WO2000048619A1 - Combined therapy of diterpenoid triepoxides and trail for synergistic killing of tumor cells - Google Patents

Abstract

A synergistic combination of TRAIL or ligands that interact with TRAIL receptors, and diterpenoid triepoxides is used to increase tumor cell killing by induction of apoptosis. Ligands useful in the invention include TRAIL, analogs thereof, stabilized multimers of TRAIL, TRAIL mimetics, etc. Of particular interest are combined therapy with the diterpenoid triepoxides triptolide and derivatives and analogs thereof.

Description

COMBINED THERAPY OF DITERPENOID TRIEPOXIDES AND TRAIL FOR SYNERGISTIC KILLING OF TUMOR CELLS

BACKGROUND The cellular growth of normal tissue is maintained in homeostasis. This balance is determined by cellular proliferation and renewal on one hand, and cell death on the other. Neoplasia can result from aberrant regulation of this homeostasis, through somatic genetic abnormalities that cause cancer initiation and progression. One approach to tumor therapy is to determine agents that act to initiate programmed cell death, or apoptosis. Apoptosis is a form of cell suicide that is critical for differentiation during embryogenesis and regulation of cell numbers. It can also be induced in neoplastic cells, so that they self-destruct. A growing body of evidence suggests that the intracellular "death program" activated during apoptosis is similar in different cell types and conserved during evolution. Apoptosis involves two essential steps. The Bcl-2 family of proteins that consists of different anti- and pro-apoptotic members is important in the "decision" step of apoptosis. In contrast, the "execution" phase of apoptosis is mediated by the activation of caspases, cysteine proteases that induce cell death via the proteolytic cleavage of substrates vital for cellular homeostasis. Bcl-2-related proteins act upstream from caspases in the cell death pathway.

Tumor necrosis factor (TNF) is the prototypic member of a family of cytokines, which interact with their receptors to carry out diverse functions. Some TNF-receptor family members, termed the "death receptors" and which comprise a "death domain", have the unique ability to transmit an intracellular death signal. These receptors include TNF-R1 , Fas (CD95), TRAMP (wsl/Apo-3/DR-3), TRAIL-R1 (DR-4) and TRAIL-R2 (DR-5, TRICK2, KILLER). The ligands for these receptors are capable of inducing apoptosis in tumor cells. However, the potential utility of systemically administered TNF, Fas ligand or lymphotoxin has been limited by their acute toxic effects on normal tissues in vivo. Many cancer cell lines are sensitive to the cytotoxic effects of TRAIL whereas most normal, non-transformed cells are resistant. Walczak et al. (1999) Nature Medicine 5:157-163, fused the receptor-binding region of TRAIL to a leucine zipper motif that favors trimerization. Trimers of leucine zipper TRAIL efficiently killed cultured mammary adenocarcinoma cells but not normal, non-transformed mammary epithelial cells. Systemic administration was able to enhance survival of animals when challenged with this tumor cell line, without detectable adverse effects on viability, tissue integrity, or red and white blood cell counts. The tumoricidal effect of TRAIL was shown to be due to the rapid induction of tumor cell apoptosis.

Strategies to treat tumors with this family of proteins are of great interest. It may be possible to 'sensitize' resistant tumors to apoptosis, for example by modulating the expression or function of FLIP, which blocks the activity of caspases. Other strategies that have been suggested include manipulation of p53 expression. Methods of sensitization may permit the use of lower levels of the proteins, thereby reducing side effects. It may also permit the treatment of otherwise resistant tumors. Approximately 50% of the tumors tested have been resistant to killing by TRAIL (Griffith & Lynch (1998) Curr. Qpin. Immunol. 10:559-563).

