Interaction of Aripiprazole With Human α1-Acid Glycoprotein

We recently reported that aripiprazole binds strongly to human albumin. In continuing our investigations, we investigated the mechanism responsible for the binding and the related interactions of aripiprazole with α1-acid glycoprotein (AGP). The extrinsic Cotton effects for the binding of aripiprazole and its derivatives to AGP were generated, but the magnitudes of the induced circular dichroism intensities did not correlate with those for the binding affinities. It therefore appears that the binding mode of aripiprazole with AGP is somewhat complicated, compared with that of albumin. Isothermal titration calorimetry data obtained for the binding of aripiprazole with AGP were different from that for albumin systems in that the 3 driving reactions, entropy-driven, enthalpy-driven, and the entropy-enthalpy mixed type, were all found for the AGP system, but not albumin. Moreover, the weak binding mode of aripiprazole with the 2 proteins were supported by a molecular docking model analysis. The concentration of albumin in plasma is about 50 times higher than those of AGP, but AGP levels in plasma are increased by about 10 times under inflammatory disease. Therefore, the involvement of these 2 plasma proteins should be considered in more depth for understanding the pharmacokinetics of aripiprazole.     

A Site-Directed Mutagenesis Study of Drug-Binding Selectivity in Genetic Variants of Human α1-Acid Glycoprotein

Human α₁-acid glycoprotein (AGP), a major carrier of many basic drugs in circulation, consists of at least two genetic variants, namely A and F1*S variant. Interestingly, the variants of AGP have different drug-binding properties. The purpose of this study was to identify the amino acid residues that are responsible for the selectivity of drug binding to genetic variants of AGP using site-directed mutagenesis. First, we screened amino acid residues in the region proximal to position 100 that are involved in binding of warfarin and dipyridamole, which are F1*S-specific ligands, and of propafenone, which is an A-specific ligand, using ultrafiltration. In the F1*S variant, His97, His100, and Trp122 were involved in either warfarin- or dipyridamole-binding, while Glu92, His100, and Trp122 participated in the binding of propafenone in the A variant. Exchange of the residue at position 92 between AGP variants reversed the relative strength of propafenone binding to the two variants, but had a markedly different effect on binding of warfarin and dipyridamole. These findings indicate that the amino acid residue at position 92 plays a significant role in drug-binding selectivity in AGP variants, especially for drugs that preferentially bind to the A variant. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4316–4326, 2009     

Biantennary Glycans as Well as Genetic Variants of α1-Acid Glycoprotein Control the Enantioselectivity and Binding Affinity of Oxybutynin

Purpose The purpose of this study was to investigate the role of biantennary branching glycans of α₁-acid glycoprotein (AGP) and its genetic variants in the enantioselective binding of oxybutynin (OXY). Method Human native AGP was separated using imminodiacetate–copper (II) affinity chromatography into two fractions, the A variant and a mixture of the F1 and S variants (F1–S). These fractionated AGPs were further separated by concanavalin A affinity chromatography into two fractions, with and without biantenarry glycans. An on-line high-performance liquid chromatography (HPLC) system consisting of a high-performance frontal analysis column, an extraction column, and an analytical HPLC column was developed to determine the binding affinities of OXY enantiomers for respective AGP species. Results The total binding affinity as well as the enantiomeric selectivity of OXY in the F1–S mixed variant was significantly higher than that for the A variant, indicating that the chiral recognition ability of native AGP for the OXY enantiomers highly depends on the F1–S mixed variant. Furthermore, not only the genetic variants but also bianntenary glycans of AGP affect the binding affinity of OXY and are also responsible for the enantioselectivity. Conclusions Both genetic variants and glycan structures significantly contribute to the enantioselectivity and the binding affinity of OXY.     

