KR20080080539A - Use of angeloyl-substituted ingenanes in combination with other agents to treat cancer - Google Patents

Abstract

The present invention relates generally to therapeutic protocols for the treatment of cancer in subjects including humans. More particularly, the present invention provides a method for treating cancer comprising the administration of an angeloyl-substituted ingenane or a pharmaceutically acceptable salt, derivative, homolog or analog thereof and at least one other agent. Preferably, the latter agent is an anti-cancer agent or has or induces an anti-cancer effect.

Description

USE OF ANGELOYL-SUBSTITUTED INGENANES IN COMBINATION WITH OTHER AGENTS TO TREAT CANCER} Combined with Other Agents to Treat Cancer

The present invention generally relates to a treatment protocol for the treatment of cancer in a subject, including humans. More specifically, the present invention relates to a method of treating cancer comprising administering angeloyl-substituted ingenane, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof and one or more other agents. Preferably, the agent is an anticancer agent or has or induces an anticancer effect.

Bibliographic details of the publications cited by the present inventors are summarized at the end of this specification.

Any prior art citation herein is not, and should not be considered, an admission or any form of implication that such prior art forms part of the general general knowledge in any country.

Screening of natural products is a term applied to the screening of the natural environment for bioactive molecules. Bioactive molecules with the potential as particularly useful therapeutic agents are being studied. Natural environments include plants, microorganisms, soils, corals and marine animals. The study of potential therapeutic agents for the treatment of cancer is an important focus.

The Euphorbia family of plants includes a variety of plants, including the weeds of Euphorbia species. The most in-depth study in this group was Euphorbia. pilulifera L) ( syn . E. hirta L., E. capitata Lam.), generic names of which include Peel-Bearing Spurge, Snaketail, Cat's Hair, Queensland Asma Weed and Flowery-Headed Spurge. The plant is widely distributed in the tropics, including India, and northern Australia, Queensland. Euphorbia peplus is another species from which ingenol angelate with anticancer properties has been isolated (see US Pat. Nos. 6,432,452, 6,787,161 and 6,844,013). PEP005 is this. Ingenol angelate extracted and purified from E. peplus and is particularly useful for the treatment of actinic keratosis and non-melanoma skin cancer (NMSC) by short-term topical administration. Cytotoxicity of PEP005 has been found in vitro for many cell lines, and its in vivo efficacy has been clinically proven. The chemical name of PEP005 is ingenol-3-angelate.

Cancer is a cellular disease that occurs when the cell population overproliferates. In the United States alone, 2,604,650 people died of cancer between 1990 and 1994, with men (53%) more affected than women (47%). The most common cancer deaths are lung cancer (about 30%), colon and rectal cancer (about 11%), breast cancer (about 8%) and prostate (about 6.5%). The most common cancers in women are breast (about 31%), lung (about 12%), colon and rectum (about 12%), uterus (about 6%) and ovary (about 4%). It is estimated that 570,280 people died of certain types of cancer from 2005 to 2006.

Cancer treatment generally requires a treatment protocol that includes one or more of surgery, radiation and / or chemotherapy. Chemotherapy is a particularly common and well established treatment for cancer. However, despite the fact that anticancer therapies offer significant benefits for patients, their use may be limited due to the problems of toxic and harmful reactions. In fact, patients with terminal illness often choose not to be actively treated because they have a serious impact on quality of life.

In the treatment of malignant diseases, low molecular weight (LMW) anticancer drugs exhibit a lack of therapeutic efficacy, highlighting the increasing need for a holistic approach for cancer therapy (Jain, J Natl Cancer Inst 81: 570, 1989). Jain, Science 271: 1079, 1996). New concepts have been created for modulating the pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity, biorecognition and efficacy of drugs, based on an interdisciplinary approach in polymer engineering, pharmacy, biobinder chemistry and molecular biology. A new drug delivery system has been developed. The combination of drug and macromolecule has created a new type of therapeutics based on water-soluble polymer carriers, micelle-forming block copolymers, nanospheres, microspheres, liposomes, dendrimers and hydrogels (Kim and Lim, Arch Pharm Res) 25: 229, 2002). However, unlike LMW counterparts, macromolecular drugs often encounter significant transmission limitations that can limit the achievement of these desirable properties. Thus, tumor selectivity is often emphasized due to the passive accumulation of macromolecules in malignant masses, a phenomenon known for poorly formed vasculature of tumors and improved permeability and retention (EPR) effects (Maeda Adv Enzyme Regul 41: 189, 2001), tumor cell membranes may constitute potent blockers for macromolecules that must first enter their target cells in order to cause cell death. In addition, increased intratumoral pressure may compromise the delivery of macromolecular drugs to deep regions in malignant masses when poor lymphatic drainage of tumors is formed (Swartz et al., J Biomech 32: 1297, 1999). ).

Another important drug delivery system includes a combination therapy in which multiple agents are administered to treat various indications or to enhance the activity of one or more agents. The ability to reduce the toxicity of anticancer agents by coadministration with other compounds has been a long term goal in cancer therapy.

There is a need to develop formulations and protocols for the treatment of cancers with improved efficacy.

Summary of the Invention

The present invention provides a treatment protocol for the treatment of cancer. This protocol includes administering angeloyl-substituted ingenane (ie, ingenol angelate), or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and at least one other agent. The agent may relate to anticancer properties or may induce or promote a reduction in the size, growth and / or spread of cancer cells or tissues. Therefore, the present invention provides a property having anticancer activity; Stimulating neutrophils or other immune cells or immune effector molecules (eg, antibodies) in an immune response against cancer cells; Properties with the ability to mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells; Properties that promote the production of antibodies that can bind to cancer cells; And / or Angeloyl-substituted ingenans, or pharmaceutically acceptable salts, derivatives, homologs or analogs thereof, with or associated with agents exhibiting at least one of the properties having cytotoxic T-cell activity against cancer cells. It relates to a combination therapy for the treatment of cancer comprising administering.

The present invention provides a combined chemotherapeutic protocol or combined chemotherapeutic and immunotherapeutic efficacy (ie chemoimmune effect) for inducing anticancer activity for treating a subject with cancer. The combined treatment protocol has been proposed to enhance the anticancer activity or efficacy of the protocol compared to the use of ingenol angelate or other agents alone.

"Anti-cancer activity" includes apoptosis, necrosis, lysis, death, senescence and / or cell cycle arrest of cancer cells. It also includes inhibition of cancer metastasis and induction of immune memory against cancer cells. "Anti-cancer efficacy" includes not only anti-cancer activity, but also other properties such as, in particular, the rate, timing and efficiency of inducing cancer cell cytotoxicity, apoptosis, necrosis, senescence and / or cell cycle arrest; Properties with the ability to treat or inhibit metastasis; Induces or stimulates an immunological response to cancer cells; Induces immune memory against recurrence of cancer cells; Reducing the individual content of the required angeloyl-substituted ingenane or one or the other of the agent; Reducing the toxic side effects of anticancer therapy in the subject; And a property of reducing cancer growth after a remission period.

"Cancer" includes solid and hematologic cancers such as tumors, leukemias, sarcomas or carcinomas. Combination of ingenol angelate and other agents may be presented in a single formulation or in separate formulations mixed together prior to administration or may be administered simultaneously or sequentially from separate formulations. The terms "cancer" and "tumor" may be used interchangeably herein. A "tumoral" disease is also a form of cancer or tumor.

Thus, one embodiment of the present invention relates to a treatment protocol for treating a subject having cancer or a subject suspected of having cancer, wherein the protocol is

Administering to said individual an angeloyl-substituted ingenan, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and

The subject has the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Administering an agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone. Examples of T-cell mediated inhibition or death of cancer cells include cytotoxic T-cell mediated inhibition or cell death.

The present invention further provides a method of treating a subject with cancer or a subject suspected of having cancer, the method comprising

Administering to said individual an angeloyl-substituted ingenan, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and

 The subject has the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

Administering an agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

Thus, when the formulations are administered together, the total amount of all of the formulations may be less than when administered separately or, although nevertheless, additionally in terms of improved or enhanced anticancer activity or efficacy, nevertheless therapeutically more May be valid.

The present invention further provides a therapeutic protocol for treating cancer in an individual, the protocol comprising ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and the following properties:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Administering at least one other agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone,

Incorporation of ingenol angelate with the agent results in a merger index (CI) of less than 1, where CI is determined using the following equation:

CI = [(C) ₁ / (C _x ) ₁ ] + [(C) ₂ / (C _x ) ₂ ] + [α (C) ₁ (C) ₂ / (C _x ) ₁ (C _x ) ₂ ]

Wherein (C _x ) ₁ is the concentration of one of the ingenol angelates or agents that produces the x% effect of the drug alone, and (C) ₁ is the other of the ingenol angelates or agents (C) _Is the concentration of one of the ingenol angelates or agents that, in combination with ₂ , produces the same x% effect, and α is a constant if the modes of anticancer activity of the ingenol angelates and agents are mutually exclusive or non-excluded and α are 0 and 1, respectively.

The present invention also provides ingenol angelate and (1) a second anticancer agent; (2) anticancer T-cells, such as anticancer cytotoxic T-cells; (3) neutrophil-stimulating agents; (4) ADCC-promoting agents (including neutrophils); (5) anticancer cell antibodies; And / or (6) administering one or more of the agents that induce, stimulate, or cause the production of one or more of (1) to (5).

Another embodiment of the invention is a first portion comprising ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and a second portion comprising an agent or a pharmaceutically acceptable salt thereof Wherein the moiety optionally provides a multipart pharmaceutical formulation further comprising one or more pharmaceutically acceptable carriers, diluents and / or excipients, the formulation having the following properties:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Wherein at least one of the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

The present invention further provides combinations comprising ingenol angelate, or pharmaceutically acceptable salts, derivatives, homologs or analogs thereof and agents or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers, diluents and / or excipients. The formulation provides the following properties:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Wherein at least one of the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

Another embodiment of the present invention relates to the use of ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and an agent or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer, The formulation has the following properties:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Wherein at least one of the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

With regard to formulations and uses, in a preferred embodiment, the CI of the combination of ingenol angelate and the formulation is less than one. CI values less than 1 mean 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.91, 0.92, 0.93, 0.94 , 0.95, 0.96, 0.97, 0.98 and 0.99, as well as numerical values between them.

In a preferred embodiment, the ingenol angelate is ingenol-3-angelate, referred to herein as "PEP005". “Ingenol angelate”, “ingenol-3-angelate”, “PEP005” or anticancer agent means naturally isolated forms, chemically modified forms, chemically synthesized forms, as well as pharmaceutically acceptable salts, derivatives thereof, Homologues or analogs.

Anticancer agents contemplated by the present invention include cytotoxic, necrotic, apoptotic, cell cycle arrest and static agents useful for the treatment of tumorous diseases such as solid and hematological cancers. Examples of specific anticancer agents are shown in Table 1 below. In addition, the term "anticancer agent" refers to cytotoxic T-cells and other T-cell types and other immune cells, as well as antibodies and neutrophil-stimulating agents and ADCC promoting agents, and agents causing any one or more of the above. Include.

The subject can be any animal, including a mammal, such as a human.

The invention further relates to a delivery system for the formulation in question. In one embodiment, the ingenol angelate and the formulation are maintained separately and mixed together prior to administration. In such embodiments, administration may be via a single body tissue-invasive inlet device or through multiple body tissue-invasive inlet devices such as, but not limited to, syringes and needles. Thereafter, simultaneous or sequential administration of ingenol angelate and the agent is part of the present invention in any order.

Examples of Agents for Use as Co-Pharmaceuticals with Angeloyl-Substituted Ingenans for the Treatment of Cancer Alkylating agents Alkeran-Melphalan Alkeran Injection-Melphalan BiCNU-Carmustine CeeNU-Romustine Cycloblastin-Cyclophosphamide Endoxic Acid with Sodium Chloride-Cyclophosphamide Endoxic Acid (new formulation without sodium chloride) -Cyclophosphamide riadel implant-Carmustine holoxane-Iposamide leukeran-Chlorambucil muporan-Potemustine milleran-Sulfonated temodal-Temozolomide thiotepa-Tiotepa Antibiotic Cytotoxic Adriamycin Solution-Doxorubicin Hydrochloride Blenmax-Bleomycin Sulfate Bleoxane-Bleomycin Sulfate Bleomycin Sulfate for Injection (DBL)-Bleomycin Sulfate Kaelix-Doxorubicin Hydrochloride Cosmegen-Dactinomycin Daunorubicin Injection-Daunorubicin Injection Doubnoxine-Daunorubicin Hydrochloride Docetaxel Doxorubicin Hydrochloride Injection (DBL)-Doxorubicin Hydrochloride Doxorubicin Hydrochloride Injection USP-Doxorubicin Hydrochloride Epirubicin Hydrochloride Injection (DBL)-Epirubicin Hydrochloride Fludara-Fludarabine Phosphate Mitomycin C Mitomycin Mitozantron Injection-Mitozantron Hydrochloride Novantrone-Mitozantron Hydrochloride Oncotrone-Mitozantron Hydrochloride Pamorrubicin-Epirubicin Hydrochloride Javedos-Idarubicin Hydrochloride

Anti-metabolite Cytarabine (DBL)-Cytarabine Cytarabine Injection-Cytarabine Efudix-Fluorouracil (5-Fluorouracil) Fluorouracil Injection BP-Fluorouracil Fluorouracil Injection BP-Fluorouracil Gemzar-Gemcitabine Hydrochloric Acid Hytopanthin-Topotecan Hydrochloride Hydrea-Hydroxyurea Ranvis-Thioguanine Ledertrexate-Methotrexate Leuase-Colastase Lustatin-Cladribine Metoblastine-Methotrexate Methotrexate Injection and Tablet (DBL)-Methotrexate Methotrexate Injection BP-Methotrexate Puri-Netol-Mercaptopurine Tomusdex-Ralti Trexed Xceloda-Capecitabine

Hormone antitumor Anandron-Nilutamide Androcur-100-Cyproterone Acetate Arimidex-Anastrozole Aromacin-Exemestane Chemmat Tamoxifen-Tamoxifen Citrate Cosudex-Bicalutamide Cyprone-Cyproterone Acetate Ciprostat- 100-Cyproterone Acetate Citadren-Aminoglutetimide Depot-Provera-Medoxyprogesterone Acetate Depo-Lalvera-Medroxyprogesterone Acetate Yulexin-Flutamide Parestone-Torremifen Citrate Femara-Letrozole Flutamine-Flutamide Fugerel-Flutamide Genox-Tamoxifen Citrate GenRx Tamoxifen-Tamoxifen Citrate HealthSense Tamoxifen-Tamoxifen Citrate Lucrine-Luperolin Acetate Lucrine Depot-Lupproline Acetate Medroxoxal-Med Roxyprogesterone Acetate Megase-Meges Troll Acetate Nolvadex, Nolvadex-D-Tamoxifen Citrate Procur-Cyproterone Acetate Provera-Medroxyprogesterone Acetate Valrobera-Methoxyprogesterone Acetate Sandodostatin-Octreotide Tamoxin-Tamoxifen Citrate Tamoxen-Tamoxifen Citrate Tamoxifen Hexal-Tamoxifen Citrate Tamoxifen-BC-Tamoxifen Citrate Terry White Chemsut Tamoxifen-Tamoxifen Citrate Zoladex 10.8 mg Implant-Goserelin Acetate Zoladex 3.6 mg Implant-Goserelin Acetate

Other antitumor agents Anzatax Injection-Paclitaxel Agriline-Anagrelide Hydrochloride Amcidyl-Amsacrine Camptosar-Irinotecan Hydrochloride Carboplatin Injection-Carboplatin Carboplatin Injection (DBL)-Carboplatin Cisplatin Injection-Cisplatin Cisplatin Injection Solution (DBL)-Cisplatin DTIC -Dacarbazine for injection of dacarbazine (DBL)-dacarbazine eloxatin-oxaliplatin etophosce-etoposide phosphate etoposide injection-etoposide etoposide injection (DBL)-etoposide glibeck-imatinib mesyl Late Herceptin-Trastuzumab Hexylene-Altretamine Mabthera-Rituximab Natlan-Procarbazine Hydrochloride Proleukin-Aldesleukin (rbe) Sodium Iodide (131I) Capsule (Treatment)-Sodium Iodide (131I) ) Taxol-Paclitaxel Taxotere-Docetaxel Bepeside-Etoposide Besanoid-Tretinoin Bumon-Teniposide Vinca Alkaloids Eldicin-Bindesin Sulfate Navelin-Vinorelvin Tartrate Oncovin-Vincristine Sulfate Velve-21 Vinblastine Sulfate Injection (DBL)-Vinblastine Sulfate Vincristine Sulfate Injection-Vincristine Sulfate Vincristine Sulfate Injection (DBL) Vincristine sulfate vinorelbine Other Formulations Antibodies T-Cell Cytotoxic T-Cell Cancer Antigen (Vaccine) Agents that Promote, Activate, or Cause the Production of Anticancer Antibodies, Cytotoxic T-Cells, or Neutrophils That Promote or Mediate Neutrophil ADCC

Thus, the formulations set forth in Table 1 may be useful for chemotherapeutic compounds and molecules other than antibodies to combinations of antigens specific for cancer cells or for cancer-specific antigens, as well as various other biologically- or chemically-synthesized cytotoxicities. Apoptotic, necrotic, static and cell cycle arrest agents.

Throughout this specification, unless the context specifically requires, the term "comprises" or derivatives thereof, such as "comprising" or "comprising", includes inclusion of a specified element or integer or group of elements or integers. It is to be understood that it is not intended to exclude any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to as sequence identifier numbers (SEQ ID NOs). Sequence numbers correspond to the sequence identifiers <400> 1 (SEQ ID NO: 1), <400> 2 (SEQ ID NO: 2), and the like. A summary of sequence identifiers is shown in Table 2 below. The sequence listing is presented following the claims.

Summary of Sequence Identifiers Sequence number Explanation One Primer for IL-1β-5 ' 2 Primer for IL-1β-3 ' 3 Primer for TNFα-5 ' 4 Primer for TNFα-3 ' 5 Primer for Glyceraldehyde-3-phosphate dehydrogenase-5 ' 6 Primer for Glyceraldehyde-3-phosphate dehydrogenase-3 ' 7 Primer for Phagocyte Inflammatory Protein 2-5 ' 8 Primer for Phagocytic Inflammatory Protein 2-3 '

Brief description of the drawings

Some drawings include colored drawings or entities. Color photographs are available from the patentee or from the Office, if desired. Fees may be charged from the JPO.

1 shows a graph showing the effects during the experiment with PEP005 provided for 1 hour, 24 hours and 48 hours in a cell line panel.

2 shows a graph showing PEP005 cytotoxicity in two leukemia cell lines, K562 and CCRF-CEM after administration for 24, 48 and 72 hours.

3 shows photos and graphs of cell cycle changes over 0, 1, 5, 12, 24 and 48 hours of exposure to PEP005.

4A and 4B are diagrams of apoptosis induction (A) sub-Gl DNA content, PI staining and (B) PI-annexin V staining.

5 shows a graph showing comparative analysis of PEP005 cytotoxicity with other anticancer drugs in a panel of cell lines. The data are HT29 (1), HCT1116 (2), HCC2998 (3), COLO205 (4), MCF7 (5), MDA435 (6), HOP62 (7), HOP92 (8), IGROV1 (9) and OVCAR3 (10). ) (IC ₅₀ -IC ₅₀ mean) for the cell line.

6 is a graph showing the interaction of PEP005 and 5FU in human COLO205 cancer cell line. PEP-5FU; PEP005 followed by 5FU; 5FU-PEP: 5FU followed by PEP005; PEP + 5FU: simultaneous administration of PEP005 and 5FU. The merge index theory below 0.8 indicates a synergistic effect, above 1.2 indicates antagonism, and a merge index between 0.8 and 1.2 corresponds to an additive effect.

7 is a graph showing the interaction of PEP005 and doxorubicin in human COLO205 cancer cell lines. PEP-doxorubicin: PEP005 followed by doxorubicin; Doxorubicin-PEP: doxorubicin followed by PEP005; PEP + doxorubicin: simultaneous administration of PEP005 and doxorubicin.

8A and 8B are graphs showing the interaction of PEP005 and oxaliplatin in (A) human COLO205 cancer cell line. PEP-24h-oxa: PEP005 24 hours followed by 24 hours in R10 medium followed by 24 hours in oxaliplatin; PEP-72h-oxa; PEP005 was post-cultured for 1 hour, 5 hours, 24 hours followed by 72 hours in R10 medium followed by 24 hours oxaliplatin. (B) PEP-oxaliplatin merge without time intervals. Efficacy of efficacy showing interaction of PEP005 and oxaliplatin in human COLO205 cancer cell line.

9 shows a graph showing the interaction of PEP005 and gemcitabine in human COLO205 cancer cell line. PEP-gemcitabine: PEP005 followed by gemcitabine; Gemcitabine-PEP: gemcitabine followed by PEP005; PEP + kimcitabine: simultaneous administration of PEP005 and gemcitabine.

10 depicts a graph showing the interaction of PEP005 and vinorelbine in human COLO205 cancer cell line. PEP-vinorelbine: PEP005 followed by vinorelbine; Vinorelbine-PEP: vinorelbine followed by PEP006; PEP + vinorelbine: simultaneous administration of PEP005 and vinorelbine.

11 depicts a graph showing the interaction of PEP005 and docetaxel in human COLO205 cancer cell line. PEP-docetaxel: PEP005 followed by docetaxel; Docetaxel-PEP: docetaxel followed by PEP005; PEP + docetaxel: simultaneous administration of PEP005 and docetaxel.

12 shows a graph showing the interaction of PEP005 and cisplatin in human COLO205 cancer cell line. PEP-cisplatin: PEP005 followed by cisplatin; Cisplatin-PEP: Cisplatin followed by PEP005; PEP + cisplatin: simultaneous administration of PEP005 and cisplatin.

