バイオポリマーを用いたシャペロン （Hsp90）標的型抗癌剤ナノ粒子の設計と開発

東洋大学201

Expression of Tryptophan 2,3-Dioxygenase in Metastatic Uveal Melanoma

Uveal melanoma (UM) is the most common primary eye malignancy in adults and up to 50% of patients subsequently develop systemic metastasis. Metastatic uveal melanoma (MUM) is highly resistant to immunotherapy. One of the mechanisms for resistance would be the immune-suppressive tumor microenvironment. Here, we have investigated the role of tryptophan 2,3-dioxygenase (TDO) in UM. Both TDO and indoleamine 2,3-dioxygenase (IDO) catalyze tryptophan and produce kynurenine, which could cause inhibition of T cell immune responses. We first studied the expression of TDO on tumor tissue specimens obtained from UM hepatic metastasis. High expression of TDO protein was confirmed in all hepatic metastasis. TDO was positive in both normal hepatocytes and the tumor cells with relatively higher expression in tumor cells. On the other hand, IDO protein remained undetectable in all of the MUM specimens. UM cell lines established from metastasis also expressed TDO protein and increasing kynurenine levels were detected in the supernatant of MUM cell culture. In TCGA database, higher TDO2 expression in primary UM significantly correlated to BAP1 mutation and monosomy 3. These results indicate that TDO might be one of the key mechanisms for resistance to immunotherapy in UM

Formulation, characterization and evaluation of morusin loaded niosomes for potentiation of anticancer therapy.

Morusin, a water-insoluble prenylated flavonoid is known for its numerous medicinal properties. It manifests its anticancer potential by suppression of genes involved in tumor progression. However, poor solubility of the drug results in low bioavailability and rapid degradation thus hindering its clinical utilization. In order to overcome this, we have synthesized a niosome system composed of non-ionic surfactant span 60 and cholesterol using a thin-layer evaporation technique to improve the aqueous-phase solubility of the drug. Highly cytocompatible niosomes of 479 nm average size with smooth and uniform spherical morphology were synthesized in a facile manner. Unlike free morusin, nanomorusin was found to be freely dispersible in aqueous media. Having an extremely high drug entrapment efficiency (97%), controlled and sustained release of morusin resulting in enhanced therapeutic efficacy was observed in cancer cell lines of 4 different lineages. The results demonstrate that the morusin-niosome system is a promising strategy for enhanced anti-cancer activity against multiple cancer types and could be an indispensable tool for future targeted chemotherapeutic strategies

Noninvasive amniotic fluid sampling to establish PK of azithromycin in pregnancy

Background PK studies to guide dosing of azithromycin (AZ) for pregnancy specific conditions, such as preterm premature rupture of membranes (PPROM), and data on accumulation of AZ in fetal compartment are lacking. We aim to evaluate feasibility of non-invasive collection of amniotic fluid (AF), validate an assay for AZ in AF, and describe concentration of AZ in the amniotic cavity over one week following a single maternal dose. Methods Patients with PPROM treated with 1g AZ PO once and wore underwear lining pads to collect AF as it leaked. AF strained from each pad, up to 10cc collected, centrifuged and frozen. Calibration curve established using range of 1 to 200 ng/mL, with Azithromycin-D3 as internal standard. Spiked standards and samples were extracted with plasma to ACN ratio as 1:2, and centrifuged. The clean supernatant was subjected to LCMS runs using Thermo-Orbitrap coupled with Dionex 3000 UHPLC system under +ve ion mode and sample 5µL injection volume. The chromatographic separations were done using HSS XSelect C18 reverse phase column using 50:50 water and ACN with 0.1% FA as mobile phase, flow rate of 0.250 mL/min. The linearity equation (y= 10945x, r2\u3e0.99) established using average of 8 injections over 4 days; 2 injections per day. AZ from AF samples was quantitated in duplicate and expressed as concentration/time profile. Results Five patients were enrolled. Mean gestational age on admission with PPROM was 27.5 ±2.3wk with a median latency of 7 days [IQR 4-13]. A median of 2 samples/day [IQR 1-3] were collected per participant. Azithromycin was quantified in duplicate; intra-assay coefficient of variation was 17%. Azithromycin concentration was \u3c60ng/ml after day 3. Azithromycin concentration was positively correlated with IL-8 (r=0.38, p=0.03), IL1a (r=0.39, p=0.03), and IL-1b (r=0.36, p=0.04) in amniotic fluid. Conclusion This simple technique for noninvasive collection of AF allows for precise quantification of AZ in AF with LCMS. AZ persists in the fetal compartment for at least seven days after a single maternal dose, although not necessarily at an adequate inhibitory concentration.https://jdc.jefferson.edu/obgynposters/1014/thumbnail.jp

