Pharmaceutical Use of Oligopeptides in Combination with Antiandrogens - Patent application

Abstract

The present disclosure relates to methods and medicaments for inhibiting the proliferation of cancer cells by contacting the cancer cells with an antiandrogen drug in combination with a formulation comprising an oligopeptide capable of increasing expression of ferritin heavy chain 1 (FTH1) by epithelial cells. The methods and medicaments are suitable for treating prostate cancer. The isolated oligopeptide of the present disclosure consists of an amino acid sequence of Xm(R / D)EES(G / D)(E / K)Xn (SEQ ID NO: 9), where m and n are integers independently selected from the range of 0 to 10, and each X, when present, is independently selected from any amino acid.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 306,979, filed February 4, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] (Reference to Electronic Sequence Listing) The contents of the electronic sequence listing (197732000840SEQLIST.xml; size: 12,028 bytes; and creation date: February 2, 2023) are incorporated herein by reference in their entirety.

[0003] (Technology) The present disclosure relates to a method and a medicament for inhibiting the proliferation of cancer cells by contacting the cancer cells with an anti-androgen drug in combination with a formulation comprising an oligopeptide that can increase the expression of ferritin heavy chain 1 (FTH1) by epithelial cells.These methods and medicaments are suitable for treating prostate cancer. [Background technology]

[0004] (background) Prostate cancer (PC) is one of the most common male-specific cancers and the third leading cause of death worldwide. Current treatment options for PC are prostatectomy, hormonal therapy, chemotherapy or radiation therapy, which have low success rates, with tumors developing resistance and cancer recurring soon after treatment. Approximately 10% of men treated with curative intent develop metastases over time. Current treatments for metastatic disease are based on androgen ablation and / or androgen receptor (AR) inhibition (e.g., treatment with bicalutamide). Unfortunately, virtually all patients exposed to long-term treatment become resistant to antiandrogen therapy. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a continuing unmet need for more effective therapies for treating locally advanced and metastatic prostate cancer. Additionally, combination therapies to enhance the effects of antiandrogens are desirable. [Means for solving the problem]

[0006] (overview) The present disclosure relates to a method and a medicament for inhibiting the proliferation of cancer cells by contacting the cancer cells with an anti-androgen drug in combination with a formulation comprising an oligopeptide that can increase the expression of ferritin heavy chain 1 (FTH1) by epithelial cells.These methods and medicaments are suitable for treating prostate cancer. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a plot of clonogenic assay results showing the effect of treatment of LNCaP cells (an androgen receptor positive cell line) with salmon protein hydrolysate (SPH) alone or in combination with an antiandrogen drug (bicalutamine = BIC).

[0008] [Diagram 2] FIG. 2 is a plot of clonogenic assay results showing the effect of treatment of PC3 cells (an androgen receptor negative cell line) with salmon protein hydrolysate (SPH) alone or in combination with an antiandrogen drug (bicalutamine = BIC).

[0009] [Diagram 3] FIG. 3 is an alignment of the amino acid sequences of the eight major peptides derived from the three bioactive fractions FRP18, FRP20 and FRP30, along with the shared "EES" motifs, consensus sequences and sequence identifiers.

[0010] [Figure 4]FIG. 4 is a plot of clonogenic assay results showing the effect of treatment of VCaP cells (an androgen receptor positive cell line) with salmon protein hydrolysate (SPH) alone or in combination with an anti-androgen drug (enzalutamide = ENZ).

[0011] [Diagram 5] FIG. 5 is a plot of clonogenic assay results showing the effect of treatment of VCaP-EnzR cells, an androgen receptor positive cell line that is relatively resistant to treatment with the antiandrogen drug (enzalutamide), with salmon protein hydrolysate (SPH) alone or in combination with the antiandrogen drug (enzalutamide = ENZ).

[0012] [Figure 6] FIG. 6 is a plot of clonogenic assay results showing the effect of treatment of LNCaP cells (an androgen receptor positive cell line) with an antiandrogen drug (bicalutamine = BIC) alone or in combination with either SPH or one of the eight oligopeptides.

[0013] [Figure 7] FIG. 7 is a plot of clonogenic assay results showing the effect of treatment of PC3 cells (an androgen receptor negative cell line) with an antiandrogen drug (bicalutamine = BIC) alone or in combination with either SPH or one of the eight oligopeptides.

[0014] [Figure 8] FIG. 8 is a plot of clonogenic assay results showing the effect of treatment of VCaP cells (an androgen receptor positive cell line) with an antiandrogen drug (enzalutamide = ENZ) alone or in combination with either SPH or one of the eight oligopeptides.

[0015] [Figure 9]FIG. 9 is a plot of clonogenic assay results showing the effect of treatment of VCaP-EnzR cells, an androgen receptor positive cell line that is relatively resistant to treatment with the antiandrogen drug (enzalutamide), with the antiandrogen drug (enzalutamide = ENZ) alone or in combination with either SPH or one of the eight oligopeptides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] (Detailed Description) The present disclosure relates to a method and a medicament for inhibiting the proliferation of cancer cells by contacting the cancer cells with an anti-androgen drug in combination with a formulation comprising an oligopeptide that can increase the expression of ferritin heavy chain 1 (FTH1) by epithelial cells.These methods and medicaments are suitable for treating prostate cancer.

[0017] Iron homeostasis has been shown to be a key regulator of prostate cancer (PC) development and progression. Cellular iron redox is maintained by an equilibrium between the storage protein ferritin, the iron transporter, and the efflux transporter ferroportin. For example, direct iron supplementation has recently been shown in cellular and mouse models to be toxic to prostate cancer cells (Bordini et al., Clin Cancer Res, 26:6387-6398, 2020). Furthermore, iron supplementation in combination with the androgen inhibitor bicalutamide inhibited the growth of androgen receptor (AR)-negative (-ve) PC cell lines more than either treatment alone. However, the direct administration of large amounts of iron is considered clinically infeasible due to multiple side effects.

[0018] Furthermore, ferritin heavy chain 1 (FTH1) transcripts and multiple FTH1 pseudogenes were identified as targets of oncogenic microRNAs. Increasing FTH1 expression by impairing competing endogenous RNA crosstalk led to reduced carcinogenesis in vitro and in vivo (Chan et al., Nucleic Acids Research, 46(4):1998-2011, 2018). Furthermore, both AR+ve (LNCaP, VCaP) and AR-ve (PC3, DU145) prostate cancer cells were found to overexpress transferrin receptor 1 (TFR1) and underexpress FTH1 protein compared to normal prostate epithelial cells (Keer et al., J Urol, 143(2):381-385, 1990; and Chan et al., supra, 2018). Other reports have described the role of iron homeostasis dysfunction and tumor growth through the p53-ISCU pathway (Funaguchi et al., Sci Rep, 5:16497, 2015) and the hepcidin pathway (Tesfay et al., Cancer Res, 75(11):2254-2263, 2015), both of which control iron homeostasis and were found to be significantly upregulated in PC cells.

[0019] Previously, prostate cancer cells were found to retain higher levels of metabolically active iron (known as the labile iron pool) than benign prostate epithelial cells (Funaguchi et al., Sci Rep, 5:16497, 2015). Two proteins important for modulating intracellular iron levels are the iron uptake protein, TFR1, and the iron storage protein, ferritin, which is a globular protein complex composed of FTH1 and ferritin light chain (FTL) subunits.

[0020] TFR1 and FTH1 protein levels were measured by Western blot analysis of lysates from normal prostate epithelial cells (PrEC) and two prostate cancer cell lines (LNCaP (AR+ve cell line); and PC3 (AR-ve cell line)). TFR1 protein expression was significantly increased in both prostate cancer cell lines compared to normal prostate epithelial cells. In contrast, FTH1 protein levels were reduced to barely detectable levels in both prostate cancer cells, while it was faintly expressed in PrEC cells. Increased TFR1 expression and decreased FTH1 expression are associated with high iron uptake capacity and low iron storage capacity.