13A-13D show graphs showing the effect of PEP005 on endothelial cell and neutrophil peroxide production and antibody production. (A) Activation of human endothelial by PEP005. Endothelial cells were treated for 4 hours with PEP005 prior to the addition of neutrophils and the adhesion to the endothelial cell layer was assessed. 100 U / ml TNFα was used as a positive control. Data is mean ± SE of 5 separate experiments. ANOVA analysis illustrates the significant effect of the dose of PEP005 on the level of neutrophil adhesion (p = 0.003). (B) Killing melanoma cells by PEP005-activated human neutrophils in vitro. Human MM96L melanoma cells were co-cultured with neutrophils and treated with PEP005 for 24 hours. Neutrophils and PEP005 were removed and melanoma cell survival was measured after 6 days. Killing is expressed as the survival of tumor cells in the absence of PEP005 and neutrophils, which was 100% (□) at a neutrophil: MM96L ratio of 0: 1. (C) Peroxide production by PEP005-treated neutrophils. Human neutrophils were incubated with PEP005 for 2 hours and the production of peroxides was measured using lucigenin assay. (D) Antibody response after PEP005 treatment of B16 tumors. After growing B16 tumors in C57BL / 6 mice reached 30-60 mm 3 (1 tumor per mouse), they were treated with PEP005 treatment and B16-specific IgG1 and IgG2a antibody titers started treatment after day 11 (n = 6). ) Blood was collected from (■) or 135 days post treatment start (n = 5) (▲) and measured by standard ELISA. In addition, antibody titers against placebo-treated animals in which tumors reached 121.5 ± SD 20.1 (n = 4) at day 11 (o). Naive mice (n = 5) have no tumor (□).

14A and 14B show graphs of PEP005 rescued IL-2-deficient activated CD8 ⁺ T-cells from apoptosis. (A) Anterior / lateral scattering diagram of untreated cells and cells incubated with IL-2 (25 U / ml). Cells gated at R1 are shown in the lower panel for their active caspase-3 expression as indicated by FITC fluorescence. Thick black lines represent unrelated controls. The percentage of apoptotic cells was calculated as the percentage of cases under marker 1 (M1) from the total gated cells and subtracted the same ratio under M1 in an unrelated control. (B) Percentage of apoptosis in cells given IL-2 (black bars) and cells lacking IL-2 (white bars) are plotted against increasing concentrations of PEP005. Data was obtained from three separate experiments. Values are shown as mean ± SD. Statistical analysis was carried out using the hole bilateral Student's t-test and presented p values. CI = 95% was taken and * indicates significance level.

15A-15D show graphs of PEP005 induced proliferation and apoptosis rescue at 0.01 μg / ml (20 nM). Cells that received different treatments as described in the method section were pulsed with BrdU and stained for BrdU and caspase. (A) the percentage of proliferating cells at 0, 24 and 48 hours of the experiment, (B) the percentage of total apoptotic cells analyzed on day 2, and (C) the percentage of proliferating cells at day 2 The percentage of apoptotic cells, (D) The percentage of apoptotic cells in the non-proliferating at the indicated time point was analyzed on day 2.

Detailed description of the invention

In the description and claims of the invention, the following terms are used in the definitions given below.

Unless otherwise stated, it is to be understood that the present invention is not limited to the specific combination, preparation method, dosing regimen, etc. of the ingredients as they may be changed. 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 be limiting.

The terms "a" and "the" (the articles "a", "an" and "the") include plural embodiments unless the context clearly dictates otherwise. Thus, for example, "cancer" includes a single cancer or two or more cancers; “Angeloyl-substituted ingenane” or “engenol angelate” includes not only a single compound but also two or more compounds; "Combination" includes a single blend or two or more blends.

The terms "compound", "formulation", "active agent", "chemical agent", "pharmacologically active agent", "pharmaceutical", "active" and "drug" are used interchangeably herein, which means that certain pharmacological and / Or chemical compounds, proteins (eg, antibodies) or cells that induce physiological effects. The term also includes pharmaceutically acceptable and pharmacologically active ingredients of the active agents specifically referred to herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, and the like. When the terms "compound", "formulation", "active agent", "chemical agent", "pharmacologically active agent", "pharmaceutical", "active" and "drug" are used, not only the active agent itself, but also the pharmaceutical agent It is to be understood to include acceptable and pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs and the like. Certain pharmacological or physiological effects are anticancer activity or efficacy, including cytotoxicity, apoptosis, necrosis, senescence and / or cell cycle arrest of cancer cells or cells that are likely to become cancer. The present invention relates to the incorporation of two or more agents that yield greater anticancer efficacy compared to the use of the agent alone. Such greater anticancer efficacy may in particular be due to the reduced toxicity of the anticancer agent, the greater efficacy in inducing cancer cell death or growth arrest, the reduced side effects on the subject, the reduced metastasis or treatment thereof and / or the immunological response to cancer cells. Or immune memory.

The terms (ie, "compound", "agent", "active agent", "chemical agent", "pharmacologically active agent", "pharmaceutical", "active" and "drug" are anticancer chemotherapeutic agents, antibodies and T It is broadly referred to as a "formulation" comprising cells (such as cytotoxic T-cells) and other immune cells, as well as neutrophil-stimulating agents and ADCC-promoting agents, as well as agents that cause any of these effects. Thus, agents used for incorporation with angeloyl-substituted ingenans include chemotherapeutic agents that induce at least one of cytotoxicity, apoptosis, necrosis, senescence or cell cycle arrest of cancer cells; T-cells (eg, cytotoxic T-cells) or agents capable of inducing T-cell mediated inhibition or death of cancer cells; Antibody specificity for antigens in cancer cells or the incorporation of antigens predominantly or exclusively in cancer cells; Or neutrophils or agents that induce neutrophil-promoted antibody-dependent cellular cytotoxicity (ADCC) of cancer cells.

"Compound", "formulation", "active agent", "chemical agent" "pharmacologically active agent", "pharmaceutical", "active" and "drug" are combinations having two or more activities, such as two or more ingenol angelates Or two or more agents. "Merge" also includes multipart compositions, such as two-part compositions, which separately provide or separately provide or dispense an agent or mix prior to dispensing. For example, a multipart pharmaceutical pack may contain two or more active agents that are kept separate. It also applies to the terms “anticancer agent” and “chemotherapy agent” and other agents such as cells (eg, cytotoxic T-cells or neutrophils) or antibodies.

As used herein, the terms "effective amount" and "therapeutically effective amount" of an agent refer to reduced relapse, reduced or treated metastasis of cancer growth after remission, immune system anticancer activity against cancer cells, and / or to an individual. Agents that provide any therapeutic or physiological effect or result, such as anti-cancer activity or efficacy of cancer cells with reduced toxicity, and in particular death or resection or apoptosis or senescence or cell cycle arrest or necrosis (eg, ingenol angel) Rate or formulation). Undesirable effects, such as side effects, sometimes occur with certain therapeutic effects; The skilled person thus balances the potential benefits against the potential risk in determining what an appropriate "effective amount" is. The exact amount required depends on the individual, depending on the species, age, general condition of the individual, mode of administration and the like. Thus, it may not be possible to specify the correct "effective amount". However, in any individual case the appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

Thus, as used herein, "effective amount" refers to the amount of active agent that provides the desired effect of reducing cancer cell growth upon administration by an appropriate dosing regimen. The combined content of the administered active agent is preferably an amount that provides an indication of the desired cytotoxicity, senescence, cell cycle arrest, necrosis and / or resection or other anticancer efficacy of the cancer cells. Administration can be at intervals of minutes, hours, days, weeks or months. Appropriate dosages and dosing regimens may be determined by the attending physician or veterinarian.

In one embodiment, an effective amount of incorporate angelate and agent incorporation has been suggested to be less when used in the absence of other. In another embodiment, the coalescing content is additive even though the therapy is more effective. Thus, the incorporation of angeloyl-substituted ingenans has been proposed to provide greater anticancer activity or efficacy compared to the activity or efficacy of the formulation alone. Anticancer activity or efficacy may include the rate, time and efficacy of causing cancer cell cytotoxicity, apoptosis, necrosis, senescence, and / or cell cycle arrest; Ability to treat or inhibit metastasis; Induction or stimulation of an immunological response to cancer cells; Induction of immune memory for the recurrence of cancer cells; Reduction in the amount of angeloyl-substituted ingenane or one or the other of the required formulations; Reduction of toxic side effects of anticancer therapy in the subject; And reduced cancer growth after the remission period.

In one embodiment, the effective amount can be calculated such that the merge index (CI) is less than 1, and the CI is determined using the following equation:

CI = [(C) ₁ / (C _x ) ₁ ] + [(C) ₂ / (C _x ) ₂ ] + [α (C) ₁ (C) ₂ / (C _x ) ₁ (C _x ) ₂ ]

Wherein (C _x ) ₁ is the concentration of one of the ingenol angelates or agents that produces the x% effect of the drug alone, and (C) ₁ is the other of the ingenol angelates or agents (C) _Is the concentration of one of the ingenol angelates or agents that, in combination with ₂ , produces the same x% effect, and α is a constant if the modes of anticancer activity of the ingenol angelates and agents are mutually exclusive or non-excluded and α are 0 or 1, respectively.

CI values less than 1 mean 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.91, 0.92, 0.93, 0.94 , 0.95, 0.96, 0.97, 0.98 and 0.99, as well as values between them.

A “pharmaceutically acceptable” carrier, excipient or diluent may be administered to a subject with a pharmaceutical vehicle consisting of an undesirable or non-biological substance, ie, the substance with the active agent selected without causing any or substantial side effects. It means to be. Carriers include excipients and other additives such as diluents, detergents, colorants, wetting agents or emulsifiers, pH buffers, preservatives and the like.

Similarly, “pharmacologically acceptable” salts, esters, amides, prodrugs or derivatives of compounds as provided herein are salts, esters, amides, prodrugs or derivatives which are undesirable or non-biological.

As used herein, the terms "treating" and "treatment" refer to a reduction in the severity and / or frequency of signs of the condition being treated, the exclusion of signs and / or intrinsic causes, the signs of conditions and / or inherent causes thereof. Prevention of occurrence and improvement or correction or amelioration of damage after a condition.

"Treating" an individual may include the treatment of an individual having clinical signs by improving the condition signs, as well as preventing the condition or other harmful physiological situation in the individual in question. The term "treating" also applies to secondary or metastatic cancers other than the primary cancer, the cancer that was originally treated. It also includes the induction of immune memory against cancer cells. Treatment may be referred to as a treatment protocol to induce improved or enhanced anticancer activity or efficacy as defined above.

As used herein, "individual" refers to an animal, preferably a mammal, more preferably a human, who can benefit from the pharmaceutical combinations and methods of the present invention. There is no limit to the type of animal that can benefit from the pharmaceutical combinations and methods presented herein. Regardless of human or non-human animals, an individual can be referred to as an individual, patient, animal, host or receptor. The compounds and methods of the present invention have applications in human medicine, veterinary medicine, as well as livestock or wild animal husbandry.

As set forth above, preferred animals include humans or other primates, such as orangutans, gorillas, silk monkeys, livestock, laboratory experimental animals, companion animals or caged wildlife, as well as birds.

Examples of experimental animals include mice, rats, rabbits, ape animals, guinea pigs and hamsters. Rabbits, rodents, and apes animals provide convenient test systems or animal models. Domestic animals include sheep, cattle, pigs, goats, horses, and donkeys.

The present invention relates in part to a treatment protocol comprising administering two or more agents to an individual with or suspected of having cancer, wherein the one or more agents comprise angeloyl-substituted ingenan (ie, ingenol). Angelate) or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, wherein the one or more other agents have the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Wherein at least one of the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

Therefore, the treatment protocol may include ingenol angelate and (1) another anticancer agent in the subject; (2) cancer-specific T-cells (such as cytotoxic T-cells); (3) neutrophil-stimulating agents; (4) ADCC-promoting agents (including neutrophils); (5) anticancer cell antibodies; And / or (6) administering one or more of the agents that induce, stimulate, or cause the production of one or more of (1) to (5).

The present invention extends to a formulation having one or more of the properties of one of (i) to (iv) or having two or more or three or more of all four properties.

The therapeutic protocol of the present invention further extends to the use of ingenol angelate to lower the neutrophil limit prior to ADCC induction of cancer cells. Examples of neutrophil-stimulating agents include ingenol angelate (eg PEP005) or interleukin (eg IL-6).

Examples of T-cells in part (ii) are cytotoxic T-cells.

An example of an ADCC-promoting agent is an antibody. Also included in the present invention are agents that modulate complement-mediated cell death.

"Ingenol angelate" is not only a compound isolated from a species such as a plant, such as the counterpart, but also derivatives, homologues, analogs or functional equivalents thereof, as well as its chemically synthesized forms, as well as pharmaceutically acceptable salts thereof Can be mentioned. This also applies to PEP005.

As part of the counter electrode is in Oh Khalifa (Acalypha), Aki also tone (Acidoton), evil Martino Ste driven (Actinostemon), ahdelriah (Adelia), adeno-clean (Adenocline), adenovirus Cri-piece (Adenocrepis), adenovirus par la ( Adenophaedra , Adisca , Agrostistachys , Alchornea , Alchorneopsis , Alcinaeanthus , Alcoceria , Alureceres Aleurites , Amanoa , Andrachne , Angostyles , Anisophyllum , Antidesma , Aphora , Aporosa , Aporosa Cellar (Aporosella), are gitam California (Argythamnia), Astro nose Syracuse (Astrococcus), Astro Guinea (Astrogyne), bakan Leah (Baccanrea), Bali OSU Pere drought (Baliospermum), Bernard Guardia (Bernardia), beret sharply option system ( Beyeriopsis), biscotti Pia (Bischofia), Blythe Ah (Blachia), Blue Meo money drones (Blumeodondron), Bo Narnia (Bonania), Bra s Leia (Bradleia), Bray California (Breynia), brainy option System (Breyniopsis), Delia (Briedelia) in Bree, bra Ah Buraeavia , Caperonia , Caryodendron , Celianella , Cephalocroton , Chaenotheca , Chaetocarpus , Cama the price (Chamaesyce), cheil Rosa (Cheilosa), key Petaling Room (Chiropetalum), nose Rio pilrum (Choriophyllum), Chica (Cicca), kaok Ceylon (Chaoxylon), clay money (Cleidon), clay Afghanistan tooth (Cleistanthus), Cluytia , Cnesmone , Cnidoscolus , Coccoceras , Codiaeum , Coelodiscus , Conami , Conami , Conce Conceveiba , Conceveibastrum , Conceveibu m), Corey Te (Corythea), croissants and Corsica (Croizatia), croton (Croton), croissants tonop system (Crotonopsis), Black Joe Fora (Crozophora), Cuban tooth (Cubanthus), Equus Nuria (Cunuria), stereo with daktil Montserrat (Dactylostemon), month recham Pia (Dalechampia), dendeuro ku Cynthia (Dendrocousinsia), Dias pereusu's (Diaspersus), Didier linen seutuseu (Didymocistus), di not know the focal Riggs (Dimorphocalyx), disco carboxylic crispus (Discocarpus), ditak system (Ditaxis), dodeca Stigma (Dodecastingma), laundry page test (Drypetes), disop system (Dysopsis), Ella Te Rios Fernand drought (Elateriospermum), enda Denny Stadium (Endadenium), endo's Pere drought (Endospermum), but Ellis Tooth (Erismanthus), erythromycin carboxylic crispus (Erythrocarpus), erythromycin seven Ruth (Erythrochilus), Aust mekan tooth (Eumecanthus), yuporeubiah (Euphorbia), oil FORT non Oden drones (Euphorbiodendron), exo-nose car Ria (Excoecaria), flu Flueggea , knife Les Aria (Calearia), Garcia (Garcia), GABA re-thiazol (Gavarretia), gel, titanium (Gelonium), starvation La (Giara), notation thiazole (Givotia), article Rocky dione (Glochidion), claw key radio knobs sheath (Clochidionopsis ), glycidyl dendron (Glycydendron), gimnan Tess (Gymnanthes), Kim North Faria (Gymnosparia), under Matos Pere drought (Haematospermum), Coca Deccan drive (Hendecandra), Hebe Ah (Hevea), Hebrews to Lorna forehead ( Hieronima), Nima (Hieronyma to Hie), Hippo Crescent plate drive (Hippocrepandra), Homa Lantus (Homalanthus), Hime no carboxylic Dia (Hymenocardia), Johnny wave (Janipha), Jatropha (Jatropha), line crotonic (Julocroton), Lashio Croton (Lasiocroton), ray Oh carboxylic crispus (Leiocarpus), Leonardo Boutique (Leonardia), Les century egg tooth (Lepidanthus), Les wooko Croton (Leucocroton), Matt Javea (Mabea), Maca ranga (Macaranga), Malo tooth ( Mallotus ), Manihot , Mappa , Maprounea , Melan Melanthesa , Mercurialis , Mettenia , Micrandra , Microdesmis , Microelus , Microstachy , Maocroton Mona Denny Titanium (Monadenium), all over (Mozinna), Neos cor MindTouch California (Neoscortechinia), Omar Lantus (Omalanthus), Omphale O (Omphalea), Opel lanthanide (Ophellantha), ohreubi Kula Liao (Orbicularia), agarose Sat des (Ostodes), oxy deck test (Oxydectes), palrenga (Palenga), Fanta denier (Pantadenia), para give peptidase test (Paradrypeptes), Pau Sandra (Pausandra), Phedi Lantus (Pedilanthus), Blow (Pera), Perry Stadium (Peridium), stigmasterol a swallowtail (Petalostigma), Phil Lantus (Phyllanthus), dendeuro (Picrodendro), L'Dia (Pierardia), Phil Reno pitum (Pilinophytum), pimel Leo dendron (Pimeleodendron) to avoid a peak, pyran Gea ( Piranhea), platinum group or (Platygyna), Luque four thiazole (Plukenetia), grape knife Riggs (Podocalyx), Poinsettia (Poinsettia), Fora Silesia (Poraresia), Pro Sar theme (Prosartema), syudan tooth (Pseudanthus), peak no coma (Pycnocoma), Quadra cyano (Quadrasia ), Reverchonia , Richeria , Richeriella , Ricinella , Ricinocarpus , Rottlera , Sagotia , San Witia ( Sanwithia ), Sapium , Savia , Sclerocroton , Sebastiana , Securinega , Senefeldera , Senefeldera , Senefilderopsis , Serophyton , Siphonia , Spathiostemon , Spixia , Stillingia , Strophioblachia , Synadenium ), Tetracoccus , Tetraplandra ), Tet L'keys Stadium (Tetrorchidium), Tyr mountain Tara (Thyrsanthera), Titi late Ruth (Tithymalus), Trapani gay ah (Trageia), Tre WIA (Trewia), teurigo noseute Montserrat (Trigonostemon), tiriah (Tyria) and size And species from Xylophylla .

