WO2017103625A1 - Traitement contre le cancer - Google Patents

Traitement contre le cancer Download PDF

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Publication number
WO2017103625A1
WO2017103625A1 PCT/GB2016/053991 GB2016053991W WO2017103625A1 WO 2017103625 A1 WO2017103625 A1 WO 2017103625A1 GB 2016053991 W GB2016053991 W GB 2016053991W WO 2017103625 A1 WO2017103625 A1 WO 2017103625A1
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Prior art keywords
fabp5
inactivated
cancer
variant
fragment
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PCT/GB2016/053991
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English (en)
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Youqiang KE
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The University Of Liverpool
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Publication of WO2017103625A1 publication Critical patent/WO2017103625A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Definitions

  • the present invention relates to an inactivated FABP5 protein, or fragment or variant thereof, for use as a medicament.
  • the invention also relates to an inactivated FABP5 protein, or fragment or variant thereof, for use in the treatment of a FABP5 mediated disorder, or for use in the treatment of cancer. Further, the invention relates to methods of treating FABP5 mediated disorders, and methods of treating cancer using such proteins, fragments or variants.
  • the invention also relates to an inactivated FABP5 protein, or fragment or variant thereof, for use in therapeutically reducing PPARy activation, as well as to a method of therapeutically reducing PPARy activation using an inactivated FABP5 protein, or fragment or variant thereof.
  • the invention also relates to a pharmaceutical composition comprising an inactivated FABP5 protein, or a fragment or variant thereof.
  • Prostate cancer is a serious health threat to man, particularly in the developed countries.
  • High dietary fat intake has been shown to have a significant correlation with a higher risk of prostate cancer.
  • high levels of trans-isomers of oleic and linoleic acids (long chain fatty acids) in blood are also associated with an increased risk of prostate tumours.
  • Fatty acids are not only a source of energy, they are also potent signalling molecules involved in metabolic regulation. Their regulatory effects on enzymatic and transcriptional networks can lead to alterations in gene expression, cell growth and survival pathways, as well as alterations in inflammatory responses.
  • Fatty acid binding proteins are known as intracellular chaperones of lipids. They reversibly bind hydrophobic ligands (such as saturated and unsaturated fatty acids) with high affinity and transport them into the cells. The transportation of these hydrophobic ligands into the cells may result in increased activation of nuclear receptors, such as peroxisome proliferator-activated receptor gamma (PPARy).
  • FABP5 is a 15 kDa cytosolic protein which belongs to the FABP family. In addition to the skin, FABP5 is detected in endothelial cells of placenta, heart, skeletal muscle, small intestine, renal medulla and in the lung's Clara and goblet cells.
  • FABP5 has been implicated in inflammatory and metabolic diseases, as well as in malignancies of the prostate, bladder, pancreas, breast, and glioblastoma.
  • An overexpression of FABP5 has been demonstrated in malignant prostate and breast cell lines compared to their benign counterparts and the increased level of FABP5 has been shown to induce metastasis in vivo. It has also been shown that metastasis-inducing activity of FABP5 is achieved by up-regulating VEGF.
  • Suppression of FABP5 expression in a highly malignant prostate cancer cell line PC3-M significantly reduced their invasiveness in vitro and inhibited their tumourigenicity in vivo by reducing the level of VEGF and micro-vessel densities.
  • increasing FABP5 expression in the weakly malignant prostate cancer cell line LNCaP promoted their invasiveness and proliferation rate in vitro and increased their tumourigenicity in vivo.
  • the invention relates to an inactivated FABP5 protein, or fragment or variant thereof, for use as a medicament.
  • the invention relates to an inactivated FABP5 protein, or fragment or variant thereof, for use in the treatment of a FABP5 mediated disorder.
  • the invention relates to an inactivated FABP5 protein, or fragment or variant thereof, for use in the treatment of cancer.
  • the invention in a fourth aspect, relates to a method of treating a FABP5 mediated disorder in a subject in need of such treatment, the method comprising providing to a subject a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof.
  • the invention in a fifth aspect, relates to a method of treating cancer in a subject in need of such treatment, the method comprising providing to a subject a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof.
  • the invention relates to an inactivated FABP5 protein, or fragment or variant thereof, for use in therapeutically reducing PPARy activation.
  • the invention relates to a method of therapeutically reducing PPARy activation, the method comprising providing to a subject in need of such therapeutic reduction of PPARy activation, a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof.
  • the invention provides a pharmaceutical composition comprising an inactivated FABP5 protein, or a fragment or variant thereof.
  • an inactivated FABP5 protein, or fragment or variant thereof for use in accordance with the first, second, third or sixth aspect of the invention, may be provided in the form of a pharmaceutical composition of the eighth aspect of the invention.
  • a pharmaceutical composition of the eighth aspect of the invention provides suitable means by which a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof, may be provided in a method of treatment of the fourth, fifth or seventh aspect of the invention.
  • a cancer to be treated in accordance with the third or fifth aspect is a FABP5 expressing cancer.
  • the cancer is selected from the group consisting of: prostate cancer, breast cancer, colon cancer, skin cancer, liver cancer, ovarian cancer, urinary bladder cancer, oesophageal cancer and pancreatic cancer.
  • the methods, uses, and pharmaceutical compositions of the invention may be of use in the treatment of FABP5 mediated disorders, such us inflammatory diseases (for example rheumatoid arthritis), or metabolic diseases (for example obesity).
  • the methods, uses and pharmaceutical compositions of the invention are of particular use in the treatment of cancer, and especially castration resistant prostate cancer. This is of great significance, since to date there are very few effective treatments for this type of cancer and the survival rate of patients diagnosed with this type of cancer is very poor.
  • Figure 1 A comparison of fatty acid uptake between different types of prostate cancer cell lines.
  • FIG. 3 The graphs illustrate the proliferation of PC3-M cells upon treatment with a FABP5 inhibitor compared to the proliferation of PC3-M cells upon treatment with an inactivated FABP5 protein.
  • Figure 4 Results of a cell migration assay comparing the migration of untreated PC3-M cells to the migration of PC3-M cells treated with a FABP5 inhibitor or inactivated FABP5 protein; B A graph comparing the average size of the cell culture wound size in untreated PC3-M cells and PC3-M cells treated with an inhibitor, or inactivated FABP5 protein.
  • Figure 5 A picture illustrating the results of a soft agar colony formation assay performed on untreated PC3-M cells and PC3-M cells treated with a FABP5 inhibitor, or inactivated FABP5 protein; B A graph showing the results of a soft agar colony formation assay performed on untreated PC3-M cells and PC3-M cells treated with a FABP5 inhibitor, or inactivated FABP5 protein.
  • Figure 6 A picture showing the results of a PC3-M cell invasion assay in a Boyden chamber system performed on untreated PC3-M cells and PC3-M cells treated with a FABP5 inhibitor, or inactivated FABP5 protein; B A graph showing the results of a PC3-M cell invasion assay in a Boyden chamber system performed on untreated PC3-M cells and PC3-M cells treated with a FABP5 inhibitor, or inactivated FABP5 protein.
  • Figure 7 A picture showing the results of a triple negative MDA-MB-231 breast cancer cell invasion assay in a Boyden chamber system performed on untreated MDA-MB-231 cells and MDA-MB-231 cells treated with a FABP5 inhibitor, or inactivated FABP5 protein; B A graph showing the results of a triple negative MDA-MB-231 breast cancer cell invasion assay in a Boyden chamber system performed on untreated MDA-MB-231 cells and MDA-MB-231 cells treated with a FABP5 inhibitor, or inactivated FABP5 protein.
  • Figure 8 A A graph showing the effects of a FABP5 inhibitor on tumour volume.
  • “Inhibitor first week” refers to treatment provided to a mouse on the second day after inoculation with a PC3-M cell tumour.
  • “Inhibitor second week” refers to treatment provided to a mouse on the seventh day after inoculation with a PC3-M cell tumour;
  • B A graph showing the effects of an inactivated FABP5 protein on tumour volume.
  • “Inhibitor first week” refers to treatment provided to a mouse on the second day after the mouse was inoculated with a tumour.
  • “Inhibitor second week” refers to treatment provided to a mouse on the seventh day after the mouse was inoculated with a tumour.
  • Figure 9 A graph comparing tumour volume in a mouse at the end of 31 day treatment with either the inhibitor or the inactivated FABP5 protein; B A graph comparing tumour weight in a mouse at the end of 31 day treatment with either the inhibitor or the inactivated FABP5 protein.
  • Figure 10 A graph comparing tumour volume in mice at the end of a 31 day treatment with either the FABP5 inhibitor or the inactivated FABP5 protein, or a combination of the inhibitor and the inactivated FABP5 protein; B A graph comparing tumour weight of a tumour removed from a mouse treated for 31 days with either the FABP5 inhibitor or the inactivated FABP5 protein, or combination of the two.
