Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

Definitions

• the present invention is generally directed to vitamin B J2 receptor modulating agents that bind TcII cell surface receptors and affect the receptor trafficking pathway and methods related thereto.
• Cell surface receptors constitute a class of proteins that are responsible for receptor-mediated endocytosis of specific ligands. Basically, the receptors serve as escorts for ligand delivery to intracellular destinations.
• Coated pits These pits continually invaginate and pinch off, forming “coated vesicles” in the cytoplasm. Coated pits and vesicles provide a pathway for receptor mediated endocytosis of specific ligands.
• the ligands that bind to specific cell surface receptors are internalized via coated pits, enabling cells to ingest large numbers of specific ligands without taking in correspondingly large volume of extracellular fluid.
• the internalized coated vesicles may or may not lose their coats and bind with other vesicles to form larger vesicles called "endosomes.”
• endosomes In the endosome the ligand and the receptor are separated or “sorted.” Endosomes that sort ligands and receptors are known as “compartment of uncoupling of receptor and ligand” or “CURL.” Endosomes may fuse with primary lysosomes, where their contents are digested, or they may be delivered to other intracellular destinations.
• the receptor proteins are generally not digested, but are rather recycled to the cell membrane surface through a process called "exocytosis,” or transferred to early or late endosomes via multivesticular bodies. The entire pathway is referred to as the "receptor trafficking pathway.”
• Some receptors deliver their ligand directly to the cytoplasm or other specific intracellular locations.
• a serum carrier protein, transferrin binds iron and transports it to transferrin receptors on the plasma membrane surface.
• transferrin receptors After binding and internalization, via coated pits, the resulting vesicle combines first with early endosomes and then with late endosomes. This process results in the gradual drop in pH in the vesicle. The drop in pH causes the transferrin carrier protein to lose its affinity to iron. When this occurs, the iron translocates through the membrane of the vesicle and joins the intracellular pool of enzymes. The transferrin receptor may then recycle to the cell surface where it may repeat the process.
• EGF epidermal growth factor
• the EGF receptor may recycle to the cell surface depending on its state of phosphorylation (Cancer Treat. Rep. 61:139-160, 1992; J. Cell. Biol. 116:321-330, 1992).
• a single receptor may utilize more than one receptor trafficking pathway within the same cell.
• membrane trafficking is distinct between apical and basal sides of the cell (Sem. Cell. Biol. 2:387-396, 1991).
• non-polarized epithelial cells may simultaneously follow two separate sorting pathways.
• the control or regulation of cell surface receptors may be achieved by a variety of techniques. Regulation of cell surface receptors may be accomplished, at a very basic level, by the binding of naturally occurring ligands. As discussed above, receptor binding of a ligand will generally trigger the internalization of the ligand- receptor complex. Such internalization may desensitize the cell to further ligand binding.
• This type of regulation is transient in nature and does not result in diminution of biologic response.
• Receptor antagonists are organic protein or peptide ligands generally derived through empirical structure-function studies, or through the use of detailed knowledge of ligand and receptor interaction. Essentially, an antagonist may constitute any molecule with similar binding activity to a natural ligand, but incapable of producing the biological response normally induced by the natural ligand. Thus, the antagonist competitively blocks receptor activity. With a competitive antagonist, the regulation of receptor activity is dependent upon both the antagonist's affinity for the receptor, as well as its extracellular concentration over time.
• Receptor agonists are protein or peptide ligands derived in a similar manner as antagonists.
• an agonist may constitute any molecule that binds to the receptor in a manner superior to that ofthe natural ligand.
• One receptor of particular interest is the vitamin B 12 receptor.
• vitamin B 12 is a co-enzyme necessary in cell division, as well as cellular metabolism, in proliferating normal and neoplastic cells. Insufficient vitamin B ]2 causes cellular division to be held in abeyance and ultimately may result in apoptosis.
• the nutrient is generally derived from dietary intake and is transported throughout the body complexed to transport proteins. The complex of transport protein and vitamin B I2 is recognized by a cellular receptor that internalizes the complex and releases the vitamin intracellularly.
• Vitamin B ]2 is taken in through the diet. Binding proteins in the saliva (R-binder) and gut (intrinsic factor-(IF)) complex vitamin B 12 after release from endogenous binding proteins by action of enzymes and low pH in the stomach. Vitamin B 12 is transferred across the intestinal epithelium in a receptor specific fashion to transcobala in ⁇ (TcII). The vitamin B ]2 /transcobalamin II complex is then transported throughout the body and recognized by receptors present on dividing cells, internalized and released within the cell where it is utilized by certain enzymes as a co-factor.
• the high affinity receptor in dividing tissues or cells responsible for internalization of vitamin B 12 recognizes transcobalamin II complexed with vitamin B 12 .
• the vitamin B 12 /TcII receptor recognizes only the vitamin B 12 /TcII complex and not the serum transport protein or the vitamin alone.
• the receptor is undetectable on non-dividing cells; the mechanism for supplying non-dividing cells with vitamin B J2 is poorly understood.
• more vitamin B J2 is required during cell division than during metabolism, and that the vitamin B 12 /TcII receptor is the only high affinity means for cellular uptake of vitamin B 12 during cell division. When stimulated to divide, cells demonstrate transient expression of this receptor leading to vitamin B 12 uptake that precedes actual DNA synthesis (J. Lab. Clin. Med.
• Vitamin B 12 receptor levels may be measured by binding of ⁇ Co-vitarnin B J2 complexed to transcobalamin II (present in serum) on replicate cultures grown in chemically defined medium without serum. No receptor mediated uptake occurs in the absence of carrier protein. Dividing cells, induced to differentiate, lose receptor expression and no longer take up vitamin B 12 . More importantly, leukemic cells, deprived of vitamin B J2 , will stop dividing and die (Acta Haemat. 81:61, 1989). In a typical experiment, leukemic cell cultures were deprived of serum for 3 days, and then supplemented either with serum (a source of vitamin B 12 ) or a non-metabolizable analogue of vitamin B J2 and cultured up to five days. Cell cultures supplemented with vitamin B 12 continued to grow, whereas those deprived ofthe active nutrient stopped growing and die.
• vitamin B 12 may be useful in the treatment of cancer or other disorders characterized by uncontrolled growth of cells.
• vitamin B J2 deprivation may be used in combination .with chemotherapeutic drugs that inhibit cellular replication.
• chemotherapeutic drugs that inhibit cellular replication.
• the two modalities together were more efficient in depleting folate levels in leukemic cells than either alone (FASEB J. 4: 1450, 1990; Arch. Biochem. Biophvs. 270:729, 1989; Leukemia Research 15:165, 1991).
• Floats are precursors in the production of DNA and proteins.
• cultures of leukemic cells were exposed to nitrous oxide for several hours to convert the active form of endogenous vitamin B ]2 to an inactive form.
• Replicate cultures were then left without further treatment, or additionally treated with methotrexate.
• Cellular folate levels were measured three days later.
• Cells treated with the combination i.e., both methotrexate and inactive vitamin B 12
• this approach was applied to the treatment of highly aggressive leukemia/lymphoma in animal models (Am. J. Haematol.
• vitamin B 12 can act synergistically with chemotherapeutic drugs (such as methotrexate and 5-FU) to inhibit tumor growth and treat animals with leukemia/lymphoma.
• chemotherapeutic drugs such as methotrexate and 5-FU
• This combination therapy was demonstrated in multiple animal models. Observations in patients have indicated that long-term (months to years) vitamin B 12 depletion is required to produce significant normal tissue toxicity. Even in those cases, subsequent infusion of vitamin B 12 can readily reverse symptomology (Br. J. Cancer 5:810, 1989).
• receptor- controlling agents have emerged as a class of pharmaceutical drugs.
• receptor- controlling agents have emerged as a class of pharmaceutical drugs.
• the production of receptor- controlling drugs has been significantly enhanced.
• the present invention provides a vitamin B 12 receptor modulating agent, comprising a vitamin B ]2 molecule coupled to a rerouting moiety by a linker.
• the invention further provides vitamin B 12 receptor modulating agents wherein the linker is selected from a water-solubilizing linker or a non-water solubilizing linker and embodiments wherein the linker is covalently coupled to a vitamin B 12 coupling site selected from b-, d-, and e- coupling sites or the ribose 5'-OH coupling site.
• FIGURE 1 is a schematic illustrating a mechanism of action of a receptor modulating agent of the present invention.
• a healthy receptor will internalize when bound by the appropriate ligand, release the ligand within the cell and then recycle to the cell surface.
• Receptor modulating agents of the present invention impede the receptor trafficking pathway by inhibiting the recycling of receptors to the cell surface.
• the targeting moiety on receptor modulating agents binds the receptor and the rerouting moiety redirects the receptor/receptor modulating agent complex to other points within the cell, where it may be retained or degraded. (Not shown in this schematic are receptors synthesized de novo):
• FIGURE 1 illustrates a formula representing a vitamin B J2 (cyanocobalamin) molecule and identifies a preferred coupling site suitable for use in the present invention for derivatization and conjugation;
• FIGURE 2 is a schematic depicting a representative reaction scheme for the synthesis of a vitamin B 12 -GABA adduct
• FIGURE 3A is a schematic depicting a representative reaction scheme for the synthesis of a vitamin B 12 derivative comprising a vitamin B J2 molecule with a diaminododecane linker arm coupled to any one of coupling sites d-, e-, or _>-;
• FIGURE 3B is a schematic depicting a representative reaction scheme for coupling a succinic anhydride to a vitamin B ⁇ 2 diaminododecane adduct in preparation for coupling the adduct to a rerouting moiety, or other molecule, with an amino reaction site;
• FIGURE 4 is a schematic depicting a representative reaction scheme for the synthesis of a vitamin B 12 derivative comprising a vitamin B 12 molecule and a diaminododecane linker arm coupled to a ribose coupling site;
• FIGURE 5 is a schematic depicting a representative reaction scheme for coupling vitamin B or a vitamin B 12 -GAB A adduct to amikacin
• FIGURE 6 is a schematic depicting a representative reaction scheme for coupling vitamin B 12 or a vitamin B 12 -GABA adduct to streptomycin;
• FIGURE 7 is a schematic depicting a representative reaction scheme for coupling a vitamin B 12 carboxylate derivative or a vitamin B 12 -GABA adduct to acridine;
• FIGURE 8 is a schematic depicting a representative reaction scheme for the synthesis of a bivalent receptor modulating agent, a vitamin B J2 dimer, using a trifunctional linker.
• the trifunctional linker allows for coupling with additional compounds (e.g., R-NH2) such as, by way of example, aminoglucosides, aminoacridines, glycosylation inhibitors and biotin;
• FIGURE 9 is a schematic depicting a representative reaction scheme for the synthesis ofa vitamin B J2 dimer using a homobifunctional or homotrifunctional cross ⁇ linking reagent
• FIGURE 10 is a schematic depicting a representative reaction scheme for the synthesis of a vitamin B J2 dimer using a heterobifunctional cross-linker
• FIGURES 11 are schematics depicting representative reaction schemes for the synthesis of various receptor modulating agents generally comprised of a rerouting moiety, designated by the reactive group and R, and a vitamin B ]2 molecule or derivative thereof as a targeting moiety;
• FIGURE 12 is a graph illustrating the binding curve of Transcobalamin II to the cyanocobalamin monocarboxylic acids produced in Example 1.
• FIGURE 13 is a graph illustrating the binding curve of Transcobalamin ⁇ to the cyanocobalamin diaminododecane adducts produced in Example 3 and 4.
• FIGURE 14 is a graph illustrating the binding curve of transcobalamin ⁇ to a series of vitamin B 12 dimers.
• Dimer X ⁇ -acid dimer with isophthaloyl dichloride
• FIGURE 15 is a graph illustrating the binding curve of transcobalamin II to a series of biotinylated vitamin B 12 molecules.
• AA cyanocobalamin ⁇ -monocarboxylic acid conjugate diaminododecane and biotin (17);
• AB cyanocobalamin e- monocarboxylic acid conjugate diaminododecane and biotin (18);
• AC cyanocobalamin ⁇ /-monocarboxylic acid conjugate diaminododecane and biotin (19);
• AF cyanocobalamin ribose-succinate conjugate diaminododecane (13); and AG - cyanocobalamin ribose-succinate conjugate diaminododecane and biotin (20).
• biotinylated molecules were prepared as set forth in Examples below, (see Example 8.)
