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WO2000071563A2 - Agents de stabilisation de microtubules de taccalonolide - Google Patents

Agents de stabilisation de microtubules de taccalonolide Download PDF

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Publication number
WO2000071563A2
WO2000071563A2 PCT/US2000/013795 US0013795W WO0071563A2 WO 2000071563 A2 WO2000071563 A2 WO 2000071563A2 US 0013795 W US0013795 W US 0013795W WO 0071563 A2 WO0071563 A2 WO 0071563A2
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Prior art keywords
lower alkyl
hydroxy
present
acyloxy
taccalonobde
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WO2000071563A3 (fr
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Thomas K. Hemscheidt
Susan L. Mooberry
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University of Hawaii at Manoa
University of Hawaii at Hilo
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University of Hawaii at Manoa
University of Hawaii at Hilo
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Priority to US09/979,447 priority Critical patent/US6878699B1/en
Priority to AU52763/00A priority patent/AU5276300A/en
Publication of WO2000071563A2 publication Critical patent/WO2000071563A2/fr
Anticipated expiration legal-status Critical
Publication of WO2000071563A3 publication Critical patent/WO2000071563A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • 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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring

Definitions

  • the present invention relates to methods for inhibiting cell proliferation and compounds useful therefor.
  • Neoplastic diseases or cancers characterized by the proliferation of cells not subject to normal growth regulation, are a major cause of death in humans. An estimated 1,221,800 new cases and 561,000 deaths are expected to occur in 1999. Lung cancer remains the leading cause of cancer-related deaths in the United States; the estimated 158,900 deaths would account for 28% of the total.
  • microtubule system of eucaryotic cells is a major component of the cytoskeleton and is in a dynamic state of assembly and disassembly; that is, heterodimers of tubulin are polymerized to form microtubules, and microtubules are depolymerized to their constituent components.
  • Microtubules play a key role in the regulation of cell architecture, metabolism, and division, and the dynamic state of the microtubules is critical to their normal function. With respect to cell division, tubulin is polymerized into microtubules that form the mitotic spindles. The microtubules are then depolymerized when the mitotic spindle's role has been fulfilled. Accordingly, agents which disrupt the polymerization or depolymerization of microtubules, and thereby inhibit cell growth, comprise some of the most effective chemotherapeutic agents in clinical use.
  • Such anti-mitotic agents or poisons all kinetically inhibit the normal dynamics of microtubules.
  • antimicrotubule agents There are subtle differences between certain classes of antimicrotubule agents based on their molecular mechanism of action. Colchicine binds to soluble tubulin and then is incorporated into a growing microtubule, vinblastine binds to the microtubule end and thereby suppresses microtubule dynamics. At high concentrations, both colchicine and vinblastine cause the loss of cellular microtubules. Pachtaxel and related taxanes also inhibit microtubule dynamics, yet at high concentrations these agents cause an increase in polymerized tubulin in the cell and thick microtubule bundles are formed.
  • Pachtaxel was first isolated in 1971 in the bark of the Pacific yew tree (Taxus brevifolia), and was approved in 1992 by the US Food and Drug Administration for treatment of metastatic ovarian cancer and later for breast cancer. Pachtaxel has attracted unusually strong scientific attention, not only because of its unique antiproliferative mechanism of action, but also because it is active against a broad range of tumors. The discovery of the effectiveness of the natural product pachtaxel lead to the production and testing of semisynthetic congeners including docitaxel (Taxotere). These compounds, taxanes, are now recognized as a new class of anticancer compounds.
  • pachtaxel can be administered effectively only in a solvent including cremophor (the commercially available formulation marketed as Taxol), which combination can provoke severe hypersensitive immune responses.
  • cremophor the commercially available formulation marketed as Taxol
  • neoplastic cell displaying drug resistance (DR) or multiple-drug resistance (MDR) is through the over-expression of P-glycoprotein. Compounds which are poor substrates for transport by P-glycoprotein should be useful in circumventing such DR or MDR phenotypes.
  • epothilones A and B discodermohde. eleutherobm and related sarcodictyms A and B, and lauhmalides
  • the epothilones were isolated from the myxobacte ⁇ um Sorangium cellulosum as a result of a large-scale screening effort. The epothilones have generated significant interest, as they retain activity against drug-resistant cell lines.
  • Discodermohde was pu ⁇ fied from the ma ⁇ ne sponge Dtscodemia dtssoluta as an immunosuppressant and was screened for antimitotic activity on the basis of a predictive structure-activity relationship when compared with other tubuhn- interacting drugs. Discodermohde promotes tubulin assembly more potently than Taxol and it is an effective inhibitor of cell growth in paclitaxel-resistant cells
  • Eleutherobm a potent cytotoxin from the soft coral eleutherobm sp., promotes tubulin polymerization but exhibits cross-resistance to paclitaxel-resistant cell lines
  • Epothilones have been isolated from a species of bacte ⁇ a found m soil samples collected from the banks of the Zambesi River in the Republic of South Africa, and have been recently synthesized. Lauhmahdes have been isolated from the ma ⁇ ne sponge C m ⁇ ofijtensts Strong pachtaxel-hke microtubule-stabihzmg activity has been found in the lauhmahdes
  • Taccalonobde is an effective inhibitor of proliferation against both SK-OV-3 and MDA-MB-435 cells with IC o values of 2 3 ⁇ M and 2 l ⁇ M, respectively
  • taccalonobde is effective against the multidrug resistant SKNLB-1 cell line and thus appears to be a poor substrate for P-glycoprotem- mediated transport
  • taccalonobde is almost equipotent with pachtaxel m its effects on cellular microtubules, it is much less potent than pachtaxel in its abihty to initiate the polymerization of pu ⁇ fied tubuhn and microtubule protein
  • Taccalonobde A is the first microtubule stabilizing agent to be discovered from a plant since the identification of the mechanism of action of pachtaxel and it is the first natural product steroid identified to have these cellular effects
  • BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the effect of taccalonobde A on two different cancer cell lines as
  • Figure 2 illustrates the effect of taccalonobde A on the drug resistant cell line SKVLB-1 (a sub-line of SK-OV-3 selected for resistance to vinblastine).
