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Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. Laromustine generates two types of reactive intermediates: 90CE and methylisocyanate. When incubated with rat, dog, monkey, and human liver microsomes, [(14)C]laromustine was converted to 90CE (C-8) and seven other radioactive components (C-1-C-7). There was little difference in the metabolite profile among the species examined, in part because the formation of most components (C-1-C-6 and 90CE) did not require NADPH but involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Laromustine caused direct inhibition of CYP2B6 and CYP3A4/5 (the two enzymes involved in C-7 formation) as well as of CYP2C19. K(i) values were 125 microM for CYP2B6, 297 muM for CYP3A4/5, and 349 microM for CYP2C19 and were greater than the average clinical plasma C(max) of laromustine (25 microM). There was evidence of time-dependent inhibition of CYP1A2, CYP2B6, and CYP3A4/5. Treatment of primary cultures of human hepatocytes with up to 100 microM laromustine did not induce CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5, but the highest concentration of laromustine decreased the activity and levels of immunoreactive CYP3A4. The results of this study suggest the laromustine has 1) negligible victim potential with respect to metabolism by cytochrome P450 enzymes, 2) negligible enzyme-inducing potential, and 3) the potential in some cases to cause inhibition of CYP2B6, CYP3A4, and possibly CYP2C19 during and shortly after the duration of intravenous administration of this anticancer drug, but the clinical effects of such interactions are likely to be insignificant.

A direct anion exchange ion chromatography (IC)-based method for the detection of the chemical warfare (CW) agent degradation product, O-isopropyl methylphosphonic acid (IMPA) in agar medium has been developed. This is the first report of the development and validation of an IC-based method for the analysis of IMPA in a microorganism and agar matrix. In these experiments, IMPA served as a surrogate for the CW agent GB (Sarin) in a development program for living micro-organism-based CW agent destruction systems. Petri dishes containing the medium with 10, 100 and 1000 μg/mL IMPA were used. Samples were prepared by dilution of the agar medium with deionized water followed by sonication and then filtration before the analysis by IC/conductivity detection. Using this method, the amount of IMPA in the Petri dishes was measured. Excellent separation efficiency and freedom from interference due to common anions such as chloride and sulfate was obtained in the developed method. Retention time precision w...

Quantitative methods have been developed for the analysis of chemical warfare agent degradation products in reaction masses using capillary electrophoresis (CE). This is the first report of a systematic validation of a CE-based method for the analysis of chemical warfare agent degradation products in agent neutralization matrixes (reaction masses). After neutralization with monoethanolamine/water, the nerve agent GB (isopropyl methylphosphonofluoridate, Sarin) gives isopropyl methylphosphonic acid (IMPA) and O-isopropyl O'-(2-amino)ethyl methylphosphonate (GB-MEA adduct). The nerve agent GD (pinacolyl methylphosphonofluoridate, Soman), [pinacolyl = 2-(3,3-dimethyl)butyl] produces pinacolyl methylphosphonic acid (PMPA) and O-pinacolyl O'-(2-amino)ethyl methylphosphonate (GD-MEA adduct). The samples were prepared by dilution of the reaction masses with deionized water before analysis by CE/indirect UV detection or CE/conductivity detection. Migration time precision was less than 4.0% RSD for IMPA and 5.0 RSD for PMPA on a day-to-day basis. The detection limit for both IMPA and PMPA is 100 micrograms/L; the quantitation limit for both is 500 micrograms/L. For calibration standards, IMPA and PMPA gave a linear response (R2 = 0.9999) over the range 0.5-100 micrograms/mL. The interday precision RSDs were 1.9, 1.0, and 0.7% for IMPA at 7.5, 37.5 and 75.0 micrograms/mL, respectively. Corresponding values for PMPA (again, RSD) were 2.9, 1.1, and 1.0% at 7.5, 37.5 and 87.5 micrograms/mL, respectively, as before. Analysis accuracy was assessed by spiking actual neutralization samples with IMPA or PMPA. For IMPA, the seven spike levels used ranged from 20 to 220% of the IMPA background level, and the incremental change in the found IMPA level ranged from 86 to 99 % of the true spiking increment (R2 = 0.9987 for the linear regression). For PMPA, the five spike levels ranged from 10 to 150% of the matrix background level, and similarly, the accuracy obtained ranged from 95 to 97% of the true incremental value (R2 = 0.9999 for the linear regression).

