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Canada Peptide’s Blog – News, Research & Events

  • Peptide Therapeutics: Emerging Peptide Areas and Technologies

    It is estimated that the total number of naturally occurring peptides that have been successfully identified is about 7000. These peptides play a crucial role in human physiology including acting as growth factors, neurotransmitters, anti-infective, ion channel ligands, and hormones. Generally, peptides are very selective and highly effective signaling molecules that have the ability to bind to specific cell surface receptors where they may trigger a variety of intracellular effects.

    Due to their attractive pharmacological profiles as well as some of their very intrinsic properties, peptides are seen as a perfect starting point for designing a variety of novel therapeutics. Since they have proven to possess exceptional specificity, they also come with excellent safety, tolerability, and very high efficacy in humans. These features and attributes are the main factors that differentiate peptides from the bulk of most of the traditional small molecules.

    Also, peptide therapeutics are mostly associated with low production complexities, unlike other protein-based biopharmaceuticals that naturally have high production complexities. As such, the production costs of peptides are relatively low, just like that of most small peptides, making them very attractive in the biopharmaceutical arena.

    Peptides that occur naturally are not normally preferred for use in various therapeutics, owing to some of their certain intrinsic weaknesses such as poor physical and chemical stability, and short plasma half-life. Before they can be considered suitable candidates for the design and development of a variety of medicines, some of these weaknesses have to be addressed and proper mitigation measures put in place to circumvent them.

    Though some of the intrinsic weaknesses have been successfully resolved, others are still a work in progress. Apart from the traditional peptide designs, a wide range of peptide technologies has been on the rise in recent years, and these technologies have provided a lifeline for the future development of peptide-based therapies. Some of these promising emerging technologies include the discovery and applications of cell-penetrating peptides, multifunctional peptides, and the development of peptide drug conjugates and technologies that focus on using alternative routes of administration.

    In this piece, we focus on some of these emerging peptide areas and technologies and their relevance as far as the formulation of peptide therapies is concerned.

    A Brief on Traditional Peptide Technologies

    Over the years, peptide technologies have evolved, leading to the development of highly potent signal transduction molecules, with very powerful physiological effects. They are primarily characterized by their short circulating plasma half-life, and their suboptimal chemical and physical properties when being considered for application in the development of various types of medicines. As such, most of the traditional rational design of peptides has always evolved around techniques designed to mitigate some of the known weaknesses of peptides.

    Rational Design of Peptide Therapeutics

    In ration design of peptide therapeutics, the starting point for the therapeutic can be a known crystal structure that is then further developed into secondary and tertiary structures. Once this is accomplished, what then follows is that inputs from various analyses such as those obtained from alanine substitution and small focused libraries are designed in sequential steps which ultimately leads to the identification of essential amino acids as well as potential sites for possible substitution. During this process, especially during the process of liquid drug formation of the desired final product, it is vital to identify the amino acids that are chemically labile and prone to events such as oxidation, glycosylation, and isomerization – processes which should always be avoided.

    Another vital aspect of the rational design of peptide drugs is the need to improve the physicochemical properties of the natural peptides. This is usually considered as a way of aggregating the peptides which are sometimes water-soluble. Some of the chemical design strategies that can be used to avoid aggregation may include corrupting hydrophobic patches. It is possible to achieve this through substitution or N-methylation of certain specific amino acids. If some of the peptide candidates are faced with solubility issues, then the point of focus would be on the charge distribution and the isoelectric point of the peptide, with regards to the pH of the desired formulation of the final product.

    It is also possible to improve certain physiochemical properties of peptides by introducing stabilizing a-helixes or a salt bridge formation or other kinds of chemical modifications. Any modification introduced during the rational design of a peptide with the aim of improving the physicochemical properties of that peptide must be in tandem with the overall design of the desired pharmacological properties of the peptide therapeutic in question. With the current application of predictive IT software tools in the facilitation of such rational peptides, design has shown immense potential in leveling the playing field, and it is believed that it will lead to an increase of design prospects in the future, hence creating more room for the design of additional therapeutics.

    Owing to the challenges represented by the intrinsic physicochemical properties of peptides, it has been possible to somehow get suboptimal solutions that have been incredibly useful to both physicians and patients. One good example worth considering is the use of glucagon used in treating severe hypoglycemia for patients suffering from diabetes. Presently, the glucagon rescue kit in the market for treating unconscious hypoglycemic patients features a lyophilized peptide powder in a sterile vial that can be quickly constituted and then administered to unconscious patients.

    There is a general feeling that second-generation peptide drugs made through rational design may lead to more user-friendly peptide therapeutics. One prime example of this approach is the invention and the subsequent design of a glucagon analog that has displayed immense stability in liquid dosage form and is considered as an application that is ready for use in the form of a rescue pen.