  • Figure 3 illustrate the cell cycle distribution of cells treated with taccalonobde.
  • MDA-MB-435 cells in log phase growth were treated with vehicle (Figure 3 A) or lO ⁇ m taccalonobde for 6 hr ( Figure 3B), 12 hr ( Figure 3C), or 24 hr ( Figure 3D) and then fixed, stained and analyzed by flow cytometry.
  • Figure 4 shows the formation of the slower migrating form of Bcl-2 in cell lysates from cells treated with taccalonobde for the indicated times.
  • the slower migrating form of Bcl-2 is caused by protein phosphorylation.
  • Figure 5 illustrates caspase 3 activity of cell lysates prepared from taccalonolide-treated cells.
  • Cell lysates (30 ⁇ g protein) were evaluated for the ability to cleave the caspase 3 substrate DEVD-pNA.
  • Figure 6 illustrates the effect of taccalonobde on tubulin polymerization.
  • the polymerization of purified, glycerol-free bovine brain tubulin was measured spectrophotometrically in a temperature-controlled (35°C) microplate spectrophotometer in the presence of vehicle control (closed circles), 2 ⁇ M pachtaxel (open circles), or taccalonobde (2 ⁇ M-closed triangles; 200 ⁇ M-open triangles).
  • a hyperproliferative mammalian cell having a multiple drug resistant phenotype comprising contacting the cell with an amount of a taccalonobde compound effective to disrupt the dynamic state of microtubule polymerization and depolymerization to arrest cell mitosis, thereby inhibiting the proliferation of the cell.
  • taccalonobde agents are that they stabilize microtubules and turn on a "cellular suicide" switch which leads to cancer cell death.
  • Taccalonobdes contemplated for use in the practice of the present invention include compounds having the structure:
  • R 1 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 2 H, OH, or R 2 and R ? cooperate to form an epoxide at C-2/C-3,
  • R 3 H, OH, or R 3 cooperates with R 2 to form an epoxide at C-2/C-3,
  • R 6 H, OH, or a carbonyl oxygen if R 6 is not present,
  • R 6 when present, is H or OH
  • R 7 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 8 H or lower alkyl
  • R ⁇ H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 12 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 15 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 16 H or lower alkyl
  • R ,8 H or lower alkyl
  • R H or lower alkyl
  • R- H or lower alkyl
  • R H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 27 when present, is H or OH
  • R 28 H or lower alkyl; wherein the F ring is optionally present, and when present,
  • R x O, NR X or CR X 2 , wherein each R x is independently H or lower alkyl, or when the F ring is not present, the substituent at
  • C23 is H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • C24 is H, lower alkyl, hydroxy, lower alkoxy or acyloxy, and wherein the dashed lines between C2/C3, C6/C7, Cl I/O 2 and C22/23 represent optional double bonds at each position.
  • lower alkyl refers to straight or branched chain alkyl groups having 1 up to 6 carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, t-butyl, and the like.
  • lower alkoxy refers to oxygen-substituted alkyl radicals having 1-6 carbon atoms.
  • acyloxy refers to the moiety -O-C(O)-R, wherein R is an alkyl radical having 1-6 carbon atoms, optionally hydroxy and/or amino substituted, e.g., methyl, ethyl, propyl, aminomethyl, hydroxymethyl, and the like.