  • FIG 11 A Western blot to determine of the optimal experimental time point at which the maximum amount of recombinant protein was synthesized in bacterial cells.
  • DAUDA fluorescent fatty acid
  • Figure 12 A graph showing stable PC3-M colonies expressing strong bioluminescence signals by pGL4.50 vector transfection. Relative light units (mean ⁇ SE) of the PC3-M parental cells and 33 colonies derived from PC3-M cells were obtained from 3 separate measurements.
  • B Images showing the intensities of the bioluminescence images of the serially-diluted (20-100000) parental PC3-M cells and 3 representative PC3M-Luc transfectants. The colour bar on the right indicates the signal intensity range (photons/second/cm2).
  • C A graph demonstrating the correlation between the bioluminescence flux intensity (photons/second) and the number of cells derived from 3 different PC3-M-Luc colonies.
  • D A Graph showing whole body tumour bioluminescence flux produced by each group of nude mice after orthotopic implantation of luciferase-labelled PC3-M cells and treated with PBS (control), SBFI26 (1 mg/kg), dmrFABP5.
  • E A graph showing the number of mice that developed 1 or more metastases (or metastasis incidence, detected by bioluminescence signal) in the control and experimental groups, 15 and 25 days after treatment.
  • F Images demonstrating ventral bioluminescence of primary tumours and metastases in all 4 groups of experimental mice 25 days after treatment. The colour bar on the right indicates the signal intensity range (photons/second/cm2).
  • FIG. 13 A Western blot of PPARy expression in benign and malignant prostate epithelial cells.
  • B A graph showing quantitative assessment of levels of PPARy in benign and malignant prostate epithelial cells. The level of PPARy in the benign prostate PNT2 cells was set at 1 ; levels in the other prostate cell lines were obtained by comparison with that in PNT2.
  • C Western blot analysis of p-PPARy1 and p-PPARv2 in benign and malignant prostate epithelial cells.
  • E Effect of 24 h treatments with SBFI26, PPARy antagonist (GW9662), PPARy agonist (Rosiglitazone), dmrFABP5 and a combination of SBFI26 and dmrFABP5 on levels of p- PPARyl and 2 in PC3-M cells.
  • F A graph showing a quantitative assessment of p-PPARy1 and 2 levels in PC3-M cells after treatments with SBFI26, GW9662, Rosiglitazone, dmrFABP5, and a combination of SBFI26 and dmrFABP5.
  • Levels of both p-PPARy1 and 2 in untreated PC3-M cells were set at 1 ; levels in the other treated cells were obtained by comparison with those in untreated PC3-M.
  • G Western blot showing the effect of 24h treatments with wtrFABP5 and a combination of wtrFABP5 and dmrFABP5 on levels of p- PPARyl and 2 in LNCaP cells.
  • H A graph showing quantitative assessment of levels of p- PPARyl and 2 in control (untreated) and in treated LNCaP cells. Levels of p-PPARy1 and 2 in control LNCaP cells were set at 1 ; levels in the other treated cells were obtained by comparison with those in control.
  • the present invention is based upon the inventor's surprising finding that individuals with FABP5 mediated disorders, such as certain types of cancer may benefit from treatment with inactivated FABP5 proteins, or fragments or variants thereof.
  • FABP5 mediated disorders include cancer (for example prostate cancer), inflammatory diseases (for example rheumatoid arthritis) and metabolic diseases (for example obesity).
  • the inventors have shown through in vivo and in vitro studies that providing an inactivated FABP5 protein to a subject with cancer, or to a cancer cell line, brings about a significant reduction in cancer. Reduction in cancer may be seen by, for example, a reduction in tumour size and/or a reduction in tumour weight. Importantly, and surprisingly, the inventors have also shown that an inactivated FABP5 protein may reduce or completely eliminate cancer metastasis. Metastasis of prostate cancer is the major factor in deaths due to this disease.
  • LCFAs long chain fatty acids
  • Wild-type FABP5 proteins are known to bind LCFAs and facilitate their transport into proximity with PPARy.
  • the present invention makes use of the inventor's surprising finding that inactivated FABP5 proteins, or fragments or variants thereof, may reduce the activation of PPARy. This ability renders the inactivated FABP5 proteins, or fragments or variants thereof as useful therapeutic agents in the treatment of various FABP5 mediated disorders, and in particular FABP5 mediated disorders which are characterised by increased PPARy activation.
  • inactivated FABP5 proteins may suppress these conditions (such as cancer) by inhibiting the FABP5-PPARv- VEGF signalling axis.
  • wild-type FABP5 proteins bind LCFAs and facilitate their transport to PPARy. The binding of LCFAs to PPARy results in the activation of PPARy and other downstream signalling pathways.
  • inactivated FABP5 proteins of the invention, or fragments or variants thereof compete with wild-type FABP5, resulting in a reduction of LCFAs delivered to the site of PPARy activation, thereby reducing the activation of PPARy.
  • the invention therefore provides a new and surprising approach to treating FABP5 mediated diseases such as cancer, as well as a new approach of therapeutically reducing PPARy activation, by using inactivated FABP5 proteins or fragments or variants thereof. This is unexpected, since it may previously have been thought that, since FABP5 proteins unable to bind LCFAs would not interfere with the binding of wild-type FABP5 proteins to LCFAs, they would therefore not represent promising agents for use in therapeutic applications.
  • FABP5 mediated disorder refers to a disorder which is associated with increased activity of FABP5 within the cell.
  • increased activity of FABP5 within the cell may be as a result of increased expression of FABP5, or increased biological activity of FABP5 (for example due to a gain of function mutation).
  • increased activity of FABP5 may, in turn, increase the amount of LCFAs transported into the cell and into proximity with PPARy, thus ultimately resulting in increased activation of PPARy.
  • FABP5 mediated disorder encompasses a disorder in which FABP5 causes increased levels of LCFAs within the cell and/or increased activation of PPARy.
  • the FABP5 mediated disorder may be selected from the group consisting of cancer (for example prostate cancer), inflammatory disease (for example rheumatoid arthritis or atherosclerosis) and metabolic disease (for example obesity, diabetes, hypertension, dyslipidaemia, or metabolic syndrome).
  • cancer for example prostate cancer
  • inflammatory disease for example rheumatoid arthritis or atherosclerosis
  • metabolic disease for example obesity, diabetes, hypertension, dyslipidaemia, or metabolic syndrome.
  • cancer refers to a single or cluster of overgrowing cells, characterised by upregulated cell growth, and replication, reduced cell differentiation, Such cells frequently have the ability to metastasize to other parts of the body.
  • a cancer may be a solid cancer or a haematological cancer.
  • a suitable solid cancer can be selected from a list consisting of: prostate cancer, breast cancer (for example triple negative estrogen receptors (ER-), progesterone receptors (PR-), and HER2 (HER2-) breast cancer), colon cancer, skin cancer, liver cancer, ovarian cancer, urinary bladder cancer, oesophageal cancer, pancreatic cancer, cervical cancer, and oral cancer (in particular oral squamous cell carcinoma).
  • a solid cancer of particular interest in the context of the invention is prostate cancer.
  • inactivated FABP5 proteins, or fragments or variants thereof may be of particular relevance for use in treatment of castration resistant prostate cancer.
  • this type of cancer is extremely difficult to treat, and patients have generally very poor survival rates.
  • the tendency of such cancers to metastasise is a major cause of their lethality, and so the ability of inactivated FABP5 proteins, or fragments or variants thereof, to reduce or even entirely block such metastasis is an important advantage offered by the invention.
  • a suitable haematological cancer may be selected from the list consisting of: acute and chronic leukaemia, lymphoma, myeloma and chronic myeloproliferative diseases.
  • a haematological cancer of particular interest in the context of the invention is leukaemia.
  • a suitable cancer is one that expresses FABP5 protein.
  • the methods or uses according to the second or fourth aspect may comprise the step of assaying a sample indicative of gene expression in the subject, and providing the subject with a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof, if FABP5 expression has been detected in the sample.
  • the methods or uses according to the third or fifth aspect may comprise the step of assaying a sample indicative of gene expression in the cancer obtained from the subject, and providing the subject with a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof, if FABP5 expression has been detected in the sample.
  • the methods or uses according to the sixth or seventh aspect may comprise the step of assaying a sample to determine PPARy activation levels in a subject, and providing the subject with a therapeutically effective amount of an inactivated FABP5 protein, or fragment or variant thereof, if increased PPARy activation has been detected in the sample.