    Also, products of this nature may be useful when used as part of a closed-loop dual-chamber artificial pancreatic device for the delivery of both glucagon and insulin in therapeutics designed to be released centrally using an automatic pump in tandem with continuous blood glucose sensors. With the fast development, as well as the rapid miniaturization of devices and pumps, it may be possible for a pulsatile delivery system to be developed in the future, and this could be used for the smart delivery of peptides.

    How to extend plasma half-life for peptides

    Natural peptides generally come with a relatively short circulating plasma half-life. As such, there are various techniques that are currently under exploration in an attempt to increase the plasma half-life of the peptides. One of the approaches under serious consideration is to try and limit the enzymatic degradation of the peptide. This is achieved by identifying certain molecular cleavage sites where the relevant amino acid is substituted. It is possible to achieve the protection of certain enzymatic cleavages by enhancing the secondary structure of the peptide. With this approach, there will be a need for the insertion of a structure-inducing probe, a lactam bridge as well as clipping or stapling of the peptide sequence or through cyclization.

    Most of the strategies which are currently under exploration use binding to the circulating protein albumin as the main driving factor for extending the plasma half-life which makes it possible for the peptide therapeutics to be administered less frequently. Some of these strategies include the insertion of albumin-binding peptide elements in the peptide backbone, peptide acylation, and peptide conjugation to albumin-binding antibody fragments.

    Polyethylene glycol (PEG)-ylation may sometimes be used as a means of slowing down or limiting globular filtration, which in turn helps to increase the plasma half-life by simply limiting the elimination of peptides. Due to increasing concerns over safety and tolerability surrounding the use of PEG as one of the constituents for injectable therapies, however, PEGylation is no longer one of the strongly preferred methods.

    Emerging Peptide Areas and Technologies

    Currently, there is an immensely large pool of natural peptides, some of which have shown great potential as the perfect starting point for the development of various peptide therapeutics. The metabolic arena, such as the gut, for example, has attracted a lot of interest due to its diverse bacteria that could be helpful in identifying new peptides from sources such as degradation products, protein sources, and signaling molecules. With the ongoing microbiome research, there is hope that there will be new and significant opportunities for peptide therapeutics as solutions for a variety of metabolic diseases.

    However, when it comes to innovative peptides for drug development, it is important for chemists and researchers to widen their scope and not focus on the traditional peptide technologies alone. One of the emerging technologies and approaches involves the use of multifunctional peptides which represent more than one pharmacological activity such as triple or dual agonism. This approach comes with a lot of sense based on the information from genomics. As such, it can be seen that knock-out animals often present with no distinct phenotype.

    Additionally, although there are tremendous industrial efforts in the GPCR field that have made it possible to successfully identify several selective agonists and antagonists, only a few ligands have made it all the way to approval for use as medicines. All these tend to indicate some kind of redundancy in the biological systems and they also tend to favor multi-target approaches when it comes to the development of peptide medicines. Another method that involves the use of polypharmacology is the ability to use more personalized and individual treatments of differentiated patient groups.

    Some of the multifunctional peptides that are currently in development include various antimicrobial peptide drug candidates that feature additional biological functions such as wound healing and the stimulation of the immune system. Additionally, there is a general trend gravitating towards multifunctional peptides, and this is evident in the fields such as GLP-1 agonists that have been used in the establishment of a drug class which has seen the introduction of various drugs in the market today, including Bydureon, Lyxumia, and Victoza. Since the introduction of these drugs into the market, they have grown in popularity, making them be commercially successful for developers as well as manufacturers.

    One of the chemical strategies used in the development of multifunctional peptides may feature a hybrid of two peptides which are then bound together like modules. The binding can be directly or indirectly where a linker or a chimera may be used. Recently, GLP-1-GIP-GCG triple incretin agonist was exhaustively described as an anti-obesity agent following the strong beneficial effects it showed on model rats.

    However, the development of multifunctional peptides could still present a challenge since the prediction of the in vivo outcomes of the drug candidates tends to be more complex when dealing with dual-target pharmacological compared to dealing with just a single target pharmacological. One of the challenges likely to be encountered with this is the translation of in vitro to in vivo where the potentially biased signaling may be encountered from novel ligands targeting two or more receptors.

    Additionally, the process of translating results from animal models to human beings comes with many risks for multifunctional peptides when the same is compared to a single receptor peptide. The main reason for this challenge is the uncertainty associated with two or more targets. Within the antibody field, such challenges have also been encountered in the development of certain specific antibodies targeted for treating cancer. Due to these and other reasons, it is realistic to expect that established paradigms may be the ultimate source for the origins of multifunctional peptides.

