Methods of treating CNS cancers

Abstract

The present disclosure generally relates to CNS cancers and methods of treating CNS or brain cancers, in particular CNS or brain cancers that express or are associated with hGIIA such as glioblastoma.

Description

[0001] Methods of treating CNS cancers

[0002] Cross-reference to related applications

[0003] The present application claims priority from Australian Provisional Patent Application No. 2024902565 filed on 16 August 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] Technical Field

[0005] The present disclosure generally relates to CNS cancers and methods of treating CNS or brain cancers, in particular CNS or brain cancers that express or are associated with hGIIA such as glioblastoma.

[0006] Background

[0007] Glioblastoma multiforme (GBM) is the most aggressive primary brain tumour in adults. Despite a multidisciplinary approach including surgery, concomitant radiation therapy, and chemotherapy to treat GBM, and extensive efforts to identify new therapeutic approaches, most patients experience poor prognosis limiting the median survival to under 15 months. This is mainly due to factors such as tumour heterogeneity, tumour microenvironment, blood-brain barrier (BBB), and DNA damage repair mechanism of the O6-methylguanine-DNA methyltransferase (MGMT) protein which leads to drug resistance and tumour recurrence. Accordingly, there remains a need for effective therapies for treating CNS cancers like glioblastoma.

[0008] Summary

[0009] The present disclosure is based, in part, on the surprising discovery that existing cyclic peptides that inhibit hGIIA can cross the blood-brain barrier and also exert an anti-cancer effect in 2D and 3D cell culture models of glioblastoma. Accordingly, such therapeutic agents may be effective in treating CNS cancers, and more particularly hGIIA-dependent CNS cancers.

[0010] In a first aspect, the present disclosure provides a method of treating or preventing a CNS cancer or a disease, disorder or condition associated therewith in a subject, said method including the step of administering a therapeutically effective amount of a hGIIA inhibitor to the subject to thereby treat or prevent the CNS cancer, wherein the hGIIA inhibitor is a cyclic peptide.

[0011] Suitably, the CNS cancer expresses or is associated with hGIIA. In some examples, the CNS cancer is glioblastoma. Suitably, the cyclic peptide is capable of crossing the blood-brain barrier of the subject. According to certain examples, the cyclic peptide is cyclo-((2-Nal)-Leu-Ser-(2-Nal)-Arg) or an analogue or derivative thereof.

[0012] For certain examples, the cyclic peptide is administered at a dose about 5 mg / day to about 1500 mg / day. More particularly, the cyclic peptide can be administered at a dose of about 5 mg / day to about 100 mg / day.

[0013] Suitably, the cyclic peptide is administered orally, intravenously, intranasally or by inhalation. In some examples, the cyclic peptide is administered orally.

[0014] Referring to some examples, the subject is receiving a further anti-cancer agent for treatment of the CNS cancer. Accordingly, the present method may include the further step of administering a further anti-cancer agent for treatment of the CNS cancer to the subject. For such examples, the further anti-cancer agent can be selected from the group consisting of a chemotherapeutic agent, a radiation therapy, a molecularly targeted agent, a T cell expressing a chimeric antigen receptor (CAR-T cell), an antibody or antigen-binding fragment thereof, an antibody-drug conjugate (ADC), an angiogenesis inhibitor, an immunotherapeutic agent and any combination thereof.

[0015] In a second aspect, the present disclosure relates to a method of identifying, designing or producing an agent for use in treating or preventing a CNS cancer, said method including the step of determining whether the candidate agent crosses a blood-brain barrier, wherein the candidate agent is a cyclic peptide and wherein the cyclic peptide is a hGIIA inhibitor.

[0016] Suitably, the present method includes the steps of:

[0017] (a) administering a candidate agent to a subject;

[0018] (b) determining whether the candidate agent crosses the blood-brain barrier of the subject.

[0019] In particular examples, the candidate agent is administered orally, intravenously, intranasally or by inhalation to the subject.

[0020] In various examples, the subject has or is at risk of developing the CNS cancer. For such examples, the present method may further include the step of determining whether the candidate agent treats or prevents the CNS cancer in the subject.

[0021] Suitably, the candidate agent is a variant or a derivative of cyclo-((2-Nal)-Leu-Ser-(2-Nal)- Arg).

[0022] Referring to certain examples, the present method further includes the step of isolating and / or purifying an agent that crosses the blood brain barrier. More particularly, the present method may further include the step of isolating and / or purifying an agent that crosses the blood brain barrier and treats or prevents the CNS cancer of the subject. Suitably, the present method further includes the step of formulating the isolated and / or purified agent into a pharmaceutically acceptable formulation. The method can further include the step of sterilising the formulation. Furthermore, the method may further include the step of filling the formulation into a container. For such examples, the container can be any one or more of a vial, an ampoule, a bag, a blister pack, a bottle, a cartridge, an inj ection needle, an inj ection syringe, a single dose container, a strip of multiple single dose containers, or a tube.

[0023] In a third aspect, the present disclosure provides an agent for use in treating or preventing a CNS cancer in a subject, obtained by the method of the second aspect.

[0024] In a fourth aspect, the present disclosure provides a kit for use in the method of the first aspect, said kit comprising a hGIIA inhibitor, wherein the hGIIA inhibitor is a cyclic peptide, optionally a further anti-cancer therapy and optionally instructions for use.

[0025] Brief description of the drawings

[0026] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

[0027] Figure 1. Workflow overview for 3D bioprinting using the RASTRUM platform.

[0028] The RASTRUM bioprinting platform was utilised to bioprint a 3D cell culture model for the growth of U251, LN229, and T98G cell lines. The first step in identification of a suitable matrix was done on the RASTRUM cloud. Next, the PEG-based inert base was printed. This was followed by encapsulating cell models within the scaffold. The growth was monitored using the Incucyte imaging system for a total of eight days. The workflow image was created with BioRender.com.

[0029] Figure 2. Dose-response curves for c2 and TMZ on GBM cell lines A) U251, B) LN229 and C) T98G. Cell viability at 72 h was determined for cells seeded at 5xl03cells / well, using the ratio of the absorbance of treated cells to the no-drug DMSO control. Dose-response curves represent thirteen concentrations of each drug and were fitted to a non-linear regression [Inhibitor] vs. response model. Data are mean ± SEM of three biological replicates.

[0030] Figure 3. Incucyte images at t= 0, 24, 48, and 72 h post drug treatment in U251. These images represent changes in scratch area (indicated by a red box) for 72 h post treatment with c2 and TMZ (200pM), DMSO (1%) and fresh media. Incucyte images were obtained at 10 x objective. n=l

[0031] Figure 4. Incucyte images at t= 0, 24, 48, and 72 h post drug treatment in LN229. These images represent changes in scratch area (indicated by a red box) for 72 h post treatment with c2 and TMZ (200pM), DMSO (1%) and fresh media. Incucyte images were obtained at 10 x objective. n=l

[0032] Figure 5. Incucyte images at t= 0, 24, 48, and 72 h post drug treatment in T98G.

[0033] These images represent changes in scratch area (indicated by a red box) for 72 h post treatment with c2 and TMZ (200pM), DMSO (1%) and fresh media. Incucyte images were obtained at 10 x objective. n=l

[0034] Figure 6. Changes in wound confluence for 72h post drug treatment in U251, LN229, and T98G. Wound confluence as a percentage of wound confluence at t=0 h post drug treatment for U251 cells (A), LN229 cells (B), and T98G cells (C). Comparison of % wound confluence between treatments at 48 hrs are plotted for U251 cells (D), LN229 cells (E), and T98G cells (F). Comparison of % wound confluence at 72 hrs between treatments are plotted for U251 cells (G), LN229 cells (H), and T98G cells (I). A one-way ANOVA was performed to compare % wound confluence of wells treated with of c2 (200pM), TMZ (200pM), and DMSO (1%), * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001, ns — not significant, n = 3. Data are mean ± SEM.

[0035] Figure 7. Cellular morphology in 3D models of A) U251, B) LN229, and C) T98G cells. In the 3D spheroid model created using RASTRUM, the cells embedded in the PEG-hydrogel matrix. The LN229 and T98G cells formed clustered spheroid-like structures within 5 days after printing, unlike U251, which grew in a mesh-like network seemingly similar to 2D cell culture.

[0036] Figure 8. Growth curves of U251, LN229, and T98G cell lines, when subjected to A) 400 pM c2 and B) 1000 pM TMZ. Growth curves display changes in % cell confluency with time. Drug treatment was done at t=120 h. Data are mean ± SEM of three technical replicate wells.

[0037] Figure 9. Dose-response curves for c2 and TMZ on glioblastoma cell lines A) U251, B) LN229, and C) T98G. Cell viability 72 h post drug treatment on 3D bioprinted cells was determined using the ratio of the absorbance of treated cells to the no-drug DMSO control. Dose-response curves represent eight concentrations of each drug and were fitted to a non-linear regression [Inhibitor] vs. response model. Data are mean ± SEM of three technical replicates.

[0038] Figure 10. A. Vimentin is highly expressed in GBM patient derived cells and GBM cell lines; B. c2 treatment disrupts the vimentin network and reduces its expression.

[0039] Figure 11. LC-MS / MS and MALDI-MSI workflow. Tissues and serum samples extracted from mice intraperitoneally injected by c2 and cF were analysed using LC-MS / MS for the presence of the analytes and AP-MALDI for the distribution of these analytes. The workflow image was created with BioRender.com.

[0040] Figure 12. Retention time of c2 and cF. The [M+H]Tprecursor ions for A) c2 (m / z = 751.4) eluted from the column at 2.62 min while B) cF m / z:=:667.3) eluted from the column at 1.72 min. These peaks are shown in blue, while the internal standard peaks of c2 and cF are shown in pink. Figure 13. c2 and cF concentration in brain, liver and kidney tissues in ng / g fresh weight.

[0041] A) The highest concentration of c2 was detected as 995 ± 46 ng / g in liver (n=3), followed by 322 ± 132 ng / g in kidneys (n=5) and 50 ± 14 ng / g in brain (n:=:4). B) The highest concentration of cF was detected as 650 ± 294 ng / g in liver (n=3), followed by 210 ± 24 ng / g in kidneys (n=5) and 25 ± 5 ng / g in brain (n=4). Data shows concentration in each mouse sample and the mean concentration in each tissue type.

[0042] Figure 14. Flow cytometric analysis of cell death mediated by C2, and TMZ. U251 cells were treated with the complex and analysed at 72 h as described in Method 1. (A). Representative dot plots and (B). Bar graphs demonstrating percent viable, apoptotic, and necrotic cells. Each treatment condition has been assigned a specific colour, as indicated to the right of the bar graph. Data points represent mean ± SEM. n = 3 from three separate experiments with samples run in triplicate. * p < 0.05, *** p < 0.001 and **** p < 0.0001 compared to control group, as measured by one-way ANOVA.

[0043] Figure 15. Flow cytometric analysis of cell death mediated by C2, and TMZ. T98G cells were treated with the complex and analysed at 72 h as described in Method 1 . (A). Representative dot plots and (B). Bar graphs demonstrating percent viable, apoptotic, and necrotic cells. Each treatment condition has been assigned a specific colour, as indicated to the right of the bar graph. Data points represent mean ± SEM. n = 3 from three separate experiments with samples run in triplicate. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to control group, as measured by one-way ANOVA.

[0044] Figure 16. Flow cytometric analysis of cell death mediated by C2, and TMZ. LN229 cells were treated with the complex and analysed at 72 h as described in Method 1. (A). Representative dot plots and (B). Bar graphs demonstrating percent viable, apoptotic, and necrotic cells. Each treatment condition has been assigned a specific colour, as indicated to the right of the bar graph. Data points represent mean ± SEM. n = 3 from three separate experiments with samples run in triplicate. * p < 0.05, ** p < 0.01 and **** p < 0.0001 compared to control group, as measured by one-way ANOVA.

[0045] Figure 17. Flow cytometric analysis of cell cycle mediated by C2 and TMZ. U251 cells were treated with 50, 100 or 200 uM concentration of C2 or TMZ and analysed at 48 h as described in Method 2. (A) Representative histogram plots and (B) Bar graphs show percent Sub Gl, G0 / G1 , S and G2+M phases of cell cycle. Data points represent mean ± SEM. n = 3 from three separate experiments where samples were run in triplicate. * p < 0.05 and ** p < 0.01 compared to control group, as measured by one-way ANOVA.

[0046] Figure 18. Flow cytometric analysis of cell cycle mediated by C2 and TMZ. T98G cells were treated with 50, 100 or 200 uM concentration of C2 or TMZ and analysed at 48 h as described in Method 2. (A) Representative histogram plots and (B) Bar graphs show percent Sub Gl, G0 / G1, S and G2+M phases of cell cycle. Data points represent mean ± SEM. n = 3 from three separate experiments where samples were run in triplicate. * p < 0.05 and ** p < 0.01 compared to control group, as measured by one-way ANOVA.

[0047] Figure 19. Flow cytometric analysis of cell cycle mediated by C2 and TMZ. LN229 cells were treated with 50, 100 or 200 pM concentration of C2 or TMZ and analysed at 48 h as described in Method 2. (A) Representative histogram plots and (B) Bar graphs show percent Sub Gl, G0 / G1, S and G2+M phases of cell cycle. Data points represent mean ± SEM. n = 3 from three separate experiments where samples were run in triplicate. * p < 0.05 and ** p < 0.01 compared to control group, as measured by one-way ANOVA.

