Rapid detection of the aspergillosis biomarker triacetylfusarinine C using interference-enhanced Raman spectroscopy.

Triacetylfusarinine C (TAFC) is a siderophore produced by certain fungal species and might serve as a highly useful biomarker for the fast diagnosis of invasive aspergillosis. Due to its renal elimination, the biomarker is found in urine samples of patients suffering from Aspergillus infections. Accordingly, non-invasive diagnosis from this easily obtainable body fluid is possible. Within our contribution, we demonstrate how Raman microspectroscopy enables a sensitive and specific detection of TAFC. We characterized the TAFC iron complex and its iron-free form using conventional and interference-enhanced Raman spectroscopy (IERS) and compared the spectra with the related compound ferrioxamine B, which is produced by bacterial species. Even though IERS only offers a moderate enhancement of the Raman signal, the employment of respective substrates allowed lowering the detection limit to reach the clinically relevant range. The achieved limit of detection using IERS was 0.5 ng of TAFC, which is already well within the clinically relevant range. By using an extraction protocol, we were able to detect 1.4 µg/mL TAFC via IERS from urine within less than 3 h including sample preparation and data analysis. We could further show that TAFC and ferrioxamine B can be clearly distinguished by means of their Raman spectra even in very low concentrations.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

Review of bioanalytical assays for the quantitation of various HDAC inhibitors such as vorinostat, belinostat, panobinostat, romidepsin and chidamine.

Histone deacetylase inhibitors (HDAC inhibitors) are used to treat malignancies such as cutaneous T cell lymphoma and peripheral T cell lymphoma. Only four drugs are approved by the US Food and Drug Administration, namely vorinostat, romidepsin, panobinostat and belinostat, while chidamide has been approved in China. There are a number of bioanalytical methods reported for the measurement of HDAC inhibitors in clinical (human plasma and serum) and preclinical (mouse plasma, rat plasma, urine and tissue homogenates, etc.) studies. This review covers various HDAC inhibitors such as vorinostat, romidepsin, panobinostat, belinostat and chidamide. In addition to providing a comprehensive review of the available methods for the above mentioned HDAC inhibitors, it also provides case studies with perspectives for chosen drugs. Based on the review, it is concluded that the published methodologies using either HPLC or LC-MS/MS are well suited for the quantification of HDAC inhibitors in various biological fluids to delineate pharmacokinetic data.

Investigation of metabolism and disposition of GSK1322322, a peptidase deformylase inhibitor, in healthy humans using the entero-test for biliary sampling.

GSK1322322 (N-((R)-2-(cyclopentylmethyl)-3-(2-(5-fluoro-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-methylpyrimidin-4-yl)hydrazinyl)-3-oxopropyl)-N-hydroxy-formamide) is an antibiotic in development by GlaxoSmithKline. In this study, we investigated the metabolism and disposition of [(14)C]GSK1322322 in healthy humans and demonstrated the utility of the Entero-Test in a human radiolabel study. We successfully collected bile from five men using this easy-to-use device after single i.v. (1000 mg) or oral administration (1200 mg in a solution) of [(14)C]GSK1322322. GSK1322322 had low plasma clearance (23.6 liters/hour) with a terminal elimination half-life of ∼4 hours after i.v. administration. After oral administration, GSK1322322 was readily and almost completely absorbed (time of maximal concentration of 0.5 hour; bioavailability 97%). GSK1322322 predominated in the systemic circulation (>64% of total plasma radioactivity). An O-glucuronide of GSK1322322 (M9) circulated at levels between 10% and 15% of plasma radioactivity and was pharmacologically inactive. Humans eliminated the radioactive dose in urine and feces at equal proportions after both i.v. and oral doses (∼45%-48% each). Urine contained mostly unchanged GSK1322322, accounting for 30% of the dose. Bile contained mostly M9, indicating that glucuronidation was likely a major pathway in humans (up to 30% of total dose). In contrast, M9 was found in low amounts in feces, indicating its instability in the gastrointestinal tract. Therefore, without the Entero-Test bile data, the contribution of glucuronidation would have been notably underestimated. An unusual N-dehydroxylated metabolite (a secondary amide) of GSK1322322 was observed primarily in the feces and was most likely formed by gut microbes.

