Unbiased Phenotype-Based Screen Identifies Therapeutic Agents Selective for Metastatic Prostate Cancer.

In American men, prostate cancer is the second leading cause of cancer-related death. Dissemination of prostate cancer cells to distant organs significantly worsens patients' prognosis, and currently there are no effective treatment options that can cure advanced-stage prostate cancer. In an effort to identify compounds selective for metastatic prostate cancer cells over benign prostate cancer cells or normal prostate epithelial cells, we applied a phenotype-based in vitro drug screening method utilizing multiple prostate cancer cell lines to test 1,120 different compounds from a commercial drug library. Top drug candidates were then examined in multiple mouse xenograft models including subcutaneous tumor growth, experimental lung metastasis, and experimental bone metastasis assays. A subset of compounds including fenbendazole, fluspirilene, clofazimine, niclosamide, and suloctidil showed preferential cytotoxicity and apoptosis towards metastatic prostate cancer cells in vitro and in vivo. The bioavailability of the most discerning agents, especially fenbendazole and albendazole, was improved by formulating as micelles or nanoparticles. The enhanced forms of fenbendazole and albendazole significantly prolonged survival in mice bearing metastases, and albendazole-treated mice displayed significantly longer median survival times than paclitaxel-treated mice. Importantly, these drugs effectively targeted taxane-resistant tumors and bone metastases - two common clinical conditions in patients with aggressive prostate cancer. In summary, we find that metastatic prostate tumor cells differ from benign prostate tumor cells in their sensitivity to certain drug classes. Taken together, our results strongly suggest that albendazole, an anthelmintic medication, may represent a potential adjuvant or neoadjuvant to standard therapy in the treatment of disseminated prostate cancer.

Determination of erythromycin in medicated salmonid fish feed by liquid chromatography and UV spectroscopy.

A simple, robust LC-UV method was developed to assay erythromycin in medicated salmonid feed. In this method, erythromycin was extracted from feed with acetonitrile and water, cleaned up by SPE, evaporated to dryness, reconstituted, and analyzed by LC-UV. The resulting method produced high accuracy, 82-90%, for both salmon and trout feed that represented varied pellet sizes and ingredient amounts. The intraday and interday precisions, at < or = 6 and 5%, respectively, indicated the method's good repeatability. Calibration was linear over the range of drug concentrations typically used in medicated feed. An independent analyst validation further demonstrated the repeatability of the method. The developed method could support the drug approval process for erythromycin in medicated salmonid fish feed.

Marker residue determination of tritium-labeled ivermectin in the muscle of aquacultured largemouth bass, hybrid striped bass, and yellow perch following oral treatment.

The residue depletion profiles of tritium-labeled ivermectin and its metabolites in the muscle of aquacultured largemouth bass (LMB), hybrid striped bass (HSB), and yellow perch (YP) following oral treatment are reported. Fish were administered ³H-ivermectin at the dose level of 0.1 mg/kg body weight (7-9 µCi) in a gel capsule via stomach tube. At each postdose withdrawal time, six fish of each species were sedated with buffered MS-222 and blood samples taken. Fish were then euthanized, and fillets with adhering skin (scales removed) and bile samples were collected. The muscle fillets were homogenized in dry ice to a fine powder. Aliquots of tissue, plasma, and bile were assayed for total radioactive residue (TRR). The homogenized muscle was extracted in acetonitrile or methanol followed by high-performance liquid chromatographic (HPLC) analysis to determine the presence of parent ivermectin and its potential metabolites. The highest TRR concentrations (ivermectin equivalents) of 53, 45, and 44 ng/g (ppb) were obtained on postdose day 1 for HSB, LMB, and YP, respectively. The TRR depleted most slowly in HSB to 25 ppb at day 91, followed by YP to 19 ppb at day 42 and then by LMB to 22 ppb at day 35. The total residue of ivermectin and its metabolites by HPLC analysis followed the same depletion pattern in the three species. Additionally, the depletion rate of TRR of ³H-ivermectin in the three species followed the pattern bile > plasma > muscle. The results further indicate that one of the polar metabolites of ivermectin could serve as a potential marker residue as an indication of use, rather than the parent ivermectin.

Development of a method to determine albendazole and its metabolites in the muscle tissue of yellow perch using high-performance liquid chromatography with fluorescence detection.

An HPLC method was developed for the determination of albendazole (ABZ) and its metabolites, a sulfoxide (ABZSO), a sulfone (ABZSO2), and albendazole-2-aminosulfone (ABZ-2-NH2SO2), from yellow perch muscle tissue with adhering skin. The muscle tissue samples were made alkaline with potassium carbonate and extracted with ethyl acetate, followed by a series of liquid-liquid extraction steps. After solvent evaporation, the residue was reconstituted in the initial mobile phase combination of the gradient. The mobile phase consisted of a buffer, 50 mM ammonium acetate (pH = 4.0) in 10% methanol-water, and 100% acetonitrile. The gradient was from 20% acetonitrile to 85% acetonitrile. The analytes were chromatographed on an RP Luna C18(2) column and detected by fluorescence with excitation and emission wavelengths of 290 and 330 nm, respectively. The average recoveries from fortified muscle tissue for ABZ (20-100 ppb), ABZ-SO (20-200 ppb), ABZSO2 (8-100 ppb), and ABZ-2-NH2SO2 (20-100 ppb) were 85, 95, 101, and 86%, respectively, with corresponding CV values of 9, 3, 6, and 4%, respectively. Their LOQ values were 10, 10, 1, and 10 ppb, respectively. The procedure was applied to determine ABZ and its major metabolites in the incurred muscle tissue of yellow perch obtained after orally dosing the fish with ABZ.

