The intractable and devastating nature of pancreatic ductal adenocarcinoma (PDAC) necessitates an urgent need for novel therapies. This study presents the development of a novel polymer prodrug system for the combination treatment of PDAC, based on an optimized pharmacologically active anti-metastatic macromolecular carrier, PCQ, conjugated with gemcitabine (GEM). Structure-activity relationship evaluations showed that random PCQ copolymers exhibited superior anti-migratory activity compared to the gradient PCQ analogs. GEM was incorporated into the random PCQ copolymers using disulfide linker to prepare a reduction-responsive prodrug, PCQ (r)6-SS-GEM12. The resultant therapeutic system presents a pharmacologically active delivery strategy that targets both the proliferative and the metastatic phenotype in PDAC. The PCQ(r)6-SS-GEM12 prodrug demonstrated a selective release of GEM under the reductive tumor environment leading to a significant inhibition of tumor growth with pronounced anti-metastatic effect. Collectively, our data show that the combination of antimetastatic PCQ and cytotoxic GEM-based reduction-responsive prodrug polymer offers an innovative strategy to treat PDAC.

We discovered dibenzannulated medium-ring keto lactams (11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-diones) as a new antimalarial chemotype. Most of these had chromatographic LogD7.4 values ranging from <0 to 3 and good kinetic solubilities (12.5 to >100 μg/mL at pH 6.5). The more polar compounds in the series (LogD7.4 values of <2) had the best metabolic stability (CLint values of <50 μL/min/mg protein in human liver microsomes). Most of the compounds had relatively low cytotoxicity, with IC50 values >30 μM, and there was no correlation between antiplasmodial activity and cytotoxicity. The four most potent compounds had Plasmodium falciparum IC50 values of 4.2 to 9.4 nM and in vitro selectivity indices of 670 to >12,000. They were more than 4 orders-of-magnitude less potent against three other protozoal pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani) but did have relatively high potency against Toxoplasma gondii, with IC50 values ranging from 80 to 200 nM. These keto lactams are converted into their poorly soluble 4(1H)-quinolone transannular condensation products in vitro in culture medium and in vivo in mouse blood. The similar antiplasmodial potencies of three keto lactam-quinolone pairs suggest that the quinolones likely contribute to the antimalarial activity of the lactams.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with a five-year survival rate of around 10 %. CXCR4 and STAT3 display crucial effects on proliferation, metastasis, angiogenesis, and formation of immunosuppressive microenvironment in pancreatic tumors. Here, we have tested the hypothesis that conjugation of α-tocopherol (TOC) to a polycation (PAMD), synthesized from CXCR4-antagonist AMD3100, will improve delivery of therapeutic siRNA to silence STAT3 in PDAC tumors. PAMD-TOC/siSTAT3 nanoparticles showed superior anti-cancer and anti-migration performance compared to the parent PAMD/siSTAT3 nanoparticles in both murine and human PDAC cell lines. The biodistribution of the nanoparticles in orthotropic mouse KPC8060 and human PANC-1 models, indicated that tumor accumulation of PAMD-TOC/siRNA nanoparticles was improved greatly as compared to PAMD/siRNA nanoparticles. This improved cellular uptake, penetration, and tumor accumulation of PAMD-TOC/siSTAT3 nanoparticles, also contributed to the suppression of tumor growth, metastasis and improved survival. Overall, this study presents a prospective treatment strategy for PDAC.

Local pulmonary administration of therapeutic siRNA represents a promising approach to the treatment of lung fibrosis, which is currently hampered by inefficient delivery. Development of perfluorooctylbromide (PFOB) nanoemulsions as a way of improving the efficiency of pulmonary polycation-based delivery of siRNA is reported. The results show that the polycation/siRNA/PFOB nanoemulsions are capable of efficiently silencing the expression of STAT3 and inhibiting chemokine receptor CXCR4-two validated targets in pulmonary fibrosis. Both in vitro and in vivo results demonstrate that the nanoemulsions improve mucus penetration and facilitate effective cellular delivery of siRNA. Pulmonary treatment of mice with bleomycin-induced pulmonary fibrosis shows strong inhibition of the progression of the disease and significant prolongation of animal survival. Overall, the study points to a promising local treatment strategy of pulmonary fibrosis.