Vorinostat with sustained exposure and high solubility in poly(ethylene glycol)-b-poly(DL-lactic acid) micelle nanocarriers: characterization and effects on pharmacokinetics in rat serum and urine.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid, known as vorinostat, is a promising anticancer drug with a unique mode of action; however, it is plagued by low water solubility, low permeability, and suboptimal pharmacokinetics. In this study, poly(ethylene glycol)-b-poly(DL-lactic acid) (PEG-b-PLA) micelles of vorinostat were developed. Vorinostat's pharmacokinetics in rats was investigated after intravenous (i.v.) (10 mg/kg) and oral (p.o.) (50 mg/kg) micellar administrations and compared with a conventional polyethylene glycol 400 solution and methylcellulose suspension. The micelles increased the aqueous solubility of vorinostat from 0.2 to 8.15 ± 0.60 and 10.24 ± 0.92 mg/mL at drug to nanocarrier ratios of 1:10 and 1:15, respectively. Micelles had nanoscopic mean diameters of 75.67 ± 7.57 and 87.33 ± 8.62 nm for 1:10 and 1:15 micelles, respectively, with drug loading capacities of 9.93 ± 0.21% and 6.91 ± 1.19%, and encapsulation efficiencies of 42.74 ± 1.67% and 73.29 ± 4.78%, respectively. The micelles provided sustained exposure and improved pharmacokinetics characterized by a significant increase in serum half-life, area under curve, and mean residence time. The micelles reduced vorinostat clearance particularly after i.v. dosing. Thus, PEG-b-PLA micelles significantly improved the p.o. and i.v. pharmacokinetics and bioavailability of vorinostat, which warrants further investigation.

Enantioselective separation and determination of adrafinil and modafinil on Chiralcel OJ-H column in rat serum and urine using solid-phase extraction followed by HPLC.

A simple and rapid normal-phase HPLC method for enantiospecific separation of a psychostimulant, adrafinil (ADL), and its metabolite modafinil (MDL) in rat serum and urine was developed. The separation was accomplished on a normal-phase polysaccharide stationary phase Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase at a flow rate of 1.0 mL/min. Detection was carried out at 225 nm using a photo diode array (PDA) detector. The elution order of the enantiomers was determined by a polarimeter connected in series with the PDA. ADL and its metabolite were recovered from rat serum and urine by solid phase extraction using Oasis HLB cartridges and the mean recoveries were >or=80%. The enantiomers were eluted within 15 min without any interference from endogenous substances. The calibration curves were linear (r(2) > 0.998) in the concentration range of 1.20-500 microg/mL for ADL and MDL. The assay was specific, accurate, precise and reproducible (intra- and inter-day precisions RSDs <7.2%). ADL in rat serum was stable over three freeze-thaw cycles at ambient temperature for 4 h. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Doping control analysis for adrafinil and its major metabolites in human urine.

A new and reliable two-step liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in combination with gas chromatography/mass spectrometry (GC/MS) for the screening and confirmation of adrafinil and its major metabolites, modafinil and modafinil acid, in human urine has been developed and validated. The method involved reversed-phase C18 solid-phase extraction (SPE) cartridge extraction and MS analysis by means of LC/MS/MS and GC/MS. The study illustrated that the ESI capillary temperature played a key role in the formation of the protonated molecule. The limits of detection (LODs) of the developed method for the three compounds were lower than the minimum required performance limit (MRPL) of the World Anti-Doping Agency (WADA). The human urine samples obtained after the oral administration of modafinil and from the Beijing 2008 Olympic Games were analyzed by using the described method, which has also been successfully applied to routine analyses and the WADA Proficiency Test.

Metabolism distribution and excretion of a matrix metalloproteinase-13 inhibitor, 4-[4-(4-fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439), in rats and dogs: assessment of the metabolic profile of CP-544439 in plasma and urine of humans.

