N‐Acetylcysteine as a chemical scavenger for sulfur mustard: New insights by mass spectrometry

The vesicant sulfur mustard (SM) is a banned chemical warfare agent. Although, SM has been used in combat since WWI, there is no causal therapy currently available. Accordingly, development and investigation of antidotes and scavengers targeting SM are of high clinical relevance. N‐acetylcysteine (NAC) was shown to mitigate symptoms of SM intoxications in vitro and in vivo. However, it is still unclear whether the beneficial effects of NAC are only due to physiological processes or also due to chemical scavenging of SM. Therefore, in this study, we examined the scavenging potential of NAC toward SM. Co‐incubations of SM and different NAC concentrations in human serum were performed to monitor diverse adducts (covalent reaction products) of human serum albumin (HSA), NAC, and SM. After proteolytic cleavage of HSA with proteinase K the alkylated tripeptide hydroxyethylthioethyl‐CysProPhe (HETE‐CPF) and the disulfide bridged tripeptide NAC‐CPF were detected. Samples were analyzed by microbore liquid chromatography–electrospray ionization–high‐resolution tandem‐mass spectrometry (μLC‐ESI MS/HR MS). Furthermore, degradation kinetics of SM in phosphate buffered saline were measured in the presence and absence of NAC. Although NAC‐CPF was identified and characterized for the first time by mass spectrometry and reaction products of NAC and SM were detected and identified by MS/HR MS, analyses clearly documented minor reactivity not significantly contributing to reduction of SM concentrations. Therefore, we conclude that chemical scavenging of SM by NAC does not play the key role in NAC therapy of SM poisoning.     

Evidence of sulfur mustard poisoning by detection of the albumin-derived dipeptide biomarker C(-HETE)P after nicotinylation

Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent that was frequently used in recent years and led to numerous poisoned victims who developed painful erythema and blisters. Post-exposure analysis of SM incorporation can be performed by the detection of human serum albumin (HSA)-derived peptides. HSA alkylated by SM contains a hydroxyethylthioethyl (HETE)-moiety bound to the cysteine residue C³⁴ yielding the dipeptide biomarker C(-HETE)P after pronase-catalyzed proteolysis. We herein present a novel procedure for the selective precolumn nicotinylation of its N-terminus using 1-nicotinoyloxy-succinimide. The reaction was carried out for 2 h at ambient temperature with a yield of 81%. The derivative NA-C(-HETE)P was analyzed by micro liquid chromatography-electrospray ionization tandem-mass spectrometry working in the selected reaction monitoring mode (μLC-ESI MS/MS SRM). The derivative was shown to be stable in the autosampler at 15°C for at least 24 h. The single protonated precursor ion (m/z 428.1) was subjected to collision-induced dissociation yielding product ions at m/z 116.1, m/z 137.0, and m/z 105.0 used for selective monitoring without any plasma-derived interferences. NA-C(-HETE)P showed a mass spectrometric response superior to the non-derivatized dipeptide thus yielding larger peak areas (factor 1.3 ± 0.2). The lower limit of identification corresponded to 80 nM SM spiked to plasma in vitro. The presented procedure was applied to real case plasma samples from 2015 collected in the Middle East confirming SM poisoning.     

Alkylated glutamic acid and histidine derived from protein-adducts indicate exposure to sulfur mustard in avian serum

Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals. For legal reasons, bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins, for example, serum albumin (SA), are valuable long-lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck, and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant, and re-dissolution analysis were performed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry in the selected reaction monitoring mode, μLC-ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE]⁺ at m/z 105.0. After in vitro incubation with SM and pronase-catalyzed proteolysis, the alkylated amino acids Glu(-HETE) and His(-HETE) were detected. Both borne the SM-characteristic HETE-moiety bound to their side chain. The eightfold deuterated SM analog (d8-SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50°C), and time to maximize the yield of Glu(-HETE) (30 min) and His(-HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein-adducts were stable in serum at −30°C for at least 33 days and for three freeze-and-thaw cycles. At the body temperature of chicken (+40°C), Glu(-HETE) was degraded in serum (period of half-change 3 days), whereas His(-HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM.     

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys¹⁰-residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.     

