EP1189607A2 - Activite anti-tumeur de derives de vitamine e, de cholesterol, de taxol et d'acide betulinique - Google Patents

Activite anti-tumeur de derives de vitamine e, de cholesterol, de taxol et d'acide betulinique

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Publication number
EP1189607A2
EP1189607A2 EP00923141A EP00923141A EP1189607A2 EP 1189607 A2 EP1189607 A2 EP 1189607A2 EP 00923141 A EP00923141 A EP 00923141A EP 00923141 A EP00923141 A EP 00923141A EP 1189607 A2 EP1189607 A2 EP 1189607A2
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EP
European Patent Office
Prior art keywords
ester
ether
cancer
anionic
tris
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Withdrawn
Application number
EP00923141A
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German (de)
English (en)
Inventor
Marc Fariss
J. Doyle Smith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Washington
Virginia Commonwealth University
Washington State University Research Foundation
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University of Washington
Virginia Commonwealth University
Washington State University Research Foundation
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Publication of EP1189607A2 publication Critical patent/EP1189607A2/fr
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Classifications

    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention generally relates to the use of derivatives of Vitamin E (tocopherol and tocotrienol), cholesterol, taxol and betulinic acid as antitumor agents for the treatment of and prevention of cancers of the liver, lung, colon, prostate and breast as well as melanomas and leukemias.
  • Vitamin E tocopherol and tocotrienol
  • cholesterol tocotrienol
  • taxol tocotrienol
  • betulinic acid as antitumor agents for the treatment of and prevention of cancers of the liver, lung, colon, prostate and breast as well as melanomas and leukemias.
  • TS ⁇ -tocopheryl hemisuccinate, free acid
  • CS cholesteryl hemisuccinate
  • CSE cholesteryloxybutyric acid
  • TS, TSE, CS and CSE have been shown to be cytotoxic for several tumor cell types such as leukemias and breast (hormone-dependent), the activity of related compounds in other tumor cell types is unknown and cannot be predicted from previous studies.
  • TS, TSE, CS or CSE treatments do not appear to adversely affect some cell types such as murine-derived bone marrow cells (6) and numerous other normal cell types (2,3,13).
  • d- ⁇ - Tocopheryl 2,2-dimethyl glutarate d- ⁇ -Tocopheryl 2,2-dimethyl glutarate.
  • d- ⁇ -Tocopheryl 2.2-dimethyl succinate d-Tocopheryl ⁇ - dimethyl aminobutyrate methiodide, d- ⁇ -Tocopheryl ⁇ -dimethyl aminobutyrate, d- ⁇ -Tocopheryl succinate polyethylene glycol 1000, tocotrienol rich fraction succinate ester, tocotrienol rich fraction succinate tris salt, d- ⁇ -Tocopheryloxybutyric acid polyethylene glycol 1000, ⁇ - Tocotrienol succinate, cholesteryl succinate tris salt, cholesteryl succinate polyethylene glycol 1000, ⁇ -cholesteryloxybutyric acid tris salt, cholesterol hydrogen phthalate, taxol monosuccinate tris salt, taxol disuccinate tris salt, and betulinic acid succinate.
  • the derivatives of the present invention may be used alone or in combination with one another, or with other antitumor agents.
  • d- ⁇ - tocopheryl 2.2-dimethylglutarate d- ⁇ -tocopheryl 2.2 dimethyglutarate tris salt: d- ⁇ -tocopheryl ⁇ -(N,N-dimethylamino)ethyl ether (free base); d- ⁇ -tocopheryl ⁇ -(N,N-dimethylamino)ethyl ether oxalate salt; and d- ⁇ -tocopheryl ⁇ -N.N-(dimethylamino)ethyl ether methiodide.
  • FIG. 1A-1F Effect of indicated compounds on the growth of Hep3B liver cells.
  • Fig.lA ⁇ -TS (•) and ⁇ TS (O);
  • Fig.lB TSE;
  • Fig.lC ⁇ -TS-tris;
  • Fig.lD ⁇ -T-MS (•) and TS (O);
  • Fig.lE T-DMAB-Q;
  • Fig.lF ⁇ -T.
  • Figure 2A-2F Effect of indicated compounds on the growth of Hep3B liver cells.