Spectrofluorimetric Determination of α-Tocopherol in Capsules and Human Plasma

A simple, sensitive and rapid spectrofluorimetric method for determination of α-tocopherol in pharmaceutical capsule and human plasma was developed and validated. The native fluorescence of α-tocopherol was measured at 334 nm with excitation at 291 nm, after extraction of α-tocopherol from human plasma hexane:dichloromethane mixture. The calibration curves were linear (R≥0.9993) in the concentration range of 0.25-2.5 μg/ml of α-tocopherol in both standard solutions and plasma samples. The developed method was directly and easily applied for determination of α-tocopherol in the plasma of healthy volunteers and different type of bladder cancer and stomach cancer patients and also pharmaceutical capsule.     

Role of Biantennary Glycans and Genetic Variants of Human α1-Acid Glycoprotein in Enantioselective Binding of Basic Drugs as Studied by High Performance Frontal Analysis/capillary Electrophoresis

Purpose. To establish a clear understanding of the role of biantennary branching glycans and genetic variants of ₁-acid glycoprotein (AGP) in enantioselective bindings of basic drug. Methods. Human native AGP was separated using concanavalin A affinity chromatography into two subfractions, the unretained fraction (UR-AGP, defect of biantennary glycan) and the retained fraction (R-AGP, possessing biantennary glycan(s)). Imminodiacetate-copper (II) affinity chromatography was used to separate human native AGP into A variant and a mixture of F1 and S variants (F1*S variants). The mixed solutions of the (R)- or (S)-isomer of the model drugs (15 M disopyramide (DP) or 30 M verapamil (VER)) and 40 M of respective AGP species were subjected to high-performance frontal analysis/capillary electrophoresis (HPFA/CE) to determine the unbound drug concentrations. Results. The unbound concentrations (Cu) of DP in UR-AGP solutions were lower than those in R-AGP solutions, whereas there was no significant difference in the enantiomeric ratios (Cu(R)/Cu(S)) of DP between UR- and R-AGP solutions. In case of genetic variant, the Cu(R)/Cu(S) values of DP in F1*S and A solutions were 1.07 and 2.37, respectively. On the other hand, the enantiomeric ratio of VER in F1*S and A variant solutions were 0.900 and 0.871, respectively. Conclusions. The biantennary glycan structures are related to binding affinity of DP to AGP, but not responsible for the enantioselectivity. Genetic variants give significant effect on the enantioselectivity in DP binding, but not in VER binding.     

Tryptophan Residues Play an Important Role in the Extraordinarily High Affinity Binding Interaction of UCN-01 to Human α-1-Acid Glycoprotein

PURPOSE: To investigate the factors that contribute to the exceptionally high affinity binding of UCN-01 to human alpha1-acid glycoprotein (hAGP). METHODS: Interactions between UCN-01, UCN-02, and staurosporine with native and chemically modified hAGPs were examined using ultracentrifugation and spectroscopic analysis. RESULTS: The binding affinity of staurosporine, as well as UCN-02, to hAGP was lower than that of UCN-01 by 20- and 100-fold respectively. The percentage of UCN-01 that binds to hAGP was low at acidic pH but increased with increasing pH, reaching a maximum at pH 7.4. The binding of UCN-01 to desialylated hAGP was comparable to that of hAGP. No significant difference was found for the binding of UCN-01 to F1*S and A variants of hAGP. Chemical modification of the His, Lys, Trp, and Tyr residues caused a decrease in percentage of bound UCN-01. Trp-modified hAGP showed the largest decrease in binding. Tryptophanyl fluorescence quenching results indicate that Trp residues play a prominent role in the binding of UCN-01 to hAGP. CONCLUSIONS: A substituent at position C-7 of UCN-01 appeared to influence the binding specificity of the drug, and Trp residues in hAGP play a prominent role in the high affinity binding of UCN-01 to hAGP.     