Chemical reactivity of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) in vitro

Ethyl (6R)‐6‐[N‐(2‐chloro‐4‐fluorophenyl)sulfamoyl]cyclohex‐1‐ene‐1‐carboxylate (TAK‐242) was metabolized to cyclohexene and phenyl ring moieties in non‐clinical pharmacokinetic studies and it was suggested that the cyclohexene ring moiety of TAK‐242 is tightly bound to endogenous macromolecules. After incubation of TAK‐242 and glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C, TAK‐242 reacted with GSH to produce a glutathione conjugate of the cyclohexene ring moiety of TAK‐242, which had been observed as a metabolite (M‐SG) in non‐clinical pharmacokinetic studies. Formation of M‐SG was time dependent with a first order reaction and M‐I, a metabolite from the phenyl ring moiety of TAK‐242, was also produced in parallel. The formation of M‐SG was accelerated with increasing pH, therefore it was indicated that TAK‐242 reacted with GSH by a nucleophilic substitution reaction. Because glutathione transferase (GST) enhanced M‐SG formation in vitro, it is expected that the conjugation of TAK‐242 with GSH is also facilitated by GST in vivo in addition to a spontaneous chemical reaction. When radio‐labeled TAK‐242 ([cyclohexene ring‐U‐¹⁴ C]TAK‐242) was incubated with rat serum albumin (RSA) or human serum albumin (HSA) in vitro, the radioactive material was covalently bound to RSA and HSA, and M‐I was generated simultaneously in the reaction mixture. The chemical structure of the TAK‐242 adduct covalently bound to HSA was characterized by the accurate mass spectra that cyclohexene ring moiety of TAK‐242 was covalently bound to the lysine residue in HSA. The adduct was also detected in the plasma of rats and humans after single i.v. dosing of TAK‐242 (in vivo). Copyright © 2011 John Wiley & Sons, Ltd.     

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin

Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long‐term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high‐resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2‐(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met‐Pro‐Cys‐DPAET, MPC‐DPAET and Asp‐Ile‐Cys‐DPAET, DIC‐DPAET). MPC‐DPAET was shown to undergo partial in‐source decay during electrospray ionization for MS detection thereby losing the N ‐terminal Met residue. This results in the more stable Pro‐Cys‐DPAET (PC‐DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC‐DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd.     

In Vitro Regioselective Stability of β‐1‐O‐ and 2‐O‐Acyl Glucuronides of Naproxen and Their Covalent Binding to Human Serum Albumin

β‐1‐O‐ (NAG) and 2‐O‐glucuronides (2‐isomer) of (S)‐naproxen (NA) were prepared to determine which positional isomer‐(s) of the acyl glucuronide of NA is responsible for forming covalent adducts with human serum albumin (HSA). Their comparative stability and covalent binding adduct formation with HSA were investigated at pH 7.4 and at 37 °C. NA and its acyl glucuronides were simultaneously determined by HPLC. Three positional isomers were formed successively after incubation of NAG in the buffer only. However, when NAG was incubated with HSA (30 mg/mL), isomers other than the 2‐isomer were formed in little or negligible quantities. In HSA solution, NAG (k_d = 2.08 ± 0.08 h⁻¹) was four times less stable than 2‐isomer (k_d = 0.51 ± 0.02 h⁻¹). NAG was degraded by hydrolysis (k_hyd = 1.01 ± 0.10 h⁻¹) and isomerization (k_iso = 1.07 ± 0.07 h⁻¹) to the same extent; however, hydrolysis was predominant for the 2‐isomer (k_d = 0.51 ± 0.02 h⁻¹). The incubation of both NAG and 2‐isomer with HSA led to the formation of a covalent adduct; however, the adduct formation from the 2‐isomer proceeded more slowly than that from NAG. The present results suggest that the covalent binding of NA to HSA via its acyl glucuronides proceeds through both transacylation (direct nucleophilic displacement) and glycation mechanisms; NAG rapidly forms an adduct that may be unstable, and the protein adduct from the 2‐O‐acyl glucuronide is as important for the covalent binding as those from the 1‐O‐acyl glucuronides.     

Identification of creatine kinase and alpha-1 antitrypsin as protein targets of alkylation by sulfur mustard

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide–thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys²⁸² of CK and Cys²³² of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.     

