Thymus differentiation and T-cell specificity in nu/nu +/+ mouse aggregation chimaeras

Thymus development and T cell differentiation were studied in mouse chimaeras produced by aggregating pre-implantation embryos of thymus-deficient nude BALB/c (nu/nu) and wild-type C57BL/6 (+/+) mice and vice versa. Chimaeras showed mosaic distribution of skin and coat pigmentation, of hair follicles, of glucosephosphate isomerase within all tested organs and of lymphocytes expressing the different major transplantation antigens (H-2). When tested for their capacity to generate vaccinia virus-specific and self-H-2 specific cytotoxic T cells, all chimaeras of BALB/c (nu/nu) H-2d in equilibrium C57BL/6 (+/+) H-2b type generated T cells of one or both parental origins that were specific for virus and for self-H-2 of the +/+ (H-2b) type only. In contrast, some BALB/c (+/+) H-2d in equilibrium C57BL/6 (nu/nu) H-2b chimaeras generated vaccinia virus-specific cytotoxic T cells specific for either H-2d (+/+) type or for H-2b (nu/nu) type. These asymmetrical results can be interpreted to indicate the following: (i) The +/+ thymus part alone is functional, but because of asymmetrical cross-reactivities of anti-self-H-2 specificities, the observed T cell restriction phenotypes differ. (ii) Both nu/nu and +/+ thymus parts are functional but immune response defects may be exaggerated in such chimaeras producing unexpected non-responsiveness to vaccinia virus linked to H-2d in H-2b (+/+) in equilibrium H-2d (nu/nu).     

A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen.

Spleen cells from athymic nu/nu mice contain two kinds of physically separable active cells that can have very different effects on the generation of CL (cytotoxic lymphocytes) by normal LN cells in an in vitro response against allogeneic stimulator cells. They can provide an accessory cell required for the activation of CLP (cytotoxic lymphocyte precursor cells) which need not be H-2 identical to the CLP and will function normally even when H-2 identical to the stimulator cells. They can also provide a suppressor cell that prevents the activation of CLP that can recognize the H-2 of the nu/nu mouse. Thus, with A, B, and C to represent three H-2 differnt mouse strains, a culture containing CLP from strain A and nu/nu spleen cells from strain B or strain (A x B)F1 will produce CL against strain C or (A x C)F1 stimulator cells but not against strain B or strain (A x B)F1 stimulator cells unless the suppressor cell is first removed. It is proposed that the in vivo role of the suppressor cell in a normal mouse is to prevent the activation of CLP reactive against self.     

Development and validation of a liquid chromatography/tandem mass spectrometry method for the determination of DMXAA in human and mouse plasma

A rapid, sensitive, and specific LC/MS/MS-based method was developed for determining the concentration of DMXAA in human and mouse plasma. Sample preparation involved a single protein precipitation step using acetonitrile. Separation of DMXAA and 6-isopropoxy-9-oxoxanthene-2-carboxylic acid, the internal standard, was achieved on a Waters X-Terra C(18) (50 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (55:45, v/v) containing 0.1% formic acid and isocratic flow at 0.2 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 5-3000 ng/mL. The values for precision and accuracy were <9.6%, except at the LLOQ (5 ng/mL) level, which was within 16.8%. Recovery of DMXAA in mouse plasma was >65%. DMXAA was stable through 2 freeze/thaw cycles, to 2h in mouse plasma or 50% acetonitrile, and on the autosampler to 5.1h. This method was subsequently used to measure concentrations of DMXAA in mice following intraperitoneal administration.     