[0021] As described in Example 2 herein, salmon protein hydrolysate (SPH) has now been found to inhibit the proliferation of AR+ve and AR-ve prostate cancer cells when cultured in vitro in the presence of bicalutamide.Bicalutamide is an AR antagonist type nonsteroidal antiandrogen drug, which is used to treat metastatic prostate cancer and locally advanced prostate cancer.Increasing the effect of bicalutamide and other antiandrogens in vivo by combining with oligopeptide therapeutic agents (e.g., SPH or bioactive peptide(s) of SPH) is believed to improve its usefulness in treating prostate cancer and other indications for which bicalutamide is prescribed off-label.

[0022] In detail, as shown in Figure 1 and Figure 2 and described in Example 2, SPH as a single treatment had no effect on LNCaP (AR+ve) and PC3 (AR-ve) cell proliferation.However, co-treatment of both low (40μg / ml) and high (160μg / ml) concentrations of SPH together with bicalutamide (BIC) at minimum inhibitory concentration (MIC) of 1μM for LNCaP and 10μM for PC3 significantly increased the therapeutic effect of bicalutamide treatment in both cell lines.In LNCaP AR+ve cells, SPH co-treatment (160μg / ml) reduced the relative cell survival percentage of 0.5μM BIC treatment from 85% to 19%, and reduced the relative cell survival percentage of 1.0μM BIC treatment from 25% to 2%. Similarly, in PC3 AR-ve cells, SPH co-treatment (160 μg / ml) reduced the relative cell viability percentage of 10 μM BIC treatment from 52% to 32%.

[0023] Furthermore, as shown in Figures 4 and 5 and described in Example 2, SPH as a single treatment had little effect on VCaP (AR+ve) and VCaP-ENZR (AR+ve) cell proliferation. However, co-treatment of high concentration (160 μg / ml) SPH with sub-minimal inhibitory concentration (MIC) enzalutamide (ENZ) increased the therapeutic effect of enzalutamide treatment in both cell lines. In VCaP cells, SPH co-treatment (160 μg / ml SPH) reduced the relative cell viability percentage of 0.1 μM ENZ treatment from 77% to 52%. Similarly, in VCaP-ENZR cells, SPH co-treatment (160 μg / ml SPH) reduced the relative cell viability percentage of 10 μM ENZ treatment from 64% to 56%.

[0024] Furthermore, gene expression was attenuated in both LNCaP and PC3 prostate cancer cell lines when treated with SPH and bicalutamide (BIC). Similarly, gene expression was attenuated in both VCaP and VCaP-ENZR prostate cancer cell lines when treated with SPH and enzalutamide (ENZ). In particular, FTH1 was upregulated more than two-fold and TFRC was downregulated more than two-fold compared to the housekeeping gene ACTB in prostate cancer cells cultured in the presence of both SPH and BIC or ENZ, but not BIC or ENZ alone (see Tables 2-1 and 2-2).

[0025] The desirable properties of SPH treatment in combination with BIC or ENZ were also found when prostate cancer cells were treated with BIC or ENZSPH in combination with synthetic oligopeptides having amino acid sequences identified from the active fraction of SPH. The most potent antiproliferative effects were observed when prostate cancer cells were treated with oligopeptides FT-002 or FT-005 in combination with BIC or ENZ (see Example 3 and Figures 6-9). In addition, in prostate cancer cells cultured in the presence of oligopeptides in combination with BIC or ENZ, FTH1 was upregulated and TFRC was downregulated to the same or greater extent than observed when prostate cancer cells were cultured in the presence of SPH and BIC or ENZ (see Tables 3-1 and 3-2).

[0026] (definition) As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural references unless the context dictates otherwise. For example, "an excipient" includes one or more excipients.

[0027] It is understood that aspects and embodiments described herein as "comprising" include aspects and embodiments "consisting of" and / or "consisting essentially of."

[0028] The term "about" as used herein with respect to a value describes 90% to 110% of that value. For example, about a 2-fold change in FTH1 mRNA expression includes from 1.8-fold to 2.2-fold change, including a 2.0-fold change in FTH1 mRNA.

[0029] As used herein, the term "androgen receptor" refers to a receptor that is activated by binding to testosterone or dihydrotestosterone (androgen hormone).Androgen receptor is also known as "nuclear receptor subfamily 3, group C, member 4" and "NR3C4".The amino acid sequence of human androgen receptor is shown under GenBank accession number NP_000035 (isoform 1), and the nucleic acid sequence of human androgen receptor is shown under GenBank accession number NM_000044 (variant 1).

[0030] As used herein, the terms "ferritin heavy chain 1" and "FTH1" refer to a nucleic acid sequence encoding the "ferritin heavy chain" protein (also known as the "ferritin H subunit"). The amino acid sequence of human ferritin heavy chain is set forth under GenBank Accession No. NP_002023, its mRNA sequence is set forth under GenBank Accession No. NM_002032, and its coding sequence spans nucleotides 210-761.

[0031] The term "isolated" as used herein with respect to a molecule (e.g., an oligopeptide) refers to a molecule that has been removed from its natural or synthetic environment or otherwise purified. A substantially "isolated" molecule is at least 75% free, preferably at least 90% free, and more preferably at least 95%, 96%, 97%, 98% or 99% free from other components. For example, an "isolated oligopeptide consisting of the amino acid sequence of SEQ ID NO:9" is at least 75% free of peptides and proteins that do not contain the amino acid sequence of SEQ ID NO:9.

[0032] The term "increasing" and grammatical equivalents as used herein with respect to expression or levels of FTH1 mRNA refers to causing the amount of FTH1 mRNA to increase. Preferably, the increase in FTH1 mRNA includes a statistically significant increase. Preferably, the increase in FTH1 mRNA includes an increase of about 2-fold to about 200-fold, about 2-fold to 20-fold, or about 2-fold to 4-fold, or more preferably at least 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, or 3.5-fold over baseline FTH1 mRNA levels.

[0033] The term "reducing" and grammatical equivalents as used herein with respect to TFRC mRNA expression or levels refer to causing the amount of TFRC mRNA to decrease. Preferably, the decrease in TFRC mRNA includes a statistically significant decrease. Preferably, the decrease in TFRC mRNA includes about a 2-fold to about a 200-fold, about a 2-fold to about a 20-fold, or about a 2-fold to about a 4-fold decrease from baseline TFRC mRNA levels, or more preferably, at most 0.8, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 decrease from baseline TFRC mRNA levels.

[0034] As used herein, the terms "treating" and "treatment" refer to an approach to obtain beneficial or desired results (including clinical results). Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, amelioration or alleviation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to the expected survival in the absence of treatment. Thus, as used herein, the terms "treating" and "treatment" do not require complete alleviation of signs or symptoms, do not require cure, and in particular include protocols that have a modest effect on an individual.

[0035] An "effective amount" of an agent (e.g., an isolated oligopeptide or formulation thereof) disclosed herein is an amount sufficient to carry out a specifically described purpose. An "effective amount" can be empirically determined for that described purpose. An "effective amount" or "sufficient amount" of an agent is an amount sufficient to produce a desired biological effect (e.g., a beneficial outcome (including a beneficial clinical outcome)). The term "therapeutically effective amount" refers to an amount of an agent (e.g., an isolated oligopeptide or formulation thereof) effective to "treat" a disease or disorder in a subject (e.g., a mammal, e.g., a human). An "effective amount" or "sufficient amount" of an agent can be administered in one dose or in multiple doses.

[0036] Administration "in combination with" or "in addition to" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0037] The terms "individual" and "subject" refer to mammals, including, but not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0038] I. Salmon Protein Hydrolysates and Isolated Oligopeptides Salmon protein hydrolysate (SPH) is a complex mixture of over 500 peptides obtained by enzymatic hydrolysis of salmon protein material. When properly prepared, such as in ProGo™ (manufactured by Hofseth BioCare ASA, Alesund, Norway), SPH contains more than 97% protein content (w / w) and is composed primarily of oligopeptides and peptides with molecular weights of about 1000 to less than 3000 daltons. SPH (e.g., ProGo™, or other formulations prepared as described (see, e.g., Example 1 herein; and Examples 1, 4, and 5 of US2021 / 0252099)) has previously been shown to be "bioactive" in that it modulates the expression of oxidative stress-related genes. Specifically, SPH has been shown to upregulate FTH1 mRNA and heme oxygenase 1 (HMOX1) mRNA expression and downregulate arachidonate 12-lipoxygenase (ALOX12) mRNA in normal epithelial cells (see, e.g., Example 8 of US2021 / 0252099).