Human serum albumin interaction with oxaliplatin studied by capillary isoelectric focusing with the whole column imaging detection and spectroscopic method

Capillary isoelectric focusing (CIEF) with whole column imaging detection (WCID) was used to investigate drug-protein interactions. This study was designed to examine the interaction between the platinum-based anticancer drug, oxaliplatin, with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 10 to 100 μM and a constant concentration of HSA (5.0 × 10⁻⁵ M). The reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48 and 72 h at 37 °C in a water bath. The CIEF results indicate that with increasing the drug concentration, the complex formation of protein adducts increased compared to low-drug concentrations and major structural changes were observed as the incubation time progressed. The altered CIEF profile demonstrated the possible conformation change due to the binding of the drug. Results also showed a significant protein's pI shift for higher HSA–oxaliplatin incubation ratios. Furthermore, spectroscopic evidence shows that oxaliplatin caused the fluorescence quenching of HSA by formation of HSA–oxaliplatin complex. Using the Stern–Volmer equation, the quenching constants were calculated in the linear range. The quenching rate constants K_q at three different temperatures indicating the presence of static quenching mechanism in the interactions of oxaliplatin with HSA. This paper describes the validity of the CIEF-WCID technique for the study of protein–drug interactions and provides useful information and insight into the interaction of anticancer drugs with HSA.     

Anti‐doping analytes in serum: A comparison of SST and SST‐II Advance blood collection tubes

Serum insulin‐like growth factor‐1 (IGF‐1), procollagen type III N‐terminal peptide (PIIINP), and human growth hormone (hGH) isoforms were analyzed in identical serum samples collected into BD Vacutainer^® SST and BD Vacutainer^® SST‐II Advance serum separator tubes. Comparing the serum collected into each tube, measurement correlation was high (R² > 0.83) and percent bias was minimal (<|3.2%|) for all analytes measured using World Anti‐Doping Agency (WADA)‐approved tests. As such, it is recommended that both SST and SST‐II Advance tubes can be used interchangeably for anti‐doping purposes.     

Solid‐phase organic synthesis of chiral,non‐racemic 1,2,4‐trisubstituted 1,4‐diazepanes with high σ1 receptor affinity

The aim of this work was to transfer the established chiral‐pool synthesis of 1,2,4‐trisubstituted 1,4‐diazepanes in solution on the solid phase. For this purpose, (S)‐configured amino acids, (S)‐alanine, and (S)‐leucine, with a small methyl and a larger isobutyl moiety were attached to the solid support 9 by reductive amination. After five reaction steps on the solid support, the 1,4‐diazepanes (S)‐ 19a , b were cleaved off and reductively alkylated to afford the 1,2,4‐trisubstituted 1,4‐diazepanes (S)‐ 20a and (S)‐ 21b , respectively. Both compounds show high σ₁ affinity and selectivity over the σ₂ subtype.     

A Synthetic Analogue of 20‐HETE, 5,14‐HEDGE,Reverses Endotoxin‐Induced Hypotension via Increased 20‐HETE Levels Associated with Decreased iNOS Protein Expression and Vasodilator Prostanoid Production in Rats

Abstract: Nitric oxide (NO) produced by inducible NO synthase (iNOS) is responsible for endotoxin (ET)‐induced hypotension and vascular hyporeactivity and plays a major contributory role in the multiorgan failure. Endotoxic shock is also associated with an increase in vasodilator prostanoids as well as a decrease in endothelial NO synthase (eNOS) and cytochrome P450 4A protein expression, and production of a vasoconstrictor arachidonic acid product, 20‐hydroxyeicosatetraenoic acid (20‐HETE). The aim of this study was to investigate the effects of a synthetic analogue of 20‐HETE, N‐[20‐hydroxyeicosa‐5(Z),14(Z)‐dienoyl]glycine (5,14‐HEDGE), on the ET‐induced changes in eNOS, iNOS and heat shock protein 90 (hsp90) expression as well as 20‐HETE and vasodilator prostanoid (6‐keto‐PGF_1α and PGE₂) production. ET‐induced fall in blood pressure and rise in heart rate were associated with an increase in iNOS protein expression and a decrease in eNOS protein expression in heart, thoracic aorta, kidney and superior mesenteric artery. ET did not change hsp90 protein expression in the tissues. ET‐induced changes in eNOS and iNOS protein expression were associated with increased 6‐keto‐PGF_1α and PGE₂ levels and a decrease in 20‐HETE levels, in the serum and kidney. These effects of ET on the iNOS protein expression and 6‐keto‐PGF_1α, PGE₂ and 20‐HETE levels were prevented by 5,14‐HEDGE. Furthermore, a competitive antagonist of vasoconstrictor effects of 20‐HETE, 20‐hydroxyeicosa‐6(Z),15(Z)‐dienoic acid, prevented the effects of 5,14‐HEDGE on the ET‐induced changes in systemic and renal levels of these prostanoids and 20‐HETE. These data are consistent with the view that an increase in systemic and renal 20‐HETE levels associated with a decrease in iNOS protein expression and vasodilator prostanoid production contributes to the effect of 5,14‐HEDGE to prevent the hypotension during rat endotoxemia.     

