A novel phosphodiesterase inhibitor for the treatment of chronic liver injury and metabolic diseases.

BACKGROUND AND AIMS: Chronic liver disease leads to ~2 million deaths annually. Cyclic AMP (cAMP) signaling has long been studied in liver injury, particularly in the regulation of fatty acid (FA) ß-oxidation and pro-inflammatory polarization of tissue-resident lymphocytes. Phosphodiesterase 4B inhibition has been explored as a therapeutic modality, but these drugs have had limited success and are known to cause significant adverse effects. The PDE4 inhibitor 2-(4-([2-(5-Chlorothiophen-2-yl)-5-ethyl-6-methylpyrimidin-4-yl]amino)phenyl)acetic acid) (known as A-33) has yet to be explored for the treatment of metabolic diseases. APPROACH AND RESULTS: Herein, we evaluated the efficacy of A-33 in the treatment of animal models of alcohol-associated liver disease and steatotic liver disease. We demonstrated that A-33 effectively ameliorated the signs and symptoms of chronic liver disease, resulting in significant decreases in serum alanine aminotransferase and aspartate aminotransferase levels, decreased overall fat and collagen deposition in the liver, decreased intrahepatic triglyceride concentrations, and normalized expression of genes related to ß-oxidation of fatty acids, inflammation, and extracellular matrix deposition. We also designed and synthesized a novel analog of A-33, termed MDL3, which inhibited both phosphodiesterase 4B and PDE5A and was more effective in ameliorating pathophysiological signs and symptoms of liver injury and inflammation. In addition, MDL3 re-sensitized obese mice to glucose and significantly inhibited the pathological remodeling of adipose tissue, which was not observed with A-33 administration. CONCLUSIONS: In conclusion, we synthesized and demonstrated that MDL3, a novel phosphodiesterase 4B and PDE5A inhibitor, presents a promising avenue of exploration for treating chronic liver disease.

Anti-CLL1 liposome loaded with miR-497-5p and venetoclax as a novel therapeutic strategy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a lethal hematologic malignancy. Chemotherapy resistance results in a dismal survival rate of 1-2 years in older adults with AML. Therefore, novel therapies are urgently required. In this context, microRNA (miRNA)-based treatments remain an untapped strategy in AML. Using patient-derived specimens, we found increased inflammatory cytokines, including interleukin-6 (IL-6) in the serum of older adults with AML, and decreased miR-497-5p in CD34+ leukemic blasts. Target prediction revealed that miR-497-5p could directly target mitogen-activated protein kinase-1 (MAP2K1) mRNA to indirectly target cytokines and the JAK/STAT signaling pathway through the p38-MAPK signaling pathway, potentially inhibiting leukemic growth and overcoming chemoresistance from venetoclax. To improve miRNA delivery and minimize off-target effects, which represent key barriers to clinical translation, we developed liposomes for co-delivery of miR-497-5p and venetoclax. We decorated our liposomes with a peptide targeting CLL1, which is present on 92% of leukemia blasts while being absent in normal hematopoietic cells. This targeted approach demonstrated high efficacy in inhibiting AML growth in mice with minimal toxicity, as well as reduced exposure to chemoresistance. Our findings suggested that anti-CLL1-decorated, miR-497-5p, and venetoclax-loaded liposomes represent a promising novel miRNA-based therapeutic, which should be investigated further as a strategy to reduce venetoclax resistance in AML.

A Nationwide Nepalese Study to Establish Reference Intervals for Major Biochemical Analytes with Elucidation of Nepalese Features of Reference Values.