[0048] Key to the Sequence Listing

[0049] SEQ ID NO: 1 Amino acid sequence of hGIIA protein with signal sequence

[0050] SEQ ID NO: 2 Amino acid sequence of hGIIA protein without signal sequence

[0051] SEQ ID NO: 3 Amino acid sequence of vimentin

[0052] Detailed Description

[0053] General

[0054] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.

[0055] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0056] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0057] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

[0058] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary7skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0059] Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory' Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory', (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0060] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0061] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0062] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0063] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0064] Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5. 5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0065] The term “about” in relation to a numerical value x is optional and means, for example, any number within (i.e., plus or minus) 1%, 5% or 10% of the referenced number. In certain examples, the term “about” encompasses the exact number recited.

[0066] The term “substantially” does not exclude “completely” (e.g., a composition which is “substantially free” from Y may be completely free from Y).

[0067] All computer programs, algorithms, gene and protein accession numbers and their associated sequences, patents, patent applications and scientific literature referred to herein is incorporated herein by reference.

[0068] Methods of treating CNS cancers

[0069] The inventors have surprisingly shown for the first time that cyclic peptide hGIIA inhibitors like c2 can readily cross the blood-brain barrier in vivo, whilst also demonstrating significant anti-cancer activity in relation to GBM cell lines. This in vitro anti-cancer effect was also surprisingly observed to be significantly greater than that of the standard of care chemotherapeutic agent for GBM, temozolomide. Such cyclic peptide hGIIA inhibitors therefore offer promise as a treatment for CNS cancers that are associated with hGIIA expression and / or activity.

[0070] Accordingly, there is provided herein a method of treating or preventing a CNS cancer or a disease, disorder or condition associated therewith in a subject, said method including the step of administering a therapeutically effective amount of a hGIIA inhibitor to the subject to thereby treat or prevent the CNS cancer, wherein the hGIIA inhibitor is a cyclic peptide.

[0071] In a related form, the present disclosure relates to the use of a hGIIA inhibitor in the manufacture of a medicament for the prevention or treatment of a CNS cancer in a subject, wherein the hGIIA inhibitor is a cyclic peptide.

[0072] In yet another form, there is provided a hGIIA inhibitor or a composition comprising same for use in the prevention or treatment of a CNS cancer in a subject, wherein the hGIIA inhibitor is a cyclic peptide.

[0073] As used herein, the terms “treating”, “treat” or “treatment” and variations thereof, refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. The term “ameliorating”, with, reference to such CNS cancers, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0074] As used herein, the terms “prevent”, “prevented”, or “preventing”, refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.

[0075] As used herein, the term “subject” refers to any animal, for example, a mammalian animal, including, but not limited to humans, non-human primates, livestock (e.g., sheep, horses, cattle, pigs, donkeys), companion animals (e.g., pets such as dogs and cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), performance animals (e.g., racehorses, camels, greyhounds) or captive wild animals. In various examples, the “subject” is a human, such as a male human or a female human. Typically, the terms “subject” and “patient” are used interchangeably, particularly in reference to a human subject.

[0076] The term “therapeutically effective amount” describes a quantity of a specified agent, such as a cyclic peptide like c2 or a derivative or analogue thereof, sufficient to achieve a desired effect in a subject being treated with that agent or composition. For example, this can be the amount of the agent and optionally one or more further therapeutic agents (e.g., one or more further anticancer agents), necessary to reduce, alleviate and / or prevent a cancer, such as an hGIIA-dependent cancer. Suitably, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of a cancer, such as an hGIIA-related cancer. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression, such as cancer metastasis or recurrence, or overcome resistance to and / or enhance the anti -cancer activity of a further anticancer agent.

[0077] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent useful for reducing, alleviating, ameliorating and / or preventing the diseases, disorders and conditions described herein will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition (e.g., disease progression), and the manner of administration of the therapeutic composition. Suitably, a therapeutically effective amount of the agent is administered orally to the subject.

[0078] As generally used herein, the terms “cancer”, “tumour”, “malignant” and “malignancy” refer to diseases or conditions, or to cells or tissues associated with the diseases or conditions, characterized by aberrant or abnormal cell proliferation, differentiation and / or migration often accompanied by an aberrant or abnormal molecular phenotype that includes one or more genetic mutations or other genetic changes associated with oncogenesis, expression of tumour markers, loss of tumour suppressor expression or activity and / or aberrant or abnormal cell surface marker expression.

[0079] The terms “cancer of the CNS” and “CNS cancer” are used interchangeably herein and refer to cancers of the brain, such as glioma, neuroblastoma, glioblastoma, other astrocytomas, oligodendroglial tumours, meningiomas, ependymomas, and medulloblastomas, cancers of the spinal cord, and cancers of the meninges surrounding the brain and spinal cord.

[0080] Suitably, the CNS cancer is a glioma. A glioma is a tumour that arises from glial cells or their precursors of the brain or spinal cord. Gliomas are histologically defined based on whether they exhibit primarily astrocytic or oligodendroglial morphology, and are graded by cellularity, nuclear atypia, necrosis, mitotic figures, and microvascular proliferation - all features associated with biologically aggressive behaviour. For some examples, the glioma is an astrocytoma, a brainstem glioma, an ependymoma, a mixed glioma, an oligodendroglioma, or an optic nerve glioma. In certain examples, the glioma includes, but is not limited to, an acoustic neuroma, such as an astrocytoma: Grade I - hairy astrocytoma, Grade II - fibrillar astrocytoma, Grade III - anaplastic astrocytoma, Grade IV - glioblastoma. Other types of glioma include, but are not limited to, chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic neural glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumours, oligodendroglioma, pituitary neural), schwannomas, juvenile pilocytic astrocytoma (JPA), pineal tumour and / or rhabdoid tumour.

[0081] Referring to various examples, the CNS cancer is a glioblastoma. The terms “glioblastoma”, “glioblastoma multiforme” and “GBM” refer to a type of cancer that is a subset of astrocytomas, such as grade IV astrocytoma. GBM includes variants of GBM: giant cell GBM and gliosarcoma, as well as four subtypes of GBM: classical, neural, proneural, and mesenchymal. GBM cancers can be primary (de novo) or secondary (metastatic).

[0082] Suitably, the CNS cancer expresses, and more particularly overexpresses, or is associated with hGIIA. According to certain examples, the CNS cancer is associated with an elevated level or activity of hGIIA. In this regard, the CNS cancer may be considered to be a hGIIA-related, hGIIA-associated or hGIIA-dependent cancer. The terms “hGIIA-dependent cancer”, “hGIIA- related cancer”, “hGIIA-associated cancer” and the like are used interchangeably herein and refer to cancers associated with a disease state, where increased levels of hGIIA expression and / or activity and / or increased activation of cellular cascades or downstream signalling pathways involving hGIIA are found (e.g., those mediated by prostaglandins and / or leukotrienes). Accordingly, such CNS cancers may be associated with increased or elevated levels of non- catalytic activity and / or catalytic activity of hGIIA. In particular examples, however, these terms are intended to encompass CNS cancers associated with an increased or elevated level of non- catalytic activity of hGIIA. In general, these terms refer to any cancer, the onset, progression or the persistence of the symptoms of which requires the participation, expression and / or activity of hGIIA.

[0083] In view of the above, the CNS cancer described herein may be at least partly mediated by, associated with or dependent on vimentin, such as by way of its interaction with hGIIA. To this end, the CNS cancer may be considered to be a vimentin-related, vimentin-associated or vimentin- dependent cancer, which generally refer to any cancer, the onset, progression or the persistence of the symptoms of which requires the participation, expression and / or activity of vimentin, with or without the need for hGIIA activity or binding thereto.

[0084] Suitably, the present methods further including the earlier or initial step of identifying whether the subject’s CNS cancer, such as from a sample (e.g., a biopsy sample or a biological sample, such as a blood, plasma, serum, urine, CSF etc.) obtained from the subject (e.g., from the subject’s cancer), is an hGIIA-related cancer (e.g., an hGIIA-dependent cancer) or a vimentin- related cancer (e.g., a vimentin-dependent cancer). In such instances, the present method may include the step of determining a level of hGIIA and / or vimentin expression and / or activity, such as a level of circulating hGIIA and / or an expression level of vimentin of the subject’s CNS cancer. Well known assays, such as those described herein, can be used for determining the hGIIA and / or vimentin expression, activity and / or signalling status of the subject to be treated if such prior determination is desired.

[0085] Accordingly, the methods disclosed herein may further include including the step of determining the level of circulating hGIIA before and / or after administration of the cyclic peptide to the subject. In some examples, the level of circulating hGIIA may be determined before administration of the cyclic peptide to the subject. For other examples, the level of circulating hGIIA may be determined after administration of the cyclic peptide to the subject. In particular examples, the level of circulating hGIIA may be determined before and after administration of the cyclic peptide to the subject.

[0086] Suitable methods to determine the level of circulating hGIIA are known in the art. For example, western blot, spectrophotometry and / or ELISA methods may be utilised in this regard. In one example, the level of circulating hGIIA may be measured in a blood sample obtained from the subject. In another example, the level of circulating hGIIA may be measured in a plasma sample obtained from the subject. In still further examples, the level of circulating hGIIA is measured in a serum sample obtained from the subject. In yet another example, the level of circulating hGIIA is measured, such as indirectly measured, in a urine sample obtained from the subject. In various examples, the level of circulating hGIIA is measured, such as indirectly measured, in a CSF sample obtained from the subject.

[0087] In various examples, the level of circulating hGIIA in the subject may be elevated prior to administering the therapeutically effective amount of the cyclic peptide to the subject.

[0088] As used herein, the level of circulating hGIIA is considered “elevated” or “increased” when it is higher (including relatively or absolutely higher) than a predetermined reference, control or threshold level thereof. The level of circulating hGIIA may also be compared to a reference or threshold level of circulating hGIIA. Thus, any of the methods disclosed herein may comprise a step of establishing a reference or threshold level of hGIIA. Suitable threshold levels can then be determined according to the particular methodology used to measure the levels of hGIIA, which threshold levels can establish a significant difference in the levels of hGIIA and a healthy control (e.g., a median or mean level of circulating hGIIA in a healthy or cancer-free population of subjects), such a difference then being indicative of an increase in the level of circulating hGIIA in the subject. It will be appreciated that the precise threshold levels will vary depending on the samples used to establish those threshold levels of circulating hGIIA and according to the particular analytical methodology used in each instance.

[0089] In broad terms, a threshold level for hGIIA in a biological sample, such as blood, plasma, serum, urine or CSF, may be about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL or any range therein. In particular examples, a threshold level for hGIIA in a biological sample, such as blood, plasma, serum, urine or CSF, is at least about 1 ng / mL, more particularly at least about 2.5 ng / mL, even more particularly at least about 5 ng / mL or still even more particularly at least about 10 ng / mL, such as measured by one or more of those methods described herein. By way of example, a hGIIA level in a biological sample, such as blood, plasma, serum, urine or CSF, from the subject that is less than about 10 ng / mL, more particularly less than about 5 ng / mL, still more particularly less than about 2.5 ng / mL or even more particularly less than about 1 ng / mL, such as measured by one or more of those methods described herein, in the subject is considered within the normal range. Similarly, a hGIIA level in a biological sample, such as blood, plasma, serum, urine or CSF, from the subject that is greater than about 5 ng / mL, more particularly greater than about 10 ng / mL, still even more particularly greater than about 20 ng / mL or even more particularly greater than about 30 ng / mL, such as measured by one or more of those methods described herein, may be considered an elevated level of hGIIA and by extension could be indicative of the CNS cancer of the subject being at least partly associated with hGIIA.

[0090] An “elevated” or “increased” level of circulating hGIIA is suitably a level that is greater than the reference, control or normal level of circulating hGIIA. The “normal” level of circulating hGIIA may be determined by selecting any suitable biological sample or samples from which to derive the circulating level of hGIIA in a non-disease state (e.g., a mean or median level, such as in a healthy or cancer-free population of subjects). The “elevated” or “increased” level of circulating hGIIA may be determined by selecting any suitable biological sample from which to derive the level hGIIA in a disease state. The level of hGIIA may be measured in any one or more cells, tissues, organs or biological samples. The biological sample may be or may comprise a bodily fluid. The bodily fluid may blood, serum, plasma, CSF or urine. For example, the level of hGIIA may be measured in plasma. In another example, the level of hGIIA may be measured in serum. In another example, the level of hGIIA may be measured in urine. In various examples, the level of hGIIA is measured in cerebrospinal fluid (CSF). The biological sample may be subject to any suitable pre-treatment steps before the measurement is performed, in order to improve the accuracy and / or efficiency of the measurement. Thus, any of the methods disclosed herein may comprise a step of taking a biological sample from a subject and determining the level hGIIA in the sample. Alternatively, any of the methods disclosed herein may not comprise a step of taking a biological sample from a subject and determining the level hGIIA in the sample. Instead, the level of hGIIA in the sample may have been determined previously.