  • Fig.2A ⁇ - T
  • Fig.2B PEG 1000:
  • Fig.2C T-P-Na :
  • Fig.2D TS-PEG
  • Fig.2E TSE-PEG:
  • Fig.2F TRF.
  • FIG.3A-3F Effect of indicated compounds on the growth of Hep3B liver cells.
  • Fig.3A TRF-S-T
  • Fig.3B TRF-S
  • Fig.3C Rice bran oil
  • Fig.3D Palm oil
  • Fig.3E ⁇ -T3
  • Fig.3F ⁇ - T3 succinate.
  • FIG.4A-4F Effect of indicated compounds on the growth of Hep3B liver cells.
  • Fig.4A ⁇ - T3 acetate:
  • Fig.4B CS-tris (•) and CSE-tris (O);
  • Fig.4C CS-PEG;
  • Fig.4D Choles-h-p;
  • Fig.4E Btl-acid succinate;
  • Fig.4F Btl-acid.
  • Figure 5A-5F Effect of indicated compounds on the growth of Hep3B liver cells.
  • Fig.5A TS-tris
  • Fig.5B TSE-tris
  • Fig.5C CSE-tris: Fig.5D: 2.2-Dim-TS
  • Fig.5E T-DMAB-Q
  • Fig.5F T-DMAB-T.
  • Figure 7A-7F Effect of indicated compounds on the growth of Dul45 prostate cancer cells.
  • Fig.7A ⁇ T-MS (O) and ⁇ TS ( ⁇ );
  • Fig.7B TSE;
  • Fig.7C T-DMAB-Q;
  • Fig.7D PEG 1000;
  • Fig.7E TS-PEG;
  • Fig.7F TSE-PEG.
  • FIG.8A TRF
  • Fig.8B TRF-S
  • Fig.8C ⁇ -T3
  • Fig.8D ⁇ -T3-S
  • Fig.8E CS-tris
  • Fig.8F CS- PEG.
  • Figure 9A-9D Effect of indicated compounds on the growth of Dul45 prostate cancer cells.
  • Fig.9A TS-tris
  • Fig.9B 2,2-Dim-TS
  • Fig.9C T-DMAB-T
  • Fig.9D choles-h-p.
  • Figure 10A-10F Effect of indicated compounds on the growth of NIH-H69 small cell lung cancer cells.
  • Fig.lOA ⁇ -T-MS
  • Fig.lOB ⁇ -TS-tris
  • Fig.lOC ⁇ -TS
  • Fig.lOD ⁇ TS
  • Fig.lOE TSE: Fig.lOF: PEG 1000.
  • Figure 11A-11F Effect of indicated compounds on the growth of NIH-H69 small cell lung cancer cells.
  • Fig.llA ⁇ -T
  • Fig.llB T-P-Na.
  • Fig.llC ⁇ -T
  • Fig.llD T-DMAB-Q
  • Fig.llE TSE-PEG
  • Fig.llF TS-PEG.
  • FIG. 12A-12E Effect of indicated compounds on the growth of NIH-H69 small cell lung cancer cells.
  • Fig.l2A TRF-S-T:
  • Fig.l2B TRF-S;
  • Fig.l2C TRF;
  • Fig.l2D Rice bran oil;
  • Fig.l2E Palm oil.
  • Figure 13A-13F Effect of indicated compounds on the growth of NIH-H69 small cell lung cancer cells.
  • Fig.l3A ⁇ -T3
  • Fig.l3B ⁇ -T3-S
  • Fig.l3C ⁇ -T3 acetate
  • Fig.l3D CSE-tris
  • Fig.l3E CS-PEG
  • Fig.l3F CS-tris.
  • Figure 14A-14E Effect of indicated compounds on the growth of NIH-H69 small cell lung cancer cells.
  • Fig.l4A TS-tris: Fig.l4B: TSE; Fig.l4C: T-DMAB-T; Fig.l4D: choles-h-p; Fig.l4E: 2,2-Dim-TS.
  • Figure 15A-15F Effect of indicated compounds on the growth of HT29 colon cancer cells.
  • Fig.l5A ⁇ -T-MS
  • Fig.l5B TS
  • Fig.l5C 2,2-Dim-TG
  • Fig.l5D TSE
  • Fig.l5E PEG 1000
  • Fig.l5F TS-PEG.
