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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/222—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/739—Lipopolysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2006—IL-1
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to a method of treating cancer and to compositions of use in such a method.
• DMXAA 5,6-dimethylxanthenone-4-acetic acid
• TNF cytokine tumour necrosis factor
• DMXAA has shown evidence of marginal clinical anti-cancer activity in humans.
• DMXAA amplifies the induction of TNF by cultured human peripheral blood cells in response to a variety of agents capable of inducing a second signal that by itself modulates TNF production.
• agents capable of inducing a second signal that by itself modulates TNF production include ligands that occupy external cellular receptors connected with the TNF induction pathway and compounds that modulate cellular biochemical pathways connected to TNF induction.
• the present invention provides a method of treating cancer, the method including the step of administering to a mammal in need of such treatment, either simultaneously or sequentially:
• Ri , R 2 and R 3 are each independently selected from the group consisting of H, -C ⁇ alkyl, halogen, CF 3 , CN, NO 2 , NH 2 , OH, OR, NHCOR, NHSO 2 R, SR, SO 2 R or NHR, wherein each R is independently C t -C ⁇ alkyl optionally substituted with one or more substituents selected from hydroxy, amino and methoxy, and wherein each of R l5 R 2 and R 3 may be present at any of the available positions 1 to 8;
• the mammal is a human.
• the compound (ii) is a ligand that binds to the CD14 receptor of cells, such as bacterial LPS, deacylated LPS and CD14 receptor antibodies.
• the compound (ii) is a ligand that binds to a surface receptor of cells connected with TNF production other than the CD 14 receptor, such as interleukin-1 alpha.
• the compound (ii) is a compound that induces protein kinase C, such as phorbol myristate ester.
• the compound (ii) is a compound that can decrease the activity of protein phosphatases, preferably protein phosphatase 2A, such as okadaic acid.
• compound (i) is of the formula (la):
• the compound of formula (I) or (la) is 5,6-dimethylxanthenone-4- acetic acid, having the formula
• the present invention provides the use of a compound (i) of the formula (I) as defined above, or a pharmaceutically acceptable salt or ester thereof, in the manufacture of a medicament for treating cancer in a mammal by sequential or simultaneous co-administration of the medicament and a compound (ii) selected from compounds which modulate TNF production and compounds which act on biochemical pathways leading to TNF synthesis.
• the compound (i) is DMXAA.
• compound (ii) is selected from a ligand that binds to the CD 14 receptor of cells; a ligand that binds to a surface receptor of cells connected with TNF production other than the CD 14 receptor; a compound that induces protein kinase C; or a compound that can decrease the activity of protein phosphatases.
• the present invention provides the use of a compound (ii) selected from compounds which modulate TNF production and compounds which act on biochemical pathways leading to TNF synthesis, in the manufacture of a medicament for treating cancer in a mammal by sequential or simultaneous co- administration of the medicament and a compound (i) of the formula (I) as defined above, or a pharmaceutically acceptable salt or ester thereof.
• the compound (i) is DMXAA.
• compound (ii) is selected from a ligand that binds to the CD 14 receptor of cells; a ligand that binds to a surface receptor of cells connected with TNF production other than the CD 14 receptor; a compound that induces protein kinase C; or a compound that can decrease the activity of protein phosphatases.
• the present invention provides a pharmaceutical composition suitable for treating cancer, the composition including a compound (i) of the formula
• the compound (i) is DMXAA.
• compound (ii) is selected from a ligand that binds to the CD 14 receptor of cells; a ligand that binds to a surface receptor of cells connected with TNF production other than the CD 14 receptor; a compound that induces protein kinase C; or a compound that can decrease the activity of protein phosphatases.
• composition is formulated for co-administration of compounds (i) and (ii), or is formulated for sequential administration of compounds (i) and (ii) in any order.
• Figure 1 shows the effect of DMXAA on LPS-induced TNF production in HPBL in vitro.
• HPBL were incubated (8 h) with the indicated concentrations of LPS alone (no shading) or in combination with DMXAA (shading). Supernatants were then removed and assayed for TNF content;
• Figure 2 shows the effect of DMXAA on dLPS-induced TNF production in HPBL in vitro.