Despite immense importance of reference intervals (RIs) for clinical diagnosis, there have been no reliable RIs available for Nepalese. Hence, this nationwide study was organized to establish RIs for 30 common biochemical parameters. This study was conducted following the harmonized protocol provided by IFCC Committee on Reference Interval and Decision Limits (C-RIDL) with recruitment of 617 apparently healthy volunteers (18 - 65 years) by near-equal gender balance from 5 major cities. Fasting blood were collected, serum was separated and measured collectively using Beckman-Coulter/Olympus AU480 chemistry analyzer. The sources of variations of reference values (RVs) were evaluated by multiple regression analysis and nested ANOVA. Latent abnormal values exclusion (LAVE) method was applied to reduce influence of latent diseases. RIs were standardized based on a value-assigned serum panel provided by C-RIDL. By ANOVA, no between-city differences were observed, while sex-related changes were typically noted for urate, creatinine, iron, γ-glutamyl transferase (GGT), immunoglobulin M, and transferrin, but not for high-density lipoprotein cholesterol. Age-related changes were observed for total cholesterol (TC), triglyceride, low-density lipoprotein cholesterol, and C-reactive protein (CRP). RIs were successfully derived all parametrically. The LAVE procedure was effective in lowering upper limits for aspartate aminotransferase, alanine aminotransferase (ALT), and CRP. Compared to other collaborating countries, Nepalese RIs were low for urea, cholesterols, ALT, and high for triglyceride, GGT, CRP, immunoglobulin G, and complements. The RIs for major chemistry analytes were derived and standardized for nationwide use in Nepal. This study distinctly elucidated sources of variation and international features of Nepalese RIs. Supplementary Information: The online version supplementary material available at 10.1007/s12291-023-01123-6.

G6PD deficiency in malaria endemic areas of Nepal.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is currently a threat to malaria elimination due to risk of primaquine-induced haemolysis in G6PD deficient individuals. The World Health Organization (WHO) recommends G6PD screening before providing primaquine as a radical treatment against vivax malaria. However, evidence regarding the prevalence and causing mutations of G6PD deficiency in Nepal is scarce. METHODS: A cross-sectional, population-based, prevalence study was carried out from May to October 2016 in 12 malaria-endemic districts of Nepal. The screening survey included 4067 participants whose G6PD status was determined by G6PD Care Start™ rapid diagnostic test and genotyping. RESULTS: The prevalence of G6PD deficiency at the national level was 3.5% (4.1% among males and 2.1% among females). When analysed according to ethnic groups, G6PD deficiency was highest among the Janajati (6.2% overall, 17.6% in Mahatto, 7.7% in Chaudhary and 7.5% in Tharu) and low among Brahman and Chhetri (1.3%). District-wise, prevalence was highest in Banke (7.6%) and Chitwan (6.6%). Coimbra mutation (592 C>T) was found among 75.5% of the G6PD-deficient samples analysed and Mahidol (487 G>A) and Mediterranean (563 C>T) mutations were found in equal proportions in the remaining 24.5%. There was no specific geographic or ethnic distribution for the three mutations. CONCLUSIONS: This study has identified populations with moderate to high prevalence of G6PD deficiency which provides strong evidence supporting the WHO recommendations to screen G6PD deficiency at health facility level before the use of primaquine-based radical curative regimen for Plasmodium vivax.

Factors associated with quality of life among chronic kidney disease patients in Nepal: a cross-sectional study.

BACKGROUND: Chronic kidney disease (CKD) leads to decreased quality of life (QOL) by increasing the risk of death during the progression of its pathogenesis. However, many factors can be improved to support QOL. This study aimed to assess QOL among CKD patients in Nepal and to determine the factors associated with their QOL. METHOD: A cross-sectional study was used for data collection. CKD cases receiving medical attention in the Bir Hospital in Mahaboudh, Kathmandu; Tribhuvan University Teaching Hospital in Maharajgunj, Kathmandu; Sumeru Hospital in Dhapakhel, Lalitpur; and Shahid Dharma Bhakta National Transplant Centre in Bhaktapur between August and October 2019 were invited to participate in the study. A validated questionnaire and the kidney disease quality of life short form (KDQOL-SF™ 1.3) were used to assess QOL. A questionnaire was completed by the researcher in face-to-face interviews. Logistic regression was used to detect the associations between variables at the significance level of α = 0.05. RESULTS: A total of 440 participants were recruited into the study: 56.59% were males, 74.32% were aged between 31 and 70 years, 25.68% were illiterate, and 82.95% were unemployed. The prevalence of good QOL among CKD in the domains of the physical component summary (PCS), mental component summary (MCS), and kidney disease component summary (KDCS) with and without hemodialysis were 53.64, 22.05, 21.28, and 13.19%, respectively. After controlling for all potential confounding factors, eight variables were found to be associated with good QOL in the domain of PCS: age, education, stage of CKD, hemodialysis, transporting oneself to a hospital, health insurance, medical expenses, and perceived lack of difficulty in handling medical expenses. Six variables were associated with good QOL in the domain of MCS after controlling for all potential confounding factors: residence, stage of CKD, transporting oneself to a hospital, health insurance, medical expenses, and perceived lack of difficulty in handling medical expenses. CONCLUSIONS: Public health interventions should be developed and implemented to improve QOL among CKD patients in Nepal by focusing on older female patients who have low education, live in rural areas and no health insurance.

