De Novo Construction of Fluorophores via CO Insertion-Initiated Lactamization: A Chemical Strategy toward Highly Sensitive and Highly Selective Turn-On Fluorescent Probes for Carbon Monoxide.

Extensive studies in the last few decades have led to the establishment of CO as an endogenous signaling molecule and subsequently to the exploration of CO's therapeutic roles. In the current state, there is a critical conundrum in CO-related research: the extensive knowledge of CO's biological effects and yet an insufficient understanding of the quantitative correlations between the CO concentration and biological responses of various natures. This conundrum is partially due to the difficulty in examining precise concentration-response relationships of a gaseous molecule. Another reason is the need for appropriate tools for the sensitive detection and concentration determination of CO in the biological system. We herein report a new chemical approach to the design of fluorescent CO probes through de novo construction of fluorophores by a CO insertion-initiated lactamization reaction, which allows for ultra-low background and exclusivity in CO detection. Two series of CO detection probes have been designed and synthesized using this strategy. Using these probes, we have extensively demonstrated their utility in quantifying CO in blood, tissue, and cell culture and in cellular imaging of CO from exogenous and endogenous sources. The probes described will enable many biology and chemistry labs to study CO's functions in a concentration-dependent fashion with very high sensitivity and selectivity. The chemical and design principles described will also be applicable in designing fluorescent probes for other small molecules.

Toward "CO in a Pill": Silica-Immobilized Organic CO Prodrugs for Studying the Feasibility of Systemic Delivery of CO via In Situ Gastrointestinal CO Release.

Carbon monoxide (CO), an endogenous signaling molecule, is known to exert a range of pharmacological effects, including anti-inflammation, organ protection, and antimetastasis in various animal models. We have previously shown the ability of organic prodrugs to deliver CO systemically through oral administration. As part of our efforts for the further development of these prodrugs, we are interested in minimizing the potential negative impact of the "carrier" portion of the prodrug. Along this line, we have previously published our work on using benign "carriers" and physically trapping the "carrier" portion in the gastrointestinal (GI) tract. We herein report our feasibility studies on using immobilized organic CO prodrugs for oral CO delivery while minimizing systemic exposure to the prodrug and the "carrier portion." In doing so, we immobilize a CO prodrug to silica microparticles, which are generally recognized as safe by the US FDA and known to provide large surface areas for loading and water accessibility. The latter point is essential for the hydrophobicity-driven activation of the CO prodrug. Amidation-based conjugation with silica is shown to provide 0.2 mmol/g loading degree, effective prodrug activation in buffer with comparable kinetics as the parent prodrug, and stable tethering to prevent detachment. One representative silica conjugate, SICO-101, is shown to exhibit anti-inflammation activity in LPS-challenged RAW264.7 cells and to deliver CO systemically in mice through oral administration and GI CO release. We envision this strategy as a general approach for oral CO delivery to treat systemic and GI-specific inflammatory conditions.

Synthesis and biological evaluation of tert-butyl ester and ethyl ester prodrugs of L-γ-methyleneglutamic acid amides for cancer.

In cancer cells, glutaminolysis is the primary source of biosynthetic precursors. Recent efforts to develop amino acid analogues to inhibit glutamine metabolism in cancer have been extensive. Our lab recently discovered many L-γ-methyleneglutamic acid amides that were shown to be as efficacious as tamoxifen or olaparib in inhibiting the cell growth of MCF-7, SK-BR-3, and MDA-MB-231 breast cancer cells after 24 or 72 h of treatment. None of these compounds inhibited the cell growth of nonmalignant MCF-10A breast cells. These L-γ-methyleneglutamic acid amides hold promise as novel therapeutics for the treatment of multiple subtypes of breast cancer. Herein, we report our synthesis and evaluation of two series of tert-butyl ester and ethyl ester prodrugs of these L-γ-methyleneglutamic acid amides and the cyclic metabolite and its tert-butyl esters and ethyl esters on the three breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 and the nonmalignant MCF-10A breast cell line. These esters were found to suppress the growth of the breast cancer cells, but they were less potent compared to the L-γ-methyleneglutamic acid amides. Pharmacokinetic (PK) studies were carried out on the lead L-γ-methyleneglutamic acid amide to establish tissue-specific distribution and other PK parameters. Notably, this lead compound showed moderate exposure to the brain with a half-life of 0.74 h and good tissue distribution, such as in the kidney and liver. Therefore, the L-γ-methyleneglutamic acid amides were then tested on glioblastoma cell lines BNC3 and BNC6 and head and neck cancer cell lines HN30 and HN31. They were found to effectively suppress the growth of these cancer cell lines after 24 or 72 h of treatment in a concentration-dependent manner. These results suggest broad applications of the L-γ-methyleneglutamic acid amides in anticancer therapy.