Preferred genera and particularly suitable for the practice of the present invention are Euphorbia . Particularly useful species of this genus include Euphorbia aaron-rossii , Euphorbia abbreviata , Euphorbia acuta , Euphorbia alatocaulis , Euphorbia albocaulis . ( Euphorbia albicaulis ), Euphorbia algomarginata , Euphorbia aliceae , Euphorbia alta , Euphorbia anacampseros , Euphorbia andromedae angu, Euphorbia and romedae Euphorbia angusta , Euphorbia anthonyi , Euphorbia antiguensis , Euphorbia apocynifolia , Euphorbia arabica , Euphorbia auriansis Euphorbi a arizonica ), Euphorbia arkansana , Euphorbia arteagae , Euphorbia arundelana , Euphorbia astroites , Euphorbia atrococ bar, Euphorbia atrococca bar, Euphorbia atrococca Euphorbia baselicis , Euphorbia batabanensis , Euphorbia bergeri , Euphorbia bermudiana , Euphorbia bicolor , Euphorbia biformis , Euphorbia bifurcata , Euphorbia bilobata , Euphorbia biramensis , Euphorbia biuncialis , Euphorbia blephavia blepharostipula rod, Euphorbia bilobata Euphorbia blodgetti ), Euphorbia Euphorbia boerhaavioides , Euphorbia boliviana , Euphorbia bracei , Euphorbia brachiata , Euphorbia brachycera , Euphorbia brande Euphorbia brand ), yuporeubiah debris toniyi (Euphorbia brittonii), yuporeubiah car to a cyano (Euphorbia caesia), yuporeubiah calcineurin coke (Euphorbia calcicola), yuporeubiah Kam paste-less (Euphorbia campestris), yuporeubiah candela Broome (Euphorbia candelabrum), yuporeubiah copy Tel rata (Euphorbia capitellata , Euphorbia carmenensis , Euphorbia carunculata , Euphorbia cayensis , Euphorbia celastroides , Euphorbia calicophyla , Euphorbia chalicophila Rhodes (Euphorbia chamaerrhodos), yuporeubiah Atacama in San Pedro (Euphorbia chamaesula), yuporeubiah key Appenzell system (Euphorbia chiapensis), yuporeubiah keys come Noi Death (Euphorbia chiogenoides), yuporeubiah kine Las sense (Euphorbia cinerascens), yuporeubiah climb Rio linen sheath (Euphorbia clarionensis ), Euphorbia colimae , Euphorbia colorata , Euphorbia commutata , Euphorbia consoquitlae , Euphorbia conbobuloides Euphorbia convolvuloides Euphorbia corallifera , Euphorbia creberrima , Euphorbia crenulata , Euphorbia cubensis , Euphorbia cuspidata , Euphorbia symbiformis , Euphorbia cymbiform Euphorbia Darlingtoni (Euphorbia darlingtonii), yuporeubiah depot Ria other (Euphorbia defoliata), yuporeubiah scald Neri (Euphorbia degeneri), yuporeubiah Del Toro IDEA (Euphorbia deltoidea), yuporeubiah den tartar (Euphorbia dentata), yuporeubiah to frame wrap (Euphorbia depressa), yuporeubiah dikti OSU Fermat (Euphorbia dictyosperma), yuporeubiah dikti agarose Fermat (Euphorbia dictyosperma), car in yuporeubiah video (Euphorbia dioeca), yuporeubiah disco two months less (Euphorbia discoidalis), yuporeubiah D'when vent LAL-less (Euphorbia dorsiventralis), yuporeubiah Drew driven diimide (Euphorbia drumondii ), Euphorbia duclouxii , Euphorbia dussii , Euphorbia eanophylla , Euphorbia eggersii , Euphorbia egglandulosa , Euphorbia eglandulosa (Euphorbia elata), oil PORTO Carried to O (Euphorbia enalla), noise yuporeubiah coming Erie des (Euphorbia eriogonoides), yuporeubiah Erie pillar (Euphorbia eriophylla), yuporeubiah S. Kula in Fort miss (Euphorbia esculaeformis), yuporeubiah Espirito N-Sys (Euphorbia espirituensis), yuporeubiah Esu La (Euphorbia esula), yuporeubiah exciter current events (Euphorbia excisa), yuporeubiah exciter crew four (Euphorbia exclusa), yuporeubiah X typhimurium tartar (Euphorbia exstipitata), yuporeubiah X T furanyl other (Euphorbia exstipulata), yuporeubiah pens Larry (Euphorbia fendleri), yuporeubiah Euphorbia filicaulis , Euphorbia filiformis , Euphorbia florida , Euphorbia fruticulosa , Euphorbia garber , Euphorbia gaumerii , Euphorbia gaumerii Euphorbia gerardiana , Euphorbia geyeri , Euphorbia glyptosperma , Euphorbia gorgonis , Euphorbia gracilior , Euphorbia grasilligra , Euphorbia gracillima Euphorbia gradyi , Euphorbia graminea , Euphorbia graminiea, Euphorbia grisea , Euphorbia guadalajarana , Euphorbia guanarensis , Euphorbia guanarensis Euphorbia gymnadenia , Euphorbia haematantha , Euphorbia hedyotoides , Euphorbia heldrichii , Euphorbia helenae , Euphorbia hellelieri , Euphorbia helleri Euphorbia Helweigii Euphorbia helwigii , Euphorbia henricksonii , Euphorbia heterophylla , Euphorbia hexagona , Euphorbia hexagonoides , Euphorbia hincobia hintobia horphorbia Euphoror Euphorbia hintonii , Euphorbia hirtula , Euphorbia hirta , Euphorbia hooveri , Euphorbia humistrata , Euphorbia hypericifolia , Euphorbia hypericifolia Euphorbia inundata , Euphorbia involuta , Euphorbia jaliscensis , Euphorbia jejuna , Euphorbia johnston , Euphorbia jutae , Euphorbia juttae Euphorbia knuthii ) , Euphorbia lasiocarpa , Euphorbia lata , Euphorbia latazi , Euphorbia latericolor , Euphorbia laxiflora , Euphorbia lephorbia lecheoid , Euphorbia ledienii , Euphorbia leucophylla , Euphorbia lineata , Euphorbia linguiformis , Euphorbia longgecornia , Euphorbia longecornuta Euphorbia longepetiolata), yuporeubiah Ron Gera Simulation (Euphorbia longeramosa), yuporeubiah Ron ginsul Ricola (Euphorbia longinsulicola), yuporeubiah rongi Pilar (Euphorbia longipila), yuporeubiah Lou Puri or (Euphorbia lupulina), yuporeubiah Metalurgs is (Euphorbia lurida), yuporeubiah receiver cucumber Death ( Euphorb ia lycioides), yuporeubiah mark podoyi Death (Euphorbia macropodoides), yuporeubiah Marc Bauer Guiana (Euphorbia macvaughiana), yuporeubiah Salamanca (Euphorbia manca), yuporeubiah Mando Chania or (Euphorbia mandoniana), yuporeubiah mangle Retiro (Euphorbia mangleti), yuporeubiah Mango (Euphorbia mango ), Euphorbia marylandica , Euphorbia mayana , Euphorbia melanadenia , Euphorbia melanocarpa , Euphorbia meridensis , Euphorbia meridento ( Euphorbia mertonii ), Euphorbia mexiae , Euphorbia microcephala , Euphorbia microclada , Euphorbia micromera , Euphorbia misella , Euphorbia misella Euphorb ia missurica , Euphorbia montana , Euphorbia montereyana , Euphorbia multicaulis, Euphorbia multiformis , Euphorbia multinodis , Euphorbia multinodis Euphorbia multiseta), yuporeubiah mousse when coke (Euphorbia muscicola), yuporeubiah neo Mexicana (Euphorbia neomexicana), yuporeubiah four plastic denier (Euphorbia nephradenia), ANA (Euphorbia niqueroana), yuporeubiah O musician Kana (Euphorbia oaxacana) in the yuporeubiah Nicu, yuporeubiah Occidental less (Euphorbia occidentalis), yuporeubiah O donto denier (Euphorbia odontodenia), yuporeubiah Oliva years old child (Euphorbia olivacea), yuporeubiah OLLO and Rua or (Euphorbia olowaluana), yuporeubiah off deionized mica (Euphorbia opthalmica), yuporeubiah Obata (Euphorbia ovata ), Euphor Via Parkinson poda (Euphorbia pachypoda), yuporeubiah Parkinson Manager (Euphorbia pachyrhiza), yuporeubiah Fadi polyamic (Euphorbia padifolia), yuporeubiah eight Mary (Euphorbia palmeri), yuporeubiah par Rudy Cola (Euphorbia paludicola), yuporeubiah Pardo City Flora (Euphorbia parciflora), Euphorbia parishii , Euphorbia parryi , Euphorbia paxiana , Euphorbia pediculifera , Euphorbia peplidion , Euphorbia pepliides Euphorbia peplus , Euphorbia pergamena , Euphorbia perlignea , Euphorbia petaloidea , Euphorbia petaloidea , Euphorbia petaloidea Euphorbia petrina), blood yuporeubiah Ken System (Euphorbia picachensis), yuporeubiah Philo Peninsula (Euphorbia pilosula), yuporeubiah blood rulri Blow (Euphorbia pilulifera), yuporeubiah blood and Leona (Euphorbia pinariona), yuporeubiah Pinero torum (Euphorbia pinetorum), yuporeubiah Pio North Fermat (Euphorbia pionosperma), yuporeubiah Euphorbia platysperma , Euphorbia plicata , Euphorbia poeppigii , Euphorbia poliosperma , Euphorbia polycarpa , Euphorbia polycnmoides ), Euphorbia polyphylla , Euphorbia portoricensis, Euphorbia portulacoides , Yuporeubiah Fort tool Lana (Euphorbia portulana), yuporeubiah peureseulriyi (Euphorbia preslii), yuporeubiah Pro Strata (Euphorbia prostrata), yuporeubiah program for interrogating four Ura (Euphorbia pteroneura), yuporeubiah peak I Theme (Euphorbia pycnanthema), yuporeubiah la Simulation (Euphorbia ramosa) , yuporeubiah rapul room (Euphorbia rapulum), yuporeubiah remiyi (Euphorbia remyi), yuporeubiah retro Scar bra (Euphorbia retroscabra), yuporeubiah Lebo rutile (Euphorbia revoluta), yuporeubiah Lee ing less (Euphorbia rivularis), yuporeubiah robusta (Euphorbia robusta), yuporeubiah Euphorbia romosa , Euphorbia rubida , Euphorbia rubrosperma , Euphorbia rupicola , Euphorbia sanmartensis , Euphorbia saxatilis M. Probe in a non-(Euphorbia saxatilis M. Bieb), yuporeubiah seukijo Rover (Euphorbia schizoloba), yuporeubiah's Crescent articulation Titanium (Euphorbia sclerocyathium), room (Euphorbia scopulorum) to yuporeubiah Scoring pool when in, yuporeubiah Senigallia less (Euphorbia senilis), yuporeubiah Euphorbia serpyllifolia , Euphorbia serrula , Euphorbia setiloba Engelm, Euphorbia sonorae , Euphorbia soobyi , Euphorbia sparflora sparflora sparcia ), Euphorbia sphaerosperma , Euphorbia syphilitica , Euphorbia spruceana , Euphorbia subcoerulea , Euphorbia stellata sub, Euphorbia stellata sub Euphorbia submammilaris , Euphorbia Euphorbia subpeltata , Euphorbia subpubens , Euphorbia subreniforme , Euphorbia subtrifoliata , Euphorbia succedanea , Euphorbia tamaparanata (Euphorbia tamaulipasana), yuporeubiah terephthalate piohyi des (Euphorbia telephioides), yuporeubiah Te sister Shima (Euphorbia tenuissima), yuporeubiah tetrahydro Fora (Euphorbia tetrapora), yuporeubiah Tirupati potassium (Euphorbia tirucalli), yuporeubiah tomen telra (Euphorbia tomentella), yuporeubiah Sat mentor Euphorbia tomentosa , Euphorbia torralbasii , Euphorbia tovariensis , Euphorbia trachysperma , Euphorbia tricolor , Euphorbia troyana , Euphorbia troana Fort Oh pitcher Kirk H. already (Euphorbia tuerckheimii), yuporeubiah Turkmen party Nino weaving (Euphorbia turczaninowii), yuporeubiah Stadium Belo Rata (Euphorbia umbellulata), yuporeubiah undul Rata (Euphorbia undulata), yuporeubiah Vere US Forde Miss (Euphorbia vermiformis), yuporeubiah Berger Euphorbia versicolor , Euphorbia villifera , Euphorbia violacea , Euphorbia whitei , Euphorbia xanti Engelm, Euphorbia xylopoda green Greenm.), Euphorbia yayalesia Urb . ), Euphorbia yungasensis , Euphorbia zeravschanica , and Euphorbia zinniiflora .

Particularly preferred species of the genus Synadenium include Synadenium grantii and Synadenium compactum .

In a particularly preferred species of the genus bichwimok (Monadenium) it may include jade tower (Monadenium lugardae) and advisory Dragon (Monadenium guentheri).

Yen preferred species of dade nyumsok (Endadenium) is enda denyum Goss wale Lenny (Endadenium gossweileni).

Euphorbia peplus is particularly useful in that it provides a source of ingenol angelate in the practice of the present invention. As used herein, the term " Euphorbia peplus " or the abbreviation " E. peplus " refers to various variants, strains, cell lines, hybrids or derivatives of such plants, as well as botanical or horticultural. Include relatives. In addition, the present invention may be practiced using whole counter plants or parts thereof, including sap or seeds or other reproductive material. In general, in the case of seeds or reproductive materials to be used, plants or seedlings are necessary for reproduction.

Largest species, or two. E. peplus further comprises a plant genetically modified. Genetically modified plants include transgenic plants or downregulated, mutated or modified plants in which the trait is eliminated or the endogenous gene sequence includes a modification or introduction of genetic material that exhibits a regulatory effect on a particular gene. Thus, the counter plant or the counter species or two. Plants exhibiting properties that do not exist naturally in Peplus (E. peplus ) are nevertheless included in the present invention and are included within the scope of the above-mentioned terms.

Ingenol angelate is commonly found in extracts of antipodes. Thus, the extract may comprise a sap or liquid or semi-liquid material that flows out or is present between leaves, stems, flowers, seeds, bark, or between bark and stem. Most preferably, the extract is obtained from sap. In addition, the extract may comprise liquid or semi-liquid substances present in the fractions extracted from the sap, leaves, stems, flowers, bark or other plant material of the counterpart plant. For example, plant materials can be treated with physical manipulations that disrupt plant fiber and extracellular matrix material, and can be extracted between tissues and intra-tissues into solvents, including in an aqueous environment. All sources of such ingenol angelate, including compounds obtained by chemical synthetic routes, are included in the present invention.

The most preferred compounds of the present invention are chemically referred to as ingenol-3-angelate and referred to herein as "PEP005". "Ingenol-3-angelate" or "PEP005" includes natural forms as well as chemically synthetic forms and pharmaceutically acceptable salts, derivatives, homologs and analogs thereof.

Examples of agents used in combination with angeloyl-substituted ingenane include chemotherapeutic agents such as anti-metabolic agents, anti-tumor antibiotics, mitosis inhibitors, steroids, sex hormones, alkylating agents, nitrogen mustards, nitrosoureas, hormones Agonists and microtubule inhibitors.

Antimetabolic compounds interfere with the body's chemical processes, such as the synthesis of proteins or DNA required for cell growth and reproduction. Antimetabolic drugs can interfere with cell division, which is essential in cancer treatment. Examples thereof include azaserine, D-cycloserine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C) and fludarabine.

Antitumor antibiotics interfere with DNA by stopping enzymes and mitosis or by modifying the membrane surrounding the cells. These agents act in all phases of the cell cycle. Thus, they are widely used for various cancers. Examples of antitumor antibiotics include dactinomycin, daunorubicin, doxorubicin (Adriamycin), idarubicin and mitoxantrone.

Mitosis inhibitors are vegetable alkaloids and other compounds derived from natural products. They can inhibit or stop, mitosis or inhibit enzymes for producing the proteins necessary for reproduction of the cell. They act in the M phase of the cell cycle. Examples of mitotic inhibitors include paclitaxel, docetaxel, etoposide (VP-16), vinblastine, vincristine and vinorelbine.

Steroids are natural hormones and hormone-like drugs that are useful for treating certain types of cancer, including but not limited to lymphomas, leukemias, and multiple myeloma. When these drugs are used to delay the death or growth of cancer cells, they are considered anticancer drugs. In addition, they can be combined with other types of chemotherapeutic drugs to enhance their effectiveness. Examples thereof include prednisone and dexamethasone.

Sex hormones, or hormone-like drugs, alter the action or production of female or male hormones. They are used to delay the growth of breast, prostate and endometrial (endometrial) cancers that normally grow in response to hormone levels in the body. Examples include antiestrogens (tamoxifen, fulvestrant), aromatase inhibitors (anastrozol, letrozole), progestins (megestrol acetate), anti-androgens (bicalutamide, flutamide) and LHRH agonists (Leuprolide, goserelin).

Alkylating agents act directly on DNA to interfere with cancer cell reproduction. As a type of drug, these agents are not group-specific (ie they act in all groups of the cell cycle). These drugs are active against chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma and certain cancers of the lung, breast and ovaries. Examples of alkylating agents include busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechloretamine (nitrogen mustard) and melphalan.

Nitrogen mustard in crystalline hydrochloride form is used as a drug in Hodgkin's disease, non-Hodgkin's lymphoma and brain tumors. Nitrogen mustard causes mutations in the genetic material of the cell, disrupting mitosis or cell division. Cells vary in susceptibility to nitrogen mustard and most rapidly and rapidly propagate tumor and cancer cells; In addition, the bone marrow producing red blood cells is sensitive, and inhibition of red blood cell production is a common side effect of nitrogen mustard therapy. Nitrogen mustard also suppresses the immune response. Examples of other types include aromatic mustard melphalan and chlorambucil.

Nitrosoureas act in a similar manner to alkylating agents. They interfere with enzymes that help repair DNA. These agents can migrate in the brain and are used to treat brain tumors, as well as non-Hodgkin's lymphoma, multiple myeloma and malignant melanoma. Examples of nitrosoureas include carmustine (BCNU) and romustine (CCNU).

Examples of hormonal agonists include leuprolides (lupron, viadur, eligarde) for prostate cancer, goserelin (zoladex) for breast and prostate cancer, and tryptorelin (trrel) for ovarian and prostate cancer Star) and naparelin acetate (cinarel).

Examples of microtubule inhibitors include “vinca” alkaloids, taxoids and benzimidazoles. Examples of such anticancer agents include anticancer drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mapphosphamide, ifosfamide, cytosine arabinoside , Bis-chloroethylnitrosourea, busulfan, mitomycin C, actinomycin D, mitomycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mi Toxanthrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxy Urea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), kohl Kitchen, Tak , Vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES) It doesn't happen.

In addition, the agents of the present invention include antibodies or other immunointeracting molecules. For example, an antigen can be administered that provides antibodies against a specific cancer-specific antigen or provides an antigen that elicits an antibody combination against a cancer-specific antigen. Antibodies represent examples of agents provided with angeloyl-substituted ingenans in an anticancer protocol. In addition, an individual's T-cells or other immune cells are isolated, expanded in culture, and returned to the individual. In the latter case, the present invention selects cancer patients, isolates T-cells or samples comprising T-cells, expands T-cells in in vitro culture, and optionally interleukin (eg, IL-6) cells. ) Or other cytokines or ingenol angelates (eg, PEP005) and augmented with protocols that include sending an expanded T-cell population to the subject. In addition, the subject undergoes cancer resection with angeloyl-substituted ingenan (eg PEP005). In another embodiment, T-cells are augmented or delivered to the patient by vaccination or other immune enhancing agent. PEP005 or other ingenol angelates are used to improve T-cell activity.

In the former case, the antibody may be selected to enhance ADCC of cancer cells or to promote cytotoxic T-cell responses or other responses from immune cells.

In the context of antibodies, polyclonal antibodies can be used conveniently, but the use of monoclonal antibodies is particularly preferred due to the homogeneity of the product and the ability to produce them in large quantities. Generation of hybridoma cell lines for monoclonal antibody production is induced by fusing sensitized immortal cell lines and lymphocytes against immunogenic agents (ie, including cancer antigens).

The invention further provides for the application of biochemical techniques such that antibodies derived from one animal or avian animal are almost non-immunogenic in another or avian animal of the same or different species. Biochemical processes are referred to herein as "immune clearance". "Immunoremoval" includes, for example, grafting of complement determinant site (CDR) grafting "reforming" with respect to skeletal sites of immunointeracting molecules and variable (v) site mutations, all of which are specific host (eg , To reduce the immunogenicity of immuno-interacting molecules (eg antibodies) in human subjects). In such cases, preferred immunointeracting molecules are antibodies with specificity for cancer cells, such as polyclonal or monoclonal antibodies. In the most preferred embodiment, the immunointeracting molecule is derived from one animal or avian animal, and reduced immunogenicity in another animal or avian animal from the same or different species, including but not limited to humans, for example. It is a monoclonal antibody.

"Almost non-immunogenic" includes reduced immunogenicity relative to the parent antibody, i.e., the antibody prior to exposure to the immunodeficiency process. The term “immunogenic” includes the ability to elicit, induce or promote a humoral and / or T-cell mediated response in a host animal. Particularly convenient immunogenic criteria include the ability of amino acid sequences derived from variable (v) regions of an antibody to interact with MHC type II molecules to stimulate or promote T-cell mediated responses, including T-cell-assisted humoral responses. do.

By "antibody" is meant a protein of the family of immunoglobulins that can bind, interact, or be linked to an antigen. Therefore, antibodies are antigen-binding molecules. "Antibodies" are examples of immunointeracting molecules and include polyclonal or monoclonal antibodies. Preferred immunointeracting molecules of the invention are monoclonal antibodies. The term “antibody” also includes engineered antibodies, such as bispecific antibodies against two different antigens in cancer cells.

As used herein, the term “antigen” refers to a substance that can respond to and / or induce an immune response. "Antigen" includes antigenic determinants or epitopes or cancer cells.

By "antigen-binding molecule" is meant any molecule having a binding affinity for a target antigen. These terms are understood to extend protein backbones that exhibit antigen-binding activity to induced non-immunoglobulins, immunoglobulin segments and immunoglobulins (eg, polyclonal or monoclonal antibodies). The terms "antibody" and "antigen-binding molecule" include immunodepleted forms of these molecules.

By "antigenic determinant" or "epitope" is meant a portion of an antigenic molecule that involves a specific immune response and comprises hapten. Typically, in animals, antigens present several or even multiple antigenic determinants simultaneously. A "hapten" is a substance that can specifically bind an antibody, but which induces no immune response or only poorly when not bound to a carrier. The hapten typically contains a single antigenic determinant or epitope.

Although the preferred antibodies of the invention are immunodepleted forms of mouse monoclonal antibodies for use in humans, the invention is immunized for use with any host and extends to antibodies from any source. Examples of animal and bird sources and hosts include humans, primates, livestock animals (e.g. sheep, cattle, horses, pigs, donkeys), laboratory laboratory animals (e.g. mice, rabbits, guinea pigs, hamsters), companion animals (e.g., Dogs, cats), poultry (e.g. chickens, ducks, geese, turkeys) and birds (e.g. pheasants).

Immunization and subsequent production of monoclonal antibodies is described, for example, in Kohler and Milstein, Kohler et al., Nature 256: 495-499, 1975 and Kohler et al., Eur. J. Immunol . 6 (7): 511-519, 1976, Coligan et al., Current Protocols in Immunology , 1991-1997 or Toyama et al., Monoclonal Antibody, Experiment Manual , published by Kodansha Scientific, 1987. It can be used. In practice, animals are immunized with antigen-containing (eg cancer antigens) or fractions thereof by standard methods of producing antibody-producing cells, in particular antibody-producing somatic cells (eg B lymphocytes). Such cells can then be removed from the immunized animal for immortalization. The antigen may first have to be associated with a carrier.

By "carrier" is generally meant any polymeric material that non- or poorly immunogenic material (eg, hapten) is naturally or artificially combined to improve its immunogenicity.

Immortalization of antibody-producing cells can be carried out using methods known in the art. For example, immortalization can be achieved by the conversion method using Epstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140, 1986). In a preferred embodiment, antibody-producing cells are immortalized using cell fusion methods (described in Colingan et al., Hom., 1991-1997), which are widely used for the production of monoclonal antibodies. In this method, body antibody-producing cells, in particular B cells, which have the potential of producing antibodies are fused with myeloma cell lines. Such somatic cells may be derived from lymph nodes, spleen and peripheral blood of primary sensitized animals, preferably rodents such as mice and rats. In an exemplary embodiment of the mouse of the present invention, splenocytes were used. However, rat, rabbit, sheep or goat cells or cells from other animal species can be used instead.