[0039] However, salmon protein hydrolysates and other fish protein hydrolysates are very complex compositions that contain hundreds of peptides, many of which may be benign inactive peptides, while others may have undesirable activity.Therefore, in some embodiments, isolated oligopeptides that retain the desirable bioactivity of SPH (e.g., the ability to cause an increase in FTH1 mRNA expression) are preferred over SPH for use in the methods and medicaments of the present disclosure.

[0040] The isolated oligopeptides of the present disclosure consist of an amino acid sequence of Xm(R / D)EES(G / D)(E / K)Xn (SEQ ID NO:9), where m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid. Specifically, in some embodiments, the number m and the number n are each selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Thus, the claimed oligopeptides are between 6 and 26 residues in length. In some embodiments, the oligopeptide is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more residues in length; and / or The oligopeptide is 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, or 7 or less, the lower limit being less than the upper limit. In some embodiments, the isolated oligopeptide comprises the amino acid sequence of REESGE (SEQ ID NO:1). In some embodiments, the isolated oligopeptide comprises the amino acid sequence of REESGEP (SEQ ID NO:2). In some embodiments, the isolated oligopeptide comprises the amino acid sequence of KEEDEESGE (SEQ ID NO:3). In some embodiments, the isolated oligopeptide comprises the amino acid sequence of KPREESGE (SEQ ID NO:4). In some embodiments, the isolated oligopeptide comprises the amino acid sequence of LDEESGEP (SEQ ID NO:5). In some embodiments, the isolated oligopeptide comprises the amino acid sequence of REESDKPMY (SEQ ID NO:6). In some embodiments, the isolated oligopeptide comprises the amino acid sequence of PREESDKP (SEQ ID NO:7).In some embodiments, the isolated oligopeptide comprises the amino acid sequence of REESGEL (SEQ ID NO: 8). In some embodiments, the isolated oligopeptide comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to any one of SEQ ID NOs: 1-8.

[0041] In some embodiments, an isolated oligopeptide of the disclosure consists of an amino acid sequence of Xp(R / D)EESGEPXq (consensus number 2 / SEQ ID NO: 10), where p is an integer selected from the range of 0-10, q is an integer selected from the range of 0-9, and each X, if present, is independently selected from any amino acid. Thus, in some embodiments, the claimed oligopeptide is 7 or more residues long, 8 or more residues long, 9 or more residues long, 10 or more residues long, 11 or more residues long, 12 or more residues long, 13 or more residues long, 14 or more residues long, 15 or more residues long, 16 or more residues long, 17 or more residues long, 18 or more residues long, 19 or more residues long, 20 or more residues long, 21 or more residues long, 22 or more residues long, 23 or more residues long, 24 or more residues long, or 25 or more residues long; and / or The oligopeptide is 26 or less residues long, 25 or less residues long, 24 or less residues long, 23 or less residues long, 22 or less residues long, 21 or less residues long, 20 or less residues long, 19 or less residues long, 18 or less residues long, 17 or less residues long, 16 or less residues long, 15 or less residues long, 14 or less residues long, 13 or less residues long, 12 or less residues long, 11 or less residues long, 10 or less residues long, 9 or less residues long, or 8 or less residues long, with the lower limit being less than the upper limit. In some embodiments, the oligopeptide comprises an amino acid sequence of Xr(R / D)EESGEP (consensus number 3 / SEQ ID NO: 11), where Xr is leucine or absent. In some embodiments, the isolated oligopeptide comprises an amino acid sequence of REESGEP (SEQ ID NO: 2). In some embodiments, the isolated oligopeptide comprises an amino acid sequence of LDEESGEP (SEQ ID NO: 5).

[0042] In a preferred embodiment, the isolated oligopeptide is capable of increasing the expression of ferritin heavy chain 1 (FTH1) mRNA by cancer cells contacted with the oligopeptide in the presence of an antiandrogen. The increase in expression of FTH1 mRNA as a result of contact with the oligopeptide is compared to cancer cells contacted with the androgen in the absence of the oligopeptide, compared to cancer cells cultured under the same conditions except for the absence of the oligopeptide, or compared to cancer cells cultured under the same conditions except for the presence of a negative control oligopeptide (e.g., an oligopeptide of approximately the same length but not including SEQ ID NO:9). In some embodiments, the cancer cell is a mammalian cell. In some preferred embodiments, the mammalian cell is a human cell. In an exemplary embodiment, the cancer cell is a LNCaP cell. In another exemplary embodiment, the cancer cell is a PC3 cell.

[0043] In a preferred embodiment, the isolated oligopeptide is capable of reducing the expression of transferrin receptor 1 (TFRC) mRNA by cancer cells contacted with the oligopeptide in the presence of an antiandrogen drug. The reduction in expression of TFRC mRNA as a result of contact with the oligopeptide is compared to cancer cells contacted with the androgen in the absence of the oligopeptide, compared to cancer cells cultured under the same conditions except for the absence of the oligopeptide, or compared to cancer cells cultured under the same conditions except for the presence of a negative control oligopeptide (e.g., an oligopeptide of approximately the same length but not including SEQ ID NO: 9). In some embodiments, the cancer cell is a mammalian cell. In some preferred embodiments, the mammalian cell is a human cell. In an exemplary embodiment, the cancer cell is a LNCaP cell. In another exemplary embodiment, the cancer cell is a PC3 cell.

[0044] In some preferred embodiments, the oligopeptide is made synthetically. In an exemplary embodiment, the oligopeptide is made by solid phase synthesis as known in the art and purified by high performance liquid chromatography.

[0045] (II. Oligopeptide Formulations) The oligopeptide formulations used in the methods and medicaments of the present disclosure comprise at least one isolated oligopeptide of the above paragraph and at least one pharma- ceutically acceptable excipient and / or oral delivery agent.In some embodiments, the formulations may further comprise enteric coating, liposome, microsphere, or microparticle / nanoparticle.For example, in some embodiments, the isolated oligopeptide is encapsulated in enteric coating, liposome, microsphere, or microparticle / nanoparticle.

[0046] The amount of the oligopeptide of the present disclosure that is effective in treating a particular disorder or condition disclosed herein will depend on the nature of the disorder or disease, and the amount can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays can be used, as necessary, to help identify optimal dosage ranges. In some embodiments, the dose of the oligopeptide of the present disclosure is about 0.1 mg to about 1000 mg, about 1.0 mg to about 100 mg, or about 10 mg per kg of body weight of the subject to be treated. In some embodiments, the dose of the oligopeptide is 0.1 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 5.0 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 60 mg / kg or more, 70 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, or 500 mg / kg, and / or The dose of the oligopeptide is 1000 mg / kg or less, 500 mg / kg or less, 100 mg / kg or less, 90 mg / kg or less, 80 mg / kg or less, 70 mg / kg or less, 60 mg / kg or less, 50 mg / kg or less, 40 mg / kg or less, 30 mg / kg or less, 20 mg / kg or less, 10 mg / kg or less, 5.0 mg / kg or less, 1.0 mg / kg or less, or 0.5 mg / kg, with the lower limit being less than the upper limit.

[0047] A. Excipients Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, bulking agents, buffers, tonicity adjusters, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the formulation may include an excipient that functions as one or more of a solvent, bulking agent, buffer, and tonicity adjuster (e.g., sodium chloride in saline may serve as both an aqueous medium and a tonicity adjuster).

[0048] In some embodiments, the formulation comprises an aqueous medium as a solvent. Suitable vehicles include, for example, sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the formulation is isotonic.