[0091] Any of the methods disclosed herein may comprise a step of establishing a reference level of hGIIA. Alternatively, any of the methods disclosed herein may comprise a step of comparing the level of hGIIA to a predetermined reference level. For example, the predetermined reference level may be stored in a database including such information. Suitable threshold levels can then be determined according to the particular methodology used to identify and / or measure the level of hGIIA. It will be appreciated that the precise thresholds will vary depending on the samples used to establish those threshold levels and according to the particular analytical methodology used in each instance. Thus, a “low” level of hGIIA is a level of hGIIA that is decreased relative to a reference level of hGIIA. Conversely, a “high or “elevated” or “increased” level of hGIIA is a level that is greater than a reference level of hGIIA. A “reference” level can be determined by selecting any suitable sample from which to derive the level of hGIIA. That sample may be from any biological sample obtained from one or more subjects, such as a population of control subjects who do not have cancer or a control sample obtained from the subject prior to administration of the cyclic peptide.

[0092] In view of the foregoing, the present method may include the initial step of selecting a subject as being suitable for treatment with the cyclic peptide by determining a level of circulating hGIIA in the subject. Suitably, an elevated level of circulating hGIIA is indicative of the subject being suitable for administration of the cyclic peptide. hGIIA inhibitors

[0093] Suitably, the cyclic peptide disclosed herein is a hGIIA inhibitor. As used herein, the term “hGIIA inhibitor” shall be taken to mean an agent which hinders, reduces, restrains or prevents hGIIA expression and / or activity relative to a level of hGIIA expression and / or activity in a subject or cell to which a hGIIA inhibitor has not been administered or contacted. In this regard, the hGIIA inhibitor may inhibit or prevent an increase or promote a decrease in a level of hGIIA, such as circulating (e.g., blood, serum, plasma, urine or CSF level) or tissue (e.g., CNS, cancer cells or tissues) hGIIA levels, associated with the CNS cancer. According to certain examples, the hGIIA inhibitor inhibits a catalytic activity and / or a non-catalytic activity of hGIIA. More particularly, the hGIIA inhibitor inhibits a non-catalytic activity of hGIIA. hGIIA activity may be inhibited by the cyclic peptide in any measurable amount. Inhibition of hGIIA activity may be complete or may be partial. Thus, the methods disclosed herein may comprise at least partial inhibition of hGIIA activity. For example, the activity of hGIIA may be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% following administration of the cyclic peptide (e.g., relative to the same measurement of activity before administration of the cyclic peptide). hGIIA activity may be measured through any suitable means known in the art, or any of the methods disclosed herein. For example, hGIIA activity may be measured through assays which measure the level of one or more inflammatory markers, such as inflammatory cytokines or inflammatory prostaglandins, the proliferation of prostate or GBM cancer cells or circulating levels of hGIIA.

[0094] Suitably, the cyclic peptide may be a hGIIA inhibitor of the following formula:

[0095] A1-A2-A3-A4-A5 in which: Al is F or Y or W or 2NapA;

[0096] A2 is L or I;

[0097] A3 is S or T;

[0098] A4 is F or Y or W or 2NapA; and

[0099] A5 is R or K.

[0100] In one example, the hGIIA inhibitor is selected from the group consisting of cFLSYK, cFLSYR and c(2NapA)LS(2NapA)R. When used herein the term “cFLSYK” means “cyclic FLSYK”, “cFLSYR” means “cyclic FLSYR” and “c(2NapA)LS(2NapA)R” means “cyclic (2NapA)LS (2NapA)R”. The terms “2NapA” and “2-Nal” are abbreviations for 2- naphthylalanine. In various examples, the hGIIA inhibitor is cyclo-((2-Nal)-Leu-Ser-(2-Nal)-Arg) (FLM-c2, KS-c2 or c2), as provided in Formula I below.

[0101] Formula I

[0102] In some examples, the hGIIA inhibitor is cFLSYR, as provided in Formula II below.

[0103] Formula II

[0104] In other examples, the hGIIA inhibitor is a derivative or a variant of cyclo-((2-Nal)-Leu- Ser-(2-Nal)-Arg) (FLM-c2, KS-c2 or c2). Such a derivative or variant for use according to the present disclosure is suitably a functional c2 peptide. By “functional c2 peptide” is meant a peptide able to: bind to vimentin, and thereby prevent or inhibit binding of hGIIA; and / or bind to EGFR, and thereby prevent or inhibit binding of EGF ligands thereto and / or inhibit downstream EGFR signalling. Determining whether a c2 peptide derivative or variant is functional may be assessed by any method or means known in the art.

[0105] In various examples, the peptide inhibitor or cyclic peptide provided herein is not cFLSYR. In other examples, the peptide inhibitor or cyclic peptide provided herein is not cFLSYK.

[0106] As used herein, “derivatives” refers to proteins or peptides, inclusive of fragments or variants thereof, that have been altered, for example by conjugation or complexing with other chemical moieties, such as by post-translational modification (e.g., phosphorylation, acetylation, ubiquitination, glycosylation and the like), modification of glycosylation (e.g., adding, removing or altering glycosylation), lipidation and / or inclusion of additional amino acid sequences as would be understood in the art. Derivatives contemplated by the disclosure include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and / or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the cyclic peptides of the disclosure. In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

[0107] Further derivatives may include conjugates of the cyclic peptide, such as c2. The term “conjugated” can be used in the context of the present disclosure to describe the cyclic peptide conjugated to another compound or structure, such as a label or carrier molecule or protein. Accordingly, in one example, the cyclic peptide is “conjugated”. The cyclic peptide may be modified via conjugation or complexing with other chemical moieties, such as chemical modification (e.g., cross-linking, acetylation, biotinylation, oxidation or reduction) and / or conjugation with labels (e.g., fluorophores, enzymes, radioactive isotopes) and / or other functional elements (e.g., a half-life extender, a CNS targeting moiety), as are known in the art.

[0108] In one example, the cyclic peptide derivative may be a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0109] The cyclic peptide may be utilized per se or in the form of a pharmaceutically acceptable salt, ester, amide, solvate, prodrug, or isomer, as appropriate. For example, it may be provided as a pharmaceutically acceptable salt. If used, a salt of the cyclic peptide should be both pharmacologically and pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare the free active compound or pharmaceutically acceptable salts thereof and are not excluded from the scope of this disclosure. Such pharmacologically and pharmaceutically acceptable salts can be prepared by reaction of the cyclic peptide with an organic or inorganic acid, using standard methods detailed in the literature.

[0110] The cyclic peptide may be in the form of any pharmaceutically acceptable salt. The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable nontoxic bases or acids including inorganic or organic bases and inorganic or organic acids. Examples of salts derived from inorganic bases can include aluminium, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Exemplary salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0111] When the cyclic peptide, such as c2, disclosed herein is basic, acid addition salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic acid, TFA, and the like. Acid addition salts of the cyclic peptide, are prepared in a suitable solvent from the peptide and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, TFA, citric, tartaric, maleic, succinic or methanesulfonic acid.

[0112] In particular examples, the cyclic peptide, or more particularly c2, is in the form of an acetate salt.

[0113] The cyclic peptide can also exist in solvated forms, including solvates of the free peptide or solvates of a salt of the compound, as well as unsolvated forms. The term “solvate” is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term “hydrate” is employed when said solvent is water. It is to be understood that all polymorphs, including mixtures of different polymorphs, are included within the scope of the cyclic peptide analogues or derivatives described herein.

[0114] Esters of the cyclic peptide may be prepared through functionalization of hydroxyl and / or carboxyl groups that may be present within the compound. Amides and prodrugs may also be prepared using techniques known to those skilled in the art. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. Moreover, esters and amides of compounds of the invention can be made by reaction with a carbonylating agent (e.g., ethyl formate, acetic anhydride, methoxyacetyl chloride, benzoyl chloride, methyl isocyanate, ethyl chloroformate, methanesulfonyl chloride) and a suitable base (e.g., 4- dimethylaminopyridine, pyridine, triethylamine, potassium carbonate) in a suitable organic solvent (e.g., tetrahydrofuran, acetone, methanol, pyridine, N,N-dimethylformamide) at a temperature of 0 °C to 60 °C.

[0115] As used herein, the term “prodrug” means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a cyclic peptide described herein. Prodrugs may become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated for the present disclosure include, but are not limited to, cyclic peptides that comprise biohydrolyzable moieties, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs include derivatives of compounds described herein that comprise — NO, — NO2, — ONO, or — ONO2 moieties. Prodrugs can typically be prepared using well-known methods, such as those described by BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed).

[0116] The protein sequence of hGIIA is publicly available (e.g., UniProt Accession No. P14555) and exemplary amino acid sequences are set forth in SEQ ID NO: 1 and SEQ ID NO: 2 below. Thus, the hGIIA amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or a fragment or derivative thereof. The sequence in SEQ ID NO: 1 includes a signal peptide at the beginning of the protein (i.e., amino acid residues 1 to 20 of SEQ ID NO: 1), with the sequence for the mature hGIIA protein beginning at Asn 21 of SEQ ID NO: 1, which is absent in the mature protein of SEQ ID NO: 2. hGIIA protein sequence with signal sequence (SEQ ID NO: 1)

[0117] MKTLLLLAVIMIFGLLQAHGNLVNFHRMIKLTTGKEAALSYGFYGCHCGVGGRGSPKD ATDRCCVTHDCCYKRLEKRGCGTKFLSYKFSNSGSRITCAKQDSCRSQLCECDKAAATC FARNKTTYNKKYQYYSNKHCRGSTPRC hGIIA protein sequence without signal sequence (SEQ ID NO: 2) NLVNFHRMIKLTTGKEAALSYGFYGCHCGVGGRGSPKDATDRCCVTHDCCYKRLEKR

[0118] GCGTKFLSYKFSNSGSRITCAKQDSCRSQLCECDKAAATCFARNKTTYNKKYQYYSNK

[0119] HCRGSTPRC

[0120] Terms used generally herein to describe sequence relationships between respective nucleotides or polypeptides include “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleotides or polypeptides may each comprise (i) only one or more portions of a complete nucleotide or polypeptide sequence that are shared by the nucleotides or amino acids, and (ii) one or more portions which are divergent between the nucleotides or amino acids, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0121] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0122] Without wishing to be bound by any theory, the inventors have found previously that c2 binds to amino acid residues within the amino acid residues numbered 347 to 357, and 342 to 350 which are located on vimentin coil 2. The cyclic peptide may bind to any one or more amino acids within coil 2 as defined herein. The cyclic peptide may bind to residues 347 to 357 of vimentin. The cyclic peptide may bind to residues 342 to 350 of vimentin. The cyclic peptide may bind to any one or more of the following residues on vimentin: M347, N350, F341, V353, E354 andN357. Previous molecular modelling analysis has indicated that these residues may directly contact an effective hGIIA or vimentin inhibitor. The cyclic peptide may bind to any one or more of the following residues on vimentin: M347, F341 and E354. The cyclic peptide may at least bind to residues M347, F341 and E354 on vimentin.

[0123] In view of the foregoing, the cyclic peptide provided herein is suitably (or may be alternatively referred to as) a vimentin inhibitor. As used herein, the term “vimentin inhibitor” shall be taken to mean an agent which hinders, reduces, restrains or prevents vimentin expression and / or an activity of a vimentin protein relative to a level of vimentin expression and / or activity in a subject to which a vimentin inhibitor has not been administered.

[0124] Vimentin is a type III intermediate filament (IF) protein that is expressed in mammalian cells including mesenchymal cells. IF proteins are found in all animal cells and bacterial cells. IFs, along with tubulin-based microtubules and actin-based microfilaments, comprise the cytoskeleton. Vimentin is the major cytoskeletal component of mesenchymal cells.

[0125] Vimentin has been shown to play a role in supporting and anchoring the position of organelles in the cytosol. Vimentin may be attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. Amongst other functions, vimentin is responsible for maintaining cell shape, integrity of the cytoplasm, and stabilizing cytoskeletal interactions.

[0126] The protein sequence of vimentin is publicly available (e.g., UniProt Accession No. P08670) and an exemplary amino acid sequence is set forth in SEQ ID NO: 3 below. Thus, the vimentin amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3, or a fragment or derivative thereof.

[0127] Vimentin protein sequence (SEQ ID NO: 3)

[0128] MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVY ATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDK VRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNL AEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKL HEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKF ADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFA VEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRI SLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLE

[0129] The vimentin protein comprises a number of different domains. For example, the structural organization of a vimentin monomer comprises a central, mostly a-helical “rod” domain flanked by intrinsically disordered non-a-helical N-terminal (“head”) and C-terminal (“tail”) domains. The rod consists of two equally sized a-helical subdomains termed coil 1 (146 amino acids) and coil 2 (140 amino acids), which are connected by the 16 amino acid-long non-a-helical linker segment L12. Coil 1 is divided into a short coil 1A and a longer coil IB segment. Linker LI connecting the coil 1A and B subdomains is 8 amino acids long and evolutionarily highly conserved; similar to other intrinsically disordered domains, it may optionally form a distinct structure. Indeed, in the crystal of a larger fragment derived from coil 1, linker LI adopts an a- helical fold without being involved in the coiled-coil formation of coil 1A and coil IB. Coil 2 represents a continuous a-helix in which the first 35 amino acids form hendecad repeats establishing a right-handed helix with a very large pitch. Hence, in the dimer, the two chains essentially form parallel helices, which are designated as “paired bundle” or pb (Premchandar et al., 2016 J Biol Chem 291(48):24931-24950).

[0130] Vimentin has also been suggested as interacting with human group IIA-secreted phospholipase A2 (hGIIA).