  • taccalonobde A i.e., compounds according to the steroid structure set forth above wherein:
  • R 1 is - O-acetyl
  • R" and R cooperate to form an epoxide, R° is a carbonyl oxygen and R 6 is not present, R is hydroxy, R 8 is H,
  • R n is - O-acetyl
  • R 12 is - O-acetyl
  • R 15 is - O-acetyl
  • R 16 is H
  • R 18 is methyl
  • R 19 is methyl
  • R 21 is methyl
  • R 26 is methyl
  • R 26' is methyl
  • R 27 is a carbonyl oxygen and R 27 is not present
  • R 28 is methyl, X is O, and there is a double bond between C22 and C23; (i.e., taccalonobde A); or
  • R 1 is - O-acetyl
  • R 2 and R 3 cooperate to form an epoxide, R 6 is a carbonyl oxygen and R 6 is not present,
  • R 7 is hydroxy
  • R 8 is H
  • R 1 1 is - O-acetyl
  • R 12 is - O-acetyl
  • R 15 is hydroxy
  • R 16 is H
  • R 18 is methyl
  • R 19 is methyl
  • R 21 is methyl
  • R 26 is hydroxy
  • R 26' is methyl
  • R 27 is a carbonyl oxygen and R 27 is not present, R 28 is methyl,
  • X is O, and there is a double bond between C22 and C23 (i.e., taccalonobde B); or
  • R 1 is - O-acetyl
  • R and R cooperate to form an epoxide
  • R 6 is a carbonyl oxygen and R 6 is not present
  • R 7 is - O-acetyl
  • R 8 is H
  • R 1 ' is - O-acetyl
  • R 12 is - O-acetyl
  • R 15 is hydroxy
  • 16 is H
  • R 18 is methyl
  • R 19 is methyl
  • R 21 is methyl
  • R 26 is hydroxy
  • R 26' is methyl
  • R 27 is a carbonyl oxygen and R 27 is not present, R 28 is methyl,
  • X is O, and there is a double bond between C22 and C23 (i.e., taccalonobde D).
  • taccalonobde C variants are known to exist in the basic taccalonobde structure, including taccalonobde C. These variants differ structurally from taccalonobde A and D as follows:
  • taccalonobde compounds of the present invention were previously isolated from plants of the Tacca spp., herbaceous plants found in the Pacific region, and particularly from Tacca plantaginea a plant found in China. More recently, the structures of a number of taccalonobdes have been investigated, and taccalonobde A has been reported to display antimalarial and cytotoxic activity against p-388 leukemia in cell culture. The basis for this cytotoxic activity was not elucidated.
  • Taccalonobde A has also been isolated from Tacca chantrieri obtained in a cultivated state. Lyophibzed tubers were ground to a powder and extracted by stirring with dichloromethane/isopropanol for 24 hours at room temperature. The extraction was repeated under identical conditions with fresh solvent, and the combined extracts were evaporated in vacuo below 40°C. The residue was dissolved in 90% (v/v) aqueous methanol and extracted three times with equal volumes of hexanes, and hexane fractions were discarded. The aqueous methanol phase was diluted with water to 80%) (v/v) aqueous methanol and extracted three times with equal volumes of toluene.
  • taccalonobdes A, B and D The chemical structure of the taccalonobdes as a class is amenable to chemical synthesis and the production of analogs, by techniques known in the art.
  • the structure for taccalonobdes A, B and D referred to above includes a methyl group attached to carbon 24, which is structurally unusual for steroids.
  • taccalonobde analogs which do not include this unusual methyl group, while preserving the activity of the class of compounds are also contemplated.
  • novel taccalonobde analogs having the following structure are also considered to be within the scope of the present invention:
  • R H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 2 H, OH, or R 2 and R 3 cooperate to form an epoxide at C-2/C-3,
  • R 3 H, OH, or R 3 cooperates with R 2 to form an epoxide at C-2/C-3,
  • R 6 H, OH, or a carbonyl oxygen if R 6 is not present,
  • R 6 when present, is H or OH
  • R 7 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 8 H or lower alkyl
  • R 11 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 12 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 15 H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • R 16 H or lower alkyl
  • R , 8 H or lower alkyl
  • R , 9 H or lower alkyl
  • R 21 H or lower alkyl
  • R H, OH, or a carbonyl oxygen if R is not present
  • C23 is H, lower alkyl, hydroxy, lower alkoxy or acyloxy
  • C24 is H, lower alkyl, hydroxy, lower alkoxy or acyloxy, and wherein the dashed lines between C2/C3, C6/C7, Cl 1/C12 and C22/23 represent optional double bonds at each position.
  • Taccalonobdes contemplated for use in the practice of the present invention can be obtained from a variety of natural sources, or they can be prepared synthetically employing synthetic techniques known in the art.
  • methods described herein can be carried out in the further presence of at least one additional anti-neoplastic agent.
  • cells subjected to invention methods are mammalian cells, including human cells.
  • the present invention also provides methods of alleviating a pathological condition caused by hyperproliferating, multiple drug resistant mammalian cells comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition disclosed herein to inhibit proliferation of the cells.
  • the mammalian cells are human.
  • the method further comprises administering to the subject at least one additional therapy directed to alleviating the pathological condition.
  • the pathological condition is characterized by the formation of neoplasms.
  • the neoplasms are mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, pancreatic adenocarcinoma, central nervous system (CNS), ovarian, prostate, sarcoma of soft tissue or bone, head and neck, gastric (which includes pancreatic and esophageal), stomach, myeloma, bladder, renal, neuro endocrine (which includes thyroid), non-Hodgkin's disease, Hodgkin's disease neoplasms, and the like.
  • neoplastic pertains to a neoplasm, which is an abnormal growth, such growth occurring because of a proliferation of cells not subject to the usual limitations of growth.
  • anti-neoplastic agent is any compound, composition, admixture, co-mixture or blend which inhibits, eliminates, retards or reverses the neoplastic phenotype of a cell.
  • Chemotherapy surgery, radiation therapy, therapy with biologic response modifiers, and immunotherapy are currently used in the treatment of cancer.
  • Each mode of therapy has specific indications which are known to those of ordinary skill in the art, and one or all may be employed in an attempt to achieve total destruction of neoplastic cells.