[0049] The formulation may include a buffering agent. The buffering agent controls the pH to inhibit degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffers include, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids, such as arginine, glycine, histidine, and lysine. The buffer may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the formulation within the range of 6-9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is within the range of about 6-9, and the lower limit is less than the upper limit.

[0050] The formulation may contain a tonicity adjuster. Suitable tonicity adjusters include, for example, dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0051] The formulation may include a bulking agent.The bulking agent is particularly useful when the pharmaceutical formulation should be lyophilized before administration.In some embodiments, the bulking agent is a protective agent that helps stabilize and prevent degradation of active agent during lyophilization or spray drying and / or storage.Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0052] The formulation may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the formulation is prepared under sterile conditions and is in a single-use container, and therefore does not need to contain a preservative.

[0053] B. Oral Delivery Agents Oral delivery agents of the present disclosure include, for example, absorption enhancers, fatty acids, enzyme inhibitors, polyethylene glycol, mucoadhesive polymers, and cell-penetrating peptides (Dan et al., Children, 7:307, 2020).

[0054] Commonly utilized administration routes for therapeutic peptides and proteins include intravenous (IV), intraperitoneal (IP), and intramuscular (IM) injections. However, oral administration is preferred by patients, and oral drugs are typically less expensive to manufacture, deliver, and administer. Unfortunately, the development of orally available dosage forms of therapeutic peptides and proteins is complicated for a variety of reasons, including but not limited to poor stability in physiological conditions, short biological half-life, and low permeability through the epithelial barrier in the small intestine. Thus, in some embodiments, the formulations of the present disclosure are designed to protect the isolated oligopeptide from proteolytic enzymes and the acidic environment found in the stomach, such that the formulation retains its bioactivity when absorbed into the bloodstream (see, e.g., Dan et al., Children, 7:307, 2020).

[0055] (III. Nonsteroidal Antiandrogens) Antiandrogens are compounds that inhibit the activity of androgens. Antiandrogens are classified as steroidal antiandrogens or nonsteroidal antiandrogens (NSAA). The methods and medicaments of the present disclosure include an NSAA. In some preferred embodiments, the NSAA is an androgen receptor antagonist that blocks the effects of testosterone and dihydrotestosterone. NSAA is commonly used in combination with castration as combined androgen blockade for the treatment of prostate cancer (Crawford et al., J Urol, 200(5):956-966, 2018). In some embodiments, the NSAA is selected from the group consisting of bicalutamide, apalutamide, enzalutamide, flutamide, nilutamide, topirutamide, darolutamide, proxalutamide, and combinations thereof. In some embodiments, the nonsteroidal antiandrogen is bicalutamide.

[0056] (IV. How to use) The salmon protein hydrolysate, isolated oligopeptide and formulation thereof of the present disclosure finds use in combination with non-steroidal antiandrogen drugs in methods and medicaments for inhibiting cancer cell proliferation. In some embodiments, the cancer cell is an adenocarcinoma cell. In some embodiments, the cancer cell is a prostate cancer cell. In some embodiments, the prostate cancer cell secretes prostate specific antigen. In some embodiments, the prostate cancer cell is androgen receptor positive. In some embodiments, the prostate cancer cell is androgen receptor negative. In some embodiments, the cancer cell is a human cell. In some preferred embodiments, the salmon protein hydrolysate, isolated oligopeptide and formulation thereof of the present disclosure finds use in combination with non-steroidal antiandrogen drugs in methods and medicaments for treating prostate cancer in human subjects in need of treating prostate cancer.

[0057] In some in vivo embodiments, the formulation is administered orally. For example, the formulation can be administered enterally. In some embodiments, the formulation is administered buccal, sublabial, or sublingual. In some embodiments, the nonsteroidal antiandrogen is administered orally.

[0058] In some embodiments, the isolated oligopeptide is capable of increasing expression of ferritin heavy chain 1 (FTH1) mRNA by cancer cells when contacted with the oligopeptide in the presence of a nonsteroidal antiandrogen. In some embodiments, the isolated oligopeptide is capable of increasing expression of FTH1 mRNA, and the increase in FTH1 mRNA is compared to cancer cells contacted with a nonsteroidal antiandrogen in the absence of the oligopeptide. In some embodiments, the isolated oligopeptide is capable of increasing expression of FTH1 mRNA, and the increase in FTH1 mRNA is compared to cancer cells cultured under the same conditions except for the absence of the oligopeptide. In some embodiments, the isolated oligopeptide is capable of increasing expression of FTH1 mRNA, and the increase in FTH1 mRNA is compared to cancer cells cultured under the same conditions except for the absence of the oligopeptide. In some embodiments, the isolated oligopeptide is capable of increasing expression of FTH1 mRNA, and the increase in FTH1 mRNA is compared to cancer cells cultured under the same conditions except for the presence of a negative control oligopeptide.

[0059] In some embodiments, the isolated oligopeptide is capable of reducing the expression of transferrin receptor 1 (TFRC) mRNA by cancer cells when contacted with the oligopeptide in the presence of a nonsteroidal antiandrogen. In some embodiments, the isolated oligopeptide is capable of reducing the expression of TFRC mRNA, and the reduction in TFRC mRNA is compared to cancer cells contacted with a nonsteroidal antiandrogen in the absence of the oligopeptide. In some embodiments, the isolated oligopeptide is capable of reducing the expression of TFRC mRNA, and the reduction in TFRC mRNA is compared to cancer cells cultured under the same conditions except for the absence of the oligopeptide. In some embodiments, the isolated oligopeptide is capable of reducing the expression of TFRC mRNA, and the reduction in TFRC mRNA is compared to cancer cells cultured under the same conditions except for the absence of the oligopeptide. In some embodiments, the isolated oligopeptide is capable of reducing the expression of TFRC mRNA, and the reduction in TFRC mRNA is compared to cancer cells cultured under the same conditions except for the presence of a negative control oligopeptide.

[0060] In some aspects, the present disclosure provides methods and medicaments for inhibiting the proliferation of cancer cells, comprising contacting the cancer cells with an effective amount of a nonsteroidal antiandrogen drug and an effective amount of an oligopeptide (or a formulation comprising the oligopeptide). In some embodiments, the contacting is performed in vivo.

