Jason Yarbrough

Structure–activity relationships for reactivity of carbonyl-containing compounds with glutathione

For toxicological-based structure–activity relationships to advance, will require a better understanding of molecular reactivity. A rapid and inexpensive spectrophotometric assay for determining the reactive to glutathione (GSH) was developed and used to determine GSH reactivity (reactGSH) data for 21 aliphatic derivatives of esters, ketones and aldehydes. From these data, a series of structure–activity relationships were evaluated. The structure feature associated with reactGSH was an acetylenic or olefinic moiety conjugated to a carbonyl group (i.e. polarized α,β-unsaturation). This structure conveys the capacity to undergo a covalent interaction with the thiol group of cysteine (i.e. Michael- addition). Quantitatively reactGSH of the α,β-unsaturated carbonyl compounds is reliant upon the specific molecular structure with several tendencies observed. Specifically, it was noted that for α,β-unsaturated carbonyl compounds: (1) the acetylenic-substituted derivatives were more reactive than the corresponding olefinic-substituted ones; (2) terminal vinyl-substituted derivatives was more reactive than the internal vinylene-substituted ones; (3) methyl substitution on the vinyl carbon atoms diminishes reactivity and methyl-substitution on the carbon atom farthest from the carbonyl group causes a larger reduction; (4) derivatives with carbon–carbon double bond on the end of the molecule (i.e. vinyl ketone) were more reactive than one with the carbon–oxygen bond at the end of the molecule (i.e. aldehyde) and (5) the ester with an additional unsaturated vinyl groups were more reactive than the derivative having an unsaturated ethyl group.

Screening of Potentially Hormonally Active Chemicals Using Bioluminescent Yeast Bioreporters

Saccharomyces cerevisiae bioluminescent bioreporter assays were developed previously to assess a chemicals estrogenic or androgenic disrupting potential. S. cerevisiae BLYES, S. cerevisiae BLYAS, S. cerevisiae BLYR, were used to assess their reproducibility and utility in screening 68, 69, and 71 chemicals for estrogenic, androgenic, and toxic effects, respectively. EC(50) values were 6.3 +/- 2.4 x 10(-10)M (n = 18) and 1.1 +/- 0.5 x 10(-8)M (n = 13) for BLYES and BLYAS, using 17beta-estradiol and 5alpha-dihydrotestosterone over concentration ranges of 2.5 x 10(-12) through 1.0 x 10(-6)M, respectively. Based on analysis of replicate standard curves and comparison to background controls, a set of quantitative rules have been formulated to interpret data and determine if a chemical is potentially hormonally active, toxic, both, or neither. The results demonstrated that these assays are applicable for Tier I chemical screening in Environmental Protection Agencys Endocrine Disruptor Screening and Testing Program as well as for monitoring endocrine-disrupting activity of unknown chemicals in water.

Trends in structure-toxicity relationships for carbonyl-containing alpha,beta-unsaturated compounds.

Using toxicity data for 30 aliphatic polarized α,β-unsaturated derivatives of esters, aldehydes, and ketones, a series of six structure–toxicity relationships were evaluated. The structure feature of all assessed compounds, an acetylenic or olefinic moiety conjugated to a carbonyl group, is inherently electrophilic and conveys the capacity to exhibit enhanced toxicity. However, the toxic potency of α,β-unsaturated carbonyl compounds is dependent on the specific molecular structure with several trends being observed. Specific observations include: (1) between homologues, the acetylenic-substituted derivative was more toxic than the corresponding olefinic-substituted one, respectively; (2) between olefinic-homologues, terminal vinyl-substituted derivative was more toxic than the internal vinylene-substituted one; (3) within α,β-unsaturated ketones, methyl substitution on the vinyl carbon atoms reduces toxicity with methyl-substitution on the carbon atom farthest from the carbonyl group exhibiting the greater inhibition; (4) between α,β-unsaturated carbonyl compounds with the carbon–carbon double bond on the end of the molecule (vinyl ketones) and those with carbon–oxygen double bonds on the end of the molecule (aldehydes), the ketones are more toxic than the aldehydes; (5) between homologues of α,β-unsaturated esters, those with additional unsaturated moieties (allyl, propargyl, or vinyl groups) were more toxic than homologues having relevant unsaturated moieties (propyl or ethyl groups); (6) between α,β-unsaturated carbonyl compounds with different shaped alkyl-groups (i.e. different degrees of branching), homologues with straight-chain hydrocarbon moieties were more toxic than those with branched groups.

Disposition of firocoxib in equine plasma after an oral loading dose and a multiple dose regimen

The objective of this study was to determine if a single loading dose (LD), 3× the label dose of firocoxib oral paste, followed by nine maintenance doses at the current label dose achieves and maintains near steady state concentrations. Six healthy, adult mares were administered 0.3mg/kg of firocoxib on Day 0, and 0.1 mg/kg 24 h later on Day 1, and at 24 h intervals from Day 2 to Day 9, for a total of 10 doses. Blood samples were collected throughout the study. The mean firocoxib maximum plasma concentration and standard deviation was 199±97 ng/mL, 175±44 ng/mL and 183±50 ng/mL after the LD, and first and last maintenance doses, respectively. The minimum mean concentration (C(min)) increased from 100±23 ng/mL after the LD to 132±38 ng/mL at Day 7. Then, the C(min) remained constant until Day 9. The average concentration at steady state (C(avg)) was 150±45 ng/mL, which compares well to the C(avg) (130±36 ng/mL) reported after multiple daily doses at 0.1 mg/kg. The administration of the single LD allowed achievement of the average steady state drug concentrations faster than a multi-dose regimen without a loading dose. After the LD, firocoxib at 0.1 mg/kg every 24 h was able to maintain a relatively constant average drug concentration which should produce less variability in onset of action and efficacy.

