Tetrahydrohyperforin and Octahydrohyperforin Are Two New Potent Inhibitors of Angiogenesis

Background

We have previously shown that hyperforin, a phloroglucinol derivative found in St. John''s wort, behaves as a potent anti-angiogenic compound. To identify the reactive group(s) mainly involved in this anti-angiogenic effect, we have investigated the anti-angiogenic properties of a series of stable derivatives obtained by oxidative modification of the natural product. In addition, in the present work we have studied the role of the four carbonyl groups present in hyperforin by investigating the potential of some other chemically stable derivatives.

Methodology/Principal Findings

The experimental procedures included the analysis of the effects of treatment of endothelial cells with these compounds in cell growth, cell viability, cell migration and zymographic assays, as well as the tube formation assay on Matrigel. Our study with hyperforin and eight derivatives shows that the enolized β-dicarbonyl system contained in the structure of hyperforin has a dominant role in its antiangiogenic activity. On the other hand, two of the tested hyperforin derivatives, namely, tetrahydrohyperforin and octahydrohyperforin, behave as potent inhibitors of angiogenesis. Additional characterization of these compounds included a cell specificity study of their effects on cell growth, as well as the in vivo Matrigel plug assay.

Conclusions/Significance

These observations could be useful for the rational design and chemical synthesis of more effective hyperforin derivatives as anti-angiogenic drugs. Altogether, the results indicate that octahydrohyperforin is a more specific and slightly more potent antiangiogenic compound than hyperforin.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Sensitive and specific liquid chromatographic-tandem mass spectrometric assay for dihydroergotamine and its major metabolite in human plasma

A sensitive and specific procedure for the simultaneous determination of dihydroergotamine (DHE) and its 8'-hydroxylated metabolite (8'-OH-DHE) in human plasma was developed and validated. The analytes were extracted from plasma samples by liquid-liquid extraction, separated through a Zorbax C18 column (50x2.1 mm I.D.) and detected by tandem mass spectrometry with an electrospray ionization interface. Caroverine was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 10.0 and 11.0 pg/ml for DHE and 8'-OH-DHE, respectively. The intra- and inter-run precision was measured to be below 9.1% for both DHE and 8'-OH-DHE. The inter-run accuracy was within 4% for the analytes. The overall extraction recoveries of DHE and 8'-OH-DHE were determined to be about 58 and 52% on average, respectively. The chromatographic run time was approximately 2.5 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of DHE and 8'-OH-DHE in a pharmacokinetic study of volunteers who received DHE orally.     

High-performance liquid chromatographic determination of methoxsalen in plasma after liquid—solid extraction

An improved method suitable for the determination of 8-methoxypsoralen in the range 50–1500 ng/ml in the plasma of psoriatic patients undergoing PUVA (psoralens and long-wave ultraviolet light) therapy is proposed. A 5-ml aliquot of plasma containing sodium citrate as anticoagulant was centrifuged, griseofulvin was added as internal standard and the sample was denatured with acetonitrile. The supernatant was applied to C₁₈ cartridges and 8-methoxypsoralen was eluted with methanol. The evaporated eluate was reconstituted in the mobile phase for high-performance liquid chromatography (HPLC) and applied to the HPLC column: mobile phase, acetonitrile—0.01 M phosphoric acid (34:66); flow-rate, 1 ml/min; temperature, 40°C; column, Spherisorb 5 ODS, 100 mm × 4.6 mm I.D., 5 μm particle size; UV detection at 248 nm; detection limit, 15 ng/ml of plasma.     

Determination of a novel substance P inhibitor in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometric detection using single and triple quadrupole detectors

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid-liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific's Javelin BDS C-8 2 mm x 4.6 mm 3 microm guard column coupled to BDS C-8 50 mm x 4.6 mm 3 microm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor --> ion combinations of m/z 535 --> 277 and 503 --> 259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.     

Development and validation of an enantioselective liquid-chromatography/tandem mass spectrometry method for the separation and quantification of eslicarbazepine acetate, eslicarbazepine, R-licarbazepine and oxcarbazepine in human plasma