Roles of microRNAs in T cell immunity: Implications for strategy development against infectious diseases.

T cell immunity plays a vital role in pathogen infections. MicroRNA (miRNAs) are small, single-stranded noncoding RNAs that regulate T cell immunity by targeting key transcriptional factors, signaling proteins, and cytokines associated with T cell activation, differentiation, and function. The dysregulation of miRNA expression in T cells may lead to specific immune responses and can provide new therapeutic opportunities against various infectious diseases. Here, we summarize recent studies that focus on the roles of miRNAs in T cell immunity and highlight miRNA functions in prevalent infectious diseases. Additionally, we also provide insights into the functions of extracellular vesicle miRNAs and attempt to delineate the mechanism of miRNA sorting into extracellular vesicles and their immunomodulatory functions. Moreover, methodologies and strategies for miRNA delivery against infectious diseases are summarized. Finally, potential strategies for miRNA-based therapies are proposed.

Design of Hedgehog pathway inhibitors for cancer treatment.

Hedgehog (Hh) signaling is involved in the initiation and progression of various cancers and is essential for embryonic and postnatal development. This pathway remains in the quiescent state in adult tissues but gets activated upon inflammation and injuries. Inhibition of Hh signaling pathway using natural and synthetic compounds has provided an attractive approach for treating cancer and inflammatory diseases. While the majority of Hh pathway inhibitors target the transmembrane protein Smoothened (SMO), some small molecules that target the signaling cascade downstream of SMO are of particular interest. Substantial efforts are being made to develop new molecules targeting various components of the Hh signaling pathway. Here, we have discussed the discovery of small molecules as Hh inhibitors from the diverse chemical background. Also, some of the recently identified natural products have been included as a separate section. Extensive structure-activity relationship (SAR) of each chemical class is the focus of this review. Also, clinically advanced molecules are discussed from the last 5 to 7 years. Nanomedicine-based delivery approaches for Hh pathway inhibitors are also discussed concisely.

Polymeric Micellar Delivery of Novel Microtubule Destabilizer and Hedgehog Signaling Inhibitor for Treating Chemoresistant Prostate Cancer.