  • Figure 16A-16E Effect of indicated compounds on the growth of HT29 colon cancer cells.
  • Fig.l6A TRF (•) and TRF-S (O);
  • Fig.lOB ⁇ -T3 (•) and ⁇ -T3-S (O);
  • Fig.lOC CSE-PEG;
  • Fig.l6D CS-PEG;
  • Fig.lOE CS-tris.
  • Figure 17A-17F Effect of indicated compounds on the growth of HT29 colon cancer cells.
  • Fig.l7A TS-tris
  • Fig.l7B TSE-tris
  • Fig.l7C CSE-tris
  • Fig.l7D T-DMAB-Q
  • Fig.l7E T-DMAB-T
  • Fig.l7F choles-h-p.
  • FIG. 18A TSE-PEG: Fig. 18B: 2.2-Dim-TS.
  • FIG.l9A PEG-1000
  • Fig.l9B ⁇ -T-MS (•) and TS (O)
  • Fig.l9C TSE
  • Fig.l9D TRF-S (•) and TRF (O)
  • Fig.l9E TSE-PEG (•) and TS-PEG (O).
  • FIG.20A Effect of indicated compounds on the growth of MCF-7 breast cancer cells.
  • Fig.20A CS-tris;
  • Fig.20B CS-PEG;
  • Fig.20C ⁇ -T3-S (•) and ⁇ -T3 (O).
  • FIG.21A TRF-tris
  • Fig.21B TRF
  • Fig.21C TS-tris
  • Fig.21D TS.
  • FIG.22A-2D Effect of indicated compounds on the growth of MDA-231 breast cancer cells.
  • Fig.22A TSE
  • Fig.22B TS-PEG
  • Fig.22C 2.2-Dim-TS
  • Fig.22D T-DMAB-T.
  • Figure 23A-23D Effect of indicated compounds on the growth of HL-60 leukemia cancer cells.
  • Fig.23A ⁇ -T3 (•), ⁇ -T3-S (O), ⁇ -T3-S-tris (T) and ⁇ -T3-acetate (V);
  • Fig.23B TRF (•), TRF-S (O) and TRF-S-T (T);
  • Fig.23C Choles-h-p;
  • Fig.23D T-DMAB-T.
  • Figure 24A-24D Effect of indicated compounds on the growth of A375 cutaneous melanoma cancer cells.
  • Fig.24A ⁇ -T-MS (•) and TS (O);
  • Fig.24B TSE;
  • Fig.24C TRF (•) and TRF- S (O);
  • Fig.24D TS-PEG (•) and TSE-PEG (O).
  • FIG.25A Effect of indicated compounds on the growth of A375 cutaneous melanoma cancer cells.
  • Fig.25A CS-PEG;
  • Fig.25B CS-tris:
  • Fig.25C ⁇ -T3-S (•) and ⁇ -T3 (O).
  • Figure 26A-26D Effect of indicated compounds on the growth of A375 cutaneous melanoma cancer cells.
  • Fig.26A TS-tris;
  • Fig.26B TSE-tris;
  • Fig.26C 2.2-Dim-TS;
  • Fig.26D T-DMAB- T.
  • Figure 27A-27E Effect of indicated compounds on the growth of OCM1 ocular melanoma cancer cells.
  • Fig.27C 2,2-Dim-TG;
  • Fig.27D TS-PEG;
  • Fig.27E TSE-PEG.
  • Figure 28A-28F Effect of indicated compounds on the growth of OCM1 ocular melanoma cancer cells.
  • Fig.28A T-DMAB-Q
  • Fig.28B PEG 100 ⁇ l
  • Fig.28C ⁇ -T3-S
  • Fig.28D ⁇ -T3
  • Fig.28E TRF
  • Fig.28F TRF-S.
  • Figure 29A-29F Effect of indicated compounds on the growth of OCM1 ocular melanoma cancer cells.
  • Fig.29A 2.2-Dim-TS
  • Fig.29B Btl-acid (•) and Btl-acid succinate (O)
  • Fig.29C T-DMAB-T
  • Fig.29D CS-tris
  • Fig.29E CS-PEG
  • Fig.29F Choles-h-p.
  • FIG.30A Effect of indicated compounds on the growth of OCM1 ocular melanoma cancer cells.
