WO2015123392A1 - Compositions et procédés d'inhibition de l'angiogenèse et la lymphangiogenèse - Google Patents

Compositions et procédés d'inhibition de l'angiogenèse et la lymphangiogenèse Download PDF

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WO2015123392A1
WO2015123392A1 PCT/US2015/015569 US2015015569W WO2015123392A1 WO 2015123392 A1 WO2015123392 A1 WO 2015123392A1 US 2015015569 W US2015015569 W US 2015015569W WO 2015123392 A1 WO2015123392 A1 WO 2015123392A1
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rifamycin
derivative
rifabutin
drug
cancer cell
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PCT/US2015/015569
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English (en)
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James Sacchettini
Deeann WALLIS
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The Texas A&M University System
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • 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

Definitions

  • the present disclosure relates to compositions for inhibition of angiogenesis, lymphangiogenesis, and related pathologies.
  • compositions including rifamycin and rifamyein derivatives, such as rifabutin or rifabutin derivatives, or rifampicin and rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, or prodrugs thereof.
  • the present disclosure also relates to methods of inhibition of angiogenesis or lymphangiogenesis, by rifamycin, rifamycin derivatives, such as rifabutin or rifabutin derivatives, or rifampicin and rifampicin derivatives, and/or pharmaceutically acceptable salts, hydrates, or prodrugs thereof, or combinations thereof to an organism,
  • Angiogenesis is the process of formation of new blood vessels from pre-existing blood vessels.
  • Lymphangiogenesis is the process of new lymphatic vessels from pre-existing lymphatic vessels.
  • Angiogenesis is a normal and necessary process in development and wound healing, but is also associated with a number of pathologies, including cancer, age-related macular degeneration, tumorigenesis, proliferative diabetic retinopathy, atherosclerosis, peripheral arterial disease, and rheumatoid arthritis.
  • pathologies including cancer, age-related macular degeneration, tumorigenesis, proliferative diabetic retinopathy, atherosclerosis, peripheral arterial disease, and rheumatoid arthritis.
  • angiogenesis is a fundamental step in the transformation of tumors from benign to malignant, and inhibition of angiogenesis is an important field of study.
  • lymphangiogenesis is associated with progression of lymphatic malignancies.
  • VEGF Vascular Endothelial Growth Factor
  • MP2 Matrix Metalloproteinases
  • bFGF Basic Fibroblast Growth Factor
  • lymphangiogenesis is known to be mediated by many of the same molecular mechanisms as angiogenesis, such models are also useful predictors of anti- lymphangiogenic effect of target compounds.
  • Rifabutin is a member of the rifamycin class of antibiotics. Rifabutin was approved for use as an antibiotic in the United States in 1992. Although rifabutin has been tested for other antibiotic and anti-inflammatory uses, its most common use remains the treatment of tuberculosis and other Mycobacterium infections. Rifampicin, another member of the rifamycin class of antibiotics, was introduced in 1967 and is also used to treat tuberculosis and similar infections.
  • compositions including rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicin or a rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof!, or a combination thereof.
  • the compositions are operable to inhibit angiogenesis and/or lymphangiogenesis in a tissue.
  • the compositions are further operable to induce drug-sensitization in a cancer cell and/or to inhibit a cancer cell in the tissue.
  • the present disclosure is directed to methods of inhibiting angiogenesis in a tissue.
  • Such methods include administering a composition comprising rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicin or a rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof, to the tissue in an amount and for a time sufficient to inhibit angiogenesis within the tissue.
  • the present disclosure is directed to methods of inhibiting lymphangiogenesis in a tissue.
  • the methods include administering a composition comprising rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicin or a rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof, to the tissue in an amount and for a time sufficient to inhibit lymphangiogenesis within the tissue.
  • the present disclosure provides methods of inhibiting angiogenesis and lymphangiogenesis in a tissue.
  • the methods include administering a composition comprising rifamycm or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicin or a rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof!, or a combination thereof, to the tissue in an amount and for a time sufficient to inhibit angiogenesis and lymphangiogenesis within the tissue.
  • methods of inhibiting angiogenesis within a tissue and sensitizing a cancer cell within the tissue to a drug comprise administering a composition comprising rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicm or a rifarnpiein derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof, to the tissue in an amount and for a time sufficient to inhibit angiogenesis within the tissue, and administering a composition comprising rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifarnpiein or a rifarnpiein derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof or a combination thereof, to the cancer cell in an amount and for a duration sufficient to sensitize the cancer cell to the
  • susceptibility of the cancer cell to the drug is increased.
  • the composition is admininstered to the cancer ceil in an amount and for a time sufficient to increase the amount of a chemotherapeutic in the drag.
  • the composition is admininstered to the cancer cell in an amount and for a time sufficient to decrease activity of or inhibit a p-glycoprotein (P-gp) efflux pump in the cell.
  • the composition is administered to the cancer eel! in an amount and for a time sufficient to increase reactive oxygen species (ROS) in the cancer ceil.
  • the drag is administered to the cancer cell in an amount and for a time sufficient to inhibit the cancer cell.
  • the present disclosure provides methods of inhibiting lymphangiogenesis within a tissue and sensitizing a cancer cell within the tissue to a drug. In further related aspects, the present disclosure methods of inhibiting angiogenesis and lymphangiogenesis within a tissue and sensitizing a cancer cell within the tissue to a drug.
  • methods of inhibiting angiogenesis within a tissue and inhibiting a cancer cell within the tissue with a drag include administering a composition comprising rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicm or a rifarnpiein derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof!, or a combination thereof, to the tissue in an amount and for a time sufficient to inhibit angiogenesis within the tissue, and administering a composition comprising rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, or rifampicm or a rifarnpiein derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof to the tissue in an amount and for a time sufficient to inhibit the cancer cell in the tissue to the
  • the composition is administered to the cancer cell in an amount and for a time sufficient to increase reactive oxygen species (ROS) in the cancer cell.
  • ROS reactive oxygen species
  • the present disclosure provides methods of inhibiting lymphangiogenesis within a tissue and inhibiting a cancer cell within the tissue with a drug.
  • NHL non-Hodgkin's lymphoma
  • RTI-x - designates a rifamycin derivative in which "x" is replaced by an identification number used in the present specification to designate a particular composition
  • FIGURE 1 illustrates a cellular network via which rifamycin and rifamycin derivatives, such as rifabutin or rifabutin derivatives, or rifampicm and rifampicm derivatives, or pharmaceutically acceptable salts, hydrates, or prodrugs thereof, can cause drug-sensitization and an example drug-sensitization effect in CHOP-resistant DLBCL cells;
  • rifamycin and rifamycin derivatives such as rifabutin or rifabutin derivatives, or rifampicm and rifampicm derivatives, or pharmaceutically acceptable salts, hydrates, or prodrugs thereof
  • FIGURE 2 A illustrates the effects of rifabutin on growth of CHOP-sensitive (CRL2631) NHL ceils and CHOP-resistant (G3) NHL cells in the presence or absence of CHOP as demonstrated by resazurin fluorescence;
  • FIGURE 2B illustrates the effects of rifabutin on growth of CHOP-resistant (G3) NHL cells in the absence of CHOP (top panel) as compared to a control drug as demonstrated by resazurin fluorescence and in the presence of varying dilutions of CHOP for 24 hrs (bottom panel).;
  • FIGURE 2C illustrates the effects of rifabutin on growth of another CHOP- resistant NHL cell line (SUDHL10-R) and the parental CHOP-sensi ive NHL cell line (SUDHL10-S) after 24 hrs (top panel) and 48 hrs (bottom panel) of treatment as demonstrated by resazurin fluorescence;
  • FIGURE 3 A illustrates the effects of rifabutin or rifabutin derivatives RTI-79 and RTI-176 on cell growth of primary human dermal fibroblasts both with and without 2 uM Dox;
  • FIGURE 3B illustrates the effects of doxorubicin and rifabutin on cell growth of primary human dermal fibroblasts
  • FIGURE 4 illustrates the effects of rifabutin on growth of CHOP-resistant lymphoma cells obtained by aspiration from a dog as demonstrated by resazurin fluorescence;
  • FIGURE 5 illustrates the effects of rifabutin in combination with CHOP or CHOP alone on tumor burden in mm over time in SCID mice injected with CHOP-resistant (G3) NHL cells;
  • FIGURE 6 illustrates the effects of CHOP or control solution with no CHOP on tumor burden in mm 3 over time in SCID mice injected with CHOP-sensitive (CRL2631) NHL cells;
  • FIGURE 7 illustrates the effects of reduced dosages of CHOP+rifabutin or control solution with no CHOP or rifabutin on tumor burden in mm 3 over time in SCID mice injected with CHOP-resistant (G3) NHL cells;
  • FIGURE 8 illustrates a Kaplan-Meier curve showing average life span for SCID mice injected with CHOP-resistant (G3) cells when treated with either doxorubicin alone (DOX) or doxonibicin i-rifabutin derivative R.TI-81 (DQX+NZ);
  • FIGURE 9 illustrates the average tumor volume of chemo-resistant SK-OV-3 xenografts in mice after control treatment with saline, treatment with 33 mg/kg DOX, and treatment with 3.3 mg/kg DOX + 25 mg/kg rifabutin over time.
  • FIGURE 10 illustrates the average tumor volume of multi-drug resistant cancer cell line (NCI/ADR -RES) xenografts in mice after control treatment with saline, treatment with 7 mg/kg DOXI L® and treatment with 7mg/kg DQXIL®+ 25 mg/kg RTI-79 over time.
  • NCI/ADR -RES multi-drug resistant cancer cell line
  • FIGURE 11 illustrates the average tumor volume of multi-drag resistant cancer cell line (NC I/ADR- ES) xenografts in mice with multiple, large tumors after control treatment with saline, treatment with 7 mg/kg DOXIL®, and treatment with 7 mg/kg DOXIL® + 25 mg/kg RTI-79 over time,
  • NC I/ADR- ES multi-drag resistant cancer cell line
  • FIGURE 12 illustrates the effects of rifabutin or RTI-79 on growth of CHOP- resistant (G3) NHL cells;
  • FIGURE 13 illustrates the effects of rifabutin or RTI-176 on growth of CHOP- resistant (G3) NHL cells;
  • FIGURE 14 illustrates the effects of rifabutin or RTI-81 on growth of CHOP- resistant (G3) NHL cells;
  • FIGURE 15 illustrates the interaction of rifabutin and doxorubicin on CHOP- sensitive (CRL2631) NHL cells;
  • FIGURE 16 illustrates the interaction of RTI-79 and doxorubicin on CHOP- sensitive (CRL2631) NHL cells.