Identification of reactive toxicants: structure-activity relationships for amides

A diverse series of amides were evaluated for aquatic toxicity (IGC50) assessed in the Tetrahymena pyriformis population growth impairment assay and for reactivity (EC50) with the model soft nucleophile thiol in the form of the cysteine residue of the tripeptide glutathione. All alkylamides along with some halo-substituted amides are well predicted by the simple hydrophobicity (log Kow)–electrophilicity (Elumo) response-surface model [log(IGC−150) = 0.45(log Kow) − 0.342(Elumo) − 1.11]. However, 2-halo amides with the halogen at the end of the molecule and α,β-unsaturated primary amides are among those derivatives identified as being more toxic than predicted by the model. Amides, which exhibit excess toxicity, were capable of forming covalent bonds through an SN2 displacement or a Michael addition. Moreover, only those amides exhibiting excess toxicity were reactive with thiol, suggesting that the reactivity with model nucleophiles such as the thiol group may provide a means of accurately defining reactive toxicants.

Determination of carboplatin in canine plasma by high‐performance liquid chromatography

Carboplatin is an antineoplastic drug administered to treat different tumoral conditions in canine oncology. The objective of this study was to validate a high-performance chromatographic (HPLC) method which could be applied in canine pharmacokinetic studies. Following ultrafiltration using a Centrifree device, standards, quality controls and plasma samples were separated by isocratic reversed-phase HPLC on an Inertsil ODS-2 (250 x 4.6 mm i.d.) analytical column and quantified using UV detection at 220 nm. The mobile phase was potassium phosphate (pH 4.5), with a flow-rate of 1.0 mL/min. The procedure produced a linear curve (r(2) > 0.999) over the concentration range 1-200 microg/mL. The lower limit of quantification was 1 microg/mL. The intra-assay and inter-assay precision was approximately 90%. The overall recovery was approximately 90%. The method was illustrated with a preliminary pharmacokinetic analysis on nine dogs treated with carboplatin at our hospital. Carboplatin disposition followed a monocompartmental structure in dogs and was characterized by a short half-life (50 min).

Determination of Propofol Using High Performance Liquid Chromatography in Whole Blood with Fluorescence Detection

High-performance liquid chromatography method for the determination of propofol has been developed and validated. Following a liquid extraction using ethyl acetate and hexane, samples were separated by reverse-phase high-performance liquid chromatography on an XBridge C(18) column and quantified using fluorescence detection at an excitation of 276 nm and an emission of 310 nm. The mobile phase was a mixture of water (pH 4.0) and acetonitrile, with a flow rate of 1.5 mL/min. The standard curve ranged from 5-2000 ng/mL. Intra- and inter-assay variability for propofol was less than 10%, and the average recovery was greater than 95%. This assay is suitable for use in pharmacokinetic studies.

Determination of firocoxib in equine plasma using high performance liquid chromatography

A new method of analysis has been developed and validated for the determination of firocoxib, a new nonsteroidal anti-inflammatory drug (NSAID) approved for use in horses and dogs to control pain and inflammation associated with osteoarthritis. Following a liquid extraction using ethyl acetate:hexane (40:60), samples were separated by isocratic reversed-phase HPLC on a Sunfire C(18) column and quantified using UV detection at 290 nm. The mobile phase was a mixture of water with 0.025% trifluoroacetic acid and acetonitrile, with a flow-rate of 1.1 ml/min. The procedure produced a linear curve over the concentration range 5-1500 ng/ml with a lower limit of quantification of 5 ng/ml. Intra- and inter-assay variability was less than 7%. The average recovery was 98%. The method is suitable for the analysis of clinical samples from pharmacokinetic studies and can also be used for small volume sample sizes.

Aquatic toxicity and abiotic thiol reactivity of aliphatic isothiocyanates: Effects of alkyl-size and -shape.

Aquatic toxicity data in the TETRATOX assay and reactivity data in an abiotic thiol assay were collected for a series of aliphatic isothiocyanates. These compounds can act as Michael-type acceptors with N-hydro-C-mercapto-addition to cellular thiols as a molecular mechanism of action. Comparison of both toxicity and reactivity among the analogues revealed that derivatives with a branch hydrocarbon moiety, especially branched in the β-position were less toxic and less reactive. In contrast, the di-isothiocyanate and the allyl and propargyl derivatives are more toxic than their 1-alkyl homologues. The toxicity and reactivity differences are consistent but except for the tert-butyl-derivative not remarkable. The differences are due to variations in steric hindrance at the reaction center. For the mono-isothiocyanates compounds toxicity (IGC(50)) is linearly related to thiol reactivity (EC(50)): log(1/IGC(50))=1.33(log(1/EC(50)))-0.41; n=23, s=0.24, r(2)=0.911, q(2)=0.907, F=215.

DETERMINATION OF METRONIDAZOLE IN ADULT ARTEMIA USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A new HPLC procedure for the determination of metronidazole, a nitroimidazole, in adult Artemia (live brine shrimp) has been developed and validated. Following a liquid extraction using methanol, samples were separated by isocratic reversed-phase HPLC on a Symmetry C18 column and quantified using UV detection at 324 nm. The mobile phase was a mixture of water and methanol, with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range 1-500 µg/gm with a lower limit of quantification of 0.25 µg/gm. Intra and inter-assay variability was less than 10%. The development of the assay allowed the determination of metronidazole in a live brine shrimp feeding study.