  • Fig.30A TS-tris
  • Fig.30B TSE-tris
  • Fig.30C T-DMAB-T
  • Fig.30D T-DMAB-Q.
  • FIG.31A Effect of indicated compounds on the growth of OCM1 ocular melanoma cancer cells.
  • Fig.31A CSE-tris;
  • Fig.31B choles-h-p;
  • Fig.31C 2.2-Dim-TS.
  • Figure 32A-32D Effect of indicated compounds on the growth of OCM1 ocular melanoma cancer cells.
  • Fig.32A TS-tris suspension (•) and TS-tris pellet (O);
  • Fig.32B Taxol (•), Taxol 50xTS-tris (O) and Taxol 50xTS-tris pellet (T);
  • Fig.32C Taxol-S (•), Taxol-S with 50xTS-tris (O) and Taxol-S with 50xTS-tris pellet ( ⁇ );
  • Fig.32D Taxol-DS (•), Taxol-DS 50xTS-tris (O), and Taxol-DS 50xTS-tris pellet (T).
  • FIG. 33A-33E Compounds representing new compositions of matter.
  • 33A 2.2-Dim-TG; 33B: 2,2-Dim-TG-tris;
  • 33C T-DMAE-ether;
  • 33D T-DMAE-ether oxalate;
  • 33E T-DMAE- Q-ether methiodide.
  • the tris salt of TSE did not improve the antitumor activity of TSE towards human melanoma cells.
  • the succinate ester of betulinic acid was a potent antitumor agent for cutaneous melanomas but not for other tumor cell types.
  • Our findings suggest that the active antitumor agents studied in these experiments are probably active against most tumor cell lines.
  • the tocopherol amine containing compounds e.g. T- TDAB-T. T-TDAB-Q, and T-TDAE-ether).
  • the PEG ester compounds, the taxol succinate Tris compounds and choles-h-p were the most potent compounds in terms of cytotoxic abilities toward human tumor cell lines derived from liver, lung, colon, prostate, breast, cutaneous melanoma, ocular melanoma and leukemia.
  • selectivity there is also some basis for selectivity in that certain compounds may prove advantageous with respect to the treatment of certain types of cancer.
  • specific examples of compounds that are the most effective in inhibiting specific tumor cell growth are summarized in each Example.
  • Vitamin E is a generic term that includes, in nature, eight substances, d- ⁇ -. d- ⁇ -, d- ⁇ -, d- ⁇ -tocopherol and d- ⁇ -. d- ⁇ -, d- ⁇ -. d- ⁇ -tocotrienol.
  • Succinate esters for each of these substances can be made.
  • a succinate ester of TRF was prepared (TRF-S) in the form of both a free acid and a tris salt.
  • TRF-S is a mixture of vitamin E compounds containing the succinate ester of d- ⁇ - tocopherol (28%) and d- ⁇ - (35%), d- ⁇ -(22%), and d- ⁇ -tocotrienol (16%).
  • the presence of the succinate ester of each of these vitamin E derivatives in the TRF-S preparation was confirmed by HPLC analysis using base hydrolysis.
  • Our findings as shown in the examples below indicate that a mixture of tocopherol and tocotrienol esters (as. for example, TRF-S-tris or TRF-S free acid) are potent antitumor agents.
  • Implementation of the claimed invention will generally involve identifying patients suffering from tumors and administering the compounds in an acceptable form by an appropriate route.
  • the dosage to be administered may vary, depending on the age, gender, weight and overall health status of the individual patient, as well as the nature of the cancer itself. The exact dosage will thus be determined on a case by case basis by the attending physician or other appropriate professional, but will generally be in the range of 1 to 100 mg/kg of body weight.
  • the compound may be administered prophylactically to patients identified as being at risk for the development of cancer, either due. for example, to a genetic predisposition or as a result of exposure to a carcinogenic substance.
  • the compounds may be administered by injection either intravenously or parenterally.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispensing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1.3-butanediol.
  • the compounds may be administered by inhalation of an aerosol.
  • This method has the advantage of delivering the antitumor compound directly to the lungs where it could, for example, provide protection against carcinogens such as those found in cigarette smoke and atmospheric pollutants, or effectively kill cancer cells located at this site.
