This invention relates to an enhanced molecular-targeted approach that targets treatment-induced or native hypoxia present within cancers, specifically, but not limited to the treatment of metastatic prostate cancer with androgen deprivation therapy, such as anti-androgens. The invention describes the utility of combined inhibition of IL-8 and VEGF signalling to effect a combined therapeutic response of malignant disease, which is magnified under conditions of hypoxia.
Inhibition of androgen signalling, through the use of anti-androgens or androgen receptor (AR)-targeted agents, remains the mainstay of therapeutic intervention in castrate-resistant prostate cancer (CRPC). The AR-targeted agent Enzalutamide (MDV3100) blocks androgen binding, inhibits AR nuclear translocation and prevents androgen-driven gene expression, yet provides only a modest overall survival benefit in “castrate-resistant” patients. The development of resistance has been attributed to the retention of active AR signalling, even in androgen-depleted conditions, as a result of amplification/over-expression of the AR gene, elevated levels of intra-prostatic androgen synthesis, or through the acquisition of mutations or expression of AR splice variants. However, the impact of the tumour microenvironment has rarely been considered in the context of castrate-resistance.
Anti-androgen therapy profoundly influences the microenvironment of the prostate gland. Loss of androgen signalling rapidly decreases prostatic blood flow and reduces microvessel density (MVD) of the prostate gland in rats. Rapid vascular atrophy and reduced MVD have also been observed in engrafted human prostate tumours following androgen withdrawal, caused-in-part by vascular endothelial cell apoptosis. Similarly, we previously demonstrated that administration of Bicalutamide, a clinically-approved AR antagonist induces a rapid and sustained hypoxia, followed by a secondary period of neo-angiogenesis in xenograft models.
Although treatment with AR-targeted agents effectively reduces proliferation of prostate cancer cells, treatment with Bicalutamide and Enzalutamide has been shown to increase their rate of invasion in vitro and potentiate the development of distant metastases in vivo. We have adopted the hypothesis that the development of treatment-associated hypoxia and its ensuing intracellular signalling may define a further mechanism of relapse to AR-targeted therapy and accelerate the outgrowth and dissemination of CRPC.
Therefore, the present invention has been developed with a view to providing an improved AR-targeted therapy by also targeting hypoxia-induced pro-angiogenic and/or pro-survival factors.
Accordingly, in one aspect, the invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
The anticancer therapy may be therapy with a chemotherapeutic agent (chemotherapy) and/or radiotherapy. Optionally, the anticancer therapy is a chemotherapeutic agent and/or radiotherapy which cause one or more areas of hypoxia within a cancer treated by said chemotherapeutic agent and/or radiotherapy.
Optionally, the anticancer therapy is androgen deprivation therapy. Optionally, the androgen deprivation therapy causes one or more areas of hypoxia within a cancer treated by said androgen deprivation therapy. Optionally, the androgen deprivation therapy comprises treatment with an anti-androgen and/or an androgen signalling inhibitor. Preferably, the anti-androgen is an androgen receptor antagonist, such as enzalutamide (MDV3100).
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
In another aspect, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
In another aspect, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
In another aspect, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a method of treating cancer, comprising administering a pharmaceutical combination comprising:
In another aspect, the present invention provides a method of treating cancer in a patient receiving an anticancer therapy, optionally a chemotherapeutic agent, radiotherapy or an androgen deprivation therapy, the comprising:
Optionally, the present invention provides a method of treating cancer, the comprising:
Optionally, the present invention provides a method of treating cancer, the comprising:
Optionally, the method comprises administering an androgen receptor antagonist to antagonise the androgen receptor. Optionally, the method comprises administering a VEGF signalling inhibitor to inhibit the signalling effects of VEGF. Optionally, the method comprises administering an IL-8 signalling inhibitor to inhibit the signalling effects of IL-8.
In another aspect, the present invention provides a use of a pharmaceutical combination comprising:
Optionally, the present invention provides a use of a pharmaceutical combination comprising:
Optionally, the present invention provides a use of a pharmaceutical combination comprising:
Optionally, the present invention provides a use of a pharmaceutical combination comprising:
In another aspect, the present invention provides a use of a pharmaceutical combination comprising:
Optionally, the present invention provides a use of a pharmaceutical combination comprising:
Optionally, the present invention provides a use of a pharmaceutical combination comprising:
Optionally, the present invention provides a use of a pharmaceutical combination comprising:
In another aspect, the present invention provides a pharmaceutical combination for use in potentiating a therapeutic effect of an anti-cancer therapy in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in potentiating a therapeutic effect of a chemotherapeutic agent in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in potentiating a therapeutic effect of radiotherapy in the treatment of cancer, the pharmaceutical combination comprising:
Optionally, the present invention provides a pharmaceutical combination for use in potentiating a therapeutic effect of an androgen deprivation therapy, optionally an androgen receptor antagonist such as enzalutamide (MDV3100), in the treatment of cancer, the pharmaceutical combination comprising:
In another aspect, the present invention provides a method for potentiating a therapeutic effect of an anti-cancer therapy in the treatment of cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a method for potentiating a therapeutic effect of a chemotherapeutic agent in the treatment of cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the present invention provides a method for potentiating a therapeutic effect of an androgen deprivation therapy, optionally an androgen receptor antagonist such as enzalutamide (MDV3100), in the treatment of cancer, comprising administering a pharmaceutical combination comprising:
Optionally, the cancer is a cancer characterised by one or more areas of hypoxia within the cancer, i.e. within the tumour mass. Without wishing to be bound by theory, it is understood that the hypoxic zones within the cancer may arise as a result of uncontrolled proliferation outstripping nutrient supply from the vasculature or may arise from disruption to the tumour vasculature following administration of a therapeutic agent or a vascular-disrupting drug. Optionally, the cancer is a cancer characterised by increased expression of VEGF and/or IL-8. Optionally, the cancer is selected from one or more of prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, glioblastoma, lung cancer or gastric cancer. In particular, the cancer is selected from prostate cancer or breast cancer.