  • Chemotherapy utilizing one or more taccalonobdes is provided by the present invention.
  • combination chemotherapy, chemotherapy utilizing taccalonobdes in combination with other neoplastic agents is also provided by the subject invention as combination therapy is frequently more effective than the use of single anti-neoplastic agents.
  • compositions containing a therapeutically effective amount of at least one taccalonobde compound of the present invention can also be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants, excipients, and the like.
  • physiologically tolerable liquid, gel or solid carriers diluents, adjuvants, excipients, and the like.
  • Such carriers, diluents, adjuvants and excipients may be found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII. U.S. Pharmacopeia Convention, Inc., Rockville, MD (1989), the contents of which are incorporated herein by reference. Additional modes of treatment are provided in AHFS Drug Information. 1993 ed. by the American Hospital Formulary Service, pp. 522-660, the relevant contents of which are incorporated herein by reference.
  • the pharmaceutical composition used to treat neoplastic disease contains at least one taccalonobde compound and at least one additional anti-neoplastic agent.
  • Anti- neoplastic compounds which may be utilized in combination with taccalonobdes include those provided in The Merck Index. 11 th ed. Merck &Co., Inc. (1989) pp. Ther 16-17, the contents of which are hereby incorporated by reference.
  • anti-neoplastic agents may be antimetabobtes which include methotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, hydroxyurea, 2-chlorodeoxyadenosine, and the like.
  • the anti-neoplastic agents contemplated are alkylating agents which include cyclophosphamide, melphalan, busulfan, paraplatin, chlorambucil, nitrogen mustard, and the like.
  • the anti-neoplastic agents are plant-derived natural products which include vincristine, vinblastine, taxol, etoposide, and the like.
  • the anti-neoplastic agents contemplated are antibiotics which include doxorubicin (adriamycin), daunorubicin, mitomycin c, bleomycin, and the like.
  • the anti-neoplastic agents contemplated are hormones which include calusterone, diomostavolone, propionate, epitiostanol, mepitiostane, testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate, flutamide, nilutamide, trilotane, and the like.
  • the anti-neoplastic agents contemplated include enzymes such as L-Asparaginase, aminoacridine derivatives, (e.g., amsacrine), and the like. Additional anti-neoplastic agents include those provided in Skeel, Roland T., "Antineoplastic Drugs and Biologic Response Modifier: Classification, Use and
  • the present taccalonobde compounds and compositions can be administered to mammals for veterinary use, such as for domestic animals, and clinical use in humans in a manner similar to other therapeutic agents.
  • the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts. Ordinarily, dosages will range from about 0.001 to lOOOmg/kg, more usually 0.01 to lOmg/kg, of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.
  • drug dosage as well as route of administration, must be selected on the basis of relative effectiveness, relative toxicity, growth characteristics of tumor and effect of taccalonobdes on cell cycle, drug pharmacokinetics, age, sex, physical condition of the patient, and prior treatment.
  • the taccalonobde compounds may be formulated into therapeutic compositions as natural or salt forms.
  • Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like.
  • Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII. U.S. Pharmacopeia Convention, Inc., Rockville, MD (1989), the relevant contents of which is incorporated herein by reference.
  • the suitability of particular carriers for inclusion in a given therapeutic composition depends on the preferred route of administration.
  • anti- neoplastic compositions may be formulated for oral administration.
  • Such compositions are typically prepared either as liquid solution or suspensions, or in solid forms.
  • Oral formulations usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain 1 %o-95 % of active ingredient, preferably 2%>-70%>.
  • compositions of the present invention may also be prepared as injectable, either as liquid solutions, suspensions, or emulsions; solid forms suitable for solution in, or suspension in, liquid prior to injection may be prepared.
  • injectables may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intrathecally, intrapleurally, and the like .
  • the active ingredient or ingredients are often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient(s). Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
  • the invention further provides methods for using taccalonobde compounds encompassed by the generic structure set forth above to inhibit the proliferation of mammalian cells by contacting these cells with a taccalonobde compound in an amount sufficient to inhibit the proliferation of the mammalian cell.
  • a preferred embodiment embraces inhibiting the proliferation of hyperprobferative mammalian cells.
  • hyperprobferative mammalian cells are mammalian cells which are not subject to the characteristic limitations of growth, e.g., loss of growth control and insensitivity to normal programmed cell death (apoptosis).
  • a further preferred embodiment is when the mammalian cell is human.
  • the invention further provides for contacting the mammalian cell with at least one taccalonobde compound and at least one additional anti-neoplastic agent.
  • the types of anti- neoplastic agents contemplated are the same as those disclosed hereinabove.
  • the invention further provides methods for using taccalonobde compounds encompassed by the generic structure set forth above to inhibit the proliferation of hyperprobferative cells with drug-resistant phenotypes, including those with multiple drug-resistant phenotypes, by contacting said cell with a taccalonobde compound in an amount sufficient to inhibit the proliferation of a hyperprobferative mammalian cell.
  • a prefe ⁇ ed embodiment is when the mammalian cell is human.
  • the invention further provides contacting the mammalian cell with a taccalonobde compound and at least one additional anti-neoplastic agent.
  • the types of anti-neoplastic agents contemplated the same as those disclosed hereinabove.