[0131] Vimentin activity may be inhibited in any measurable amount by the cyclic peptide. Inhibition of vimentin activity may be complete or may be partial. Thus, the methods disclosed herein may comprise at least partial inhibition of vimentin activity. For example, the activity of vimentin may be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% following administration of a vimentin inhibitor, such as the cyclic peptide, (e.g., relative to the same measurement of activity before administration of the vimentin inhibitor).

[0132] Vimentin activity may be measured through any suitable means known in the art, or any of the methods disclosed herein. For example, vimentin activity may be measured through assays which measure the level of one or more inflammatory markers, such as inflammatory cytokines, inflammatory prostaglandins or the proliferation of prostate cancer cells. In another example, the level of binding of vimentin to hGIIA and / or a level of hGIIA concentration and / or activity (e.g., blood or tissue levels) may be measured as a proxy of vimentin activity. Any suitable methods for determining the nature of the binding interaction between vimentin and hGIIA and / or hGIIA levels may be used.

[0133] In another example, the cyclic peptide inhibits or prevents an inflammatory response associated with the CNS cancer. Suitably, the inflammatory response is at least partly mediated or facilitated by hGIIA.

[0134] Suitably, the hGIIA inhibitor or cyclic peptide described herein is capable of crossing the blood-brain barrier of the subject. The “blood-brain barrier” or “BBB” refers to the physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain. The blood-brain barrier within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS, and are herein collectively referred to as the bloodbrain barrier or blood-brain barrier. The blood-brain barrier also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.

[0135] The brain is considered the most perfused human organ with over 100 billion blood capillaries (Pardridge, 2005). The BBB, which is the physical frontier separating the brain tissue from blood supply, ensures efficient oxygen and nutrient supply to the brain as well as protection against pathogens and neurotoxic compounds (van Tellingen et al., 2015). The BBB is made up of an extensive network of non-fenestrated brain endothelial cells (BECs) present in the lining of the capillary walls (Wang et al., 2019), interacting with surrounding cells such as astrocytes, perivascular macrophages and pericytes (van Tellingen et al., 2015, Abbott et al., 2006). BECs, localised in the inner layer of capillaries, are closely connected by intercellular tight junctions (TJs) (van Tellingen et al., 2015). The lack of fenestration due to these TJs regulates the passage of substances into and out of the brain via the transendothelial passage (Mo et al., 2021). Whilst this selectivity is widely beneficial to minimise foreign substances entering the brain, it also limits the transport of chemotherapy drugs into the brain. It has been discovered that the BBB prevents almost 100% of larger drugs and over 98% of small molecule drugs from entering the brain (Pardridge, 2005). Additionally, efflux transporters such as P-gly coprotein 1, located in the apical membrane of brain capillary endothelial cells can actively transport any drugs crossing the BBB back into the bloodstream (van Tellingen et al., 2015). Hence, the BBB can be considered the bottleneck in brain drug development and one of the most crucial factors limiting the growth of glioma neurotherapeutics. Small molecules typically cross endothelial barriers via different mechanisms including passive diffusion, but the efficiency of this process is dependent upon physicochemical properties, such as the molecular weight, degree of hydrogen bonding with solvent water, lipid solubility and electrical charge. Pardridge (2005) reports that pharmacologically significant amounts of drug are transported across the BBB when the weight of the small molecule is less than 500 daltons (Da) and it forms fewer than 10 hydrogen bonds with water.

[0136] Assays for evaluating the uptake of systemically administered cyclic peptides through the blood-brain barrier (i.e., distribution to the CNS) can be performed by any method known in the art (see, e.g., Wang et al., 2019, Scientific Reports volume 9, Article number: 6117; Carpenter et al., Biophys J. 2014 Aug 5; 107(3): 630-641; Nicolazzo et al., Journal of Pharmacy and Pharmacology, 2006, 58: 281-293), such as logBB and logPS. By way of example, measuring the concentration within the parenchymal space of the CNS or CSF (and optionally the systemic circulation) after a known dose may be used. Such pharmacokinetic methods may also combined with, for example, ELISA or mass spectrometry of the cyclic peptide or radioactivity / fluorescence measurements of a labelled version thereof.

[0137] Suitably, the concentration of the cyclic peptide in the cerebrospinal fluid of the subject in question is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or any range therein of the concentration thereof in the blood plasma of said subject after systemic administration (e.g., administered orally, intravenously, intranasally by inhalation) of, for example, a therapeutically effective dose of the cyclic peptide, such as those provided herein.

[0138] Referring to other examples, the cyclic peptide suitably demonstrates a logBB value (i.e., the concentration of drug in the brain divided by the concentration of drug in the blood) of between about -3.0 and about 1.2 (e.g., about -3.0, -2.5, -2.0, -1.5, -1.0, -0.5, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 and any range therein) or more particularly between about -2.0 and about 1.0. The measure of logBB may be predicted or experimentally determined, such as by using the Clark equation or commercially available software, such as Qikprop, as are known in the art.

[0139] Further anti-cancer treatments

[0140] In any of the methods disclosed herein, the subject may be receiving a further anti-cancer treatment, and more particularly a further anti -cancer agent, in addition to the cyclic peptide. The skilled person will appreciate that cancer treatments for use in the methods described herein may include drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiation therapy, surgery, nutritional therapy, relaxation or meditational therapy and other natural or holistic therapies, although without limitation thereto. Generally, drugs, biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA), chemotherapeutic agents and the like are referred to herein as “anti-cancer therapeutic agents” or “anti -cancer agents”.

[0141] The further anti-cancer treatment may be selected from the group consisting of surgery, a chemotherapeutic agent, a molecularly targeted agent, a T cell expressing a chimeric antigen receptor (CAR-T cell), an antibody or antigen-binding fragment thereof, an antibody-drug conjugate (ADC), an angiogenesis inhibitor, a hormone therapy, a radiation therapy, inclusive of a chemoradiation therapy, an immunotherapeutic agent and any combination thereof. In particular examples, the further anti-cancer treatment is an anti-cancer agent that is or comprises one or more of a chemotherapeutic agent, a molecularly targeted agent, a radiation therapy, an immunotherapeutic agent and any combination thereof. For some examples, the further anti -cancer agent is capable of crossing the blood-brain barrier of the subject.

[0142] The further anti-cancer treatment may be administered sequentially in any order, at the same time (i.e., simultaneously) or at different times. For example, the subject may have received a further anti-cancer treatment prior to receiving the hGIIA inhibitor described herein (e.g., c2). In another example, the subject may receive a further anti -cancer treatment after receiving the hGIIA inhibitor described herein (e.g., c2). In yet another example, the subject may receive a further anti-cancer treatment at the same time as the hGIIA inhibitor described herein (e.g., c2).

[0143] In any of the methods disclosed herein, the method may include the further step of administering a further anti-cancer treatment, such as a further anti-cancer agent, to the subject. The further anti-cancer treatment may be administered simultaneously with or before or after treatment with the cyclic peptide. In one example, the further anti -cancer treatment is administered to the subject simultaneously with the cyclic peptide. In another example, the further anti-cancer treatment is administered to the subject before the cyclic peptide. In another example, the further anti-cancer treatment is administered to the subject after the cyclic peptide.

[0144] Referring to some examples, the further anti-cancer agent is or comprises a chemotherapeutic agent. As generally used herein, the term “chemotherapy” or “chemotherapeutic agent” broadly refers to a treatment or agent with a cytostatic or cytotoxic agent (i.e., a compound) to reduce or eliminate the growth or proliferation of undesirable cells, such as cancer cells. Accordingly, the terms can refer to a cytotoxic or cytostatic agent used to treat a proliferative disorder, for example cancer. The cytotoxic effect of the agent can be, but is not required to be, the result of one or more of nucleic acid intercalation or binding, DNA or RNA alkylation, inhibition of RNA or DNA synthesis, the inhibition of another nucleic acid-related activity (e.g., protein synthesis), or any other cytotoxic effect.

[0145] Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., imidazotetrazine derivatives, such as temozolomide; nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin and lipoplatin; bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6- thioguanine, fludarabine, pentostatin; asparginase; and ribonucleotide reductase inhibitors such as hydroxyurea); and tubulin interactive agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol)). In particular examples, the chemotherapeutic agent is or comprises temozolomide.

[0146] In other examples, the further anti-cancer agent is or comprises a molecularly targeted therapy. As used herein, “molecularly targeted therapy” or “molecularly targeted therapeutic agent” refers to a therapy that targets a particular class of proteins involved in cancer growth or signalling. It is envisaged that such agents may include antibodies or fragments thereof and small molecules.

[0147] In some examples, the further anti-cancer agent described herein is or comprises an inhibitor of a kinase, such as a tyrosine kinase or a serine-threonine kinase. The term “tyrosine kinase” refers to enzymes which are capable of transferring a phosphate group from ATP to a tyrosine residue in a protein. Phosphorylation of proteins by tyrosine kinases is an important mechanism in signal transduction for regulation of enzyme activity and cellular events such as cell survival or proliferation. Tyrosine kinases include receptor tyrosine kinases and non-receptor tyrosine kinases. AATK, ABL, ABL2, ALK, AXL, BLK, BMX, BTK, CSF1 R, CSK, DDR1, DDR2, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN, GSG2, HCK, IGF1R, ILK, INSR, INSRR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR, KIT, KSR1, LCK, LMTK2, LMTK3, LTK, LYN, MATK, MERTK, MET, MLTK, MST1R, MUSK, NPR1, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PLK4, PTK2, PTK2B, PTK6, PTK7, RET, ROR1, ROR2, ROS1, RYK, SGK493, SRC, SRMS, STYK1, SYK, TEC, TEK, TEX14, TIE1, TNK1, TNK2, TNNI3K, TXK, TYK2, TYR03, YES1, and ZAP70. For various examples, the further anti-cancer agent is or comprises an inhibitor of a transcription factor. More particularly, the further anti-cancer agent is or comprises an inhibitor of NFKB, such as Withaferin A.

[0148] According to various examples, the further anti-cancer agent is or comprises an immunotherapeutic agent. Insofar as they relate to cancer, immunotherapy or immunotherapeutic agents use or modify the immune mechanisms of a subject so as to promote or facilitate treatment of a cancer. In this regard, immunotherapy or immunotherapeutic agents used to treat cancer include cell-based therapies, antibody therapies (e.g., anti-PDl antibodies, anti-CTLA4 antibodies or anti-PDLl antibodies) and cytokine therapies. These therapies all exploit the phenomenon that cancer cells often have subtly different molecules termed cancer antigens on their surface that can be detected by the immune system of the cancer subject. Accordingly, immunotherapy is used to provoke the immune system of a cancer patient into attacking the cancer's cells by using these cancer antigens as targets.

[0149] Non-limiting examples of immunotherapy or immunotherapeutic agents include adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, BMS-936559, brentuximab, certolizumab, cituximab, daclizumab, eculizumab, ibritumomab, infliximab, ipilimumab, lambrolkizumab, mepolizumab, MPDL3280A muromonab, natalizumab, nivolumab, ofatumumab, omalizumab, pembrolizumab, pexelizumab, pidilizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, abatacept, alefacept and denileukin diftitox. In particular preferred embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor, such as an anti-PDl antibody (e.g., pidilizumab, nivolumab, lambrolkizumab, pembrolizumab), an anti- PDLl antibody (e.g., BMS-936559, MPDL3280A) and / or an anti-CTLA4 antibody (e.g., ipilimumab). In particular examples, the immunotherapeutic agent is or comprises bevacizumab.

[0150] For certain examples, the further anti-cancer agent is or comprises an antibody or a fragment thereof, such as those described herein. The term “antibody” is used in the context of the present disclosure to refer to immunoglobulin molecules immunologically reactive with a particular antigen and includes both polyclonal and monoclonal antibodies. The term also includes native and genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term “antibody” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rlgG as discussed in Pierce Catalogue and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York (1998). The term antibody also includes multispecific molecules, such as bivalent or bispecific molecules. Examples of bivalent and bispecific molecules are described in Kostelny et al. (1992) J Immunol 148: 1547; Pack and Pluckthun (1992) Biochemistry 31 : 1579; Hollinger et al., 1993, supra, Gruber et al. (1994) J. Immunol. :5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[0151] An “antibody fragment” or “antigen binding fragment” of an antibody comprises one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; triabodies; tetrabodies; dimerisation-activated demibodies (e.g., WO / 2007 / 062466); linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments. For example, the term antigen binding fragment may be used to refer to recombinant single chain Fv fragments (scFv), as well as divalent (di-scFv) and trivalent (tri-scFV) forms thereof.

[0152] Referring to other examples, the further anti-cancer agent is or comprises a CAR-T cell. A “CAR-T cell” is a T cell which has been transduced with at least one CAR. CAR-T cells can be mono, dual, or tandem CAR-T cells. CAR-T cells can be autologous, meaning that they are engineered from a subject's own cells, or allogeneic, meaning that the cells are sourced from a healthy donor, and in many cases, engineered so as not to provoke a host-vs-graft or graft-vs-host reaction.

[0153] In various examples, the further anti-cancer agent is or comprises an ADC. The term “ADC” or “antibody-drug conjugate” has its usual scientific meaning known to those skilled in the art, and here refers to a class of biopharmaceutical drugs designed as a targeted therapy to treat, for example, cancer. Unlike chemotherapy, ADCs are intended to attack and destroy cancer cells without affecting healthy cells of the patient. ADCs are composed of an antibody or fragment thereof linked to a biologically active cytotoxic (anti-cancer) payload or drug. ADCs combine the targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs. They are designed with the intention of discriminating between healthy cells and diseased tissues, for example, cancer cells in a cancer.

