Cmax of midazolam to increase by 87% compared with control. After 10 days of the danshen extract intake, the mean AUC0–12, Cmax, and t1/2 of midazolam were decreased by 79.9%, 66.6%, and 43.8%, respectively. The mean clearance of midazolam was increased by 501.6% compared with control. The in vitro study showed that dihydrotanshinone I in the extract could inhibit CYP3A, while tanshinone IIA and cryptotanshinone could induce CYP3A. In conclusion, a single-dose administration of the danshen extract can inhibit intestinal CYP3A, but multidose administration can induce intestinal and hepatic CYP3A.The aim of this study was to investigate the effect of single- and multidose administration of the ethanol extract of danshen on in vivo CYP3A activity in healthy volunteers. A sequential, open-label, and three-period pharmacokinetic interaction study design was used based on 12 healthy male individuals. The plasma concentrations of midazolam and its metabolite 1-hydroxymidazolam were measured. Treatment with single dose of the extract caused the mean  Salvia miltiorrhiza, has been used for hundreds of years to treat coronary heart and cerebrovascular disease [Danshen, the dried root of  disease . The her disease –6. Dansh disease . Thus, sCYP3A4 gene [CYP enzymes (CYPs) play an important role in detoxification and systemic clearance of xenobiotics. Of >55 human CYP isozymes presently known, CYP3A is considered to be the most important drug-metabolizing enzyme. It participates in metabolism of >60% of all marketed drugs. As the critical role of CYP3A in drug metabolism, inhibition or induction of this enzyme often leads to drug interactions . By usin3A4 gene . It was 3A4 gene . The aim of this study was to investigate the effect of single- and multi-dose administration of the ethanol extract of danshen on the in vivo CYP3A activity in healthy volunteers. The constituent(s) inhibited or induced to CYP3A was also investigated using human liver microsomes and human cryopreserved hepatocytes. It will provide valuable information for using the danshen preparation in clinical practice.Tanshinone I, cryptotanshinone, tanshinone IIA, and dihydrotanshinone I were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . NADPH, midazolam, and 1-hydroxymidazolam were purchased from Sigma-Aldrich . HPLC-grade acetonitrile, methanol, and ethyl acetate were obtained from Merck . Deionized water was purified using a Milli-Q system . Midazolam tablets  were manufactured by Jiangsu Nhwa Pharma Corporation. The ethanol extract of danshen in the form of capsule  was manufactured, and the quality control was established and enforced strictly by Hebei Xinlong XiLi Pharmaceuticals Ltd according to state drug standard . The lipophilic components  and fingerprint of the ethanol extract of danshen were determined by HPLC on a C18 column and with a mobile phase of 20 mmol ammonium acetate : acetonitrile . The detection wavelength was set to 270 nm. For determination of fingerprint of the ethanol extract of danshen, a gradient mobile phase was used. The fingerprint was showed in Human liver microsomes  and human cryopreserved hepatocytes  used in this study were provided by the Research Institute for Liver Disease Co. . The clinical protocol and informed consent form were approved by the independent ethics committee of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine. 12 healthy male volunteers were enrolled in the study after obtaining written informed consent.All subjects were nonsmokers and judged to be healthy according to their medical histories, complete physical examinations, electrocardiograms, and routine laboratory test results. Subjects abstained from consuming herbal and citrus fruit products for 2 weeks before the study and from alcohol and medications for 2 weeks before and during the study period; caffeine-containing foods and beverages were also excluded during the study period. The study design was a sequential, open-label, and three-period trial  conducte18 column  with water phase (ammonium acetate 4 mmol/L and methanoic acid 0.08%) : methanol  as the mobile phase at a flow rate of 0.80 mL/min. The MS/MS system was operated in positive ion electrospray ionization. The multiple reaction monitoring (MRM) detection mode was applied to midazolam (m/z: 326.4→291.2), 1-hydroxylmidazolam (m/z: 341.8→324.0), and diazepam (m/z: 285.2→193.1). The collision energy (CE), declustering potential (DP), and collision cell exit potential (CXP) were set as follows: midazolam: 35.88 V, 105.14 V, and 13.00 V, respectively; 1-hydroxymidazolam: 33.00 V, 91.05 V, and 17.94 V, respectively; and diazepam: 43.00 V, 98.06 V, and 11.45 V, respectively. Plasma samples were spiked with an internal standard (diazepam) and extracted with ethyl acetate. After evaporation of the organic solvent under nitrogen, reconstituted residues of the organic phase were analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system . Chromatographic separation of the compounds was accomplished using a CThe quantitative range for measuring midazolam and 1-hydroxymidazolam was 0.1 ng/mL to 150 ng/mL. The accuracy, precision, recovery, and stability tests all met the requirements for quantitative determination in biological samples. No matrix effect existed in this LC-MS/MS method.m/z 277.1→249.0 for tanshinone I, 297.3→251.2 for cryptotanshinone, 295.2→249.2 for tanshinone IIA, 279.2 →233.2 dihydroxytanshinone I, and 285.2→193.1 for diazepam. CE, DP, and CXP were set as follows: tanshinone I: 29.00 V, 98.06 V, and 16.32 V, respectively; cryptotanshinone: 43.00 V, 123.00 V, and 15.12 V, respectively; tanshinone IIA: 27.36 V, 103.66 V, and 15.00 V, respectively; dihydrotanshinone I: 28.32 V, 112.07 V, and 6.92 V, respectively; and diazepam: 43.00 V, 98.06 V, and 11.45 V, respectively.For tanshinones, tanshinone I, cryptotanshinone, tanshinone IIA, and dihydrotanshinone I levels were determined by LC-MS/MS as previously described –13. The HLMs used in this study were provided by the Research Institute for Liver Disease Co. . The microsomes were prepared from ten individual human donor livers.μL as follows: HLMs (1 mg/mL) 40 μL, 1 mM NADPH 10 μL, 100 mM phosphate buffer (pH 7.4) 130 μL, 70 μM midazolam 10 μL, and a range of concentrations of tested compound 1 μL. There was a 5 min preincubation period at 37°C before the reaction was initiated by adding the NADPH. The reactions were conducted for 5 min. Triplicate samples were run to generate IC50 value by incubating midazolam at 3.5 μM in the presence of five concentrations of ethanol extract of danshen  and dihydrotanshinone I  in the incubation mixture. Ethanol extract of danshen and dihydrotanshinone I were dissolved in DMSO .The CYP3A enzymatic activities were characterized based on reaction of midazolam 1-hydroxylation. Incubation mixtures were prepared in a total volume of 200 5 viable cells in 0.25 mL of hepatocyte plating medium. Viability as determined by trypan blue exclusion was 85% or better for this study. The cells were maintained at 37°C in a humidified incubator with 90% atmospheric air and 5% CO2. Twenty-four hours after isolation, plating, and incubation, hepatocytes were treated with vehicle, which contained the same amount of DMSO (0.1%), tanshinones (2 μM), and rifampicin (25 μM) for 72 hours. All drugs were dissolved in DMSO and then added to the culture medium . Incubation medium containing 0.1% DMSO served as the vehicle control. After daily treatment for 3 days, the medium was removed, and the cells were washed with saline. Total RNA was isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer-supplied protocol. Quantitative real-time PCR was performed using gene-specific primers and the SYBR Green PCR kit (Invitrogen) in an ABI 7900 system (Applied Biosystems). The relative quantity of the target CYP3A4 gene compared with the endogenous control  was determined by the ΔΔCT method. The following primer sets were used in this study: CYP3A4 (5′-AGAAAGTCGCCTCGAAGATACA-3′ and 5′-GCTGGACATCAGGGTGAGTG-3′).Human cryopreserved hepatocytes were thawed in hepatocyte thawing medium and were seeded in collagen I precoated 24-well plates, with each well having a cell density of 3.0 × 10Cmax) and time to Cmax (Tmax) were directly obtained from the plasma concentration-time data. The elimination half-life (t1/2) was calculated as 0.693/Ke, where Ke, the elimination rate constant, was calculated via semilog regression on the terminal phase of the plasma concentration-time curve. The AUC from time 0 to infinity (AUC∞0–) was estimated as AUCt0– + Ct/Ke, where Ct is the plasma concentration of the last measurable sample, and AUCt0– was calculated according to the linear trapezoidal rule. Total plasma clearance (CL/F) was calculated as dose/AUC∞0–. The AUC metabolic ratio was calculated by dividing the AUCt0– of 1-hydroxymidazolam by the AUCt0– of midazolam. The plasma concentration-time data of analytes were analyzed by compartment-independent approaches. The maximum plasma drug concentration  were calculated using Graphpad Prism 5.04 . For comparison of several groups against one control group, a one-way analysis of variance followed by Dunnett's test was performed. A P value < 0.05 was considered to be significant.ICAll volunteers completed the study of three periods. 12 healthy male Chinese subjects with a mean age of 28 years , a mean weight of 66.4 kg , and a mean height of 173 cm  participated in this study. The mean plasma concentration-time profiles of midazolam and 1′-hydroxymidazolam before and after single or multidose administration of the danshen extract are presented in Figures Cmax of midazolam and 1′-hydroxymidazolam was increased by 80.7% (163.57 ± 86.36 ng/mL versus 95.17 ± 39.01 ng/mL) and 68.2% (45.04 ± 15.09 ng/mL versus 26.78 ± 11.08 ng/mL), compared with the control, respectively. Regarding treatment with a single dose of the danshen extract, the 0–12 of midazolam was decreased by 79.9% (42.24 ± 15.74 ng·h/mL versus 219.86 ± 64.67 ng·h/mL) compared with the control, and the clearance of midazolam was increased by 501.59% (393.71 ± 157.14 L/h versus 67.64 ± 20.05 L/h). After 10 days of treatment, the Cmax of midazolam was decreased by 66.6% compared with the control; t1/2 of midazolam was decreased by 43.8% (2.20 ± 0.90 h versus 4.20 ± 0.76 h). After 10 days of treatment, AUC0–12 of 1′-hydroxymidazolam was decreased by 44.50% (30.88 ± 15.09 ng·h/mL versus 56.21 ± 22.75 ng·h/mL) compared with the control, and t1/2 of 1′-hydroxymidazolam was decreased by 47.97% (1.72 ± 0.40 h versus 3.30 ± 0.62 h). However, the Cmax of of 1′-hydroxymidazolam was not significantly affected by 10-day treatment of the danshen extract.After 10 days of the danshen extract intake, AUCAfter 10-day treatment with the danshen extract, AUC metabolic ratio of midazolam was increased by 35.64% compared with the control (0.77 ± 0.53 versus 0.26 ± 0.15).After administration of a single dose (1 g) and multidose  of the danshen extract, the pharmacokinetic parameters of tanshinones were listed in 50 values of 8.6 μg/mL and 1.2 μM (To investigate whether the ethanol extract of danshen and which component(s) of the extract affected the catalytic activity of CYP3A, midazolam 1′-hydroxy reaction assays were conducted with various concentrations of ethanol extract of danshen and dihydrotanshinone I. The results showed that ethanol extract of danshen and dihydrotanshinone I had inhibition against CYP3A activities in HLMs with ICd 1.2 μM . μM), and rifampicin (25 μM) for 72 hours. After treatment, expressions of CYP3A4 mRNA were determined. Levels of CYP3A4 transcripts were