  • inhalers appropriate for the practice of the invention are available, including those with various dose metering chambers, various plastic actuators and mouthpieces, and various aerosol holding chambers (e.g. spacer and reservoir devices) so that an appropriate dose of the Vitamin E compound can be delivered.
  • non-ozone depleting (non-chlorofluorocarbon) propellants such as various hydrofluoroalkanes (e.g. HFA 134a and HFA 227) are available.
  • the dose may be less than for other methods and will vary according to the exact delivery technology that is employed.
  • Administration may also be achieved transdermally using a patch impregnated with the compound, by ocular administration (eye drops), sublingual administration, nasal spray administration and rectal administration (suppository).
  • Administration may also be oral.
  • absorption of those compounds which are susceptible to inactivation by digestive enzymes may be accomplished by coating the compounds with an impermeable polymer membrane that is not susceptible to the action of digestive enzymes (duodenal esterases) or is biodegraded very slowly.
  • amino acid polymers such as polylysine could be used.
  • Impermeable polymer films would be degraded by microflora found in the colon. Thus, the compound would be released in a part of the intestine devoid of secreted digestive enzymes.
  • other methods for preventing the hydrolysis of the compounds and promoting their absorption following oral administration can also be used in the practice of the present invention.
  • the compounds may be administered in any of several forms, including tablets, pills, powders, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosol, soft or hard gelatin capsules, or sterile packaged powders.
  • the compounds administered in according to the methods of the present invention may be administered as a composition which also includes a pharmaceutically acceptable carrier.
  • the compounds may be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier.
  • the carrier is a diluent, it may be a solid, semisolid or liquid material which acts as a vehicle, excipient or medium for the compound.
  • Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • the formulations can also include lubricating agents, wetting agents, emulsifying agents, preservatives, and sweetening or flavoring agents.
  • the compounds can be administered in the pure form or in a pharmaceutically acceptable formulation including suitable elixirs, binders, and the like or as pharmaceutically acceptable salts or other derivatives.
  • pharmaceutically acceptable formulations and salts include liquid and solid materials conventionally utilized to prepare injectable dosage forms and solid dosage forms such as tablets and capsules.
  • Water may be used for the preparation of injectable compositions which may also include conventional buffers and agents to render the injectable composition isotonic.
  • Solid diluents and excipients include lactose, starch, coatings and the like. Preservatives such as methyl paraben or benzalkium chloride may also be used.
  • the active composition will consist of 1-99% of the composition and the vehicular '"carrier" will constitute 1-99% of the composition.
  • one advantage of the present invention involves the oral administration of TSE, 2,2-Dim-TS. 2.2-Dim-TG and T-DMAE ether.
  • TSE 2,2-Dim-TS. 2.2-Dim-TG and T-DMAE ether.
  • the derivatives TSE, 2.2-Dim-TS, 2,2-Dim-TG and T-DMAE ether are non-hydrolyzable and thus high levels of bioavailability are maintained until they are taken up by cells (see Example 9).
  • chemotherapeutic agent * means any chemical agent or drug used in chemotherapy treatment which selectively affects tumor cells, including but not limited to such agents as taxanes (e.g. taxol), adriamycin, amscrine, etoposide, cisplatinum, vincristine, vinblastine and methotrexate. Other such agents are well-known in the art. It is anticipated that the compounds of the present invention may be administered either in combination with or separately from the chemotherapeutic agent(s). The dose will vary depending on a variety of factors including the route of administration, choice of chemotherapeutic agent, etc.
  • Another aspect of the present invention is to administer compounds by the methods of the present invention in combination with treating the patient by exposing the patient to ionizing radiation.
  • the protocols for traditional radiation therapy e.g. ⁇ -radiation. are known, readily available, and routinely practiced by those of skill in the art. These include established protocols for the administration of drugs in combination with radiation therapy [Wobst et al. (1998) Ann.Oncol. 9. 951-962].
  • the dose of ionizing radiation will vary depending on a variety of factors including intensity, source of radiation, etc.
  • Another aspect of the present invention is the administration of compounds by the methods of the present invention in order to prevent metastasis of cancer.
  • the compounds may be administered following the surgical removal of a tumor.
  • the compound may also be administered prophylactically to patients identified as being at risk for the development of cancer.