• HPBL were incubated (8 h) with the indicated concentrations of dLPS alone (light bars) or in combination with DMXAA (shaded bars). Supernatants were then removed and assayed for TNF content. Horizontal lines represent the SEM;
• Figure 3 shows the effect of anti-CD 14 antibodies on DMXAA- and LPS-induced TNF production in HPBL in vitro.
• HPBL were incubated (8 h) with LPS (1 ng/ml or 1 ⁇ g/ml), DMXAA (800 ⁇ g/ml) or flavone acetic acid (FAA) (800 ⁇ g/ml) in the absence (no shading) or the presence (shading) of anti-CD14 antibodies.
• Supernatants were then removed and assayed for TNF content.
• Horizontal lines represent the SEM;
• Figure 4 shows the effect of DMXAA on TNF production in HPBL in vitro in response to interleukin-1 alpha.
• HPBL were incubated (8 h) with the indicated concentrations of drug either alone (filled symbols) or in combination with DMXAA (unfilled symbols). Supernatants were then removed and assayed for TNF content. Vertical lines represent the SEM;
• Figure 5 shows the effect of DMXAA on TNF production in HPBL in vitro in response to phorbol-12-myristate- 13 -acetate.
• HPBL were incubated (8 h) with the indicated concentrations of drug either alone (unshaded) or in combination with DMXAA (shaded). Supernatants were then removed and assayed for TNF content. Horizontal lines represent the SEM; and
• Figure 6 shows the effect of DMXAA on TNF production in HPBL in vitro in response to okadaic acid.
• HPBL were incubated (8 h) with the indicated concentrations of drug either alone (unshaded) or in combination with DMXAA (shaded). Supernatants were then removed and assayed for TNF content. Horizontal lines represent the SEM.
• Figure 7 shows the effect of anti-CD14 antibodies and dLPS on TNF production in response to LPS and DMXAA in murine leucocytes in vitro.
• Murine leucocytes were pre-incubated for 15 minutes with our without either an9-CD14 antibodies (10 ⁇ l/well) or dLPS (500 ⁇ g/ml) before the addition of DMXAA (800 ⁇ g/ml), DMXAA (800 ⁇ g/ml) or LPS (1 ng/ml). Cultures were incubated for 8 hours and the TNF content of the supernatant was measured.
• Figure 8 shows the effect of antibiotic treatment on in vivo TNF production. Mice were treated orally for three days with an antibiotic combination to reduce the bacterial flora in the gut. Mice were then treated with DMXAA (25 mg/kg) and TNF was measured 24 hours later.
• the present invention relates to a method of treating cancer and to compositions of use in such a method.
• the invention resides in the applicant's unexpected finding of a very large synergistic interaction in cultured human peripheral blood cells between compounds of the xanthenone acetic acid class having the formula (I) as defined below and compounds capable of contributing to the control pathway that modulates TNF (tumour necrosis factor) synthesis in human cells, that is, compounds that themselves modulate TNF production or compounds which are capable of acting on pathways leading to TNF synthesis.
• Ri , R 2 and R 3 are each independently selected from the group consisting of H, Ci-C ⁇ alkyl, halogen, CF 3 , CN, NO 2 , NH 2 , OH, OR, NHCOR, NHSO 2 R, SR, SO 2 R or NHR, wherein each R is independently Ci-C 6 alkyl optionally substituted with one or more substituents selected from hydroxy, amino and methoxy, and wherein each of Ri , R and R 3 may be present at any of the available positions 1 to 8;
• the simultaneous administration of a compound of formula (I) 5,6- dimethylxanthenone-4-acetic acid (DMXAA) and a compound capable of contributing to the control pathway that modulates TNF synthesis in human cells shows greater induction of TNF in cultured human peripheral blood cells than either agent alone.
• TNF has recognised anticancer activity and can act either directly on cancer cells or indirectly on the cancer's blood supply.
• a particularly preferred compound is 5,6-dimethylxanthenone-4-acetic acid (DMXAA).
• DMXAA 5,6-dimethylxanthenone-4-acetic acid
• the compounds capable of contributing to the control pathway that modulates TNF synthesis in human cancer tissue described above are also well known compounds (see, for example, Philpott M, Ching LM, Baguley BC; Eur J Cancer 2001, in press) and can likewise be prepared by methods known to those skilled in the art. As will be readily apparent, more than one of those compounds can be combined with the compound(s) of formula (I) or (la). Reference to "a compound” should not be seen to be restrictive to only one such compound.
