WO1996021440A9 - Methods for increasing the bioavailability of biological active agents - Google Patents

Methods for increasing the bioavailability of biological active agents

Info

Publication number
WO1996021440A9
WO1996021440A9 PCT/US1995/016161 US9516161W WO9621440A9 WO 1996021440 A9 WO1996021440 A9 WO 1996021440A9 US 9516161 W US9516161 W US 9516161W WO 9621440 A9 WO9621440 A9 WO 9621440A9
Authority
WO
WIPO (PCT)
Prior art keywords
compound
inositol
phosphate
groups
therapeutic
Prior art date
Application number
PCT/US1995/016161
Other languages
French (fr)
Other versions
WO1996021440A1 (en
Filing date
Publication date
Application filed filed Critical
Priority to AU45159/96A priority Critical patent/AU4515996A/en
Publication of WO1996021440A1 publication Critical patent/WO1996021440A1/en
Publication of WO1996021440A9 publication Critical patent/WO1996021440A9/en

Links

Definitions

  • This Invention relates to methods for enhancing the bioavailability of nonpeptide biologically active molecules.
  • a potential medicinal agent may exhibit desirable in vitro and in vivo biological activity, yet not be therapeutically useful because of low absorption, poor solubility, or inability to reach the appropriate target site in a concentration sufficient for effectiveness. Poor bioavailability may result from one or more of such characteristics.
  • Flavonoids are an example of compounds with desirable biological activities whose use as therapeutic agents is limited by poor bioavailability. Flavonoids are flavone (2-phenylchrome; 2- phenyl- -benzopyrene; 2-phenyl-l, 4-benzopyron) derivatives constituting a class of naturally occurring molecules isolated from various plants.
  • flavonoids include apigenin (4', 5-7-trihydroxyflavone) ; genistein (4', 5, 7, trihydroxy-isoflavone) ; guercetin (3, 3', 4 ' , 5-7 pentahydroxy-flavone) ; rutin (guercetin-3-rutinoside) ; conjugated forms such as guercetin pentaacetate, and guercetin 3-D-galactoside; and phenolic and lipophilic poly ethoxylated flavonoids, such as nobiletin and tangeretin (Kandaswami et. al (1991) Cancer Lett. 56:147-52). Examples of naturally occurring flavonoids are shown in Figure 1.
  • flavonoids kaempferol and luteolin also blocked the increase in teleocidin-induced epidermal ornithine decarboxylase (ODC) , although in that model quercetin was reported to have an insignificant effect (Fujiki (1986) Prog. Clin. Biol. Res. 213:429-440).
  • Other flavonoids such as morin (2', 3, 4 ' , 5,7- pentahydroxyflavone) , fisetin, and kaempferol reduced significantly the increase in epidermal ODC activity in mice induced by TPA treatment (Nakadate et al. (1984) Gann 74:214-222).
  • the isoflavonoid genistein is a potent, and specific inhibitor of tryosine protein kinases (Akiyama et al. (1987) J. Biol. Chem. 262; 5592-5595.
  • This in vivo study confirmed previous data in cell culture which demonstrated the inhibitory effects of other flavonoids such as guercetin, on the proliferation of human leukemic cells in culture, including human leukemic cell line K562 (Hoffman et al. (1988) Brit. J. Cancer 59: 347-348); acute yeloid and lymphoid leukemia progenitors (Larocca et al. (1991) Brit. J. Haematol.
  • Flavonoids are relatively insoluble in water. Pharmokinetic studies in humans suggest that less than 1% of orally administered quercetin (4 grams) was absorbed unchanged (Gugler et al. (1975) Eur. J. Clin. Pharmacol. 9:229).
  • camptothecin into a water soluble form (1 mg/ml H20) by first modifying the molecule through the attachment of a hydroxyl group at position 9 or 10
  • the invention features methods for enhancing the bioavailability of therapeutic compounds.
  • a conjugate is formed by covalently attaching to the agent a chemical moiety which is highly bioavailable, resulting in increased bioavailability of the agent.
  • a linker element As shown in our examples given below we were capable of conjugating the native, unmodified hydroxyl groups of therapeutic agents to moieties via a linker element, a process yielding molecules of high water solubility ( ⁇ 100mg/ml H20 vs. Img/ml H20 for the alkylated camptothecin) , and retaining full biologic activity.
  • agent or “potential medicinal agent” is meant a compound having desirable in vitro and/or in vivo biological activity in experimental animals which could potentially be used in the treatment of a disease or malignancy.
  • the agent is a flavonoid or flavonoid derivative.
  • the agent is a nonflavonoid.
  • bioavailability is meant the extent at which an agent is water soluble and therefore injectable, or capable of trespassing the intestinal wall so that it can be absorbed into a living system or is made available at the site of physiological action.
  • An increase in bioavailability means that a larger amount of the administered agent reaches a relevant target site.
  • the chemical moiety is meant a compound possessing high bioavailability and which confers enhanced bioavailibity to an agent as a conjugate.
  • the chemical moiety to be covalently attached to the agent will vary depending on the specific agent used.
  • the chemical moiety increased bioavailabity to an agent is inositol phosphate.
  • inositol phosphate is meant inositol having one or more phosphate groups, including mono- phosphorylated inositol, di-phosphorylated inositol, or poly-phosphorylated inositol.
  • the invention may also be practiced with other carbohydrates, phosphorylated carbohydrates or other chemical groups, including phosphate group(s) , sulfate group(s) , carboxyl group(s) , acyl groups(s) , a ino acids, peptides, proteins, glutathione, glyceraldehyde or derivatives, nucleoside, nucleotides, polyols, phosphonates, including bisphosphonates, aminoalkyl molecules, and/or polyamines, or combinations of these groups.
  • phosphate group(s) phosphate group(s) , sulfate group(s) , carboxyl group(s) , acyl groups(s) , a ino acids, peptides, proteins, glutathione, glyceraldehyde or derivatives, nucleoside, nucleotides, polyols, phosphonates, including bisphosphonates, aminoalkyl molecules, and/or polyamines
  • conjugate is meant a synthetic chimeric molecule formed by a covalent bond or bonds between a chemical moiety and a potential medicinal agent, or between a chemical moiety, a linking element, and the agent molecule.
  • the potential medicinal agent possesses enhanced bioavailability relative to the unconjugated agent.
  • the increased bioavailability of an agent results from an increase in its solubility in a biological fluid. Solubility of the agent in the desired medium results from modifying the polarity of the agent. Accordingly, in one aspect, the bioavailability of an agent is increased by covalent attachment of a chemical moiety that alters the polarity of the agent, increasing its solubility in the desired medium.
  • a third method of increasing the bioavailability of an agent is to effectively increase the concentration of the agent at the target cell.
  • the bioavailability of an agent is increased by covalent binding to a chemical moiety which binds a specific target cell.
  • the resulting conjugate binds the target cell, increasing the concentration of the agent at the target cell.
  • the chemical moiety is actively transported into the target cell.
  • the resulting conjugate is transported into the target cell, resulting in delivery of the bound agent to the target cell.
  • An example of a chemical moiety actively transported into a target cell is inositol phosphate, which is transported into cells having an active inositol phosphate transport system.
  • the conjugate may be formed by direct covalent attachment to a chemical moiety to the agent, or by indirect attachment to a chemical moiety via a linking element.
  • linking element is meant a chemical element connecting the agent and chemical moiety.
  • the linking element is a carbon chain having between l to 5 carbons in length and having one or more reactive moieties such that other groups may be attached to the linking element.
  • the linking element may increase the aqueous or lipid solubility of the agent.
  • the linking element may function to enhance the bioavailability of the agent or may be utilized to facilitate conjugate synthesis.
  • the linking element is succinate.
  • the linking element connects multiple agent molecules to a cell targeting chemical moiety, this increasing the effective concentration of the agent at the target site.
  • the invention encompasses various methods for attachment of the chemical moiety to the potential medicinal agent.
  • a conjugate is formed by attachment of inositol phosphate to a flavonoid molecule.
  • the conjugate is formed by attachment of inositol phosphate to a flavonoid through the phosphate group of the inositol (for example, see Fig. 3A) .
  • the molecules are coupled via a linking element through the phosphate linkage (for example, see Fig. 3B) .
  • the molecules are coupled via a linking element at a position on the inositol other than the phosphorylated position (for examples, see Figs. 3C and 3D) .
  • the invention features flavonoid-inositol phosphate conjugates where the conjugated flavonoid has enhanced bioavailability relative to the unconjugated flavonoid, resulting from improved water-solubility and/or cellular delivery.
  • Inositol phosphate may be conjugated to flavonoids through a covalent bond or series of covalent bonds between inositol phosphate and one or more hydroxyl and/or amino residues of the flavonoid.
  • the flavonoids useful in this invention may be naturally occurring or synthetic.
  • the therapeutic agent is covalently bound to a phosphonate, and preferably to a bisphosphonate, through a linking element.
  • Said bisphosphonate may include, but is not limited to, alendronate, pamidronate, etidronate, clodromate, tiludronate, ibandronate or residronate ( Figure 4 ) . Additional examples of bisphosphonate structures are given in Shinoda et al. (1983) Calcified Tissue International, Springer Verlag, 35: 87-99.
  • Bisphosphonates have been used in the treatment of several clinical conditions, namely ectopic calcification, ectopic bone formation, Paget's disease, osteoporosis and increased osteolysis of malignant origin (Dunn et al. (1994) Drugs and Aging 5: 446-474; Licata
  • Bisphosphonates may be conjugated to a flavonoid or to a nonpeptide nonflavonoid therapeutic agent having one or more hydroxyl groups and/or one or more amino groups.
  • the bisphosphonates and the therapeutic compounds are coupled via a linking element at a position on the bisphosphonate other than the phosphorylated one(s) .