Immulina as an Immunostimulatory Supplement: Formulation and Pharmacological Studies.

Immulina is a commercially available extract of Arthrospira platensis enriched with bacterial lipoproteins that acts as a potent Toll-like receptor 2 agonist. However, the immunostimulatory effect of Immulina is not well understood in vivo. Here, to devise an Immulina formulation suitable for in vivo oral gavage dosing, Immulina nanosuspension was prepared and freeze-dried to yield lyophilized nano-Immulina, which had an average particle size of around 300 nm and fully retained the bioactivity as a Toll-like receptor 2 agonist. Compared to the regular Immulina powder, lyophilized nano-Immulina notably accelerated the dissolution in aqueous media. Immulina nanosuspension was found to stimulate the production of proinflammatory cytokines in murine bone marrow-derived dendritic cells and macrophages. The immune response to Immulina was investigated in healthy mice by longitudinally monitoring the phagocytic activity of circulating neutrophils as a surrogate marker. Following daily oral ingestion of Immulina nanosuspension (10 mg/mouse/day), the phagocytic activity of circulating neutrophils was significantly elevated, suggesting an important mechanism for Immulina to enhance innate immunity.

Subcutaneous Delivery of Albumin: Impact of Thermosensitive Hydrogels.

Albumin demonstrates remarkable promises as a versatile carrier for therapeutic and diagnostic agents. However, noninvasive delivery of albumin-based therapeutics has been largely unexplored. In this study, injectable thermosensitive hydrogels were evaluated as sustained delivery systems for Cy5.5-labeled bovine serum albumin (BSA-Cy5.5). These hydrogels were prepared using aqueous solutions of Poloxamer 407 (P407) or poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), which could undergo temperature-triggered phase transition and spontaneously solidify into hydrogels near body temperature, serving as in situ depot for tunable cargo release. In vitro, these hydrogels were found to release BSA-Cy5.5 in a sustained manner with the release half-life of BSA-Cy5.5 from P407 and PLGA-PEG-PLGA hydrogels at 16 h and 105 h, respectively. Without affecting the bioavailability, subcutaneous administration of BSA-Cy5.5-laden P407 hydrogel resulted in delayed BSA-Cy5.5 absorption, which reached the maximum plasma level (Tmax) at 24 h, whereas the Tmax for subcutaneously administered free BSA-Cy5.5 solution was 8 h. Unexpectedly, subcutaneously injected BSA-Cy5.5-laden PLGA-PEG-PLGA hydrogel did not yield sustained BSA-Cy5.5 plasma level, the bioavailability of which was significantly lower than that of P407 hydrogel (p < 0.05). The near-infrared imaging of BSA-Cy5.5-treated mice revealed that a notable portion of BSA-Cy5.5 remained trapped within the subcutaneous tissues after 6 days following the subcutaneous administration of free solution or hydrogels, suggesting the discontinuation of BSA-Cy5.5 absorption irrespective of the formulations. These results suggest the opportunities of developing injectable thermoresponsive hydrogel formulations for subcutaneous delivery of albumin-based therapeutics.

Inducing Apoptosis through Upregulation of p53: Structure-Activity Exploration of Anthraquinone Analogs.

We previously reported a series of p53-elevating anthraquinone compounds with considerable cytotoxicity for acute lymphatic leukemia (ALL) cells. To further develop this class of compounds, we examined the effect of a few key structural features on the anticancer structure-activity relationship in ALL cells. The active analogs showed comparable cytotoxicity and upregulation of p53 but did not induce significant downregulation of MDM2 as seen with the lead compound AQ-101, indicating the importance of the anthraquinone core scaffold for MDM2 regulation. The result from the current study not only contributes to the SAR framework of these anthraquinone derivatives but also opens up new chemical space for further optimization work.

