CA2264592C - Bioconjugates and delivery of bioactive agents - Google Patents

Abstract

The present invention relates to bioconjugates and the delivery of bioactive agents which are preferably targeted for site-specific release in cells, tissues or organs. More particularly, this invention relates to bioconjugates which comprise of bioactive agent and an organocobalt complex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom of the oragnocobalt complex. The bioactive agent is released from the bioconjugate by the cleavage of the covalent bond between the bioactive agent and the cobalt atom in the oragnocobalt complex. The cleavage may occur as a result of normal displacment by cellular nucleophiles or enzymatic action, but is preferably caused to occur selectively at a predetermined release site by application of an external signal. The external signal may be light or photoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if the photolysis takes places in the presence of a magnetic field surrounding the release site, the release of the bioactive agent into surrounding healthy tissue is minimized.

Description

10202530CA 02264592 1999-02-25WO 98/08859 PCT /U S97! 14140T T FBIOCONJUGATES AND DELIVERY OF BIOACTIVE AGENTSThis invention was made in part with Government support under Grant No. ES05728 awardedby the National Institutes of Health, Bethesda, Maryland. The United States Government hascertain rights in the invention. The present invention relates to bioconjugates and the delivery of bioactive agents whichare preferably targeted for site—specific release in cells, tissues or organs. More particularly, thisinvention relates to bioconjugates which comprise a bioactive agent and an organocobaltcomplex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom ofthe organocobalt complex. The bioactive agent is released from the bioconjugate by thecleavage of the covalent bond between the bioactive agent and the cobalt atom in theorganocobalt complex. The cleavage may occur as a result of normal displacement by cellularnucleophiles or enzymatic action, but is preferably caused to occur selectively at apredetermined release site by application of an external signal. The external signal may be lightor photoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if thephotolysis takes place in the presence of a magnetic field surrounding the release site, the releaseof the bioactive agent into surrounding healthy tissue is minimized.The publications and other materials used herein to illuminate the background of theinvention, and in particular, cases to provide additional details respecting the practice, areincorporated by reference, and for convenience are referenced in the following text by authorand date and are listed alphabetically by author in the appended bibliography.The focus of a substantial body of research has been the development of a systemwhereby a pharmaceutical agent can be selectively delivered to a desired anatomic location;namely the site in need of treatment. In spite of the great progress which has been achieved inthis regard, many pharmaceutical delivery systems for the treatment of various diseases or healthrisks, e.g., the treatment of cancer, impart substantial risk to the patient. With respect to thetreatment of cancer, drugs which are effective in attacking malignant cells to destroy them, or atleast limit their proliferation, have a tendency to attack benign cells also. Therefore, it is highlydesirable to limit the location of their action to that of the malignancy, and to ensure that at anyparticular time effective, but not excessive, amounts of such drugs are used.1015202530CA 02264592 1999-02-25W0 98I08859 PCT/US97/14140-2-Although it is desired to concentrate a cytotoxic agent at a targeted site, current cancertreatment protocols for administering these cytotoxic agents typically call for repeatedintravenous dosing, with careful monitoring of the patient. The drugs are often used incombination to exert a multi-faceted assault on neoplastic cells. The dose is selected to be justbelow the amount that will produce acute (and sometimes chronic) toxicity that can lead tolife-threatening cardiomyopathy, myelotoxicity, hepatic toxicity, or renal toxicity. Alopecia(hair loss), mucositis, stomatitis, and nausea are other common, but generally notlife-threatening, side effects at these doses. Since many of these compounds are potentvesicants, tissue necrosis will occur if localized extravasation (loss of the drug from blood to thesurrounding tissue) occurs. These effects occur since the blood generally attains a specifiedconcentration of that drug before becoming effective. Because the blood is transportedthroughout the body of the host being treated, so is the pharmaceutical agent. Following thistechnique provides an even distribution of the drug throughout the body, rather thanconcentrating it at the treatment site. Moreover, such systemic treatment methods expose thehealthy cells to the cytotoxic agent concurrent with the treatment of the unhealthy or diseasedcells besides limiting the concentration of the drug at the site where it is most needed.Previous attempts to administer such drugs by direct injection into the location of theorgan having the malignancy are only partially effective, because of migration of the drug fromthat location and as a result of extensive tissue necrosis from extravasation. Such dispersioncannot be totally prevented, with the result that excessive quantities of drug need to beadministered to attain a desired result. Although careful clinical monitoring may preventextensive damage or loss of viable tissue, the providing of a pharmaceutical agent-carrier systemwhich is actively transported through standard biological systems to the treatment site prior toactivation of the pharmaceutical agent would be highly desirable not only in optimizingutilization of the drug but also in the reduction of side effects and/or the minimization of thedestruction of healthy cells. The direct injection of cytotoxic agents into solid tumors of thebreast, bladder, prostate and lung using conventional cytotoxic chemotherapeutic agents asadjuvants to surgery and/or radiotherapy has been of limited success in prolonging the lives ofpatients. This is partially due to the dose limitations imposed by the acute and chronic toxicityto tissues or organ systems beyond those that are targeted.10202530CA 02264592 1999-02-25PCT/US97/14140WO 98/08859-3-As it relates to the administration of cytotoxic or antineoplastic drugs, the effectiveresolution of concerns relating to modes of administration, to the limitation of dosage size andfrequency of administration, and to side effects would certainly be of benefit to the treatment ofcancer.Oligonucleotides that specifically interfere with gene expression at the transcriptional ortranslational levels have the potential to be used as therapeutic agents to control the synthesis ofdeleterious proteins associated with viral, neoplastic or other diseases. It is possible to selectsingle-stranded oligonucleotides that recognize and bind to the major groove of a stretch ofdouble-stranded DNA in a sequence-specific manner to form a triple helix (Le Doan et al., 1987;‘Moser and Dervan, 1987). Triple helix-forrning oligonucleotides targeted to the promoter regionof certain genes have been used to physically block RNA synthesis in cell-free transcriptionassays (Cooney et al., 1988; Postel et al., 1992; Skoog et al., 1993; Rando et al., 1994).Similarly, in vitro translation assays have been used to demonstrate that antisenseoligonucleotides can bind mRNA targets and prevent protein synthesis (Uhlmann and Peyman,1990; Cohen and Hogan, 1994).Although antisense oligonucleotides have shown great efficacy in the selective inhibitionof gene expression (Stein and Cohen, 1988; Szczylik et al., 1991; Gray et al., 1993), thetherapeutic applications of such antisense oligonucleotides are currently limited by their lowphysiological stability, slow cellular uptake, and lack of tissue specificity. The instabilityobstacles have been largely overcome by use of backbone-modified oligonucleotides that aremore resistant to nucleases. Methylphosphonates, protein-nucleic acid conjugates, andphosphorothioates all appear to resist enzymatic digestion better than the corresponding naturaloligonucleotides (Chang and Miller, 1991; Wickstrom et al., 1992; Letsinger, 1993; Zon, 1993).Problems with cellular uptake of antisense oligonucleotides have been more difficult tosolve. Endogenous uptake pathways that rely on pinocytosis and related processes generallyhave insufficient capacity to deliver the quantities of antisense of oligonucleotides required tosuppress gene expression (Vlassov et al., 1994). Hydrophobic modifications have also beenundertaken to improve membrane permeability, but such derivatization strategies are most usefulonly for short oligonucleotides (Vlassov et al., 1994). Although complexes of antisense constructswith cationic liposomes or immunoliposomes (Gao and Huang, 1991; Bennett et al., 1992, Ma andWei, 1996) and polylsine (Trubetskoy et al., 1992; Bunnell et al., 1992) have significantly1015202530CA 02264592 1999-02-25WO 98/08859 PCT/US97/14140-4- "-enhanced intracellular delivery, they have simultaneously introduced new disadvantages of theirown. Thus, both methods exhibit some carrier cytotoxicity, and like other protocols, neitherstrategy allows for any tissue or cell targeting. In short, intracellular delivery and tissuespecificity remain major obstacles to the implementaiton of antisense drugs in the treatment ofhuman disorders.Other techniques for the delivery of oligonucleotides to cells include the use of: (a)folate-PEG-liposome constructs for the delivery of antisense DNA against growth factorreceptor (Wang et al., 1995); (b) folic acid-polylysine constructs for the delivery of c-mycantisense DNA (Ginobbi et al., (1997); (c) tris(N-acetylgalactosamine aminohexyl glycoside)amide of tyrosyl(g1utamyl)-glutamate (YEE(GalNAcAH)3) linked to polylysine for the deliveryof DNA to cells via the asialoglycoprotein receptor (Merwin et al., 1994); and (d) water-solubleblock polycations (Kabanov et al., 1995).It has been known for some time that a pharmaceutically active agent can be attached to acarrier or molecule. The term "prodrug“ is often associated with such systems wherein theactive agent is bonded to another molecule for purposes of administration. The drug is usuallyinactive in the prodrug state and the bond is later cleaved releasing the drug at a site where it canbe effective. However, such systems are not as useful as might be desired for various reasons,site specificity being one. Also, the release of the drug from its carrier requires the presence ofsome agent or event to separate the active drug from its carrier or molecule and, as such, mayrely on factors such as the presence of a specific enzyme, pH conditions, time release and thelike, which may be variable from host to host and which may not be effectively implemented.For example, transmembrane transport of nutrient molecules is a critical cellularfunction. Because practitioners have recognized the importance of transmembrane transport tomany areas of medical and biological science, including drug therapy, peptide therapy and genetransfer, there have been significant research efforts directed to the understanding andapplication of such processes. Thus, for example, transmembrane delivery of nucleic acids hasbeen encouraged through the use of protein carriers, antibody carriers, liposomal deliverysystems, electroporation, direct injection, cell fusion, vital carriers, osmotic shock, and calcium-phosphate mediated transformation. However, many of those techniques are limited both by thetypes of cells in which transmembrane transport is enabled and by the conditions of use forsuccessful transmembrane transport of exogenous molecular species. Further, many of these1015202530CA 02264592 1999-02-25WO 98/08859 PCT/US97/14140-5-known techniques are limited in the type and size of exogenous molecule that can be transportedacross a membrane without loss of bioactivity.One method for transmembrane delivery of exogenous molecules _having a wideapplicability is based on the mechanism of receptor-mediated endocytotic activity. Unlike manyother methods, receptor-mediated endocytotic activity can be used successfully both in vivo andin vitro. Receptor-mediated endocytosis involves the movement of ligands bound to membranereceptors into the interior of an area bounded by the membrane through invagination of themembrane. The process is initiated or activated by the binding of a receptor-specific ligand tothe receptor. Many receptor-mediated endocytotic systems have been characterized, includingthose recognizing galactose, marmose, marmose 6-phosphate, transferrin, asialoglycoprotein,transcobalamin (Vitamin B12), on-2—macroglobulins, insulin, and other peptide growth factorssuch as epidermal growth factor (EGF).Receptor-mediated endocytotic activity has been utilized for delivering exogenousmolecules such as proteins and nucleic acids to cells. Generally, a specified ligand is chemicallyconjugated by covalent, ionic or hydrogen bonding to an exogenous molecule of interest (i.e. theexogenous compound), forming a conjugate molecule having a moiety (the ligand portion) thatis still recognized in the conjugate by a target receptor. Using this technique, the phototoxicagent psoralen has been conjugated to insulin and internalized by the insulin receptorendocytotic pathway (Gasparro, 1986); the hepatocyte-specific receptor for galactose terminalasialoglycoproteins has been utilized for the hepatocyte-specific transmembrane delivery ofasialoorosomucoid—poly-L-lysine non-covalently complexed to a DNA plasmid (Wu, 1987); thecell receptor for epidermal growth factor has been utilized to deliver polynucleotides covalentlylinked to EGF to the cell interior (Myers, 1988); the intestinally situated cellular receptor for theorganometallic Vitamin B12-intrinsic factor complex has been used to mediate delivery to thecirculatory system of a vertebrate host a drug, hormone, bioactive peptide or immunogencomplexed with Vitamin B12 and delivered to the intestine through oral administration (Russell-Jones et al., 1995); the marmose-6—phosphate receptor has been used to deliver low densitylipoproteins to cells (Murray and Neville, 1980); the cholera toxin binding subunit receptor hasbeen used to deliver insulin to cells lacking insulin receptors (Roth and Maddox, 1983); thehuman chorionic gonadotropin receptor has been employed to deliver a ricin a-chain coupled toHCG to cells with the appropriate HCG receptor in order to kill the cells (Oeltmarm and Heath,1015202530CA 02264592 1999-02-25wo 93/03359 PCT/US97/14140-5-1979); the transferrin receptor has been used to deliver mitomycin C to sarcoma cells (Tanaka etal., 1996) or to deliver doxorubicin to multidrug-resistant cells (Fritzer et al., 1996);the biotinreceptor has been employed to deliver hypoxanthine-guanine phosphoribpsyl transferase(HGPRT) by biotinylating the HGPRT to restore growth to HGPRT deficient cells (Low etal.,1995); and the folic acid receptor has been used to deliver antisense DNA to src-transformedfibroblast cells (Low et al., 1995).Russell-Jones et al. (1995), describes a system which involves the formation of acovalent bond between the pharmaceutical agent one wishes to deliver and a modified VitaminB12 to form a conjugate molecule. The conjugate is orally administered and is then transportedfrom the intestinal lumen to the circulation. Importantly, the pharmaceutical agent and thevitamin are bound through an amide linkage which is prone to acid hydrolysis. Russell-Jones etal. found that many biologically active pharmaceutical agents can be bound to B12 for facilitatingthe introduction of the drug into the blood stream through oral administration. Importantly, nomethod was provided whereby the drug-B12 bond could be selectively cleaved, nor couldlocation of the active pharmaceutical agent be controlled once activated. Instead, Russell—.loneset al. relied on biochemical degradation of the drug-B12 bond to release the drug in its activeform. Importantly, under this method the drug could be released in its active form anywherewithin the circulation system, diminishing the importance of the active transport of B12 intocancer tissue. Moreover, the conjugates formed under this method require the modification ofthe structure of the corrin ring of the B12 molecule, which modification can have serious effectson receptor interactions.Thus, there exists a need for a drug delivery system which can be utilized for the deliveryof bioactive agents, including pharmaceuticals, peptides and oligonucleotides. There is also aneed for a drug delivery system which can be used for site-specific release of the bioactive agentin the cells, tissues, or organs in which a therapeutical effect is desired to be effected. The present invention relates to bioconjugates and the delivery of bioactive agents whichare preferably targeted for site specific release in cells, tissues or organs. More particularly, thisinvention relates to bioconjugates which comprise a bioactive agent and an organocobaltcomplex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom of10152030CA 02264592 1999-02-25wo 93/03359 PCT/US97l14140-7-the organocobalt complex. The bioactive agent is released from the bioconjugate by thecleavage of the covalent bond between the bioactive agent and the cobalt atom in theorganocobalt complex, as described herein.The bioactive agent is any agent which is desired to be delivered to cells, tissues ororgans for nutrient or therapeutic effects. In accordance with the present invention, bioactiveagents include, but are not limited to, nutrients, pharmaceuticals, drugs, peptides andoligonucleotides.The organocobalt complex is any organic complex containing a cobalt atom havingbound thereto 4-5 nitrogen and/or chalcogens such as oxygen, sulfur, etc., as part of a multiple‘unsaturated heterocyclic ring system. In accordance with the present invention, suitableorganocobalt complexes include, but are not limited to, cobalamin, Co[SALEN], organo-(pyridine)bis(dimethylglyoximato)cobalt, corrinoids, derivatives thereof and analogues thereof.The organocobalt complexes may be unsubstituted or substituted with conventional organicfunctional groups which will not alter the basic nature of the organocobalt complex. The basicnature of the organocobalt complex is to directly or indirectly bind the bioactive agentcovalently to the cobalt such that the cobalt-bioactive agent bond is readily cleavable asdescribed herein. The organocobalt complex may also be covalently bound directly or indirectlyto a targeting molecule. The targeting molecule is a molecule for which the desired cell, tissueor organ has a requirement or a receptor, as described herein.The bioconjugate according to the present invention is administered to a subject in needof therapeutic treatment. The bioconjugate concentrates in a targeted cell, tissue or organ site asa result of the organocobalt complex. As an example, a bioconjugate containing achemotherapeutic