[0061] In some aspects, the present disclosure provides a method and medicament for treating prostate cancer in a mammalian subject in need thereof, comprising administering to the subject an effective amount of a nonsteroidal antiandrogen drug and an effective amount of an oligopeptide (or a formulation comprising the oligopeptide). In some embodiments, the prostate cancer cells secrete prostate specific antigen. In some embodiments, the prostate cancer is a metastatic cancer. In some embodiments, the prostate cancer cells are androgen receptor positive. In some embodiments, the prostate cancer cells are androgen receptor negative. Enumerated Embodiments 1. A method for inhibiting the proliferation of cancer cells, comprising: The method includes contacting the cancer cells with an effective amount of a nonsteroidal antiandrogen and an effective amount of a formulation containing an oligopeptide consisting of an amino acid sequence of Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), wherein m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid. 2. A method for inhibiting the proliferation of cancer cells, comprising: contacting the cancer cells with an effective amount of a nonsteroidal antiandrogen and an effective amount of a formulation comprising an oligopeptide consisting of the amino acid sequence of Xp(R / D)EESGEPXq (SEQ ID NO: 10), wherein p is an integer selected from the range of 0 to 10, q is an integer selected from the range of 0 to 9, and each X, if present, is independently selected from any amino acid. 3. The method of embodiment 2, comprising the amino acid sequence of Xr(R / D)EESGEP (sequence number 11), wherein Xr is leucine or absent. 4. The method of embodiment 3, wherein the oligopeptide comprises the amino acid sequence of REESGEP (sequence number 2). 5. The method of embodiment 3, wherein the oligopeptide comprises the amino acid sequence of LDEESGEP (sequence number 5). 6. The method of embodiment 1, wherein the oligopeptide comprises the amino acid sequence of REESGE (sequence number 1), the amino acid sequence of REESGEP (sequence number 2), the amino acid sequence of KEEDEESGE (sequence number 3), the amino acid sequence of KPREESGE (sequence number 4), the amino acid sequence of LDEESGEP (sequence number 5), the amino acid sequence of REESDKPMY (sequence number 6), the amino acid sequence of PREESDKP (sequence number 7), or the amino acid sequence of REESGEL (sequence number 8). 7. A method according to any one of embodiments 1 to 6, wherein the oligopeptide is capable of increasing expression of ferritin heavy chain 1 (FTH1) mRNA by cancer cells contacted with the oligopeptide in the presence of an antiandrogen drug. 8. The method of embodiment 1, wherein the formulation comprises a fish protein hydrolysate, optionally wherein the fish protein hydrolysate is a salmon protein hydrolysate. 9. The method of any one of embodiments 1-8, wherein the nonsteroidal antiandrogen is selected from bicalutamide, apalutamide, enzalutamide, flutamide, nilutamide, topirutamide, darolutamide, proxalutamide, and combinations thereof. 10. The method of embodiment 9, wherein the nonsteroidal antiandrogen is bicalutamide. 11. The method of embodiment 9, wherein the nonsteroidal antiandrogen is enzalutamide. 12. The method of any one of embodiments 1 to 11, wherein the cancer cells are adenocarcinoma cells. 13. The method of any one of embodiments 1 to 11, wherein the cancer cells are prostate cancer cells. 14. The method of embodiment 13, wherein the prostate cancer cells are androgen receptor positive. 15. The method of embodiment 13, wherein the prostate cancer cells are androgen receptor negative. 16. The method of any one of embodiments 1 to 15, wherein the cancer cells are human cells. 17. The method of any one of embodiments 1 to 16, wherein the contacting is performed in vivo. 18. The method of any one of embodiments 1 to 17, wherein the formulation comprising the oligopeptide further comprises at least one pharma- ceutically acceptable excipient. 19. The method of any one of embodiments 1 to 18, wherein the formulation comprising the oligopeptide further comprises an oral delivery agent. 20. The method of embodiment 19, wherein the oral delivery agent comprises an absorption enhancer, a fatty acid, an enzyme inhibitor, polyethylene glycol, a mucoadhesive polymer, a cell-penetrating peptide, or a combination thereof. 21. The method of embodiment 19 or embodiment 20, further comprising an enteric coating, liposomes, microspheres, and / or microparticles / nanoparticles. 22. A method for treating prostate cancer in a mammalian subject in need thereof, comprising: The method comprises administering to the subject i) an effective amount of a nonsteroidal antiandrogen and ii) an effective amount of a formulation comprising an oligopeptide consisting of an amino acid sequence of Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), wherein m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid. 23. The method of embodiment 22, wherein the antiandrogen and the formulation are administered orally. 24. The method of embodiment 23, wherein the formulation is administered enterally. 25. The method of embodiment 24, wherein the formulation is administered by the buccal, sublabial, or sublingual route. 26. The method of any one of embodiments 22-25, wherein administration of the antiandrogen drug and the formulation results in a reduction in the volume of the prostate cancer compared to the volume before the treatment. 27. The method of any one of embodiments 22 to 26, wherein the mammalian subject is a human subject. 28. The method of any one of embodiments 22 to 27, wherein the oligopeptide comprises the amino acid sequence of REESGEP (sequence number 2). 29. The method of any one of embodiments 22 to 27, wherein the oligopeptide comprises the amino acid sequence of LDEESGEP (sequence number 5). 30. The method of any one of embodiments 22 to 27, wherein the oligopeptide comprises the amino acid sequence of REESGE (sequence number 1), the amino acid sequence of REESGEP (sequence number 2), the amino acid sequence of KEEDEESGE (sequence number 3), the amino acid sequence of KPREESGE (sequence number 4), the amino acid sequence of LDEESGEP (sequence number 5), the amino acid sequence of REESDKPMY (sequence number 6), the amino acid sequence of PREESDKP (sequence number 7), or the amino acid sequence of REESGEL (sequence number 8). 31. The method of any one of embodiments 22 to 27, wherein the formulation comprises a fish protein hydrolysate, optionally wherein the fish protein hydrolysate is a salmon protein hydrolysate. 32. The method of any one of embodiments 22-31, wherein the nonsteroidal antiandrogen is selected from bicalutamide, apalutamide, enzalutamide, flutamide, nilutamide, topirutamide, darolutamide, proxalutamide, and combinations thereof. 33. The method of embodiment 32, wherein the nonsteroidal antiandrogen is bicalutamide. 34. The method of embodiment 32, wherein the nonsteroidal antiandrogen is enzalutamide. 35. Administering gonadotropin-releasing hormone antagonists The method of any one of embodiments 22 to 34, further comprising: 36. The method of any one of embodiments 22 to 35, wherein the prostate cancer is androgen receptor positive. 37. The method of any one of embodiments 22 to 35, wherein the prostate cancer is androgen receptor negative. 38. The method of any one of embodiments 22 to 37, wherein the prostate cancer is a metastatic cancer. EXAMPLES

[0062] (Example) Abbreviations: ACTB (beta actin); AR (androgen receptor); BIC (bicalutamide); ENZ (enzalutamide); FTH1 (ferritin heavy chain 1); MIC (minimum inhibitory concentration); SPH (salmon protein hydrolysates), TFRC or TFR1 (transferrin receptor 1); -ve (negative); +ve (positive).

[0063] Example 1 Preparation of Bioactive Salmon Protein Hydrolysates Salmon protein hydrolysate (SPH) powder was produced by enzymatic hydrolysis of salmon (Salmo salar) heads and backbones after filleting as described (US 2021 / 0252099). Briefly, 1000 grams of crushed heads and backbones were added to 1000 ml of water and the mixture was heated to 50°C. 10 g of endopeptidase enzyme (pepsin) was added and the mixture was stirred for 30 minutes. Then, 10 g of exopeptidase enzyme (carboxypeptidase) was added and the mixture was stirred for 15 minutes. Next, 5 grams of Flavourzyme® (a mixture of endo- and exoproteases derived from Aspergillus oryzae, commercially available by Novozymes A / S, Bagsvaerd, Denmark) was added and the mixture was stirred for 10 minutes. The endopeptidase- and exopeptidase-treated salmon protein mixture was then heated to 85° C. for 15 min to inactivate the proteases. After filtration, the hydrolysate fraction was concentrated to 30% dry matter in a conventional evaporator and spray-dried to obtain salmon protein hydrolysate powder.

[0064] Example 2 Determining the effect of salmon protein hydrolysates in combination with antiandrogens on androgen receptor positive and androgen receptor negative cell lines This example describes the effects of salmon protein hydrolysates (SPH) and the antiandrogen bicalutamide on the in vitro growth and gene expression of two prostate cancer cell lines.

[0065] (material and method) Test Solutions. SPH test solutions were prepared by adding 10 μg, 40 μg, or 160 μg of SPH powder to 1 ml of DMEM with 0.3% FBS + 2% DMSO and sonicating for 10 minutes immediately before use. Bicalutamide (herein referred to as BIC) (Sigma-Aldrich, Cat. No. B9061) test solutions were prepared by adding 0.43 μg or 4.3 μg (1 μM and 10 μM) to 1 ml of DMEM with 0.3% FBS + 2% DMSO and sonicating for 10 minutes immediately before use. Enzalutamide (herein referred to as ENZ) (Intas Pharmaceuticals, India), test solutions were prepared by serial dilution of 1 mM stock solution (3% DMS in water) into culture medium.