Special myeloma cell lines were formed from lymphocyte tumors for use in hybridoma-producing fusion procedures [Kohler et al., Hom., 1976; Kozbor et al., Homology, 1986; And VoIk et al., J. Virol . 42 (1): 220-227, 1982. These cell lines were formed for at least three reasons. The first is to promote the selection of fused hybridomas from myeloma cells that are not fused and similarly indefinite self-proliferating. In general, this is achieved using enzyme deficient myeloma which renders growth impossible in certain selective media that support the growth of hybridomas. Secondly, lymphocyte tumor cells result from the inherent ability to produce their own antibodies. In order to exclude the production of tumor cell antibodies by hybridomas, myeloma cell lines that are unable to produce endogenous light or heavy chain immunoglobulins are used. The third reason for the selection of these cell lines is suitability and efficiency for fusion.

Many myeloma cell lines are fused, including, for example, P3X63-Ag8, P3X63-AG8.653, P3 / NSl-Ag4-1 (NS-1), Sp2 / 0-Ag14, and S194 / 5.XXO.Bu.1. It can be used to generate cell hybrids. P3X63-Ag8 and NS-1 cell lines are described in Kohler and Milstein, Kohler et al., Hom., 1976. Shulman et al., Nature 276: 269-270, 1978 produced Sp2 / 0-Ag14 myeloma strains. S194 / 5.XXO.Bu.1, see Rowbridge, J. Exp. Med. 148 (1): 220-227, 1982.

Methods of generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells generally have a ratio of 10: 1 each in the presence of an agent or agents (chemical, viral or electrical) that promote fusion of the cell membrane (their ratio being about 20: 1 to about 1: 1, but including) mixing somatic cells with myeloma cells. Fusion methods are described in Kohler et al., Hom., 1975; Kohler et al., Homology, 1976; Gefter et al., Somatic Cell Genet . 3: 231-236, 1977; And Volk et al., Homology, 1982. Fusion-promoting agents used by these investigations are Sendai virus and polyethylene glycol (PEG).

Since the fusion procedure produces viable hybrids at very low frequency (e.g., when the spleen is used as a source of somatic cells, one hybrid is obtained for 1x10 ⁵ splenocytes). It is desirable to have a means for selecting fused cell hybrids from fused myeloma cells. There is also a need for means for detecting certain antibody-producing hybridomas in other fused cell hybrids. In general, the selection of fused cell hybrids is achieved by culturing the cells in a medium that supports the growth of hybridomas but inhibits the growth of unfused myeloma vesicles and normally divides indefinitely. Somatic cells used for fusion do not maintain prolonged viability in in vitro culture and thus do not pose a problem. In the examples of the present invention, myeloma cells lacking hypoxanthine phosphoribosyl transferase (HPRT-negative) were used. Selection for these cells is made in hypoxanthine / aminopterin / thymidine (HAT) medium, where the fused cell hybrid will survive due to the HPRT-positive genotype of spleen cells. It is also possible to use myeloma cells with various genetic deficiencies (such as drug sensitivity) that can be selected in media that support the growth of genotype qualified hybrids.

Several weeks are required to selectively culture the fused cell hybrid. At the beginning of this period, the hybrids that produce a given antibody must be identified and can later be cloned and propagated. Generally, about 10% of the resulting hybrids produce the desired antibody, although ranges from about 1 to about 30% are typical. Detection of antibody-producing hybrids can be accomplished by any one of a number of standard assay methods, including, for example, enzyme linked immunoassays and radioimmunoassay techniques, as described, for example, in US Pat. No. 6,056,957.

Once a given fused cell hybrid is selected and cloned into each antibody-producing cell line, each cell line can be propagated in two standard ways. Suspensions of hybridoma cells can be injected into histocompatible animals. The injected animals then form tumors secreted by specific monoclonal antibodies produced by fused cell hybrids. Animal fluids, such as serum or ascites fluid, can be tapped to produce high concentrations of monoclonal antibodies. Alternatively, individual cell lines can be propagated in vitro in laboratory culture vessels. Culture medium containing high concentrations of single specific monoclonal antibodies can be purified after harvesting by decantation, filtration or centrifugation.

The cell line is tested for specificity of detecting the antigen of interest by any suitable immunodetection means. For example, cell lines were divided into multiple wells, cultured, and the supernatants from each well were analyzed by enzyme-linked immunocapture assay (ELISA), indirect fluorescent antibody techniques, and the like. Identifying cell line (s) that produce a monoclonal antibody capable of recognizing the target antigen but not the non-target epitope, and then culturing directly in vitro or injecting into a tissue compatible animal to form a tumor, and then Antibodies are generated, harvested and purified.

Thus, the present invention provides polyclonal and monoclonal antibodies that specifically interact with cancer cells and specifically induce ADCC of cancer cells or promote cell death or identity.

Thereafter, monoclonal antibodies are generally treated with immunosuppressive means. Such methods may take any of a number of forms, including the generation of chimeric antibodies having the same or similar specificities as the monoclonal antibodies produced by the present invention. Chimeric antibodies are antibodies that typically comprise light and heavy chain genes from immunoglobulin variable and constant region genes belonging to various species by genetic engineering. Thus, according to the present invention, when a hybridoma producing a given monoclonal antibody is obtained, an interspecies monoclonal antibody is produced in which the binding site of one species is coupled with the non-binding site of an antibody of another species. Technique is used (Liu et al., Proc. Natl. Acad. Sci . USA 84: 3439-3443, 1987). For example, CDRs from non-human (eg, mouse) monoclonal antibodies can be grafted with human antibodies to “humanize” mouse antibodies (European Patent Publication No. 0 239 400, Jones et al., Nature 321: 522-525, 1986; Verhoeyen et al., Science 239: 1534-1536; 1988 and Richmann et al., Nature 332: 323-327, 1988). In such cases, the immune elimination method has specificity for humans. More specifically, CDRs can be grafted into human antibody variable regions with or without human constant regions. Non-human antibodies that provide CDRs are commonly referred to as “donors” and human antibodies that provide a backbone are commonly referred to as “receptors”. The constant regions need not be present, but once present, they should be about the same, ie at least about 85-90%, preferably at least about 95%, identical to the human immunoglobulin constant regions. Thus, possibly all parts of a humanized antibody except the CDRs will be substantially identical to that part of the natural human immunoglobulin sequence. Thus, “humanized antibodies” are antibodies comprising humanized light chains and humanized heavy chain immunoglobulins. Donor antibodies have been found to be "humanized" by the "humanization" process, since the resulting humanized antibodies are expected to bind to the same antibodies as the donor antibodies that provide the CDRs. As used herein, "humanized" refers to an antibody that has been immunodeficient against a particular host, in this case a human host.

It is understood that an immunodepleted antibody may have additional conservative amino acid substitutions that have little effect on antigen binding or other immunoglobulin action.

Exemplary methods that can be used to generate immunodepleted antibodies according to the invention are described, for example, in Chou et al., Hom., 1988; US Patent No. 6,056,957; Queen et al. (US Pat. No. 6,180,377); Morgan et al. (US Pat. No. 6,180,377) and Chothia et al., J. Mol. Biol . 196: 901, 1987.

Thus, certain formulations intended for use with ingenol angelate can be listed in Table 1. However, the present invention

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells;

(ii) mediate T-cell (eg, cytotoxic T-cell) mediated inhibition or death of cancer cells or stimulate or activate such T-cells;

(iii) exhibit the properties of or produce such an anticancer cell antibody;

(iv) is expanded to any agent that can mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells.

It has been suggested that the anticancer activity or efficacy of the combination of angeloyl-substituted ingenan with the agent is greater than the angeloyl-substituted ingenan or the agent alone.

Thus, the present invention relates to a protocol for treating a subject with cancer or a subject suspected of having cancer, the protocol

Administering to said individual an angeloyl-substituted ingenan, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and

The subject has the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell (eg, cytotoxic T-cell) mediated inhibition or death of cancer cells or be capable of stimulating or activating such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Administering an agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

The invention further provides a method of treating an individual with cancer, the method comprising

Administering to said individual an angeloyl-substituted ingenan, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and

The subject has the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell (eg, cytotoxic T-cell) mediated inhibition or death of cancer cells or be capable of stimulating or activating such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Administering an agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone.

The content provided may be less than each content added together or may be additive. In such cases, therapeutic results have been suggested to be more effective. It is a cancer cell; Cytotoxic T-cells or agents capable of inducing T-cell mediated inhibition or death of cancer cells; Antibodies specific for the incorporation of an antigen in cancer cells or an antigen mainly or entirely in cancer cells; Or as "anticancer efficacy" comprising one or more of the cytotoxicity, apoptosis, necrosis, senescence or cell cycle arrest of neutrophils or agents that induce neutrophil-promoted antibody-dependent cellular cytotoxicity (ADCC) of cancer cells. do.

"Anti-cancer effect" includes cytotoxicity, necrosis, stagnation, senescence, cell cycle arrest and resection of cancer cells. This term also extends to antibodies, neutrophils and cytotoxic T-cells or agents that induce anticancer activity.

In addition, the present invention provides a therapeutic protocol for treating cancer in a subject, the protocol comprising ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof and one or more other agents or pharmaceutically acceptable thereof. Possible salt administration, wherein incorporation of the ingenol angelate with the agent has a merger index (CI) of less than 1, where CI is determined by the formula:

CI = [(C) ₁ / (C _x ) ₁ ] + [(C) ₂ / (C _x ) ₂ ] + [α (C) ₁ (C) ₂ / (C _x ) ₁ (C _x ) ₂ ]

Wherein (C _x ) ₁ is the concentration of one of the ingenol angelates or agents that produces the x% effect of the drug alone, and (C) ₁ is the other of the ingenol angelates or agents (C) _Is the concentration of one of the ingenol angelates or agents that, in combination with ₂ , produces the same x% effect, and α is the case where the modes of anticancer activity or efficacy of the ingenol angelates and agents are mutually exclusive or non-excluded Constant and α is 0 or 1, respectively.

Ingenol angelate and the formulation may be administered separately, sequentially or simultaneously, or combined in a single composition to be administered to an individual. Ingenol-substituted ingenans and agents can have the same or different modes of administration, such as all of which are provided, for example, by intravenous, subcutaneous, topical, interstitial or intralesional, intraperitoneal or intranasal administration. Or they may be provided by various routes of administration.

In a particularly preferred embodiment, the present invention provides a protocol for treating a subject with cancer or a subject suspected of having cancer, the protocol comprising

Administering to the individual ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and

The subject has the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell (eg, cytotoxic T-cell) mediated inhibition or death of cancer cells or be capable of stimulating or activating such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

  Administering an agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of ingenol-3-angelate and the agent is greater than ingenol-3-angelate or the agent alone.

Another embodiment of the invention provides a method of treating a subject with cancer, the method comprising

Administering to the individual ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and

The subject has the following characteristics:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell (eg, cytotoxic T-cell) mediated inhibition or death of cancer cells or be capable of stimulating or activating such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

 Administering an agent exhibiting at least one of

Wherein the anticancer activity or efficacy of the combination of ingenol-3-angelate and the agent is greater than ingenol-3-angelate or the agent alone.

The present invention provides a therapeutic protocol for the treatment of cancer in a subject, wherein the protocol is selected from ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and those set forth in Table 1 Administering one or more other agents wherein the combination of ingenol-3-angelate and the selected agent has a merger index (CI) of less than 1, where CI is determined using the following equation:

CI = [(C) ₁ / (C _x ) ₁ ] + [(C) ₂ / (C _x ) ₂ ] + [α (C) ₁ (C) ₂ / (C _x ) ₁ (C _x ) ₂ ]

Wherein (C _x ) ₁ is the concentration of one of ingenol-3-angelate or agent that produces the x% effect of the drug alone, and (C) ₁ is the ingenol-3-angelate or agent Is the concentration of one of the ingenol-3-angelates or agents that merge with the other (C) ₂ to produce the same x% effect, and α is the mode of anticancer activity of the ingenol-3-angelates and agents A constant in the case of exclusive or non-excluded, α is 0 or 1, respectively.

In another embodiment, the present invention is directed to a combination comprising ingenol angelate and an agent with anticancer effect, wherein the combination further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

In particular, the present invention provides ingenol angelate and the following properties:

(i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells,

(ii) mediate T-cell (eg, cytotoxic T-cell) mediated inhibition or death of cancer cells or be capable of stimulating or activating such T-cells,

(iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies,

(iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;

A formulation comprising an agent having at least one property selected from wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenane with the agent is greater than ingenol angelate or the agent alone, wherein the combination is 1 It further comprises the above pharmaceutically acceptable carrier, diluent and / or excipient.

In another embodiment, the present invention provides a multi-part formulation, wherein the combination comprises at least one portion comprising ingenol angelate, and at least one other portion comprising the formulation. These portions or additional portions may further comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients.

Although anti-cancer effects or anti-cancer treatments include selectivity within the scope of the present invention, the agent (or combination of angeloyl-substituted ingenol and agent) is not considered to be selectively cytotoxic to cancer cells alone.

The agent may be a chemically synthesized or isolated molecule or may be a biological molecule such as an antibody or a cell such as a cytotoxic T-cell or other immune cell or a vaccine that induces an antibody against cancer cells. Can be or a molecule that induces any desired property.

The term “pharmaceutically acceptable salts, derivatives, homologues or analogs” refers to any that can provide (directly or indirectly) the compound or physiological (eg analgesic) active compound, metabolite or residue thereof when administered to an individual. It is intended to deliver pharmaceutically acceptable tautomers, salts, prodrugs, hydrates, solvates, metabolites or other compounds. Examples of suitable derivatives include OH or SH groups and suitable carboxylic acids such as C ₁ -C ₃ alkyl-CO ₂ H and HO ₂ C- (CH ₂ ) _n -CO ₂ H, where n is from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 but preferably 1 to 4) and esters formed from the reaction of CO ₂ H—CH ₂ phenyl.

Thus, the active compound may be in crystalline form as a free compound or as a solvate (eg hydrate). Solvation methods are generally known in the art.

Salts of the active compounds of the present invention are preferably pharmaceutically acceptable, but non-pharmaceutically acceptable salts are also to be understood as included within the scope of the present invention, which are useful as intermediates in the preparation of pharmaceutically acceptable salts. . Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; Pharmaceutically acceptable inorganic acids such as acid addition salts of hydrochloric acid, orthophosphoric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid and hydrobromic acid; Or pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, citric acid, lactic acid, music acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, tri Of halomethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valeric acid Salts;

The term "prodrug" is used herein in the broad sense to include compounds that can be converted in vivo to the compound of interest (eg, by enzymatic or hydrolytic cleavage). Examples thereof include esters such as acetates of hydroxy or thio groups, as well as phosphates and sulfonates and the like. Methods of acylating hydroxy or thio groups are known in the art, for example by reacting alcohols (hydroxy groups) or thio groups with carboxylic acids. Other examples of suitable prodrugs are described in Design of Prodrugs , H. Bundgaard, Elsevier, 1985, which is incorporated herein by reference.

The term “metabolite” includes any compound that can be converted in vivo when the active agent is administered to a subject. Examples of such metabolites include glucuronide, sulfates and hydroxylates.

It is understood that the active agents as described herein may exist in the form of tautomers. The term "tautomer" is used herein in its broad sense to include a compound that may exist in equilibrium between two isomeric forms. Such compounds may differ in the position of these atoms or groups in the compound and in the bond connecting the two atoms or groups. Specific examples are keto-enol tautomers.

The compounds of the present invention may take the form of polyanions with anions, related to electrical neutrality, or may be electrically neutral. Examples of suitable related anions include sulphate, tartarate, citrate, chloride, nitrate, nitrite, phosphate, perchlorate, halosulfonate or trihalomethylsulfonate and the like.

The active agent may be administered for the therapy by any suitable route. Suitable routes of administration include oral, rectal, nasal, aerosol inhalation or particulates, topical (including buccal and sublingual), transdermal, vaginal, intra bladder, intralesional and parenteral (subcutaneous, intramuscular, intravenous, intraoral, Meninges, epidural and intradermal).

As used herein, "cancer" includes solid and hematologic cancers, leukemias, sarcomas, and carcinomas. Examples of cancers that can be treated using the protocol of the present invention include ABL1 oncogenes, AIDS-related cancers, auditory neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic cancer, adrenal cortex cancer, idiopathic myeloid, alopecia, bonwa Sexual soft sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, capillary dilated ataxia, basal cell carcinoma (bladder), bladder cancer, bone cancer, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors , Carcinoid tumor, cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, bumpy skin Fibrosarcoma, connective tissue formation-small-circular-cell-tumor, catheter cancer, endocrine cancer, endometrial cancer, cerebral hepatocellular carcinoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal-carcinoma, genitourinary cancer, germ cell tumor, gestational-nutrient membrane-disease, glioma, gynecologic cancer, blood tumor, hairy cell leukemia, head and neck cancer, hepatocellular Cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, respiratory trait, hypercalcemia, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Lee-Fraumeny syndrome, cleft lipoma, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant hepatic tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, Mesothelioma, metastatic cancer, oral cancer, complex endocrine neoplasia, mycelial sarcoma, myelodysplastic syndrome, myeloma, myeloproliferative disease, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nibegain hand Syndrome, non-melanoma skin cancer (NMSC), non-small cell lung cancer (NSCLC), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid cancer, penile cancer, peripheral Neuroectodermal tumor, pituitary cancer, true polycythemia, prostate cancer, rare cancer and related diseases, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rossmond-thomson syndrome, salivary adenocarcinoma, sarcoma, Schwanno, sezary syndrome ), Skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional cell cancer (bladder), transitional cell cancer (kidney) Pelvic-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, europlakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia or Wilms' tumor, and the like. The terms "cancer" and "tumor" may be used interchangeably herein. "Tumor disease" is also a form of cancer or tumor.

The present invention relates to a composition comprising ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and an agent together with one or more pharmaceutically acceptable additives and optionally other agents. Pharmaceutically acceptable additives may be in the form of carriers, diluents, adjuvants and / or excipients and include all conventional solvents, dispersing agents, fillers, solid carriers, coating agents, antifungal or antibacterial agents, skin penetrating agents, surfactants, isotonics Sex and absorbent formulations and slow or controlled release substrates. The active agent may be presented in the form of a kit of ingredients to allow simultaneous, separate or sequential administration of the active agent. Each carrier, diluent, adjuvant and / or excipient must be "pharmaceutically acceptable" in that it is compatible with the other ingredients of the composition and is physiologically acceptable by the individual. The composition may be presented simply in unit dosage form and may be produced by methods known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, the composition is produced by allowing the active ingredient to be homogeneously and fully combined with the liquid carrier, diluent, adjuvant and / or excipient or finely divided solid carrier, and then molding into the product if necessary.

Compositions of the invention suitable for oral administration may be presented as discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of active ingredient; As a powder or granules; As a liquid or suspension in an aqueous phase or a non-aqueous liquid; Or as an oil-in-water liquid emulsion or a water-in-oil emulsion. In addition, the active ingredient may be presented as a macrocyclic, soft medicine or paste.

Tablets may be produced by compression or molding, optionally with one or more accessory ingredients. Compressed tablets are active ingredients in free flowing form, such as powders or granules, optionally mixed with a binder (e.g., inert diluent, antiseptic disintegrant, sodium powder glycolate, crosslinked povidone, crosslinked sodium carboxymethyl cellulose) surfactant or dispersant. Can be produced by compressing the Molded tablets may be produced by molding in a suitable device a mixture of powdered compounds moistened with an inert liquid diluent. Tablets may be optionally coated or scored and may be formulated to provide slow release or controlled release of the active ingredient, for example using hydroxypropylmethyl cellulose in various proportions, to provide a desired release profile. The tablet may optionally provide enteric skin to provide release to a portion of the intestine but not the stomach.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which may include antioxidants, buffers, bacteriostatics, and solutes to make the blood and the composition of the intended individual isotonic; And aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit dose or multi-dose closed containers, such as ampoules and vials, and in a lyophilized (freeze-dried) state requiring only the addition of a sterile liquid carrier such as water for injection immediately before use. Can be stored. Temporary injection solutions and suspensions may be produced from sterile powders, granules, and tablets of the type described above.

Compositions suitable for topical administration to the skin, ie transdermal administration, may comprise the active agent dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gels, creams, pastes, ointments and the like. Examples of suitable carriers include mineral oil, propylene glycol, waxes, polyoxyethylene and long chain alcohols. Transdermal devices such as patches may also be used and may include microporous membranes produced from suitable materials such as cellulose nitrate / acetate, propylene and polycarbonate. The patch may also include suitable skin adhesives and backing materials.

In addition, the active compounds of the present invention may be presented as implants, which may include drug bearing polymer devices, wherein the polymer is biocompatible and nontoxic. Suitable polymers can include hydrogels, silicones, polyethylenes, and biodegradable polymers.

The compounds of the present invention may be administered in sustained release (ie, controlled) or slow release formulations. Sustained release formulations are those in which the active ingredient is slowly released in the body of an individual upon administration to maintain a predetermined drug concentration over a minimum period of time. The preparation of sustained release formulations is known to those skilled in the art. Examples of dosage forms include oral, implant and transdermal formulations. For slow release administration, the active ingredient can be suspended, for example, as a slow release particle or in liposomes.

The pharmaceutical compositions of the present invention may be packaged for commercial sale with other active agents, or alternatively other active agents may be combined with ingenol angelate and the agent or pharmaceutical salts, derivatives, homologs or analogs thereof. Can be.