[0154] Referring to certain examples, the further anti-cancer agent is or comprises an angiogenesis inhibitor. The term “angiogenesis inhibitor,” as used herein, refers to any agent or molecule that reduces or inhibits the formation of new blood vessels in a mammal. With regard to cancer therapy, the angiogenesis inhibitor suitably reduces or inhibits the formation of new blood vessels in or on a tumour, preferably in or on a solid tumour.

[0155] In view of the above, therapeutic advantages of the presently disclosed methods and uses may be realised through combination regimens in conjunction with conventional therapy, such as radiotherapy, chemotherapy, surgery, or other forms of medical intervention.

[0156] In some examples, the conventional therapy is radiotherapy, which may be administered with or without temozolomide or any other chemotherapeutic agent, such as those described above. The temozolomide or any other chemotherapeutic agent may be administered sequentially in any order, at the same time or at different times as the cyclic peptide, so as to provide the desired effect. Alternatively, the temozolomide or any other chemotherapeutic agent may be formulated to be administered in the same formulation as the cyclic peptide.

[0157] It will be appreciated that the therapeutic effect of the various anti -cancer agents described herein (e.g., the cyclic peptide and / or the further anti -cancer agent) need not be absolute to elicit a biological effect, such as treatment of the subject’s CNS cancer. Accordingly, therapeutic efficacy provided by an anti-cancer agent described herein can be partial or incomplete (e.g., a readout, indicator or biomarker of cancer progression in the subject is reduced by about 20%, 30%, 40%, 50%, 60% or 70%, 80%, 90%, 95%, 96%, 97%, 98% and 99%, including any intermediate value therebetween with administration of an anti -cancer agent as described herein).

[0158] Dosage and administration

[0159] The cyclic peptide, such as c2, and the compositions disclosed herein can be administered to a patient suffering from a CNS cancer, in an amount sufficient to cure, or at least partially arrest the CNS cancer and its complications.

[0160] In other examples, the methods, compositions and uses disclosed herein also include or relate to prophylactic or preventative application of a suitable effective dose of the cyclic peptide or the compositions described herein. In some embodiments, the cyclic peptide or the compositions provided herein, in an appropriate effective dose, is used as a maintenance therapy.

[0161] The therapeutically effective dose level for any particular patient or subject, will depend upon a variety of factors familiar to one skilled in the art, including, for example: the CNS cancer being treated and / or the severity of the cancer, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the duration of the treatment, drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, a patient's body surface area, whether the patient is undergoing therapy, and any specific contraindications.

[0162] In one example, the cyclic peptide described herein is administered, such as orally administered, at a dose of between about 1.0 mg / kg to about 100 mg / kg (e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg or any range therein). In some examples, the cyclic peptide is administered at a dose of about 1 mg / kg to about 100 mg / kg. In other examples, the cyclic peptide is administered at a dose of about 1 mg / kg to about 50 mg / kg. In other examples, the cyclic peptide is administered at a dose of about 1 mg / kg to about 25 mg / kg. In other examples, the cyclic peptide is administered at a dose of about 1 mg / kg to about 10 mg / kg. In further examples, the cyclic peptide is administered at a dose of about 5 mg / kg to about 25 mg / kg. In related examples, the cyclic peptide is administered at a dose of about 10 mg / kg to about 15 mg / kg. In specific examples, the cyclic peptide is orally administered at a dose of about 5 mg / kg to about 50 mg / kg. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 5 mg / kg to about 100 mg / kg.

[0163] Referring to certain examples, the cyclic peptide is administered, such as orally administered, at a dose of about 5 mg / day to about 1500 mg / day, or about 5 mg / day to about 1200 mg / day, or about 5 mg / day to about 1000 mg / day, or about 5 mg / day to about 900 mg / day, or about 5 mg / day to about 800 mg / day, or about 5 mg / day to about 700 mg / day, or about 5 mg / day to about 600 mg / day, or about 5 mg / day to about 500 mg / day, or about 5 mg / day to about 400 mg / day, or about 5 mg / day to about 300 mg / day, or about 5 mg / day to about 200 mg / day, or about 5 mg / day to about 100 mg / day or about 5 mg / day to about 50 mg / day or about 5 mg / day to about 25 mg / day or about 5 mg / day to about 20 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 100 mg / day to about 1500 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 125 mg / day to about 1500 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 125 mg / day to about 1000 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 125 mg / day to about 500 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 200 mg / day to about 1500 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 200 mg / day to about 1000 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 200 mg / day to about 500 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 250 mg / day to about 1500 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 250 mg / day to about 1000 mg / day. In other examples, the cyclic peptide is administered, such as orally administered, at a dose of about 250 mg / day to about 500 mg / day.

[0164] According to various examples, the cyclic peptide is administered, such as orally administered, at a dose, such as a daily dose, of about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 mg or any range therein.

[0165] It is further contemplated that the cyclic peptide may be administered daily or alternatively every 2nd, 3rd, 4th, 5th, 6thor 7thday, such as with those dosages hereinbefore provided. In particular examples, the cyclic peptide is administered once, twice, three times, four times or five times per week over the course of the treatment regime. For some examples, the cyclic peptide are administered at a dosage of about 5 mg to about 1500 mg, about 5 mg to about 1200 mg, about 5 mg to about 1000 mg, about 5 mg to about 900 mg, about 5 mg to about 800 mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 5 mg to about 500 mg, about 5 mg to about 400 mg, about 5 mg to about 300 mg, about 5 mg to about 200 mg, about 5 mg to about 100 mg, or about 0.5 mg to about 50 mg, two, three, four or five times per week.

[0166] Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of dosing may depend on the CNS cancer being treated, the form, route and site of administration, and the nature of individual dosages will be determined by the nature and extent the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

[0167] In any of the methods disclosed herein, the cyclic peptide may be administered daily for a period of at least about 1 day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

[0168] 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 days or any range therein), or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, or at least about 12 days, or at least about 13 days or at least about 2 weeks or any range therein. Moreover, the cyclic peptide may be administered daily for a period of at least about 2 weeks (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 weeks or any range therein), or at least about 3 weeks, or at least about 4 weeks, or at least about 5 weeks, or at least about 6 weeks. In certain examples, the cyclic peptide or the analogue or derivative thereof is orally administered daily for a period of at least about 2 weeks. In another example, the cyclic peptide is administered daily for a period of at least about 6 weeks.

[0169] Those skilled in the art will appreciate that in accordance with the presently disclosed methods and uses, the cyclic peptide or the compositions disclosed herein may be administered alone or in conjunction with one or more additional agents, such as one or more further anti-cancer agents described herein, as part of a combination therapy. Any safe route of administration may be employed for providing a patient with the composition of the present disclosure. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intranasal, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Referring to various examples, the cyclic peptide is administered orally, intravenously, intranasally or by inhalation. In various examples, the cyclic peptide is administered intravenously. In particular examples, the cyclic peptide is administered intranasally. In other examples, the cyclic peptide is administered by inhalation. In certain examples, the cyclic peptide is administered orally. The terms “oral administration”, “orally administering” and the like represent any method of administration in which an active agent, such as c2, can be administered by swallowing, chewing, sucking or drinking an oral dosage form of said active agent. Such solid or liquid oral dosage forms are traditionally intended to substantially release and or deliver the active agent in the gastrointestinal tract beyond the mouth and / or buccal cavity. Examples of solid dosage forms include conventional tablets, multi-layer tablets capsules, caplets, etc., which do not substantially release the drug in the mouth or in the oral cavity.

[0170] Referring to other specific examples, the cyclic peptide is administered by inhalation. To this end, the cyclic peptide can be included in a pharmaceutical composition suitable for being aerosolised. In other examples, the cyclic peptide is administered to the subject by injection, infusion or the like into the parenchyma or CSF via the intracerebroventricular or intrathecal (cisternal or lumbar) route. In alternative examples, the cyclic peptide is not administered into the parenchyma or CSF of the subject via the intracerebroventricular or intrathecal route. To this end, the cyclic peptide is required to cross the subject’s blood-brain barrier to exert an anti-cancer effect on the CNS cancer.

[0171] Methods of screening

[0172] In light of the present disclosure, the person skilled in the art may conduct screening assays to identify, design or produce an agent for use in treating or preventing CNS cancer.

[0173] Accordingly, in one form there is provided a method of identifying, designing or producing an agent for use in treating or preventing a CNS cancer, said method including the step of determining whether the candidate agent crosses a blood-brain barrier, such as that of a subject, wherein the candidate agent is a cyclic peptide and wherein the cyclic peptide is a hGIIA inhibitor.

[0174] Thus, the present disclosure provides methods to identify, design or produce an agent for use in treating or preventing a CNS cancer when administered to a subject. It is envisaged that any method known in the art to assess whether the candidate agent crosses the blood-brain barrier of the subject, such as those described herein, may be utilised for the present method. Such methods may be carried out using in silico, in vitro and / or ex vivo assays, such as in in vitro models of the blood-brain barrier, as are known in the art, such as a computational model (e.g., an in silico model), a 2D transwell model, a 3D organoid model, and a microfluidic model (see, e.g., Chaulagain et al., Int J Mol Sci. 2023 Feb; 24(3): 2710). Alternatively, the present method may be performed in vivo, for example, in animal subjects to assess the permeability of candidate agents in respect of the blood-brain barrier (e.g., permeability of labelled, such as fluorescently labelled or radiolabelled, conjugates of the candidate agent, pharmacokinetic studies), such as by those methods described herein.

[0175] As such, in a related form there is provided a method of identifying, designing or producing an agent for use in treating or preventing a CNS cancer, said method including the steps of:

[0176] (a) administering a candidate agent to a subject, wherein the candidate agent is a cyclic peptide and wherein the cyclic peptide is a hGIIA inhibitor;

[0177] (b) determining whether the candidate agent crosses a blood-brain barrier of the subject.

[0178] Referring to particular examples, the subject is a non-human animal (i.e., not a human), or more particularly, a non-human mammal. For such examples, the subject may include laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), more particularly a rodent or even more particularly a rat or a mouse.

[0179] Suitably, the candidate agent is administered to the subj ect through any appropriate delivery route, such as those described herein, so as to assess its ability to cross the blood-brain barrier. For some examples, the candidate agent is administered orally, intravenously, intranasally or by inhalation to the subject. The candidate agent is suitably not administered to the subject by injection, infusion or the like into the parenchyma or CSF via the intracerebroventricular or intrathecal (cisternal or lumbar) route.

[0180] The anti-cancer effect of the respective candidate agents may also be assessed in the subject. To this end, the subject has, is suffering from or is at risk of developing the CNS cancer in question. As such, the subject can be an animal model, or more particularly a rodent model, of a CNS cancer, such as a mouse model of glioblastoma, as are known in the art (see, e.g., Fang Jin et al., Gliomas: Chapter 2 “Mouse Models of Experimental Glioblastoma”, Debinski W, editor. Brisbane (AU): Exon Publications; 2021 Apr 30).

[0181] In view of the foregoing, the present method may further include the step of determining whether the candidate agent treats or prevents a CNS cancer in the subject, such as by those methods well known in the art. Again, such methods may be carried out using in vitro and / or ex vivo assays, such as in relation to cancer cell lines or tumoroids derived from the CNS cancer (e.g., a 2D culture (e.g., a cancer cell line), a 2D co-culture (e.g., cancer cells with stromal cells), a 2.5D culture (e.g., cells growing on top of a layer of an extracellular matrix (ECM) protein or a hydrogel), a 3D culture (e.g., cancer spheroids, organoids or tumoroids) or a patient-derived xenograft (PDX)), or additionally or alternatively in in vivo models (e.g., rodent models) of the CNS cancer.

[0182] Indicators of therapeutic efficacy of the candidate agent in relation to the CNS cancer may be assessed by any means or method known in the art, including, but not limited to, flow cytometry (e.g., trypan blue, annexin V staining), fluorescent based cell detection assays (e.g., Calcein AM, Mitotracker Red), luminescent based detection assays (e.g., Cell-Titer Gio), spectrophotometry based detection assays (e.g., crystal violet, MTS / MTT assays such as Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay), a scratch wound assay, a boyden chamber assay and a cell invasion assay using fluorescent detection of cell invasion (e.g., activin, serum).

[0183] In another example, the effect of the presence of the candidate agents on inflammation may also be assessed or monitored. This may be achieved, for example, by determining a level of expression and / or activity of one or more markers of inflammation (e.g., a prostaglandin, cytokine, or any other known marker of inflammation, such as hGIIA), such as a circulating and / or CSF level of such markers of inflammation. It is further envisaged that such an anti-inflammatory effect or levels thereof may be utilised as an indicator of the anti-cancer effect of the candidate agent in relation to the CNS cancer (e.g., a decrease or reduction in a level of circulating hGIIA in the subject may indicate or correlate with a positive therapeutic effect in relation to the subject’s cancer). Moreover, the present methods may include the initial or earlier step of determining whether the candidate agent is a hGIIA inhibitor and / or a vimentin inhibitor.

[0184] Suitably, the candidate agent is a derivative or analogue of c2, cFLSYK or cFLSYR. In some examples, the candidate agent is a derivative or analogue of c2.