• the present invention is generally directed to a vitamin B J2 receptor modulating agent that is capable of binding to a vitamin B J2 cell surface receptor to form a receptor modulating agent/receptor complex ("agent/receptor complex").
• agent/receptor complex a receptor modulating agent/receptor complex
• the binding of a suitable receptor modulating agent to a cell surface receptor generally results in invagination of the agent/receptor complex into the cell into the vesicular system in the same manner as the natural ligand.
• a receptor modulating agent of the present invention affects the receptor trafficking pathway by effectively impeding, preventing, or delaying the receptor from recycling to the surface, thus depriving the cell of receptors able to engage in binding the cell's natural ligand and triggering related biological responses.
• affecting the receptor trafficking pathway refers to impeding the receptor trafficking pathway in such a manner as to affect biological response. This would include trapping, delaying, retaining, redirecting, or degrading the cell surface receptor.
• a “receptor modulating agent” is comprised of at least one targeting moiety covalently attached to at least one rerouting moiety.
• a “targeting moiety,” as described in detail below, is a moiety capable of specifically binding to a vitamin B 12 cell surface receptor to yield an agent/receptor complex and, in a preferred embodiment, has an affinity for the cell surface receptor of within a hundredfold, and more preferably, within tenfold, of the affinity of the natural ligand for the receptor.
• a preferred targeting moiety is a vitamin B 12 molecule.
• a "rerouting moiety” is a moiety that redirects an agent/receptor complex, resulting in prolonged retention, degradation, and/or modulation of the receptor within the interior of a cell or on the cell surface, including, by way of example, retaining the receptor in the cell membrane or directing the receptor to a lysosome within the cell. Suitable rerouting moieties are described in detail below.
• a targeting moiety is coupled to a rerouting moiety to yield the receptor modulating agent by any suitable means known in the art, including direct covalent linkage of an appropriate chemical linker or through a very tight association in non- covalent attachment.
• coupling is accomplished through the combination of an avidin or streptavidin conjugate with a vitamin B 12 /biotin conjugate.
• Coupling of the targeting moiety and the rerouting moiety should be of a nature that resists cleavage by the enzymatic and low pH conditions normally encountered within the internal portion of the cell, including endosomes and lysosomes. Suitable linkers are noted below.
• the ability to resist cleavage may be detected by any means known in the art, including exposing the receptor modulating agent to enzymes at low pH and measuring release of the targeting or rerouting moiety using techniques known in the art.
• Coupling of a targeting moiety and a rerouting moiety should not significantly hinder the ability ofthe targeting moiety to specifically bind the cell surface receptor.
• the receptor modulating agent may also include additional moieties, so long as they do not interfere with either the targeting or the rerouting moieties.
• moieties may be coupled to the receptor modulating agent through the use of a trifunctional linker or they may be coupled to a rerouting or targeting moiety.
• Optimal attachment ofthe two moieties may be determined by comparing the affinity of binding of the receptor modulating agent with free targeting moiety in assays of inhibition of binding.
• targeting moieties of a receptor modulating agent include any moiety that specifically binds to a vitamin B 12 cell surface receptor. Suitable targeting moieties include a vitamin B 12 molecule.
• Vitamin B 12 is an essential nutrient for dividing cells. By inhibiting its uptake, the growth of dividing cells can be halted.
• the cell surface receptor for vitamin B 12 is the transcobalamin ⁇ /vitamin B 12 ("TcH/B 12 ") receptor, that is characterized by a high affinity for the carrier protein, transcobalamin II (TcII), when complexed with vitamin B 12 ("TcH/B 12 complex").
• TcH/B 12 receptor does not recognize vitamin B 12 alone, but does recognize the carrier protein TcII with reduced affinity when not complexed with vitamin B 12 .
• vitamin B 12 refers to the class of compounds known as cobalamins and derivatives thereof, including, by way of example, cyanocobalamin.
• vitamin B 12 is used interchangeably with the term cyanocobalamin.
• Suitable vitamin B 12 molecules include any vitamin B J2 capable of coupling to another molecule while maintaining its ability to form a TcH B 12 complex.
• a preferred vitamin B J2 targeting moiety is generally comprised of a vitamin B 12 molecule, such as a cyanocobalamin, and a linker, described in detail below.
• the linker may be coupled to any one of several sites on a vitamin B ]2 molecule, including potential carboxyl coupling sites a- through g-, an alcohol (ribose) coupling site ("coupling site A”), or a benzimidazole coupling site ("coupling site /").
• a linker is coupled to coupling sites b-, d- or e- on a vitamin B 12 molecule. Even more preferably, a linker is coupled to coupling site d- or e-.
• This embodiment of the present invention includes compounds represented by the following formula:
• Rj , R2, R3, R4, R5, Rg, and R7 is a linker.
• Rj , R2, R3, R4, R5, Rg, and R7 is a linker.
• Coupling sites that are not occupied by a linker may have a variety of chemical moieties attached thereto, including an amino, secondary amino, tertiary amino, hydroxy, lower alkyl, lower alkoxy, alkoxyalkyl, alkoxyalkoxy, cycloalkylalkoxy, and thioalkyl groups.
• R j , R2, or R 4 is a linker and the remaining R groups are -NH 2 , with the exception of R7, that is preferably -OH.
• R 2 is a linker
• R ls R3-R6 are -NH 2
• R7 is -OH.
• R7 is a linker and Rj-Rg are -NH 2 .
• Suitable linkers include any one of several linkers, preferably containing at least two coupling or reactive groups, allowing the linker to bind to both vitamin B 12 and a rerouting moiety.
• the terms "coupling group” and "reactive group” are used interchangeably.
• a linker may be homobifunctional, heterobifunctional, homotrifunctional, or heterotrifunctional.
• Homobifunctional agents may facilitate cross-linking, or dimerization of vitamin B 12 molecules in a single step, hence a coupling reaction using these agents should be performed with an excess of homobifunctional agents, unless dimerization is the desired result, as in the synthesis of dimers described in detail below.
• Suitable homobifunctional agents include, but are not limited to: disuccinimidyl suberate (DSS)*; bis(sulfosuccinimidyl) suberate (BS 3 )*; disuccinimidyl suberate (DSS)*; bis(sulfosuccinimidyl) suberate (BS 3 )*; disuccinimidyl tartarate (DST)*; disulfosuccinimidyl tartarate (Sulfo-DST)*; bis[2- (succinimidooxycarbonyloxy)ethyl] sulfone BSOCOES)*; bis[2-
• Heterobifunctional agents facilitate cross-linking in a stepwise method, allowing more than one linker to be incorporated and a variety of targeting agents, such as vitamin B 12 molecules, to be linked.
• Suitable heterobifunctional agents include, but are not limited to:
• Homo- and heterotrifunctional linkers are coupled to a rerouting moiety and a vitamin B J2 molecule as described above, with the additional advantage of a third coupling site on the linker.
• markers such as radiolabeled and fluorescent molecules
• proteins and peptides such as antibodies
• conjugating molecules such as biotin.
• Suitable trifunctional linkers include, but are not limited to:
• 5-N-Boc amino isophthoyl ditetrafluorphenyl ester 3,5-diamino methyl benzoate; 5-(p-iodobenzoyl)amino-l,3-isophthaloyl ditetrafluorophenyl ester; 5-(p- tri-N-butylisomylbenzoyl)-amino-l,3-isophthaloyl ditetrafluorophenyl ester;
• linker The preferred length of a linker is dependent upon a number of empirical factors based upon the nature of the receptor modulating agent, including its component targeting and rerouting moieties. In general, a linker should have a length sufficient such that the targeting moiety and the rerouting moiety of the receptor modulating agent may perform their designed functions free from steric inhibition.
• a linker should have a length sufficient to facilitate the specific binding of a targeting moiety to a cell surface receptor to yield an agent/receptor complex. Additionally, a linker should also have a length sufficient to permit a rerouting moiety to redirect an agent/receptor complex so as to interfere with the receptor trafficking pathway.
• linkers for vitamin B ⁇ _ receptor modulating agents should have a length sufficient to allow for binding of a vitamin B 12 derivative to transcobalamin II to form a TcH B ⁇ 2 complex and, subsequently, to permit the binding of a TcH/B ⁇ _ complex to a TcH B ⁇ 2 cell surface receptor.
• Linkers for receptor modulating agents including a biotin moiety should be of a length sufficient to facilitate binding ofthe receptor modulating agent to avidin (or streptavidin).
• Suitable linkers are generally relatively linear molecules greater than 4 atoms in length, typically between 6 and 50 atoms in length, and preferably are 8 to 35 atoms in length. In one preferred embodiment, the linker is a linear molecule of 12-15 atoms in length.
• atom refers to a chemical element such as, by way of example, C, N, O, or S.
• linker may be linear, branched, and even contain cyclical elements.
• the linker is a water-solubilizing linker.
• water-solubilizing linker refers to any linker that, when covalently coupled to a rerouting and or targeting moiety, increases the water solubility of either the components or the receptor modulating agent.
• water solubility refers to solubility in water or any other aqueous medium. In general, the solubility of a compound may be determined as described in "Handbook of Solubility Parameters and Other Cohesion Parameters" by A.F.M. Benton, CRC Press, 1983.
• the water- solubilizing linkers may also enhance the water solubility of the receptor modulating agents.
• the water-solubilizing linkers are composed of hydrophilic moieties (e.g., polar functional groups) including electronically neutral and charged (i.e., ionic) moieties.
• Suitable hydrophilic moieties include electronically neutral moieties containing polar functional groups (i.e., groups that contain atoms of differing electronegativities such as organic compounds containing nitrogen, oxygen, and sulfur) that increase their hydrophilicity.
• these neutral hydrophilic moieties contain functional groups that hydrogen bond with water.
• Such hydrogen bonding groups include ether (-O-), hydroxy (-OH), amino (-NR 2 , -NHR, -NH 2 ), and to a lesser extent thioether (-S-), and thiol (-SH) groups.
• Moieties that contain multiple polar functional groups are more hydrophilic than those moieties that contain a single polar functional group.
• Suitable moieties that contain multiple polar groups mclude, by way of example, polyhydroxy, polyamino, polyether, polyphosphoric acid, polyalcohol and polyamine moieties.
• Polyhydroxy moieties include, by way of example, glycols, glycerols, and polysaccharides including glucose, fructose, galactose, idose, inositol, mannose, tagatose, and N-methylglucamine.
• Polyalcohol moieties include, by way of example, N-methylglucamine and glucose derivatives.
• Polyether moieties include, by way of example, polyethylene glycol, ethoxy ethanol, and ethoxy ethoxy ethanol.
• Polyamine moieties include, by way of example, spermine or spermidine.
• Suitable charged hydrophilic moieties include those moieties that become either formally negatively or positively charged in water.
• Suitable negatively charged moieties include acid anions resulting from the dissociation of acids in water.
• carboxylic acids (-CO 2 H) dissociate to form negatively charged carboxylate ions (-CO ⁇ ) at pH greater than about 5.
• Other stronger acids such as phosphoric (-PO 3 H 2 ) and sulfonic (-SO 3 H) acids ionize to form phosphonate (-PO 3 2 ") and sulfonate (-SO 3 ') anions, respectively, at pH greater than about 2.
• More weakly acidic moieties such as phenols and thiols, may also dissociate to form their corresponding anionic derivatives that are also water solubilizing.
• basic moieties may become formally positively charged moieties in water. These moieties become highly water soluble through protonation in aqueous solution. For example, at pH about 5, amines (-NR 2 , -NHR, -NH 2 ) become ammonium ions (-NHR 2 + , -NH 2 R + , -NH 3 + ), all of that are highly water solubilizing moieties.
• Quaternary ammonium moieties are extremely water-solubilizing at all pHs. Suitable charged solubilizing moieties also include polylysine groups.
• the water solubility of a vitamin B J2 derivative may be evaluated by any one of several means, including, by way of example, simply combining the derivative with an aqueous medium and observing the solubility at various temperatures. Alternatively, solubility may be ascertained by dissolving the derivative in water, stirring the solution, and allowing the solution to stand at room temperature for about 24 hours. The solution is then centrifuged and the resultant aqueous layer analyzed using high-pressure liquid chromatography ("HPLC"). The HPLC analysis was conducted isocratically using acetonitrile as the solvent on a LiChrospher 100, C-18 column (5 ⁇ M, 125 x 4 mm) using a flow rate of 2 mL/min.
• the quantitation of a vitamin B ⁇ 2 containing solution may be accomplished by HPLC using UV detection.