  • FIGURE 17 illustrates the effects of rifabutin or RTI-82 on multidrug-resistant breast cancer (MDA-MB-231) cells;
  • FIGURE 18 illustrates the interaction of rifabutin with actinomycin D on multidrug resistant sarcoma (MES-SA-Dx5) cells;
  • FIGURE 19 illustrates the interaction of rifabutin with menadione on dexamethasone resistant multiple myeloma (MM.1 ) cells;
  • FIGURE 20 illustrates the interaction of rifabutin and RTI-79 with and without doxorubicin at an 8: 1 rifabutin or RTI-79: doxorubicin molar ratio on multi-drug resistant cancer cell line (NCI/ADR-RES) cells;
  • FIGURE 21 illustrates the interaction of RTI-79 and doxorubicin on multi-drug resistant T lymphoblastoid leukemia (MOLT-4) cells;
  • FIGURE 22 illustrates the effects of rifabutin and RTI-79 with and without doxorubicin at an 8: 1 rifabutin or RTI-79 doxorubicin molar ratio on ovarian carcinoma (OVCAR8) cells;
  • FIGURE 23 illustrates the effects of rifabutin and actinomycin D on multi-drug resistant sarcoma (MES-SA-Dx5) cells.
  • FIGURE 24 illustrates the effects of rifabutin and menadione on dexamethasone resistant multiple myeloma ( M.1 ) cells;
  • FIGURE 25 illustrates the interaction of rifabutin and mitoxantrone on osteosarcoma (U-2 OS) cells
  • FIGURE 26 illustrates the interaction of rifabutin with gemcitabine on multi-drug resistant breast cancer (MDA-MB-231) cells;
  • FIGURE 27 illustrates the interaction of rifabutin with paclitaxel on myeloid leukemia cells (HL-60) cells;
  • FIGURE 28 illustrates the interaction of rifabutin and camptothecin on ovarian cancer (OVCAR-8) cells;
  • FIGURE 29 illustrates the number of viable cells present after re-exposure to CHOP of CHOP-sensitive (CRL2631) cells to a full or half dose of CHOP in the presence or absence of rifabuti ;
  • FIGURE 30A illustrates a Western blot for phosphorylated Akt (pAkt) Akt, 14-3- 3 ⁇ , and an actin control in CHOP-sensitive (CRL2631) and CHOP-resistant (G3) cells
  • FIGURE 30B illustrates the effect of varying amounts of Akt Inhibitor VIII on growth of G3 cells as demonstrated by resazurin fluorescence
  • FIGURE 30C illustrates a Western blot for phosphorylated Akt (pAkt) Akt, !4-3 ⁇ 3 ⁇ , and a Vimentin control in G3 cells exposed or not exposed to Akt Inhibitor VIII;
  • FIGURE 32 illustrates the ROS levels in distinct populations of cells in CHOP- sensitive (CRL2631) cells purified by flow cytometry;
  • FIGURE 34 il lustrates the effect on cell growth of varying amounts of CHOP in the presence or absence of 10 uM rifabutin on low-ROS CRL2631 cells as demonstrated by resazurin fluorescence;
  • FIGURE 35 illustrates the effect of 10 uM rifabutin on ROS in CHOP-resistant (G3) cells over time;
  • FIGURE 36 A provides a western-blot showing different ABCB1 protein levels in si-ABCBl and si-NCl (control si-RNA) treated ADR-RES cells, as well as the untreated ADR- RES cells and its parental drag-sensitive strain OVCAR8;
  • FIGURE 36B shows the effects of rifabutin (RBT) on calcein-AM efflux in OVCAR8 than in ADR-RES cells.
  • FIGURE 36C shows the effects of 5 ⁇ rifabutin (RBT), RTI-79, and rifampin (RMP) on calcein-AM efflux and the further effects of ABCB1 RNA-silencing;
  • FIGURE 37A shows dose-response curves of various RTIs on calcein-AM efflux.
  • FIGURE 37B shows the effects of various RTIs on luM doxorubicin's toxicity in G3 cells
  • FIGURE 37C shows the correlation between efflux inhibition effect and drag sensitizing ability for various RTI-x rifamycin derivatives
  • FIGURE 37D shows the comparison of doxorubicin fluorescence intensity in the NCI/ ADR-RES cells with rifabutin treatment or dimethyl sulfoxide (DMSO) control.
  • FIGURE 38 A shows the effects of MDR/P-gp inhibitors and two control drugs (carboxin, nifazoxinide) on ROS in doxorabiein-sensitive OVCAR8 cells;
  • FIGURE 38B shows the effects of MDR/P-gp inhibitors and two control drugs (carboxin, nifazoxinide) on ROS in doxorubicin-resistant ADR-RES cells;
  • FIGURE 38C shows the effects of MDR/P-gp inhibitors and two control drugs (carboxin, nifazoxinide) on ROS in doxorubicin-resistant G3 cells;
  • FIGURE 39 shows staining of ADR-RES cells treated with RTI-79; ADR-RES cells were infected 24 hrs with a baculovirus expressing a recombinant GFP protein fused with a mitochondrial localization signal (green); cells were stained with CellROX to detect ROS (red) or DAPI to detect nuclei (blue);
  • FIGURE 40 shows the effects of cell-permeable calcium modulators (BAPTA, Verapamil) and a Complex I inhibitor (Rotenone) on ROS in G3 cells;
  • FIGURE 41 A shows the effects of P-gp inhibitors (I eserpine, Elacridar) on ROS levels in ADR and OVCAR8 ceils
  • FIGURE 41B shows the effects of RTI-79 on ROS and calcium mobilization in doxorubicin-sensitive lymphoma (CRL2631, 10S, WSU) and ovarian carcinoma (OVCAR8) cells and doxorubicin-resistant lymphoma (G3R, ! OR, WSUR) cells;
  • FIGURE 41C shows the levels of ROS and calcium mobilization in more CHOP- sensitive lymphoma (CRL2631, I OS, WSU) compared to the more resistant derivative cell lines (G3, 10R, WSU-R), and in the more doxorubicin-sensitive OVC.AR8 versus the more resistant derivative cell line ADR,
  • FIGURE 42 shows a time course of RTI-79 induction of ROS and calcium mobilization in G3 cells.
  • FIGURE 43 shows the effects of siRNA knockdown of P-gp on induction of ROS and mobilization of calcium.
  • FIGURE 44 shows the effects of rifabutin on G3 and CRL2631 cells in a collagen invasion 3D assay.
  • FIGURE 45 shows the effects of rifabutin on G3 and CRL2631 cells in a modified Boyden chamber assay.
  • FIGURE 46A shows the effect of RTI-79, rifabutin, and rifampicin on the secretion of MMP2 in human umbilical vein endothelial cells ("HUVECs"); and
  • FIGURE 46B shows the effect of RTI-79, rifabutin, and rifampicin on the secretion of MMP2 in human umbilical vein endothelial cells (''HUVECs''),
  • FIGURE 47 shows the effect of RTI-79 and rifabutin on HUVEC invasion in a cell invasion assay compared to negative and positive controls.
  • FIGURE 48 shows the qualitative effects of RTI-79 and rifabutin on tube formation in a tube formation assay at 4 hours, 8 hours, and 24 hours compared to negative and positive controls.
  • FIGURE 49A shows the qualitative effect of RTI-79 and rifabutin on HUVEC invasiveness in a three-dimensional collagen invasion assay
  • FIGURE 49B shows the quantitative effect of RTI-79 on HUVEC invasiveness in a three-dimensional collagen invasion assay.
  • FIGURE 50 shows the effect of RTI-79 on angiogenesis in a chick chorioallantoic membrane ("CAM") model compared to cell media alone and to cell media containing pro- angiogemc factors.
  • CAM chick chorioallantoic membrane
  • FIGURE 51A shows dextran-FITC immunohistochemical staining of endothelium formed during an in vivo matrigei plug assay wherein RTI-79 treated or untreated mice were injected with matrigei plugs containing matrigei alone, matrigei and positive control (bFGF), matrigei and negative control (sorafinib), or matrigei and RTI-79; and
  • FIGURE 51 B shows relative F1TC fluorescence observed in combined blood, plasma, and dissolved matrigei plugs after removal following the in vivo matrigei plug assay.
  • FIGURE 52A shows immunohistochemical staining of CD-31 in tumors removed from mice 14 days or 54 days after xenografting of ADR-RES ovarian cancer cells and 5 days or 45 days after twice- weekly treatment with saline or RTI-79;
  • FIGURE 52B shows pixel areas of CD-31 stained capillaries in the tumors.
  • the present disclosure relates to compositions and methods of inhibition of angiogenesis and lymphangiogenesis.
  • such compositions and methods can be employed simultantaneously and/or sequentially for drug- sensitization of a cancer cell and/or inhibiting a cancer cell.
  • the present disclosure relates to compositions and methods for inhibition of angiogenesis and drug- sensitization of a cancer cell, as well as compositions and methods for inhibition of angiogenesis and inhibiting a cancer cell.
  • the present disclosure related to compositions and methods for inhibition of lymphangiogenesis and drug-sensitization of a cancer cell, as well as compositions and methods for inhibition of lymphangiogenesis and inhibiting a cancer cell.
  • the present disclosure relates to compositions and methods inhibition of angiogenesis and lymphangiogenesis and drug- sensitization of a cancer ceil and/or inhibiting a cancer cell. It will be understood by those of ordinary skill in the art that these and other aspects of the present disclosure are non-limiting and can also be related or overlapping. Where support for a specific combination of various aspects of the present disclosure is not expressly recited, it will nonetheless be understood that these aspects can be combined by one of ordinary skill in the art with reference to this disclosure. These compositions and methods are described in further detail below.
  • angiogenesis refers to the formation of new blood vessels in an organism.
  • Angiogenesis includes the formation of new blood vessels associated with or due to one or more pathologies or disease states within the organism.
  • Inhibition of angiogenesis refers to partial or total inhibition of angiogenesis, and/or to a relative reduction in total angiogenesis or a rate or progression of angiogenesis, as demonstrated by, for example and not limitation, decreased density of blood vessel formation in a region within a patient, such as a tissue, an organ, or a tumor, or within the substantial!)' the entire body of a patient, as well as in vivo and/or in vitro assays evidencing same.
  • the patient can be any animal.
  • the patient can be a mammal, such as a human, a pet mammal such as a dog or cat, an agricultural mammal, such as a horse, cow, pig, sheep, or goat, or a zoo mammal.
  • a mammal such as a human
  • a pet mammal such as a dog or cat
  • an agricultural mammal such as a horse, cow, pig, sheep, or goat, or a zoo mammal.
  • lymphangiogenesis refers to the formation of new lymphatic vessels in an organism.
  • Lymphangiogenesis includes the formation of new lymphatic vessels associated with or due to one or more pathologies or disease states within the organism.
  • Inhibition of lymphangiogenesis refers to partial or total inhibition of lymphangiogenesis, and/or to a relative reduction in total lymphangiogenesis or a rate or progression of lymphangiogenesis, as demonstrated by, for example and not limitation, decreased density of lymphatic vessel formation in a region within a patient, such as a tissue, an organ, or a tumor, or within the substantially the entire body of a patient, as well as in vivo and/or in vitro assays evidencing same.
  • the patient can be any animal.
  • the patient can be a mammal, such as a human, a pet mammal such as a dog or cat, an agricultural mammal, such as a horse, cow, pig, sheep, or goat, or a zoo mammal.