Synthesis of morphine‐[N‐methyl‐14C]‐6‐β‐D‐glucuronide

Protected morphine‐6‐glucuronide was converted into morphine‐[N‐methyl‐¹⁴C]‐6‐glucuronide by a three‐step procedure. Methyl (3‐pivaloylmorphin‐6‐yl 2,3,4‐tri‐O‐isobutyryl‐β‐D‐glucopyranosid)uronate was N‐demethylated by treatment with 1‐chloroethyl chloroformate to afford protected normorphine‐6‐glucuronide as its hydrochloride salt. The normorphine‐6‐glucuronide derivative was alkylated with iodomethane‐[¹⁴C] in the presence of potassium carbonate to produce C‐14 labelled protected morphine‐6‐glucuronide. Finally, hydrolysis of the protecting groups using 5% sodium hydroxide solution gave morphine‐[N‐methyl‐¹⁴C]‐6‐β‐D‐glucuronide with a specific activity of 41.8 mCi mmol^?1 and radiochemical purity of 99.2% (HPLC). Copyright © 2002 John Wiley & Sons, Ltd.     

Biotin/Folate‐decorated Human Serum Albumin Nanoparticles of Docetaxel: Comparison of Chemically Conjugated Nanostructures and Physically Loaded Nanoparticles for Targeting of Breast Cancer

Docetaxel (DTX) is a widely used chemotherapeutic agent with very low water solubility. Conjugation of DTX to human serum albumin (HSA) is an effective way to increase its water solubility. Attachment of folic acid (FA) or biotin as targeting moieties to DTX‐HSA conjugates may lead to active targeting and specific uptake by cancer cells with overexpressed FA or biotin receptors. In this study, FA or biotin molecules were attached to DTX‐HSA conjugates by two different methods. In one method, FA or biotin molecules were attached to remaining NH₂ residues of HSA in DTX‐HSA conjugate by covalent bonds. In the second method, HSA‐FA or HSA‐biotin conjugates were synthesized separately and then combined by DTX‐HSA conjugate in proper ratio to prepare nanoparticles containing DTX‐HSA plus HSA‐FA or HSA‐biotin. Cell viability of different nanoparticle was evaluated on MDA‐MB‐231 (folate receptor positive), A549 (folate receptor negative), and 4T1 (biotin receptor positive) and showed superior cytotoxicity compared with free docetaxel (Taxotere^®). In vivo studies of DTX‐HSA‐FA and DTX‐HSA‐biotin conjugates in BULB/c mice, tumorized by 4T1 cell line, showed the conjugates prepared in this study were more powerful in the reduction in tumor size and increasing the survival rate when compared to free docetaxel.     

Assessing the Validity of Normalizing Aflatoxin B1-Lysine Albumin Adduct Biomarker Measurements to Total Serum Albumin Concentration across Multiple Human Population Studies

The assessment of aflatoxin B₁ (AFB₁) exposure using isotope-dilution liquid chromatography-mass spectrometry (LCMS) of AFB₁-lysine adducts in human serum albumin (HSA) has proven to be a highly productive strategy for the biomonitoring of AFB₁ exposure. To compare samples across different individuals and settings, the conventional practice has involved the normalization of raw AFB₁-lysine adduct concentrations (e.g., pg/mL serum or plasma) to the total circulating HSA concentration (e.g., pg/mg HSA). It is hypothesized that this practice corrects for technical error, between-person variance in HSA synthesis or AFB₁ metabolism, and other factors. However, the validity of this hypothesis has been largely unexamined by empirical analysis. The objective of this work was to test the concept that HSA normalization of AFB₁-lysine adduct concentrations effectively adjusts for biological and technical variance and improves AFB₁ internal dose estimates. Using data from AFB₁-lysine and HSA measurements in 763 subjects, in combination with regression and Monte Carlo simulation techniques, we found that HSA accounts for essentially none of the between-person variance in HSA-normalized (R² = 0.04) or raw AFB₁-lysine measurements (R² = 0.0001), and that HSA normalization of AFB₁-lysine levels with empirical HSA values does not reduce measurement error any better than does the use of simulated data (n = 20,000). These findings were robust across diverse populations (Guatemala, China, Chile), AFB₁ exposures (10⁵ range), HSA assays (dye-binding and immunoassay), and disease states (healthy, gallstones, and gallbladder cancer). HSA normalization results in arithmetic transformation with the addition of technical error from the measurement of HSA. Combined with the added analysis time, cost, and sample consumption, these results suggest that it may be prudent to abandon the practice of normalizing adducts to HSA concentration when measuring any HSA adducts—not only AFB₁-lys adducts—when using LCMS in serum/plasma.     