Thus, certain further embodiments of the present invention provide a system for controlled release of all one or more of the active agent or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, wherein the system

(a) a repository-core comprising an effective amount of an active substance and having a predetermined geometry; and

(b) a support-platform applied to the reservoir-core

Where the storage-core is

(i) an expandable polymeric material or aqueous liquid and gelled polymeric material in contact with water, wherein the ratio of expandable polymeric material to gelling polymeric material is in the range from 1: 9 to 9: 1; and

 (ii) homopolymer materials having both swelling and gel forming properties

At least one component and an active substance selected from the group consisting of

The support-platform is an elastic support applied to the reservoir-core to partially coat the surface of the reservoir-core and allow it to change due to hydration of the reservoir-core so that it is slowly soluble and / or slowly gels in the aqueous fluid. .

Support-platforms may include polymers such as hydroxypropylmethylcellulose, plasticizers such as glycerides, binders such as polyvinylpyrrolidone, hydrophilic agents such as lactose and silica, and / or hydrophobic agents such as magnesium stearate and glycerides Can be. The polymer (s) typically form 30 to 90% by weight of the support-platform, for example about 35 to 40%. The plasticizer may form at least 2% by weight, for example about 15-20%, of the support-platform. The binder (s), hydrophilic agent (s) and hydrophobic agent (s) typically form up to about 50% by weight of the total of the support-platform, for example about 40-50%.

Tablets may include one or more water insoluble or poorly soluble hydrophobic excipients. Such excipients include alkylcelluloses such as ethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose and derivatives thereof; Polymethacryl polymers, polyvinyl acetate and cellulose acetate polymers; Fatty acids or esters or salts thereof; Long chain fatty alcohols; Polyoxyethylene alkyl ethers; Polyoxyethylene stearate; Sugar esters; And any known hydrophobic cellulose derivatives and polymers, including lauroyl macrogol-32 glyceryl, stearoyl macrogol-32 glyceryl, and the like. The hydroxypropylmethyl cellulose material is preferably selected from low Mw and low viscosity materials such as E-Type methocel and 29-10 type as defined in USP.

Other agents or excipients that provide hydrophobic properties to the coating can be selected from any waxy material for use as tablet excipients. Preferably the HLB value is less than 5, more preferably about 2. Suitable hydrophobic agents include wax materials such as carnauba wax, paraffin, microcrystalline wax, beeswax, cetyl ester wax, and the like; Or non-fatty hydrophobic materials such as calcium phosphate salts such as dibasic calcium phosphate and the like.

Preferably the coating comprises calcium phosphate salts, glyceryl behenate and polyvinyl pyrrolidone or mixtures thereof and at least one adjuvant, diluent, lubricant or filler.

Preferred components in the coating are as follows and generally the appropriate proportions are given as weight percent of the coating.

Polyvinyl pyrrolidone (povidone) is preferably present in an amount of about 1 to 25% by weight, more particularly 4 to 12%, for example 6 to 8% of the coating.

Glyceryl behenate is an ester of glycerol and behenic acid (C ₂₂ fatty acid). Glyceryl behenate may be present in its monoester, diester or triester form or as a mixture thereof. Preferably the HLB value is less than 5, more preferably about 2. It may be present in an amount of about 5 to 85 weight percent, more particularly 10 to 70 weight percent of the coating, in certain preferred embodiments 30 to 50 percent.

The calcium phosphate salt may be a dibasic calcium phosphate dihydrate and may be present in an amount of about 10 to 90% by weight of the coating, preferably 20 to 80%, for example 40 to 75%.

Coatings may include other conventional tablet excipients such as lubricants, colorants, binders, diluents, glidants and taste masking agents or flavoring agents.

Examples of excipients include colorants such as iron oxides such as yellow iron oxides; Lubricants such as magnesium stearate; And glidants such as silicon dioxide such as colloidal silicon dioxide and the like. Yellow iron oxide may be used in an amount of about 0.01 to 0.5 weight percent based on the coating; Magnesium stearate may be present in an amount of from 1 to 20% by weight, more preferably from 2 to 10%, for example from 0.5 to 1.0% of the coating; Colloidal silica can be used in an amount of 0.1 to 20% by weight, preferably 1 to 10%, more preferably 0.25 to 1.0% of the coating.

The core comprises, in addition to the drug substance, a disintegrant or mixture of disintegrants used in the immediate release formulation, which is known in the art. Disintegrants useful in the practice of the present invention may be materials that boil and / or swell in the presence of an aqueous medium to provide the force necessary to mechanically rupture the coating material.

Preferably, the core comprises, in addition to the drug substance, crosslinked polyvinyl pyrrolidone and croscarmellose sodium.

The following presents a preferred core material. The content is given in weight percent based on the weight of the core.

Crosslinked polyvinyl pyrrolidone is defined above and is useful as a disintegrant and can be used in the core in the amounts disclosed in relation to the core.

Croscarmellose sodium is an internal crosslinked sodium carboxymethyl cellulose (also known as Ac-Di-Sol) useful as a disintegrant.

Disintegrants can be used in amounts of 5 to 30 weight percent based on the core. However, higher content of certain disintegrants can swell to form a substrate that can control the release of the drug substance. Thus, particularly when rapid release is required after a delay time, it is preferred that the disintegrant be used in an amount of up to 10% by weight, for example about 5 to 10% by weight.

The core may further comprise conventional tablet excipients, such as those described above with respect to the coating material. Suitable excipients include lubricants, diluents and fillers, including but not limited to lactose (eg, monohydrate), iron oxide, magnesium stearate, and colloidal silica.

Lactose monohydrate is a disaccharide consisting of one glucose and one galactose. It may act as a filler or diluent in the tablets of the present invention. It may be present in the range of about 10-90%, preferably 20-80%, in certain preferred embodiments 65-70%.

As mentioned above, in embodiments of the present invention, it is important to correctly place the cores in the coating to ensure that the tablets have the appropriate coating thickness.

In this way, lag time is reliable, reproducible, and intra- and inter-subject variations in bioavailability can be ruled out.

In another embodiment, a multiparticulate release ingenol angelate / formulation composition is provided for oral administration. The combination is produced by complexing the active agent or pharmaceutically acceptable salts, derivatives, homologues or analogs thereof, optionally in the form of small particles, typically ion exchange resins of less than 150 microns. To produce a multiparticulate blend, (a) immediate release particles produced by coating the drug containing particles with a polymer that is insoluble in the saliva's neutral medium but soluble in the acidic environment of the stomach; (b) enteric-coated particles produced by coating the drug-containing particles with a polymer insoluble in the acidic environment of the stomach but soluble in the neutral environment of the small intestine; (c) extended release particles produced by coating the drug containing particles with a polymer that forms a water-insoluble but permeable membrane; (d) enteric skin-extended release particles produced by coating the extended release drug particles with enteric skin; (e) The drug-containing particles are coated with the polymer in the final dosage form of one or more of the delayed release particles produced by coating the polymer with a polymer which is insoluble in the stomach and upper small intestine but which dissolves in the lower or upper small intestine. .

In the case of the treatment of cancer, the combination may comprise a compound which reduces the resistance to the therapeutic agent. Examples of such compounds include inhibiting P-glycoprotein or other cellular mechanisms associated with mediating intracellular accumulation of drugs.

Formulations may also include carriers, diluents and excipients. Details of pharmaceutically acceptable carriers, diluents and excipients, and methods of making and combining pharmaceutical compositions are given in Remmingtons Pharmaceutical Sciences 18th Edition, 1990, Mack Publishing Co., Easton, Pennsylvania, USA.

Thus, the present invention relates to angeloyl-substituted ingenans, such as PEP005 and agents, such as antibodies, cancer antigens and T-cells (eg, cytotoxic T-cells) or other immune cells for the treatment of individuals with cancer. And the use of the agents set forth in Table 1, including. "Treatment" may result in cancer cell death or may arrest cancer cell growth or may help prevent metastasis to cancer and / or trigger immune memory. Therefore, treatment can result in cancer cell death, necrosis, cytotoxicity, senescence, cell cycle arrest or stagnation.

The present invention is now further described with reference to the following examples, which are presented by way of illustration only and are not intended to limit the scope of the invention as set forth above.

In the examples, the following materials and methods were used:

Cell line

All cell lines were from ATCC (Rockville, MD). Cells were monolayered in RPMI5 DMEM or McCoy medium supplemented with 10% v / v fetal bovine serum (Invitrogen, Sergis-Mongtoise, France), 2 mM glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin. Proliferated. All cells were split twice weekly using trypsin / EDTA (0.25% w / v and 0.02% w / v, Invitrogen, Sergis-Morttoise, France, respectively) and 2.5 × 10 ⁴ cells / ml Sowing was carried out at a concentration. All cell lines were tested periodically for mycoplasma contamination by PCR using the Stratagene kit (La Jolla, Calif.).

Twelve cell lines were analyzed and their characteristics are summarized in Table 3 below (data from the NCI cell line screen).

Cell line PEP005 p53 MDR MMR HER2 / neu EGFR colon HT29 R mut lowness height lowness height HCT116 R mut lowness lowness lowness height COLO205 S mut lowness height height height HCC2998 S mut lowness lowness height height breast MCF7 R wt lowness lowness lowness lowness MDA-MB-435 S mut lowness lowness height lowness ovary OVCAR3 R mut lowness height height IGROV1 R wt lowness lowness height height lungs Hop62 S mut height height lowness lowness Hop92 R mut lowness lowness height height leukemia K562 R mut lowness height lowness lowness CCRF-CEM S mut height lowness lowness lowness wt: wild type; mut: mutation

drug

Purified PEP005 (Ingenol-3-angelate) is supplied by Peplin Limited (Brisbane, Australia). 60 mM stock solution was generated in DMSO and stored in 20 ° C. dark.

In vitro growth inhibition assay (MTT assay)

MTT analysis was performed as previously described (Hansen et al., J Immunol Methods 119 (2): 203-210, 1989). Briefly, cells were seeded at a density of 2 × 10 ³ cells / well in 96-well tissue culture plates. Cell viability was obtained by the addition of yellow water-soluble tetrazolium MTT (brominated 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium; Sigma, Palavie Saint-Jean, France) Measurement was carried out after 120 hours of incubation by colorimetric conversion with marzan. This reaction is catalyzed by mitochondrial dehydrogenase and used to assess the relative number of viable cells (Mosmann, J. Immunol. Methods 65 (1-2): 55-63, 1983). Cells were incubated at 37 ° C. for 0.4 h with 0.4 mg / ml MTT. After incubation, the supernatant was discarded and the cell pellet was resuspended in 0.1 ml of DMSO and absorbance was measured at 560 nm using a microplate reader (Dynatec, Michigan, USA). Wells with untreated cells or drug containing medium without cells were used as positive and negative controls, respectively. Growth inhibition curves are shown as percentage of untreated control cells.

Single agent experiment

Cells were seeded in 96-well plates at 2 × 10 ³ cells / well and treated with increasing PEP005 concentration after 24 hours. After 1 hour, 24 hours or 48 hours of incubation, cells were washed and post-cultured for 72 hours in drug free medium. Growth inhibition was then measured by MTT assay.

Concurrent Exposure of PEP005 with Other Drugs

For simultaneous drug exposure, cells are seeded in 96-well plates at 2 × 10 ³ cells / well and at a concentration of PEP005 alone which increases after 24 hours or at various concentrations corresponding to IC ₂₀ , IC ₄₀ or IC ₆₀ values. It was treated with another drug. After about four double times (120 hours), growth inhibition effect was measured by MTT assay.

Sequential Exposure to PEP005 and Other Drugs

Cells were seeded in 96-well plates at 2 × 10 ³ cells / well and allowed to proliferate for 24 hours. Cells were then exposed to various concentrations of the first drug for 1 hour / 24 hours / 48 hours, the drug was removed, the cells washed and a second drug added. After further drug exposure, the second drug was removed, cells were washed and post-cultured for 72 hours in drug free medium. Growth inhibition was then measured by MTT assay.

Statistical analysis and measurement of synergistic activity

The effect of drug consolidation was evaluated using the Chou and Talalay method based on the Intermediate Effect Principle (Chou and Talalay, Adv Enzyme Regul 22: 27-55, 1984). The dose-effect on the merging of the ratios is shown using the equation f _a / f _u = (C / C _m ) ^m , where C is drug concentration and IC ₅₀ is half the maximum effect (ie cell growth). F _a is the fraction of cells affected by drug concentration C (eg 0.9 if cell growth is inhibited by 90%), f _u is the fraction unaffected, and m is S-sigmodicity coefficient of the concentration-effect curve Based on the slope of the curve for each drug in the merge, whether the drugs have mutually non-exclusive effects (eg, independent or interaction mode of action) The merge index (CI) is then It was decided:

CI = [(C) ₁ / (C _x ) ₁ ] + [(C) ₂ / (C _x ) ₂ ] + [α (C) ₁ (C) ₂ / (C _x ) ₁ (C _x ) ₂ ]

In the above equation, (C _x ) ₁ is the concentration of drug 1 required to produce the x% effect of drug 1 alone, and (C) ₁ is the drug 1 required to produce the same x% effect in combination with (C) ₂ Concentration. If the mode of action of the drugs is mutually exclusive or non-exclusive, α is 0 or 1, respectively. The CI value is calculated by obtaining the release of the equation for the various values of f _a (ie, for different degrees of cell growth inhibition). CI values of <1 indicate synergism, values of 1 indicate additive effects, and values of> 1 indicate antagonism. Data was analyzed using concentration-effect analysis on microcomputer software (Biosoft, Cambridge, UK) on an IBM-PC computer. Statistical Analysis and Graphs Instat and Prism software (graphpad, San Diego, USA) were used. Dose-effect relationships for tested drugs alone or for paired consolidations were subjected to a mid-effect plot analysis to determine the relative potential (IC ₅₀ ), shape (m) and agreement (r) in each selected cell line. It was. As described above, IC ₅₀ and m values were used to calculate synergy and antagonism based on CI equations, respectively. The results are expressed as mean ± standard deviation of two or more experiments performed in duplicate. In each experiment, cells were exposed for 38 hours to paired merges as described above. Mean and standard deviation were compared using Student's t-test (2-sided p value).

Cell Cycle Analysis and Apoptosis

Cell cycle analysis and measurement of apoptotic cell rate were evaluated by flow cytometry. Briefly, cells were seeded in 25 cm 3 flasks and treated with or without PEP005 at various concentrations. At various time points adherent and non-adherent cells were recovered, washed with PBS, fixed in 70% v / v ethanol and stored at + 4 ° C. until use. The cells were dehydrated in PBS and incubated with 250 μg / ml RNAse A containing Triton X-100 for 20 minutes at room temperature and 50 μg / ml propiodide iodide at + 4 ° C. in 20 minutes in the dark. Cell cycle distribution and percentage of apoptotic cells were measured using a FACScan flow cytometer.

Example 1

Single agent experiment

1 shows the effect of time course experiments using PEP005 provided for 1 hour, 24 hours and 48 hours in a panel of cell lines. Based on the data, 48 hour exposure was selected for further evaluation of PEP005 in the panel of cancer cell lines. This exposure period was shown to be optimal for observing the antiproliferative effect of PEP005 in the most sensitive human cancer cell lines. Each point is the average of three or more individual experiments each conducted in duplicate. PEP005 shows cytotoxic effects on human colon COLO205 and HCC2998 cells, with breast cancer cell lines MDA-MB-435 and COLO205 being the most sensitive.

IC ₅₀ for 48 hour exposure is shown in Table 4 below.

Cell line IC ₅₀ (μM) COLO205 0.01 ± 0.002 MDA435 2.6 ± 0.52 HCC2998 30 ± 6 HOP92 85 ± 17 HOP62 110 ± 22 HCT116 120 ± 24 HT29 140 ± 28 MCF7 180 ± 36 OVCAR3 200 ± 40 IGROV1 200 ± 42

As a control, a leukemia cell line previously found to be sensitive to PEP005 was used.

PEP005 was found to inhibit K562 and CCRF-CEM leukemia cell line survival (FIG. 2) and IC ₅₀ was 0.006 ± 0.001 μM and 60 ± 14 μM, respectively.

Two different schedules of PEP005 administration were tested, 3 hours 3 days and 3 hours 5 days. The results of these experiments have been shown to increase the sensitivity of COLO205 and MDA under certain conditions. IC ₅₀ for 3 hours × 3 days is 0.1 (COLO205) and 1 μM (MDA). For 3 hours × 5 days, it was 0.002 and 0.0015 μM for COLO205 and MDA, respectively. These two modes of administration did not alter the sensitivity profile for PEP005 of other cell lines.

Example 2

Alteration of Cell Cycle and Apoptosis Induction of PEP005 in COLO205 Cells

The effect of PEP005 on cell cycle progression was tested by flow cytometry to evaluate the segmented DNA eluted from apoptotic cells when permeated with PI. In time course experiments, COLO205 cells were incubated with 0.03 μM, 0.3 μM and 1 μM PEP005 for 1 hour, 5 hours, 12 hours, 24 hours and 48 hours. Constant PEP005 exposure resulted in dose dependent accumulation of cells in the G0-G1 phase, with the S phase almost completely blocked (FIG. 3).

In addition, the effect of PEP005 results in the gradual production of particles corresponding to the low fold DNA content (sub-G1 fraction), which is regarded as a feature of induction of apoptosis. Quantification of apoptotic fractions is shown as the percentage of total cell number (FIG. 4). This result was confirmed by Annexin V-staining. After 24 hours of PEP005 treatment, it was no longer apoptosis but was found to be necrosis.

Example 3

Comparison of PEP005 Cytotoxicity with Other Anticancer Drugs

5 shows a comparative analysis of PEP005 cytotoxicity with oxaliplatin, cisplatin, doxorubicin, gemcitabine, ara-C and 5FU in a panel of cell lines. Results are presented as (IC ₅₀ -IC ₅₀ average).

Example 4

Combined Formulation Experiment

The effect of sequential and simultaneous exposure to PEP005 with oxaliplatin, cisplatin, doxorubicin, docetaxel, gemcitabine, vinorelbine, and 5FU was determined using a consolidation index indicating the fraction affected for the concentration of drug corresponding to IC ₅₀ . Decided. A merge index calculated below 0.8 is a synergistic effect, at least 1.2 is antagonistic, and a merge index of 0.8 to 1.2 corresponds to an additive effect.

Example 5

Merge 5FU-PEP005

In COLO205, the combination of PEP005 and 5FU yielded varying degrees of synergistic activity when 5FU was administered before or after PEP005 (FIG. 6). Each dot represents one experiment conducted in triplicate. 5FUs provided before or after PEP005 result in a merge index value of <1, indicating a synergistic effect between the two compounds.

Example 6

Docetaxel-PEP005 Merged

As shown in FIG. 11, addition and synergistic effects were observed when docetaxel was given before or after PEP005. Antagonistic effects were observed when docetaxel was administered simultaneously with PEP005.

Example 7

Doxorubicin-PEP005 Merged

Synergy was obtained by sequential exposure immediately. Other schedules were mostly antagonists (FIG. 7).

Example 8

Oxaliplatin-PEP005 Merged

Oxaliplatin and PEP005 combination (simultaneous or immediate sequential exposure) was antagonistic (FIG. 8A). This antagonism between oxaliplatin and PEP005 is reversed when COLO205 is exposed to oxaliplatin for 24 to 72 hours after PEP005, in which case the sequence is highly synergistic (FIG. 8B).

Example 9

Gemcitabine-PEP005 merge

Following the sequence gemcitabine, PEP005 exhibits a synergy opposite to antagonism when the drug is administered in the co-set or prior to gemcitabine. This antagonism is reversed when gemcitabine is administered 24 hours after the end of PEP005 exposure, in which case the merger is synergistic with CI <1 for most concentrations (FIG. 9).

Example 10

Merged vinorelbine-PEP005

This merging yields the same results observed when merging PEP005 with docetaxel. Synergistic activity was obtained using sequence vinorelbine followed by PEP005. All other schedules are antagonistic (FIG. 10).

Example 11

Cisplatin-PEP005 Merged

In contrast to oxaliplatin, the antagonism observed in simultaneous or immediate sequential exposure was maintained with a 24-hour washout between the two drugs. The results are shown in FIG.

Example 12

Anticancer Effect of PEP005 in Combination with Other Agents

The above examples illustrate that PEP005 shows antiproliferative effects in human colon COLO205 and breast MDA435 cancer cells. In human sensitive cancer cells, the antiproliferative effects of PEP005 are associated with cell cycle changes (reduction of cells in S phase) and apoptosis is associated with caspase 3 activation and cellular necrosis. This effect appears to be optimal between 24 and 48 hours of exposure to the drug.

PEP005 shows a unique cytotoxicity profile. Incorporation with other anticancer agents exhibits additive and / or synergistic effects with various common cytotoxic drugs such as 5FU doxorubicin, oxaliplatin, gemcitabine, vinorelbine, docetaxel and cisplatin. Some evidence of schedule dependency was observed. This suggests that one drug is somewhat protected from the cytotoxic effects of the other. Interestingly, considering the cell cycle blockade induced by PEP005, consider introducing a 24 hour lag time between administrations of PEP005 into the experiment. In most cases, it was observed that the schedule where PEP005 was separated for 24 hours from administration of the second drug was shown to be more effective in inducing cytotoxic effects. Although preliminary, the time / schedule dependency on PEP005-merge needs to be explicitly investigated.

Example 13

Anticancer Effect of PEP005 in Combination with Various Chemotherapy Agents

In this example, a combination application of PEP005 with three (for cell line U937) or four (for cell line K562) chemotherapeutic agents (provided at the same time) was investigated. Chemotherapeutic agents were applied at 10 different concentrations in half-log increments, and PEP005 was added simultaneously at two fixed concentrations each.

The experimental design is shown in Table 5 below.

Experiment design Cell line PEP005 (ng / ml) Standard Formulation (10 conc. Half-log increments) U937 (lymphoma) 3, 10 Daunocruvicin, cytarabine, etoposide K562 (CML) 0.3, 1, 3 Adriamycin, activated cyclophosphphosphamide, vincristine, etoposide

The purpose of this example is to investigate the in vitro anticancer activity of PEP005 in combination with various chemotherapeutic agents in lymphoma cell line U937 and chronic myeloid leukemia (CML) cell line K562.