Development and validation of a LC-ESI-MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics

A new LC-ESI-MS/MS assay method has been developed and validated for the quantification of swertiamarin, a representative bioactive substance of Swertia plants, in rat plasma using gentiopicroside, an analog of swertiamarin on chemical structure and chromatographic action, as the internal standard (IS). The swertiamarin and IS were extracted from rat plasma using solid-phase extraction (SPE) as the sample clean-up procedure, and they were chromatographed on a narrow internal diameter column (Agilent ZORBAX ECLIPSE XDB-C(18) 100 mm × 2.1 mm, 1.8 μm) with the mobile phase consisting of methanol and water containing 0.1% acetic acid (25:75, v/v) at a flow rate of 0.2 mL/min. The detection was performed on an Agilent G6410B tandem mass spectrometer by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M+CH(3)COO](-)→179 and m/z 415 [M+CH(3)COO](-)→179 for swertiamarin and IS, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL within a linear range of 5-1000 ng/mL (n=7, r(2)≥0.994), and the limit of detection (LOD) was demonstrated as 1.25 ng/mL (S/N≥3). The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day), accuracy, recovery, freeze/thaw, long-time stability and dilution integrity. This method was successfully applied to determination of the pharmacokinetic properties of swertiamarin in rats after oral administration at a dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration, 1920.1 ng/mL; time to reach maximum plasma concentration, 0.945 h; elimination half-time, 1.10h; apparent total clearance, 5.638 L/h/kg; and apparent volume of distribution, 9.637 L/kg.     

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.     

Mechanisms in removal of tumor by antibody

We report two preliminary trials of antibody treatment of B-cell lymphoma. Advanced lymphoma was treated with chimeric FabFc₂, in which mouse Fab'γ is linked to two human Fcγ1 fragments so as to recruit natural effectors to tumor targets. Terminal lymphoma was treated with bispecific antibody (BsAb) which recruits the ribosome-inactivating protein saporin. These different mechanisms led to interesting differences in patterns of tumor clearance. Eight patients were treated with chimeric antibody of two specificities, each at 12 mg/kg: anti-CD37, plus either anti-CD38 or anti-CD19 according to tumor phenotype. On completion of the 3-wk treatment, residual plasma antibody had a half-life exceeding 10 d. Tumor cells in blood disappeared rapidly. However significant reductions in solid masses occurred in only three patients, becoming apparent 3–4 wk after beginning treatment and then continuing slowly. Five patients were treated with preformed immune complexes of saporin and F(ab'γ)₂ BsAb. Although doses of saporin reached 10 mg weekly, contact with the tumor can only have been fleeting: plasma antibody was undetectable (<0.5 μg/mL) 48 h after infusion, whereas the saporin disappeared even faster and was undetectable (<4 ng/mL) at 24 h. Tumor cells disappeared from the blood more slowly than occurred with chimeric antibody. In contrast shrinkage of extravascular tumor was more rapid, and occurred in all patients, but proved less durable.     

Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin

Up-regulation of the dolichol pathway, a "hallmark" of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF(165). Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF(165)-specific phosphotyrosine kinase activity; and (iii) Matrigel(TM) invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in Matrigel(TM) implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in Matrigel(TM) implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ～50-60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ～65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.     

Metabolic and pharmacokinetic studies of curcumin, demethoxycurcumin and bisdemethoxycurcumin in mice tumor after intragastric administration of nanoparticle formulations by liquid chromatography coupled with tandem mass spectrometry

This paper aims to investigate the metabolism and pharmacokinetics of curcumin, demethoxycurcumin and bisdemethoxycurcumin in mice tumor. To improve water solubility, nanoparticle formulations were prepared as curcuminoids-loaded solid lipid nanoparticles (curcuminoids-SLNs) and curcumin-loaded solid lipid nanoparticles (curcumin-SLNs). After intragastric administration to tumor-bearing ICR mice, the plasma and tumor samples were analyzed by liquid chromatography with ion trap mass spectrometry. We discovered that curcuminoids were mainly present as glucuronides in plasma, whereas in free form in tumor tissue. A validated LC/MS/MS method was established to determine the three free curcuminoids in tumor homogenate. Samples were separated on a Zorbax SB-C(18) column, eluted with acetonitrile-water (containing 0.1% formic acid), and detected by TSQ Quantum triple quadrupole mass spectrometer in selected reaction monitoring mode. The method showed good linearity (r(2)=0.997-0.999) over wide dynamic ranges (2-6000 ng/mL). Variations within- and between-batch never exceeded 11.2% and 13.4%, respectively. The extraction recovery rates ranged from 78.3% to 87.7%. The pharmacokinetics of curcuminoids in mice tumor fit two-compartment model and first order elimination. For curcumin-SLNs group, the dosing of 250 mg/kg of curcumin resulted in AUC((0-48 h)) of 2285 ngh/mL and C(max) of 209 ng/mL. For curcuminoids-SLNs group, the dosing equivalent to 138 mg/kg of curcumin resulted in higher tumor concentrations (AUC=2811 ngh/mL, C(max)=285 ng/mL). It appeared that co-existing curcuminoids improved the bioavailability of curcumin.     

