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Novel Claudin Targeted C-terminal Clostridium Perfringens Enterotoxin Conjugated Anticancer Nanotherapeutics

Dr. Sushil Y. Raut , Assistant Professor and Head of Research Promotion Cell, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research

Dr. Tanaji D. Nandgude , Associate Professor, Dr. D. Y. Patil institute of Pharmaceutical Sciences and Research

Cancers of epithelial origin account for the majority of cancer-related deaths. Tight Junctions (TJs) are the principal barrier to paracellular solute transport between cells, as well as a fundamental component of epithelial cell polarity to protein and lipid mobility inside the plasma membrane. Carcinogenesis comes from any qualitative or quantitative dysregulation of TJ properties. Structural and functional integrities of epithelial TJs is maintained by claudins and occludin receptor families. Claudin receptors found to be upregulated during cell proliferation and metastasis. As claudins are the most highly up-regulated genes in tumor cells, but at very low levels in normal tissues, these claudins may represent useful cancer markers for early detection, diagnosis, or therapeutic targets. Claudins overexpressed in many tumors like pancreatic, ovarian, breasts and colon due to undesirable phosphorylation. The effective binding ligand for claudin is Clostridium Perfringens Enterotoxin (CPE), which causes toxic effects in human, like diahorrea. CPE is a polypeptide having 319 amino acids monomeric units. The effective binding domain of CPE to claudin is only terminal 30 amino acids having C-terminal for receptor binding. Use of CPE peptide only with terminal 30 amino acids found to be effective for targeted delivery of nanomedicines to cancer cells without any harmful effects. Nanosized carrier system like polymeric nanoparticles, liposomes, lipid nanoparticles, polymeric micelles, found to be effective for targeting of chemotherapeutics to tumor cells by passive as well as active strategies. These nanocarriers can help to increase the therapeutic efficacy of drug by enhanced permeation and retention effect (EPR) and reduces side effects. They also have great potential to intrude with proliferating and metastatic tumor cells at molecular level. Thus, the CPE peptide conjugated nanomedicines have a great potential for targeted treatment of variety of tumors.

Active targeting with nanomedicines can result in effective drug delivery to targeted cells, maximising therapeutic efficacy while decreasing systemic side effects. The main application of this approach is paying attention toward cancer therapy. Cancer threats were found in worldwide population due to increasing adoption of cancer-causing behaviors. According to the GLOBOCAN 2020 forecasts, there will be 19.3 million cancer cases and 10.0 million cancer deaths in 2020. These figures of global cancer burden necessitated the advancement in strategies of cancer treatment. Current chemotherapeutic methods suffer from a number of drawbacks that commonly result in treatment failure. Non-specific biodistribution and insufficient targeting of therapeutic agents, lack of aqueous solubility, poor oral bioavailability, low therapeutic indices, dose-dependent toxicity to healthy tissues, and, most importantly, almost always emerging drug resistance are the main causes of disappointment.  Drug resistance continues to be a major impediment to the effectiveness of current cancer treatment. Although, the response of patient to current cancer chemotherapy reduced timely even at high dosing frequencies. Appropriate carrier system defines the success of chemotherapy. Nanomedicine is a new carrier method that has the ability to infiltrate cancer at the molecular level and deliver powerful dosages of therapeutic medicines to cancer cells with greater specificity and fewer side effects. Nanomedicine refers to the advancement of nanoparticles such as polymeric micelles, liposomes, dendrimers, carbon nanotubes, nanocrystals, and fullerenes as drug carriers. Specificity of nanomedicines towards tumor cells can be improved by adopting the approach of active targeting by pointing the tumor cell surface receptors as well as tumor vasculature. During proliferation and early metastasis process, the many tumor cell surface receptors found to be over expressed. These receptors were also present in normal cells, but their expression may be upto a limited extent. The demand for the excessive nutrients may be the one of reason for overexpression of tumor cell surface receptor. These receptors having affinity for their specific ligand for binding, such ligand when conjugated with drug loaded nanocarrier, the successful targeting of tumor cells can be possible which serves as one of the advanced strategies in cancer treatment. The  specific  and  selective  affinity  of  ligand  towards  its  receptor,  describes  the  bio-distribution  of  anti-cancer  agents  and also control over the pharmacokinetic properties of the drug. Long circulation durations will allow nanoparticles to reach the tumor location more effectively due to the increased permeability and retention effect (EPR) and targeting receptors will boost nanoparticle endocytosis. Already nanomedicines were well explored as an effective carrier system in wide variety of diseases, but ligand directed nanomedicines in cancer treatment to target the tumor cells pose strange attention nowadays. This approach can encounter the problems associated with conventional anti-cancer therapy. But nanomedicine engineering is not quite easy, as there are several hurdles in their path such as their designing, administration, biodistribution and clearance also with their main aim of site specificity and accumulation for delivering active agent. Biological safety of nanomedicines is an important issue in the context of their designing which is dependant of the physical, chemical and biological properties.