• an aqueous solution of a vitamin B 12 derivative is prepared and analyzed by HPLC as described above.
• a series of vitamin B 12 aqueous solutions of known concentration are prepared and analyzed by HPLC.
• the results of these HPLC analyses are then used to construct a standard curve where the concentration of the vitamin B J2 standard is plotted against the HPLC signal for the standard. Once such a standard curve has been constructed, aqueous solutions of various vitamin B 12 derivatives may be similarly analyzed and the concentration ofthe derivative in the solution determined.
• the water solubility of a vitamin B 12 derivative may be determined directly by absorbance spectroscopy. Briefly, a known amount of vitamin B 12 is dissolved in a known amount of water to provide an aqueous solution of known concentration (e.g., 10 mg derivative/ 10 mL water). The absorbance of this solution (or dilutions of the solution) is then measured by a UV absorbance spectrophotometer. The absorbance of the solution of known concentration provides the vitamin B derivative's absorptivity.
• the concentration (or the amount of the vitamin B ]2 derivative in the solution) of subsequent aqueous solutions of the vitamin B 12 derivative may be determined by measuring the absorbance ofthe solution.
• the water-solubilizing linker is a polyether or a polyhydroxy linker.
• the water-solubilizing linker is the polyether linker such as a 4,7,10-trioxa-l,13-tridecanediamine linker or a 3,6-dioxa-l,8-octanediamine linker.
• the synthesis of a representative vitamin B ⁇ 2 derivative having water-solubilizing linker is described in Example 23.
• the synthesis of a representative vitamin B 12 -biotin conjugate having a water-solubilizing linker is described in Example 24.
• the syntheses of representative vitamin B 12 dimers having water-solubilizing linkers are described in Example 25.
• Coupling or reactive groups include any functional group capable of coupling a linker to a vitamin B. 2 molecule.
• Suitable coupling groups include, nucleophilic and electrophilic functional groups.
• Suitable nucleophilic groups include hydroxy groups, amino groups, and thio groups.
• Suitable electrophilic groups include carboxylic acid groups and carboxylic acid derivatives including acid halides, acid anhydrides, and active esters, such as NHS esters.
• a preferred linker is a diaminododecane.
• a linker may be coupled to the preferred _>-, d-, or e- coupling sites (see Structure I above) by any one of several suitable means, including, by way of example, activating a vitamin B 12 molecule by hydrolyzing its propionamide groups to produce monocarboxylates, purifying the resulting monocarboxylates, and coupling a linker to a selected coupling site. Hydrolysis of the coupling sites may be accomplished by exposing vitamin B J2 to aqueous acid for a period of time and under suitable conditions to hydrolyze the desired propionamide groups.
• hydrolysis is performed by exposure ofthe amide to dilute aqueous acid for a period of about 6 to 12 days, typically about 9 to 11 days, and most preferably about 10 days at room temperature.
• Suitable aqueous acids include, by way of example, 0.1N hydrochloric acid, 0.5N phosphoric acid or 0.5N sulfuric acid.
• Purification of _>-, d-, and e- monocarboxylates can be accomplished by any one of several means, including column chromatography, such as gel-permeation chromatography, adsorption chromatography, partition chromatography, ion- exchange chromatography, and reverse-phase chromatography.
• column chromatography is preparative reverse-phase liquid chromatography.
• LC purification may be conducted at a flow rate of 0.15 mlJmin. on a 5 ⁇ m, 4.6 X 250 mm propylamine column (RAININ microsorb-MV amino column) eluting with 58 ⁇ M pyridine acetate, pH 4.4 in H 2 O:THF (96:4) solution. Even more preferably, the coupling reaction is monitored using analytical high pressure liquid chromatography (HPLC).
• HPLC analytical high pressure liquid chromatography
• Reverse-phase HPLC chromatography is preferably carried out using an analytical version of above-noted propylamine column using a gradient solvent system at a flow rate of 1 mlJrnin.
• the d- isomer is identified as the longest retained peak (third)
• the e- isomer is identified as the second retained peak
• the b- isomer is identified as the shortest retained peak (first) eluanted from the LC column.
• the e-isomer may also be identified as that vitamin B 12 derivative demonstrating the greatest biological activity, as noted below.
• a ribose coupling site may be activated by any one of several suitable means including activating a hydroxyl group at coupling site h by reaction with a suitable reagent (e.g., succinic anhydride) to yield a ribose derivative that bears a reactive group (e.g., a carboxylate group).
• a suitable reagent e.g., succinic anhydride
• ribose coupling site and "coupling site A” are used interchangeably. This technique is described in detail in Toraya, Bioinorg. Chem. 4:245-255, 1975. Separation and purification ofthe activated molecule may be accomplished on a C18 column as noted below.
• a 5'-OH ribose coupling site is activated using any one of several suitable reagents including esterifying agents and ether forming reagents.
• suitable reagents provide a cyanocobalamin derivative having a reactive group for further coupling reactions.
• Esterification ofthe ribose 5 -OH may be accomplished with esterifying agents including, by way of example, carboxylic acid derivatives such as anhydrides, acid halides, and reactive esters including TFP and NHS esters.
• esterifying agents including, by way of example, carboxylic acid derivatives such as anhydrides, acid halides, and reactive esters including TFP and NHS esters.
• succinic anhydride provides a 5'-O-ribose ester derivative having a carboxylic acid group as a reactive group for subsequent coupling reactions.
• a suitable N-protected reactive ester of 4-aminobutyric acid (GABA) yields a 5'-O-ribose ester derivative having, after N-deprotection, an amino group as a reactive group for subsequent coupling reactions.
• GABA 4-aminobutyric acid
• Suitable 5'-O-ribose ether derivatives may be prepared by any one of several
• the 5'-OH group may be first converted to a good leaving group (e.g., a -toluenesulfonic acid group) followed by displacement with a suitable nucleophile.
• suitable nucleophiles include alcohol, amine, and sulfhydryl derivatives that produce S'-O-ribose ether, amine, and thioether derivatives, respectively.
• Suitable alcohol, amine, and sulfhydryl derivatives include those derivatives having a reactive group, such as a carboxylic acid or amine group, protected as necessary, for subsequent coupling reactions.
• suitable ether forming reagents include N-protected alcohols, monoprotected diamines, and N-protected thioamines. Accordingly, depending upon the selection of the ether forming reagent, ether linkages including alkyl ether and benzyl ether linkages may be formed.
• Suitable 5'-O-ribose ether derivatives may also be prepared by 5'-OH alkylation with suitable alkylating agents.
• suitable alkylating agents include alkylating agents having a reactive group, such as a carboxylic acid or amine group, protected as necessary, for subsequent coupling reactions.
• Preferred alkylating agents include active halide compounds such as haloacetates, benzyl halides, and silyl halides.
• Alkylation with a haloacetate such as methyl bromoacetate or trimethylsilyl bromoacetate, or a benzyl halide such as methyl 4-(bromomethyl)benzoate provide 5 -O-ribose ethers (i.e., alkyl and ether linkages) having, after ester hydrolysis, a carboxylic acid group for subsequent coupling reactions.
• a haloacetate such as methyl bromoacetate or trimethylsilyl bromoacetate
• a benzyl halide such as methyl 4-(bromomethyl)benzoate
• Alkylation with a silyl halide such as methyl l l-(chlorodimethylsilyl)undecanoate provides a 5'-O-ribose silyl ether (i.e., a silyl ether linkage) having, after hydrolysis, a carboxylic acid group for subsequent coupling reactions.
• a silyl halide such as methyl l l-(chlorodimethylsilyl)undecanoate
• linkers may be coupled to a vitamin B 12 molecule to form a vitamin B J2 linker adduct using any one of several means, including, by way of example, an amide forming reaction, employing an amine group on the linker and a carboxylate coupling site on a vitamin B 12 molecule.
• a linker may be coupled to a vitamin B molecule through an amide forming reaction, employing a carboxylate group on the linker and an amino group on a B ]2 molecule.
• the amide forming reaction may include the use of a coupling agent.
• Suitable coupling agents include carbodiimide coupling agents, such as, by way of example, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), l-benzyl-3-(3-dimethylaminopropyl) carbodiimide (BDC), l-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbod ⁇ mide (CMC), and 1,3- dicyclohexylcarbodiimide (DCC).
• EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
• BDC l-benzyl-3-(3-dimethylaminopropyl) carbodiimide
• CMC l-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbod ⁇ mide
• DCC 1,3- dicyclohexylcar
• the amide forming reaction coupling the linker to a B ]2 molecule may employ a reactive carboxylic acid group and an amine.
• Suitable reactive carboxylic acid groups include carboxylic acid derivatives that yield an amide upon reaction with an amine.
• Such reactive groups include, by way of example, any reactive carboxylic acid derivative, including, by way of example, carboxylic acid halides, such as acid chlorides and bromides; carboxylic acid anhydrides, such as acetic anhydrides and trifluoroacetic anhydrides; esters, such as p-nitrophenyl esters and N-hydroxysuccinimide esters.
• carboxylic acid halides such as acid chlorides and bromides
• carboxylic acid anhydrides such as acetic anhydrides and trifluoroacetic anhydrides
• esters such as p-nitrophenyl esters and N-hydroxysuccinimide esters.
• a linker may be coupled to a benzimidazole (coupling site i, see Structure I) using techniques described in detail in Jacobsen. Anal. Biochem. 113 : 164- 171. 1981.
• Vitamin B J2 linker adducts may be separated and purified using any suitable means, including column chromatography, such as gel-permeation chromatography, adsorption chromatography, partition chromatography, ion-exchange chromatography, and reverse-phase chromatography.
• column chromatography is preparative LC. These techniques are described in detail in Lim, HPLC of Small Molecules. IRL Press, Washington, D C, 1986.
• the vitamin B 12 receptor modulating agents of the present invention must be capable of binding transcobalamin II.
• a receptor modulating agent to bind TcII may be ascertained using any one of several means known in the art, including competitive binding assays with the receptor modulating agent competing with native vitamin B J2 .
• Rerouting moieties of the present invention include any moiety that is capable of affecting the receptor trafficking pathway. This characteristic can be assessed by employing a receptor modulating agent having a radiolabeled targeting moiety and following its path through the cell. This is accomplished using techniques known in the art, including using radiolabeled, biotinylated, or FITC labeled targeting moiety followed by binding assays, ELISA, or flow cytometry.
• a preferred receptor modulating agent is one that results in the removal of the highest percent of receptor for the longest period of time.
• Suitable rerouting moieties of this invention do not significantly detract from the selectivity ofthe targeting moiety. Whether a rerouting moiety detracts from the selectivity of a targeting moiety may be determined by any one of several methods known in the art, including comparing binding of the receptor modulating agent on receptor positive and receptor negative cells, as assessed by ELISA, flow cytometry, or other binding assays.
• Rerouting moieties cause the retention/degradation of an agent/receptor complex within at least one cell type, but not necessarily in all cells.
• a rerouting moiety causes retention of an agent/receptor complex in some cells, but not necessarily other agent/receptor complexes in other cells.
• Different rerouting moieties may also distinguish between receptor species, for example, as in polarized epithelium where the same receptor may independently traffic on the apical, basal, or basolateral sides ofthe cell.
• a rerouting moiety is covalently attached to the targeting moiety, and the resulting receptor modulating agent is compared for receptor modulation on different receptor-bearing cells using binding or functional assays known in the art.
• Suitable rerouting moieties of this invention may be categorized into five different functional classes: (1) lysosomotropic moieties; (2) intracellular polymerizing moieties; (3) protein sorting signals or sequences; (4) conditional membrane binding peptides; and (5) bi- or multivalent receptor cross-linking moieties. While such rerouting moieties may have different functional mechanisms of action, all promote retention of the agent/receptor complex within the intracellular vesicular system. All of these classes of rerouting moieties will impart the ability to affect the receptor trafficking pathway.
• lysosomotropic moieties refers to moieties that route the agent/receptor complex to the lysosomes. Numerous suitable lysosomotropic moieties are known, and are reviewed in Biochem. Pharmacol. 23:2495-2531, 1974.
• a preferred lysosomotropic moiety includes an aminoglycoside antibiotic marked by the characteristic ability to accumulate in lysosomes after intracellular protonation. Intracellular protonation occurs in the increasingly acidic conditions that occur during the transfer from early to late endosomes and, finally, to the lysosome.
