High performance liquid chromatographic analysis and preclinical pharmacokinetics of the heteroarotinoid antitumor agent, SHetA2.

BACKGROUND: SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methane-thione], NSC 726189} is a sulfur-containing heteroarotinoid, which selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective was to develop and validate a HPLC/UV method for the determination of SHetA2, and study the pharmacokinetics of SHetA2 in the mouse. METHODS: SHetA2 and the internal standard, methylated XK469 (MeXK469) were isolated from 0.2 ml of mouse plasma by solid phase extraction. The analytes were separated on a narrow-bore C18 column, with the mobile phase consisting of 60% acetonitrile in water at a flow rate of 0.2 ml/min. UV detection was set at 341 nm. Pharmacokinetic studies of SHetA2 were carried out in mice following i.v. bolus dose at 20 mg/kg and oral administrations at 20 and 60 mg/kg. RESULTS: The standard curves were linear between 25 and 2,500 nM and the lower limit of quantification (LLOQ) was 25 nM. The within-run coefficients of variation (CVs) were 11.1% at 10, 9.4% at 100, and 5.2% at 2,500 nM and the respective between-run CVs were 10.9, 3.1, and 1.5% (all n=5). The recovery was 85.8% for SHetA2 and 80.6% for MeXK469. Following i.v. bolus dose, plasma concentrations of approximately 10 microM were achieved at 5 min in mice and declined biexponentially with detectable levels at 60 h. The data were fitted with a two-compartment model, which gave a mean initial t1/2 of 40 min and terminal t1/2 of 11.4 h (n=6). The total body clearance was approximately 1.81 l/h/kg. The volume of distribution at steady state (Vdss) was 20.8 l/kg. Plasma protein binding was found to be 99.3-99.5% at low micromolar concentrations. Plasma concentration data for the i.v. and p.o. doses were also fitted interactively to a two-compartment deconvolution model. From this result, oral bioavailability values of 15% at 20 mg/kg and 19% at 60 mg/kg were obtained. CONCLUSIONS: A highly sensitive HPLC/UV method for the quantification of SHetA2 in plasma has been developed to support pharmacokinetics of SHetA2 in the mouse. Pharmacokinetic behaviors of this drug appear to be favorable for future development.

A Means of Establishing and Justifying Binary Ethanol/Water Mixtures as Simulating Solvents in Extractables Studies.

UNLABELLED: Ethanol/water mixtures are frequently used as simulating solvents in extractables studies. However, the basis for determining and justifying the right ethanol proportion in a simulating solvent for a particular drug product or solution has not been previously established.A solvent strength model has been developed in this study, based on the correlation between the levels of a model compound, di-(2-ethylhexyl) phthalate (DEHP), extracted from a reference source material, plasticized poly-(vinyl chloride) (PVC) resin, and the proportion of ethanol in ethanol/water extractions solvents. This model was established by experimentally investigating DEHP leaching and takes the form: [Formula: see text] If the level of DEHP extracted from the standard source PVC resin by a drug product is measured, then the level can be input into the above equation and the proper ethanol content of the appropriate simulating solvent can be determined.The model has been applied to certain drug products and additives used in drug products, and the proper ethanol/simulating solvents for these products have been established. Additionally, the leaching behavior revealed in this study has been established to be consistent with previously published research and a mechanism for the observed behavior has been proposed. LAY ABSTRACT: Although ethanol/water mixtures are frequently used as simulating solvents in extractables studies, it is difficult to establish and justify what the right proportion of ethanol is for a particular drug product. A solvent strength model has been developed based on the leaching behavior of a model compound, di-(2-ethylhexyl) phthalate (DEHP), from a reference source material, plasticized poly-(vinyl chloride) (PVC). By measuring the level of DEHP leached into a drug product from the reference source material, one can use the model to calculate the correct ethanol proportion in the simulating solvent.Using this approach, ethanol/water proportions have been obtained for certain drug products. Additionally, the leaching profiles for DEHP obtained in this study were noted to be consistent with such profiles for other extractables from the PVC reference source material and with other investigations of ethanol/water as model stimulants.

Subnanogram on-line column-switching liquid chromatographic-tandem mass spectrometric quantification method for nelfinavir and methoxyphenol metabolite M1 in rat plasma.