Determination of α1-adrenoceptor antagonists in plasma by radioreceptor assay

A simple, rapid and sensitive radioreceptor assay (RRA) for the quantification of α₁-adrenoceptor antagonists such as prazosin in plasma is described. The method involves the use of an RRA based on [³H]prazosin displacement in rat cerebral cortical membranes. The method is reliable, with intra-assay and inter-assay RSDs ranging from 5.9 to 9.2%. The limit of detection is 0.2 (prazosin hydrochloride), 0.05 (tamsulosin hydrochloride) and 0.3 (bunazosin hydrochloride) pmol per assay. Using this method the plasma levels of prazosin hydrochloride were determined in beagle dogs administered orally 2.39 μmol kg⁻¹ of this drug. The plasma levels of prazosin in beagle dogs are in good agreement with those obtained using a high-performance liquid chromatography (HPLC). This RRA proved to be applicable to the monitoring of plasma prazosin levels in patients with essential hypertension and/or benign prostatic hypertrophy receiving therapy with this drug with the therapeutic dosage schedule. Thus, the concentrations of α₁-adrenoceptor antagonists in plasma can be adequately monitored by RRA as well as by HPLC.     

Reversal of Signs of Induced Cotton Effects of Dicumarol-α1-Acid Glycoprotein Systems by Phenothiazine Neuroleptics Through Ternary Complexation

The interaction of dicumarol and phenothiazine neuroleptics binding to ₁-acid glycoprotein (AGP) was investigated by circular dichroism (CD) and equilibrium dialysis. The induced CD spectra of the dicumarol–AGP complex were affected differently by the different substituents of the phenothiazine molecule. The sign of the induced Cotton effect of dicumarol bound to AGP was reversibly changed with the introduction of the propyldimethylamine substituent at position 10 or chloride group at position 2 of the phenothiazine molecule. Chlorpromazine, which contains both of these substituents reversed the sign of the induced Cotton effect with the highest intensity. The addition of trifluoperazine, fluphenazine, and promethazine containing neither of the two substituents generated a new negative CD band. However, the addition of opromazine, which contains sulfoxide at position 5, decreased the CD intensity of the dicumarol–AGP complex without changing the shape of the CD spectra. Equilibrium dialysis studies revealed that the interaction of dicumarol–AGP with phenothiazine derivatives occurred simultaneously, and the interaction followed a cooperative and anticooperative binding model. Further, among the six phenothiazine derivatives that reversed the signs of the induced Cotton effects of the dicumarol–AGP complex, a linear relationship was observed between coupling constants and the difference in the induced optical ellipticity. The opromazine and dicumarol interaction was competitive for a common binding site on the AGP molecule. Removal of sialic acid did not have any effect on this interaction. These data support the hypothesis that the acidic and the basic drug binding sites overlap each other.     

Simple Spectrophotometric Methods for the Determination of Meloxicam in Presence of Its Degradation Products

To develope two simple and accurate spectrophotometric methods for the determination of meloxicam( I )in presence of its degradation products, 5 -methyl -2 -aminothiazole ( II )and benzothiazine carboxylic acid( Ill ). Method:Both methods are based on the formation of chelate complexes of the studied drug with uranyl acetate and ferric chloride at room temperature in a methanolic medium. Results:The resulting complexes are stable for 24 hrs and show absorption maxima at 406 nm and 580 nm for uranyl and ferric complexes respectively. These methods are applicable over the concentration ranges of 10 -100 and 37.5 -300 p~g ~ mL-1 with mean recoveries of (99.44 ~ 0. 48 ) % and (99. 42 ~ 0. 45 ) %, and molar absorptivity of 4.67 x 103 and 1. 029 x 103 respectively. Conclusion:Both methods are proved to be stability indicating as no interference was observed with the degradation products. The proposed methods were successfully applied to the determination of the frug in bulk powder, laboratory prepared mixtures containing different percentages of degradation products of the drug in bulk powand pharmaceutical dosage forms     

The Measurement of the β/α Anomer Composition Within Amorphous Lactose Prepared by Spray and Freeze Drying Using a Simple 1H-NMR Method

Purpose  

Reports of the anomeric composition of amorphous lactose are rare and state a highly variable range of composition (between 0% and 60% w/w β content). We aimed to develop a quantitative measurement by ¹H-NMR of α and β anomer content in amorphous lactose produced by different production methods.     