• Aminoglycoside antibiotics are similar in structure, but are divided into structurally related families of compounds based upon the sugar units. These families include gentamycin, kanamycin, neomycin, and streptomycin.
• the gentamycin family includes gentamycin C ⁇ , gentamycin C 2 , gentamycin C la , sisomicin, and netilmicin;
• the kanamycin family includes kanamycin A, tobramycin, and amikacin;
• the neomycin family includes neomycin B, paromomycin, ribostamycin, and bytirosin B;
• the streptomycin family includes streptomycin A and streptomycin B.
• the rerouting moiety is gentamycin, that accumulates in lysosomes in concentration as much as three-hundredfold that of the extracellular concentration (J. Pharmacol. Exp. Ther. 255:867-74, 1990: Ren. Fail. 14:351-7. 1992).
• Suitable aminoglycosides have reactive amine groups capable of being coupled through peptide or other chemical linkers.
• a targeting moiety may be readily attached via covalent linkage to these rerouting moieties using any one of several techniques known in the art to form covalent bonds, for example, using thioether, disulfide, ether, ester, and peptide bonds. Since many of the aminoglycoside antibiotics have several amines that could be derivatized in a conjugation procedure, a primary amine contained in these compounds can be selectively reacted to favor covalently attachment to the targeting moiety through the 5' amine.
• covalent attachment to the targeting moiety may be accomplished by converting the aldehyde moiety to an amine, and attaching to the targeting moiety using carbodiimide or other suitable activated carboxylic acid.
• Aminoglycosides are water soluble and do not readily bind to other proteins, and thus do not impart nonspecific binding to a receptor modulating agent.
• Particularly preferred aminoglycosides include those that allow for preferential derivation of a selected amine.
• preferred aminoglycosides include those compounds to that protective groups can be added to various nitrogen atoms thereof and, subsequently, selectively deprotected to yield a single free amine.
• the free amine can be further derivatized, for example, by addition of a peptide linker or covalently attached directly to the targeting moiety.
• These rerouting moieties include ribostamycin, kanamycin, amikacin, and streptomycin.
• Ribostamycin is particularly preferred, due to its relative low toxicity and its derivatization chemistry, allowing an acyl migration reaction to be effected on a hydroxyl protected ribostamycin to yield a single amine adduct.
• Kanamycin may also be used in a selective protection/acylation reaction;
• amikacin is commercially available in a form that allows attachment without deprotecting its amines or alcohol groups; and streptomycin can also be readily derivatized by protonating guanidinium groups under physiologic conditions to provide the polycations necessary for cellular or lysosomal retention.
• nonaminoglycoside lysosomotropic compoimds that may accumulate after intracellular protonation are also suitable rerouting moieties.
• Suitable nonaminoglycoside compounds exhibiting this characteristic are known in the art, a series of aminoacridine and amino quinoline dyes, typified by cholquinine and quinacrine; a group of amino naphthalenes, typified by dansyl cadaverine; and derivatives thereof. Such dyes are characterized by cellular retention and low toxicity. All of these compounds have characteristic sites for covalent attachment to a targeting moiety via the amine and may be attached thereto as described above.
• Another aspect of the present invention utilizes a lysosomotropic peptide subject to charge modification under intracellular conditions as a rerouting moiety.
• the rerouting peptide acts to retain an agent/receptor complex in the intracellular vesicular system until membrane flow delivers it to the lysosome for degradation.
• these peptides are capable of being phosphorylated by intracellular protein kinases. When phosphorylated by the intracellular enzymes, such peptides would be highly anionic.
• Charge-based retention can be an inherent property ofthe rerouting peptide or can be imparted by intracellular modification.
• Intracellular modification may be accomplished by any of several means known in the art, including phosphorylation of certain residues of some receptors (e.g., the EGF receptor) may cause intracellular rerouting (Cancer Treat. Res. 61:139-160. 1992: J. Cell. Biol. 116:321-30. 1992).
• the rerouting peptides may be covalently attached to a targeting moiety by any means, including, for example, covalently linking the peptide directly to the targeting moiety, or by use of an appropriate linker moiety, such as G-G-G, that may be derivatized and covalently attached to the targeting moiety.
• Preferred rerouting peptides include protein kinase-substrate peptides that incorporate serine. These peptides are particularly preferred for enhancement of receptor rerouting in tumor target cells, that have increased levels of protein kinase activity for serines or tyrosines. Increased levels of kinase activity within tumor cells may be attributed to the presence of oncogene products, such as H-ras, on the * cytoplasmic side of tumor cell plasma membranes (CI B A. Found. Symp. 164:208- 18, 1992).
• Suitable rerouting peptides also include protein kinase substrates and peptides that possess a single positive charge.
• the latter type of rerouting peptide may form an ion pair with a "glutamate-like" residue of an attached or closely associated residue(s) of the receptor.
• Particularly preferred rerouting peptides may be derived, using technologies known in the art, from the proteins and the amino acid sequences identified in Table 1.
• the rerouting moiety is a lysosomotropic amino acid ester that, in high concentration, can cause the lysis of granule-containing cells, such as NK cells, cytolytic T cells and monocytes.
• concentration must generally be maintained below 100 mM to avoid lysis.
• Suitable lysosomotropic amino acid esters and their sources are presented in Table 2.
• the lysosomotropic amino acid esters identified in Table 2 can be used to retain the agent/receptor complex in lysosomes after intracellular cleavage of the ester.
• such amino acid esters may be utilized as the C-terminal portion of a larger peptide containing a linker sequence and/or a phosphorylation substrate sequence, and with suitable residues, such as cysteine, for covalent attachment to a targeting moiety, such as a sequence encoding a peptide or protein ligand for a given cell surface receptor.
• a second functional class of rerouting moieties is disclosed.
• This class includes peptides that undergo polymerization within endosomes or lysosomes, inhibiting their passage through intracellular membranes.
• Intracellular polymerizing compounds can be incorporated into a larger peptide containing the targeting moiety and a linker.
• Suitable peptides include the dipeptide ester referenced in Table 2 (i.e., L-Leucyl-L-Leucine-O-Me). When transported into cells, these dipeptide esters preferentially accumulate in lysosomes and secondary granules of cytotoxic cells. These dipeptides also undergo self- association and polymerization, that result in trapping at low concentrations, and membrane rupture at higher concentrations.
• Suitable intracellular polymerizing compounds also include peptides that can self-associate into alpha-helical structures termed "leucine zippers". In the context of this invention, such structures may be used to form intracellular polymers that are incapable of exiting intracellular vesicles. Such sequences can be selected by observing self-association of the compounds in solution, and the formation of polymers capable of binding to DNA. Suitable peptide sequences that can self- associate into alpha-helical structures are presented in Table 4. TABLE 4 LEUCINE ZIPPERS
• a third functional class of rerouting moieties is disclosed.
• This class includes moieties that can be recognized by intracellular receptors. Such sequences are identified by their ability to stop movement of endogenously synthesized proteins to the cell surface. Suitable peptides include certain peptide sequences (such as sorting or signal sequences) associated with the trafficking of endogenously synthesized proteins (Cur. Opin. Cell. Biol. 3:634-41, 1991). Such peptide sequences, when covalently attached to the C-terminus of an exogenously added targeting moiety, result in the retention of the agent/receptor complexes in the endoplasmic reticulum ("ER"), Golgi apparatus, or lysosomes.
• ER endoplasmic reticulum
• Such peptide sequences are recognized by intracellular receptors, examples of that include both mammalian and bacterial versions of ER receptors described in detail in J. Cell Biol 120:325-8, 1993; Embo. J. 11:4187-95, 1992; Nature 348:162-3, 1990. Further exemplary peptide sequences and variants thereof (shown in parentheses) that can be recognized by intracellular receptors are set forth in Table 5, Sections A and B.
• REDLK is a preferred sequence recognized by prokaryotic cells and to a lesser degree by eukaryotic cells (see Table 5, section C).
• receptor modulating agents can be constructed to selectively inhibit a receptor-mediated process in bacteria, while having little effect on mammalian cells.
• PEPTIDE SEQUENCES THAT BIND INTRACELLULAR RECEPTORS
• HDEL HVEL, HNEL, J. Biol. Chem. 268:7728-32. 1993
• a further class of peptide sequences of this invention termed “internalization signals,” function by binding to clathrin, both in the coated pits, as well as those intracellular vesicles that maintain a clathrin coat.
• Representative examples of such clathrin-binding peptides (CBP) are disclosed in Table 5, section D. The CBP binds clathrin in the coated pits initially located on the cell surface causing retention of the targeting moiety to that it is conjugated.
• a further class of moieties capable of recognizing intracellular receptors includes carbohydrates.
• Suitable carbohydrates include any carbohydrate that is capable of binding to intracellular carbohydrate (CHO) receptors but not to cell surface CHO receptors.
• Such carbohydrates include: mannose-6-phosphate and glucose-6-phosphate.
• Suitable carbohydrate moieties include those that bind to the insulin-like growth factor H/mannose-6-phosphate (IGF H M6P) receptor, including analogs of mannose-6-phosphate, as well as other phosphorylated saccharides (Carbohydrate Res. 213:37-46. 1991: FEBS Lett. 262: 142-4. 1990).
• the affinity of the rerouting moiety can be varied by changes in the chemical nature of the phosphorylated saccharides (J. Biol Chem. 264:7970-5, 1989; J. Biol Chem. 264:7962-9, 1989) (monosaccharides bind with the lowest affinity, while di- or tri-saccharides bind with increasingly higher affinity). Clustering of phosphorylated saccharides on protein carriers can dramatically increase affinity to the intracellular receptor.
• mannose-6-phosphate receptor expression is primarily intracellular, expression also occurs on cell surfaces.
• covalent attachment of a targeting moiety with a carbohydrate that binds the mannose-6-phosphate receptor should be constructed so as to give at least a hundredfold difference in binding affinity between the targeting moiety and the rerouting moiety.
• a vitamin B 12 /transcobalamin ⁇ receptor targeting moiety in this case vitamin B 12 , would have a binding affinity for the carrier protein, transcobalamin ⁇ (TcE), of > 10 *10 M and an affinity for the IGF ⁇ M-6-P receptor of IO" 8 M or less. This will maintain the specificity of the vitamin B 12 binding (via TcH), while allowing transfer of the receptor modulating agent from serum M-6-P soluble receptor to cell surface receptor.
• TcH transcobalamin ⁇
• other carbohydrate-based rerouting moieties also promote retention of the modulating agent/receptor complex in the ER or Golgi complex.
• Such moieties are based on the recognition by various glycosyl transferases of carbohydrate moieties, either as a natural substrate or as an inhibitor. Such moieties are reviewed in Sem. Cell Biol 2:289-308, 1991.
• saccharide recognition moieties include penultimate sugars, such as glucose and N-acetyl glucosamine (that are natural substrates). More preferred, however, are glycosylation inhibitors that are recognized by glycosyl transferases, but cannot serve to append further carbohydrate residues on growing chains (Sem. Cell. Biol 2:309- 318, 1991).
• a fourth functional class of rerouting moieties is disclosed.
• This class is generally comprised of rerouting moieties that anchor the receptor to the cell membrane.
• this class includes membrane-binding peptides that exhibit conditional pH-dependent membrane binding. Such peptides exhibit ⁇ -helical character in acid but not neutral pH solutions. When a conditional membrane-binding peptide assumes a helical conformation at an acidic pH, it acquires the property of amphiphilicity, (e.g., it has both hydrophobic and hydrophilic interfaces). More specifically, within a pH range of approximately 5.0-5.5, such a peptide forms an alpha-helical, amphiphilic structure that facilitates insertion of the peptide into a target membrane.
• An alpha-helix- induced acidic pH environment may be found, for example, in the low pH environment present within cellular endosomes or lysosomes.
• a conditional, membrane-binding peptide is unfolded (due to strong charge repulsion among charged amino acid side chains) and is unable to interact with membranes.
• conditional membrane-binding peptide sequences include the charged ' amino acids glutamate, aspartate, and histidine.
• a preferred conditional membrane- binding peptide includes those with a high percentage of helix-forming residues, such as glutamate, methionine, alanine, and leucine.
• conditional membrane- binding peptide sequences include ionizable residues having pKas within the range of pH 5-7, so that a sufficiently uncharged membrane-binding domain will be present within the peptide at pH 5 to allow insertion into the target cell membrane.
• Conditional membrane-binding peptides can be incorporated through covalent bonds to a chemical or peptide targeting moiety or synthesized as an entire peptide sequence including a linker and peptide targeting moiety.