  • a FABP5 mediated disorder for example cancer, inflammatory disease or metabolic disease
  • improvement of the symptoms may include reduced inflammation (which may manifest itself through a reduction in pain or stiffness) or reduced obesity, respectively.
  • the terms “treat”, “treating” or “treatment” refer to a clinical improvement of cancer in a subject with this disease. Such a clinical improvement may be demonstrated by an improvement of the pathology and/or symptoms associated with the cancer.
  • effective treatment may be demonstrated by preventing the development of the disease in a subject, slowing or halting the progression of the disease in the subject, or reversing the disease.
  • the disease may be reversed partially, or completely. In some embodiments, complete reversal of the disease may be sufficient to result in curing of the disease.
  • Clinical improvement of the pathology may be demonstrated by one or more of the following: reduced biomarker levels in the subject, increased time to regrowth of cancer upon stopping of treatment, lack of regrowth of cancer upon stopping treatment, decreased tumour invasiveness, reduction or complete elimination of metastasis, increased cancer cell differentiation, or increased survival rate.
  • Effective treatment may be demonstrated by the establishment, and optionally maintenance, of at least one of these indications, such as reduced biomarker levels.
  • anti-tumour effects may be demonstrated by inhibition of tumour growth, delay in tumour growth, reduced speed of tumour growth, or a partial or complete reduction in tumour mass.
  • the ability of the inactivated FABP5 protein, or fragment or variant thereof to reduce or completely eliminate metastasis is particularly beneficial in the context of prostate cancer, and especially castration resistant prostate cancer, in which approximately 90% of patients develop metastasis.
  • the average mean survival for subjects with castration resistant prostate cancer is between 1 to 2 years, and death is generally caused by metastatic cancer as opposed to the primary prostate cancer.
  • the ability of the inactivated FABP5 protein of the invention, or fragment or variant thereof to reduce metastasis is an important and highly desirable characteristic.
  • a reduction of metastasis may be demonstrated by a reduction in the occurrence of metastasis in subjects with cancer.
  • the inactivated FABP5 protein, or fragment or variant thereof may reduce the occurrence of metastasis by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.
  • the inactivated FABP5 protein, or fragment or variant thereof may completely eliminate metastasis, i.e. reduce occurrence of metastasis in subjects with cancer by 100%. Suitable experimental models by which the occurrence of metastasis with or without treatment with inactivated FABP5 proteins, or fragments or variants thereof are described further in the Examples.
  • a reduction of metastasis may be demonstrated by increasing the mean survival for subjects with cancer.
  • the inactivated FABP5 protein, or fragment or variant thereof may increase the mean survival of a subject with cancer by at least 6 month, at least 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or more.
  • reduced biomarker levels may be demonstrated by reduced levels of prostate specific antigen (PSA). Relevant PSA levels may be determined in the subject's serum.
  • PSA prostate specific antigen
  • Clinical improvement of symptoms associated with cancer may be, but are not limited to partial or complete alleviation of pain and/or swelling, increased appetite, reduced weight loss and reduced fatigue.
  • clinical improvement of symptoms associated with prostate cancer may be demonstrated by reduced urinary obstruction, reduced pain and/or reduced haematuria.
  • the term "subject" refers to an individual suffering from a FABP5 mediated disorder, such as cancer.
  • the subject has a FABP5 mediated disorder (for example cancer), or is identified to be at risk of developing a FABP5 mediated disorder.
  • a subject may be identified as being at risk of developing cancer if the subject has a familial history of cancer, or if the subject has a genetic predisposition to cancer.
  • the subject may have a genetic predisposition to prostate cancer if the subject has mutations in genes selected from the group consisting of: BRCA1 , BRCA2, and HOXB13. Other genes which result in a predisposition to cancer will be known to the skilled person.
  • the subject may be a mammal.
  • the subject may be a selected from a group consisting of: a human, a primate, a dog, a cat, a rat and a mouse.
  • the subject may be provided an inactivated FABP5 protein, or fragment or variant thereof, as a first line treatment for a FABP5 mediated disorder (such as cancer).
  • a FABP5 mediated disorder such as cancer
  • the subject would have not been provided any other treatment for a FABP5 mediated disorder, such as cancer, prior to treatment in accordance with the present invention.
  • an inactivated FABP5 protein is able to treat relapsed or refractory cancer, such as castration resistant prostate cancer
  • inactivated FABP5 proteins, or fragments or variants thereof may also be used to treat cancers in which other treatments were found ineffective.
  • the subject may have received another treatment for cancer prior to treatment in accordance of the present invention.
  • the subject may have received another treatment such as radiotherapy, chemotherapy, hormone therapy, vaccine treatment, bone-directed treatment, or surgical removal of the cancer.
  • a surgical removal of cancer may be, for example, carried out by cryosurgery.
  • an inactivated FABP5 protein, or fragment or variant thereof may be employed in the use or method of the invention as a second line treatment for a FABP5 mediated disorder, such as cancer.
  • a FABP5 mediated disorder such as cancer.
  • an inactivated FABP5 protein, or fragment or variant thereof may be used in a third, or further, line treatment for a FABP5 mediated disorder, such as cancer.
  • the treatment in accordance with the present invention may make use of the inactivated FABP5 protein, or fragment or variant thereof, in conjunction with a second treatment.
  • a suitable second treatment may be selected from a group consisting of: radiotherapy, chemotherapy, hormone therapy, vaccine treatment, bone-directed treatment, surgery and FABP5 inhibitor therapy other than the inactivated FABP5 protein, or fragment or variant itself.
  • an inactivated FABP5 protein, or fragment or variant thereof, in conjunction with a second treatment may have a synergistic effect on treatment of a FABP5 mediated disorder, such as cancer.
  • the FABP5 inhibitor therapy may be: SBFI26 (a-truxillic acid 1-naphthyl mono-ester).
  • an inactivated FABP5 protein, or fragment or variant thereof is for use as an adjuvant in the treatment of a FABP5 mediated disorder, such as cancer.
  • An adjuvant is an agent provided to a patient together with, or after a "main” therapy for a FABP5 mediated disorder.
  • an adjuvant is an agent provided to a patient together with, or after a "main” anti-cancer therapy, such as surgical removal of cancer, in order to prevent the return of cancer after the main therapy.
  • An inactivated FABP5 protein, or fragment or variant thereof may be used as an adjuvant for a patient who has undergone surgical treatment of cancer and/or radiotherapy for cancer.
  • an inactivated FABP5 protein, or fragment or variant thereof is provided for use as a neoadjuvant in the treatment of cancer.
  • a neoadjuvant is an agent provided to a patient in order to reduce the size of a tumour prior to a "main" anti-cancer therapy, such as surgical removal of cancer.
  • An inactivated FABP5 protein, or fragment or variant thereof may be used as a neoadjuvant therapy for a patient who will subsequently undergo, for example, surgical treatment of cancer and/or radiotherapy for cancer.
  • Methods according to some embodiments of the invention utilise a sample that provides an indication as to the gene expression in the subject, or in the subject's cancer.
  • the sample may be a solid sample or a body fluid sample.
  • the sample may be a fluid sample selected from the group consisting of: a blood sample (for example, a whole blood sample, a blood plasma sample, or a serum sample), a urine sample, and an interstitial fluid sample.
  • a solid sample may be particularly relevant in the case of solid cancers.
  • a suitable solid sample may be a biopsy or a smear sample.
  • Target molecules when required by some embodiments of the invention, are any molecules which are representative of the expression of FABP5 in the sample, or in the cancer.
  • Target molecules may be representative of FABP5 expression either directly or indirectly.
  • a suitable target molecule which is directly representative of gene expression may comprise an RNA transcript encoding FABP5.
  • a suitable target molecule which is indirectly representative of FABP5 gene expression may comprise the FABP5 protein.
  • assaying refers to the determination of the presence or absence of target molecules representative of FABP5 gene expression.
  • a suitable method by which such assaying may be carried out is selected based on the type of target molecule selected.
  • gene expression can be detected directly by techniques that allow the assaying of RNA target molecules.
  • techniques may include for example RT-PCR, realtime PCR, Northern blot, RNA sequencing (RNA-seq), and RNA microarray.
  • gene expression can be detected indirectly, by techniques that allow assaying of protein target molecules.
  • techniques may include for example as ELISA, radioimmunoassay, immunoprecipitation, Western blot and mass spectrometry.
  • providing encompasses any techniques by which the subject receives a therapeutically effective amount of the inactivated FABP5 protein, or fragment or variant thereof.
  • the subject may be provided directly with the inactivated FABP5 protein, or fragment or variant thereof.
  • the subject may be provided with a pharmaceutical composition comprising the inactivated FABP5 protein, or fragment or variant thereof.
  • the subject may be provided indirectly with the inactivated FABP5 protein or fragment or variant thereof.