induced 18.2-fold by rifampicin (25 μM). At 2 μM, levels of CYP3A4 transcripts by tanshinone I, cryptotanshinone, tanshinone IIA, and dihydrotanshinone I increased by 1.4-, 2.8-, 5.2-, and 1.5-fold, respectively (Hepatocytes were treated with DMSO (0.1%), tanshinones  is a widely accepted probe drug for CYP3A phenotype . MDZ is Cmax of midazolam occurred with single-dose administration of the ethanol extract of danshen. This increase was not reflected in the AUC0–12, CL/F, or t1/2. Although midazolam is classified into BCS class I, with high membrane permeability [Cmax could attribute to inhibition of CYP3A enzymes in the small intestine. In this study, an 80.7% increase in the eability , its oraeability . Gorski eability . So, theμM to activate midazolam 1-hydroxylation. In contrast, dihydrotanshinone I absorbed into liver cannot reach the concentration with inhibition against CYP3A4. Therefore, the inhibition of CYP3A4 in intestine can be offset by cryptotanshinone in liver activation effect. It can be one of reasons why the AUC of midazolam was not obviously changed after single-dose treatment of the danshen extract. An in vitro study found that the content of the ethanol extract of danshen had a significant inhibitory effect on CYP3A-mediated 1-hydroxymidazolam metabolism with IC50 8.6 μg/mL in human liver microsomes. Although tanshinone IIA, tanshinone I, and cryptotanshinone in the danshen extract had no significant inhibitory effect individually [50 1.20 μM. The results were consistent with Wang's report [50) toward intestinal CYP3A. The danshen extract contains cryptotanshinone and dihydrotanshinone I, and the content of dihydrotanshinone I was 5 times lower than cryptotanshinone in the preparation. We reported that cryptotanshinone could activate midazolam 1-hydroxylation in human liver microsomes . After avidually , 20, dihs report . After tCmax,  AUC∞0–, and t1/2 were decreased compared with baseline. It suggests that both presystemic processes and systemic elimination of midazolam are altered by prolonged intake of the ethanol extract of danshen. And it indicates that intestinal and hepatic CYP3A are induced by multi-dose of the extract administration. After 10 days of treatment, t1/2 of 1-OHMDZ was also reduced by 54.1% compared with control, suggesting that UGTs could be induced to enhance the glucuronidation of 1-OHMDZ by the danshen extract. This is also evidenced by metabolic ratio of midazolam. By contrast, after 10-day intake of the danshen extract, the clearance was increased, and the CYP3A4 mRNA which does not reach the cutoff value. The cutoff value may vary among different laboratories because of the variability among hepatocyte lots. However, tanshinone I and dihydrotanshinone I at 2 μM do not cause significant increase of CYP3A4 mRNA. The results were consistent with Yu's findings in the reporter gene [It was the first report to study induction of tanshinones on CYP3A4 using primary human hepatocytes. A cutoff value of 4-fold increase in mRNA levels for induction compared with those found in negative control was applied for assessing CYP3A4 mRNA expression . The firter gene . After pWe reported that administration of danshen tablets for 2 weeks  in healthy volunteers according to one-sequence crossover design caused 35.4% increase in apparent oral clearance. In this study, the clearance was increased by 501.5%. The extent CYP3A induction in intestine by the danshen extract rich in tanshinones in this study was 14.2 times higher than that of the danshen extract with low tanshinones content which contains cryptotanshinone 1.2 mg and tanshinone IIA 1.6 mg in each dose . The conUGT gene. Since more than 40% of clinically used drugs are catalyzed by CYP3A and with further biotransformation by UGT, the two drug metabolising enzymes can be induced by the danshen extract to promote their substrates conversion to more polar derivatives which can be readily excreted. PXR has also been shown to play key roles in the regulation of several other inducible CYP2C9, CYP2C19, P-gp, MRP2, sulfate transferase (ST), and other drug metabolisming enzymes and transporter genes [In addition to CYP3A, the nuclear receptors of UGT are also targets of PXR –26. So, er genes –26. So, Drugs that are substrates for CYP3A-mediated metabolism are likely to be potential candidates for drug-herb interactions . The dur


Micrurus laticollaris  venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1–30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered . In mouse phrenic nerve-diaphragm preparations, MLV (1–10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min  to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.In this work, we have examined the neuromuscular activity of  Micrurus, Leptomicrurus and Micruroides) constitute a taxonomic assemblage of more than 120 species and subspecies [Pelamis platurus, the American representatives of the family, Elapidae [Micrurus venoms contain phospholipase A2, which can induce myotoxicity [Coral snakes  caused irreversible time- and concentration-dependent neuromuscular blockade in indirectly stimulated biventer cervicis (BC) preparations, with complete blockade occurring from 20 min at the highest concentration (30 µg/mL) (n = 4 each)  A. The ti 4 each) B. FigureM. laticollaris venom  also produced irreversible time- and concentration-dependent blockade, although less active than that of biventer cervicis (BC) preparations, with complete neuromuscular blockade occurring just at the highest concentration  after 80 min of incubation, whereas 39% ± 11% and 88% ± 1% blockade for one and 3 µg/mL, respectively, were observed after 120 min. . This also agrees with studies for other neurotoxins that discriminate between these two populations [et al., 1973; Chang and Su, 1975). Chemical differences between junctional and extrajunctional receptors obviously could also be a clue to the molecular mechanisms involved in generating and maintaining these differences. Studies using chimeric subunits and site-direct mutagenesis have demonstrated the importance of amino acids located within loops D, E and F in determining selectivity for nicotinic agonists and antagonists [2-neurotoxins in these venoms induces a triphasic effect, i.e., the increase of the muscle twitch-tension amplitude before the irreversible neuromuscular blockade [M. laticollaris venom/mL (see Micrurus venoms could be a result of an increase in neurotransmitter release [In mouse phrenic-nerve diaphragm (PND) preparations, the 120 min. . Figure l toxins ,31, but l toxins . Furtherl toxins ,34, i.e.ulations ,36  induced a significant increase in the frequency of miniature end-plate potentials (MEPPs/min) in mouse diaphragm after 15 min and then a decrease after 60 min  to 28 ± 2.5 (t15) and 12 ± 2 (t60); n = 4) , t5, t15, t30, t45 and t60 min, respectively, n = 4). As discussed above, this effect could contribute to an increase of neurotransmitter release before the onset of neuromuscular blockade. This conclusion also exposes the presence of presynaptic neurotoxins in M. laticollaris venom, which are probably related to PLA2-neurotoxin actions [Indeed, ; n = 4) , with no actions ,22,42,43t0) to −17 ± 3.7 mV (t120); n = 4) (t0) to −81 ± 1.3 mV (t120); n = 4). However, preparations pretreated with d-Tc (5 µg/mL) for 10 min and then exposed to venom (10 µg/mL) also exhibited important membrane depolarization from 30 min of venom incubation (from−75 ± 1.7 mV (t0) to −22 ± 3 mV (t120); n = 4)  caused a significant membrane depolarization from 15 min of incubation in PND preparations  was provided, in a lyophilized form, by Instituto de Biotecnología of the Universidad Nacional Autónoma de México . When required, the venom was dissolved in Krebs or Tyrode solution prior to use. Male Swiss mice (25–30 g) obtained from the Multidisciplinary Center for Biological Investigation (CEMIB/Unicamp) were housed 10/cage at 23 °C on a 12 h light/dark cycle with lights on at 6 a.m. Male HY-LINE chicks (4–8 days old) were supplied by Globo Aves Agricola Ltda.  and were housed in metal cages with a sawdust substrate. The mice and chicks had free access to food and water.2 1.9, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25 and glucose 11.7, pH 7.5 at 37 °C, after equilibration with 95% O2/5% CO2) and allowed to stabilize for 20 min prior to use, as described elsewhere [Chick biventer cervicis nerve-muscle preparations (BC) were mounted (resting tension: 1 g) in Krebs solution , as described elsewhere [Mouse phrenic nerve-diaphragm preparations (PND) were mounted in Tyrode solution  and venom-treated preparations (t5–60t). For RP measurements, different regions of the muscle were monitored at various intervals from control (t0) and venom-treated preparations (t5−t120 for RP); some preparations were pre-incubated with d-tubocurarine , a non-depolarizing compound, to verify the venom activity on nicotinic receptors and/or the muscle membrane.The effect of the venom on the frequency of miniature end-plate potentials (MEPPs) and membrane resting potential (RP) was recorded using mouse hemidiaphragm muscle mounted in a Lucite chamber containing Tyrode solution (composition shown above), as described previously elsewhere ,48. The t-test or ANOVA, followed by the Tukey test, with p < 0.05 indicating significance. All data analyses were done using Microcal Origin software .Changes in the twitch-tension responses of BC and PND preparations and the membrane resting potential of PND preparations were expressed as a percentage relative to baseline (time zero) values. The results were expressed as the mean ± SEM, and statistical comparisons were done using the Student’s Micrurus laticollaris venom causes neuromuscular blockade in avian and mammalian preparations in vitro essentially by a combined postsynaptic action between nicotinic receptor blockade and muscle membrane resting potential depolarization, since contracture responses to exogenous ACh in BC preparations was completely inhibited by all venom concentrations tested, and there was a significant depolarization of the muscle fiber membrane caused by venom in PND preparations. The venom-induced blockade was preceded by a presynaptic effect, which initially facilitates neurotransmitter release, as shown by the increase in twitch-tension amplitude and the frequency of MEPPs in PND preparations, suggesting also the presence of β-neurotoxins in the crude venom.Based on these results, we conclude that