Two cell lines wet atmosphere (95% v) in RPMI 1640 medium (PAA, Germany) supplemented with 10% v / v fetal bovine serum (PAA, Germany) and 0.1 mg / ml gentamicin (Invitrogen, Karlsruhe, Germany). / v air, 5% v / v CO ₂ ) at 37 ° C. The cells typically passed once per week. They are maintained in no more than 20 passages in culture.

All cell lines were purchased from ATCC (Rockville, USA). U937 was formed from patients with histiocytoma (Sundstrom and Nilsson, Int. J. Cancer 77: 565-577, 1976). Continuous cell line K562 was established by Lozzio and Lozzio from a pleural effusion of a 53 year old woman with chronic myelogenous leukemia in terminal acute evaporation (Lozzio and Lozzio, Blood 45: 321-334, 1975).

A modified propidium iodide (PI) assay (Dengler et al., Anticancer Drugs 6: 522-532, 1995) was used to assess the effect of PEP005 on the activity of chemotherapeutic agents in two test cell lines. Briefly, cells were harvested from logarithmic culture medium by centrifugation, counted, and plated in microtiter plates with a flat bottom of 96 wells at a cell density of 20,000 cells / well. After a 24 hour recovery period to allow the cells to resume log growth, 10 μl of culture medium (6 control wells / plates) and a culture medium comprising chemotherapeutic agents (6 wells / concentration) were added to the cells. Immediately after, 10 μl of culture medium containing PEP005 was added at one fixed concentration. In each plate only PEP005 (at fixed concentration) was given to 6 wells. The cells were then cultured continuously for a period of 4 days.

50 μl of aqueous PI solution was then added to the wells (final PI concentration 7 μg / ml). After a 30 minute incubation period, fluorescence (FUl) was measured using a Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm), which provides a direct measurement of dead cells. The microplates were then stored at −20 ° C. for 24 hours to kill all cells. After thawing of the plate and the second fluorescence measurement (FU2), the amount of viable cells was calculated by subtracting FU1 from FU2.

In each experiment, treatment groups (PEP005 alone as well as merged) and untreated control cells were measured six times. For the calculations, the average of six data sets was used. Growth inhibition was expressed as test / control × 100 (% T / C). The IC ₅₀ and IC ₇₀ values, the drug concentrations required to inhibit cell growth by 50% (T / C = 50%) and 70% (T / C = 30%), respectively, based on the T / C value, are compared to the compound concentration. Cell viability (T / C) is shown and measured, and IC values are calculated based on the principle of point-to-point curve conjugation. To illustrate the effect on the dose-response relationship, the concentration-response curves of the chemotherapeutic agent alone and the combined application of two fixed PEP005 concentrations were applied together in one graph.

PEP005 is provided as a dry powder by Peplin Limited, Brisbane, Australia. 10 mg / ml stock solution was generated in DMSO and aliquots were stored at -20 ° C. Aliquots of stock solutions were thawed on the day of use and stored at room temperature prior to and during dosing. The intermediate dilution step was carried out using RPMI 1640 cell culture medium to obtain a solution 15 times the final concentration. For the final dilution step (1:15), 10 μl of solution was added directly to 140 μl medium in wells on top of the cultured test cells.

Duanorubicin (# D8809), cytarabine (# C1768), etoposide (E1383) and vincristine (V8879) were supplied by Sigma (Taufkirken, Germany). 4-hydroperoxycyclophosphamide (activated cyclophosphamide) was supplied by Asta Medica (Frankfurt, Germany) and adriamycin was used as a clinical preparation from a pharmacist (Magda Hamburg, Germany).

The results of the combined experiments are summarized in Table 6 (cell line U937) and Table 7 (cell line K562) below.

Cell line U937 was treated with PEP005 in combination with topoisomerase II inhibitors daunorubicin and etoposide (VP16), as well as with the DNA damaging agent cytarabine. Table 6 shows the results of three independent experiments. PEP005 was applied at fixed concentrations of 3 and 10 ng / ml respectively. The effect of PEP005 alone is in the range of T / C = 62% to T / C = 89%. Positive interaction of PEP005 is detected with cytarabine and VP16. PEP005 at a concentration of 10 ng / ml resulted in IC ₇₀ values ranging from 0.74 μg / ml (cytarabine alone) to 0.037 μg / ml (reduced to 5% compared to cytarabine alone) in Experiment GF578 (GF624). ) From 0.24 μg / ml to 0.074 μg / ml (reduced to 31%) and in the third experiment (GF769) from 0.3 μg / ml to 0.173 μg / ml (58%). PEP005 shifted the concentration-response curve to the left, which resulted in a decrease in IC ₅₀ and IC ₇₀ values. Incorporation of PEP005 with VP16 at a concentration of 10 ng / ml resulted in IC ₇₀ values from 1.67 μg / ml (VP16 alone) to 0.24 μg / ml in IC GF578 (IC ₇₀ value decreased to 14%) GF624) from 1.15 μg / ml to 0.19 μg / ml (reduced to 17%) and in the third experiment (GF769) from 2.22 μg / ml to 1.32 μg / ml (reduced to 59%).

No significant positive interactions were found in CML cell line K562 (Table 7). K562 cells were PEP005 in combination with the chemotherapeutic agent adriamycin (topoisomerase II inhibitor), activated cyclophosphamide (alkylation agent), vincristine (tubulin binder) and VP16 (topoisomerase II inhibitor). Treated with. Three independent experiments were conducted and PEP005 alone was run at test / control values of 79% to 108%. No significant reduction of IC ₇₀ values by PEP005 (less than 50% of IC ₇₀ values of chemotherapeutic agents alone) was detected in the cell line.

This example illustrates that PEP005 causes a significant increase in in vitro antitumor activity in the combination with cytarabine and VP16 in lymphoma cell line U937.

Evaluation of Positive Interactions Between PEP005 and Chemotherapy

-, IC ₇₀ of the IC ₇₀ alone chemotherapy (merge)> 50%

+, IC ₇₀ (combined) of chemotherapeutic agent alone ≤50%

++, IC _7O (combined) of chemotherapeutic agent ≤30%

+++, IC _7O (combined) of chemotherapeutic agent ≤10%

Evaluation of Positive Interactions Between PEP005 and Chemotherapy

-, IC ₇₀ of the IC ₇₀ alone chemotherapy (merge)> 50%

+, IC ₇₀ (combined) of chemotherapeutic agent alone ≤50%

++, IC _7O (combined) of chemotherapeutic agent ≤30%

+++, IC _7O (combined) of chemotherapeutic agent ≤10%

Example 14

Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC)

This example provides evidence that neutrophils are needed to prevent recurrence of skin tumors following topical treatment with PEP005. Topical PEP005 treatment induces primary necrosis of tumor cells, potentially activates protein kinase C, and is associated with an acute T-cell-independent inflammatory response characterized by significant neutrophil infiltration. In neutrophil deficient Foxnl ^nu mice and in CD18 deficient mice (severely worsening neutrophil extravasation), PEP005 treatment is associated with a> 70% increase in tumor recurrence rate. NK cell or monocyte / macrophage deficiency has no effect on relapse rate. In both in vitro and mice, PEP005 induces MIP-2 / IL-8, TNFα and IL-1β, which are all mediators of neutrophil recruitment and activation. In vitro PEP005 activates human endothelial cells resulting in neutrophil adhesion and also causes human neutrophils to produce tumor cell lethal reactive oxygen intermediates. Treatment of tumors with PEP005 significantly increases the level of anticancer antibodies, which can promote neutrophil mediated antibody dependent cellular cytotoxicity (ADCC) in vitro. In addition, PEP005 treatment of tumors grown in SCID mice is associated with a> 70% increase in tumor recurrence rate. Putting these data together suggests a pivotal role for neutrophil-mediated ADCC in preventing relapse. Therefore, PEP005-mediated healing of tumors appears to involve early chemotherapy followed by neutrophil-dependent ADCC-mediated eradication of the rest of the disease, which may lead to neutrophils mediating important anticancer activity using specific chemotherapeutic agents. Illustrate that it can.

Substances and Methods

Cell and Cell Culture

B16 mouse melanoma strain (ATCC CRL-6322), LK2 UV-induced mouse squamous cell carcinoma (SCC) strain (Cavanagh and Halliday, Cancer Res 56: 2607-2615, 1996) and human melanoma strain MM96L (Parsons and Hayward , Photochem Photobiol 42: 287-293, 1985) and Me1O538 (Larizza et al., Clin Exp Metastasis 7: 633-644, 1989) with 10% v / v Fetal Bovine Serum (FCS) (CSL Biosciences, Parkville, Australia). RPMI 1640 medium (Life Technologies, Inc.) supplemented with 100 μg / ml streptomycin and 100 IU / ml penicillin (Life Technologies, Inc., Rockville, MD, USA) (complete media) , Rockville, MD) at 37 ° C. and 5% CO ₂ . Human epidermal keratinocytes were isolated from neoplastic foreskin and cultured in the presence of mitomycin C-treated 3T3 feeder cell layers (Hotchin and Watt, J Biol Chem 267: 14852-14858, 1992). Human dermal fibroblasts were isolated from tissue digests taken during joint replacement surgery and grown for several weeks in defined media as previously described (Parsonage et al., Thromb Haemost 90: 688-697, 2003). Human neutrophils are isolated from peripheral blood by percoll density centrifugation as described above (de Boer and Roos, J. Immunol 136: 3447-3454, 1986) and purity is assessed by Giemsa staining, which is typically> 95 It was%.

PEP005 Treatment and Histology

LK2 cells (10 ⁶⁾ a 6-10 week old ^nu Foxnl (BALB / c nu ^- / nu ^-) mice were subcutaneously injected into the flanks of the (animal resources System Center, Australia Perth material) (two tumor sites / mouse, and 1 per group 2 mice). Two patches of normal skin and tumors in the opposite flank were treated once topically with PEP005 or placebo (isopropanol-based gel). PEP005 is dissolved in 100% v / v acetone, diluted in isopropanol-based gel (pH 4-6) consisting of 25% v / v isopropyl alcohol and 25% w / w propyl alcohol in water and 10 μg of PEP005 It was applied to 10 μl of gel. PEP005 powder was obtained from Peplin Limited (Brisbane, Australia) with purity greater than 98.5%. Mice were euthanized with post-PEP005 treatment at 1, 2, 6, 24 and 48 hours, the treated site was dissected, fixed with formalin, embedded in paraffin wax and subjected to histology using standard H & E staining. Treated. Slides were inspected using a ScanScopeT2 slide scanner (Aperio Technologies).

PEP005 treatment in neutrophil deficient mice

LK2 cells (10 ⁶ ) were injected subcutaneously into the flanks of 6-10 week old Foxnl ^nu mice (n = 3 mice and n = 12 tumors per group) (4 tumor sites / mouse). Two weeks later (when the tumors were about 14 mm 3), six mice received rat anti-Ly-6G anti-granulocyte antibodies (100 μg in PBS) (RB6-8C5; BD Biosciences, San Diego, CA, USA). ) Was injected intraperitoneally. Another six mice were injected intraperitoneally with homozygous control antibody (100 μg of rat IgG2b in PBS) (A95-1; BD Biosciences). Antibodies were injected at day −2, day 0 and day 2 or placebo treatment at day 0 for the onset of PEP005. Six additional control mice were not injected with antibodies. Tumors in a total of nine mice (n = 3 no antibody, n = 3 A95-1 treated and n = 3 RB6-8C5 treated) were topically treated daily with PEP005 for 3 days as above. Another nine mice divided into groups as above were treated with the same volume of placebo (isopropyl gel). Tumor and erythema size were measured with a caliper. Blood was collected twice a week from the tail tip, smeared, air dried on glass slides and stained with Quick Dip (Pronion Laboratories, Melbourne, Australia). In all tumor experiments, mice were euthanized when the accumulated tumor load per mouse exceeded 1,000 mm 3.

PEP005 Treatment in CD18 Deficient Mice

B6.129S7- Itgb2 ^tmlBay / J mice (The Jackson Laboratories, Bar Harbor, Maine, USA) are CD18 low-order mice on B6 background and 2-16% of normal CD18 expression in granulocytes (Wilson et al. al., J Immunol 157: 1571-1578, 1993). B16 cells (5 × 10 ⁵ ) were injected subcutaneously into the flanks of 6-10 week old mice (n = 9 mice per group) (one tumor site / mouse) and 6 days later (when the tumors were about 14 mm). , Mice were treated with PEP005 or placebo. Tumor and erythema size were measured as above.

PEP005 Treatment in NK Deficient Mice

LK2 cells (10 ⁶ ) were injected subcutaneously into the flanks of 6-10 week old Foxnl ^nu mice (n = 3 mice and n = 12 tumors per group) (4 tumor sites / mouse). Two weeks after tumors were about 14 mm 3, mice were treated with PEP005 or placebo (day 0, day 1 and day 2) and tumor volume was monitored as above. Mice were injected with anti-asialo GM1 polyclonal anti-serum (50 μL intraperitoneal) (Celdaran, Ontario, Canada) on day 2 and day 2 (initiation of PEP005 treatment). NK deficiency (> 90%) was confirmed 2 days after antibody administration using splenocytes from parallel groups of animals and FAC analysis (using Pan NK antibody DX5, BD Biosciences Farmingen).

PEP005 Treatment in op / op Mice

B16 cells (5 × 10 ⁵ ) were injected subcutaneously into the flanks of 6-10 week old op / op low-order mice (Hurston Medical Research Center, Brisbane, Australia) (n = 3 and n = 12 tumors per group). (4 tumor sites). Tumors were treated with PEP005 or placebo and monitored as above.

PEP005 Treatment in SCID Mice

LK2 cells (10 ⁶ ) were injected subcutaneously into the flanks of 6-10 week old SCID mice (animal resource center) (n = 3 mice and n = 12 tumors per group) (4 tumor sites / mouse). Tumors were treated with PEP005 or placebo and monitored as above.

Statistical analysis

SPSS 13.0 for Windows (Release 13.0) was used for statistical analysis. Cox regression analysis was used to compare relapse rates in various groups and to control for mouse effects when using 4 tumors per mouse. Analysis shows that the influence of mice on the time of relapse is not significant (p = 0.88 and p = 0.315), which illustrates that each tumor behaves almost independently.

In vivo cytokine response

LK2 cells were injected into the flanks of 6-10 week old Foxnl ^nu mice (2 tumor sites / mouse) as above. Two weeks later (when the tumor was about 14 mm 3), the tumor and two skin regions on the opposite flank of the animal were treated once with PEP005 as above. After treatment, parallel mice (n = 2 per time point) are euthanized at 0, 1, 2 and 6 hours, and tumor and skin sites are dissected. Total RNA was extracted and purified from skin and tumor tissue according to manufacturer's instructions (RNeasy Protect-midi kit; Kiagen, Clifton Hill, Australia). Total RNA (4 μg) was reverse transcribed using (Oligo d (T ₁₅ )) and Superscript III (Life Technologies). PCR product intensity after gel electrophoresis and staining was measured linearly with respect to the number of cycles used, and the analysis appears to be semiquantitative. PCR reactions were performed using GeneAmp PCR System 9700 (Perkin Elmer, Norwalk, CT), DyNAZyme II DNA polymerase (Finzyme, Espoo, Finland) and Hot Star Taq DNA polymerase (Kiagen) as described above. (Mateo et al., Intervirology 43: 55-60, 2000). Primers were IL-1β, 5 'CAG GAT GAG GAC ATG AGC ACC [SEQ ID NO: 1], 3' CTC TGC AGA CTC AAA CTC CAC [SEQ ID NO: 2]; TNFα 5 'CCA GAC CCT CAC ACT CAG AT [SEQ ID NO: 3], 3' GGT AGA GAA TGG ATG AAC AC [SEQ ID NO: 4]; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5 'TGA AGG TCG GTG TGA ACG GAT TTG GC [SEQ ID NO: 5], 3' CAT GTA GGC CAT GAG GTC CAC CAC [SEQ ID NO: 6]; Macrophage inflammatory protein 2 (MIP-2) 5 ′ TCC AGA CTC CAG CCA CAC TTC AGC [SEQ ID NO: 7], 3 ′ TCT CAG ACA GCG AGG CAC ATC AGG [SEQ ID NO: 8].

Cytokine Production by PEP005-treated Human Cells

Me10538 cells, keratinocytes, fibroblasts (75% fusion in about 5 × 10 ⁵ cells per well in a 24 well plate) and neutrophils (10 ⁴ cells per well in a 96 well plate) were treated with PEP005 (1-100 ng / ml). ) Was incubated for 6 hours with or without. The supernatant was harvested and a multiphase detection kit (bio) for the presence of cytokines that were TNFα, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and GM-CSF Source International, Nibel, Belgium).

PEP005-induced activation of vascular endothelial

The adhesion of neutrophils to the vascular endothelium was measured using standard static adhesion assays (Butler et al., Exp Cell Res 310: 22-32, 2005). First-Pass Human Umbilical Vein Endothelial Cells (HUVECs) contain 20% v / v Fetal Bovine Serum (FBS) (Sigma, St. Louis, MO), 1 ng / ml Epidermal Growth Factor (Sigma, St. Louis, MO) ), 1 μg / ml hydrocortisone (Sigma, St. Louis, MO), 28 μg / ml gentamycin (David Bull Laboratories, Wick, UK) and 2.5 μg / ml amphotericin B (Sigma, USA Medium 199 (Life Technologies, Edinburgh, UK) supplemented with St. Louis, Missouri, was used to grow from glass cover slip to fusion in wells of 24 well plates. PEP005 was added to the culture wells at 1-100 ng / ml and TNFα was used as a positive control at 100 units / ml. Endothelial cells were exposed to TNFα or PEP005 for 4 hours and then thoroughly washed to remove PEP005 or TNFα. Human neutrophils (7.5 × 10 ⁵ / well) were then added to endothelial cells and allowed to adhere at 37 ° C. for 5 minutes. Cells were then washed, fixed in 2% w / v glutaraldehyde and examined by phase control microscopy to determine the number of neutrophils adhered to endothelial cell monolayers.

MM96L Cell Death by In Vitro Neutrophils

Human neutrophils were added to MM96L human melanoma cells (5,000 cells per 96 U-well plate, run 3 times) with or without 10 ng / ml PEP005. After 24 hours, the cultures were washed with PBS to remove neutrophils and PEP005 and then maintained in complete medium for an additional 6 days. Cells were washed with phosphate buffered saline (PBS), fixed in methanol, and the total protein of adherent MM96L cells was measured using sulfo-rhodamine B as described above (Skehan et al., J Natl Cancer Inst 82: 1107-1112, 1990). Cell viability is expressed as the percentage of total protein measured from wells containing melanoma cells only.

Lysogenic Media Release by In Vitro Neutrophils

Neutrophils (10 ⁷ ) were treated with 10 ng / ml PEP005 for 2 hours, and (i) peroxide measured using lucigenin assay (Gyllenhammar, J Immunol Methods 97: 209-213, 1987), (ii) commercially available. The release of soluble TRAIL measured by ELISA using a kit (BioSource International) and (iii) measured by ELISA using a commercial kit (Cell Sciences Inc., Canton, Mass., USA). The production of release of pencine 1-3 was evaluated.

Anticancer Antibody Measurement

B16 cells (10 ⁶ ) were injected subcutaneously into the flanks of 6-10 week old C57BL / 6 mice (animal resource center) (1 tumor / mouse) and when the tumors were about 30-60 mm 3, As well as PEP005. Serum was collected at Day 11 and Day 135 and analyzed by ELISA for antibody specificity of B16. Groups of animals with B16 not treated with PEP005 and naïve groups were included. B16 cells were sonicated in carbonate buffer (pH = 9), absorbed overnight in Immuno Maxisorp 96 well plates (Nunc) and dried. Plates were blocked with 0.01% v / v Tween, 5% v / v FBS in PBS for 1 hour at 37 ° C. The test serum was serially diluted twice, followed by rat anti-mouse biotinylated primary antibody (BD Biosciences Farmingen) and HRP-labeled streptavidin (BioSource International, Camarillo, CA) followed by ABTS Probe treated with substrate (Sigma) and OD measured at 405 nm.

In vitro neutrophil ADCC analysis

LK2 tumor (approximately 14 mm 3) is Foxnl ^nu Formed in mice (4 tumor sites / mouse) and treated with PEP005 as described above. On day 11 after the start of treatment, antiserum was collected by cardiac puncture and pooled. Parallel groups of mice were not treated, and serum was also collected on day 11, when tumors grew to about 50 mm 3. Serum from naïve animals was taken simultaneously (n = 3 per group). LK2 cells were seeded three times (4 × 10 ³ / well) in 96 round bottom wells and incubated with 1/3 dilution of antiserum (13 μl total volume) at 4 ° C. for 2 hours. Guinea pig complement (Gibco) (final dilution 1/10), PEP005 (final concentration 10 / ng / ml) or medium (control) with mouse neutrophils at 100: 1 effector: target ratio (final total volume 200 μl) Was added. The plate was kept at 4 ° C. and spun at 50 g for 5 minutes. Plates were then incubated with changing medium twice for 4 days at 37 ° C. and total cellular proteins of adherent LK2 cells were measured using crystal violet staining as described previously (Antalis et al., J. Exp. Ed . 187: 1799-1811, 1998). Mice neutrophils were generated as described above, except two injections of casein isolated from 18 hours prior to intraperitoneal lavage in mice and did not lyse red blood cells (Bergman et al., Cancer Immunol. Immunother). 49: 259-266, 2000).

result

Neutrophil recruitment and tissue morphology after PEP005 treatment

UV-induced mouse squamous cell carcinoma line, LK2, grew as a subcutaneous tumor in Foxnl ^nu mice. Histological examination of normal skin and tumor sites topically treated with placebo (isopropanol gel) using H & E staining revealed normal morphology with a small number of white blood cells present. However, the skin obtained 6 hours after topical application of PEP005 was shown to not only reduce the integrity of hair follicles and sebaceous glands, but also increased local vasodilation. Appropriate increase in neutrophil number was observed at the treated site. In addition, similar results were obtained at the PEP005-treated tumor sites. At 24 hours after treatment with PEP005, a large number of neutrophils and some macrophages were found in the treated skin and within and around the treated tumor site, with neutrophils surrounding the tumor. In addition, some bleeding of local vessels was apparent at this time. Higher magnification burns of the area surrounding the tumor mass clearly showed a number of cells with multi-lobal nuclei and red stained cytoplasm that are characteristic of neutrophils. Similar patterns of neutrophil infiltration were also observed when the above experiments were repeated using C57BL / 6 mice and B16 melanoma (data not shown). Therefore, the acute inflammatory response following PEP005 treatment is characterized by marked neutrophil infiltration.

