Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats. (C) 2001 Elsevier Science Inc. All rights reserved.

Conventional and novel strategies in the treatment of adrenocortical cancer

Adrenocortical carcinoma is a highly malignant neoplasm with an incidence of two per million people per year. Several treatment strategies have resulted in temporary or partial tumor regression but very few cases have attained long survival. Surgical resection of the primary tumor and metastases is most effective. Several chemotherapeutic protocols have been employed with variable success. Mitotane (o,p'-DDD) is an adrenalytic drug effective in inducing a tumor response in 33% of patients treated. Mitotane requires metabolic transformation for therapeutic action. Tumors may vary in their ability to metabolize mitotane and the ability of tumors to transform mitotane may predict the clinical response to the drug. Preliminary data show a possible correlation between metabolic activity of neoplastic adrenocortical tissue and response to mitotane. We have attempted to develop mitotane analogs with enhanced adrenalytic effect. Compared to mitotane, a di-chloro compound, the bromo-chloro and di-bromo analogs appear to have a greater effect. Future approaches to the treatment of adrenocortical carcinoma are likely to be based on blocking or reversing the biological mechanisms of tumorigenesis. Angiogenic and chemotactic mechanisms may play a role in adrenal tumor growth and inhibition of these mechanisms may result in inhibition of tumor growth. New mitotane analogs with greater adrenalytic potential could be a promising approach to developing more effective and selective therapies for adrenal cancer. Alternative approaches should attempt to suppress tumor growth by means of compounds with anti-angiogenic and anti-chemotactic activity.

Disposition of naproxen, naproxen acyl glucuronide and its rearrangement isomers in the isolated perfused rat liver

1. An isolated perfused rat liver (IPRL) preparation was used to investigate separately the disposition of the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP), its reactive acyl glucuronide metabolite (NAG) and a mixture of NAG rearrangement isomers (isoNAG), each at 30 mug NAP equivalents ml(-1) perfusate (n = 4 each group). 2. Following administration to the IPRL, NAP was eliminated slowly in a log-linear manner with an apparent elimination half-life (t(1/2)) of 13.4 +/-4.4 h. No metabolites were detected in perfusate, while NAG was the only metabolite present in bile in measurable amounts (3.9 +/-0.8%, of the dose). Following their administration to the IPRL, both NAG and isoNAG were rapidly hydrolysed (t(1/2) in perfusate=57 +/-3 and 75 +/- 14min respectively). NAG also rearranged to isoNAG in the perfusate. Both NAG and isoNAG were excreted intact in bile (24.6 and 14.8% of the NAG and isoNAG doses, respectively). 3. Covalent NAP-protein adducts in the liver increased as the dose changed from NAP to NAG to isoNAG (0.20 to 0.34 to 0.48% of the doses, respectively). Similarly, formation of covalent NAP-protein adducts in perfusate were greater in isoNAG-dosed perfusions. The comparative results Suggest that isoNAG is a better substrate for adduct formation with liver proteins than NAG.

Rearrangement of diflunisal acyl glucuronide into its beta-glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta -glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5 +/- 2.5, 28.8 +/- 8.3 and 11.0 +/- 1.6 mug DF/ml respectively, obtained at 3.8 +/- 0.3, 3.6 +/- 1.8 and 7.5 +/- 0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2 +/- 3.3, 19.8 +/- 0.8 and 42.9 +/- 10.1% of the doses respectively were recovered in feces, with less than or equal to 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine. whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degreesC. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 mug DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 mug DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta -glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon. (C) 2001 Elsevier Science Inc. All rights reserved.

The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.