Effect of fasting on nychthemeral concentrations of plasma growth hormone (GH), insulin-like growth factor I (IGF-I), and cortisol in channel catfish (Ictalurus punctatus)

This experiment was conducted to characterize the effect of fasting versus satiety feeding on plasma concentrations of GH, IGF-I, and cortisol over a nychthemeron. Channel catfish fingerlings were acclimated for two weeks under a 12L:12D photoperiod, then fed or fasted for 21 d. On day 21, blood samples were collected every 2 h for 24 h. Weight of fed fish increased an average of 66.2% and fasted fish lost 21.7% of body weight on average. Average nychthemeral concentrations of plasma GH were not significantly different between fed (24.7 ng/mL) and fasted (26.8 ng/mL) fish, but average nychthemeral IGF-I concentrations were higher in fed (23.4 ng/mL) versus fasted (17.8 ng/mL) fish. An increase in plasma IGF-I concentrations was observed in fasted fish 2 h after a peak in plasma GH, but not in fed fish. Average nychthemeral plasma cortisol concentrations were higher in fed (14.5 ng/mL) versus fasted (11.0 ng/mL) fish after 21 d. Significant fluctuations and a postprandial increase in plasma cortisol were observed in fed fish and there was an overall increase in plasma cortisol of both fasted and fed fish during the scotophase. The present experiment indicates little or no effect of 21-d fasting on plasma GH levels but demonstrates fasting-induced suppression of plasma IGF-I and cortisol levels in channel catfish.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.     

Artesunate: developmental toxicity and toxicokinetics in monkeys

BACKGROUND: The developmental toxicity, toxicokinetics, and hematological effects of the antimalarial drug, artesunate, were previously studied in rats and rabbits and have now been studied in cynomolgus monkeys. METHODS: Groups of up to 15 pregnant females were dosed on Gestation Days (GD) 20–50 or for 3–7‐day intervals. RESULTS: At 30 mg/kg/day, 6 embryos died between GD30 and GD40. Histologic examination of 3 live embryos (GD26–GD36) revealed a marked reduction in embryonic erythroblasts and cardiomyopathy. At 12 mg/kg/day, 6 embryos died between GD30 and GD45. Four surviving fetuses examined on GD100 had no malformations, but long bone lengths were slightly decreased. At the developmental no‐adverse‐effect‐level (4 mg/kg/day), maternal plasma AUC was 3.68 ng.h/mL for artesunate and 6.93 ng.h/ml for its active metabolite, dihydroartemisinin (DHA). No developmental toxicity occurred with administration of 12 mg/kg/day for 3 or 7 days, GD29–31 or GD27–33 (maternal plasma AUC of 9.84 ng.h/mL artesunate and 16.4 ng.h/mL DHA). Exposures at embryotoxic doses were substantially lower than human therapeutic exposures. However, differences in monkey and human Vss for artesunate (0.5 L/kg vs. 0.18 L/kg) confound relying solely on AUC for assessing human risk. Decreases in reticulocyte count occur at therapeutic doses in humans. Changes to reticulocyte counts at embryotoxic doses in monkeys (≥12 mg/kg/day) were variable and generally minor. CONCLUSIONS: Artesunate was embryolethal at ≥12 mg/kg/day when dosed for at least 12 days at the beginning of organogenesis, but not when dosed for 3 or 7 days, indicating that developmental toxicity of artesunate is dependent upon duration of dosing in cynomologus monkeys. Birth Defects Res (Part B) 83:418–434, 2008. © 2008 Wiley‐Liss, Inc.     

