc-4,7,10- rioxa-tridecaneamine trifluoroacetic acid salt: N-BOC-trioxa-biotinamide, 1.25 g (2.28 mmol), was dissolved in 10 mL trifluoroacetic acid and stirred for 30 min at ambient temperature.
  • This example sets forth a general methodology for preparing a biotin compound comprising an w-terminal carboxylate.
  • the method described can be applied in a general way to prepare biotin moieties with any number of linker molecules attached. While in the example described a diamino-ether linker is used, examples wherein the linker has an amine and an alcohol at the termini are also provided for with this method.
  • Biotin-4,7,10-trioxa-1,13-tridecanediamine (1 g, 2.24 mmol) was added to a dry flask and was dissolved in anhydrous CH 3 CN/DMF (100/25 mL) To this solution was added 0.4 mL (2.9 mmol) triethylamine, followed by 0.31 g (2.7 mmol) diglycolic anhydride. The reaction was stirred at room temperature for 1 h, and the solvent was removed under vacuum. The product was triturated in 100 mL ether and was filtered.
  • This example sets forth a general methodology for preparing a water soluble aryl stannyl biotin compound, which can be used to incorporate any number of halogens/radiohalogens into a biotin compound.
  • the radiohalogen, iodine-125 is used, and the corresponding stable iodoaryl biotin is prepared for an HPLC standard.
  • TFP ester of tributyltinbenzoate (0.628 g, 1.12 mmol) was added in a dry flask and was dissolved in 10 mL of anhydrous DMF.
  • TFP ester of tributyltinbenzoate was added dropwise to the biotin-trioxatridecanediamine solution over the period of 10-15 min.
  • Biotin - 4,7,10-trioxa- 1, 13-tridecane-diamine (0.5 g, 1.12 mmol) was added to a dry flask and was dissolved in 25 mL anhydrous DMF.
  • Triethylamine (0.4 mL, 2.9 mmol) was added to the solution followed by 0.266 g (1.1 mmol) P-iodobenzoyl chloride.
  • the reaction was stirred overnight at room temperature, and the solvent was removed under vacuum. The residue was extracted with 200 mL chloroform and the chloroform was washed with (2 ⁇ 15 mL) water.
  • the chloroform solution was dried over anhydrous sodium sulfate, then the chloroform was removed under vacuum. The resultant residue was dried under vacuum to yield 0.6 g (80%) of the desired product as a colorless solid, mp 107-109°C.
  • Radioiodination procedure A 50 ⁇ L quantity of a 1 mg/mL solution of biotin-trioxadiamine-tributyltinbenzoate in MeOH/5% HOAc was placed in a small vial. To that solution was added (via syringe), 1 ⁇ gL of Na 125 I solution (275 ⁇ Ci in 0.1N NaOH), followed by 10 ⁇ L of a 1 mg/mL solution of N-chlorosuccinimide in MeOH. After 5 min. at r.t., 10 ⁇ L of sodium metabisulfite (1 mg/mL in water) was added to quench the reaction.
  • the entire reaction mixture was then injected on the HPLC and 275 ⁇ Ci* was collected (peak at 4.2 min).
  • the HPLC conditions were: an Alltima C18 column (Alltech); a gradient that began with 70% MeOH / 30% water for 2 min; increased to 100% MeOH over the next 13 minutes; then was held at 100% MeOH for 10 min. [Note: safety precautions for handling of radioiodine must be followed; The quantity of iodine-125 measured can vary by the thickness of vessel walls that it is measured in]
  • the UV active dansyl molecule activated towards reaction as the sulfonyl chloride derivative, is reacted with 4,7,10-1,13-tridecanediamine to yield the dansyl adduct, then this adduct is reacted with the TFP ester of biotin to yield a water solubilized UV active dansyl-biotin derivative, 33.
  • biotin-linker adduct 8 is reacted with commercially available fluorescein isothiocyanate.