  • the invention further provides a method for alleviating pathological conditions caused by hyperproliferating mammalian cells, for example, neoplasia, by administering to a subject in need thereof an effective amount of a taccalonobde as described herein to inhibit the proliferation of the hyperproliferating cells.
  • pathological condition refers to any pathology arising from the proliferation of mammalian cells that are not subject to the normal limitations of cell growth.
  • Such proliferation of cells may be due to neoplasms, including mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, central nervous system (CNS), ovarian, prostate, sarcoma of soft tissue or bone, head and neck, gastric (which includes pancreatic and esophageal), stomach, myeloma, bladder, renal, neuroendocrine (which includes thyroid), lymphoma, non-Hodgkin's, Hodgkin's disease neoplasms, and the like.
  • the neoplastic cells are human.
  • the present invention further provides methods of alleviating such pathological conditions utilizing taccalonobde in combination with other therapies, as well as other anti-neoplastic agents.
  • therapies and their appropriateness for different neoplasia may be found in Cancer Principles and Practice of Oncology. 4 th ed., Editors DeVita, V., Hellman, S., and Rosenberg, S., Lippincott Co. (1993), the contents of which are incorporated herein by reference.
  • taccalonobde compounds are shown to potently stabilize the microtubule structure in cultured cells.
  • taccalonobde compounds appear to be a poor substrate for the drug-efflux pump P-glycoprotein.
  • Reagents 4,6-Diamidino-2-phenybndole (DAPI), sulforhodamine B (SRB), antibodies against ⁇ -tubulin, and Basal Medium Eagle containing Earle's salts (BME) were obtained from the Sigma Chemical Company (St. Louis, MO). Richter's medium was obtained from BioWhittaker (Walkersville, MD) and Fetal Bovine Serum (FBS) was obtained from Hyclone Laboratories (Logan, UT).
  • Taccalonobde A was obtained as a bpophibc extract of Tacca chantrieri obtained from the Lyon Arboretum (Honolulu, HI), and subjected to bioassay-directed purification. Lyophibzed tubers of Tacca chantrieti were ground to a fine powder and extracted twice by stirring with dichloromethane/isopropanol (7/3 v/v) for 24 hours at room temperature. The extraction was repeated under identical conditions with fresh solvent, solids were filtered off, and the combined extracts were evaporated in vacuo below 40°C.
  • the residue was dissolved in 90%> (v/v) aqueous methanol and extracted three times with equal volumes of hexanes, and the hexane fractions were discarded.
  • the aqueous methanol phase was diluted with water to 80%> (v/v) aqueous methanol and extracted three times with equal volumes of toluene.
  • the combined toluene fractions were evaporated in vacuo below 40°C to dryness.
  • the residue was dissolved in a minimum amount of dichloromethane and applied to a silica gel column equilibrated in ether, and the column was eluted with ether. Active fractions were combined and rechromatographed over silica gel using a solvent system of 75% methyl-tert-butyl ether/25 % hexanes (v/v) containing 1%> isopropanol.
  • A- 10 rat aortic smooth muscle cells, SK-OV-3 human ovarian carcinoma cell line and HeLa cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in BME containing 10%> FBS and 50 ⁇ g/mL gentamycin sulfate.
  • a sub-line of SK-OV-3 selected for resistance to vinblastine (SKVLB-1) was provided by Dr. Victor Ling (British Columbia Cancer Center, Vancouver, British Columbia) and was maintained in BME containing 10% FBS and 50 ⁇ g/mL gentamycin sulfate.
  • the A- 10, HeLa, SK-OV-3, and SKVBL-1 cells were grown in Basal Medium Eagle containing Earle's salts, 50 ⁇ g/ml gentamycin, and 10%) fetal bovine sorum (Hyclone, Logan UT). Vinblastine was added to a final concentration of l ⁇ g/mL to SKVLB-1 cells 24 hours after passage to maintain selection pressure for P-glycoprotein-overexpressing cells.
  • the MDA-MB-435 human mammary adenocarcinoma cell line was obtained Dr. Mai Higazi
  • the IC 5 o for inhibition of cell proliferation was determined by measuring cell- associated protein after drug treatment using the sulforhodamine B assay (see, for example, J. Natl. Cancer Inst. 82:1107-1112 (1990) and Drug Development Res. 34:91-109 (1995)).
  • EXAMPLE 4 Immunofluorescence Assays
  • A- 10 cells were grown to 70-85 % confluency on glass coverslips in BME/10% FBS. Drug compounds in PBS were added to the indicated final concentrations and cells were incubated for an additional 24 hours.
  • the cells were fixed with cold methanol for 5 minutes, blocked for 20 minutes with PBS containing 10% calf serum to block nonspecific binding sites, and incubated at 37°C for 60-90 min with monoclonal anti- ⁇ -tubulin at the dilutions recommended by the manufacturer. Bound primary antibodies were subsequently visualized by a one hour incubation with fluorescein (FITC)-conjugated sheep antimouse IgG (F-3008; Sigma).
  • FITC fluorescein
  • the coverslips were washed, stained with O.l ⁇ g/mL DAPI for 10 minutes, mounted on microscope slides and the fluorescence patterns were examined and photographed using a Zeiss Axioplan microscope equipped with epifluorescence optics for fluorescein and DAPI.