    References

    1. C. Delgado, et al.
      The uses and properties of PEG-linked proteins
      Crit. Rev. Ther. Drug Carr. Syst., 9 (1992), pp. 249-304

    2. R.R. Henry, et al.
      Continuous subcutaneous delivery of exenatide via ITCA 650 leads to sustained glycemic control and weight loss for 48 weeks in metformin-treated subjects with type 2 diabetes
      J. Diabetes Complicat., 28 (2014), pp. 393-398

    3. M. Hattori, T.D. Taylor
      The human intestinal microbiome: a new frontier of human biology
      DNA Res., 16 (2009), pp. 1-12

    4. Pocai, et al.
      Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice
      Diabetes, 58 (2009), pp. 2258-2266

    5. K. Fosgerau, et al.
      The new glucagon-GLP-1 dual agonist ZP2929 in combination with long-acting insulin improves glycemic control without causing weight loss in db/db mice
      Diabetes, 60 (2011), pp. A408-A442

    6. B. Finan, et al.
      Unimolecular dual incretins maximize metabolic benefits in rodents, monkeys, and humans
      Sci. Transl. Med., 5 (2013), p. 209ra151

    7. K. Fosgerau, et al.
      The novel GLP-1-gastrin dual agonist, ZP3022, increases beta-cell mass and prevents diabetes in db/db mice
      Diabetes Obes. Metab., 15 (2013), pp. 62-71

    8. R. Just, et al.
      The novel GLP-1-GLP-2 dual agonist ZP-GG-72 increases intestinal growth and improves insulin sensitivity in DIO mice
      Diabetes, 63 (2014), pp. A1-A102

    9. Pocai
      Unraveling oxyntomodulin, GLP1's enigmatic brother
      J. Endocrinol., 215 (2012), pp. 335-346

    10. V.A. Gault, et al.
      A novel glucagon-like peptide-1 (GLP-1)/glucagon hybrid peptide with triple-acting agonist activity at glucose-dependent insulinotropic polypeptide, GLP-1, and glucagon receptors and therapeutic potential in high fat-fed mice
  • Peptides in Cancer Research: Therapies and Vaccines

    Cancer refers to the uncontrolled division of cells and the ability of those cells to attack cells in neighboring organs to form tumor masses before spreading to other parts of the body. Although angiogenesis, or the development of new blood vessels from existing blood vessels, is a vital process for growth and development, it is also one of the steps used by tumors in transitioning from a tumor-dormant state to a malignant state. Therefore, one of the processes that have been explored in suppressing the growth of tumor cells is the smart use of angiogenesis inhibitors.

    Chemotherapy is one of the popular approaches currently used in the treatment of cancer. With this approach, a cytotoxic agent is usually delivered to the cancer cells, with the hope that the agent will kill the cancer cells and stop their further growth, development, and spread within the body.

    However, one of the challenges that comes with the classic use of chemotherapy is the lack of the ability to administer the right amount of drugs directly to the cancer cells without any adverse effects on the normal, healthy cells. The other challenges faced with conventional chemotherapies include premature clearance, biotransformation, altered blood distribution, and drug resistance.

    To overcome such challenges associated with chemotherapies, targeted chemotherapies, as well as targeted drug delivery systems, are currently being explored. If these approaches used to circumvent the adverse side-effects of chemotherapy are successful, then it will be possible to select and effectively localize the drugs at pre-defined sites, while at the same time reducing the chances of the drug affecting other healthy normal cells.

    The discovery of additional receptors that are abnormally overexpressed in cancer cells as well as tumor-related peptides have led to the design and development of a new era of more effective and selective anti-cancer drugs.

    Some of the biologics approaches to cancer therapies feature the use of proteins, peptides, and monoclonal antibodies. The use of monoclonal antibodies (mAb), as well as large protein ligands, come with two major challenges when compared to the use of peptides.

    Firstly, monoclonal antibodies and large protein ligands are poorly delivered to the tumors because of their large sizes.

    Secondly, they come with dose-limiting toxicity to the liver as well as the bone marrow because of their non-specific uptake into the reticuloendothelial system. Due to these limitations, their applications have thus been restricted to vascular targets found in the luminal side of the tumor vessel, and to hematological malignancies.

    Peptides, on the other hand, boast many advantages, including ease of modification, ease of synthesis, and their small sizes. Also, peptides are biocompatible and have the ability to successfully penetrate tumor tissues.

    It is also possible to prevent the proteolytic degradation of peptides through various chemical interventions such as the inclusion of D-amino acid or processes such as cyclization. It is also worth pointing out that bicyclic peptides come with even better properties when compared to those present in antibody drugs. Currently, there are two main classifications of the peptide drugs in the market: analogs and antagonists of peptides hormones or tumor-targeting agents carrying radionuclides.