The purpose of this study was develop and validate a sensitive and specific enantioselective liquid-chromatography/tandem mass spectrometry (LC-MS/MS) method, for the simultaneous quantification of eslicarbazepine acetate (ESL), eslicarbazepine (S-Lic), oxcarbazepine (OXC) and R-licarbazepine (R-Lic) in human plasma. Analytes were extracted from human plasma using solid phase extraction and the chromatographic separation was achieved using a mobile phase of 80% n-hexane and 20% ethanol/isopropyl alcohol (66.7/33.3, v/v). A Daicel CHIRALCEL OD-H column (5 μm, 50 mm × 4.6 mm) was used with a flow rate of 0.8 mL/min, and a run time of 8 min. ESL, S-Lic, R-Lic, OXC and the internal standard, 10,11-dihydrocarbamazepine, were quantified by positive ion electrospray ionization mass spectrometry. The method was fully validated, demonstrating acceptable accuracy, precision, linearity, and specificity in accordance with FDA regulations for the validation of bioanalytical methods. Linearity was proven over the range of 50.0-1000.0 ng/mL for ESL and OXC and over the range of 50.0-25,000.0 ng/mL for S-Lic and R-Lic. The intra- and inter-day coefficient of variation in plasma was less than 9.7% for ESL, 6.0% for OXC, 7.7% for S-Lic and less than 12.6% for R-Lic. The accuracy was between 98.7% and 107.2% for all the compounds quantified. The lower limit of quantification (LLOQ) was 50.0ng/mL for ESL, S-Lic, OXC and R-Lic in human plasma. The short-term stability in plasma, freeze-thaw stability in plasma, frozen long-term stability in plasma, autosampler stability and stock solution stability all met acceptance criteria. The human plasma samples, collected from 8 volunteers, showed that this method can be used for therapeutic monitoring of ESL and its metabolites in humans treated with ESL.     

反相高效液相色谱法对大鼠体内菲达司他的药代分析

建立反相高效液相色谱（RP—HPLC）测定大鼠血浆中菲达司他浓度的方法,在此基础上对菲达司他在大鼠体内的药代动力学进行初步研究。菲达司他的血浆样品利用乙酸乙酯提取法进行处理,色谱检测条件为用安捷伦Zorbax Eclipse XDB C18色谱柱,紫外检测波长200nm,流动相中v（水）：v（甲醇）为72．28,流速1mL／min,柱温30℃。建立的RP—HPLC法线性范围为0．8～400ug／mL（R=0．9997）。提取回收率大于95％,日内、日间精密度相对标准差（RSD）小于2％。本法简便、准确,适用于菲达司他药代动力学的研究;菲达司他在大鼠体内的药代动力学过程二室模型．     

Quantitative and selective assay of 5-methylindirubine, an inhibitor of cyclin-dependent kinases, in murine plasma using coupled liquid chromatography and electrospray tandem mass spectrometry

A sensitive and rapid LC-MS/MS assay for the quantitative determination of 5-methylindirubine (5-MI) in murine plasma is described. A 50-microL-murine plasma aliquot was spiked with an internal standard, indirubine-3-monoxime (IMO), and extracted with 1.25 mL diethyl ether. Dried extracts were reconstituted in methanol-water (8:2, v/v) and 10 microL-volumes were injected onto the HPLC system. Separation was achieved on a Gemini C18 column (150 mm x 2.1 mm ID, particle size 5 microm) using an alkaline eluent (10 mM ammonium hydroxide-methanol (5:95, v/v)). Detection was performed by negative ion electrospray followed by tandem mass spectrometry. The assay quantifies 5-MI in a range from 1 to 500 ng/mL using 50 microL of murine EDTA plasma samples. Validation results demonstrate that 5-MI concentrations can be accurately and precisely quantified in murine plasma. This assay is used to support pre-clinical pharmacologic studies with 5-MI.     

Development and validation of a simple liquid chromatographic method with ultraviolet detection for the determination of imatinib in biological samples

The aim of this study was to develop a rapid and sensitive HPLC method with UV detection for the estimation of imatinib from the plasma of patients with chronic myeloid leukemia (CML). The robustness of the method was checked by conducting first dose pharmacokinetics on blood samples from four patients who had been administered Gleevec (100 mg) in an oral dose. Samples were prepared in a simple and single step by precipitating the plasma proteins with methanol and injecting 50 microl aliquot from supernatant was subjected for analysis. Assay was conducted using a C8 column (250 mm x 4.6 mm, 5 microm particle size) under isocratic elution with 0.02 M potassium dihydrogen phosphate-acetonitrile (7:3, v/v) at a flow rate of 1 ml/min and detected using photodiode array at 265 nm. Calibration plots in spiked plasma were linear in a concentration range of 0.05-25 microg/ml. The inter and intra-day variation of standard curve was <4% (R.S.D.). This method could be a simple and quick method for the estimation of imatinib from the patient's plasma.     