• the compound capable of contributing to the control pathway that modulates TNF synthesis is a ligand that binds to the CD 14 receptor of cells.
• ligands are bacterial lipopolysaccharide (LPS), deacylated lipopolysaccharide (dLPS), and antibodies to the CD14 receptor for LPS and dLPS.
• the compound capable of contributing to the control pathway that modulates TNF synthesis is a compound that acts on surface receptors, other than CD 14 receptors, that are connected with TNF production.
• a compound is interleukin-1 alpha (IL-1).
• the compound is capable of contributing to the control pathways that modulate TNF synthesis by inducing the enzyme protein kinase C.
• examples of such compounds are phorbol myristate esters such as phorbol myristate acetate.
• the compound is capable of decreasing the activity of protein phosphatases, preferably protein phosphatase 2A.
• protein phosphatases preferably protein phosphatase 2A.
• An example of such a compound is okadaic acid.
• the therapeutic methods of the present invention therefore include the step of administering to a patient, simultaneously or sequentially, an agent capable of contributing to the control pathway that modulates TNF synthesis, and a compound of the formula (I) as defined above or a pharmaceutically acceptable salt or ester thereof.
• the compound of formula (I) and the compound capable of contributing to the control pathway that modulates TNF synthesis can be administered to a patient in any suitable form.
• the compounds may conveniently be administered intravenously, using formulations for each compound already known in the art.
• the formulation of medicaments for use in cancer treatment utilising combinations of the compounds referred to herein, together with pharmaceutically acceptable carriers, vehicles and excipients would be well within the abilities of a person skilled in this art.
• One known precaution would be to protect solutions of the highly water soluble DMXAA compound from light.
• the compounds of formula (I) and the compound capable of contributing to the control pathway that modulates TNF synthesis can be administered either simultaneously or sequentially, i.e. the compound capable of contributing to the control pathway that modulates TNF synthesis can be administered either before or after the compound of formula (I) is administered. Simultaneous co-administration, in most cases, is likely to be preferred.
• Partially purified buffy coats were purchased from Auckland Blood Centre and divided into 15-ml aliquots in 50-ml centrifuge tubes (2070 Conical Tubes, Becton Dickinson Labware, New Jersey, USA). HPBL in tissue culture dishes (10 ml; 10 7 cells/ml) were incubated overnight in ⁇ -MEM culture medium supplemented with FCS (10% v/v), streptomycin sulphate (100 ⁇ g/ml) and penicillin-G (100 units/ml). All extraction operations were carried out at 7°C to prevent clotting. Unsupplemented ⁇ -MEM medium was added to 30 ml and a 10-ml layer of Ficoll- Paque PLUS was slowly added to the bottom of the tubes.
• TNF standards were prepared by making serial • dilutions of the TNF stock solution in supplemented culture media (concentration range 10 - 10,000 pg/ml).
• ELISA plates were made using the OptEIA Human TNF- alpha Set (Pharmingen, San Diego, CA, USA). TNF standards and samples were added to the ELISA plates and the assays were carried out according to the makers' directions.
• LPS bacterial cell wall lipopolysaccharide
• TNF TNF-derived neurotrophic factor
• modified bacterial cell wall components which by themselves do not stimulate TNF production, may be stimulated by DMXAA.
• Such components are included in genetically modified bacteria that might localise in tumour tissue but produce an attenuated systemic response, thus eliminating endotoxic shock as a side effect of such therapy (Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, Ash O, Carmichael E, Chakraborty A, Fischer J, Lin SL, Luo X, Miller SI, Zheng LM, King I, Pawelek JM, Bermudes D, Nature Biotechnology, 1999, 17, 37-41.
• dLPS deacylated LPS
• DMXAA alone 800 ⁇ g/ml
• DMXAA 800 ⁇ g/ml
• cytokine IL-1 is an inflammatory cytokine that itself has been reported to have experimental antitumor activity (Braunschweiger PG, Johnson CS, Kumar N, Ord V, Furmanski P, Cancer Res. 1988 48, 6011-6016).
• IL-1 alone is capable of inducing TNF in human peripheral blood leukocytes (HPBL).