[0185] In one example, the methods disclosed herein may further include the step of isolating and / or purifying an agent that crosses the blood-brain barrier. Suitably, the agent also demonstrates therapeutic efficacy (i.e., an anti-cancer effect) against the CNS cancer in question.

[0186] In one particular example, the method further includes the step of formulating the isolated and / or purified agent into a pharmaceutically acceptable formulation.

[0187] It is also contemplated that the candidate agent or inhibitor may be rationally designed or engineered de novo based on desired or predicted structural characteristics or features that indicate the candidate agent could inhibit hGIIA and / or vimentin and / or cross the blood-brain barrier (e.g., be of a particular size or molecular weight predicted to cross the blood-brain barrier). In other examples, the candidate agent may be identified by screening a library of molecules without initial selection based on desired or predicted structural characteristics or features that indicate the candidate agent could possess such characteristics. Such libraries may comprise randomly generated or directed libraries of proteins or peptides, and more particularly cyclic peptides, libraries of naturally-occurring molecules and / or combinatorial libraries of synthetic organic molecules.

[0188] Non-limiting examples of techniques applicable to the design and / or screening of candidate agents may employ X-ray crystallography, NMR spectroscopy, computer assisted screening of structural databases, computer-assisted modelling or biochemical or biophysical techniques which detect molecular binding interactions, as are well known in the art.

[0189] Biophysical and biochemical techniques which identify molecular interactions include competitive radioligand binding assays, co-immunoprecipitation, fluorescence-based assays including fluorescence resonance energy transfer (FRET) binding assays, electrophysiology, analytical ultracentrifugation, label transfer, chemical cross-linking, mass spectroscopy, microcalorimetry, surface plasmon resonance and optical biosensor-based methods, such as provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan ei 1., (John Wiley & Sons, 1997) Biochemical techniques such as two-hybrid and phage display screening methods are provided in Chapter 1 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997).

[0190] Accordingly, an earlier step of the method may include identifying a plurality of candidate agents that are selected according to broad structural and / or functional attributes, such as an ability to bind coil 2 of vimentin.

[0191] Additionally, the present method may further include one or more of the steps of: selecting a candidate agent that crosses the blood-brain barrier; isolating or purifying the candidate agent; formulating the candidate agent into a pharmaceutical formulation; and adding the candidate agent or the pharmaceutical formulation to packaging and / or a container, such as a vial, ampoule, bag, blister pack, bottle, cartridge, injection needle, injection syringe, single dose container, strip of multiple single dose containers, or tube.

[0192] Compositions

[0193] Compositions comprising a cyclic peptide that inhibits hGIIA or a compound that disrupts the vimentin-hGIIA interaction (e.g., a vimentin inhibitor), together with an acceptable carrier or diluent, can be useful in the methods disclosed herein. Therapeutic compositions can be prepared by mixing the desired compounds having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions. Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0194] Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.

[0195] Therapeutic compositions to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The composition may be stored in a lyophilized form or in solution if to be administered systemically. If in a lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use. An example of a liquid formulation is a sterile, clear, colourless unpreserved solution filled in a single-dose vial for subcutaneous injection.

[0196] Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. The dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease or condition, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference (56th ed., 2002). Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

[0197] EXAMPLES Example 1. Using cell-based models of GBM to compare the therapeutic effectiveness of c2 relative to temozolomide

[0198] There is a clinical need for novel chemotherapeutic drugs for GBM due to the suboptimal clinical effectiveness of temozolomide (TMZ), the current first-choice drug, due to its low efficacy and side effect profile (Petrenko et al., 2022). The demonstrated pre-clinical efficacy of the novel experimental compound c2 against prostate cancer cell lines LNCaP-Al (ICso 3-5 pM) and PC-3 (ICso 50-100 nM)), together with the c2’s surprising ability to cross the BBB (as demonstrated in Example 2), makes it a potential drug candidate for GBM therapy.

[0199] In this Example, the in vitro anticancer activity of these compounds was determined using a standard cell viability assay and the cell migration parameters for c2 and TMZ were compared using a wound healing assay.

[0200] Materials and methods

[0201] Cell lines

[0202] Three brain cancer cell lines were used in this study, and these were carefully selected to represent GBM at an in vitro level. Details of these cell lines are outlined in Table 1 below.

[0203] Table 1: Origin, morphological details and culture conditions of the cell lines.

[0204] Cell culture

[0205] Frozen vials (stored at - 80 °C) containing identity confirmed, low passage (passage number less than 10), and mycoplasma-free U251, LN229, and T98G were provided by the Medical Oncology group at Ingham Institute for Medical Research. U251 cells were grown in minimum essential media (MEM) (Sigma Aldrich, Australia), while LN229 and T98G were grown in Dulbecco’s modified eagle medium (DMEM) (Sigma Aldrich, Australia), and these were all supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Australia), L-glutamine (4 mM) (Gibco, Australia), and penicillin-streptomycin (100 I.U. / mL) (Gibco, Australia). All cell culture media were phenol red free, unless stated otherwise. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 (HERAcell 150i CO2 incubator, ThermoFisher Scientific, Australia) until 80% cell confluency was reached as determined by light microscopy (Nikon Eclipse TS 100, Nikon, Australia). Confluent cells were passaged as required.

[0206] Drug preparation

[0207] TMZ was purchased from Selleck Chemicals LLC, USA. c2 was synthesized by Bachem, Switzerland. The chemicals were weighed, in grams, to 4 decimal places using an electronic balance (Mettler Toledo, Australia) and dissolved in DMSO (Sigma Aldrich, Australia) to make up stock concentrations shown in Table 2.

[0208] Table 2: Stock and working concentrations of drugs.

[0209] On the day of treatment, frozen aliquots of drugs were thawed at room temperature, and diluted to the required concentration using serial dilutions in warmed growth media, ensuring the DMSO concentration was kept below 2%.

[0210] Cell seeding and treatment

[0211] Cultured cells were washed with PBS, trypsinised, and centrifuged under regular cell culture conditions. Cell pellets were resuspended in 5 mL of media, and uniformly dispersed by pipetting up and down a few times. 10 pL of this cell solution was added to a Neubauer haemocytometer (Assistent, Germany) for cell counting and then plated at a density of 5xl03cells / well (100 pL per well) in Greiner 96-well plates (CELLSTAR®, USA). Well plates were incubated for 24 h to allow cells to adhere to the plate surface as a monolayer.

[0212] 24 h post-seeding, drugs at a series of concentrations were added into wells in triplicate. Drugs were added to wells ensuring the maximum DMSO % was maintained for all concentrations of each drug. A no drug DMSO control matching the maximum DMSO % was also added to triplicate wells. Plates were returned to the incubator for a further 72 h and were left undisturbed until the next step. MTS method

[0213] 20 pL of 96® Aqueous solution reagent (MTS reagent) was directly added to each well, plates were covered with aluminium foil, and were placed in the incubator for 3 h. The absorbance was read on a Spectramax M2 spectrophotometer (Molecular Devices, USA) at a wavelength of 490 nm.

[0214] Data analysis and half maximal inhibitory concentration ICso calculation

[0215] All raw absorbance values were corrected for background absorbance by subtraction of the mean blank (no MTS) absorbance value and the triplicate readings of absorbance were used to calculate a mean absorbance. The percentage viability was determined using the formula below (Kamiloglu et al., 2020). These calculations were done via spreadsheet (Microsoft Excel).

[0216] Mean absorbance (drug concentration)

[0217] % viability = — - -; ;- - - - - - — x 100

[0218] Mean absorbance (no drug control)

[0219] Three biological replicate trials were conducted for each treatment group and the results were exported to GraphPad PRISM vlO.1.1. Thirteen- point dose-response curves were generated for all treatments using this software. Data was analysed using non-linear regression; [Inhibitor] vs. response and were fit to four parameter model (Y = Bottom + (Top - Bottom) / (1 + (ICso / X)ASlope). Half maximal inhibitory concentration for each compound was calculated across all three cell lines using GraphPad PRISM. Variation among the biological repeats were presented as a standard error of mean (SEM). Differences in ICso values were assessed by ordinary one-way Analysis of variance (ANNOVA) tests on GraphPad PRISM, unless otherwise indicated.

[0220] Cell migration assay

[0221] Cultured cells were washed with PBS, trypsinised, and centrifuged under regular cell culture conditions. Cell pellets were resuspended in 5 mL of media, and uniformly dispersed. 10 pL of this homogenate was added to a Neubauer haemocytometer for cell counting and then plated at a density of 2xl04cells / well (100 pL per well) in incorporated 96 well assay plates (Corning, USA). Once cells reached 90-100% confluence, a 96-well wound maker tool (Essen Bioscience, USA) was used to create a homogeneous, 700-800 pm wide wound in each cell monolayer on 96- well plates. Media in wells was gently removed and wells were carefully washed with PBS before replenishing with 200 pL media containing c2 (200 pM) or TMZ (200 pM) or 1% DMSO (vehicle) or a blank containing fresh media only. Drugs were added in triplicate wells per treatment. Well plates were incubated in an Incucyte Zoom imaging system (ThermoFisher Scientific, Australia) where wells were imaged by phase-contrast microscopy every 8 h for 72 h, at which time the wound was fully closed in most of the blank groups. Images were exported for analysis on ImageJ-FIJI vl.53k, public domain software. An ImageJ-FIJI plugin with open source wiki was downloaded and set up manually (https: / / github.com / AlejandraArnedo / Wound-healing-size-tool / wiki) ( Suarez- Arnedo et al., 2020). The plugin automatically picked up the wound area in inch2and the percentage wound confluence. For images of c2 treated wells, a bandpass filter to filter down pixels was applied as the plugin did not pick up the wound sizes on its normal resolution. The percentage wound confluence at each time point was then converted to a percentage of confluence at t=0 time point.

[0222] 3D culture

[0223] RASTRUM bioprinting platform

[0224] A Px01.71, ~0.7 kPa matrix, containing fibronectin, collagen type IV, and laminin was used. Printer sterility was ensured by performing a sterility test as per the manufacturer’ s protocols, approximately 3-5 days before the print run.

[0225] All reagents and cartridges were purchased from Inventia Life Sciences. The first step in bioprinting was designing the plate map using predefined models on the RASTRUM cloud. Next, start of day greenlighting was done following RASTRUM protocol before proceeding. All RASTRUM reagents were stored at -20°C and thawed on demand. 1500 pL of F23 and 200 pL of F320 bioinks, together with 1500 pL of F3 activator, 16 mL sterile filtered 70% ethanol, and 40 mL sterile filtered milli-Q water was added to the cartridge. Cartridge containing the reagents and a Nuncleon 96-well plate (ThermoFisher Scientific, Australia) was placed inside the bioprinter to print the base matrix. Next, IxlO6cells (measured using a haemocytometer) was suspended in 200 pL of the activator Fl 76 and placed in the cartridge to print cells. Once complete, 96-well plate was topped up with phenol red free media (MEM for U251 and DMEM for LN229, and T98G). Figure 1 summarises the bioprinting procedure.

[0226] Incucyte live cell analysis

[0227] Well plates were incubated in an Incucyte Zoom imaging system (ThermoFisher Scientific, Australia) where wells were imaged by phase-contrast microscopy every 8 h for 5 days. Media was replaced every three days.

[0228] On day 5, seven different concentrations of c2 (100 - 400 pM), TMZ (200 - 1000 pM), WFA (0.5 - 10 pM), and TQ (10 - 100 pM) were added to wells, each in triplicate. DMSO (2% for c2, 1.05% for TMZ, 0.1 % for WFA, and 0.3% for TQ) was added as a no drug control, each in triplicate. Different DMSO concentrations were added as the DMSO percentage in the drugs were different due to differences in stock concentrations. Plates were returned to the Incucyte for a further 72 h after which and MTS viability assay was performed by addition of MTS reagent as described below.

[0229] Cell viability assay

[0230] 20 pL of 96® Aqueous solution reagent was directly added to each well, plates were covered with aluminium foil, and were placed in the incubator for three hours. The absorbance was read on a Spectramax M2 spectrophotometer (Molecular Devices, USA) at a wavelength of 490 nm.

[0231] Data analysis

[0232] Incucyte data for growth of spheroids was exported an Excel file containing confluence percentage. This data was plotted on an XY graph to visualise the rate of growth of spheroids. Spectrophotometer data was exported as spreadsheet (Microsoft Excel) containing absorbance values. The percentage viability was determined using the formula above using a spreadsheet.

[0233] Results of three technical replicates were exported to GraphPad PRISM vlO.1.1. Eightpoint dose-response curves were generated for all treatments using this software. Data was analysed using non-linear regression; [Inhibitor] vs. response model. ICso values were generated, and the variations were presented as a SEM. Differences in ICso values between 2D and 3D cell culture were assessed by ordinary one-way ANOVA tests on GraphPad PRISM.

[0234] Results and discussion

[0235] Cell viability studies in glioblastoma cell lines

[0236] Cell viability 72 h post treatment was determined using an MTS assay, and dose-response curves generated using the absorbance values at 490 nm were fitted to a non-linear regression; [Inhibitor] vs. response model as shown in Figure 2.

