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.

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.

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.

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.

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.

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.

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.

"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.

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.

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.

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.

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.

Redox-responsive nanoplatform for codelivery of miR-519c and gemcitabine for pancreatic cancer therapy.

Desmoplastic and hypoxic pancreatic cancer microenvironment induces aberrant expression of miRNAs and hypoxia-inducible factor-1α (HIF-1α) responsible for gemcitabine (GEM) resistance. We demonstrated that miR-519c was down-regulated in pancreatic cancer and transfection of miR-519c in GEM-resistant pancreatic cancer cells inhibited HIF-1α level under hypoxia. We synthesized redox-sensitive mPEG-co-P(Asp)-g-DC-g-S-S-GEM polymer, with GEM payload of 14% (w/w) and 90% GEM release upon incubation with l-glutathione. We synthesized mPEG-co-P(Asp)-g-TEPA-g-DC for complex formation with miRNA. Chemical modification of miR-519c with 2'-O-methyl phosphorothioate (OMe-PS) at 3' end enhanced its stability and activity without being immunogenic. Epidermal growth factor receptor targeting peptide GE11 decoration increased tumor accumulation of micelles after systemic administration and significantly inhibited orthotopic desmoplastic pancreatic cancer growth in NSG mice by down-regulating HIF-1α and genes responsible for glucose uptake and cancer cell metabolism. Our multifunctional nanomedicine of GEM and OMe-PS-miR-519c offers a novel therapeutic strategy to treat desmoplasia and hypoxia-induced chemoresistance in pancreatic cancer.

Towards "CO in a pill": Pharmacokinetic studies of carbon monoxide prodrugs in mice.

Carbon monoxide (CO) is a known endogenous signaling molecule with potential therapeutic indications in treating inflammation, cancer, neuroprotection, and sickle cell disease among many others. One of the hurdles in using CO as a therapeutic agent is the development of pharmaceutically acceptable delivery forms for various indications. Along this line, we have developed organic CO prodrugs that allow for packing this gaseous molecule into a dosage form for the goal of "carbon monoxide in a pill." This should enable non-inhalation administration including oral and intravenous routes. These prodrugs have previously demonstrated efficacy in multiple animal models. To further understand the CO delivery efficiency of these prodrugs in relation to their efficacy, we undertook the first pharmacokinetic studies on these prodrugs. In doing so, we selected five representative prodrugs with different CO release kinetics and examined their pharmacokinetics after administration via oral, intraperitoneal, and intravenous routes. It was found that all three routes were able to elevate systemic CO level with delivery efficiency in the order of intravenous, oral, and intraperitoneal routes. CO prodrugs and their CO-released products were readily cleared from the circulation. CO prodrugs demonstrate promising pharmaceutical properties in terms of oral CO delivery and minimal drug accumulation in the body. This represents the very first study of the interplay among CO release kinetics, CO prodrug clearance, route of administration, and CO delivery efficiency.

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.

Exploiting Exosomes in Cancer Liquid Biopsies and Drug Delivery.

Exosomes are cell-derived nanovesicles that transfer molecular cargo from donor to recipient cells and mediate intercellular communication. Advancement in elucidating the biological capabilities and functionalities of exosomes has revealed the striking roles of exosomes as conveyors of bioactive molecules across the biological barriers. Tumor-derived exosomes hold great promise to serve as a liquid biopsy tool for cancer diagnosis and prognosis, as large quantities of exosomes are excreted by tumor cells continuously into the circulation, carrying the molecular cargo (DNA, RNA, proteins) reflective of the genetic and signaling alterations in tumor cells. Two inherent characteristics of exosomes offer important opportunities for drug delivery: their superb transcellular permeability and biocompatibility. Exosomes are uniquely capable of encapsulating a variety of payloads and deliver them to the target tissues. This review discusses the potential of tumor-derived exosomes in cancer liquid biopsies as well as the underlying mechanisms. Furthermore, the recent progress of developing exosomes as highly versatile and efficient drug carriers is also summarized.

Pharmacokinetics and biodistribution of polymeric micelles containing miRNA and small-molecule drug in orthotopic pancreatic tumor-bearing mice.

Rationale: Successful treatment of pancreatic cancer remains a challenge due to desmoplasia and prevalence of KRAS mutation. While hedgehog (Hh) ligand levels are upregulated in pancreatic cancer cells and contribute to desmoplasia, there is significant downregulation of tumor suppressor let-7b, which targets mutant KRAS, C-MYC and several other genes involved in pancreatic cancer progression, invasion, and metastasis. We recently explored combination therapy of GDC-0449 (Hh inhibitor) and let-7b mimic using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) micelles in pancreatic tumor mouse model. Here, our objective was to determine the biodistribution (BD), pharmacokinetics (PK), therapeutic efficacy and toxicity of this micellar formulation. Methods: We determined the PK of micelles encapsulating Cy5.5-let-7b and GDC-0449 following intravenous injection in orthotopic pancreatic tumor-bearing NSG mice at doses of 2 mg/kg and 10 mg/kg, respectively. Mice were scanned for fluorescence by IVIS to determine the biodistribution of Cy5.5-let-7b at the whole-body level, and its concentration in plasma and major organs was determined by measuring fluorescence using a fluorimeter and by real-time RT-PCR. GDC-0449 concentration was determined by LC/MS/MS. Therapeutic efficacy and toxicity of the micellar formulation of let-7b and GDC-0449 was also determined after two weeks of treatment. Results: The use of a micellar formulation markedly prolonged the elimination half-life (t1/2, e) of Cy5.5-let-7b in plasma from 0.49 ± 0.19 h to 2.65 ± 0.46 h and increased the area-under-the-curve (AUC 0-∞ ) by 7-fold, while t1/2,e and AUC 0-∞ of GDC-0449 were increased by 1.78-fold and 3.2-fold, respectively. The micelles significantly decreased the clearance of both encapsulated let-7b mimic and GDC-0449 compared to the emulsion formulation. Compared to the emulsion counterpart, the micellar formulation elevated the delivery of Cy5.5-let-7b and GDC-0449 to the orthotopic pancreatic tumor tissue by 7.8- and 4.2-fold, respectively. Furthermore, there was a significant reduction in tumor volume and negligible systemic toxicity as evident by hematological parameters and histological evaluation. Conclusion: PEG-b-PCC-g-DC-g-TEPA micelles carrying GDC-0449 and let-7b mimic have great potential to improve drug delivery for pancreatic cancer treatment.

BAI1 Suppresses Medulloblastoma Formation by Protecting p53 from Mdm2-Mediated Degradation.

Adhesion G protein-coupled receptors (ADGRs) encompass 33 human transmembrane proteins with long N termini involved in cell-cell and cell-matrix interactions. We show the ADGRB1 gene, which encodes Brain-specific angiogenesis inhibitor 1 (BAI1), is epigenetically silenced in medulloblastomas (MBs) through a methyl-CpG binding protein MBD2-dependent mechanism. Knockout of Adgrb1 in mice augments proliferation of cerebellar granule neuron precursors, and leads to accelerated tumor growth in the Ptch1+/- transgenic MB mouse model. BAI1 prevents Mdm2-mediated p53 polyubiquitination, and its loss substantially reduces p53 levels. Reactivation of BAI1/p53 signaling axis by a brain-permeable MBD2 pathway inhibitor suppresses MB growth in vivo. Altogether, our data define BAI1's physiological role in tumorigenesis and directly couple an ADGR to cancer formation.