Direct, reversible, electron transfer has been demonstrated between electrodes and native cytochrome P450 cam (cyt P450 cam ) in lipid films. Electron injection directly from electrodes into the haem Fe III of cyt P450 cam has been confirmed by the influence of the known reaction of cyt P450 cam Fe II with CO on the voltammetric midpoint potential. Analysis of square wave voltammograms suggested a distribution of enzyme formal potentials in the films. With oxygen present, more than one electron was injected into the enzyme in films, mimicking in vivo electron acceptance by cyt P450Fe III and cyt P450Fe II –O 2 during catalytic oxidations. Cyt P450 cam films also catalysed electrochemically driven reduction of trichloroacetic acid in anaerobic solutions. These stable enzyme–lipid films may find applications in fundamental biochemical and toxicity studies, biocatalysis and biosensors.

Electrochemical parameters, formal potentials (E0′), electron-transfer rate constants (k0′), and diffusion coefficients (Dct) have been determined for myoglobin (Mb) embedded in the Langmuir–Blodgett (LB) and cast films of five synthetic lipids including two types of polymeric lipids bearing ammonium or poly(ethylene glycol) as a head group. The heterogeneous electron-transfer rate constants of MbFeIII/MbFeII in these films were ca. 102–103-fold larger than those for Mb in solution at an indium tin oxide electrode. Myoglobin-lipid LB films on basal-plane pyrolytic graphite (PG) electrodes gave 3–10-fold larger k0′ values compared with cast films of the same lipid. The Mb–lipid films showed gel to liquid-crystal phase transitions consistent with bilayer structures. The peak current of the square wave voltammograms for the cast Mb–lipid films showed breaks in the phase-transition temperature regions of the corresponding lipid films. Dependence of k0′ on lipid structure was not significant. A variety of synthetic lipids provided suitable microenvironments for the enhanced electron transfer of Mb. Immobilized Mb in the lipid films was stable for more than a month. The specific orientation of Mb in all Mb–lipid films was shown by linear dichroism. The orientation does not depend on the type of the lipids.

Oxidation of ferrocene in acetonitrile resulted in films on Pt electrodes under voltammetric conditions. Films were more readily formed with tetrabutylammonium tetrafluoroborate as the electrolyte than with perchlorate salts. No films were detected when ferrocene was oxidized in aqueous 0.05 M cetyltrimethylammonium bromide (CTAB). Analysis of the films by FT-IR and Auger spectroscopy confirmed iron-containing oxidation products on Pt, presumably from chemical reactions of ferricinium ions. Oxidation of 50-100 mM ferrocene in acetonitrile on Pt yielded insoluble precipitates. Analyses by MS, FT-IR, and UV-visible (water extract), suggested a mixture of oligomeric material and a small fraction of ferricinium ions. Film formation had much less influence on voltammograms on 12.5-micron-radius Pt microdisks than on 0.5-mm-radius Pt. This is consistent with the smaller sensitivity of microelectrodes to chemical reactions following charge transfer. The smaller apparent heterogeneous rate constants (k zero') found for ferrocene on macroelectrodes than on microelectrodes could possibly be influenced by film formation on larger Pt electrodes. Correlations between macro- and microelectrode kinetic data suggest that macroelectrode k zero' values may be valid in a relative sense when ohmic drop is negligible. Bias in k zero's on conventional-sized electrodes should be small in solutions giving minimal film formation, such as micellar CTAB.

The Fe(III)/Fe(II) redox couple of the heme protein myoglobin (Mb) gave standard electron-transfer rate constants about 1000-fold larger in liquid crystal films of didodecyldimethyl ammonium bromide (DDAB) on pyrolytic graphite (PG) electrodes than in aqueous solutions. Electron-transfer rates of Mb were also enhanced in films of soluble cationic and anionic surfactants adsorbed on PG. Results suggest a role for strongly adsorbed surfactant at electrode-film interfaces, which may prevent adsorption of macromolecular impurities which can block electron transfer. Mb-DDAB films were prepared by spontaneous insertion of Mb from solution into water-insoluble cast films of DDAB

Following the recent withdrawal of several prominent drugs from US and European markets because of detrimental drug-drug interactions, metabolic drug interactions have received considerable attention in the pharmaceutical industry. In turn, the question of drug safety has received significant legal, regulatory and commercial emphasis, bringing this issue to the forefront of both industry and government drug agendas. The value of predicting the drug interactions of compounds as early as possible in the drug discovery process for all therapeutic areas cannot be underestimated. From 1964 to 1999, approximately 8% of the drugs approved by the FDA were later withdrawn from the US market. Pharmaceutical companies are facing increasing pressure to prove the long-term safety of their products, and this is complicated by the fact that animal models are not perfectly predictive of human responses, and may provide contradictory information. The failure to address safety concerns successfully during the drug optimization process may result in companies withdrawing any approved drugs from the market; drug safety issues not only present human health consequences, but also have a negative economic and public relations impact on the pharmaceutical industry. This paper discusses the significance of drug interactions, and addresses strategies to evaluate the potential of a drug candidate for drug interactions.