  • the compounds which are administered in the practice of the present invention may be administered singly (i.e. only one derivative compound administered at a time) or in combination (i.e. more than one derivative compound administered at a time).
  • the present invention provides five novel compounds(see Figure 33), namely
  • the compounds may be used in the practice of the present invention as antitumor agents. Details of the syntheses of the five new compounds are given in Examples 10-14 below. Those of skill in the art will recognize that certain modifications of these compounds may be made in order to, for example, optimize bioavailability, solubility, potency, and the like, or for any other reason. All such modifications of these compounds and the derivative compounds which result from such modifications are intended to be encompassed by the present application.
  • the human cells lines used in the present investigation and their origins are as follows: Hep 3B (liver); NIH-H69 (lung); HT29 (colon); Dul45 (prostate); MCF7 and MD-231 (breast); A375 (cutaneous melanoma); OCMl (ocular melanoma); HL60 (leukemia). All cell types except OCMl are available from the American Type Culture Collection (ATCC). OCMl cells were obtained from Dr. June Kan-Mitchell at the U. of California, San Diego.
  • ATCC American Type Culture Collection
  • the anti-tumor agents of the present invention were synthesized by Dr. J. Doyle Smith, Virginia Commonwealth Univ.. Richmond, VA.
  • the succinate esters, PEG esters, butyrate ethers and the tris salts were prepared by procedures similar to those described in U.S. Patent 5,610,180 to Fariss.
  • T-DMAB-T was prepared as follows: In a flask equipped with a magnetic stirrer and reflux condenser was placed 553mg (3.3mM) of ⁇ -(dimethylamino)butyric acid and 2 ml (18.9 mM) of POCl 3 . The mixture was stirred and heated at 93°C for 1 hour to give a clear, colorless solution. The excess POCl 3 was removed under reduced pressure with warming to give an oil, which is the crude ⁇ -(dimethylamino)butyryl chloride. To this was added 1.02g of d- ⁇ -tocopherol and 3 ml of CHC1 3 . The mixture was stirred and warmed to 53°C for 4 days.
  • test compounds except tris salts and betulinic acid were dissolved in 100% ethanol and administered to cells such that the final concentration of ethanol in medium was less the 0.2%.
  • Tris salt derivatives were administered in distilled water, following two 15 second sonications.
  • Betulinic acid was administered in dimethylsulfoxide (DMSO).
  • Alamar blue dye was used to evaluate cell survival and proliferation. Living cells metabolize the non-fluorescent dye to a fluorescent metabolite which can be detected by a fluorescence plate reader. There is a positive correlation between the level of fluorescence and the number of living cells. The fluorescence intensity of the cells treated with a test compound was compared to that of a control group which has no added test compound (vehicle only). The result was expressed as "Cell number (% control)". A reduction in the cell number indicates inhibition of cell growth, or an increase in cell death.
  • Fluroescent values from blank cells were subtracted from fluorescent values of cells treated with test compounds. The resulting values were then divided by the corresponding values obtained from the control samples to give the number of viable cells (% control).
  • the IC 50 values concentration required to inhibit cell growth by 50%
  • mice Male Sprague-Dawley rats from Simonsen Labs (Gilroy, CA) weighing 175-225 g were used throughout the course of this study. Animals received water and food (Purina Rat Chow 5001 , Ralston Purina, St. Louis, MO) ad libitum for at least three days prior to the onset of the experiment. Powdered TS-tris, TSE-tris and TS-PEG were suspended in saline with brief sonication (30 sec) and were given intraperitoneally at a dose of 0.19 mmol/kg body weight. Saline was given to rats at a dose of 4 ml/kg. received CC1 4 by oral lavage 6 or 18 h after tocopherol administration.
  • Purina Rat Chow 5001 Powdered TS-tris, TSE-tris and TS-PEG were suspended in saline with brief sonication (30 sec) and were given intraperitoneally at a dose of 0.19 mmol
  • rats were anesthetized with diethyl ether, and blood samples (4-5 ml) were withdrawn from the inferior aorta. Blood samples were immediately mixed with 15 mg tripotassium EDTA, and aliquots were centrifuged at low speed to prepare plasma samples. All procedures were approved by the Washington State University Animal Care and Use Committee.
