EP1677803A2 - Methode destinee a inhiber l'expression de genes de multiresistance aux medicaments et a inhiber la production de proteines resultant de l'expression de ces genes en vue d'ameliorer l'efficacite d'agents chimiotherapeutiques pour le traitement des cancers - Google Patents

Methode destinee a inhiber l'expression de genes de multiresistance aux medicaments et a inhiber la production de proteines resultant de l'expression de ces genes en vue d'ameliorer l'efficacite d'agents chimiotherapeutiques pour le traitement des cancers

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Publication number
EP1677803A2
EP1677803A2 EP04761853A EP04761853A EP1677803A2 EP 1677803 A2 EP1677803 A2 EP 1677803A2 EP 04761853 A EP04761853 A EP 04761853A EP 04761853 A EP04761853 A EP 04761853A EP 1677803 A2 EP1677803 A2 EP 1677803A2
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EP
European Patent Office
Prior art keywords
cholesterol absorption
phenyl
absorption inhibitor
azetidinone
hydroxy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP04761853A
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German (de)
English (en)
Inventor
Kristina Sachs-Barrable
Tatjana Lukic
David John Stewart
Kishor M. Wasan
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Forbes Medi-Tech Inc
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Forbes Medi-Tech Inc
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Publication of EP1677803A2 publication Critical patent/EP1677803A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • TITLE METHOD OF INHIBITING THE EXPRESSION OF MULTI-DRUG
  • This present invention relates to the field of cancer treatment.
  • MDR1 encodes P-glycoprotein, a 170 kDa multispanning transmembrane protein belonging the ATP Binding Cassette (ABC) Transporter protein superfamily 2 . It is suggested that one mechanism by which tumor cells acquire MDR is by over expression of P-glycoprotein (Pgp) 3,4,5,6
  • P-glycoprotein likely acts by rapidly pumping hydrophobic chemotherapeutic agents out of the tumor cells, thereby decreasing intracellular accumulation of certain chemotherapeutic agents below their cytostatic concentrations.
  • One in vitro solution is to increase the chemo drug concentration.
  • cancer chemotherapeutic agents are already administered at their maximally tolerated range in vivo, increasing the doses is an unacceptable solution leading, on most cases to extreme toxicity 7,8 .
  • P-glycoprotein is structurally similar to the cystic fibrosis transporter protein, the major histocompatibility complex-linked peptide transporter, and a non-P-glycoprotein-related multidrug resistance protein (MRP) 9,10,11 ' 12 .
  • MRP multidrug resistance protein
  • P-glycoprotein is expressed in diverse sites including the normal human adrenal cortex, the luminal aspect of bile canaliculi and colonic epithelium, the renal tubular epithelium and the end ⁇ thelial cell of the blood- brain and blood testicular barriers. The function of the P-glycoproteins at these sites is unclear but appears to function as an energy dependent pump of broad specificity possibly related to secretion of hormones and protection against toxins.
  • P-glycoprotein can actively efflux a large number of hydrophobic, and heterocyclic cancer chemotherapeutic agents including adriamycin (doxorubicin), colchicine, colcemid, etoposide, paclitaxel, vincristine, vinblastine as well as others.
  • adriamycin doxorubicin
  • colchicine colchicine
  • colcemid etoposide
  • paclitaxel paclitaxel
  • vincristine vinblastine as well as others.
  • P-glycoproteins are encoded by a highly conserved family of genes 13 .
  • the MDR-1 gene encodes class I P-glycoprotein that confers multidrug resistance in humans 14 .
  • the pgp-1 and pgp-2 genes in hamsters and the mdr-3 and MDR-1 genes in mice encode the class I and II proteins, both of which confer multidrug resistance in rodents 15 .
  • Structural features of the protein are characteristic of an energy-dependent efflux pump 16 . Over expression of the protein is associated with multidrug resistance 17 .
  • relapse or disease progression following the initial chemotherapy regime is refractory to treatment, and frequently involves MDR and expression of elevated levels of MDR-1 18 .
  • in vitro studies often show amplification of MDR-1 as the cause for drug resistance 19 , elevated gene copy number is apparently rare in human tumors 20 .
  • MDR-1 gene product or its mRNA have been associated with poor prognosis in a number of human tumor studies.
  • Clinical studies in breast carcinoma have shown that a significant percentage of patients expressed large levels of P- glycoprotein and the low P-glycoprotein expressing patients had significantly better rates of response to chemotherapy and progression-free survival than did high expressing groups 21,22 .
  • Sixty-eight percent of the tumors in a retrospective study of colon carcinoma expressed high levels of P-glycoprotein and over expression correlated with aggressiveness measured by blood cell invasion and metastasis 23 .
  • the MDR-1 negative groups all had higher rates of complete remission and longer durations of disease-free survival than the MDR-1 positive groups 24,25,26 .
  • Drugs that are alleged to reverse MDR have been shown to have mechanisms by decreasing drug efflux (e.g., calcium channel blockers verapramil, diltiazem and nicardipidine 32 , calmodulin inhibitors reserpine, quinidine or quineine 33 , cyclosporin A 34 , cyclosporin derivatives 35 , or FK506 and rapamycin 36 .
  • Drugs that are alleged to reverse MDR have been shown to have mechanisms by decreasing drug efflux (e.g., calcium channel blockers verapramil, diltiazem and nicardipidine 32 , calmodulin inhibitors reserpine, quinidine or quineine 33 , cyclosporin A 34 , cyclosporin derivatives 35 , or FK506 and rapamycin 36 .
  • Clinical reversal of resistance for most of these agents has been limited, mainly due to clinically toxic concentration levels that were needed to achieve
  • the present invention provides, in one aspect, a method of inhibiting the expression of a multi-drug resistance gene in an animal cell which comprises administering to an animal an effective amount of at least one cholesterol absorption inhibitor.
  • the present invention provides, in another aspect, a method of enhancing the effectiveness of a chemotherapeutic agent in an animal having cancer, which comprises administering to said animal an effective amount of at least one chemotherapeutic agent and at least one cholesterol absorption inhibitor.
  • the present invention provides, in yet another aspect, a method of reversing a multi- drug resistance phenotype exhibited by an animal cell which comprises exposing the cell to an effective amount of at least one cholesterol absorption inhibitor.
  • the present invention provides, in yet another aspect, a method of inhibiting the production of a protein expressed by a multiple drug resistance gene in an animal cell which comprises administering to an animal an effective amount of at least one cholesterol absorption inhibitor.
  • the present invention provides, in yet another aspect, a composition for use in cancer treatment which comprises at least one chemotherapeutic agent and at least one cholesterol absorption inhibitor.
  • the present invention provides, in yet another aspect, a kit comprising at least two separate components: a) a composition comprising at least one cholesterol absorption inhibitor; and b) a composition comprising at least one chemotherapeutic agent; along with instructions describing the administration of each composition.
  • the crux of the present invention is the provision and co-administration (though not necessarily concurrently or proximally consecutively) of cholesterol absorption inhibitors and chemotherapeutic agents. It has been found that cholesterol absorption inhibitors effectively inhibit the expression of multi-drug resistance genes. It is suggested that the reduction in production of proteins expressed by these genes prevents the efflux of cancer chemotherapeutic agents from animal cells, maximizing their effectiveness. This is important due to the documented problems relating to the rendering virtually ineffective of heretofore promising chemotherapeutic agents by the multiple drug resistance mechanism.
  • Some of the preferred cholesterol absorption inhibitors of the present invention (those depicted below including those in formulae (i) through (iv) comprise an ascorbyl moiety. These particular compounds have numerous additional advantages. In particular, solubility in aqueous solutions such as water is improved by the ascorbyl moiety thereby allowing oral administration perse. Likewise, other modes of administration are facilitated. Accordingly, these selected compounds of the present invention can be prepared and used as such or they can be easily incorporated into pharmaceutical preparations, optionally in conjunction with the selected chemotherapeutic agent, regardless of whether such preparations are water-based. This enhanced solubility generally translates into lower administration dosages of the compounds in order to achieve the desired therapeutic effect.