Inhibition of UVB‐Induced Skin Damage by Exopolymers from Aureobasidium pullulans SM‐2001 in Hairless Mice

Because antioxidants from natural sources may be an effective approach to the treatment and prevention of UV radiation‐induced skin damage, the effects of purified exopolymers from Aureobasidium pullulans SM‐2001 (‘E‐AP‐SM2001’) were evaluated in UVB‐induced hairless mice. E‐AP‐SM2001 consists of 1.7% β‐1,3/1,6‐glucan, fibrous polysaccharides and other organic materials, such as amino acids, and mono‐ and di‐unsaturated fatty acids (linoleic and linolenic acids) and shows anti‐osteoporotic and immunomodulatory effects, through antioxidant and anti‐inflammatory mechanisms. Hairless mice were treated topically with vehicle, E‐AP‐SM2001 stock and two and four times diluted solutions once per day for 15 weeks against UVB irradiation (three times per week at 0.18 J/cm²). The following parameters were evaluated in skin samples: myeloperoxidase (MPO) activity, cytokine levels [interleukin (IL)‐1β and IL‐10], endogenous antioxidant content (glutathione, GSH), malondialdehyde (MDA) levels, superoxide anion production; matrix metalloproteases (MMP‐1, ‐9 and ‐13), GSH reductase and Nox2 (gp91phox) mRNA levels, and immunoreactivity for nitrotyrosine (NT), 4‐hydroxynonenal (HNE), caspase‐3, and cleaved poly(ADP‐ribose) polymerase (PARP). Photoageing was induced by UVB irradiation through ROS‐mediated inflammation, which was related to the depletion of endogenous antioxidants, activation of MMPs and keratinocyte apoptosis. Topical treatment with all three doses of E‐AP‐SM2001 and 5 nm myricetin attenuated the UV‐induced depletion of GSH, activation of MMPs, production of IL‐1β, the decrease in IL‐10 and keratinocyte apoptosis. In this study, E‐AP‐SM2001 showed potent inhibitory effects against UVB‐induced skin photoageing. Thus, E‐AP‐SM2001 may be useful as a functional ingredient in cosmetics, especially as a protective agent against UVB‐induced skin photoageing.     

N‐[3‐[4‐(2‐methoxyphenyl) piperaziny‐1‐yl]propyl]cyclam: synthesized as a potential 5‐HT1A receptor ligand and labelled with 99mTc‐nitrido core

This paper reports the synthesis of new potential 5‐HT_1A receptor ligand N‐[3‐[4‐(2‐methoxyphenyl)piperaziny‐1‐yl]propyl]cyclam (MPPC) and radiolabelling of it with ^99mTc‐nitrido core. The novel neutral complex ^99mTcN‐MPPC combines 1,4,8,11‐tetraazacyclotetradecane (cyclam) ligand as chelate moiety for ^99mTc‐nitrido with a 1‐(2‐methoxyphenyl)piperazine moiety derived from WAY 100635 via a 3‐carbon alkyl chain. This provided a reliable and reproducible method for attaching the technetium to the pharmacophore moiety of WAY 100635. ^99mTcN‐MPPC was prepared by a two‐step procedure and the radiochemical purity was found to be greater than 95%. It was hydrophilic and stable for at least 4 h at room temperature. In vivo stability study in normal rats showed that no degradation of ^99mTcN‐MPPC was found in deproteinated blood samples at 2 h post‐injection. This effective ^99mTc‐labelling strategy for obtaining neutral ^99mTc nitrido complexes would be a useful tool to prepare new SPECT agents to image 5‐HT_1A receptor with cyclam conjugated ligands. Copyright © 2008 John Wiley & Sons, Ltd.     