  • Conjugation of therapeutic agents of low water solubility to bisphosphonates may increase their bioavailability by increasing both their water solubility and their localized delivery to bone. This is desirable in the treatment of osteosarcomas when the agent is cytotoxic for human malignant osteosarcoma cells.
  • apigenin, quercetin, and camptothecin exhibit strong antiproliferative effects on primary human osteosarcoma cells in culture.
  • Agents conjugated to bisphosphonate which are capable of inhibiting tyrosine protein kinases, such as flavonoids (Hagiwaza ewt. al. (1977) ibid; Grazian et. al. (1983) ibid; Akiyama et. al.
  • osteoporotic lesions may be delivered by the bisphosphonates to osteoporotic lesions in the bones of human patients with osteoporosis. Inhibition of tyrosine kinase activity in osteoclasts at the site of osteoporotic lesions may result in a decrease activity of bone resorption which is promoted by osteoclasts. Osteoclast proliferation has been shown to be sensitive to tyrosine kinase activity C. Hall et al. (1994) Bioch. Bioph. Res. Commun. , 199: 1237-1244; Yoneda et al. (1993) J. Clin. Invest., 91: 2791-2795; Lowe et al. (1993) Proc. Nat'l. Acad. Sci. USA, 90: 4485-4489.
  • CD19-receptor of the leukemic cells may be used to increase the water solubility and site delivery of other non-flavonoid compounds.
  • the solubility of a compound in water may be increased by formation of a conjugate between a chemical moiety and a non-flavonoid compound having one or more hydroxyl groups and/or one or more amino groups.
  • the conjugate of the invention may be administered to a patient in a number of ways known to the art, including, intravenous, parenteral, intranasal, oral, topical, transdermal, and subcutaneous sustained release injectable implant formulations.
  • Formulations may be prepared by any of the techniques known in the pharmaceutical arts. Such techniques are described, for example in Remington's Pharmaceutical Sciences ((1980)
  • Formulations for parenteral administration may contain common excipient such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated napthalenes, and others of similar nature.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated napthalenes, and others of similar nature.
  • biocompatible, biodegradable lactate polymers, lactate/glycoside copolymers, or polyoxethylene-polyoxy-propylene copolymers may be useful excipient to control the release of the conjugate of the invention.
  • Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for administration by inhalation may contain excipient such as, for example, lactose.
  • Inhalation formulas may be aqueous solutions containing excipient such as, for example, polyoxyethylene 9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasal.
  • Compositions for parenteral administration may also include glycocholate for buccal administration, or cikic acid for vaginal administration.
  • Topical administration may utilize slow releasing patches.
  • Fig. 2 provides examples of the chemical structures of active synthetic flavonoids.
  • Fig. 3 is a diagram illustrating four methods for the synthetic coupling of inositol phosphate to a flavonoid derivative.
  • Fig. 3A shows coupling of the components through the phosphate group.
  • Fig. 3B shows coupling via a linking element through the phosphate linkage.
  • Figs. 3C and 3D show coupling via a linking element at a position on the inositol other than the phosphorylated one(s)..
  • Fig. 4 contains examples of bisphosphonates.
  • Fig. 5 and Fig. 6 are synthetic pathways for preparation of a phosphoinositol-succinate linked quercetin.
  • Fig. 7 is a synthetic pathway for preparation of a phosphoinositol-succinate linked apigenin.
  • Fig. 8 is a synthetic pathway for preparation of a phosphoinositol-succinate linked genistein.
  • Fig. 9 is a synthetic pathway for preparation of an inositol phosphate linked regioselectively to quercetin via a glycerol linker.
  • Fig. 10 is a synthetic pathway for preparation of a phosphoinositol quercetin linked through the phosphate group.
  • Fig. 11 Antiproliferative activity of quercetin and phosphoinositol-succinate linked quercetin on a primary human adenocarcinoma cell line (SW 480) in culture.
  • the biologic activity of the water- soluble conjugated quercetin (6c) is similar to that of unconjugated quercetin.
  • the unconjugated quercetin was dissolved in dimethylsulfoxide (DMSO) .
  • DMSO dimethylsulfoxide
  • Fig. 12 Antiproliferative activity of quercetin and phosphoinositol-succinate linked quercetin on a primary human osteosarcoma cell line (MG 63) in culture.
  • the biologic activity of the water-soluble conjugated quercetin (6c) is similar to that of unconjugated quercetin.
  • Fig.13 A bisphosphonate-coupled flavonoid.
  • the invention features methods for increasing the bioavailability of a potential medicinal agent such that the agent becomes therapeutically useful.
  • the methods of the invention are particularly useful with a biologically active agent whose therapeutic use is limited by low bioavailability.
  • the invention may be used with any agent for which increased bioavailability is desired.
  • the bioavailability of any agent may be low due to a variety of factors, including low solubility and/or low rate of transport to its target cell.
  • the bioavailability of an agent may be low due to its inability to reach an effective concentration at the site of the target cell.
  • the invention provides methods for overcoming these problems, thus increasing the medicinal usefulness of a variety of biologically active agents.
  • flavonoid derivatives have antiproliferative effects on malignant cells in vitro and in experimental animals in vivo. Their therapeutic use is precluded, however, by their poor water solubility.
  • the present invention provides for increasing the water solubility of the therapeutic agents.
  • the methods of the invention may also be used to increase the cellular delivery of any agent by allowing the agent to be actively transported into cells by the cell membrane inositol phosphate transport system. (See, Saltiel and Sorbara-Cazan (1987) Biochem. Biophys. Res. Commun. 149:1084-1092).
  • Bisphosphonate-linked flavonoid and nonflavonoid therapeutic compounds may be delivered to bone lesions of patients with osteosarcomas or osteoporosis.
  • the methods of the invention are preferably used with agents having biological activates which would be useful in the treatment of diseases, such as the antiiproliferative effect of flavonoid in tumors, leukemia, and non alignant proliferative disorders such as psoriasis.
  • flavonoid is meant any flavone molecule containing one or more hydroxyl groups and/or one or more amino groups.
  • a linking element at a position on the inositol other than the phosphorylated one(s) (for examples, see Figs. 3C and 3D) .
  • Each synthetic method of the invention may be varied in the position(s) of attachment to the inositol and the flavonoid, in the number and position of phosphate groups, in the length and chemical nature of the linking unit, in the flavonoid derivative coupled, and in the inositol isomer used (e.g., yo- or chiro-, etc.).
  • Linking elements include ⁇ uccinate diester, 4-hydroxybutyrate ester phosphate diester, and glycerol diester.
  • flavonoids are conjugated to inositol-2 phosphate via a succinate linker.
  • suitable linkers include alkyl chains, mono or poly alcohol units, mono- or polyether units, thioethers, mono- or polyester units, mono- or polyamine units, mono- or polysulfates, mono- or polyarenes, mono- or polyphosphates, mono- or polyamides, peptides, mono- or polysulfides, mono- or polysaccharide, or a combination of these structures.
  • the invention is shown herein in what is considered to be the most practical and preferred embodiments.
  • Example l Preparation of (quercetin-5-yl) (myo-inositol-2- phosphate-1-yl) succinate.
  • Compound 6c (quercetin-5-yl) (myo-inositol-2-phosphate-l-yl) succinate) may be prepared as illustrated in Figs. 5 and 6. 3,4,5,6-Tetra-benzyl-l-(1,1,1,-trichloroethylchloro- formyl)-Myo inositol.
  • reaction mixture was then cooled to -42-C (MeCN/C02) and 2.1 ml of 1 M 85% MCPBA in CH2C12 was added. The ice bath was immediately removed and the reaction mixture allowed to warm to room temperature. After ten minutes, 5 ml of 0.1 M NH4 HC03 buffer (Ph 7.87) and 5 ml of 10% Na2S204 were added. The crude reaction mixture was extracted with
  • p-methoxybenzylated diol 7a may be elaborated by a method analogous to that used in Example 1, to produce the acid chloride 7d.
  • p-Methoxybenzylation of apigenin produces flavonoid 7e, which may be coupled with 7a in analogy to Example 1, and finally deprotected oxidatively to produce the title compound 7g.
  • Example 3
  • An inositol phosphate linked to genestein via a succinate linker may be prepared according to Figure 8.
  • p-Methoxybenzylation of genestein may produce flavonoid
  • An inositol-phosphate-quercetin derivatives may be prepared as shown in Fig. 10. Generally, 3,4,5,6-tetrabenzyl myo-inositol is treated with benzyl bis(disopropylamino-phosphoramidite followed by a peracid to produce the 1,2-cyclic phosphate. Subsequently, the cyclic phosphate is coupled with quercetin (or a protected quercetin) , which after catalytic hydrogenation produces the desired compounds.
  • Example 5 1-(myo-inositol-2-phosphate-l-y)-2-(guercetin-5yl) glycerol.
  • Fig. 9 illustrates a synthetic pathway to a coupled quercetin with a glycerol linker.
  • 3,4,5,6- tetrabenzyl myo-inositol is treated with allyl bromide, then phosphorylated.
  • the peracid generates concomitantly an epoxide at the allyl group.
  • Subsequent treatment with a protected quercetin, followed by deprote ⁇ tion provides the product.
  • Biologic Activity of Water-Soluble Conjugated Flavonoids was determined in vitro in cultures of a primary human colon adenocarcino a cell line (SW 480) , and a human osteosarcoma cell line (MG-63) .
  • SW 480 primary human colon adenocarcino a cell line
  • MG-63 human osteosarcoma cell line
  • the malignant cells were plated in 24-well culture plates, and their population was measured after 6 or 9 days of culture in the presence or absence of the test material.
  • the unconjugated flavonoid was dissolved in dimethylsulfoxide (DMSO) and then diluted to the desired concentration with the culture media.
  • DMSO dimethylsulfoxide
  • the water-soluble conjugated flavonoid was dissolved in a minimum volume of distilled water and diluted to the desired concentration with the culture media.

