3‐Acetyl‐11‐keto‐β‐boswellic acid loaded‐polymeric nanomicelles for topical anti‐inflammatory and anti‐arthritic activity

Objectives The aim of this study was to develop 3‐acetyl‐11‐keto‐β‐boswellic acid (AKBA)‐loaded polymeric nanomicelles for topical anti‐inflammatory and anti‐arthritic activity. Methods Polymeric nanomicelles of AKBA were developed by a radical polymerization method using N‐isopropylacrylamide, vinylpyrrolidone and acrylic acid. The polymeric nanomicelles obtained were characterized by Fourier transform infrared (FTIR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). In‐vitro and in‐vivo evaluations of AKBA polymeric nanomicelles gel were carried out for enhanced skin permeability and anti‐inflammatory and anti‐arthritic activity. Key findings TEM and DLS results demonstrated that polymeric nanomicelles were spherical with a mean diameter approximately 45 nm. FTIR data indicated a weak interaction between polymer and AKBA in the encapsulated system. The release of drug in aqueous buffer (pH 7.4) from the polymeric nanomicelles was 23 and 55% after 2 and 8 h, respectively, indicating sustained release. In‐vitro skin permeation studies through excised abdominal skin indicated a threefold increase in skin permeability compared with AKBA gel containing the same amount of AKBA as the AKBA polymeric nanomicelles gel. The AKBA polymeric nanomicelle gel showed significantly enhanced anti‐inflammatory and anti‐arthritic activity compared with the AKBA gel. Conclusions This study suggested that AKBA polymeric nanomicelle gel significantly enhanced skin permeability, and anti‐inflammatory and anti‐arthritic activity.     

A novel Tc‐99m and fluorescence‐labeled arginine‐arginine‐leucine‐containing peptide as a multimodal tumor imaging agent in a murine tumor model

We developed a Tc‐99m and TAMRA‐labeled peptide, Tc‐99m arginine‐arginine‐leucine (RRL) peptide (TAMRA‐GHEG‐ECG‐RRL), to target tumor cells and evaluated the diagnostic performance of Tc‐99m TAMRA‐GHEG‐ECG‐RRL as a dual‐modality imaging agent for tumor in a murine model. TAMRA‐GHEG‐ECG‐RRL was synthesized using Fmoc solid‐phase peptide synthesis. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with PC‐3 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc‐99m, Tc‐99m TAMRA‐GHEG‐ECG‐RRL complexes were prepared in high yield (>96%). The K_d of Tc‐99m TAMRA‐GHEG‐ECG‐RRL determined by saturation binding was 41.7 ± 7.8 nM. Confocal microscopy images of PC‐3 cells incubated with TAMRA‐GHEG‐ECG‐RRL showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc‐99m TAMRA‐GHEG‐ECG‐RRL in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of RRL. Specific uptake of Tc‐99m TAMRA‐GHEG‐ECG‐RRL was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In conclusion, in vivo and in vitro studies revealed substantial uptake of Tc‐99m TAMRA‐GHEG‐ECG‐RRL in tumors. Tc‐99m TAMRA‐GHEG‐ECG‐RRL has potential as a dual‐modality tumor imaging agent.     

Platelet lysate formulations based on mucoadhesive polymers for the treatment of corneal lesions

Objectives Growth factors contained in platelet α‐granules initiate and modulate tissue repair and are proposed for the treatment of soft and hard‐tissue surgical conditions and in the management of non‐healing wounds. Platelet lysate is a hemoderivative obtained from platelet‐rich plasma and is capable of releasing a pool of growth factors. Many medical and surgical techniques have been proposed for the treatment of corneal lesions; management of these conditions remains problematic and healing with standard protocols is unattainable. The aim of this study was to develop formulations suitable for prolonging the contact of platelet lysate with the damaged cornea for the time necessary to exert a therapeutic effect. Methods Two vehicles, one based on polyacrylic acid and one based on chitosan, were autoclaved and loaded with platelet lysate and the resultant formulations were characterized for rheology, mucoadhesion, vehicle compatibility and stability. The proliferation effect was tested on two cell culture types (rabbit corneal epithelial cells and fibroblasts). An in‐vitro wound‐healing test was performed on fibroblasts. In both cases the formulations were compared with platelet lysate diluted with saline at the same concentration. Findings Both formulations maintained the rheological and mucoadhesive properties of the vehicles and the proliferative activity of platelet lysate. The chitosan formulation was able to significantly enhance epithelial cell growth even after storage of up to 2 weeks (in‐use conditions), while the polyacrylic acid formulation was less efficient, probably due to the characteristics of the cell model used. Conclusions The in‐vitro wound‐healing test performed on fibroblasts confirmed the differences between the two vehicles. The effect induced by the platelet lysate and chitosan formulation was faster than that of the polyacrylic acid formulation and complete in‐vitro wound repair was achieved within 48 h.     