  • the subject may, for example, be provided with a nucleic acid encoding such a protein.
  • Such suitable routes may be selected from the group consisting of: oral, parenteral, intravenous, intraperitoneal, intramuscular, intravascular, intranasal, rectal, subcutaneous, transdermal and percutaneous. More suitably, the subject may be provided a therapeutically effective amount of the inactivated FABP5 protein, or fragment or variant thereof by intravenous route.
  • the inactivated FABP5 protein, or fragment or variant thereof may be provided by injection directly into the tumour.
  • the inactivated FABP5 protein, or fragment or variant thereof may be provided by injection directly into the mammary fat pad, for example in the treatment of breast cancer.
  • terapéuticaally effective amount refers to the amount of inactivated FABP5 protein, or fragment or variant thereof, that when provided to the subject, is sufficient to treat a FABP5 mediated disorder, such as cancer, or reduce the activation of PPARy.
  • a therapeutically effective amount is an amount of inactivated FABP5 protein, or fragment or variant thereof, which will result in a clinical improvement of the FABP5 mediated disorder, such as cancer, in a subject with this disease.
  • a clinical improvement may be demonstrated by an improvement of the pathology and/or symptoms associated with the FABP5 mediated disorder (for example cancer).
  • the therapeutically effective amount will vary depending on various factors, such as the subject's body weight, sex, diet and route by which the inactivated FABP5 protein, or fragment or variant thereof is provided.
  • Such a therapeutically effective amount may be provided to the subject in a single or multiple doses.
  • the therapeutically effective amount will vary depending on the FABP5 mediated disorder in question.
  • the therapeutically effective amount may vary depending on the type of cancer.
  • the therapeutically effective amount will vary depending on the severity, or stage of the FABP5 mediated disorder, for example cancer.
  • the therapeutically effective amount of inactivated FABP5 protein, or fragment or variant thereof may be between 0.1 and 100 mg/kg of body weight.
  • the therapeutically effective amount may be between 1 and 90 mg/kg, between 5 and 80 mg/kg, between 7.5 and 70 mg/kg, between 10 and 60 mg/kg, or between 12 and 50 mg/kg. More suitably, the therapeutically effective amount may be between 15 and 40 mg/kg, or between 20 and 30 mg/kg. Suitably, the therapeutically effective amount may be around 20 mg/kg. It will be appreciated that a therapeutically effective amount of inactivated FABP5 protein, or fragment or variant thereof, may be determined in vitro or in vivo, using techniques known to the skilled person.
  • FABP5 (Fatty acid binding protein 5) protein
  • FABP5 is also known as C-FABP5, E-FABP, K-FABP5, PA- FA BP or PAFABP. Other names of this protein may be known to the skilled person.
  • FABP5 is a 15kDa protein, with a defined sequence of 135 amino acids as shown in SEQ ID NO: 1.
  • FABP5 is a fatty acid binding protein found in the epidermal cells. It is a cytoplasmic protein which binds LCFAs and may facilitate the transport of LCFAs to various proteins, such as PPARy.
  • Inactivated FABP5 proteins are FABP5 proteins which have reduced biological activity compared to wild-type FABP5 proteins.
  • a reduction in the biological activity of the inactivated FABP5 proteins may be demonstrated by a reduction in binding of the inactivated FABP5 to LCFAs and/or reduction of activation of PPARy as compared to wild-type FABP5 proteins.
  • the ability of inactivated FABP5 proteins, or fragments or variants thereof to reduce the activation of PPARy gives rise to the sixth and seventh aspects of the invention.
  • the inactivated FABP5 protein, fragment or variant thereof may reduce activation of PPARy directly or indirectly.
  • the inactivated FABP5 protein, fragment or variant thereof reduces the activation of PPARy indirectly. Such indirect reduction may be as a result of reduced binding of the inactivated FABP5 to LCFAs. The inventors also believe, that the inactivated FABP5 protein, fragment or variant thereof may reduce the amount of LCFAs transported into the cell as compared to wild type FABP5 proteins, and thereby indirectly reduce the activation of PPARy. In a suitable embodiment, inactivated FABP5 proteins have reduced binding to LCFAs as compared to wild type FABP5 proteins.
  • the inactivated FABP5 proteins bind to LCFAs by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% less than wild-type FABP5 proteins.
  • inactivated FABP5 proteins of the invention do not bind LCFAs at all.
  • the binding of inactivated FABP5 proteins to LCFAs may be quantified, for example, using a 1 l-(Dansylamino) undecanoic acid (DAUDA) displacement assay, as described in more detail in the Examples section.
  • DAUDA 1 l-(Dansylamino) undecanoic acid
  • Other assays suitable for determining the binding of a protein (such as an inactivated FABP5 protein) to LCFAs, will be known to those skilled in the art.
  • a FABP5 protein of the invention may have reduced binding to LCFAs selected from the group consisting of linoleic acid, oleic acid and palmitic acid.
  • the inactivated FABP5 proteins may reduce the activation of PPARy as compared to wild type FABP5 proteins.
  • the inactivated FABP5 protein may reduce the activation of PPARy by less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90%, as compared to wild type FABP5 proteins.
  • a proteins ability to reduce the activation of PPARy may be determined by measuring the relative amount of phosphorylated to non-phosphorylated PPARy upon exposure to said protein. This, in turn, may be measured by methods known in the art. As shown in the Examples section, activation of PPARy may be determined, for example, by Western blot.
  • Reduced binding of the inactivated FABP5 protein to LCFAs, and/or reducing the activation of PPARy may be as a result of one or more mutations in the amino acid sequence encoding the FABP5 protein (such mutations being assessed with reference to the wild type amino acid sequence set out in SEQ ID NO: 1).
  • the one or more mutations in SEQ ID NO: 1 may be in the region conserved among the FABP family proteins.
  • the one or more mutations in SEQ ID NO: 1 may be in the fatty acid-binding motif.
  • the inactivated FABP5 protein, or fragment or variant thereof comprises mutations in at least one, at least two, or at least three amino acids, selected from the list consisting of: Arg 109, Arg 129, and Tyr 131.
  • the mutations are non- synonymous mutations.
  • An example of an inactivated FABP5 protein suitable for use in accordance with the invention (and as a starting point for the generation of fragments or variants) containing a single substitution mutation at Arg 109 is referred to herein as smrFABP5, and the sequence of this protein is set out in SEQ ID NO: 3.
  • the inactivated FABP5 protein, or fragment or variant thereof comprises mutations in Arg 109 and Tyr 131.
  • the inactivated FABP5 protein comprises mutations in Arg 129 and Tyr 131.
  • the inactivated FABP5 protein comprises mutations in Arg 109 and Arg 129 (SEQ ID NO: 2). This inactivated FABP5 protein has been shown to be particularly effective (see the Examples, in which it is referred to as dmrFABP5), and represents an example of an inactivated FABP5 protein from which fragments or variants may be derived.
  • inactivated FABP5 proteins, fragments, and variants, described herein, and particularly those inactivated proteins considered in the preceding paragraphs do not occur in nature. Instead, these inactivated FABP5 proteins, fragments, and variants arise as a result of the hand of man.
  • Fragments of the inactivated FABP5 protein are polypeptides that consist of a truncation in the amino acid sequence of the inactivated FABP5 protein. Fragments of the inactivated FABP5 protein share 100% identity with the corresponding portion of the amino acid sequence of the inactivated FABP5 protein. Similarly to the inactivated FABP5 protein of the invention, the inactivated FABP5 protein fragments also have a reduction in biological activity. Such a reduction in biological activity may be demonstrated by a reduction in binding of the inactivated FABP5 to LCFAs and/or reduction of activation of PPARy as compared to wild-type FABP5 proteins or their fragments.
  • the biological activity of the inactivated FABP5 protein fragments may be approximately the same as the biological activity of the inactivated FABP5 protein, which is discussed elsewhere in this specification.
  • fragments of the inactivated FABP5 protein are polypeptides that consist of a truncation of the amino acid of SEQ ID NO: 2 or SEQ ID NO: 3. Fragments of the inactivated FABP5 protein may share 100% identity with the portion of SEQ ID NO: 2 or SEQ ID NO: 3 that they correspond to.