Abstract

Methods for increasing the bioavailability of a potential nonpeptide medicinal agent by covalent attachment to a chemical moiety resulting in formation of a conjugate having increased bioavailability and water solubility relative to the unconjugated agent. Medicinal agents include biologically active flavonoid and nonflavonoid compounds whose water solubility and bioavailability is increased through covalent attachment to moieties that include inositol phosphate and bisphosphonate molecules.

Description

METHODS FOR INCREASING THE BIOAVAILABILITY OF BIOLOGICAL ACTIVE AGENTS
Background of the Invention
This Invention relates to methods for enhancing the bioavailability of nonpeptide biologically active molecules.
Many potential medicinal agents are not therapeutically useful because of their poor bioavailability. A potential medicinal agent may exhibit desirable in vitro and in vivo biological activity, yet not be therapeutically useful because of low absorption, poor solubility, or inability to reach the appropriate target site in a concentration sufficient for effectiveness. Poor bioavailability may result from one or more of such characteristics.
Flavonoids are an example of compounds with desirable biological activities whose use as therapeutic agents is limited by poor bioavailability. Flavonoids are flavone (2-phenylchrome; 2- phenyl- -benzopyrene; 2-phenyl-l, 4-benzopyron) derivatives constituting a class of naturally occurring molecules isolated from various plants. Some of the naturally occurring flavonoids include apigenin (4', 5-7-trihydroxyflavone) ; genistein (4', 5, 7, trihydroxy-isoflavone) ; guercetin (3, 3', 4 ' , 5-7 pentahydroxy-flavone) ; rutin (guercetin-3-rutinoside) ; conjugated forms such as guercetin pentaacetate, and guercetin 3-D-galactoside; and phenolic and lipophilic poly ethoxylated flavonoids, such as nobiletin and tangeretin (Kandaswami et. al (1991) Cancer Lett. 56:147-52). Examples of naturally occurring flavonoids are shown in Figure 1.
In vitro studies in cell culture have demonstrated that certain flavonoids have antiproliferative properties, and can inhibit the growth of cultured human malignant cells derived from primary human colon, gastric, ovarian, breast and sguamous cell carcinomas, human osteosarcomas and leukemias. (Edwards et al. (1979) J. Nat. Prod. 42:85-91; Smolina et al. (1975) Cancer Res. 35:1865-1872; Molnar et al. (1981) Neoplasma 28:11-18; Catillo et al. (1989) supra; Hirano et. al (1989) Res. Commun. Chem. Pathol. Pharmacol. 64:69-78; Mori et al. (1988) Phytochemistry 27:1017-1020; Yoshida et al. (1990) FEBS Lett 260:10-13; Bracke et. al. (1989) Clin. Exp. Metastasis 7:283-300; Kandaswa i et al. (1991) supra; Scambia et al. (1991) Cancer chemoth. and Pharmac. 28:255-258; Ver a et al. (1988) Cancer Res. 48:5754); Hoffman et al (1989), Brit. J. Cancer 59:347; Larocca et. al. (1991), Brit, J. Haemat. 79:562; Yoshida et al.,
(1992), Cancer Res. 52:6676; Ranelletti et al. (1992), Int. J. Cancer 50:486) .
In vivo studies in rats demonstrated that intraperitoneal injection of quercetin inhibited significantly the growth of squamous cell carcinomas in i plantable cell growth chambers in rats (Castillo (1989) supra) , and inhibited the phosphorylation activity of the Rous sarcoma virus transforming gene product in vitro and In vivo (Graziani et. al. (1983) Eur. J. Biochem. 135:583- 589). The activity of several enzymes, and the release of hista ine from rat mast cells and human basophils, are affected by certain flavonoids (reviewed by Middleton (1984) TIPS 5:335).
The in vivo antoproliferative effects of flavonoids have been studied in mice treated with various tumor promoters. Quercetin was shown to reduce skin tumors initiated with 7,12- dimethylbenz (a) anthracene (DMBA) and promoted by teleocidin (Nashio et al. (1984) Gann 75:113- 116) or 12-0-tetracanoylphorbol-13-acetate (TPA) (Nashio et al. (1984) Oncology 14:120-123). Similar results were obtained with apigenin which, like guercetin, exhibited antipromoter activity in mice treated with DMBA and promoted with TPA (Wei et al. (1990) Cancer Res. 50:499-502). The flavonoids kaempferol and luteolin also blocked the increase in teleocidin-induced epidermal ornithine decarboxylase (ODC) , although in that model quercetin was reported to have an insignificant effect (Fujiki (1986) Prog. Clin. Biol. Res. 213:429-440). Other flavonoids such as morin (2', 3, 4 ' , 5,7- pentahydroxyflavone) , fisetin, and kaempferol reduced significantly the increase in epidermal ODC activity in mice induced by TPA treatment (Nakadate et al. (1984) Gann 74:214-222). Both quercetin and myricetin inhibited polycyclic aromatic hydrocarbon- (PAH) and N-methyl-N- nitrosourea-induced skin tumorigenesis in mice (Mukhtar et al. (1988) Cancer Res. 48:2361-2365). Application of catechin, epicatechin, and their semisynthetic flavonoids catechin dialkyl ketals and epicatechin-4-alkylsulphides, inhibited TPA-induced OCD activity in mouse epidermis in vivo (Gali et. al. (1993) Cancer Lett: 73:149-156).
The inhibitory potencies of several flavonoids on tyrosine protein kinases and serine/threonine kinases has been reported (Hagiwaze et. al. (1977) Biochem. Pharmacol. 37:2987-2992; Graziani et al. (1983) supra;
Akiya a et al. (1987) J. Biol. Chem. 262:5592-5595). The inhibitory effects of 41 synthetic flavonoid analogues on protein tyrosine kinase activity have been reported (Cusman et al. (1991) J. Med. Chem. 34:798-806). Examples of active synthetic flavonoids are shown in Fig. 2.
Inhibition of cellular kinases may lead to "rapid apoptotic cell death" as demonstrated recently by UcKun et. al. (1995), Science 167: 886-891. These investigators targeted the flavonoid genistein to B-cell precursor leukemic cells in mice. Targeting was achieved through the conjugation of genistein with a specific antibody that binds to the CD19 receptors which are on the surface of the leukemic cells. "More than 99.999 percent of human B-cell precursor leukemia cells were killed, which led to 100 percent long-term event-free survival (in mice) from an otherwise invariably fatal leukemia" -. As shown previously, the isoflavonoid genistein is a potent, and specific inhibitor of tryosine protein kinases (Akiyama et al. (1987) J. Biol. Chem. 262; 5592-5595. This in vivo study confirmed previous data in cell culture which demonstrated the inhibitory effects of other flavonoids such as guercetin, on the proliferation of human leukemic cells in culture, including human leukemic cell line K562 (Hoffman et al. (1988) Brit. J. Cancer 59: 347-348); acute yeloid and lymphoid leukemia progenitors (Larocca et al. (1991) Brit. J. Haematol. 79: 562-566); and human leukemic T-cells (Yoshida et al. (1992) Cancer Res. 52: 6676-6681) . Anti-inflammatory, anti-allergic, anti¬ viral, and anti-hemorrhagic effects of flavonoids have been reported (Middleton (1984) supra) .
The efficacy of both naturally occurring and synthetic flavonoid derivatives depends, in part, on the extent of their absorption, distribution, metabolism, and excretion (Middleton et al. (1984) supra) . Flavonoids are relatively insoluble in water. Pharmokinetic studies in humans suggest that less than 1% of orally administered quercetin (4 grams) was absorbed unchanged (Gugler et al. (1975) Eur. J. Clin. Pharmacol. 9:229).
The therapeutic use of certain non-flavonoid compounds is also limited by poor bioavailability due to their low water solubility. An example is the alkaloid camptothecin (Wall et. al. (1966) J. Am. Chem. Soc. 88: 3858), which has been shown to have efficiency in animal tumor models. Camptothecin contains an intact hydroxyl residue at position 20 of the E ring which is essential for its antitumor activity (Wall et. al. (1977) Annu. Rev.
Pharmacal. Toxicol. 17: 117; Hertz et al (1989) J. Med. Chem. 32: 715; Kingsbury et. al. (1991) J. Med. Chem. 34: 98-107; Hutchison (1981) 1065, review). As noted by Kingsbury et. al. (1991) 37: 1047 - ibid, "The poor water solubility of camptothecin precluded its development as a clinical agent and necessitated the use of the water- soluble sodium camptothecin". These investigators converted camptothecin into a water soluble form (1 mg/ml H20) by first modifying the molecule through the attachment of a hydroxyl group at position 9 or 10
(10-hydroxycamptothecin) and then conjugating covalently the added hydroxyl with a number of aminoalkyl groups. This conjugation did not utilize a linker element between the hydroxyl group at position 9 or 10 and the aminoalkyl compound, and it avoided the conjugation of the native hydroxyl group of camptothecin at position 20 (E ring) .