Combined Delivery of Let-7b MicroRNA and Paclitaxel via Biodegradable Nanoassemblies for the Treatment of KRAS Mutant Cancer.

In the present study, we synthesized a novel cationic copolymer composed of polyethylene glycol 5000 (PEG5K), vitamin E (VE), and diethylenetriamine (DET) at 1:4:20 molar ratio. The resulting PEG5K-VE4-DET20 copolymer formed nanoassemblies when mixed with the neutral PEG5K-VE4 copolymer at 1:8 weight ratio, which were investigated as the nanocarriers for combined delivery of paclitaxel and let-7b mimic. We found that the PEG5K-VE4-DET20 nanoassemblies could entrap paclitaxel for an extended period and burst release the drug in the presence of cathepsin B, demonstrating the biodegradability of the copolymers. At N/P ratio of 12:1, the PEG5K-VE4-DET20 nanoassemblies formed stable polyplexes with let-7b mimic, which were efficiently taken up by tumor cells and underwent endosomal escape. In non-small cell lung cancer A549 cells that harbor mutant KRAS, paclitaxel and let-7b mimic-loaded nanoassemblies (N-PTX/let-7b) markedly potentiated the cytotoxicity of paclitaxel, induced apoptosis, and diminished the invasiveness of tumor cells. In mice bearing subcutaneous A549 xenografts, intravenous administration of N-PTX/let-7b retarded tumor growth more efficaciously than Taxol. Our study demonstrates the promise of the PEG5K-VE4-DET20 nanoassemblies for concurrent delivery of hydrophobic drugs and miRNA mimics.

Impact of CYP2C19 polymorphism on the pharmacokinetics of nelfinavir in patients with pancreatic cancer.

AIM: This study evaluated the influence of CYP2C19 polymorphisms on the pharmacokinetics of nelfinavir and its metabolite M8 in patients with pancreatic cancer. METHODS: Nelfinavir was administered orally to patients for over 10 days. The plasma concentrations of nelfinavir and M8 were measured by HPLC. The genotypes of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Pharmacokinetic profiles of nelfinavir and M8 were characterized by wide interindividual variability. The mean Cmax of nelfinavir in CYP2C19*1/*1 patients was 3.89 ± 0.40 (n = 3) and 5.12 ± 0.41 (n = 30) µg ml(-1) , while that of CYP2C19*1/*2 patients was 3.60 (n = 1) and 6.14 ± 0.31 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. For the M8 metabolite, the mean Cmax of CYP2C19*1/*1 patients was 1.06 ± 0.06 (n = 3) and 1.58 ± 0.27 (n = 30) µg ml(-1) , while those of CYP2C19*1/*2 patients were 1.01 (n = 1) and 1.23 ± 0.15 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The area under the plasma concentration-time curve (AUC(0,12 h)) values of nelfinavir for CYP2C19*1/*1 patients were 28.90 ± 1.27 and 38.90 ± 4.99 µg ml(-1) ·h and for CYP2C19*1/*2 patients, AUC(0,12 h) was 28.20 (n = 1) and 40.22 ± 3.17 (n = 5) µg ml(-1) ·h at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The Cmax of nelfinavir was significantly higher (P <0.05) in CYP2C19*1/*2 patients but there was no statistical difference in AUC(0,12 h). CONCLUSION: CYP2C19*1/*2 genotype modestly affected the pharmacokinetic profiles of nelfinavir and M8 in patients with locally advanced pancreatic cancer.

Reduction-responsive crosslinked micellar nanoassemblies for tumor-targeted drug delivery.