Synthesis, identification, characterization, stability, solubility, and protein binding of ester derivatives of salicylic acid and diflunisal

O-Acyl esters were prepared from salicylic acid and diflunisal by esterification with the appropriate acyl anhydride (in the presence of sulfuric acid at 80 degrees C) or acyl chloride (in the presence of pyridine at 0 degrees C). Synthesis, identification and characterization of these compounds is described. In vitro hydrolysis, solubility and protein binding studies of these O-acyl esters were performed. For the diflunisal esters, the melting points fell as the side chain was increased from ethyl to pentyl. The melting points showed no significant difference as the length of the side chain was increased from pentyl to heptyl. The aspirin analogues showed a similar trend, The relationship between solubility and carbon chain length agreed closely with that for the melting points with carbon chain length. In vitro non-enzymatic hydrolysis studies concluded that: (1) hydrolysis rate constants generally decreased with carbon chain length; (2) the diflunisal esters have shorter half lives compared with their salicylate counterparts; and (3) the in vitro hydrolysis of these compounds was retarded by the presence of bovine serum albumin. Protein binding experiments showed that the strength of binding of the aspirin and diflunisal analogues to bovine serum albumin increased with carbon chain length. (C) 1997 Elsevier Science B.V.

Efficient synthesis of selenol esters from acid chlorides mediated by indium metal

This article describes an efficient and easy one-pot route for the synthesis of a wide range of selenol esters from acyl chloride with diselenides in the presence of indium metal. A variety of functional groups can be tolerated within the diorgano diselenide and the acyl chloride coupling partner. (C) 2009 Elsevier Ltd. All rights reserved.

Evaluation of the chemical interaction between carbon nanotubes functionalized with TGDDM tetrafunctional resin and hardener DDS

In this work, the chemical interaction between carbon nanotubes (MWCNT) functionalized with acyl chloride (SOCl2) and polymer chain tetrafuncional N,N,N′,N′-tetraglycidyl-4,4′- diaminodiphenylmethane (TGDDM) and hardener 4,4′diaminodiphenyl sulfone (DDS) has been monitored by Fourier transform infrared spectroscopy (FTIR) with a attenuated total reflectance (ATR) coupled. MWCNT were obtained from the pyrolysis of a mixture of camphor and ferrocene into a oven. The functionalization process was done by oxidative treatment in order to incorporate carboxylic group over the walls of MWCNT, before to be used SOCl2. The functionalized carbon nanotubes were evaluated by X-ray photoelectron spectroscopy (XPS), Raman and transmission electron microscopy (TEM). Nanostructured composites were processed by using epoxy resin with MWCNT in varying percentages. In this work it was observed that different percentages of functionalized nanotubes modify the interaction between the composite matrix and curing agent, where can be observed that in specimens with content less than 1 wt% MWCNT the chemical bond occurs preferentially from the opening of the SO double bond of the hardener and when is used MWCNT content higher than 1 wt% there is little chemical interaction with the SO bond of the hardener and most MWCNT binds to amine. © 2013 Elsevier Ltd.

Generation of nitrogen functionalities on activated carbons by amidation reactions and Hofmann rearrangement: Chemical and electrochemical characterization

Nitrogen functionalization of a highly microporous activated carbon (BET surface area higher than 3000 m2/g) has been achieved using the following sequence of treatments: (i) chemical oxidation using concentrated nitric acid, (ii) amidation by acyl chloride substitution with NH4NO3 and (iii) amination by Hoffman rearrangement. This reaction pathway yielded amide and amine functional groups, and a total nitrogen content higher than 3 at.%. It is achieved producing only a small decrease (20%) of the starting microporosity, being most of it related to the initial wet oxidation of the activated carbon. Remarkably, nitrogen aromatic rings were also formed as a consequence of secondary cyclation reactions. The controlled step-by-step modification of the surface chemistry allowed to assess the influence of individual nitrogen surface groups in the electrochemical performance in 1 M H2SO4 of the carbon materials. The largest gravimetric capacitance was registered for the pristine activated carbon due to its largest apparent surface area. The nitrogen-containing activated carbons showed the highest surface capacitances. Interestingly, the amidated activated carbon showed the superior capacitance retention due to the presence of functional groups (such as lactams, imides and pyrroles) that enhance electrical conductivity through their electron-donating properties, showing a capacitance of 83 F/g at 50 A/g.