Summary of the Invention The invention features methods for enhancing the bioavailability of therapeutic compounds. A conjugate is formed by covalently attaching to the agent a chemical moiety which is highly bioavailable, resulting in increased bioavailability of the agent. As shown in our examples given below we were capable of conjugating the native, unmodified hydroxyl groups of therapeutic agents to moieties via a linker element, a process yielding molecules of high water solubility (~100mg/ml H20 vs. Img/ml H20 for the alkylated camptothecin) , and retaining full biologic activity.
By the term "agent" or "potential medicinal agent" is meant a compound having desirable in vitro and/or in vivo biological activity in experimental animals which could potentially be used in the treatment of a disease or malignancy. In one embodiment of the invention, the agent is a flavonoid or flavonoid derivative. In another embodiment, the agent is a nonflavonoid.
By the term "bioavailability" is meant the extent at which an agent is water soluble and therefore injectable, or capable of trespassing the intestinal wall so that it can be absorbed into a living system or is made available at the site of physiological action. An increase in bioavailability means that a larger amount of the administered agent reaches a relevant target site.
By the term "chemical moiety" is meant a compound possessing high bioavailability and which confers enhanced bioavailibity to an agent as a conjugate. The chemical moiety to be covalently attached to the agent will vary depending on the specific agent used. In one embodiment of the invention, the chemical moiety increased bioavailabity to an agent is inositol phosphate. By the term "inositol phosphate" is meant inositol having one or more phosphate groups, including mono- phosphorylated inositol, di-phosphorylated inositol, or poly-phosphorylated inositol. The invention may also be practiced with other carbohydrates, phosphorylated carbohydrates or other chemical groups, including phosphate group(s) , sulfate group(s) , carboxyl group(s) , acyl groups(s) , a ino acids, peptides, proteins, glutathione, glyceraldehyde or derivatives, nucleoside, nucleotides, polyols, phosphonates, including bisphosphonates, aminoalkyl molecules, and/or polyamines, or combinations of these groups.
By the term "conjugate" is meant a synthetic chimeric molecule formed by a covalent bond or bonds between a chemical moiety and a potential medicinal agent, or between a chemical moiety, a linking element, and the agent molecule. In conjugate form, the potential medicinal agent possesses enhanced bioavailability relative to the unconjugated agent.
In one embodiment, the increased bioavailability of an agent results from an increase in its solubility in a biological fluid. Solubility of the agent in the desired medium results from modifying the polarity of the agent. Accordingly, in one aspect, the bioavailability of an agent is increased by covalent attachment of a chemical moiety that alters the polarity of the agent, increasing its solubility in the desired medium.
A third method of increasing the bioavailability of an agent is to effectively increase the concentration of the agent at the target cell. Accordingly, in one aspect, the bioavailability of an agent is increased by covalent binding to a chemical moiety which binds a specific target cell. The resulting conjugate binds the target cell, increasing the concentration of the agent at the target cell. In one embodiment, the chemical moiety is actively transported into the target cell. The resulting conjugate is transported into the target cell, resulting in delivery of the bound agent to the target cell. An example of a chemical moiety actively transported into a target cell is inositol phosphate, which is transported into cells having an active inositol phosphate transport system. Another example of chemical moieties that target to specific sites are the bisphosphonates which are taken up by the skeleton (Gertz et al. (1993) Osteoporosis International 3 suppl. 3: S 13-16; Fleisch (1991) Drugs 42: 919-944).
The conjugate may be formed by direct covalent attachment to a chemical moiety to the agent, or by indirect attachment to a chemical moiety via a linking element. By "linking element" is meant a chemical element connecting the agent and chemical moiety. Preferably, the linking element is a carbon chain having between l to 5 carbons in length and having one or more reactive moieties such that other groups may be attached to the linking element. Depending on the length of the carbon chain and the groups attached, the linking element may increase the aqueous or lipid solubility of the agent. Thus, the linking element may function to enhance the bioavailability of the agent or may be utilized to facilitate conjugate synthesis. In a preferred embodiment, the linking element is succinate. In another embodiment, the linking element connects multiple agent molecules to a cell targeting chemical moiety, this increasing the effective concentration of the agent at the target site. The invention encompasses various methods for attachment of the chemical moiety to the potential medicinal agent. In one embodiment of the invention, a conjugate is formed by attachment of inositol phosphate to a flavonoid molecule. In a specific embodiment, the conjugate is formed by attachment of inositol phosphate to a flavonoid through the phosphate group of the inositol (for example, see Fig. 3A) . Alternatively, the molecules are coupled via a linking element through the phosphate linkage (for example, see Fig. 3B) . In a third embodiment, the molecules are coupled via a linking element at a position on the inositol other than the phosphorylated position (for examples, see Figs. 3C and 3D) . The invention features flavonoid-inositol phosphate conjugates where the conjugated flavonoid has enhanced bioavailability relative to the unconjugated flavonoid, resulting from improved water-solubility and/or cellular delivery. Inositol phosphate may be conjugated to flavonoids through a covalent bond or series of covalent bonds between inositol phosphate and one or more hydroxyl and/or amino residues of the flavonoid. The flavonoids useful in this invention may be naturally occurring or synthetic.
In another embodiment the therapeutic agent is covalently bound to a phosphonate, and preferably to a bisphosphonate, through a linking element. Said bisphosphonate may include, but is not limited to, alendronate, pamidronate, etidronate, clodromate, tiludronate, ibandronate or residronate (Figure 4 ) . Additional examples of bisphosphonate structures are given in Shinoda et al. (1983) Calcified Tissue International, Springer Verlag, 35: 87-99. Bisphosphonates have been used in the treatment of several clinical conditions, namely ectopic calcification, ectopic bone formation, Paget's disease, osteoporosis and increased osteolysis of malignant origin (Dunn et al. (1994) Drugs and Aging 5: 446-474; Licata
(1993) Cleveland Clinic J. of Med. 60: 284-290; Fitton and McTavish (1991) Drugs 41: 289-318; Green et al.
(1994) J. Bone and Mineral Res. 9: 745-751; Gertz et al. (1993) 3: S13-16 - ibid; Fleisch (1994) Seminars in
Arthritic and Rheumatism 23: 261- 2; Fleisch (1991) 42: 919-944 - ibid) . Bisphosphonates may be conjugated to a flavonoid or to a nonpeptide nonflavonoid therapeutic agent having one or more hydroxyl groups and/or one or more amino groups. The bisphosphonates and the therapeutic compounds are coupled via a linking element at a position on the bisphosphonate other than the phosphorylated one(s) .
Conjugation of therapeutic agents of low water solubility to bisphosphonates may increase their bioavailability by increasing both their water solubility and their localized delivery to bone. This is desirable in the treatment of osteosarcomas when the agent is cytotoxic for human malignant osteosarcoma cells. For example, apigenin, quercetin, and camptothecin exhibit strong antiproliferative effects on primary human osteosarcoma cells in culture. Agents conjugated to bisphosphonate which are capable of inhibiting tyrosine protein kinases, such as flavonoids (Hagiwaza ewt. al. (1977) ibid; Grazian et. al. (1983) ibid; Akiyama et. al. (1987) ibid; Cushman etl al. (1991) ibid; UcKun et. al. (1995) ibid) , may be delivered by the bisphosphonates to osteoporotic lesions in the bones of human patients with osteoporosis. Inhibition of tyrosine kinase activity in osteoclasts at the site of osteoporotic lesions may result in a decrease activity of bone resorption which is promoted by osteoclasts. Osteoclast proliferation has been shown to be sensitive to tyrosine kinase activity C. Hall et al. (1994) Bioch. Bioph. Res. Commun. , 199: 1237-1244; Yoneda et al. (1993) J. Clin. Invest., 91: 2791-2795; Lowe et al. (1993) Proc. Nat'l. Acad. Sci. USA, 90: 4485-4489.
