An Update on Pharmaceutical Strategies for Oral Delivery of Therapeutic Peptides and Proteins

Peptides and proteins are imperative for the human body and play crucial roles in governing various bio-chemical processes. Recent advances in molecular biology and biochemistry helped in understanding the role of these endogenous macromolecules in different pathological and disease conditions. Currently, small molecule drugs (\u3c 900dalton) in comparison to the therapeutic peptides and proteins-based drugs (TPP) dominate pharmaceutical market. However, the game is changing with the recent advances of biotechnological tools like recombinant DNA technology, solid phase protein synthesis etc., which enabled large-scale production of therapeutic peptides and proteins. The Success of Human Insulin, the first FDA approved commercial recombinant protein based therapeutic in 1982, revolutionized the field of TPPs. The number of FDA approved TPPs reached about to ~239 in 2017 compared to where it was only ~130 in 2008. Rapid progress in this sector can be attributed to several advantages of proteins and peptides over small molecule drugs both financially and clinically. From a clinical perspective, proteins and peptides are inherently more specific to the target site than the small molecules drugs, which lead to less interferences with normal biological system of the patient and caused minimal off-target side effects. A handful of proteins which are used for different clinical complications are less immunogenic because they are produced in the body naturally. Furthermore, proteins and peptides also take part in several complex and complicated biological processes, which is difficult to be to be mimicked by the small molecule drugs. From a financial standpoint, median total pre-market development times were shorter for biologics (10.6 years) than the small molecules drugs (12.6 years) estimated using Merck Index. In 2009, US Congress passed the Biologics Price Competition and Innovation Act (BPCIA) which gave new biologics 12 years of guaranteed exclusivity. The most commonly utilized routes for administering TPPs are I.V, I.P or I.M injections, which largely suffer from patient compliances. There are ~350 TPPs under clinical development and among them only 2 are given orally which is Interferon-α and Human growth hormone. Currently, most efforts in both industry and academia are centered around enhancing bioavailability of orally administered TPPs which typically are less than 1%. Oral administration is the non-invasive, most preferred route of drug administration for the patients. Furthermore, oral dosage forms are cheaper to manufacture as well as to administer, because they do not need to be produced under sterile conditions or administered in clinics. However, unfavorable physicochemical characteristics of TPPs like high molecular weight, hydrophilicity, poor stability in the physiological conditions, short biological half-life, low permeability through the epithelial barrier in the small intestine put up a massive barrier in the development of orally available dosage forms of TPPs. In this review, we will discuss the challenges associated with oral delivery of TPPs and the ongoing efforts to solve them

Development of Targeted Drug Delivery System To Improve Immunotherapy In Pancreatic Cancer

Introduction: About 95% of tumor arises from epithelial cell lining ducts known to be pancreatic ductal adenocarcinomas, with less than 5-7% survival rate. Unfortunately, little progress has been seen in the outcomes of patients with PDAC as tumor develops high desmoplasia and chemo-resistance to chemotherapeutic drugs, such as gemcitabine (Gem). Immunotherapy has shown promising results in other cancers but limited response in pancreatic cancer due to desmoplasia and fibrotic tumor microenvironment. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas. Due to its high membrane expression, MUC13 may serve as an excellent target for PanCa treatment. Our recently published studies demonstrate a unique ability of our novel patented superparamagnetic iron oxide nanoparticles (SPIONS) of curcumin (Indian spice with high medicinal value) to inhibit desmoplasia and make the drug more bioavailable (1,2). Therefore, our objective is to conjugate SPION -curcumin with MUC13 antibody to directly target the pancreatic tumors and to improve immunotherapies by targeting tumor stroma. Methodology: Patented SPION1 particle was used for loading curcumin and later conjugated with MUC13 for directly targeting the pancreatic tumor. We characterized (size, zeta potential, charge and Dynamic light scattering), optimized and validated the uptake (Prussian blue staining and flow cytometer) of the formulation in invitro using ASPC1, HPAF, Panc 1, Panc M13 cell lines. Targeting efficiency of MNP-Anti-MUC13 particles in Panc-1-M13 and Panc-1 pancreatic cancer cells was done by immunofluorescence using flow cytometer and confocal microscopy. Cells treated with formulation were investigated for effect on PDAC cells and desmoplasia using Western blotting, qPCR, and immunofluorescence. Results: Our results demonstrate optimal particle size and zeta potential of SPION formulation. MUC13 conjugated SPIONS can efficiently internalize the PDAC cells and target immune checkpoint inhibitors, PDL-1 and CTLA4. MUC13-SPION formulation led to an enhanced uptake in MUC13 positive (MUC13+) PanCa cells as compared with MUC13 null (MUC13-) cells as demonstrated by immunofluorescence, Prussian blue staining and flow cytometry experiments. Interestingly, the formulation resulted in sustained delivery of CUR, enhanced inhibition of cell proliferation, migration and invasion in MUC13+ cells as compared with MUC13- cells, which suggests the targeting efficacy of the formulation. Additionally, the treatment of cells with the formulation inhibited the tumor spheroid formation and growth. The formulation softens up the tumors for therapies that can result in improved response to checkpoint immunotherapies. Conclusion: This study indicates high therapeutic significance of MUC13-SPIONS for achieving pancreatic tumor specific delivery of drugs. Therefore, the efficient MUC13 conjugated SPION-curcumin can potentiate checkpoint immunotherapies, inhibit tumor growth and its progression. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients

Novel therapy targeting Mutant-KRASG12D and Galectin-1 in Pancreatic Cancer

Introduction: Although, surgical resection and chemotherapy are the gold standard for treating Pancreatic Ductal Adenocarcinoma (PDAC), low patient survival rate remains the problem. The activating point mutation of the KRAS on codon-12 is present in 70–95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis. Recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both pancreatic cancer and stromal cells, which modulate tumor microenvironment and metastasis. Additionally, genetic deletion of gal1 inhibits metastasis and improves survival in KRAS mouse model of PDAC (1). Therefore, our objective is to develop a novel combination therapy for PDAC by targeting mutated KRASG12D point mutation and Gal1. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: ASPC1/Panc-1 (human), KPC (mouse) cells were used. Our patented SPION nano-formulation (2) has been used to deliver siKRASG12D and investigated in conjunction with Gal-1 for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated using FAM labelled siRNA and Prussian blue assay. KRASG12D silencing was confirmed at both mRNA and protein levels. Anti-cancer efficacy of the formulation was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in nude mice. Results: Our results demonstrate optimal particle size and zeta potential of SP-siKRAS formulation. SPsiKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. The formulation inhibited chemoresistance, cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility, suggesting its potential of silencing KRAS without being toxic to the body. Additionally, the formulation was efficiently delivered in nude mice to exhibit KRasG12D silencing and inhibit tumor growth. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target pancreatic cancer growth and improve patient survivability

Antibody-Drug Conjugates for Cancer Therapy: Chemistry to Clinical Implications

Chemotherapy is one of themajor therapeutic options for cancer treatment. Chemotherapy is often associated with a low therapeutic window due to its poor specificity towards tumor cells/tissues. Antibody-drug conjugate (ADC) technology may provide a potentially new therapeutic solution for cancer treatment. ADC technology uses an antibody-mediated delivery of cytotoxic drugs to the tumors in a targeted manner, while sparing normal cells. Such a targeted approach can improve the tumor-to-normal tissue selectivity and specificity in chemotherapy. Considering its importance in cancer treatment, we aim to review recent efforts for the design and development of ADCs. ADCs are mainly composed of an antibody, a cytotoxic payload, and a linker, which can offer selectivity against tumors, anti-cancer activity, and stability in systemic circulation. Therefore, we have reviewed recent updates and principal considerations behind ADC designs, which are not only based on the identification of target antigen, cytotoxic drug, and linker, but also on the drug-linker chemistry and conjugation site at the antibody. Our review focuses on site-specific conjugation methods for producing homogenous ADCs with constant drug-antibody ratio (DAR) in order to tackle several drawbacks that exists in conventional conjugation methods