Relevant Literature

A review of the TNF receptor superfamily may be found in Baker and Reddy (1998) Oncoqene 17(25):3261-70. The tumor necrosis factor receptor (TNFR) superfamily represents a growing family, with over 20 members having been identified thus far in mammalian cells. These proteins share significant homologies in their extracellular ligand binding domains and intracellular effector (death) domains. Death signals seem to be associated with the activation of both the caspase and JUN kinase pathways. Gravestein and Borst (1998) Semin Immunol 10(6):423-34 also review this receptor superfamily. The use of TRAIL as an anti-tumor agent is discussed in Walczak et al. (1999) Nature Medicine 5:157-163. Yang et al. (1998) Immunopharmacology 40(2): 139-49 provide evidence that suggests the immunosuppressive agent triptolide inhibits antigen or mitogen-induced T cell proliferation, and induces apoptotic death of T cell hybridomas and peripheral T cells. Shamon et al. (1997) Cancer Lett 112(1 ):113-7 evaluate the antitumor potential of triptolide. The isolation, purification, and characterization of immunosuppressive compounds from tripterygium: triptolide and tripdiolide is reported by Gu et al. (1995) Int J Immunopharmacol 17(5):351-6. SUMMARY OF THE INVENTION Methods are provided for improved killing of tumor cells, by increasing the sensitivity of the cells to apoptosis induced by ligands of the TRAIL receptor. The use of TRAIL itself, or fragments and derivatives thereof, is of particular interest. The tumor cells are contacted with diterpenoid triepoxides, e.g. triptolide, tripdiolide, etc., or prodrugs that convert to such compounds under physiological conditions. The killing of the tumor cells by induction of apoptosis through death domain ligands in greatly enhanced by the synergistic action of the diterpenoid compounds and the TRAIL receptor ligands.

DETAILED DESCRIPTION OF THE EMBODIMENTS Methods are provided for enhanced killing of tumor cells through the synergistic action of triptolide and triptolide related compounds, when administered in conjunction with apoptosis inducing ligands of the TRAIL receptor. These ligands interact with TRAIL receptors having a death domain, which then causes the responding cell to undergo apoptosis. Molecules of interest include TNF-related apoptosis-inducing ligand (TRAIL), antibodies that specifically bind to the TRAIL receptor, peptidomimetics, polypeptide fragments, etc. Although these compounds are able to kill tumor cells when administered alone, the concentrations required for a killing dose may create unacceptable side effects. The potent synergy between the diterpenoids and the TRAIL receptor ligands provide increased killing at equivalent or lower doses, and can sensitize otherwise resistant cells.

Definitions It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

diterpenoid triepoxide sensitizing agent: compounds of interest for use as sensitizing agents include compounds having the structure:

wherein Xi is OH, =0; or OR¹;

X₂ and X₃ are independently OH, OR¹ or H; R¹ is -C(O)-Y-Z, wherein Y is a branched or unbranched C₁ to C₆ alkyl or alkenyl group; and Z is COOR², NR³R^3', or +NR⁴R^4'R^4", where R² is a cation; R³ and R^3' are independently H or .branched or unbranched C₁ to Cβ alkyl, hydroxyalkyl, or alkoxyalkyl, or R³ and R^3' taken together form a 5- to 7-member heterocyclic ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen and sulfur, wherein the ring atoms include 2 to 6 carbon atoms, or more nitrogen atoms, and optionally one or more oxygen or sulfur atoms, and wherein the ring is unsubstituted or is substituted with one or more groups selected from R⁵, OR⁵, NR⁵R⁶, SR⁵, NO₂, CN, C(O)R⁵, C(O)NR⁵R⁶, and halogen (fluoro, chloro, bromo, or iodo), where R⁵ and R⁶ are independently hydrogen, lower alkyl or lower alkenyl; and R⁴, R^4' and R^4" are independently branched or unbranched C₁ to C₆ alkyl, hydroxyalkyl or alkoxyalkyl. Examples of such molecules may be found in International Patent application WO98/52951 , and WO97/31921 , herein incorporated by reference.

Compounds of particular interest include triptolide, tripdiolide, triptonide, tripterinin, 16-hydroxytriptolide, triptriolide, and tripchloride; as well as derivatives of triptolide, 16-hydroxytriptolide and tripdiolide (2-hydroxytriptolide) that are derivatized at one or more hydroxyl groups. Such derivatives may be ester derivatives, where the attached ester substituents include one or more amino or carboxylate groups. Prodrugs of particular interest include triptolide succinate sodium salt and triptolide succinate tris(hydroxy-methyl)aminomethane salt.

The compounds of the invention may be prepared from triptolide, tripdiolide, or 16-hydroxytriptolide obtained from the root xylem of the Chinese medicinal plant Tripterygium wilfordii or from other known sources. Methods for preparing triptolide and related compounds are known in the art.