As described above, inhibition of tyrosine kinase activity on B-cell precursor leukemia cells by the flavonoid genistein resulted in the "rapid apoptotic cell death" of the leukemic cells in mice (UcKun et. al. (1995) ibid). In that study the flavonoid genistein (which is a strong inhibitor to tyrosine kinase and is practically insoluble in water) was coupled to a specific antibody which targeted the flavonoid to the
CD19-receptor of the leukemic cells. The methods of the invention may be used to increase the water solubility and site delivery of other non-flavonoid compounds. For example, the solubility of a compound in water may be increased by formation of a conjugate between a chemical moiety and a non-flavonoid compound having one or more hydroxyl groups and/or one or more amino groups.
The conjugate of the invention may be administered to a patient in a number of ways known to the art, including, intravenous, parenteral, intranasal, oral, topical, transdermal, and subcutaneous sustained release injectable implant formulations. Formulations may be prepared by any of the techniques known in the pharmaceutical arts. Such techniques are described, for example in Remington's Pharmaceutical Sciences ((1980)
Mack Pub. Co., Easton, PA). Formulations for parenteral administration may contain common excipient such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated napthalenes, and others of similar nature. In particular, biocompatible, biodegradable lactate polymers, lactate/glycoside copolymers, or polyoxethylene-polyoxy-propylene copolymers may be useful excipient to control the release of the conjugate of the invention. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for administration by inhalation may contain excipient such as, for example, lactose. Inhalation formulas may be aqueous solutions containing excipient such as, for example, polyoxyethylene 9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasal. Compositions for parenteral administration may also include glycocholate for buccal administration, or cikic acid for vaginal administration. Topical administration may utilize slow releasing patches. The concentration of the conjugate in a physiologically acceptable formulation will vary depending on a number of factors, including the dosage to be administered, the route of administration and the condition being treated. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Brief Description of Figures and Drawings Fig. 1 provides examples of the chemical structures of a number of flavonoid compounds.
Fig. 2 provides examples of the chemical structures of active synthetic flavonoids.
Fig. 3 is a diagram illustrating four methods for the synthetic coupling of inositol phosphate to a flavonoid derivative. Fig. 3A shows coupling of the components through the phosphate group. Fig. 3B shows coupling via a linking element through the phosphate linkage. Figs. 3C and 3D show coupling via a linking element at a position on the inositol other than the phosphorylated one(s)..
Fig. 4 contains examples of bisphosphonates. Fig. 5 and Fig. 6 are synthetic pathways for preparation of a phosphoinositol-succinate linked quercetin.
Fig. 7 is a synthetic pathway for preparation of a phosphoinositol-succinate linked apigenin.
Fig. 8 is a synthetic pathway for preparation of a phosphoinositol-succinate linked genistein. Fig. 9 is a synthetic pathway for preparation of an inositol phosphate linked regioselectively to quercetin via a glycerol linker.
Fig. 10 is a synthetic pathway for preparation of a phosphoinositol quercetin linked through the phosphate group.
Fig. 11 Antiproliferative activity of quercetin and phosphoinositol-succinate linked quercetin on a primary human adenocarcinoma cell line (SW 480) in culture. The biologic activity of the water- soluble conjugated quercetin (6c) is similar to that of unconjugated quercetin. In all biologic studies the unconjugated quercetin was dissolved in dimethylsulfoxide (DMSO) .
Fig. 12 Antiproliferative activity of quercetin and phosphoinositol-succinate linked quercetin on a primary human osteosarcoma cell line (MG 63) in culture. The biologic activity of the water-soluble conjugated quercetin (6c) is similar to that of unconjugated quercetin. Fig.13 A bisphosphonate-coupled flavonoid.
Detailed Description Generally, the invention features methods for increasing the bioavailability of a potential medicinal agent such that the agent becomes therapeutically useful. The methods of the invention are particularly useful with a biologically active agent whose therapeutic use is limited by low bioavailability. However, the invention may be used with any agent for which increased bioavailability is desired. The bioavailability of any agent may be low due to a variety of factors, including low solubility and/or low rate of transport to its target cell. In some cases, the bioavailability of an agent may be low due to its inability to reach an effective concentration at the site of the target cell. The invention provides methods for overcoming these problems, thus increasing the medicinal usefulness of a variety of biologically active agents.
For example, flavonoid derivatives have antiproliferative effects on malignant cells in vitro and in experimental animals in vivo. Their therapeutic use is precluded, however, by their poor water solubility. The present invention provides for increasing the water solubility of the therapeutic agents. The methods of the invention may also be used to increase the cellular delivery of any agent by allowing the agent to be actively transported into cells by the cell membrane inositol phosphate transport system. (See, Saltiel and Sorbara-Cazan (1987) Biochem. Biophys. Res. Commun. 149:1084-1092). Bisphosphonate-linked flavonoid and nonflavonoid therapeutic compounds may be delivered to bone lesions of patients with osteosarcomas or osteoporosis.
The methods of the invention are preferably used with agents having biological activates which would be useful in the treatment of diseases, such as the antiiproliferative effect of flavonoid in tumors, leukemia, and non alignant proliferative disorders such as psoriasis. By the term "flavonoid" is meant any flavone molecule containing one or more hydroxyl groups and/or one or more amino groups.
Examples presented in this application demonstrate that conjunction of flavonoids in inositol phosphate via a linking element (Figures 6-9) resulted in a dramatic increase of their water solubility, retaining their biologic activity. In these examples, apigenin, guercetin, and genistein, which are practically insoluble in water, were converted into highly soluble conjugates with water solubility of over 100 mg per milliliter at room temperature. The examples provided herein illustrate formation of a conjugate by covalent bonding of inositol phosphate to a flavonoid. The covalent bond may be formed in a number of ways, including coupling through the phosphate group (for example, see Fig 3A) , coupling via a linking element through the phosphate linkage (for example, see
Fig 3B) , and coupling via a linking element at a position on the inositol other than the phosphorylated one(s) (for examples, see Figs. 3C and 3D) . Each synthetic method of the invention may be varied in the position(s) of attachment to the inositol and the flavonoid, in the number and position of phosphate groups, in the length and chemical nature of the linking unit, in the flavonoid derivative coupled, and in the inositol isomer used (e.g., yo- or chiro-, etc.). Linking elements include βuccinate diester, 4-hydroxybutyrate ester phosphate diester, and glycerol diester. In one preferred embodiment, flavonoids are conjugated to inositol-2 phosphate via a succinate linker. Other suitable linkers include alkyl chains, mono or poly alcohol units, mono- or polyether units, thioethers, mono- or polyester units, mono- or polyamine units, mono- or polysulfates, mono- or polyarenes, mono- or polyphosphates, mono- or polyamides, peptides, mono- or polysulfides, mono- or polysaccharide, or a combination of these structures. The invention is shown herein in what is considered to be the most practical and preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention and that obvious modifications will occur to one skilled in the art upon reading this disclosure. The examples provided below describe the direct and indirect attachment of inositol phosphate to flavnoids and flavonoid derivatives. These examples are for illustration purposes only, and are not intended to limit the scope of the invention.