PURPOSE: The purpose of the study was to devise and evaluate crosslinked nanoassemblies to achieve enhanced drug delivery to tumors. METHODS: A novel copolymer comprised of polyethylene glycol 5000 (PEG114), Vitamin E (VE) and thioctic acid (TA) with a molar ratio of PEG114:VE:TA at 1:4:4 was synthesized. The resulting PEG114-VE4-TA4 copolymer self-assembled into micelles, which formed polydisulfide crosslinks catalyzed by dithiothreitol. Employing paclitaxel as a model drug, the crosslinked PEG114-VE4-TA4 micelles were characterized for the physicochemical and biological properties. The pharmacokinetics and anticancer efficacy of paclitaxel-loaded crosslinked PEG114-VE4-TA4 micelles were assessed in a human ovarian cancer xenograft murine model. RESULTS: The crosslinked PEG114-VE4-TA4 micelles demonstrated markedly improved thermodynamic and kinetic stability. The disulfide crosslinks were responsive to the intracellular level of glutathione, which caused rapid disassembly of the micelles and accelerated drug release. Intravenous administration of paclitaxel-loaded crosslinked PEG114-VE4-TA4 micelles yielded approximately 3-fold and 5-fold higher plasma concentration than the non-crosslinked micelles and Taxol®, respectively, leading to increased drug accumulation in the tumor. Importantly, paclitaxel-loaded crosslinked micelles exerted superior tumor growth repression compared to the non-crosslinked counterparts and Taxol®. CONCLUSIONS: These results suggest that the crosslinked PEG114-VE4-TA4 nanocarrier system is a promising platform for the delivery of hydrophobic anticancer agents.

Enhanced tumor delivery of gemcitabine via PEG-DSPE/TPGS mixed micelles.

Gemcitabine is a potent anticancer drug approved for the treatment of pancreatic, non-small-cell lung, breast, and ovarian cancers. The major deficiencies of current gemcitabine therapy, however, are its rapid metabolic inactivation and narrow therapeutic window. Herein, we employed polyethylene glycol-b-distearoylphosphatidylethanolamine (PEG-DSPE)/tocopheryl polyethylene glycol 1000 succinate (TPGS) mixed micelles as a delivery system, to improve the pharmacokinetic characteristics of gemcitabine and enhance its antitumor efficacy. By conjugating stearic acid to gemcitabine and subsequently encapsulating stearoyl gemcitabine (GemC18) within PEG-DSPE/TPGS mixed micelles, the deamination of gemcitabine was delayed in vitro and in vivo. Importantly, compared to free gemcitabine, GemC18-loaded micelles pronouncedly prolonged the circulation time of gemcitabine and elevated its concentration in the tumor by 3-fold, resulting in superior antitumor efficacy in mice bearing human pancreatic cancer BxPC-3 xenografts. Our findings demonstrate the promise of PEG-DSPE/TPGS mixed micelles as a nanocarrier system for the delivery of gemcitabine to achieve safer and more efficacious therapeutic outcomes.

Liposomal Delivery of MIW815 (ADU-S100) for Potentiated STING Activation.

Stimulator of interferon genes (STING) agonists can improve the anticancer efficacy of immune checkpoint blockade by amplifying tumor immunogenicity. However, the clinical translation of cyclic dinucleotides (CDNs) as STING agonists is hindered by their poor drug-like properties. In this study, we investigated the design criteria for DOTAP/cholesterol liposomes for the systemic delivery of ADU-S100 and delineated the impact of key formulation factors on the loading efficiency, serum stability, and STING agonistic activity of ADU-S100. Our findings demonstrate that the cationic liposomal formulation of ADU-S100 can be optimized to greatly potentiate STING activation in antigen-presenting cells.

Systemic Protein Delivery via Inhalable Liposomes: Formulation and Pharmacokinetics.

The enormous and thin alveolar epithelium is an attractive site for systemic protein delivery. Considering the excellent biocompatibility of phospholipids with endogenous pulmonary surfactant, we engineered dimyristoylphosphatidylcholine (DMPC)-based liposomes for pulmonary administration, using Cy5.5-labeled bovine serum albumin (BSA-Cy5.5) as a model protein payload. The level of cholesterol (Chol) and surface modification with PEG in inhalable liposomes were optimized iteratively based on the encapsulation efficiency, the release kinetics in the simulated lung fluid, and the uptake in murine RAW 264.7 macrophages. The plasma pharmacokinetics of BSA-Cy5.5-encapsulated liposomes with the composition of DMPC/Chol/PEG at 85:10:5 (molar ratio) was studied in mice following intratracheal aerosolization, in comparison with that of free BSA-Cy5.5 solution. The biodisposition of BSA-Cy5.5 was continuously monitored using whole-body near-infrared (NIR) fluorescence imaging for 10 days. We found that the systemic bioavailability of BSA-Cy5.5 from inhaled liposomes was 22%, which was notably higher than that of inhaled free BSA-Cy5.5. The mean residence time of BSA-Cy5.5 was markedly prolonged in mice administered intratracheally with liposomal BSA-Cy5.5, which is in agreement with the NIR imaging results. Our work demonstrates the great promise of inhalable DMPC-based liposomes to achieve non-invasive systemic protein delivery.