is administered to a patient and the bioconjugate concentrates in neoplasticcells where the active chemotherapeutic is released from the bioconjugate by cleavage.Similarly, other pharmaceuticals, drugs, peptides or oligonucleotides are administered to asubject as part of the bioconjugate which is concentrated in the desired cells, tissues or organs.The pharmaceuticals, drugs, peptides or oligonucleotides are released by cleavage. In oneembodiment, the cleavage may occur as a result of normal displacement by cellular nucleophilesor enzymatic aciton. In a second embodiment, the cleavage is caused to occur selectively at therelease site by an external signal. The external signal may be light or photoexcitation, i.e.photolysis, or it may be ultrasound, i.e. sonolysis. Further, if the photolysis takes place in the10202530CA 02264592 1999-02-25wo 98/03359 PCT/US97/ 14140-3-presence of a magnetic field surrounding the release site, the release of the drug, such as acytotoxic agent, into surrounding healthy tissue can be minimized.D TIFigure 1 shows the structure and absorption spectrum of methylcobalamin (B12).Figure 2 shows the structure and absorption spectrum of ethyl-Co[SALEN] (cobalt-bis-[salicylidene]-ethylenediamine.Figure 3A shows a sequential absorption spectra of aqueous CH3-Cblm as a function ofanaerobic sonolysis (pH 7.38, 100 mM Hepes, saturating Ar).Figure 3B shows the change in absorbance spectra following aerobic sonolysis in theabsence of organic buffer.Figure 4A shows a sequential absorption spectra of aqueous compound 3 (Example 6) asa function of anaerobic sonolysis at pH 7.4, 100 mM Hepes, saturating Ar.Figure 4B shows the change in absorbance spectra following aerobic sonolysis of acompound 3 (Example 6) solution containing phosphate buffer.Figure 5 shows the effect of a chlorambucil bioconjugate on cell viability for the HCT-l16 cell line. The results are shown for chlorambucil (I), the chlorambucil bioconjugate withphotolysis (O), the chlorambucil bioconjugate with no photolysis (A) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis (V).Figure 6 shows the effect of a chlorambucil bioconjugate on cell viability for the HL-60cell line. The results are shown for chlorambucil (I), the chlorambucil bioconjugate with nophotolysis (A) and the chlorambucil bioconjugate plus 10 equivalents of hydroxycobalaminwith no photolysis (V).Figure 7 shows the effect of a chlorambucil bioconjugate on cell viability for the B-16cell line. The results are shown for chlorambucil (I), the chlorambucil bioconjugate withphotolysis (O), the chlorambucil bioconjugate with no photolysis (A) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis (V).Figure 8 shows the effect of a chlorambucil bioconjugate on cell viability for the Meth—Acell line. The results are shown for chlorambucil (I), the chlorambucil bioconjugate withphotolysis (O), the chlorambucil bioconjugate with no photolysis (A) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis (V).10202530CA 02264592 1999-02-25WO 93/08859 PCT/US97/ 14140-9- ’Figure 9 shows the effect of a chlorambucil bioconjugate on cell viability for the RD-995cell line. The results are shown for chlorambucil (I), the chlorambucil bioconjugate withphotolysis (O), the chlorambucil bioconjugate with no photolysis (A) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis (V). The present invention relates to bioconjugates and the delivery of bioactive agents whichare preferably targeted for site-specific release in cells, tissues or organs. More particularly, thisinvention relates to bioconjugates which comprise a bioactive agent and an organocobaltcomplex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom ofthe organocobalt complex. The bioactive agent is released from the bioconjugate by thecleavage of the covalent bond between the bioactive agent and the cobalt atom in theorganocobalt complex. The cleavage may occur as a result of normal displacement by cellularnucleophiles or enzymatic action, but is preferably caused to occur selectively at apredetermined release site by application of an external signal. The external signal -may be lightor photoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if thephotolysis takes place in the presence of a magnetic field surrounding the release site, the releaseof the bioactive agent into surrounding healthy tissue is minimized.The bioconjugate according to the present invention is administered to a subject in needof therapeutic treatment. The bioconjugate concentrates in a targeted cell, tissue or organ site asa result of the organocobalt complex. The bioactive agent is released from the bioconjugate bycleavage. In one embodiment, the cleavage may occur as a result of normal displacement bycellular nucleophiles or enzymatic aciton. In a second embodiment, the cleavage is caused tooccur selectively at the release site by an external signal. The external signal may be light orphotoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if the photolysistakes place in the presence of a magnetic field surrounding the release site, the release of thedrug, such as a cytotoxic agent, into surrounding healthy tissue is minimized.As one example, the bioconjugate contains a chemotherapeutic agent and is administeredto a patient having cancer. In this example, a therapeutically effective amount of thebioconjugate is administered intravenously to a patient such that the bioconjugate concentratesin the neoplastic cells. The chemotherapeutic agent is released from the bioconjugate by natural10202530CA 02264592 1999-02-25wo 9s/03359 PCT/US97/14140-10- ‘ameans (e.g., cellular nucleophiles or enzymatic action) or preferably by means of an externalsignal (e.g., light or ultrasound).As a second example, the bioconjugate contains a cytotoxic agent and is_ administered toa patient having psoriasis. In this example, a therapeutically effective amount of the bioconjugateis administered to an afflicted skin site. The cytotoxic agent is released by natural means orpreferably by means of an external signal.As a third example, the bioconjugate contains the enzymatic domain of diphtheria toxin(Nichols et al., 1997) and is administered to a patient having cancer. In this example, atherapeutically effective amount of the bioconjugate is administered intravenously to a patientI such that the bioconjugate concentrates in the neoplastic cells. The enzymatic domain ofdiphtheria toxin is released from the bioconjugate by natural means (e.g., cellular nucleophiles orenzymatic action) or preferably by means of an external signal (e.g., light or ultrasound) andproceeds to kill the cancer cells. AAs a fourth example, the bioconjugate contains an antisense oligonucleotide againsthepatitis B virus (Yao et al., 1996; Madon and Blum, 1996) and is administered to a subjecthaving hepatitis B. In this example, a therapeutically effective amount of the bioconjugate isadministered intravenously to a patient such that the bioconjugate concentrates in the liver. Theantisense oligonucleotide is released from the bioconjugate by natural means (e.g., cellularnucleophiles or enzymatic action) or preferably by means of an external signal (e.g., light orultrasound) and proceeds to inhibit gene expression and replication of hepatitis B virus.The present invention employs the following definitions:Bioactive agent: any agent which is desired to be delivered to cells, tissues or organs formodulating or otherwise modifying cell function, including for therapeutic effects. In accordancewith the present invention, bioactive agents include, but are not limited to, pharmaceuticallyactive compounds or diagnostic compounds. Bioactive agents include, but are not limited to,peptides, oligopeptides, proteins, apoproteins, glycoproteins, antigens and antibodies or antibodyfragments thereto, receptors and other membrane proteins, retro-inverso oligopeptides, proteinanalogs in which at least one non-peptide linkage replaces a peptide linkage, enzymes,coenzymes, enzyme inhibitors, amino acids and their derivatives, hormones, lipids,phospholipids, liposomes, ricin or ricin fragments; toxins such as aflatoxin, digoxin,1015202530CA 02264592 1999-02-25PCT/US97/14140-11- -»WO 98108859xanthotoxin, rubratoxin; antibiotics such as cephalosporins, penicillin and erythromycin;analgesics such as aspirin, ibuprofen and acetaminophen, bronchodilators such as theophyllineand albuterol; beta~blockers such as propranolol, metoprolol, atenolol, la_betolol, timolol,penbutolol and pindolol; antimicrobial agents such as those described above and ciprofloxacin,cinoxacin and norfloxacin; antihypertensive agents such as clonidine, methyldopa, prazosin,verapamil, nifedipine, aptopril and enalapril; cardiovascular agents including antiarrhythmics,cardiac glycosides, antianginals and vasodilators, central nervous system agents includingstimulants, psychotropics, antimanics and depressants; antiviral agents; antihistamines such aschlorphenirmine and brompheniramine; cancer drugs including chemotherapeutic agents, such aschlorambucil, carboplatin, deratives of busulfan, doxorubicin, etoposide, topotecan (TPT);tranquilizers such as diazepam, chordiazepoxide, oxazepam, alprazolam and triazolam, anti-depressants such as fluoxetine, amitriptyline, nortriptyline and imipramine; H-2 antagonists suchas nizatidine, cimetidine, famotidine and ranitidine, anticonvulsants; antinauseants;prostaglandins; muscle relaxants; anti-inflammatory substances; stimulants; decongestants;antiemetics; diuretics; antispasmodics; antiasthmatiics; anti-Parkinson agents; expectorants;cough suppressants, mucolytics; vitamins; and mineral and nutritional additives. Othermolecules include nucleotides; oligonucleotides; polynucleotides; and their art—recognized andbiologically functional analogs and derivatives including, for example, methylatedpolynucleotides and nucleotide analogs having phosphorothioate linkages; plasmids, cosmids,artificial chromosomes, other nucleic acid vectors; antisense polynucleotides including thosesubstantially complementary to at least one endogenous nucleic acid or those having sequenceswith a sense opposed to at least portions of selected viral or retroviral genomes; promoters;enhancers; inhibitors; other ligands for regulating gene transcription and translation. In addition,the bioactive agent can be any other biologically active molecule that can form a conjugate withan organocobalt complex. The bioactive agent may further contain a spacer which provides acovalent bond with the cobalt atom of the organocobalt complex, but which does not adverselyaffect the biological activity of the bioactive agent.Bioconjugate: a conjugate containing a bioactive agent and an organocobalt complex inwhich the bioactive agent is covalently bound directly to the cobalt atom or is covalenly boundindirectly to the cobalt atom via a spacer.10202530CA 02264592 1999-02-25wo 93/03359 PCT/US97/14140-12- —-Non-reactive atom: an atom in the bioactive agent that will not lead to rearrangement ordestruction of the bioactive agent under conditions of ligand exchange during receptor-mediatedendocytosis, but that instead will reproduce the original form of the bioactive agent (or bioactiveagent and spacer) and thereby unmask an active bioactive agent. The non—reactive atom may bea carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, a selenium atom or a siliconatom. A carbon atom (e. g. from an alkyl, acyl or aryl group) is particularly preferred. Such non-reactive atoms are also used in forming the covalent bond between the cobalt and the spacer.Organocobalt complex: an organic complex containing a cobalt atom having boundthereto 4-5 calcogens as part of a multiple unsaturated heterocyclic ring system. In accordancewith the present invention, suitable organocobalt complexes include, but are not limited to,cobalamin (coenzyme B12), Co[SALEN] (which is a cobalamin analogue), organo(pyridine)-bis(dimethyl'glyoximato)cobalt, corrinoids (such as disclosed by Brown et al., 1996) andderivatives or analogues of any of the preceding, as well as pharmaceutically acceptable salts.The organocobalt complexes may be unsubstituted or substituted with conventional organicfimctional groups which will not alter the basic nature of the organocobalt complex. The basicnature of the organocobalt complex is to bind the bioactive agent covalently to the cobalt suchthat the cobalt—bioactive agent bond is readily cleavable as described herein. Examples ofsubstituents which may be found on the organocobalt complex include amino, nitro, halogen(bromine, chlorine), sulfito, CM-alkene and CM alkyne. For example, the organocobalt complexcan be formed having a nitro and/or halo (e.g., bromo) derivative of the corrin ring or having anextended conjugation with exocyclic olefin or alkylene groups. Other derivatives includecobalamin lactone, cobalamin lactame and those in which the benzimidiazole ring (e.g., ofcobalamin, green corrinoid, and the like) are substituted with e.g., one or more halogen(bromine, chlorine), hydroxy or C,_6 alkyl. Such substituents are useful for increasing the km“to be used for cleavage of the bioconjugate as described herein. Further derivatives includeanilide, ethylamide, mono-, di- or tri-carboxylic acid or proprionamide derivatives of cobalaminof Vitamin B12. In one embodiment, the organocobalt complex is any organic complexconaining cobalt which is bound by transcobalamin and transported into a cell by a receptor-mediated process involving transcobalamin. In a second embodiment, the organocobalt complexmay also be covalently bound directly or indirectly (through a spacer) to a targeting molecule,wherein said targeting molecule is bound by its receptor and the complex is transported into a10152025CA 02264592 1999-02-25W0 98I08859 PCT/U S97/ 14140-13-cell by a receptor—mediated process. Co[SALEN] and its derivatives or analogues can berepresented by the general formulawherein the substitutents may be included or omitted to modulate physical properties of themolecule, e.g., water solubility, stability or km” -- the wavelength at which the complex absorbs.Thus, the substituents are as follows: R is H, a group which increases water solubility and/orstability or a group for attachment of a targeting molecule and W, W’, X, X’, Y, Y’, Z and Z’ areindependently H, a group which increases water solubility and/or stability, a group forattachment of a targeting molecule or a group for modified absorbance of energy, or W and Xtogether and W’ and X’ together are a 4-6 member cyclic or heterocyclic ring, or Y and Ztogether and Y’ and Z’ together are a 4-6 member cyclic or heterocyclic aromatic ring.Examples of groups for enhancing water solubility include amino, C,_6 alcohol, C,_6 carboxyl forany substitutent, or also SO3- for the substitutents other than R. Examples of groups forattachment of a targeting molecule include amino, CH5 alcohol and CH, carboxyl for anysubstitutent. Examples of groups for modifying absorbance include CH2OH, COZH, SO3—,amino and nitro for the substitutents other than R. Such groups are useful for increasing thewavelength of light to be used for cleavage of the bioconjugate as described herein, whiletargeting molecules are useful in selectively targeting the bioconjugate to the desired tissue.Therefore, when used in the context of the present application, the term organocobalt complex,unless specifically identified, shall be inclusive of B” in all its embodiments, includingcoenzyme B12, Co[SALEN] and other B12 or B12-like molecules, the organocobalt complexesdefined herein, as well as any derivatives and analogues thereof.Spacer: an atom or molecule which covalently binds together two components. In thepresent invention, a spacer is intended to include atoms and molecules which can be used tocovaltently bind a bioactive agent to the cobalt atom of an organocobalt complex or tocovalently bind a targeting molecule to an organocobalt complex. The spacer must not preventthe binding of the organocobalt complex or the targeting molecule with its appropriate receptor.1015202530CA 02264592 1999-02-25WO 98/08859 PCT/U S97/ 14140-14-Examples of suitable spacers include, but are not limited to, polymethylene [-(CH2),,, where n is1-10], ester [bioactive agent attached to O and Co to C = O], carbonate, ether, acetal or anycombination of two or more of these units. A skilled artisan will readily recognize other spacerswhich can be used in accordance with the present invention.Several of these spacers are useful as a “self-destructing” linker group. That is, some orall of the linkage would be consumed in a fragmentation reaction. This means that, followingcleavage of the C-Co bond by photolysis or sonolysis, an additional cleavage will take placeseveral bonds away, leading to the formation of a small, unsaturated (and typically volatile)molecule made up of atoms of the fonner linker. This is shown schematically below:linker atoms . L_ L_ Drug L: L . DrugL-L — Drug \Co(l|l) ———-— _umocuousvolatile speciesThe most typical scenario is the subsequent cleavage of a second bond, two bonds removed fromthe first. Thus, most self-destructive linkers would contain a two-atom unit whose extrusion as asmall, gaseous molecule is favorable. Another design feature is to have the new radical specieswhich is generated after the second cleavage step be an especially stable kind of radical.Examples of self-destructing linkers are shown below:H2 'C°(m),C\C,Dru9 __.__... Cow) H2C~C/Drug —-——>- CH2=CH3+-Drug2 H2C°(m),O\C,Drug ._._..._p.. Cow) (LC/DVUG -Z——l-- CO2+-DrugH ”O 0it’ 9co(m)’C‘o’D"’9 "“"“" Co(I|) 'C‘o’D'”g “‘—* °°2+'D’“9Targeting Molecule: a molecule which is bound by a receptor and transported into acell by a receptor-mediated process. Examples of suitable targeting molecules include, but arenot limited to, glucose, galactose, mannose. mannose 6-phosphate, transferrin.1015202530CA 02264592 1999-02-25W0 98/08859 PCT/US97/14140-15-asialoglycoprotein, ot—2—macroglobulins, insulin, a peptide growth factor, cobalamin, folic acidor derivatives, biotin or derivatives, YEE(GalNAcAH)3 or derivatives, albumin, texaphyrin.metallotexaphyrin, porphyrin, any vitamin, any coenzyme, an antibody, an antibody fragment(e.g., Fab) and a single chain antibody variable region (scFv). A skilled artisan will readilyrecognize other targeting molecules (ligands) which bind to cell receptors and which aretransported into a cell by a receptor-mediated process. The present invention is intended toinclude all such targeting molecules.The present invention takes advantage of the cellular properties of cobalamin and4 cobalarnin analogues or derivatives, as well as the cellular properties of other targetingmolecules. For example, studies have shown that the absorption of physiological amounts ofvitamin B12 by the gut requires that it be complexed with a naturally occurring transport proteinknown as intrinsic factor (IF). (Castle, 1953; Fox and Castle, Allen and Majerus. 