Optionally, the cancer is refractory, or substantially refractory, to treatment with a chemotherapeutic agent, radiotherapy and/or androgen deprivation therapy, such as androgen receptor antagonists and/or androgen signalling inhibitors. Optionally, the cancer is refractory, or substantially refractory, to treatment with enzalutamide (MDV3100).
Optionally, the prostate cancer is hormone-naïve, hormone-sensitive or castrate-resistant prostate cancer. Optionally, the prostate cancer is castrate-resistant prostate cancer, which cancer is known to be treated with androgen receptor antagonists and/or androgen signalling inhibitors. Alternatively, the prostate cancer is non-castrate prostate cancer. With regard to non-castrate prostate cancer, earlier treatment of this cancer with androgen signalling inhibitors has been undertaken in view of results from the STAMPEDE and LATITUDE Clinical Trials. Optionally, the prostate cancer is refractory, or substantially refractory, to treatment with a chemotherapeutic agent, radiotherapy and/or androgen deprivation therapy, such as androgen receptor antagonists and/or androgen signalling inhibitors. Optionally, the prostate cancer is refractory, or substantially refractory, to treatment with enzalutamide (MDV3100). Optionally, the present invention has utility in the treatment of localized prostate cancer, as well as in patients with no confirmed evidence of distant metastasis. Optionally, the pharmaceutical combination of the invention is administered to prostate cancer within primary site of the cancer or to an extra-prostatic site.
Optionally, the chemotherapeutic agent is selected from one or more of FOLFOX (folinic acid, fluorouracil and oxaliplatin) combination therapy, which chemotherapeutic agent is suitable for the treatment of metastatic colorectal cancer, Sunitinib or Lenalidomide, which chemotherapeutic agents have been used in the treatment of castrate-resistant prostrate cancer.
Optionally, the radiotherapy is selected from one or more of external beam radiation therapy, brachytherapy (sealed source radiotherapy), unsealed source radiotherapy (systemic radioisotope therapy), intraoperative radiotherapy, deep inspiration breath-hold radiotherapy or radionuclide therapy (e.g. radium-223).
Optionally, the androgen receptor antagonist is selected from one or more of enzalutamide (MDV3100), Apalutamide (ARN-509), Bicalutamide (Casodex), or Darulutamide.
Optionally, the androgen signalling inhibitor is selected from abiraterone-acetate, finasteride, dutasteride, leuprolide or gooserelin.
Optionally, the androgen receptor antagonist, such as enzalutamide, is to be administered at a pharmaceutically effective amount. Optionally, the androgen receptor antagonist, such as enzalutamide, is to be administered at a pharmaceutically effective amount of about 120-200 mg/day, optionally about 160 mg/day, via oral ingestion.
Optionally, radiotherapy is used in combination with the androgen deprivation therapy.
Administration of the androgen receptor antagonist may be by any suitable method known in the art, including subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intranasal, or oral routes of administration. Preferably, the androgen receptor antagonist, such as enzalutamide, is for administration by the oral route of administration.
Optionally, the VEGF signalling inhibitor described herein comprises an antibody suitable for binding VEGF. Optionally, the antibody suitable for binding VEGF is a neutralising anti-VEGF antibody (VEGF nAb), exemplified by Avastin® (bevacizumab). It will be understood that a neutralising anti-VEGF antibody is an antibody that is capable of binding, optionally specifically binding, to a VEGF molecule and preventing or inhibiting the biological activity of the VEGF molecule. The preventing or inhibiting the biological activity of the VEGF molecule includes preventing or inhibiting the interaction of the VEGF molecule with its corresponding VEGF receptor, and/or preventing or inhibiting the signalling activity of the VEGF molecule such as through blockade of VEGR receptor (VEGFR1 and/or VEGFR2) activation. Activation of the receptor may be prevented by use of an antibody that binds, optionally specifically binds, to one or more epitopes on the receptor that prevent the binding of the natural (VEGF) ligand to its binding pocket on the receptor to promote activation.