Activated charcoal dispersion of carbon monoxide prodrugs for oral delivery of CO in a pill.

A novel orally bioavailable solid formulation to deliver a gaseous signaling molecule, carbon monoxide (CO), was developed by adsorbing oxalyl saccharin, a newly developed organic CO prodrug, in activated charcoal (AC). The resulting solid dispersion formulation addresses key developability issues of this CO prodrug. By taking advantage of the large surface area of AC, the paradoxical problem of low water solubility of the prodrug and the requirement of hydrolysis to release CO is resolved, and the need for an organic cosolvent is completely circumvented. The AC formulation also mitigates the adverse effect of low pH on the CO release yield, allowing steady CO release in simulated gastric and intestine fluids. This formulation allows encapsulation in normal and enteric-coated gel capsules, which enables controllable CO delivery to the upper or lower GI system. It also features an advantage of trapping CO prodrug and CO release product in the AC, therefore lowering systemic absorption of these chemicals. Through in-vivo pharmacokinetic studies in mice, the AC formulation showed better efficiency of delivering CO through oral administration compared to the prodrug dosed with an organic cosolvent. The AC formulation has also been applied to address similar developability issues of another cheletropic reaction-based CO prodrug. We envision the wide applicability of this formulation in facilitating the future development of CO-based therapeutics.

Evaluating the regulation of transporter proteins and P-glycoprotein in rats with cholestasis and its implication for digoxin clearance.

BACKGROUND: Cardiac and hepatic functionality are intertwined in a multifaceted relationship. Pathologic processes involving one may affect the other through a variety of mechanisms, including hemodynamic and membrane transport effects. AIM: To better understand the effect of extrahepatic cholestasis on regulations of membrane transporters involving digoxin and its implication for digoxin clearance. METHODS: Twelve adult rats were included in this study; baseline hepatic and renal laboratory values and digoxin pharmacokinetic (PK) studies were established before evenly dividing them into two groups to undergo bile duct ligation (BDL) or a sham procedure. After 7 d repeat digoxin PK studies were completed and tissue samples were taken to determine the expressions of cell membrane transport proteins by quantitative western blot and real-time polymerase chain reaction. Data were analyzed using SigmaStat 3.5. Means between pre-surgery and post-surgery in the same experimental group were compared by paired t-test, while independent t-test was employed to compare the means between sham and BDL groups. RESULTS: Digoxin clearance was decreased and liver function, but not renal function, was impaired in BDL rats. BDL resulted in significant up-regulation of multidrug resistance 1 expression in the liver and kidney and its down-regulation in the small intestine. Organic anion transporting polypeptides (OATP)1A4 was up-regulated in the liver but down-regulated in intestine after BDL. OATP4C1 expression was markedly increased in the kidney following BDL. CONCLUSION: The results suggest that cell membrane transporters of digoxin are regulated during extrahepatic cholestasis. These regulations are favorable for increasing digoxin excretion in the kidney and decreasing its absorption from the intestine to compensate for reduced digoxin clearance due to cholestasis.

Sulfonamides as a new scaffold for hypoxia inducible factor pathway inhibitors.

Solid tumors generally grow under hypoxic conditions, a pathophysiological change, which activates the expression of genes responsible for malignant, aggressive, and treatment-refractory properties. Hypoxia inducible factor (HIF) is the chief transcription factor regulating hypoxia-driven gene expression. Therefore, the HIF pathway has become a critical target for cancer therapeutics development. We screened a privileged library of about 10,000 natural-product-like compounds using a cell-based assay for HIF-dependent transcriptional activity and identified several arylsulfonamide HIF pathway inhibitors. Among these compounds, the most potent ones showed an IC(50) of ∼0.5 µM in the hypoxia-responsive element (HRE)-luciferase reporter system. Further studies are needed to fully elucidate the mechanism of action of this class of compounds and their structure-activity relationship.