1972b). Thisprotein is released into the lumen of the stomach by parietal cells in the fundus. Once bound tointrinsic factor, the B12-IF complex interacts with a membrane bound receptor for IF located onthe terminal ileum of the small intestine. The receptor-IF-B12 complex is then internalized by aprocess of receptor-mediated endocytosis (RME). Allen and Majerus demonstrated that it ispossible to chemically modify B12, couple it to a resin and use the B12-resin to affinity purify IF(Allen and Majerus, 1972a). This finding suggests the possibility of coupling a largemacromolecule (such as the resin used by Allen and Majerus, 1972a) to B12 while stillpreserving its ability to interact specifically with intrinsic factor and thus be part of the activetransport system. By coupling molecules to B12 in such a way as to preserve the ability of B12 tointeract with intrinsic factor, it was found that the natural uptake mechanism for orallyadministered B12 could be used to deliver various proteins, drugs or other pharmaceuticallyactive molecules from the intestinal lumen to the circulation. It has been found that B12 isnaturally concentrated in cancer tissue through a similar transport mechanism.In mammals, B12 is transported in the blood by transcobalamin proteins TC-I, TC-ll. andTC—lll. The major form of B12 in the blood is methylcobalamin and the largest store of B12 isadenosylcobalamin in the liver. Rapidly dividing cells, including cancer cells, require coenzymeB12 for thymidine production during DNA synthesis. It has been reported by Carmel (1975) that,in some patients with tumors, up to 50-fold increases in the major cobalarnin transport proteinsTC-I and TC-II have been observed. Waxman et al. (1972), report the finding of tumor specificH202530WO 98/08859CA 02264592 1999-02-25PCT/US97/14140_16_ _..B12 binding proteins that circulate in the blood. In each instance, these increases in TC transportproteins and the corresponding systemic depletion of B12 were not the result of megaloblastosis,granulocyte proliferation, or any other pathogenic Bu deficiency.In a second example of receptor-mediated endocytosis, folate receptors that mediateendocytotic activity have previously been identified in bacterial cells (Kumar et al., 1987) andused for delivery of biologically active materials (Low et al., l995). Folic acid, folinic acid,pteropolyglutamic acid, and folate receptor—binding pteridines such as tetrahydropterins,dihydrofolates, tetrahydrofolates and their deaza and dideaza analogs are useful as targetingmolecules in accordance with the present invention. The terms “deaza” and “dideaza” analogsrefer to the art—recognized analogs having a carbon atom substituted for one or two nitrogenatoms in the naturally-occurring folic acid structure. For example, the deaza analogs include the1-deaza, 3-deaza, 5-deaza, 8-deaza, and l0—deaza analogs. The dideaza analogs include, forexample, 1,5-dideaza, 5,10-dideaza, 8,l0—dideaza, and 5,8-dideaza analogs. The foregoing folicacid deriatives are conventionally termed “folates,” reflecting their capacity to bind with folate-receptors, and such ligands when complexed with exogenous molecules are effective to enhancetrans-membrane transport. Other folates useful as complex forming ligands for this inventionare the folate receptor binding analogs aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as l—deazamethopterin or 3-deazamethopterin, and 3’5’—dichloro-4-amino-4-deoxy-NI0-methylpteroyl-glutamic acid(dichloromethotrexate). Other suitable ligands capable of binding to folate receptors to initiatereceptor-mediated endocytotic transport of the complex include anti—idiotypic antibodies to thefolate receptor. An exogenous molecule in complex with an anti—idiotypic antibody to a folatereceptor is used to trigger trans-membrane transport of the complex. Such molecules are used inaccordance with the present invention as a targeting molecule.In a further example of receptor-mediated endocytosis, biotin receptors have been used tomediate endocytotic activity (Low et al., 1995). Biotin analogs such as biocytin, biotinsulfoxide, oxybiotin and other biotin receptor-binding compounds are ligands that may also beused as suitable targeting molecules to promote the trans-membrane transport of exogenousmolecules in accordance with this invention. Other compounds capable of binding to biotinreceptors to initiate receptor-mediated endocytotic transport of the complex are alsocontemplated. These can include other receptor—binding ligands such as, for exmple, anti—idiotypic10202530CA 02264592 1999-02-25wo 93/03359 PCT/US97/14140-17- '-antibodies to the biotin receptor. An exogenous molecule complexed with an anti-idiotypicantibody to a biotin receptor could be used to trigger trans-membrane transport of the complex.Such molecules are used in accordance with the present invention as a targeting rnolecule.Other examples of targeting molecules include glucose, galactose, mannose, mannose 6-phosphate, hormones (e.g., insulin, growth hormone, and the like), growth factors or cyokines(e.g., T GF-B, EGF, insulin-like growth factor, and the like), YEE(GalNAcAH)3 or derivatives,cobalamin, on-2 macroglobulins, asialoglycoprotein, albumin, texaphyrin, metallotexaphyrin,antibodies, antibody fragments (e.g., Fab), single-chain antibody variable region (scFv),transferrin, any vitamin and any coenzyme.As previously described, a bioconjugate of the present invention comprises a bioactiveagent conjugated directly or indirectly via a covalent bond to the cobalt atom of an organocobaltcomplex. The bioactive agent is conjugated directly to the cobalt atom through a non-reactiveatom in the bioactive agent or is conjugated indirectly to the cobalt atom through the use of aspacer. Therefore, in contrast to the conjugates formed under U.S. Patent 5,428,023, theattachment of a bioactive agent to the cobalt atom in the axial position does not interfere withreceptor-mediated endocytosis from the blood into cells.The unusually weak cobalt-non-reactive atom bond (e.g., C-Co bond) of the bioconjugateprovides a readily addressable trigger for the controlled in vivo release of the bioactive agent fromthe organocobalt complex. The bond dissociation energy (BDE) of Co-non-reactive atom bond inthe-bioconjugate is in the range of 30 to 50 kcal/mol (e.g., 30-40 kcal/mol range for a Co-Cbond) which make them among the weakest covalent bonds known, yet the bond is relativelystable in aqueous solution.A common strategy will employ the modification of the anticancer drug so that itpossesses an electrophilic site which can react with the highly nucleophilic Co(I) intermediategenerated upon treatment of hydroxycobalamin with NaBH4. This structural modification willbe sufficiently far removed from the active site (pharmacophore) to preclude any interferencewith the desired biological activity. Approaches used in the case of chlorambucil are typical:the carboxylic acid group of chlorambucil is converted to either an acid chloride or a bromoethylester, either of which can be efficiently coupled with cob(I)alamin.101520CA 02264592 1999-02-25W0 98/08859 PCT/US97/ 14140General Strategy:(X=electrophiIic _site, such as alkyl highly reactive cobalamin orhalide, acid halide, I nucleophilic form synthetic cobaltDrugepoxide, Michael of cobalt complexacceptor, etc.)VDrugCo(l|l)For example, reduced Cbll is prepared by NaBH4 or zinc dust reduction, e.g. of hydroxo—cob(III)alamin. In the above scheme, the drug can be a cytotoxic agent, other drug or otherbioactive agent as described herein. In other schemes, a spacer containing a carbon atom orother atom such as that specified for the non-reactive atom for binding to the cobalt atom andwhich also contains a reactive grouping, e.g. -OH or -CN, which is further reacted with thebioactive agent, is introduced. Other reactive groups, e.g. -NH2, -SH, -COOH, etc., can also beutilized for coupling to a bioactive agent. It is important to note that, in some cases (e.g.,chlorambucil, doxorubicin), the small organic molecule released is not the parent drug, but ratherretains some of the modification installed to allow coupling. In other cases (e.g., topotecan), thestructure of the released drug may correspond to the parent molecule.More specific details of the synthesis of representative bioconjugates according to thepresent invention are as follows, using a “drug” which can be replaced by any suitable bioactiveagent and cobalamin which can be replaced by any suitable organocobalt complex. In thissynthesis, all procedures are under argon. Hydroxocob(III)alamin is dissolved in aqueousCH3OH (l:1 v/v) at 25°C. A 2-10 fold excess of NaBH4 is added. The solution slowly changescolor from red to brown and gradually green (Cbll). After approximately 15 min. theelectrophilic drug ligand (dissolved in the same deoxygenated solvent) is added, e.g., as an alkyl,acyl or aryl chloride. Strictly anaerobic conditions are maintained and the reaction mixture isstirred gently at 25°C. The color gradually changes back to red as Cbl is converted to alkyl-,acyl-, or aryl—Cblm. After about 1.5 h, the solution is acidified to pH 3.0 with dilute HC1.Methanol is removed under reduced pressure by rotary evaporation at less than 40°C. TheCA 02264592 1999-02-25W0 98/08859 PCT/U S97/ 14140-19- 'resulting aqueous solution is diluted with an equal volume of H20 and loaded onto a DowexAG-50-X2 (200-400 mesh) cation exchange column. The column is washed sequentially withH20 and 0.1 M NaOAc, pH 6.4. Fractions containing drug-cob(lII)alamins appear red and arecollected appropriately. Unreacted hydroxocob(III)alamin is retained on the column. Combinedfractions of drug-cob(III)alamin are extracted with phenol and concentrated by rotaryevaporation. Drug—cob(III)alamins can often be crystallized by the addition of acetone to aconcentrated aqueous solution. Characterization of the alkyl-, acyl-, or aryl-cobalaminconjugates is by NMR, mass spectrometry (FAB, C1, or electrospray), and IR methods.A methotrexate—containing bioconjugate can be synthesized by the following methods.In method one utilizing the above procedure, methotrexate (MTX) is converted to itscorresponding acyl chloride and reacted with cobalarnin and/or Co(III)[SALEN] and/or otherdisclosed organocobalt complexes to yield methotrexate-cobalamin and methotrexate-Co(III)[SALEN] according to the following reaction scheme 1. In the alternative method two,the C-Co bond is first formed from an acyl chloride having a protected amino group. The aminogroup is then deprotected, followed by formation of the amide bond to an arninobenzoylpterinaccording to the following reaction scheme II.CA 02264592 1999-02-25PCT/US97/ 14140WO 98108859-20- oofoo 6:2o1EuEU-N,E2Nmz_mzo /z zL/ zwmNmzmOm $2Z IO£2 =Z, z/z zL/ zmm. 0 A 6o 1 ofoo MIZ\NmO moo mzxmuo oomzniom .m.A|l|.||$2 .NEm: ._5Allll o O Q _foo:2O 6£2zz \ /2mo z zmmZSUBSTITUTE SHEET (RULE 25)10H202530CA 02264592 1999-02-25WO 98/08859 PCT/U S97/ 14140-21-An aminopterin-containing bioconjugate can be synthesized by the following method.The des-methyl derivative of methotrexate (aminopterin) is coupled to cobalt as shown by thefollowing reaction formula, in which the iminium ion is either reacted with Co(I) directly, or theiminium ion is converted to the aminonitrile and then slowly unmasked to reveal the iminiumcation.H N N N H N N N2 \H/ \ \ CHFO 2 Y \ \ fit-12N / / ll ——"j’ N / N/ NN cat. H +. NH2 R NH2 RR = Or 0 ' OR = HN—CH—CH2CH2CO2MelCO2MeH2N\’/N\ N\ HZC/icon. 7JJN/IvaNH; ROA topotecan-containing bioconjugate can be synthesized by the following method. Thecytotoxic activity of topotecan (TPT) or Camptothecin (CPT) arises from their ability to freezetopoisomerase I-DNA “cleavable complexes.” (Pommier et al.. 1995) Since some tumor typesdisplay greatly elevated levels of topo I (Giovanella et al.. 1989), topoisomerase poisons ofthistype are likely to have a higher therapeutic index in the treatment of those cancers. However,treatment with Camptothecin derivatives could be made more general if used in conjunction withthe targeted delivery approach.Topotecan is conjugated to cobalamin, Co[SALEN] and other organocobalt complexesaccording to the following reaction schemes. Camptothecin is conjugated in a similar manner.Preparation of 10a and 10b involves similar chemistry to that discussed above for 8a,b.Selective generation of the phenyl chloroformate (25) of topotecan (5) and acylation of Co(I)gives 1021. Exposure of 25 to 18 or treatment of S with the previously discussed chloroformate19 furnishes 10b. Conjugates 10c,d will require somewhat longer routes, as they cannot beprepared directly from 5. However. the established synthetic route for conversion of the naturalCA 02264592 1999-02-25W0 98/08859 PCT/US97/14140-22- “product camptothecin to 5 can be modified at the appropriate point to allow for attachment of thecobalt complex. The first three steps to prepare phenolic intermediate 26 are known (Mulliez etal., 1994). Mannich-type substitution with formaldehyde/dimethylamine then gives 5. Use ofmethylamine gives the corresponding secondary amine 27. At this point, linkage to Co via amethylene to give 10c is possible via Co(I) trapping of a second, in situ generated imminum salt.Alternatively, N-alkylation with 23 gives 10d. Cleavage of 10a and 10b provides 5 directly viafragmentative pathway or indirectly via other products. Cleavage of 10c with hydrogenextraction yields 5. Cleavage of 10d yields the product 5 having an ethylmethylamino group inplace of the dimethylamino group.CA 02264592 1999-02-25PCT/US97/ 14140OI 2W0 98l08859-23-Coll!101520CA 02264592 1999-02-25WO 98/08859 PCT/US97/14140-24- -iA busulfan-containing bioconjugate can be synthesized by the following method.Busulfan is an alkylating agent used therapeutically against chronic myelogenous leukemia(CML). The preferred point for attaching busulfan to the organocobalt complex is on one of thealkanesulfonate units. A slight change in the structure of the sulfonate portion of the ester is willnot exert a large effect on the ability of the released drug to crosslink DNA. Cleavage of 7afollowed by hydrogen abstraction furnishes the mixed ethanesulfonate/methanesulfonate 2b.Trapping of the carbon radical under oxidative conditions produces mixed bis(sulfonate) 2c,which is also a competent crosslinking agent. Cleavage of 7b results in the release of the parentdrug 221 after hydrogen abstraction.Bis-methylsulfonate busulfan is conjugated to cobalamin, Co[SALEl\l] and otherorganocobalt complexes according to the following reaction schemes. For the preparation of 7a,the commercially available sodium salt of bromoethanesulfonic acid (11) serves as the startingpoint. Heating with phosphorus pentachloride furnishes the corresponding sulfonyl chloride 12as a distillable liquid. Treatment with Co(I) leads to preferential displacement of the bromide tofumish 13, which is converted to 7a by sequential treatment with 1,4-butanediol and mesylchloride. The order of the final three steps can be changed; for example, treatment of 12 withexcess butanediol, followed by mesyl chloride gives the mixed bis(sulfonate) 14. Selectivedisplacement of the primary bromide by Co(I) then gives 7a. In the case of conjugate 71),treatment of 2-bromobutane-1,4-diol (which is readily available from malic acid diester) withCo(I) gives adduct 15. Bis(mesylation) gives 7b. Alternatively, 7b is prepared from 16 (X = Bror I) with selective displacement of the halide.PCT/US97l 14140CA 02264592 1999-02-25-25-WO 98/08859mpx:0 .m N22Nomo\/\_/\ ommz ms. oo/\Fo~oms_an 2 :-._<m_o i\\. 3% .:mo A ____oo 3 __.s5ms_Nowo\/\f\momw_>_ awomfo :o)\f\o: E :o\/\_/\o:3 EE AI} o 5E m_2~omo\/\/\ Mm//\/\ %_Nomo\/\/\oum/\/\n___ooVo o \amomfo a«/ o /oN M A 3823 xozmzovoz :_o om :o N S N.A C.10CA 02264592 1999-02-25PCT/US97/ 14140-26- -WO 98/08859A chlorambucil-containing bioconjugate can be synthesized by the following methods.Chlorambucil is a relatively stable nitrogen mustard with attenuated alkylating ability,presumably as a consequence of the less-basic aniline nitrogen.Method (Inc: In this procedure, chlorarnbucil is converted to the acid chloride followedby reaction with cob(I)alamin or Co(I)[SALEN] according to reaction sequence I. In situationswhere the acyl linkage to the organocobalt complex is too labile towards serum nucleophiles,two alternate bioconj ugation procedures can be utilized.Method Two: The procedure involves bromination of a carbon atom adjacent to thecarboxyl group under standard Hel1—Vo1lhardt-Zelinski conditions to pennit attachment of theCo complex in the ot—position according to reaction sequence II. In scheme II, replacement ofthe C-Co with C-H provides chlorambucil. The reactant stoichiometry, temperature, anddilutions conditions can be manipulated to avoid competing displacement of one of thechloroethyl groups, or of the C1 by SN2 attack.Method Three: The BOC-protected p-aminophenylacetaldehyde can be conjugated to theCo moiety, followed by formation of the active nitrogen mustard product according to thefollowing reaction sequence III.PCT/US97/14140N N: _oN:oN:o,_O 012 O ONTT \Z _oN:oN:oN /\ _oN:oN:o\ /_oNIoNIo /\_ mm_Eqow_o=: E.Anl ,2 o |||.I|YQ 2. _oN:oN:o. |©l/\4 a_=__8o CA 02264592 1999-02-25-27-WO 98/08859.oN:oNxo . _ N N, 6: N255 ooo _o :0 moN N Nz o§_m Alnllull uzl©lN:oN:oN:ooNo: HO6: .N_<_2o .80___oo A _oNxoN:o ___8 NxoN:o.m § H_O«T_0~T_0:Z O/L &Z ~IO~IO~IOO~OI IO/L IO_oN:oN:o_oN_._oN:o.10CA 02264592 1999-02-25WO 98/08859 PCT/US97l14l40-28- -'A chlorambucil, ethyl ester—containing bioconjugate can be synthesized by the followingmethods. When conjugating a drug via a carboxyl group, as in the case of chlorambucil, linkingthe drug to the cobalamin via a hydroxyethyl tether may be desirable. This can be accomplishedby one of two "convenient routes, both of which are schematically illustrated below. First,2-hydroxyethyl-cob(III)ala:nin can be readily prepared from cob(l)alamin and bromoethanol.Esterification is carried out under standard conditions, i.e. by reaction of a carboxylic acid(chlorambucil) with an alcohol (2-hydroxyethylcob(III)alamin) in the presence ofdicyclohexylcarbodiimide (DCC) (or water-soluble derivatives such as EDCI) and a catalyticamount of 4-N,N-dimethylaminopyridine (DMAP) and its hydrochloride salt (DMAP-HCI) indichloromethane or toluene. Alternatively, the ester-linked conjugate can be prepared by firstforming the 2-bromoethyl ester of chlorambucil and then reacting the ester with cob(l)alamin toprovide the same product. The reaction schemes (1, II) are shown below. With this mode ofattachment, cleavage from the bioconjugate leads to release of the ethyl ester of chlorambucilaccording to reaction scheme III.CA 02264592 1999-02-25PCT/U S97/ 14140W0 98l08859-29- [A viz,_o_o~:oN:o.m 8zuooflo 8 ammooHA. 