As reported previously, the cosmetic effect after PEP005 treatment is very desirable. At about three weeks after PEP005 treatment, the skin at the treatment site is similar to untreated skin and has normal elasticity, and by 2 to 3 months there is very little any signs of scarring or erythema (Ogbourne et al., Cancer Res 64: 2833-2839, 2004).

Tumor Recurrence in Neutrophil-deficient Mice After Topical Chemotherapy with PEP005

It has been found that many of the various tumor types grown in Foxnl ^nu mice can be treated without significant recurrence, illustrating that T-cells are not needed for effective treatment (Ogbourne et al., Homology 2004). Neutrophils are fully active in Foxnl ^nu mice, and the importance for anticancer activity of PEP005 was investigated using anti-Ly-6G antibody (RB6-8C5) to deplete these cells in mice with LK2 tumors. Tumors grew only slightly slower in control animals (without antibodies) and animals that provided control antibodies (A95-1) compared to animals that provided anti-Ly-6G (RB6-8C5) antibodies.

In a separate experiment, LK2 tumors were formed in control animals, animals that provided a control antibody, and animals that provided an anti-Ly-6G antibody. Once the tumors were about 14 mm 3, PEP005 was topically treated daily for 3 days. Initial chemoablation of the tumors was evident in all groups, but after 25 days the tumors began to reappear in the anti-Ly-6G antibody treated group. Data from these experiments are presented as tumor recurrence rate in each group over time. In control animals, only 1/12 (8.3%) of tumors recurred, and in animals that provided a control antibody, no tumors recurred. However, a significant increase in relapse rate to 83% was observed in animals lacking neutrophils with anti-Ly-6G antibody (see below) (Cox regression analysis p = 0.005, Wald statistic = 7.92, versus anti-Ly-6G antibody versus For crude antibodies). These data suggest that neutrophils are needed to prevent relapse after PEP005 treatment.

Together with PEP005-treated animals, parallel groups of animals treated with placebo (instead of PEP005) were formed. Acute erythema was not present in all placebo-treated animals, but was apparent in all PEP005-treated tumor sites. However, on day 2 of peak erythema, the mice that received anti-Ly-6G antibody were about 50% lower than the control antibody-treated animals (p <0.001, Hall Student's t-test). Thus, neutrophil infiltration appears to contribute significantly to the PEP005-induced inflammatory response.

Neutrophil counts in blood were measured for each animal group. The leukocyte counts in placebo-treated mice, mice treated with PEP005 and mice treated with the control antibody are nearly identical. In contrast, the proportion of neutrophils in the blood was drastically reduced from about 80% of total leukocytes to about 3% after anti-Ly-6G antibody treatment in both placebo and PEP005 treated animals. Neutrophil counts returned to normal values about 10-12 days after the last antibody injection.

Rapid recurrence after PEP005 treatment of B16 tumors in CD18 deficient mice

Neutrophil extravasation into the inflamed area was significantly impaired in CD18 deficient mice (Schruefer et al., J Vasc Res 43: 1-11, 2005; Wilson et al., Hom. 1993; Arnaout, Immunol Rev 114: 145 -180, 1990), antibodies and some T-cell responses remained intact (Lee et al., Nat Med 9: 1281-1286, 2003). B16 tumors grew faster in CD18 deficient mice compared to C57BL / 6 mice, so PEP005 treatment initiated post tumor cell transplantation earlier in the former so that tumor size was comparable in CD18 deficient and C57BL / 6 mice. Although 100% of B16 tumors with an average volume of about 14 mm 3 were treated with three topical applications of PEP005 in C57BL / 6 mice, all tumors of similar size treated in CD18 deficient mice rapidly recurred following the same PEP005 treatment. Acute erythema is evident after PEP005 treatment, with much less intensity in the former animals, but the area is similar in CD18 deficient and wild type mice. Erythema in CD18 deficient mice was slightly reddened mainly in some mice, but very red in all treated sites in wild type mice.

Thus, in the second neutrophil defect model, PEP005 treated tumors recurred and the intensity of PEP005-induced infections decreased. Although multiple leukocyte interactions were affected by CD18 deficiency, these data support that an acute inflammatory response with predominant neutrophil infiltration is required for the antitumor efficacy of PEP005.

Role of NK Cells and Macrophages

NK cells and macrophages are present in Foxnl ^nu mice, and these cell types can be activated by PKC activating agents (Borrego et al., Immunology 97: 159-165, 1999; Martin and Edwards, J Immunol 150: 3478 -3486, 1993). To determine the potential contribution to the antitumor effect of PEP005, experiments were repeated using polyclonal anti-assial GM1 polyclonal antibodies to deplete NK cells. (Tumor grew 1.5 to 2 times faster in NK-deficient animals compared to control). Erythema and recurrence rate are not significantly different in these groups (p = 0.47). Thus, NK cells do not appear to play a significant role in preventing relapse after PEP005 treatment.

Experiments with B16 tumors were repeated in Csfm (op) / Csfm (op) (op / op) mice lacking functional macrophage-colon stimulating factors and thus severely reduced monocytes (Schonlau et al., J Leukoc Biol 73 : 564-573, 2003). PEP005 treatment can treat 100% of tumors without relapse, indicating that macrophages do not play an important role in preventing relapse after PEP005 treatment.

Recurrence of PEP005 Treated Tumors in SCID Mice

To determine whether successful PEP005 treatment without relapse requires B-cell and antibody production, LK2 tumors were grown in SCID mice, treated with PEP005, and monitored for relapse. The recurrence rate of PEP005-treated LK2 tumors grown in SCID mice was very similar to that seen in PEP005-treated LK2 tumors grown in granulocyte deficient Foxnl ^nu mice, with> 80% of tumors recurring within 40 days. Conversely, nearly all tumors were treated when PEP005 treated with similar size LK2 tumors grown in Foxnl ^nu mice (Cox regression analysis p = 0.006, Wald statistic = 7.68, for relapse rate in SCID for Foxnl ^nu mice). LK2 tumors grew in proportional numbers in SCID and Foxnl ^nu mice, and PEP005-induced erythema was similar in the two mouse strains. High relapse rates in SCID mice indicate that anti-cancer antibody production after PEP005 treatment is necessary to prevent relapse.

Induction of MIP-2, TNFα and IL-1β after PEP005 Treatment in Vivo

To determine whether the rapid inflammation induced by PEP005 is associated with the production of pro-inflammatory mediators, IL-6, in PEP005-treated tumor sites and PEP005-treated normal skin using RT-PCR KC / Groα, MIP-2, TNFα and IL-1β mRNA expression was evaluated. MRNA isolated from mouse skin of full thickness treated with PEP005 increased about 250-fold in MIP-2 mRNA, about 8-fold in TNFα and about 2-fold in IL-1β mRNA within 6 hours of treatment. Tumor tissue was isolated from percutaneous and dermal tissue and within about 6 hours of treatment increased about 17-fold in MIP-2 mRNA, about 2-fold in TNFα, and about 1.5-fold in IL-1β mRNA. No change was detected in IL-6 and KC / Groα transcription. Thus, in both skin and tumor tissues, in vivo PEP005 treatment induced MIP-2, TNFα, to a lesser extent IL-1β; All mediators are involved in neutrophil migration and activation (Ferrante, Immunol Ser 57: 417-436, 1992; Cataisson et al., J Immunol 174: 1686-1692, 2005).

Pro-inflammatory cytokine induction in human keratinocytes, fibroblasts, neutrophils and melanoma cells after in vitro PEP005 treatment

In vitro evaluation of cytokine and chemokine induction following PEP005 treatment of human keratinocytes, neutrophils and human melanoma cell lines (Me10538) to determine whether inflammatory mediator profiles induced in mice after PEP005 treatment were applied to human cells. It was. Culture medium was analyzed for cytokines and chemokines 6 hours after treatment with PEP005. IL-8, the human counterpart of MIP-2, was induced in all cells tested, and keratinocytes and neutrophils were produced at maximum levels at 5 ng / ml PEP005 (Table 8). The reduced response of neutrophils (relative to 5 ng / ml) at 10 ng / ml PEP005 is probably due to apoptosis, because PEP005 is an effective inducer of neutrophil activation (see below), which is activation-induced cell death. Results in. TNFα levels were induced about 60-fold in keratinocytes in the presence of 1 ng / ml PEP005 and IL-6 was induced slightly in fibroblasts after treatment with> 10 ng / ml PEP005 (Table 8). IL-2, IL-4, IL-10, IL-12 or GM-CSF was not detected in any cell type tested after PEP005 treatment. Thus, PEP005 induces the production of neutrophil chemoattractant IL-8 in keratinocytes, fibroblasts, neutrophils and tumor cells and potentially induces TNFα production in keratinocytes.

Activation of Human Endothelial Cells by PEP005

Recruitment of neutrophils to the site of inflammation requires activation of vascular endothelial to promote neutrophil binding, which is a prerequisite for extravascular outflow and tissue infiltration (Jutila et al., Transplantation 48: 721-731, 1989). PEP005 can activate vascular endothelial cells in a dose dependent manner, with significant neutrophil binding occurring at 10 ng / ml PEP005. TNFα was known to activate vascular endothelial and neutrophil binding induced by TNFα, which appeared as a positive control (FIG. 13A).

Direct Antitumor Activity of PEP005-Activated Neutrophils

To determine whether neutrophils recruited to PEP005 treated tumor sites had direct antitumor activity, human neutrophils were co-cultured with MM96L human melanoma cells in the presence and absence of PEP005 (10 ng / ml). Neutrophils and drugs were removed after 24 hours and cell cultured for an additional 6 days. In the absence of PEP005, neutrophils had no effect on the growth of MM96L cells. However, in the presence of PEP005, neutrophils can reduce the viability of MM96L cells to about 50% at 3: 1 neutrophil to target ratio and> 90% at 100: 1 neutrophil to target ratio (FIG. 13B). ).

The production of potential antitumor agents by PEP005-stimulated human neutrophils was investigated. PEP005 concentrations> 10 ng / ml can induce pronounced peroxide production by human neutrophils (FIG. 13C). At 100 ng / ml (but not 10 ng / ml) PEP005 induces the moderate release of defensin, a neutrophil granule protein reported to have certain anticancer activity (Lichtenstein et al., Cell Immunol 114: 104-116, 1988 ). Soluble TNF-associated apoptosis-inducing ligand (sTRAIL / Apo-2 ligand) (Tecchio et al., Blood 103: 3837-3844, 2004) production was not induced at PEP005 concentrations of 1-100 ng / ml. Thus, while PEP005 itself does not effectively induce neutrophil degranulation, PEP005 can induce the production of reactive oxygen species.

MM96L human melanoma cells are known to be sensitive to reactive oxygen species (Parsons et al., Cancer Res . 42: 3783-3788, 1982) and, when secreted by neutrophils, these mediators may be cytotoxic to tumor cells. (Lichtenstein, Blood 67: 657-665, 1986; Di Carlo et al., Chem Immunol Allergy 83: 182-203, 2003). To determine whether PEP005-induced reactive oxygen species mediate antitumor activity, human neutrophils are co-cultured with MM96L cells in the presence and absence of PEP005 (10 ng / ml). Neutrophils and drugs were removed after 24 hours and cells were cultured for an additional 6 days. In the absence of PEP005, neutrophils had no effect on the growth of MM96L cells. However, in the presence of PEP005 neutrophils can reduce the viability of MM96L cells by about 50% at a ratio of neutrophils to target of 3: 1 and> 90% at a ratio of neutrophils to target of 100: 1.

Increased Anticancer Antibody Levels After PEP005 Treatment of Tumors

Adoptive delivered anti-tumor antibodies are known to promote neutrophil-mediated ADCC in tumor cells (Hernandez-Ilizaliturri et al., Clin Cancer Res 9: 5866-5873, 2003; Niitsu et al., Clin Cancer Res 11: 697 -702, 2005; De Carlo et al., Homology 2003). To determine whether PEP005 treatment produced elevated antitumor antibody levels, B16 tumors growing in C57BL / 6 mice were treated with PEP005. At day 11 of post-PEP005 treatment of 30-60 mm 3 tumors, anti-cancer antibody levels were increased and the response exceeded significantly at this point than in untreated animals with large tumor loads (FIG. 13D) [1/100 dilution] P = 0.045, Hall Student's t-test]. Thus, PEP005-mediated treatment of cancer appears to increase the levels of anticancer antibodies.

Mouse Neutrophil-Mediated ADCC of LK2 Cells in Vitro

The data suggest that tumor recurrence after PEP005 treatment of LK2 tumors in Foxnl ^nu mice is prevented by neutrophil-mediated ADCC. Foxnl ^nu mice cannot produce significant IgG responses, but they produce IgM responses that can fix complement, thus initiating neutrophil degranulation by the complement receptor (Nielsen et al., Eur. J. Immunol . 27: 2914-2919, 1997). Serum from Foxnl ^nu mice treated with LK2 tumor with PEP005 was intended to determine whether ADCC could be mediated in vitro. Anti-serum from such mice can significantly reduce the viability of LK2 cells (p = 0.006, Hall Student's t-test) in the presence of mouse neutrophils and added complement. Anti-serum from Foxnl ^nu mice not treated with LK2 tumors or anti-serum from naïve Foxnl ^nu mice cannot significantly reduce the viability of LK2 cells under the same conditions. Lower levels of complement present in mouse serum (not heat inactivated) are insufficient to activate anticancer activity of neutrophils under these assay conditions. However, when 10 ng / ml PEP005 was added, anticancer activity was again shown for PEP005 LK2 anti-serum, but not in LK2 or naïve serum. In the absence of PEP005 or added complement, no antiserum can significantly reduce the viability of LK2 cells. Thus, anti-serum from Foxnl ^nu mice that healed LK2 tumors with PEP005 treatment can induce neutrophil-mediated ADCC of LK2 cells in vitro in the presence of added complement or low levels of PEP005.

Example 15

Enhanced immune response with PEP005

The anti-apoptotic effect of PEP005 on effector T-cells represents a potential use for the use of PEP005 to enhance vaccine response to tumors or T-cell responses in immunotherapy. The purpose of this experiment is to further understand this observation and to determine the potential use of systemic PEP005 as a T-cell immunopotentiating agent.

Experimental plan :

The lifetime of PEP005 treatment effect on CD8 T-cell survival in vitro is measured.

Investigate whether the T-cell subset is mainly affected by PEP005 (ie is PEP005 active during the enlarger or effector phase).

Test the effect of PEP on killing of target cells by CTL.

There are four purposes of this embodiment:

1) to test if PEP005 can inhibit apoptosis of CD8 ⁺ activated T-cells;

2) to investigate the efficacy and duration of the anti-apoptotic effect of PEP005;

3) to test for differences in the effect of PEP005 on a subset of CD8 ⁺ T-cells; And

4) To test how PEP005 affects the cytotoxicity of CD8 ⁺ T-cells.

In vitro assays are used to test the effect of PEP005 on cytokine atrophy-induced apoptosis of CD8 ⁺ T-cells. Then, a comparison of the time course of the effect of PEP005 on T-cell apoptosis, as well as the effect on T-cells derived from isolated naïve and memory CD8 ⁺ T-cells. For this purpose, cytotoxicity assays using T-cell clones and antigen-targeted target cells were used.

Dry stored at −70 ° C., divided by weight into 1-2 mg aliquots, and PEP005 reconstituted in DMSO was used at an intensity of 0.0001 to 10 μg / ml. A single dose into the in vitro system was used.

CD8 ⁺ T-cell lines were generated by isolation of CD8 ⁺ T-cells from the peripheral blood of healthy human donors. Such cells are dependent on the presence of anti-apoptotic cytokines such as IL2. If they are absent, they quickly die. In the assay system used, cells are cultured in the absence and presence of IL-2 using various concentrations of PEP005 to test the effect on apoptosis.

CD8 ⁺ T-cells were isolated by negative selection using a MACS based system. Purified cells were stimulated with PHA at 10 μg / ml and the irradiated EBV transformed B-cells were used to provide costimulatory signals. Cells maintained in the presence of 25 U / ml IL2 were re-stimulated at 7 day intervals and frozen in aliquot with passage 3. For each experiment it was obtained from liquid nitrogen, reconstituted and used in the survival assay on day 7 to maintain a reproducible cell source. Cells were incubated at a density of 1 × 10 ⁶ / ml with or without IL2 (25 U / well) for survival analysis and treated with 0.01-1 μg / ml PEP005 for 48 hours. The percentage of apoptotic cells was determined by flow cytometry detection of activated caspase-3 as a marker for apoptosis. The experiment was conducted three times.

Cell proliferation detection by analysis of bromodeoxyuridine (BrdU) incorporation was linked to survival analysis. BrdU is an analog of thymidine and can be incorporated into DNA when the cell is in the S (synthetic) phase of the cell cycle. Using fluorochrome-conjugate antibodies that recognize BrdU-incorporated DNA epitopes, flow cytometry can be used to monitor the proportion of cells circulating at different time points during a survival assay, which typically takes two days.

Pass-through 5 (day 7 post-stimulation) CD8 ⁺ T-cells separated by Ficoll-Paque were washed twice in RPMI 1640 and incubated at 2 × 10 ⁶ / ml in 96-well plates in the same manner as the survival assay set. It was. Cells were given in PEP005 or medium at 0.01 μg / ml (20 nM). At 0, 24 hours and 48 hours from the start of each experiment, each well of cells was pulsed at 37 ° C. for 2 hours with 10 μM BrdU. Cells were then washed twice and resuspended in medium in the presence / absence of IL2 and PEP005 (hereinafter referred to as supplemental medium) corresponding to the original culture conditions. Supplemental media was generated at the beginning of each experiment and incubated concurrently with cell culture. After BrdU-wave treatment at 48 hours, cells are resuspended, washed once in PBS, and subjected to BrdU and active caspase-3 using the BrdU flow kit (BD Biosciences, UK) according to the manufacturer's instructions Stained. FACS data were analyzed using CellQuest Pro (BD Biosciences, UK).

At the start of the CTL assay, medium containing various concentrations of PEP005 diluted in RPMI 1640 with 5% CO ₂ , 10% HIFCS at 37 ° C. for 30 minutes in a 15 ml test tube (RPMI 1640 + 10% heat inactivated ( HI) Fetal Bovine Serum (HIFCS)) was resuspended effector CD8 ⁺ T-cells at 1 × 10 ⁶ cells / ml. During PEP005-treatment of effector cells, lymphoblast cell line (LCL) target cells were labeled with 5 μCi Na ⁵¹ CrO ₄ , sensitized with latent or lytic EBV antigenic peptides dissolved in DMSO, and 1.5 hours at RPMI 1640. Dilute with 5% CO ₂ at 37 ° C .; Dilution of DMSO solvent was used as a control. PEP005-treated effector cells were once washed in RPMI 1640, resuspended in RPMI + 10% HIFCS, diluted to various effector / target ratios and transferred to 96 V-well assay plates. ⁵¹ Cr-labeled peptide sensitized target cells were washed twice with RPMI 1640, resuspended in RPMI + 10% HIFCS and transferred to 96 V-well assay plates containing effector cells at 2,500 cells / well. In addition, target cells were cultured either alone or in medium with 1% w / v sodium dodecyl (SDS) for spontaneous lysis and maximal lysis control. The plates were then incubated at 37 ° C. for 5% CO ₂ , 4 hours. After 4 hours of incubation, the plates were centrifuged at 1,300 rpm for 5 minutes to pellet the cells. 100 μl of each well was transferred to a fluorescent activated cell sorting (FACS) test tube and radioactivity was measured. Specific targets were used to calculate the specific dissolution rate of the sample:

The control and test three counts were averaged and compared using Student's t-test.

One of the aims of this experiment was to test whether apoptosis of activated CD8 ⁺ T-cells was inhibited. Activated CD8 ⁺ T-cells normally die in the absence of survival-inducing cytokines. This is indicated by flow cytometry analysis of caspase-3 staining of cells in survival assays. The effect of PEP005 on CD8 ⁺ T-cell survival was titrated using various concentrations of PEP005 for Day 5 and 7 post-stimulated CD8 ⁺ T-cells. Increasing concentrations of PEP005 significantly reduced the rate of apoptosis of IL-2-deficient CD8 ⁺ T-cells (FIG. 14B, white bars). Apoptosis rate of IL2-deficient CD8 ⁺ T-cells was reduced to at least 19% with PEP005 at 0.01 μg / ml compared with 42.47% in untreated cells.

From the results of the survival assay, PEP005 was observed to induce a small degree of apoptosis in CD8 ⁺ T-cells treated with IL-2 (FIGS. 14A and 14B). Since IL-2 has a direct effect on stimulating proliferation, it was investigated whether cell proliferation induced by IL-2 was induced and apoptosis by PEP005. The costaining technique of BrdU and active caspase-3 allows tracking whether apoptotic cells found on day 2 are already proliferating at any given time in the course of a survival assay. The proportion of apoptotic cells proliferated at different time points in the total proliferating population is shown in FIG. 15 for different treatments and the non-proliferating counterparts are shown in FIG. 15D.