  • a cancer cell includes a cell of any type of cancer. Furthermore, it includes a cancer cell in a patient, either in a cancerous growth, such as a tumor, or in isolation from other cancer cells, such as during metastasis.
  • the patient can be any animal.
  • the patient can be a mammal, such as a human, a pet mammal such as a dog or cat, an agricultural mammal, such as a horse, cow, pig, sheep, or goat, or a zoo mammal.
  • certain embodiments herein are expressed in terms of a cancer cell, the same or similar effects can be seen in groups of cancer cells in a patient.
  • Drug-sensitization includes increased sensitivity to a drug, decreased resistance to a drug, or potentiation of a drug's activity or efficacy. Any effect can be measured using any methods accepted in the art.
  • drug-sensitization can be determined by an increased ability of the dmg to inhibit a cell. Cellular inhibition can include killing the cell, such as via apoptosis or necrosis, reducing the growth of the cell, thus reducing the growth of the cancer containing the cell, rendering the cell more susceptible to the immune system, preventing or reducing metastasis, reducing the size of a tumor containing the cell, or otherwise negatively affecting a cancer ceil.
  • An increased ability of the dmg to inhibit a cancer cell can be demonstrated by an ability to inhibit the cell with a reduced amount of drug or in a shorter period of time than in the absence of dmg-sensitization.
  • drug-resistant cancer cells which include cells with inherent or acquired resistance
  • dmg-sensitization can result in a renewed or newly acquired ability of the dmg to inhibit a cancer cell or type of cancer ceil.
  • the compositions include rifamycin, rifamycin derivatives, such as rifabutin or rifabutin derivatives, rifampicin and rifampicin derivatives, pharmaceutically acceptable salts, hydrates, and prodrugs thereof, and combinations thereof. Additional rifamycin derivates include rifapentine and rifalazil.
  • the present disclosure provides derivatives of rifabutin according to one of the following general structures:
  • R can be an aikyi, aryl, or hetero aryl group.
  • the present disclosure provides enantiomers of the general structures. In certain embodiments, it provides enantiomers with the following general chiral structures:
  • R can be an aikyi, aryi, or hetero aryl group.
  • R in certain embodiments having general structures I or II or general chiral structures la or lia, R can be one of the following structures:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • X and R can include the following combinations:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • X is a C, O, or N and R is an alkyl, aryl, or hetero-aryi group.
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl group or wherem X and R are as follows:
  • composition of the general formula above can be the following enantiomer:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • X is a C, O, or N and R can include the structures listed below:
  • the present disclosure provides derivatives of rifabutin according to the following formula, wherein is a C, O, or N:
  • composition with the general formula above can be the following enantiomer:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl group or wherem X and R are as follows:
  • the present disclosure provides derivatives of rifabutin according to the following formula:
  • the present disclosure provides a drug-sensitization composition comprising a series of 3,4-cyclo-rifamycm derivatives.
  • a drug-sensitization composition comprising a series of 3,4-cyclo-rifamycm derivatives. Examples of such compositions are as follows:
  • X can be CH, S, SO, SO 2 or N.
  • Y can be H or an acetyl group.
  • R.1 can be hydrogen.
  • R2 can be a hydroxyl or an amino (-NH?) group.
  • R 1 and R2 together can be an oxo or imine group.
  • R3 can be one of the following groups: hydrogen, a!k !, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocycioalkyl groups that can be additionally substituted with from zero to four substituents chosen independently from halogen, hydroxy, alkox.y-aik.yi, ⁇ CN, nitro, -8-al.kyl, amino, alkylaraino, diaikylami.no, dialkylammoalkyl, carboxy, carboalkoxy, acyl, carboxamido, alky] sulfoxide, acylamino, phenyl, benzyl, phenoxy, and benzyloxy.
  • compositions of the following structure :
  • Y is H or an acetyl group and R4 can be selected from alkyl, aikenyi, alkynyl, cycloalkyl, aryi, heteroaryl and heterocycloalkyl groups that can be additionally substituted with from zero to four substituents chosen independently from halogen, hydroxy, alkoxy-alkyl, -CN, nitro, -S-aikyi, amino, alkyiamino, dialkylamino, dialkylarmnoalkyl, carboxy, carboalkoxy, acyl, carboxamido, alkylsulfoxide, acyiamino, phenyl, benzyl, phenoxy and benzyloxy.
  • Y is H, or acetyl group
  • Z is carbon, oxygen or nitrogen atom
  • R4 is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryi, heteroaryl and heterocycloalkyl groups that can be additionally substituted with from zero to four substituents chosen independently from halogen, hydroxy, alkoxy-alkyl, -CN, nitro, -S-alkyl, amino, alkylamino, diaikyiamino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzy!ox)'.
  • compositions for inhibition of angiogenesis, inhibition of lymphangiogenesis, drug sensitization, inhibition of a cancer cell, and for combinations of these various applications in accordance with certain aspects of the present disclosure can include those listed in Table 1 .
  • Compositions of Table 1 are designated by like names throughout this specification.
  • compositions also mcludes pharmaceutically acceptable salts, hydrates, prodrugs, and mixtures of any of the above compositions.
  • pharmaceutically acceptable salt refers to salts whose counter ion derives from pharmaceutically acceptable nontoxic acids and bases,
  • the 3,4-cyclo-rifamycin derivatives which contain a basic moiety can form salts with a variety of organic and inorganic acids.
  • Suitable pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) base addition salts for the compounds of the present invention include inorganic acids and organic acids.
  • Examples include acetate, adipate, alginates, ascorbates, aspartates, benzenesulfonate (besylate), benzoate, bicarbonate, bisulfate, borates, butyrates, carbonate, camphorsuifonate, citrate, digluconates, dodecylsulfates, ethanesulfonate, fumarate, gluconate, glutamate, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrobromides, hydrochloride, hydroiodides, 2-hydiOxyethanesulfonates, isethionate, lactate, maleate, malate, mandelate, methanesulfonate, 2-naphthalenesulfonates, nicotinates, raucate, nitrate, oxalates, peetinates, persulfates, 3-phenyl
  • the 3,4-cyclo-rifamycin derivatives which contain an acidic moiety can form salts with variety of organic and inorganic bases.
  • Suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, ammonium salts, metallic salts made from calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, ⁇ , ⁇ -dialkyl amino acid derivatives (e.g.
  • ⁇ , ⁇ -dimethylglycine piperidine- 1 -acetic acid and morpholine-4- acetic acid
  • ⁇ , ⁇ '-dibeiizylethyienediamine chloroprocaine
  • choline diethanolamine
  • ethylenediamine meglumine (N-methylglucamine)
  • t-butylamine dicyclohexylamine, hydrabamine, and procaine.
  • the 3,4-cyc!o-rifamycin derivatives, and salts thereof, can exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.
  • the compounds described herein can contain asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center can be defined, in terms of absolute stereochemistry, as (R)- or (S)-.
  • the present invention is meant to include all such possible isomers, as well as, their racemic and optically pure forms.
  • Optically active (R) ⁇ and (SV, or (D) ⁇ and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • compositions of the present disclosure can also include a pharmaceutically acceptable carrier, in particular a carrier suitable for the intended mode of administration, or salts, buffers, or preservatives.
  • a pharmaceutically acceptable carrier in particular a carrier suitable for the intended mode of administration, or salts, buffers, or preservatives.
  • Rifamycin and many of its derivatives, such as rifabutin and rifabutin derivatives are poorly soluble in water.
  • aqueous compositions of the present disclosure can include solubility enhancers.
  • Compositions for oral use can include components to enhance intestinal absorption.
  • the overall formulation of the compositions can be based on the intended mode of administration. For instance, the composition can be formulated as a pill or capsule for oral ingestion. In other examples, the composition can be encapsulated, such as in a liposome or nanoparticle.
  • compositions can also be formulated for injection or infusion or for implantation in a biocompatible carrier.
  • compositions of the present disclosure can contain a sufficient amount of rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to inhibit angiogenesis when the composition is delivered to a region susceptible to angiogenesis in the body of a patient, such as a tissue, a blood vessel, a complex of blood vessels, an organ, or a tumor.
  • a rifamycin derivative such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof.
  • the amount can vary depending on other components of the composition and their effects on drug availability in a patient, the intended mode of administration, the intended schedule for administration, any drug toxicity concerns, drug-drug interactions, such as interactions with other medications used by the patient, or the individual response of a patient.
  • Many compositions can contain an amount well below levels at which toxicity to normal cells or to the patient overall becomes a concern.
  • compositions of the present disclosure can contain a sufficient amount of rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to inhibit lymphangiogenesis when the composition is delivered to a region susceptible to angiogenesis in the body of a patient, such as a tissue, a lymphatic vessel, a lymph node, an organ, or a tumor.
  • the amount can vary depending on other components of the composition and their effects on drug availability in a patient, the intended mode of administration, the intended schedule for administration, any drug toxicity concerns, drag-drag interactions, such as interactions with other medications used by the patient, or the individual response of a patient.
  • Many compositions can contain an amount well below levels at which toxicity to normal ceils or to the patient overall becomes a concern.
  • compositions of the present disclosure can further contain a sufficient amount of rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to cause drug-sensitization or inhibition of a cancer cell to occur when the composition is administered to a cancer cell.
  • a rifamycin derivative such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof
  • the amount can vary depending on other components of the composition and their effects on drug availability in a patient, the type of drag or drugs to which the cancer cell is sensitized, the amount of drug otherwise required to inhibit the cancer cell, the intended mode of administration, the intended schedule for administration, any drug toxicity concerns, drug-drug interactions, such as interactions with other medications used by the patient, or the individual response of a patient.
  • Many compositions can contain an amount well below levels at which toxicity to normal cells or to the patient overall becomes a concern.
  • compositions of the present disclosure can further include other therapeutic agents.
  • they can include one or more of the anti-angiogenic agents listed herein, particularly those described below in connection with Angiogenesis Inhibition Methods.
  • They can additionally or alternatively contain any one or more of the chemotherapeutic agents listed herein, and, for example, can include combinations of chemotherapeutic agents and anti- angiogenic agents.
  • the amounts of those anti-angiogenic agents and/or chemotherapeutic agents in compositions of the present disclosure can be reduced as compared to normal doses of such agents administered in a similar fashion.
  • compositions of the present disclosure can also con tain one or more drugs for which the rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof, causes drug-sensitization.
  • Example drugs are described in the current specification.
  • compositions of the present disclosure can contain one or more other drugs commonly used in combination with the drug for which sensitization occurs.
  • certain compositions can include rifabutin or a rifabutin derivative with any CHOP drug, regardless of whether rifabutin causes drug-sensitization for that drug.
  • the composition can contain another dmg that also causes drug sensitization, such as a drug that affects the amount or ROS, particularly superoxide, in a ceil.
  • a drug that affects the amount or ROS, particularly superoxide in a ceil.
  • the composition can contain superoxide dismutase inhibitors.