[0066] (Cell Culture). The C4 subline (CRL-3313™, RRID: CVCL 4783), derived from the human prostate cancer cell line LNCaP, was obtained from ATCC (Manassas, VA). C4 (herein referred to as LNCaP) is a representative androgen receptor positive (AR+ve) cell line with epithelial-like morphology and low metastatic potential. The PC-3 cell line (CRL-1435, RRID: CVCL_0035) was obtained from ATCC (Manassas, VA). PC-3 is a representative androgen receptor negative (AR-ve) cell line with epithelial morphology and high metastatic potential. The Vertebral-Cancer of the Prostate (VCaP) cell line was obtained from Sigma-Aldrich (USA) (catalog number 06020201). VCaP is a human cell line with epithelial morphology established from a metastatic prostate cancer lesion (Korenchuk et al., In Vivo, 15(2):163-168, 2001). VCaP cells are androgen receptor positive and express high levels of prostate-specific antigen. The VCaP-EnzR cell line was also obtained from Sigma-Aldrich (USA) (catalog number SCC421). VCaP-EnzR is a human cell line derived from VCaP after long-term culture in the presence of the androgen receptor antagonist enzalutamide (Kregel et al., Oncotarget, 7(18):26529-26274, 2016). VCaP-EnzR cells are enzalutamide resistant and therefore a suitable model to study castration-resistant prostate cancer. All cell lines were cultured in RPMI 1640 supplemented with 2 mmol / L L-glutamine, 100 U / mL penicillin, 100 mg / mL streptomycin, and 10% heat-inactivated fetal bovine serum. All cells were certified negative for Mycoplasma.

[0067] (Clonogenic assay). Confluent cells were trypsinized and seeded at cell densities ranging from 3000 to 5000 cells in 10 cm Petri dishes depending on the cell line, as recommended in the ATCC or Sigma-Aldrich product sheets. Cells were allowed to adhere for 24 to 48 h before application of treatment with either SPH alone (10 μg / ml to 160 μg / ml), bicalutamide alone (LNCaP and PC3 cells) or enzalutamide alone (VCaP or VCaP-EnzR cells), or a combination of SPH with BIC or ENZ. In the combined treatment, the dose of bicalutamide and the dose of enzalutamide were fixed by defining the minimum inhibitory concentration (MIC) that starts to block colony formation. Treatments were applied daily for 5 days without medium changes. The colonies were allowed to develop for 12 days, stained with crystal violet, and counted automatically using a TC20 Automated Cell Counter (Bio Rad, Hercules, CA). Untreated cells (cell culture medium + 0.1% DMSO) were used as an internal control for each assay. Relative plating efficiency was expressed as a percentage compared to that of untreated cells and reported as percent relative colony survival values. All experiments were performed in triplicate.

[0068] (Gene expression). LNCaP, PC-3, VCaP, and VCaP-EnzR cells were seeded in 24-well plates and incubated in RPMI 1640 medium supplemented with 2 mmol / L L-glutamine, 100 U / mL penicillin, 100 mg / mL streptomycin, and 10% heat-inactivated fetal bovine serum at 37 °C in a humidified room temperature at 37 °C for 1 h at 4 °C. 2 The cells were maintained at 37° C. in an ambient atmosphere. After 24-48 hours, the cells were incubated with the following concentrations of SPH and bicalutamide (BIC) or enzalutamide (ENZ), which showed a significant decrease in the relative percent colony viability in both cell lines: LNCaP cells: 40 μg / ml and 160 μg / ml SPH with 1 μM BIC; PC3 cells: 40 μg / ml and 160 μg / ml SPH together with 10 μM BIC; VCaP cells: 160 μg / ml SPH together with 1.4 μM ENZ; and VCaP-EnzR cells: 160 μg / ml SPH together with 47 μM ENZ. Wells without SPH (DMSO 0.04%) were used as negative controls. All experiments were performed in triplicate.

[0069] Samples were studied for expression of the ferritin heavy chain 1 (FTH1) and transferrin receptor 1 (TFRC) genes. Total RNA was extracted from cell pellets using UPzol reagent (Biotechrabbit) followed by DNAse treatment (DNAse TURBO) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using random hexamers. Gene expression levels were measured by qRT-PCR.

[0070] One μl of cDNA (corresponding to 50 ng of reverse transcribed RNA) was amplified by Qpcr (QuantStudio™ 6 Flex Real-Time PCR System) using TaqMan™ Universal PCR Master Mix (cat. no. 4304437) and TaqMan™ assays (Roche Molecular Systems, Inc., Pleasanton, CA). Gene expression of FTH1 and TFRC was compared to that of the housekeeping gene β-actin (ACTB) and estimated according to the standard formula: 2-ΔCt. TaqMan probe IDs used include: (i) FTH1, Hs01694011_s1; (ii) TFRC, Hs00951083_m1; and (iii) ACTB, Hs01060665_g1.

[0071] Statistical analysis. Statistical significance was determined by paired two-tailed Student's t-test between treatment groups in each cell line.

[0072] (result) (Cell proliferation). Combining SPH with bicalutamide significantly reduced plating efficiency in AR+ve and AR-ve prostate cancer cell lines, as shown in Figure 1 (LNCaP AR+ve cells) and Figure 2 (PC3 AR-ve cells), respectively. In contrast, exposure to 10 μg / ml to 160 μg / ml SPH alone did not result in a significant dose-dependent reduction in plating efficiency in either LNCaP or PC3 cells. Similarly, combining SPH with enzalutamide reduced plating efficiency in VCaP cells, as shown in Figure 4, and in VCap-EnzR cells, as shown in Figure 5, despite higher enzalutamide concentrations in VCap-EnzR cultures. In contrast, exposure to 40 μg / ml to 160 μg / ml SPH alone did not result in a substantial reduction in plating efficiency in VCaP and VCaP-EnzR cells.

[0073] In AR+ve LNCaP cell lines, exposure to bicalutamide caused a significant decrease (P<0.001) in colony formation efficiency at 1 μM concentration but not at 0.5 μM concentration (Figure 1). Combining 40 μg / ml and 160 μg / ml SPH with the MIC dose of 0.5 μM and the effective dose of 1.0 μM bicalutamide resulted in a significant decrease in colony formation. A significant attenuation of the activity of the MIC bicalutamide was observed with both 40 μg / ml and 160 μg / ml SPH cotreatment. 40 μg / ml SPH combined with 0.5 μM bicalutamide reduced colony survival from 85% to 68%, and increasing the SPH dose from 40 μg / ml to 160 μg / ml further reduced colony survival to 19% (P<0.001). 40 μg / ml SPH in combination with 1 μM bicalutamide reduced colony survival from 25% to 4%, and increasing the SPH dose from 40 μg / ml to 160 μg / ml further reduced colony survival to 2% (P<0.001).

[0074] In AR-ve PC3 cell lines, treatment with 10 μM bicalutamide caused a significant decrease in plating efficiency to 52% (P<0.001), while treatment with 1 μM bicalutamide did not result in a significant decrease in the percent colony survival (Figure 2). Combining 40 μg / ml and 160 μg / ml SPH with the non-effective dose of 1 μM bicalutamide did not result in a decrease in the percent plating efficiency in PC3 AR-ve cells. However, combining 40 μg / ml and 160 μg / ml SPH with 10 μM bicalutamide resulted in a significant decrease in the percent plating efficiency in PC3 cells. 40 μg / ml SPH in combination with 10 μM bicalutamide reduced colony survival from 52% to 41%, and increasing the SPH dose from 40 to 160 μg / ml further reduced colony survival to 32% ( P < 0.01).

[0075] Gene Expression. Two iron homeostasis genes, FTH1 and TFRC, were examined for changes in expression in LNCaP AR+ve and PC3 AR-ve cells upon treatment with bicalutamide alone or in combination with SPH. In both cell lines, co-treatment with SPH and bicalutamide resulted in changes in gene expression, with FTH1 mRNA expression being upregulated by more than two-fold and a concomitant downregulation of TFRC mRNA expression by more than two-fold.

[0076] FTH1 and TFRC gene expression levels were also measured in VCaP and VcaP-ENZR cells when treated with enzalutamide alone or in combination with SPH. In both cell lines, co-treatment with SPH and enzalutamide resulted in increased FTH1 mRNA expression and decreased TFRC expression.

[0077] Numerical differences between cell lines (PC3 cells showed greater gene expression changes) and [SPH] dose-response were noted, but no statistically significant conclusions could be drawn. (Table 2-1. Effects of SPH and bicalutamide on gene expression) [Table 2-1] (Table 2-2. Effects of SPH and Enzalutamide on Gene Expression) [Table 2-2]

[0078] Example 3 (Effects of oligopeptides in combination with antiandrogens on human prostate cancer cell lines) This example describes the effects of oligopeptides and antiandrogens (bicalutamide or enzalutamide) on the in vitro proliferation and gene expression of human prostate cancer cell lines.