  • Figure 1 is a bar graph showing the level of MDR-1 expression (normalized ratio of MDR-1/GAPDH; "GAPDH” or glyceraldehyde-3-phosphate dehydrogenase) in CaCo2 cells after treatment for one week with one of the cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4";
  • Figure 2 is a graph showing the titration for primer drop of GAPDH
  • Figure 3 depicts a polymerase chain reaction (1.5% agarose gel) gel electrophoresis results for MDR-1, GAPDH
  • Figure 4 is a bar graph showing an MTS- and LDH-Assay of cell viability after treatment of CaCo2 cells with one of the cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4";
  • Figure 5 a bar graph showing a BCA-Assay of protein concentration after treatment of CaCo2 with one of the cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4";
  • Figure 6 a bar graph showing the level of ABCC1 (MRP-1) expression (normalized ratio of MRP-1/GAPDH) in CaCo2 cells after treatment for one week with one of the cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4";
  • Figure 7 depicts a polymerase chain reaction (1.5% agarose gel) gel electrophoresis results for MDR-1, GAPDH; RNA isolation with TRIZOL ⁇ RT-PCR ⁇ PCR after treatment of CaCo2 cells with liposomal formulations of one of cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4;
  • Figure 8 is a bar graph showing the level of MDR-1 expression (normalized ratio of MDR-1/GAPDH) in CaCo2 cells after treatment for one week with one of the cholesterol absorption inhibitors described herein: a liposomal formulation of an ascorbyl stanyl phosphate ester called "FM-VP4" at 2.5, 5 and 10um as compared to a control and empty liposomes;
  • FM-VP4 liposomal formulation of an ascorbyl stanyl phosphate ester
  • FIG. 9 is a Western Blot analysis of P-glycoprotein in CaCo2 cells after incubation with one of cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4";
  • Figure 10 is an electrophoretic gel profile of sample PCR products (measuring expression profile of mdr-1 gene in Caco-2 cells as an effect of a time-dependent treatment with 10uM FM-VP4);
  • Figure 11 is a bar graph showing the fluorescent bands of the PCR products of Figure 10 imaged under UV light (UV-Epi Chem II) and quantified with the UVP- Labworks software;
  • Figure 12 is a bar graph of the effect of pre-incubation with FM-VP4 for 1 week on accumulation of Rh123 in Caco-2 monolayer. Data represents the average of ⁇ SD. * P ⁇ 0.002; **P ⁇ 0.0001 ;
  • Figure 13 is a bar graph showing the effect of pre-incubation with FM-VP4 for 1 week on P-gp transport of Rh123 across Caco-2 monolayer (basolateral to apical). Data represents the average of 3 ⁇ SD. *P ⁇ 0.07; **P ⁇ 0.009.
  • animal means any member of the animal kingdom, including all mammals and most preferably humans. Veterinary use is also contemplated.
  • the terms "effective” or “therapeutically effective”, are intended to qualify the amount of the compound(s) or composition administered to an animal, in particular a human, in order to elicit a biological or medical response of a cell, tissue, system, animal or mammal that is being sought by the person administering the compound(s) or composition and which amount achieves one or more of the following goals: a) treating or alleviating a cancer; b) preventing, treating or alleviating tumour growth; c) inhibiting or reducing the expression of one or more multiple drug resistance genes; d) inhibiting or reducing the production of one or more proteins expressed by multiple drug resistance genes; e) enhancing the effectiveness of a chemotherapeutic agent in treating a cancer; and f) sensitizing a cell to one or more chemotherapeutic agents.
  • ileal bile acid transporter or "IBAT” is synonymous with apical sodium co-dependent bile acid transporter, or ASBT.
  • benzothiepine IBAT inhibitor means an ileal bile acid transport inhibitor which comprises a therapeutic compound comprising a 2,3,4,5-tetrahydro- 1 -benzothiepine 1,1 -dioxide structure.
  • a multiple drug resistance gene refers to one or more of the following genes: ABCB1 (MDR-1); ABCA2 (ABC2); ABCB2 (TAP); ABCB3 (TAP); ABCC1 (MRP-1); ABCC3 (MRP-3).
  • prodrug refers to compounds that are drug precursors, which, following administration to a patient, release the drug in vivo via some chemical or physiological process (for example, a prodrug, on being brought to physiological pH or through enzyme action is converted to the desired drug form).
  • solvate refers to a molecular or ionic complex of molecules or ions of solvent with those of solute (for example the compounds of formulae a) to f) or prodrugs of compounds a) to f)).
  • useful solvents include polar, protic solvents such as water and/or alcohols (for example, methanol).
  • sterol includes all sterols without limitation, for example: (from any source and in any form: ⁇ , ⁇ and y) sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers.
  • stanol refers to, for example: (from any source and in any form: ⁇ , ⁇ and y) saturated or hydrogenated sterols including all natural or synthesized forms and derivatives thereof, and isomers, including sitostanol, campestanol, stigmastanol, brassicastanol (including dihydrobrassicastanol), desmostanol, chalinostanol, poriferastanol, clionastanol, ergostanol, coprostanol, codistanol, isofucostanol, fucostanol, clerostanol, nervistanol, lathostanol, stellastanol, spinastanol, chondrillastanol, pepostanol, avenastanol, isoavenastanol, fecostanol, and pollinastastanol.
  • the sterols and stanols for use in forming derivatives in accordance with this invention may be procured from a variety of natural sources or they may be artificially synthesized. For example, they may be obtained from the processing of plant oils (including aquatic plants) such as corn oil and other vegetable oils, wheat germ oil, soy extract, rice extract, rice bran, rapeseed oil, sunflower oil, sesame oil and fish (and other marine-source) oils. They may also be derived from yeasts and fungi, for example ergosterol. Accordingly, the present invention is not to be limited to any one source of sterols.
  • US Patent Serial No. 4,420,427 teaches the preparation of sterols from vegetable oil sludge using solvents such as methanol.
  • phytosterols and phytostanols may be obtained from tall oil pitch or soap, by-products of forestry practises as described in US Patent Serial No.5,770,749, incorporated herein by reference.
  • a further method of extracting sterols and stanols from tall oil pitch is described in Canadian Patent Application Serial No. 2,230,373 which was filed on February 20, 1998 (corresponding to PCT/CA99/00150 which was filed on February 19, 1999) and US Patent Application Serial No 10/060,022 which was filed on January 28, 2002 the contents of all of which are incorporated herein by reference.
  • sterol and “stand” as used herein, including, but not limited to: free sterols and stanols, esterified sterols and stanols with aliphatic or aromatic acids (thereby forming aliphatic or aromatic esters, respectively), phenolic acid esters, cinnamate esters, ferulate esters, phytosterol and phytostanol glycosides and acylated glycosides or acylglycosides.
  • sterols and “stanols” encompasses all analogues, which may further have a double bond at the 5-position in the cyclic unit as in most natural sterols, or one or more double bonds at other positions in the rings (for example, 6, 7, 8(9), 8(14), 14 5/7) or no double bonds in the cyclic unit as in stanols. Further, there may be additional methyl groups as, for example, in ⁇ Vsitosterol.
  • Sterols are naturally occurring compounds that perform many critical cellular functions. Sterols such as campesterol, stigmasterol and beta-sitosterol in plants, ergosterol in fungi and cholesterol in animals are each primary components of cellular and subcellular membranes in their respective cell types. Phytosterols, in particular, have received a great deal of attention due to their ability to decrease serum cholesterol levels when fed to a number of mammalian species, including humans.