Targeted gene delivery to hepatocytes with galactosylated amphiphilic cyclodextrins

Objectives Achieving targeted delivery of gene medicines is desirable to maximise activity. Here, galactosylated amphiphilic cyclodextrins (CDs) are examined in terms of their ability to transfect asialoglycoprotein receptor‐bearing HepG2 cells. Methods Cationic amphiphilic CDs were synthesised as well as amphiphilic CDs bearing galactose‐targeting ligands with different linker lengths. Binding of galactosylated CDs to a galactose‐specific lectin was examined by surface plasmon resonance. CDs were formulated with and without the helper lipid DOPE and complexed with plasmid DNA. Transfection was evaluated by luciferase assay. Intracellular trafficking was assessed by confocal microscopy. Key findings Binding of targeted CDs to a galactose‐specific lectin was achieved. Binding decreased with linker length between the galactosyl group and the CD core. Contrary to the lectin binding results, transfection levels increased with an increase in linker length from 7 atoms to 15. Compared to non‐targeted formulations, a significant increase in transfection was observed only in the presence of the helper lipid DOPE. Confocal microscopy revealed that DOPE caused a pronounced effect on cellular distribution. Conclusions The galactose‐targeting ligand induced substantial increases in transfection over non‐targeted formulations when DOPE was included, indicating the potential for targeted gene delivery using CD‐based delivery systems.     

2‐Hydroxypropyl‐β‐cyclodextrin‐modified SLN of paclitaxel for overcoming p‐glycoprotein function in multidrug‐resistant breast cancer cells

Objectives This study aimed to evaluate the potential of solid lipid nanoparticles (SLNs) of paclitaxel (PTX) modified with a 2‐hydroxypropyl‐β‐cyclodextrin system to enhance cellular accumulation of PTX into p‐glycoprotein (p‐gp)‐expressing cells. Methods The PTX‐loaded‐SLNs consisted of lipid (stearic acid) and surfactants (lecithin and poloxamer 188) and were then modified with 2‐hydroxypropyl‐β‐cyclodextrin by a sonication method. Key findings In terms of cytotoxicity, PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin showed higher cytotoxicity than other formulations. In particular, the cellular uptake of PTX from PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin was about 5.8‐ and 1.5‐fold higher than that from PTX solution and unmodified PTX‐loaded SLNs in MCF‐7/ADR cells, respectively. After a 4‐h incubation, clear fluorescence images inside cells were observed over time. When PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin were incubated with MCF‐7/ADR cells for 4 h, cellular uptake of PTX increased 1.7‐fold versus that of PTX in the presence of verapamil. Conclusions These results suggest that optimized SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin may have potential as an oral drug delivery system for PTX.     

The association of serum FGF23 and non‐alcoholic fatty liver disease is independent of vitamin D in type 2 diabetes patients

Recent studies have shown that circulating fibroblast growth factor (FGF) 23 and vitamin D levels are closely correlated with insulin resistance. The aim of this study was to investigate the relationship among serum FGF 23 levels, serum 25‐hydroxyvitamin D [25(OH)D] levels, and non‐alcoholic fatty liver disease (NAFLD) in Chinese patients with type 2 diabetes mellitus (T2DM). This study enrolled 331 hospitalized T2DM patients (209 patients with NAFLD and 122 patients without NAFLD). Serum FGF23 levels were measured using a sandwich enzyme‐linked immunosorbent assay. Serum 25(OH)D levels were determined by an electrochemiluminescence immunoassay. NAFLD was diagnosed by hepatic ultrasound, and the fatty liver index (FLI) was calculated to quantify hepatic steatosis. Results showed that T2DM patients with NAFLD had significantly higher serum FGF23 levels (44.17 [37.92‐51.30] pg/mL vs 40.21 [34.07‐48.33] pg/mL, P = .002), but lower serum 25(OH)D levels (16.43 [12.70‐21.37] ng/mL vs 19.59 [13.78‐26.26] ng/mL, P = .002) than those without NAFLD. Moreover, the incidence rate of NAFLD increased with increasing serum FGF23 levels and decreased with increasing 25(OH)D levels (both P < .05). Logistic regression analysis showed that both serum FGF23 and 25(OH)D levels were independent factors for NAFLD (both P < .05). Furthermore, a multiple stepwise regression analysis also revealed that both serum FGF23 and 25(OH)D levels were independently correlated with FLI (both P < .01). In conclusion, both high FGF23 and low vitamin D levels showed an independent relationship with NAFLD in Chinese T2DM patients, indicating that FGF23 and vitamin D function via different regulatory pathways in the liver.     