Optionally, the VEGF signalling inhibitor described herein comprises an inhibitor or antagonist of the VEGF receptor (such as VEGFR1 and/or VEGFR2). Optionally, this may be an agent selected from one or more of Cediranib, Lenvantinib, Pazopanib, or Regorafenib. In the case of Pazopanib, this may be administered at a dosing range of about 100 mg/day to 1000 mg/day, optionally about 200 mg/day to 800 mg/day, through oral administration (e.g. tablet).
Optionally, the VEGF signalling inhibitor is to be administered at a pharmaceutically effective amount. In the case of Avastin® (bevacizumab), the VEGF signalling inhibitor may be administered at a pharmaceutically effective amount of about 10-20 mg/kg every 2-4 weeks, optionally about 15 mg/kg every 3 weeks, as an intravenous infusion.
Administration of alternate VEGF signalling inhibitors may be by any suitable method known in the art, including subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intranasal, or oral routes of administration.
Optionally, the IL-8 signalling inhibitor described herein comprises an antibody suitable for binding IL-8. Optionally, the antibody suitable for binding IL-8 is a neutralising anti-IL-8 antibody (IL-8 nAb).
It will be understood that a neutralising anti-IL-8 antibody is an antibody that is capable of binding, optionally specifically binding, to an IL-8 molecule and preventing or inhibiting the biological activity of the IL-8 molecule. The preventing or inhibiting the biological activity of the IL-8 molecule includes preventing or inhibiting the interaction of the IL-8 molecule with its corresponding IL-8 receptor, and/or preventing or inhibiting the signalling activity of the IL-8 molecule such as through blockade of IL-8 receptor activation. Activation of the receptor may be prevented by use of an antibody that binds, optionally specifically binds, to one or more epitopes on the receptor that prevent the binding of the natural (IL-8) ligand to its binding pocket on the receptor to promote activation.
Optionally, the IL-8 signalling inhibitor described herein comprises an inhibitor or antagonist of the IL-8 receptor (such as CXCR1 and/or CXCR2), optionally a chemical species that acts as an inhibitor or antagonist of the IL-8 receptor, with unspecified selectivity to optionally block activation of the CXCR1 (IL8RA) and/or the CXCR2 (IL8RB) receptor. Several species have been developed including the use of agents like AZD5069 or SCH527123. Optionally, the inhibitor or antagonist of the IL-8 receptor comprises a selective and/or non-selective small molecule or peptide/peptidomimetic inhibitor of CXCR1 and/or CXCR2.
Optionally, the IL-8 signalling inhibitor is suitable to prevent or inhibit the activation of the CXCR1 (IL8RA) or CXCR2 (IL8RB) receptor. Optionally, the IL-8 signalling inhibitor comprises an antibody suitable for binding CXCR1 and/or CXCR2. It will be understood that a neutralising antibody to either CXCR1 or CXCR2 is an antibody that is capable of binding, optionally specifically binding, to the receptor protein and preventing or inhibiting the biological activity of the receptor. The preventing or inhibiting the biological activity of the specified receptors includes preventing or inhibiting the interaction of the IL-8 molecule or its associated ligands (CXCL1, CXCL2, CXCL5, CXCL6, CXCL8) with its corresponding receptor, and/or preventing or inhibiting the signalling activity of any of the associated ligands (CXCL1, CXCL2, CXCL5, CXCL6, CXCL8).
Optionally, the IL-8 signalling inhibitor is to be administered at a pharmaceutically effective amount and through acceptable routes of administration.
Administration of the IL-8 signalling inhibitor may be by any suitable method known in the art, including subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intranasal, or oral routes of administration. Preferably, the IL-8 signalling inhibitor, such as a neutralising anti-IL-8 antibody, is for administration by the oral route of administration.
Optionally, the androgen receptor antagonist, VEGF signalling inhibitor, and IL-8 signalling inhibitor, of the pharmaceutical combination may be administered concurrently, consecutively, simultaneously, or at different times. Optionally, the androgen receptor antagonist, VEGF signalling inhibitor, and IL-8 signalling inhibitor, of the pharmaceutical combination may be administered together in a single pharmaceutical composition, or separately in separate pharmaceutical compositions.
In another aspect, the present invention provides a method of treating cancer, comprising administering a chemotherapeutic agent used in the treatment of the cancer, in combination with a vascular endothelial growth factor (VEGF) signalling inhibitor, and an interleukin-8 (IL-8) signalling inhibitor, to a patient suffering from cancer. Optionally, the treatment schedule may be realised by administration of an anti-IL-8 signalling inhibitor and an anti-VEGF signalling inhibitor, as described above, at the level to prevent inhibit signalling by the IL-8 and VEGF ligands, or sequester the IL-8 and VEGF ligands, or by preventing or inhibiting the activation of the corresponding receptors for the IL-8 and VEGF ligands.
In another aspect, the present invention provides a method of treating cancer, comprising administering radiotherapy in the treatment of the cancer, in combination with a vascular endothelial growth factor (VEGF) signalling inhibitor and an interleukin-8 (IL-8) signalling inhibitor, to a patient suffering from cancer. Optionally, the treatment schedule may be realised by administration of an anti-IL8 signalling inhibitor and an anti-VEGF signalling inhibitor, as described above, at the level to prevent inhibit signalling by the IL-8 and VEGF ligands, or sequester the IL-8 and VEGF ligands, or by preventing or inhibiting the activation of the specified receptors for the IL-8 and VEGF ligands.