Castration-resistant prostate cancer that has become resistant to docetaxel (DTX) represents one of the greatest clinical challenges in the management of this malignancy. There is an urgent need to develop novel therapeutic agents to overcome chemoresistance and improve the overall survival of patients. We have designed a novel microtubule destabilizer (2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (QW-296) and combined it with a newly synthesized hedgehog (Hh) signaling pathway inhibitor 2-chloro-N 1-[4-chloro-3-(2-pyridinyl)phenyl]-N 4,N 4- bis(2-pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) to treat taxane-resistant (TXR) prostate cancer. The combination of QW-296 and MDB5 exhibited stronger anticancer activity toward DU145-TXR and PC3-TXR cells and suppressed tumor colony formation when compared with single-drug treatment. Because these drugs are hydrophobic, we synthesized the mPEG-p(TMC-MBC) [methoxy-poly(ethylene glycol)-block-poly(trimethylene carbonate-co-2-methyl-2-benzoxycarbonyl-propylene carbonate)] copolymer, which could self-assemble into micelles with loading capacities of 8.13% ± 0.75% and 9.12% ± 0.69% for QW-296 and MDB5, respectively. Further, these micelles provided controlled the respective drug release of 58% and 42% release of QW-296 and MDB5 within 24 hours when dialyzed against PBS (pH 7.4). We established an orthotopic prostate tumor in nude mice using stably luciferase expressing PC3-TXR cells. There was maximum tumor growth inhibition in the group treated with the combination therapy of QW-296 and MDB5 in micelles compared with their monotherapies or combination therapy formulated in cosolvent. The overall findings suggest that combination therapy with QW-296 and MDB5 has great clinical potential to treat TXR prostate cancer, and copolymer mPEG-p(TMC-MBC) could serve as an effective delivery vehicle to boost therapeutic efficacy in vivo.

Therapeutic Potential of OMe-PS-miR-29b1 for Treating Liver Fibrosis.

Trans-differentiation of quiescent hepatic stellate cells (HSCs) into active myofibroblasts secretes excess amounts of extracellular matrix (ECM) proteins. miR-29b1 has the potential to treat liver fibrosis, because it targets several profibrotic genes. We previously demonstrated that miR-29b1 and the hedgehog (Hh) pathway inhibitor GDC-0449 could, together, inhibit the activation of HSCs and ECM production in common bile-duct-ligated (CBDL) mice. Herein, we determined the effect of chemical modifications of miR-29b1 on its stability, immunogenicity, and Argonaute-2 (Ago2) loading in vitro, after modifying its antisense strand with phosphorothioate (PS-miR-29b1), 2'-O-methyl-phosphorothioate (OMe-miR-29b1), locked nucleic acid (LNA-miR-29b1), and N,N'-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN-miR-29b1). Chemical modifications significantly improved stability of miR-29b1 in 50% FBS. Among all the modified miRNAs tested, OMe-PS-miR-29b1 showed the highest stability with low immunogenicity, without the loss of efficacy in vitro. Therefore, OMe-PS-miR-29b1 was complexed with poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-tetraethylenepentamine (mPEG-b-PCC-g-DC-g-TEPA) cationic micelles, and anti-fibrotic efficacy was evaluated in CBDL mice. There was a significant improvement in liver histology and decrease in the levels of injury markers. Further, mRNA/protein levels of collagen, α-SMA, and TIMP-1 were significantly lower for the OMe-PS-miR-29b1-loaded micelles compared to miR-29b1-loaded micelles. In conclusion, micellar delivery of OMe-PS-miR-29b1 is a promising strategy to treat liver fibrosis.

Self-assembling lisofylline-fatty acid conjugate for effective treatment of diabetes mellitus.

Lisofylline is an anti-inflammatory agent with proven anti-diabetic activity. Its high solubility and rapid metabolism results in poor bioavailability and short half-life, limiting its clinical utility. We have synthesized Lisofylline-Linoleic acid (LSF-LA) conjugate which self-assembled into micelles (156.9 nm; PDI 0.187; CMC 1 µg/mL; aggregation number 54) without any surfactant and showed enhanced cellular uptake. It protected MIN6 insulinoma cells from cytokine induced cell death and enhanced insulin production under inflammatory conditions. It also suppressed the proliferation of activated peripheral blood mononuclear cells and reduced the production of inflammatory cytokines, IFN-γ and TNF-α. LSF-LA micelles exhibited reduced protein binding, significantly higher half-life (5.7-fold) and higher apparent volume of distribution (5.3-fold) than free LSF. In T1D animals, reduced blood glucose levels were observed at a reduced dose (~15 mg/kg, once daily of LSF-LA micelles vs. 25 mg/kg, twice daily of free LSF) that was further confirmed by immunohistochemical analysis.