• a particularly preferred conditional membrane-binding peptide is aal-aa2-aa3- EAALA(EALA) -EAI_EALAA-amide, that represents a modification of a published peptide sequence (Biochemistry 26:2964, 1987).
• the first amino acid residue (aal) is preferably a unique residue such as cysteine or lysine, that facilitates chemical conjugation ofthe conditional membrane-binding peptide to a targeting protein.
• the peptide can also be incorporated into a fusion protein with a protein or peptide targeting moiety (see Example 7).
• Amino acid residues 2-3 may be selected to modulate the affinity of the translocating peptide for different membranes. For instance, if both residues 2 and 3 are lysine or arginine, the peptide will have the capacity to bind to membranes or patches of lipids having a negative surface charge. If residues 2-3 are neutral amino acids, the peptide will insert into neutral membranes.
• conditional membrane-binding peptide can be derived from sequences of apo-lipoprotein A-1 and B; peptide toxins such as melittin, bombolittin, delta hemolysin and the pardaxins; antibiotic peptides, such as alamethicin; peptide hormones, such as calcitonin, corticotrophin releasing factor, beta endorphin, glucagon, parathyroid hormone, and pancreatic polypeptide.
• peptides normally bind membranes at physiologic pH but through attachment of substituents the peptides can be enhanced in their ability to form alpha-helices at acidic pH and reduced in their membrane-binding at physiologic pH.
• a modified peptide having pH-dependent membrane binding at acidic pH is fully succinylated melittin.
• a peptide (melittin) that normally binds to membranes at physiological pH is converted to a pH-dependent peptide through succinylation of lysines.
• succinylation the peptide displays an amphipathic character only at acidic pHs.
• Helix stabilization may be achieved: (1) by adding repeating "EALA" units to form a longer peptide; (2) by placing an amide at the C-terminus of the peptide, in order to counteract the helical dipole; (3) by polymerizing the peptide; (4) by substituting a natural helix-former for one or more ofthe stacked glutamates; or (5) by attaching the peptide to a targeting moiety through use of a longer linker, in order to provide sufficient distance between the membrane binding peptide and the targeting moiety for the peptide to contact and interact with the target cell intracellular membranes.
• a fifth functional class of rerouting moieties is disclosed.
• the rerouting moiety merely functions as a modulating agent in that the moiety disables the receptors by crosslinking the same.
• This class includes bi- or multivalent receptor crosslinking moieties formed from monovalent binding targeting moieties. Cross-linking of receptors in some receptor systems is sufficient to cause a rerouting of cell surface receptors to lysosomes for degradation, rather than their normal pathway of receptor recycling.
• the synthesis of a bivalent receptor modulating agent is exemplified in greater detail in the examples below.
• a preferred cross-linking receptor modulating agent is a vitamin B J2 dimer.
• each vitamin B 12 molecule acts as a targeting agent and a rerouting agent; cross-linking the B 12 dimer will cross-link the vitamin B 12 receptors, thus impeding the receptor trafficking pathway.
• a preferred vitamin B ]2 dimer is generally comprised of two vitamin B ]2 molecules, such as cyanocobalamin, coupled by one or more linkers through coupling sites independently selected from a-g, h (ribose), and i (benzimidazole).
• linkers Preferably, cross-linking occurs between b- or e- coupling sites on both molecules.
• the dimer must be capable of forming a B 12 /TcII complex. As noted above, this characteristic may be assayed using any one of several techniques known in the art, including competitive binding assays.
• a vitamin B J2 may be coupled to a second vitamin B 12 molecule in the same manner as described in detail for conjugation of rerouting moieties to vitamin B targeting moieties.
• dimers may be synthesized using one or more linkers of various lengths, solubilities, and any combination of homobifunctional, heterobifunctional, homotrifunctional, or heterotrifunctional linkers.
• the use of a trifunctional linker allows for coupling with any number of additional moieties.
• ' number of atoms comprising the linker between the vitamin B ]2 molecules should generally be greater than 10 atoms, typically be in the range of 30 to 55 atoms and, preferably be about 45.
• the number of atoms is calculated relative to a linear chain of atoms, linear chain, branched chain, and cyclical chain linkers or combinations thereof would be suitable.
• the structure ofthe atom chain in a linker would include, by way of example, alkyl, heteroalkyl, alkylaryl, and heteroalkyl aryl.
• a dimer may be synthesized by combining two different vitamin B 12 linker adducts in the presence ofa coupling agent.
• the linkers couple and dimers may then be separated and purified using the same methods outlined above.
• activated vitamin B J2 may simply be combined with a homobifunctional or homotrifunctional linker.
• the ratio of vitamin B 12 to linker should be in the range of 2: 1.
• a 1:1 ratio is used in preparation of mixed dimers (e.g., b- and e-acid derivatives) or mixed ligands (e.g., B 12 and hormone). Dimers may be separated and purified as noted above.
• vitamin B 12 linker adducts may be coupled by a third linker.
• the third linker a "cross-linker” serves to bridge the linkers on the vitamin B J2 linker adducts.
• Suitable cross-linkers mclude those noted above. The synthesis of representative vitamin B J2 dimers having bridging cross-linkers is described in Example 25.
• vitamin B 12 dimers may also be prepared utilizing a cross-linker that functions through high-affinity specific binding interactions.
• the receptor modulating agents ofthe present invention include vitamin B J2 dimers and vitamin B J2 tetramers bound through the avidin-biotin interaction.
• An vitamin B 12 /avidin conjugate may be readily prepared by the addition ofa suitable vitamin B 12 /biotin conjugate to avidin (or streptavidin).
• Avidin and streptavidin are tetrameric proteins having four biotin binding sites situated in pairs on either side of the protein.
• vitamin B 12 /avidin conjugates may be prepared in that the number of vitamin B 12 molecules or derivatives per avidin may be varied from one to four.
• the vitamin B J2 /avidin conjugate has four vitamin B 12 molecules or derivatives per conjugate.
• these tetrameric vitamin B 12 conjugates may be prepared by binding a suitable vitamin B 12 /biotin conjugate (i.e., a vitamin B 12 /biotin conjugate composed of
• Suitable vitamin B J2 /biotin conjugates include those conjugates in that the biotin is covalently linked to a vitamin B J2 coupling site with a linker of a length sufficient to permit binding of the vitamin B J2 conjugate to avidin.
• suitable linkers are in the range of about 12 to 50 atoms in length, and preferably about 25 to 45.
• the vitamin B, 2 /avidin conjugate is a vitamin B 12 dimer having two vitamin B 12 molecules or derivatives per conjugate.
• These vitamin B 12 /avidin conjugates may be prepared using a suitable vitamin B 12 /biotin conjugate having two biotins linked to a single vitamin B ]2 molecule or derivative.
• suitable vitamin B ]2 /biotin conjugates include conjugates in that the biotins ofthe conjugate may both bind to each of one pair of avidin binding sites.
• the length of the linker between the vitamin B 12 and the biotins in the vitamin B 12 /biotin conjugate may be of a length sufficient to permit binding to a pair of avidin binding sites, but not so lengthy as to be capable of binding to one binding site on one side of the avidin, and another binding site on the opposite side of the avidin.
• the vitamin B 12 /avidin conjugate prepared from avidin and two vitamin B 12 /biotin conjugates (having two biotins per vitamin B 12 ) has each pair of biotin binding sites occupied by a vitamin B 12 /biotin conjugate.
• Polymerization of peptides for use in the present invention may be accomplished by placing a cysteine residue at each end of a peptide, followed by oxidation using dissolved oxygen or other mild oxidizing agent, such as oxidized glutathione.
• the average length of a polymerized peptide may be controlled by varying the polymerization reaction conditions.
• the amino acid sequence of any of the peptides of this invention may be selected to include all L-amino acids or all D-amino acids having a side chain pK a from 5.0 to 9.0.
• D-amino acids may be advantageously used to form nonproteolyzable peptides, since the D-amino acids are not metabolized within the cell.
• the peptides of the present invention may include a combination of L- and D-amino acids, wherein D-amino acids are substituted for L-amino acids on either side of a proteolytic cleavage site.
• Yet another preferred noncleavable peptide incorporates peptide bond analogs that are not susceptible to proteolytic cleavage by cellular enzymes.
• the receptor modulating agents of this invention comprise a targeting moiety coupled to the rerouting moiety.
• the rerouting moieties identified above may be covalently attached to the targeting moiety by any one of several techniques known in the art, including (a) by chemical modifications such as a disulfide formation, thioether formation, amide formation or a reduced or nonreduced Schiffs base, (b) by direct peptide bond formation as in a fusion protein, or (c) by use ofa chemical and peptide linker.
• Suitable peptide linkers in this regard correspond to two or more amino acid residues that allow the rerouting peptide to assume its active conformation independent of its interaction with the targeting moiety, and that allows sufficient distance for rerouting moiety access to, for example, intracellular membranes from the peptide attachment site on the targeting moiety.
• a rerouting moiety may be conjugated to a vitamin B 12 targeting moiety by any one of several means, including, by way of example, coupling a rerouting moiety to a reactive group on a vitamin B ]2 linker adduct; coupling a vitamin B 12 to a reactive group on a rerouting moiety linker adduct or an appropriate side chain thereof; coupling a vitamin B J2 linker adduct to a rerouting moiety linker adduct or an appropriate side chain thereof; coupling a rerouting moiety/biotin binding protein conjugate to a vitamin B 12 /biotin conjugate; or coupling a rerouting moiety biotin conjugate to a vitamin B 12 /biotin binding protein conjugate.
• Coupling of a rerouting moiety to a vitamin B 12 linker adduct, or a vitamin B 12 to a rerouting moiety linker adduct may be accomplished using the same techniques noted above for coupling a vitamin B 12 molecule with a linker.
• the only critical consideration of this aspect ofthe invention is that the total linker length must be sufficient to avoid steric hindrance.
• the total linker length is at least 6 atoms.
• B 12 /biotin conjugate may be accomplished using any one of several means described in detail in Avidin-Biotin Chemistry: A Handbook, ed. D. Savage, Pierce Chemical Co., 1992. Briefly, a biotin binding protein conjugate is prepared using a rerouting moiety or, as in a second embodiment, a vitamin B 12 molecule. Suitable biotin binding proteins include avidin or streptavidin. In some circumstances, a linker may be utilized to distance the molecules. For example, when coupling a vitamin B 12 to an avidin, a linker of at least 6 atoms is preferred.
• a biotin conjugate is prepared using a vitamin B 12 molecule or, as in a second embodiment, a rerouting moiety.
• a vitamin B 12 molecule is combined with an NHS ester of biotin.
• the vitamin B ⁇ 2 molecule is a vitamin B 12 linker adduct as described above. Even more preferably, the vitamin B 12
• molecule is a vitamin B 12 linker adduct characterized by a 14-atom linear linker coupled to the b- or e- coupling site. The synthesis of a representative vitamin
• B 12 /biotin conjugate is described in Example 24. Once formulated, coupling between the biotin conjugates and biotin binding protein conjugates is easily accomplished by combining the complementing conjugates, i.e., a vitamin B 12 /biotin conjugate with a rerouting moiety/avidin conjugate.
• the complementing conjugates i.e., a vitamin B 12 /biotin conjugate with a rerouting moiety/avidin conjugate.
• a B 12 /biotin conjugate is utilized to couple a vitamin B J2 to any number of compounds through biotin binding protein conjugates.
• any compound that is capable of coupling a biotin binding protein may be coupled to a vitamin B J2 and thereby internalized into cells expressing the vitamin B J2 receptor.
• Such compounds include, in addition to the rerouting moieties described in detail below, hormones, enzymes, antibodies or fragments thereof, markers, or therapeutics. Coupling any of these compounds to a biotin binding protein, such as avidin or streptavidin, may be accomplished using techniques described in detail in Avidin-Biotin Chemistry: A Handbook, ed. D. Savage, Pierce Chemical Co., 1992.
• a vitamin B 12 /biotin conjugate is coupled to a therapeutic/avidin conjugate directed at neoplastic disorders.
• Neoplastic disorder therapeutics that may be coupled to a vitamin B ]2 /biotin conjugate through avidin include doxorubicin, daunorubicin, etoposide, teniposide, vinblastine, vincristin, cyclophophamide, cisplatin, and nucieoside antimetabolites such as arabinosylcytosine, arabinosyladenine, and fludarabine.