Example l. Preparation of (quercetin-5-yl) (myo-inositol-2- phosphate-1-yl) succinate. Compound 6c (quercetin-5-yl) (myo-inositol-2-phosphate-l-yl) succinate) may be prepared as illustrated in Figs. 5 and 6. 3,4,5,6-Tetra-benzyl-l-(1,1,1,-trichloroethylchloro- formyl)-Myo inositol. To 772.7 mg of compound 5a (1.43 mmol) (co-evaporated with toluene) in 4.0 ml of pyridine was added 197 El (1.43 mmol) 1,1,1-trichloroethylchloroformate at O-C. The reaction was stirred at 0-C for 1 h. The reaction mixture was diluted with 2 ml of dichloromethane. The organic phase was washed with water (3 x 2 ml) , dried, and evaporated. The crude products were co-evaporated with heptane until pyridine was no longer detected by smell and their chromatographed on silica gel with hexane: ether (3:1) (Rf=0.15) providing product as an oil with a 79% yield. 1H NMR (CDC13) I 2.35 (s, 1H, OH), 3.51- 3.57 (m, 2H, H3, H6) , 3.97 (yt, J-9.5 Hz,, 1H, H4, or H5) , 4.14 (yt, J=9.8 Hz, 1H H4 or H5), 4.34-4.39 (m, 1H, H2) , 4.61-4.79 (m, 6H) , 4.84-4.91 (m, 5H) , 7.24-7.38 (m) .
3,4,5,6-Tetra-benzyl-2-dibenzylphophoryl-l-(1,1,1- trichloroethylchloroformyl)-myo inositol (5b). To 414 mg of the product from above (0.578 mmol) (co-evaporated with tolune) and 122 mg of lH-tetrazole (1.74 mmol) was added 2.5 ml acetonitrile and 578 El of N,N-diethyl dibenzylphosphoramidite (1.74 mmol). The reaction was stirred at room temperature for 20 min. The reaction mixture was then cooled to -42-C (MeCN/C02) and 2.1 ml of 1 M 85% MCPBA in CH2C12 was added. The ice bath was immediately removed and the reaction mixture allowed to warm to room temperature. After ten minutes, 5 ml of 0.1 M NH4 HC03 buffer (Ph 7.87) and 5 ml of 10% Na2S204 were added. The crude reaction mixture was extracted with
CH2C12. The organic layer was washed with water (1 x 5 ml) , dried, and evaporated. The crude products were chromatographed on silica gel with benzene:ethyl acetate (19:1) (Rf.33) providing 5b as an oil in a 62% yield. 31P NMR (CDC13)I 1.41 1H NMR (CDC13)I 3.54-3.63 (m, 2H, H6, H3) , 3.90 (yt, J=9.5 Hz, 1H, H4, or H5) , 3.97 (yt, J=9.6 Hz, 1H, H4 or H5) , 4.59 (d, J=10.9 Hzz, 1H, 1/2 CH2Ph) , 4.63 (d, J=9.8 Hz, 1H, 1/2 CH2Ph) , 4.67 (d, J-10.9 Hz, 1H, 1/2 CH2Ph) , 4.72-4.92 (m,9H), 4.96-5.00 (m, 2H, CH2Ph) , 5.12- 5.16 (m, 2H, CH2Ph) , 5.37 (d, J=2.3, 8.6 HZ, IH, H2), 7.12-7.43 (m) .
3,4,5,6-Tetra-benzyl-2-dibenzylphosphoryl-myo inositol. To 664 mg of 5b (0.669 mmol) was added to 10 ml THF/HOAC/H20 (5:1:1) and 1.108 g of zinc dust was added. The reaction was stirred at room temperature for lh. An additional 1.108 g of zinc dust was added and the reaction was stirred at room temperature for lh. The reaction mixture was filtered through Celite, diluted with CH2C12. The organic layer was washed with water (1 x 5 ml) , dried, and evaporated. The crude products were chromatographed on silica gel with benzene:ethyl acetate (8:1) (Rf=0.009) providing product as an oil in a 73% yield. 31%P NMR (CDC13) I 0.95 IH NMR (CDC13)I 2.86 (d, J=5.0 Hz, IH, OH), 3.47-3.53 (m, 2H, H3, H6) , 3.54-3.59 (m, IH, HI), 3.73, (yt, J=9.4Hz, IH, H4, H5) , 4.55 (d, J-ll.l HZ, IH, 1/2 CH2Ph) , 4.71 (d, J=11.2 Hz, IH, 1/2 CH2Ph) 4.73 (d, J=10.7 Hz, IH, 1/2 CH2Ph) , 4.80-5.12 (m, 10H) , 7.16-7.39 (m) . 3,4,5,6-Tetra-benzyl-2-dibenzylphosphoroyl-
1-succinyl-myo inositol (5c) . To 200 mg of the product from above (0.234 mmol) (coevapora ed with toluene) (was added 122.6 mg off succinic anhydride (1.23 mmol), 150.3 mg of 4-dimethylamionopyride (1.23 mmol), and 5 ml 1,2- dichloroethane. The reaction was stirred at room temperature for 2h. The reaction mixture was cooled to 0-C and 12 ml of 0.5 M HS04/S04 buffer (pH - 1.0) was added. The crude mixture was stirred for 5 min at 0-C. The mixture was extracted with CH2C12. The organic layer was washed with water (3 x 5 ml) , dried, and evaporated. The crude products were chromatographed on silica gel with benzene:ethyl acetate (5:2) (Rf=0.05) providing 5c as an oil in a 82% crude yield. 31P NMR (CDC13)I 2.13. IH NMR (CDC13) I 2.37- 2.64 (m, succinate and impurity), 3.53-3.61 (m, 2H, H3, H6) , 3.88 (yt, J=9.5 Hz, IH, H4 or H5) , 3.93 (yt. J=9.7 Hz, IH, H4 or H5) , 4.58 (d, J=10.9 Hz, IH, 1/2 CH2Ph) , 4.63 (d, J=11.3 Hz, IH, 1/2 CH2Ph) , 4.72-4.96 (m, 9H) , 5.05 (m, 2H, CH2Ph) , 5.20 (yt, J-2.1, 8.5 Hz, IH, HI), 7.08-7.15 (m,2H), 7.18-7.38 (mm). (3,6,3', 4')-Tetra-benzylquercetin-5-yl)
(3,4,5,6-tetrabenzyl-myo-inossitol-2-phosphate-l-yl) succinate (6b) . To 88.2 mg of crude 5c (0.0963 mmol) was added 6 ml of benzene and 200 El of oxalyl chloride. The reaction was stirred at room temperature for 2.5 h. The reaction mixture was anhydrously evaporated then dissolved in 2 ml 1,2- dichloroethane. To the reaction mixture was added 128 mg of 3', 4', 3,
7-tetra-benzylguercetin (0.193 mmol) (coevaporated with toluene) and 23.6 mg of 4-dimethylaminopyridine (0.193 mmol) in 1.5 ml 1,2-dichloroethane. The reaction was stirred for 5 min. The mixture was extracted with CH2C12. The organic layer was washed with water (3 x 1.5 ml) , dried, and evaporated. The crude products were chromatograped on silica get with benzene:ethyl acetate (8:1) (Rf = 0.22) providing 6b as an oil in a 63% yield. 31P NMRI1.51. IH NMR (CDC13)I 2.38-2.80 (m, 2H, succinate), 2.99-3.17 (m succinate), 3.52-3.61 (m, 2H, H3 and H6 inositol), 3,91 (yt, J-9.5 Hz, IH, H4 or H5) , 3.96 (yt, J=9.5 Hz, IH, H4 or H5) , 4.55 (d, J=10.9 Hz, IH, 1/2 CH2Ph) , 4.66 (d, J-11.2 Hz, IH, 1/2 CH2Ph) , 4.70 (d,
J=11.9 HZ, IH, 1/2 CH2Ph) , 4.74 (d, J=11.0 Hz, IH, 1/2 CH2Ph) 4.81-4.86 (m,7H), 5,00-5.06 (m, 2H, CH2Ph) , 5.13 (S, 2H, CH2PΪ1) , 5.18-5.25 (m, 3H) , 6.71 (d, J=2.6 Hz, IH, H6 or H8 quercetin), 6.82 (d, J=2.4 Hz, IH, H6 or H8 quercetin), 6.93 (d, J-8.7 Hz, IH, H5' quercetin)
7.10-7.49 (m) , 7.64 (d, J=.l Hz, IH, , H2' quercetin). Quercetin-5-yl (myo inositol-2-phosphate-l-yl) succinate (6c). To 47.3 mg of 6b (0.030 mmol) was added 2 ml of ethanol/acetone (1:1 and 0.5 mg of 10% Pd/C. The reaction mixture was placed into a Parr apparatus under 36 psi of hydrogen for 11 h. The reaction mixture was filtered through Celite and the filtrate washed consecutively with 3 ml each of acetone, acetone/water (1:1), and water. The collected solvent was evaporated providing 6c. This product is found to be extensively soluble in water) (solubility exceeds 10 g per 100 ml) UV (compound 6c in water): 357, 264, 205 nm.
Example 2. (Apigenin-5-yl)-(myo-inositol-2-phosphate-l-yl)-succinate
(7g).
An inositol phosphate linked to apigenin via a succinate linker may be prepared according to Figure 7.
Briefly, p-methoxybenzylated diol 7a may be elaborated by a method analogous to that used in Example 1, to produce the acid chloride 7d. p-Methoxybenzylation of apigenin produces flavonoid 7e, which may be coupled with 7a in analogy to Example 1, and finally deprotected oxidatively to produce the title compound 7g. Example 3.
(Genestein-5-yl)-(myo-inositol-2-phosphate-l-yl)-succinat e (8c) .
An inositol phosphate linked to genestein via a succinate linker may be prepared according to Figure 8. p-Methoxybenzylation of genestein may produce flavonoid
8a, which may be coupled with 7d in analogy to Examples 1 and 2, and finally deprtotected oxidatively to produce the title compound 8c.
Example 4.
Inositol-l-yl-quercetin-5-yl-phosphate.
An inositol-phosphate-quercetin derivatives may be prepared as shown in Fig. 10. Generally, 3,4,5,6-tetrabenzyl myo-inositol is treated with benzyl bis(disopropylamino-phosphoramidite followed by a peracid to produce the 1,2-cyclic phosphate. Subsequently, the cyclic phosphate is coupled with quercetin (or a protected quercetin) , which after catalytic hydrogenation produces the desired compounds.
Example 5. 1-(myo-inositol-2-phosphate-l-y)-2-(guercetin-5yl) glycerol.
Fig. 9 illustrates a synthetic pathway to a coupled quercetin with a glycerol linker. Generally,
3,4,5,6- tetrabenzyl myo-inositol is treated with allyl bromide, then phosphorylated. The peracid generates concomitantly an epoxide at the allyl group. Subsequent treatment with a protected quercetin, followed by deproteσtion provides the product.
Biologic Activity of Water-Soluble Conjugated Flavonoids. Biologic activity was determined in vitro in cultures of a primary human colon adenocarcino a cell line (SW 480) , and a human osteosarcoma cell line (MG-63) . The malignant cells were plated in 24-well culture plates, and their population was measured after 6 or 9 days of culture in the presence or absence of the test material. In these studies, the unconjugated flavonoid was dissolved in dimethylsulfoxide (DMSO) and then diluted to the desired concentration with the culture media. The water-soluble conjugated flavonoid was dissolved in a minimum volume of distilled water and diluted to the desired concentration with the culture media. During the culture period the media of the cultured cells, with or without the test material, were changed every two days, and at the end of the culture period the cell population was counted. As seen in the examples shown in Figures Hand 12, the antiproliferative activity of the phosphoinositol-succinate linked quercetin (hydroxyl position 5) was similar to that of the unconjugated quercetin in both the colon adenocarcinoma (Fig. 11) and osteosarcoma (Fig. 12) cultures.
What is claimed is:

Claims

Claims
1. A method for increasing the bioavailability of a non-peptide therapeutic compound, said method comprising reacting said compound with a phosphate-containing chemical moiety to form a chemical bond between said compound and said moiety whereby bioavailability of said compound is increased.
2. A method for increasing the bioavailability of a non-peptide therapeutic compound, said method comprising reacting said compound at a native amino or hydroxyl group with a chemical moiety to form a chemical bond between said compound and said moiety whereby bioavailability of said compound is increased.
3. The method of claims 1 and 2, wherein said chemical moiety that contributes to increased bioavailability is inositol, an inositol phosphate, a carbohydrate, or a carbohydrate phosphate.
4. The method of claim 3 wherein said inositol phosphate is myo-inositol phosphate, L-myo-inositol phosphate, D-myo-inositol phosphate, L-chiro-inositol phosphate, or D-chiro-inositol phosphate.
5. The method of claim 3, wherein said inositol phosphate is selected from the group consisting of mono-phosphorylated inositol, diphosphorylated inositol, poly-phosphorylated inositol, and cyclic phosphates of inositol.
6. The method of claims 1 and 2, wherein said chemical moiety contains a sulfate group, a carboxyl group, an acyl group, a carbamate group, an amino acid, a peptide, a protein, glutathione, glyceraldehyde or derivatives, a nucleoside, a nucleotide, a polyol, an amino alkyl molecule, and/or a polyamine.
7. The method of claim 6 wherein said chemical moiety contains more than one of said groups or a combination of said groups.
8. The method of claims 1 and 2 wherein said chemical moiety is a phosphonate or bisphosphonate.
9. The method of claim 8 wherein said bisphosphonate is alendronate, etidronate, clodronate, pamidronate, tiludronate, residronate, or ibandronate.
10. The method of claim 1 and 2, wherein said chemical moiety is attached indirectly to the therapeutic compound via a linking element which links covalently the therapeutic compound and the chemical moiety, having the following general structure:
TC-L-M where TC is a therapeutic compound L is a linking compound, and M is the chemical moiety.
11. The method of claim 10, wherein said linking compound is a succinate diester, a 4-hydroxybutyrate ester, a phosphate diester, a glycerol diester, an alkyl chain, a mono or poly alcohol unit, a mono or polyether unit, a mono or polyester unit, a mono or polyamine unit, a mono or poly sulfate, a mono or polyarene, a mono or poly phosphate, a mono or polyamide, a peptide, a mono or polysulfide, a mono or polysaccharide, or a combination of these structures.
12. The method of claim 10, wherein said linking compound connects a plurality of said therapeutic compounds to each chemical moiety.
13. The method of claims 1 and 2, wherein said chemical moiety binds covalently directly, or through a linker compound, to one or more hydroxyl and/or amino groups of the therapeutic compound.
14. The method of claim l or claim 2, wherein said therapeutic compound is a flavonoid or isoflavonoid, containing one or more hydroxyl or amino residues.
15. The method of claim 1 or claim 2, wherein said therapeutic compound is a nonflavonoid compound containing one or more hydroxyl or amino residues.
16. The method of claim 10 or claim 13, wherein said therapeutic compound is a nonflavonoid compound which is conjugated covalently to a linking compound through a native hydroxyl and/or amino residue, the linking compound also being bound covalently to said moiety.
17. A method for increasing the solubility of a therapeutic compound which contains one or more hydroxyl or amino groups, said method comprising reacting said therapeutic compound with a solubility-increasing moiety to bond covalently said therapeutic compound to said moiety directly or through a linking compound via said hydroxy or amino residues of the therapeutic compound.
18. The method of claim 17, wherein said increased solubility is increased aqueous solubility.
19. The method of claim 17 wherein said chemical moiety comprises one or more inositol phosphate groups.
20. The method of claim 19, wherein said conjugate is formed by covalent reaction of said inositol phosphate directly or through a linking compound with one or more hydroxyl groups of the flavonoid or nonflavonoid compounds.
21. The method of claim 19, wherein said conjugate is formed by covalent reaction of said inositol phosphate directly or through a linking compound with one or more amino groups of the flavonoid or nonflavonoid compounds.
22. A method for increasing the solubility of a non-peptide therapeutic compound which contains one or more hydroxy groups or amino groups, said method comprising reacting said agent with an inositol phosphate to bond covalently said agent to said inositol phosphate directly or through a linking compound via said hydroxy and or amino groups of the therapeutic compound.
23. The method of claim 22, wherein said increased solubility is increased aqueous solubility.
24. A method for increasing the cellular delivery of a biologically active non-peptide agent which has one or more hydroxyl groups or amino groups, said method comprising reacting said agent with an inositol phosphate to bond covalently said therapeutic agent to said inositol phosphate directly or through a linking compound via said hydroxy or amino group to form a conjugate, which is transported into cells by a transport system for inositol phosphate or inositol phosphate-containing molecules.
25. The method of claims 22 and 24, where in said conjugate said inositol phosphate is substituted by at least one other non-inositol carbohydrate phosphate molecule.
26. The method of claims 10, and 13 , wherein said therapeutic compound is a flavonoid or a nonflavonoid, and said chemical moiety comprises one or more bishosphonate groups of claims 8 or 9.
27. A conjugate formed by covalent reaction of a bishosphonate directly or through a linking compound with one or more hydroxyl groups of a flavonoid or non-flavonoid therapeutic non-peptide compound.
28. The conjugate of claim 27 wherein said conjugate is formed by covalent reaction of said bisphosphonate directly or through a linking compound with one or more amino groups of the flavonoid or non-flavonoid compound.
29. A method for increasing the solubility of a non-peptide therapeutic compound which contains one or more hydroxy groups or amino groups, said method comprising reacting said agent with a bisphosphonate directly or through a linking compound via said hydroxy or amino groups.
30. The method of claim 29, wherein said increased solubility is increased aqueous solubility.
31. A method for increasing the localized delivery to bone of a non-peptide therapeutic agent which has one or more hydroxyl groups or amino groups, said method comprising reacting said therapeutic agent with a bisphosphonate to bond covalently said therapeutic agent to said bisphosphonate directly or through a linking compound via said hydroxy or amino groups to form a conjungate which is targeted to bone by the bisphosphonate groups of the conjugate.
32. The method of claim 31 in which said phosphate-containing moiety is linked to a native hydroxyl or amino group of said therapeutic compound.
PCT/US1995/016161 1995-01-09 1995-12-08 Methods for increasing the bioavailability of biological active agents WO1996021440A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45159/96A AU4515996A (en) 1995-01-09 1995-12-08 Methods for increasing the bioavailability of biological active agents