Residue depletion of albendazole and its metabolites in the muscle tissue of large mouth and hybrid striped bass after oral administration.

The residue depletion profiles of albendazole (ABZ) and its major metabolites: albendazole sulfoxide (ABZ-SO), albendazole sulfone (ABZ-SO(2)) and albendazole aminosulfone (ABZ-2-NH(2)SO(2)) were studied in the muscle tissues of large mouth (LMB) and hybrid striped bass (HSB). A single oral dose of 10mg/kg albendazole was given to the two fish species via intra-gastric tube. The muscle tissues with adhering skin were collected at 8, 16, 24, 48, 72, 96 and 120h post dose from both species. The samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final sample extracts were analyzed by high performance liquid chromatography. The results indicate that both ABZ and its pharmacologically active metabolite ABZ-SO were retained longer in LMB than in HSB after oral treatment. Albendazole was detectable until 8h or 6.7 degree days ( degrees D) and 48h (40 degrees D) in HSB and LMB, respectively. However, ABZ-SO was detectable up to 48h (40 degrees D) and 96h (80 degrees D) in HSB and LMB, respectively. Among the inactive metabolites, ABZ-SO(2) was present in both fish species; however, ABZ-2-NH(2)SO(2) was detected only in LMB.

Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography-tandem mass spectrometry.

In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M-H](-)m/z 128 ion resulting in the product ions m/z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10-100 microgkg(-1) of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% (n=107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5 microgkg(-1). An internal standard, (13)C(3)-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D.=15%, n=18) with an MDL of 7.4 microgkg(-1). Average recovery of CYA from shrimp was 85% (R.S.D.=10%, n=13) with an MDL of 3.5 microgkg(-1).

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs.

OBJECTIVE: To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS: 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES: Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS: All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE: Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.

Determination of albendazole and its metabolites in the muscle tissue of hybrid striped and largemouth bass using liquid chromatography with fluorescence detection.

A liquid chromatographic method was developed for the determination of albendazole and its metabolites albendazole sulfoxide, albendazole sulfone, and albendazole-2-aminosulfone from largemouth and hybrid striped bass muscle tissue with adhering skin. The muscle tissue samples were made alkaline with potassium carbonate and extracted with ethyl acetate. The extracts were further subjected to cleanup by using a series of liquid-liquid extractions. After solvent evaporation, the residue was reconstituted in mobile phase and chromatographed. The chromatography was carried out on a reversed-phase Luna C18 column, using acetonitrile-methanol buffer as the mobile phase. The analytes were detected by fluorescence with excitation and emission wavelengths of 290 and 330 nm, respectively. The average recoveries from the fortified muscle tissue of the 2 fish species for albendazole (25-100 ppb), albendazole sulfoxide (8.75-52.5 ppb), albendazole sulfone (1-10 ppb), and albendazole-2-aminosulfone (10-100 ppb) were 89, 82, 99, and 74%, respectively. The coefficient of variation for each compound was <20% in all cases. The procedure was applied to the determination of albendazole and its 3 metabolites in the muscle tissue of the 2 fish species after orally dosing them with albendazole.

Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry.

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues.

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.

Determination of albendazole and its major metabolites in the muscle tissues of Atlantic salmon, tilapia, and rainbow trout by high performance liquid chromatography with fluorometric detection.

A liquid chromatographic procedure for the determination of albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its major metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in rainbow trout, tilapia, and salmon muscle with adhering skin tissue is described. The muscle tissue samples are made alkaline with potassium carbonate and extracted with ethyl acetate. The extracts are further subjected to cleanup by utilizing a number of liquid-liquid extraction steps. After solvent evaporation, the residue is reconstituted in mobile phase and chromatographed. The chromatography is carried out on a reversed phase Luna C(18) column, using acetonitrile/methanol/buffer as a mobile phase and a fluorescence detector. The average recoveries from the fortified muscle tissue of the three fish species for albendazole (25-100 ppb), albendazole sulfoxide (15.5-62 ppb), albendazole sulfone (1-10 ppb), and albendazole-2- aminosulfone (10-100 ppb) were 94, 77, 82, and 67%, respectively. The average CV for each compound was < or =10%. The procedure was validated and then applied to the determination of albendazole and its three major metabolites in the muscle tissue of the three fish species obtained after orally dosing with albendazole.