1 i ,_zsm (©«zoom E_oz \|\ . V\ \/\/ A 5 z 2_o E ._oowzaJ a 3m:__O:DEm._O_£05-II %_5_._o Efio :15CA 02264592 1999-02-25WO 98/08859 PCT/U S97] 14140-30-An etoposide-containing bioconjugate can be synthesized by the following method.Etoposide is a semisynthetic derivative of the natural product epipodophyllotoxin that is widelyused against a variety of tumors, especially small cell lung carcinoma and germ cell tumors (DeJong et al., 1995). It has also shown considerable promise in the treatment of refractory cases ofovarian and breast cancer. Etoposide appears to function as a topoisomerase II poison.Etoposide is conjugated to cobalamin. Co[SALEN] and other organocobalt complexesaccording to the following reaction schemes. Bioconjugates 8a and 8b require conversion ofthe free phenol of etoposide (3) to the corresponding c_hloroforrnate 17. Direct acylation withCo(I) gives acylCo(III) derivative 821, while treatment with the previously describedhydroxyethylCo(III) derivative 18 furnishes carbonate 8b. This derivative is also available viaacylation of 3 with the chloroforrnate 19 derived from 18. Preparation of acetal-modifiedconjugate 8c may be more challenging. The ethylene acetal of 3 can be hydrolyzed and then theacetal reformed using aldehyde 20a or dimethyl acetal 20b (Keller—Jusl et al., 1971). Compound20:: may also be accessed via careful, selective oxidation of 18. while 20b should be availablevia alkylation of the Co(I) derivative with commerically available bromoacetaldehyde dimethylacetal. In addition. the acetal of glucose can be formed and then the secondary alcohol of 21 canbe glucosylated . Cleavage of 8a or 8b either give 3 directly via fragmentative pathways. orfurnish products which canundergo eventual hydrolysis to 3. Trapping with H0 followinghomolysis of 8c would then furnish 3.CA 02264592 1999-02-25PCT/U S97! 14140WO 98/08859-3]-~m_o...mmOOIOnew,..mo.m_m . Nfizofo/\A__ooVA]8SN~55 .OIo/\___oo IO 101520CA 02264592 1999-02-25wo 93/03359 PCT/US97/14140-32-A doxorubicin-containing bioconjugate can be synthesized by the following method.Doxorubicin (4) is the most widely used of the anthracycline antibiotics, and is clinically usefulagainst a broad spectrum of solid and hematological tumors. Like etoposide, doxorubicinappears to target topoisomerase II, ultimately leading to growth arrest and nonapoptotic celldeath (Fornari et al., 1996; Ling et al., 1996). The clinical usefiilness of doxorubicin is limitedby nonspecific toxicity, especially cardiotoxicity. Thus, it would appear to be a particularlygood candidate for selective delivery. This is confirmed by its frequent use in liposome-basedmethods (Hu et al., 1996; Longman et al., 1995; Hosada et al., 1995), as part ofimmunoconjugates (Johnson et al., 1995; Sivam et al., 1995), or in prodrug approaches(Svensson et al., 1995). ZDoxorubicin conjugated to cobalamin, Co[SALEN] and other organocobalt complexesaccording to the following reaction schemes. For the synthesis bioconjugate 9a, thecondensation of the daunosamine amino group with acyl-Co(III) complex 22 is performed. Thisreaction forms the 2-pyrroline ring in analogy to published routes using 4-iodobutyraldehydeand 5-iodo-2-pentanone.(9b) The acyclic tertiary amine derivative 9b is available from 4 viainitial reductive amination with acetaldehyde, then alkylation of the resulting secondary aminewith the mesylate 23 derived from 18. Alternatively, treatment of 4 with chloroformate 19provides carbamate 9c. If alternative points of attachment are desired, hydrazone-linkedderivatives such as 9d can be used using simple cobalamin alkyl hydrazides such as 24,obtainable from 23. The cleavage of these bioconjugates is shown in the reaction scheme below.CA 02264592 1999-02-25PCT/US97/14140WO 98/088598zomxofi\....rO om+1 m___oo_/\/“O \=_._zIOAem8NFIKJ/\/zzzf8%momfoJ5+ IZ|.L.mmmo‘o ms. uzofmmz E dzofoO 925.2.3 02mm ___oo/\/OL_/Nzzzo _.O m_)_CA 02264592 1999-02-25PCT/US97/14140WO 98/08859-34- zomzo101520CA 02264592 1999-02-25“'0 93’°3359 PCT/US97/14140-35-A carboplatin containing bioconjugate can be synthesized as shown in the followingreaction scheme./\/\/\/\B’ onCH3SO2Clpyridine/‘\HZN NH3/\/\/\/\ Em“ /\/\/\/\Br OSO;>_CH3 ——?——> Br H'N[_\N'§-{ZCO1CO»*\/\/\/\ NH2 <—————— 3F1//. r }flQ\\ //‘Hi7/ \ /\Cl ‘C1 cl (:1A peptide-containing bioconjugate can be synthesized by the following methods: (1)The C-terminal carboxyl group of the peptide can be activated and used to acylate Co(I), inanalogy to the acylation of Co with chlorambucil acid chloride. (2) The C-terminal carboxylgroup can be esterified with bromoethanol, in analogy to the other chlorambucil route. and thebromide displaced with Co(I). (3) The N-terminal amino group of the peptide can be treatedwith CH3=O and Co(I) to form a Co(III)- CH2-NH-peptide linkage, in analogy to the synthesisof the topotecan bioconjugate. (4) A Co(III)-amino acid complex can be prepared, and used in acoupling step with the remainder of the peptide. These methods can involve attachment of theCo at either the N- or C-terminus, or via a side-chain. A longer linker may be employed in anyof these routes, if it is desirable to keep the cobalt complex further removed from the peptidechain.(2) ° H R ° ° H. BrCH CH OHN NHR’ 2 2 B N NHR’HOJJ\,/ N ‘“"“‘—‘> '\/\o/U\( NH EDAC/DMAP HR o RnCA 02264592 1999-02-25W0 98l08859 PCT/US97/14140 _‘-36-(1) O H R O O O H R O. (R"CO) O .HO/u\’/N u NHR _j_2__> uRJJ\o)l\|/N N NHR _j_?_>R 0 R base R R _n mixed anhydride nRHNHR‘O R O R-O R OHN NHR’R O RHCo(l||)(3)I20 RHN‘R0R O Rn“H2 ————CH2O'H > 'R0)J\‘/N N N~‘ >H CH2R o Rniminium salt0 R oH HN N\‘R0 fl CH2R o RI1Co(|Il)CA 02264592 1999-02-25W0 98l08859 PCT/US97/ 141400/UR/NHR'> Co(|||) RO . .H ziirnmo acidNROJY-R0 NH2 :_______> R \| > Co(lll)CH 20' H4 RNHR'NHR' -.RO/U\[ _______ R0”“\(° °7 Co(lIl) JO H R‘ N NH2amino acid R0 {fl 0 H R O H' j _ R o R , N N\ . .con“) activate _ RO Q ammo acidT 7R O R°a'b°xy] amino acid n| C0(lll)o R o Cw") o R oH - HH JKKN NHR' a°t“'ate , , , yd/U\l/N N NHR'0 fig carboxyl ammo ac: Hl R o RnR O R" Co(Hl)An oligonucleotide or nucleic acid containing bioconjugate can be synthesized by theWoso 2CH 3Onfollowing methods. In both methods, a phosphate ester is used to link the end of the nucleotideand a hydroxyethyl-Co group. This linkage can be accomplished by either directly coupling Co-CH2CH2—OH and Nucl—OPO32' , or by esterifying Nucl-OPO32' with Br-CH2CH2—OH, thendisplacing the Br with Co(I), as above.1015CA 02264592 1999-02-25W0 98/08859 PCT/US97/ 14140-33-Nucleotide N ,\ Nucleotide| ‘/ N— N I<2 - Q C 9 -O:P—O ——""'_""* O:P—O(l) ._ BF-CH ZCH 2-OH ('3\/\ Br\ NucleotideI~ 9O=ll°—OOWCo(l||)NucleotideHON¢\N—CNBnCH2CHyOH ‘\> Co(|l|)An unsymmetrically substituted Co[SALEN] complex can be prepared from 5-amino-salicylaldehyde and the glycolate ether of 2,5-dihydroxybenzaldehyde. The amino group, whichis prepared from commercially available 5-nitrosalicylaldehyde, functions as the attachment forthe targeting molecule (binding domain, BD) by way of EDCI-catalyzed amide formation. Theother molecule has a carboxylic acid unit attached for solubility enhancement. Coupling of thesetwo molecules with ethylenediamine and Co(II) acetate furnishes a mixture of three complexes:the two symmetrical complexes and the mixed one. All of these are useful, although the onelacking a BD-unit attached to either side of the SALEN is less preferred.With regard to binding domains, two possiblities are shown: a cobalamin derivative, anda peptide. In the former case, the known carboxylic acid is used to attach cobalamin to theamino group of the SALEN. This bioconjugate still usees cobalamin-based receptor—mediatedendocytosis to get into the cell, but the drug is attached through the SALEN instead of thecobalamin. The latter case uses a peptide known to bind to cell surface receptors of tumor’ cells(e.g., a fragment of epidermal growth factor), with the carboxyl terminus attached to the aminogroup on the SALEN. Alternatively, one of the glutamate carboxyl groups of folate is used toCA 02264592 1999-02-25WO 98/08859 PCT /U S97/14140-39-obtain a folate-based bioconjugate. In addition to connecting the binding domain via an amidelinkage, one could use reductive amination if the targeting molecule contained an aldehyde (BD-CHO + SALEN-NH2 + NaBH4), or one could use the carboxyl group on the other piece to forman amide or ester linkage. Many other approaches (e.g., ether formation, olefination by Wittigreaction, attachment via a diester or diamide linker, etc.) are also possible.CA 02264592 1999-02-25WO 98/08859 PCT/US97/ 14140-40-cH2co2HH2N CHO C02“ @ 3-“ CH0 OHC ('3OH on HO1»H2NCH2CH2NH2Co(OAc)2 COZH = for example, <______ reactive ‘HZNOC CONH2 HZNOC 0 con 0 R 1HN ‘> CONH2H|\( O 0o\P,°1020CA 02264592 1999-02-25W0 98I08859 PCT/US97/14140-41-Extended benzenoid systems of the SALEN ligands are shown below.R R R R_N\ ,N._ - w _N\ ,N__ w-G O O O X 0 O X‘v 2 2 vi 0 0As a starting point in their synthesis, any of the commercially available naphthalene diolscan be used. The diols undergo formylation reactions to furnish the molecules shown below.These moleucles are then be coupled with Co(II) acetate and various diamines to give theextended Co[SALEN] complexes. The OH groups on the second ring can be left intact, used toattach the binding domain, or modified to enhance water solubility through attachment of a polargroup, such as polyamine, polycarboxylic acid, or carbohydrate moiety.--O9 OH 2,7-dihydroxynaphthalene -—>HO1,5-dihydroxynaphthalene —--->HOHO —O9 OH 2.3-dihydroxynaphthalene ——> ‘'01.3-dihydroXYnaPhthalene '--> v S OHOH-0<6. 0,—O6 0H 2,5~dihydroxynaphtha|ene —> O1,6-dihydroxynaphthalene —-—>' OHHOHOCA 02264592 1999-02-25WO 98/08859 PCT/US97/ 14140-42-Modification of quinolines and isoquinolines are also carried out to give pyridine—fusedSALENS.I \ I \ I \ OH I \ CH0/5 N CH0 N/ H N/ CHO N/ OHCH CHOI \ I \ OH I \ CH0CH0 N / H N / CHO N / OHCHO 10 Along similar lines, SALENS derived from monocyclic heteroyclic hydroxyaldehydes are made,examples of which are shown below.CHOI \ OH \ OH/ IN CHO N’J J CHCHOI \ I \ CHO_N N__ / .._N.‘ N_ /\ / \ ’ ‘ / \O \ 0C/Q _ J N / ‘N l10152025CA 02264592 1999-02-25WO 98/08859 PCT/US97ll4l40-43-A bioconjugate containing the “green corrinoid” (Brown et a1., 1996) can be synthesizedas follows. The “green corrinoid” can be reduced in analogy to cobalamin, and that the reducedcorrinoid will react with iodomethane to form the methylCo(III) corrinoid. Since themethylCo(III) corrinoid exhibits similar behavior to natural cobalamin, similar conjugationprocedures with the electrophilic drug derivatives described above can be carried out.CH3NaBH4 CH 3|-———-——> >Co(|ll)"green corrinoid"The bioconjugates of the present invention have the improved property of being capableof targeted, selective release of the bioactive agent from the bioconjugate. The bioactive agent isis released from the bioconjugate by cleavage. In one embodiment, the cleavage may occur as aresult of normal displacement by cellular nucleophiles or enzymatic aciton. In a secondembodiment, the cleavage is caused to occur selectively at the release site by an external signal.The external signal may be light or photoexcitation, i.e. photolysis, or it may be ultrasound, i.e.sonolysis. Further, if the photolysis takes place in the presence of a magnetic field surroundingthe release site, the release of the drug, such as a cytotoxic agent, into surrounding healthy tissuecan be minimized.Although it is desired to cause the bioactive agent to be released at the desired cells,tissue or organs, e.g., at the site of the tumor or other cancer cells, it is also desirable to protectadjacent tissues from the negative side effects of such potent agents. The bioactive agent isreleased from the bioconjugate at the targeted site preferably by application of an external signal,such as light or ultrasound. The photolysis of the bioconjugates of the present invention occursthough cleavage of the Co—C bond to produce a solvent-caged radical pair consisting of Co(II)and the bioactive agent radical (R-). Lott et al. (1995) demonstrate that alkylcob(III)alarninphotolysis can undergo magnetic field dependent recombination. A magnetic field application of100-3000 gauss can be used to enhance radical pair recombination in surrounding tissue wheredrug release from the conjugate is not desired, leading up to at least a 2-fold decrease inphotochemically-triggered drug release in such surrounding healthy tissue.202530CA 02264592 1999-02-25WO 98/08859 PCT/U S97/ 14140-44-The sonolysis of the bioconjugates of the present invention occurs through cleavage ofthe Co-non-reactive atom bond in aqueous solution to produce the bioactive agent and a Co(lI)(e.g., cob(II)alamin _(Cbl")) under anaerobic conditions or the bioactive agent and aquoCo-(III)(e.g., aquocob(III)alarnin (H20-Cblm)) under aerobic conditions. In either event, sonolysis fromthe focused application of ultrasound, results in Co-non-reactive atom bond cleavage and theirreversible release of the bioactive agent from the organocobalt complex.The bioconjugates of the present invention can undergo natural cleavage as follows.Bioconjugates may be cleaved by natural means such as by serum nucleophiles. Once inside thecell, cobalamin-drug bioconjugates (utilized here only as an example and not intended to limitthe invention) can undergo cleavage by a variety of mechanisms. Standard B12 ligand exchangemechanisms permit the displacement of the drug. Alternatively, cellular nucleophiles can attackat either the carbon or the cobalt atoms of the Co-C bond. Cyanide is the most common exampleof a nucleophile which attacks at cobalt. leading to cyanocob[lII]alamin and a free drug in whichthe former C-Co bond has been replaced with a C-H bond. Thiols (such as are found in cysteineor glutathione) can attack at carbon. leading to a reduced cob[I]alamin and a free drug whichincorporates the former thiol group._(e.g., R—Co[lll] + R‘-SH + base -> R—S-R‘ + Co[l] + base-H.)Hydroxide and other basic agents can also cleave organic ligands from Co[III] complexes,although this typically occurs via an elimination process which alters the structure of the ligandthrough incorporation of a new double bond. In addition, B12 metabolic enzymes present in cellscan result in the cleavage of the bioactive agent from the co-atom.The bioconjugates of the present invention can undergo cleavage by photoactivation orphotolysis as follows. The photochemical release of the bioactive agent from the organocobaltcomplex can be triggered by the application of visible light at 400-800 nm. It is preferred to useorganocobalt complexes which require longer wavelengths of visible light (600—800 nm). morepreferably red light (600 to 750 nm). When photolysis is utilized for the release of the bioactiveagent form the bioconjugate, it is particularly preferred that the non-reactive atom of thebioactive agent or the atom of the spacer bound to the cobalt atom be a carbon atom.The vitamin B12 cofactor occurs naturally in two forms: adenosylcob(III)alamin.(AdoCblm), also known as coenzyme 13,2, and methylcob(III)alamin (CH3Cb'"). The remarkablyweak C-Co bond from the corrin ring to the 5'-deoxyadenosyl or methyl ligand imparts a mostunusual chemistry to cobalamins. The C-Co bond energy in AdoCblm and CH3Cblm is152030CA 02264592 1999-02-25W0 98/08859 PCT/US97/14140-45-estimated to be as low as 31 and 37 kcal/mole, respectively. This makes the C-Co bond one ofthe weakest covalent bonds known and allows photocleavage of the bond by visible light. Theinitial product of the photocleavage of AdoCblm is the geminate radical pair {CH2-Ado : Cbln}.Brackets { : } indicate the radical pair is geminate (born of the same parent molecule) and heldin close proximity by solvent interactions. Picosecond laser flash photolysis experiments ofAdoCblm have shown this to be a reversible process with a geminate recombination rate constantof km z 1 x 109 s], following photolysis. Recent nanosecond laser flash photolysis studies haveprobed a slower radical pair recombination that occurs in the solvent and is limited by diffusion.The TI-713* electronic transition of the corrin ring of cobalamin produces a long wavelengthabsorption maximum at 525 nm, as shownby Figure 1. Irradiation of alkylcobalamins at thiswavelength leads to cleavage of the C-Co bond with a photolysis quantum yield of 0.1-0.3. Thein viva photolysis of bioconjugates according to the present invention is preferably accomplishedby delivering the light with a fiber optic probe because of the strong absorption of hemoglobinnear this wavelength. To avoid potential problems with the absorption spectrum of cobalaminwhile maintaining a photolabile C-Co bond, Co[SALEN], which is a five-coordinate analogue ofcoenzyme B12_ can be used. Alkyl-Co[SALEN] complexes have absorption maxima near 650nm, with significant absorption beyond 700 nm as shown by Figure 2. This is a distinctadvantage for photoactivatable drug release, as human tissue becomes increasingly transparentabove 610 nm. Other synthetic or naturally occurring B12 derivatives are, or may become.available that have absorption maxima above 600 nm.. For example, a green cobalt corrinoidhaving an absorption maximum at about 624 nm is reported by Brown et al. (1996). Humantissue becomes transparent to depths of up to about 5.7 cm at wavelengths of between about 600and 800 nm. The use of longer wavelengths enables the more selective irradiation of limitedportions of a subject’s body, with consquent release in a small target region.The bioconjugates of the present invention can undergo cleavage by photoactivation orphotolysis in the presence of a magnetic field as follows. The use of the magnetic field furtherlimits the release of thebioactive agent to the desired site. For example, because of the toxicityof antineoplastic agents to healthy tissues, it is incumbent upon any effective site specificdelivery system to limit damage to cells other than at the site of activity. In the photohomoylsiscleavage reaction, the electrons in the broken covalent bond start out with their spins paired ’T‘\l/(singlet state) from having participated in the covalent bond. During the early lifetime of the1015202530CA 02264592 1999-02-25PCT/US97/14140W0 98I08859-45-radical pair, the spins retain their original orientation R4‘ + \l/R’ until the electron spinsrandomize over time. During the time the spins are paired, the radicals can recombine and revertto the starting material. If either of these paired radical spins should intersystem cross (ISC) tothe triplet spin state (R4‘ + ’I‘R’) they can no longer recombine until their spins are once againpaired. That situation is preferred to release the bioactive agent from the bioconjugate.However, in healthy tissue, it is desired to prevent, or at least minimize, the cleavage of theconjugate. It that event, it is desirable to alter the rate of ISC by introducing an externalmagnetic field that increases the gap between the triplet state energy levels, thus encouraging therecombination of the original bioconjugate in the healthy tissues.The recombination rate can be increased by application of a magnetic field in the rangeof 100-3000 gauss to the healthy tissues leading to a net decrease in the photochemical quantumyield and a decrease in drug release into healthy tissues by a factor of at least 2. Application ofabout 300 to 1000 gauss is considered to be optimal. See, Grissom (1995).The bioconjugates of the present invention can undergo cleavage by ultrasound orsonolysis as follows. Although any non-reactive atom can be bound to the cobalt atom in thebioconjugate and cleaved by sonolysis, it is preferred that the atom be a carbon atom. Thevitamin B12 cofactor occurs naturally in two forms: adenosylcob(lII)alamin, (Adocblm), alsoknown as coenzyme B12 and methy1cob(III)alamin, (CH3Cblm). The remarkably weak C-Cobond from the corrin