A simple bioanalytical assay for determination of montelukast in human plasma: application to a pharmacokinetic study

An analytical method based on high-performance liquid chromatographic (HPLC) was developed for the determination of montelukast in human plasma using mefenamic acid as an internal standard. After precipitation of plasma proteins with acetonitrile, chromatographic separation was carried out using a Zorbax Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) with mobile phase consisted of methanol-acetonitrile-0.04M disodium hydrogen orthophosphate (22:22:56, v/v, pH 4.9). The wavelengths of fluorescence detection were set at 350 nm for excitation and 450 nm for emission. The linearity was confirmed in the concentration range of 5-1000 ng/ml in human plasma. Intra- and inter-day accuracy determined from quality control samples were 101.50 and 107.24%, and 97.15 and 100.37%, respectively. Intra- and inter-day precision measured as coefficient of variation were < or =4.72 and < or =9.00%, respectively. Extraction recoveries of drug from plasma were >48.14%. The protocol herein described was employed in a pharmacokinetic study of tablet formulation of montelukast in healthy Thai male volunteers.     

Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of valsartan in human plasma is reported. The assay is based on protein precipitation with methanol and reversed-phase chromatography with fluorimetric detection. The preparation of a batch of 24 samples takes 20 min. The liquid chromatography was performed on an octadecylsilica column (50 mm x 4 mm, 5 microm particles), the mobile phase consisted of acetonitrile -15 mM dihydrogenpotassium phosphate, pH 2.0 (45:55, v/v). The run time was 2.8 min. The fluorimetric detector was operated at 234/374 nm (excitation/emission wavelength). The limit of quantitation was 98 ng/ml using 0.2 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Ovarian follicular dynamics and hormonal profiles in heifer and mixed-parity Mediterranean Italian buffaloes (Bubalus bubalis) following an estrus synchronization protocol

The primary objective was to elucidate ovarian follicular dynamics and hormonal profiles in nulliparous heifer (HE; n = 11 ) and mixed-parity (MP; n=10 ) Mediterranean Italian water buffaloes (Bubalus bubalis) following an estrus synchronization protocol. Both groups received a progesterone releasing intravaginal device (PRID) implant for 10 days; a luteolytic dose of synthetic prostaglandin was given 7 days after PRID insertion. Daily ultrasound monitoring and collection of blood to determine plasma concentrations estradiol and progesterone started 1 day after PRID removal and lasted for 55 and 65 days in HE and MP buffaloes, respectively. Data analysis was restricted to the first 5 days after PRID removal and to one estrus cycle following induced ovulation. The HE buffaloes were not inseminated and only one ovulated within 5 days after PRID removal; the remainder ovulated between 8 and 48 days after PRID removal (except one in which ovulation was never detected). All HP buffaloes were inseminated 72, 96 and 120 h after PRID removal; seven buffaloes ovulated within 5 days after PRID removal and two were pregnant. Mean diameter of the largest follicle was significantly smaller in HE than MP buffaloes the first 4 days after PRID removal. There was a parity by time interaction ( P=0.0047 ) for plasma progesterone concentrations; progesterone was higher in HE than MP buffaloes 1 day after PRID removal, but the converse was true 2 days after PRID removal. After induced ovulation, HE buffaloes exhibited a one-wave ( n=5; length of cycle, 8-12 days), two-wave ( n=4; range: 20-26 days) or three-wave cycle ( n=1; 25 days). In contrast, all non-pregnant MP buffaloes ( n=8 ) had a two-wave cycle (range: 19-25 days). For buffaloes with two-wave cycles, the growth rate and diameter of the largest follicle was significantly smaller in HE than MP buffaloes for both the first follicular wave (1.3mm versus 1.7 mm per day and 10.5 mm versus 13.3 mm, respectively) and the second follicular wave (1.0 mm versus 1.3 mm per day and 11.0 mm versus 13.8 mm). In conclusion, there were many significant morphological and endocrine differences between HE and MP buffaloes.     

A sensitive LC-MS/MS method for determination of levamisole in human plasma: application to pharmacokinetic study

A sensitive and rapid LC-MS/MS method was developed and validated for the determination of levamisole in human plasma. The assay was based on liquid-liquid extraction of analytes from human plasma with ethyl ether. Chromatographic separation was carried on an Agilent HC-C(8) column (150 mm × 4.6 mm, 5 μm) at 40°C, with a mobile phase consisting of acetonitrile-10 mM ammonium acetate (70:30, v/v), a flow rate of 0.5 mL/min and a total run time of 6 min. Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 205.1→178.2 for levamisole, and m/z 296.1→264.1 for mebendazole (internal standard). The assay was linear over a concentration range of 0.1-30 ng/mL with a lower limit of quantification of 0.1 ng/mL. The coefficient of variation of the assay precision was less than 8.5%. The assay was successfully used to analyze human plasma samples in a pharmacokinetic study where levamisole was administered as a liniment.     

Systemic but not intraovarian concentrations of insulin-like growth factor-I are affected by short-term fasting.