TRAIL Receptor Ligand: Trail receptor ligands (TRL), as defined herein, refer to compounds, usually polypeptide compounds, that bind to mammalian cell surface receptors corresponding to TRAIL-R1 or TRAIL-R2, or homologs or orthologs thereof, and that, by binding so deliver a signal for apoptosis to the cell. The intracellular protein interactions triggered by the death-inducing TRAIL receptors can be attributed to binding interactions of the death domain, which is homologous to an approximately 80 amino acid domain near the C-terminus of TNF-R1 , and is responsible for signaling cytotoxicity (Huang et al. (1996) Nature 384:372-5). Exemplary is the TRAIL-R1 receptor. The sequence of the human homolog is provided herein for convenience as SEQ ID NO:3 and SEQ ID NO:4. The use of TRAIL as a ligand is of particular interest. The sequence of the human homolog is provided herein for convenience as SEQ ID NO:1 and SEQ ID NO:2. Identification of non-human homologs is accomplished by conventional screening methods of DNA libraries or biological samples for DNA sequences having a high degree of similarity to known TRAIL sequences.

The sequence of the TRAIL polypeptide may be altered in various ways known in the art to generate targeted changes in sequence. The sequence changes may be substitutions, insertions or deletions, such as deletions of a domain or exon, providing for active peptide fragments of the protein. Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc. Such alterations may be used to alter properties of the protein, by affecting the stability, specificity, etc. An alteration of particular interest stabilizes multimers of TRAIL (for examples see Walczak et al. (1999) Nature Medicine 5:157-163; Wiley et al. (1995) Immunity 3:673-682; Piti et al. (1996) J. Biol. Chem. 271:12687-12690; and Fanslow et al. (1994) Semin. Immunol. 6:267-278. The protein may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post-expression modifications may be achieved.

Where targeting is desired, the active domain of TRAIL may be produced as a fusion protein with an antibody that is specific for a target cell of interest, thereby providing for an antitumor antibody composition. The antibody may be produced as a single chain, instead of the normal multimeric structure. Single chain antibodies are described in Jost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine. The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.

TRAIL DNA sequences may be employed for synthesis of the complete protein, or polypeptide fragments thereof, particularly fragments corresponding to functional domains; binding sites; etc.; and including fusions of the subject polypeptides to other proteins or parts thereof. For expression, an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducibie or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. Various transcriptional initiation regions may be employed that are functional in the expression host.

The polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells. In some situations, it may be desirable to express the TRAIL gene in mammalian cells, where the protein will benefit from native folding and post-translational modifications.

The subject peptides may also be prepared by synthesis. Various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Foster City, CA, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

Other molecules that interact with the TRAIL receptors may be used in the subject methods. Such ligands will specifically bind to the extracellular domain of the TRAIL-R1 , TRAIL-R2 receptors, and compete with TRAIL for binding. Ligands will also activate TRAIL signaling through the death domain to activate apoptosis. Candidate ligands are screened for their ability to meet this criteria. Assays to determine affinity and specificity of binding are known in the art, including competitive and non-competitive assays. Assays of interest include ELISA, RIA, flow cytometry, etc. Binding assays may use purified or semi-purified TRAIL protein, or alternatively may use cells that express TRAIL, e.g. cells transfected with an expression construct for TRAIL, etc. As an example of a binding assay, purified TRAIL receptor protein is bound to an insoluble support, e.g. microtiter plate, magnetic beads, etc. The candidate ligand and soluble, labeled TRAIL are added to the cells, and the unbound components are then washed off. The ability of the ligand to compete with for TRAIL receptor binding is determined by quantitation of bound, labeled TRAIL. A functional assay that detects apoptosis may be used for confirmation.

Suitable ligands in addition to TRAIL and variants thereof, include peptides, small organic molecules, peptidomimetics, antibodies, or the like. Antibodies may be polyclonal or monoclonal; intact or truncated, e.g. F(ab')₂, Fab, Fv; xenogeneic, allogeneic, syngeneic, or modified forms thereof, e.g. humanized, chimeric, etc.

In many cases, the ligand will be an polypeptide, e.g. TRAIL, an antibody or fragment thereof, etc., but other molecules that provide relatively high specificity and affinity may also be employed. Combinatorial libraries provide compounds other than oligopeptides that have the necessary binding characteristics. Generally, the affinity will be at least about 10"⁶, more usually about 10^-8 M, i.e. binding affinities normally observed with specific monoclonal antibodies.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, sulfhydryl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

Suitable antibodies for use as ligands are obtained by immunizing a host animal with peptides comprising all or a portion of TRAIL protein. Suitable host animals include mouse, rat sheep, goat, hamster, rabbit, etc. The origin of the protein immunogen may be mouse, human, rat, monkey etc. The host animal will generally be a different species than the immunogen, e.g. mouse TRAIL used to immunize hamsters, human TRAIL to immunize mice, etc. Methods to generate monoclonal antibodies are well known in the art and need not be further elaborated.