    Agonists and Antagonists

    Luteinizing-hormone-releasing hormone analog, or LHRH, is one example of the peptide drugs that have been introduced into cancer therapies. The first GnRH agonist was developed by Schally et al., which was later to be used in the treatment of prostate and breast cancers.

    Following these developments, a variety of peptides have been developed and approved for use as therapies against various forms of cancer.
    Some of the notable examples of these peptides include goserelin, leuprolide, buserelin, and triptorelin, among others. The depot formulation of these peptides makes it possible for them to be more efficacious and to conveniently treat patients suffering from prostate cancer.

    Through several studies, it has been observed that the use of these peptides has a way of affecting the down-regulation of GnRH receptors found in the pituitary. This down-regulation leads to the inhibition of the release of follicle-stimulating hormones – FSH and LH. This is in addition to the concomitant release of the production of testosterone.

    With the introduction of LHRH antagonists, it was observed that there was a therapeutic improvement over agonists. This is because they have the ability to induce immediate and dose-related inhibition of FSH and LH through the competitive blockage of the LHRH receptors. Presently, most of the highly potent GnRH antagonists are available for therapeutic use for patients suffering from prostate cancer.

    Somatostatin Analogs in Cancer Therapies

    In addition to using peptidic LHRH agonists and antagonists in the treatment and the development of cancer therapies, somatostatin analogs are currently the only cancer-therapeutic peptides that have been approved on the market.

    Very powerful and potent agonists of somatostatin such as Sandostatin were developed to be used specifically for the treatment of acromegaly, thyrotropinoma, and gigantism, which are usually associated with diarrhea, and with carcinoid in patients suffering from vasoactive intestinal tumors. Lanreotide is another long-acting analog of somatostatin that is currently being used in managing acromegaly as well as other symptoms associated with neuroendocrine tumors.

    The bulk of neuroendocrine tumors, or NETs, are known to display very strong overexpression of somatostatin receptors. At the moment, five somatostatin receptor subtypes (SST) have been clearly identified. It has been observed that the density of these receptors is usually abnormally high on tumor tissues compared to those on healthy tissues. As such, SST is currently being considered as an attractive target for delivering radionuclides that use well-modified somatostatin analogs.

    For instance, Sandoz was introduced in the late 1980s, and it has since become one of the best benchmarks for diagnosing SST-positive NETs. The past decades have also seen the development of numerous peptide-based tumor-imaging agents developed with the sole purpose of targeting SST.

    Currently, there are only two radiopeptide tracers in the market that have been approved by the FDA; they include NeoTect and Octreoscan. These radiopeptides tracers employ the use of scintigraphic methods in the detection of carcinoids as well as other types of tumors and for localizing sarcoidosis. DTPA-Octreotide is usually injected into a vein after radio-labeling with Indium-111 to get transported into the bloodstream. This is so that it can easily attach to the tumor cell that has receptors for somatostatin.

    Following the injection of the DTPA-Octreotide in the vein, a device that is used to detect the radioactive octreotide is then used to generate images indicating the actual location of the tumor in the body. It is possible to also apply this principle in cancer therapies, specifically peptide receptor radionuclide therapy – PRRT.

    This therapy works by combining modified octreotide with a radionuclide that also has the ability to bind to carcinoid tumor cells where there is an overexpression of somatostatin receptors. Following the binding, it is possible for the targeted radiation to destroy the malignant cells that the peptides are bound to. For this to be successful, however, it is important that the complex being used between the peptide and the radionuclide be very stable, especially if it will be subjected to radiopeptide therapy.

    Peptide Vaccines

    One of the most promising approaches for the treatment of cancer seems to be the use of active immunization featuring the employment of immune cells or immune molecules. With these methods of killing cancer cells, there is great reliance on vaccines that have peptides derived from amino acid sequences associated with certain types of tumors or antigens.

    Tumor-associated antigens (TAA) are the agents normally expressed by tumor cells, and it is possible to recognize their presence with the use of T cells found within the host’s immune system. Currently, it is possible to identify a considerable number of TAAs and to characterize them as well.

    It is possible to inject TAAs into cancer patients as a method of inducing a systematic immune response with the hopes that the response may lead to the destruction of the cancer cells. Any protein or peptide that has been produced in the tumor cell and has a mal structure owing to some certain form of mutation has the ability to act as a tumor antigen.

    The production of such abnormal proteins or peptides is usually a result of mutations that are occurring and correspond to certain genes. Consequently, there are several clinical studies that have been started with the main goal of assessing the therapeutic potential of active immunization or the potential of active vaccination using TAA peptides in patients that have already developed metastatic cancer.