Synthesis and evaluation of Tc‐99m and fluorescence‐labeled elastin‐derived peptide,VAPG for multimodal tumor imaging in murine tumor model

We developed a Tc‐99m and fluorescence‐labeled peptide, Tc‐99m TAMRA‐GHEG‐ECG‐VAPG to target tumor cells and evaluated the diagnostic performance as a dual‐modality imaging agent for tumor in a murine model. TAMRA‐GHEG‐ECG‐VAPG was synthesized by using Fmoc solid‐phase peptide synthesis. Radiolabeling of TAMRA‐GHEG‐ECG‐VAPG with Tc‐99m was done by using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with SW620 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry by using confocal microscopy. After radiolabeling procedures with Tc‐99m, Tc‐99m TAMRA‐GHEG‐ECG‐VAPG complexes were prepared in high yield (>96%). The K_d of Tc‐99m TAMRA‐GHEG‐ECG‐VAPG determined by saturation binding was 16.8 ± 3.6 nM. Confocal microscopy images of SW620 cells incubated with TAMRA‐GHEG‐ECG‐VAPG showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc‐99m TAMRA‐GHEG‐ECG‐VAPG in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of VAPG. Specific uptake of Tc‐99m TAMRA‐GHEG‐ECG‐VAPG was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In vivo and in vitro studies revealed substantial uptake of Tc‐99m TAMRA‐GHEG‐ECG‐VAPG in tumor cells. Tc‐99m TAMRA‐GHEG‐ECG‐VAPG has potential as a dual‐modality tumor imaging agent.     

Reduced Nrf2 activation in PI3K phosphorylation‐impaired vitiliginous keratinocytes increases susceptibility to ROS‐generating chemical‐induced apoptosis

Keratinocytes in affected epidermis of vitiligo patients are known to have impaired activation of the PI3K/AKT pathway. Based on critical roles of keratinocytes and oxidative stress in vitiligo development, this study examined whether keratinocytes with impaired PI3K activation were more vulnerable to apoptosis caused by oxidative stress from phenolic compounds, p‐tert‐butylphenol (4‐TBP) and hydroquinone (HQ). Cell viability assay, FACS analysis, ELISA for TNF‐α or IL‐1a, ROS assay, Western blot analysis for Nrf2 expression, and confocal microscopy with anti‐Nrf2 and phospho‐PI3K antibodies were done on primary cultured normal human keratinocytes with or without PI3K knockdown in the presence or absence of chemical treatment or antioxidant. Immunofluorescence staining using anti‐Nrf2, phospho‐PI3K, TNF‐ɑ, and IL‐1ɑ antibodies, ROS assay using dihydroethidium, and TUNEL assay were performed on sets of depigmented and normally pigmented skin from vitiligo patients. Results showed that 4‐TBP or HQ treatment increased apoptosis and the expression levels of TNF‐ɑ, IL‐1ɑ, and ROS in PI3K‐knockdown keratinocytes which reduced Nrf2 nuclear translocation compared to control keratinocytes. These changes were significantly recovered by an antioxidant treatment. Depigmented epidermis of vitiligo patients also showed lower levels of Nrf2 and phospho‐PI3K but higher levels of ROS, TNF‐ɑ, IL‐1ɑ, and ROS with more TUNEL‐positive cells. Therefore, impaired PI3K activation in keratinocytes in depigmented epidermis of vitiligo patients are vulnerable to apoptosis caused by ROS‐generating chemicals due to reduced Nrf2 activation.     