Modulation of motor activity by α1 and α2 stimulation in mice

Summary The influence of two -adrenoceptor agonists, clonidine and B-HT 920, on motor activity was tested in mice. Both, clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine) in the dose range 30–300 g/kg s.c. equieffectively inhibited exploratory activity. On the other hand only clonidine, which stimulates ₂- and ₂-adrenoceptors increased locomotor activity in mice treated with reserpine (5 mg/kg) and apomorphine (3 mg/kg) in the doses of 0.3 and 1 mg/kg i.p. The highly selective ₂-agonist B-HT 920 was ineffective under these conditions up to 30 mg/kg i.p. It is concluded, that in mice sedative -adrenoceptors are of the ₂- and excitatory of the ₁-type.     

Determination of Molecular Mobility of Lyophilized Bovine Serum Albumin and γ-Globulin by Solid-State 1H NMR and Relation to Aggregation-Susceptibility

Purpose. Feasibility of solid-state ¹H NMR for determining the mobility of protein molecules in lyophilized cakes was considered. The mobility in cakes with various levels of water content was studied in relation to aggregation-susceptibility. Methods. Spin-spin relaxation time (T₂) of protons in lyophilized bovine serum albumin (BSA) and -globulin (BGG) was measured as a function of hydration level by solid state ¹H NMR using a solid-echo pulse sequence. Moisture-induced aggregation of the lyophilized proteins was also determined by high performance size exclusion chromatography. Results. Lyophilized BSA and BGG became susceptive to aggregation when water content exceeded about 0.2 g/g of protein. T₂ of protein protons in the lyophilized cakes started to increase at lower water contents. The increase in aggregation susceptibility observed with increasing water content appears to follow the increase in T₂ of protein protons. For lyophilized BGG, both aggregation and T₂ of protein protons decreased at water contents above 0.5 g/g protein. Conclusions. Mobility of protein molecules in lyophilized cakes was successfully detemined by solid-state ¹H NMR. The aggregation susceptibility of proteins was strongly related to their molecular mobility as indicated by T₂.     

Physiological Modeling of Altered Pharmacokinetics of a Novel Anticancer Drug,UCN-01 (7-Hydroxystaurosporine), Caused by Slow Dissociation of UCN-01 from Human α1-Acid Glycoprotein

Purpose. The extremely low clearance and small distribution volumeof UCN-01 in humans could be partly due to the high degree of bindingto hAGP (1,2). The quantitative effects of hAGP on the pharmacokineticsof UCN-01 at several levels of hAGP and UCN-01 were estimatedin rats given an infusion of hAGP to mimic the clinical situation anda physiological model for analysis was developed. Methods. The plasma concentrations of UCN-01 (72.5–7250 nmol/kgiv) in rats given an infusion of hAGP, 15 or 150 nmol/h/kg, weremeasured by HPLC. Pharmacokinetic analysis under conditionsassuming rapid equilibrium of protein binding and incorporating thedissociation rate was conducted. Results. The Vdss and CLtot of UCN-01 (725 nmol/kg iv) in ratsgiven an infusion of hAGP, 150 nmol/h/kg, fell to about 1/250 and 1/700that in control rats. The Vdss and CLtot following 72.5–7250nmol/kg UCN-01 to rats given 150 nmol/h/kg hAGP were 63.9–688ml/kg and 3.18–32.9 ml/h/kg, respectively, indicating non-linearitydue to saturation of UCN-01 binding. The CLtot estimated by thephysiological model assuming rapid equilibrium of UCN-01 bindingto hAGP, was six times higher than the observed value while the CL_totestimated by the model incorporating k_off, measured using DCC, wascomparable with the observed value. Conclusions. These results suggest that the slow dissociation ofUCN-01 from hAGP limits its disposition and elimination.     