Smart carriers and nanohealers:A nanomedical insight on natural polymers

Biodegradable polymers are popularly being used in an increasing number of fields in the past few decades. The popularity and favorability of these materials are due to their remarkable properties, enabling a wide range of applications and market requirements to be met. Polymer biodegradable systems are a promising arena of research for targeted and site-specific controlled drug delivery, for developing artificial limbs, 3D porous scaffolds for cellular regeneration or tissue engineering and biosensing applications. Several natural polymers have been identified, blended, functionalized and applied for designing nanoscaffolds and drug carriers as a prerequisite for enumerable bionano technological applications. Apart from these, natural polymers have been well studied and are widely used in material science and industrial fields. The present review explains the prominent features of commonly used natural polymers (polysaccharides and proteins) in various nanomedical applications and reveals the current status of the polymer research in bionanotechnology and science sectors

LC-MS based stability-indicating method for studying the degradation of lonidamine under physical and chemical stress conditions

Background and purpose: Lonidamine is a hexokinase II inhibitor, works as an anticancer molecule, and is extensively explored in clinical trials. Limited information prevails about the stability-indicating methods which could determine the forced degradation of lonidamine under stressed conditions. Hence, we report the use of a rapid, sensitive, reproducible, and highly accurate liquid chromatography and mass spectrometry method to analyze lonidamine degradation. Experimental approach: The Xbridge BEH shield reverse phase C18 column (2.5 μm, 4.6 × 75 mm) using isocratic 50:50 water: acetonitrile with 0.1% formic acid can detect lonidamine with help of mass spectrometer in tandem with an ultraviolet (UV) detector at 260 nm wavelength. Findings/ Results: A linear curve with r2\u3e 0.99 was obtained for tandem liquid chromatography-mass spectrometry (LC-MS)-UV based detections. This study demonstrated (in the present set up of isocratic elution) that LC-MS based detection has a relatively high sensitivity (S/N (10 ng/mL): 220 and S/N (20 ng/mL): 945) and accuracy at lower detection and quantitation levels, respectively. In addition to developing the LC-MS method, we also report that the current method is stability-indicating and shows that lonidamine gets degraded over time under all three stress conditions; acidic, basic, and oxidative. Conclusion and implications: LC-MS based quantitation of lonidamine proved to be a better method compared to high-performance liquid chromatography (HPLC)-UV detections for mapping lonidamine degradation. This is the first report on the stability-indicating method for studying the forced degradation of lonidamine using LC-MS method

Heat Shock Protein 90 (Hsp90)-Inhibitor-Luminespib-Loaded-Protein-Based Nanoformulation for Cancer Therapy