短肽支架纤维对创面表皮碱性成纤维生长因子及表皮细胞生长因子的促进作用

目的：探讨自组装纤维支架肽RADA16在皮肤烫伤中的治疗作用。方法：在SD大鼠背部以电力机械方法制造深Ⅱ度烧伤模型,对照治疗组以0.9%NaCl溶液代替,烧伤后按时换药,并在不同时间段（伤后7、10、14d分别取烧伤修复创面的皮肤组织进行免疫组化染色,记录创面碱性成纤维细胞生长因子（bFGF）及表皮细胞生长因子（EGF）的表达,并以图像处理系统软件记录生长因子表达的半定量检测。比较了短肽处理组及空白对照组创面生长因子表达的灰度值。结果：免疫组织化学结果显示,在烧伤修复的不同修复期,短肽处理组bFGF和EGF均明显表达在新生表皮组织中,与空白对照组比较,差异均有统计学意义（P〈0.05）。结论：自组装纤维支架肽对烧伤皮肤生长因子的表达具有促进作用。     

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC‐4 cells

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti‐cancer activities. Herein, we investigated the anti‐oral cancer activity of casticin on SCC‐4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca²⁺ production, levels of ΔΨ_m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G₂/M phase arrest in SCC‐4 cells. Casticin promoted ROS and Ca²⁺ productions, decreases the levels of ΔΨ_m, promoted caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC‐4 cells. In conclusion, casticin decreased cell number through G₂/M phase arrest and the induction of cell apoptosis through caspase‐ and mitochondria‐dependent pathways in SCC‐4 cells.     

Topical delivery of fluorescence (6‐Cf) labeled and radiolabeled (99m‐Tc) cisplatin and imiquimod by a dual drug delivery system

The present investigation deals with the development of topical (top.) formulation for co‐delivery of cisplatin and imiquimod to enhance the antitumor efficacy of the drug for skin‐cited malignancies even in immune compromised patient. Cisplatin (CDDP) and imiquimod‐loaded protransfersome gel (CDDP‐Imi‐Pts gel) formulation was characterized for entrapment efficiency, pH, and viscosity. Further, fluorescence‐labeled (6‐carboxyfluorescin) and radiolabeled (^99mtechnetium) drug‐loaded formulations were compared with respect to biodistribution and pharmacokinetic studies. Gamma scintigraphy of mice following radiolabeled formulation administrations was performed to accomplish the localization of drugs in various organs. The percentage entrapment efficiency of cisplatin and imiquimod in the protransfersome gel formulations were found to be 36.22 ± 6.41 and 63.11 ± 3.73. The skin/blood localization ratio of 1.096, 120.13, 0.174, and 349.88 was found for intraperitoneal radiolabeled drug solution (^99m‐Tc‐CDDP‐Imi‐Sol), top. radiolabeled drug‐loaded protransfersome gel formulation (^99m‐Tc‐CDDP‐Imi‐Pts gel), intraperitoneal 6‐carboxyfluorescin labeled drug solution (6‐Cf‐CDDP‐Imi‐Sol), top. 6‐carboxyfluorescin labeled drug‐loaded protransfersome gel formulation (6‐Cf‐CDDP‐Imi‐Pts gel), respectively after 0.5h of administration. CDDP‐Imi‐Pts gel has a potential for site specific delivery and reduces the systemic toxicity of anti cancer drugs. These findings suggest that the cisplatin–imiquimod co‐delivery offers an attractive, novel approach for treatment of skin‐cited malignancies.     

Titanium dioxide nanoparticles translocate through differentiated Caco‐2 cell monolayers,without disrupting the barrier functionality or inducing genotoxic damage

The widespread use of titanium dioxide nanoparticles (TiO₂NPs) in commercial food products makes intestinal cells a suitable target. Accordingly, we have used the human colon adenocarcinoma Caco‐2 cells to detect their potential harmful effects. Caco‐2 cells can differentiate in to enterocytic‐like cells, forming consistent cell monolayers and are used as a model of the intestinal barrier. Using both undifferentiated and differentiated Caco‐2 cells, we have explored a set of biomarkers, aiming to evaluate undesirable effects associated to TiO₂NP exposure. Results indicate non‐toxic effects in exposures ranging 1‐200 μg ml^?1. Significant differences were observed in cell uptake, with a higher amount of incorporated TiO₂NPs in undifferentiated cells, as visualized using confocal microscopy. In well‐established monolayers, translocation was detected using both confocal microscopy and transmission electron microscopy with energy‐dispersive X‐ray spectroscopy. In spite of the observed uptake and translocation, TiO₂NP exposures did not modify the integrity of the monolayer, as measured using the transepithelial electrical resistance and Lucifer yellow methods. The potential genotoxic effects in differentiated cells were evaluated in the comet assay, with and without formamidopyrimidine DNA glycosylase enzyme to detect oxidatively the damaged DNA bases. Although some changes were detected at the lower dose (10 μg ml^?1), no effects were observed at higher doses.     