  • the composition can contain another drug that affects drug resistance or a property causing drug resistance in cancer cells.
  • it can contain dmgs affecting the apoptotic pathway, such as the apoptotic pathway inhibitors for Bel-XL or mimetics for BH3 proteins.
  • the amount of rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof, present in a com ostion can be measured in any of a number of ways.
  • the amount may, for example, express concentration or total amount. Concentration can be for example, weight/weight, weight/volume, moles/weight, or moles/volume. Total amount can be total weight, total volume, or total moles. Typically, the amount can be expressed in a manner standard for the type of formulation or dosing regimen used.
  • the present disclosure further includes methods of identifying whether a rifamycin derivative, such as a rifabutin derivative, or a rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof, is able to inhibit angiogenesis.
  • Such methods include preparing or obtaining such a derivative, applying it to a tissue, an organ, a blood vessel, or a model thereof, or a cell that expresses or is responsive to pro-angiogenic factors, and identifying that the derivative inhibits angiogenesis, mhibits proxies or markers of angiogenesis, and/or inhibits expression of or response to pro-angiogenic factors.
  • the present disclosure further includes methods of identifying whether rifamycin, a ri amycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, is able to sensitize a cancer cell or inhibit a cancer cell.
  • Such methods include preparing or obtaining such a derivative, applying it to a cancer cell, and identifying that the derivative renders the cancer ceil more susceptible to a chemotherapeutic in any manner described herein.
  • the disclosure provides methods of inhibiting angiogenesis in a patient by administering a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • the disclosure provides methods of inhibiting lymphangiogeneis in a patient by administering a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • the present disclosure provides methods of inhibiting both angiogenesis and lymphangiogenesis in a patient by administering a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • the disclosure provides methods of reducing the amount of an anti-angiogenic agent administered to a patient by also administering a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient.
  • a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof.
  • a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and
  • methods of the present disclosure can be employed to reducing the amount of another anti-angiogenesis agent administered, including, for example, anti-VEGF antibodies such as bevacizumab, aflibercept, soluble VEGF receptors, growth factors such as angiopoeitin 2, protease inhibitors such as Tissue Inhibitor of Metalloproteinase ("TIMP") 1-4, cytokines such as interferon-a, - ⁇ , and - ⁇ , inter le kin-4, -12, and -18, and platelet factor 4, clotting proteins such as prothrombin and antithrombin III.
  • anti-VEGF antibodies such as bevacizumab, aflibercept, soluble VEGF receptors
  • growth factors such as angiopoeitin 2
  • protease inhibitors such as Tissue Inhibitor of Metalloproteinase (“TIMP") 1-4
  • cytokines such as interferon-a, - ⁇ , and - ⁇ , inter le kin-4
  • reducing the amount of another anti-angiogenesis agent administered by administering a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient will reduce unwanted effects of the anti-angiogenesis agent or reduce the risk of occurrence of unwanted effects.
  • compositions disclosed herein can be delivered to a region of a patient susceptible to angiogenesis and/or lymphangiogenesis, such as a tissue, an organ, a tumor, one or more lymph nodes, and one or more blood vessels, by delivering the composition to the patient.
  • the mode of delivery can be selected based on a number of factors, including metabolism of the rifamycin or rifamycin derivative, such as the rifabutin or rifabutin derivative, the angiogenesis- related pathology to be treated, the health of the patient, ability or inability to use particular dosing forms or schedules with the patient, preferred dosing schedule, including any adjustment to dosing schedules due to side effects of additional medications or treatments, and ease of administration.
  • the mode of administration can be enteral, such as orally or by introduction into a feeding tube.
  • the mode of administration can be parenteral, such as intravenously.
  • the dosage amounts and schedule of administration of the composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof, to the patient can vary depending on other components of the composition and their effects on bioavailability in a patient, the intended mode of administration, the intended schedule for administration, whether other drugs, such as other anti-angiogenic agents and chemotherapeutic drugs, are administered, any drug toxicity concerns, and the patient's response to the composition.
  • the dosage amount and frequency of administration of the composition can be such that levels of the rifamycin, rifamycm derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof, to the patient in the patient remain well below levels at which toxicity to the patient becomes a concern.
  • the amount and frequency can also be such that levels of the rifamycin, rifamycm derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof in the region susceptible to angio genesis remain continuously at a level sufficient to inhibit angiogenesis in the susceptible region.
  • levels of the rifamycin, rifamycm derivative such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof in the region susceptible to angio genesis remain continuously at a level sufficient to inhibit angiogenesis in the susceptible region.
  • the rifamycin, rifamycm derivatives such as rifabutin or a rifabutin derivatives, rifampicin or rifampicin derivativse, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof, can exhibit their anti-angiogenic effects on a patient by directly or indirectly inhibiting expression and release of pro-angiogenic growth factors, including bFGF, VEGF, and angiogenesis- associated enzymes, including MMP2.
  • pro-angiogenic growth factors including bFGF, VEGF, and angiogenesis- associated enzymes, including MMP2.
  • the rifamycin, rifamycin derivatives such as rifabutin or a rifabutin derivatives, rifampicin or rifampicin derivativse, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof can additionally or alternatively inhibit angiogenesis by the induction of intracellular ROS species, such as superoxide species, that modulate downstream signalling pathways involved with angiogenesis.
  • the angiogenesis related to cancer can be vascularization of a tumor.
  • the vascularization of the tumor can be prevented, reduced, delayed, or reversed.
  • the tumor can decrease in size, the rate of growt of the tumor can decrease, the growth of the tumor can be arrested, or the tumor can die, and malignant transformation of the tumor can be arrested, delayed, reversed, or reduced .
  • compositions including rifamycin, rifamycin derivatives, such as rifabutin or a rifabutin derivatives, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof can be administered to a patient systemicalfy, such as by oral administration or intravenous infusion, or can be administered locally, such as by injection into a tumor, into the proximity of the tumor, or to the blood vessels supplying the region (such as a tissue or organ) of the pati ent bearing the tumor.
  • the angiogenesis related to diseases of the eye can be angiogenesis associated with age-related macular degeneration or diabetic retinopathy, or both.
  • Vascularization of the eye can be prevented, reduced, delayed, or reversed.
  • the retinopathy or macular degeneration can be delayed, arrested, or reversed.
  • compositions including rifamycin, rifamycin derivatives, such as rifabutin or a rifabutin derivatives, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof can be administered to a patient systemicaiiy, such as by oral administration or intravenous infusion, or can be administered locally, such as by intra-vitreal injection.
  • the injection can occur weekly, or semi-monthly, or once every three weeks, or monthly, or once every six weeks, or every two months, or every three months, or as needed.
  • the amount by weight of the rifamycin, rifamycin derivatives, such as rifabutin or a rifabutin derivatives, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof, administered is between about 0.01 nig and about 10 mg. In certain embodiments, the amount by weight administered is between about 0.1 mg and about 2.5 mg.
  • the amount by weight is about 0.1 mg, or about 0.2 mg, or about 0.3 mg, or about 0.4 mg, or about 0.5 mg, or about 0.6 mg, or about 0.7 mg, or about 0.8 mg, or about 0.9 mg, or about 1.0 mg, or about 1.1 mg, or about 1.2 mg, or about 1.3 mg, or about 1.4 mg, or about 1.5 mg, or about 1.6 mg, or about 1.7 mg, or about 1.8 mg, or about 1.9 mg, or about 2.0 mg, or about 2.1 mg, or about 2.2 mg, or about 2.3 mg, or about 2.4 mg, or about 2.5 mg.
  • the present disclosure further includes drug-sensitization and cancer cell inhibition methods in which a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof is administered to a cancer cell in order to sensitize the cancer cell to another drug.
  • Such methods can augment or complement the methods of inhibition of angiogenesis and lymphangiogenesis disclosed herein. For example, unwanted angiogenesis and/or lymphangiogenesis is commonly observed in cancer and cancer-related pathologies.
  • the methods described herein can be employed to both inhibit angiogenesis and lymphangiogenesis and drug-sensitize and/or inhibit a cancer cell.
  • the compositions and methods can prevent or reduce metastasis.
  • Metastasis from solid tumors is a complex, multistep process whereby cancer cells must breach the basement membrane and migrate away from the primary tumor environment to invade the surrounding stroma and enter the vasculature directly or via the lymphatics. The cancer cells must then also invade another area of the body.
  • the compositions disclosed herein can prevent or reduce metastais by preventing or reducing any of these movements or activities of the cancer cells.
  • Rifamycin or a rifamycin derivative such as rifabutin or a rifabutin derivative, can also decrease the levels of metastasis-associated cellular factors in or around cancer cells.
  • MMP matrix metalloproteinase
  • VEGF vascular endothelial growth factor
  • composition can be any composition described above.
  • the compostion can be administered with any other drug which can alternatively be present in a pharmaceutical composition as desribed herein.
  • the other drug can include DOXIL®.
  • the drug can be any drug for rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof! or a combination thereof, increases drug-sensitization.
  • the drag can be a chemotherapeutic.
  • chemotherapeutics include alkylating agents, antimetabolites, anti-tumor antibiotics, hormonal agents, targeted therapies, differentiating agents and other drugs.
  • Example alkylating agents include nitrogen mustards such as mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide, ifosfamide, and melphalen.
  • Example alkylating agents further include nitrosoureas, such as streptozocin, carmustine (BCNU), and iomustine.
  • Example alkylating agents further include alkyl sulfonates such as busulfan, triazmes, such as procarbazine and dacarbaz ne (DTiC) and temozolomide, and ethyl en [mines, such as thiotepa and altretamine (hexamethylmelamine).
  • Example alkylating agents further include platinum drugs, such as cisplatin, carboplatin, and oxalapiatin.
  • Example antimetabolites include purine antagonists such as mercaptopurine (6- MP), thioguanine (6-TG), fludarabine phosphate, clofarabine, cladribine, and pentostatin.
  • Example antimetabolites also include pyrimidine antagonists such as fluorouracil (5 -FIT), floxuridine, capecitabine, cytarabine, gemcitabine and azacitidine.
  • Example antimetabolites further include plant alkaloids.
  • Some plant alkaloids include topoisomerase inhibitors such as topoisomerase I inhibitors such as camptothecin, topotecan and irinotecan, or topoisomerase II inhibitors such as amsacrine, etoposide, and tenyposide.
  • Other plant alkyloids include mitotic inhibitors such as taxanes, including paclitaxel and docetaxel, epothilones, including ixabepilone, vinca alkaloids, including vinblastine, vincristine, and vmore!bine, as well as estramustine.
  • Example antimetabolites further include folate antimetabolites such as methotrexate and pemetrexed. Other antimetabolites include hydroxyurea.
  • Example anti-tumor antibiotics include anthracyc lines or anthracycline analogs such as daunorubicin, doxorubicin, epirubicin, mitoxantrone, and idarubicin.
  • Other anti-tumor antibiotics include daetinomycin, plicamycin, mitomycin, bleomycin, apicidin, and actinomycin.
  • Example hormonal agents include gonadotropin-releasing hormone agonists such as leuprolide and goserelin.
