99mTc‐anti‐epidermal growth factor receptor nanobody for tumor imaging

Nanobodies are important biomolecules for tumor targeting. In this study, we synthesized and labeled anti‐epidermal growth factor receptor (EGFR) nanobody OA‐cb6 with ^99mTc(CO)₃⁺ and evaluated its characteristics for targeting the EGFR in the A431 human epidermal carcinoma cell line. Nanobody radiolabeling was achieved with high yield and radiochemical purity, and the radioconjugate was stable. Biodistribution results in nude mice exhibited a favorable tumor‐to‐muscle ratio at 4‐hr postinjection, and tumor location was visualized at 4 hr after injection of radiolabeled nanobody. Our result showed that the OA‐cb6‐^99mTc‐tricarbonyl radiolabeled nanobody is a promising radiolabeled biomolecule for tumor imaging in cancers with high EGFR overexpression.     

VEGF Trap-Eye for the treatment of neovascular age-related macular degeneration

Background: Age-related macular degeneration (AMD) affects > 14 million individuals worldwide. Although 90% of patients with AMD have the dry form, neovascular AMD accounts for the vast majority of patients who develop legal blindness. Until recently, few treatment options existed for treatment of neovascular AMD. The advent of anti-VEGF therapy has significantly improved the safe and effective treatment of neovascular AMD. In addition to two anti-VEGF drugs currently in widespread use, ranibizumab and bevacizumab, a number of medications that interrupt angiogenesis are currently under investigation. One promising new drug is aflibercept (VEGF Trap-Eye), a fusion protein that blocks all isoforms of VEGF-A and placental growth factors-1 and -2. Objective: To review the current literature and clinical trial data regarding VEGF Trap-Eye for the treatment of neovascular AMD. Methods: Literature review. Results/conclusion: VEGF Trap-Eye is a novel anti-VEGF therapy, with Phase I and II trial data indicating safety, tolerability and efficacy for the treatment of neovascular AMD. Two Phase III clinical trials (VIEW-1 and VIEW-2) comparing VEGF Trap-Eye to ranibizumab are currently continuing and will provide vital insight into the clinical applicability of this drug.     

Preparation of the iodine‐124 derivative of the Bolton–Hunter reagent ([124I]I‐SHPP) and its use for labelling a VEGF antibody as a PET tracer

This study describes the radioiodination of an antibody specific to the vascular endothelial growth factor (VEGF), VG76e, with [¹²⁴I]iodine to obtain a novel PET tracer for measurement of angiogenesis. In vitro binding assays showed a significantly higher immunoreactive fraction with the protein labelling reagent N‐succinimidyl 3‐(4‐hydroxy‐5‐[¹²⁵I]iodophenyl) propionate ([¹²⁵I]Bolton–Hunter reagent, [¹²⁵I]I‐SHPP) (34.0±4.0%) as compared with N‐succinimidyl 3‐[¹²⁵I]iodobenzoate (10.9±6.4%) or direct radioiodination using [¹²⁵I]iodide and IodoGen (3.1±3.0%). Consequently, the cyclotron–produced positron–emitting [¹²⁴I]iodine (T_1/2=4.2 days) was employed to prepare [¹²⁴I]I‐SHPP. Using an improved radioiodination methodology, [¹²⁴I]I‐SHPP was prepared from sodium [¹²⁴I]iodide with IodoGen at pH 6.5. The [¹²⁴I]Bolton–Hunter reagent was isolated with 25–58% (n=3) radiochemical yield and 88–95% (n=3) radiochemical purity by the conventional extraction procedure. The conjugate of VG76e with [¹²⁴I]I‐SHPP was prepared with 17–18% (n=3) labelling efficiency and 98% radiochemical purity. The immunoreactive fraction was determined to be 33.5% (n=2). Copyright © 2002 John Wiley & Sons, Ltd.     

Novel formulation of solid lipid microparticles of curcumin for anti‐angiogenic and anti‐inflammatory activity for optimization of therapy of inflammatory bowel disease