Inhalable liposomes for treating lung diseases: clinical development and challenges.

Inhalation delivery of liposomal drugs has distinct advantages for the treatment of pulmonary diseases. Inhalable liposomes of several drugs are currently undergoing clinical trials for a range of indications in the lungs. Herein, general principles of pulmonary delivery as well as the clinical development of inhalable liposomal drugs are reviewed.

"CO in a pill": Towards oral delivery of carbon monoxide for therapeutic applications.

Along with the impressive achievements in understanding the endogenous signaling roles and mechanism(s) of action of carbon monoxide (CO), much research has demonstrated the potential of using CO as a therapeutic agent for treating various diseases. Because of CO's toxicity at high concentrations and the observed difference in toxicity profiles of CO depending on the route of administration, this review analyzes and presents the benefits of developing orally active CO donors. Such compounds have the potential for improved safety profiles, enhancing the chance for developing CO-based therapeutics. In this review, the difference between inhalation and oral administration in terms of toxicity, CO delivery efficiency, and the potential mechanism(s) of action is analyzed. The evolution from CO gas inhalation to oral administration is also extensively analyzed by summarizing published studies up to date. The concept of "CO in a pill" can be achieved by oral administration of novel formulations of CO gas or appropriate CO donors.

Therapeutic targets, novel drugs, and delivery systems for diabetes associated NAFLD and liver fibrosis.

Type 2 diabetes mellitus (T2DM) associated non-alcoholic fatty liver disease (NAFLD) is the fourth-leading cause of death. Hyperglycemia induces various complications, including nephropathy, cirrhosis and eventually hepatocellular carcinoma (HCC). There are several etiological factors leading to liver disease development, which involve insulin resistance and oxidative stress. Free fatty acid (FFA) accumulation in the liver exerts oxidative and endoplasmic reticulum (ER) stresses. Hepatocyte injury induces release of inflammatory cytokines from Kupffer cells (KCs), which are responsible for activating hepatic stellate cells (HSCs). In this review, we will discuss various molecular targets for treating chronic liver diseases, including homeostasis of FFA, lipid metabolism, and decrease in hepatocyte apoptosis, role of growth factors, and regulation of epithelial-to-mesenchymal transition (EMT) and HSC activation. This review will also critically assess different strategies to enhance drug delivery to different cell types. Targeting nanocarriers to specific liver cell types have the potential to increase efficacy and suppress off-target effects.

Adapting decarbonylation chemistry for the development of prodrugs capable of in vivo delivery of carbon monoxide utilizing sweeteners as carrier molecules.

Carbon monoxide as an endogenous signaling molecule exhibits pharmacological efficacy in various animal models of organ injury. To address the difficulty in using CO gas as a therapeutic agent for widespread applications, we are interested in developing CO prodrugs through bioreversible caging of CO in an organic compound. Specifically, we have explored the decarboxylation-decarbonylation chemistry of 1,2-dicarbonyl compounds. Examination and optimization of factors favorable for maximal CO release under physiological conditions led to organic CO prodrugs using non-calorific sweeteners as leaving groups attached to the 1,2-dicarbonyl core. Attaching a leaving group with appropriate properties promotes the desired hydrolysis-decarboxylation-decarbonylation sequence of reactions that leads to CO generation. One such CO prodrug was selected to recapitulate the anti-inflammatory effects of CO against LPS-induced TNF-α production in cell culture studies. Oral administration in mice elevated COHb levels to the safe and efficacious levels established in various preclinical and clinical studies. Furthermore, its pharmacological efficacy was demonstrated in mouse models of acute kidney injury. These studies demonstrate the potential of these prodrugs with benign carriers as orally active CO-based therapeutics. This represents the very first example of orally active organic CO prodrugs with a benign carrier that is an FDA-approved sweetener with demonstrated safety profiles in vivo.