The metabolism and disposition of 4-[4-(4-fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439), a selective inhibitor of matrix metalloproteinase-13, was investigated in rats and dogs following oral administration of [(14)C]CP-544439. Both species showed quantitative recovery of the radiolabel, and feces was the major route of excretion. Whole-body autoradioluminography study in rats suggested distribution of CP-544439 in all tissues except central nervous system. The radiolabel was rapidly eliminated from most tissues except the periodontal ligament. Metabolism of CP-544439 was extensive in both species. Only 8.4 and 1.5% of the total dose constituted unchanged CP-544439 in the rat and dog, respectively. Similarly, pharmacokinetic analysis of [(14)C]CP-544439 and unchanged CP-544439 indicated that the exposure of the parent drug was 16 and 6.5% of the total radioequivalents in rat and dog, respectively. Metabolic profiling revealed that CP-544439 was primarily metabolized via glucuronidation, reduction, and hydrolysis. Glucuronidation was the primary route of metabolism in dogs, whereas reduction of the hydroxamate moiety was the major pathway in rats. Human plasma and urine obtained from a dose escalation study in healthy human volunteers were also analyzed in this study to assess the metabolism of CP-544439 in humans and ensure that selected animal species were exposed to all major metabolites formed in humans. Analysis suggested that CP-544439 was metabolized via all three pathways in humans consistent with rat and dog; however, the glucuronide conjugate M1 was the major circulating and excretory metabolite in humans. Preliminary in vitro phenotyping studies indicated that glucuronide formation is primarily catalyzed by UGT1A1, 1A3, and 1A9.

Stability studies of vorinostat and its two metabolites in human plasma, serum and urine.

Effects of storage time and freeze-thaw procedure on the stability of vorinostat (suberoylanilide hydroxamic acid) and its two metabolites, vorinostat O-glucuronide (M1) and 4-anilino-4-oxobutanoic acid (M2), in human plasma, serum and urine have been examined using high turbulence liquid chromatography (HTLC) online extraction and tandem mass spectrometry (MS/MS) [L. Du, D.G. Musson, A.Q. Wang, Rapid Commun. Mass Spectrom. 19 (2005) 1779-1787]. Vorinostat was demonstrated not to be stable in human plasma during the process of sample processing and storage. Acidifying the plasma sample to prevent possible enzymatic hydrolysis and using plasma with different anticoagulants were evaluated to increase the stability of vorinostat, but neither of these approaches improved stability. Human serum was then used as an alternative to plasma to monitor drug concentration, and vorinostat and its two metabolites maintained consistent concentrations in human serum after 3 freeze-thaw cycles and more than 1 year storage at -70 degrees C. By comparing the stability results of serum, EDTA plasma and heparin plasma, it was deduced that clotting proteins of plasma might be a major cause of vorinostat degradation. The stability of the three analytes during the process of serum sample collection was verified indicating that prolonged sample collection (up to 180 min) has no effect on the integrity of these analytes.

Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, suppresses the growth of carcinogen-induced mammary tumors.

Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been shown to inhibit the development of N-methylnitrosourea (NMU)-induced rat mammary tumors when fed in the diet continuously for the duration of the carcinogenic process. The present study was designed to determine whether the inhibitory effects of SAHA occur during the initiation process or at subsequent stages in the carcinogenic process. In addition, animals with established NMU tumors were administered SAHA to determine whether SAHA could inhibit the continued growth of established mammary tumors. It was found that SAHA fed at 900 ppm in the diet inhibited tumor yields when administered from 14 days prior to NMU administration to termination (-14 to +130) and from +14 and +28 days to termination. However, SAHA had no effect on tumor yields when administered from -14 to +14 or from -14 to +50 days and then returned to the control diets for the remainder of the experimental period (130 days). These results indicate that the inhibitory effects of SAHA are not exerted at the initiation phase of NMU-induced mammary tumorigenesis and appear, instead, to inhibit the subsequent stages in tumor development. Of most interest was the ability of SAHA to inhibit the growth of established mammary tumors. Administration of SAHA in the diet at 900 ppm resulted in significant inhibition of established tumor growth. Thirty-two percent of SAHA-treated tumors exhibited partial regression compared to 12% of controls, growth was stabilized in 24% of treated tumors compared to 12% of controls while 11% exhibited complete regression compared to 0% of controls. Collectively, SAHA-treated tumors exhibited a 7-fold reduction in growth compared to untreated tumors over the test period. The results of this animal model study indicate that SAHA, when fed in the diet, serves as both a chemopreventive and chemotherapeutic agent in the absence of any detectable side effects.