    Up to now, just a handful of TAA peptides, the bulk of them being ones that can be recognized by CD8 (+), have undergone clinical testing. There are a lot of melanoma TAAs that have also been identified and are currently being evaluated for their potential as peptide-based cancer vaccines in a myriad of clinical trials around the globe.

    With the recent developments in the field of molecular biology, it has been possible to rapidly identify dozens of candidates of TAAs that may be used to target a wide variety of cancers troubling the human population.

    Current and Future State of Peptide-Based Anti-Cancer Therapies

    There has been tremendous growth in the use of peptides in target drug delivery, diagnostic tools, and as direct therapeutic agents in cancer biology. Among the developments in controlled and targeted delivery of peptide therapeutics, there has been a significant rise in the use of binding peptides as a non-immunogenic approach used for targeting cancer cells.

    For instance, the RGD peptide comes with the ability to efficiently recognize and penetrate cancerous tumors without any effect on the adjacent cells. Following the development of these and other similar peptides that boast extraordinary tumor penetrating capabilities, a significant improvement has been witnessed in the design and development of cancer therapies for the future.

    For example, chlorotoxin has received a lot of attention recently since it has a very high affinity for glioma cells compared to normal brain cells and non-neoplastic cells. This ability to preferentially bind has made possible the development of new treatments as well as a new diagnosis for brain cancer. It is also worth observing that antiangiogenesis has sparked new interests in cilengitide. This is an integrin inhibitor that is currently being studied and evaluated for its potential as a non-small cell lung cancer therapeutic agent in numerous clinical trials around the globe.

    Gastrin/bombesin releasing peptide (BN/GRP) peptides have also demonstrated the ability to bind selectively on the G-protein-coupled receptors on the surface of the cells, leading to the stimulation of the growth of various malignancies in both murine and human cancer models. Consequently, there have been proposals that the secretion of BN/GRP by neuroendocrine cells may be the main reason for the development and the progression of prostate cancer.

    It has been observed that GRP is present in great numbers in the lungs and the gastrointestinal tracts. It is normally produced in the small cell lung cancer – SCLC, prostatic, breast, and pancreatic cancers. It primarily functions as a growth factor. Since bombestin-like peptides are very active in the pathogenesis of a wide variety of human tumors, and also due to their paracrine tumoral growth factors and the high presence of BN/GRP receptors in a myriad of human cancers, researchers have opted to create a design and synthesis of BN/GRP receptor antagonists such as RC-3950 and RC-3095.

    Presently, there are numerous researchers and studies that are solely focused on the development of GHRH (growth-hormone-releasing hormone) antagonists as one of the highly potent anti-cancer therapies. This is because the growth hormone-releasing hormone is usually produced by various human tumors and has the ability to exert paracrine stimulatory effects on the tumors.

    There is still a lot of work underway to discover angiogenesis inhibitors. Numerous ongoing clinical trials in this area are focused on peptides derived from sources such as growth factors, growth factor receptors, and extracellular matrix proteins. It is hoped that the result of the studies will be a major breakthrough in developing more viable cancer treatments and therapies.

    References

    1. L.K. Kvols and E.A. Woltering
      Role of somatostatin analogs in the clinical management of non-neuroendocrine solid tumors.
      Anticancer Drugs 17, 601-608 (2006)

    2. R.T. Dorsam and J.S. Gutkind
      G-protein-coupled receptors and cancer.
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    3. S. Fister et al.
      Gonadotropin-releasing hormone type II antagonists induce apoptotic cell death in human endometrial and ovarian cancer cells in vitro and in vivo.
      Cancer Res. 67, 1750-1756 (2007)

    4. L. Vujanovic and L.H. Butterfield
      Melanoma cancer vaccines and anti-tumor T cell responses.
      J. Cell. Biochem. 102, 301-310 (2007)

    5. F.G. Rick et al.
      Agonists of luteinizing hormone-releasing hormone in prostate cancer.
      Expert Opin. Pharmacother. 14, 2237-2247 (2013)

    6. F. Goel et al.
      LHRH agonists for adjuvant therapy of early breast cancer in premenopausal women.
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    7. Hackshaw
      Luteinizing hormone-releasing hormone (LHRH) agonists in the treatment of breast cancer.
      Expert Opin. Pharmacother. 10, 2633-2639 (2009)

    8. F. Klug et al.
      Characterization of MHC ligands for peptide based tumor vaccination.
      Curr. Pharm. Des. 15, 3221-3236 (2009)

    9. G. Mezö and M. Manea
      Luteinizing hormone-releasing hormone antagonists.
      Expert Opin. Ther. Pat. 19, 1771-1785 (2009)

    10. R.M. Myers et al.
      Cancer, chemistry, and the cell: molecules that interact with the neurotensin receptors.
      ACS Chem. Biol. 4, 503-525 (2009)