Towards the kinetic resolution of mechanically planar chiral rotaxanes

In this work, we reported the synthesis and characterization of two [2]rotaxanes endowed with a central ammonium group and two triazolium recognition stations on either side, acting as complexation sites for a dibenzo-24-crown-8 ether macrocycle. These mechanically interlocked architectures were obtained through the interlocking of a functionalized achiral macrocycle with Cs symmetry (where the symmetry element is a mirror plane corresponding to plane of the ring) and a C∞v symmetric axle (where a mirror plane and a C∞ principal axis are aligned along the axle length). We took advantage of the reversible acid/base triggered molecular shuttling of the ring between two lateral triazolium units to switch the rotaxanes between prochiral and mechanically planar chiral forms, which exists as two rapidly-interconverting co-conformers. We exploited the reactivity of the central amino group to attach an optically pure chiral substituent, with the goal of demonstrating the enantiomeric nature of the co-conformers and to obtain a non-zero diastereomeric excess in the resulting diastereomeric products through a dynamic kinetic resolution. To this end, two enantiopure reagents were chosen that could perform clean and fast reaction with amines: a sulfonyl chloride and an acyl chloride. Only the acyl chloride successfully produced an amide in high yield with the deprotonated rotaxane. The group added to the central amine station acted as a stopper against the shuttling of the macrocycle along the axis, thus preventing the fast interconversion of the two mechanically planar enantiomers. We analysed the results through static and dynamic NMR spectroscopic techniques by varying temperature and solvent used. Indeed, the presence of diastereomers was recorded alongside the configurational isomers resulting from the slow rotation of the CN-CO bond of the amide moiety, thus paving the way for a dynamic kinetic resolution.

Hepatic structure-pharmacokinetic relationships: The hepatic disposition and metabolite kinetics of a homologous series of O-acyl derivatives of salicylic acid

1 The hepatic disposition and metabolite kinetics of a homologous series of O-acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single-pass, in-sills rat liver preparation. 2 The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non-parametric moments analysis was used to determine the area under the curve (AUC'), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3 Pregenerated SA ([C-14]-salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4 The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5 The metabolite AUC' value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC' decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6 The two-compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUG', MITT and CV2 for the parent ester and metabolite. The moments values obtained using the two-compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non-parametric approach. 7 The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirin's selectivity as an anti-thrombotic agent has been postulated to be due to selective anti-platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti-thrombotic agents than aspirin.

Hepatic disposition and metabolite kinetics of a homologous series of diflunisal esters

The hepatic disposition and metabolite kinetics of a homologous series of diflunisal O-acyl esters (acetyl, butanoyl, pentanoyl, anti hexanoyl) were determined using a single-pass perfused in situ rat liver preparation. The experiments were conducted using 2% BSA Krebs-Henseleit buffer (pH 7.4), and perfusions were performed at 30 mL/min in each liver. O-Acyl esters of diflunisal and pregenerated diflunisal were injected separately into the portal vein. The venous outflow samples containing the esters and metabolite diflunisal were analyzed by high performance liquid chromatography (HPLC). The normalized outflow concentration-time profiles for each parent ester and the formed metabolite, diflunisal, were analyzed using statistical moments analysis and the two-compartment dispersion model. Data (presented as mean +/- standard error for triplicate experiments) was compared using ANOVA repeated measures, significance level P < 0.05. The hepatic availability (AUC'), the fraction of the injected dose recovered in the outflowing perfusate, for O-acetyldiflunisal (C2D = 0.21 +/- 0.03) was significantly lower than the other esters (0.34-0.38). However, R-N/f(u), the removal efficiency number R-N divided by the unbound fraction in perfusate f(u), which represents the removal efficiency of unbound ester by the liver, was significantly higher for the most lipophilic ester (O-hexanoyldiflunisal, C6D = 16.50 +/- 0.22) compared to the other members of the series (9.57 to 11.17). The most lipophilic ester, C6D, had the largest permeability surface area (PS) product (94.52 +/- 38.20 mt min-l g-l liver) and tissue distribution value VT (35.62 +/- 11.33 mL g(-1) liver) in this series. The MTT of these O-acyl esters of diflunisal were not significantly different from one another. However, the metabolite diflunisal MTTs tended to increase with the increase in the parent ester lipophilicity (11.41 +/- 2.19 s for C2D to 38.63 +/- 9.81 s for C6D). The two-compartment dispersion model equations adequately described the outflow profiles for the parent esters and the metabolite diflunisal formed from the O-acyl esters of diflunisal in the liver.