[0079] The four human prostate cancer cell lines used in this example and the experimental design are as described in Example 2. The tested oligopeptides were produced by solid phase synthesis and obtained from Biomatik (Canada). The amino acid sequences of the oligopeptides are shown in Table 3-1 below, and the alignment and consensus of the sequences are shown in Figure 3. (Table 3-1. Oligopeptide numbers, sequences and identifiers) [Table 3-1]

[0080] All oligopeptides were tested in combination with BIC or ENZ in clonogenic assays. The effect on gene expression of the more active oligopeptides from those clonogenic assays was also tested. Treatment conditions for each cell line included the following: LNCaP cells: (i) 0.4 μM BIC with 10 μM oligopeptide, (ii) 0.4 μM BIC alone, or (iii) 0.4 μM BIC with 160 μg / ml SPH; PC3 cells: (i) 10 μM BIC together with 10 μM oligopeptide, (ii) 10 μM BIC alone, or (iii) 10 μM BIC together with 160 μg / ml SPH; VCaP cells: (i) 1.4 μM ENZ with 10 μM oligopeptide, (ii) 1.4 μM ENZ alone, or (iii) 1.4 μM ENZ with 160 μg / ml SPH; and VCaP-EnzR cells: (i) 47 μM ENZ with 10 μM oligopeptide, (ii) 47 μM ENZ alone, or (iii) 47 μM ENZ with 160 μg / ml SPH. Wells without SPH (DMSO 0.04%) were used as negative controls. All experiments were performed in triplicate.

[0081] (result) (Cell survival). When 10 μM of FT-002 or FT-005 oligopeptides were co-administered with 0.4 μM bicalutamide (IC50), LNCaP cells were found to exhibit a significant reduction in survival (p<0.001) of more than 70% compared to 0.4 μM bicalutamide (IC50) treatment alone, as shown in FIG. 6. Similarly, PC3 cells exhibited a reduction in survival of approximately 70% compared to 10 μM bicalutamide (IC50) treatment alone, and a reduction in survival of more than 50% (p<0.001) compared to the combination of bicalutamide and 160 μM SPH, as shown in FIG. 7. FT-002 or FT-005 in combination with bicalutamide also reduced colony survival by more than 50% compared to the combination of bicalutamide and 160 μM SPH.

[0082] VCaP cells treated with 10 μM FT-002 or FT-005 oligopeptides in combination with 1.4 μM enzalutamide (IC50) showed a significant greater than 50% decrease in cell viability compared to 1.4 μM enzalutamide alone (p<0.001) and approximately 40% decrease compared to the combination of 1.4 μM enzalutamide and 160 μM SPH treatment (p<0.01), as shown in Figure 8. The relative viability of VCaP-EnzR cells was significantly decreased from 52% (47 μM enzalutamide alone) to 11% when treated with 10 μM FT-002 or FT-005 oligopeptides in combination with 47 μM enzalutamide, as shown in Figure 9. FT-002 or FT-005 oligopeptides in combination with enzalutamide also significantly reduced colony survival (>50%, p<0.001) compared to treatment with enzalutamide in combination with 160 μM SPH treatment.

[0083] (Gene Expression). Two iron homeostasis genes, FTH1 and TFRC, were examined for changes in expression in LNCaP, PC3, VCaP, and VCaP-EnzR cells treated with bicalutamide or enzalutamide in the presence of oligopeptides. Differential gene expression analysis revealed a greater than two-fold upregulation of FTH1 gene expression and a consistent downregulation of the transferrin receptor gene in all cell lines. (Table 3-2. Effects of oligopeptides and bicalutamide on gene expression) [Table 3-2] (Table 3-3. Effects of oligopeptides and enzalutamide on gene expression) [Table 3-3-1] [Table 3-3-2]

[0084] Example 4 (Evaluation of oligopeptides in combination with antiandrogens against human prostate cancer xenografts) This example describes the effect of salmon protein hydrolysates (SPH) or oligopeptides in combination with antiandrogen drugs (bicalutamide or enzalutamide) on tumor growth in vivo.

[0085] LNCaP and PC3 xenograft mouse models are used to study the efficacy of oligopeptides and antiandrogens to treat prostate cancer. Mice used in this study are male NSG™ (NSG™; NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ, Jackson Laboratory, Cat. No. 005557) mice (6-8 weeks old), or other immunodeficient mouse strains. Briefly, 1.0 × 10 6 pieces, 2.0×10 6 pcs. or 5.0×10 6 Mice are administered LNCaP or PC3 cells by subcutaneous injection into one flank on day 0. Mice are weighed daily and monitored for general health. Tumor growth over time is monitored using digital calipers and animals are scored several times a week for physical condition and activity level. The average tumor volume is approximately 150 mm 3 ~250mm 3 Upon reaching 2500 mm, mice are randomly divided into study groups (6-10 mice per group). The study groups may include some or all of the following: (i) SPH alone, (ii) bicalutamide alone, (iii) FT-002 alone, (iv) FT-005 alone, (v) bicalutamide and FT-002, and (vi) bicalutamide and FT-005. Bicalutamide is administered orally at 15 mg / kg once daily. Oligopeptides are administered first by intraperitoneal injection, followed by daily doses of 1 mg / kg, 10 mg / kg, or 100 mg / kg intraperitoneally or orally. Treatment continues for 3-6 weeks. However, if the mice reach 2500 mm 3 Mice are humanely euthanized if they exhibit tumors larger than 100 μg / kg, ulcerated tumors, or a wellness score less than 3. All remaining animals are euthanized on day 24 (D24) or at the end of the study. Blood may be collected prior to tumor implantation and then tested weekly for serum PSA levels until euthanasia.

[0086] VCaP and VCaP-EnzR xenograft rat models are used to study the efficacy of oligopeptides and antiandrogens to treat prostate cancer. The VCaP and VCaP-EnzR xenograft models use OncoRat animals (Hera BioLabs) because growth is difficult and variable in immunocompromised mouse models. On day 0, rats are inoculated with VCaP or VCaP-EnzR cells by subcutaneous injection into one flank. Rats are weighed daily and monitored for overall health. Tumor growth over time is monitored using digital calipers, and animals are scored several times a week for physical condition and activity level. Treatment begins approximately 3 weeks after inoculation. The study arms may include some or all of the following: (i) SPH alone, (ii) enzalutamide alone, (iii) FT-002 alone, (iv) FT-005 alone, (v) enzalutamide and FT-002, and (vi) enzalutamide and FT-005. Enzalutamide is administered orally once daily. The oligopeptide is administered first by intraperitoneal injection, followed by daily doses of 1 mg / kg, 10 mg / kg, or 100 mg / kg intraperitoneally or orally. Treatment continues for 3-6 weeks. Blood may be collected prior to tumor implantation and then tested weekly for serum PSA levels until euthanasia.

[0087] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications can be implemented in light of the above teachings. Therefore, the above examples should not be construed as limiting the scope of the present disclosure, the scope of which is clearly indicated by the appended claims.

Claims

1. A combination for use in a method for inhibiting the growth of cancer cells, comprising: The combination comprises a non-steroidal antiandrogen and a formulation comprising an oligopeptide consisting of the amino acid sequence of Xp(R / D)EESGEPXq (SEQ ID NO: 10), where p is an integer selected from the range of 0 to 10, q is an integer selected from the range of 0 to 9, and each X, if present, is independently selected from any amino acid; and the method comprises contacting the cancer cells with the non-steroidal antiandrogen and the formulation.