Nucleic acid (NA) therapy has gained importance over the past decade due to its high degree of selectivity and minimal toxic effects over conventional drugs. Currently, intravenous (IV) or intramuscular (IM) formulations constitute majority of the marketed formulations containing nucleic acids. However, oral administration is traditionally preferred due to ease of administration as well as higher patient compliance. To leverage the benefits of oral delivery for NA therapy, the NA of interest must be delivered to the target site avoiding all degrading and inhibiting factors during its transition through the gastrointestinal tract. The oral route presents myriad of challenges to NA delivery, making formulation development challenging. Researchers in the last few decades have formulated various delivery systems to overcome such challenges and several reviews summarize and discuss these strategies in detail. However, there is a need to differentiate between the approaches based on target so that in future, delivery strategies can be developed according to the goal of the study and for efficient delivery to the desired site. The goal of this review is to summarize the mechanisms for target specific delivery, list and discuss the formulation strategies used for oral delivery of NA therapies and delineate the similarities and differences between local and systemic targeting oral delivery systems and current challenges.

Alcohol-associated liver disease (AALD) is a major cause of liver disorders worldwide. Current treatment options are limited, especially for AALD-associated fibrosis. Promising approaches include RNA interference for miR-155 overexpression in Kupffer cells (KCs), as well as the use of CXCR4 antagonists that inhibit the activation of hepatic stellate cells (HSCs) through the CXCL12/CXCR4 axis. The development of dual-functioning nanoparticles for the effective delivery of antifibrotic RNA together with a CXCR4 inhibitor thus promises to improve the treatment of AALD fibrosis. In this study, cholesterol-modified polymeric CXCR4 inhibitor (Chol-PCX) was synthesized and used to encapsulate anti-miR-155 or non-coding (NC) miRNA in the form of Chol-PCX/miRNA nanoparticles. The results indicate that the nanoparticles induce a significant miR-155 silencing effect both in vitro and in vivo. Treatment with the Chol-PCX/anti-miR-155 particles in a model of moderate alcohol consumption with secondary liver insult resulted in a significant reduction in aminotransferase enzymes as well as collagen content in the liver parenchyma. Overall, our data support the use of Chol-PCX as a carrier for anti-miR-155 for the combined therapeutic inhibition of CXCR4 and miR-155 expression as a way to improve fibrotic damage in the liver.

In the past few decades, nanotechnology has made tremendous progress. Materials exhibit novel physicochemical properties in the nanoscale size range. Hence, nanomaterials are promising for various applications including catalysis, energy storage, electronics, sensors, semiconductors, drug delivery, bioimaging, and tissue engineering. However, because of a lack of biological properties, their use is limited. In advanced biomedical engineering, new functional materials are being developed by simply mimicking the characteristics of natural materials either chemically, physically, or biologically. In this chapter, we discuss such biomimetic nanocomposites, their synthesis strategies, and their biomedical applications such as drug delivery and tissue engineering. We provide a brief overview of various strategies for synthesizing biomimetic nanomaterials, including solvent casting and particulate leaching, phase separation/inversion, gas foaming, electrospinning, self-assembly, and rapid prototyping. We comprehensively describe biomimetic drug-delivery systems using hydrogels, liposomes, micelles, dendrimers, and polymeric nanocomposites. Biomimetic-nanocomposite-based scaffolds composed of cells can be used in tissue engineering because they have appropriate mechanical properties, recapitulate the extracellular matrix, and provide a microenvironment that enhances tissue regeneration. We also systematically explain tissue engineering based on biomimetic membranes, hydrogels, fibers, and three-dimensionally printed nanocomposites. However, despite the tremendous progress in biomimetic nanocomposites described herein, further studies are needed before these materials reach clinical use.

RNA interference (RNAi) is an emerging therapeutic modality for cancer, which remains in critical need of effective delivery vectors due to the unfavorable biopharmaceutical properties of small RNAs. Polyamines are essential for functioning of mammalian cells. Dysregulated polyamine metabolism is found in many cancers and has been an attractive therapeutic target in combination therapies. Combination therapies based on drugs that affect polyamine metabolism and nucleic acids promise to enhance anticancer activity due to a cooperative effect on multiple oncogenic pathways. Here, we report bioactive polycationic prodrug (F-PaP) based on an anticancer polyamine analogue bisethylnorspermine (BENSpm) modified with perfluoroalkyl moieties. Following encapsulation of siRNA, F-PaP/siRNA nanoparticles were coated with hyaluronic acid (HA) to form ternary nano particles HA@F-PaP/siRNA. The presence of perfluoroalkyl moieties and HA reduced cell membrane toxicity and improved stability of the particles with cooperatively enhanced siRNA delivery in pancreatic and colon cancer cell lines. We then tested a therapeutic hypothesis that combining BENSpm with siRNA silencing of polo-like kinase 1 (PLK1) would result in cooperative cancer cell killing. HA@F-PaP/siPLK1 induced polyamine catabolism and cell cycle arrest, leading to enhanced apoptosis in the tested cell lines. The HA-coated nanoparticles facilitated tumor accumulation and contributed to strong tumor inhibition and favorable modulation of the immune tumor microenvironment in orthotopic pancreatic cancer model.