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US37020895A 1995-01-09 1995-01-09
US08/370,208 1995-01-09
US45568195A 1995-05-31 1995-05-31
US08/455,681 1995-05-31

Publications (2)

Publication Number Publication Date
WO1996021440A1 WO1996021440A1 (en) 1996-07-18
WO1996021440A9 true WO1996021440A9 (en) 1996-10-03

Family

ID=27004859

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/016161 WO1996021440A1 (en) 1995-01-09 1995-12-08 Methods for increasing the bioavailability of biological active agents

Country Status (2)

Country Link
AU (1) AU4515996A (en)
WO (1) WO1996021440A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9168216B2 (en) 2005-06-17 2015-10-27 Vital Health Sciences Pty. Ltd. Carrier comprising one or more di and/or mono-(electron transfer agent) phosphate derivatives or complexes thereof

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458772B1 (en) 1909-10-07 2002-10-01 Medivir Ab Prodrugs
ATE487456T1 (en) * 2001-12-13 2010-11-15 Vital Health Sciences Pty Ltd TRANSDERMAL TRANSPORT OF COMPOUNDS
AU2002950713A0 (en) 2002-08-09 2002-09-12 Vital Health Sciences Pty Ltd Carrier
EP1786400B1 (en) * 2004-08-12 2009-03-11 Quest Pharmaceutical Services Pharmaceutical compositions for controlled release delivery of biologically active compounds
FR2878850B1 (en) * 2004-12-02 2008-10-31 Cis Bio Internat Sa DERIVATIVES OF INOSITOL-1-PHOSPHATE
CA2601028C (en) * 2005-03-11 2014-05-27 Howard Florey Institute Of Experimental Physiology And Medecine Flavonoid compounds and uses thereof
WO2007016380A2 (en) * 2005-07-28 2007-02-08 (Osi) Eyetech, Inc. Cyclitol linker polymer conjugate
US7763650B2 (en) * 2006-03-15 2010-07-27 Medipharma S.A. Pharmaceutical compound and method
AU2006233256B2 (en) * 2006-10-30 2012-01-19 Armaron Bio Pty Ltd Improved flavonols
CN101969959A (en) 2007-07-31 2011-02-09 利默里克生物制药公司 Phosphorylated pyrone analogs and methods
JP2012505251A (en) * 2008-10-10 2012-03-01 リメリック バイオファーマ， インコーポレイテッド Pyrone for the treatment of metabolic diseases
EP2531047A4 (en) 2010-02-05 2014-03-19 Phosphagenics Ltd Carrier comprising non-neutralised tocopheryl phosphate
EP2552486B1 (en) 2010-03-30 2020-08-12 Phosphagenics Limited Transdermal delivery patch
WO2012122586A1 (en) 2011-03-15 2012-09-20 Phosphagenics Limited New composition
WO2017096427A1 (en) 2015-12-09 2017-06-15 Phosphagenics Limited Pharmaceutical formulation
CN110662733A (en) 2016-12-21 2020-01-07 埃维科生物技术有限公司 Method of producing a composite material

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9168216B2 (en) 2005-06-17 2015-10-27 Vital Health Sciences Pty. Ltd. Carrier comprising one or more di and/or mono-(electron transfer agent) phosphate derivatives or complexes thereof

Similar Documents

Publication Publication Date Title
WO1996021440A9 (en) Methods for increasing the bioavailability of biological active agents
WO1996021440A1 (en) Methods for increasing the bioavailability of biological active agents
EP1999136B1 (en) Peptidomimetic inhibitors of psma,compounds comprising them, and methods of use
AU770617B2 (en) Prodrug activation using catalytic antibodies
EP2223929A1 (en) Gambogic glycoside derivatives and analogs, the preparation and the application thereof
Golik et al. Synthesis and antitumor evaluation of paclitaxel phosphonooxymethyl ethers: a novel class of water soluble paclitaxel pro-drugs
US6541613B2 (en) Isoflavone derivatives
KR20080035687A (en) Phosphonated rifamycins and uses thereof for the prevention and treatment of bone and joint infections
ES2375060T3 (en) BISPHOSPHONIC ACID DERIVATIZED OF LIPIDS.
WO2009056952A1 (en) Geminal bisphosphonates, their preparation and their use in the field of oncology
US6699900B2 (en) Hydrophilic and lipophilic silibinin pro-forms
EP2678039B1 (en) Bisphosphonate-prodrugs
Ueda et al. Novel, water-soluble phosphate derivatives of 2′-ethoxy carbonylpaclitaxel as potential prodrugs of paclitaxel: Synthesis and antitumor evaluation
HU228618B1 (en) Medical compositions comprising pyridylbisphosphonate compounds and their use
JP4162927B2 (en) Carbacyclic phosphatidic acid derivatives
WO1999063995A1 (en) Isoflavone derivatives
WO2023047399A1 (en) Cannabinoid-lipid conjugates, methods for producing the same and uses thereof
EP3648797A1 (en) Multifunctional conjugates
US4874779A (en) Mitomycin phosphate derivatives
US6207655B1 (en) Bis-phosphonate confugates with alkylating moieties having antitumor activity
MXPA96006180A (en) Piridilbisphosphonates to be used as a therapist agent
CZ2001861A3 (en) O-Phosphonomethyl choline and alkyl esters thereof, process of their preparation and use
FI117053B (en) New pyridinyl-amino:methylidene bis:phosphonic acid derivs. - used to treat bone disease e.g. osteolytic bone diseases due to malignancy, Paget's disease and osteoporosis
CN110785175A (en) Ibandronate conjugates of nucleoside antimetabolites
UA23324U (en) The 8-bromotheofiline potassium salt (brotheofin), having antitumor and antimetastasis activity