Objectives This project was undertaken with a view to optimize the treatment of inflammatory bowel disease through a novel drug delivery approach for localized treatment in the colon. Curcumin has poor aqueous solubility, poor stability in the gastrointestinal tract and poor bioavailability. The purpose of the study was to prepare and evaluate the anti‐inflammatory activity of solid lipid microparticles (SLMs) of curcumin for the treatment of inflammatory bowel disease in a colitis‐induced rat model by a colon‐specific delivery approach. Methods We have developed a novel formulation approach for treating experimental colitis in the rat model. SLMs of curcumin were prepared with various lipids, such as palmitic acid, stearic acid and soya lecithin, with an optimized percentage of poloxamer 188. The SLMs of curcumin were characterized for particle size, drug content, drug entrapment, in‐vitro release, surface morphology and infrared, differential scanning calorimetry and X‐ray studies. The colonic delivery system of SLM formulations of curcumin were further investigated for their anti‐angiogenic and anti‐inflammatory activity using chick embryo and rat colitis models. Key findings Particle size, drug content, drug entrapment and in‐vitro release studies showed that formulation F4 containing one part stearic acid and 0.5% surfactant had the smallest diameter of 108 μm, 79.24% entrapment and exhibited excellent in‐vitro release characteristics when compared with other formulations and pure curcumin. SLMs of curcumin (F4) proved to be a potent angio‐inhibitory compound, as demonstrated by inhibition of angiogenesis in the chorioallantoic membrane assay. Rats treated with curcumin and its SLM complex showed a faster weight gain compared with dextran sulfate solution (DSS) control rats. The increase in whole colon length appeared to be significantly greater in SLM‐treated rats when compared with pure curcumin and DSS control rats. An additional finding in the DSS‐treated rats was chronic cell infiltration with predominance of eosinophils. Decreased mast cell numbers in the mucosa of the colon of SLMs of curcumin and pure curcumin‐treated rats was observed. Conclusions The degree of colitis caused by administration of DSS was significantly attenuated by colonic delivery of SLMs of curcumin. Being a nontoxic natural dietary product, curcumin could be useful in the therapeutic strategy for inflammatory bowel disease patients.     

Renal function in HIV/HBV co‐infected and HBV mono‐infected patients on a long‐term treatment with tenofovir in real life setting

The human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co‐infection is likely to be associated with an increased risk of kidney disease, due to the additional factors that may affect renal function in the HIV population. We aimed to evaluate renal toxicity in HIV/HBV and HBV mono‐infected patients on long‐term therapy with tenofovir (TDF) and to explore the association of polymorphisms in ATP‐binding cassette (ABCC)2, ABCC4, ABCC10 with the development of renal dysfunction. From September 2006 to November 2014, 44 HIV/HBV co‐infected and 34 HBV mono‐infected patients were commenced on TDF. Data of renal safety were retrospectively collected and analyzed. ABCC2, ABCC4 and ABCC10 genotypes were identified by real‐time PCR. Over 60 months of observation, there was a significant increase in mean creatinine levels from baseline (P<.01) that was not significantly different between the two study groups. Moreover, a significant decline in estimated glomerular filtration rate (eGFR) was observed from baseline (P<.01), and it was significantly greater in HBV mono‐infected than co‐infected patients (P=.03). The distribution of ABCC2, ABCC4 and ABCC10 genotypes among a subgroup of 34 patients did not show significant association with eGFR decline <90 mL/min per 1.73 m². Although our findings showed a statistically significant decrease in eGFR with long‐term use of TDF, its clinical impact seems to be modest. The role of genetic factors to identify patients at greater risk for developing tenofovir‐induced renal toxicity needs to be further investigated.     

Development and validation of a disease‐specific quality of life questionnaire for gastro‐oesophageal reflux disease: the GERD‐QOL questionnaire

Aliment Pharmacol Ther 31 , 452–460

Summary

Background A simple and meaningful health‐related quality of life (HRQoL) questionnaire for gastro‐oesophageal reflux disease (GERD) patients is lacking. Aim To develop and validate a disease‐specific HRQoL instrument (GERD‐QOL) for GERD patients. Methods An 18‐item questionnaire was generated to measure the impact of GERD on sleep, exercise, diet, need for medication, sex life, work, social activity and psychological well‐being. GERD patients were invited to complete the GERD‐QOL, a visual analogue scale (VAS) and a validated Chinese generic QoL (SF‐36) questionnaire before and after esomeprazole treatment. Factor analysis was performed for item selection and psychometric properties were measured. An English version was developed by a forward‐backward translation process. Results A final 16‐item GERD‐QOL questionnaire was developed. The items were grouped into four subscales (Daily activity, Treatment effect, Diet, and Psychological well‐being) after factor analysis. GERD‐QOL had good item‐internal consistency (Cronbach’s alpha: 0.64–0.88), high test‐retest reliability (intraclass correlation coefficient: 0.73–0.94, P < 0.001). Its subscale scores were correlated with SF‐36 and VAS, which demonstrated high construct validity (P < 0.001). Discriminant validity was verified by correlating GERD‐QOL scores with symptom severity (P < 0.001). Responsiveness after esomeprazole treatment was significant (paired‐t‐test P < 0.001). An English version of GERD‐QOL was developed. Conclusion The instrument, GERD‐QOL, is valid and reliable.     