Graphene-based biomimetic materials targeting urine metabolite as potential cancer biomarker: Application over different conductive materials for potentiometric transduction

This work presents a novel surface Smart Polymer Antibody Material (SPAM) for Carnitine (CRT, a potential biomarker of ovarian cancer), tested for the first time as ionophore in potentiometric electrodes of unconventional configuration. The SPAM material consisted of a 3D polymeric network created by surface imprinting on graphene layers. The polymer was obtained by radical polymerization of (vinylbenzyl) trimethylammonium chloride and 4-styrenesulfonic acid (signaling the binding sites), and vinyl pivalate and ethylene glycol dimethacrylate (surroundings). Non-imprinted material (NIM) was prepared as control, by excluding the template from the procedure. These materials were then used to produce several plasticized PVC membranes, testing the relevance of including the SPAM as ionophore, and the need for a charged lipophilic additive. The membranes were casted over solid conductive supports of graphite or ITO/FTO. The effect of pH upon the potentiometric response was evaluated for different pHs (2-9) with different buffer compositions. Overall, the best performance was achieved for membranes with SPAM ionophore, having a cationic lipophilic additive and tested in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 5.1. Better slopes were achieved when the membrane was casted on conductive glass (-57.4 mV/decade), while the best detection limits were obtained for graphite-based conductive supports (3.6 × 10−5mol/L). Good selectivity was observed against BSA, ascorbic acid, glucose, creatinine and urea, tested for concentrations up to their normal physiologic levels in urine. The application of the devices to the analysis of spiked samples showed recoveries ranging from 91% (± 6.8%) to 118% (± 11.2%). Overall, the combination of the SPAM sensory material with a suitable selective membrane composition and electrode design has lead to a promising tool for point-of-care applications.

Occupational exposure to diisononyl phthalate (DiNP) in polyvinyl chloride processing operations

PURPOSE: Diisononyl phthalate (DiNP) is primarily used as a plasticizer in polyvinyl chloride (PVC) materials. While information is available on general population exposure to DiNP, occupational exposure data are lacking. We present DiNP metabolite urinary concentrations in PVC processing workers, estimate DiNP daily intake for these workers, and compare worker estimates to other populations. METHODS: We assessed DiNP exposure in participants from two companies that manufactured PVC materials, a PVC film manufacturer (n = 25) and a PVC custom compounder (n = 12). A mid-shift and end-shift urine sample was collected from each participant and analyzed for the DiNP metabolite mono(carboxy-isooctyl) phthalate (MCiOP). Mixed models were used to assess the effect on MCiOP concentrations of a worker being assigned to (1) a task using DiNP and (2) a shift where DiNP was used. A simple pharmacokinetic model was used to estimate DiNP daily intake from the MCiOP concentrations. RESULTS: Creatinine-adjusted MCiOP urinary concentrations ranged from 0.42-80 μg/g in PVC film and from 1.11-13.4 μg/g in PVC compounding. PVC film participants who worked on a task using DiNP (n = 7) had the highest MCiOP geometric mean (GM) end-shift concentration (25.2 μg/g), followed by participants who worked on a shift where DiNP was used (n = 11) (17.7 μg/g) as compared to participants with no task (2.92 μg/g) or shift (2.08 μg/g) exposure to DiNP. The GM end-shift MCiOP concentration in PVC compounding participants (4.80 μg/g) was comparable to PVC film participants with no task or shift exposure to DiNP. Because no PVC compounding participants were assigned to tasks using DINP on the day sampled, DiNP exposure in this company may be underestimated. The highest DiNP intake estimate was 26 μg/kg/day. CONCLUSION: Occupational exposure to DiNP associated with PVC film manufacturing tasks were substantially higher (sixfold to tenfold) than adult general population exposures; however, all daily intake estimates were less than 25% of current United States or European acceptable or tolerable daily intake estimates. Further characterization of DiNP occupational exposures in other industries is recommended.