To determine whether systemic and/or intraovarian concentrations of insulin-like growth factor-I (IGF-I) are affected by short-term fasting, 24 heifers were blocked by weight and, within block, were assigned to one of three treatments: fasted for 0 h (controls; n = 8), fasted for 24 h (n = 8), or fasted for 48 h (n = 8). Blood plasma was collected every 8 h from -64 h to 0 h before ovariectomy (OVEX). OVEX was performed per vagina under local anesthesia during the follicular phase of an estrous cycle (36-42 h after synchronization with prostaglandin-F2 alpha). Follicular fluid (FFL) and granulosa cells were collected individually from follicles greater than or equal to 6 mm (large), and FFL was pooled from follicles 1.0-5.9 mm (small) in diameter. Fasting did not affect (p greater than 0.20) the number (mean +/- SE) of small (52 +/- 7) or large (1.5 +/- 0.4) follicles per heifer, specific binding of 125I-hCG to granulosa cells of follicles greater than or equal to 8 mm in diameter, or concentrations of progesterone in FFL of small follicles. At OVEX, body weight was less (p less than 0.01) for 24 h- and 48 h-fasted heifers (412 +/- 7 kg and 399 +/- 7 kg, respectively) than for 0 h-fasted heifers (442 +/- 7 kg). At OVEX, plasma concentrations of IGF-I were lower (p less than 0.05) in the 48 h-fasted group (105 +/- 8 ng/ml) than in the 0 h-fasted group (140 +/- 8 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatiotemporal dynamics of contact activation factors of blood coagulation

A new in vitro model is proposed for studying the spatiotemporal distributions of activated clotting factors, in which clotting is activated in a thin layer of non-stirred plasma supplemented with a fluorogenic substrate and is monitored by fluorescence from its cleavage product. Analysis of the spatiotemporal dynamics of factor XIa and kallikrein in glass-activated human plasma provides evidence that both contact factors remain restricted to the glass surface and possibly a narrow boundary zone (<0.1 mm). The kinetics of factor XIa and kallikrein studied by a new method (in non-stirred plasma) coincided with those studied fluorimetrically with full stirring: their concentrations rapidly rose for the first few minutes after activation and then slowly declined. Factor XI and prekallikrein activation is likely to be restricted by the limited number of sites available for binding to the surface. The maximum concentration of the active factors was estimated at 2 x 10(8) molecules per mm(2) at the glass surface (irrespective of stirring). At the plastic surface, this value was 15--30 times lower.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral-chiral liquid chromatography

A coupled achiral-chiral liquid chromatographic assay has been developed to determine the concentrations of metyrapone and the enantiomers of its chiral metabolite metyrapol in plasma and urine. The chromatographic system consisted of a silica precolumn (75 × 4.6 mm I.D.) coupled in-line to a 250 × 4.6 mm I.D. column containing cellulose tris(4-methylbenzoate) coated on silica gel (Chiralcel OJ-CSP). When plasma samples were analyzed, the mobile phase was hexane-ethanol (92:8, v/v) modified with 0.1% diethylamine and when urine samples were analyzed the mobile phase was hexane-ethanol (94:6, v/v) modified with 0.2% diethylamine. Under these chromatographic conditions the chromatographic retentions [expressed as capacity factors (k′)] for metyrapone were k′ = 2.35 (plasma) and 2.52 (urine); for (−)-metyrapol k′ = 4.22 (plasma) and 4.62 (urine); for (+)-metyrapone k′ = 5.16 (plasma) and 5.86 (urine); enantioselectivities (α) were 1.09 (plasma) and 1.13 (urine). The assay has been validated for use in metabolic studies. The analyses of plasma and urine samples from one subject following oral administration of 750 mg of metyrapone indicated that the enzymatic reduction of myterapone by aldo-keto reductase was enantiospecific.     

Determination of 5-chloro-7-iodo-8-quinolinol (clioquinol) in plasma and tissues of hamsters by high-performance liquid chromatography and electrochemical detection

This paper describes a method of determining clioquinol levels in hamster plasma and tissue by means of HPLC and electrochemical detection. Clioquinol was separated on a Nucleosil C18 300 mm x 3.9 mm i.d. 7 microm column at 1 ml/min using a phosphate/citrate buffer 0.1M (400 ml) with 600 ml of a methanol:acetonitrile (1:1, v/v) mobile phase. The retention times of clioquinol and the IS were, respectively, 11.6 and 8.1 min; the quantitation limit (CV>8%) was 5 ng/ml in plasma and 10 ng/ml in tissues. The intra- and inter-assay accuracies of the method were more than 95%, with coefficients of variation between 3.0 and 7.7%, and plasma and tissue recovery rates of 72-77%. There was a linear response to clioquinol 5-2000 ng/ml in plasma, and 10-1000 ng/g in tissues. The method is highly sensitive and selective, makes it possible to study the pharmacokinetics of plasma clioquinol after oral administration and the distribution of clioquinol in tissues, and could be used to monitor plasma clioquinol levels in humans.     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.