Comparative study of liposomes,transfersomes, ethosomes and cubosomes for transcutaneous immunisation: characterisation and in vitro skin penetration

Objectives Lipid colloidal vaccines, including liposomes, transfersomes, ethosomes and cubosomes, were formulated, characterised and investigated for their ability to enhance penetration of a peptide vaccine through stillborn piglet skin in vitro. Methods Liposomes and transfersomes were formulated using a film‐hydration method, ethosomes using a modified reverse phase method and cubosomes using a lipid precursor method. The size, zeta potential, peptide loading and interfacial behaviour of the formulations were characterised. Skin penetration studies were performed using Franz diffusion cells with piglet skin as the membrane. The localization of peptide in the skin was examined using confocal laser scanning microscopy. Key finding The various formulations contained negatively charged particles of similar size (range: 134–200 nm). Addition of the saponin adjuvant Quil A to the formulations destabilised the monolayers and reduced peptide loading. Cubosomes and ethosomes showed superior skin retention compared with the other systems. Confocal laser scanning microscopy showed greater peptide penetration and accumulation in the skin treated with cubosomes and ethosomes. With the other systems peptide was only located in the vicinity of the hair follicles and within the hair shaft. Conclusions We conclude from the in‐vitro studies that cubosomes and ethosomes are promising lipid carriers for transcutaneous immunisation.     

The role of the fibroblast growth factor family in bone‐related diseases

Fibroblast growth factor (FGF) family members are important regulators of cell growth, proliferation, differentiation, and regeneration. The abnormal expression of certain FGF family members can cause skeletal diseases, including achondroplasia, craniosynostosis syndrome, osteoarthritis, and Kashin–Beck disease. Accumulating evidence shows that FGFs play a crucial role in the growth and proliferation of bone and in the pathogenesis of certain bone‐related diseases. Here, we review the involvement of FGFs in bone‐related processes and diseases; FGF1 in the differentiation of human bone marrow mesenchymal stem cells and fracture repair; FGF2, FGF9, and FGF18 in osteoarthritis; FGF6 in bone and muscle injury; FGF8 in osteoarthritis and Kashin–Beck disease; and FGF21 and FGF23 on bone regulation. These findings indicate that FGFs are targets for novel therapeutic interventions for bone‐related diseases.     

Attenuation of the LPS‐induced,ERK‐mediated upregulation of fibrosis‐related factors FGF‐2, uPA,MMP‐2, and MMP‐9 by Carthamus tinctorius L in cardiomyoblasts

Severe and potentially fatal hypotension and cardiac contractile dysfunction are common symptoms in patients with sepsis. LPS was previously found to dramatically upregulate expression of fibrosis‐related factors FGF‐2, uPA, MMP‐2, and MMP‐9 in primary cardiac fibroblasts. MMPs are capable of denaturing and degrading fibrillar collagens and other components of the extracellular matrix (ECM). Studies have shown that dysregulation of expression of MMPs is associated with development of myocardial extracellular matrix remodeling and cardiac fibrosis, which contribute to progression of heart failure. In this study, H9c2 cells and cardiac fibroblasts were divided into five treatment groups: control, LPS (1 μg/mL) and three concentrations of FC_EtOH (Carthami Flos ethanolic extract) (31.25, 62.5, and 125 μg/mL). Phosphorylation of ERK‐1/2 was observed to be rapidly induced upon treatment with LPS. In contrast, it was significantly suppressed by the administration of FC_EtOH (125 μg/mL). Effects of FC_EtOH on LPS‐induced MMP‐2 and MMP‐9 expression in H9c2 cells occurred directly through ERK1/2 were determined. H9c2 cells were therefore pretreated with EGF‐R to activate ERK pathway. Both protein levels of MMP‐2 and MMP‐9 and immunefluorescent signals of MMP‐9 were significantly enhanced by EGFR. In contrast, MMP‐2 and MMP‐9 were significantly reduced after FC_EtOH administration. Based on these findings, the authors concluded that FC_EtOH elicits a protective effect against LPS‐induced cardio‐fibrosis through the ERK1/2 pathway. Carthamus tinctorius L may potentially serve as a cardio‐protective agent against LPS‐ induced cardiac fibrosis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 754–763, 2017.     