In another aspect, the present invention provides a method of treating cancer, comprising administering androgen deprivation therapy in the treatment of the cancer, in combination with a vascular endothelial growth factor (VEGF) signalling inhibitor and an interleukin-8 (IL-8) signalling inhibitor, to a patient suffering from cancer. Optionally, the treatment schedule may be realised by administration of an anti-IL8 signalling inhibitor and an anti-VEGF signalling inhibitor, as described above, at the level to prevent inhibit signalling by the IL-8 and VEGF ligands, or sequester the IL-8 and VEGF ligands, or by preventing or inhibiting the activation of the specified receptors for the IL-8 and VEGF ligands.
In another aspect, the present invention provides a method of treating cancers that have zones of hypoxia, comprising treatment with a chemotherapeutic agent, radiotherapy or an androgen deprivation therapy, in combination with a vascular endothelial growth factor (VEGF) signalling inhibitor and an interleukin-8 (IL-8) signalling inhibitor, to a patient suffering from cancer. Optionally, the treatment schedule may be realised by administration of an anti-IL8 signalling inhibitor and an anti-VEGF signalling inhibitor, as described above, at the level to prevent inhibit signalling by the IL-8 and VEGF ligands, or sequester the IL-8 and VEGF ligands, or by preventing or inhibiting the activation of the specified receptors for the IL-8 and VEGF ligands.
The term “pharmaceutical combination” includes a combination of two or more therapeutic compositions or procedures suitable for the treatment of cancer. Thus, it will be understood, that administration of said therapeutic compositions to a patient, or carrying out said therapeutic procedures on a patient, may occur concurrently, consecutively, simultaneously, or at different times.
The term “antibody” includes a molecule from the subgroup of gamma globulin proteins which is also referred to as the immunoglobulins (Ig). Antibodies can, preferably, be of any subtype, i.e. IgA, IgD, IgE, IgM or, more preferably, IgG. Antibodies immobilised on particle carriers as described herein can be prepared by well-known methods using a purified polypeptide or a suitable fragment derived therefrom as an antigen. A fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Such fragments may be obtained either by proteolytic digestion from target protein(s) or may be a synthetic peptide(s). Preferably, the antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human or humanized antibody or primatized, chimerized or fragment thereof. Optionally, the antibody is a an antibody fragment, such as Fab, Fab′, (Fab′)2, Fv, scFv, Bis-scFv, minibody, Fab2, or Fab3 fragments, or a chemically modified derivative of any of these. An antibody of the present invention preferably binds specifically (i.e. does not cross react with other polypeptides or peptides) to a target protein(s) such as VEGF and/or IL-8. Specific binding can be tested by various well known techniques.
The terms “specific binding,” “specifically binds,” and similar may be understood to mean that an antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant cross-reactivity with other antigens and epitopes. “Appreciable” or preferred binding includes binding with an affinity of at least (KD equal to or less than) 10−6 M, 10−7 M, 10−8 M, 10−9 M, 10−10 M, or 10−11 M. An antibody that “does not exhibit significant crossreactivity” is one that will not appreciably bind to an undesirable entity (e.g., an undesirable proteinaceous entity). An antibody specific for a particular epitope will, for example, not significantly crossreact with other epitopes on the same protein or peptide. Specific binding can be determined according to any well known means for determining such binding. In some embodiments, specific binding is determined according to Scatchard analysis and/or competitive binding assays.
The term “inhibit” may be understood to mean a decrease of a biological activity. “Biological activity” includes inter- and intra-cellular signalling, transduction of a signal, etc. Inhibiting biological activity includes decreases the activity by 50%, 60%, 70%, 80%, 90%, 95% or 100% of the normal, uninhibited activity.
“Therapeutically effective amount” may be understood to mean a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). Efficacy can be measured in conventional ways, depending on the condition to be treated. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP), or determining the response rates (RR). Therapeutically effective amount also refers to a target serum concentration, such as a trough serum concentration, that has been shown to be effective in suppressing disease symptoms when maintained for a period of time.
The phrase “treatment of cancer”, and the like, includes treatment of cancer to reduce tumour volume and/or reduce the rate of tumour growth. Tumour volume may be measured before or at the point of initial treatment and then again at any time after the treatment has begun, e.g. at day 28.
“Potentiate” may be understood to mean that administration of the VEGF inhibitor, and optionally IL-8 inhibitor, enhances or extends the therapeutic activity of the androgen receptor antagonist and/or results in a decreased amount of androgen receptor antagonist being required to produce a therapeutic effect. Thus, as will be understood, the therapeutically effective concentration of androgen receptor antagonist included in the pharmaceutical combinations of the present invention may be decreased as compared to an established effective, or ineffective, concentration for the androgen receptor antagonist when administered alone.
The term “patient” can include human and other mammalian subjects that receive the therapeutic treatment disclosed herein.
“Cancer” can include tumours, or neoplasms, which are benign, pre-malignant, or malignant. “Prostate cancer” can include carcinomas, including, carcinoma in situ, invasive carcinoma, metastatic carcinoma and pre-malignant conditions.