[0237] In the U251 cell line, the ICso for c2 was calculated as 168.3 ± 3.2 pM and for TMZ as 417.3 ± 5.5 pM. In the selected concentration range, cells reached ~ 20% viability at 350 pM, while TMZ seemed to reach ~ 40% viability at the highest concentration (1000 pM). Results for LN229 followed a similar pattern those for U251, with the ICso for c2 calculated as 199.6 ± 5.5 pM and for TMZ as 430.2 ± 6.1 pM. Similar pattern of results were also observed in the T98G cell line. c2 reported an ICso of 330.8 ± 33.3 pM, with cells reaching ~ 25% viability at 400 pM. TMZ showed poor potency even at concentrations as high as lOOpM, and the ICso was greater than 1000 pM. The ICso values are summarised in Table 3. Table 3. Summary of IC50 values for c2 and TMZ calculated using MTS viability assay.

[0238] Cell migration studies in glioblastoma cell lines

[0239] The wound healing assay is a straightforward test used to determine cell migration in response to a drug. Here, the present Example compared the migratory potential of U251, LN229, and T98G cells when subjected to c2, TMZ, DMSO and a no treatment control. Incucyte images obtained at 10 x objective for the U251 cell line indicated wound closure in the no treatment wells within 24 h, while DMSO and TMZ treated wells reaching almost close wounds between 48 to 72 h. c2 on the other hand inhibited wound closure completely over the 72-h timeframe of the experiment (Figure 3). A similar trend was observed with LN229 and T98G cells, where the wound size decreased with time in all treatment groups except for c2, where little to no change was observed within the 72 h. Figures 4 and 5 show Incucyte images for LN229 and T98G cells respectively.

[0240] Results, as shown in Figure 6, consolidated the qualitative data seen in Figures 3 to 5. In all three cell lines, the wound size remained constant for c2, while TMZ and DMSO treated wells had similar rate of wound closure in LN229 and T98G. In U251, the effect of DMSO standalone seemed to be more inhibitory for cell migration than TMZ. As anticipated, wounds in untreated wells closed quickest, where U251 showed cells migrating the fastest of the three cell lines.

[0241] When the mean confluence percentages were compared, a significant difference (p<0.05) between the percentage wound confluence of c2 treated wells and DMSO vehicle treated wells at 72 h, was observed in all cell lines. However, the TMZ results were not significantly different compared to DMSO.

[0242] 3D culture experiments

[0243] Both LN229 and T98G cells formed clustered spheroid-looking structures in the matrix. However, the spherical shape and size were inconsistent within and between cell lines. U251 cells on the other hand grew much faster, but did not grow into spheroids. They appeared to grow in an extensive mesh-like network as shown in Figure 7.

[0244] The rate of spheroid formation in LN229 and T98G appeared similar, while U251 cells grew at a faster rate. All three cell lines demonstrated cell growth in the first five days, starting from a confluence of about 40%. After drug treatment on day 5 (t = 120 hrs), a drop or a plateau in confluence was observed in 72 h post drug treatment. This also suggests that unlike with 2D cell culture, 3D spheroids may need to be exposed to the treatment for a longer duration to see effects. With all drugs, U251 cells displayed the biggest drop in its cell confluence. Figure 8 represents growth curves of the 3D cell culture models. Only the growth curves for the highest concentration of each drug treatment are shown in this figure.

[0245] Dose-response curves (Figure 9) were similar to the 2D dose-response curves. The ICso for c2 was calculated as 184.6 ± 13.0 pM in U251, 336.3 ± 83.0 pM in LN229, and 512.3 ± 702.2 pM in T98G. TMZ showed poor potency even at concentrations as high as lOOOpM, and the ICso was greater than 1000 pM in T98G. A comparison of ICso values between 2D and 3D cell culture is summarised in Table 4.

[0246] Table 4. ICso values for 3D cell culture using MTS viability assay.

[0247] * Data are mean ± SD of three technical replicates

[0248] Further experiments in 2D and 3D cultures with quantitative immunofluorescence analysis, demonstrated reduced expression of the cell proliferation marker KI67 in GBM cells treated with c2. Additionally, vimentin, a known marker for cell migration, was examined in GBM primary and immortal cell lines using immunofluorescence assays. This confirmed higher expression of vimentin in both 2D and 3D GBM cell cultures (Figure 10A and 10B). Interestingly, c2 treatment was shown to modulate the dynamic distribution and expression of vimentin in GBM cells (Figure 10A), potentially contributing to the observed reduction in GBM migration in scratch assays. As such, and without being bound by any theory, it is hypothesised that c2 inhibits wound healing by modulating and inhibiting vimentin structure and expression.

[0249] To further understand the effect of c2 on various cancer proteins, the present inventors next screened the protein levels of 80 different oncogenes using Invitrogen oncopanels. c2 was found to inhibit the down regulation of HIF1 protein and EGF protein. Recent studies suggested the crosstalk and common stimuli for the activation of NF-KB and HIF1 signalling. To enhance the potential of c2 in inhibiting the mechanism of cancer cell resistance, c2 was tested in combination with various phytochemicals modulating NF-KB environment. From this, c2 was found to work at a significantly lower concentration when combined with Withaferin A (WFA) suggestive of a synergistic mechanism of action in killing GBM cells. The combination of c2 with WFA may therefore provide a combination treatment strategy for GBM which inhibits inflammation mediated by sPLA2IIA, vimentin mediated cell invasion and simultaneously modulating NF-KB- mediated regulation of immune resistance of cancer cells.

[0250] Example 2. Blood-brain barrier pharmacokinetics of c2

[0251] In this Example, the inventors sought to determine whether the cyclic peptide c2 is capable of crossing the blood-brain barrier.

[0252] Materials & methods

[0253] Drug preparation and administration

[0254] 40 mg / kg of c2 (Bachem, Switzerland) and cF (Bachem, Switzerland) were prepared in DMSO (Sigma Aldrich, USA) ensuring that the DMSO was at 0.5 v / v% in isotonic saline (Braun, Australia). Each of the three groups of mice (n:=:5) was weighed on the day of experiment and their weights were recorded with an average of 24 ± 2 g. Drugs were admini stered intraperitoneally into the caudal right quadrant of the abdomen using a 27G needle. Upon IP injections of 40 mg / kg of cF, c2 or DMSO control, the animals were returned to the cages and were monitored at 30- minute regular intervals for three hours.

[0255] Cardiac bleed, and tissue harvest

[0256] Three hours post IP injection, mice were anaesthetised with 5% inhalant isoflurane in a sealed chamber. Each thorax was swabbed with 70% ethanol and a 25G needle mounted onto a 1 mL syringe that was used to draw blood (0.5 - 1.0 mL) from the heart. Mice were transcardially perfused to remove all the blood from tissues. The heart, lung, liver, both kidneys, and brain tissues were removed from each animal. The brain was sectioned into the left and right hemispheres using a scalpel, and all tissues were immediately snap frozen in liquid nitrogen before storing them at - 80° C. Blood samples from the cardiac bleed were allowed to coagulate at room temperature for 30 minutes, and centrifuged for 5 minutes at 16,000 x g (Heraeus Multifuge X3R Centrifuge, ThermoFisher, Australia) before storing the serum at -80° C.

[0257] Tissue homogenisation

[0258] Animal tissues were weighed prior to transferring them to 2.0 mL polypropylene screw cap tubes with 1.000 mL room temperature Milli-Q water (Milli-Q® Lab Water Systems, Merck, Australia) and five 1.4 mm ceramic beads (ThermoFisher Scientific, USA). The zirconium oxide beads were added to ensure that the tissues are well ground and to mix all cells homogenously. Their high chemical inertness ensured that the beads resisted any form of chemical reaction with the cells. The tissues were homogenised cryogenically in the presence of liquid nitrogen, using a Precellys 24 high-throughput homogeniser (Bertin Technologies, France) at 6500 rpm for 30 seconds.

[0259] Liquid chromatography-tandem mass spectrometry

[0260] Preparation of stock solutions

[0261] Stock solutions of c2 and cF for both the analyte and internal standard (IS) were prepared separately in LC-MS grade ACN (Honeywell Burdick and Jackson, USA) at a stock concentration of 1.00 mg / mL and aliquoted for storage at -80 °C. Working solutions of c2 and cF were prepared by diluting the stock solutions with a 1% FA (Schlarau, Australia) in ACN solution.

[0262] Analytes for calibration standards were prepared using a serial dilution of the stock solutions as outlined in Table 5. ISs of both c2 and cF were made up to 25 ng / mL.

[0263] Table 5 Concentrations of calibration standards of c2 and cF.

[0264] * Solutions were spiked with the concentration on the left-hand side of the arrow to achieve the final concentration on the right-hand side of the arrow

[0265] Serum samples

[0266] Each of the frozen tubes containing mouse serum were thawed, vortexed for 20 seconds and sonicated for 10 minutes (Daihan Scientific, South Korea). 100 pL of serum from each of the five DMSO treated mice were mixed to make up a “pool” solution, and vortexed for 20 seconds. This was later used as the matrix for the standards. Nine separate 5 pL aliquots of serum from the “pool” and 5 pL of serum from each of the treated animals were dispensed into 0.5 mL LoBind (Eppendorf, USA) tubes. To each tube, 5pL of “spike” was added as outlined in Table 6 into eight tubes containing the “pool.” The “spike” was added to one of the tubes containing the “pool” and 5 pL of milli-Q water was added to the rem aining serum samples. 40 pL of 25 ng / mL of IS standard was added ensuring that the IS was c2 for the cF treated samples and its standards, and cF for the c2 treated samples and its standards. Addition of IS also accomplished a protein dump. Lo-bind tubes were vortexed for 20 seconds, sonicated for 10 minutes and centrifuged (10 minutes, 14,000 rpm, 4 °C) using a Velocity 14R Refrigerated Benchtop Centrifuge (Dynamica, United Kingdom). 15 pL of supernatant was dispensed into TRVs, followed by the addition of 15 pL of milli-Q water and vortexed. These vials were stored at 4 °C until they were analysed.

[0267] Another 15 pL of supernatant was aspirated from each sample into fresh LoBind tubes. The samples were then rotary evaporated (1400 rpm, 4 mBar, 25 °C) using a vacuum concentrator (Christ, Germany) coupled with a P30001 Vario vacuum pump (Vacuubrand, USA) for about 2 h until dry. 30 pL of 50% aqueous ACN containing 1% FA was added and samples were then vortexed for 20 seconds, sonicated for 10 minutes and centrifuged for 10 minutes at 14,000 rpm, 4 °C. Roughly 20 pL of the supernatant was aspirated into TRV. All the samples were then analysed using LC-MS / MS, the peak areas for cF and c2 were obtained, and a standard curve was plotted.

[0268] Table 6. Analyte stock concentrations nsed to spike the 5pL serum matrices.

[0269] Brain, liver, and kidney tissues

[0270] Forbrain and liver tissue homogenates, a “pool” was made using the respective tissues from the vehicle (DMSO) treated animals. Similar to the serum samples, standards made in brain and liver homogenates were spiked with c2 and cF as outlined in Table 6. The procedure was slightly altered to spike the cF treated kidneys with the c2 standards and vice versa. Here, 20 pL of “spike” and 160 pL of IS were added to 20 pL of homogenate. Samples were then vortexed for 20 seconds, sonicated for 10 minutes and centrifuged twice for 10 minutes at 14,000 rpm, 4 °C. 100 pL of supernatant was transferred into fresh LoBind tubes and evaporated for 3 h similar to the serum samples. The analyte was reconstituted in 25 pL of 50% aqueous ACN containing 1% FA prior to transfer to TRV. MS and LC conditions Waters ACQUITY UPLC I-Class system was coupled to a SCIEX triple quadrupole 7500 QTRAP mass spectrometer fitted with a OptiFlow® Pro Ion Source was utilised as the UPLC- MS / MS system. Ionisation was achieved using the OptiFlow® Pro Ion Source in positive scanning mode at 450°C with an ion spray voltage of 2000 V. The ion source parameters were optimised as follows: curtain gas 40 psi, ion source gas 1 150 psi, ion source gas 270 psi, CAD gas 9. Scheduled multiple reaction monitoring (MRM) was performed for analytes using optimised entrance potential (EP), collision energies (CE), collision cell exit potentials (CXP). The analyte parent ions, [M+H were fragmented by CID and three of their product ion fragments were monitored to quantify and confirm the identity of the cF and two fragments were monitored to quantify and confirm the identity of c2 (Table 7).

[0271] Table 7 The MRM parameters for c2 and cF.

[0272] 5 pL of each sample solution in a TRY was injected in partial loop mode using a Waters ACQUITY™ UPLC I-Class autosampler. Separation of the analyte was achieved using a Waters ACQUITY™ UPLC C18 HSS T3 (1.8 pm, 2.1 x 100 mm) column at 35 °C and flow rate of 0.200 mL / min. Mobile phase A (Milli-Q water with 0.1% FA) and B (ACN with 0.1% FA) were run with an initial linear gradient of 40% B to 60% over 5.0 min, then immediately 100% B until 6.0 min at which point the gradient changed to initial conditions for equilibration until 11 min. Needle washes were completed using a 1 :9 solution of ACN and milli-Q water for the weak needle wash (600 pL) and a 9: 1 solution of ACN and milli-Q water for the strong needle wash (200 pL).

[0273] Duplicate injections were made for each sample to observe any variation due to the instrument, while also considering the time available for the project.