  • Other example hormonal agents include aromatase inhibitors such as aminoglutethimide, exemestane, letrozole and anastrozole.
  • Other hormonal agents include tamoxifen and flutamide.
  • Still other example hormonal agents include anti-estrogens such as fulvestrant, tamoxifen, and toremifene or anti-androgens such as bicalutamide, flutamide, and nilutamde.
  • Example hormonal agents further include progestins such as megestrol acetate, and estrogens.
  • Example targeted therapies include antibodies or other therapeutics that act on a molecular level such as imatinib, gefitinib, sunitinib, and bortezomib.
  • Example differentiating agents include retinoids such as tretinoin, bexarotene, and arsenic trioxide.
  • chemotherapeutics include L-asparaginase, phenoxodiol, rapamycin, and menadione.
  • the cancer cell can be sensitized to a dr g already known to inhibit the cancer cell, or it can be sensitized to a drug not previously used with that type of cancer cell. If the cancer cell is a drug-resistant cancer cell that has acquired resistance, it can be sensitized to a drug that previously exhibited a decreased ability to inhibit the cancer cell or cancer cells of the same type.
  • the composition can directly mhibit the cancer ceil instead of or in addition to causing drug-sensitization.
  • the cancer ceil that undergoes drug-sensitization or inhibition can be any type of cancer cell. It may, for instance, be a carcinoma, a sarcoma, a leukemia, a lymphoma, or a glioma. It can also be a soft cancer or a hard cancer.
  • an immuiiologicai-related cancer such as leukemia, lymphoma, including on-Hodgkin's lymphoma, or Hodgkin's disease, myeloma, including multiple myeloma, sarcoma, lung cancer, breast cancer, ovarian cancer, uterine cancer, including endometrial cancer, testicular cancer, intestinal cancer, including colon cancer, rectal cancer, and small intestinal cancers, stomach cancer, esophageal cancer, oral cancer, pancreatic cancer, liver cancer, prostate cancer, glandular cancers such as adrenal gland cancer and pituitary tumor, bone cancer, bladder cancer, brain and other nervous tissue cancers, including glioma, eye cancer, including retmoblasoma, skin cancer, including basal cell carcinoma and melanoma, and kidney cancer.
  • an immuiiologicai-related cancer such as leukemia, lymphoma, including on-Hodgkin's lymphoma, or Hodgkin's disease
  • the composition can be delivered to the cancer ceil in a patient by delivering the composition to the patient.
  • the mode of deliver ⁇ ? can be selected based on a number of factors, including metabolism of the rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof, or another drug in the composition, mode of administration of other drugs to the patient, such as the drug to which the cancer ceil is sensitized, the location and type of cancer cell to be drug-sensitized, health of the patient, ability or inability to use particular dosing forms or schedules with the patient, preferred dosing schedule, including any adjustment to dosing schedules due to side effects of chemotherapeutics, and ease of administration, in certain embodiments, the mode of administration can be enteral, such as orally or by introduction into a feeding tube. In certain embodiments, the mode of administration
  • the dosage amounts and administration schedule of the composition can vary depending on other components of the composition and their effects on drug availability in a patient, the type of drug or drugs to which the cancer ceil is sensitized, the intended mode of administration, the intended schedule for administration, when other drugs are administered, any drug toxicity concerns, and the patient's response to the drug.
  • the amount and frequency of rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof or combination thereof that is delivered can be such that levels in the patient remain well below levels at which toxicity to norma! cells or to the patient becomes a concern.
  • the amount and frequency can also be such that the levels in the cancer cell remain continuously at a level sufficient to induce drug-sensitization or are at a level sufficient to induce-drug sensitization when or shortly after the drug to which the cancer cell is sensitized is delivered to it.
  • the composition can be taken or administered on a regular basis during treatment with the drug to which the cancer cell is sensitized or it can be taken only a set time before, at the same time, or a set time after the drug to which the cancer cell is sensitized.
  • the disclosure provides methods of inhibiting a cancer cell using a drug to which the cancer cell is resistant by administering a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof!, to the cancer cell.
  • a composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or a pharmaceutically acceptable salt, hydrate, and prodrug thereof, or a combination thereof!, to the cancer cell.
  • the disclosure provides methods of reducing the amount of a drug administered to a patient by also administering the composition. Such methods may, in particular, be employed with drugs that have other harmful effects.
  • alkylating agents such as topoisomerase inhibitors
  • methods of the present disclosure can be used to reduce the amount of mitotic inhibitors administered, reducing the chance or amount of resulting peripheral nerve damage, or the methods can be used to reduce the amount of anti-tumor antibiotics administered, reducing the chance or amount of resulting hearing damage.
  • anti-tumor antibiotics for which there is a total lifetime dosage limit, methods of the present disclosure can allow a patient to be treated with the drug for a longer time, increasing life expectancy or improving quality of life.
  • Methods of the present disclosure can also allow amounts of some chemotherapeutics administered to remain sufficiently low as to allow the patient to have children after cancer treatment. Methods of the present disclosure can further allow amounts of the chemotherapeutics administered to be lowered into a range where a drug approved for use in adults might also be used in children.
  • composition comprising rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof or combination thereof directly inhibits a cancer cell alone or in addition to causing drug-sensitization, the dosage and administration can be adequate to allow this inhibition.
  • it can consist of regular administration of an amount of rifamycin, a rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof, to maintain a certain level in the patient, the blood, a tissue, or a tumor.
  • dosage amounts and the administration schedule can be adjusted based on other components of the composition and their effects on drug availability in a patient, the intended mode of administration, the intended schedule for administration, when other drugs are administered, any drug toxicity concerns, and the patient's response to the drug.
  • rifamycin, a rifamycin derivative such as rifabutin or a rifabutin derivative, rifanipicm or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, and prodrug thereof, or combination thereof
  • an efflux pump such as the ATP-binding cassette sub-family B member 1 (ABCB1) pump.
  • This glycoprotein is found in the cell membrane and actively transports certain ehemotherapeutics, such as doxorubicin, out of cancer cells, reducing efficacy of the drug.
  • the amount of chemotherapeutic present in a cancer cell can be increased and thus the killing effect on the cancer cell can be increased.
  • rifamycin, rifamycin derivatives such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof, suppress ABCB1 activity, increasing the effective amount of a chemotherapeutic within a cancer cell.
  • rifamycin, rifamycin derivatives such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof, can exhibit drug-sensitization effects on a cancer cell by acting on the Akt (protein kinase B)/14-3-3 1 ⁇ 2itochondrial electron transport chain (ETC)/reactive oxygen species (ROS) signaling network within a cell.
  • Akt protein kinase B
  • ETC excreactive oxygen species
  • FIGURE 1 An example of how the rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof, or combination thereof can effect this pathway in a drug-resistant cancer cell is shown in FIGURE 1.
  • a CHOP-resistant cancer cell such as a CHOP-resistant diffuse large B-cell lymphoma (DLBCL) cell, undergoes cellular changes such that Akt is constitutively activated. This constitutively activated Akt phosphorylates mitochondrial GSK-3B.
  • DLBCL CHOP-resistant diffuse large B-cell lymphoma
  • This phospborylated GSK-3B then binds the 14-3-3 ⁇ protein, rendering the GSK-3B unavailable to bind to mitochondrial ETC Complex 1.
  • GSK-3B binding to ETC Complex 1 inhibits the complex activity, so the overall result of constitutive Akt activation is that ETC Complex 1 is not inhibited when it otherwise should be.
  • Downregulation of Complex I activity by GSK-3B can lead to increased electron leakage from the ETC 1 , resulting in increases in ROS.
  • ETC Complex 1 acts to reduce the amount of electron spillage from the ETC during mitochondrial activity. Electrons spilled in such a manner react with oxygen to produce reactive oxygen species (ROS). Thus, increased ETC Complex 1 activity and the resultant reduction in electron leakage decrease the amount of ROS in the cell. Low levels of ROS can lead to an intracellular environment that inhibits the ability of chemotherapeutics such as CHOP to induce cancer cell death by apoptosis. Thus, one effect of constitutive Akt activation is a decrease in ROS, making the cancer cell harder to kill.
  • ROS reactive oxygen species
  • rifamycin, rifamycin derivatives such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof or combinations thereof, suppress ETC Complex 1 activity, restoring it to a more normal level .
  • rifamycin, rifamycin derivatives such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof or combinations thereof.
  • the present disclosure also includes methods of inducing drug-sensitization in a cancer cell by administering an amount of rifamycin, rifamycin derivatives, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof, sufficient to decrease activity of ETC Complex 1 or increase cellular levels of ROS.
  • the disclosure includes methods of administering an amount sufficient to increase cellular levels of ROS to an amount sufficient to allow a drug to which a cancer cel l is sensitized to kill, reduce the growth of, or negatively affect the cancer ceil.
  • the same methods are applicable to cancer cells that exhibit low ROS levels for other reasons. Furthermore, the same methods can be used for drug- sensitization in cancer cells that have no ROS abnormality by increasing ROS to an abnormal level if the cancer cells then become sensiti ve to the drug at the abnormal ROS level .
  • Effects mediated by ROS described above may, in particular, be mediated by superoxide species and superoxide species can be the particular form of ROS affected.
  • compositions and methods of the present disclosure can act via other cellular pathways alternatively to or in addition to the Akt/14-3-3y ' ETC ROS network. This can be particularly true with respect to drug-sensitization to chemotherapeutics that operate in a different manner than CHOP.
  • ROS can affect the mitoehondrial-directed Bcl-2 apoptosis pathway as well.
  • the effect of rifabutin on ROS induction has been shown to be very rapid, whereas the effect on Akt has been shown to take at least 18 hours.
  • Akt is constitutive ly active thereby increasing Complex I activity resulting in decreases in ROS. induction of ROS by compositions and methods of the present disclosure will further promote drug sensitivity in the resistant cancer cell by downregulating the Akt pathway.
  • rifamycin, rifamycin derivatives such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivatives, or pharmaceutically acceptable salts, hydrates, and prodrugs thereof, or combinations thereof, can exhibit drug-sensitization effects on a cancer ceil by mobilizing calcium within the cell. Increased calcium mobilization correlates with increased ROS amounts. Drug-sensitive cells often exhibit both increased levels of calcium and increased ROS levels as compared to drug-resistant cells. Typically, ROS levels rise first in such cells, followed by calcium mobilization. Accordingly, rifamycin or a rifamycin derivative, such as rifabutin or a rifabutin derivative, can directly inhibit efflux pump activity, which then causes a burst of ROS followed by calcium mobilization.
  • compositions disclosed herein can inhibit a cancer cell through more than one activity. For instance, it can both decrease efflux pump activity and increase ROS. In certain embodiments, these multiple activities can have synergistic effects.
  • CRL2631 was established from peripheral blood leukocytes (PBL) of a patient with DLBCL.