• a vitamin B ]2 /biotin conjugate is coupled to a marker conjugated with a biotin binding protein.
• Suitable markers include, by way of example, fluorescent molecules or radiolabeled molecules. This combination may be utilized as a detection system incorporated into a screening device to identify patients with low receptor bearing cells or in the evaluation of receptor up-regulation, for example, following treatment of patients for any one of a wide variety of receptor modulation disorders.
• a vitamin B 12 /biotin conjugate is coupled to a radioisotope conjugated to a biotin binding protein.
• Suitable radioisotopes include, any high energy emitting radioisotopes capable of conjugating a biotin binding protein. This combination may be utilized as a targeted radiodiagnostic or radiotherapeutic.
• a vitamin B 12 /biotin conjugate is used to immobilize vitamin B 12 to a solid matrix or avidin-coated substrate.
• this would enable one to isolate TcII, TcII receptors, and evaluate coupling sites on the Vitamin B 12 .
• the receptor modulating agents of this invention regulate receptor-dependent biological responses through alterations in the receptor trafficking pathway.
• cell surface receptors are often associated with clathrin-coated pits.
• the coated pits invaginate to form vesicles.
• the vesicles are then directed by the rerouting agent to lysosomes for receptor degradation or delivered to endosomes where the rerouting agent securely binds or delays the agent/receptor complex.
• the receptor modulating agents can incapacitate the receptors normally undergoing recycling. Newly synthesized receptors will eventually replace the internalized receptor on the cell surface.
• Biological activity of receptor modulating agents of the present invention may be ascertained in vitro by any one of several means known in the art, including competition binding assays or cell proliferation studies. These techniques are described in detail in Laboratory Techniques in Biochemistry and Molecular Biology: An Introduction to Radioimmunoassay and Related Techniques. 3rd Edition, ed. Burdon and van Knippenberg, Elsevier, 1987.
• a receptor modulating agent may be cultured with a suitable cell line, such as K562 cells (ATCC CCL 243), under conditions representing in vivo conditions.
• Such conditions would include the provision of a human source of TcII (such as human serum), vitamin B 12 , and, preferably, by careful removal by chromatography, of all TcII from other medium supplements such that proliferation is solely dependent on a known amount of exogenous TcII.
• a human source of TcII such as human serum
• vitamin B 12 such as human serum
• chromatography of all TcII from other medium supplements
• proliferation is solely dependent on a known amount of exogenous TcII.
• Cell cultures deprived of vitamin B 12 gradually lose their proliferative capacity, eventually resulting in cell death.
• Biological activity may be evaluated in vivo using techniques described in detail in Shieh et al., J. Immunol 152(2):859-866. 1994 in that human tumor cell lines are injected into nude mice, followed by therapy with receptor modulating agents. Next, tumor cells are removed, single-cell suspensions prepared and TcII cell surface receptor density may be evaluated by flow cytometry and bio
• the receptor modulating agent ofthe present invention may be administered in a therapeutically effective amount to treat a variety of disorders characterized in that control ofthe disease process or symptoms can be achieved by modulation of one or more receptor systems and the associated biological responses.
• disorders include neoplastic disorders, autoimmune diseases, rheumatic arthritis, cardiovascular disease, and neurodegenerative diseases.
• non-neoplastic disease processes is a stage in that the disease process itself, or its symptoms, can be halted or ameliorated by the use of an antiproliferative agent such as vitamin B 12 /TcII receptor modulating agents.
• an antiproliferative agent such as vitamin B 12 /TcII receptor modulating agents.
• These commonly recognized stages include a sensitization or eiicitation phase in that immune cells responsible for the disease become turned on by antigen specific or nonspecific means, followed by a proliferative phase in that the immune cells expand in number, and finally a symptomatic phase in that the expanded immune cells create tissue damage directly or indirectly.
• Neoplastic disorders include, by way of example, leukemia, sarcoma, myeloma, carcinoma, neuroma, melanoma, cancers of the breast, limg, liver, brain, colon, cervix, prostate, Hodgkin's disease, and non-Hodgkin's lymphoma. Because of this, antiproliferative chemotherapeutic drugs are commonly utilized in the treatment of many diseases other than cancer, but are limited in use to life-threatening situations due to their associated toxicity. Antiproliferative agents, such as the ones of the present invention (with little of the direct toxicity of chemotherapeutic drugs), may be used more widely.
• the vitamin B ⁇ receptor modulating agents ofthe present invention are not destructive to plasma membrane processes (e.g., ion transport).
• the antiproliferative activity is reversible by administration of vitamin B 12 .
• the agents of this invention may not be mutagenic, teratogenic, or carcinogenic since they act at the level of the plasma membrane, and not at the level of the nucleus, and DNA by intercalation or cross-linking (as many chemotherapeutic drugs act).
• Tumor Cells Tumor Assoc. Ags. Tumor Therapy
• Ki67 (alone and in combination
• T-cells Proliferating and activated T-cells can cause a wide variety of diseases ranging from the chronic inflammation of Crohn's disease to more acute organ graft rejection. In all of these diseases, the T-cell may serve a central pathogenic role or a more accessory role. Antiproliferative chemotherapeutic drugs serve to reduce symptomotology and in some cases lead to long-term remission. Similarly, proliferating fibroblasts and epithelial cells may give rise to diseases characterized by cell overgrowth. Vitamin B 12 receptor modulating agents may be used to replace or used in combination with existing chemotherapeutic regimens in these diseases.
• antiproliferative vitamin B 12 receptor modulating agents in these diseases is not to apply it so aggressively or with improper timing such that normal healing (adhesions, scarring) or cell renewal (psoriasis) processes are also inhibited.
• low doses of receptor modulating agents may be used during healing and higher doses used once healing is completed.
• receptor modulating agents may not be administered at all until after healing is completed.
• B 12 /TcII receptor modulating agents can be used to deprive neoplastic cells of vitamin B J2 . It has already been shown that sufficient deprivation leads to the death of rapidly proliferating lymphoid neoplasms such as leukemia and lymphoma. Moreover, short-term treatment to reduce cellular availability of this nutrient, combined with existing chemotherapeutic agents, markedly improves therapeutic efficacy.
• vitamin B 12 depletion may induce cytostasis and differentiation as well as cell death.
• B 12 /TcII receptor modulating agents may be used to induce differentiation in hormonally responsive solid tumors.
• An increase in the number of cells expressing a differentiated phenotype should translate into an increase in expression of hormone receptors.
• the hormone receptor status of tumors, such as breast and prostate cancer are directly correlated with their response to hormonal therapy.
• B 12 TcII receptor modulating agents can be used to increase the number of receptor positive tumor cells or increase receptor density in order to enhance efficacy of subsequent hormonal therapy.
• Vitamin B 12 receptor modulating agents may affect both replicating neoplastic and normal cells. However, bone marrow progenitors demonstrate differential sensitivity or response.
• B J2 receptor modulating agents can be used to modulate sensitivity of bone marrow progenitors so as to enhance their resistance to the toxic effects of chemotherapeutic agents.
• chemotherapeutic drugs act primarily on replicating cells, with nonreplicating cells being much less sensitive. Decreasing the sensitivity of progenitors to toxic drugs would increase the bone marrow reserves and enhance subsequent response to colony stimulating factors, and enable higher doses of chemotherapy or reduce the interval to reconstitution. It should also be recognized that such positive effects on bone marrow progenitors, as a natural consequence of B 12 receptor therapy for cancer, are an additional mechanism by that the therapeutic index of chemotherapeutic drugs other than 5-FU and methotrexate can be improved.
• Methotrexate is one such drug commonly used to treat symptoms associated with rheumatoid arthritis.
• the drug acts to reduce both localized (e.g., synovium) and generalized inflammation associated with disease progression.
• Methotrexate acts synergistically with vitamin B ⁇ depletion in therapy of leukemia.
• B J2 receptor modulating agents can therefore be combined with methotrexate to enhance efficacy in rheumatoid arthritis.
• Other methotrexate applications include treating the destructive inflammation associated with chronic heart disease and colitis.
• treatment refers to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, inhibition or elimination of the causative agent, or prevention of the infection or disorder in a subject who is free therefrom.
• treatment of infection includes destruction of the infecting agent, inhibition of or interference with its growth or maturation, neutralization of its pathological effects and the like.
• a disorder is "treated” by partially or wholly remedying the deficiency that causes the deficiency or that makes it more severe.
• the receptor modulating agents ofthe present invention are administered in a therapeutically effective dose.
• a therapeutically effective dose may be determined by in vitro experiment followed by m vivo studies.
• Pharmaceutical compositions containing the receptor modulating agents in an admixture with a pharmaceutical carrier or diluent can be prepared according to conventional pharmaceutical compounding techniques.
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration (e.g., intravenous, oral, topical, aerosol, suppository, parenteral or spinal injection). Preferably, administration is via stereotactical injection.
• HPLC separations of compounds were obtained on Hewlett-Packard quaternary 1050 gradient pumping system with a UV detector. Analysis ofthe HPLC data was obtained using Hewlett-Packard HPLC Chemstation software.
• HPLC for Monomers HPLC separations were conducted at a flow rate of
• Preparative LC was conducted to separate the mixture of monocarboxylic acids using RAININ Rabbit-plus peristaltic pumping system with a DYNAMAX (model UV-1) UV-visible absorbance detector at a flow rate of 0.15 mlJmin.
• An ID column (Alltech, 150 psi), (1000 mm X 25 mm) packed with aminopropyl silica (40-63 mm) was used.
• This example serves to demonstrate the hydrolysis of b-, d- and e-propionamide sites on a vitamin B 12 molecule using dilute acid in preparation for coupling of a linker to the sites.
• the hydrolysis of the _»-, d- and e-propionamides is selective over the hydrolysis of a-, c- and jj-acetamides, or the /-amide in the heterocyclic chain connecting the benzimidazole.
• An optimal yield of monocarboxylate to di- and tri-carboxylate derivatives was obtained at room temperature in 0.1 N HCl over a 10-day period.
• the nonhydrolyzed vitamin B J2 and the di- and tri-carboxylates produced were readily isolated from the desired monocarboxylates by preparative liquid chromatography.
• cyanocobalamin (1) (3.7 mmol, 5 g) was dissolved in 500 mL of 0.1 N HCl and stirred at room temperature for 10 days under argon atmosphere. The solution was then neutralized with 6 N NaOH and the cobamides were desalted by extraction into phenol and applied to a 200 g (60 x 4 cm, 200-400 mesh) Dowex Cl" x 2 column (acetate form; prepared by washing with saturated sodium acetate until it was free from Cl", then washing with 200 mL water). The column was eluted with water to remove unreacted cyanocobalamin and then eluanted with 0.04 M sodium acetate (pH 4.67).
• the first fraction of the elution contained three monocarboxylic acids. These were desalted by extraction into 100 mL of 90% (w/w) phenol, twice with 25 mL and once with 10 mL of phenol. Three volumes of ethyl ether (3 x 160 mL) and one volume of acetone (160 mL) were added to the combined phenol extracts. Monocarboxylic acids were removed from the organic phase by extraction with water (2 x 100 mL). The combined aqueous phases were extracted twice with 20 mL of ether to remove residual phenol. The aqueous solution of monocarboxylic acids was evaporated to dryness. Yield: 2.5 g (50%).
• the mixture of three acids (0.350 g) was then applied to a 200 g (1000 mm x 25 mm) column of aminopropyl-coated silica (40-63 mm) and was eluanted with 58 mM pyridine acetate pH 4.4 in H 2 O:THF(96:4); the eluant was collected with an automatic fraction collector.
• the first eluanted acid was found to be ->-monocarboxylic acid (2), the second eluanted acid was e-monocarboxylic acid (3), and the third eluanted acid was ⁇ -monocarboxylic acid (4).
• the acid fractions were desalted by phenol extraction. The solids obtained were crystallized from aqueous acetone.
• This example serves to demonstrate the activation of the ribose coupling site coupling site h (see structure I) with succinic anhydride.
• Cyanocobalamin (1) (0.15 mmol, 200 mg) was dissolved in 40 mL of dimethylsulfoxide (DMSO) containing 8 g (80 mmol) of succinic anhydride and 6.4 mL of pyridine. After 14-16 h at room temperature, the excess of succinic anhydride was destroyed by adding 500 mL of water and keeping the pH ofthe reaction mixture at 6 with 10% KOH. KCN was then added at a final concentration of 0.01 M and the pH of the solution was readjusted to 6 with 3 N HC1.