By “refractory”, and “substantially refractory”, it may understood that a cancer, optionally a prostate cancer, does not respond to (or is resistant to) treatment with an anti-cancer therapy, such as an androgen receptor antagonist and/or an androgen signalling inhibitor, or becomes unresponsive over time.
The androgen receptor (AR), also known as NR3C4 (nuclear receptor subfamily 3, group C, member 4), is a type of nuclear receptor that is activated by binding either of the androgenic hormones, testosterone or dihydrotestosterone, in the cytoplasm and then translocating into the nucleus. AR expression is maintained throughout prostate cancer progression, and the majority of androgen-independent or hormone refractory prostate cancers express AR.
The androgen receptor antagonist, a VEGF signalling inhibitor, and an IL-8 signalling inhibitor which may be comprised in the pharmaceutical combination disclosed herein may further comprise a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of, for example, the USA or EU. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Pharmaceutical compositions comprising the androgen receptor antagonist, a VEGF signalling inhibitor, and/or an IL-8 signalling inhibitor can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, or intramuscular administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. If a composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. If a composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Actual dosage levels of the androgen receptor antagonist, VEGF signalling inhibitor, and IL-8 signalling inhibitor in the pharmaceutical combinations provided herein may be varied so as to obtain an amount of each of these components which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required. For example, the physician or veterinarian could start doses of the androgen receptor antagonist, VEGF, inhibitor, and IL-8 signalling inhibitor at levels lower than that required to achieve the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable daily dose of compositions provided herein will be that amount of the androgen receptor antagonist, VEGF signalling inhibitor, and IL-8 signalling inhibitor which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day.
By “about”, as used herein, it is meant that may be ±20% of the recited value, optionally±10% of the recited value, optionally±5% of the recited value, further optionally the value may be precisely the recited value.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, a reference to “an inhibitor” includes one or more inhibitors.
The embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:
Authenticated PC3 (ATCC CRL-1435), LNCaP (ATCC CRL-1740) and 22Rv1 (ATCC CRL-2505) cells were cultured as described in Seaton et al. (Carcinogenesis 2008; 29: 1148-56). C4-2B cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS). MDV3100-sensitive (LNCaP-Par) and resistant (LNCaP-EnzR) cells were obtained from Prof. Vander Griend, University of Chicago, Chicago, Ill., and were cultured as described in Kregel et al. (Oncotarget 2016; 7: 26259-74). HUVEC (ATCC CRL-1730) cells were maintained in Endothelial Cell Growth Medium (#CC-3162, Lonza, UK). For experiments involving hypoxia (0.1% 02), cells were cultured as previously reported (Maxwell et al., Oncogene 2007; 26: 7333-45). Human IL-8 monoclonal antibody (#MAB208), human VEGF mAb (#AB293NA) and isotype-matched IgG (#MAB002) were obtained from R&D Systems (UK). MDV3100 (#S1250) was obtained from Selleckchem (Germany). Cells were regularly tested to ensure they were free of mycoplasma contamination.
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (Maxwell et al., Oncogene 2007; 26: 7333-45).
AR activity was measured by luciferase reporter assay, as previously described (Seaton et al., Carcinogenesis 2008; 29: 1148-56).
siRNA Transfection
HIF1α (#M-004018-05) or RelA (#M-003533-02) were targeted using oligonucleotide pools (GE Dharmacon, CO, USA) as previously described (Maxwell et al., Oncogene 2007; 26: 7333-45). Non-targeting-(NT) transfections were at the same concentrations as the siRNAs.
Nucleic acid samples were prepared and analyzed as previously described (Maxwell et al., Oncogene 2007; 26: 7333-45; Maxwell et al., Eur Urol 2013; 64: 177-88). Primer sequences were as follows: AR: Forward, 5′-CGGAAGCTGAAGAAACTTGG-3′ (SEQ ID NO: 1); Reverse, 5′-CGTGTCCAGCACACACTACA-3′ (SEQ ID NO: 2); PSA/KLK3: Forward, 5′-TGAGCCTCCTGAAGAATCGA-3′ (SEQ ID NO: 3); Reverse, 5′-TTGCGCACACACGTCATT-3′ (SEQ ID NO: 4). Primer sequences for 18s, BCL2, CAIX, IL-8, and VEGF have been previously reported (Maxwell et al., Oncogene 2007; 26: 7333-45; Lekas et al., Urol Res 1997; 25: 309-14).
Immunoblotting was performed as previously described (McCourt et al., Clin Cancer Res 2012; 18:3822-33). Antibodies were obtained as follows: AR, Millipore (#PG-21, UK); AR-V7 (#ab198394) & PARP (#14-6667-82), Abcam (UK); Bcl-2 (#4223), Cell Signalling Technology (The Netherlands); c-FLIP (#AG-20B-0056), Adipogen (Switzerland); RELA(p65) (#sc-372), Santa Cruz (CA, USA); GAPDH (#MCA4740), Biorad (UK).