  • the compounds of the present invention are formed of naturally-derived or artificially synthesized beta-sitosterol, campestanol, sitostanol, and campesterol and each of these compounds so formed is then admixed in a pharmaceutical composition prior to delivery in various ratios.
  • the compound of the present invention comprises a chemical linkage between one or more disodium ascorbyl phytostanyl phosphates (referred to herein as "FM-VP4") which comprises two major components: disodium ascorbyl campestanyl phosphate (“DACP”) and disodium ascorbyl sitostanyl phosphate (“DASP").
  • cholesterol absorption inhibitor refers to any compound having a negative effect on cholesterol transport, uptake or absorption, by whatever mechanism and includes any compound which inhibits bile acid reabsorption or transport.
  • the cholesterol absorption inhibitor comprises one or more sterols, stanols or mixtures thereof or derivatives thereof as described herein.
  • this includes all free sterols and stanols, and all sterol and stanol aliphatic and aromatic esters, sterol and stanol phenolic acid esters, sterol and stanol cinnamate esters, sterol and stanol ferulate esters, sterol and stanol glycosides, sterol and both stanol acylated glycosides or acylglycosides.
  • the cholesterol absorption inhibitor comprises one or more derivatives or compounds comprising a sterol or stanol, including biologically acceptable salts thereof, having one or more of the following formulae:
  • R is a sterol or stanol moiety
  • the cholesterol absorption inhibitor is a disodium ascorbyl stanyl phosphate composition which comprises a mixture of disodium ascorbyl campestanyl phosphate and disodium ascorbyl sitostanyl phosphate.
  • the compounds of formulae i) to iv) can be prepared by known methods, for example those described below and in PCT/CA00/00730, which was filed on June 20, 2000 and claims priority back to US Patent Application 09/339,903 filed on June 23, 1999, the entire contents of which are incorporated herein by reference..
  • compounds of formulae i) to iv) can be prepared as follows: the selected sterol or stanol (or halophosphate, halocarbonate or halo-oxalate derivatives thereof) and ascorbic acid are mixed together under reaction conditions to permit condensation of the "acid" moiety with the "alcohol” (sterol).
  • reaction conditions are the same as those used in other common esterification reactions such as the Fisher esterification process in which the acid component and the alcohol component are allowed to react directly or in the presence of a suitable acid catalyst such as mineral acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid.
  • organic solvents generally employed in such esterification reactions are ethers such as diethyl ether, tetrahydrofuran, or benzene, toluene or similar aromatic solvents and the temperatures can vary from room to elevated temperatures depending on the reactivity of the reactants undergoing the reaction.
  • the process to form the ester comprises "protecting" the hydroxyl groups of the ascorbic acid or derivatives thereof as esters (for example, as acetate esters) or ethers (for example, methyl ethers) and then condensing the protected ascorbic acid with the sterol/stanol halophospahte, halocarbonate or halo- oxalate under suitable reaction conditions.
  • condensation reactions are conducted in an organic solvent such as diethyl ether, tetrahydrofuran, or benzene, toluene or similar aromatic solvents.
  • the reaction temperatures may vary from low (-15°C) to elevated temperatures.
  • ascorbic acid is initially protected from decomposition by the formation of 5,6-isopropylidene-ascorbic acid. This can be achieved by mixing acetone with ascorbic acid and an acidic catalyst such as sulfuric acid or hydrochloric acid under suitable reaction conditions.
  • Phytostanol chlorophosphate is prepared by forming a solution of phytostanol in toluene and pyridine (although other nitrogen bases such as aliphatic and aromatic amines may alternatively be used) and treating this solution with a phosphorus derivative such as phosphorus oxychloride.
  • the residue so formed after filtration and concentration of the mother liquor is phytostanol chlorophosphate.
  • the latter is then mixed with 5,6- isopropylidene-ascorbic acid and, after the addition of a suitable alcohol such as ethanol and HCI, concentrated.
  • a suitable alcohol such as ethanol and HCI
  • pyridine/THF may be added and the product concentrated. After final washing and drying, the resultant novel product a stanol-phosphate-ascorbate.
  • ascorbic acid is protected at the hydroxyl sites not as 5,6-isopropylidene-ascorbic acid but as esters (for example as acetates, phosphates and the like..).
  • esters for example as acetates, phosphates and the like..
  • the latter may then be condensed with sterols or stands, derivatized as described above, using known esterification methods ultimately to produce the compounds.
  • the formation of mono and diphosphates of ascorbic acid is described thoroughly in the literature. For example, US Patent Serial No. 4,939,128 to Kato et al., the contents of which are incorporated herein by reference, teaches the formation of phosphoric acid esters of ascorbic acid. Similarly, US Patent Serial No.
  • the cholesterol absorption inhibitor of the present invention comprises one or more disodium ascorbyl phytostanyl phosphates (referred to as "FM- VP4") which comprises two major components: disodium ascorbyl campestanyl phosphate (“DACP”) and disodium ascorbyl sitostanyl phosphate (“DASP").
  • FM- VP4 disodium ascorbyl phytostanyl phosphates
  • DASP disodium ascorbyl sitostanyl phosphate
  • the cholesterol absorption inhibitor comprises a compound from the family of hydroxy substituted azetidinones.
  • this azetidonone is a hydroxy substituted azetidinone compound represented by the formula: R R2
  • An and Ar 2 are independently selected from the group consisting of aryl and R 4 -substituted aryl;
  • Ar 3 is aryl or R 5 -substituted aryl;
  • X, Y and Z are independently selected from the group consisting of -CH 2 -, - CH(lower alkyl)- and -C(dilower alkyl)-;
  • R and R 2 are independently selected from the group consisting of --OR 6 , -O(CO)R 6 , -O(CO)OR 9 and «O(CO)NR 6 R 7 ;
  • Ri and R 3 are independently selected from the group consisting of hydrogen, lower alkyl and aryl;
  • R 4 is 1-5 substituents independently selected from consisting of lower alkyl, ⁇ OR 6 , - O(CO)R 6 , -O(CO)OR 9 , -O(CH 2 ) 1-5 OR 6 , -O(CO)NR 6 R 7 , --NR 6 R 7 , -NR 6 (CO)R 7 , - NR 6 (CO)OR 9 , -NR 6 (CO)NR 7 R 8 , -NR 6 SO 2 R 9 , -COOR 6 , -CONR 6 R 7 , -COR 6 , - SO 2 NR 6 R 7 , S(O)o- 2 Rg, -O(CH 2 ) ⁇ - ⁇ 0 -COOR 6 , --O(CH 2 ) 1-10 CONR 6 R , -(lower alkylene)COOR 6 , -CH.dbd.CH-COOR 6 , -CF 3 , -CN, -NO 2 and halogen;
  • Re is 1-5 substituents independently selected from the group consisting of -OR 6 , - O(CO)R 6 , -O(CO)OR 9 , -O(CH 2 ) ⁇ -5 OR 6 , -O(CO)NR 6 R 7 , -NR 6 R 7 , -NR 6 (CO)R 7 , - NR 6 (CO)ORg, -NR 6 (CO)NR 7 R 8 , -NR B SO 2 R 9 , -COOR 6 , -CONR 6 R 7 , --COR 6 , - SO 2 NR 6 R 7 , S(O) 0-2 R 9 , --O(CH 2 ) ⁇ - ⁇ o --COOR 6 , -O(CH 2 ) 1-10 CONR 6 R 7l -(lower alkylene)COOR 6 and -CH.dbd.CH-COOR 6 ;
  • R 6 , R ⁇ and R 8 are independently selected from the group consisting of hydrogen, lower alkyl, aryl and aryl-substituted lower alkyl;
  • R 9 is lower alkyl, aryl or aryl-substituted lower alkyl.