Development of a novel,fibrin‐specific PET tracer

Fibrin deposition is observed in several diseases such as atherosclerosis, deep vein thrombosis, and also tumors, where it contributes to the formation of mature tumor stroma. The aim of this study was to develop a gallium‐labeled peptide tracer on the basis of the fibrin‐targeting peptide Epep for PET imaging of fibrin deposition. For this purpose, the peptide Epep was modified with a NOTA moiety for radiolabeling with ⁶⁷Ga and ⁶⁸Ga and compared with the earlier validated ¹¹¹In‐DOTA‐Epep tracer. In vitro binding assays of ⁶⁷Ga‐NOTA‐Epep displayed an enhanced retention as compared to previously published data showing binding of ¹¹¹In‐DOTA‐Epep to human (84.0 ± 0.6 vs 66.6 ± 1.4 %Dose) and mouse derived fibrin clots (83.5 ± 1.7 vs 74.2 ± 2.4% Dose). In vivo blood kinetics displayed a bi‐phasic elimination profile (t_1/2,_α = 2.6 ± 1.0 minutes and t_1/2,_β = 15.8 ± 1.3 minutes) and ex vivo biodistribution showed low blood values at 4 hours post injection and a low uptake in nontarget tissue (<0.2 %ID/g; kidneys, 1.9%ID/g). In conclusion, taking into account the ease of radiolabeling and the promising in vitro and in vivo studies, gallium‐labeled Epep displays the potential for further development towards a PET tracer for fibrin deposition.     

Development of a Korean‐specific virtual population for physiologically based pharmacokinetic modelling and simulation

Physiologically based pharmacokinetic (PBPK) modelling and simulation is a useful tool in predicting the PK profiles of a drug, assessing the effects of covariates such as demographics, ethnicity, genetic polymorphisms and disease status on the PK, and evaluating the potential of drug–drug interactions. We developed a Korean‐specific virtual population for the SimCYP® Simulator (version 15 used) and evaluated the population's predictive performance using six substrate drugs (midazolam, S‐warfarin, metoprolol, omeprazole, lorazepam and rosuvastatin) of five major drug metabolizing enzymes (DMEs) and two transporters. Forty‐three parameters including the proportion of phenotypes in DMEs and transporters were incorporated into the Korean‐specific virtual population. The simulated concentration–time profiles in Koreans were overlapped with most of the observed concentrations for the selected substrate drugs with a < 2‐fold difference in clearance. Furthermore, we found some drug models within the SimCYP® library can be improved, e.g., the minor allele frequency of ABCG2 and the fraction metabolized by UGT2B15 should be incorporated for rosuvastatin and lorazepam, respectively. The Korean‐specific population can be used to evaluate the impact of ethnicity on the PKs of a drug, particularly in various stages of drug development.     

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass‐spectrometry‐based approach

The ongoing shift from small molecule drugs to protein therapeutics in the pharmaceuticals industry presents a considerable challenge to generic drug developers who are increasingly required to demonstrate biosimilarity for biological macromolecules, a task that is decidedly more complex than doing the same for small molecule drugs. In this work, we demonstrate a multipronged mass‐spectrometry‐based workflow that allows rapid and facile molecular characterization of antibody‐based protein therapeutics, applied to biosimilars development. Specifically, we use a combination of native mass spectrometry (MS), ion mobility spectrometry (IMS), and global time‐resolved hydrogen deuterium exchange (HDX) to provide an unambiguous assessment of the structural, dynamic, and chemical similarity between Avastin (bevacizumab) and a biosimilar in the late stages of pre‐clinical development. Minor structural and dynamic differences between the biosimilar and Avastin, and between lots of the biosimilar, were tested for functional relevance using Surface Plasmon Resonance‐derived kinetic and equilibrium binding parameters.     