Interaction studies of a novel Co(II)-based potential chemotherapeutic agent with human serum albumin (HSA) employing biophysical techniques

Novel macrocyclic cobalt(II) complex C₃₆H₂₄N₈O₄S₄CoCl₂ with a butterfly topology was synthesized and characterized by spectroscopic (IR, ¹H, ¹³C NMR, EPR, UV-Vis, ESI-MS) and analytical methods. The complex exhibits distorted octahedral geometry around Co(II) metal ion, which was confirmed by EPR measurements with g_a, g_b, and g_c values (8.01, 2.20, and 1.66), respectively, and molar extinction coefficient ε = 58 M⁻¹cm⁻¹. The Interaction studies with human serum albumin (HSA) in phosphate buffer (0.1 M, pH 7.0) were studied by electronic absorption titration, fluorescence titration, circular dichroism, and cyclic voltammetry. Hyperchromism in fluorescence intensity indicates binding of complex with HSA near tryptophan residue in IIA subdomain leading to less polar microenvironment around tryptophan and more at tyrosine. The intrinsic binding constant K _b obtained from absorption spectral titrations was found to be 9.3 × 10⁴ M⁻¹, suggesting medium binding affinity of HSA with complex. CD spectrum indicates α-helical structure up to β-pleated secondary structure. CV data confirmed medium reversible binding with HSA. The binding of complex with HSA shows typical reversible mode of binding, which enables the delivery of drug candidate to the tissue enzymes and receptors in an efficient manner, and thereby affects the uptake of the drug.     

Preparation and evaluation of 188Re sulfide colloidal nanoparticles loaded biodegradable poly (L‐lactic acid) microspheres for radioembolization therapy

Radioembolization with radioactive microspheres has been an effective method for the treatment of liver lesions. The aim of this study was to prepare carrier‐free ¹⁸⁸Re loaded poly (L‐lactic acid) (PLLA) microspheres through ¹⁸⁸Re sulfide colloidal nanoparticles (¹⁸⁸Re‐SC nanoparticles). The formation of ¹⁸⁸Re‐SC nanoparticles was confirmed by ultraviolet‐visible spectrophotometry. The labeling yield of ¹⁸⁸Re‐SC nanoparticles was verified using the RTLC method. Effects of synthesis parameters on morphology and size of prepared ¹⁸⁸Re‐sulfide colloidal‐PLLA microspheres (¹⁸⁸Re‐SC‐PLLA microspheres) were studied by scanning electron microscopy. In vitro stability of ¹⁸⁸Re‐SC‐PLLA microspheres was investigated in normal saline at room temperature and in human serum at 37°C. In vivo distribution studies and gamma camera imaging were performed in healthy BALB / c mice. The microspheres could be prepared with sizes between 13 and 48 μm (modal value 29 μm) and radiolabeling efficiency >99%. After incubation, the microspheres were found stable in vitro up to 72 hours. The biodistribution after intravenous injection in healthy BALB / c mice showed high accumulation in lung as a first capture pathway organ for microsphere followed by great retention over 48 hours for these microspheres. These data show that ¹⁸⁸Re‐SC‐PLLA microspheres are suitable candidate for clinical studies.     

Reviewing the binding of a series of parabens to human serum albumin

To better understand the factors that contribute to the accumulation of unmetabolized parabens (p‐hydroxybenzoic acid esters) in breast cancer tissue, the binding of a series of parabens (methyl‐, ethyl‐, butyl‐, benzyl‐paraben) to human serum albumin (HSA) was investigated by fluorescence spectroscopy and also their ability to modify the binding parameters of albumin site markers. Emission spectra of HSA upon fluorescence excitation of Trp 214 residue at 295 nm were recorded at different molar ratios of PB/HSA and data were corrected for the inner‐filter effect. A significant inner‐filter effect was obtained for molar ratios of 2.0 and above. For lower molar ratios, a slight increase in fluorescence of HSA was detected. p‐Hydroxybenzoic acid, the main metabolite of parabens, did not modify the fluorescence of HSA whatever the molar ratio used. Binding parameters for compounds that are markers of site I, bilirubin and warfarin, were determined in the absence and presence of methyl, butyl and benzyl paraben at molar ratios of PB/HSA of 0, 1 and 2. No variation of the binding constants of these markers was observed. The results indicate that parabens weakly interact with HSA thus suggesting that they are in a free form in blood and therefore more available to reach tissues. Copyright © 2013 John Wiley & Sons, Ltd.