The degree of apoptosis of both non-proliferating and proliferating cells not treated with IL-2 is as high as about 75%. The proportion of non-proliferating apoptotic cells (FIG. 15D) and proliferating cells (FIG. 15C) among the cells treated with PEP005 experienced apoptosis in similar proportions. These data suggest that high apoptosis in cells treated with both IL-2 and PEP005 shown in previous survival assays is not due to proliferation caused by IL2 in this system itself.

In most experiments, the rate of apoptosis in the survival assay was analyzed on day two. However, in order to assess the duration of the anti-apoptotic effect, one must examine how long the survival improvement implemented by PEP005 can last in a survival assay. To study this goal, survival assays were extended to day 7 and apoptosis rates were measured daily using caspase-3 staining. Apoptosis of cells cultured with IL-2, PEP005 or a combination thereof was significantly delayed by 2-3 days. This observation is similar to that done with other surviving enhanced cytokines in the group.

Prior literature suggests that the PKC activator PMA can increase CD8 ⁺ T-cell cytotoxicity under suboptimal conditions, including cytotoxicity to target cells with or without antigen-specificity (Russell, J.). Immunol. 136 (1): 23-7, 1986). We tested whether PEP005 shares this effect with PMA. To this end, cytotoxicity assays used antigen-specific CD8 ⁺ T-cell clones (SY30C23, IM106 and SY3 / 2) and peptide wave treated LCL cell lines. After titrating the decreasing concentration of HLA-limited EBV antigenic peptide added to target cells, the effect of PEP005 at various concentrations on the cytotoxicity of CD8 ⁺ T-cells was tested.

Peptide addition concentrations at 5 μg / ml allow untreated effector cells to provide optimal specific lysis of target cells. When 1 μg / ml peptide is used for target cell sensitization, PMA-treated CD8 ⁺ T-cell clones show higher cytotoxicity compared to untreated cells, and this observation shows that PMA is only cytotoxic under suboptimal conditions. This confirms the consideration in the conventional literature that it can increase. At 0.1 μg / ml peptide, there is only a low degree of cytotoxicity under all conditions. Concentrations of PEP005 in the range of 2 nM to 2 μM do not affect the cytotoxicity of CD8 ⁺ clones.

The data show that PEP005 extends the lifespan of both CD4 ⁺ and CD8 ⁺ activated T-cells. The anti-apoptotic effect acted to the same extent on proliferating and non-proliferating cells. No increase or decrease in cytotoxic activity was detected. Therefore, the conclusion from in vitro work is that increased in vivo antitumor activity is due to the anti-apoptotic effect of PEP005 rather than an increase in cytotoxicity.

Those skilled in the art will appreciate that modifications and variations of the invention described herein are possible in addition to those specifically described. It is to be understood that the present invention includes all such modifications and variations. In addition, the present invention includes all steps, features, compositions and compounds, and any and all combinations of any two or more of the above steps or features, individually or collectively referred to or indicated herein.

Bibliography

Antalis et al., J. Exp. Med . 187: 1799-1811, 1998

Arnaout, Immunol Rev 114: 145-180, 1990

Bergman et al., Cancer Immunol. Immunother . 49: 259-266, 2000

Borrego et al., Immunology 97: 159-165, 1999

Butler et al., Exp Cell Res 310: 22-32, 2005

Cataisson et al., J Immunol 174: 1686-1692, 2005

Cavanagh and Halliday, Cancer Res 56: 2607-2615, 1996

Chou and Talalay, Adv Enzyme Regul 22: 27-55, 1984

Chothia et al., J. Mol Biol . 196: 901, 1987

Chou et al., US Patent No. 6,056,957

Coligan et al., Current Protocols in Immunology , 1991-1997

de Boer and Roos, J Immunol 136: 3447-3454, 1986

Dengler et al., Anticancer Drugs 6: 522-532, 1995

Di Carlo et al., Chem Immunol Allergy 83: 182-203, 2003

European Patent Publication No. 0 239 400

Ferrante, Immunol Ser 57: 417-436, 1992

Gefter et al., Somatic Cell Genet . 3: 231-236,1977

Gyllenhammar, J Immunol Methods 97: 209-213, 1987

Hansen et al., J Immunol. Methods 119 (2): 203-210, 1989

Hernandez-Ilizaliturri et al., Clin Cancer Res 9: 5866-5873, 2003

Hotchin and Watt, J Biol Chem 267: 14852-14858, 1992

Jain, J Natl Cancer Inst 81: 570, 1989

Jain, Science 271: 1079, 1996

Jones et al., Nature 321: 522-525, 1986

Jutila et al., Transplantation 48: 727-731, 1989

Kim and Lim, Arch Pharm Res 25: 229, 2002

Kohler et al., Nature 256: 495-499, 1975

Kohler et al., Eur. J. Immunol . 6 (7): 511-519, 1976

Kozbor et al., Methods in Enzymology 121: 140, 1986

Larizza et al., Clin Exp Metastasis 7: 633-644, 1989

Lee et al., Nat Med 9: 1281-1286, 2003

Lichtenstein, Blood 57: 657-665, 1986

Lichtenstein et al., Cell Immunol 114: 104-116, 1988

Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987

Lozzio and Lozzio, Blood 45: 321-334, 1975

Martin and Edwards, J Immunol 150: 3478-3486, 1993

Mateo et al., Intervirology 43: 55-60, 2000

Morgan et al., US Patent No. 6,180,377

Mosmann, J. Immunol. Methods 65 (1-2): 55-63, 1983

Nielsen et al., Eur. J. Immunol . 27: 2914-2919, 1997

Niitsu et al., Clin Cancer Res 11: 697-702, 2005

Ogbourne et al., Cancer Res 64: 2833-2839, 2004

Parsonage et al., Thromb Haemost 90: 688-697, 2003

Parsons et al., Cancer Res . 42: 3783-3788, 1982

Parsons and Hayward, Photochem Photobiol 42: 287-293, 1985

Queen et al., US Patent No. 6,180,377

Richmann et al., Nature 332: 323-327, 1988

Russell, J Immunol . 136 (1): 23-7, 1986

Schonlau et al., J Leukoc Biol 73: 564-573, 2003

Schruefer et al., J Vasc Res 43: 1-11, 2005

Shulman et al., Nature 276: 269-270, 1978

Skehan et al., J Natl Cancer Inst 82: 1107-1112, 1990

Sundstrom and Nilsson, Int. J. Cancer 17: 565-571, 1976

Swartz et al., J Biomech 32: 1297, 1999

Tecchio et al., Blood 103: 3837-3844, 2004

Toyama et al., Monoclonal Antibody, Experiment Manual , published by Kodansha Scientific, 1987

Trowbridge, J Exp. Med . 148 (1): 220-227, 1982

Verhoeyen et al., Science 239: 1534-1536, 1988

Volk et al., J Virol 42 (1): 220-227, 1982

Wilson et al., J Immunol 151: 1571-1578, 1993

                         SEQUENCE LISTING <110> PEPLIN RESEARCH PTY LTD        SUHBRIER, Andreas (US ONLY)        AYLWARD, Jim (US ONLY)        PARSONS, Peter (US ONLY)        OGBOURNE, Steven (US ONLY)        LORD, Janet (US ONLY)        SCHEEL-TOELLNER, Dagmar (US ONLY)   <120> Therapeutic formulations <130> 30127093 / EJH <150> AU 2005906295 <151> 2005-11-14 <160> 8 <170> PatentIn version 3.2 <210> 1 <211> 21 <212> DNA <213> murine <400> 1 caggatgagg acatgagcac c 21 <210> 2 <211> 21 <212> DNA <213> murine <400> 2 ctctgcagac tcaaactcca c 21 <210> 3 <211> 20 <212> DNA <213> murine <400> 3 ccagaccctc acactcagat 20 <210> 4 <211> 20 <212> DNA <213> murine <400> 4 ggtagagaat ggatgaacac 20 <210> 5 <211> 26 <212> DNA <213> murine <400> 5 tgaaggtcgg tgtgaacgga tttggc 26 <210> 6 <211> 24 <212> DNA <213> murine <400> 6 catgtaggcc atgaggtcca ccac 24 <210> 7 <211> 24 <212> DNA <213> murine <400> 7 tccagactcc agccacactt cagc 24 <210> 8 <211> 24 <212> DNA <213> murine <400> 8 tctcagacag cgaggcacat cagg 24  

Claims (60)

A treatment protocol for treating an individual with cancer or a suspected cancer Administering to said individual an angeloyl-substituted ingenan, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and The subject has the following characteristics: (i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells, (ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells, (iii) exhibit the properties of, or be capable of producing, an anticancer cell antibody, (iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;  Administering an agent exhibiting at least one of Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone. The method of claim 1, wherein the incorporation of ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof with the agent results in a CI of less than 1 when the merging index (CI) is determined by the formula Treatment protocol: CI = [(C) ₁ / (C _x ) ₁ ] + [(C) ₂ / (C _x ) ₂ ] + [α (C) ₁ (C) ₂ / (C _x ) ₁ (C _x ) ₂ ] Wherein (C _x ) ₁ is the concentration of one of the ingenol angelates or agents that produces the x% effect of the drug alone, and (C) ₁ is the other of the ingenol angelates or agents (C) _Is the concentration of one of the ingenol angelates or agents that, in combination with ₂ , produces the same x% effect, and α is a constant if the modes of anticancer activity of the ingenol angelates and agents are mutually exclusive or non-excluded and α are 0 or 1, respectively. The therapeutic protocol of claim 1 or 2, wherein the ingenol angelate is ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof. The treatment protocol of claim 3, wherein the ingenol angelate is ingenol-3-angelate. The treatment protocol of claim 4, wherein the ingenol- 3- angelate is obtainable from Euphorbia peplus . The therapeutic protocol of claim 4, wherein the ingenol-3-angelate is produced by chemical synthetic means. The treatment protocol of claim 1, wherein the agent is selected from those set forth in Table 1. 8. The treatment protocol of claim 1, wherein the agent is an anticancer chemotherapeutic agent. The therapeutic protocol of claim 7 wherein the agent is cytotoxic T-cells or capable of stimulating or activating cytotoxic T-cells against cancer cells. 8. The therapeutic protocol of claim 7, wherein the agent is an anticancer cell antibody or can stimulate the production of anticancer cell antibodies. The treatment protocol of claim 10, wherein the agent is an anti-cancer cell antigen vaccine. The therapeutic protocol of claim 10, wherein the agent mediates antibody-dependent cellular cytotoxicity of cancer cells. The therapeutic protocol of claim 12, wherein the agent comprises neutrophils or can stimulate or activate neutrophils. The treatment protocol of claim 1, wherein the subject is a human. The cancer according to claim 1 or 14, wherein the cancer is ABL1 oncogene, AIDS-related cancer, auditory neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic cancer, adrenal cortex cancer, idiopathic myeloid metaplasia, alopecia, and bony soft tissue. Sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, capillary ataxia, basal cell carcinoma (skin), bladder cancer, bone cancer, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, breast cancer , Cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, ridged skin fibrosarcoma, connective tissue formation Bovine-circular-cell-tumor, catheter cancer, endocrine cancer, endometrial cancer, cerebral hepatocellular carcinoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, FA. , Gallbladder cancer, stomach cancer, Intestinal cancer, gastrointestinal-carcinoma, genitourinary cancer, germ cell tumor, gestational-nutrient membrane-disease, glioma, gynecological cancer, blood tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, Human papillomavirus, respiratory morphology, hypercalcemia, hypopharyngeal cancer, intraocular melanoma, islet cancer, Kaposi's sarcoma, kidney cancer, Langerhans' cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeny syndrome, cleft lipoma, liposarcoma, Liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant hepatic tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, complex endocrine neoplasia, Fungal carcinomas, myelodysplastic syndromes, myeloma, myeloproliferative diseases, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nibegain injury syndrome, non-melanoma skin cancer (NMSC), non-small bovine Cell lung cancer (NSCLC), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, true polycythemia, prostate Cancer, rare cancers and related diseases, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rosmond-Thompson syndrome, salivary adenocarcinoma, sarcoma, Schwanno, sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, Soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell cancer (bladder), transitional cell cancer (kidney-pelvic-ureter), trophoblastic cancer, urethral cancer, urinary The treatment protocol is selected from the group consisting of mechanical cancer, europlakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor. A treatment protocol for treating an individual with cancer or a suspected cancer Administering to the individual ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof; and The subject has the following characteristics: (i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells, (ii) mediate T-cell mediated inhibition or death of cancer cells or be capable of stimulating or activating such T-cells, (iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies, (iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;  Administering an agent exhibiting at least one of Wherein the anticancer activity or efficacy of the combination of ingenol-3-angelate and the agent is greater than ingenol-3-angelate or the agent alone. The treatment protocol of claim 16, wherein the agent is selected from those set forth in Table 1. 17. The treatment protocol of claim 17, wherein the agent is an anticancer chemotherapeutic agent. The therapeutic protocol of claim 17, wherein the agent is cytotoxic T-cells or capable of stimulating or activating cytotoxic T-cells against cancer cells. The therapeutic protocol of claim 17, wherein the agent is an anticancer cell antibody or can stimulate the production of anticancer cell antibodies. The therapeutic protocol of claim 20, wherein the agent is an anti-cancer cell antigen vaccine. The therapeutic protocol of claim 20, wherein the agent mediates antibody-dependent cellular cytotoxicity of cancer cells. The therapeutic protocol of claim 22, wherein the formulation comprises neutrophils or can stimulate or activate neutrophils. The treatment protocol of claim 16, wherein the subject is a human. The cancer according to claim 16 or 24, wherein the cancer is ABL1 oncogene, AIDS-related cancer, auditory neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic cancer, adrenal cortex cancer, idiopathic myeloid metaplasia, alopecia, cellulitis Sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, capillary ataxia, basal cell carcinoma (skin), bladder cancer, bone cancer, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, breast cancer , Cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, ridged skin fibrosarcoma, connective tissue formation Bovine-circular-cell-tumor, catheter cancer, endocrine cancer, endometrial cancer, cerebral hepatocellular carcinoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, FA. , Gallbladder cancer, stomach cancer, Gastrointestinal cancer, gastrointestinal-carcinoma, genitourinary cancer, germ cell tumor, gestational-nutrient membrane-disease, glioma, gynecological cancer, blood tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's Illness, human papilloma virus, respiratory disease, hypercalcemia, hypopharyngeal cancer, intraocular melanoma, islet cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumen's syndrome, cleft lip cancer, fat Sarcoma, Liver Cancer, Lung Cancer, Lymphedema, Lymphoma, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Male Breast Cancer, Malignant Hepatic Tumor of the Kidney, Medulloblastoma, Melanoma, Merkel Cell Cancer, Mesothelioma, Metastatic Cancer, Oral Cancer, Complex Endocrine Renal Organism, mycelial sarcoma, myelodysplastic syndrome, myeloma, myeloproliferative disease, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nibegain injury syndrome, non-melanoma skin cancer (NMSC), non- Cell lung cancer (NSCLC), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, true polycythemia, prostate Cancer, Rare Cancers and Related Diseases, Renal Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Rosmond-Thompson Syndrome, Salivary Adenocarcinoma, Sarcoma, Schwanoma, Sedentary Syndrome, Skin Cancer, Small Cell Lung Cancer (SCLC), Small Intestine Cancer, Soft Tissue Sarcoma, Spinal Cord Tumors, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell cancer (bladder), transitional cell cancer (kidney-pelvic-ureter), trophoblastic cancer, urethral cancer, urinary tract cancer, euro A treatment protocol selected from the group consisting of plakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor. As a method of treating an individual with or suspected of having cancer, Administering to said individual an angeloyl-substituted ingenan, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and The subject has the following characteristics: (i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells, (ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells, (iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies, (iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;  Administering an agent exhibiting at least one of Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone. The method of claim 26, wherein the formulation is selected from those set forth in Table 1. The method of claim 27, wherein the agent is an anticancer chemotherapeutic agent. The method of claim 27, wherein the agent is cytotoxic T-cells or capable of stimulating or activating cytotoxic T-cells against cancer cells. The method of claim 27, wherein the agent is capable of stimulating the production of anticancer cell antibodies or anticancer cell antibodies. The method of claim 30, wherein the agent is an anticancer cell antigen vaccine. The method of claim 30, wherein the agent mediates antibody-dependent cellular cytotoxicity of cancer cells. The method of claim 32, wherein the agent comprises neutrophils or can stimulate or activate neutrophils. The method of claim 26, wherein the subject is a human. 35. The method of claim 26 or 34, wherein the cancer is an ABL1 proto-oncogene, AIDS-related cancer, auditory neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adrenal cortex, idiopathic myeloid metaplasia, alopecia, cellulitis Sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, capillary ataxia, basal cell carcinoma (skin), bladder cancer, bone cancer, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, breast cancer , Cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, ridged skin fibrosarcoma, connective tissue formation Bovine-circular-cell-tumor, catheter cancer, endocrine cancer, endometrial cancer, cerebral hepatocellular carcinoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, FA. , Gallbladder cancer, stomach cancer, Gastrointestinal cancer, gastrointestinal-carcinoma, genitourinary cancer, germ cell tumor, gestational-nutrition membrane-disease, glioma, gynecological cancer, blood tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease , Human papillomavirus, Reptile status, Hypercalcemia, Hypopharyngeal cancer, Guided melanoma, Islet cancer, Kaposi's sarcoma, Kidney cancer, Langerhan's cell histiocytosis, Laryngeal cancer, Leiomyosarcoma, Leukemia, Li-prameni syndrome, Cleft lipoma, Liposarcoma , Liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant hepatic tumor of kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, complex endocrine neoplasia , Fungal carcinoma, Myelodysplastic syndrome, Myeloma, Myeloproliferative disease, Non-cancer, Nasopharyngeal cancer, Kidney blastoma, Neuroblastoma, Neurofibromatosis, Nivegein damage syndrome, Non-melanoma skin cancer (NMSC), Non- Cell lung cancer (NSCLC), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, true polycythemia, prostate Cancer, Rare Cancers and Related Diseases, Renal Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Rosmond-Thompson Syndrome, Salivary Adenocarcinoma, Sarcoma, Schwanoma, Sedentary Syndrome, Skin Cancer, Small Cell Lung Cancer (SCLC), Small Intestine Cancer, Soft Tissue Sarcoma, Spinal Cord Tumors, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell cancer (bladder), transitional cell cancer (kidney-pelvic-ureter), trophoblastic cancer, urethral cancer, urinary tract cancer, Europlakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor. Having a first portion comprising ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof, and a second portion comprising an agent or a pharmaceutically acceptable salt thereof, wherein the portion is optionally 1 It further comprises a pharmaceutically acceptable carrier, diluent and / or excipient, wherein the formulation has the following properties: (i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells, (ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells, (iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies, (iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;  Wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone. Ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analogue thereof and agent or pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents and / or excipients, wherein the agent has the following properties field: (i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells, (ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells, (iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies, (iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;  Wherein the anticancer activity or efficacy of the combination of ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone. 38. The combination of claim 36 or 37, wherein the ingenol angelate is ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof. The combination of claim 38, wherein the ingenol angelate is ingenol-3-angelate. 40. The combination of claim 38 or 39, wherein ingenol- 3- angelate is obtainable from Euphorbia peplus . 40. The combination according to claim 38 or 39, wherein the ingenol-3-angelate is produced by chemical synthesis means. 38. The combination according to claim 36 or 37, wherein the formulation is selected from Table 1. The combination of claim 42, wherein the agent is an anticancer chemotherapeutic agent. The combination of claim 42, wherein the agent is a cytotoxic T-cell or capable of stimulating or activating cytotoxic T-cells against cancer cells. The combination of claim 42, wherein the agent is an anticancer cell antibody or can stimulate the production of anticancer cell antibodies. 46. The combination of claim 45, wherein the agent is an anticancer cell antigen vaccine. The combination of claim 45, wherein the agent mediates antibody-dependent cellular cytotoxicity of cancer cells. 48. The combination of claim 47, wherein the formulation comprises neutrophils or can stimulate or activate neutrophils.  As the use of ingenol angelate, or a pharmaceutically acceptable salt, derivative, homologue or analogue thereof and preparation or pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer,  The formulation has the following properties: (i) induces apoptosis, necrosis, senescence, cytotoxicity and / or cell cycle arrest of cancer cells, (ii) mediate T-cell mediated inhibition or death of cancer cells or that can stimulate or activate such T-cells, (iii) exhibit the properties of, or be capable of producing, anticancer cell antibodies, (iv) the ability to mediate or produce neutrophil-promoted antibody-dependent cellular cytotoxicity of cancer cells;  Or at least one of wherein the anticancer activity or efficacy of the combination of the angeloyl-substituted ingenan and the agent is greater than the angeloyl-substituted ingenan or the agent alone. The use of claim 49, wherein the ingenol angelate is ingenol-3-angelate, or a pharmaceutically acceptable salt, derivative, homologue or analog thereof. 51. The use of claim 50, wherein the ingenol angelate is ingenol-3-angelate. 52. The use of claim 50 or 51, wherein ingenol- 3- angelate is obtainable from Euphorbia peplus . 52. The use of claim 50 or 51, wherein the ingenol-3-angelate is produced by chemical synthesis means. The use of claim 49, wherein the formulation is selected from Table 1. 55. The use of claim 54, wherein the agent is an anticancer chemotherapeutic agent. 55. The use of claim 54, wherein the agent is a cytotoxic T-cell or capable of stimulating or activating cytotoxic T-cells against cancer cells. 55. The use of claim 54, wherein the agent is an anticancer cell antibody or can stimulate the production of anticancer cell antibodies. 58. The use of claim 57, wherein the agent is an anticancer cell antigen vaccine. The use of claim 57, wherein the agent mediates antibody-dependent cellular cytotoxicity of cancer cells. 60. The use of claim 59, wherein the formulation comprises neutrophils or can stimulate or activate neutrophils.

Images