Pancreatic cancer does not respond to a single-agent imatinib therapy. Consequently, multimodality treatments are contemplated. Published data indicate that in colorectal cancer, imatinib and radioimmunotherapy synergize to delay tumor growth. In pancreatic cancer, the tumor response is additive. This disparity of outcomes merited further studies because interactions between these modalities depend on the imatinib-induced reduction of the tumor interstitial fluid pressure. The examination of human and murine PDGFr-β/PDGF-B pathways in SW1990 pancreatic cancer xenografts revealed that the human branch is practically dormant in untreated tumors but the insult on the stromal component produces massive responses of human cancer cells. Inhibition of the stromal PDGFr-β with imatinib activates human PDGFr-β/PDGF-B signaling loop, silent in untreated xenografts, via an apparent paracrine rescue pathway. Responses are treatment-and time-dependent. Soon after treatment, levels of human PDGFr-β, compared to untreated tumors, are 3.4×, 12.4×, and 5.7× higher in imatinib-, radioimmunotherapy + imatinib-, and radioimmunotherapy-treated tumors, respectively. A continuous 14-day irradiation of imatinib-treated xenografts reduces levels of PDGFr-β and phosphorylated PDGFr-β by 5.3× and 4×, compared to earlier times. Human PDGF-B is upregulated suggesting that the survival signaling via the autocrine pathway is also triggered after stromal injury. These findings indicate that therapies targeting pancreatic cancer stromal components may have unintended mitogenic effects and that these effects can be reversed when imatinib is used in conjunction with radioimmunotherapy. Of these, the most extensively studied is the transduction of extracellular growth stimulating signals to the cell nucleus by the HER family of kinases -5. The iin vivo studies in various mouse models suggesting that imatinib may be useful in the treatment of solid cancer [The expression of c-Kit  and PDGFd cancer -27, mostd cancer ,19-23. Rd cancer . The expStudies described below show that the pretreatment status of PDGFr-β and its ligand PDGF-B in human pancreatic cancer xenografts is radically altered during therapeutic intervention with imatinib, radioactivity or both. Unlike in the case of the LS174T colorectal cancer , imatini2.2.1.i.e., whether of the human origin derived from tumor cells or of the mouse origin derived from mesenchymal cells of the host (131ICC49 + imatinib-treated (black bar), and 131ICC49-treated mice (dark gray bar), respectively, as compared to PBS-treated control mice (white bar). The receptor phosphorylation followed along the same lines with the treated tumors showing on average 3.1×, 10.3×, and 6.6× higher levels of phosphorylation than tumors from PBS-treated mice demonstrating that the primary response of the tumor tissue to the insult by imatinib or by radiation from 131ICC49 is the increased phosphorylation of the human PDGFr-β receptor. PDGFr-β associated with the stroma, i.e., receptors of the mouse origin, do not seem to respond to any of applied treatments in a major way. No significant deviations in levels of mouse PDGFr-β in treated tumors as compared to those measured in tumors from control mice were detected . Mouse PDGFr-β was suppressed in the 131ICC49 + imatinib-and 131ICC49-treated tumors by ∼30% (P = 0.009), whereas imatinib-treated tumors showed only a minor decrease, ∼5%, in mouse PDGFr-β levels . The imatinib treatment at 5 d had more effect than at 14 d. The level of human PDGFr-β was ∼18% higher in the 14 d sample . The corresponding Western immunoblotting data is shown in The ELISA examination of samples recovered 14 d after treatment revealed further significant alterations in the PDGFr-β expression. Phosphorylation of PDGFr-β was suppressed in reatment . Imatiniβ levels . The expd sample . 131ICC42.2.P = 0.048), 131ICC49 + imatinib- (P = 0.015), and 131ICC49-treated (P = 0.0008) tumors, respectively. In contrast, human PDGF-B was elevated from ∼55 pg/mg protein before treatment to 184.4 ± 26.9, 128.4 ± 15.8, and 143.3 ± 30.0 pg/mg in the corresponding day 5 tumor samples from mice treated with imatinib (P = 0.034), 131ICC49 + imatinib (P = 0.046), and 131ICC49 (P = 0.107). After 14 days, mouse PDGF-B in imatinib-treated tumors persisted at essentially unchanged levels, 239.7 ± 10.4 pg/mg (P = 0.219). 131ICC49-treated tumors showed mouse PDGF-B of 332.0 ± 13.6 pg/mg at 14 d, an increase of ∼74 pg/mg (P = 0.010). In 131ICC49 + imatinib-treated tumors, the amount of mouse PDGF-B increased by 20 pg/mg protein and was registered at 171.6 ± 8.6 pg/mg (P = 0.033). The amount of human PDGF-B in imatinib-treated tumors measured 14 d after treatment was still higher than levels measured in untreated tumors but it declined to 91.4 ± 8.83 pg/mg (P = 0.065). 131ICC49 treatment resulted in 119.3 ± 15.0 pg/mg , ∼36 pg/mg higher than in 5 d samples, and nearly 3× higher than in tumors from PBS-treated control mice . Imatinib was generously provided by Novartis Pharma AG . All radioiodinations were performed on site as described previously .3.2.2. Female 4–6 weeks old athymic NCr-nu/nu mice  were allowed to acclimate for no less than 5 days. All protocols involving animals were approved by the Institutional Animal Care and Use Committee. Mice were housed in micro-isolator cages with free access to sterilized standard rodent diet and water. SW1990 cells in 0.2 mL of serum-free medium were implanted SQ at 5 × 106 cells/mouse. One week later identification transponders were implanted, also SQ. Body weights and tumor sizes were monitored twice weekly, and approximately 5–7 weeks after the cell implant, mice were randomized for various therapy studies.The SW1990 cell line derived from spleen metastasis of a grade II human pancreatic ductal adenocarcinoma  was purc3.3.131ICC49 as described previously [131ICC49 group, mice receiving i.p. injections of PBS (b.i.d.) on days −2, −1, and 0; and group 4: 131ICC49 plus imatinib group, mice receiving imatinib as the i.p. doses of 100 mg/kg/day in PBS (b.i.d.) on days −2, −1, and 0. On day 0, mice in groups 3 and 4 received via a tail vein i.v. doses of 131ICC49 2 h after the last dose of imatinib. Sections of tumors harvested during necropsy were frozen in liquid N2, and stored at −80 °C until ready for ELISA and Western blotting analyses.Mice were treated with imatinib and eviously . Briefly3.4.3VO4, and 1μg/mL leupeptin, and was supplemented just before the use with phenylmethylsulfonyl fluoride (PMSF) to the final concentration of 1 mM. Minced tumor fragments were sonicated on ice using a Vibra Cell Model VC 375 ultrasonic processor  for 14 s with a 14-s break between sonications for the total of 3 minutes at a 40% duty cycle. The homogenate was transferred into the microcentrifuge tubes and centrifuged at 14,000× g for 10 min at 4 °C. Supernatants were aliquoted and stored at −80 °C after the total protein content was determined using the micro BCA protein assay kit .Frozen tumors were minced and transferred into a volume of ice-cold lysis buffer equivalent to two tumor weights. The lysis buffer contained 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM ethylene diaminetetraacetic acid (EDTA), 1 mM ethylene glycolbis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate, 1 mM activated Na3.5.The assay was purchased from Cell Signaling Technology, Inc. . All procedures were conducted according to the vendor's instructions for the detection of endogenous levels of phospho-PDGFr-β (Tyr751). To quantify the total PDGFr-β and human PDGFr-β, the general principle of the assay was applied with the appropriate modification in the selection of the primary and secondary antibodies. The capture antibody coated onto all wells and provided by the manufacturer was rabbit mAb 28E1, which binds non-phosphorylated and phosphorylated PDGFr-β proteins of human and mouse origin. Primary antibodies were as follows: mouse mAb 3166 anti-phospho-PDGFr-β (Tyr751) for detection of the total phospho-PDGFr-β; mouse mAb (ab 10847) anti-human PDGFr-β for detection of human PDGFr-β ; and rat mAb (clone APB5) anti-mouse PDGFr-β for detection of the murine PDGFr-β . Horseradish peroxidase-linked (HRP) anti-mouse antibody (7076) provided with the assay was used as a secondary antibody for phospho-PDGFr-β and human PDGFr-β detection. Donkey HRP-linked anti-rat polyclonal Ab  was used to detect murine PDGFr-β. Stabilized tetramethylbenzidine (TMB) was used as a substrate to produce color. The absorbance was read at λ = 450 nm within 30 min after the addition of the “stop” solution provided with the kit.3.6.Tumor lysates were prepared as described above. Protein concentration was measured and aliquots of 50 μg total proteins (25 μg for human PDGFr-β studies) from each tumor lysate were added to the non-reducing sample buffer. Protein samples were denatured at 95 °C for 5 min, cooled and loaded onto 4–20% gradient Tris-HCl SDS-polyacrylamide minigels . Proteins were transferred overnight onto a Hybond™-P 0.45-μm PVDF membrane  in a cold room using a constant voltage of 14 mV for 18 h. Kaleidoscope Prestained Standards  were used to monitor the efficiency of protein transfer. The membrane was incubated in 5% BSA in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20 overnight at 4 °C to reduce nonspecific binding. The blocked membrane was incubated in the same buffer containing primary antibodies overnight at 4°C. The membrane was first probed simultaneously with rabbit anti-human/anti-mouse phospho-PDGFr-β (Tyr751) polyclonal Ab 3161 and with rabbit anti-β-actin mAb 13E5  to detect total phosphorylated PDGFr-β and β-actin, respectively, both at 1:5,000 dilution. The membrane was washed in PBS/0.5% Tween-20 for 30 min at rt with the buffer exchange every 5 min, then incubated in the blocking buffer containing HRP-linked goat anti-rabbit IgG secondary antibody 7074 at a 1:40,000 dilution for 1 h at rt . The membrane was washed again in PBS/0.5% Tween-20 and antigens were detected using the chemiluminescence substrate ChemiGlow® according to the manufacturer instructions . Before probing for the total expression of PDGFr-β, the membrane was stripped 2 × 10 min in 200 mM glycine, 3.5 mM SDS, and 1% Tween-20 pH adjusted to 2.2 with HCl, washed 2 × 10 min in PBS, 1 × 5 min in 0.05 M Tris, pH 8.0, 0.138 M NaCl, 0.0027 M KCl, 0.5% Tween-20, followed by a 1-min methanol rinse and a final 5-min wash in 0.05 M Tris, pH 8.8, 0.138 M NaCl, 0.0027 M KCl, 0.5% Tween-20. Membrane was then re-probed with rabbit anti-human/anti-mouse PDGFr-β polyclonal Ab 3162  to determine total PDGFr-β. The subsequent gels were analyzed in the same manner to determine the expression of human PDGFr-β. Primary antibodies were mouse monoclonal anti-human PDGFr-β from Abcam (#ab10847) used at 1:55,000 dilution. ImmunoPure goat anti-mouse IgG (H + L) conjugated with horseradish peroxidase from Pierce (31430) was used as a secondary antibody at 1:300,000 dilution. Protein band intensity was measured using the ImageJ software  and norm3.7.Quantikine human and mouse PDGF-BB assays were purchased from R&D Systems, Inc. . Human PDGF-BB assay uses the PDGFr-β/Fc chimera pre-coated onto a microplate to capture PDGF-BB. In the mouse PDGF-BB assay, anti-PDGF-BB MAb is pre-coated onto a microplate. R&D Systems report cross-reactivity with human PDGF-BB to be 38.1%. Both assays were performed according to the manufacturer instructions. The average total protein load per well was 0.7 mg. Reported concentrations of mouse PDGF-BB were adjusted for the cross-reactivity with human PDGF-BB.3.8.2O2 (5 min). After a gentle rinse in PBS, slides were washed for 5 min in PBS and blocked for 15 min with normal goat serum  for goat secondary antibodies or Serum Blocking Reagent G  for rabbit secondary antibodies. The slides were drained and the excess serum wiped off. The avidin blocking reagent (R&D) was applied for 15 min. Slides were gently rinsed in PBS and the biotin blocking reagent (R&D) was applied for 15 min. Slides were again gently rinsed in PBS. Slides were placed in a humidified chamber and the primary antibodies were applied. For the detection of PDGF-B, rabbit polyclonal IgG sc-127  at a concentration of 1 μg/mL was used. PDGFR-β was detected with either goat polyclonal IgG sc-1627  at a concentration of 1 μg/mL or rabbit polyclonal IgG 3162  was used at a 1:50 dilution of the stock solution provided by vendor. After the overnight incubation, slides were washed 3 × 5 min in PBS. The appropriate biotinylated secondary antibodies (KPL) were applied for 60 min. Slides were washed 3 × 5 min in PBS and exposed to the peroxidase-labeled streptavidin (KPL) for 30 min. After a gentle rinse and 3 × 2 min washes with PBS, the DAD peroxidase substrate solution was applied for 20 min. Stained slides were rinsed and washed in distilled water (5 min) and counterstained with hematoxylin using standard protocols. Sections were photographed with a Nikon DXM 1200 digital camera mounted on a Nikon Optiphot microscope.Paraffin sections were rehydrated and the antigen retrieved using the standard protocol. The endogenous peroxidase activity was blocked with 3% H3.8.In all summary statistics Student's t-test was used to compare the averages.4.cancer cells must be undertaken before any therapies targeting the stromal component of pancreatic cancer are contemplated.Data reported here suggest that in view of the massive activation of autocrine and paracrine PDGFr-β signaling pathways, some stroma-directed therapies may have minimal or no benefit in pancreatic cancer. Based on the compelling data presented here it is apparent that a scrupulous determination of PDGFr-β and PDGF-B status in