ring to the 5'-deoxyadenosyl or methyl ligand imparts a most unusualchemistry to cobalamins. The C-Co bond energy in AdoCblm and CH3Cblm is estimated to beas low as 31 and 37 kcal/mole, respectively. This makes the C-Co bond one of the weakestcovalent bonds known and allows sonolysis of the bond by the application of ultrasound in therange of about 20 kHz - 500 MHZ, preferably 20 kHz - 100 MHZ, more preferably 20 kHz to10 MHz.Sonolysis of aqueous solutions produces a high concentration of hydroxyl radicals andhydrogen atoms according to the equation:H—OH ---- --> H- + -OHThese reactive oxidizing and reducing species are responsible for initiating most reactions inaqueous solvents. Ultrasound irradiation and sonochemistry are often not described as high-energy processes, but during sonolysis, the development, growth and implosion of bubbles in aliquid create extreme reaction environments on a microscopic scale. The collapse of cavitation1015202530CA 02264592 1999-02-25PCT/US97/14140-47- “W0 98/1188597 bubbles produces pressures >500 atm. and temperatures >5000°C. The radicals formed survivethe collapse of the bubbles. The formation of hydroxyl radicals in vivo has been the focus ofseveral investigations because of the potentially deleterious effects of oxidizing free radicals inhuman tissue. However, such radicals do not present an unacceptable health risk, as clinicalexperience has demonstrated that diagnostic ultrasound is a benign procedure. The ability toform these radicals by sonolysis in vivo provides a mechanism for the triggered release of activeneoplastic and other agents from conjugation with B”, Co[SALEN] or other suitable cobalaminor cobalamin like substrates.In the sonolysis of drug-Bu conjugates (utilized here only as an example and notintended to limit the invention), the C-Co bond is cleaved in aqueous solution to produce thedrug and a cob(II)alamin (Cbln) under anaerobic conditions or the drug and aquocob(Ill)alamin(H20-Cblm) under aerobic conditions. The cleavage is not a direct breaking of the C-C0 bond.Rather, under anaerobic conditions the predominant pathway for C-Co bond cleavage bysonolysis is through reduction of drug-Cblm to the labile drug-Cbl" by H-, followed bydissociation to the closed-shell drug and Cbl". The reaction of HO- with drug-Cbl" leads toH2O—Cblm, as well as degradation of the corrin ring. Under aerobic conditions, the pathway forthe C-Co bond cleavage by sonolysis is through reduction of the drug-Cblm to produce drug andCbl", but 02 oxidizes Cbl" to H20-Cblm. In either event, sonolysis from the focused applicationof ultrasound, results in C-Co bond cleavage and the irreversible release of the drug from thecobalamin. Therefore, sonolysis-triggered release can occur under aerobic and hypoxicconditions alike.The present invention is useful in the treatment of (including, but not limited to) cancer,hepatitis, psoriasis and other localized diseases, as well as for gene therapy and peptide therapy.The bioconjugates according to the present invention can be administered by any route,including intravenously, parenterally, orally, intramuscularly, intrathecally or as an aerosol. Themode of delivery will largely depend on the biological effect desired to be achieved. A skilledartisan will readily know the best route of administration for a particular treatment in accordancewith the present invention. The appropriate dosages will depend on the route of administrationand the treatment indicated, and can be readily determined by a skilled artisan, such as byextrapolation from current treatment protocols. If the organocobalt complex of the bioconjugate10202530CA 02264592 1999-02-25WO 98108859 PCT/US97/14140-43-is cobalamin or derivative or analogue, it is preferred to administer orally a bolus of vitamin B12prior to administration of the bioconjugate to reduce or eliminate potential hepatotoxicity whichmight otherwise result from the administration of the bioconjugate. The oral dose of B” willsaturate the enterohepatic shuttle system and load hepatocytes with cobalamin. It is preferredthat 0.1 mg to 100 mg, more preferably 1.0 mg to 10 mg, of vitamin B12 be administered prior tothe administration of the bioconjugate containing cobalamin. In addition, vitamin B12 can beadministered, preferably intravenously, following the selective cleavage of bioconjugate to washout all bioconjugate which has not been cleaved, and thus further reduce potential systemiceffects. It is preferred that 0.1 mg to 100 mg, more preferably 10 mg to 100 mg, of vitamin B12in saline be administered intravenously over 4-5 hrs. Finally, prior to the administration of acoba1amin—based bioconjugate, nitrous oxide can be administered to the subject in order todeplete body stores of cobalamin in its various forms, such as methylcobalamin. Administrationof nitrous oxide has the effect of creating a greater body deficit of cobalamin beforeadministration of the cobalamin-based bioconjugate. 0Pharmaceutical compositions containing a compound of the present invention as the activeingredient can be prepared according to conventional pharmaceutical compounding techniques. See,for example, Remington's Pharmaceutical Sciences, 17th Ed. (1985, Mack Publishing Co., Easton,PA). Typically, an antagonistic amount of active ingredient will be admixed with a pharmaceuticallyacceptable carrier. The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., intravenous, oral, parenteral, intrathecal, transdermal,or by aerosol.For oral administration, the compounds can be formulated into solid or liquid preparationssuch as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. In preparingthe compositions in oral dosage form, any of the usual pharmaceutical media may be employed,such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents,suspending agents, and the like in the case of oral liquid preparations (such as, for example,suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents,lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as,for example, powders, capsules and tablets). Because of their ease in administration, tablets andcapsules represent the most advantageous oral dosage unit form, in which case solid pharmaceuticalcarriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by10202530CA 02264592 1999-02-25WO 98/08859 PCTIU S97/ 14140-49-standard techniques. The active agent must be stable to passage through the gastrointestinal tract.If necessary, suitable agents for stable passage can be used, and may include phospholipids orlecithin derivatives described in the literature, as well as liposomes, microparticles (includingmicrospheres and macrospheres).For parenteral administration, the compound may dissolved in a pharmaceutical carrier andadministered as either a solution or a suspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. Thecarrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administered intracerebroventricularlyor intrathecally, they may also be dissolved in cerebrospinal fluid. —In the treatment of cancer (sarcomas, carcinomas or leukemias), a distinction can be‘made between the types of systemic adjuvant chemotherapies that are typically used in concertwith the more extreme methods of surgical excision and radiation therapy. There are two broadclasses of chemotherapeutic adjuvants: (1) endocrine and antivasogenic therapeutic agents whichare aimed at altering the body's physiology; and (2) cytotoxic chemotherapeutic agents which aretypically administered systemically to kill or inhibit the growth of transformed cells.Cytotoxic or antineoplastic agents are represented by a number of drug classes.Alkylating agents undergo chemical reactions that generate highly reactive electrophiliccarborationsnium ions that readily form covalent linkages (alkylate) with various nucleophilicbiologically important moieties such as nucleic acid bases and phosphate, amine, sulthydryl,hydroxyl, carboxyl and imidazole groups. These agents, among other functions, have cytotoxicactions that disturb the fundamental mechanisms concerned with cell growth, mitotic activity,differentiation and fimction. Chlorambucil, modified busulfan, cyclophosphamide, ifosfamideand cisplatin and its structural analogs are representative alkylating agents.Antimetabolites such as folic acid analogs (e.g. methotrexate) and pyrimidine analogs(e.g. fluorouracil and fluorodeoxyuridine) exert cytotoxic activity by blocking or preventingmetabolic pathways leading to neoplastic cell destruction. Methotrexate is also known to beuseful in the treatment of psoriasis by inhibiting the proliferation of epidermal cells.Another potent cytotoxic class is mitotic inhibitors such as the paclidaxel or the alkaloidscamptothecin, vincristine and vinblastine.10202530CA 02264592 1999-02-25WO 98/08859 PCT /U S97/14140-50-Also, certain antibiotics, such as doxorubicin and daunorubicin, (tetracyclic aglyconeglycosides), intercalcate with DNA and inhibit nucleic acid synthesis.In accordance with the present invention, bioconjugates for the treatment of cancer areformed preferably using chemotherapeutics selected from the group consisting of alkylatingagents, antimetabolites and mitotic inhibitors. For example, methotrexate is an antimetabolite;chlorambucil, cisplatin and modified busulfan are alkylating agents, and camptothecin and itsderivatives are alkaloids. The bioconjugates formed from these cytotoxic agents can beadministered intravenously for the treatment of the specific classes of cancer for which they areknown to be effective, e.g. cancer of the colon, lung, kidney, breast, prostate, melanoma,nasopharyngeal, T-cell leukemia, myelogenous leukemia, lymphocytic leukemia and the like.When delivered intravenously to the blood stream and the bioconjugates contain cobalamin, thenatural affinity of cancer cells for B12 will target the bioconjugates to these tissues or cell sites.Alternatively, the bioconjugates can be engineered to be selective for the delivery of thechemotherapeutic agent to the desired cancer cell by the incorporation of a suitable targetingmolecule (such as those set forth above) on the organocobalt complex.In accordance with the present invention, solid tumors are treated as follows, with use ofa drug-B12 bioconjugate as an example. This example is not intended to limit the presentinvention in any manner, and a skilled artisan could readily determine other bioconjugates of thepresent invention which could be utilized for the treatment of solid tumors. The drug-B12bioconjugate is administered, preferably intravenously, to a cancer patient to target metastaticcancer when the cancer cell has a significant requirement for cobalamin. This propensity ofcobalamins to migrate to the cancer cells significantly reduces cardiotoxicity, myelotoxicity,hepatotoxicity and similar side effects that limit the size and frequency of effective dosing ofantineoplastic agents. Furthermore, problems associated with toxicity to non-targeted cells isminimized. Delivery is further enhanced by the triggering of the release of the antineoplasticagent from the bioconjugate by the mechanism of photolysis or sonolysis which provides for ahigh degree of spatial and temporal control of the drug release at a localized area over a shorttime. The application of a magnetic field with photolysis further serves to protect health cells byrecombination of the bioconjugate and limit the release of active agent to -the specific cancercell-containing site(s).10H202530CA 02264592 1999-02-25W0 98/08859 PCT/US97/14140-51-Although chemotherapy is generally reserved for targeting metastasized cells after thesurgical excision of the primary tumor mass. the triggered release of a bioactive agent drawn tothe tumor site allows for treatment of the primary tumor, as well as metastatic neoplasms thathave spread to a limited and known area. The bioconjugate dosage, length of treatment, degreeof photoactivation, and other treatment parameters can be determined by one skilled in the artbased on the type of cancer, antineoplastic agent administered, specific cobalamin used,condition of the patient and other factors which are variable and best determined on a case-by-case basis.In accordance with the present invention, leukemia is treated as follows, with use of adrug-B12 bioconjugate as an example. . This example is not intended to limit the presentinvention in any manner. and a skilled artisan could readily determine other bioconjugates of thepresent invention which could be utilized for the treatment of leukemia. At least two forms ofleukemia, chronic myeloid leukemia (CML) and acute promyeloctyic leukemia (APL), producehigh levels of B12 binding proteins that result in a 3- to 36-fold increase in the unsaturated B13binding capacity in the blood. The increased concentration of B” binding proteins is consistentwith the rapid turnover of immature blood cells and provides an opportunity to target thedelivery of antileukemic drugs, such as bis-alkylating agents derived from busulfan. to thetransformed hematopoietic cells responsible for the leukemic condition. The bioconjugates ofthis invention provide a means for the effective administration of such alkylating agents to cellsites from which the active agent can be released from the conjugate. This targeted delivery andrelease provides a significant advance in the treatment of CML and APL, for which currentchemotherapy methods sometimes provide incomplete remission.The present invention is also useful for the treatment of psoriasis. Psoriasis is a primetarget for the transdermally or orally controlled delivery of antimetabolites activated byphotolytically induced cleavage. Although not life-threatening, psoriasis can significantlydiminish the quality of life of patients who experience severe exfoliation associated withpsoriatic and rheumatoid arthritis. Antimetabolites, such as methotrexate and 5-fluorouracil, areeffective in controlling severe cases of skin proliferation. Effective oral therapy is limited byhepatotoxicity in spite of low dosing, and the risk of cumulative liver damage requires suchtherapy to be reserved for only the most severe episodes during a patient’s life. The delivery of10202530CA 02264592 1999-02-25W0 98/08859 PCTIUS97/14140-52-such agents to the skin improves the appearance and psychological quality of life of patientswhose lives have been dominated by severe psoriasis.The present invention is further useful for peptide therapy. One example of peptidetherapy is as a cytotoxic agent, for example as an antineoplastic agent. In this example, abioconjugate containing the enzymatic domain of diphtheria toxin (DT) is administered to asubject such as described above for solid tumors or leukemia. The targeted release of the DTpeptide results in the inhibition of protein synthesis and eventual cell death.The present invention is also useful for gene therapy. One example of gene therapy isthe delivery of an antisense oligonucleotide to inhibit viral gene expression and viral replication.In this example, a bioconjugate containing an antisense oligonucleotide against hepatitis B virusis administered to a patient having a hepatitis B infection. The accumulation of the bioconj ugateand release of the antisense oligonucleotide in the liver inhibits hepatitis B virus gene expressionand replication.The present invention is further described by reference to the following Examples, whichare offered by way of illustration and are not intended to limit the invention in any manner.Standard techniques well known in the art or the techniques specifically described below wereutilized.EXAMPLE 1Bho_tglxsL_'s Q_.Bf g A protocol for a typical anaerobic continuous-wave photolysis of methylcob(IIl)alaminor cH,cb1‘“ is as follows. An aqueous solution of 200 uM cH3cb1‘“ and 50 mM K* Hepes pH7.3 is placed in a 1 cm quartz cuvette. Samples that are to be photolyzed under anaerobicconditions are either subjected to repeated freeze-pump—thaw cycles, or purged with Ar for 40minutes immediately prior to photolysis and sealed. Continuous-wave visible light irradiation isaccomplished at the desired wavelength with an Arl pumped dye laser. The incident light on theface of the cuvette is reduced to 12 mW crniz with neutral density filters. The light flux isdetermined by potassium ferrioxalate actinomety and by a Scientech surface-reading therrnopile.The cuvette is placed in a therrnostated cell holder at 25-3 7°C. For quantum yield measurementsas a function of magnetic field, the cuvette is placed in the gap of a GMW Associates152030CA 02264592 1999-02-25“'0 98’"3359 PCT/US97/14140-53-electromagnet with 7.5 cm diameter cylindrical poles. The magnetic field within the area of thecuvette is homogeneous to within 2% and the long term stability is better than 0.5% asmonitored by a transverse Hall probe and digital teslameter.Absorption spectra from 300-600 nm are recorded in one second with a diode arrayspectrophotometer at variable time intervals from 10 seconds to 2 minutes (depending upon thefluence of the photolyzing light source) for a total of 3 1%. Exposure to light during analysis iskept to a minimum. The concentration of CH3CbIm is determined using the measuredabsorbance at 350 or 520 nm by the method of Chen and Chance (1993). The plot of [CH3Cbl'"]vs. time (t) appears zero-order in all cases. The absorption spectra for CI-l3Cblm and ethyl-Co[SALEN] are shown in Figures 1 and 2. For CH3Cblm the 1t—7t* electronic transitions thatlead to cleavage of the C-Co bond are maximal at 377 and 528 nm. Much preliminary work withB12 photolysis has been carried out with 514 nm light from an Argon-ion (A?) laser. This isclose to the long-wavelength maximum absorbance and gives a quantum yield of about 0.3 forcH3ch1‘“.The absorbance of blood and tissue is significant at this optimal wavelength forcob(III)alamin excitation. Blood has a low (partial) transmittance window near 514 nm. Thisabsorbance is sufficient to ouickly pyrolize whole bovine blood placed in the light path of a 20W/cmz beam of514 nm light.It would therefore be beneficial to provide a cobalamin for conjugation wherein the ‘It-Tt*electronic transitions that lead to cleavage of the C-Co bond are maximal at a wavelength wherethere is minimal or no interference. Above about 610 nm blood becomes partially transparentand losses beyond 50% transmittance are largely due to light scattering from the erythrocytes.Heparinized bovine blood placed in the light path of a 20 W/cmz beam of 630 nm light showsonly minor heating over long exposure times. There is also demonstrated a high transmittance oftissue at 610-800 nm.This suggests the use of an organocobalt complex for conjugation having an absorptionwavelength where tissue and blood are relatively transparent. Figure 2 shows thatethyl-Co[SALEN] complexes have absorption maximums near 650 rim, with significantabsorption beyond 700 nm. An Ar+-pumped dye laser or a Krypton-ion (Kr') laser can be asuitable high-intensity source of photons in the region of 6l0’ nm. Ar+-pumped dye lasers are1015202530CA 02264592 1999-02-25W0 98l08859 PCT/U S97/ 14140-54-often used for photodynamic therapy with hematoporphyrins. Also, an inexpensive He-Ne laser,having a principal line at 633 nm might be used. However, such