Studies on defense mechanisms against Candida albicans infection in congenitally athymicnude (nu/nu) mice

The defense mechanisms against Candida albicans infection were studied by using a mouse thigh lesion model in congenitally athymic nude (nu/nu) mice and their normal littermates (nu/+). Nu/nu mice were more resistant to C. albicans infection than nu/+ mice judging from the course of the thigh lesion, the results of CFUs (colony-forming units) of C. albicans in the lesion, and histopathological observations. Histopathological and serological studies revealed that granulocytic cellular infiltration was predominant, and there were few indications of development of cell-mediated immunity to protect Candida infection in Candida-infected nu/nu and nu/+ mice. These results confirmed that lower susceptibility of nu/nu mice to C. albicans infection as compared with nu/ + mice was due to accelerated non-specific defense mechanisms in nu/nu mice, and that cell-mediated or humoral immunity played a minor role in the defense against Candida infection in this experimental model.Furthermore, treatment with high titer of rabbit anti-C. albicans serum was effective to control the number of Candida cells in thigh lesions of BALB/c mice.Above experimental results seem to clearly indicate the great variability of defense manifestation according to the experimental model exployed.     

Zinc concentrations in mouse embryo and maternal plasma

The effect of a single teratogenic dose of the antiepileptic drug valproic acid and its nonteratogenic metabolite, 2-en-valproic acid, on zinc concentrations in mouse plasma, embryo, and decidua on d 9 of gestation was investigated. The substances were injected subcutaneously (sc) as their sodium salts. In this mouse model, valproic acid induced between 20% (400 mg/kg dose) and 60% (600 mg/kg dose) incidence of exencephaly in living fetuses; 2-en-valproic acid was not teratogenic at these dose levels. The zinc concentrations in plasma were significantly increased 1 and 2 h after administration of both substances. The embryonic zinc concentrations were increased 2 and 4 h after application of both substances. The concentrations of zinc in the decidua were not affected. The similarity of effects of valproic acid and its nonteratogenic analog on zinc concentrations in maternal plasma and embryo suggests that the teratogenicity of a single administration of valproic acid in the mouse is not owing to interference with the zinc metabolism in this species.     

Synthetic methods for the preparation of ARQ 501 (beta-Lapachone) human blood metabolites

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione), a synthetic version of beta-Lapachone, is a promising anti-cancer agent currently in multiple Phase II clinical trials. Promising anti-cancer activity was observed in Phase I and Phase II trials. Metabolism by red blood cells of drugs is an understudied area of research and the metabolites arising from oxidative ring opening (M2 and M3), decarbonylation/ring contraction (M5), and decarbonylation/oxidation (M4 and M6) of ARQ 501 offer a unique opportunity to provide insight into these metabolic processes. Since these metabolites were not detected in in vitro incubations of ARQ 501 with liver microsomes and were structurally diverse, confirmation by chemical synthesis was considered essential. In this report, we disclose the synthetic routes employed and the characterization of the reference standards for these blood metabolites as well as additional postulated structures, which were not confirmed as metabolites.     

Kinetic difference of berberine between hippocampus and plasma in rat after intravenous administration of Coptidis rhizoma extract