Conformationally restrained polyamine analog PG11047 is a well-known drug candidate that modulates polyamine metabolism and inhibits cancer cell growth in a broad spectrum of cancers. Here, we report a structure–activity relationship study of the PG11047 analogs (HPGs) containing alkyl chains of varying length, while keeping the unsaturated spermine backbone unchanged. Synthesis of higher symmetrical homologues was achieved through a synthetic route with fewer steps than the previous route to PG11047. The amphiphilic HPG analogs underwent self-assembly and formed spherically shaped nanoparticles whose size increased with the hydrophobic alkyl group’s increasing chain length. Assessment of the in vitro anticancer activity showed more than an eight-fold increase in the cancer cell inhibition activity of the analogs with longer alkyl chains compared to PG11047 in human colon cancer cell line HCT116, and a more than ten-fold increase in human lung cancer cell line A549. Evaluation of the inhibition of spermine oxidase (SMOX) showed no activity for PG11047, but activity was observed for its higher symmetrical homologues. Comparison with a reference SMOX inhibitor MDL72527 showed nine-fold better activity for the best performing HPG analog.

Pancreatic ductal adenocarcinoma (PDAC) is a growing medical problem associated with extensive metastasis and high mortality. Intraperitoneal (IP) administration of therapeutics promises to help the treatment of cancers originated from organs in the peritoneal cavity. In this study, we evaluated how physicochemical properties of self-assembled polycation/siRNA nanoparticles affect their IP delivery efficacy in an orthotopic PDAC model. We have examined the effect of covalent polycation modification with lipophobic and hydrophobic tetrafluoro-p-toluic acid (TFTA), hydrophobic cholesterol, and hydrophilic poly(ethylene glycol) respectively. The surface charge of the three different nanoparticles was also modulated by coating the surface with serum albumin. We found that positively charged fluorine-containing particles with lipophobic properties based on a mixture of positively charged polymeric AMD3100 CXCR4 antagonist (PAMD) and PAMD modified with TFTA (mPAMD-TFTA)/siRNA displayed the best cell uptake and transfection efficacy in vitro. Biodistribution evaluation of the nanoparticles in a syngeneic orthotopic PDAC model revealed that the fluorine-containing formulation also achieved the highest PDAC tumor accumulation after IP administration. With a combination of CXCR4 inhibition by PAMD and PLK1 downregulation by siRNA, the treatment with mPAMD-TFTA/siPLK1 showed significant inhibition of both primary and metastatic PDAC tumors. Overall, our study provides insights into and guides the design of the nanoparticles for improved IP delivery of siRNA in PDAC.

Pancreatic ductal adenocarcinoma is a deadly disease with limited treatment options due to late diagnosis and resistance to conventional chemotherapy. Among emerging therapeutic targets, the CXCR4 chemokine receptor and polo-like kinase 1 (PLK1) play critical roles in the progression, metastasis, and chemoresistance of pancreatic cancer. Here, we tested the hypothesis that combining CXCR4 inhibition by a polymeric CXCR4 antagonist PAMD-CHOL with PLK1 knockdown by siRNA will enhance the therapeutic effect of gemcitabine (GEM) in the orthotopic model of metastatic pancreatic cancer. We formulated nanoparticles with cholesterol-modified PAMD and siPLK1 and found strong synergism when combined with GEM treatment in vitro in both murine and human pancreatic cancer cell lines. The biodistribution of the nanoparticles in orthotopic pancreatic cancer models revealed strong accumulation in primary and metastatic tumors, with limited hepatic disposition. The cholesterol-containing nanoparticles showed not only increased tumor accumulation than the cholesterol-lacking control but also deeper penetration into the tumors. In a therapeutic study in vivo, the triple combination of PAMD-CHOL/siPLK1 and GEM showed superior anticancer activity when compared with single and dual combination controls. In conclusion, PAMD-CHOL/siPLK1 nanoparticles synergistically enhance anticancer activity of GEM in pancreatic cancer and represent a promising addition to the treatment arsenal.