  • EXAMPLE 5 Cell Cycle Analysis MDA-MB-435 cells were treated with the IC 85 concentration of taccalonobde, lO ⁇ M, or vehicle control for 6,12, 18 or 24 hr. The cells were fixed in 70%> ethanol, treated with RNAse A and stained with propidium iodide as described by Mooberry et al., in Int. J. Cancer 73:440-448 (1997). The DNA content was analyzed using a Coulter EPICS XL-MCL flow cytometer and the data were plotted as number of events vs. propidium iodide fluorescence intensity. See Figure 3A-3D.
  • Caspase-3 activity was measured by cleavage of DEVD-pNA using the BIOMOL Caspase-3 Cellular Activity Kit Plus (AK-703). MDA 435 cells were incubated with lO ⁇ M taccalonobde, the IC 85 concentration, for up to 24 hr. Following the incubation the cells were harvested, washed, and lysed and the cell lysates immediately frozen at -70°C. The protein concentration of the lysates was determined (Pierce Reagent, Rockford IL) and samples containing 30 ⁇ g protein were incubated in duplicate with the substrate DEVD-pNA at 37°C.
  • Controls consisted of samples containing the caspase inhibitor Ac-DEVD-CHO, caspase-3 positive controls, with and without Ac-DEVD-CHO and reagent blanks. Enzymatic cleavage of DEVD-pNA was measured spectrophotometrically as an increase in absorbance at 405 nm in a microtiter plate reader.
  • MDA-MB-435 cells were treated with taccalonobde at the IC85 concentration for inhibition of proliferation for 0-24 h. Following drug exposure the cells were harvested and cellular proteins extracted in radioimmunoprecipitation buffer in the presence of protease inhibitors as described previously (see Mooberry et al., Supra). The protein concentrations of the samples were determined (Pierce Reagent, Rockford, IL) and cell lysate aliquots containing equal concentrations of protein were separated, transferred, probed with specific antibodies, and detected as previously described (see Mooberry et al., in Cancer Res. 59:653-660 (1999)). The Bcl-2 and caspase 3 antibodies were purchased from PharMingin (San Diego, CA).
  • the assembly of purified tubulin was monitored spectrophotometrically by the change in absorbance at 340nm.
  • Purified glycerol-free bovine brain tubulin (TL-238, Cytoskeleton, Denver, CO) was solubilized in ice cold G-PEM buffer (80mM PIPES, pH 6.9, ImM MgCl 2 , ImM EGTA and lmM GTP) and taccalonobde, Taxol or vehicle control was added and mixed well.
  • the final concentration of tubulin was 1 mg/ml and ethanol concentration was 1%> or less.
  • An aliquot of the mixture was transferred to a room temperature 96 well plate and tubulin polymerization monitored for 60 min. at 35°C in a temperature controlled microplate spectrophotometer.
  • CytoDYNAMIX Screen 01 (Cytoskeleton, Denver CO).
  • MAP-rich tubulin (ML113) was reconstituted to lmg/ml with 80mM G-PEM buffer containing vehicle control (0.2%) ethanol), taccalonobde (200 ⁇ M), or Taxol (2 ⁇ M).
  • the assay used a 96 well format in a temperature controlled microplate reader. Microtubule protein polymerization was measured by change of absorbance at 340nm at 37°C for 60 min.
  • EXAMPLE 10 Effects of Taccalonobde and Paclitaxel on Cellular Microtubules Strong paclitaxel-like microtubule-stabilizing activity was found in the crude bpophilic extract from the plant T. chantrieri. Bioassay-directed purification of the extract yielded the microtubule active-compound taccalonobde A. Studies with the purified compound show that taccalonobde A caused dramatic reorganization of cellular microtubules in mterphase and mitotic cells.
  • A- 10 cells were treated with taccalonobde A, or paclitaxel for 18-24 hours and the drug's effects on microtubules examined by indirect immunofluorescence.
  • the control cells exhibited normal microtubules arrays.
  • Treatment of the cells with taccalonobde A disrupted the normal microtubule array with the microtubules radiating from the central regions of the cell, the microtubule organizing center, to the cell periphery. Although the microtubule network is extensive it does not occupy the entire cytosol.
  • Taccalonobde at concentrations of 0.1-0.5 ⁇ M, caused a slight increase in the density of cellular microtubules, but at concentrations of 5 ⁇ M-50 ⁇ M, significant alterations of the normal microtubules network occurred.
  • a lO ⁇ M concentration of taccalonobde caused at least two effects in the A- 10 cells. Some cells had a much higher density of long thick microtubules that filled more of the cytoplasm, similar to the effects of paclitaxel, while other adjacent cells showed the appearance of short thick tufts of microtubules that appear to nucleate independent of the microtubule organizing center. More of the cells exhibited the formation of the short thick microtubules and at concentrations higher than lO ⁇ M all cells contained thick short tufts of microtubules that were more prevalent in the cell periphery.
  • Paclitaxel at concentrations between l-20 ⁇ M initiated the formation of a highly organized array of microtubules, some of which formed thick microtubule bundles. Long microtubule bundles often surrounded the nucleus. The extensive long hoops and bundles that formed after treatment with 2 ⁇ M paclitaxel were not seen with taccalonobde.