  • Tocopherol and tocopherol ester levels were measured according to the methods described by Fariss et al. (11). TSE levels were measured according to the procedures of Tirmenstein et al. (12). Samples were analyzed by reversed-phase high-performance liquid chromatography equipped with fluorimetric detection. Retention times for d- ⁇ -tocopherol (internal standard), ⁇ -T and TSE were 8.0. 11.5 and 13.4 min respectively.
  • TS-tris suspensions were prepared by adding 1 ml of water or saline to 30 mg of TS-tris in a microfuge tube and sonicating for 15 sec, twice.
  • taxol-TS-tris suspensions 1 mg of taxol or taxol monosuccinate tris salt or taxol disuccinate tris salt) was added to 30 mg of TS-tris prior to the addition of water or saline and sonication. (Therefore on a molar basis there is 50 times more TS-tris than taxol).
  • washed liposomal suspensions To prepare washed liposomal suspensions, the suspensions mentioned above were spun at lO.OOOrpm for 15 min and the resulting pellet (liposomes) was washed with saline or water. This procedure (spinning and washing) was conducted 3 times and the final washed liposomal pellet was resuspended by sonication in the original volume of water or saline. The presence of liposomes in each suspension was confirmed by light microscopy.
  • Hep3B human liver tumor
  • the compounds that appear to be most effective in inhibiting the growth of human Hep 3B cells are T-DMAB-T, T-DMAB-Q, T-DMAE-ether, TS-PEG, TSE-PEG, TRF-S, TRF-S-T, ⁇ -T3. Choles-h-p, and Btl-acid. Antitumor activity was also observed for all of the compounds tested except ⁇ -T, ⁇ -T. T-P-Na 2 , PEG 1000, and ⁇ -T3 acetate.
  • the compounds that appear to be most effective in inhibiting the growth of human prostate cancer (Du 145) cells are T-DMAB-T, T- DMAB-Q, ⁇ -TS-T, TS-PEG. TSE-PEG, ⁇ -T3 and Choles-h-p. Antitumor activity was also observed for all of the compounds tested except PEG1000. Table 2. IC 50 ( ⁇ M) of Test Compounds in Human Prostate Cancer (Du 145) Cells
  • the compounds that appear to be most effective in inhibiting the growth of human small cell lung cancer (NIH-H69) cells are ⁇ -T-MS, T-DMAB-Q, T-DMAB-T, TS-PEG, TSE-PEG, TRF-S, ⁇ -T3. choles-h-p, 2,2-Dim-TS, and CS-tris. Antitumor activity was observed for all compounds tested except ⁇ -T. ⁇ -T. PEG 1000. ⁇ -T3-acetate and T-P-Na,.
  • the compounds that appear to be most effective in inhibiting the growth of human colon cancer (HT29) cells are ⁇ -TS-T, T-DMAB-Q, T-DMAB-T. TS-PEG, TRF-S. and choles-h-p. Antitumor activity was also observed for all of the compounds tested except TSE and PEG 1000.
  • the compounds that appear to be most effective in inhibiting the growth of hormone-dependent human breast cancer (MCF7) cells is TS-PEG.
  • Antitumor activity was, however, also observed for all of the compounds tested except PEG 1000.
  • the most effective compounds for inhibiting the growth of hormone-independent MD- 231 human breast cells were TS-PEG, TRF-S, and DMAB-T.
  • the compounds that appear to be most effective in inhibiting the growth of human leukemia (HL-60) cells are TS-PEG, Choles-h-p and ⁇ -T3. Antitumor activity was also observed for all of the compounds tested except ⁇ -T, PEG 1000, and CS- PEG.
  • the compounds that appear to be most effective in inhibiting the growth of human cutaneous melanoma (A375) cells are TS. TS-T, ⁇ T-MS, TS-PEG. TSE-PEG, TRF-S. Btl-acid. Btl-acid-S. T-DMAB-T, and ⁇ -T3. Antitumor activity was also observed for all of the compounds tested.
  • OCMl human ocular melanoma cancer
  • the compounds that appear to be most effective in inhibiting the growth of human ocular melanoma (OCM-1) cells are choles-h-p. Btl-acid. TS-PEG, T-DMAB-Q, and T-DMAB-T.