  • PBL peripheral blood leukocytes
  • G3 CHOP-resistant NHL cell lines
  • FIGURE 2 A The effects of rifabutin on cell growth of both CHOP-sensitive (CRL2631) and CHOP-resistant (G3) cells in the presence or absence of CHOP are shown in FIGURE 2 A.
  • a reduction in cell growth is demonstrated by a reduction in fluorescence emitted by the ceil growth indicator dye, resazurin.
  • Rifabutin was confirmed to have drug-sensitization activity in clinically derived CHOP resistant cell lines. As shown in FIGURE 2A, CHOP inhibited growth of CHOP- sensitive (CRL2631 ) cells but had little effect on G3 cells. Rifabutin did not affect the growth of cells in the absence of CHOP, indicating low toxicities (FIGURE, 2A). Rifabutin enhanced the sensitivity of CHOP-sensitive cells to CHOP as shown in FIGURE 2B, relati ve to a control drug. FIGURE 2C shows similar effects in another CHOP-resistant NHL line.
  • Swiss mice were dosed with levels equal to and exceeding that of intended doses. Swiss mice were given repeated weekly oral doses of rifabutin at 180 mg/kg, RTI-79 at 250 mg/kg or RTI-81 at 30 mg/kg in conjunction with intravenous 3.3 mg/kg DOX. No overt toxicity or weight loss was seen over several weeks time. Further, no significant differences between mice treated with RTI-79 with or without DOX were observed after both histological analysis of heart tissue by hematoxylin and eocin (H&E) and analysis of blood and serum for complete blood count and manual differential.
  • H&E hematoxylin and eocin
  • Intravenous rifabutin or RTI-81 were also given repeatedly both at 75 mg/kg in conjunction with intravenously administered 3.3 mg/kg DOX and no overt toxicity or weight loss was seen over several weeks time. Further data in Example 4 below shows treatment efficacy using less than one-fifth the above oral dose of 33 mg/kg rifabutin with intravenous 3.3 mg/kg doxorubicin.
  • FIGURE 4 shows that growth of aspirated lymphoma cells was resistant to CHOP at doses up to 640 ng/ml, but significant growth inhibition was observed at a dose of 1280 ng/ml CHOP.
  • the inclusion of 5 ⁇ rifabutin significantly enhanced the sensitivity of the aspirated lymphoma cells to CHOP such that significant growth inhibition was observed at 320 and 640 ng/ml CHOP.
  • Rifabutin had no effect on cell viability in the absence of CHOP.
  • Rifabutin in an amount of 100 mg kg was administered on the day of each CHOP treatment and 24-hours later by gavage.
  • Mouse body weight and tumor size were monitored every two days and tumor size measured by caliper.
  • the tumor volume formula (L*W*W)/2 was used to calculate tumor mass.
  • mice were treated before the appearance of palpable tumors.
  • one week after transplantation of CHOP- resistant G3 cells one group (7 mice) was treated with CHOP-only and a second group (8 mice) was treated with CHOP + rifabutin.
  • mice received a second treatment and tumors began to appear in the CHOP-only group.
  • the two treatment groups differed not only in the tumor size but also in the number of tumors developed. More tumors appeared and grew at a significantly higher rate in CHOP-only mice compared to CHOP+rifabutin mice.
  • the CHOP only treatment group developed tumors at 12 of 14 (85.7%) injection sites.
  • the CHOP+rifabutin treatment group developed fewer tumors at only 6 of 16 (37.5%) injection sites.
  • SCID mice developed G3 tumors at 35 of 42 (83.3%) injection sites when receiving no treatment; this is similar to the CHOP only treatment group. Significance was analyzed by the T test yielding a highly significant difference between the means of the tumor burdens of the two groups (p ⁇ .01) at Day 7. Thus, rifabutin actually reduces the tumor take rate which could translate into more complete responses when humans are treated early with this combination.
  • CHOP+rifabutin was administered weekly beginning one week post-inoculation.
  • Tumor load was significantly less in mice that received even reduced CHOP dosages as compared to imtreated mice, demonstrating that rifabutin can allow the use of lower dosages of CHOP without a significant decrease in therapeutic effect (FIGURE 7).
  • NCI/ADR-RES multi-drug resistant ovarian cancer cell line
  • RTI-79 was administered by oral gavage 24 and 48 hours after each DOXIL® administration. Tumor size was monitored. As shown in FIGURE 1 1 , after 46 days the tumor volume in the RTI-79-treated mice was 55% lower than in mice receiving only DOXIL®, Furthermore, tumor volume in RTI-79-treated mice was reduced by 50% during the course of the study.
  • compositions of the present disclosure were tested and their effects on cell growth were measured. A reduction in cell growth is demonstrated by a reduction in fluorescence emitted by the cell growth indicator dye, resazurin.
  • Compositions were tested on CHOP-resistant G3 NHL cells that had been cultivated in RPMI medium for five days. Prior to assay, the ceils were counted by haemocytometer and cell concentration standardized to 625,000 cells/ml. Test drugs were solubilized in 100% DMSO and then diluted to final assay concentration with 0.1 phosphate buffered saline (PBS) and a final DMSO concentration of 0.5%).
  • PBS 0.1 phosphate buffered saline
  • Table 2 IC50S for selected rifamycin analogs on lymphoma cell line G3.
  • Example data for RTI-79 and rifabutin is shown in FIGURE 12.
  • Example data for RTI-176 and rifabutin is shown in FIGURE 13.
  • Example data for RTI-81 and rifabutin is shown in FIGURE 14.
  • Example data for interaction of rifabutin and doxorubicin on CRL263 I cells is shown in FIGURE 15.
  • Example data for interaction of RTI-79 and doxorubicin on CRL2631 cells is shown in FIGURE 16.
  • RBT rifabutin; + potentiation between 1.2 to 2.0 fold increase; ++ potentiation between 2.1 to 5 fold increase; +-;--+- potentiation greater than 5 fold increase
  • Example data for rifabutin or TI-82 on MDA-MB-231 cells is presented in FIGURE 17.
  • Example data for rifabutin or RTI-79 with or without doxorubicin on SK-OV3 cells is presented in FIGURE 18.
  • Example data for rifabutin or RTI-81 on MES-SA ⁇ Dx5 cells is presented in FIGURE 19.
  • Example data for interaction of rifabutin or RTI-79 and doxorubicin on ADR-RES cells is shown in FIGURE 20.
  • Example data for interaction of RTI-79 and doxorubicin on MOLT-4 cells is shown in FIGURE 21.
  • Example data for the interation of rifabutin or RTI-79 and doxorubicin on ovarian carcinoma OVCAR-8 cells is shown in FIGURE 22. These results establish that rifabutin and rifabutin analogs are able to induce drug- sensitization for a variety of types of cancer.
  • Chemotherapeutics include: the targeted therapy bortezomib ( eleade®), the pyrimidine antagonist gemcitabine, the platinum drug cis-piatin, the anti-tumor antibiotic actinomycin D, the anti-tumor antibiotic apicidm, the topoisomerase I inhibitor camptothecm, the anti-tumor antibiotic doxorubicin, the mitotic inhibitor vinblastine, the nitrogen mustard alkyating agent melphalen, the hormonal agent tamoxifen, the folate antimetabolite methotrexate, the toposimerase II inhibitor etoposide, phenoxodiol, the antibiotic rapamycin, and menadione.
  • the targeted therapy bortezomib eleade®
  • the pyrimidine antagonist gemcitabine gemcitabine
  • the platinum drug cis-piatin the anti-tumor antibiotic actinomycin D
  • the anti-tumor antibiotic apicidm the topo
  • Additional cell lines used include: ovarian cancer OVCAR-8, T lymphoblastoid leukemia MOLT-4, dexamethasone-resistant multiple myeloma MM. IR, myeloid leukemia cells HL-60, osteosarcoma cells U-2 OS, and myeloma RPMI 8226. Results are showin in Table 4.
  • Table 4 ICsos for selected cancer cell lines and clinically relevant cancer therapeutics in interaction with Rifabutin
  • Ovarian carcinoma OVCAR-8 Mitoxantrone 14.0 3.0 4.7
  • Example data for interaction of rifabutin with actinomycin D on MES ⁇ S A ⁇ Dx5 cells is shown in FIGURE 23.
  • Example data for interaction of rifabutin with menadione on MM.1R cells is shown in FIGURE 24.
  • Example data for interaction of rifabutin and mitoxantrone on U-2 OS cells is shown in FIGURE 25.
  • Example data for interaction of rifabutin andgemcitabiiie on MDA-MB-231 cells is shown in FIGURE 26.
  • Example data for interaction of rifabutin with paclitaxel on HL-60 cells is shown in FIGURE 27.
  • Example data for interaction of rifabutin and camptothecin on OVCAR-8 cells is shown in FIGURE 28. These results demonstrate the ability of rifabutin and rifabutin derivatives to induce drug-sensitivity for a wide variety of chemotherapeutics in a wide variety of cancers.
  • a I X CHOP dose in this experiment corresponds to final concentrations of the following components: 0.83 ⁇ 4-hydroxycyclophosphamide [4HC, a pre-activated form of cyclophosphamide], 0.057 ⁇ doxorubicin, 0.01 ⁇ vincristine, and 0.186 ⁇ prednisone.
  • FIGURE 3 OA A Western blot of CHOP-sensitive (CRL2631) or CHOP-resistant (G3) lymphoma cells revealed that Akt, phosphorylated Akt, and 14 ⁇ 3-3 ⁇ levels were consistent with the model proposed in FIGURE 1 (FIGURE 3 OA) in that Akt was markedly more active in CHOP-resistant G3 cells than in CRL2631.
  • the model was further confirmed by treatment of CHOP-resistant (G3) cells with Akt Inhibitor VIII, which caused a dose-dependent reversal of CHOP resistance (FIGURE 30B).
  • the inhibitory effect of Akt inhibitor VIII on the expression of phosphorylated Akt and total 14-3-3 ⁇ protein was confirmed by Western blot (FIGURE 30C).
  • Rifabutin and its derivatives showed clear inhibition of efflux pumps in NCl/ADR-RES and G3 ceils when tested in calcein-AM assays. This inhibitory effect was unambiguously due to inhibition of ABCB1 pumps.
  • the difference in pump activity between ADR- RES cells and its drug-sensitive parental strain, OVCAR-8, can be seen in FIGURES 34A and 34B
  • Doxorubicin-sensitive (OVCAR.8 ovarian) and Doxorubicm-resistant (G3 lymphoma; ADR-RES ovarian) cells were treated for 2 hrs with 10 uM RTI-79, p-glycoprotein (P-gp) inhibitors (reserpine, elacridar), or control drugs (DMSO, carboxin, nifaz.oxi.dine). Cells were then stamed with the fluorescent ROS mdicator, CellROX, and subjected to flow cytometry to quantitate total intracellular ROS.
  • P-gp p-glycoprotein
  • DMSO carboxin, nifaz.oxi.dine
  • FIGURE 38 shows, RTI-79 induced ROS in ovarian carcinoma and lymphoma cell lines, as did the MDR/P-gp inhibitors, reserpine, elacridar. This suggests that RTFs ability to induce ROS was the result of inhibition of efflux pumps.