• co- administration of DMXAA greatly increases (up to 56-fold in this case) the induction of TNF as compared to that by IL-1 alone.
• TNF was measured by enzyme-linked immunosorbent assay after 8 h.
• DMXAA sodium salt (this laboratory) was dissolved in medium and protected from light (9).
• LPS and deacylated LPS (Sigma Chemical Co., MO) were dissolved in ⁇ - MEM, filter-sterilised and used immediately.
• the MEM- 18 mouse anti-human CD 14 IgG antibody was obtained from Sanbio bv, am Uden, Netherlands, and was freed from azide before use by ultrafiltration and was LPS-free (Endospecy ES-50M LPS quantitation system, Seikagaku Corporation, Tokyo, Japan).
• Blood samples for extraction of leucocytes were taken by cardiac puncture of halothane-anaesthetised mice into 1-ml syringes containing ACD-A anticoagulant (0.1 ml). All extraction operations were carried out at 7°C to prevent clotting. Samples were pooled and unsupplemented ⁇ -MEM was added to 30 ml and a 10-ml layer of Ficoll-Paque PLUS was slowly added to the bottom of the tubes. After centrifugation at 300xg for 30 min the upper layer was removed and the leucocyte layer was carefully drawn off into a fresh 50-ml centrifuge tube.
• the volume was adjusted to 50 ml with unsupplemented growth medium, the cells were centrifuged at 300xg, and the leucocytes were resuspended at 10 7 cells/ml in ⁇ -MEM supplemented with foetal bovine serum (10% v/v), streptomycin sulphate (100 ⁇ g/ml) and penicillin-G (100 units/ml).
• control cells cells treated with anti-CD 14 antibodies alone, or cells treated with dLPS alone produced very low concentrations of TNF.
• LPS significantly increased TNF production and this increase was abolished by co- incubation with anti-CD 14 antibody or dLPS.
• DMXAA alone did not significantly increase TNF production, but co-incubation with anti-CD 14 antibody resulted in a high level of TNF production that was even greater than that caused by LPS.
• Co- incubation with dLPS also caused a significant (p ⁇ 0.001) elevation of TNF production, although the magnitude of the effect was less than that caused by anti- CD 14 antibody.
• mice The possible role of LPS for the in vivo production of TNF in mice was reviewed by pre-treating mice with a combination of antibiotics designed to sterilise the gut. The results support the concept that DMXAA acts as a co-stimulator with other inducers of TNF in both murine and human mononuclear cells.
• mice were treated orally with antibiotics for 3 days and then treated with 25 mg/kg DMXAA. TNF levels were measured 24 hours later.
• C57BL mice were either untreated, or treated for 4 days prior to DMXAA with a mixture of antibiotics (cephalocin 2 g/1 and neomycin 2 g/1 in the drinking water).
• mice received a single i.p. dose of DMXAA (25 mg/kg) and blood was collected by cardiac puncture of halothane-anaesthetised mice after 3 hours. Blood from each mouse was transferred to individual microcentrifuge tubes and allowed to clot overnight on ice before centrifugation at 2000xg for 20 minutes at 4°C. If clotting was not complete the sample was allowed to stand on ice for a further 2 hours, after which it was re-centrifuged. Serum was drawn off the top of the blood samples and stored at -20°C until assay of TNF content.
• TNF standards were prepared by making serial dilutions of the TNF stock solution in supplemented culture media (concentration range 10 - 10,000 pg/ml).
• ELISA enzyme-linked immunosorbent assay
• ELISA plates were made using the OptEIA Human TNF-alpha-Set (Pharmingen, San Diego, CA, USA). TNF standards and samples were added to the ELISA plates and the assays were carried out according to the manufacturer's directions.
• TNF concentrations were low in control mice and in mice treated with antibiotics alone. As shown in Figure 8, treatment with DMXAA substantially increased serum TNF concentrations in mice not receiving antibiotics. Administration of DMXAA to mice following antibiotic treatment also increased serum TNF but the increase was significantly smaller (p ⁇ 0.001) than that in mice not receiving antibiotic treatment.
• the present invention provides an improved method of cancer therapy that is expected to find widespread clinical utility.
• the invention also provides compositions of use in such methods of cancer therapy.

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