  • Biotin-trioxa-amine adduct 8 (0.5 g, 1.12 mmol) is dissolved in 25 mL anhydrous DMF and 0.4 mL triethylamine is added. To that solution 0.48 g (1.23 mmol) fluorescein isothiocyanate is added. The reaction mixture is stirred at room temperature for 20 h and the solvent is removed under vacuum. The residue is extracted with chloroform, and the chloroform solution is washed with water. The chloroform solution is then dried over sodium sulfate and the chloroform is removed under vacuum. The resultant solid is dried under vacuum. Purification is accomplished by silica gel column chromatography.
  • biotin-linker adduct 8 is reacted with the rigidized cyanine dye, prepared by the method of Lipowska (Synth. Commun. 3087-3094, 1993)
  • Biotin-trioxa-amine adduct 8 (0.5 g, 1.12 mmol) is dissolved in 25 mL anhydrous DMF and 0.4 mL triethylamine is added. To that solution 1.25 mmol rigidized cyanine dye is added. The reaction mixture is stirred at room temperature for 30 min and the DMF is removed under vacuum, keeping the bath temperature ⁇ 50°C. The residue is triturated with ether to give the desired biotin-cyanine adduct, 26. Purification is accomplished by column chromatography. Example 9
  • Step 1 A 2.0 g (1.47 mmol) quantity of a cyanocobalamin monocarboxylic acid, 2 or 4, and 0.68 g (5.9 mmol) of N-hydroxysuccinimide (NCS) were dissolved in 100 mL of water. To that mixture was added 1.46 g (29 mmol) of NaCN, then 16 g (36 mmol) of 4,7,10-trioxa-1,13-tridecanediamine was added, and the pH was adjusted to 6 with 1 N HCl.
  • NCS N-hydroxysuccinimide
  • the remaining solid was dissolved in 50 mL of H 2 O and applied to an Amberlite XAD-2 (200 g; 4 cm ⁇ 60 cm) column.
  • the column was eluted with 1 L water, then the desired product was eluted with 500 mL methanol.
  • the methanol fractions were evaporated to dryness, and the residue was dissolved in 25 mL of water and was applied to a ion exchange column (100 g; 2.5 cm ⁇ 60 cm; acetate form; 200-400 mesh).
  • the final product was eluted using 250 mL water, thereby leaving non-converted acid bound to the column, which was later eluted with 0.04 mol/L sodium acetate buffer pH 4.7.
  • the following two examples set forth a general methodology for preparing water soluble dimers of biotin moieties.
  • the length between the two biotin carboxylates (when fully extended) is important, and the examples therefore describe reactions containing two different linker lengths.
  • the same reaction conditions provide dimers of different biotin moieties, and provide dimers wherein the terminus of the linker are hydroxyls or one hydroxyl and one amine.
  • the biotin dimer has a distance between the carboxylates which allows cross-linking (and polymerization) of biotin-binding proteins.
  • the following two examples set forth a general methodology for preparing water soluble trimers of biotin moieties.
  • the length between any two of the three biotin carboxylates is important and the examples therefore describe reactions utilizing two different linker lengths.
  • the same reaction conditions provide trimers of modified biotins, and provide biotin trimers wherein the terminus of the linker are hydroxyls or one hydroxyl and one amine.
  • the desired trimer is obtained by reaction of the water solubilizing linker with the trifunctional reagent, then addition of the biotin to that adduct.
  • the biotin adduct of the water solubilizing linker e.g. 8 or 9
  • the trimesic acid chloride is reacted with the trimesic acid chloride to obtain the desired biotin trimer.
  • Reaction Step 1 Preparation of N,N',N"-tris-(13-N-BOC-4,7,10-trioxatridecanyl)benzene-1,3,5-tricarboxyamide: To a solution of benzene-1,3,5-tricarbonyl trichloride (0.28 g, 1.05 mmol) in 10 mL of CH 2 Cl 2 at 0 °C was added 0.96 g (2.99 mmol) of 13-N-BOC-4,7,10-trioxa-tridecaneamine [see Example 3] and 1.45 g (14.34 mmol) triethylamine in 5 mL CH 2 Cl 2 .
