Automated sample preparation and purification of homogenized brain tissues.

A robotic homogenized tissue sample transferring method has been developed by using a Packard MultiProbe II 8-tip system. It enables robotically transferring homogenized tissue samples from individual test tubes into a 96-well format plate for further sample purification and analysis. Extensive validation has been made to establish the accuracy and variability of this method. This automatic tissue sample transferring approach combined with automatic tissue homogenization, has significantly increased the throughput of tissue sample preparation in screening of drug candidates using liquid chromatography coupled with highly sensitive and selective tandem mass spectrometry (LC-MS/MS).

Anthrax lethal factor inhibition.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Systemic efficacy of nodulisporamides against fleas on dogs.

Nodulisporic acid A (NSA) has been shown previously to be safe in dogs and to deliver >90% flea control for 4 days following a single oral administration. Three newly prepared nodulisporamide derivatives were subsequently identified from an artificial membrane flea feeding system as exhibiting potency substantially greater than NSA. To determine if they have superior in vivo activity, these 3 nodulisporamides, as well as NSA, were evaluated in dogs at 15 mg/kg/os. Parasite challenges were made by placing 100 live Ctenocephalides felis fleas onto the dorsum of dogs every 48 hr and examining efficacy at each of those intervals over a 22-day period. Results showed that NSA produced >90% efficacy at day 2 and 81% efficacy at day 4, and its residual flea killing fell to approximately 50% by day 6 posttreatment. All dogs treated with the 3 new experimental nodulisporamides were 100% protected from flea challenges to day 8 posttreatment, and 2 of the compounds continued to produce >90% residual activity to 2 wk posttreatment. Pharmacokinetic analysis showed that plasma profiles and half-lives of NSA and these 3 new compounds correlated closely with flea efficacy. These results demonstrate that specific substitutions to the pharmacophore of NSA can substantially increase the duration of activity against fleas.

Synthesis of nodulisporic acid 2' '-oxazoles and 2' '-thiazoles.

[reaction--see text] The semisynthetic conversion of nodulisporic acid A (1) into a set of three heterocyclic side chain derivatives provided compounds, highlighted by 6, with an improved spectrum of ectoparasiticidal activity and pharmacokinetic profile relative to the natural product.

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.

Potent, orally absorbed glucagon receptor antagonists.

The SAR of 2-pyridyl-3,5-diaryl pyrroles, ligands of the human glucagon receptor and inhibitors of p38 kinase, were investigated. This effort resulted in the identification of 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrr ole 49 (L-168,049), a potent (Kb = 25 nM), selective antagonist of glucagon.

Characterization of a technique for rapid pharmacokinetic studies of multiple co-eluting compounds by LC/MS/MS.

A method for rapid pharmacokinetic screening of multiple potential drug candidates has been developed. This technique, based on the ability of liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to independently monitor multiple components, enables the quantification of substances which may or may not be chromatographically resolved. Our results indicate that the limit of quantitation and accuracy of this multiple-compound LC/MS/MRM quantitation method are comparable to a single-compound LC/MS/MRM quantitation method. No apparent ion suppression due to the existence of extraneous compounds in the analytical solution and biological matrix effect are observed in the range of the calibration curve. The issue of potential residual molecule cross-talk interference existing in the multiple-reaction monitoring mode has been discussed. This multiple-compound LC/MS/MRM quantitation method can be used for high throughput pharmacokinetic screening and to assay mixtures that have co-eluting analytes or similar m/z of precursor/product ion pairs.

A tacrolimus-related immunosuppressant with reduced toxicity.

BACKGROUND: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS: Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS: Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS: Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

Radical-induced oxidation of oxahydrindene (hexahydrobenzofuran) portion of 22,23-dihydroavermectin B1a: identification of autooxidation products.

Three products resulting from free-radical-induced oxidation of the oxahydrindene portion of 22,23-dihydroavermectin B1a (H2B1a) are 5-oxo-H2B1a, 8a-oxo-H2B1a, and 5,8a-bisoxo-H2B1a. The last of these compounds has not been reported previously.

Liquid chromatographic determination of ivermectin in animal plasma with trifluoroacetic anhydride and N-methylimidazole as the derivatization reagent.

Ivermectin is a potent anthelmintic agent which was detected at low concentrations in cattle plasma by LC after conversion to a fluorescent derivative. This was accomplished by reaction with acetic anhydride (AA) and pyridine for 24 h at 100 degrees C or with AA and N-methylimidazole (NMIM) for 1 h at 95 degrees C. Substituting trifluoroacetic anhydride (TFAA) for AA reduced the reaction time to less than 30 s at 25 degrees C, yielding an intensely fluorescent derivative with substantially fewer reagent by-products. The need for further sample preparation after derivatization with TFAA-NMIM was thereby eliminated, and detection limits of less than 20 pg ml-1 ivermectin could be achieved with 1 ml of plasma by a considerably simpler analytical procedure.