Cholesterol and Morpholine Grafted Cationic Amphiphilic Copolymers for miRNA-34a Delivery.

miR-34a is a master tumor suppressor playing a key role in the several signaling mechanisms involved in cancer. However, its delivery to the cancer cells is the bottleneck in its clinical translation. Herein we report cationic amphiphilic copolymers grafted with cholesterol (chol), N, N-dimethyldipropylenetriamine (cation chain) and 4-(2-aminoethyl)morpholine (morph) for miR-34a delivery. The copolymer interacts with miR-34a at low N/P ratios (∼2/1) to form nanoplexes of size ∼108 nm and a zeta potential ∼ +39 mV. In vitro studies in 4T1 and MCF-7 cells indicated efficient transfection efficiency. The intracellular colocalization suggested that the copolymer effectively transported the FAM labeled siRNA into the cytoplasm within 2 h and escaped from the endo-/lysosomal environment. The developed miR-34a nanoplexes inhibited the breast cancer cell growth as confirmed by MTT assay wherein 28% and 34% cancer cell viability was observed in 4T1 and MCF-7 cells, respectively. Further, miR-34a nanoplexes possess immense potential to induce apoptosis in both cell lines.

Coadministration of Polymeric Conjugates of Docetaxel and Cyclopamine Synergistically Inhibits Orthotopic Pancreatic Cancer Growth and Metastasis.

PURPOSE: The aim of this study was to determine whether co-administration of hedgehog (Hh) pathway inhibitor cyclopamine (CYP) and microtubule stabilizer docetaxel (DTX) as polymer-drug conjugates, methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-cyclopamine) (P-CYP) and methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-docetaxel) (P-DTX) could synergistically inhibit orthotopic pancreatic tumor growth in NSG mice. METHODS: P-DTX and P-CYP were synthesized from mPEG-b-PCC through carbodiimide coupling reaction and characterized by 1H-NMR. The micelles were prepared by film hydration and particle size was measured by dynamic light scattering (DLS). Cytotoxicity, apoptosis and cell cycle analysis of P-DTX and P-CYP were evaluated in MIA PaCa-2 cells. In vivo efficacy of P-DTX and P-CYP were evaluated in NSG mice bearing MIA PaCa-2 cells derived orthotopic pancreatic tumor. RESULTS: P-CYP and P-DTX self-assembled into micelles of <90 nm and their combination therapy efficiently inhibited the proliferation of MIA PaCa-2 cells, induced apoptosis and cell cycle arrest at M-phase more efficiently than P-CYP and P-DTX monotherapies. Furthermore, the combination therapy of P-CYP and P-DTX significantly reduced Hh component expression compared to P-CYP alone as determined by Western blot analysis. Lastly, the combination therapy induced greater inhibition of orthotopic pancreatic tumor growth in NSG mice compared to their monotherapies. CONCLUSION: Combination of polymer conjugated anticancer drug (P-DTX) with polymer conjugated Hh inhibitor (P-CYP) enhanced pancreatic cancer cell killing, apoptosis as well as in vivo tumor growth inhibition with no obvious toxicities.

Biodistribution of Self-Assembling Polymer-Gemcitabine Conjugate after Systemic Administration into Orthotopic Pancreatic Tumor Bearing Mice.

Therapeutic efficacy of gemcitabine (GEM) is severely limited due to its rapid metabolism by enzymatic deamination in vivo. We recently determined its therapeutic efficacy before (F-GEM) and after conjugation to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (mPEG-b-PCC-g-GEM-g-DC, abbreviated as P-GEM) in subcutaneous and orthotopic pancreatic tumor bearing mice. In this study, pharmacokinetic (PK) parameters and biodistribution profiles of F-GEM and P-GEM were determined after intravenous injection into orthotopic pancreatic tumor bearing NSG mice. To assess the short-term toxicity, the levels of hematological, hepatic, and renal injury markers were measured after 24 h postadministration into these mice. P-GEM was distributed to all the major organs, with higher accumulation in the liver, spleen, and tumor compared to F-GEM. Area under the curve (AUC), elimination half-life (t1/2), and mean residence time (MRT) of P-GEM treated group were significantly higher compared to those of F-GEM treated group: 246,425 ± 1605 vs 83,591 ± 1844 ng/mL × h as AUC, 5.77 ± 2.02 vs 1.99 ± 0.09 h as t1/2, and 4.45 ± 0.15 vs 1.12 ± 0.13 h as MRT. Further, P-GEM exhibited negligible systemic toxicity as evidenced by almost similar alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for both P-GEM and F-GEM. These results suggest that P-GEM protects GEM from degradation and provides sustained drug release, resulting in enhanced GEM delivery to the tumor by more than 2.5-fold compared to F-GEM. Hence, P-GEM is a promising gemcitabine conjugated polymeric micelle for treating pancreatic cancer.