  • N,N',N"-tris-(13-amino-4,7,10-trioxa-tridecanyl)benzene-1,3,5-tricarboxamide trifluoroacetic acid salt The N-BOC protected triamide (0.25 g, 0.23 mmol) was dissolved in 5 mL trifluoroacetic acid at ambient temperatuie and stirred for 30 min.
  • Reaction Step 1 Preparation of 8-N-BOC-3,6-dioxa-octaneamine: To a solution of 163 g (1100 mmol) of 3,6-dioxa-1,8-octaneamine in 700 mL CHCl 3 was added 8.00 g (36.65 mmol) of di-tert-butyl dicarbonate in 100 mL CHCl 3 with stirring at ambient temperature over 30 min.
  • This experiment was designed to test streptavidin cross-linking with biotin monomer, three biotin moiety dimers comprising water soluble linkers, and th ree biotin moiety trimers comprising water soluble linkers.
  • biotin dimers and trimers used experimentally was water soluble, that is, each biotin compound had a water solubility in excess of 0.2 mg/mL at neutral pH and ambient temperature.
  • Biotin dimers illustrated herein as compounds 39 and 40 and trimers illustrated herein as compounds 48 and 49 were used in this experiment.
  • a Reacti-Bind Streptavidin Coated Polystyrene Strip Plate (Pierce) was washed with 100 mM PBS prior to use. To lane one (12-wells) was added 100 ⁇ L PBS. To lanes 2-7 were added 100 pmole of biotin dimer or biotin trimer to be tested in 100 ⁇ L PBS. Lane 8 was treated in the same manner with 100 pmoles of biotin monomer. The plate was then incubated at 37 °C with shaking for 10 minutes. All the wells were then emptied and rinsed with 100 ⁇ L PBS.
  • the PBS was removed and 25 pmoles of 125 I labeled streptavidin (specific activity of 1 ⁇ Ci/ug) was added to each well in 100 ⁇ L PBS. The plate was then incubated with shaking for another 10 minutes. The streptavidin was removed and all the wells were washed with 100 ⁇ L PBS. After removal of the PBS, the first 3 wells of each lane were again filled with 100 ⁇ L PBS. The remaining wells were then filled with 100 ⁇ L of their respective biotin dimer or biotin trimer as in the first step. The plate was then incubated again for 10 minutes, and all wells were rinsed with PBS as before.
  • the Figure illustrates the percentage streptavidin binding of the biotin monomer and the dimers and trimers as a function of sequential biotin compound additions.
  • Each of the biotin trimers used demonstrated increased streptavidin binding with each sequential biotin trimer addition.
  • Neither the biotin monomer nor any of the biotin dimers tested demonstrated appreciable increases in streptavidin binding with sequential biotin compound additions.
  • biotin trimers of the present invention can be successfully used to provide amplification of binding sites for complementary binding moieties, such as streptavidin.
  • the water solubility of such biotin dimers, trimers and multimers is important in in vivo applications for such compounds, such as amplification of binding sites at a preselected site, e.g. a tumor, by repeated administration of a biotin dimer, trimer, multimer compound, or combinations of such compounds.
  • the TFP ester, 24 is prepared by esterification of the biotin-trioxa-amido- glycolate, 13, with tetrafluorophenol. Under the same reaction conditions any number of activated esters can be prepared by substitution of different phenols, or other alcohols (e.g. N-hydroxysuccinimide).
  • Biotin - 4,7,10-trioxa-1, 13-tridecanediamine - diglycolic carboxylate (1.0 g, 1.78 mmol) was added in a dry flask and was dissolved in anhydrous CH 3 CN/DMF (75/25 mL).