MicroRNA-145 replacement as a therapeutic tool to Improve TRAIL therapy

Pancreatic cancer (PanCa) is a third leading cause of cancer related deaths in US. Unlike other cancers, PanCa is highly resistant to TNF-related apoptosis-inducing ligand (TRAIL) that emerges as one of the most-promising therapy in clinical trials. Our group has previously identified microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. In this study, we have observed that miR-145 restoration in PanCa cells renders them sensitive to TRAIL treatment. Therefore, we have engineered unique superparamagnetic nanoparticles (SPs) for co-delivering miR-145 and TRAIL in PanCa for improving their therapeutic response to TRAIL. The results in this study demonstrate that acquired resistance to TRAIL in PanCa cells can overcome with the replacement of lost levels of miR-145 expression. Our SP nanoparticles were engineered to co-deliver miR-145 and TRAIL to PanCa cells, which resulted in simultaneous restoration of miR-145 and inhibition of acquired resistance to TRAIL. Combined actions of miR-145 and TRAIL markedly improve TRAIL-induced apoptotic effects in PanCa cells through the activation of an extrinsic apoptosis pathway pathway as indicated by activation of DR5, FLIP, FADD and enhanced expression of caspase-8/3. The co-delivery of miR-145 and TRAIL using SP nanoparticles inhibited tumorigenic characteristics of PanCa cells, which include proliferation, invasion, migration and clonogenicity. The results were reciprocated and got further confirmed with the inhibition of tumorsphere formation and in vivo tumorigencity in xenograft mice. Immunohistochemical staining of excised tumor tissues demonstrate an activation of death receptor pathway and subsequent expression of apoptotic markers. The study provides novel insights on two facades- how resistance of cancer cells to TRAIL-based pro-apoptotic therapies can be tackled, and how efficient intracellular delivery of TRAIL can be achieved. Our results suggest that acquired resistance to TRAIL can be overcome by co-delivery of miR-145 and pEGFP-TRAIL using SP nanoparticles

Tannic acid inhibits lipid metabolism and induce ROS in prostate cancer cells

Prostate cancer (PCa) cells exploit the aberrant lipid signaling and metabolism as their survival advantage. Also, intracellular storage lipids act as fuel for the PCa proliferation. However, few studies were available that addressed the topic of targeting lipid metabolism in PCa. Here, we assessed the tannic acid (TA) lipid-targeting ability and its capability to induce endoplasmic reticulum (ER) stress by reactive oxygen species (ROS) in PCa cells. TA exhibited dual effects by inhibiting lipogenic signaling and suppression of lipid metabolic pathways. The expression of proteins responsible for lipogenesis was down regulated. The membrane permeability and functionality of PCa were severely affected and caused nuclear disorganization during drug exposure. Finally, these consolidated events shifted the cell’s survival balance towards apoptosis. These results suggest that TA distinctly interferes with the lipid signaling and metabolism of PCa cells

MicroRNA-145 replacement as a therapeutic tool to Improve TRAIL therapy

Pancreatic cancer (PanCa) is a third leading cause of cancer related deaths in US. Unlike other cancers, PanCa is highly resistant to TNF-related apoptosis-inducing ligand (TRAIL) that emerges as one of the most-promising therapy in clinical trials. Our group has previously identified microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. In this study, we have observed that miR-145 restoration in PanCa cells renders them sensitive to TRAIL treatment. Therefore, we have engineered unique superparamagnetic nanoparticles (SPs) for co-delivering miR-145 and TRAIL in PanCa for improving their therapeutic response to TRAIL. The results in this study demonstrate that acquired resistance to TRAIL in PanCa cells can overcome with the replacement of lost levels of miR-145 expression. Our SP nanoparticles were engineered to co-deliver miR-145 and TRAIL to PanCa cells, which resulted in simultaneous restoration of miR-145 and inhibition of acquired resistance to TRAIL. Combined actions of miR-145 and TRAIL markedly improve TRAIL-induced apoptotic effects in PanCa cells through the activation of an extrinsic apoptosis pathway pathway as indicated by activation of DR5, FLIP, FADD and enhanced expression of caspase-8/3. The co-delivery of miR-145 and TRAIL using SP nanoparticles inhibited tumorigenic characteristics of PanCa cells, which include proliferation, invasion, migration and clonogenicity. The results were reciprocated and got further confirmed with the inhibition of tumorsphere formation and in vivo tumorigencity in xenograft mice. Immunohistochemical staining of excised tumor tissues demonstrate an activation of death receptor pathway and subsequent expression of apoptotic markers. The study provides novel insights on two facades- how resistance of cancer cells to TRAIL-based pro-apoptotic therapies can be tackled, and how efficient intracellular delivery of TRAIL can be achieved. Our results suggest that acquired resistance to TRAIL can be overcome by co-delivery of miR-145 and pEGFP-TRAIL using SP nanoparticles

Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer

Introduction: In pancreatic ductal adenocarcinoma (PDAC), low patient survival rate remains a problem. The activating point mutation of KRAS on codon-12 is present in 70–95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis; recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both PDAC and stromal cells, which modulate tumor microenvironment and metastasis. Therefore, we have developed a novel combination therapy for PDAC by targeting mutated KRASG12D and Gal-1 to target both proliferation and metastasis in PDAC. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a mouse model. Results: Our results demonstrate optimal particle size/zeta potential of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. It inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility and stability suggesting its potential of silencing KRAS without being toxic to the body. The formulation efficiently exhibited KRasG12D silencing and inhibited tumor growth and metastasis in nude mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability

VERU-111 suppresses tumor growth and metastatic phenotypes of cervical cancer cells through the activation of p53 signaling pathway

In this study, we investigated the therapeutic efficacy of VERU-111 in vitro and in vivo model systems of cervical cancer. VERU-111 treatment inhibited cell proliferation and, clonogenic potential, induce accumulation of p53 and down regulated the expression of HPV E6/E7 expression in cervical cancer cells. In addition, VERU-111 treatment also decreased the expression of phosphorylation of Jak2 (TyR1007/1008) and STAT3 at Tyr705 and Ser727. VERU-111 treatment arrested cell cycle in the G2/M phase and modulated cell cycle regulatory proteins (cyclin B1, p21 p34cdc2 and pcdk1). Moreover, VERU-111 treatment induced apoptosis and modulated the expression of Bid, Bcl-xl, Survivin, Bax, Bcl2 and cleavage in PARP. In functional assays, VERU-111 markedly reduced the tumorigenic, migratory, and invasive potential of cervical cancer cells via modulations of MMPs. VERU-111 treatment also showed significant (P\u3c0.05) inhibition of orthotopic xenograft tumor growth in athymic nude mice. Taken together, our results demonstrate the potential anti-cancer efficacy of VERU-111 in in vitro and in vivo. VERU-111 can be explored as a potent therapeutic agent for the treatment of cervical cancer

Antibody-Drug Conjugates for Cancer Therapy: Chemistry to Clinical Implications

Chemotherapy is one of the major therapeutic options for cancer treatment. Chemotherapy is often associated with a low therapeutic window due to its poor specificity towards tumor cells/tissues. Antibody-drug conjugate (ADC) technology may provide a potentially new therapeutic solution for cancer treatment. ADC technology uses an antibody-mediated delivery of cytotoxic drugs to the tumors in a targeted manner, while sparing normal cells. Such a targeted approach can improve the tumor-to-normal tissue selectivity and specificity in chemotherapy. Considering its importance in cancer treatment, we aim to review recent efforts for the design and development of ADCs. ADCs are mainly composed of an antibody, a cytotoxic payload, and a linker, which can offer selectivity against tumors, anti-cancer activity, and stability in systemic circulation. Therefore, we have reviewed recent updates and principal considerations behind ADC designs, which are not only based on the identification of target antigen, cytotoxic drug, and linker, but also on the drug-linker chemistry and conjugation site at the antibody. Our review focuses on site-specific conjugation methods for producing homogenous ADCs with constant drug-antibody ratio (DAR) in order to tackle several drawbacks that exists in conventional conjugation methods