    11. E.D. Deeks
      Histrelin: in advanced prostate cancer.
      Drugs 70, 623-630 (2010)

    12. F. Hohla and A.V. Schally
      Targeting gastrin releasing peptide receptors: New options for the therapy and diagnosis of cancer.
      Cell Cycle 9, 1738-1741 (2010)

    13. P.J. Pommerville and J.G. de Boer
      GnRH antagonists in the treatment of advanced prostate cancer.
      Can. J. Urol. 17, 5063-5070 (2010)

    14. P. Whelan
      Triptorelin embonate: a 6-month formulation for prostate cancer.
      Expert Opin. Pharmacother. 11, 2929-2932 (2010)

    15. D. Wild et al.
      First clinical evidence that imaging with somatostatin receptor antagonists is feasible.
      J. Nucl. Med. 52, 1412-1417 (2011)

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      Lutetium-labelled peptides for therapy of neuroendocrine tumours.
      Eur. J. Nucl. Med. Mol. Imaging 39 Suppl. 1, S103-112 (2012)

    17. K.P. Koopmans and A.W. Glaudemans
      Rationale for the use of radiolabelled peptides in diagnosis and therapy.
      Eur. J. Nucl. Med. Mol. Imaging 39 Suppl 1, S4-10 (2012)

    18. P. Limonta et al.
      GnRH receptors in cancer: from cell biology to novel targeted therapeutic strategies.
      Endocr. Rev. 33, 784-811 (2012)

    19. P.W. Moody et al.
      Pituitary adenylate cyclase-activating polypeptide causes tyrosine phosphorylation of the epidermal growth factor receptor in lung cancer cells.
      J. Pharmacol. Exp. Ther. 341, 873-881 (2012)

    20. P.K. Nanda et al.
      Bombesin analogues for gastrinreleasing peptide receptor imaging.
      Nucl. Med. Biol. 39, 461-471 (2012)

    21. J. Thundimadathil
      Cancer treatment using peptides: Current therapies and future prospects.
      J. Amino Acids 2012, 13 (2012)

    22. F. Barbieri et al.
      Peptide receptor targeting in cancer: the somatostatin paradigm.
      Int. J. Pept. 2013, 926295 (2013)

    23. R.A. Feelders et al.
      Pasireotide, a multi-somatostatin receptor ligand with potential efficacy for treatment of pituitary and neuroendocrine tumors.
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      The potential for substance P antagonists as anti-cancer agents in brain tumours.
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    25. O. Keskin and S. Yalcin
      A review of the use of somatostatin analogs in oncology.
      Onco Targets Ther. 6, 471-483 (2013)

    26. F.G. Rick et al.
      Agonists of luteinizing hormone-releasing hormone in prostate cancer.
      Expert Opin. Pharmacother. 14, 2237-2247 (2013)

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      New considerations for ADT in advanced prostate cancer and the emerging role of GnRH antagonists.
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      Six-month leuprorelin acetate depot formulations in advanced prostate cancer: a clinical evaluation.
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      (68)Ga-labeled bombesin analogs for receptor-mediated imaging.
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      An update of radiolabeled bombesin analogs for gastrin-releasing peptide receptor targeting.
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      Somatostatin receptor expression in hepatocellular carcinoma: prognostic and therapeutic considerations
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  • Antimicrobial Peptides’ Applications in the Treatment of Liver Cancer

    Antimicrobial peptides can be found in almost all species of plants, invertebrates and vertebrates. Most of the already identified antimicrobial peptides feature less than 100 amino acid residues as well as a myriad of common features, the most notable ones being hydrophobicity, amphipathic structure, and cationicity. The peptides can be found inducibly or constitutively in a variety of tissue and organs that witness constant exposure to microbial pathogens like the cells in the epithelial tissues of the skin, the respiratory tracts, and the gastrointestinal lining. Antimicrobial peptides display a wide range of antimicrobial activities against fungi, bacteria, viruses and protozoa. Presently, they have been heavily studied and their uses in the treatment of multi-drug resistant pathogens have been well-documented.

    There are two major mechanisms through which the actions of the function of cationic antimicrobial peptides can take: the first mechanism is through the inhibition of the function and the synthesis of intracellular RNA, DNA and protein. The second is through the disruption of the microbial membrane integrity through interacting with the negatively charged components. Apart from their direct antimicrobial activities, antimicrobial peptides are also known to have immunomodulatory properties like chemotactic activities to the immune cells. The duality in the nature of the functions of antimicrobial peptides have made it nearly impossible or difficult for microbes to develop resistance against antimicrobial peptides. There are several research findings that suggests that antimicrobial peptides are also loaded with anticancer activity. In the past, there have been studies focused on peptides that possesses both anticancer and antimicrobial activities. Owing to the characteristics as well as the observed anticancer activities of the studied peptides, it may be possible to select anticancer peptides or that the anticancer peptides could be designed and used in the development of therapeutic agents for the treatment of various types of cancers.