• DMSO dimethylsulfoxide
• This example serves to demonstrate the coupling of a linker to a cyanocobalamin monocarboxylate.
• Coupling of the monocarboxylates (2, 3, 4) with diaminododecane was first attempted using N-ethyl-N -dimethylamino-propyl- carbodiimide hydrochloride (EDC) in H 2 O according to Yamada and Hogenkamp, J. Biol Chem. 247. 6266-6270, 1972.
• EDC N-ethyl-N -dimethylamino-propyl- carbodiimide hydrochloride
• the products obtained did not have a reactive amino group.
• Alteration of the reaction conditions by changing the reaction rn ture to DMF H 2 O and adding NaCN/N-hydroxysuccinimide (see Example 4) to the reaction mixture gave the desired diaminododecane adducts.
• Cyanocobalamin monocarboxylic acid (2, 3, 4) (0.370 mmol, 500 mg) and N-hydroxysuccinimide (1.48 mmol, 170 mg) were dissolved in a mixture of DMF:H 2 O(l:l) (18.4 mL) and 363 mg of NaCN was added.
• 1,12-Diaminododecane was dissolved in a mixture of DMF : H 2 O (1: 1) (18.4 mL) and the pH was adjusted to 6 with 1 N HCl. The diaminododecane solution was then added in one portion to the cyanocobalamin solution.
• EDC (285 mg) was added and the pH ofthe solution was readjusted to 5.5.
• reaction mixture was then stirred overnight in the dark at room temperature. In five intervals of 6-14 h, 170 mg of N-hydroxysuccinimide and 285 mg of EDC were added to the solution, readjusting the pH value 5.5 each time. After a total reaction time of 4 days (reaction followed by HPLC), the solution was evaporated to dryness. The residue was digested with 100 mL of acetone and the solvent was decanted. The solid residue was dissolved in 50 mL of H 2 O and applied to an 200 g Amberlite XAD-2 (60 x 4 cm) column. The column was eluanted with 1
• Pr 3 CH 3 1.27 (m, 15H); 1.35 (br s, 9H); 1.42 (s, 3H); 1.53 ( , 2H); 1.6 (m, 4H);
• GABA GAMMA-AMINOBUTYRIC ACID
• GABA Gamma-aminobutyric acid
• cyanocobalamin monocarboxylates (2, 3, 4)
• EDC N-ethyl-N 1 - dimethylammopropylcarbodiimide hydrochloride
• a cyanocobalamin-GABA adduct (12) was purified. Reverse-phase HPLC chromatography is carried out as described above.
• a cyanocobalamin-GABA adduct (12) can be further activated with a carbodiimide and coupled to a moiety as described below.
• This example serves to demonstrate modification of an arnino terminus linking moiety to a carboxylate terminus. Such a modification may be necessary for conjugating amino containing rerouting agents (e.g., aminosugars) to cyanocobalamin derivatives containing a linker.
• amino containing rerouting agents e.g., aminosugars
• CYANOCOBALAMIN MODIFIED ON MONOCARBOXYLIC ACID DIAMINODODECANE-BIOTIN CONJUGATES
• Biotin conjugates (17, 18, 19) were obtained by reaction of activated cyanocobalamin monocarboxylic acid diaminododecane (14), (15), and (16) with the NHS ester of biotin (Sigma Chemical Co.).
• Streptomycin (21) is conjugated with cyanocobalamin monocarboxylate (2, 3, 4) or a diaminoalkylsuccinate derivative (14, 15, 16) through the use of an oxime coupled linking moiety (FIGURE 7).
• the linking group is conjugated with cyanocobalamin monocarboxylate (2, 3, 4) or a diaminoalkylsuccinate derivative (14, 15, 16) through the use of an oxime coupled linking moiety (FIGURE 7).
• COMPOUND (ACRIDINE) RECEPTOR MODULATING AGENT This example demonstrates the coupling of the vitamin B 12 to acridine.
• Chloroquine, quinacrine and acridine are lysosomotropic dyes that are relatively nontoxic and concentrated as much as several hundredfold in lysosomes.
• Acridine derivatives may be covalently attached to a targeting moiety (such as cyanocobalamin) by the reaction scheme illustrated in FIGURE 8, method A, or similarly as described in method B. Both reaction schemes produce a cyanocobalamin-acridine conjugate.
• MethodA A diamine side chain is first synthesized in a manner analogous to the side chain of quinacrine. Specifically, mono-phthaloyl protected 1,4- diaminobutane (27) is reacted with 6,9-dichloro-2-methoxyacridine (28) in phenol (J.
• Example 12 SYNTHESIS OF A CYANOCOBALAMIN LYSOSOMOTROPIC COMPOUND (AMIKACIN) RECEPTOR MODULATING AGENT
• a reaction scheme for the conjugation is depicted in FIGURE 6.
• chemical moieties that are retained subcellularly within lysosomes are termed lysosomotropic.
• Aminoglycosides are lysosomotropic compounds, and thus may be used as rerouting moieties of this invention.
• amikacin The primary long chain amine on the hydroxyaminobutyric acid side chain ofthe aminoglycoside, amikacin, is preferentially reactive. Specifically, amikacin (33) (Sigma Chemical Co., St. Louis), is reacted with a vitamin B 12 monocarboxylate (2, 3, 4) in the presence of EDC. A cyanocobalamin-amikacin conjugate (34) is then separated and purified by reverse-phase LC chromatography under conditions noted above.
• amikacin (33) Sigma Chemical Co., St. Louis
• a cyanocobalamin-amikacin conjugate (34) is then separated and purified by reverse-phase LC chromatography under conditions noted above.
• This example demonstrates the production of a cyanocobalamin dimer suitable for use as a cross-linking receptor modulating agent.
• Cross-linking of receptors in some receptor systems is sufficient to cause a rerouting of cell surface receptors to lysosomes for degradation, rather than their normal pathway of receptor recycling.
• b-acid dimer (36) yield 96 mg (30%), mp 217-220°C with decomposition, *H NMR (D 2 O, ⁇ ) 0.43 (s, 6H, C-20 CH 3 ); 1.18 (s, 8H); 1.3 (m, 36H); 1.37 (m, 12H); 1.46 (s, lOH); 1.6 (m, 8H); 1.9 (d, 12H); 2.05 (m, lOH); 2.2 (d, 16H, B10 & B11 CH 3 ); 2.35 (m, 8H); 2.6 (d, 18H); 2.8-3.0 (m, 16H); 3.15 (m, 6H); 3.3 (s, 8H); 3.37 (m, 14H); 3.6 (m, 4H); 3.76 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H); 4.12 (m, 2H); 4.18 (m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.6 (s, 2H);
• This example serves to illustrate synthesis of a bivalent receptor modulating agent using a heterotrifunctional cross-linker.
• the reaction scheme for this synthesis is depicted in FIGURE 9.
• the heterotrifunctional cross-linker is formed an ETAC reagent (Bioconiugate Chem. 1:36-50, 1990; Bioconjugate Chem. 1:51-59, 1990; J. Am. Chem. Soc. 101:3097-3110, 1979).
• Bivalency in addition to enhancing affinity of binding, also imparts the ability to cross-link neighboring receptors and trigger endocytosis.
• the bivalent "arms" ofthe agent may be lengthened with peptide or other linking molecules to enable simultaneous binding of both "arms". In the case of vitamin B 2 this may be assessed by gel filtration.
• carboxy-ETAC (39) is prepared by the method of Liberatore et al. (Bioconjugate Chem. 1:1990).
• the carboxy-ETAC is converted to its acid chloride by reaction in thionyl chloride.
• Addition of amine (40) gives the amine-ETAC adduct (41).
• Reaction of amine-ETAC (1 mmol) in CH 3 CN with 1 M aqueous cysteamine (10 mmol) is conducted by stirring at room temperature for 24 h. This compound is reduced with NaCNBH 3 under acidic conditions.
• the crude arnine- ETAC-cysteamine adduct (42) is purified by reverse-phase LC, using conditions noted above.
• a vitamin B J2 monocarboxylate (2, 3, 4) is conjugated with tyramine- ETAC-cystearnine compound by reaction with EDC in H O.
• the resultant vitamin B 12 -ETAC-tyramine dimer (43) is purified by reverse-phase LC, using conditions described above.
• Reaction Step D TFP ester of biotin-caproic acid (48) (0.67 mmol, 0.35 g) was dissolved in DMF (40 mL). Triethylamine (80 ⁇ L) was added followed by aminoisophthalic acid (1.005 mmol, 0.182 g). The reaction was stirred at room temp, for 8 days (HPLC monitored) while adding triethylamine (80 ⁇ L) every after 24 h. It was then evaporated to dryness. The residue was then applied to a column of silica and was initially eluanted with acetonitrile (450 mL).
• Reaction Step E Biotin-caproic acid-isophthalic acid (49) (0.376 mmol, 200 mg) was dissolved in DMF (30 mL) under argon atmosphere. TFP acetate (0.94 mmol, 241 mg) was added by double-ended needle, followed by triethylamine (112 ⁇ L). The reaction was then stirred at room temp, for 24 h (HPLC monitored). It was then evaporated to dryness. The light brownish oil was taken in ether, solid was filtered and was washed with ether (50 mL) (50) to yield 250 mg (86%).
• Reaction Step F In a solution of cyanocobalamin carboxylic acid - diaminododecane conjugate (8, 9, 10) (0.130 mmol, 0.2 g) in a mixture of DMF:H O (3:1) (40 mL) triethylamine (12 ⁇ L) was added. DiTFP ester of biotin-caproic acid- isophthalic acid (50) (0.065 mmol, 0.050 g) was added over a period of 5-10 min. The reaction mixture was stirred at room temperature for 3 h (HPLC monitored). It was then evaporated to dryness. The residue was digested with 100 mL of acetone and the solvent was decanted to yield 230 mg (62%) (51) mp 195-198°C with decomposition.
• CYANOCOBALAMIN MONOCARBOXYLIC ACID DIAMINODODECANE CONJUGATE D ⁇ MER ISOPHTHALATE CROSS-LINKING WITH PARA-IODOBENZOYL MODETY
• Reaction Step A A 5g (28 mmol) quantity of 5-aminoisophthalic acid (52) was dissolved in 30 mL IN NaOH and placed in an ice/water bath. To the cold solution was added 7.5g (28 mmol) 4-iodobenzoyl chloride (52) in 60 mL of acetonitrile, dropwise.
• the thick white precipitate was then stirred for 10 minutes before removing the ice/water bath and allowing the mixture to stir an additional 10 minutes.
• the reaction mbcture was adjusted to pH 4 with acetic acid and the resulting solid collected. This solid was then dissolved in 30 mL IN NaOH and washed with ether (2 x 50 mL). The resulting aqueous solution was filtered and acidified to pH 4 with acetic acid. The white precipitate was then collected and dried on high vacuum to yield .6 g (99+% ) of (54).
• Reaction Step B A 5g (12.2 mmol) quantity of 5-[N-(p-iodobenzoyl)amino]- isophthalic acid (54) was suspended in 100 mL anhydrous ethyl acetate. To this was added 12.5g (73 mmol) 2,3,5,6-tetrafluorophenol (55) followed by 5g (24.2 mmol) 1,3-dicyclohexylcarbodiimide. This suspension was then stirred at room temperature for 3 days before filtering off the solid and washing with an additional 20 mL of ethyl acetate. The filtrate was then evaporated to dryness. The resulting sticky white solid was suspended in 50 mL acetonitrile and stirred for 30 minutes.
• Reaction Step C To a solution of cyanocobalamin carboxylic acid- diaminododecane conjugate (56) (0.192 mmol, 0.3 g) in a mixture of DMF:H 2 O (3:1) (40 mL) was added triethylamine (0.018 mL). To this solution, DiTFP ester of 5-[N- (p-iodobenzoyl)amino]-isophthalic acid (57) (0.096 mmol, 0.068 g) was added over a period of 5-10 min. The reaction mixture was stirred at room temperature for 4-5 h (HPLC monitored). It was then evaporated to dryness.
• Reaction Step A A 2 g (2.8 mmol) quantity of the diTFP ester of 5-[N-(p- iodobenzoyl)amino]-isophthalic acid (57) (as prepared above) was dissolved in 20 mL dry toluene under argon. To this was added 2.8 mL ( 5.5 mmol) of ⁇ i tributyltin) (61) followed by 40 mg (0.04 mmol) tetrakis(triphenylphosphine)palladium (62). The rnixture was stirred at room temperature for 15 minutes before heating to 80 C for 2 h. Since the mixture only darkened slightly over the 2 h period, an additional 40 mg of palladium catalyst was added.