  • taccalonobde-treated cells An interesting difference between the taccalonobde-treated cells and the pacbtaxel-treated cells occurred with respect to cell shape.
  • the taccalonobde treated cells tended to retain a rounder shape like untreated or vehicle-treated controls while the pacbtaxel-treated cells were more elongated than either control or taccalonobde treated cells.
  • Microtubules in cells treated with taccalonobde were resistant to the microtubule depolymerizing effects of vinblastine, consistent with the effects of paclitaxel.
  • a common characteristic of antimicrotubule agents is their ability to disrupt normal mitosis due to inhibition of the highly dynamic mitotic spindles.
  • the effects of taccalonobde on mitotic spindles were examined in A- 10 and HeLa cells. In both cell lines taccalonobde caused the appearance of abnormal multi-polar mitotic spindles. Normal mitotic spindles are bipolar. The mitotic spindles formed in taccalonobde-treated cells contained 3 or more spindle poles that did not show coordinated orientation. These abnormal mitotic spindles were unable to align the DNA in metaphase.
  • the IC o values for taccalonobde were determined in two drug-sensitive cell lines, MDA-MB-435 and SK-OV3, and in a multidrug-resistant cell line, SKVLB-1 See Table 1. Cells were treated with a range of concentrations of taccalonobde for 48hr and the inhibition of proliferation determined using the SRB assay. The IC o for each cell line was calculated from the dose response curves. The resistance factor was calculated by dividing the IC 5 o of the drug sensitive cell line by the IC 5 o of the drug resistant cell line. The values represent the mean of 3 or 4 experiments ⁇ the SD. Table 1
  • Taccalonobde A is found to be a potent inhibitor of cell proliferation with IC 5 ⁇ values in the low ⁇ M range. In contrast to paclitaxel, Taccalonobde A is able to completely inhibit the proliferation of the SKVLB-1 cell line that overexpresses the drug efflux pump P-glycoprotein.
  • the resistance factor (which is calculated by dividing the IC 5 o of the sensitive cell line by the IC 5 o in the resistant cell line) between the parental, drug-sensitive line (SK-OV-3) and the drug resistant cell line (SKVLB- 1) is 285, the resistance factor for paclitaxel with these cell lines is greater than 58,000 (see Mooberry et al., in Cancer Res. 59:653-660 (1999)).
  • Taccalonobde appears to be a poor substrate for p-glyprotein mediated drug transport. Paclitaxel did not achieve >70%> inhibition of the SKVLB-1 cell line with concentrations of up to lOO ⁇ M. Thus, taccalonobde A was significantly more effective against the SKVLB-1 cell line than paclitaxel. These data confirm that these taccalonobde agents are poor substrates for transport by P-glycoprotein.
  • Taccalonobde initiated the breakdown of the nucleus into micronuclei, a characteristic of apoptosis and a sub Gl peak was measured by flow cytometry in taccalonobde-treated cells.
  • a common feature of apoptosis induced by agents that target microtubules is the phosphorylation of Bcl-2, a cellular regulator of apoptosis.
  • the effects of taccalonobde on Bcl-2 were investigated by immunoblotting techniques. The appearance of a slower migrating form of Bcl-2 is consistent with Bcl-2 phosphorylation. The slower migrating form of Bcl-2 is apparent within about 18 hours of treatment, and persists at 24 hours (see Figure 4).
  • the apoptotic pathway induced by taccalonobde A like paclitaxel, involves Bcl-2 phosphorylation.
  • apoptosis One characteristic of apoptosis is the activation of the caspase cascade of proteinases that are responsible for the initiation and execution phases of apoptosis.
  • Caspase 3 activation is a hallmark of late events in apoptosis.
  • Caspsase 3 activity of cell lysates prepared from cells treated with lO ⁇ M taccalonobde (the IC 8 ) showed activation of the caspase over the time course of 8-24 hr (see Figure 5).
  • Immunoblotting techniques with caspase 3 antibody confirmed the activation of the caspase by the appearance of the pi 7 subunit of the activated caspase. The pi 7 product was detected 18 and 24 hrs following taccalonobde treatment. The data suggest that taccalonobde initiates apoptotic cell death in the MDA-MB-435 cell line.
  • Microtubule stabilizing agents are able to stimulate the polymerization of purified tubulin under conditions where very little tubulin polymerization occurs.
  • the effect of taccalonobde on tubulin polymerization was measured spectrophotometrically by the absorbance at 350nm. Increased turbidity of the solution, constant with tubulin polymerization is measured over time.
  • Taccalonobde stimulated the polymerization of tubulin, consistent with its effects in cells but it was much less potent than Taxol (Figure 6). Electron microscopy revealed that the polymers formed in the presence of taccalonobde resembled tubules and at high magnification were indistinguishable from Taxol-initiated polymers.
  • Taccalonobde A has been shown to inhibit the proliferation of drug sensitive and drug resistant cancer cell lines. (See Figures 1 and 2). Taccalonobde agents have been shown to be pacbtaxel-like stabilizers of microtubules that cause alterations of both interphase and mitotic microtubules. For example, Taccalonobde A is a potent inhibitor of cell proliferation and initiates micronuclei formation. Taccalonobde A is superior to paclitaxel in the ability to circumvent P-glycoprotein-mediated drug resistance. The taccalonobdes therefore represent a new class of pacbtaxel-like micro tubule-stabilizing agents with properties that may provide advantages over the taxanes.