In the current study, the effects of ethanol (EtOH) on toxicokinetics of methamphetamine (MA) and its metabolite amphetamine (AP) were investigated. A single dose of MA hydrochloride at 15 mg/Kg was given intragastrically, either alone  or in conjunction with 3 g/Kg EtOH  to rabbits. In placebo group, normal saline only was given . Plasma and urine samples were collected and analyzed by gas chromatography/mass spectrometry (GC/MS) for MA and AP. Toxicokinetic parameters of MA and AP were determined using WinNonlin. Our results showed that toxicokinetic profiles of MA and AP in the two experimental groups were both fitted to an open two-compartment model with first-order kinetics. These were not affected by co-administration of EtOH. However, concomitant intake of EtOH significantly increased MA plasma absorption constant (Ka) and maximum concentration (Cmax). The Ka of MA was increased from 0.679/h ± 0.023/h to 0.964/h ± 0.033/h , whereas the Tmax was significantly decreased from 1.620 h ± 0.062 h to 1.259h ± 0.033h (P < 0.05). In contrast, no significant difference was observed on MA elimination. Furthermore, the plasma AP area under the curve (AUC0-30 h) increased from 5.281 mg/h/L ± 0.264 mg/h/L to.13.052 mg/h/L ± 0.956 mg/h/L and Cmax increased from 0.315 mg/L ± 0.010 mg/L to 0.423 mg/L ± 0.042 mg/L (P < 0.01). Taken together, co-administration of EtOH with MA significantly accelerated MA absorption and subsequent metabolism to AP, but did not have significant effect on MA elimination.  Methamphetamine (MA) is a psychostimulant, which was FDA approved for treatment of ADHD and obesity. However, methamphetamine has high potential for abuse and addiction. Methamphetamine is rapidly absorbed via gastrointestinal tract and metabolized in liver. The major routes of metabolism are N-demethylation and aromatic hydroxylation. Amphetamine (AP) and MA abuse is often accompanied with recreational consumption of ethanol (EtOH). It is unclear whether EtOH may change the absorption, distribution, metabolism, and/or excretion of MA. Past studies indicated that ethanol does not seem to significantly affect pharmacokinetic parameters of intravenously administered MA, except an apparent decrease of volume of distribution at the steady state , 2. HoweIn the current study, the effects of EtOH on the pharmacokinetics of orally administered MA were characterized in rabbits. Insights from the current study may reveal potential mechanisms underlying drug metabolism and provide valuable reference for blood and urine drug testing.Chemicals and analysis softwares525A, IS) were obtained from Chinese National Laboratory of Narcotics, and 1.0 g/L methanol stock solutions were prepared. The derivatization reagent trifluoroacetic anhydride  was purchased from Sigma. All other chemicals and solvents were of analytical grade.MA hydrochloride, AP sulfate and propyl adiphenin .SPSS11.5 statistical software was purchased from SPSS Co. (USA).Animals Twenty white male rabbits, each weighing 2.0 Kg ± 0.1 Kg, were bred by the Laboratory Animal Center of Hebei Medical University. Sixteen rabbits were randomly divided into two experimental groups, receiving a single, oral dose of MA hydrochloride alone (MA group) or with EtOH (MA + EtOH group). The remaining four rabbits received normal saline as the placebo-controlled group. The handling and use of animals were in accordance to the institutional guidelines and all experiments were carried out in accordance with current and ethical guidelines for the care and use of laboratory animals.Administration protocol After overnight fasting, a single dose of 15 mg/Kg body weight of MA hydrochloride solution was administered intragastrically in the MA group or with 3 g/Kg EtOH in the MA + EtOH group. The same volume of normal saline was administered intragastrically to the placebo-controlled group. Changes in vital signs including ECG, blood pressure and respiration rate were monitored and recorded for 3 hours by “BL-Physiological Function Experimental System .ExperimentsBlood samples were collected before administration and at 0.5, 1, 1.5, 2, 2.5, 3, 5, 8, 12, 16, 20, 24 and 30 h after drug administration. Likewise, urine samples were collected at 0.5, 1, 2, 5, 8, 12, 16, 20, 24 and 30 h. The plasma and urine samples were stored at -20 °C until analysis.525A. The samples were processed by liquid-liquid extraction. To each sample, 5 mL diethyl ether was added and vortexed for 5 min. After centrifugation (3000 r/min) for 10 min, the organic layer was transferred into a clean test tube and all these extraction steps were repeated once again. After a drop of acidic methanol was added, the organic layer was evaporated dry by water bath at 40 ºC. MA and AP in the residue were subjected to acylation derivatization by 25 µL TFAA and 25 µL ethyl acetate in a microwave oven for 2 min, and evaporated dry again by a stream of nitrogen. The residue was reconstituted with methanol. Aliquots of 1.0 µL were injected into the GC system. One milliliter of plasma and urine from each animal was diluted with 2 mL boric acid buffer solution and subsequently mixed with 200 ng of the internal standard SKFAnalytical methods 525A , from 9.5 min to 10.2 min. The dynode was shut off between 6.3 min and 9.5 min.A GC-MS  system equipped with a splitless injection port, and a capillary column DB-5MS (30 m×0.25 mm×0.25 μm) was used for analysis. Carrier gas was high-purity helium (99.999%) at a constant flow of 1.0 mL/min. The temperature for GC was raised from 70 ºC to 200 ºC at 20 ºC/min, and then to 280 ºC at 30 ºC/min, remaining at 280 ºC for 1min. The temperatures for the injector port, the transfer line heater and the ion source (EI) were all set at 250 ºC. The electron energy was 70 eV. At the automatic tuning mode, the multiplier voltage was 1557 V and the current intensity was 100 µA. The mass spectrometer was operating in the full scan mode and the scanning range of 50 amu to 350 amu for qualitative analysis. Quantitative analysis employed the selected ion monitoring (SIM) mode at different time windows: AP-TFAA , from 4.0 min to 5.2 min; MA-TFAA , from 5.2 min to 6.3 min; SKFMethod validationPlasma and urine samples from controlled group were used as negative controlls. The calibration curves were constructed using linear regression analysis based on the concentration of drugs and the ratio of the peak area of MA or the amphetamine metabolite to that of the internal standard SKF525A. Pharmacokinetic parameters and statistical analysis max (peak plasma concentration), tmax (time to peak concentration), t1/2 , AUC0-30h (area under the plasma concentration- time curve), CL  and V/F (apparent volume of distribution) for MA and its main metabolite AP, were calculated based on the compartment model using the WinNonlin Pro computer program. Statistical analysis was carried out using SPSS 11.5.Pharmacokinetic parameters of each drug, including CThe data were analyzed by mean ± standard deviation and the level of statistical significance was P < 0.05. Accuracy and precision Concentrations of MA and AP were determined by using liquid–liquid extraction and gas chromatography–mass spectrometry (GC-MS) The lower limits of detection (LOD) and lower limits of quantification (LOQ) for plasma and urine are presented in MA and AP in plasma The mean plasma concentrations of MA and its metabolite AP in both experimental groups are presented in The pharmacokinetic profiles of MA fitted to an open two-compartment model with first-order kinetics. Co-administration with EtOH did not change its pharmacokinetic model. The pharmacokinetic parameters for MA and AP are listed in max of 1.62 h. Concomitant intake of EtOH caused a significant increase in the plasma MA absorption constant (Ka) and maximum concentration (Cmax). The mean MA Ka values increased from 0.679 h ± 0.023 h to 0.964 h ± 0.033 h (P < 0.05), and the mean Cmax from 1.408 mg/L ± 0.072 mg/L to 1.676 mg/L ± 0.135 mg/L (P < 0.05), whereas plasma MA Tmax appeared to be significantly decreased from 1.620 h ± 0.062 h to 1.259 h ± 0.033 h (P < 0.05). Finally, no significant difference was observed on MA elimination. When MA was administered intragastrically to rabbits, it was rapidly absorbed, with a short T0-30h) and Cmax increased from 5.281 mg/h/L ± 0.264 mg/h/L to.13.052 mg/h/L ± 0.956 mg/h/L and from 0.315 mg/L ± 0.010 mg/L to. 0.423 mg/L ± 0.042 mg/L, respectively (P < 0.01). Furthermore, when EtOH was co-administered, the plasma AP area under the curve  induced changes in blood pressure, respiration and heart rate of rabbits in 20 to 30 min, lasting at least for 1 h. Previous research has demonstrated that a single intraperitoneal administration of MA (10 mg/Kg) induced stereotypic behavior such as continuous sniffing, circling and nail-biting in male ICR mice . These bToxicokinetic profiles of MA in rabbitsOur results showed that the apparent volume of distribution (Vd) of both MA and its metabolite AP exceeded the total body fluid in rabbits (1.2 L or 0.6 L/Kg), indicating that MA readily accumulated in tissues and the MA content was lower in blood than that in tissues.et al. investigated the pharmacokinetics of MA hydrochloride after oral administration of sustained-release tablets to human at a dose of 10 mg and 20 mg  can cause toxic symptoms in rabbits, and EtOH accelerated absorption and subsequent metabolism of MA, but not elimination. Since EtOH appears to increase the toxicity of MA, we suggest that the interaction between EtOH and MA should be considered in MA and EtOH mixed abuse.Our results indicated that oral administration of MA (15 mg/Kg,