Secreted IL-8 (#M1918, Pelikine, Mast Group, UK), and VEGF (#DY293B, R&D Systems, UK) were measured by ELISA as per manufacturer's instructions.
Cells were seeded on coverslips, cultured under normoxic or hypoxic conditions, fixed, permeabilised, and blocked overnight. AR was visualized using primary anti-AR antibody (#PG-21, Millipore, UK) and Alexa fluor-568 secondary antibody (#A11036, Molecular Probes, UK). ProLong Gold Antifade Reagent with DAPI (#P36941, Thermo Scientific, UK) was used to visualize the nuclei. Cells were viewed under a Nikon Eclipse Ti—S fluorescent microscope and images captured using NIS-Elements software.
RNA-Seq analysis was performed as previously described (Yamamoto et al., Clin Cancer Res 2015; 21: 1675-87).
Cell cycle analysis was carried out by PI staining as previously described (Wilson et al., J Pharmacol Exp Ther 2008; 327: 746-59).
Immunohistochemistry was performed for AR (ab9474; Abcam, UK) and CD31 (ab28364; Abcam, UK) on 4 μm tissue microarray sections of formalin-fixed paraffin-embedded prostate cancer specimens. Standard processing steps for each antibody were in accordance with manufacturer's instructions. Briefly, heat-induced antigen retrieval with epitope retrieval ER1 solution (#AR9961, Leica Biosystems, UK) was performed prior to incubation with primary antibody. Slides were washed with Bond washing buffer (#AR9590, Leica Biosystems, UK) and incubated with secondary antibody (Bond Polymer Refine kit #DS9800; Leica Biosystems, UK). Subsequently chromogenic detection was achieved by incubation with 3,30-diaminobenzidine (DAB) followed by Bond DAB enhancer (#AR9432, Leica Biosystems, UK). All slides were counterstained with haematoxylin and dehydrated through ethanol to xylene before mounting.
The V2A angiogenesis assay (#ZHA-4000, Cellworks, UK) was carried out according to the manufacturer's instruction. Cells were allowed to settle for 4 days prior to treatment. Treatments were carried out in duplicate and were replenished every 2 days for 10 days. For treatments involving conditioned media, PC3 or LNCaP cells were treated as required for 24 h, media harvested and stored at −20° C. Vessels were visualized at 4× magnification. Vessel density was measured in four fields per well using AngioSys 2.0 software (Cellworks, UK).
Experimental metastasis assays were performed in chick embryos as described (Deryugina et al., Histochem Cell Biol 2008; 130: 1119-30). On day 12 of incubation, PC3 cells were injected directly into the blood circulation through an allantoic vein (1×105 cells in 0.1 mL serum-free medium per embryo). MDV3100 was delivered i.v. on days 1 and 2 after cell inoculations (0.1 mL of a 20 μM solution per embryo). On day 5, portions of the CAM were harvested to quantify the number of human tumour cells by human-specific Alu-qPCR as previously described (Deryugina et al., Cancer Res 2005; 65: 10959-69), using a standard curve generated by serial dilutions of human tumour cells within a constant number (1×106) of chick embryo fibroblasts.
In vivo experiments were conducted in accordance with the Animal (Scientific Procedures) Act 1986 and the UKCCCR Guidelines (2010) for the welfare of animals in experimental neoplasia.
MDV3100 (in 0.1% DMSO in corn oil) was administered orally (p.o). Vehicle control (VC) or MDV3100 (4 mg/kg, equivalent to 100 mg/day in men) was administered daily. Human IL-8 and VEGF nAbs were administered 3×/week via intraperitoneal (i.p.) injection, at a final concentration of 50 μg/ml and 100 μg/ml, respectively. IgG control was at a final concentration of 150 μg/ml.
LNCaP cells (1×107 in Matrigel) were implanted on the rear dorsum of 8-10-week-old male Balb/c SCID mice. When tumour volume was 150-200 mm3, mice were randomly assigned to treatment groups (3-5 per group). Tumour dimensions were measured as previously described (9). Mice were sacrificed at day 28 or day 35. Tumour growth over 28 days (% T/C) was calculated using the equation: % T/C=(mean tumour volume on day 28−mean starting volume)/(mean control tumour volume on day 28−mean control starting volume)×100.
A viewing chamber was attached to a raised skin flap on the dorsal surface of the mouse (Balb/c SCID) and a fragment (˜0.5 mm) of LNCaP tumour was placed on the microvascular as previously described (9). Tumour vasculature was imaged weekly/4 weeks using a stereomicroscope. Image analysis was carried out using Touptek software (Touptek Photonics, China).
Tumour oxygenation was measured once weekly as previously described (Ming et al., Int J Cancer 2013; 132:1323-32).
Data was analyzed using GraphPad Prism software. Statistical significance between groups was determined using a two-tailed Student's t-test, Mann Whitney U-test or 2-way ANOVA with Bonferroni post-tests, as appropriate.
Anti-androgen therapy is associated with reductions in MVD and tumour oxygen levels while expression of the pro-angiogenic factors IL-8 and VEGF-A (VEGF) is altered in response to Bicalutamide-promoted hypoxia in vivo. The intent of this study was to examine the importance of these treatment-associated, hypoxia-inducible factors in modulating the oxygen tension and the vascularity of the microenvironment following Enzalutamide (MDV3100) therapy.