Drugs targeting heat shock protein 90 (Hsp90) have been extensively explored for their anticancer potential in advanced clinical trials. Nanoformulations have been an important drug delivery platform for the anticancer molecules like Hsp90 inhibitors. It has been reported that bovine serum albumin (BSA) nanoparticles (NPs) serve as carriers for anticancer drugs, which have been extensively explored for their therapeutic efficacy against cancers. Luminespib (also known as NVP-AUY922) is a new generation Hsp90 inhibitor that was introduced recently. It is one of the most studied Hsp90 inhibitors for a variety of cancers in Phase I and II clinical trials and is similar to its predecessors such as the ansamycin class of molecules. To our knowledge, nanoformulations for luminespib remain unexplored for their anticancer potential. In the present study, we developed aqueous dispensable BSA NPs for controlled delivery of luminespib. The luminespib-loaded BSA NPs were characterized by SEM, TEM, FTIR, XPS, UV-visible spectroscopy and fluorescence spectroscopy. The results suggest that luminespib interacts by non-covalent reversible interactions with BSA to form drug-loaded BSA NPs (DNPs). Our in vitro evaluations suggest that DNP-based aqueous nanoformulations can be used in both pancreatic (MIA PaCa-2) and breast (MCF-7) cancer therapy

Pharmacokinetics of Ketamine at Dissociative Doses in an Adult Patient With Refractory Status Asthmaticus Receiving Extracorporeal Membrane Oxygenation Therapy.

PURPOSE: First-line management of severe asthma exacerbations include the use of inhaled short-acting β-agonists, anticholinergics, and systemic corticosteroids. Continuous intravenous ketamine given at dissociative doses may be a pharmacologic option in patients who are intubated with life-threatening severe bronchospasm unresponsive to standard therapy. We describe the case of a 44-year-old man admitted to the intensive care unit for status asthmaticus requiring intubation and mechanical ventilation. METHODS: The patient developed severe refractory hypercapnic respiratory failure necessitating additional respiratory support with veno-venous extracorporeal membrane oxygenation (ECMO) therapy. Ketamine treatment was initiated at 0.5 mg/kg/h continuous infusion on the day of admission for pain control and required up-titration to 2 mg/kg/h by intensive care unit day 4 for bronchodilation. Whole blood samples were obtained for pharmacokinetic analysis of ketamine during ECMO. FINDINGS: The plasma concentration at steady state was 1018.7 ng/mL, with an estimated clearance of 1.96 L/kg/h after up-titration. The Vd was 14.18 L/kg, the ke was 0.14 hr-1, and the t½ was 5 hours. IMPLICATIONS: Compared with healthy adults, there was a 6.5-fold increase in the Vd. However, the Vd was similar compared with critically ill patients not receiving ECMO. Further studies should focus on the effect of ECMO on ketamine pharmacokinetic properties

Clinical Proteomics of the Neglected Human Malarial Parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings

Development of a Dual Drug-Loaded, Surfactant-Stabilized Contrast Agent Containing Oxygen

Co-delivery of cancer therapeutics improves efficacy and encourages synergy, but delivery faces challenges, including multidrug resistance and spatiotemporal distribution of therapeutics. To address these, we added paclitaxel to previously developed acoustically labile, oxygen-core, surfactant-stabilized microbubbles encapsulating lonidamine, with the aim of developing an agent containing both a therapeutic gas and two drugs acting in combination. Upon comparison of unloaded, single-loaded, and dual-loaded microbubbles, size (~1.7 µm) and yield (~2 × 109 microbubbles/mL) (~1.7) were not statistically different, nor were acoustic properties (maximum in vitro enhancements roughly 18 dB, in vitro enhancements roughly 18 dB). Both drugs encapsulated above required doses calculated for head and neck squamous cell carcinoma, the cancer of choice. Interestingly, paclitaxel encapsulation efficiency increased from 1.66% to 3.48% when lonidamine was included. During preparation, the combination of single drug-loaded micelles gave higher encapsulation (µg drug/g microbubbles) than micelles loaded with either drug alone (lonidamine, 104.85 ± 22.87 vs. 87.54 ± 16.41), paclitaxel (187.35 ± 8.38 vs. 136.51 ± 30.66). In vivo intravenous microbubbles produced prompt ultrasound enhancement within tumors lasting 3-5 min, indicating penetration into tumor vasculature. The ability to locally destroy the microbubble within the tumor vasculature was confirmed using a series of higher intensity ultrasound pulses. This ability to locally destroy microbubbles shows therapeutic promise that warrants further investigation