Structure-activity relationship for two lipoxygenase inhibitors and their potential for inducing nephrotic syndrome.

In a study of structure-activity relationship with drug-induced nephropathy two lipoxygenase inhibitors, the N-hydroxyurea derivative 70C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxyurea) and the N-hydroxamic acid analogue 360C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxamic acid), were administered to rats. 70C and 360C were dosed to female Wistar rats at 100 mg/kg po daily for 7 days. Another group of rats was given a single intravenous bolus dose of puromycin aminonucleoside (PAN) at 100 mg/kg. Urine samples were collected from all groups during the study and plasma samples were collected after 7 days. Kidneys were excised and fixed for examination by electron microscopy. 70C- and PAN-treated groups both showed early changes in the glomeruli, in which the visceral cells appeared enlarged and showed varying degrees of foot process loss. This foot process loss was associated with decreases in total plasma protein and albumin and increases in the plasma cholesterol, triglycerides, creatinine, and urea were recorded. Marked proteinuria was observed in both the 70C and PAN groups. The foot process loss together with increased proteinuria, hypoalbuminemia, hypercholesterolemia, and lipemia are all characteristic of the human condition, Minimal Change Nephrotic Syndrome. All the biochemical and morphological investigations showed that 360C-treated rats were similar to the control group, suggesting that the hydroxyurea moiety of 70C is responsible, either directly or indirectly, for the induction of the nephrotic syndrome seen in rats.

Rapid quantitative determination of a collagenase inhibitor and its major metabolite by on-line liquid chromatography with ionspray tandem mass spectrometric detection.

A rapid, sensitive and specific analytical method has been developed and validated to quantify the collagenase inhibitor N2-(2(5)-[(hydroxycarbamoyl)methyl)-4-methylvaleryl]-N1, 3-dimethyl-L-valin-amide (I) and its major metabolite (II) from plasma and urine. This method employs an automated solid-phase extraction procedure to isolate the analytes and the internal standard from the biological matrix. Reconstituted extracts were analyzed by HPLC-ionspray MS-MS. Chromatography was performed on a 4.6 mm I.D. reversed-phase guard column. The retention times of the analytes and the internal standard were approximately 1.3 min. The assay has a limit of quantification of 5 ng/ml plasma and a limit of detection of 1 ng/ml, based on 100-micro l plasma aliquots. No sample-drying step was required. The standard curves were linear form 5 to 5000 ng/ml using weighted linear regression analysis (1/y2). The intra- and inter-assay precision were better than + or - 10% with intra- and inter-assay accuracies between 95 and 105%. this new HPLC-MS-MS assay procedure for I and II in plasma and urine has proven to be specific, sensitive, accurate and robust, and is being used routinely for the analysis of I in pre-clinical and clinical trial samples. Up to 200 unknowns may be analyzed each 24 h per analyst.

S.E.M. study of urease-induced crystalluria in the presence of hydroxamic derivatives.

The effect of 3 hydroxamic acid derivatives on urease-induced crystalluria was studied in vitro. Acetohydroxamic acid is more effective than salicylhydroxamic and gentisohydroxamic acids in inhibiting the kinetic of urease enzymatic reaction and in retarding the formation of struvite and apatite. Crystal deposits studied by scanning electron microscope (S.E.M.) and X-ray microanalysis indicated that acetohydroxamic acid favours the formation of large crystals of struvite, and less aggregation is observed. Salicyl and gentiso derivatives favour the formation of brushite together with struvite and apatite.