  • a suitable fragment of the inactivated FABP5 protein consists up to 134 contiguous amino acids of SEQ ID NO: 2, for example up to 133 contiguous amino acids of SEQ ID NO: 2, up to 132 contiguous amino acids of SEQ ID NO: 2, up to 131 contiguous amino acids of SEQ ID NO: 2, up to 130 contiguous amino acids of SEQ ID NO: 2, up to 129 contiguous amino acids of SEQ ID NO: 2, up to 128 contiguous amino acids of SEQ ID NO: 2, up to 127 contiguous amino acids of SEQ ID NO: 2, up to 126 contiguous amino acids of SEQ ID NO: 2, up to 125 contiguous amino acids of SEQ ID NO: 2, up to 124 contiguous amino acids of SEQ ID NO: 2, up to 123 contiguous amino acids of SEQ ID NO: 2, up to 122 contiguous amino acids of SEQ ID NO: 2, up to 121 contiguous amino acids of SEQ ID NO: 2, up to contiguous amino acids of SEQ ID NO: 2, up
  • Variants of the inactivated FABP5 protein are polypeptides that share a partial sequence identity with the amino acid encoding the inactivated FABP5 protein and which have a reduced biological activity as compared to the biological activity of a wild-type FABP5 protein.
  • reduced biological activity may be demonstrated by a reduction in binding of the inactivated FABP5 variants to LCFAs and/or reduction of activation of PPARy as compared to wild-type FABP5 proteins or their fragments.
  • the biological activity of the inactivated FABP5 protein variants may be approximately the same as the biological activity of the inactivated FABP5 protein or fragments thereof.
  • variants of the inactivated FABP5 protein and the amino acid sequence encoding the inactivated FABP5 protein, or fragments thereof, may be determined based on:
  • a variant of the inactivated FABP5 protein may share at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, or at least 80% identity with an amino acid sequence encoding an inactivated FABP5 protein or with a fragment thereof.
  • variant of the inactivated FABP5 protein may share at least 81 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 96% identity, at least 98% identity, at least 99% identity with an amino acid sequence encoding an inactivated FABP5 protein or with a fragment thereof.
  • a variant of the inactivated FABP5 protein may share at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, or at least 80% identity with SEQ ID NO: 2 or with a fragment thereof.
  • variant of the inactivated FABP5 protein may share at least 81 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 96% identity, at least 98% identity, at least 99% identity with SEQ ID NO: 2 or with a fragment thereof.
  • variants of an amino acid encoding an inactivated FABP5 protein, or fragments thereof may have sequences longer than the amino acid sequences of the inactivated FABP5 proteins or fragments thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 50% identity with the amino acid encoding the inactivated FABP5 protein, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, or at least 75% identity with the amino acid encoding the inactivated FABP5 protein, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, or at least 90% identity with the amino acid encoding the inactivated FABP5 protein, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 96% identity, at least 98% identity, at least 99% identity, or more identity with the amino acid encoding the inactivated FABP5 protein, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 50% identity with SEQ ID NO: 2, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, or at least 75% identity with SEQ ID NO: 2, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, or at least 90% identity with SEQ ID NO: 2, or fragment thereof.
  • a variant of the inactivated FABP5 protein may have an amino acid sequence, which, when taken as a whole, shares at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 96% identity, at least 98% identity, at least 99% identity, or more identity with SEQ ID NO: 2, or fragment thereof.
  • compositions comprising an inactivated FABP5 protein, or a fragment or variant thereof.
  • the composition is a composition comprising the FABP5 protein (or fragment or variant thereof) and a pharmaceutically acceptable diluent, carrier or excipient.
  • Such compositions may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • compositions may also include antioxidants and/or preservatives.
  • antioxidants may be mentioned thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid.
  • Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.
  • the FABP5 protein (or fragment or variant thereof) may be presented as solids in finely divided solid form, for example they may be micronised. Powders or finely divided solids may be encapsulated.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • compositions described above may be suitable for use in treating cancer, and particularly those various forms of cancer described herein.
  • the FABP5 protein (or fragment or variant thereof) may be for administration to the subject by any suitable route by which a therapeutically effective amount of the FABP5 protein (or fragment or variant thereof) may be provided.
  • the inactivated FABP5 protein may be administered via nasal delivery.
  • Methods of administering pharmaceuticals to the nose are well- known to those skilled in the art.
  • the design of suitable nasal delivery devices is described, for example, in WO/1999/058180, incorporated herein by reference.
  • the inactivated FABP5 protein may be administered via inhalation.
  • Methods of administering pharmaceuticals to the lung by inhalation are well known to those skilled in the art.
  • the design of suitable inhaler devices is described, for example in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985, p. 181 -182, incorporated herein by reference.
  • the inactivated FABP5 protein (or fragment or variant thereof) is for oral administration to treat cancer.
  • suitable oral administration forms that may be used in such embodiments include solid dosage forms.
  • Solid dosage forms for oral administration include capsules, tablets (also called pills), powders and granules.
  • the inactivated FABP5 protein (or fragment or variant thereof) is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.
  • oral formulations may contain a dissolution aid.
  • the dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable.
  • examples include nonionic surface agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorb
  • the inactivated FABP5 protein (or fragment or variant thereof) is for administration in liquid dosage form.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol
  • the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents.
  • Suspensions in addition to the inactivated FABP5 protein (or fragment or variant thereof), may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.
  • the FABP5 protein (or fragment or variant thereof) may be for administration to the subject by intravenous route.
  • a sterile pharmaceutical composition may be especially desirable.
  • a sterile pharmaceutical composition may be created, for example, by filtration through sterile filtration membranes, prior to or following lyophilisation and reconstitution of the inactivated FABP5 protein, or fragment or variant thereof.
  • the inactivated FABP5 protein (or fragment or variant thereof) may be stored in lyophilised form or in solution.
  • a pharmaceutical composition comprising the inactivated FABP5 protein (or fragment or variant thereof) may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierce-able by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierce-able by a hypodermic injection needle.
  • a sterile pharmaceutical composition comprising the inactivated FABP5 protein (or fragment or variant thereof) suitable for intravenous delivery may be formulated according to conventional pharmaceutical practice as described in Remington: The Science and Practice of Pharmacy (20 th ed, Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
  • the pharmaceutical composition comprising the inactivated FABP5 protein (or fragment or variant thereof) may be for the sustained release of the inactivated FABP5 protein.
  • a pharmaceutical composition may comprise semipermeable matrices of solid hydrophobic polymers containing the inactivated FABP5 protein (or fragment or variant thereof), which matrices are in the form of shaped articles, films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels, copolymers of L- glutamic acid and gamma ethyl- L-g I utamate , non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DepotTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-(-)-3-hydroxybutyric acid examples include polyesters, hydrogels, copolymers of L- glutamic acid and gamma ethyl- L-g I utamate , non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid
  • compositions for sustained release of inactivated FABP5 protein, or fragment or variant thereof may comprise crystals of the inactivated FABP5 protein suspended in suitable formulations capable of maintaining crystals in suspension.
  • Such pharmaceutical compositions when injected intravenously, subcutaneously or intraperitoneally may produce a sustained release effect.
  • Wild-type FABP5 cDNA and cDNA encoding an inactivated FABP5 protein were cloned into PQE-32 expression vectors using Kpnl and Pstl restriction sites. Successful insertions and correct orientation of the cDNA was confirmed by DNA sequencing.
  • E. coli (DH5a) cells Upon construction of the expression vectors in E. coli (DH5a) cells, a large amount of DNA from each construct was produced and transformed into E. coli BL21 cells to produce recombinant proteins. The optimal time point, at which the maximum amount of protein was produced, was determined by Western blot analysis of samples extracted from E. coli cultures at hourly intervals from the time of induction (using 1 mM of IPTG).
  • the recombinant proteins were purified from the bacterial cells by a gravity-flow chromatography on a Ni-NTA agarose column. Then affinity purification was performed with an antibody against the 6xHis-tag which was conjugated with FABP5 proteins using Qiagen Ni-NTA Fast Start Kit. After cell lysis with Native lysis buffer, 6xHis-tagged antibody- bounding proteins were washed with native wash buffer, and then eluted with native elution buffer into two separate tubes.
  • the purity of the 6xHis-tagged FABP5 proteins in the first and second elution was analyzed by SDS-PAGE gel (Coomassie Blue staining) and Western blot with the use of Penta-His antibody and monoclonal rabbit anti-human C-FABP as the primary antibody.
  • Fatty acid uptake assay offers a sensitive and simple method for measuring cellular uptake of LCFAs.
  • a red fluorescent label, BODIPY 558/568C12 was used to fluorescently label the fatty acids, according to the manufacturer's guidelines. Since the label does not interfere with cellular uptake of fatty acids, the amount of LCFAs taken up by the cells can be easily determined by flow cytometry.
  • the amount of fatty acid taken up by four different prostate derived cell lines was assayed. These were cell lines from normal epithelium (PNT2) and cell lines from progressively more malignant prostate cancers, LNCaP being weekly malignant, 22RV1 being moderately malignant and PC3-M being highly malignant. The results were represented as a percentage of the total amount of fatty acid provided to each cell line.