  • the degree of ROS induced by RTI-79 and P-gp inhibitors was much greater in the doxorubicm- resistant ADR-RES and G3 ceil lines than in the doxorubicin-sensitive OVCAR8 cell line. Control drugs established that this effect is specific to MD /P-gp inhibitors and RTI-79.
  • the electron transport chain (ETC) is known to be a primary generator of ROS in the cell. Most of the ROS is generated by Complexes I and III of the ETC. Inhibition of Complex I results in electrons piling up and leaking to react with oxygen to produce ROS.
  • Dox-resistant G3 lymphoma cells were treated 10 uM RTI-79, BAPTA-AM (cell permeable calcium chelator), verapamil (a calcium channel blocker and P-gp inhibitor), a Complex I inhibitor (Rotenone), a Complex III inhibitor (antimycin A), or control drags (oxaloacetate, carboxin, nifazoxinide). Rotenone, but not antimycin A, induced ROS, suggesting that RTI-79-induced and efflux pump inhibitor-induced ROS originate at Complex I of the ETC.
  • MDR/P-gp activity is closely associated with calcium status in the cell, so calcium modulators were tested for effects on ROS, As shown in FIGURE 40, both a cell- permeable calcium chelator (BAPTA-AM) and a calcium channel blocker (and efflux pump inhibitor) induced ROS in G3 cells. As shown in FIGURE 41 A and 39B, P ⁇ gp inhibitors (Reserpine, Elacridar) induced ROS relative to control drags (Carboxin, Nifazoxinide). As shown in FIGURE 4 I B, a P-gp inhibitor (Elacridar) induced calcium in a similar manner as RTI- 79.
  • BAPTA-AM cell- permeable calcium chelator
  • Ca channel blocker and efflux pump inhibitor
  • Dox-resistant cells were also treated with 10 uM RTI-79 for 2 hrs (G3R+RTI79; 10R+RTI-79, WSUR+RTI-79).
  • Cells were co-stained with the cell-permeable red fluorescent ROS indicator, CellROX, and cell-permeable green fluorescent calcium indicator, Fluo-4AM, and then subjected to flow cytometry to quantitate changes in ROS and calcium levels.
  • FIGURE 41C levels of both ROS and calcium in doxorubicin-sensitive cells were much higher than in the resistant lines, and RTI-79 induced both ROS and calcium mobilization in resistant cells.
  • the ability of RTI-79 to sensitize doxorabi cm-resistant ceils was closely correlated with the inhibition of efflux pumps, induction of ROS, and mobilization of calcium.
  • RTI-79 might inhibit MDR/P-gp by inducing ROS, which then increase calcium mobilization that then inhibits efflux pump activity.
  • RTI-79 can first directly inhibit efflux pump activity, which then causes a burst of ROS followed by calcium mobilization.
  • ADR-RES Dox-resistant
  • OVCAR.8 Dox-sensitive ovarian carcinoma cells were transfected with siRNA to knockdown efflux pumps to determine the effect on ROS and calcium. Cells were then co- stained with CeliROX and Fluo-4AM for I hour. Some cells were treated with RTI-79 for I hour and controls (no RTI-79) were treated with DMSO.
  • a collagen invasion 3D assay allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells. Malignant ceil lines with high metastatic potential in vivo show a higher rate of invasion than non-metastatic tumor cells and normal ceils showed little or no ability to penetrate the barrier.
  • the CHOP-resistant G3 cell line is much more invasive in a collage invasion 3D assay than its CHOP-sensitive parent cell line (C L2631 ).
  • Collagen matrices (1 mg/ml) were prepared as previously described in Su, S.C., et ai.. Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing. Am. J. Physiol. Cell Physiol, 295(5): C1215-29 (2008), incorporated in material part by reference herein, with the inclusion of either DMSO control or 10 uM Rifabutin (Rif). Ceils were allowed to invade for 24 hours.
  • a modified Boyden chamber assay was used as an independent method to evaluate rifabutin's ability to suppress invasion/metastasis.
  • G3 and CRL2631 cells were grown in the presence of 10 ⁇ . ⁇ rifabutin or dose volume equivalent DMSO for 24 hours at 37 °C. Cell invasion was assessed with a Chemieon QCM Collagen Invasion Assay (Millipore).
  • the assay is a 96-well plate assay wherein each well is equipped with a suspended insert. Inserts contain an 8-micron membrane coated with a thin layer of polymerized collagen. Invading cel ls migrate through the collagen layer and attach to the bottom of the membrane. Cells were detached from the membrane and lysed prior to detection via CyQuant dye.
  • Fluorescence intensity is proportional to number of invading cells. As shown in FIGURE 45, the presence of rifabutin resulted in decreased relative fluorescence from 170,374 to 114,395 RLU in G3 cells. In CRL2631 cells RLU decreases from 39,356 to 27,432 RLU in the presence of rifabutin (p ⁇ 0.05).
  • Scheme 1 illustrates the general preparation of 1 1-deoxo-l l-imino-3,4-spiro- piperidyl-rifamycms (lc) and l I-deoxo-l l-amino-3,4-spiro-piperidyl-rifamycms (Id).
  • the compounds of (1 c) are synthesized by condensation of 3-amino-4-deoxy-4-imino-rifamycin S (la) with a substituted piperidone or hexanon-type of ketone (lb) at a temperature range from 10°C to 70°C in organic solvent, such as THF or ethanol, in the presence of an excess of ammonium salt, such as ammonium acetate, in a sealed reaction tube.
  • Reduction of 1 1-imino- rifamycin (lc) with reducing reagent, such as NaBH , in organic solvent, such as THF and EtOH at a temperature range from 0°C to room temperature produces 1 1-amino-rifamycin (Id).
  • the tbioether could be oxidized to sulfoxide (-SO-) or suifone (-S02-) depending upon the ratio of compound lc and oxidizing agents.
  • product is obtained by de-protection of Boc-propected-piperidine or Fmoc-protected-piperidine.
  • Scheme 2 illustrates the general preparation of 3,4-spiro-piperidyl-rifamycins (2c) and 1 ] -deoxo-1 l-hydroxy-3,4-spiro-piperidyl-rifamycms (2d).
  • the compounds of (2c) are synthesized by condensation of 3-amino-4-deoxy-4-immo-rifamycm S (l a) with a substituted piperidone or hexanon-type of ketone (lb) at a temperature range from 10°C to 70°C in organic solvent, such as THF or ethanol, in the presence or absence of a catalyst, such as Zinc.
  • the intermediate of (la) is commercially available or can be obtained from the rifamycin S.
  • Scheme 3 illustrates the general preparation of 11 -deoxo-1 l-hydroxyimino-3 ,4- spiro-piperidyi-rifarnycins (3c).
  • the compounds of (3c) are synthesized from the reaction of 11- oxy-rifamycin compound (2c) with hydroxyiamme (or its HCl salt) at a temperature range from 10°C to 70°C in organic solvent, such as THF or methanol, in the presence or absence of base, such as pyridine.
  • I H-NMR Proton nuclear magnetic resonance
  • 13C- NMR spectra were recorded on Varian Inova 500 MHz spectrometer operating at 125 MHz and Chemical shifts were reported in ppm and referenced to residual solvent signals (CHCl;r :: d 77.23 for carbon)
  • HRMS high resolution mass spectra
  • Biotin-glycine-substituted rifabutin derivative RTI-173 contains a substitution at the 21 -hydroxy site, yet has a similar activity as rifabutin on G3 cells when combined with doxorubicm, suggesting that this site can be modified without affecting drug-sensitization or cancer inhibition effects.
  • Biotin-glycine-linked rifabutin derivative (RTI-173) has the following formula:
  • FfUVECs Human umbilical vein endothelial cells
  • endothelial growth medium containing Ml 99 supplemented with 15% FBS, 400 ng/ml bovine brain extract, 100 ng/ml heparin, and antibiotics. Cells were maintained for no more than 6 passages.
  • HUVECs were plated in a 24-weIl plate at 100,000 cells per well and treated with either dimethylsulfoxide ("DM SO") as a negative control, Sorafenib, a known inhibitor of M P2 and VEGF secretion in osteosarcoma, at a concentration of ⁇ . ⁇ as a positive control, rifabutin at a concentration of 10 ⁇ , rifampicin at a concentration of 10 ⁇ , or RTI-79 at concentrations of 10, 5, 1, and 0.5 uM. Treatments were added to the media. Media supematents were harvested after 48 hours and assayed for MMP2 and VEGF.
  • DM SO dimethylsulfoxide
  • Enzyme-linked immunosorbent assays for secreted MMP2 and VEGF were performed and quantitated. As illustrated in FIGURE 46A and FIGURE 46B, respectively, secretion of MMP2 and VEGF was reduced by RTI-79, rifabutin, and rifampicin to levels below or comparable to positive control.
  • HUVEC cells were grown in the presence of RTI-79 at a concentration of ⁇ , 5 ⁇ , or ⁇ , rifabutin 1 ⁇ , ⁇ , 5 ⁇ . ⁇ , or 1 ⁇ , a dose equivalent volume of DM80 as a negative control, or resveratrol at a concentration of 40 ⁇ for 24 hours at 37°C in a Chemieon® QCM Collagen Invasion Assay (Millipore).
  • the Chemieon® assay apparatus is a modified 96-well plate wherein each well is equipped with a suspended insert containing an 8 ⁇ membrane coated with a thin layer of polymerized collagen. Invading cells migrate through the collagen coating and attach to the bottom of the membrane.
  • Relative fluorescence intensity for the cell invasion assays is shown in FIGURE 47. As shown, RTI-79 and rifabutin inhibited cell invasion relative to negative control, while ⁇ RTI-79 inhibited cell invasion to levels comparable to inhibition with 40 ⁇ resveratrol. Note that this dose of resveratrol has been observed to be slightly toxic to HUV EC cells after 72 hours.
  • Tube formation assays measure the ability of endothelial cells plated at subconfiuent densities with extracellular matrix support to form capillary-like structures. After plating in the tube formation, the endothelial cells attach and generate mechanical forces on the surrounding extracellular support matrix to form tracks that facilitate cellular migration. The resulting cords of cells ultimately arrange into hollow lumens.
  • FIGURE 48 Representative 3X magnification micrographs of the tube formation assays are provided in FIGURE 48. These micrographs illustrate that while tubular networks form for each treatment, including positive control, the networks observed with RTI-79 treatment and rifabutin treatment are not as branched and are less organized than those observed with DMSO treatment, indicative of disrupted sprouting angiogenesis. A dose response is apparent for RTI-79 treatment. Treatment effect is pronounced at 8 hours, and while all tubular networks are deteriorating at 24 hours, this effect is more pronounced in the treated cells.
  • a three-dimensional collagen invasion assay was employed to assess the effects of RTI-79 and rifabutin on cell invasion as a proxy for angiogenesis.
  • Three-dimensional culture system modeling can more closely reflect the microenvironment of tumors and metastases more closely than two-dimensional models.