Roles of phosphoinositide-dependent kinase-1 in α1B-adrenoceptor phosphorylation and desensitization

The role of phosphoinositide-dependent protein kinase-1 (PDK-1) activity on α(1B)-adrenoceptor phosphorylation and function was explored using pharmacological inhibitors and expression of a dominant-negative mutant of this enzyme. Noradrenaline-, phorbol myristate acetate-, lysophosphatidic acid- and epidermal growth factor-mediated α(1B)-adrenoceptor phosphorylation were markedly reduced by the two inhibitors used: UCN-01 [(7-hydroxystaurosporine; (3R*,8S*, 9R*, 10R*,12R*)-2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)-8,12-epoxy-1H, 8H-2,7b,12a-triazadibenzo[a,g]-cyclonona[cde]triden-1-one)] and OSU-03012 [(2-amino-N-[4-[5-(2-phenanthrenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide)]. A similar effect was observed in cells expressing a PDK-1 dominant-negative mutant. Phosphorylated PDK-1 (S241) and protein kinase C α (T497) were associated with cell membranes in the basal state which increased in response to the hormonal stimuli mentioned previously. UCN-01 essentially abolished phospho-PDK-1 membrane-association and markedly attenuated that of protein kinase C α. Consistent with the findings, UCN-01 reduced lysophosphatidic acid- and epidermal growth factor-induced α(1B-)adrenoceptor desensitization. Our data suggest that PDK-1 plays a permissive role in α(1B)-adrenoceptor desensitization and phosphorylation and participates in the formation of signaling complexes, which delicately modulate receptor function and regulation.     

A Dual Wavelength Differential First Derivative Spectrophotometric Method for Identification and Determination of Carbon Monoxide Generated During the Microsomal Metabolism of Xenobiotics in vitro

本文研究了双波长差示一阶导数分光光度法运用于作为对照品的一氧化碳（ＣＯ）饱和水浓度标定及定性与定量测定微粒体代谢中生成的一氧化碳的方法。本法的优点在于能显著消除试样本底干扰，大大提高了定量准确性及灵敏度。在ＣＯ浓度２～１０μｍｏｌ·Ｌ１范围内与导数光谱峰（４１５ｎｍ）和谷（４２６ｎｍ）之间距离（Ｄ）呈良好线性关系，ｒ＝０．９９９９（ｎ＝５），回归方程Ｃ（ｍｍｏｌ·Ｌ１）＝１７．６Ｄ０．４。最低检测浓度低于０．１μｍｏｌ·Ｌ１ＣＯ。系统回收率和加样回收率（Ｘ±ＲＳＤ）分别为１０２．１±２．９％（ｎ＝７）和７９．７±６．８％（ｎ＝１２）；日内、日间精密度（ＲＳＤ）分别为４．４％（ｎ＝１８）和６．１％（ｎ＝１６）。将本法用于４个三卤苯胺和一个三卤苯的体外代谢测定，结果表明，仅２，４，５三氟苯胺在体外能被大鼠肝微粒体、ＮＡＤＰＨ和分子氧代谢生成一氧化碳。苯巴比妥和地塞米松等肝药酶诱导剂能显著提高一氧化碳的代谢转化速率，它们分别为空白对照组的３或８倍。     

Determination of β-Endorphin and Fragments Thereof in Human Plasma Using High-Performance Liquid Chromatography and a Multiple Radioimmunoassay System

A method for the determination of -endorphin and -endorphin fragments in human plasma was developed. -Endorphin-related peptides were extracted from plasma using octadecasilyl-silica cartridges. Extracts were subjected to re versed-phase high-performance liquid chromatography (HPLC). Extracts as well as HPLC column eluates were assayed using a multiple radioimmunoassay system; several antibodies directed against various distinct regions of the -endorphin molecule were employed. Using this method, evidence for the presence of multiple -endorphin fragments in the plasma of healthy young volunteers (under normal conditions) was obtained.     