Susceptible tumors: The host, or patient, may be from any mammalian species, e.g. primate sp., particularly humans; rodents, including mice, rats and hamsters; rabbits; equines, bovines, canines, felines; etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.

Tumors known susceptible to induction of apoptosis include carcinomas, e.g. colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma, etc.; neurological malignancies, e.g. neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, efc; and the like.

Some cancers of particular interest include non-small cell lung carcinoma. Non-small cell lung cancer (NSCLC) is made up of three general subtypes of lung cancer. Epidermoid carcinoma (also called squamous cell carcinoma) usually starts in one of the larger bronchial tubes and grows relatively slowly. The size of these tumors can range from very small to quite large. Adenocarcinoma starts growing near the outside surface of the lung and may vary in both size and growth rate. Some slowly growing adenocarcinomas are described as alveolar cell cancer. Large cell carcinoma starts near the surface of the lung, grows rapidly, and the growth is usually fairly large when diagnosed. Other less common forms of lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignant mesothelioma.

The majority of breast cancers are adenocarcinomas subtypes. Ductal carcinoma in situ is the most common type of noninvasive breast cancer. In DCIS, the malignant cells have not metastasized through the walls of the ducts into the fatty tissue of the breast. Infiltrating (or invasive) ductal carcinoma (IDC) has metastasized through the wall of the duct and invaded the fatty tissue of the breast. Infiltrating (or invasive) lobular carcinoma (ILC) is similar to IDC, in that it has the potential metastasize elsewhere in the body. About 10% to 15% of invasive breast cancers are invasive lobular carcinomas.

Melanoma is a malignant tumor of melanocytes. Although most melanomas arise in the skin, they also may arise from mucosal surfaces or at other sites to which neural crest cells migrate. Melanoma occurs predominantly in adults, and more than half of the cases arise in apparently normal areas of the skin. Prognosis is affected by clinical and histological factors and by anatomic location of the lesion. Thickness and/or level of invasion of the melanoma, mitotic index, tumor infiltrating lymphocytes, and ulceration or bleeding at the primary site affect the prognosis. Clinical staging is based on whether the tumor has spread to regional lymph nodes or distant sites. For disease clinically confined to the primary site, the greater the thickness and depth of local invasion of the melanoma, the higher the chance of lymph node metastases and the worse the prognosis. Melanoma can spread by local extension (through lymphatics) and/or by hematogenous routes to distant sites. Any organ may be involved by metastases, but lungs and liver are common sites. Pharmaceutical Formulations: The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. They may also be used in appropriate association with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Implants for sustained release formulations are well-known in the art.

Implants are formulated as microspheres, slabs, etc. with biodegradable or non- biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing sensitizer is placed in proximity to the site of the tumor, so that the local concentration of active agent is increased relative to the rest of the body.

The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. Dosage: The combined used of diterpenoid triepoxides and TRL compounds has the advantages that the required dosages for the individual drugs is lower, and the effect of the different drugs complementary. Depending on the patient and condition being treated and on the administration route, the diterpenoid triepoxides will generally be administered in dosages of 0.001 mg to 5 mg/kg body weight per day. The range is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses typically being 20, 30 or even 40 times smaller (per unit body weight) in man than in the rat. Similarly the mode of administration can have a large effect on dosage. Thus for example oral dosages in the rat may be ten times the injection dose. The dosage for the TRAIL receptor ligand will vary substantially with the compound. For example, it has been found that dosages of up to 5 mg/kg are tolerated of TRAIL polypeptides in rodents (Walczak et al., supra.), although dosages in humans may be substantially lower, e.g. from about 0.05 mg/kg to about 0.5 mg/kg/day. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenous administration; a tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient, etc. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

For use in the subject methods, the TRL and diterpenoid triepoxides may be formulated with other pharmaceutically active agents, particularly other anti- metastatic, anti-tumor or anti-angiogenic agents. Angiostatic compounds of interest include angiostatin, endostatin, carboxy terminal peptides of collagen alpha (XV), etc. Cytotoxic and cytostatic agents of interest include adriamycin, alkeran, Ara-C, BICNU, busulfan, CNNU, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, hydrea, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, velban, vincristine, vinblastine, VP-16, carboplatinum, fludarabine, gemcitabine, idarubicin, irinotecan, leustatin, navelbine, taxol, taxotere, topotecan, etc.