  • Biotin-4,7,10-trioxa-1,13-tridecanediamine (1.0 g, 2.2 mmol) was dissolved in 12 mL of saturated aqueous sodium bicarbonate and was cooled with ice-water N- Methoxycarbonylmaleimide (4.5 mmol, 0 696 g) was added and the reaction was stirred at
  • a solution of 0.5 g of biotin-trioxamido-glycolate TFP ester in 50 mL anhydrous THF is added dropwise to a solution containing 5 mL anhydrous hydrazine in THF at 0 °C. After the addition is complete, the reaction solution is allowed to come to room temperature over a 1 h period. The THF and excess hydrazine are removed under vacuum. The residue is triturated with 100 mL ether and the solid product is collected by filtration
  • N-Methylglyclbiotinamide methyl ester was hydrolized in a mixture of 31.5 mL
  • N-(13'-Amino-4',7',10' -trioxatridecanyl)-4-iodobenzamide A 2.0 g (5.05 mmol) quantity of 3 dissolved in 100 mL anhydrous DMF was added dropwise over 1 h to a mixture of 11 g (50 mmol) 4,7,10 -trioxa-1,13-tridecanediamine and 2 mL Et 3 N. The reaction was stirred at rt for 30 min and solvent was removed under vacuum with rotary evaporation. The resulting oil was triturated in 200 mL of ether. The produce was extracted with CH 3 Cl (2 ⁇ 100 mL).
  • N-(13'-Amino-4',7',10'-trioxatridecanyl)-4-tributylstannylbenzamide A 2.0 g quantity (3.57 mmol) of 4 dissolved in 50 mL anhydrous DMF was added dropwise over 1 h to a mixture containing 8 g (35.7 mmol) 4,7,10-trioxa-1,13-tridecanediamine and 1 mL Et 3 N. The reaction mixture was stirred at rt for 30 min and solvent was removed under vacuum. The resulting oil was triturated in 200 mL of ether. The product was extracted with CH 3 Cl (2 ⁇ 100 mL).
  • N-(13-(p-tri-n-butylstannylbenzamido)-4,7,10-trioxatridecanyl)-N- methylglycylbiotin-amide N-(13-(p-tri-n-butylstannylbenzamido)-4,7,10-trioxatridecanyl)-N- methylglycylbiotin-amide.
  • 0.25 g (0.8 mmol) quantity of N- methylglycylbiotinamide dissolved in 15 mL DMF under argon atmosphere was added 0.25 g (p.96 mmol) 2,3,5,6-tetraflourophenyl triflouroacetate followed by 0.119 mL (0.96 mmol) Et 3 N.
  • the reaction mixture was stirred at rt for 20 min.
  • N'-(13-(p-iodobenzamido)-4,7,10-trioxatridecanyl)-N-methylglycylbiotinamide N'-(13-(p-iodobenzamido)-4,7,10-trioxatridecanyl)-N-methylglycylbiotinamide.
  • N-methylglycylbiotinamide dissolved in 12 mL DMF under argon atmosphere was added 0.15 g (0.576 mmol) 2,3,5,6-tetraflourophenyl triflouroacetate and 71 ⁇ L (0.576 mmol) Et 3 N.
  • the reaction mixture was stirred at rt for 20 min.

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Abstract

L'invention concerne des composés contenant de la biotine et des réactifs de biotinylation hydrosolubles comprenant des fractions de liaison soluble. Les composés contenant de la biotine et les réactifs de biotinylation hydrosolubles peuvent également comprendre une ou plusieurs fractions conférant une résistance au clivage par la biotinidase ou clivable in vitro ou in vivo. Les composés contenant de la biotine et les réactifs de biotinylation peuvent comprendre une fraction réactive fournissant un site de réaction avec une autre fraction, telle qu'une fraction fonctionnelle de ciblage, diagnostique ou thérapeutique. L'invention concerne également des conjugués comprenant des fractions fonctionnelles. De plus, l'invention concerne des dimères, des trimères et des multimères de biotine comprenant des fractions de liaison hydrosolubles présentant une hydrosolubilité supérieure, des procédés d'amplification du nombre de sites destinés à la liaison de protéines de liaison de biotine sur une cible sélectionnée, à l'aide de composés trimères de biotine, des procédés de synthèse des composés contenant la biotine et des réactifs de biotinylation, ainsi que des procédés d'utilisation de ces composés et un exemple de l'invention, la réticulation de la streptavidine avec des dérivés de biotine est illustré par la figure.