In order to investigate the pharmacokinetics of berberine in Coptidis rhizoma extract in rat hippocampus and plasma, a simple and accurate high-performance liquid chromatography method was employed in this study. Berberine was determined using a Hypersil C(18) column with an isocratic mobile phase of acetonitrile-0.05 M potassium dihydrogen phosphate (containing 0.5% triethylamine, pH 3.0) and with UV detection at 236 nm. The lower limit of quantification for berberine in both hippocampus and plasma was 24 ng/ml, and the lowest concentrations of berberine determined in rat hippocampus and plasma samples were 30.7 ng/ml at 48 h and 38.5 ng/ml at 4 h, respectively. The calibration curve for berberine was linear over the concentration range 24--6000 ng/ml. At this concentration range, the overall recoveries (90.6--94.2%) for berberine were determined and the accuracy of intra- and inter-day assays from rat samples were less than 7% RSD. Following intravenous administration of C. rhizoma extract at a dose of 10.2 mg/kg containing 3 mg/kg berberine, berberine in the plasma eliminated rapidly (t(1/2 beta)=1.13 h). However, berberine in the hippocampus increased rapidly (t(1/2 alpha)=0.215 h), peaked at 3.67 h with a concentration of 272 ng/g, and had a slow elimination rate (t(1/2 beta)=12.0 h), which suggests that berberine could have a direct action on neuron and accumulate in the hippocampus. This study first showed the pharmacokinetic characteristics of berberine in rat hippocampus and the kinetic characteristics of berberine are dissimilar in the hippocampus and plasma.     

An LC-MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10-5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51-7.70% with accuracy of 95.1-104.2%. The average recovery of cTP6 for three concentration levels was 59.6-64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6-8h after intra-muscular injection. The half-life was 2.24-2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 μg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 μg/kg for 7 consecutive days in rhesus monkeys.     

Quantitative determination of pravastatin and R-416, its main metabolite in human plasma, by liquid chromatography-tandem mass spectrometry

A quantitative method was developed and validated for rapid and sensitive analysis of pravastatin and R-416, the main metabolite of pravastatin, in human plasma. The analytes were extracted from plasma samples by a solid phase extraction method using a Bond Elut C(8). The method involved the use of liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) mass spectrometry. A pravastatin analog, R-122798, was used as the internal standard (I.S.). Separation of pravastatin, R-416 and the I.S. was accomplished using a reverse-phase column (C(18)). The components eluted were ionized by the APCI source (negative ion) and subsequently detected by a highly selective triple quadrupole mass spectrometer in the SRM mode. Linear standard curves were obtained from 0.1 ng/mL (lower limit of quantification, LLOQ) to 100 ng/mL. The intra-assay precisions (coefficient of variation) for the samples at the LLOQ were 1.8% for pravastatin and 1.6% for R-416. The intra-assay accuracy values were 95.8-107.6% for pravastatin, and 92.6-109.0% for R-416, respectively. Precision and accuracy of quality control (QC) samples were determined at concentrations of 0.5, 10 and 80 ng/mL for all analytes. The intra- and inter-assay precision calculated from QC samples were within 10% for pravastatin and within 11% for R-416. The overall recoveries for pravastatin and R-416 were 75.7-82.1% and 68.6-74.3%, respectively. Pravastatin and R-416 were stable in human plasma for 3 weeks at -20 degrees C in a freezer, up to 6h at room temperature, and up to 48 h at 6 degrees C. This assay method was successfully used to evaluate the pravastatin and R-416 levels in healthy volunteers following oral administration of Mevalotin.     

The novel sensitive and high throughput determination of cefepime in mouse plasma by SCX-LC/MS/MS method following off-line μElution 96-well solid-phase extraction to support systemic antibiotic programs

A sensitive and high throughput off-line μElution 96-well solid-phase extraction (SPE) followed by strong cation exchange (SCX) liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for determination of cefepime has been developed and validated in mouse plasma. Using the chemical analog, ceftazidime as an internal standard (IS), the linear range of the method for the determination of cefepime in mouse plasma was 4–2048 ng/mL with the lower limit of quantitation level (LLOQ) of 4 ng/mL. The inter- and intra-assay precision and accuracy of the method were below 9.05% and ranged from 95.6 to 113%, respectively, determined by quality control (QC) samples at five concentration levels including LLOQ. After μElution SPE, 71.1% of cefepime was recovered. The application of the validated assay for the determination of cefepime in mouse pharmacokinetics (PK) samples after intravenous (IV) and subcutaneous (SC) doses was demonstrated.     

Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection

An HPLC-MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard (II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mmx50 mm, 3 microm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 431-->109 (I) and m/z 437-->115 (II) used for quantitation. The assay was validated over the concentration range of 10-5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.