  • A is phenyl or R 4 -substituted phenyl
  • Ar 2 is phenyl or R 4 -substituted phenyl
  • Ar 3 is R 5 -substituted phenyl.
  • Arj is R -substituted phenyl wherein R 4 is halogen
  • Ar 2 is R -substituted phenyl wherein R is halogen or ⁇ OR ⁇ , wherein R 6 is lower alkyl or hydrogen
  • Ar 3 R 5 -substituted phenyl, wherein R 5 is -OR 6 , wherein R6 is lower alkyl or hydrogen.
  • the compound is selected from the group consisting of:
  • the cholesterol absorption inhibitor within the scope of the present invention may be an inhibitor of bile acid transport or reabsorption, including, but not limited to ileal, apical and hepatic transport inhibitors.
  • IBATs ileal bile acid transport inhibitors
  • IBAT inhibitors useful in the present invention are disclosed in PCT/US95/10863, the contents of which are incorporated herein by reference. More IBAT inhibitors are described in PCT/US97/04076, herein incorporated by reference. Still further IBAT inhibitors useful in the present invention are described in U.S. application Ser. No. 08/816,065, herein incorporated by reference. More IBAT inhibitor compounds useful in the present invention are described in WO 98/40375, herein incorporated by reference. An array of additional IBAT inhibitor compounds useful in the present invention are described in U.S. Pat. No. 5,994,391, also incorporated herein by reference.
  • bile acid transport inhibitors include selected benzothiepines, such as those disclosed in WO 93/321146, PCT/US97/04076, PCT/US95/10863, EP 508425, FR 2661676, WO 99/35135, WO 92/18462, and U.S. Patent No. 5,994,391 (Lee et al.).
  • WO 92/16055 to The Wellcome Foundation Limited describes a number of suitable benzothiazepine compounds. Additional hypolipidemic benzothiazepine compounds (particularly 2,3,4,5-tetrahydrobenzo-1-thi-4-azepine compounds) are disclosed in patent application Nos. WO 96/05188, WO 96/05188 and WO 96/16051.
  • IBAT inhibitor compounds include a class of naphthalene compounds, described by T. Ichihashi et al. in J. Pharmacol. Exp. Ther., 284(1), 43-50 (1998).
  • S-8921 methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy- 6,7,8-trimethoxy-2-na phthoate
  • the structure of S-8921 is shown in formula B-20 of this publication.
  • Further naphthalene compounds or lignin derivatives are described in PCT Patent Application No. WO 94/24087.
  • IBATs include, for example, SC-635 developed by Pharmacia Corporation, IL and Monsanto, MO; 264W94 developed by GlaxoSmithKline; S-8921 developed by Shiongi) and (3R,5R)-3-butyl-3-ethyl-2,3,4,5-tetrahydro-,7,8-dimethoxy-5-phenyl-1-4- benz othiazepine 1 , 1 -dioxide.
  • the cholesterol absorption inhibitor within the scope of the present invention may be an androstane and/or androstene derivative, wherein androstane and/or androstene are coupled with ascorbic acid and represented by one or more of the general formulae:
  • Ri, R 2 , R 3 , R 4 , R5, Re may individually be chosen from hydrogen, OH, carbonyl, and an ascorbyl moiety; and R 7 may be hydrogen or any halogen.
  • the ascorbyl moiety which is coupled to the compound from the androstane or androstene family is selected individually from one or more of the following structures:
  • M+ represents any metal, alkali earth metal, or alkali metal.
  • the androstane/androstene compounds of formulae vi) to viii), incorporating one or more of the ascorbyl linkers of formulae ix) to xx), can be prepared by known methods, for example those described in PCT/CA03/00824, the contents of which are incorporated herein by reference or can be prepared by the methods described above for the preparation of compounds of formulae i) to iv), adjusted accordingly.
  • biologically acceptable salts refers any salts that retain the desired biological and/or physiological activity of the compounds as described herein and exhibit minimal undesired toxicological effects. Accordingly, reference to all compounds herein thereby includes reference to respective acidic and/or base salts thereof, formed with inorganic and/or organic acids and bases.
  • Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example trifluroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclpentanepropionates, digluconates, dodecylsulfates, heptanoates, hexanoates, hydrochlorideshyrobromides, hydroiodides, 2-hydroethanesulfonates, lactates, maleates, methanesulfonates, 2- naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfonates, 3- phenylpropionates, phosphates, picrates, pivalates, prop
  • Those compounds which contain an acid moiety may form salts with a variety or organic and inorganic bases.
  • the present invention encompasses not only the parent compounds comprising, for example, the selected sterol and/or stanol a but also, where possible (i.e. where the parent contains a free hydroxyl group), the present invention encompasses the biologically acceptable metal, alkali earth metal, or alkali metal salts of the disclosed compounds.
  • the salts, as described herein, are even more water soluble than the corresponding parent compounds and therefore their efficacy and evaluation both in vitro and in vivo may be enhanced.
  • Salt formation of the compounds of the present invention can be readily performed, for example, by treatment of any parent compound containing a free OH group with a series of bases (for example, sodium methoxide or other metal alkoxides) to produce the corresponding alkali metal salts.
  • bases for example, sodium methoxide or other metal alkoxides
  • Other metal salts of calcium, magnesium, manganese, copper, zinc, and the like can be generated by reacting the parent with suitable metal alkoxides.
  • stereoisomers of the compounds of the present invention such as those which may exist due to asymmetric carbons on various constituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons) and diastereomeric forms, are contemplated within the scope of the present invention.
  • Individual stereoisomers of the compounds of the present invention may, for example, be admixed as racemates or with all other, or other selected sterioisomers.
  • the chiral centres of the compounds can have the S or R configuration as defined by the IUPAC 1974 Recommendations.
  • Such stereoisomers can be prepared using conventional techniques, either by reacting enantiomeric starting materials, or by separating isomers of compounds of the present invention. When diastereomeric or enantomeric products are prepared, they can be separated by conventional methods, for example, chromatographic or fractional crystallization.
  • Isomers may include geometric isomers, for example cis-isomers or trans-isomers across a double bond. All such isomers are contemplated among the compounds useful in the present invention.
  • the compounds useful in the present invention also include tautomers.
  • While the present invention fully covers the inhibition of expression of the following multiple drug resistance genes: ABCB1 (MDR-1); ABCA2 (ABC2); ABCB2 (TAP); ABCB3 (TAP); ABCC1 (MRP-1); ABCC3 (MRP-3) it has been found that the cholesterol absorption inhibitors, as described herein, are particularly useful in inhibiting the expression of MDR-1 (ABCB1), which has P-glycoprotein as its gene product. In addition, it has been found that the cholesterol absorption inhibitors, as described herein, are particularly useful in inhibiting the production of P-glycoprotein, as expressed by the MDR-1 gene.
  • a method of inhibiting the expression of a multi-drug resistance gene in an animal cell which comprises administering to an animal an effective amount of at least one cholesterol absorption inhibitor as described herein.
  • composition for use in cancer treatment which comprises at least one chemotherapeutic agent and at least one cholesterol absorption inhibitor as described herein.
  • a method of enhancing the effectiveness of a chemotherapeutic agent in an animal having cancer which comprises administering to said animal an effective amount of at least one chemotherapeutic agent and at least one cholesterol absorption inhibitor as described herein.
  • a method of reversing a multi-drug resistance phenotype exhibited by an animal cell which comprises exposing the cell to an effective amount of at least one cholesterol absorption inhibitor as described herein.
  • a method of inhibiting the production of a protein expressed by a multiple drug resistance gene in an animal cell which comprises administering to an animal an effective amount of at least one cholesterol absorption inhibitor.