A comparison of the effects of TMB-8 and nifedipine on pressor responses to α1- and α2-adrenoceptor agonists in pithed rats

TMB-8 has been characterized as an inhibitor of the release of Ca⁺ from intracellular pools. We have studied the modification of the pressor responses to selective _l-adrenoceptor agonists (methoxamine and phenylephrine), and to selective ₂-adrenoceptor agonists (B-HT 920 and B-HT 933) in pithed rats, produced by TMB-8. We have compared this modification with that produced by the calcium antagonist nifedipine. Nifedipine (100 g/kg, 300 g/kg, and 1000 g/kg) inhibited in a dose-dependent manner the pressor responses to the ₁- and ₂-adrenoceptor agonists, the dose-response curves to the ₂-adrenoceptor agonists being shifted further to the right. TMB-8 at a dose of 3000 g/kg did not modify the pressor effects of the _l-adrenoceptor agonists, and neither did it reinforce the inhibition of such responses produced by nifedipine. By contrast, TMB-8 pretreatment (0.03 g/kg, 0.3 g/kg, 3 g/kg, 30 g/kg, 300 g/kg and 3000 g/kg) inhibited the responses to both ₂-adrenoceptor agonists, the inhibition being more pronounced with B-HT 920. A similar effect was obtained with 0.03 g/kg TMB-8 and 0.3 g/kg TMB-8, particularly in the case of B-HT 920. It was stronger with higher doses, but similar for all doses over 3 g/kg. The inhibition of the pressor responses mediated by the stimulation of ₂-adrenoceptors by TMB-8 was less in rats treated with the Ca²⁺ entry promoter BAY K 8644 (300 g/kg), and could also be reduced by the continuous infusion of CaCl₂ (0.25 g/min). These results suggest that in pithed rats TMB-8 may also behave as an inhibitor of the Ca⁺ influx into vascular smooth muscle.     

Binary Solvents Dispersive Liquid—Liquid Microextraction (BS-DLLME) Method for Determination of Tramadol in Urine Using High-Performance Liquid Chromatography

Background

Tramadol is an opioid, synthetic analog of codeine and has been used for the treatment of acute or chronic pain may be abused. In this work, a developed Dispersive liquid liquid microextraction (DLLME) as binary solvents-based dispersive liquid-liquid microextraction (BS-DLLME) combined with high performance liquid chromatography (HPLC) with fluorescence detection (FD) was employed for determination of tramadol in the urine samples. This procedure involves the use of an appropriate mixture of binary extraction solvents (70 μL CHCl3 and 30 μL ethyl acetate) and disperser solvent (600 μL acetone) for the formation of cloudy solution in 5 ml urine sample comprising tramadol and NaCl (7.5%, w/v). After centrifuging, the small droplets of extraction solvents were precipitated. In the final step, the HPLC with fluorescence detection was used for determination of tramadol in the precipitated phase.

Results

Various factors on the efficiency of the proposed procedure were investigated and optimized. The detection limit (S/N = 3) and quantification limit (S/N = 10) were found 0.2 and 0.9 μg/L, respectively. The relative standard deviations (RSD) for the extraction of 30 μg L of tramadol was found 4.1% (n = 6). The relative recoveries of tramadol from urine samples at spiking levels of 10, 30 and 60 μg/L were in the range of 95.6 – 99.6%.

Conclusions

Compared with other methods, this method provides good figures of merit such as good repeatability, high extraction efficiency, short analysis time, simple procedure and can be used as microextraction technique for routine analysis in clinical laboratories.     