Structural stability and electrochemistry of Chlorella ferredoxin immobilized in a cast film of molecular bilayer membranes of a biological lipid, dimyristoylphosphatidylcholine (DMPC) on a quartz substrate or a pyrolytic graphite electrode were examined. A specular reflectance spectrum of the film showed that the [2Fe-2S] cluster in ferredoxin retains its original structure even in an air atmosphere. This indicates that the lipid bilayers provide suitable microenvironments for a stable immobilization of the protein. Cast films of ferredoxin + DMPC on a graphite electrode gave well-defined, nearly reversible redox waves with a formal potential of − 0.59 V vs. Ag¦AgCl, which is close to the reported value at promoter-modified electrodes, indicating that the electrochemical communication is from native ferredoxin.

The rule of three, relating to activity-exposure-toxicity, presents the single most difficult challenge in the design and advancement of drug candidates to the development stage. Absorption, distribution, metabolism and excretion (ADME) studies are widely used in drug discovery to optimize this balance of properties necessary to convert lead compounds into drugs that are both safe and effective for human patients. Idiosyncratic drug reactions (IDRs; referred to as type B reactions, which are mainly caused by reactive metabolites) are one type of adverse drug reaction that is important to human health and safety. This review highlights the strategies for the decision-making process involving substructures that, when found in drugs, can form reactive metabolites and are involved in toxicities in humans; the tools used to reduce IDRs are also discussed. Several examples are included to show how toxicity studies have influenced and guided drug design. Investigations of reactive intermediate formation in subcellular fractions with the use of radiolabeled reagents are also discussed.

The activity–exposure–toxicity relationship, which can be described as ‘the rule of three’, presents the single most difficult challenge in the design of drug candidates and their subsequent advancement to the development stage. ADME studies are widely used in drug discovery to optimize the balance of properties necessary to convert lead candidates into drugs that are safe and effective for humans. Metabolite characterization has become one of the key drivers of the drug discovery process, helping to optimize ADME properties and increase the success rate for drugs. Various strategies can influence drug design in the decision-making process in the structural modification of drug candidates to reduce metabolic instability.

Structural information on metabolites can be a considerable asset for enhancing and streamlining the process of developing new drug candidates. Modern approaches that generate and use metabolite structural information can accelerate the drug discovery and development process by eliminating potentially harmful candidates earlier in the process and improving the safety of new drugs. This review examines the relative merits of current and potential strategies for dealing with metabolite characterization.

Idiosyncratic drug reactions (IDRs; a specific type of drug toxicity characterized by delayed onset) are a major complication of drug therapy that need to be addressed during drug discovery and development. Efforts to improve drug safety are hampered by the lack of an accepted approach to predict IDRs, which in turn is due to the low incidence of occurrence of IDRs and the various potential mechanisms involved in these reactions. The concept of the relative rarity and formation of reactive metabolite of IDRs is briefly described. Hypothetical chemical mechanisms for the formation of reactive metabolites are summarized, including a classification of adverse drug reactions and types of reactive metabolites. The relative merits of current and potential strategies for dealing with reactive intermediates in drug discovery and development are examined, and the significance of covalent binding in drug discovery/development in vitro and in vivo systems is considered. Also discussed are the merits of tools (screening methods to trap reactive intermediates, enzyme inhibition and covalent binding) and strategies for predicting which new drugs have the potential to produce reactive intermediates and IDRs; these approaches may be considered to have the potential to improve the overall safety profile of drug candidates at various stages of the drug discovery and development process.

A significant enhancement in the method detection limits is observed in the analysis of chemical warfare agent (CWA) degradation products in environmental samples by capillary electrophoresis (CE) using electrokinetic injection. The CE method uses indirect UV detection of the nonderivitized acidic analyte and a cationic surfactant, didodecyldimethylammonium hydroxide, for reversal of the electroosmotic flow. Analytes studied include the dibasic acid methylphosphonic acid (MPA) and its monoacid/monoalkyl esters, RMPA, where R = ethyl, isopropyl, and pinacolyl (2-(3,3-dimethylbutyl)). The CE method uses an attractive buffer system which is highly stable and inexpensive, and, in addition to reversing the electroosmotic flow, provides excellent separation efficiencies within a 3-min run. This CE method is also free from interference caused by carbonate, humic acids, and fluoride. Compared to pressure injection, electrokinetic injection with this CE buffer system provided substantially lower detection limits, up to 100-fold lower for samples in reagent water. However, to best realize the benefits of the electrokinetic injection enhancement for environmental samples, a prior cleanup of the sample using standard ion-exchange cartridges is necessary. This cleanup step uses sequential cartridges to remove sulfate (barium cartridge), chloride (silver cartridge), and cations (H+ cartridge). Using this approach, detection limits for these four acids were as low as 1-2 micrograms/L for water samples and 25-50 micrograms/L for aqueous leachates of soil samples (10 mL of leachate/1.5 g of soil). The utility of this method for separation of CWA degradation products by CE is discussed in terms of pressure injection versus electokinetic injection. The effects of voltage and time of injection on the separation were investigated. Results from three types of soils and four types of water (groundwater, artificial seawater, tap water, bay water) indicated that the method has potential for environmental monitoring. Quantitative CE analysis with electrokinetic injection enhancement of detection limits of these types of environmental samples requires the use of an appropriate internal standard approach. The data presented here indicate that an internal standard-based approach could be expected to give analysis results in the sub-part-per-million concentration range of 90-110% of the true value.