EGFR-Targeted Cationic Polymeric Mixed Micelles for Codelivery of Gemcitabine and miR-205 for Treating Advanced Pancreatic Cancer.

Gemcitabine (GEM), a first-line chemotherapy for pancreatic cancer undergoes rapid metabolism and develops chemoresistance after repeated administration. We previously demonstrated that the combination of GEM and miR-205 provides an effective therapeutic strategy to sensitize GEM-resistant pancreatic cancer cells. Since epidermal growth factor receptor (EGFR) is overexpressed in pancreatic cancer cells, in this study, we aimed to deliver mixed micelles containing GEM and miR-205 decorated with EGFR-targeting cetuximab (C225) monoclonal antibody for targeted therapy. Cetuximab C225 was conjugated to malemido-poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (C225-PEG-PCD) to prepare mixed micelles with mPEG-b-PCC-g-GEM-g-DC-g-TEPA for targeted codelivery of GEM and miR-205. This mixed micelle formulation showed a significant enhancement in EGFR-mediated cellular uptake in GEM-resistant MIA PaCa-2R cells. Further, an enhanced tumor accumulation of C225-micelles conjugated with near-infrared fluorescent Cy7.5 dye and Dy677-labeled miR-205 in orthotopic pancreatic tumor bearing NSG mice was evident after systemic administration. In addition, inhibition of tumor growth was also observed with increased apoptosis and reduced EMT after treatment with C225-micelles containing GEM and miR-205. Therefore, we believe that the targeted delivery of GEM and miR-205 in combination could be a novel strategy for treating advanced pancreatic cancer.

Pharmacokinetics and Biodistribution of GDC-0449 Loaded Micelles in Normal and Liver Fibrotic Mice.

PURPOSES: To determine the pharmacokinetic parameters and biodistribution of GDC-0449 loaded polymeric micelles after systemic administration into common bile duct ligation (CBDL) induced liver fibrotic mice. METHODS: We used GDC-0449 encapsulated methoxy poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (PEG-PCD) non-targeted polymeric micelles for GDC-0449 delivery to normal and liver fibrotic mice. To maximize GDC-0449 delivery to hepatic stellate cells (HSCs), mixed micelles formulations with 10, 20 and 30% w/w mannose-6-phosphate (M6P)-conjugated micelles were administered to normal and liver fibrotic mice for targeting M6P/IGF-IIR overexpressed on activated HSCs and biodistribution of GDC-0449 was determined at 30 and 120 min post systemic administration. RESULTS: GDC-0449 distributed to all major organs after systemic administration of drug loaded micelles, with higher accumulation in the liver of both normal and fibrotic mice. The plasma concentration versus time profiles suggest rapid clearance of GDC-0449 after systemic administration of drug loaded micelles in both normal and fibrotic mice, with similar plasma clearance (CL), area under the curve (AUCint) and volume of distribution at steady state (Vss). However, there is significant increase in GDC-0449 accumulation in the liver when M6P-conjugated mixed micelles were injected, with the highest GDC-0449 concentration in the liver with mixed micelles carrying 30% M6P-conjugated polymer. HSCs accounted for 14.19% of GDC-0449 accumulation for M6P-targeted micelles in fibrotic mice compared to 5.62% of non-targeted micelles in the liver uptake study. CONCLUSION: M6P-conjugated GDC-0449 loaded mixed micelles may be used as a potential drug delivery vehicle for treating liver fibrosis.