Pharmacokinetics,tissue distribution,and excretion of FGF‐21 following subcutaneous administration in rats

As one of the fibroblast growth factor (FGF) superfamily, FGF‐21 has been extensively investigated for its functions and roles since its discovery. It has been demonstrated to be one of the key regulators for glucose and lipid metabolism, and exhibits beneficial effects on cardiovascular disease. However, studies focusing on its pharmacokinetic behavior in vivo as a novel therapeutic agent have not been reported. In the present study, rapid and sensitive analytical approaches including radioactivity assay and assay after precipitation/separation by high performance liquid chromatography (HPLC) were established to determine the content of FGF‐21 tagged with ¹²⁵I in plasma, tissue, and excrement. The results indicated that FGF‐21 were quickly absorbed into systematic circulation and slowly eliminated; C_max and exposure increased in a dose‐dependent manner, exhibiting a typical linear pharmacokinetic pattern. Tissue distribution also confirmed that the kidney is the primary organ for FGF‐21 to be distributed, even though radioactivity of FGF‐21 was recovered in all tissues examined. In addition, the results also supported that urinary excretion was the critical route for FGF‐21 to be eliminated. The study fully clarifies the pharmacokinetic behavior of FGF‐21 and can provide valuable information and support further safety and toxicology development.     

Synthesis of 14C‐labelled myosmine, [2′‐14C] ‐3‐(1‐pyrrolin‐2‐yl)pyridine

¹⁴C‐Labelled myosmine ([2′‐¹⁴C]‐3‐(1‐pyrrolin‐2‐yl)pyridine) was synthesized for autoradiography studies starting from [carboxyl‐¹⁴C]‐nicotinic acid by initial esterification of the latter in the presence of 1,1,1‐triethoxyethane. Without any purification the ethyl nicotinate formed was directly reacted with N‐vinyl‐2‐pyrrolidinone in the presence of sodium hydride, yielding ¹⁴C‐labelled myosmine. The product was purified by silica gel column chromatography. The radiochemical yield was 15% and the specific activity 55.2 mCi/mmol. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of 2‐[11C]methoxy‐3,17β‐estradiol to measure the pharmacokinetics of an antitumor drug candidate, 2‐methoxy‐3,17β‐estradiol

2‐Methoxy‐3,17β‐estradiol, an endogenous estrogen metabolite, showed cytotoxicity in various cancer cell lines and also has antiangiogenic and proapoptotic activities. Clinical I and II trials of 2‐methoxy‐3,17β‐estradiol for multiple myeloma, advanced solid tumors, metastatic breast and prostate cancer are underway. We prepared 2‐[¹¹C]methoxy‐3,17β‐estradiol to measure the pharmacokinetics and organ distribution of 2‐methoxy‐3,17β‐estradiol in clinical trials. 2‐[¹¹C]Methoxy‐3,17β‐estradiol was synthesized from a precursor, 2‐hydroxy‐3,17β‐O‐bis(methoxymethyl)estradiol, in two steps with over 99% radiochemical purity. The overall reaction time was 45 min and the decay‐corrected radiochemical yield was 32.9%. The distribution coefficient (logP_7.4) of 2‐[¹¹C]methoxy‐3,17β‐estradiol at pH 7.4 was measured as 2.95. Copyright © 2006 John Wiley & Sons, Ltd.     

Detection and identification of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide in nitrite adulterated urine specimens containing morphine and its glucuronides

In vitro urine adulteration is a well‐documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine‐3‐glucuronide and morphine‐6‐glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography‐mass spectrometry (LC‐MS) when morphine and morphine‐6‐glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide, respectively. These reaction products were also formed when an authentic morphine‐positive urine specimen was fortified with nitrite. 2‐Nitro‐morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography‐mass spectrometry (GC‐MS) after forming a trimethylsilyl derivative. On the contrary, morphine‐3‐glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC‐MS and GC‐MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide as markers for the indirect monitoring of morphine and morphine‐6‐glucuronide in urine specimens adulterated with nitrite. Copyright © 2013 John Wiley & Sons, Ltd.