  • taccalonobde compounds of the present invention are potent inhibitors of cell proliferation, acting by disruption of the microtubule network.
  • the taccalonobde compounds disrupt microtubule organization and thus normal cellular functions, including those of mitosis.
  • the present invention demonstrates that taccalonobde compounds circumvent P-glycoprotein-mediated multiple drug resistance.
  • Transport by P-glycoprotein limits the ability of natural product anticancer drugs to inhibit the growth of tumor cells with acquired or de nova drug resistance.
  • Paclitaxel while very useful in the initial course of chemotherapy, is a substrate for transport by P-glycoprotein, and so is of limited usefulness against P-glycoprotein-mediated MDR tumors. Therefore, identification of agents which overcome multiple drug resistance should lead to the development of useful and novel anticancer agents.
  • the taccalonobde compounds of the present invention appear to be such agents since they are poor substrates for P-glycoprotein mediated transport. This fact is reflected in the low cell resistance factor for taccalonobde agents compared with paclitaxel and numerous other natural product drugs.

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Abstract

Selon la présente invention, on a identifié des extraits d'un plante tropicale qui promeuvent le groupement de microtubules similaires au paclitaxel. La purification dirigée de bioessais produit le taccalonolide A stéroïde. Le taccalonolide, tel que le paclitaxel, amorce la formation d'interphase anormale et de microtubules mitotiques. Des cellules traitées avec du taccalonolide présentent des groupes épais de microtubules qui germent indépendamment du centre d'organisation des microtubules. Des fuseaux mitotiques anormaux comprenant des fuseaux multipolaires sont amorcés par le taccalonolide et ressemblent à des fuseaux mitotiques anormaux trouvés en présence de paclitaxel. Comme le paclitaxel, le taccalonolide provoque la rupture du noyau en micronoyaux. Il entraîne l'arrêt de G2/M, la phosphorylation Bc 1-2, et amorce une cascade apoptotique qui inclut l'activation de la caspase 3. Le taccalonolide est un inhibiteur efficace de la prolifération des cellules SK-OV-3 et MDA-MB-435 avec des valeurs IC50 de 2.3νM et 2.1νM respectivement. Par rapport au paclitaxel, le taccalonolide est efficace contre la lignée cellulaire SKVLB-1 de résistance pléïotrope, et semble donc être un substrat médiocre en matière de transport médié par P-glycoprotéine. Bien que le taccalonolide est presque équipotent au paclitaxel concernant ses effets sur les microtubules cellulaires, il est de loin moins efficace que le paclitaxel sur le plan de sa capacité à amorcer la polymérisation de tubuline purifiée et de protéine de microtubules. Le taccalonolide A est le premier agent de stabilisation de microtubules découvert à partir d'une plante, depuis l'identification du mécanisme de l'action du paclitaxel, et il constitue le premier stéroïde de produit naturel identifié doté de ces effets cellulaires.
PCT/US2000/013795 1999-05-21 2000-05-18 Agents de stabilisation de microtubules de taccalonolide Ceased WO2000071563A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040256A1 (fr) * 1999-12-03 2001-06-07 Bayer Aktiengesellschaft Taccalonolides et leur utilisation
WO2001068068A3 (fr) * 2000-03-17 2002-06-20 Inst Nat Sante Rech Med Composes se liant au cytosquelette
WO2012170573A3 (fr) * 2011-06-06 2013-06-13 The Board Of Regents Of The University Of Texas System Nouveaux stabilisateurs taccalonolides de microtubules
CN103494975A (zh) * 2013-10-15 2014-01-08 周莉 裂果薯总皂苷的病理学用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1096215A (zh) * 1994-01-27 1994-12-14 陈德贵 治疗癌症的中草药片
CN1298877A (zh) * 1999-12-03 2001-06-13 拜尔公司 箭根酮内酯及其用途

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040256A1 (fr) * 1999-12-03 2001-06-07 Bayer Aktiengesellschaft Taccalonolides et leur utilisation
WO2001068068A3 (fr) * 2000-03-17 2002-06-20 Inst Nat Sante Rech Med Composes se liant au cytosquelette
WO2012170573A3 (fr) * 2011-06-06 2013-06-13 The Board Of Regents Of The University Of Texas System Nouveaux stabilisateurs taccalonolides de microtubules
JP2014516078A (ja) * 2011-06-06 2014-07-07 ザ ボード オブ レゲンツ オブ ザ ユニバーシティ オブ テキサス システム 新規なタッカロノリド微小管安定剤
US9340572B2 (en) 2011-06-06 2016-05-17 The Board Of Regents Of The University Of Texas System Taccalonolide microtubule stabilizers
US10501490B2 (en) 2011-06-06 2019-12-10 The Board Of Regents Of The University Of Texas System Taccalonolide microtubule stabilizers
CN103494975A (zh) * 2013-10-15 2014-01-08 周莉 裂果薯总皂苷的病理学用途
CN103494975B (zh) * 2013-10-15 2015-05-06 周莉 裂果薯总皂苷的病理学用途

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