Anti‐angiogenesis effect of β‐D‐mannuronic acid (M2000) as a novel NSAID with immunosuppressive properties under experimental model

Angiogenesis is a process through which new capillaries are formed from pre‐existing ones, which contributes significantly to the pathogenesis of numerous diseases, such as cancer and chronic inflammatory disorders. The β‐D‐mannuronic acid (M2000) is a novel non‐steroidal anti‐inflammatory drug (NSAID) with immunosuppressive effects and is a matrix metalloproteinase (MMP) inhibitor. This research aimed to study the anti‐angiogenesis effects of M2000 under in vitro and in vivo models. The cytotoxic and anti‐proliferative effects of M2000 were examined using the trypan blue method and the MTT assay, respectively. The 3D collagen‐cytodex model and the chick chorioallantoic membrane (CAM) assay were then used to evaluate the anti‐angiogenesis property of M2000. Cytotoxicity assay revealed that M2000 (at concentrations of less than 100 μg/mL) had no cytotoxic effect on human umbilical vein endothelial cells (HUVECs). It was also illustrated that M2000 had little or no anti‐proliferative effect on HUVECs. In addition, the anti‐angiogenesis effects of M2000 were shown to be marginal in the in vitro model and both significant and dose‐dependent in the in vivo status. This study showed that M2000 could be considered as an anti‐angiogenic molecule which more likely exerts its activity mainly via indirect effects on endothelial cells and its anti‐inflammatory effects may partly be attributable to its anti‐angiogenic activity. Therefore, it could be recommended as a candidate for prevention and treatment of cancer, chronic inflammatory diseases, and other angiogenesis‐related disorders.     

LC−MS/MS assay for the determination of a novel anti‐fibrotic candidate mefunidone in monkey plasma and its application to a pharmacokinetics study

Mefunidone (MFD) is a promising anti‐fibrotic candidate molecule with greater anti‐fibrotic activity than pirfenidone (PFD). However, there has been no report on the methodology used for the quantification of MFD or on any investigation of its pharmacokinetics. In this study, an efficient and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to assay MFD in monkey plasma. This assay method was validated and applied to a pharmacokinetics study in monkeys. The lower limit of quantification of this assay was 0.1 μg·mL^?1, and the linear calibration curve was acquired with R² > 0.99 between 0.1 and 60 μg·mL^?1. The intra‐day and inter‐day precision were evaluated with coefficient of variations of 1.5%–5.8%, whereas the mean accuracy ranged from 91.7% to 106.9%. A negligible matrix effect and good recovery were obtained using this assay, with average extraction recoveries of MFD and the internal standard (IS) in the range of 85.5%–124.8% and 84.1%–94.0%, respectively. The precision of the absolute matrix effect of MFD and the IS was 1.2–3.0% and 1.2–7.3%, respectively. The samples were stable under all experimental conditions. Linear pharmacokinetics were observed for MFD in monkeys, where the exposures of MFD increased proportionally with increasing MFD doses at the range of 10–90 mg·kg^?1. Moderate elimination of MFD from the body was observed, with t_1/2 of 5–7 h, and the elimination rate of MFD was stable during multiple dosing. In conclusion, this method provides an reliable analytical approach for quantification of MFD in plasma and was successfully applied to a pharmacokinetics study in monkeys.     

Synthesis of a quaternary bis derivative of imipramine as a novel compound with potential anti‐enuretic effect

Objectives Imipramine has been used for over four decades (early reports in 1960s) for the treatment of nocturnal enuresis, although the reason for its effect is not clear. Imipramine is a tertiary amine, which may act both in the periphery and/or pass through the blood‐brain barrier (BBB) in unionized form and exhibit a central effect. Since imipramine has anti‐cholinergic properties, some believe it may exert its anti‐enuretic effect by affecting peripheral cholinergic receptors, i.e. its anti‐enuretic effect may be due to peripheral anti‐cholinergic properties, whereas others think it can pass through the BBB and interact with central nervous system (CNS) receptors. If the anti‐enuretic effect of imipramine is due to its peripheral anti‐cholinergic effects, its entrance into the CNS is unnecessary. Therefore, the synthesis of a form of imipramine that can exhibit peripheral anti‐cholinergic effects but does not have CNS adverse effects would have a safer drug profile in this case. On the other hand, if the anti‐enuretic effect of imipramine is primarily due to its action on the CNS, a form of imipramine that cannot pass through the BBB has no effect on nocturnal enuresis treatment and thus may help to clarify the mechanism of action of imipramine in nocturnal enuresis treatment. Methods This article describes the synthesis and evaluation of the anti‐cholinergic effect of a new bis derivative of imipramine, which contains two imipramine units in its structure. Key findings The compound exhibited anti‐cholinergic activity comparable with that of imipramine on isolated guinea pig ileum. Conclusions Being a quaternary ammonium, this compound is not expected to be able to cross the BBB and thus would cause fewer CNS side effects.