A Convenient Fluorometric Method to Study Sulfur Mustard–Induced Apoptosis in Human Epidermal Keratinocytes Monolayer Microplate Culture

Sulfur mustard [SM; bis-(2-chloroethyl) sulfide], which causes skin blistering or vesication [(1991). Histo- and cytophatology of acute epithelial lesions. In: Papirmeister, B., Feister, A. J., Robinson, S. I., Ford, R. D., eds. Medical Defense Against Mustard Gas: Toxic Mechanisms and Pharmacological Implications. Boca Raton: CRC Press, pp. 43–78.], is a chemical warfare agent as well as a potential terrorism agent. SM-induced skin blistering is believed to be due to epidermal–dermal detachment as a result of epidermal basal cell death via apoptosis and/or necrosis. Regarding the role of apoptosis in SM pathology in animal skin, the results obtained in several laboratories, including ours, suggest the following: 1) cell death due to SM begins via apoptosis that proceeds to necrosis via an apoptotic–necrotic continuum and 2) inhibiting apoptosis decreases SM-induced microvesication in vivo. To study the mechanisms of SM-induced apoptosis and its prevention in vitro, we have established a convenient fluorometric apoptosis assay using monolayer human epidermal keratinocytes (HEK) adaptable for multiwell plates (24-, 96-, or 384-well) and high-throughput applications. This assay allows replication and multiple types of experimental manipulation insister cultures so that the apoptotic mechanisms and the effects of test compounds can be compared statistically. SM affects diverse cellular mechanisms, including endoplasmic reticulum (ER) Ca^{2 +} homeostasis, mitochondrial functions, energy metabolism, and death receptors, each of which can independently trigger apoptosis. However, the biochemical pathway in any of these apoptotic mechanisms is characterized by a pathway-specific sequence of caspases, among which caspase-3 is a key member. Therefore, we exposed 80–90% confluent HEK cultures to SM and monitored apoptosis by measuring the fluorescence generated due to hydrolysis of a fluorogenic caspase-3 substrate (acetyl- or benzyl oxycarbonyl-Asp-Glu-Val-Asp-fluorochrome, also designated as AC-or Z-DEVD- fluorochrome) added to the assay medium. Fluorescence was measured using a plate reader. We used two types of substrates, one (Sigma-Aldrich, CASP-3-F) required cell disruption and the other (Beckman-Coulter CellProbe HT Caspase-3/7 Whole Cell Assay Kit) was cell permeable. The latter substrate was useful in experiments such as determining the time-course of apoptosis immediately following SM exposure without disruption (e.g., due to cell processing). In SM-exposed HEK, fluorescence generated from the fluorogenic caspase-3 substrate hydrolysis increased in a time (0–24 h) and concentration (0.05, 0.1, 0.15, 0.2, 0.3, 0.5 mM) dependent manner. SM caused maximum fluorescence at about 0.5 mM. However, at 2 mM SM, fluorescence decreased compared with 0.5 mM, which remains to be explained. Following 0.3 mM SM exposure, which is considered to be the in vitro equivalent of a vesicating dose in vivo (Smith W. J., Sanders, K. M., Ruddle, S.E., Gross, C. L., (1993). Cytometric analysis of DNA changes induced by sulfur mustard. J. Toxicol.-Cut. Ocular Toxicol. 12(4): 337–347.), a small fluorescence increase was observed at 6 to 8 h, which was markedly higher at 12 h. At 24 h, all SM concentrations increased fluorescence. Fluorescence increase due to SM was prevented 100% by a caspase-3–specific peptide inhibitor AC-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde, 0.1 mM), but less effectively by a general caspase inhibitor Z-VAD-FMK (benzyl oxycarbonyl-Val-Ala-Asp-fluoromethylketone, 0.01 mM), indicating that the fluorescence increase was due to caspase-3–mediated apoptosis. These results suggest potential applications of this method to study apoptosis mechanisms involving caspase-3 substrates and possibly those involving other caspase substrates.