Methods of Use

A combined therapy of diterpenoid triepoxide compounds and death domain ligands are administered to a host suffering from a susceptible tumor. Administration may be topical, localized or systemic, depending on the specific disease. The compounds are administered at a combined effective dosage that over a suitable period of time substantially reduces the tumor cell burden, while minimizing any side- effects, usually killing at least about 25% of the tumor cells present, more usually at least about 50% killing, and may be about 90% or greater of the tumor cells present. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use. To provide the synergistic effect of a combined therapy, the TRL and the diterpenoid triepoxide active agents can be delivered together or separately, and simultaneously or at different times within the day. In one embodiment of the invention, the diterpenoid triepoxide compounds are delivered prior to administration of the TRL.

The susceptibility of a particular tumor cell to killing with the combined therapy may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the tumor cell is combined with a combination of a TRL and a diterpenoid triepoxide at varying concentrations for a period of time sufficient to allow the active agents to induce apoptosis, usually between about one hour and one week.. For in vitro testing, cultured cells from a biopsy sample of the tumor may be used. The viable cells left after treatment are then counted.

The dose will vary depending on the specific cytotoxic agent utilized, type of tumor, patient status, etc., at a dose sufficient to substantially ablate the tumor cell population, while maintaining patient viability. In some cases therapy may be combined with stem cell replacement therapy to reconstitute the patient hematopoietic function. Treatment will generally be continued until there is a substantial reduction, e.g. al least about 50%, decrease in the tumor burden, and may be continued until there are essentially no tumor cells detected in the body. It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the array" includes reference to one or more arrays and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the cell lines, constructs, and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

EXPERIMENTAL Members of the tumor necrosis factor family such as tumor necrosis factor-α, Fas and TNF-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, induced programmed cell death, apoptosis, in tumor cells. Recent studies show that TRAIL is more cytotoxic than TNF-α and Fas in solid tumor cell lines and TRAIL is less toxic than Fas and TNF-α to normal cells. There are, however, several tumor cell lines which are resistant or relatively resistant to the cytotoxic action of TRAIL. We have found that PG490, which contains purified triptolide, induces apoptosis in many solid tumor cell lines. Additionally, the combination of PG490 and chemotherapy is more cytotoxic than each alone. Potent synergy was observed between PG490 plus TNF-α and PG490 plus TRAIL. The combination of PG490 plus TRAIL induces apoptosis in greater than 80-99% of cells in all solid tumor cell lines tested. This combination is more effective than chemotherapy alone or chemotherapy plus TRAIL. Even cell lines that are relatively resistant to PG490 or TRAIL alone, such as ME180 cervical cancer cells, are dramatically senstive to the combination of PG490 plus TRAIL with cell death in greater than 80% of cells.

Materials and Methods

Source of triptolide. PG490 (triptolide, MW 360) was obtained from Pharmagenesis (Palo Alto, CA). The material was composed of white to off-white crystals, had a melting point of 226-240 °C, conformed to a standard triptolide preparation by Proton Nuclear Magentic Resonance (5), and was 97% pure by reverse phase HPLC evaluation using acetonitrile:methanol:water (18:9:73; J. Fidler and R.L. Jin, Pharmagenesis, private communication)

Cell Lines. A549 and H1299 (non-small cell lung cancer), ME180 (cervical carcinoma) and MDA-MB231 (mammary adenocarcinoma) cell lines were purchased from ATCC. Cells were cultured in the appropriate media with 10% FCS supplemented with L-glutamine, penicillin and streptomycin.