  • the suspension of taxol plus TS-tris (referred to as taxol, 50X TS-tris) and the washed liposomes obtained from this suspension (referred to as taxol,50X TS-tris pellet) had excellent antitumor activity [killing greater than 85% of the tumor cells at 10 nM taxol (also containing 0.5 micromolar TS, a nontoxic dose as seen in Fig 32A)].
  • taxol,50X TS-tris pellet had excellent antitumor activity [killing greater than 85% of the tumor cells at 10 nM taxol (also containing 0.5 micromolar TS, a nontoxic dose as seen in Fig 32A)].
  • the antitumor activity of these two preparations was nearly identical to that of taxol dissolved inethanol (referred to as Taxol).
  • taxol can be solubilized by TS-tris in water or saline, that taxol is contained within TS-tris liposomes (probably as part of the lipid layer) and that both the TS-tris/taxol suspension and liposomal fractions have excellent antitumor activity.
  • our data suggest that the addition of TS-tris and taxol allows for 100% kill of tumor cells that normally is not observed with taxol alone (see Fig.32B).
  • taxol monosuccinate tris salt treatment in combination with TS-tris suspension and liposomes also demonstrates the same increased solubility for this taxol derivative in the presence of TS-tris as that observed for taxol.
  • the monosuccinate tris of taxol has minimal solubilty in water (as determined by centrifugation and light microscopy).
  • the disuccinate tris salt of taxol displayed complete solubility in water at a concentration of 4 mg/ml.
  • the plasma concentrations of tocopherol analogs were assessed in vivo in rats 24 hours after administration. Rats were given either an intrapeironeal (ip) injection or an oral intubation of TS-tris salt. TSE or TS-PEG (0.19mmol/kg body weight). After 24 hours, rats were sacrificed and blood samples were obtained and extracted for HPLC analysis of concentrations of TS-PEG. TS, TSE and T.
  • Vitamin E succinate potentiates the inhibitory effect of prostoglandins on oral squamous carcinoma cell proliferation. Prostoglandins Leukot. Essential Fatty Acids, 52, 69-73.

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Abstract

La présente invention concerne des méthodes d'utilisation de dérivés de vitamine E (tocophérol et tocotriénol), de cholestérol, de taxol et d'acide bétulinique comme agents anti-tumeur dans le traitement et la prévention de cancers du foie, des poumons, du côlon, de la prostate et du sein, ainsi que de mélanomes et de la leucémie.
EP00923141A 1999-04-07 2000-04-07 Activite anti-tumeur de derives de vitamine e, de cholesterol, de taxol et d'acide betulinique Withdrawn EP1189607A2 (fr)

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US5869535A (en) 1995-03-21 1999-02-09 The Board Of Trustees Of The University Of Illinois Method and composition for selectively inhibiting melanoma
AU8494601A (en) * 2000-08-18 2002-03-04 Univ Illinois Prodrugs of betulinic acid derivatives for the treatment of cancer and HIV
JP2004538304A (ja) * 2001-07-27 2004-12-24 ユニバーシティー オブ ロチェスター アンドロゲンレセプターのビタミンｅ阻害および前立腺癌細胞中の前立腺特異的抗原の発現
RU2328273C2 (ru) * 2001-12-19 2008-07-10 Рисерч Дивелопмент Фаундейшн Липосомальная доставка соединений, основанных на витамине е
US7365221B2 (en) 2002-09-26 2008-04-29 Panacos Pharmaceuticals, Inc. Monoacylated betulin and dihydrobetulin derivatives, preparation thereof and use thereof
US20040102421A1 (en) * 2002-11-21 2004-05-27 Children's Hospital Research Center At Oakland Tocopherol and tocotrienol anti-inflammatory medicaments
CN101160132A (zh) 2005-04-12 2008-04-09 帕纳克斯医药公司 3-o-(3′,3′-二甲基琥珀酰基)桦木酸二-n-甲基-d-葡萄糖胺的多晶形物
JP2012506426A (ja) * 2008-10-23 2012-03-15 ダボス ライフ サイエンス ピーティーイー． リミテッド 癌を予防するためのトコトリエノール組成物の使用
US8969303B2 (en) 2010-02-05 2015-03-03 First Tech International Limited Tocotrienol esters
US20250345454A1 (en) * 2024-05-09 2025-11-13 Peptinovo Biopharma Inc. Chemotherapeutic paclitaxel based micellular nanoparticles

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