[0274] Data analysis All LC-MS / MS raw data were imported into the SCIEX OS software for peak assignment, data filtering, screening and obtention of analyte and IS peak areas. Quantification data were initially processed using Excel software (Microsoft 365, Microsoft, USA). Standard curves were generated from SCIEX OS software, but for the purposes of this thesis, we have also plotted the raw values of known concentration vs area of peak on Prism software version 10.1.1 (GraphPad, USA) for clearer resolution, Analyte concentrations in individual tissues were plotted in GraphPad PRISM.

[0275] The raw imaging data from Obitrap Fusion and .xml position file were time-aligned and processed into imzML format using MT imzML Converter Light 0.0.2 software (MassTech, USA). The imzML data was then imported into LipostarMSI (Molecular Horizon Sri, Italy) using the following settings: import parameters were as follows: Savitzky-Golay smoothing was performed at window size, 7 points; degree, 2; iterations, 1; for peak picking the minimum signal to noise ratio (S / N) was set at 0.00; noise window size, 0.10 amu; minimum absolute intensity at 0.00. Peaks below 0.20% of the base peak were discarded. The data set m / z tolerance was set at ±5.00 ppm; minimum peak frequency 1 .00%, mean peak intensity, 0.00, spatial chaos 0.70 and isotopic clustering abundance deviation at 30% and m / z image correlation threshold 0.50 (Tortorella et al., 2020). Figure 11 summarises the LC-MS / MS and MALDI-MSI workflow.

[0276] Results and discussion

[0277] Selection of internal standard, peak assignment and analyte concentration range

[0278] Chromatographic peaks were initially identified using their retention time (RT), which is the time at which the time between injection and appearance of the peak maximum following chromatographic separation. RT of the [M±H]+precursor ions for c2 (2.6 min) (Error! Reference source not found. Figure 12A) and cF (1.7 min) (Figure 12B) in a 50:50 solution of milli-Q water and ACN spiked with the analytes, were consistent with previous results. S / N is another important factor that was considered as it determines the limits of detection and quantification. Noise can lead to both false positive or false negative identifications of sample components by mimicking or masking the signal. A low S / N corresponds to a higher level of signal corruption superimposing on the analyte signal, and hence can be difficult to distinguish as a true peak. In determining the quantification lower concentration limit of this study, only clear and distinct chrom tographic peaks with S / N greater than 10 were used.

[0279] Serum c2 and cF concentration

[0280] The RTs of c2 and cF in tissue samples were the same as those from just solvent solution as c2 was observed at 2.62 ± 0.011 min and cF was observed at 1 .72 ± 0.013 min. The unknown concentration of analyte in serum was calculated using a standard curve (not shown). These results indicate stability of c2 and cF in the selected matrix, i.e. serum. c2 concentration in mouse serum was calculated as 115 ± 66 ng / mL (n=5) while cF concentration was calculated as 13 ± 7 ng / mL (n=5). c2 and cF concentration in various mouse tissues

[0281] The developed method involving the quantification of analytes in reconstituted samples of known analyte concentration was subsequently applied to quantify the analyte concentrations in mouse brain, liver and kidney tissue homogenates, expressing drug concentrations in tissues per gram of fresh weight.

[0282] The plot of the drug concentration-tissue type profiles of c2 and cF after IP administration of the 40 mg / kg formulation is presented in Figure 13. The highest concentration of c2 was detected as 995 ± 46 ng / g in liver (n=3), followed by 322 ± 132 ng / g in kidneys (n=5) and 50 ± 14 ng / g in brain (n=4). The highest concentration of cF was detected as 650 ± 294 ng / g in liver (n=3), followed by 210 ± 24 ng / g in kidneys (n=4) and 25 ± 5 ng / g in brain (n=4).

[0283] Furthermore, the present inventors compared the distribution of the compounds between the two kidneys, as well as the two brain hemispheres. The average concentration of c2 and cF across the two kidneys was very similar. Hemispheric symmetry was observed in both c2 and cF distribution. Average analyte concentrations in individual kidneys and brain hemispheres are summarised in Table 1.

[0284] Table 1 Summary of average c2 and cF concentrations detected in left and right kidneys and the two brain hemispheres.

[0285] In summary, the results from the present Example conclusively demonstrate that both c2 and cF cross the BBB in vivo. Further, it is important to note that the BBB may be disrupted or physiologically absent in certain vascular spaces in patients with tumours. The BBB integrity is altered in both primary and metastatic brain tumours making it more permeable to small molecules (Mo et al., 2021). Disruption of the BBB as a result of increased VEGF is observed in gliomas (Luo et al., 2020). Additionally, the blood-brain tumour barrier (BBTB) present in high grade gliomas appear to be more porous (Arvanitis et al., 2020, Dhermain et al., 2010). Whilst it is unlikely for this increased porosity to allow pharmacologically relevant amounts of drug into the brain, it may be possible to observe an increased uptake of drugs in gliomas compared to healthy brains. The present murine data thus indicate that therapeutically significant concentrations of cF and c2 should be possible in human GBM patients.

[0286] Example 3. Flow cytometry analysis of c2 in GBM cell lines

[0287] In this Example, the inventors sought to investigate the role of c2 in cell death, apoptosis and cell cycle progression in GBM cell lines.

[0288] Materials & methods

[0289] Cell Death Analysis

[0290] At 72 h after each investigated agent treatment, the cells were examined using Annexin V- fluorescein isothiocyante (Annexin V-FITC) and Propidium Iodide (PI) staining (Abeam, Cambridge, USA). In a 6-well plate, U251, T98G or LN229 cells (2 * 105cells / well) were seeded and treated with 100 or 200 pM and incubated for 72 h. The supernatant from each well was next transferred to labelled tubes. 300 pL of trypsin was then added per well. The separated cells were then transferred to designated tubes. The cells were then counted with trypan blue using an inverted microscope (Nikon Eclipse TS100, Sydney, Australia) and diluted to 500 cells / pL which was centrifuged at 500* g for 5 min at 4 °C. The pellet was immersed in 100 pL of 1 * Annexin V Binding Buffer. Each sample was transferred to round-bottomed polystyrene tubes (Interpath Services, Somerton, Australia). The study involved adding 2 pL of each Annexin V-FITC and PI to each tube, incubating for 10 min, and analysing each cell using a benchtop flow cytometer (Biosciences, Erembodegem, Belgium). The Annexin V / PI data was processed using FlowJo™ vl0.9 software, on a scatter plot of FL1-II vs. FL2-H. Three experiments were conducted in triplicate.

[0291] Cell Cycle Arrest

[0292] U251, T98G or LN229 cells were plated at 2 * 105cells / well in a 6-well plate, treated with 50, 100 or 200 pM: concentration of C2 or TMZ at 48 h, a dose that was established for each complex’s treatment. At 48 h post treatment, the supernatant from each well was transferred into correspondingly labelled tubes. A total of 300 pL of trypsin was then added per well. The separated cells were then transferred to designated tubes. Following two PBS washes, the cells were resuspended in 70% ethanol and kept at 4°C overnight. Following another two PBS washes, the fixed cells were stained for 45 min with PI diluted to 50 pg / mL in 10 mM Tris-Cl, pH 8.0, 10 mM NaCl, 0.1% Triton X-100, and 100 pg / mL RNase A. After that, the BD FACSCanto II Benchtop Flow Cytometer (Biosciences, Erembodegem, Belgium) was used to evaluate the cell-cycle profile. FlowJoTM vl0.9 was used to analyse the data. Three experiments were conducted in triplicate.

[0293] Results

[0294] Annexin V / PI staining was used to study the form of cell death after the cells were treated with C2 or TMZ (Figure 14, 15 and 16). Flow cytometry was performed at 72 h post treatment. U251 cells treated with C2 (200 pM) significantly (*** p < 0.001) underwent apoptosis (Figure 14A). An increase in necrotic U251 cells was observed by C2 and TMZ with most significant necrosis observed by C2 at 100 pM (*** p < 0.05) and at 200 pM (**** p < 0.0001) as well as at 200 pM of TMZ (* p < 0.05) (Figure 14B). T98G cells underwent significant apoptosis with 100 pM of C2 (*** p < 0.001) and 200 pM of C2 (**** p < 0.0001), as well as with TMZ at 100 pM (** p < 0.01) and at 200 pM (**** p < 0.0001) (Figure 15 A). T98G cells treated with C2 and TMZ also underwent notable necrosis. A significant increase in necrotic T98G cells was observed at 100 pM (** p < 0.01) and 200 pM (**** p < 0.0001) of C2 as well as at 100 pM (** p < 0.01 and 200 pM (**** p < 0.0001) of TMZ (Figure 15B). LN229 cells treated with C2 (100 pM and 200 pM) significantly (* p < 0.05 and **** p < 0.0001) underwent necrosis (Figure 3B). TMZ also induced significant (* p < 0.05) necrotic cells at 200 pM (Figure 16B).

[0295] The cell-cycle profiles of U251, LN229 and T98G cells treated for 72 h with c2 or TMZ, with concentrations equivalent 50, 100 or 200 pM at 48 h are shown in Figure 17, 18 and 19. In U251 treated cells, an increase in G0 / G1 phase of the cell cycle was observed across all complexes with an exceptional increase in C2 at 100 pM (p < 0.05) (Figure 17B). In T98G treated cells, a decrease in G0 / G1 and G2+M phase and increase in S phase of the cell cycle was detected across all treatments (Figure 18B). In LN229 treated cells, a decrease in G0 / G1 phase and increase in S phase of the cell cycle was observed across all complexes with a notable increase in C2 at 100 pM (p < 0.05) and TMZ at 200 pM (p < 0.001) (Figure 19B).

[0296] References

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Claims

CLAIMS:

1. A method of treating or preventing a CNS cancer or a disease, disorder or condition associated therewith in a subject, said method including the step of administering a therapeutically effective amount of a hGIIA inhibitor to the subject to thereby treat or prevent the CNS cancer, wherein the hGIIA inhibitor is a cyclic peptide.

2. The method of claim 1, wherein the CNS cancer expresses or is associated with hGIIA.

3. The method of claim 1 or claim 2, wherein the CNS cancer is glioblastoma.

4. The method of any one of the preceding claims, wherein the cyclic peptide is capable of crossing the blood-brain barrier of the subject.

5. The method of any one of the preceding claims, wherein the cyclic peptide is cyclo-((2- Nal)-Leu-Ser-(2-Nal)-Arg) or an analogue or derivative thereof.

6. The method of any one of the preceding claims, wherein the cyclic peptide is administered at a dose about 5 mg / day to about 1500 mg / day.

7. The method of any one of the preceding claims, wherein the cyclic peptide is administered at a dose of about 5 mg / day to about 100 mg / day.

8. The method of any one of claims 1 to 7, wherein the cyclic peptide is administered orally, intravenously, intranasally or by inhalation.

9. The method of claim 8, wherein the cyclic peptide is administered orally.

10. The method of any one of the preceding claims, wherein the subject is receiving a further anti-cancer agent for treatment of the CNS cancer.

11. The method of any one of claims 1 to 9, including the further step of administering a further anti-cancer agent for treatment of the CNS cancer to the subject.

12. The method of claim 10 or claim 11, wherein the further anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, a radiation therapy, a molecularly targeted agent, a T cell expressing a chimeric antigen receptor (CAR-T cell), an antibody or antigen-binding fragment thereof, an antibody-drug conjugate (ADC), an angiogenesis inhibitor, an immunotherapeutic agent and any combination thereof.

13. A method of identifying, designing or producing an agent for use in treating or preventing a CNS cancer, said method including the step of determining whether the candidate agent crosses a blood-brain barrier, wherein the candidate agent is a cyclic peptide and wherein the cyclic peptide is a hGIIA inhibitor.

14. The method of claim 13, wherein said method includes the steps of:(a) administering a candidate agent to a subject;(b) determining whether the candidate agent crosses the blood-brain barrier of the subject.

15. The method of claim 14, wherein the candidate agent is administered orally, intravenously, intranasally or by inhalation to the subject.

16. The method of claim 14 or claim 15, wherein the subject has or is at risk of developing the CNS cancer.

17. The method of claim 16, further including the step of determining whether the candidate agent treats or prevents the CNS cancer in the subject.

18. The method of any one of claims 13 to 17, wherein the candidate agent is a derivative of cyclo-((2-Nal)-Leu-Ser-(2-Nal)-Arg).

19. The method of any one of claims 13 to 18, further including the step of isolating and / or purifying an agent that crosses the blood brain barrier.

20. The method of any one of claims 16 to 18, further including the step of isolating and / or purifying an agent that crosses the blood brain barrier and treats or prevents the CNS cancer of the subject.

21. The method of claim 19 or 20, further including the step of formulating the isolated and / or purified agent into a pharmaceutically acceptable formulation.

22. The method of claim 21, further including the step of sterilising the formulation.

23. The method of claim 21 or claim 22, further including the step of filling the formulation into a container.

24. The method of claim 23, wherein the container is any one or more of a vial, an ampoule, a bag, a blister pack, a bottle, a cartridge, an injection needle, an injection syringe, a single dose container, a strip of multiple single dose containers, or a tube.

25. An agent for use in treating or preventing a CN S cancer in a subj ect, obtained by the method of any one of claims 13 to 24.

26. A kit for use in the method of any one of claims 1 to 12, said kit comprising a hGIIA inhibitor, wherein the hGIIA inhibitor is a cyclic peptide, optionally a further anti-cancer therapy and optionally instructions for use.

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