    One of the leading causes of malignant cancer deaths in the world is Hepatocellular carcinoma (HCC). Some of the already known major risks factors responsible for HCC are infections arising from hepatitis B and C viruses, intake of metabolic aflatoxin, the development of non-alcohol fatty liver diseases, and the excessive consumption of alcohol. Though there have been some very promising advances in the diagnosis of this condition, the mortality rates associated with it are still on the rise, and this is primarily due to the absence of effective therapies. Consequently, there is a need for the design and subsequent development of novel HCC treatment strategies.

    Antimicrobial peptides that have already displayed dual antimicrobial and anticancer activities are currently considered promising prospects for therapeutic agents that can be used in the treatment, as well as control, of HCC. They can be used as standalone treatments, or in conjunction with other treatments. In this piece, we will briefly discuss some of the new candidates for antimicrobial peptides for the treatment of HCC, the potential action mechanisms of the anticancer peptides, and the designs as well as the modifications of anti-HCC peptides.

    Anti-HCC Activity Peptides

    In the past few years, the number of anticancer peptides being evaluated for design for treatments against HCC has been on the rise. Most of the anti-HCC peptides are primarily obtained from sources such as terrestrial and marine animals and various types of bacteria. They can be seen as part of the superficially binding peptides that use phage-displayed selection on HCC cancer cell lines. The anticancer peptides have the ability to target ion channels, phospholipid layers, and other types of molecules in certain specific signaling pathways to induce the natural death of cancer cells.

    The Mechanisms of the Actions of Anticancer Cells against HCC

    Apart from the previously noted functions of the anticancer peptides, peptides obtained from antimicrobial peptides or any other natural source may display the following mechanisms in combating HCC: Immune modulation, direct killing, wound healing, and anti-inflammation activities. Below is a brief look into some of these mechanisms-:

    Direct Killing Activities

    The antimicrobial activity of antimicrobial peptides is triggered whenever there is an electrostatic interaction between the negatively charged bacterial component and the cationic peptide. This interaction is then followed by the insertion into, as well as the interruption of, the microbial membrane. During this process, it is important to note that the anionic phospholipid components of the cancer cell membranes are not the same as that of the normal cells. It has been observed that the cancer cell membranes usually tend to have a denser negatively charged phosphatidylserine (PS) compared to that of normal cells. This feature is responsible for making them more sensitive to the actions of the anticancer peptides.

    Anti-inflammatory Activity

    Whenever the permeability of the gut is compromised in any way, there is always the likelihood of the gut microbiota, as well as their products, disseminating directly from the intestines into the liver through the portal vein. Whenever this happens, there are always higher chances for the induction of hepatic inflammatory responses. This may then lead to the development of conditions such as cirrhosis, fibrosis and HCC. Such inflammatory reactions are believed to be a result of the toll-like receptor (TLR) signaling pathways that are normally prominent during the early stages of the development of liver cancer. TLR4 is one of the extracellular pathogen recognition receptors with the ability to bind LPS. As such, they have a significant role to play during the chronic inflammation of HCC. Through hepatocyte TLR4-mediated pathways, it is possible to induce the expression of the inflammatory molecule of Hepcidin. TLR5 present in hepatocytes works as a protection mechanism against high-fat-diet-induced liver disease through the binding actions of bacterial flagellin.

    Anticancer peptides that have been derived from antimicrobial peptides may benefit from their strong electrostatic interaction with the negatively charged LPS of gram-negative bacteria and flagellin, but only if they are both used as anti-inflammatory agents. Additionally, TLRs are known to be widely expressed in the bulk of the liver immune cells, including in macrophages, T-cells, B cell and dendritic cells. Consequently, with the modulation activities of TLRs, it may be possible to induce an anti-HCC activity within the liver, and the peptide agonists of TLRs may be vital in the development of novel therapeutics agents for treating HCC.

    Immune Modulation

    The recognition of tumor-associated antigens (TAA) is possible through the study of antigen-presenting cells which then induces the activation of tumor-responsive T lymphocytes. Several studies have suggested that TAA-derived long peptides may have the ability to elicit Th1, T cells and cytotoxic T lymphocytes (CTL)-mediated anticancer immune response. It is believed that this is possible through cross-presentation. Alpha-fetoprotein (AFP) is one of the most common TAA in HCC. It has been observed that peptides obtained from human AFP may have the ability to stimulate specific T-cell responses both in the T-cells obtained from patients suffering from HCC and in cultured peripheral blood lymphocytes of healthy individuals. A considerable number of tumor-infiltrating lymphocytes inside of a tumor have also been shown to have activity against HCC. Consequently, peptides derived from TAA may be helpful in enhancing anti-HCC treatment therapies.