Combination therapy of paclitaxel and cyclopamine polymer-drug conjugates to treat advanced prostate cancer.

Repeated treatments with chemotherapeutic agent(s) fail due to cancer stem cells (CSCs) and chemoresistance regulated by microRNAs (miRNA) whose expression alters owing to dysfunctional signaling pathways including Hedgehog (Hh) signaling. We previously demonstrated the combination of Hh inhibitor cyclopamine (CYP) and paclitaxel (PTX) effectively inhibit PTX-resistant cells and side population, a cell fraction rich in CSCs. In this study, we synthesized mPEG-b-PCC-g-PTX-g-DC (P-PTX) and mPEG-b-PCC-g-CYP-g-DC (P-CYP) polymer-drug conjugates, which they self-assembled into micelles. The combination of P-PTX and P-CYP alleviated PTX resistance and suppressed tumor colony formation. Further, combination therapy inhibited Hh signaling and up-regulated tumor suppressor miRNAs. We established orthotopic prostate tumor in nude mice and there was significant tumor growth inhibition in the group treated with the combination therapy of P-PTX and P-CYP compared with monotherapy. In conclusion, this combination therapy of P-PTX and P-CYP has the potential to treat chemoresistant prostate cancer.

Micelle Mixtures for Coadministration of Gemcitabine and GDC-0449 To Treat Pancreatic Cancer.

Hedgehog (Hh) signaling plays an important role in the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). Although gemcitabine (GEM) has been used as a first-line therapy for PDAC, its rapid metabolism and short plasma half-life restrict its use as a single chemotherapy. Combination therapy with more than one drug is a promising approach for treating cancer. Herein, we report the use of methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (mPEG-b-PCC-g-DC) copolymer for conjugating GEM and encapsulating a Hh inhibitor, vismodegib (GDC-0449), into its hydrophobic core for treating PDAC. Our objective was to determine whether the micelle mixtures of these two drugs could show better response in inhibiting Hh signaling pathway and restraining the proliferation and metastasis of pancreatic cancer. The in vivo stability of GEM significantly increased after conjugation, which resulted in its increased antitumor efficacy. Almost 80% of encapsulated GDC-0449 and 19% conjugated GEM were released in vitro at pH 5.5 in 48 h in a sustained manner. The invasion, migration, and colony forming features of MIA PaCa-2 cells were significantly inhibited by micelle mixture carrying GEM and GDC-0449. Remarkable increase in PARP cleavage and Bax proved increased apoptosis by this combination formulation compared to individual micelles. This combination therapy efficiently inhibited tumor growth, increased apoptosis, reduced Hh ligands PTCH-1 and Gli-1, and lowered EMT-activator ZEB-1 when injected to athymic nude mice bearing subcutaneous tumor generated using MIA PaCa-2 cells compared to monotherapy as observed from immunohistochemical analysis. In conclusion, micelle mixtures carrying GEM and GDC-0449 have the potential to treat pancreatic cancer.

EGFR-Targeted Polymeric Mixed Micelles Carrying Gemcitabine for Treating Pancreatic Cancer.

The objective of this study was to design GE11 peptide (YHWYGYTPQNVI) linked micelles of poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-gemcitabine-graft-dodecanol (PEG-b-PCC-g-GEM-g-DC) for enhanced stability and target specificity of gemcitabine (GEM) to EGFR-positive pancreatic cancer cells. GE11-PEG-PCD/mPEG-b-PCC-g-GEM-g-DC mixed micelles showed EGFR-dependent enhanced cellular uptake, and cytotoxicity as compared to scrambled peptide HW12-PEG-PCD/mPEG-b-PCC-g-GEM-g-DC mixed micelles and unmodified mPEG-b-PCC-g-GEM-g-DC micelles. Importantly, GE11-linked mixed micelles preferentially accumulated in orthotopic pancreatic tumor and tumor vasculature at 24 h post systemic administration. GE11-linked mixed micelles inhibited orthotopic pancreatic tumor growth compared to HW12-linked mixed micelles, unmodified mPEG-b-PCC-g-GEM-g-DC micelles, and free GEM formulations. Tumor growth inhibition was mediated by apoptosis of tumor cells and endothelial cells as determined by immunohistochemical staining. In summary, GE11-linked mixed micelles is a promising approach to treat EGFR overexpressing cancers.