  • kits comprising at least two separate components: a) a composition comprising at least one cholesterol absorption inhibitor as described herein; and b) a composition comprising at least one chemotherapeutic agent; along with instructions describing the administration of each composition.
  • compositions and kit of the present invention are suitable for use in the treatment of cancers in general including, but not limited to breast, prostate, liver, pancreatic, kidney, stomach, intestinal, colon, lung, brain, adrenal cortex, bone marrow, embryonic stem cell, parenchymatous, epithelial, lymph varieties (including Hodgkins and non-Hodgkins lymphomas), bone, testicular, cervical, uterine, esophageal, mouth, and skin.
  • cancers which frequently show high levels of P-glycoprotein at diagnosis, even though the patients have not been previously treated with chemotherapeutic agents (combinations of one or more anti cancer drugs).
  • chemotherapeutic agents combinations of one or more anti cancer drugs.
  • Such cancers include, but are not limited to: colon, kidney, breast, adrenal cortex and liver.
  • compositions and kit of the present invention are not intended to be limited to any one particular cancer chemotherapeutic agent or combination of agents.
  • preferred chemotherapeutic agents include, but are not limited to, all hydrophobic, and heterocyclic cancer chemotherapeutic agents such as adriamycin (doxorubicin), phosphates, colcemid, etoposide, paclitaxel, bisantene, vincristine, and vinblastine.
  • compositions of the present invention allow for a "combination therapy" wherein the cholesterol absorption inhibitor and the selected chemotherapeutic agent are either co-administered in a substantially simultaneous manner, for example, in a single tablet or capsule having a fixed ratio of active ingredients or in multiple, separate administrations for each moiety.
  • This separate administration includes sequential dosage forms.
  • this "combined administration" encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single dosage form having a fixed ratio of active ingredients or in multiple, separate dosage forms for each agent.
  • administration also encompasses use of each type of agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in achieving one or more of the therapeutic goals outlined herein.
  • the present invention provides various means of achieving one or more of the following therapeutic goals: a) treating or alleviating a cancer; b) preventing, treating or alleviating tumour growth; c) inhibiting or reducing the expression of a multi-drug resistance gene; d) inhibiting or reducing the production of one or more proteins expressed by multiple drug resistance genes; e) enhancing the effectiveness of a chemotherapeutic agent in treating a cancer; and f) sensitizing a cell to one or more chemotherapeutic agents;
  • This invention further comprises the use of any of the disclosed compounds and compositions for any indications described herein, more specifically, for use in achieving one or more of the therapeutic goals as defined above.
  • WO0213815 discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a select compound (having formula I therein described) which itself has no pharmacological activity but which allegedly enhances the bioavailability of other active ingredients by inhibiting p-glycoprotein present in the intestinal wall. The enables the absorption of active ingredients (ex anti-cancer drugs) which are otherwise poorly absorbed.
  • WO02034291 discloses molecules which bind to the MDR-1 gene and selectively inhibit gene expression. Delivery systems for these drugs include non-polymer systems involving lipids. Also covered is a method of inhibiting MDR gene expression which comprises contacting a nucleic encoding MDR polypeptide with a HIF-1 SUMO-1 complex blocking agent.
  • WO0160349 discloses a pharmacological agent comprising picolinic acid, fusaric acid and derivatives thereof.
  • the fusaric acid component was found to be effective in controlling growth of cells with high levels p-glycoprotein. It is suggested that fusaric acid may have some role in the treatment of tumors that are resistant to MDR-associated drugs.
  • WO0220486 also by the same inventor, discloses similar information.
  • WO0048571 discloses pharmaceutical compositions comprising n-benzoyl- staurosporine and their use as an anti-tumour, anti-proliferative agents.
  • WO0226208 discloses tocol-based particulate emulsions for the delivery of chemotherapeutic agents.
  • the emulsion comprises one or more tocols, surfactants, an optional co-solvent and the chemotherapeutic agent.
  • US Patent Serial No. 5,639,887 describes various bicyclic amines that are effective in resensitizing multiple drug resistant cells to chemotherapeutic agents such as doxorubicin, vincristine and bisantrene.
  • US Patent Serial No. 5,670,507 teaches a method for reversing a MDR phenotype in tumors insensitive to hydrophobic chemotherapeutic drugs due to over expression of mdr-1 which comprising administering an effective amount of a long chain amino alcohol compound selected from the two disclosed formulae.
  • US Patent Serial No 5,866,699 discloses synthetic oligonucleotides having a nucleotide sequence complementary to at least a portion of the MDR-1 gene, or transcripts thereof, which portion encodes a nucleoside binding site. Also disclosed is a pharmaceutical formulation containing such oligonucleotides, and methods of treating MDR cancer cells, of preventing the expression of P170 in a cell, and of preventing the induction of MDR in a cancer cell.
  • US Patent Serial No. 5,874,567 teaches a modified oligonucleotide between 15 and 30 nucleotides in length, inclusive, having a sequence that specifically hybridizes in a human cell with a complementary sequence of a human MDR-1 gene and allelic variants thereof to inhibit expression of a multidrug resistance phenotype exhibited by the cell.
  • the complementary sequence is selected from the group consisting of SEQ ID Nos: 103, 104 and 105 (as described therein), the modification comprising a backbone modification selected from the group consisting of dithioate, methylphosphonate, morpholino, polyamide, or any combination thereof.
  • US Patent Serial No. 6,162,616 discloses compositions for use in attenuating aberrant MRP-beta. Gene expression, protein production and/or protein function including MRP-beta. Nucleic acids, including probes and antisense oligonucleotides, MRP-beta. Polypeptides and antibodies, MRP-beta. Expressing host cells, and non- human mammals transgenic or nullizygous for MRP-beta.
  • cholesterol absorption inhibitors there are additional advantages to the use of cholesterol absorption inhibitors in overcoming MDR.
  • Some of the cholesterol absorption inhibitors as described herein comprise an ascorbyl moiety. These particular compounds have numerous added advantages. First and foremost, solubility of the compounds is greatly enhanced, both in aqueous solutions and non-aqueous media such as oils and fats. With this greater solubility, effective therapeutic dosages and concomitantly costs, can be reduced. Secondly, these derivatives are heat stable (stable to oxidation and hydrolysis) which is essential for some processing mechanisms.
  • the desired effects described herein may be achieved in a number of different ways.
  • the cholesterol absorption inhibitors and chemotherapeutic agents may be administered by any conventional means available for use in conjunction with pharmaceuticals i.e. with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions can comprise from about 1% to 99% of the "active" components and preferably from about 5% to 95% of the active components.
  • compositions and pharmaceutical compositions can be prepared using conventional, pharmaceutically available excipients, and additives and by conventional techniques.
  • pharmaceutically acceptable excipients and additives include non- toxic compatible fillers, binders, disintegrants, buffers, preservatives, anti-oxidants, lubricants, flavourings, thickeners, colouring agents, emulsifiers and the like.
  • the exact amount or dose of the cholesterol absorption inhibitors which is required to achieve the desired effects will, of course, depend on a number of factors such as the particular compound or composition chosen, the potency of the compound or composition administered, the formulation in which it is administered, the mode of administration and the age, weight, condition and response of the patient. All of these factors, among others, will be considered by the attending clinician with respect to each individual or patient.
  • a total daily dose the cholesterol absorption inhibitor having one of formulae i)-viii) and comprising sterols and/or stanols may be administered in a daily dosage range of from 10mg to about 20 g, more preferably 10mg to 1.5g, and most preferably up to 800 mg per day in single or multiple divided doses.