2. The oligopeptide (i) the amino acid sequence of REESGEP (SEQ ID NO: 2); or (ii) the amino acid sequence of Xr(R / D)EESGEP (SEQ ID NO: 11), wherein Xr is leucine or absent; or (iii) the amino acid sequence of LDEESGEP (SEQ ID NO: 5); or (iv) the amino acid sequence of REESGEP (SEQ ID NO: 2), the amino acid sequence of LDEESGEP (SEQ ID NO: 5), the amino acid sequence of REESGE (SEQ ID NO: 1), the amino acid sequence of KEEDEESGE (SEQ ID NO: 3), the amino acid sequence of KPREESGE (SEQ ID NO: 4), the amino acid sequence of REESDKPMY (SEQ ID NO: 6), the amino acid sequence of PREESDKP (SEQ ID NO: 7), or the amino acid sequence of REESGEL (SEQ ID NO: 8).

2. The combination of claim 1, comprising:

3. A combination for treating prostate cancer in a mammalian subject in need thereof, comprising: A combination comprising: i) a nonsteroidal antiandrogen; and ii) a formulation comprising an oligopeptide consisting of the amino acid sequence Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), where m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid.

4. (i) The antiandrogen and the formulation are administered orally; or (ii) the formulation is for enteral administration; or (iii) The formulation is administered by the buccal, sublabial, or sublingual route. The combination according to claim 3.

5. A combination described in claim 3 or 4, wherein administration of the antiandrogen drug and the formulation results in a reduction in the volume of the prostate cancer compared to the volume before the treatment, and optionally, the mammalian subject is a human subject.

6. The oligopeptide (i) the amino acid sequence of REESGEP (SEQ ID NO: 2); or (ii) the amino acid sequence of LDEESGEP (SEQ ID NO: 5); or (iii) the amino acid sequence of REESGE (SEQ ID NO: 1), the amino acid sequence of REESGEP (SEQ ID NO: 2), the amino acid sequence of KEEDEESGE (SEQ ID NO: 3), the amino acid sequence of KPREESGE (SEQ ID NO: 4), the amino acid sequence of LDEESGEP (SEQ ID NO: 5), the amino acid sequence of REESDKPMY (SEQ ID NO: 6), the amino acid sequence of PREESDKP (SEQ ID NO: 7), or the amino acid sequence of REESGEL (SEQ ID NO: 8) 5. The combination according to claim 3 or 4, comprising:

7. The combination of claim 3 or 4, wherein the formulation comprises a fish protein hydrolysate.

8. The nonsteroidal antiandrogen drug (i) enzalutamide; or (ii) bicalutamide; or (iii) selected from bicalutamide, apalutamide, enzalutamide, flutamide, nilutamide, topirutamide, darolutamide, proxalutamide, and combinations thereof; A combination according to claim 3 or 4.

9. A combination according to claim 3 or 4, characterized in that a gonadotropin-releasing hormone antagonist is administered in combination with the combination.

10. The prostate cancer is androgen receptor positive, or the prostate cancer is androgen receptor negative; A combination according to claim 3 or 4.

11. The combination described in claim 3 or 4, wherein the prostate cancer is metastatic cancer.

12. A combination for use in a method for inhibiting the growth of cancer cells, comprising: The combination comprises a nonsteroidal antiandrogen and a formulation comprising an oligopeptide consisting of the amino acid sequence Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), where m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid; and the method comprises contacting the cancer cells with the nonsteroidal antiandrogen and the formulation.

13. The combination of claim 12, wherein the formulation comprises a fish protein hydrolysate.

14. A combination described in any one of claims 1 to 2 and 12 to 13, wherein the oligopeptide is capable of increasing the expression of ferritin heavy chain 1 (FTH1) mRNA by cancer cells contacted with the oligopeptide in the presence of an antiandrogen drug.

15. The nonsteroidal antiandrogen drug (i) enzalutamide; or (ii) bicalutamide; or (iii) selected from bicalutamide, apalutamide, enzalutamide, flutamide, nilutamide, topirutamide, darolutamide, proxalutamide, and combinations thereof; A combination according to any one of claims 1 to 2 and 12 to 13.

16. A combination described in any one of claims 1 to 2 and 12 to 13, wherein the cancer cells are adenocarcinoma cells.

17. The cancer cells are prostate cancer cells, Optionally, the prostate cancer cells are androgen receptor positive; Optionally, the prostate cancer cells are androgen receptor negative; Optionally, the cancer cells are human cells. A combination according to any one of claims 1 to 2 and 12 to 13.

18. A combination according to any one of claims 1 to 2 and 12 to 13, wherein the formulation containing the oligopeptide further comprises at least one pharmaceutically acceptable excipient.

19. The formulation comprising the oligopeptide further comprising an oral delivery agent; Optionally, the oral delivery agent comprises an absorption enhancer, a fatty acid, an enzyme inhibitor, polyethylene glycol, a mucoadhesive polymer, a cell-penetrating peptide, or a combination thereof; Optionally, further comprising an enteric coating, liposomes, microspheres, and / or microparticles / nanoparticles; A combination according to any one of claims 1 to 2 and 12 to 13.

20. A composition for use in a method for inhibiting the proliferation of cancer cells, comprising: a nonsteroidal antiandrogen, the method comprising contacting the cancer cells with the nonsteroidal antiandrogen in combination with a formulation, the formulation comprising an oligopeptide consisting of an amino acid sequence of Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), where m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid.

21. A composition for use in a method for inhibiting the proliferation of cancer cells, comprising:

1. A composition comprising a formulation, the method comprising contacting the cancer cells with the formulation in combination with a nonsteroidal antiandrogen, the formulation comprising an oligopeptide consisting of an amino acid sequence of Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), where m and n are integers independently selected from the range of 0 to 10, and each X, when present, is independently selected from any amino acid.

22. A composition for use in a method for inhibiting the proliferation of cancer cells, comprising: a nonsteroidal antiandrogen, the method comprising contacting the cancer cells with the nonsteroidal antiandrogen in combination with a formulation, wherein the formulation comprises an oligopeptide consisting of an amino acid sequence of Xp(R / D)EESGEPXq (SEQ ID NO: 10), where p is an integer selected from the range of 0 to 10, q is an integer selected from the range of 0 to 9, and each X, if present, is independently selected from any amino acid.

23. A composition for use in a method for inhibiting the proliferation of cancer cells, comprising:

1. A composition comprising a formulation, the method comprising contacting the cancer cells with the formulation in combination with a nonsteroidal antiandrogen, the formulation comprising an oligopeptide consisting of an amino acid sequence of Xp(R / D)EESGEPXq (SEQ ID NO: 10), where p is an integer selected from the range of 0 to 10, q is an integer selected from the range of 0 to 9, and each X, if present, is independently selected from any amino acid.

24. A composition for treating prostate cancer in a mammalian subject in need thereof, comprising:

1. A composition comprising a nonsteroidal antiandrogen, wherein the composition is administered in combination with a formulation comprising an oligopeptide consisting of the amino acid sequence Xm(R / D)EES(G / D)(E / K)Xn (consensus number 1), wherein m and n are integers independently selected from the range of 0 to 10, and each X, if present, is independently selected from any amino acid.

25. A composition for treating prostate cancer in a mammalian subject in need thereof, comprising:

1. A composition comprising a pharmaceutical composition for use in ...

26. The oligopeptide, (i) the amino acid sequence of REESGEP (SEQ ID NO: 2); or (ii) the amino acid sequence of LDEESGEP (SEQ ID NO: 5); or (iii) the amino acid sequence of REESGE (SEQ ID NO: 1), the amino acid sequence of REESGEP (SEQ ID NO: 2), the amino acid sequence of KEEDEESGE (SEQ ID NO: 3), the amino acid sequence of KPREESGE (SEQ ID NO: 4), the amino acid sequence of LDEESGEP (SEQ ID NO: 5), the amino acid sequence of REESDKPMY (SEQ ID NO: 6), the amino acid sequence of PREESDKP (SEQ ID NO: 7), or the amino acid sequence of REESGEL (SEQ ID NO: 8) The composition of any one of claims 20 to 25, comprising:

27. The nonsteroidal antiandrogen drug, (i) enzalutamide; or (ii) bicalutamide; or (iii) selected from bicalutamide, apalutamide, enzalutamide, flutamide, nilutamide, topirutamide, darolutamide, proxalutamide, and combinations thereof; 27. The composition of claim 26.

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