  • the fatty acid uptake of PNT2 was 68 ⁇ 4.7%
  • of LNCaP was 65.97 ⁇ 5.8, of 22RV1 was 87.72 ⁇ 1.2%
  • PC3-M cells was 90.12 ⁇ 3.1 % ( Figure 1).
  • SBFI26 inhibited fatty acid uptake of FABP5 in PC3-M cells
  • the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium assay is based on the ability of mitochondrial dehydrogenase enzyme from viable cells to convert MTT into a blue coloured product known as formazan.
  • Formazan is impermeable to cell membranes and therefore, in the form of blue crystals, accumulates in live cells. Dead cells do not have the ability to convert MTT into formazan.
  • the cells were then washed with PBS and, in triplicate, treated with different doses of inhibitor ( 25 ⁇ , 50 ⁇ , 75 ⁇ , " ⁇ ⁇ , 125 ⁇ and 150 ⁇ ) or inactivated FABP5 protein (5 ⁇ , 10 ⁇ , 20 ⁇ , 40 ⁇ , 50 ⁇ , 60 ⁇ and 70 ⁇ ) and incubated overnight at 37°C, 5% C02.
  • the MTT solution was added according to the manufacturer's guidelines, and incubated at 37°C, 5% C02 for 4 hours.
  • the cells were subsequently solubilized using DMSO and the optical density was read at 570nm. The growth rate was measured against its own standard curve to obtain the dose which was most cytotoxic.
  • the cell migration assay is an efficient method of estimating cancer cell migration in vitro. This method is based on measuring the time it takes for a new artificial wound (scratch in a confluent cell monolayer) to close the gap up as a result of cell migration.
  • PC3-M cells were grown to 60-80% confluency in a 75 cm 2 flask, upon which they were harvested and suspended in RPMI complete culture medium. The assay was carried out in 24-well plates and PC3-M cells were seeded into 9 wells. When a confluent monolayer was formed, a 1000 ⁇ _ pipette tip was used to scratch and remove cells from a discrete area to form a cell free zone, which was washed twice with PBS.
  • the soft agar assay is a technique used to assess cellular tumourigenicity in vitro.
  • PC3-M cells positive controls
  • PC3-M cells treated with 50 ⁇ inactivated FABP5 protein or 100 ⁇ SB-FI-26 inhibitor were seeded, in triplicate, on 0.5% agar in routine culture medium on top of a bed of a first layer at 5 X 10 4 cells per dish.
  • approximately 200 ⁇ of medium was added to the dishes in order to avoid drying out and to ensure the cells have sufficient nutrients and treatment.
  • the Boyden Matrigel chamber assay provides conditions to measure the invasiveness of cancer cells in vitro. This assay is based on a chamber with two different medium compartments separated by an 8 micron-pore size membrane. In general, cancer cells are seeded in the upper compartment and allowed to transport to the lower compartment through the membrane pores in response to the presence of chemo-attractant agents.
  • PC3-M cells a prostate cancer cell line
  • MDA-MB- 231 a triple negative breast cancer cell line
  • MDA-MB- 2321 a triple negative breast cancer cell line
  • these cells also express high levels of FABP5.
  • mice Male Balb/C nude mice were subcutaneously injected in the right flank region with 2x10 6 PC3-M cells in 200 ml PBS to establish tumours. The mice were then subcutaneously treated with one of the following: 1) control with PBS; 2) 1 mg/kg of SBFI26 inhibitor provided one day after tumour cell inoculation; 3) 1 mg/kg of SBFI26 inhibitor provided 7 days after tumour cell inoculation (when tumours had reached 35-40mm 3 in size); 4) 20mg/kg of inactivated FABP5 was injected one day after tumour cell inoculation; 5) 20mg/kg of inactivated FABP5 was injected 7 days after tumour cell inoculation (when tumours had reached 35-40 mm 3 in size). Treatments were repeated every other day for a total of 31 days and tumour size monitored every 3-4 days.
  • mice Upon completion of the 31 day treatment, the mice were sacrificed and the tumour size was measured. It was found that the tumour size in mice treated with the inhibitor or with the inactivated FABP5 protein was significantly reduced compared to the untreated control group ( Figure 8 A and B). In the case of both treatments, commencing the treatment in the first week resulted in a slightly greater reduction in tumour size as compared to commencing the treatment in the second week ( Figure 9 A). Overall, treatment with the protein reduced the size of the tumour more than treatment with the inhibitor.
  • tumour weight was also assessed, and it was noted that the weight of the tumour was significantly reduced in the treated groups. The greatest reduction in tumour weight was noted in mice treated with the inactivated FABP5 protein, and specifically when treatment was commenced in the second week post inoculation ( Figure 9 B).
  • tumour volume and weight were also assed in mice inoculated with PC3-M cells, and treated with a combination of the inhibitor and inactivated FABP5 protein. It was found that the combination treatment reduced the tumour volume and weight slightly more than the two treatments individually ( Figure 10 A and B). 8. Production of recombinant FABP5s and testing their biological activities
  • the optimal experimental time point at which the maximum amount of recombinant protein was synthesized in bacterial cells was determined by Western blot. After the wild type and 2 mutant FABP5 cDNAs were cloned into pQE32 expression vectors, 3 recombinant FABP5s were produced in the BL21 strain of E. coli cells. Bacterial samples were removed, once per hour from culture, for up to 6 hours after IPTG induction. Before loading, the total proteins from each lysed bacterial sample was quantified using a Bradford Assay kit (Bio-Rad, Hertfordshire, UK). Distilled water was added to samples to ensure equal amount of protein were loaded. 6xHis-tag bound protein bands were recognized by the Penta-His antibody. The wtrFABP5 protein synthesized in bacterial cells at different time points is shown in 8 separate lanes
  • Fatty acid binding properties of the recombinant FABP5s was assessed by a fluorescent fatty acid (DAUDA) ligand displacement assay.
  • DAUDA fluorescent fatty acid
  • the effect of wtrFABP5 on the fluorescence emission spectra of at the excitation wavelength of 345nm was assessed.
  • the graph in Figure 1 1C/a shows that PBS (1) and wtrFABP5 in PBS (2) did not produce any emission; whereas 2 ⁇ DAUDA produced emission at 560nm (3); 2 ⁇ DAUDA in the presence of 3 ⁇ wtrFABP5 caused an increase in the fluorescence intensity and a left shift to 530nm compared to DAUDA alone.
  • the maximum affinity was detected at 530nm with a shift of wavelength of emission (Figure 11 C/a).
  • the fluorescence intensity of displaced DAUDA from each recombinant FABP5 by linoleic acid was used as an indication of their relative binding affinity.
  • Competitive inhibition of DAUDA-wtrFABP5 binding to linoleic acid was demonstrated in PBS alone (1) or 2 ⁇ DAUDA alone (2), which give the same results as Figure 11 C/a above.
  • 3 ⁇ wtrFABP5 plus 2 ⁇ DAUDA caused an increase in fluorescence intensity and a left shift to 530nm compared to DAUDA alone (3).
  • inactivated FABP5 proteins which constitute inactivated FABP5 proteins in accordance with the invention, lack the ability to deliver LCFAs to the site of PPARy activation and therefore reduce the PPARy signaling pathway.
  • the inactivated FABP5 proteins according to the present invention are suitable for use in the treatment of cancer, or other FABP5 or PPARy mediated disorders.
  • Figure 12 shows the inhibitory effect of a known chemical inhibitor, SBFI26 and inactivated FABP5 protein of the invention (dmrFABP5) on tumorigenicity and metastatic ability of PC3- M cells implanted orthotopically into the prostate gland of the nude mouse.
  • mice that developed 1 or more metastases or metastasis incidence, detected by bioluminescence signal
  • the difference between the control and each of the experimental groups was assessed by 2-tailed Fisher's Exact test *, P ⁇ 0.05.
  • In the control group 7/7 (100%) mice produced metastases.
  • In the group treated with the chemical inhibitor SBFI26 4/8 (50%) of mice produced visceral metastasis.
  • in groups treated with the inactivated FABP5 protein of the invention dmrFABP5
  • no mice with metastases were detected, showing 100% suppression of metastasis.
  • Figure 12F shows ventral bioluminescence images of primary tumours and metastases in all 4 groups of experimental mice 25 days after treatment.
  • the colour bar on the right indicates the signal intensity range (photons/second/cm 2 ).