  • the chosen a cholesterol absorption inhibitor is a sterol or stanol, whether free or as part of a compound or derivative, it may be administered in a form comprising up to 6 grams sterols and/or stanols per day. It should be recognized that the provision of much larger daily doses of sterols, stanols and their derivatives are not harmful to the animal host, as excess will simply pass through normal excretory channels.
  • the cholesterol absorption inhibitor is a substituted azetidinone
  • it may be administered in a daily dose of from about 0.1 to about 30 mg/kg of body weight, preferably about 0.1 to about 15 mg/kg, and most preferably up to 10mg/kg per day.
  • the dosage level is therefore from about 5 mg to about 1000 mg of drug per day, given preferably in a single dose or 2-4 divided doses.
  • the cholesterol absorption inhibitor is any compound which inhibits bile acid reabsorption, it may be administered in a dose from about 0.003mg — 20mg per kilogram body weight of the individual animal.
  • a total daily dose of an IBAT inhibitor can be in the range of from about 0.01 to about 1000 mg/day, preferably from about 0.1 mg to about 50 mg/day, more preferably from about 1 to about 10 mg/day.
  • the cholesterol absorption inhibitor and chemotherapeutic agent are administered separately, the number of doses and the amount of such dosage of each component given per day may not necessarily be the same. For example, it is possible that the cholesterol absorption inhibitor may require either a greater number of administrations per day than the chemotherapeutic agent and/or may require a larger dosage.
  • the daily dose of these cholesterol absorption inhibitors can be administered to an individual in a single dose or in multiple doses, as required. Sustained release dosages can be used.
  • compositions of the present invention may be administered parenterally, such as by intravenous injection.
  • pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds and compositions of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • compositions comprising one or more of the compounds of the present invention, include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • the cholesterol absorption inhibitor is administered in the form of a liposome.
  • Liposomes are hollow microspheres composed of one or more double lipid layers. They were first used more than 30 years ago as vehicles for various drug substances, and since then knowledge of their behavior in vitro has allowed a more rational design focused on the specific treatment of certain diseases.
  • Liposomes occurs formed when thin lipid films are hydrated.
  • the hydrated lipid sheets detach during agitation and self-close to form multi-lamellar vesicles.
  • Chemotherapeutic agents such as doxorubicin
  • doxorubicin are often encapsulated in liposomes using the established methods.
  • 100 nm diameter liposomes are prepared by exposing chloroformic solution of various lipid mixtures to high vacuum and subsequently hydrating the resulting lipid films (DSPC/CHOL, EPC/CHOL, DSPC/PEG-PE/CHOL) with pH 4 buffers, and extruding them through polycarbonated filters, after a freezing and thawing procedure.
  • a transmembrane pH gradient is then created by adjusting the pH of the extravesicular medium to 7.5 by addition of an alkalinization agent.
  • the selected drug is then entrapped by addition of the drug solution in small aliquots to the vesicle solution, at an elevated temperature, to allow drug accumulation inside the liposomes.
  • Trapping efficiencies are determined by separating free from liposome encapsulated drug on gel filtration columns and quantifying the two fractions for lipid and drug content by liquid scintillation counting, fluorescence spectroscopy or UV-VIS spectroscopy. These liposomes are then evaluated for size distribution (quasielastic light scattering, scanning electron microscopy), drug uptake and release studies, stability, and in vivo tumor targeting efficiency.
  • compositions of the present invention may be manufactured in any manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients include lactose, sucrose, mannitol, sorbitol, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum Dhosph, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p- hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, syrup, methyl cellulose
  • kits for such purpose.
  • a kit is contemplated wherein two separate units are combined: a pharmaceutical composition comprising at last one cholesterol absorption inhibitor, as described herein, and a separate pharmaceutical composition comprising at least one cancer chemotherapeutic agent.
  • the kit will preferably include directions for the administration of the separate components. This type of kit arrangement is particularly useful when separate components must be administered in different dosage forms (for example, oral vs. parenteral vs. intravenous) or are administered at different dosage intervals or are administered at different dosage amounts.
  • EXAMPLE 1 Formation of one of the cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester referred to herein as "FM-VP4";
  • Step 2 Attachment to Phytostanols
  • EXAMPLE 2 Formation of one of the cholesterol absorption inhibitors described herein: Disodium ascorbyl phosphate ester of dehydroisoandrosterone To a dry round bottom flask, acetone (150 ml) and L-ascorbic acid (50 g) were added at 0 °C. Acetyl chloride (7.5 ml) was added dropwise through an addition funnel in 10 minutes. The reaction mixture was stirred at 0 °C for 24 hours. The precipitate was filtered off and washed with acetone (3x20 ml). The white product, 5,6-isopropylidine ascorbic acid, was dried under vacuum for 1.5 hours to give a dry powder (52 g), yield 85%.
  • a dry three neck round bottom flask was fitted with a stirring bar, argon inlet and an addition funnel.
  • a solution of dehydroisoandrosterone (1.73 g, 6 mmol) in anhydrous THF (15 ml) and pyridine (2.4 ml) was added dropwise to the mixture of anhydrous THF (12 ml) and POCI 3 (0.7 ml, 7.5 mmol) at 0 °C over a period of 10 minutes.
  • a white precipitate formed immediately.
  • the suspension was stirred at 0 °C for 40 minutes, and at room temperature for 1 hour and 40 minutes.
  • Ascorbyl phosphate ester of dehydroisoandrosterone (0.5 g, 0.95 mmol) was dissolved in methanol (3 ml) at room temperature, and then sodium methoxide in methanol (1ml, 20%) was added. The suspension was stirred at room temperature for 30 minutes. The precipitated solid was filtered out, washed with methanol, acetone and hexanes. The mother liquor was concentrated to 2 ml, acetone was added to precipitate the product. An additional white solid was obtained. The combined solid was dried under vacuum at room temperature. Disodium ascorbyl phosphate ester of dehydroisoandrosterone (0.49 g, yield 91%) was obtained.
  • Ascorbyl phosphate ester of 5 ⁇ -androstan-3 ⁇ -ol-17-one (0.5 g, 0.95 mmol) was dissolved in methanol (3 ml) at room temperature, and then sodium methoxide in methanol (1.5 ml, 20%) was added. The suspension was stirred at room temperature for 25 minutes. The precipitated solid was filtered out, washed with methanol, acetone and hexanes. The mother liquid was concentrated to 2 ml, and then acetone was added to precipitate the product. An additional product was obtained. The combined solid was dried under a reduced pressure at room temperature to give disodium ascorbyl phosphate ester of 5 ⁇ -androstan-3 ⁇ -ol-17- one (0.38 g). The overall yield was 57% (based on 5 ⁇ -androstan-3 ⁇ -ol-17-one).
  • EXAMPLE 4 Synthesis of another of the cholesterol absorption inhibitors described herein: Disodium Ascorbyl Phosphate Ester of Androst-5-ene-3 ⁇ ,17 ⁇ -diol
  • Instrument is Waters Delta Preparative 4000 HPLC system. Column is Waters Symmetry C18, 5 ⁇ m, 30x100 mm. Mobile phases are 0.1% H 3 PO 4 in water and acetonitrile. Water and acetonitrile are HPLC grade or equivalent.
  • the crude product was purified by preparative HPLC. The product was collected and evaporated on a rotary evaporator to remove acetonitrile. The water solution was extracted with ethyl acetate twice. The ethyl acetate layer was dried over Na 2 SO 4 , concentrated and dried under a reduced pressure to give a white powder product. This product was submitted for NMR and mass spectra. Both spectra indicated the product is ascorbyl phosphate ester of androst-5-ene-3 ⁇ ,17 ⁇ -diol.
  • the crude phosphate ester was dissolved in methylene chloride (25 ml), and treated with iodine (1.27 g) for 4 hours at room temperature.