lasers are typically limited to 50mW maximum output.There are laser dyes in the 600-700 nm region that can achieve energy conversionefficiencies of up to 45%. This means that a 6 W Ar+ pump laser can yield nearly 3 W ofspatially-coherent monochromatic light in the region of 610-750 nm. The exact wavelength canbe chosen to optimize the continuous-wave quantum yield and still maintain a reasonable degreeof tissue penetration. In tests with alkyl-Co[SALEN] complexes it has been found that 690 nmA light from an Ar+-pumped dye laser operating with rhodamine 6-G dye is satisfactory.Optimization can be determined depending on the specific cobalamin chosen for animal and/orclinical trials of the bioconjugates. In addition, high-power diode lasers that emit red light of thedesirable wavelength are commercially available. These diode lasers have the advantage ofproviding up to 100 watts of optical power in a narrow region of the optical spectrum that isuseful for triggering cleavage of the bioconjugates.EXAMPLE 2Sonolysis of B E and QQISALENI BjQcgn'ugatesSonolysis was carried out with a Branson ultrasonic bath (model 3200) operating at 47kHz. The correct placement of the reaction vessel at a focal point of high- intensity ultrasoundwas determined by the oxidation of iodide to iodine in the presence of starch (Mason, 1991) andthe temperature of the bath was maintained at 21° C by a constant temperature circulator.Aerobic sonolysis was typically carried out in a test tube or Erlenmeyer flask, whereas anaerobicsonolysis was carried out in a closed reaction vessel fitted with a sidearm and quartz cuvette.Anaerobic conditions were produced by sparging with Ar for 30 min prior to sonolysis. In someexperiments, the pH was buffered by the use of 100 mM phosphate (aerobic experiments) or 100mM N—(2-hydroxyethyl)piperazine-N—2-ethanesulfonate (Hepes) (anaerobic experiments), asspecified. All procedures were carried out in the absence of light to prevent photolytic cleavageof the Co-C bond. Absorption spectra were recorded on a diode array spectrophotometer (HP8452A). The solutions were transferred to a quartz cuvette with a 1 cm light path for all opticalmeasurements and care was exercised to ensure that insignificant photolysis occured during the1 s measurement time.WO 98/0885910202530CA 02264592 1999-02-25PCT/US97/14140-55- : Sequential absorption spectra of aqueous CH3-Cblmas a function of anaerobic sonolysis (pH 7.38, 100 mM Hepes, saturating Ar) are shown inFigure 3A. The absorbance at 340, 374, and 520 nm decreases linearly as a function of sonolysistime and the absorbance at 316 and 420 nm increases linearly, thereby indicating the reaction iszero order in substrate concentration. The isosbestic points at 336, 390, and 585 nm are inagreement with those obtained through anaerobic photolysis of CH3-Cblm. Under the conditionsof sonolysis, an additional isosbestic point occurs at 476 nm, rather than at 486 nm, as typicallyobserved in the course of photolysis. This slight shift in the isosbestic point is caused by aminor product that has an absorbance maximum near 490 nm. This may be cob(I)alarnin thathas a sufficient lifetime (t.,, = 22 min at pH 6) to be observed spectrophotometrically. Theabsorption band at 374 nm is characteristic of a C—Co bond, and its disappearanceunambiguously indicates displacement of the axial carbon ligand.Under aerobic conditions, molecular oxygen scavenges H- and prevents the reduction ofCH3-Cblm via the equation02 + H- ——> -OOHIn the absence of an organic buffer with abstractable hydrogen atoms, reaction via HO- remainsto be a viable process.Figure 3B shows the change in absorbance spectra following aerobic sonolysis in theabsence of organic buffer. The decrease in absorbance at 340 and 374 nm is linear withincreasing sonolysis time indicating the reaction is zero order in substrate concentration, but theunexpected increase in absorbance at 520 nm indicates the stable product of cobalamin sonolysisis not hydroxocob(IIl)alamin, as would be expected if molecular oxygen were to reoxidizecob(II)alamin to cob(III)alamin. Aerobic photolysis under the same conditions shows theexpected decrease in absorbance at 374 nm but no change at 520 nm. This difference suggeststhat HO- is able to displace the alkyl ligand from Com, but other HO- reactions also occur(perhaps through the secondary products HOO- and -O2") to oxidize the corrin ring. Similarabsorbance spectra are obtained from sonolysis of an aerated aqueous solution containing 100mM phosphate buffer.A similar result is seen in the reaction of CH3-Cblm with H- and HO- when these radicalspecies are generatedby pulse radiolysis (Blackburn et a1., 1972). Reducing species H- reacts toproduce the same spectral changes as shown in Figure 3A. Multiple oxidizing species (HOO-1015202530CA 02264592 1999-02-25W0 98/08859 PCTIUS97/14140-55-and -02") can react with CH3-Cblm to cleave the Co-C bond, but these species also lead to theirreversible degradation of the corrin ring, as evidenced by the spectral changes similar to thoseseen in Figure‘3B. A precedent for irreversible oxidation of the corrin ring exists in thephotooxygenolysis of alkylcobalamins by singlet oxygen (Krautler and Stepanek. 1985).Aerobic sonolysis of solutions containing 100 mM Hepes or 100 mM I-butyl alcoholproduces no change in the absorption spectra over comparable time. This is because molecularoxygen quenches the l-l- reaction pathway, and t-butyl alcohol quenches the HO- reactionpathway. Although Hepes has not previously been reported to be a scavenger of HO-, manyreports indicate that organic solute molecules such as formate, can inhibit the reaction of HO-(Weissler, 1962). The absence of any spectral changes under these conditions suggests that_ direct sonolysis of the Co-C bond is not an important reaction pathway.EXAMPLE 3Biological Testing Against NCI Human Tumor Cell LinesThe efficacy of the bioconjugates is tested against tumor cell lines using existingprotocols for assessing the effectiveness of targeted coenzyme B12 antineoplastic agent-containing bioconjugates. Representative cell lines tested include HCT 116 (human colontumor). A549 (human lung), ACHN (human kidney), MCF7 (human breast). human prostate.SK5-mel (human melanoma), KB (human nasopharyngeal). CCRF-CEM (human T-cellleukemia), HL-60 (human promyelocytic leukemia), RD-995 (mouse fibrosarcoma). B-16(mouse melanoma) and Meth-A (mouse carcinoma). Drug screening is carried out with acolorimetric cell viability assay in a 96-well plate.Additionally, selected bioconjugates are radiolabeled with MC or 3H to assess the level ofuptake by human tumor cells. As noted above, the prior art reports that some tumor andleukemia cells produce high levels of B13 binding proteins in the serum and sequester B13 in highconcentrations of up to 50 fold.Tumor Cell Line Testing Protoefl The drug bioconjugates, synthesized underprocedures described herein or adaptions thereof. are diluted over 5 orders of magnitude(approximately 0.005 to 50 pg/mL). Four hours after seeding of the cell in the plate, the cellsare treated with the appropriate drug dissolved in isotonic buffered solution. In controlexperiments. without photolysis triggered drug release. the drug is left on the cells for threeWH20WO 98108859CA 02264592 1999-02-25PCT/US97/14140-57-days, as in the normal basic cancer screen. In the wells for triggered drug release, the laseroutput is focused on selected wells for varying times and with varying intensity. Alternatively, amatrix of light—emitting diodes (LEDS) is used. The cells are incubated under standardmammalian tissue culture conditions under the proper CO2 balanced atmosphere. After threedays, the cells are re-fed and the colorimetric dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) is added. The reduction of the MTT to purple formazan product isquantified in a 96 well plate reader. The concentration of the purple forrnazan dye is correlatedwith the number of viable cells. The reduction in cell survival at a given dose rate andphotolysis exposure give a quantitative estimation of cell death and drug delivery effectiveness.Care is taken not to expose portions of the plate to photolysis conditions through adventitiousspillover of radiation. This is accomplished by using 96 well plates with an opaque mask (Fishercat# 07-200-565) for photolysis.! [make gf | ggig-BQ : The uptake of drug—B,2 anddrug-Co[SALEN] bioconjugates by cultured tumor cells is monitored by radiolabeling the drugor cobalamin during synthesis. 3H-Labeled 5-fluorouracil, methotrexate and chlorambucil are purchased from DuPont/NEN (New England Nuclear). These drug bioconjugates, as well as “C-labeled methy1cob(III)alamin (synthesized from Cob(I)alamin and ”CH3I) provide an indicationof receptor-mediated uptake by the various tumor cell lines. In this study, the cells are exposedto the radiolabeled drug as described in the preceding section, except no MTT is added at the endof the three—day incubation period. Since all of the cell lines except the leukemia cells growwhile attached to the bottom of the microtiter plate well, the growth medium is aspirated toremove the unincorporated radiolabeled drug, followed by several washes with fresh medium.The labeled cells are detached from the bottom of the wells and the radioactivity quantified byscintillation counting. Growth of non—attached leukemia cell takes place in round-bottommicrotiter plates such that centrifugation sediments the cells and allows washing with freshgrowth medium before solubilizing the cells and quantifying the incorporated radiolabeled drugby scintillation counting.10202530CA 02264592 1999-02-25WO 98/08859 PCT/US97/14140 _‘-58- mEXAMPLE 4si A ESynthesis of N, N ’-bi s( salicylidene )e1;1_1yle_1;ediamine,To a stirred solution of salicylaldehyde (12.21 g/10.62 mL) in 70°C ethanol (100 mL)was added ethylenediamine (3.01 g/3.33 mL).formed, and the reaction mixture was allowed to cool to room temperature with stirring. TheA yellow crystalline material immediatelysolution was filtered, and the crystals were washed with cold ethanol. The ethanol layers wereombined and reduced to approximately 20 mL and allowed to stand at 0°C overnight. Theresulting crystals were collected by vacuum filtration and washed with water. The collectedsolids were dried in vacuo to obtain 13.15 g (98%) N,N’-bis(salicylidene)ethylenediamine asyellow platelets with a melting point of 126°C (literature value (24) 127-128°C). 1H NMR(DMSO-d6) 8 8.57 (s 2H, HC=N), 7.42 (d, 2H aromatic, J=7.3 Hz), 7.31 (t, 2H, aromatic, J=9.0Hz), 6.88 (t, 4H, aromatic, J=8.3, 16.1 Hz), 3.89 (s, 4H, CH2). BC NMR (DMSO—d6) 5 166.94(2C, CO‘), 16O.57A(2C, HC+N), 132.38 (2C, aromatic), 131.69 (4C, aromatic), 118.59 (2C,aromatic), 116.51 (2C, aromatic), 58.88 (2C, CH2).nth i f N N’-b' al' liden e lene iamine al 11 Co SALEN .To a hot (100°C) deoxygenated solution of the above product (2.68 g) in dimethylforma-mide (25 mL) was added via cannula needle an aqueous solution (10 mL) of cobalt (II) acetatetetrahydrate (2.49 g). The red precipitate which formed was collected by vacuum filtration,washed with cold dimethylformarnide, and dried in vacuo to obtain 2.6 g (80%) of N,N’-bis-(salicylidene)ethylenediaminocobalt (II) as red crystals.EXAMPLE 5S nt i d' ed Co AThe diglycolate ether of Co[SALEN] is prepared as described in Example 4, using theglycolate ether of 2,5-dihydroxybenzaldehyde in place of salicylaldehyde. An unsymmetricallysubstituted (glycolate ether/amide) complex is prepared as described in Example 4 by using amixture of the glycolate ether of 2,5-dihydroxybenzaldehyde and 5-aminosalicylaldehyde inplace of salicylaldehyde.1015202530WO 98/08859CA 02264592 1999-02-25PCTlUS97/ 14140-59-EXAMPLE 6es’ ' - b i ' c ' eSynthesis of 1-bromo-2—[4-(4'-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethane.Twenty-five mL of freshly distilled CHZCI2, 0.343 g dicyclohexylcarbodiimide (1.66mmol), 0.305 g 4-dimethylarninopyridine (2.5 mmol), and 0.263 g 4-dimethylamino—pyridiniumchloride (1.66 mmol) were added to a flame-dried 50 mL round bottom flask equipped with astir bar, reflux condenser, and Ar inlet (Boden and Keck, 1985). The solution was purged withargon and brought to reflux. While refluxing, a solution of 0.304 g chlorambucil (1.0 mmol) and‘ 0.125 g 2-bromoethanol (1.0 mmol) in 5 mL CH2Cl2 (purged under argon for 30 min.) wastransferred via cannula to the refluxing solution over a period of 30 min. After addition wascomplete, the reaction mixture was stirred for 2 h at room temperature. Precipitateddicyclohexylurea was removed by filtration and the solution was concentrated by rotaryevaporation. The resulting residue was taken up in CH2CI2 , filtered, and purified by flash silicacolumn chromatography. The desired product was eluted using 129 ethyl acetatezhexanes (v/v)to give 0.374 g ofa yellow oil in 91% yield (ester 2). ‘H NMR (CDCI3, 300 MHz) d 7.06 (d, 2H,J=8.4 Hz), 6.60 (d, 2H, J=8.7 Hz), 4.35 (t, 2H, J=6.15 Hz), 3.56-3.72 (m, 8H), 3.48 (t, 2H,.l=6.15 Hz), 2.56 (t, 2H, J=7.65 Hz), 2.35 (t, 2H, J=7.35 Hz), 1.91 (quintet, 2H, J=7.58 Hz). “CNMR (CDCI3, 75 MHz ‘H decoupled) d 173.05, 144.35. 130.37, 129.75 (2), 112.12 (2), 63.68,53.55 (2), 40.60 (2), 33.94, 33.41, 29.05, 26.72. Two hundred mg of hydroxocob(III)alamin (0.15 mmol) was dissolved in 10 mL waterand purged with Ar while stirring (Brown and Peck, 1988). The exiting gas was conducted insequence through: (1) a flask containing 0.025 g NaBH4 (0.66 mol); (2) a flask containing 5mL H20; and (3) a flask containing 0.226 g ester 2 (0.55 mmol) in 5 mL CH3OH. Afterdeaerating for 1 h, the water from flask (2) was transferred to flask (1) containing NaBH4 viacannula and swirled to promote dissolution. This solution was transferred via cannula to theaqueous cobalarnin solution. Reduction was allowed to proceed for 20 min, after which thechlorambucil bromoethylester was added to the solution. The reaction mixture was allowed tostir for an additional 5 min. and then 0.2 mL acetone was added to destroy the excessborohydride. The solution was concentrated to approximately 2 mL by rotary evaporation and10202530WO 98/08859CA 02264592 1999-02-25PCT/U S97/ 14140-50-the resulting solution was applied to a 2.5 X 30 cm column of Amberlite XAD-2 resin. Thecolumn was washed with 1 L H20 to desalt and the cobalamin was eluted with 50% aqueousacetonitrile (v/v). The eluent was reduced to approximately 2 mL by rotary evaporation and thesolution was applied to a 1 X 40 cm column of SP-Sephadex C25 (Na+ form). Elution withwater removed the major red band which was reduced to a minimal volume. Acetone was addeduntil faint turbidity persisted after swirling. The solution was allowed to stand for 1 h at 0°C andexcess acetone was added to promote further crystallization. The crystals were collected byvacuum filtration and dried in vacuo. 3 was obtained as red crystals (122.5 mg) with a yield of53%. MS (FAB+) calcd for C6,; HmN14O,6CoPCl2, 1541; found 1541.4-[4'-(bis-[2-chloroethyl]amino)phenyl]butyroylcob(lII)alamin (4) was synthesized in asimilar manner starting with the acid chloride of chlorambucil and reating it withhydroxocob(III)a1arnin as above.Synthesis of 2-[4-(4'-[bis-(2—chloroethy1)amino]pheny1)butyroxy]ethyl-Co[SALEN] and4-[4'-(bis-[2-chloroethyl]amino)pheny1]butyroyl-Co[SALEN] are synthsized in a similar mannerusing Co[SALEN] in place of hydroxocob(III)alamin.Synthesis of 2-[4-(4'-[bis-(2—chloroethyl)aniino]phenyl)butyroxy]ethyl-(Co[SALEN]-folate) and 4-[4'-(bis-[2-chloroethyl]arnino)phenyl]butyroyl-(Co[SALEN]-folate) are synthsizedin a similar manner using Co[SALEN]‘—folate in place of hydroxoc0b(III)alamin.Synthesis of 2-[4-(4'-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethyl-(green corrinoid)and 4-[4'-(bis-[2-chloroethyl]amino)phenyl]butyroyl-(green corrinoid) are synthsized in a similarmanner using CH3-Co(III) corrinoid (prepared by reacting methyliodide with the green corrinoidof Brown et al. (1996) after it had been reduced with NaBH4) in place of hydroxocob(lll)a1amin.EXAMPLE 7Sonolvsis of 2-l4-(4'-lbis-(2-chloroethvhaminolnhenvhbutvroxvlethvlcob(IIl)alamin (3)The products released by exhaustive sonolysis, as described in Example 2, of compound3 (prepared in Example 6) were isolated by reverse-phase HPLC (Rainin Microsorb C-18).Elution and separation of the sonolysis products were carried out with an increasing gradient ofacetonitrile (A) and 0.05 M phosphoric acid (B): initial condition 5% A: 95% B, increasedlinearly for 10 min to 30% A and 70% B, maintained for 2 min; followed by a linear increase to10152030WO 98/08859CA 02264592 1999-02-25PCTIUS97ll4140 ”-51-70% A and 30% B over 10 min (Rinchik et al., 1993). The solvent was evaporated from eachfraction and the products were extracted with CH2Cl2 and characterized by ‘H and BC NMR.Sequential absorption spectra of aqueous 3 as a function of anaerobic sonolysis at pH7.4, 100 mM Hepes, saturating Ar, are shown in Figure 4A. The absorbance at 374 and 520 nmdecreases linearly as a function of sonolysis time, and the absorbance at 316 and 420 nmincreases linearly, thereby indicating the reaction is zero order in substrate concentration. Theisosbestic points at 336, 390, 486 and 585 nm are in agreement with those obtained throughanaerobic photolysis of CH3-Cblm. The absorption band at 374 nm is characteristic of a Co-Cbond, and its disappearance unambiguously indicates displacement of the axial carbon ligand.Figure 4B shows the change in absorbance spectra following aerobic sonolysis of a 3solution containing phosphate buffer. Different stable products are obtained under aerobicconditions. Because of the presence of molecular oxygen, the released product was shown byNMR to be 2-[4-(4'—[bis-(2-chloroethyl)arnino]phenyl)butyroxy]etha.n-l—al. [H NMR (CDCI3,300 MHz) 9.59 (s, 1H), 7.08 (d, 2H, J=2.9 Hz), 6.62 (d, 2H, J=2.9 Hz), 4.67 (s, 2H), 3.73-3.59(m, 8H), 2.60 (t, 2H, J=7.5 Hz), 2.45 (t, 2H, J=7.4 Hz), 1.95 (quintet, 2H, J=7.4); “C NMR(CDCI3, 75 MHz ‘H decoupled) 195.85, 173.09, 144.54, 130.43, 129.92 (2), 112.29 (2), 68.73,53.74 (2), 40.69 (2), 33.99, 33.13, 26.79. The decrease in absorbance at 374 nm is linear withincreasing sonolysis time indicating the reaction is zero order in substrate concentration.The Co-C bond of CH3-Cblm can be cleaved by sonolysis in aqueous solutions toproduce the alkane and cob(II)alamin under anaerobic conditions or to produce the aldehyde andhydroxocob(III)alarnin under aerobic conditions. Unlike photolysis and thermolysis that lead todirect Co-C bond cleavage, the predominant pathway for Co-C bond cleavage by sonolysis isthrough H- mediated reduction of CH,-ch1‘" to the labile 19 e’ CH3-CbI"' species followed bydissociation to the closed-shell alkane and Cbl", or through the reaction of HO- with CH3-Cblmthat leads to formation of hydroxocob(III)alamin as well as degradation of the corrin ring.A parallel exists between the reactions of alkylcob(III)alamin under the conditions ofsonolysis and pulse radiolysis, (Blackburn et al., 1972) but without the need for expensiveequipment. Although the violent cavitation during sonolysis has sufficient energy to break theCo~C bond to produce the {R- °Cbl"} radical pair by a dissociative