  • Three-dimensional collagen matrices containing pro-angiogenic factor SIP were prepared in 96 well plates. HUVECs were plated at 40,000 cells per well in media containing pro-angiogenic factors VEGF and FGF with or without additional treatments. Treatments include: 20, 10, 5, 2.5, 1.25, 0.6, and 0.3 uM RTI-79 or DMSO as a negative control. HUVECs were allowed to sprout and invade matrices for 24 hours prior to fixation and imaging at 20X.
  • FIGURES 49A and 49B Representative micrographs of the three dimensional collagen invasion assays are provided in FIGURES 49A and 49B.
  • inclusion of RTI-79 in the collagen matrix decreased HUVEC invasion.
  • Control cells treated with DMSO appear spindle ahaped and some cell bodies have migrated out of the focal plane as indicated by blurred cells. Cells in treatment groups tend to remain in a single focal plane and have a rounded as opposed to spindle-shaped appearance.
  • FIGURE 49B inclusion of RTI-79 in the collagen matrix at 20, 10, and 5 uM resulted in decreased HUVEC cell invasion in terms of number of invading structures per 20x field (p ⁇ G.01). Inclusion of 10 um RTI-79 also resulted in decreased distance over which the cells invaded.
  • Example 18 Chick Chorioallantoic Membrane Model Assay
  • CAM chick chorioallantoic membrane
  • FIGURE 50 Representative photographs for each treatment arm of the CAM assay are provided in FIGURE 50. As shown, treatment with medium containing RTI-79 at a concentration of 5 ⁇ resulted in decreased blood vessel development relative to treatment with cell medium alone and treatment with cell medium containing the supernatant of U2-OS cells.
  • Matrigel was injected into mice to form a biocompatible "plug" in which angiogenesis could be observed.
  • ImL of Matrigel was injected into the groin region of female three-month old C57 mice.
  • Ail Matrigel treatments except the Matrigel-on!y negative control contained 150 ng/mL of bFGF to promote angiogenesis.
  • the Matrigel injections also contained sorafinib at a concentration of 10 ⁇ or RTI-79 at a concentration of 10 ⁇ .
  • Half of the mice injected with RTI-79-containing matrigel also received daily gavage of RTI-79 at a dose of 25 mg/kg/day.
  • mice were injected intravenously with FICT-dextran. 30 minutes after FITC-dextran injection, the matrigel plugs and blood were harvested from each mouse. Plasma was isolated from blood and utilized to normalize FITC concentration per mouse. The plugs were imaged and weighed, then dissolved in dispase overnight. FITC fluorescence from the dissolved plugs and the harvested plasma were measured with a fluorescent plate reader, and the observed fluorescence for each treatment arm was quantitated to determine FITC staining of the plugs normalized to the weight of the plugs and the FiTC within the plasma.
  • FIGURE 51 A Representative micrographs of the FITC -stained matrigel plugs are shown in FIGURE 51 A. Inclusion of RTI-79 in the Matrigel injection and RTI-79 gavage decreased the formation of blood vessels relative to Matrigel treated only with bFGF.
  • FIGURE 5 IB shows the relative FITC 1 fluorescence per gram matrigel plug weight relative to the FITC fluorescence observed in the plasma (RLU plug/g plug/RLU in plasma). Inclusion of RTI-79 in the Matrigel injection and RTI-79 gavage decreased relative FITC fluorenseence of the plug as compared to Matrigel treated only with bFGF.
  • Xenografts of ADR-RES ovarian cancer cells were injected into nude mice.
  • the mice receive twice weekly gavage of either saline or 25 mg/kg RTI-79 beginning 9 days after xenograft injection.
  • Tumors were harvested 5 days or 45 days after treatment initiation and fixed in fomralin, embedded in paraffin, and sectioned into 7 ⁇ sections. The sections were stained with anti-CD31 antibody and counterstained with hematoxylin.
  • FIGURE 52A Representative micrographs of the tumors are provided in FIGURE 52A.
  • FIGURE 52B showns the quantitative analysis of anti-CD-31 staining in the tumors of mice gaveged with saline and the mice gavaged with RTI-79. As shown, the tumors of saline gaveged mice exhibit greater density of CD-31 staining than did the tumors of mice gaveged with RTI-79.
  • Example 21 Example Rifabutin and Rifabutin Derivative Compositions and Methods of Administration to a Patient Exhibiting or at Risk of Pathology-Related Angiogenesis
  • compositions comprising rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof , can be prepared as described herein.
  • compositions can be formulated in tablets or capsules for oral use. These tablets or capsules can be extended release tablets or capsules to provide a more stable and continuous supply of the rifamycin or rifamycin derivative to a region exhibiting or susceptible to angiogenesis in the patient.
  • Tablets or capsules can contain at least 10 mg, at least 50 nig, at least 100 mg, at least 150 mg, or at least 200 mg of rifamycin or rifamycin derivative.
  • Combmation tablets or capsules with other drugs can be prepared, particularly if the recommended dosing schedule for those drugs is similar to that of the rifamycin or rifamycin derivative.
  • compositions can also be formulated for intravenous injection as well.
  • the amount of rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof, or combination thereof can be lower in a dose formulated for intravenous injection than in a dose formulated for oral administration because intravenous injection avoids the need for absorption through the intestines.
  • Injectable doses of the compositions can be provided in multi- use containers or in single -use containers. These containers can be compatible for use with standard intravenous needles and syringes as well as intravenous drip systems.
  • Single-use containers can contain the entire amount of rifamycin or rifamycin derivative administered. Alternatively, they can contain amounts appropriate for daily doses. Single-use containers can contain at least 1 mg, at least 5 mg, at least 10 mg, at least 50 mg, at least 100 mg, or at least 150 mg of rifamycin or rifamycin derivative. Multi-use containers can be designed to allow admin stration of these same amounts of rifamycin or rifamycin derivative. Injectable compositions can further contain other injectable agents.
  • compositions comprising rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof, or combmation thereof can be administered to patients exhibiting or at risk of pathology-related angiogenesis in the form of any compositions described in this example or elsewhere herein or any any other form.
  • the rifamycin or rifamycin derivates can be administered orally to patients exhibiting or at risk of pathology- related angiogenesis. In particular, they can be administered in the form of tablets or capsules.
  • the rifamycin or rifamycin derivative can be administered such that the patient receives at least 50 mg/adult human/week, at least 100 mg/adult human-'week, at least 1 50 mg/adult human/week, or at least 300 mg/adult human/week. Amounts can be reduced for children. For example, a child under age 5 might receive one quarter or less of an adult human dose. A child age 5 to age 10 can receive one half to one quarter the adult human dose. A child age 10 or over over can receive three quarters to one half the adult human dose.
  • the rifamycin or rifamycin derivative can be administered such that the patient receives at least 0.5 mg/kg/week, at least 1 mg/kg/week, at least 2 mg/kg/week, at least 5 mg/kg/week, at least 10 mg/kg/week, at least 20 mg/kg/week, at least 30 mg/kg/week, at least 50 mg/kg/week or at least 100 mg/kg/week.
  • compositions comprising rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof, or a combination thereof, administered oral ly in this fashion can be administered weekly, daily, or multiple times per day.
  • the dosing schedule can be adjusted so as to maintain minimal blood concentrations for a period of time, particularly with extended release formulations.
  • maintenance of minimal blood concentrations can not be necessary for some methods of treatment and dosing can instead be designed to achieve a total blood concentration for a shorter period of time, such as for four hours or less.
  • compositions do not have to be administered for a full week.
  • a patient can receive a single dose in connection with an anti-angiogenic treatment and can not receive a further dose until much later, with another anti-angiogenic treatment, or not at all.
  • the patient can be provided with a pack of varying-dose tablets or capsules labeled by day (e.g. Day 1 , Day 2, etc.), by portions of the day (e.g.
  • the composition can comprise rifabutin or RTI-79 administered orally in one to three doses of rifabutin or RTI-79 in 100 mg to 300 mg amounts over a period of up to 48 hours.
  • a single oral dose of 300 mg rifabutin causes a mean ( ⁇ SD) peak plasma concentration (Cmax) of 375 ( ⁇ 267) ng/ ' mL (range 141 to 1033 ng/mL).
  • rifabutin The plasma elimination of rifabutin is bi phasic with an initial half-life of approximately 4 hours, followed by a mean terminal half- life of 45 ( ⁇ 17) hours (range 16 to 69 hours).
  • the rifabutin derivative RTI-79 is expected to present similar results. Maximal RTI-79 plasma concentration is reached within 3 hours of administration. Accordingly, appropriate dosages for variations of this example using intravenously injected rifabutin or RTI-79 rather than orally administered forms can be calculated.
  • rifamycin or a rifamycin derivative such as rifabutin or RTI-79
  • rifamycin or a rifamycin derivative can be administered in a method that matches the pharmokinetics of the rifamycin or rifamycin derivative to that of the anti-angiogenic agent also administered to the patient.
  • composition comprising rifamycin, rifamycin derivative, such as rifabutin or a rifabutin derivative, rifampicin or rifampicin derivative, or pharmaceutically acceptable salt, hydrate, or prodrug thereof!, or combination thereof, such as rifabutin or RTI-79, can be administered in amounts similar to those described herein to reduce or prevent metastatic transformation of tumors due to angiogenesis.

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Abstract

La présente invention concerne une composition comprenant un dérivé de rifamycine ou un sel, hydrate ou promédicament pharmaceutiquement acceptables de celui-ci. La composition comprend une quantité et une formulation du dérivé de rifamycine ou d'un sel, hydrate, ou promédicament pharmaceutiquement acceptables de celui-ci suffisantes pour inhiber l'angiogenèse et/ou la lymphangiogenèse dans un organisme et/ou suffisantes pour induire une sensibilisation médicamenteuse dans une cellule cancéreuse dans l'organisme ou une inhibition de ladite cellule cancéreuse dans l'organisme.
PCT/US2015/015569 2014-02-14 2015-02-12 Compositions et procédés d'inhibition de l'angiogenèse et la lymphangiogenèse WO2015123392A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110059136A1 (en) * 2008-02-08 2011-03-10 Thomas Julius Borody Methods and Compositions for Treating Inflammatory Bowel Disease
US20140314878A1 (en) * 2012-08-30 2014-10-23 James Sacchettini Compositions and methods for drug-sensitization or inhibition of a cancer cell

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110059136A1 (en) * 2008-02-08 2011-03-10 Thomas Julius Borody Methods and Compositions for Treating Inflammatory Bowel Disease
US20140314878A1 (en) * 2012-08-30 2014-10-23 James Sacchettini Compositions and methods for drug-sensitization or inhibition of a cancer cell

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHICHIRI ET AL.: "Rifampicin as an Oral Angiogenesis Inhibitor Targeting Hepatic Cancers", CANCER RESEARCH, vol. 69, no. 11, 2009, pages 4760 - 4768, XP055009288, Retrieved from the Internet <URL:http://cancerres.aacrjournals.org/content/69/11/4760.full> [retrieved on 20150323] *

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