H2S is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression. Cocktail method was used to evaluate the influences of acute H2S poisoning on the activities of cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which were reflected by the changes of pharmacokinetic parameters of six specific probe drugs, bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide, respectively. The experimental rats were randomly divided into two groups, control group and acute H2S poisoning group . The mixture of six probes was given to rats by oral administration and the blood samples were obtained at a series of time points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. The results for acute H2S poisoning and control groups were as follows: there was a statistically significant difference in the AUC and Cmax for bupropion, metoprolol, phenacetin, and tolbutamide, while there was no statistical pharmacokinetic difference for midazolam and omeprazole. Acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 in rats.Hydrogen sulfide (H If exposure is transient, recovery is usually complete and rapid. However, in some instances, prolonged or severe exposure leads to a fatal outcome or permanent sequelae [Hydrogen sulfide  to functionalize a wide range of biologically active compounds , 17. Cyt2S toxicology mainly focuses on the central nervous system and cardiovascular system [2S. In this paper, cocktail probe drugs approach is used to evaluate the induction or inhibition effects of H2S on the activities of rats CYP450 isoforms such as CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which are reflected by the changes of pharmacokinetic parameters from 6 specific probe drugs, bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide, and then to provide a guidance for rational clinical oral administration after acute H2S poisoning.At present, the study of Hr system –32. To oBupropion, metoprolol, midazolam, phenacetin, omeprazole, tolbutamide , and the internal standard carbamazepine (IS) were purchased from Sigma-Aldrich Company . HPLC grade acetonitrile and methanol were from Merck Company . All other chemicals were of analytical grade. Ultrapure water (resistance > 18 mΩ) was prepared by a Millipore Milli-Q purification system .Male Sprague-Dawley rats (250 ± 20 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The animal license number was SCXK (Shanghai) 2012-0005. All twenty rats were housed at Wenzhou Medical University Laboratory Animal Research Center. Animals were housed under controlled conditions (22°C) with a natural light-dark cycle. All experimental procedures were conducted according to the Institutional Animal Care guidelines and approved ethically by the Administration Committee of Experimental Animals, Laboratory Animal Center of Wenzhou Medical University.Bruker Esquire HCT mass spectrometer  equipped with a 1200 Series liquid chromatograph  and controlled by ChemStation software  was used.μm particle, Agilent Zorbax SB-C18 column at 30°C. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile) as follows: 0–4.0 min (10-80% B), 4.0–8.0 min (80-80% B), 8.0–9.0 min (80-10% B), and 9.0–13.0 min (10-10% B). The flow rate was 0.4 mL/min.Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 m/z 240 for bupropion, m/z 268 for metoprolol, m/z 326 for midazolam, m/z 180 for phenacetin, m/z 198 for omeprazole, m/z 271 for tolbutamide, and m/z 237 for IS) and positive ion electrospray ionization interface. Drying gas flow was set to 7 L/min and temperature to 350°C. Nebuliser pressure and capillary voltage of the system were adjusted to 25 psi and 3,500V, respectively.The quantification was performed by the peak area method. The determination of target ions was performed in selective ion monitoring mode . Quality-control (QC) samples were prepared by the same way as the calibration standards, with three different plasma concentrations . The analytical standards and QC samples were stored at −20°C.2S poisoning group (n = 8); the rats were placed in triad infected ark which was with the H2S detector and passed into a certain concentration of H2S gases to create a model of acute H2S poisoning. The acute H2S poisoning group rats were exposed to 300 ppm H2S for 2 h. Control animals were maintained under similar conditions, but without the H2S exposure. Rats were allowed to eat and drink ad libitum except during the 2 h exposure.Twenty male Sprague-Dawley rats (250 ± 20 g) were randomly divided to control group and acute H2S poisoning and control rats were given the mixed six probe drugs with oral administration. The administration dose of the probe drugs bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide were 10 mg/kg, 10 mg/kg, 10 mg/kg, 10 mg/kg, 10 mg/kg, and 1 mg/kg, respectively.After completing the modeling, the acute HμL plasma was obtained for each sample.Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 5, 15, and 30 min and 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, and 48 h after oral administration of probe drugs. The samples were immediately centrifuged at 8000 r/min for 5 min, and 100 μL of the internal standard working solution (2.0 μg/mL) was added to 0.1 mL of collected plasma sample followed by the addition of 0.2 mL of acetonitrile-methanol . After the tube was vortex-mixed for 1.0 min, the sample was centrifuged at 15000 rmp for 10 min. The supernatant (2 μL) was injected into the LC-MS system for analysis.The plasma samples were extracted and measured by LC-MS. In a 1.5 mL centrifuge tube, an aliquot of 10 t-test inspection were analyzed by SPSS l8.0 statistical software. A P < 0.05 was considered as statistically significant.Plasma probe drugs concentration versus time data for each rat was analyzed by DAS software . The pharmacokinetic parameters of the test group and control group probe drugs with the Calibration curves for six probe drugs were generated by linear regression of peak area ratios against concentrations, respectively. The calibration plots of the probe drugs in the range of 10–2000 ng/mL were listed in 2S poisoning and control group, there was a statistically significant difference in the AUC and Cmax for bupropion, metoprolol, phenacetin, and tolbutamide (P < 0.05), while there was no statistical difference for midazolam and omeprazole (P > 0.05).The main pharmacokinetic parameters after administration of bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide from noncompartment model analysis were summarized in 2S poisoning group with the control group, the pharmacokinetic parameters of bupropion have changed; t1/2 from the 0.9 h increased to 1.0 h, but there was no statistical significance (P > 0.05); AUCt); CL from 27.3 reduced to 16.3 L/h/kg, with statistical significance (P < 0.05); Cmax varied from 276.6 to 411.3 ng/mL, with statistical significance (P < 0.05). Comparing acute H2S poisoning group with the control group, the pharmacokinetic parameters of metoprolol have changed; AUCt); CL from 9.5 reduced to 4.9 L/h/kg, with no statistical significance (P < 0.05); Cmax varied from 810.8 to 1235.7 ng/mL, with statistical significance (P < 0.01). Comparing acute H2S poisoning group with the control group, the pharmacokinetic parameters of midazolam have almost not changed; Cmax varied from 1343.6 to 1353.0 ng/mL, and there no statistical significance (P > 0.05). Comparing acute H2S poisoning group with the control group, the pharmacokinetic parameters of phenacetin have changed; t1/2 from the 0.6 h reduced to 0.4 h, and there was statistical significance (P < 0.05); AUCt)(0– from the 1239.9 increased to 1993.4 ng/mL∗h with significant difference (P < 0.05); CL from 11.1 reduced to 5.4 L/h/kg, but there was no significant difference (P > 0.05); Cmax varied from 1329.2 to 1847.7 ng/mL, and there was significant difference (P < 0.01). Comparing acute H2S poisoning group with the control group, the pharmacokinetic parameters of omeprazole have almost not changed; t1/2 from the 1.1 h reduced to 0.8 h, and there no statistical significance (P > 0.05). Comparing acute H2S poisoning group with the control group, the pharmacokinetic parameters of tolbutamide have changed; t1/2 from the 3.9 h increased to 4.2 h, but there was no statistical significance (P > 0.05); AUCt); CL from 0.10 reduced to 0.06 L/h/kg, and there was statistical significance (P < 0.05); Cmax varied from 1689.3 to 2216.5 ng/mL, and there was statistical significance (P < 0.05).As can be seen from Cmax of bupropion, metoprolol, phenacetin, and tolbutamide in acute H2S poisoning group are higher than the control group; this result is consistent with the 2S poisoning group almost coincided with control group.The representative bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide concentration versus time profiles of twelve rats were presented in 2S poisoning group, the AUC bupropion goes higher (P < 0.05), CL decreases (P < 0.05), and Cmax becomes higher (P < 0.05); this indicates that acute H2S poisoning will inhibit the activity of CYP2B6 enzyme. Similar results were found for tolbutamide; the AUCt)(0– increased (P < 0.05); CL reduced (P < 0.05); Cmax became higher (P < 0.05); this indicates that acute H2S poisoning will inhibit the activity of CYP2C9 enzyme.Compared to the control group, in acute H2S poisoning group, the AUCt)(0– of metoprolol increased (P < 0.01); Cmax became higher (P < 0.01); this indicates that acute H2S poisoning will inhibit the activity of CYP2D6 enzyme. Similar results were found for phenacetin; AUCt)(0– increased (P < 0.05); Cmax became higher (P < 0.01); this indicates that acute H2S poisoning will inhibit the activity of CYP1A2 enzyme.Compared to the control group, in the acute H2S poisoning group, the pharmacokinetic parameters of midazolam have almost not changed; this indicates that the acute H2S poisoning will not induce or inhibit the activity of CYP3A4 enzyme. Similar results were found for omeprazole; the pharmacokinetic parameters of omeprazole have almost not changed between control group and acute H2S poisoning group; this shows that the acute H2S poisoning will not induce or inhibit the activity of CYP2C19 enzyme.Compared to the control group, in the acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 of rats. The results may make sense for the clinical oral use of drugs for the people after acute H2S poisoning. Drugs are metabolized by CYP2B6, CYP2D6, CYP1A2, and CYP2C9 enzymes after acute H2S poisoning; we should pay close attention to changes in the plasma concentration to avoid drug interactions that may occur.The results demonstrated that acute H2S poisoning and control group, there was a statistically significant increase in the AUC and Cmax for bupropion, metoprolol, phenacetin, and tolbutamide, while there was no statistical pharmacokinetics difference for midazolam and omeprazole. Acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 of rats. The results may make sense for the clinical oral use of drugs for the people after acute H2S poisoning.The concentrations of probe drugs in rat plasma were successfully measured by LC-MS. In the experiment for acute H


Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains (“domain cassettes” (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection.var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS.Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples.PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis.This is the first study to show association between  Plasmodium falciparum is linked to the sequestration of parasites in the host’s deep organ vasculature caused by cytoadherence of parasite infected erythrocytes (iE) to endothelial cell receptors Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1) family expressed at the iE surface PfEMP-1 molecules have diverged to bind different endothelial receptors, including ICAM-1, VCAM, CD36, and the glycosaminoglycan Chondroitin-Sulfate A (CSA) PfEMP-1 is thought to be associated with clinical syndromes. iE binding to CSA is specifically associated with pregnancy-associated malaria (PAM) Virulence of var genes, coding for PfEMP-1 proteins, are divided into three main groups A-C, the conserved VAR2CSA variants and the intermediate groups B/A and B/C defined by their conserved 5′ upstream sequences (UPS) and complexity of the encoded domain structure PfEMP-1 molecules (i.e. DBLα-CIDRα-DBLβ/DBLγ domains) are associated with the UPS type, whereas domains in the C-terminal (i.e. DBLδ-CIDRβ/γ-DBLε-DBLζ) are not. The alignment of domain subtype architectures of 399 known PfEMP-1s revealed a set of 21 conserved domain compositions called domain cassettes (DC) The PfEMP-1 PfEMP-1 types involved in the sequestration during CM has been particularly challenging due to the great diversity of the var gene family. Nevertheless, it is well established that A and B/A var genes are over-expressed in parasites isolated from patients suffering from severe malaria var genes encoding DC8 and DC13 PfEMP-1 was associated with parasites isolated from Tanzanian children suffering from CM P. falciparum parasites expressing DC8 PfEMP-1 had superior brain endothelial cell binding capabilities over parasites expressing other PfEMP-1 PfEMP-1 with CM, it is important for future studies of pathogenesis and development of vaccine constructs in particular, to establish if these PfEMP-1 variants precipitate severe disease in other regions. We investigated the PfEMP-1 types expressed by parasites from subjects with CM, uncomplicated malaria (UM) or PAM in Benin, using both var gene transcript levels measurement and a proteomic approach.Infections causing severe malaria in children are also linked to the expression of a restricted subset of Ethical clearance was obtained from the Institutional Ethics Committee of the Faculté des Sciences de la Santé at the Abomey-Calavi, University in Benin. All patients were included after written informed consent from themselves or their guardian. The medical team of each health facility managed patients where adequate anti-malarial treatment was administered according to the national malaria program policy.Patients were enrolled at Cotonou in southern Benin over the 2011 malaria transmission season (May to mid-August). Malaria transmission in Cotonou is approaching 33 infecting bites per person per year P. falciparum infection, and absence of any severity sign as defined by the WHO P. falciparum infection with exclusion of other cause for coma, particularly meningitis. Patients and pregnant women at delivery were all screened for malaria infection by rapid diagnostic test for P. falciparum . A questionnaire was administered to the children′ guardian and woman to collect social data, disease history, and treatment received before hospital admission.Uncomplicated malaria (UM) was defined as the combination of fever (tympanic temperature ≥37.8°C), confirmed presence of P. falciparum infection, and parasitaemia was quantified by counting against 200 leucocytes assuming a mean of 8,000 leucocytes per millimeter of blood.Peripheral venous blood sample (5 ml) was collected from all study individuals in a vacutainer tube containing EDTA, and a placental tissue was obtained after delivery. Giemsa-stained thick blood film confirmed seryl-tRNA synthetase primers Ring stage parasites were conserved either in Trizol (Invitrogen) stored at −80°C or as dried spots on Whatmann 3MM filter paper stored at room temperature. Total RNA from peripheral iE was prepared from Trizol samples followed by treatment with DNAse 1 (Sigma) for 15 min at 37°C. Absence of DNA in RNA samples was confirmed by stable base fluorescence after 30 cycles of real-time PCR with var genes as well as the cassettes DC1, DC5, DC8, DC13, and DC16, we selected 21 sets of primers from the 42 sets published by Lavstsen et al., var2csa specific primers, as this gene has been documented to be the single PfEMP-1 gene expressed in PAM Var genes transcripts abundance was determined as relative quantification with the control gene (ΔCt var_primer = Ct var_primer-Ct average_control primers), Transcripts units (Tu) was calculated as Tu = 2 (5-ΔCt) according to Lavstsen et al., To target the group A, B and C in vitro cultured for less than 18 h in order to obtain mature parasite forms (trophozoites and schizonts). Sixty-two placentas positive for P. falciparum by rapid diagnostic test were collected, but only 10 were flushed as they showed sufficient parasite density. In these 31 samples  containing mature forms, parasites were depleted from uninfected erythrocytes over a MACS column et al., Eleven from CM and 10 parasites from UM were To prepare trypsin digestion peptides, 100 µg of proteins were used for UM, PAM and CM samples were reduced with 20 mM DTT during 30 min at 56°C then alkylated with 55 mM of chloroacetamide for 30 min at room temperature. The samples were precipitated in acetone for 2 h at−20°C and centrifuged for 15 min at 14,000 rpm. The lysate was transferred in digestion buffer . The samples were digested overnight at 37°C. The peptides samples were acidified with 10% of TFA for 2 h at 37°C, and centrifuged 15 min at 14,000 rpm.2O 98%, ACN 2% and TFA 0.1% at 5 µL/minute. Peptides were then separated on a C18 RP analytical column  with a 90-minute gradient from 99% A  to 40% B .Analyses were performed using an Ultimate 3000 Rapid Separation liquid chromatographic system  coupled to a hybrid Linear Trap Quadrupole-ORBITRAP Velos mass spectrometer . Briefly, acidified peptides were loaded and washed on a C18 reverse phase precolumn  using a loading buffer containing H6 Automatic Gain Control (AGC), 3.104 resolutions, 400–2000 mass-to-charge ratio (m/z) range, 1000 ms maximum ion injection time; 2/MS/MS with AGC: 1.104, 200 ms maximum injection time, 2000 minimum signal threshold, 2 Da isolation width. The fragmentation was permitted for precursor with a charge state of 2, 3 or 4.The LTQ-ORBITRAP mass spectrometer acquired data throughout the elution process and operated in a data dependent scheme as follows: full MS scans were acquired with the ORBITRAP, followed by up to 10 LTQ MS/MS CID spectra on most abundant precursors detected in the MS scan. Exclusion latency was set to 24 seconds for previously fragmented precursors. Mass spectrometer settings were: 1/Full MS with 1.10The software used to generate.mgf files was Proteome discoverer 1.2. The threshold of Signal to Noise for extraction values was 3.Plasmodium sequences from NCBI and their reverse sequences plus the var genes of sequences from Vardom (http://www.cbs.dtu.dk/services/VarDom/).MS/MS spectra were submitted to mascot (Matrix science) version 2.2 search engine p<0.05) were accepted as identified.The search parameters were set as follows: trypsin specificity, 2 ppm mass tolerance, 1 missed tryptic cleavage, oxidation (M) was set as a variable modification only those proteins with MOWSE score >40 . The concordance between genomic and proteomic data has been evaluated by the Cohen’s kappa coefficient. STATA 12 software was used for statistical analyses. P values <0.05 were considered as significant.Thirty-two children with cerebral malaria (CM), 30 patients with uncomplicated malaria (UM), and 15 pregnant women at delivery were included in the study . The avevar transcription is significantly higher in CM samples (median transcription units (interquartile range IQR) = 11.2 (1.7–27.2) vs. 1.0 (1.0–4.1); P = 0.003) whereas group B var transcript levels with B1 primer was higher in UM than in CM (4.35 (1.2–12.8) vs. 1.0 (1.0–1.0); P<10−3). The group C targeted with primer C1 and C2 was similar in two groups CM and UM (1.0 (1.0–1.0) in CM vs. 1.0 (1.0–2.0) in UM and 1.0 (1.0–1.0) in CM vs. 1.0 (1.0–1.0) in UM, respectively).Thirty-one CM samples and 22 UM samples were analyzed by RT-qPCR with a set of 21 primers vs. 1.0 (1.0–1.7); P = 0.0036; 1.0 (1.0–8.7) vs. 1.0 (1.0–1.0); P = 0.005 and 7.6 (1.3–20.1) vs. 1.0 (1.0–2.7); P = 0.002, respectively) and a higher prevalence of transcription (as defined with at least one DC8 primer) in CM than in UM (41.9 (24.5–60.9) vs. 4.3 (1.1-21.9); P = 0.0003). The DBLa1.7 primer pair targeting DC13 showed increased transcript levels in CM compared to UM samples (22.5 (9.6–41.1) vs. 4.3 (1.1–21.9); P = 0.047). Primers DBLb3 targeting a subset of var genes including genes encoding DC8 and DC13 PfEMP-1 also showed significantly higher transcript levels in CM vs. UM samples (2.5 (1.0–9.5) vs. 1.0 (1.0–1.0); P = 0.0001.All the primer pairs targeting DC8 , showed increased transcript levels in CM than in UM samples (2.6 (1.0–15.4) Var2csa transcripts were found at significantly higher levels in PAM than in CM or UM samples (173.2 (134.5–211.9) vs. 1.17 (1–4.5) vs. 5.45 (1.0–31.1); P = 0.0001 for overall comparison, all P<10−3 for paired [2×2] comparisons, data not shown).PfEMP-1 protein expression 11 CM, 10 UM and PAM protein lysates were prepared for LC–MS/MS analysis. Identified peptide sequences were annotated using a database containing human and Plasmodium genomes as well as additional available var gene sequences. Due to the extensive sequence variation of PfEMP-1, it is expected that the extracted PfEMP-1 molecules would not match 100% to the reference database sequences. Thus, the search parameters were initially varied to test the signal to noise ratio of the matched peptides to PfEMP-1 and human protein database. The analysis first focused on peptides sequences that have been detected in mass spectrometry in the aim to detect all these peptides without any limit of identification score for protein. This also allows to identify major domains and domain cassettes matching with these peptides. Then, we considered only proteins identified with at least two peptides.To investigate PfEMP-1 proteins; among those, the 440 peptides with a minimal score 10 were retained for analysis. These peptides matched with at least one of the 399 PfEMP-1 proteins from the PfEMP-1 database P. falciparum genomes PfEMP-1 groups. To avoid non-informative peptides  and/or poorly predictive peptides, we added selection criteria defined in the materials and methods section. The 440 peptides have been annotated as specific to a PfEMP-1 domain type; 196 peptides being predicted to belong to one PfEMP-1 group or one DC. Among these peptides, 107 were specific to CM samples, 56 to PAM samples, and 20 to UM samples while 13 peptides were found in two groups (PfEMP-1 were more frequently found among CM samples than in UM and PAM isolates (0.04 (0–0.3) vs. 0 (0–0), P = 0.051 and 0.04 (0–0.3) vs. 0 (0–0), P = 0.045, respectively). Conversely, cassettes DC13 and DC16 were found with similar frequency in isolates from the three clinical groups (DC13: 0 (0–0) in CM, 0 (0–0) in PAM, and 0 (0–0) in UM, P = 0.273; DC16: 0 (0–0) in CM, 0 (0–0) in PAM, and 0 (0–0) in UM, P = 0.440, respectively).The analysis on peptides without any restriction of score allowed to detect 548 peptides matching only with o groups . PeptidePfEMP-1 protein in 10/11; 6/10 and 9/10 isolates from CM, UM, and PAM, respectively, with at least two peptides that yielded a score >20. A total of 104 PfEMP1 proteins were predicted , while 72 PfEMP-1 proteins were identified specifically in CM isolates . The protein IT4var07, that has been implicated in CM PfEMP-1 proteins were identified only in UM samples .We identified at least one redicted . VAR2CSAPfEMP-1 proteins, by isolate and by clinical group (vs. 0 (0–0.25) in PAM vs. 0.42 (0.27–0.53) in UM; P = 0.139 and; 0.19 (0–0.26) in CM vs. 0 (0–0) in PAM vs. 0 (0–0.19) in UM; P = 0.422, respectively). Nevertheless, the proportion of group B proteins tended to be higher in CM than in PAM groups (P = 0.06).We compared the proportion of proteins associated with each group among all identified al group . The provs. 1 (0.25–1) in PAM vs. 0 (0–0) in UM; P = 0.004), being found more frequently in PAM than in the other two clinical groups (both <P = 0.02). Similarly, the number of UPS A PfEMP1 proteins differed among clinical groups (0.18 (0.075–0.35) in CM vs. 0(0–0) in PAM vs. 0 (0–0.07) in UM; P = 0.017), being found more frequently in CM isolates than in PAM (P = 0. 