    Additionally, cell-mediated interventions and treatments that feature the adoptive transfer of TILs are also under consideration for use as one of the immunotherapeutic strategies in the management and treatment of cancer. However, it is possible for the cancer cells to evade the immune surveillance through the alteration of their antigen presentation or through the secretion of cytokines and chemokines which will ultimately induce Treg cells to create an immunosuppressive microenvironment. Anticancer peptides that have immunostimulatory activities may be used as a means of enhancing the efficacy of cell-mediated therapy. An example for such an application would be Tyroserleutide which is an immunostimulatory peptide that features three amino acids with the ability to stimulate antitumor effects in macrophages against human HCC cell line.

    Wound Healing Activities

    Liver fibrosis is one of the known conditions linked to chronic liver injury. It will usually lead to cirrhosis and possibly HCC at the very later stages. Antimicrobial peptides such as endogenous mediators have the ability to enhance the processes of wound healing through antimicrobial activities, angiogenesis, chemotactic activities and LPS neutralization. For instance, in studies involving recombinant human AMP LL-37, it was observed that it enhanced the wound healing process by simply stopping the activation of macrophages with LPS, and also through the induction of the proliferation, as well as the migration, of endothelial cells, re-epithelization and vascularization.

    Additionally, it may be possible to modify natural antimicrobial peptides or to combine them with other short peptides to enhance their overall potential or capabilities.

    The Modification of Anticancer Peptides

    Studies have shown that host antimicrobial peptides have anticancer ability. However, most of their properties may not be directly applicable in the design and the development of anticancer agents since they have very low killing activity. The potential candidates for anticancer peptides may be modified to alter their mode of action. Also through modifications, it is possible for the novel analogs to be developed as a way of increasing the membrane-binding affinity and selectivity to cancer cells through the modulation of their amino acid components. For example, a modified peptide CB1a obtained from antimicrobial peptide Cecropin B has shown to have very promising activity against leukemia with very low cytotoxicity to non-cancer cells. Also, various strategies such as fragmentation, polymerization, hybridization, and cyclization, among others may be used to increase the efficacy as well as the stability while decreasing undesirable collateral cytotoxicity. It is also possible to design anticancer peptides to synthesis in silico with the aim of reducing the associated time and labor.

    The Potential Applications of Anticancer Peptides

    Anticancer peptides have two main killing mechanisms – modulating immune responses to kill cancer cells and binding with targets to kill cancer cells. These mechanisms make them very attractive in the development of a variety of HCC treatments. To produce peptide based anticancer therapies, it is vital to explore various delivery systems, including nanoparticles, liposomes, and peptide-derived vaccines.

    Peptide-Based Vaccines

    Most of the peptide-based vaccines are easy to store, are water soluble, and can be customized to be used to target and achieve very specific objectives. For example, Glypican-3 (GPC3) obtained from heparin sulfate proteoglycans is usually overexpressed in HCC and, as such, it is viewed as a potential target for cancer immunotherapeutic. Peptide vaccines derived from GPC3 have demonstrated the ability to lead to an increase in the peptide-specific CTLs and they are currently being evaluated in a variety of clinical trials. There are, however, certain limitations that have been associated with the use of peptide vaccines. For example, peptide vaccines are known to have low efficacy, to be unstable under certain physiological conditions, and to have poor immunogenicity. There are several strategies that have been employed as a means of overcoming certain limitations, with some of the interventions including the use of immunostimulatory adjuvants, multi-epitope approaches, and the use of new delivery systems featuring nano or micro particles. It is also possible to use antimicrobial peptides with anticancer properties as adjuvants for vaccines. Clinical trials for peptide-based vaccines for cancer therapies targeting TAAs have been reviewed in the past few years, and it has been observed that by targeting tumor-specific mutated antigens, it may be possible to reduce the off-tumor cytotoxicity, allowing room for more personalized treatments.

    Nanoparticles

    Nanoparticles like liposomes and gold nanoparticles have been used in the design and development of various delivery systems because of their properties such as enviable bioactivity, long drug half-life, and good cell selectivity. Another promising strategy in the development of HCC therapies is the possibility of delivering anticancer peptides with the help of nanocarriers. In one study, it was reported that through the use of liposomes to deliver HCC-targeting peptide, there was enhanced therapeutic efficiency as well as proper selection by phage-display in a mouse with the HCC xenograph model. Currently, there is need for more studies to help in the identification of the unique neoantigens that can be used as potential candidates for immunotherapeutic agents against HCC.

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