Strategic Need of Targeted Nanomedicine:

Present status of the cancer world-wide is very devastating and in future it may be explosive to capture more figures. Thus, many global cancer research organisations focused to combat upon the cancer. It was well explored the performance of present therapies, which was partially remedial or failed to cure the cancer. Patients with cancer found to be frustrated with present therapies due to their side effects to normal tissues. In case of chemotherapy, conventional approaches target the tumor cells as well as normal cells and thus result outcome is negative. Surgical therapy is one of the alternatives for this but associated with invasiveness, also surgical removal of every tumor tissue and metastating cells is not possible all time. Radiation therapy is also available for cancer treatment but focusing on specific tumor tissue inside the body is challenging. Thus, failure of available therapies necessitates the development of novel approaches which not only target the cancer cells but also sparing the normal cells without any systemic toxicity. Tumor cells overexpress various receptors in demand of many nutrients as well as biofactors for rapid extravasation and spreading. These receptors are also present in normal cells, but they are down regulated. Various nutrients or biofactors specifically binds to overexpressing tumor cell surface receptors, thus we can use them as a specific ligand to conjugate with chemotherapeutic loaded nanocarriers. Ligand decorated nanocarrier binds with tumor cell surface receptors specifically and internalised the drug loaded nanocarrier via receptor mediated endocytosis where it releases the loaded drug to show cell specific cytotoxicity. Claudins are the specific transmebrane receptors found to be over-expressed on variety of cancers. These claudins can be targeted with CPE peptide with terminal 30 amino acids when conjugated with nanocarriers. Use of nanocarriers such as polymeric nanoparticles, liposomes, lipid nanoparticles, polymeric micelles etc. has great potential to deliver the potent doses of chemotherapeutics specifically to tumor cells without causing any side effects. The variety of reported approaches includes:

Cocco E and his colleagues created a fluoroscene isothiocynate (FITC)-C-CPE conjugate to investigate its in vitro and in vivo binding to a number of primary chemotherapy-resistant ovarian cancer cell lines that overexpress claudin receptors. Claudin-3 and 4 expression patterns, as well as multiple primary ovarian carcinoma cell lines, were investigated using rtPCR and flow cytometry, respectively. The location and uptake of the FITC-C-CPE conjugate were assessed using confocal imaging and biodistribution tests. It was concluded that C-CPE is an effective targeting ligand for FITC.

Kakutani H and co-workers prepared a novel claudin-4 targeting molecule (DTA–C-CPE) genetically by fusing C-CPE and diphtheria toxin fragment A (DTA). Study also involved the evaluation of the specific targeting of DTA–C-CPE and examination of the cytotoxic effects of the developed conjugate in L cells. This investigation favorably explored the specific targeting potential of C-CPE.

Rajapaksa TE and his team also used C-CPE as a claudin targeting ligand of PLGA nanoparticles and specifically delivered inhibitory protein to intestinal M cell. Recombinant proteins bearing the influenza HA with or without a c-terminal targeting peptide, CPE30, were incorporated into sub-micron sized PLGA particles. Selective uptake of inhibitory protein by intestinal M cells was investigated by the in vitro and in vivo methods.

Saeki R and co-workers developed a cytotoxic molecule i.e. C-CPE-protein synthesis inhibitory factor (C-CPE-PSIF) is harmful to claudin-4-expressing cells, according to research. C-CPE-PSIF was shown to be less harmful in polarised cells than in depolarised cells, and polarised cells treated with C-CPE-PSIF from the basal but not the apical side were found to be cytotoxic. C-CPE-PSIF also has anticancer properties. C-CPE mutants with alanine substitutions were similarly shown to be more cytotoxic than C-CPE.

Conclusions and Future Scope:

Till date only little research was initiated which only explored the targeted delivery of certain toxoids and inhibitory proteins in fusion with C-CPE peptide to target the claudins. Very few researchers focused on claudin targeted delivery of cancer chemotherapeutics and diagnostic agents. Reversal of chemo-resistance of cancer cells by adopting C-CPE peptide conjugated nanocarrier systems will be a challenging area of research. Specific delivery of anticancer drugs and diagnostic agents by using dendrimers, carbon nanotubes, liposomes and polymeric nanoparticles will have a great potential to treat the epithelial cancers.

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Dr. Sushil Y. Raut

Dr. Sushil Y. Raut Assistant Professor and Head of Research Promotion Cell, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune. He has over 12 years of teaching and research experience. He has more than 25 research and review publications. He filed 3 Indian patents and over 20 national as well as international presentations in his credits. He is recipient of international research mobility fellowship and other prestigious awards.

Dr. Tanaji D. Nandgude

Dr. Tanaji D. Nandgude, Associate Professor, Dr. D. Y. Patil institute of Pharmaceutical Sciences and Research, Pimpri, Pune. He has 15 years of experience in teaching and research in pharmacy. He has more than 50 research/review papers, more than 60 research paper presentations, 5 book publications and granted projects to his credit. He is recipient of “Young Researcher Award”.

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