010) and UM isolates (P = 0.081).The number of VAR2CSA (UPSE) proteins differed between the three clinical groups (0 (0–0) in CM var gene transcripts from each var group (A-C) and the identified PfEMP-1 proteins in CM samples  is frequent in CM samples. This phenomenon was less pronounced in the genomics data, as our RT-qPCR approach targets already defined regions corresponding to the set of primers used.Finally, genomics and proteomics data were compared showing a strong concordance of the levels of  samples  with a Cvar genes and PfEMP-1 proteins in parasites isolated from children suffering from severe cerebral malaria or uncomplicated malaria as well as women diagnosed with PAM in Cotonou, Benin. We used RT-qPCR and mass spectrometry techniques to produce the first study to associate genomic data on var genes with proteomic data on PfEMP-1 expression in P. falciparum patients isolates.This study aimed to characterize the expression of var genes were associated with parasites from children with severe malaria. In addition, using a set of 21 primer pairs targeting var transcripts encoding specific PfEMP-1 domain cassettes, parasites from CM patients were found to have higher transcript levels of DC8 and DC13 encoding var transcripts PfEMP-1 than parasites from UM patients. This finding on parasite isolated from children from West Africa corroborate previous observations made using the same technology on parasites from Tanzania, East Africa PfEMP-1 and severe childhood pathogenesis is not geographically-restricted. Such finding is of utmost importance for future studies of pathogenesis and development of vaccine constructs.As expected , transcrP. falciparum proteins expressed on the membrane of the erythrocyte. Firstly, parasite proteins represent a tiny minority among the far more abundant erythrocyte proteins, and the identification of such rare proteins requires a very efficient MS protocol. Secondly, these variant parasite surface-expressed proteins are characteristically poorly soluble with a very long and highly variable extracellular domain. The use of a 50 cm-long HPLC column allowed a better separation of the peptides, and data analysis was optimized by performing three searches against the Human, Plasmodium genomes and PfEMP-1 database of 399 known PfEMP-1 sequences using different settings of 10 and 20 parameters. Next, all predicted PfEMP-1 peptide sequences were annotated with a domain subtype and PfEMP-1 group only if the sequence uniquely matched more than one domain of the said type.The proteomics approach is particularly difficult to apply to investigations of PfEMP-1 variant associated with cerebral malaria, as opposed to PAM, remains a difficult quest. In comparison with parasites from the other clinical groups, proteins and peptides identified by LC-MS/MS in CM isolates are preferentially associated with PfEMP-1 variants encoded by group A var genes, confirming our transcriptomic data, and in line with other studies PfEMP-1 that is uniquely characteristic of CM is unlikely. Conversely, the identification of specific domain cassettes expressed during CM appears to be a real alternative. Thus, we confirmed the over-expression of DC8 in CM parasite samples from West Africa, suggesting that this particular cassette is associated with CM right across sub-Saharan Africa.As shown in var genes patterns and the PfEMP-1 proteins identified by LC-MS/MS  in parasite samples. Cerebral malaria (CM), uncomplicated malaria (UM), or pregnancy-associated malaria (PAM).(DOCX)Click here for additional data file.Table S2List of PfEMP1 proteins identified by LC-MS/MS in parasite samples from different malaria syndromes. Cerebral malaria (CM), uncomplicated malaria (UM), or pregnancy-associated malaria (PAM).(DOCX)Click here for additional data file.


DT = 1.026 cm−1, ET = 0), triplet 2,6-diazido-3-chloro-5-fluoropyridyl-4-nitrene , quintet 4-azido-3-chloro-5-fluoropyridyl-2,6-dinitrene , quintet 2-azido-3-chloro-5-fluoropyridyl-4,6-dinitrene  and septet 3-chloro-5-fluoropyridyl-2,4,6-trinitrene . Preferential photodissociation of the azido groups located in ortho-positions to the fluorine atom of pyridines is associated with strong π-conjugation of these groups with the pyridine ring. On photoexcitation, such azido groups are more efficiently involved in reorganization of the molecular electronic system and more easily adopt geometries of the locally excited predissociation states.In contrast to theoretical expectations, the photolysis of 2,4,6-triazido-3-chloro-5-fluoropyridine in argon at 5 K gives rise to EPR peaks of just two triplet mononitrenes, two quintet dinitrenes, and a septet trinitrene. EPR spectral simulations in combination with DFT calculations show that observable nitrenes can be assigned to triplet 2,4-diazido-3-chloro-5-fluoropyridyl-6-nitrene ( Both these processes are widely used in modern science and technology –6. When  product  7–8]. M–8. M1a w product  9]. By . By 1a wrmediate  10]. Al. Al1a wides 1a–c . Althoug isomers –31.11  of triazide DT = 1.026 cm−1, ET = 0 cm−1 and DT = 1.122 cm−1, ET = 0.0018 cm−1, two quintet dinitrenes with DQ = 0.209 cm−1, EQ = 0.039 cm−1 and DQ = 0.215 cm−1, EQ = 0.0545 cm−1, and a septet trinitrene with DS = −0.1021 cm−1, ES = −0.0034 cm−1 in a 14:12:20:5:1 ratio  and 5c  and septet trinitrene 6c  [11 display very similar EPR spectra.The EPR spectral simulations show that the products of the reaction are two triplet nitrenes with :1 ratio . Surpris43 cm−1) . The EPR43 cm−1) –31. Quin18 since only this trinitrene has a septet spin state. According to EPR spectral simulations –Cl bond . This dinitrene can be assigned either to dinitrene 16 or to dinitrene 17. Previous EPR studies have shown that quintet dinitrenes with Θ = 121 ± 1° displayed EQ = 0.04 ± 0.001 cm−1, and dinitrenes with Θ = 125 ± 1° showed EQ = 0.035 ± 0.001 cm−1 [16 and 17 are equal to 121.6° and 125.4°, respectively . Thus, for instance, in EPR spectra of two quintet molecules with EQ/DQ = 0.039/0.209 and 0.038/0.213, the minor component with EQ/DQ = 0.038/0.213 becomes visible when its ratio to the major component exceeds 1:6 (14%). Based on these data, we do not exclude that dinitrene 17 with EQ/DQ ≈ 0.036/0.221 may be formed in ca. 5% yield along with dinitrene 16 (≈33%) during the photolysis of triazide 11.Although we do not observe diagnostic signals of dinitrene ions See . However11  was used in the spin-Hamiltonian calculations [E. The formation of such high-spin nitrenes affects significantly the line-width and intensity of some lines in the experimental EPR spectra. Similar effects were also observed in EPR spectra of nitrenes 15–18. In order to adequately reproduce the experimental EPR spectrum from E) = 75 MHz for dinitrenes 15 and 16 and Γ(E) = 40 MHz for trinitrene 18 were used in the spectral simulations. According to these values of Γ(E), the variations in the angles Θ for dinitrenes 15/16 and trinitrene 18 do not exceed 0.7° and 0.4°, respectively. Despite these very small variations in the angles Θ, all our attempts to theoretically reproduce the experimental EPR spectrum from E) were unsuccessful. EPR spectra of nitrenes 15, 16 and 18 simulated for Γ(E) = 0, 40 and 75 MHz are presented in Recent EPR studies have shown that the best EPR spectral simulations for high-spin nitrenes were obtained only when an additional line-broadening parameter Γ density . Finally, the photodecomposition of the remaining azido groups in dinitrenes 15 and 16 gives septet trinitrene 18 in just about 2% yield. The low yield of trinitrene 18 may be associated with low efficiency of the azido group excitation in quintet dinitrene 16. One also cannot exclude that the photoexcitation of quintet azidonitrenes into predissociation states is a much less efficient process in comparison with the photoexcitation of singlet azides. In contrast to singlet azides, quintet azidonitrenes already have four singly occupied orbitals, and photoexcitation of these species may lead to excited states in which the azido groups are not locally excited.Thus, the first stage of the photolysis of triazide 2 and 13 . On the 11 with light at λ = 260–320 nm occurs selectively on the azido groups located in positions 4 and 6 of the pyridine ring to give triplet mononitrenes 12 and 13 as the primary photoproducts. On further irradiation, quintet dinitrenes 15 and 16 and septet trinitrene 18 are formed. The maximum yield of the latter is about just 2%. Preferential photodissociation of the azido groups located in ortho-positions to the fluorine atom of pyridines is associated with strong π-conjugation of these groups with the pyridine ring. On photoexcitation, such azido groups are more efficiently involved in reorganization of the molecular electronic system and more easily adopt geometries of the locally excited predissociation states. Despite the lack of symmetry, trinitrene 18 shows a small parameter of ES = −0.0034 cm−1 due to a large positive value of the spin-orbit coupling parameter ESSO (≈0.005 cm−1). In overall, the ZFS parameters of 18 are very close to the ZFS parameters of previously studied C2v symmetric septet pyridyl-2,4,6-trinitrenes. The most accurate theoretical predictions of the ZFS parameters of 18 are obtained at the BLYP/EPRII level of theory. At the same time, modern DFT calculations are still too crude to be used for reliable EPR spectral identification of such structurally alike quintet isomers as dinitrenes 16 and 17. Such quintet isomers are better discriminated based on analysis of their dipolar angles Θ. Among theoretical approaches, the most accurate estimations of the ZFS parameters of dinitrene 16 are obtained from PBE/DZ calculations of its parameters DSSS and ESSS. The study also demonstrates that successful EPR spectral simulations for nitrenes 15, 16 and 18 are possible only if additional line-broadening parameters Γ(E) of 40–75 MHz along with appropriate values of g, S, D, E and ν0 are used in the spin-Hamiltonian calculations.In contrast to theoretical expectations, photodissociation of triazide 11 was synthesized according to the literature procedure [102 cavity. Solid argon matrices doped with triazide 11 were prepared by vacuum co-deposition of two separate molecular beams (Ar and triazide 11 vapor) on the tip of an oxygen-free high-conductivity copper rod  cooled at 5 K. The vapor of 11 was produced by an oven heating the polycrystalline 11 to 60 °C. The matrix-isolated samples were irradiated with a high-pressure mercury lamp, by using a filter passing the light at λ = 260–320 nm, and spectra were recorded at various irradiation times.Triazide rocedure . X-band EasySpin program package (version 4.0.0) [S = 1, 2 or 3 using the parameters ν0 = 9.605832 GHz and g = 2.0023 and line widths ΔH = 60 G for S = 1, and 30 G for S = 2 and S = 3.The computer simulations of EPR spectra were performed by using the n 4.0.0) . The simg, D, E) and orientations of the D tensor were obtained from additional single-point calculations with the ORCA program package [DSS and spin–orbit coupling (SO) DSO parts of the D tensors were performed by using the McWeeny–Mizuno and Pederson–Khanna approaches, respectively [The geometries of the molecules were optimized at the B3LYP/6-311G(d) level of theory with the Gaussian 03 program package . The nat package –41. DFT ectively .15–17 and septet trinitrene 18 as individual species and for the mixtures of quintet molecules with EQ/DQ = 0.039/0.209, 0.038/0.213 and 0.036/0.221 at different molar ratios as well as EPR spectral simulations for nitrenes 15, 16 and 18 at different values of the line-broadening parameter Γ(E).Supporting Information features EPR spectral simulations for quintet dinitrenes File 1EPR spectral simulations.