pathway analogous to.thephotolysis of CH3-Cblm, (Endicott and Netzel, 1979; Chagovetz and Grissom, 1993; Natarajanand Grissom, 1996), alkylcob(III)alamins are not sufficiently volatile to be found in the extreme10202530WO 98/08859CA 02264592 1999-02-25PCT/US97l14l40-62-environment of the collapsing bubbles. Therefore, direct Co-C bond cleavage by sonolysis isnot possible in spite of the more than 80 kcal/mol difference in bond-dissociation energybetween Co-C and H-OH.Anaerobic sonolysis of the Co-C bond is irreversible because a closed-shell alkane isformed. Under aerobic conditions, the rate of H0 reaction with O2 is on the same order ofmagnitude as the reaction of H- with CH3 , thereby suggesting the closed-shell alkane, CH4,should be one of the end products of CH3-Cblm sonolysis (Buxton et al., 1988; Baulch et al.,1992). In contrast, Co-C bond cleavage of CH3-Cblm, via anaerobic photolysis, is reversiblefrom the {CH3- -cb1“} radical pair.In summary, the ability to form cob(II)alamin and the closed-shell alkane without the useof chemical reductants and without the use of electrochemical, photochemical, or pulseradiolysis equipment may be a useful method to promote activation of drug-cobalamincomplexes in vivo.EXAMPLE 8ria an M d ‘n vitr of i c ' ti itMedia BreparatjgnAll media were purchased from Sigma and materials used to supplement the media werepurchased from Atlanta Biologicals. The HL-60 cell culture was grown in an on-MEM media.The media was completed prior to inoculation by the addition of reagents to bring the finalmedia concentration to 7.5% w/v sodium bicarbonate, 10% fetal calf serum, 100 fig/mL.penicillin and streptomycin, and 50 units/mL mystatin. McCoy’s media, with sodiumbicarbonate buffer, was used for HCT-116 cell culture. It was completed in the same mannerwith 8% newborn calf serum and 2% fetal calf serum. Completed media could be stored at 4°Cfor several weeks. The culture medium was warmed to 37°C before inoculation with cells.S k Cultur P ainte nceStock cell cultures were started from ATCC cell lines. The original ATCC cell line wasarat' n naliquoted in 10% DMSO and stored in liquid nitrogen. Stock cultures of 40 mL were grown andmaintained in collaen-treated, sterile 75 mL culture flasks purchased from Coming. The cultureswere incubated at 37°C in a 5% CO2 environment to maintain a pH of 7.1. Humidity within the10H202530WO 98/08859CA 02264592 1999-02-25PCT/US97/14140-53-incubator was maintained to prevent hypertonicity in the media by placing an open pan of waterin the bottom of the incubator.The concentration of cells within the stock culture was controlled and cell concentrationswere estimated in several ways. The media became more purple and subsequently orange as aresult of cell metabolism and metabolic byproducts that accumulated in the media. The cellswere also visually observed under microscope at 40X and IOOX power. Normal HCT-116 cellsappeared rounded, flat, and adhered strongly to the walls of the culture flask. When the cellsalmost covered the bottom of the flask, the cell concentration was reduced. Normal HL-60 cells9 appeared round, but were well differentiated and easily suspended in the media. Changes in cellmorphology were often indicative of bacterial or fungal contamination. For the accuratedetermination of cell concentrations, a Coulter Cell Counter” was employed. Stock cultureswere not allowed to grow to greater than 100,000 ‘cells/mL. Both of the cell lines were observedto have a doubling time of about 24 hrs.AssaxfirermatinnThe assays were performed in collagen-treated, sterile 96-well plates that were purchasedfrom Corning. HL-60 cells were grown in round-bottomed wells (Corning catalog #25850) andHCT-116 cells were grown in flat-bottomed wells (Coming catalog #25860). Cell concentrationswere measured by a Coulter Cell Counter“. Cells were diluted in bulk and loaded onto theplates with 200 uL in each well. The assays were performed using approximately 25,000 HL-60cells/well and 40,000 HCT-116 cells/well.Since HL-60 cells grow in suspension, the cell concentration was measured and diluteddirectly. HCT—1 16 cells, however, adhere to the walls of the flask and must be suspended bytreatment with trypsin. A 0.025 mg/mL trypsin solution was thawed immediately before use.The bulk culture medium was removed by aspiration and 2mL of the cold trypsin solution wereadded to the flask. The flask was agitated periodically to promote suspension of the cells. Carewas taken to limit cell exposure to the trypsin solution to less than five minutes, since prolongedexposure will damage the cell membrane. When the cells were suspended, as observed by amicroscope, 8 mL of media were added to inactivate the trypsin. The cell concentration in theresulting suspension was measured, the suspension diluted appropriately, and loaded onto the10202530CA 02264592 1999-02-25WO 98/08859 PCTIU S97] 14140-64-96-well plates. Once on the plates, the cells were allowed to adhere for 3 hrs before treatmentwith SFU or one of the derivatives.T et 'a' fCel ’ ‘litHL-60 cells were treated and placed in the incubator for 24 hrs. The plates werecentrifuged and the supernatant was carefully aspirated without disturbing the cell pellet. A 200pL aliquot of media was added immediately. The cells were allowed to grow for 48 hrs. HCT-116 cells were treated and allowed to grow undisturbed for 72 hrs. The culture medium wasremoved by aspiration (after centrifugation in the case of HL-60 cells). 100 mL of McCoy’smedia and 11 uL_MTT dye were added. The cells were incubated at 37°C for 3 hrs. During thistime, viable cells reduce the MTT dye to purple formazan by the action of alcoholdehydrogenase. The cells were lysed by the addition of 100 uL of a solution 1.2M Hcl‘ in 60%ethanol, thereby releasing the reduced dye into solution. The absorbance at 405 nm wasmeasured for each well using a BIO-RAD Microplate Reader (Model 45 0)““/EXAMPLE 9In Vitro Activity of Qhlorambucil-Cgbalamin Bioconugatesermal Sta ili ofB' co 'u te 'n diaIt was noted that the chlorambucil bioconjugates 3 and 4 (prepared in Example 6) havethermal lability. Thus, they are expected to thermally decompose during the assay, perhapsbefore entering the cells or before release by photolysis. Thermal decomposition of bothbioconjugates was monitored by a UV—vis diode array spectrophotometer (HP8452) at 37°C inwater, cell-free media, and filtered media in which HCT-116 cells had been grown to aconcentration of about 100,000 cells/mL. Spectra were taken hourly for a total of 8 hrs. Thepresence of intact bioconjugate was then determined by photolysis, 20 min, with a high-pressuremercury lamp. If photolysis had no effect on the spectrum, all of the bioconjugate was assumedto have decomposed.In Vigrg Assays of 3 and 4 ActivityBoth bioconjugates were assayed against HCT-116, HL-60, B-16, Meth-A, and RD-995cell lines. The assays were performed in the same manner as described in Example 8 as202530CA 02264592 1999-02-25PCT/US97/14140WO 98/08859-5 5-modified herein. The B-16, RD-995 and Meth—A cells lines are all Balb/c derived carcinomalines which were provided by Dr. R. Daynes of the University of Utah. These cell lines weregrown in RPMI media which was completed with 5% fetal bovine serum and other mediacomponents as previously described. Both the B-16 and RD-995 cell lines were suspended intrypsin, as in the case of HCT-116 cells. The Meth—A cells loosely adhere to the walls of theflask and grow both attached to the flask and suspended in solution. These cells could becompletely suspended by successive washing of the flask wall with media.Assays were performed at cell concentrations of about 40,000 cells per well, with theexception of the HL-60 assay which was performed at 25,000 cell per well. The HCT-116, B-16 and RD-995 cells were assayed in flat bottomed, 96-well plates, while the HL-60 and Meth—Acells were assayed in round bottomed plates. Chlorambucil, unconjugated, was tested prior to thebioconj ugates. The cells were treated with the bioconjugates in both non-photolytic andphotolytic conditions. The cells were incubated for three days (media was aspirated andreplaced after 24 h. in the case of the HL-60 cells) and the resulting viability measured by anMTT assay.The MTT assay was somewhat altered for this experiment. The culture medium wasaspirated after 72 hrs. The Meth—A and HL-60 cells were centrifuged prior to aspiration. Then,100 1.1L of McCoy’s media and 11 uL of the MTT solution were added as before. At the end of4 hours, the culture medium was aspirated a second time (following centrifugation in the case ofHL-60 and Meth—A cells) and 100 uL of DMSO was added. The DMSO lysed the cellsreleasing the MTT dye into solution. The absorbance of each well at 450 nm was measured asbefore. The HCl/ethanol solution previously used has a tendency to precipitate proteins from theresulting solution, which may give falsely increased absorbance measurements. Thereplacement of DMSO avoids this problem.The concentration of chlorambucil and the bioconjugates were varied from 0.04 uM to400 uM within the assay. The cells were treated with the bioconjugates under dim, red lights toavoid photolysis. Non-photolytic conditions were maintained by wrapping the 96 well plateswith foil during the incubation periods. Photolysis was performed in black plates with flat, clearbottomed wells (Costar catalog number: 3603). These plates are sterile, collagen treated, andmade of optically clear plastic. Growth of the cells in these plates did not show any differencesto those grown in the normal clear plastic plates. Photolysis was achieved by an array of high1015202530CA 02264592 1999-02-25PCT/US97/14140WO 98/08859-56-intensity green LEDs (Hewlett Packard catalog number: 782-6124). The array was constructedfrom one of the black plates in which one LED was placed in each well. The LEDs could beturned on and off as vertical rows. In each assay, two rows of cells were left untreated as growthcontrols. One of these rows was not photolyzed by the LEDS to demonstrate any unexpectedeffects of photolysis; irradiation did not demonstrate any effects on the untreated control cells.An empty plate was placed between the array and the assay plate to avoid heating the cells. Tenminutes of irradiation produced complete photolysis for the bioconjugate in cell-free media. Thecells were irradiated for 10 min during the assay. The time of irradiation, following treatmentwith drug, was determined by a timecourse assay. The entire plate was treated with one of thebioconjugates at a concentration equal to the IC50 of chlorambucil in that cell line. The rowswere irradiated separately one half hour after treatment and then hourly.Irradiation at l h after treatment demonstrated the greatest bioconjugate activity in all ofthe cell lines. Further assays were performed with irradiation one hour after treatment. In thecase of the Meth-A cell line, the cells were transferred from the round—bottomed plate into theblack plate for photolysis and then returned to the round bottomed plate. The HL-60 cell linecould not be tested under these photolytic conditions. Both bioconjugates show significant thermal decomposition in both water and cell freemedia at 37°C. At the end of 8 hrs, photolysis has no effect on the spectrum, indicating that allof the bioconjugate has decomposed. In the HCT-116 cell media both bioconjugates show fastinitial decomposition and are significantly stabilized at subsequent time. Haptocorrin, acobalamin binding protein is known to stabilize alkylcobalamins by several orders of magnitude.This protein is present in the cell-free media from the added bovine serum. However, most ofthis protein in serum is saturated with cobalamin, so binding to the bioconjugates may beinhibited. It is known that several types of tumor cells secrete high amounts of cobalaminbinding proteins, especially haptocorrin. Thus, media in which cells have been growing has ahigher concentration of apo—haptocorrin. The initial fast decomposition of the bioconjugatesrepresents the amount remaining after the saturation of haptocorrin in the media. The boundbioconjugates are significantly stabilized by haptocorrin and the haptocorrin complex associatesand dissociates in a dynamic fashion in solution, especially in the presence of significant1015202530CA 02264592 1999-02-25PCT/US97/14140WO 98/08859-67 _concentrations of other cobalamins in the media. Thus, the bioconjugates are still susceptible todecomposition when not bound to haptocorrin. While haptocorrin does stabilize thebioconjugates by several orders of magnitude, slow decomposition is still seen during the assay.However, this assay does indicate that the bioconjugates are stabilized such that a significantamount is still intact during the time of uptake and photolysis.The data from the photolysis timecourse and activity assays are summarized in Figures 5-9 for compound 3 in each of the cell lines tested. Similar results were seen for compound 4. Ingeneral, both bioconjugates showed similar uptake and photolysis behavior in the photolysistimecourse assay. The maximal photolytically induced toxicity is seen one hour after treatmentwith either of the bioconjugates. In all cases, photolysis of the bioconjugate demonstratedincreased toxicity over that of unconjugated chlorambucil. Figure 5 shows that thechemotherapeutic drug, chlorambucil, has an LD50 of about 2 uM with respect to the HCT-116cell line, whereas the bioconjugate shows no substantial toxicity at concentrations approaching100 uM. If cells treated with the bioconjugate are subjected to brief irradiation with red light 12hours after dosing, the LD5o decreases by a factor of 25 to 0.08 uM. If a 10-fold excess ofvitamin B12 is added to saturate the cell surface receptors, the bioconjugate is not taken into thecells and photolysis triggers release of the active chlorambucil in the cell culture medium. Thereleased chlorambucil now enters the cell by passive diffusion and an LD50 of 2 uM is observed—— in close agreement with the value for chlorambucil standard.Figure 6 shows that in cell line HL-60, the unconjugated chlorambucil standard exhibitsan LD50 of 0.5 p.M, but the bioconjugate is at least 2-fold better with an LD50 of 0.2 uM. Thecytotoxicity of MC-121 against the leukemia cell line is still a dramatic result when Comparedwith the absence of toxicity when the cellular uptake of the conjugate is out—competed by theaddition of 10 equivalents of vitamin B12. Similar results were obtained with Meth-A cells. TheHL-60 and Meth-A cells have a high turnover rate, and in the case of Meth-A divide morerapidly than the other cell lines. These cells may, in fact, metabolize cobalamin at a faster ratethan the other cell lines and thus release the chlorambucil in significant concentrations withoutphotolysis. In order for this to be practical, however, cobalamin metabolism must occur beforesignificant hydrolysis of chlorambucil moiety. It is reported that HL-60 cells are able to convertvitamin B” into the other cobalamin forms efficiently (more quickly than normal lymphocytes)(Quadros and Jacobsen, 1995) and thus, would be able to efficiently release the conjugated1015202530CA 02264592 1999-02-25WO 98/08859 PCTlUS97/ 14140-63-chlorambucil. In the other cell lines, however, the bioconjugates are essentially not toxic in non-photolytic conditions, which is a promising indication that these bioconjugates may not be toxicin normal somatic cells or healthy hematopoetic cells. lC50 values of the bioconjugates in bothnon-photolytic and photolytic conditions are summarized in Table 1.TABLE 1E59 values (QM)Cell Line HCT-1 1 6 HL-60 B-1 6 Meth-A Rd-995Chlorambucil 20.8 5.8 1.4 1.8 1.1Bhamlsasis3 1.7 0.6 0.2 0.24 1.1 0.3 0.3 0.3No Photolysis3 3.2 210.14 :""""_" 8.9 84.2It will be appreciated that the methods and compositions of the instant invention can beincorporated in the form of a variety of embodiments, only a few of which are disclosed herein.It will be apparent to the artisan that other embodiments exist and do not depart from the spirit ofthe invention. Thus, the described embodiments are illustrative and should not be construed asrestrictive.LIT FE NAllen, R. H. and Majerus, P. W. (1972a). J. Biol. Chem. 247:7695-7701.Allen, R. H. and Majerus, P. W. (l972b). J. Biol. Chem. 24717709-7717.Baulch, D. L., et a1., (1992). J. Phys. Chem. Ref Data 21:411.Bennett, C.F., et al. (1992). Mol. Pharmacol. 41:1023-1033.Blackburn, R., et al. (1972). J. Chem. Soc. Faraday I, 1687.Boden, E.F. and Keck, G.E. (1985). J. Org. Chem. 5022394.Brown, K.L. and Peck, S. (1988). Organomat. Syn. 4:304.102030354045CA 02264592 1999-02-25WO 98/08859 PCTIUS97/ 14140-69- —Brown et al. (1996). J. Inorg. Chem. 35:3442.Bunnell, B.A., et al. (1992). Somatic Cell Mol. Genet. 18:559-569.Buxton, G. V., et a1., (1988). .1. Phys. Chem. Ref Data 17:513.Carmel (1975). New Engl. J. Med. 292:282-284.Castle, W. B. (1953). N. Engl. J. Med. 24:603-611.Chagovetz, A. M. and Grissom, C. B. (1993). J. Am. Chem. Soc. 115212152.Chang, E.H. and Miller, P.S. (1991). In Prospects for Antisense Nucleic Acid Therapy ofCancer and AIDS, ed. Wickstrom, E., Wiley-Liss, New York, pp. 115-124.Chen and Chance (1993). Biochem. 3211480-1487.Cohen, J.S. and Hogan, M.E. (1994). Sci. Am. 76-82.Cooney, M., et al. (1988). Science 2412456-459.Endicott, J. F. and Netzel, T. L. (1979). J. Am. Chem. Soc. 10114000.Fomari, F.A., et a1. (1996). Biochem. Pharmacol. 51:931-940.Fox, H. J. and Castle, W. B. (??). Am. J. Med. Sci. 203:18-26.Fritzer, M., et al. (1996). Biochem. Pharmacol. 51:489-493.Gao, X. and Huang, L. (1991). Biochem. Biophys. Res. Commun. 1791280-285.Gasparro (1986). Biochem. Biophys. Res. Commun. 1412502-509.Ginobbi, P., et al. (1997). Anticancer Res. 17:29-36.Grissom, C.B. (1995). Chem. Rev. 95:3-25.Hosada, J., et al. (1995). Biol. Pharm. Bull. 1811234-1237.Howard, W.A., et al. (1997). Bioconj. Chem. 8:498-502.Hu, Y.-P., et a1. (1996). Cancer Chemother. Pharmacol. 37,556-560.Johnson, D. A., et al. (1995). Anticancer Res. 15:1387-1394.Kabanov, A.V., et al. (1995). Bioconjugate Chem. 6:639-643.10202530354045CA 02264592 1999-02-25WO 98108859 PCT /US97l 14140-70-Krautler, B. and Stepanek, R. (1985). Angew. Chem. Int. Ed. Engl. 24:62.Kumar et al. (1987). J. Biol. Chem. 262:717l-7179.Le Doan, T., et al. (1987). Nucl. Acids Res. 15:7749-7760.Ling, Y.-L., et al. (1996). Mol. Pharmacol. 49:832-841.Letsinger, R.L. (1993). Nucl. Acids Symp. Ser. 29:1-2.Longman, S. A., et al. (1995). Cancer Chemother. Pharmacol. 36:91-101.Lott et a1., (1995). J. Am. Chem. Soc. 117212194-12201.Low, P.S. et al. (1995). U.S. Patent No. 5,416,016.Ma, D.D.F. and Wei A-Q. (1996). Leukemia Res. 20:925-930.Madon, J. and Blum, H.E. (1996). Hepatology 24:474-481.Mason, T. J. (1991). Practical Sonochemistry: Users Guide to Applications in Chemistry andChemical Engineering Horwood, New York.Merwin, J.R., et al. (1994). Bioconfugate Chem. 5:612-620.Moser, H.E. and Dervan, P.B. (1987). Science 238:645-650.Murray, G.J. and Neville, D.M. (1980). J. Biol. Chem. 255:11942-11948.Myers (1988). European Patent Application No. 86810614.7Natarajan, E. and Grissom, C. B. (1996). Photochem. 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Claims (40)