We report the development of analyses for nerve agent degradation products or related species by the reversal of electroosmotic flow in capillary electrophoresis (CE). The developed methods were used in this laboratory for analysis of samples in the second and third official proficiency tests (International Round-Robins) for the Provisional Technical Secretariat/Preparatory Commission for the Organization for the Prohibition of Chemical Weapons, and those results are reported here. Analytes studied include methylphosphonic acid (a dibasic acid), the monoisopropyl ester of ethylphosphonic acid, and the monoalkyl esters of methylphosphonic acid (R = ethyl, isopropyl, isobutyl, pinacolyl (3,3-dimethyl-2-butyl), cyclohexyl, and 2-ethylhexyl). The cationic surfactants used here for the reversal of electroosmotic flow are didodecyldimethylammonium hydroxide and cetyltrimethylammonium hydroxide. CE methods using conductivity or indirect UV detection provide a good separation efficiency and very high sensitivity for the analysis of such compounds. The detection limits for these species were about 75 micrograms/L when using conductivity detection and about 100 micrograms/L when using indirect UV detection. Because pH plays an important role in the CE separation of the alkylphosphonic acids and their monoesters, the influence of pH on these separation systems was investigated. Electrolytes were stable for at least 3 months. Excellent separation efficiency and freedom from interference due to common anions were obtained in the developed methods which typically achieved complete separations in less than 3 min. The method was applied to aqueous leachates of soil, wipes of surfaces, and vegetation sampled from a field site known to have been exposed to nerve agents and subsequently cleaned up. The data from these environmental samples indicated that the method can be expected to be useful for environmental monitoring.

Salt concentration and pH of external solutions were shown to control the electrochemistry of the heme protein myoglobin (MbFe(III)-H2O) in stable, ordered films of didodecyldimethylammonium bromide (DDAB). Protonation of aquometmyoglobin (MbFe(III)-H2O) in these films precedes electron transfer from electrodes, causing formal potentials to shift negative as pH increases from 5 to 8. At pH8, MbFe(III)-H2O dissociates to MbFe(III)-OH, which is reduced directly at the electrode at higher rates than MbFe(III)-H2O. Correlations of voltammetric data with FT-IR spectra suggested that at pH4.6, an unfolded form of Mb resides in the films and is reduced directly. The concentration of salt in solution influences electrochemical properties of Mb-DDAB films by its influence on Mb conformation and by effects on interfacial Donnan potentials. NMR indicated strong binding of anions to Mb within DDAB films. Bound anions may neutralize positive charge on Mb's surface so that it can reside in a partly hydrophobic environment, as postulated on the basis of previous ESR and linear dichroism studies.

In previous work, greatly enhanced rates of electron transfer were found for myoglobin (Mb) in ordered films of surfactants on pyrolytic graphite (PG) electrodes. Direct electron transfer is now reported for Mb in films of didodecyldimethylammonium bromide (DDAB) on platinum, tin-doped indium oxide, and gold electrodes. Rates of electron transfer in these films were similar on all electrodes. In the absence of surfactant, electron transfer was observed on bare electrodes only when Mb was purified by chromatography, and only on hydrophilic tin-doped In 2 O 3 or PG. Treatment of tin-doped In 2 O 3 or PG electrodes with unpurified protein solutions blocked electron transfer to Mb in the purified solutions. Reflectance-absorbance infrared and X-ray photoelectron spectroscopy revealed proteinaceous adsorbates on electrodes exposed to unpurified solutions of Mb. This adsorbate blocks electron transfer to Mb and to ferricyanide in solution. Results suggest that electron transfer in the Mb-DDAB films may be facilitated partly by strong adsorption of surfactants on electrodes. Surfactant adsorbed at electrode-film interfaces appears to inhibit adsorption of macromolecules from Mb solutions which could otherwise block electron transfer between Mb and electrodes.

Chlorella ferredoxin in solution was found to be incorporated into the cast films of ammonium lipids on basal plane pyrolytic graphite (BPG) electrodes via ion exchange between the lipid counter anion and negatively charged ferredoxin and was embedded in them without denaturation in a way to undergo direct electron transfer reactions with the electrode.