PCT/US1997/002560 1996-02-08 1997-02-07 Composes contenant de la biotine, reactifs et procedes de biotinylation WO1997029114A1 (fr)

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AU20524/97A AU2052497A (en) 1996-02-08 1997-02-07 Biotin-containing compounds, biotinylation reagents and methods

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000002051A1 (fr) * 1998-07-07 2000-01-13 Dept. Of Radiation Oncology, University Of Washington Reactif trifonctionnel pour la conjugaison avec une biomolecule
EP0920623A4 (fr) * 1996-07-26 2000-03-29 Immunomedics Inc Therapie par capture de neutrons de bore dans laquelle des procedes de pre-ciblage sont utilises
WO2000033874A3 (fr) * 1998-12-04 2000-10-12 Immunomedics Inc Therapie par capture des neutrons de bore a base de techniques de preciblage
WO2001095857A2 (fr) 2000-06-16 2001-12-20 Mitra Medical Technology Ab Derives de biotine
EP1196199A4 (fr) * 1999-06-02 2002-06-19 Univ Washington Composes hydrosolubles multi-biotine
WO2002042427A3 (fr) * 2000-10-25 2003-03-06 Surromed Inc Marqueurs de masse pour analyse quantitative
WO2004062572A3 (fr) * 2003-01-03 2004-12-09 Bayer Healthcare Llc Procede permettant de determiner la densite de fragments fonctionnels sur des reactifs polymeres
EP0837696A4 (fr) * 1995-06-07 2005-09-28 Immunomedics Inc Apport ameliore d'agents diagnostiques ou therapeutiques a un site cible
RU2279896C2 (ru) * 2000-06-16 2006-07-20 Митра Медикал Текнолоджи Аб Многоразовые экстракорпоральные колонки, загруженные конъюгатами лиганд-дибиотин
JP2007507522A (ja) * 2003-10-02 2007-03-29 ザ ジェネラル ホスピタル コーポレーション 磁気共鳴画像法および薬物送達のためのポリビオチン化合物
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
CZ302510B6 (cs) * 2010-03-29 2011-06-22 Univerzita Palackého v Olomouci Efektivní zpusob biotinylace sloucenin s karboxylovou skupinou pomocí syntézy na pevné fázi pro potreby afinitní chromatografie
CZ302669B6 (cs) * 2010-03-29 2011-08-24 Univerzita Palackého v Olomouci Efektivní zpusob biotinylace aminosloucenin pomocí syntézy na pevné fázi pro potreby afinitní chromatografie
WO2013036826A3 (fr) * 2011-09-09 2014-05-15 The Trustees Of Columbia University In The City Of New York Complexes de streptavidine et leurs utilisations
US8951499B2 (en) 1996-02-08 2015-02-10 University Of Washington Trifunctional reagent for conjugation to a biomolecule
JP2015514702A (ja) * 2012-03-30 2015-05-21 ゼネラル・エレクトリック・カンパニイ Ｈｐｌｃフリー放射性ヨウ素化のためのビオチンスタナン
US10328149B2 (en) 2014-06-13 2019-06-25 Tenboron Oy Conjugates comprising an anti-EGFR1 antibody
CN113924124A (zh) * 2019-05-31 2022-01-11 D&D制药技术股份有限公司 与生物素部分结合的生理活性物质和包含所述生理活性物质的用于口服施用的组合物

Citations (1)

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US5482698A (en) * 1993-04-22 1996-01-09 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin polymer conjugates

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US5482698A (en) * 1993-04-22 1996-01-09 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin polymer conjugates

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JOURNAL OF IMMUNOASSAY, 1995, Vol. 16, No. 1, DRESSENDORFER et al., "A Non-Isotopic Immunoassay for Guanosine 3':5'-Cyclic Monophosphate Using a Cyclic GMP-Biotin Conjugate as Tracer", pages 37-53. *