• Reaction Step B In a solution of cyanocobalamin carboxylic acid- diaminododecane conjugate (8, 9, 10) (0.065 mmol, 0.1 g) in a mixture of DMF:H 2 O (3:1) (40 mL) triethylamine (0.006 mL) was added. DiTFP ester of 5-[N-(p- tributyltin benzoyl) aminoj-isophthalic acid (63) (0.0325 mmol, 0.028 g) was added over a period of 5-10 min.
• Example 18 EVALUATION OF THE ABILITY OF VITAMIN B 12 RECEPTOR MODULATING AGENTS TO BIND TO TC ⁇
• This example serves to demonstrate a competitive binding assay suitable for evaluating the ability of vitamin B 12 receptor modulating agents to bind TcII. Binding of the vitamin B J2 derivatives to recombinant transcobalamin II was conducted in picomolar concentrations and the percent bound ascertained.
• B 12 receptor modulating agents were evaluated for their ability to bind to TcII relative to radiolabeled B 12 .
• Varying concentrations of each derivative were incubated with immobilized TcII in the presence of a constant amount of radiolabeled B J2 . After incubation for 20 minutes at 37°C, the free radiolabeled B 12 was separated from the
• TcII bound tracer by removal ofthe supernatant.
• the radioactivity ofthe supernatant solution was then measured to determine the amount of free radiolabeled B 12 present at the end of each competition.
• the ability of each derivative to inhibit radiolabeled B J2 binding was determined.
• a binding curve was then constructed for each B 12 derivative where the amount of radiolabeled B 12 bound (% radiolabel bound) was correlated with the concentration of derivative present in the original mixture. The more effective the derivative is in binding to TcII, the lower the percent bound radiolabeled vitamin B J2 .
• FIGURE 15 illustrates the binding curve of transcobalamin II to the cyanocobalamin monocarboxylic acids produced in Example 1.
• AD cyanocobalamin (1)
• AL cyanocobalamin _»-monocarboxylic acid (2)
• AM cyanocobalamin e-monocarboxylic acid (3)
• AN cyanocobalamin d- monocarboxylic acid (4).
• the rf-carboxylate (3) appears to bind nearly as well as cyanocobalamin.
• Two samples of vitamin B J2 were used, one as a known standard and the other as an unknown.
• FIGURE 16 illustrates the binding curve of transcobalamin II to the cyanocobalamin diaminododecane adducts (8, 9, 10) and succinate adduct (13) produced in Examples 3 and 4 above.
• AH cyanocobalamin _>-monocarboxylic acid conjugate diaminododecane (7)
• AI cyanocobalamin e-monocarboxylic acid conjugate diaminododecane (8)
• AJ cyanocobalamin ⁇ /-monocarboxylic acid conj diaminododecane (9);
• AK cobalamin e-monocarboxylic acid conj diaminododecane
• AE cyanocobalamin ribose-succinate (11).
• the i-conjugate (17) has the least binding, whereas the ⁇ -conjugate (18) has intermediate binding, and the --conjugate (19) binds quite well
• the biotin conjugate attached to the ribose site (13) appears to bind very well, as does its precursor amino derivative (12).
• the additional compound studied is of unknown structure, but may have the amine group coordinated with the cobalt atom as the mass spectrum indicates that it has the appropriate mass for (7) minus HCN. It is clear that this unknown compound is not likely to bind TcII.
• FIGURE 17 illustrates the binding curve of transcobalamin II to a series of vitamin B J2 dimers.
• Dimer X _»-acid dimer with isophthaloyl dichloride (36);
• dimer Y e-acid dimer with isophthaloyl dichloride (37);
• dimer Z d-acid dimer with isophthaloyl dichloride (38);
• dimer A 6-acid dimer with />-iodo benzoyl isophthaloyl dichloride (58);
• dimer B e-acid dimer with »-Iodo benzoyl Isophthaloyl dichloride (59); and
• dimer C d-acid dimer with/?-iodo benzoyl isophthaloyl dichloride (60).
• FIGURE 18 illustrates the binding curve of Transcobalamin II to a series of biotinylated vitamin B 12 molecules.
• AA cyanocobalamin _>-monocarboxylic acid conjugate diaminododecane and Biotin (17);
• AB cyanocobalamin e-monocarboxylic acid conjugate diaminododecane and biotin (18);
• AC cyanocobalamin --monocarboxylic acid conjugate diaminododecane and Biotin (19);
• AF cyanocobalamin ribose-succinate conjugate diaminododecane (13); and
• AG cyanocobalamin ribose-succinate conjugate diaminododecane and biotin (20).
• This example serves to demonstrate the use of an assay to ascertain biological activity ofthe receptor modulating agents ofthe present invention.
• Receptor down-modulation involves a comparison of treatment of a target cell line such as K562; each sample is treated with vitamin B 12 or a vitamin B 12 receptor modulating agent at 4°C for 24 hours. Following this period, cells of each sample are separated from a vitamin B 12 or a vitamin B 12 receptor modulating agent by centrifugation. The cells are then washed and resuspended in phosphate buffered saline containing 2 mM EDTA for a brief period of time not to exceed 15 minutes at 4°C. Then, the cells are washed again and returned to a tissue culture medium at 4°C. The tissue culture medium contains TcII and a radiolabeled TcII/B 12 complex.
• the time course of TcH/B 12 binding to the cell receptor is determined by measuring the percent radiolabel bound to the cell at 0, 15, 30, 60, 120, and 240 minutes. Those samples exposed to the vitamin B J2 receptor modulating agents of the present invention show significantly reduced TcH/B 12 complex binding compared to cells cultured in vitamin B 12 . Trypsin treated cells reveal any nonspecific binding or uptake ofthe labeled vitamin B 12 on or within the cell.
• This example serves to demonstrate a method suitable for assessing the biological activity of a receptor modulating agent ofthe present invention.
• 0.2x10 cells/ml K562 cells were cultured in RPMI medium modified by addition of 10 ⁇ M MeTHF, 2.7 nM vitamin B 12 and 1% human serum. No folate was added. 10 ⁇ M /-diamimododecane adduct (7) was added and cultured over 9 days at
• the receptor modulating agent in this case rf-diaminododecane adduct (7), clearly demonstrates the marked biological activity ofthe receptor modulating agent.
• the synthetic peptide f-met-leu-phe is equivalent to a bacterial cell wall constituent (Biochem. Soc. Trans. 19:1127-9, 1991; Agents Actions Suppl 35:3-8,
• the peptide f-met-leu-phe-(gly) 3 -leu-O-Me is synthesized using tea-bag methodology or solid phase peptide synthesis procedures described by Merrifield et al. (Biochemistry 21:5020-31, 1982) and Houghten (Proc. Nat'l Acad. Sci. (USAi 82:5131-35, 1985), or using a commercially available automated synthesizer, such as the Applied Biosystems 430 A peptide synthesizer.
• the peptide-amide is deprotected in 45% trifluoroacetic acid-51% methylene chloride-2% ethanedithiol-2% anisole for 20 minutes, and cleaved from the 4-methylbenzhydrylamine resin using the Tam- Merrifield low-high HF procedure (J. P. Tarn et al., J. Am. Chem. Soc. 105:6442-55, 1983).
• the peptide is then extracted from the resin using 0.1 M ammonium acetate buffer, pH 8, and is lyophilized.
• the crude peptide is purified using reverse-phase HPLC on a Vydac C-4 analytical column (The Separations Group, Hesperia, Calif), and a linear gradient of 0.5-1.0%/min.
• HPLC-purified peptide is analyzed by amino acid analysis (R L Heinriksen and S.C. Meredith, Anal. Biochem. 160:65-74, 1984) after gas phase hydrolysis (N.M. Meltzer et al., Anal Biochem. 160:356-61, 1987).
• the sequence ofthe purified peptide may be confirmed by Edman degradation on a commercially available sequencer (RM. Hewick et al., J. Biol Chem. 15:7990-8005, 1981).
• the peptide amide is converted to an O-methyl ester (i.e., f-met-leu-phe-(gly) -leu-O-Me) by treatment with dimethylformamide (5g/60 mL with 1.3 equivalents of NaHCO 3 in excess methyl iodide (4 equivalents). The mbcture is stirred under argon gas at room temperature for 40 hours. If required, the peptide is extracted to dryness with 150 mL of ethyl acetate.
• the receptor for modulating agent is used to treat PMN, activated with GM-CSF (to increase expression of fMLP receptors). Loss of binding of biotinylated fMLP is compared on fMLP versus f-MLP receptor modulating agent treated cells.
• Example 22 SYNTHESIS OF A FUSION PROTEIN RECEPTOR MODULATING AGENT
• An EGF receptor modulating agent containing a genetically engineered fusion protein is hereby described. Briefly, the C-terminus of a DNA sequence encoding EGF, or its receptor binding domain, is ligated by conventional procedures (e.g., using T 4 DNA ligase) to a DNA sequence corresponding to a GGG spacer. The C-terminus of the EGF-GGG DNA sequence is then fused to the N-terminus of a DNA sequence encoding the conditional, membrane binding peptide KGEAALA(EALA)4-EALEALAA.
• peptide-spacer DNA sequences may be synthesized in vitro using standard oligonucleotide synthesis procedures (see, e.g., U.S. Patent Numbers 4,500,707 and 4,668,777).
• the recombinant EGF peptide DNA sequence is cloned in an E. coli expression vector using conventional procedures.
• E. coli strain HB101 is transformed with the fused recombinant DNA sequence and cultured to produce the EGF peptide.
• the fusion protein is purified form the transformed E. coli culture by standard methods, including anti-EGF affinity chromatography.
• the fusion protein may be eluanted from the affinity matrix using standard techniques, such as high salt, chaotropic agents, or high or low pH. Loss of EGF receptor is measured by flow cytometry and mouse monoclonal antibody to EGF receptor.
• Example 23 SYNTHESIS OF A VITAMIN B ⁇ DERIVATIVE HAVING A ATER-SOLUBILIZING LINKER
• the preparation of a vitamin B ⁇ derivative having a water-solubilizing linker is described. Briefly, the example describes a procedure for the reaction of a cyanocobalamin monoocarboxylic acid with 4,7,10-trioxa-l,13-tridecane diamine. The results for the b- and e-monoacids of cyanocobalamin are described. The reaction product for the e-isomer is shown below.
• Cyanocobalamin monocarboxylic acid (1.472 mmol, 2 g) and N- hyd ⁇ oxysuccinimide (680 mg) were dissolved in water (100 mL) and 1.456 g of sodium cyanide was added.
• 4,7,10-Trioxa-l,13-tridecanediamine (36 mmol, 16 g) was then added and the pH was adjusted to 6 with 1 N HCl.
• N-ethyl-N 1 - dimethylamino-propyl-carbodiimide-hydrochloride (EDC) (1.136 g) was added and the pH of the solution was readjusted to 5.5. The reaction mixture was then stirred overnight in the dark at room temperature.
• Example 24 SYNTHESIS OF A VITAMIN B ⁇ /BIOTIN CONJUGATE HAVING A WATER-SOLUBILIZING LINKER
• the preparation of a vitamin Bu/biotin conjugate having a water-soluble linker is described. Briefly, the vitamin B ⁇ 2 derivative having a water-solubilizing linker prepared as described in Example 23 is treated with an NHS-ester of biotin. The results for the b- and e-isomers of cobalamin are described. The reaction product for the e-isomer is shown below.
• the preparation of two vitamin B ]2 dimers having water-solubilizing linkers is described. Briefly, the dimers are prepared by coupling the vitamin B ]2 derivative of Example 23 with either a bifunctional crosslinker or a trifunctional crosslinker.
• a RAININ Rabbit-plus peristaltic pumping system was used with a DYNAMAX (model UV-1) UV visible absorbance detector; the eluant was collected with an automatic fraction collector. The fractions containing the final product (HPLC monitored) were evaporated to dryness.
• Cyanocobalamin - methylacetate derivative Cyanocobalamin (0.15 mmol, 200 mg) was dissolved in 40 mL of DMSO containing 10 g (65 mmol) of methyl bromoacetate and 6.4 mL of pyridine. After 14-16 h at 50- 55°C, 500 mL of water was added and the pH ofthe reaction mbcture was adjusted to 6 with 10% KOH.

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