We Claim:

1. A bioconjugate of a bioactive agent and an organocobalt complex, said organocobalt complex containing a cobalt atom having bound thereto 4-5 nitrogen, chalcogens or both nitrogen and chalcogens as part of a multiple unsaturated heterocyclic ring system, wherein the bloactive agent is covalently conjugated to the cobalt atom through a non-reactive atom in the bioactive agent molecule and has the property of being capable of targeted, selective release of the bioactive agent from the bioconjugate.

2. The bioconjugate of claim 1, wherein said non-reactive atom is selected from the group consisting of a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, a selenium atom and a silicon atom.

3. The bioconjugate of claim 1, wherein said non-reactive atom is a carbon atom.

4. The bioconjugate of claim 3, wherein the non-reactive carbon atom is a carbon atom from an alkyl, acyl or aryl group that will not lead to rearrangement or destruction of the bioactive agent under conditions of ligand exchange during receptor-mediated endocytosis.

5. The bioconjugate of any one of claims 1-4, wherein said bioactive agent is covalently bound directly to the cobalt atom of the organocobalt complex.

6. The bioconjugate of any one of claims 1-4, wherein said bioactive agent is covalently bound indirectly to the cobalt atom of the organocobalt complex via a spacer, said spacer being atoms and molecules which can be used to covaltently bind a bioactive agent to the cobalt atom of an organocobalt complex.

7. The bioconjugate of claim 6, wherein said spacer is a self-destructing linker.

8. The bioconjugate of any one of claims 1-6, wherein said bioactive agent is a diagnostic compound.

9. The bioconjugate of any one of claims 1-6, wherein said bioactive agent is a pharmaceutically active compound.

10. The bioconjugate of claim 9, wherein said pharmaceutically active compound is an anticancer agent.

11. The bioconjugate of any one of claims 1-6, wherein said bioactive agent is a peptide or peptide analogue.

12. The bioconjugate of any one of claims 1-6, wherein said bioactive agent is a protein or protein analogue.

13. The bioconjugate of any one of claims 1-6, wherein said bioactive agent is a nucleic acid or a nucleic acid analogue.

14. The bioconjugate of claim 13, wherein said nucleic acid or nucleic acid analogue is a polynucleotide.

15. The bioconjugate of claim 14, wherein said nucleic acid or nucleic acid analogue is an oligonucleotide.

16. The bioconjugate of claim 13, wherein said nucleic acid is antisense DNA or RNA.

17. The bioconjugate of any one of claims 1-16, wherein said organocobalt complex is cobalamin, a cobalamin lactone, a cobalamin lactam, or a cobalamin derivative, wherein the cobalamin derivative is (a) cobalamin in which the benzimidiazole ring is substituted with a halogen, hydroxy or C1-6 alkyl, (b) an anilide, ethylamide, monocarboxylic acid, dicarboxylic acid, tricarboxylic acid or proprionamide derivative of cobalamin, or (c) cobalamin substituted with an amino, a nitro, a halogen, a sulfito, a C2-6 alkylene or a C2-6 alkyne.

18. The bioconjugate of any one of claims 1-16, wherein said organocobalt complex is a compound having the following formula:

wherein R is H, amino, C1-6 alcohol, or C1-6 carboxyl, W, W', X, X', Y, Y', Z
and Z' are independently H, amino, C1-6 alcohol, C1-6 carboxyl, SO3-, CH2OH, CO2H, or nitro, or W and X together form a 4-6 member cyclic or heterocyclic ring, or W' and X' together form a 4-6 member cyclic or heterocyclic ring, or Y
and Z together form a 4-6 member cyclic or heterocyclic aromatic ring or Y' and Z' together form a 4-6 member cyclic or heterocyclic aromatic ring.

19. The bioconjugate of claim 18, which further comprises a targeting molecule covalently linked to one of said R, W, W', X, X', Y, Y', Z or Z' and is selected from the group consisting of glucose, galactose, mannose, mannose 6-phosphate, transferrin, cobalamin, asialoglycoprotein, .alpha.-2-macroglobulins, insulin, a peptide growth factor, folic acid and derivatives, biotin and derivatives, YEE(GalNAcAH)3 and derivatives, albumin, texaphyrin, metallotexaphyrin, a vitamin, a coenzyme, an antibody, an antibody fragment and a single-chain antibody variable region (scFv).

20. The bioconjugate of any one of claims 1-16, wherein said organocobalt complex is selected from the group consisting of organo(pyridine) bis(dimethylglyoximato)cobalt, a corrinoid or derivatives thereof, wherein said derivative is (a) a corrinoid in which the benzimidiazole ring is substituted with a halogen, hydroxy or C1-6 alkyl, (b) a corrinoid substituted with an amino, a nitro, a halogen, a sulfito, a C2-6 alkylene or a C2-6 alkyne, or (c) an organo(pyridine) bis(dimethylglyoximato)cobalt substituted with an amino, a nitro, a halogen, a sulfito, a C2-6 alkylene or a C2-6 alkyne.

21. A use of an effective amount of the bioconjugate of any one of claims 1-20 for delivering a bioactive agent to cells of a targeted tissue site of a subject.

22. The use of claim 21, wherein the organocobalt complex of said bioconjugate binds to cell receptors and is transported into a cell by a receptor-mediated process.

23. A use of an effective amount of the bioconjugate of any one of claims 1-18 and 20 for delivering a bioactive agent to cells of a targeted tissue site of a subject, wherein a targeting molecule of said bioconjugate binds to cell receptors and is transported into a cell by a receptor-mediated process.

24. A use of an effective amount of the bioconjugate of claim 19 for delivering a bioactive agent to cells of a targeted tissue site of a subject, wherein the targeting molecule of said bioconjugate binds to cell receptors and is transported into a cell by a receptor-mediated process.

25. The use of any one of claims 21-24, wherein said bioconjugate is for intravenous administration.

26. The use of any one of claims 21-24, wherein said bioconjugate is for parenteral administration.

27. The use of any one of claims 21-24, wherein said bioconjugate is for oral administration.

28. The use of any one of claims 21-24, wherein said bioconjugate is for intramuscular administration.

29. The use of any one of claims 21-24, wherein said bioconjugate is for intrathecal administration.

30. The use of any one of claims 21-24, wherein said bioconjugate is for administration as an aerosol.

31. The use of any one of claims 21-29, wherein said bioconjugate is for active transport into said cells of said targeted tissue site and accumulates in an inactive form until activated by cleavage of the bioactive agent from the organocobalt complex.

32. The use of claim 31, wherein the cleavage occurs as the result of cellular displacement.

33. The use of claim 31, wherein the cleavage occurs as the result of cellular B12 metabolic enzymes.

34. The use of claim 31, wherein the cleavage is caused by an external signal.

35. The use of claim 34, wherein said external signal is for application to the targeted tissue site.

36. The use of claim 34 or 35, wherein said external signal is visible light of a wavelength which causes photolysis of said bioconjugate.

37. The use of claim 36, wherein said visible light has a wavelength of 400-800 nm.

38. The use of claim 36, wherein said visible light has a wavelength of 600-800 nm.

39. The use of claim 36, wherein said visible light has a wavelength of 600-750 nm.

40. The use of any one of claims 36-39, wherein said visible light is for delivery by fiber optics.