  • FIG. 12G Representative photomicrographs of sections of tissues showing liver and lung metastases (arrows) were stained with H&E, magnification * 10 and scale bar is 100 ⁇ . Representative micrographs from each group are shown; PBS (1), SBFI26 (2), dmrFABP5 (3) and combination of SBFI26 and dmrFABP5 (4). Histological staining showed that all mice developed metastases in the control group, mainly in the liver and lung. In the SBFI26 treated group, half of the mice developed liver metastases. In contract, those what were treated with inactivated FABP5 in accordance with the invention (dmrFABP5), either alone or in combination with SBFI26 did not develop any metastases in the liver or lung. One representative stained slide from each group/organ is shown in (Figure 12G).
  • Quantitative levels of p-PPARy1 and p-PPARv2 in the cell lines were assessed. Levels of p- PPARyl and 2 in benign PNT2 cells were set at 1 ; levels in the other prostate cells were obtained by comparison with those in PNT2. Relative levels in LNCaP were 9.54 ⁇ 1.81 (p- PPARyl) and 9.5 ⁇ 0.5 (p-PPARv2), 22RV1 were 25.4 ⁇ 1.8 (p-PPARy1) and 47.0 ⁇ 1.7 (p- PPARv2), DU145 were 26.99 ⁇ 1.72 (p-PPARy1) and 85.5 ⁇ 14.5 (p-PPARv2).
  • LNCaP cells which expressed low levels of p-PPARy1 and 2 were treated with wtrFABP5 and a combination of wtrFABP5 and dmrFABP5 for 24 hours.
  • wtrFABP5 increased levels of both isoforms by 1 fold (P-PPARY1 )- and 0.9- fold (p-PPARy1).
  • p-PPARy1 p-PPAR-like isoforms by 1 fold
  • p-PPARy1 0.9- fold
  • p-PPARy1 0.9- fold
  • dmrFABP5 even reduced the level of p-PPARv2 by 5-times (Student's t test, p ⁇ 0.001) to a level lower than that obtained in pretreatment.
  • Levels of p-PPARy1 and 2 in control LNCaP cells were set at 1 ; levels in the other treated cells were obtained by comparison with those in control ( Figure 13 G, H).
  • the inactivated FABP5 protein of the invention was capable of successfully reducing the relative percentage of both isoforms of p-PPARy in the cells.
  • ADT targeting AR and circulating male hormone has been the main therapeutic method to treat prostate cancer patients during the past 4 decades.
  • the disease relapses within a period of time with a more aggressive form, called androgen-independent cancer or CRPC, this does not respond to ADT effectively.
  • CRPC a more aggressive form
  • the main theory is that the biological sensitivity of AR is amplified after the first round of ADT to such an extent that even micro-quantities of remaining hormone in peripheral blood can still promote the malignant progression of CRPC cells.
  • ADT on CRPC was a general clinical practice. However, an opposite opinion to this practice was proposed recently.
  • Androgen-independent prostate cancer cell line PC3-M expressing high levels of FABP5 and PPARy, was an extremely malignant and metastatic cell line.
  • SBFI26 is a known, effective inhibitor of FABP5 and showed efficient anti-tumour roles in the mouse model for primary tumours implanted in the prostate gland (by 4.9- fold) and inoculated in the flank (by 52%).
  • SBFI26 Compared to the control group, in which all mice (100%) developed metastases, SBFI26 treatment suppressed metastases in half of the mice of the treated group (50%).
  • SBFI26 was used to benchmark the protein of the invention (dmrFABP5).
  • the mutant protein dmrFABP5 has the same structure as wtrFABP5, but it is incapable of binding to fatty acids, as shown in Figure 1 1 C and D.
  • dmrFABP5 has a dominant negative effect on tumourigencity and metastatic ability of PC3- M cells by suppressing the biological activity of FABP5.
  • dmrFABP5 produced significant suppression of PC3-M cellular proliferation, invasiveness, migration and colony formation in vitro.
  • the inactivated FABP5 protein in accordance with the invention (dmrFABP5) was highly effective in suppressing both the primary tumour growing in the prostate gland and subcutaneous tumour growing in the flank.
  • dmrFABP5 produced a 13- fold reduction in tumour mass in the prostate gland and 3-fold reduction in subcutaneous tumours ( Figures 8, 9, 10 and 12).
  • dmrFABP5 produced a 100% suppression of metastasis in mice with CRPC cells implanted into the prostate gland ( Figure 12).
  • the suppressive effect of dmrFABP5 was 2.7-fold higher in tumours growing in the prostate gland, and 30% higher in tumours growing in the flank.
  • SBFI26 may be a competitive inhibitor for FABP5 and hence prevent intra- and extra-cellular fatty acids from being transported into the cytoplasm.
  • the reduced fatty acid uptake may result in reduction or cessation of the stimulation of PPARy by fatty acids.
  • PPARy may no longer be able to upregulate the down-stream cancer-promoting genes, such as VEGF, and to suppress apoptosis.
  • dmrFABP5 did not produce any noticeable changes in fatty acid uptake of PC3-M cells (result not show), whilst being more effective than SBFI26 in suppressing tumour growth and reducing metatsasis in nude mice ( Figures 8, 9, 10 and 12), which suggests that dmrFABP5 acts through a mechanism different from SBFI26.
  • PPARY is a fatty acid receptor localised in the nuclear membrane.
  • SBFI26 is a weak agonist of PPARY. Since SBFI26 suppressed fatty acid uptake by replacing fatty acids which bind to FABP5, it is possible that some SBFI26 may be delivered to activate PPARY in a weaker way than with the fatty acids. This may be the reason why SBFI26 is a less effective inhibitor than dmrFABP5. Since the inhibitory effects of SBFI26 and dmrFABP5 are exerted at 2 different points in the same signal transduction pathway, no further suppression is anticipated when used in combination.
  • dmrFABP5 Since the exact route of fatty-acid delivery to PPARY is not known, how dmrFABP5 inhibits PPARY is not fully understood. Since the structure of dmrFABP5 is very similar to wild type FABP5, the inventors hypothesise that the protein of the invention (dmrFABP5) may occupy the same binding site as wild type FABP5 so as to prevent it from delivering fatty acids to PPARY, thus reducing transcription of PPARy regulated genes, including pro-cancer genes, such as VEGF. Further study is needed to understand more fully the mechanisms involved in the suppressive effect of dmrFABP5 on PPARY activation.
  • inactivated FABP5 protein of the invention was effective in reducing the proportion of activated PPARy in highly malignant cells that are independent of androgen for growth.
  • Castration resistant prostate cancer CRPC
  • ADT androgen deprivation therapy
  • inactivated FABP5 protein, fragment or variant thereof in accordance with the invention offers an androgen independent therapy for the treatment of CRPC and other FABP5 mediated disorders.
  • the benign cell line PNT2, highly malignant, androgen- independent cell lines DU145, PC3 and PC3-M, the moderately malignant, androgen- responsive cell line 22RV1 , and the weakly malignant cell line LNCaP were cultured and maintained in 1640 medium (Invitrogen) supplemented with 10% FCS (Biosera), 100 U/mL penicillin and 100 ⁇ g/mL streptomycin (Invitrogen).
  • FCS Biosera
  • streptomycin Invitrogen
  • Construction of expression vectors was carried out as described previously. After single and double mutations were generated in the fatty acid- binding motif of the FABP5 cDNAs, the wild type, the single- and the double-codon mutated FABP5 cDNAs were first cloned into the pBluescript II SK (Qiagen) vector and then excised from the pBluescript plasmid with Kpn ⁇ and Pst ⁇ . The 3 cDNAs were then separately inserted into the expression vector pQE32 (Qiagen) which was linearized with Kpn ⁇ and Pst ⁇ . The constructs were transformed into competent E.
  • coli to form 3 separate pools harbouring cDNAs for wtrFABP5, smrFABP5 and dmrFABP5.
  • the correct orientations of the inserted cDNAs in the constructs were confirmed by nucleic acid sequence analysis.
  • Expression and purification of recombinant FABP5s After the initial incubation, the E coli cells were induced by 1 mM IPTG for 4 h. Bacterial cells were harvested and lysed in lysis buffer, as described previously. Recombinant FABP5s were separated from bacterial proteins by affinity chromatography on a Ni-NTA agarose column (Qiagen).
  • Isolated proteins were dialyzed against PBS at 4°C for 4 hours to remove imidazole and stored at -80°C.
  • the purity of the recombinant FABP5s was checked by Western blot using the Penta-His antibody (Qiagen) and the polyclonal rabbit anti-human FABP5 antibody (Hycult). Before ligand binding assay, possible fatty acid binding to wtrFABP5 was removed by delipidation using Lipidex-1000 (Sigma).
  • Ligand binding assay The fatty acid-binding ability of all purified recombinant FABP5s was examined by using the DAUDA displacement assay which used fatty acids to replace the fluorescently labelled fatty acid analogue DAUDA (Cayman).
  • the excitation wavelength used for DAUDA was fixed at 345 nm, emission wavelengths for other molecules were collected over the range of 450-600 nm.