Human Toll-like receptor 8 (hTLR8) is expressed in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells. Engagement by TLR8 agonists evokes a distinct cytokine profile which favors the development of type 1 helper T cells. Crystal structures of the ectodomain of hTLR8 cocrystallized with two regioisomers of a dual TLR7/8-agonistic N1-substituted imidazoquinolines showed subtle differences in their interactions in the binding site of hTLR8. We hypothesized that the potency of a previously reported best-in-class pure TLR8 agonist, 3-pentylquinoline-2-amine, could be further enhanced by "designing in" functional groups that would mimic key intermolecular interactions that we had observed in the crystal structures. We performed a focused exploration of decorating the quinoline core with alkylamino groups at all possible positions. These studies have led to the identification of a novel TLR8 agonist that was ∼20-fold more potent than the parent compound and displays prominent adjuvantic activity in a rabbit model of immunization.

Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of Thelper 1-polarizing cytokines, suggesting that TLR8-active compounds might be promising candidate vaccine adjuvants. Recently, a C2-butyl furo[2,3-c]quinoline was reported with purely TLR8 agonistic activity. This compound was successfully co-crystallized with the human TLR8 ectodomain, and the co-crystal structure revealed ligand-induced reorganization of the binding pocket of TLR8. The loss of a key hydrogen bond between the oxygen atom of the furanyl ring of the agonist and Thr574 in TLR8 suggested that the furan ring is dispensable. Employing a disconnection strategy, 3- and 4-substituted aminoquinolines were investigated. Focused structure-based ligand design studies led to the identification of 3-pentyl-quinoline-2-amine as a novel, structurally simple, and highly potent human TLR8-specific agonist (EC50=0.2M). Preliminary evaluation of this compound in exvivo human blood assay systems revealed that it retains prominent cytokine-inducing activity. Together, these results indicate the suitability of this compound as a novel vaccine adjuvant, warranting further investigation.

Toll-like receptor (TLR) 7 and 8 agonists are potential vaccine adjuvants, since they directly activate APCs and enhance Th1-driven immune responses. Previous SAR investigations in several scaffolds of small molecule TLR7/8 activators pointed to the strict dependence of the selectivity for TLR7 vis-à-vis TLR8 on the electronic configurations of the heterocyclic systems, which we sought to examine quantitatively with the goal of developing “heuristics” to define structural requisites governing activity at TLR7 and/or TLR8. We undertook a scaffold-hopping approach, entailing the syntheses and biological evaluations of 13 different chemotypes. Crystal structures of TLR8 in complex with the two most active compounds confirmed important binding interactions playing a key role in ligand occupancy and biological activity. Density functional theory based quantum chemical calculations on these compounds followed by linear discriminant analyses permitted the classification of inactive, TLR8-active, and TLR7/8 dual-active compounds, confirming the critical role of partial charges in determining biological activity.

Endotoxins (lipopolysaccharides, LPS) are one of the strongest immunostimulators in nature, responsible for beneficial effects at low, and pathophysiological effects at high concentrations, the latter frequently leading to sepsis and septic shock associated with high mortality in critical care settings. There are no drugs specifically targeting the pathophysiology of sepsis, and new therapeutic agents are therefore urgently needed. The lipopolyamines are a novel class of small molecules designed to sequester and neutralize LPS. To understand the mechanisms underlying the binding and neutralization of LPS toxicity, we have performed detailed biophysical analyses of the interactions of LPS with candidate lipopolyamines which differ in their potencies of LPS neutralization. We examined gel-to-liquid crystalline phase behavior of LPS and of its supramolecular aggregate structures in the absence and presence of lipopolyamines, the ability of such compounds to incorporate into different membrane systems, and the thermodynamics of the LPS:lipopolyamine binding. We have found that the mechanisms which govern the inactivation process of LPS obey similar rules as found for other active endotoxin neutralizers such as certain antimicrobial peptides.

Toll-like receptor (TLR)-8 agonists typified by the 2-alkylthiazolo[4,5-c]quinolin-4-amine (CL075) chemotype are uniquely potent in activating adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds could be promising candidate vaccine adjuvants, especially for neonatal vaccines. Alkylthiazoloquinolines with methyl, ethyl, propyl and butyl groups at C2 displayed comparable TLR8-agonistic potencies; activity diminished precipitously in the C2-pentyl compound, and higher homologues were inactive. The C2-butyl compound was unique in possessing substantial TLR7-agonistic activity. Analogues with branched alkyl groups at C2 displayed poor tolerance of terminal steric bulk. Virtually all modifications at C8 led to abrogation of agonistic activity. Alkylation on the C4-amine was not tolerated, whereas N-acyl analogues with short acyl groups (other than acetyl) retained TLR8 agonistic activity, but were substantially less water-soluble. Immunization in rabbits with a model subunit antigen adjuvanted with the lead C2-butyl thiazoloquinoline showed enhancements of antigen-specific antibody titers.

Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds may be promising candidate adjuvants. We synthesized and evaluated hitherto unexplored furo[2,3-c]quinolines and regioisomeric furo[3,2-c]quinolines derived via a tandem, one-pot Sonogashira coupling and intramolecular 5-endo-dig cyclization strategy in a panel of primary screens. We observed a pure TLR8-agonistic activity profile in select furo[2,3-c]quinolines, with maximal potency conferred by a C2-butyl group (EC50 = 1.6 μM); shorter, longer, or substituted homologues as well as compounds bearing C1 substitutions were inactive, which was rationalized by docking studies using the recently described crystal structure of human TLR8. The best-in-class compound displayed prominent proinflammatory cytokine induction (including interleukin-12 and interleukin-18), but was bereft of interferon-α inducing properties, confirming its high selectivity for human TLR8.

Aqueous solubilities of many drugs in current clinical use are very low, necessitating formulations that often present problems for parenteral administration, including toxicities due to the excipients used. Recognizing that pharmacologically active compounds frequently possess amines, we asked whether pyridoxal phosphate (PLP), an inoccuous, water-soluble vitamin, could be utilized to form prodrug-like complexes via the formation of imine or iminium adducts and whether the vitamin would impart solubilizing properties to such complexes. Direct spectroscopic and crystallographic data obtained using model primary and secondary amines showed that PLP forms stable imine adducts with primary amines under entirely aqueous conditions and at physiologic pH, while no reaction was observed for secondary amines; the basis of the exceptional stability appears to be a consequence of favorable H-bond interactions of the imine nitrogen with the 5-OH group of PLP. Amphotericin B and nystatin in their native forms display marked aqueous insolubility and possess lone primary amines. We were able to utilize PLP in achieving excellent solubilization of both of these antifungal agents, surpassing aqueous solubilities of 100 mg/mL. In in vitro bioassays, both polyenes in their PLP-adducted form display attenuated antifungal potencies which are attributable to “prodrug-like” complexes. These results point to the utility of excipient-free, entirely aqueous formulations of amphotericin B for parenteral use, and may also be extended to other primary amine-bearing compounds exhibiting poor aqueous solubility.

Sepsis, otherwise referred to as "blood poisoning" is a serious clinical problem, the incidence of which continues to rise in the US and worldwide despite advances in antimicrobial chemotherapy. The primary trigger in Gram-negative sepsis is endotoxin, a lipopolysaccharide (LPS) constituent of the outer membrane of all Gram-negative bacteria. The structurally highly conserved glycolipid called lipid A is the active moiety of LPS. Lipid A is composed of a hydrophilic, bis-phosphorylated di-glucosamine backbone, and a hydrophobic polyacyl domain. The bis-anionic, amphiphilic nature of lipid A enables it to interact with a variety of cationic hydrophobic ligands, including polymyxin B, a toxic peptide antibiotic which binds to lipid A and neutralizes endotoxicity. Having determined the structural basis of the interaction of polymyxin B with lipid A, our long-term goal has been to rationally design non-peptidic, nontoxic, small-molecule LPS-sequestrants. Our efforts began with defining the central pharmacophore that determined LPS-recognition and -neutralization properties in small molecules, which led to the discovery of a novel lipopolyamine lead, DS-96. DS-96 is an effective LPS-neutralizer, rivaling polymyxin B in a panel of vitro assays, as well as in protecting animals against endotoxicosis. Structure-activity relationships in our effort to rationally design endotoxin sequestering agents, preclinical assessment of hits and leads, and approaches to overcoming issues with toxicity are described in this chapter.

Amphotericin B (AmB), a well-known polyene antifungal agent, displays a marked tendency to self-associate and, as a consequence, exhibits very poor solubility in water. The therapeutic index of AmB is low and is associated with significant dose-related nephrotoxicity, as well as acute, infusion-related febrile reactions. Reports in the literature indicate that the toxicity of AmB may be related to the physical state of the drug. Reaction of AmB in dimethylformamide with bis(dimethylaminopropyl)carbodiimide yielded an unexpected N-alkylguanidine/N-acylurea bis-adduct of AmB which was highly water-soluble. The absorption spectrum of the AmB derivative in water indicated excellent monomerization, and the antifungal activities of reference AmB and its water-soluble derivative against Candida albicans were found to be virtually identical. Furthermore, the water-soluble adduct is significantly less active in engaging TLR4, which would suggest that the adduct may be less proinflammatory.