Pagodroma nivea. Extensive purification and chemical analysis revealed that the fossil samples are a mixture of glycerol derivatives. In vitro experiments showed that the Mumijo extract caused in cortical neurons a strong neuroprotective effect against the apoptosis-inducing amyloid peptide fragment β-fragment 25–35 (Aβ25–35). In addition, the fraction rich in glycerol ethers/wax esters displayed a significant growth-promoting activity in permanent neuronal PC12 cells. It is concluded that this new Mumijo preparation has distinct and marked neuroprotective activity, very likely due to the content of glycerol ether derivatives.Mumijo is a widely used traditional medicine, especially in Russia, Altai Mountains, Mongolia, Iran Kasachstan and in Kirgistan. Mumijo preparations have been successfully used for the prevention and treatment of infectious diseases; they display immune-stimulating and antiallergic activity as well. In the present study, we investigate the chemical composition and the biomedical potential of a Mumijo(-related) product collected from the Antarctica. The yellow material originates from the snow petrels,  Medium-pressure liquid chromatographic (MPLC) analyses were carried out on a Büchi 861 apparatus with SiO2 (230–400 mesh) packed columns. High-performance liquid chromatography (HPLC) separations were achieved on a Knauer 501 apparatus equipped with an RI detector. GC-MS spectra were performed with a Hewlett- Packard 5890 gas chromatograph equipped with a split/splitness injector and connected to a Mass Selective Detector (MSD) HP 5970 MS using electron impact ionization (EI) at a ionization energy of 70 eV. HPLC was achieved with a Varian Prostar 210 apparatus equipped with a Varian 350 refractive index detector or a Varian 325 UV detector.Electrospray ionization (ESI) mass spectra were obtained on an API 2000 mass spectrometer. Nuclear magnetic resonance (NMR) experiments were performed on a Varian Unity INOVA 500 spectrometer; chemical shifts refer to the residual solvent signal (CDhttp://www.bgr.bund.de/cln_011/nn_322990/DE/Themen/MeerPolar/Polarforsch-ung/Projekte/Antarktis__Projekte/GEOMAUD.html) November 1, 1995 to August 25, 2005. The location had been the Schirmacher Oasis . The yellow stony material from Antarctica  and collected during the “GeoMaud”—Geoscientific Expedition to Dronning Maud Land (Antarctica) . The concentration cited under Results section refers to the amount of solid Mumijo used for extraction.in vacuo and a crude extract (5.8 g) was obtained. This was subjected to fractionation by silica gel MPLC to give six fractions, A to F, using hexane, EtOAc and MeOH as a progressively polar solvent system series. All fractions were subjected to a preliminary spectroscopic inspection . Fractions B to D were showed to be neutral lipid mixtures and were subsequently separated and/or analyzed, as indicated.For the chemical analysis, the fossil material (8.5 g dry weight after extraction) was homogenized and extracted first with methanol (3 × 300 ml) and then with chloroform (3 × 200 ml). Combined extracts were concentrated Wax esters (fraction B): 1H NMR (CDCl3): δ 0.86 , 1.23 , 1.52–1.58 , 2.26 , 4.03  p.p.m. Fraction B (4 g) contained wax esters, whose fatty acid and alcohol compositions were determined by GC-MS in their natural state. GC-MS analysis was performed on a fused silica column . The oven temperature was programmed from 150°C to 350°C at a rate of 10°C/min and held at the final temperature for 10 min. Helium was used as carrier gas at a constant flow rate of 1.0 ml/min and a gas inlet pressure of 13.3 psi. Quantitative determination was based on the area of GLC peaks . Wax estFatty acid methyl esters (fraction C):  1H NMR (CDCl3): δ 0.85 , 1.23 , 1.58 , 2.30 , 3.65  p.p.m. Fraction C (1.1 g) was shown to be a mixture of fatty acids. These had been methylated with diazomethane and the resulting esters were analyzed by GC-MS analyses on a fused silica column as above. The temperature of the column was changed 5 min after injection from 150°C to 300°C with a slope of 5°C/min. The quantitative determination was based on the area of GLC peaks and the results of the analysis are summarized in 2O → MeOH → CHCl3), thus giving a monoglycerides fraction  eluted with H2O/MeOH 2:8, and a monoalkyl glycerol ethers fraction  eluted with 100% MeOH. An aliquot each of fractions D/2 and D/3 (20 mg each) was dissolved in pyridine (500 μl) and allowed to react with Ac2O (200 μl) for 12 h. The reaction mixtures were concentrated and the residues were purified by normal phase HPLC . Monoglyceride diacetates from fraction D/2:1H NMR (CDCl3): δ 0.86 , 1.23 , 2.04 , 2.06 , 2.07 , 2.30 , 4.11–4.16 , 4.25–4.31  5.23 , 5.33  p.p.m. ESI (positive ion mode): m/z: 407, 409, 435, 437, 463, 465, 491, 493, 521, 549 [M + Na]+ series. The quantitative estimation, reported in Glyceryl ethers diacetates from fraction D/3: 1H NMR (CDCl3): δ = 0.86 , 1.23 , 1.54 , 2.04 , 2.05 , 2.07 , 3.41 , 3.51 , 4.14  4.31 , 5.16 , 5.33  p.p.m. ESI (positive ion mode): m/z: 393, 395, 421, 423, 449, 451, 477, 479, 507, 535 [M + Na]+ series.Fraction D (0.4 g) was re-chromatographed on an RP-18 column by MPLC (H2+ and Mg2+). The cell suspension was centrifuged and the pellet was resuspended in Dulbecco's modified Eagle's medium (4500 mg of glucose/l), supplemented with 100 mU/l insulin, 2 mM glutamate and 10% fetal calf serum. After incubation for 48 h in poly-l-lysine-coated plastic 96-well plates, the medium was supplemented with 10 μM uridine, 10 μM fluorodeoxyuridine and 1 μM cytosine arabinofuranoside  for 3 days. The cultures contained >85% neurons; the other cells were glial fibrillary acidic protein-positive  contain a single molecular ion [M]+ alongside a set of dominant ions [R1CO2H2]+ deriving from a double hydrogen rearrangement fragmentation at the ester group. These ions show a difference of 28 amu and lead to the conclusion that the individual GC peaks contain wax ester isomers with the same carbon number and same degree of unsaturation, but different position of the esters moiety within the wax ester due to different chain lengths of carbonyl- and ester components. In our case, due to the relatively high abundance of [R1CO2H2]+ ions, the assignment of the individual was esters isomers was possible, as shown in 2-H]+ ions and [R1CO]+ acylium ions.The wax esters fraction was analyzed by GC-MS using EI, according to the method proposed by Reiter et al. . This ra+ peak series, indicating their composition of homologs; the high field region of the 1H-NMR spectra of both fractions revealed that all the homologs were unbranched. The assessment of fatty acid and alcohol composition of monoglycerides and monoalkyl glycerol ethers diacetates, reported in Fractions D/2 and D/3 were shown (by NMR) to be a mixture of glycerol derivatives; the identification of their components was carried out by NMR and ESIMS. An aliquot each of fractions D/2 and D/3 was acetylated by treatment with acetic anhydride and pyridine and then purified by normal phase HPLC. The fractions turned out to be mixtures of monoglyceride diacetates (fraction D/2) and glyceryl ethers diacetates (fraction D/3) by NMR analysis see . A confiβ fragment, Aβ25–35, was assessed in primary rat cortical neurons. Application of the fragment at a concentration of 1 μM caused within the 5-day incubation period a significant reduction of viable cells to 27.8 ± 6.1% (P < .001). Mumijo extract alone was found to have no effect on the viability of the neurons. However, if the neurons were pre-incubated with Mumijo extract prior to addition of the Aβ25–35, a significant higher cell viability was determined  to 98.6 ± 9.3% (10 μg/ml) and 82.4 ± 8.9% (30 μg/ml) (P < .001), respectively. The neuroprotective effect displayed by the Mumijo extract was still significant at 1 μg/ml (not shown). The toxic effect of the Atermined . At concμg/ml. However, after purification, Mumijo fraction D/3 was effective and resulted in a significant stimulation of cell growth. Already at a concentration of 0.3 μg/ml, a significant increase in the growth stimulatory activity could be measured , while the maximal growth promoting function was determined between 3.0 and 10.0 μg/ml . The permanent PC12 cell line, a model system for neuronal differentiation , was useA detailed description of the components in Mumijo from Central Asia revealed  primarilThe chemical analysis of the fossil sample of Mumijo actually revealed that its composition parallels those previously reported for other samples of non-fossil material, with some substantial differences. The organic extract of Antarctic Mumijo contained mainly wax esters (70% wt), with considerable amounts of free fatty acids (20% wt). Monoglycerides and free monoalkyl glycerol ethers were also detected in significant amounts . Monoalkyl glycerol ethers are most frequently found as alkyldiacylglycerols (similar to triacylglycerols). However, these compounds, whose occurrence in petrel stomach oils has been reported , 31, 32,O-hexadecylglycerol , 1-O-octadecylglycerol  and 1-O-octadec-9-enyl glycerol . The trivial names go back to the fish species from which they have originally been isolated. In 1948, Berger [α-substituted glycerol ethers, and of chimyl and batyl alcohols. Since then, reports have appeared claiming a number of further pharmacological activities, such as antimicrobial [α-glyceryl ethers as well as of batyl and selachyl alcohols significantly accelerated the rate of wound healing in man when the healing process had been pathologically inhibited. Burford and Gowdey [α-glyceryl ethers of fatty alcohols. These properties could explain some medical applications of Mumijo in oriental medicine, such as its use for gastric and intestinal ulcers, healing of fractures, burns and skin diseases, tuberculosis, respiratory disease and inflammations [Glyceryl ethers were identified by Tsujimoto and Toyama  in the f, Berger  reportedicrobial , tubercoicrobial  and Lacticrobial , as wellicrobial  and radiicrobial . Bodman icrobial  reportedd Gowdey  claimed d Gowdey  reportedmmations .α-glyceryl ethers of) fatty alcohols is in accordance with recent data, which demonstrate that distinct fatty alcohols present strong potential for the treatment of neurological diseases, and are able to modulate neuroinflammation via induction of differentiation of neural stem cells into mature neurons. Based on these data, it had been proposed that those compounds might represent an approach for the treatment (or cure) of neuropathies [The present finding that Mumijo is rich in (opathies . Very weopathies . In addiopathies .Taken together, our data presented here show that the Antarctic Mumijo is rich in glyceryl ether derivatives which—according to the data given—display distinct and marked neuroprotective activity. Schematically, the biomedical potential of Antarctic Mumijo is summarized in