Tumour oxygenation levels were studied in an LNCaP xenograft model subjected to MDV3100 administration. Intra-tumoral oxygen levels were measured at pre-determined intervals (
Using a dorsal skin flap (DSF) model, we assessed the percentage area covered by tumour vessels in all treatment groups (
To further understand the events contributing to MDV3100-promoted hypoxia, we studied the direct effect of MDV3100 administration upon vascular endothelial cells using two independent i assays. In agreement with previous studies, we detected AR mRNA in HUVEC endothelial cells by qPCR analysis (
The effect of MDV3100 on blood vessel formation was examined. MDV3100 significantly impaired the development of branching junctions detected by an in vitro vascular tubule formation assay conducted over 10 days (
Hypoxia Potentiates and Sustains AR Expression and Activation in Prostate Cancer Cells The effect of hypoxia in modulating the AR signalling pathway was determined in vitro. Exposure of LNCaP cells to hypoxia over a 24 h time-course increased AR mRNA (
The mechanism underpinning hypoxia-induced AR expression was investigated further. Promotion of HIF-1 and NF-κB-driven transcription is a well-established response to hypoxia. Attenuation of p65RelA-driven transcription using siRNA reduced AR mRNA expression, AR protein expression and decreased PSA/KLK3 mRNA expression in LNCaP cells (
The effect of environmental hypoxia and its potentiation of AR signalling upon the pharmacology of MDV3100 was explored by initial experiments conducted on LNCaP cells cultured under normoxic or hypoxic conditions. Cells were treated with MDV3100 for 72 h prior to measurement of cell viability. While MDV3100 reduced the viability of cells cultured in normoxia relative to the DMSO control (p<0.05), the same concentration of MDV3100 was ineffective in LNCaP cells subjected to hypoxic conditions (
We postulated that treatment-induced hypoxia would have the potential to regulate expression of multiple genes regulating diverse biological processes including angiogenesis (VEGF), metabolic adaptation (carbonic anhydrase IX(CAIX)) and cell survival (BCL2). qPCR analysis on hypoxic LNCaP cells confirmed the induction of each of these genes and demonstrated the important contribution of HIF-1α and RelA (NF-κB) signalling to their up-regulation under hypoxia (
VEGF and IL-8 represent a prototypical growth factor and chemokine, respectively, inducing a plethora of signalling responses in malignant and non-malignant cells. We conducted further experiments using neutralizing antibodies against each of these factors to determine the biochemical significance of hypoxia-induced VEGF and IL-8 in sustaining hypoxia-induced gene transcription in prostate cancer cells. Administration of either the anti-IL-8 or anti-VEGF nAbs reduced hypoxia-promoted IL-8 and VEGF mRNA expression (
The importance of these signalling factors to survival under hypoxic conditions was determined by cell viability analysis. We observed that inhibition of VEGF signalling, either alone or in combination with the anti-IL-8 nAb significantly reduced the number of viable hypoxic LNCaP cells but not 22Rv1 cells (
VEGF and IL-8 are known to be associated with promotion of angiogenesis. Using an in vitro tubule formation assay, we observed that administration of VEGF directly stimulated endothelial cell tubule formation while addition of exogenous rh-IL-8 was less potent in stimulating tubule formation in this assay (
To further characterize the importance of these specific pro-angiogenic factors in modulating the response of MDV3100-treated tumours, we examined tumour growth dynamics of an LNCaP xenograft model (
Tumours were measured 3×/week using digital calipers. The length and width of the tumour was measured and the volume calculated using the formula volume=(length×width 2)/2. *, numbers in brackets show % T/C compared to enzalutamide alone at day 28; **, control is enzalutamide alone.
Analysis was performed using a 2-way ANOVA with Bonferroni post-tests, comparing the statistical significance of measured observations between treatment groups. ****, p<0.0005
Average tumour weights were also recorded at the end of the study (
We next sought to determine whether elevated expression of IL-8 and VEGF may be detected in other experimental models of MDV3100-resistance. Elevated expression of VEGF and IL-8 mRNA expression was initially detected through analysis of a RNA-seq database characterizing gene expression in two MDV3100 resistant prostate cancer cell lines, MR49C and MR49F, originally derived through serial in vivo passaging of LNCaP xenografts treated with MDV3100 (
The use of new generation AR antagonists including MDV3100 remains a primary treatment strategy for men with de novo systemic and advanced castrate-resistant disease. However, the development of resistance presents a major clinical problem. The present study supports the proposal that resistance to AR-targeted agents including MDV3100 may arise at least in part from development of treatment-induced hypoxia. Our in vitro and in vivo experimental data demonstrate a potent cytotoxic effect of MDV3100 on AR-expressing vascular endothelial cells and the inhibition of tubule formation, which is consistent with the observed rapid reduction in intra-tumoral oxygen levels. The ensuing hypoxia reduces the therapeutic sensitivity of prostate cancer cells to MDV3100, mediated in part through augmenting and sustaining AR expression and signalling in hypoxic conditions. Moreover, it is well established that hypoxia also induces activation of the HIF and NF-κB gene transcription pathways that underpin many hallmarks of advanced, treatment-refractory cancer. We and others have demonstrated a link between HIF-1 and/or NF-κB and AR expression and activity. Therefore we propose that the activation of these pathways provides a signalling bypass and escape mechanism to any residual on-target AR inhibition afforded by MDV3100 in hypoxic cells. For example, the hypoxia-driven, NF-κB-mediated increase in expression of the anti-apoptotic protein Bcl-2 coincides with the reduced sensitivity of hypoxic cells to MDV3100 in vitro and in vivo.