Therapy for urolithiasis by hydroxamic acids. II. Urease inhibitory potency and urinary excretion rate of hippurohydroxamic acid derivatives.

The apparent I50 values of various hippurohydroxamic acids against urease activity of sword bean were mostly 0.5 to 2.0 microM regardless of hydrophobicity of their substituents. However, the marked increase of hydrophilicity caused by substitution of trimethoxy groups conspicuously decreased the inhibitory potency. Methylation at alpha-position of the hydroxamic acid group in these compounds remarkably decreased the inhibitory potency, probably owing to steric hindrance by the alpha-methyl group. Thenoyl-, furoyl- and nicotino-glycinohydroxamic acids which are bioisostereomers of hippurohydroxamic acid had I50 values of 0.64, 1.3 and 5.3 microM, respectively. Furthermore, the inhibitory potency of some substituted hippurohydroxamic acids against the ureolytic activity of intact Proteus mirabilis isolated from patients with urinary tract infection, were half to one-tenth of those against urease activity of sword bean. On the other hand, m- and p-nitro-, m- and p-methoxy-, m- and p-acetylamino-hippurohydroxamic acid and furoylglycinohydroxamic acid showed high urinary excretion rates of 14 to 16% of the doses administered orally to rats, while most of the others had excretion rates of about 3 to 5%.

Therapy for urolithiasis by hydroxamic acids. III. Urease inhibitory potency and urinary excretion rate of N-acylglycinohydroxamic acids.

Hydroxamic acid, a potent urease inhibitor, having a high urinary excretion rate is expected to be a therapeutic agent for urolithiasis caused by urea-splitting bacterial infection of the urinary tract. Twenty-one new derivatives of N-aliphatic-acylglycinohydroxamic acids (GHAs) were synthesized, and their inhibitory potencies against the urease activity of sword bean in a phosphate buffer and against the ureolytic activity of Proteus mirabilis in human urine, and their urinary excretion rates in rats were also measured for this purpose I50 values of most of GHAs against the urease activity of sword bean were about 1 to 10 microM and 2-ethyl-n-butyroyl GHA was the most potent inhibitor with the value of 0.79 microM. I50 values of most of the GHAs against the ureolytic activity of Proteus mirabilis were about 5 to 50 microM and n-nonaroyl GHA was the most potent inhibitor with the value of 3.6 microM. 2,2-Dimethylpropionyl GHA had the highest urinary excretion rate with the recovery of 11%. Routes of administration of 2,2-dimethylpropionyl GHA and sex of rats used did not affect the amount of urinary excretion at all. The results in this report suggest that DL 2-methyl-n-butyroyl, 2-ethyl-n-butyroyl and 2,2-dimethylpropionyl GHA are the most hopeful therapeutic agents for urolithiasis among them.

Rapid screening for urease inhibitors.

Two methods are described that are suitable for the rapid screening of compounds as urease inhibitors. The first utilizes an electrode sensitive to NH4+ ions; the second is dependent on pH rise. A feature of both is a direct readout of reaction rate.

Influence of acetohydroxamic acid on experimental Corynebacterium renale pyelonephritis.

The role of Corynebacterium renale urease in the establishment of pyelonephritis was studied by the oral administration of acetohydroxamic acid (AHA), a urease inhibitor, to experimentally infected rats. The bacteria were introduced by surgical insertion of a zinc disc containing 1 X 10(6) colony-forming units of C-renale into the urinary bladder whereas sterile discs were implanted in the bladders of the control animals. Daily administration of AHA via the drinking water did not halt the development of pyelonephritis. Larger doses, given by gavage, did accomplish this goal; that is, the pH of the urine was lowered, the number of colony-forming units of C. renale in the kidney was reduced drastically, and pyelonephritic lesions were observed in the kidney by light-microscopic examination. All experimental rats developed cystitis in varying degrees of severity. About 70% of the intact AHA given by gavage was excreted in the urine 24 h after administration of this compound. Rats implanted with a urease-negative mutant of C. renale displayed no signs of pyelonephritis but did develop cystitis.