  • the reaction mixture was diluted with methylene chloride (75 ml), washed with 1 N NaOH (2x50 ml) and water (2x50 ml), and dried over Na 2 SO 4 .
  • the solvent was removed, and the product (1.4 g, yield 71%) was crystallized from methylene chloride and methanol.
  • 3 ⁇ -lodoandrost-5-ene-17-one (1.27 g, 3.19 mmol) was dissolved in glacial acetic acid (40 ml) at 50-55 °C, the activated zinc dust (2.7 g) was added in one portion. The mixture was stirred at 50 °C ⁇ 55 °C for 2 hours, the zinc dust was filtered out and washed with methylene chloride. The solution was diluted with methylene chloride (120 ml), washed with water (2x100 ml), 1N NaOH (2x100 ml) and water (100 ml), and dried over Na 2 SO 4 . The solvent was removed to afford a white powder. The white powder was dried under vacuum to give androst-5-ene-17-one (0.83 g, yield: 95%).
  • the crude diascorbyl diphosphate ester of androst-5-ene-3 ⁇ ,17 ⁇ -diol (400 mg) was dissolved in methanol (5 ml). To this solution was added 2 ml of sodium methoxide in methanol (20%, w/v) under magnetic stirring. White precipitate was observed upon the addition of sodium methoxide methanol solution. The suspension was stirred for half an hour before it was filtered and washed with methanol and acetone. The solid product was dried under high vacuum, and tetrasodium diascorbyl diphosphate ester of androst-5-ene-3 ⁇ ,17 ⁇ -diol (330 mg) was obtained.
  • EXAMPLE 8 Measuring MDR-1 Expression in CaCo2 cells after treatment with FM-VP4 for one week
  • Confluent CaCo2 cells (P33-39) in T-75 flasks were treated with varying amounts of FM-VP4 (0.5um, 1.0um, 5.0um and 10.0uM) for one week. The media and treatment were changed every other day. After seven days, cells were harvested and total RNA isolated with TRIZOLTM. Analysis conducted by reverse transcription ⁇ cDNA using polymerase chain reaction.
  • PCR Program 94°C for 5 minutes, 94°C for 1 minute, 55°C for 1 minutes, 72°C for I minute 30 seconds, repeat from step 2 for 29 more times, 72°C for 10 minutes, 4°C.
  • Primer drop at cycle 0,6,9,12,16,20,23,26,28 and 30.
  • Figure 1 is a graph showing these results, from which it is clear that with increasing concentrations of the cholesterol absorption inhibitor, MDR-1 gene expression is significantly reduced. Further supporting these results are shown in Figure 2, a graph showing the titration for primer drop of GAPDH and Figure 3 depicting polymerase chain reaction (1.5% agarose gel) electrophoretic plate results for MDR-1 and GAPDH.
  • Example 8 The entire protocol as provided in Example 8 with respect to the MDR-1 gene was used to confirm the effects of a cholesterol absorption inhibitor on the expression of another multiple drug resistance gene: MRP-1.
  • Caco2-cells were seeded into 48- or 96-well plates. The growth media was aspirated every 2-3 days and replaced with fresh media. The cells were kept at 37°C in a humidified atmosphere of 5% CO 2 . Cells then were treated with media, 0.1% Triton X-100 (as positive control for toxicity), FM-VP4, the selected cholesterol absorption inhibitor. After 24 hours 50ml of each well were transferred into a new 96-well plate to perform the LDH-Assay. The MTS-Assay (CellTiter 96 AQueous One Solution kit (PromegaTM) was done on the original plate.
  • MTS-Assay CellTiter 96 AQueous One Solution kit (PromegaTM) was done on the original plate.
  • MTS-Assay is a colorimetric method for determining the number of viable cells (cytotoxicity) after treatment.
  • MTS reagent a tetrazolium salt
  • a color compound formazan
  • BCA Protein Assay This particular colorimetric assay measures total protein levels by identifying specific peptide bonds. The protein concentration is calculated from a calibration curve constructed with a protein standard (bovine serum albumin or "BSA").
  • the LDH-assay looks at the integrity of cell membranes and can be used for cytotoxicity mediated by chemicals or other agents.
  • Cell damage is associated with leakage of intracellular, cytoplasmic contents and lactate dehydrogenase or "LDH" (a stable cytosolic enzyme), can be used as a reported molecule for this event.
  • LDH lactate dehydrogenase
  • LDH released from cells into the culture medium, was measured using a kit (Cytotox96 Non-Radioactive Cytotoxicity Assay) from PromegaTM. This method is based on a series of linked enzyme reactions, the final reaction being the reduction of a tetrazolium salt to a coloured, insoluble, formazen product which can be measured at 492nm. Background absorbance from media alone and media including the treatment was substracted from the reading to correct the values.
  • FIG 4 is a bar graph of the MTS- and LDH-Assay of cell viability after treatment of CaCo2 with "FM-VP4".
  • Figure 5 a bar graph showing a BCA-Assay of protein concentration after treatment of CaCo2 with "FM-VP4".
  • the cholesterol absorption inhibitor tested, FM-VP4 does not show any toxicity (mitochondrial activity) for a concentration range up to 10OmM regardless of whether the treatment was for 24 hours (cell viability is 90.9 +/- 9.8%) or 96 hours (94.6 +/- 4.4%).
  • the highest tested concentration of FM-VP4 75mM showed a cell viability of 18.5+7-5.8% after 24 hours and 22.1+/-1.6%.
  • EXAMPLE 11 Western Blot analysis of P-glycoprotein in Caco2-cells after incubation with FM-VP4 ( ⁇ Protein expression)
  • FIG. 9 is a Western Blot analysis of P-glycoprotein in CaCo2 cells after incubation with the selected cholesterol absorption inhibitor: "FM-VP4".
  • FM-VP4 cholesterol absorption inhibitor
  • Figure 7 depicts a polymerase chain reaction (1.5% agarose gel) electrophoresis results for MDR-1 , GAPDH; RNA isolation with TRIZOL ⁇ RT-PCR ⁇ PCR after treatment of CaCo2 cells with liposomal formulations of one of cholesterol absorption inhibitors described herein: an ascorbyl stanyl phosphate ester called "FM-VP4;
  • Figure 8 is a bar graph showing the level of MDR-1 expression (normalized ratio of MDR-1/GAPDH) in CaCo2 cells after treatment for one week with liposomal FM-VP4 at 2.5, 5 and 10um as compared to a control and empty liposomes.
  • MDR-1 gene expression in Caco2-cells after treatment with liposomal FM-VP4 at lower concentrations is significantly reduced and at lower concentrations than non-liposomal formulations.
  • the dry lipid film was stored by capping it and placing it in the freezer.
  • the water bath was set to 55°C.
  • Caco-2 cells were seeded at 10,000 cells/cm 2 in T-75 flasks (Corning).
  • the growth media (Dulbecco's minimal essential medium-DMEM) containing 10% heat-activated fetal bovine serum, 292 ⁇ g/ml glutamine, 0.1 mM non- essential amino acids, 100U/ml penicillin and 100mg/ml glutamine was changed every other day.
  • the cells were kept at 37°C in a humidified atmosphere of 5% CO 2 .
  • Cells were treated with 10 ⁇ M "FM-VP4", as descbribed above, solubilized in water when they reached about 95% confluency. The control group only got media.
  • FIGS 10 and 11 Expression profile of mdr-1 gene in Caco-2 cells was examined as an effect of a time-dependent treatment with 10DM FM-VP4. A sample from each PCR product was subjected to electrophoresis on a 1.5% agarose gel (10. The fluorescent bands were imaged under UV light (UV-Epi Chem II) and quantified with the UVP-Labworks software (11).