A new on-line, rapid and sensitive column-switch LC/MS/MS method to measure nelfinavir (NFV), an HIV-1 protease inhibitor, and its major metabolite (M1) in rat plasma was developed. Rat plasma containing the analytes and the internal standard was treated with acetonitrile and the supernatant was processed through an on-line extraction and an analytical columns, with a column-switch device. ESI-LC/MS with multiple reaction monitors for appropriate analytes was performed. This assay gave a limit of quantitation (LOQ) of <1 ng/mL for the analytes with 5 min run time. The within-run and between-run precisions were <12 and <10%, respectively. This analytical method was successfully applied to a study to correlate changes in maternal and placental NFV plasma concentrations in rats following NFV exposure in utero.

Increasing the sensitivity of an LC-MS method for screening material extracts for organic extractables via mobile phase optimization.

Organic extractables (substances extracted from materials used in pharmaceutical packaging) are discovered, identified, and quantified via screening of extracts with analytical methods including liquid chromatography with mass spectrometric detection (LC-MS). Because extractables include a large number of diverse compounds that are typically present in plastic extracts at low levels, the LC-MS methods must be broad scope and sensitive. To accomplish these objectives, screening studies typically couple gradient reversed-phase separations with electrospray MS detection (both positive and negative ion modes). While such methods are generally applicable for a number of extractables, they are not optimal for some commonly encountered extractables due to either poor chromatographic performance (e.g., peak tailing) or poor MS response. Modifications to mobile phase composition (e.g., pH adjustment) were examined to improve the performance of an LC-MS screening method. The use of 0.1% acetic acid with 1 mM ammonium acetate (pH 3.6) as the aqueous portion of the mobile phase provided favorable sensitivities for a number of extractables both in positive and negative ion modes. In positive ion mode, the acidic mobile phase improved responses for moderately weak basic compounds by increasing their degree of protonation. For very weak basic compounds such as amides, ammonium ions in the mobile phase promoted proton adduct responses. In negative ion mode, an acidic mobile phase containing acetate anion improved ESI responses for acidic compounds, primarily due to gas phase effects.

Development and validation of an HPLC-MS-MS method for quantitating bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate (Tinuvin 770) and a related substance in aqueous extracts of plastic materials.

Tinuvin 770 is a light stabilizer present in numerous polymers utilized in medical or pharmaceutical applications (e.g., manufacturing, packaging, delivery systems and devices). Under conditions of use, Tinuvin 770 and its related substances may leach from the polymers and accumulate in pharmaceutical products that are administered to subjects to produce a therapeutic benefit. In order to establish the amounts of Tinuvin 770 that may be extracted from such systems and devices, sensitive and selective analytical methodologies are required. A liquid chromatographic method with tandem mass spectrometric detection (LC-MS-MS; API-ES, positive ion mode) has been developed for the purpose of quantitating Tinuvin 770 and a related substance at low concentrations [200 ng/mL (ppb) or less] in aqueous extracting media. Issues related to injection-to-injection carryover and sample matrix effects were mitigated by the addition of potassium chloride to the test samples, where the potassium ion increases Tinuvin solubility via a "salting in" effect. The developed method was validated for this application by assessing performance characteristics including accuracy, response linearity, precision, specificity, and solution stability. The validated method is suitable for the quantitation of these analytes in the concentration range of 1-200 ng/mL.

Generation of the Extractables Profile for an Elastomeric Material and Investigation of the Accumulation Behavior of Targeted Leachables Including Bis(2,2,6,6-tetramethyl-4-piperidyl) Sebacate (Tinuvin 770) and a Related Substance.

The organic extractables profile of a synthetic polyisoprene material being considered for use as a closure on a bag-type packaging system has been delineated. The predominant organic extractables associated with the test material were bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate (Tinuvin 770), several Tinuvin-related substances, fatty acids, and antioxidant-related compounds. Based on their potential product safety impact, Tinuvin and one of its related substances were chosen as target leachables. In order to establish the accumulation behavior of these target leachables under conditions that simulate the desired application, monobags (100-mL fill volume) and multichambered bags (1000-mL fill volume) were constructed with injection sites made from the test material, filled with water, and subjected to accelerated aging including multiple sterilization cycles and long-term storage at 40 °C. Even under the worse-case contact conditions, the accumulation levels of the target leachables were much less than their total available pool in the injection sites.

Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometry.

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MS(n)) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40 mg/kg.

Characterization of rat liver microsomal and hepatocytal metabolites of brevetoxins by liquid chromatography-electrospray tandem mass spectrometry.

Brevetoxins are natural neurotoxins that are produced by "red tide" algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M-H]- peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.

Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha.

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.