In proposing that the “off-target” hypoxia-driven changes may underpin escape from MDV3100 therapy, our further experiments focused on establishing the critical importance of two key hypoxia-regulated signalling factors in the acquisition of MDV3100 resistance. Evidence supporting the role of IL-8 and VEGF in treatment resistance is supported by our demonstration of elevated expression of these factors in LNCaP-derived MDV3100-resistant experimental models. Our data clearly show that concurrent inhibition of IL-8 and VEGF is superior in reducing MDV3100-induced expression of hypoxia-induced angiogenesis and pro-survival gene expression in vitro, in attenuating the restoration of oxygen tension in vivo, and in suppressing tumour growth and prolonging sensitivity of prostate cancer xenografts to MDV3100 therapy. Furthermore, we have shown that the concurrent repression of IL-8 and VEGF signalling can restore the sensitivity of hypoxic prostate cancer cells to MDV3100 in vitro, in part by decreasing Bcl-2 and FLIP expression and increasing MDV3100-associated apoptosis induction. Moreover, we have shown that the co-administration of anti-VEGF and anti-IL-8 nAbs restores the sensitivity of MDV3100-refractory cell lines of a LNCaP origin.
Phase III trials of anti-angiogenic compounds including Bevacizumab, Sunitinib and Lenalidomide have been discontinued due to poor efficacy in metastatic CRPC. The clinical failure of these highly-selective anti-angiogenic agents in heterogeneous disease such as CRPC may arise due to their narrow target profile and the subsequent inability to target other key factors that play a role in regulating the tumour response to hypoxia. Specifically, the proto-typical ELR+CXC-chemokine IL-8 is a potent mediator of angiogenesis, has been shown to underpin the angiogenic response of HIF-1α-deficient/VEGF-depleted DLD-1 colon cancer xenografts and mediate the resistance of head and neck squamous cell carcinoma to bevacizumab and sunitinib in renal cell carcinoma models. IL-8 expression is clinically-relevant in prostate cancer and correlates not only with microvessel density but also with shorter time-to-progression for patients being treated with hormone therapy. As demonstrated by our pre-clinical data, co-targeting of both VEGF and IL-8 signalling enhanced the MDV3100-mediated tumour control in the LNCaP xenograft model, and effectively retarded revascularization of MDV3100-treated tumours in vivo. Furthermore, co-targeting IL-8 and VEGF signalling afforded the greatest suppression of hypoxia-induced signalling and gene expression in vitro, and restored the sensitivity of hypoxic LNCaP and MDV3100 resistant LNCaP-EnzR cells to MDV3100. In totality, our data suggests that in addition to suppressing vascular responses within the tumour, the combination of anti-IL-8 and anti-VEGF nAbs also has direct impacts on adaptive MDV3100 resistance intrinsic to the tumour cell, including the repression of survival signals. It is important to note that this effective control of LNCaP tumour growth was observed using an anti-human IL-8 nAb, which restricts its action in this model to targeting only the IL-8 secreted from the implanted human tumour cells. Consequently, the actions of hypoxia-induced murine CXC-chemokine ligands (CXCL1, CXCL2 and CXCL5) originating from murine stromal and vascular endothelial cells remains intact in this experimental model and may provide some degree of functional compensation. Administration of pharmacological agents that provide total disruption of hypoxia-induced ELR+ve chemokine signalling within the prostate tumour microenvironment may yet provide even more impressive results. More importantly, our data reveal that the use of “broad-spectrum” and “multi-targeted anti-angiogenic therapies” that target both chemokines and growth factors is essential in order to derive the greatest impact in potentiating MDV3100 response in CRPC.
We conclude that treatment-induced intra-tumoral hypoxia upregulates the expression of pro-angiogenic and pro-survival factors VEGF and IL-8 which facilitate the acquisition and outgrowth of MDV3100-resistant prostate tumours. Our demonstration that combined VEGF/IL-8 inhibition significantly potentiates the prostate tumours to MDV3100 in vivo and increases sensitivity of MDV3100-resistant models provides new insights into the complex nature and multi-targeted therapeutic regimens that may be required to prolong the clinical benefit of next generation AR-antagonists in men with advanced, life-threatening prostate cancer.
The invention is not limited to the embodiments described herein but can be amended or modified without departing from the scope of the present invention as defined by the appended claim set.
Number | Date | Country | Kind |
---|---|---|---|
1713936.1 | Aug 2017 | GB | national |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2018/073502 | 8/31/2018 | WO | 00 |