Expression and Purification of Purified TRAIL. TRAIL codons 95-285 were amplified by polymerase chain reaction and subcloned into a pQE-9 (Qiagen, Santa Clarita, CA) bacterial expression vector downstream of a 6X-Histidine tag. TRAIL was purified by Nickel affinity chromatography according to the manufacturer's protocol. The purity of TRAIL was confirmed by Coomassie blue staining. Cell death reagents and assays. Cell viability was measured by an MTT assay. Briefly, untreated cells or cells treated with PG490 (5-100 ng/ml) and/or TRAIL (10-1000 ng/ml) in a 96-well plate were harvested at 48 h followed by the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to the cells. Cells were then solubilized with 0.1 N acidified CH₃CI-HCL. The 96-well plate was read at a wavelength of 590 nm on an iEMS Labsystems plate reader. Induction of cell death by TRAIL, PG490 and PG490 plus TRAIL was confirmed as apoptotic by Annexin staining followed by FACS analysis.

The combination of triptolide and TRAIL induced apoptosis in greater than 95% of cells in diverse solid tumor cell lines, including lung, breast and sarcoma cell lines. For example, in the non-small cell lung carcinoma H1299, triptolide or TRAIL alone induce apoptosis in less than 50% of cells, but the combination treatment induced apoptosis in greater than 95% of the cells.

Table 1

Maximal cytotoxicity was achieved with a combination of triptolide and TRAIL when cells were pre-treated with triptolide for 2-5 hours prior to the addition of TRAIL. This combination was effective in cells that lack p53, such as the H1299 cell line. The combination therapy was more cytotoxic in these cells lines than chemotherapy alone (carboplatinum, doxorubicin or taxol), or chemotherapy in combination with TRAIL.

TRAIL at 100 ng/ml induced a four fold increase in NF-κB transcriptional activity in A549 (p53 wild type) and a two fold increase in NCI-H-1299 (p53 null) non- small cell lung cancer cell lines. Triptolide inhibited TRAIL-induced NF-κB activation by 90-95%. The cytotoxicity of TRAIL alone in both cell lines was less than 30% but increased to 80-95% in combination with triptolide. Pretreatment with MG132, a proteasome inhibitor that inhibits NF-κB, also sensitized A549 and H1299 cells to TRAILOinduced apoptosis.

The data show that triptolide and TRAIL act in synergy to induce appoptosis in greater than 80% of cells in several diverse solid tumor cell lines.

Claims

WHAT IS CLAIMED IS:

1. A method for enhanced killing of tumor cells, the method comprising: contacting a susceptible tumor cell with a synergistic combination of a TRAIL receptor ligand; and a diterpenoid triepoxide having the structure:

wherein Xi is OH, =O; or OR¹; X₂ and X₃ are independently OH, OR¹ or H;

R¹ is -C(O)-Y-Z, wherein Y is a branched or unbranched Ci to C₆ alkyl or alkenyl group; and Z is COOR², NR³R^3', or +NR⁴R R^4", where R² is a cation; R³ and R^3' are independently H or branched or unbranched Ci to C₆ alkyl, hydroxyalkyl, or alkoxyalkyl, or R³ and R^3' taken together form a 5- to 7-member heterocyclic ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen and sulfur, wherein the ring atoms include 2 to 6 carbon atoms, or more nitrogen atoms, and optionally one or more oxygen or sulfur atoms, and wherein the ring is unsubstituted or is substituted with one or more groups selected from R⁵, OR⁵, NR⁵R⁶, SR⁵, NO₂, CN, C(O)R⁵, C(O)NR⁵R⁶, and halogen (fluoro, chloro, bromo, or iodo), where R⁵ and R⁶ are independently hydrogen, lower alkyl or lower alkenyl; and R⁴, R^4' and R^4" are independently branched or unbranched Ci to Cβ alkyl, hydroxyalkyl or alkoxyalkyl; in a combined dosage effective to kill at least about 50% of said tumor cells.

2. The method of Claim 1 , wherein said diterpenoid triepoxide is selected from the group consisting of triptolide, tripdiolide, 16-hydroxytriptolide; triptolide succinate, an ester derivative of triptolide, an ester derivative of tripdiolide, and an ester derivative of 16-hydroxytriptolide.

3. The method of Claim 1 , wherein said TRAIL receptor ligand is human TRAIL polypeptide or an active fragment thereof.

4. The method of Claim 3, wherein said TRAIL polypeptide is a soluble, stabilized multimer.

5. The method of Claim 1 , wherein said tumor is a human tumor.

6. The method of Claim 4, wherein said tumor is a solid tumor.

7. The method of Claim 5, wherein said tumor is a carcinoma.

8. The method of Claim 7, wherein said tumor is a mammary adenocarcinoma.

9. The method of Claim 7 wherein said tumor is a non-small cell lung carcinoma.