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Cited By (25)

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US6228362B1 (en) 1992-08-21 2001-05-08 Immunomedics, Inc. Boron neutron capture therapy using pre-targeting methods
EP0837696A4 (fr) * 1995-06-07 2005-09-28 Immunomedics Inc Apport ameliore d'agents diagnostiques ou therapeutiques a un site cible
US8951499B2 (en) 1996-02-08 2015-02-10 University Of Washington Trifunctional reagent for conjugation to a biomolecule
EP0920623A4 (fr) * 1996-07-26 2000-03-29 Immunomedics Inc Therapie par capture de neutrons de bore dans laquelle des procedes de pre-ciblage sont utilises
WO2000002050A1 (fr) * 1998-07-07 2000-01-13 Department Of Radiation Oncology, University Of Washington Reactif trifonctionnel pour la conjugaison avec une biomolecule
WO2000002051A1 (fr) * 1998-07-07 2000-01-13 Dept. Of Radiation Oncology, University Of Washington Reactif trifonctionnel pour la conjugaison avec une biomolecule
WO2000033874A3 (fr) * 1998-12-04 2000-10-12 Immunomedics Inc Therapie par capture des neutrons de bore a base de techniques de preciblage
EP1196199A4 (fr) * 1999-06-02 2002-06-19 Univ Washington Composes hydrosolubles multi-biotine
RU2279896C2 (ru) * 2000-06-16 2006-07-20 Митра Медикал Текнолоджи Аб Многоразовые экстракорпоральные колонки, загруженные конъюгатами лиганд-дибиотин
JP2004503299A (ja) * 2000-06-16 2004-02-05 ミトラ、メディカル、テクノロジー、アクチボラグ ビオチン誘導体
WO2001095857A3 (fr) * 2000-06-16 2002-03-28 Mitra Medical Technology Ab Derives de biotine
WO2001095857A2 (fr) 2000-06-16 2001-12-20 Mitra Medical Technology Ab Derives de biotine
US8124364B2 (en) 2000-06-16 2012-02-28 Glycorex Transplantation Ab Biotin derivatives
WO2002042427A3 (fr) * 2000-10-25 2003-03-06 Surromed Inc Marqueurs de masse pour analyse quantitative
WO2004062572A3 (fr) * 2003-01-03 2004-12-09 Bayer Healthcare Llc Procede permettant de determiner la densite de fragments fonctionnels sur des reactifs polymeres
US8092782B2 (en) 2003-10-02 2012-01-10 The General Hospital Corporation Polybiotin compounds of magnetic resonance imaging and drug delivery
JP2007507522A (ja) * 2003-10-02 2007-03-29 ザ ジェネラル ホスピタル コーポレーション 磁気共鳴画像法および薬物送達のためのポリビオチン化合物
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
CZ302669B6 (cs) * 2010-03-29 2011-08-24 Univerzita Palackého v Olomouci Efektivní zpusob biotinylace aminosloucenin pomocí syntézy na pevné fázi pro potreby afinitní chromatografie
CZ302510B6 (cs) * 2010-03-29 2011-06-22 Univerzita Palackého v Olomouci Efektivní zpusob biotinylace sloucenin s karboxylovou skupinou pomocí syntézy na pevné fázi pro potreby afinitní chromatografie
WO2013036826A3 (fr) * 2011-09-09 2014-05-15 The Trustees Of Columbia University In The City Of New York Complexes de streptavidine et leurs utilisations
JP2015514702A (ja) * 2012-03-30 2015-05-21 ゼネラル・エレクトリック・カンパニイ Ｈｐｌｃフリー放射性ヨウ素化のためのビオチンスタナン
US10328149B2 (en) 2014-06-13 2019-06-25 Tenboron Oy Conjugates comprising an anti-EGFR1 antibody
US10835606B2 (en) 2014-06-13 2020-11-17 Tenboron Oy Conjugates comprising an anti-EGFR1 antibody
CN113924124A (zh) * 2019-05-31 2022-01-11 D&D制药技术股份有限公司 与生物素部分结合的生理活性物质和包含所述生理活性物质的用于口服施用的组合物

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