A robotic sample preparation scheme for the high performance liquid chromatographic determination of ivermectin in animal plasma.

A lengthy sample preparation scheme for the high performance liquid chromatographic determination of the antiparasitic agent ivermectin at ppb concentrations in animal plasma is adapted to a laboratory robotic system. Sample treatment involves both liquid-liquid partitioning and solid phase extraction. Specific modifications to the manual procedure include the use of serial vortex mixings in place of batchwise lateral shaking and the substitution of small (6 mL), disposable solid phase extraction columns driven by compressed gas for large (25 mL), gravity-fed, reusable glass columns. Coupling these columns to an automated solvent dispensing device simplifies handling of the large solvent volumes prescribed in the manual procedure. A productivity gain over the manual procedure is realized when operating the robotic system in the single sample mode, and an additional gain is achieved by integrating the system for multiple sample handling. Equivalence to the manual procedure is demonstrated.

Separation and determination of the two components of glycerol formal by high-performance liquid chromatography.

The two isomeric components of glycerol formal, 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol, are marginally separated (Rs = 1.0) by polar-bonded-phase high-performance liquid chromatography (HPLC) on a cyanopropyl column with acetone-hexane as the eluent. Esterification of these components with 3,5-dinitrobenzoyl chloride produces derivatives which are, however, completely resolved (Rs > 2) by normal-phase HPLC on silica; derivatization has the added advantage of introducing an ultraviolet-absorbing chromophore into each component. Preparative scale chromatography is used to isolate each of the derivatives, which are characterized by their UV, NMR and mass spectral properties. These esters are used as reference standards for an analytical method based on derivatization and normal-phase chromatography. In this way a sample of glycerol formal is calibrated for use as a standard in the direct determination of the two components by polar-bonded-phase HPLC.

Direct determination of avermectins in plasma at nanogram levels by high-performance liquid chromatography.

22,23-Dihydroavermectin B1a (I) is determined in animal plasma over the concentration range 5-60 ng/ml by reverse-phase high-performance liquid chromatography (HPLC) with UV photometric detection. Prior to HPLC the sample is isolated by gravity-fed adsorption column chromatography on Florisil. The delta 2 isomer of I (designated as compound III) is used as an internal standard, and the conversion of I to this isomer by base hydrolysis is described. An accuracy of 2 ng/ml (mean deviation) and a precision in the range of 1-3 ng/ml (standard deviation) were observed for the method. The limit of detection is 2 ng/ml based on the background observed for normal cattle plasma. The method is applicable to bioavailability studies of I at usual therapeutic concentrations.

High pressure liquid chromatographic determination of arprinocid in feed.

Arprinocid [9 - (2 - chloro - 6 - fluorophenylmethyl)-9H-purin-6-amine] is determined in feed by high pressure liquid chromatography with a silica column and ultraviolet detection. The drug is extracted from the feed into chloroform in the presence of pH 7 phosphate buffer, transferred to 0.1N HCl, and separated from interfering substances by partitioning with hexane. The acidic solution is neutralized, and the analyte is extracted into chloroform for injection into the chromatograph. This procedure has been applied to feeds containing 0.0030--0.0090% arprinocid with a precision of less than 5% relative standard deviation at the 0.0060% formulated concentration level. The results of this chromatographic procedure also correlate with those from a colorimetric analysis.

Further studies on the colorimetric determination of ronidazole in feeds.

A previously published colorimetric method for determining ronidazole, (1-methyl-5-nitro-imidazol-2-yl)methyl carbamate, can be used as an analytical technique for measuring the stability of this drug in medicated feeds. Although the color reaction per se is not selective, elution profiles of ronidazole and its demonstrated hydrolytic degradation product, 1-methyl-2-hydroxymethyl-5-nitroimidazole, show that the chromatographic separation used in the sample preparation efficiently isolates the drug from the degradation product. No interference was found in feeds containing 0.010% ronidazole and up to 0.010% degradation product.

Colorimetric determination of arprinocid in feed.

An analytical method has been developed for the determination of arprinocid (9-(2-chloro-6-fluorophenylmethyl)-9H-purin-6-amine) in feed, based upon measurement of the absorbance of the diazo chromophore formed from a product of zinc reduction of the drug in acidic solution. The analyte is extracted from the feed into chloroform in the presence of a pH 7 phosphate buffer and isolated by adsorption chromatography on alumina, followed by partitioning between hexane and 0.15M HCl. The reduction product in the aqueous phase is then treated for colorimetric measurement. This procedure has been applied to determining 0.0010--0.0080% arprinocid in feed with a precision of less than 5% relative standard deviation near the middle of this concentration range. Of 32 feed additives examined, only zoalene and sulfamethazine were serious interferences. A study and discussion of several factors, e.g., reaction time, pH, and amount of zinc metal, that affect the analytical reactions are also included.