Synthesis and Characterization of a Novel Mycophenolic Acid-Quinic Acid Conjugate Serving as Immunosuppressant with Decreased Toxicity.

Mycophenolic acid (MPA) is one of the most commonly used immunosuppressive drugs for improving the outcome of cell and organ transplantations. However, an undesired adverse effect of MPA impedes its application in the clinics for post-transplant patients. By conjugating MPA to quinic acid (QA) via amide bonds, we synthesized a novel immunosuppressant, N-[2-[[(4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-1-oxo-4-hexen-1-yl]amino]ethyl]-(1α,3R,4α,5R)-1,3,4,5-tetrakis(acetyloxy)cyclohexanecarboxamide (abbreviated as MQ4), which exhibits improved stability demonstrated by its incubation in vitro with human plasma, suggesting its better resistance to hydrolytic degradation induced by plasma enzyme. While the immunosuppressive effect of MQ4 on human lymphocyte proliferation was partially compromised as shown by flow cytometry, significant decrease in cytotoxicity of MQ4 to insulin producing ß cells could compensate this drawback to some degree. There was a decreased level of apoptotic mediator caspase-3, which may contribute to the decreased toxicity of MQ4 to INS-1E cells. MQ4 could further improve insulin stimulation index and downregulate NFκB expression compared to physical mixing of QA to MPA. Taken together, MQ4 is a promising immunosuppressive agent for preventing and minimizing post-transplanted immune rejection.

Codelivery of small molecule hedgehog inhibitor and miRNA for treating pancreatic cancer.

Successful treatment of pancreatic ductal adenocarcinoma (PDAC) remains a challenge due to the desmoplastic microenvironment that promotes both tumor growth and metastasis and forms a barrier to chemotherapy. Hedgehog (Hh) signaling is implicated in initiation and progression of PDAC and also contributes to desmoplasia. While Hh levels are increased in pancreatic cancer cells, levels of tumor suppressor miR-let7b, which targets several genes involved in PDAC pathogenesis, is downregulated. Therefore, our overall objective was to inhibit Hh pathway and restore miR-let7b simultaneously for synergistically treating PDAC. miR-let7b and Hh inhibitor GDC-0449 could inhibit the proliferation of human pancreatic cancer cells (Capan-1, HPAF-II, T3M4, and MIA PaCa-2), and there was synergistic effect when miR-let7b and GDC-0449 were coformulated into micelles using methoxy poly(ethylene glycol)-block-poly(2-methyl- 2-carboxyl-propylenecarbonate-graft-dodecanol-graft-tetraethylene-pentamine) (mPEG-b-PCC-g-DC-g-TEPA). This copolymer self-assembled into micelles of <100 nm and encapsulated hydrophobic GDC-0449 into its core with 5% w/w drug loading and allowed complex formation between miR-let7b and its cationic pendant chains. Complete polyplex formation with miRNA was observed at the N/P ratio of 16/1. Almost 80% of GDC-0449 was released from the polyplex in a sustained manner in 2 days. miRNA in the micelle formulation was stable for up to 24 h in the presence of serum and high uptake efficiency was achieved with low cytotoxicity. This combination therapy effectively inhibited tumor growth when injected to athymic nude mice bearing ectopic tumor generated using MIA PaCa-2 cells compared to micelles carrying GDC-0449 or miR-let7b alone. Immunohistochemical analysis revealed decreased tumor cell proliferation with increased apoptosis in the animals treated with miR-let7b and GDC-0449 combination.