  • EXAMPLE 15-Effect of Cholesterol Absorption Inhibitors on cellular accumulation of Rhodamine 123 by Caco-2 cells Objective: To investigate the accumulation of Rhodamine which is an index for the activity of the multidrug efflux transporter P-glycoprotein (P-gp). Lower P-gp activity leads to an accumulation of Rhodamine 123 in the cells.
  • P-gp multidrug efflux transporter
  • Rhodamine 123 is a P-gp substrate (fluorescent dye) and has been used as a probe substrate to measure the functional activity of P-gp.
  • the excitation wavelength is 485nm, the emission wavelength 520nm.
  • Cells were incubated with "FM-VP4", as described herein, and Rhodamine 123 is added to the basolateral side. After lysis of the cells the content of Rhodamine 123 was determined.
  • Caco-2 cells were seeded on 12mm diameter polycarbonate filter inserts (Transwell, Costar) at a densitiy of 40.000 cells/cm 2 .
  • the growth media (Dulbecco's minimal essential medium-DMEM) containing 10% heat-activated fetal bovine serum, 292 ⁇ g/ml glutamine, 0.1 mM non-essential amino acids, 100U/ml penicillin and 100mg/ml glutamine was changed every other day.
  • the cells were kept at 37°C in a humidified atmosphere of 5% CO 2 .
  • FM-VP4 at different concentrations were added to the media and applied onto the apical and basolateral side of the Transwell plate.
  • Transepithelial electrical resistance (TEER) of the monolayers was measured to confirm monolayer integrity.
  • Caco-2 cells with Teer Values above 400 ⁇ /cm 2 were then used for P-gp studies.
  • the membrane was cut out, transferred into a 1.5ml Eppendorf tube and 250 ⁇ l of 1% Triton X-100 was added and vortexed. After 10 minutes at room temperature and a centrifugation step for 5 min to remove cell debris, 25 ⁇ l (in triplicates) were transferred into a 96-well plate and the fluorescence (excitation: 485nm; emission: 530nm) was measured. Finally the protein content was determined using a BCA Protein Assay (PIERCE) and Rhodamine 123 accumulation was normalized.
  • PIERCE BCA Protein Assay
  • Rh123 in Caco-2 monolayer Data represents the average of ⁇ SD. *P ⁇ 0.002;
  • the positive control for P-gp inhibition with 100 ⁇ M Verapamil results in an accumulation of 1604 pmol Rhodamine 123 per mg protein.
  • Enhanced cellular accumulation of a P-gp substrate Rhodamine 123 and therefore a decreased P-gp activity using FM-VP4 concentrations above 5 ⁇ M for 7days can be observed.
  • Caco-2 cells express P-gp on their apical membranes. A good system to measure apical and basolateral transport across monolayers and to determine the permeability of a certain substance is a Transwell Plate. Cells are grown on a semi-permeable membrane which is placed between two chambers. Caco-2 cells differentiate into highly functionalized epithelial barrier. Morphological and biochemical it is similar to the small intestinal columnar epithelium.
  • Rhodamine 123 has been used as a probe to measure the functional activity of P-gp. It is a fluorescent dye. Rhodamine 123 has a molar extinction coefficient of 85,200 M "1 cm "1 at 511nm.
  • Caco-2 cells were seeded on 12mm diameter polycarbonate filter inserts (Transwell, Costar).
  • the growth media (Dulbecco's minimal essential medium-DMEM) containing 10% heat-activated fetal bovine serum, 292 ⁇ g/ml glutamine, 0.1 mM non- essential amino acids, 100U/ml penicillin and 100mg/ml glutamine was changed every other day.
  • the cells were kept at 37°C in a humidified atmosphere of 5% CO 2 .
  • FM-VP4 at different concentrations was added to the media and applied onto apical and the basolateral side of the Transwell plate for one week.
  • the Control well contained only media.
  • a positive control for inhibition of P-gp was Verapamil (calcium channel blocker).
  • Transepithelial electrical resistance (TEER) of the monolayers was measured to confirm monolayer integrity.
  • Caco-2 cells with TEER Values above 400 ⁇ /cm 2 were then used for transport studies.
  • 5 ⁇ M Rhodamine was added into the basolateral chamber. In a time dependent manner samples were taken from the apical side and the fluorescence was determined. Immediately the samples were replaced by fresh receiver medium (media, media plus FM-VP4 or media plus P-gp inhibitor).

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Abstract

Dans un aspect, la présente invention concerne une méthode destinée à inhiber l'expression d'un gène de multirésistance aux médicaments chez un animal, et consistant à administrer à cet animal une dose efficace d'au moins un inhibiteur de l'absorption du cholestérol. Dans un autre aspect, elle concerne une méthode destinée à inhiber la production d'une protéine exprimée par un gène de multirésistance aux médicaments dans une cellule d'un animal, et consistant à administrer à cet animal une dose efficace d'au moins un inhibiteur de l'absorption du cholestérol. Dans un autre aspect, la présente invention concerne une méthode destinée à augmenter l'efficacité d'un agent chimiothérapeutique chez un animal atteint d'un cancer, et consistant à administrer à cet animal une dose efficace de cet agent chimiothérapeutique et d'au moins un inhibiteur de l'absorption du cholestérol. En outre, l'invention concerne des compositions et des trousses destinées au traitement du cancer et comprenant au moins un agent chimiothérapeutique et au moins un inhibiteur de l'absorption du cholestérol.
EP04761853A 2003-09-26 2004-09-27 Methode destinee a inhiber l'expression de genes de multiresistance aux medicaments et a inhiber la production de proteines resultant de l'expression de ces genes en vue d'ameliorer l'efficacite d'agents chimiotherapeutiques pour le traitement des cancers Withdrawn EP1677803A2 (fr)

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WO2007001975A1 (fr) 2005-06-20 2007-01-04 Schering Corporation Dérivés de la pipéridine utiles comme antagonistes du récepteur h3 de l'histamine
CN102027135B (zh) * 2008-03-14 2015-04-22 健泰科生物技术公司 与药物抗性有关的遗传变异
CN102552284A (zh) * 2011-12-13 2012-07-11 陕西师范大学 麦角甾醇在制备肿瘤多药耐药逆转药物中的应用
MX2016010998A (es) 2014-02-27 2017-03-31 Lycera Corp Terapia celular adoptiva que usa un agonista de receptor huérfano gamma relacionado con receptor de ácido retinoico y métodos terapéuticos relacionados.
CA2947290A1 (fr) 2014-05-05 2015-11-12 Lycera Corporation Sulfonamide de tetrahydroquinoline et composes apparentes destines a servir d'agonistes de rory et pour le traitement de maladies
JP2018515491A (ja) 2015-05-05 2018-06-14 リセラ・コーポレイションＬｙｃｅｒａ Ｃｏｒｐｏｒａｔｉｏｎ ＲＯＲγの作動薬及び疾患の療法として使用するジヒドロ−２Ｈ−ベンゾ［ｂ］［１，４］オキサジンスルホンアミド及び関連化合物
KR20180025894A (ko) 2015-06-11 2018-03-09 라이세라 코퍼레이션 Rory의 작용제로서 사용하기 위한 아릴 디히드로-2h-벤조[b][1,4]옥사진 술폰아미드 및 관련 화합물 및 질환의 치료
CN106420768B (zh) * 2015-08-13 2018-12-28 广州中医药大学第二附属医院 赪酮甾醇作为p-糖蛋白抑制剂的应用

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WO2004010948A2 (fr) * 2002-07-30 2004-02-05 Karykion Inc. Compositions d'ezetimibe, et procedes pour le traitement de tumeurs benignes et malignes associees au cholesterol

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CN1874780A (zh) 2006-12-06
BRPI0414812A (pt) 2006-11-14
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KR20060135615A (ko) 2006-12-29
RU2006114049A (ru) 2007-11-20

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