  • Fluorescence peak emission wavelength for wtrFABP5 was determined by set experiment as PBS, 3 ⁇ wtrFABP5, 2 ⁇ DAUDA and 3 ⁇ wtrFABP5 in 2 ⁇ DAUDA.
  • Fatty acid binding ability of the 3 FABP5s to 2 ⁇ DAUDA was tested by monitoring the change in fluorescence intensity and wavelength at the peak emission for each rFABP5 in the present or absence of 2 ⁇ linoleic acid.
  • the lead compound and best fatty acid that produced the highest binding affinity were then added to the assay at 10 ⁇ , and tested in triplicate to confirm their activity.
  • PC3-M cells (5* 10 4 ) were plated in triplicate in 96 well plates and incubated overnight. Cells were treated with different concentrations of SBFI26 (25-125 ⁇ ) and dmrFABP5 (5-70 ⁇ ) for 24 h. Cell viability was assessed using MTT assay, as described previously. The anti-proliferative effect of the best concentration for each inhibitor was determined after 6 days of treatment.
  • Wound healing migration assay was carried out to evaluate the effect of each inhibitor on the migratory rate of PC3-M cells. Wounds were generated by scratching the monolayer cells with a blue pipette tip. The floating cells were removed by washing with PBS and inhibitors were added to the culture medium. The wound was photographed under the microscope at 0, 12 and 24 h after treatment and the wound widths were assessed by quantitative analysis using ImageJ software.
  • Invasion assay PC3-M cells in serum-free medium were seeded in the upper Boyden chamber (BD Biosciences) in triplicate at a density of 2.5* 10 4 cells per well in serum-free medium. Complete medium was added to the lower chambers. After 3 hours of incubation, 50 ⁇ dmrFABP5 and 100 ⁇ SBFI26 were added to the upper chambers. After 24 h of incubation, cells that invaded the lower chambers were stained with crystal violet and counted with a cell counter.
  • Soft-agar assay Low melting agarose was seeded in 6-well plates and 5*10 4 cells/well layered on agar, followed by 200 ⁇ of medium alone or medium with different FABP5 inhibitors. Colonies larger than 300 ⁇ in each well were counted 2 weeks later in a similar way to that described previously.
  • PC3-M cells were transfected with the pGL4.50 [/i/c2/CMV/Hygro] vector (Promega) using FuGene HD transfection reagent (Promega) following the manufacturer's instructions. Individual colonies were isolated by ring cloning and 3 colonies that stably-expressed the highest bioluminescence signals were identified using D-luciferin (Promega) with a Varioskan Flash Reader (Thermo Scientific). Association of the luminescence intensity with the number of cells was assessed by an MS imaging system (Perkin Elmer).
  • Bioluminescence images was analysed using the Living Imagine software (Xenogen) and the measurement recorded was based on total photons/second (p/s) within each defined region of interest.
  • Nude mouse tumorigenecity assay PC3-M cells (2* 10 6 ) in 200 ⁇ _ PBS were subcutaneously injected into the right flank region of the mouse (6-8 week old) to test the suppressive effect of the inhibitors on tumorigenicity.
  • mice In the first round, 5 groups of mice (8 each) were used: 1) control with PBS; 2) 1 mg/kg SBFI26, injected from the 1 st day after cell inoculation; 3) 1 mg/kg SBFI26, injected from the 7 th day after cell inoculation; 4) 20mg/kg dmrFABP5, injected from the 1 st day after cell inoculation; 5) 20mg/kg dmrFABP5, injected from the 7 th day after cell inoculation.
  • mice 4 groups of mice (5 each) were used and at 7 days after the cell inoculation, each group was subjected to different intra- tumoural injections: 1) control with PBS; 2) SBFI26 plus dmrFABP5; 3) PPARy antagonist (GW9662, 1 mg/kg) (Sigma); 4) SBFI26, dmrFABP5 plus smrFABP5.
  • the injections were repeated every 2 days for 30 days, tumour size was measured every 3-4 days and the volume calculated by the formula of L* WxH xO.5236. Work was performed in accordance with UKCCCR guidelines under Home Office License PPL40/2963.
  • Fatty acid uptake assay Assay for fatty acid uptake was performed using red fluorescence-labelled BODIPY. The fluorescence intensity from cells before and 30 minutes after adding BODIPY was measured to determine fatty acid uptake. In inhibition and competition experiments, different concentrations of unlabelled lead compound SBFI26 (50- 200 ⁇ ) with the same concentration of labelled BODIPY were added directly to the highly malignant PC3-M cells.
  • inactivated FABP5 SEQ ID NO: 2
  • dmrFABP5 The protein sequence of inactivated FABP5 (SEQ ID NO: 2), also referred to as dmrFABP5, comprising mutations at Arg 109 and Arg 129 (shown in bold)
  • the restriction sites (GGATCC is the PstI restriction site and CTGCAG is the Kpnl restriction site) are indicated in bold.
  • the cDNA sequence comprises mutations in codons encoding Arg 109 and Arg 129.
  • the mutated codons are underlined below.
  • the restriction sites (GGATCC is the Pstl restriction site and CTGCAG is the Kpnl restriction site) are indicated in bold.

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Abstract

L'invention concerne l'utilisation de protéines FABP5 inactivées, ou des fragments ou variants de celles-ci, comme médicament. Celles-ci peuvent être utilisées dans le traitement de troubles induits par des FABP5, comme le cancer. Les protéines FABP5 inactivées, ou des fragments ou variants de celles-ci, peuvent également être utilisées dans la réduction thérapeutique de l'activation de PPARY. Les traitements de l'invention sont particulièrement utiles dans le traitement du cancer de la prostate et l'inhibition des métastases associées à cette maladie. Une protéine inactivée FABP5 inactivée appropriée, ou un fragment ou variant de celle-ci, peut intégrer des mutations dans des résidus d'acides aminés associés à la liaison d'acide gras à chaîne longue, comme Arg 109 et/ou Arg 129.
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* Cited by examiner, † Cited by third party
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CN107693509A (zh) * 2017-11-22 2018-02-16 中国医药集团总公司四川抗菌素工业研究所 Sb‑fi‑26在制备治疗乳腺癌药物中的应用
EP4208158A4 (fr) * 2020-09-04 2024-10-09 Aurigene Oncology Ltd Méthode pour le traitement du cancer à l'aide d'inhibiteurs de fabp5

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110237575A1 (en) * 2008-11-12 2011-09-29 Shipps Jr Gerald W Inhibitors of fatty acid binding protein (fabp)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110237575A1 (en) * 2008-11-12 2011-09-29 Shipps Jr Gerald W Inhibitors of fatty acid binding protein (fabp)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BROUARD M C ET AL: "Urinary excretion of epidermal-type fatty acid-binding protein and S100A7 protein in patients with cutaneous melanoma", MELANOMA RESEARCH, XX, XX, vol. 12, no. 6, 1 December 2002 (2002-12-01), pages 627 - 631, XP008080862 *
KIMITAKA SHIBUE ET AL: "Fatty acid-binding protein 5 regulates diet-induced obesity via GIP secretion from enteroendocrine K cells in response to fat ingestion", AMERICAN JOURNAL OF PHYSIOLOGY: ENDOCRINOLOGY AND METABOLISM., vol. 308, no. 7, 27 January 2015 (2015-01-27), US, pages E583 - E591, XP055351043, ISSN: 0193-1849, DOI: 10.1152/ajpendo.00543.2014 *
S. DHARMARAJAN ET AL: "Liver Fatty Acid-Binding Protein (L-Fabp) Modifies Intestinal Fatty Acid Composition and Adenoma Formation in ApcMin/+ Mice", CANCER PREVENTION RESEARCH, vol. 6, no. 10, 1 October 2013 (2013-10-01), United States, pages 1026 - 1037, XP055351025, ISSN: 1940-6207, DOI: 10.1158/1940-6207.CAPR-13-0120 *
Z. BAO ET AL: "A Novel Cutaneous Fatty Acid-Binding Protein-Related Signaling Pathway Leading to Malignant Progression in Prostate Cancer Cells", GENES AND CANCER, vol. 4, no. 7-8, 1 July 2013 (2013-07-01), US, pages 297 - 314, XP055350905, ISSN: 1947-6019, DOI: 10.1177/1947601913499155 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107693509A (zh) * 2017-11-22 2018-02-16 中国医药集团总公司四川抗菌素工业研究所 Sb‑fi‑26在制备治疗乳腺癌药物中的应用
EP4208158A4 (fr) * 2020-09-04 2024-10-09 Aurigene Oncology Ltd Méthode pour le traitement du cancer à l'aide d'inhibiteurs de fabp5

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