Metabolic alterations in the sera of Chinese patients with mild persistent asthma: a GC-MS-based metabolomics analysis

Aim:

To character the specific metabolomics profiles in the sera of Chinese patients with mild persistent asthma and to explore potential metabolic biomarkers.

Methods:

Seventeen Chinese patients with mild persistent asthma and age- and sex-matched healthy controls were enrolled. Serum samples were collected, and serum metabolites were analyzed using GC-MS coupled with a series of multivariate statistical analyses.

Results:

Clear intergroup separations existed between the asthmatic patients and control subjects. A list of differential metabolites and several top altered metabolic pathways were identified. The levels of succinate (an intermediate in tricarboxylic acid cycle) and inosine were highly upregulated in the asthmatic patients, suggesting a greater effort to breathe during exacerbation and hypoxic stress due to asthma. Other differential metabolites, such as 3,4-dihydroxybenzoic acid and phenylalanine, were also identified. Furthermore, the differential metabolites possessed higher values of area under the ROC curve (AUC), suggesting an excellent clinical ability for the prediction of asthma.

Conclusion:

Metabolic activity is significantly altered in the sera of Chinese patients with mild persistent asthma. The data might be helpful for identifying novel biomarkers and therapeutic targets for asthma.     

GC-TOF/MS-based metabolomic profiling of estrogen deficiency-induced obesity in ovariectomized rats

Aim:

To explore the alteration of endogenous metabolites and identify potential biomarkers using metabolomic profiling with gas chromatography coupled a time-of-flight mass analyzer (GC/TOF-MS) in a rat model of estrogen-deficiency-induced obesity.

Methods:

Twelve female Sprague-Dawley rats six month of age were either sham-operated or ovariectomized (OVX). Rat blood was collected, and serum was analyzed for biomarkers using standard colorimetric methods with commercial assay kits and a metabolomic approach with GC/TOF-MS. The data were analyzed using multivariate statistical techniques.

Results:

A high body weight and body mass index inversely correlated with serum estradiol (E2) in the OVX rats compared to the sham rats. Estrogen deficiency also significantly increased serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Utilizing GC/TOF-MS-based metabolomic analysis and the partial least-squares discriminant analysis, the OVX samples were discriminated from the shams. Elevated levels of cholesterol, glycerol, glucose, arachidonic acid, glutamic acid, glycine, and cystine and reduced alanine levels were observed. Serum glucose metabolism, energy metabolism, lipid metabolism, and amino acid metabolism were involved in estrogen-deficiency-induced obesity in OVX rats.

Conclusion:

The series of potential biomarkers identified in the present study provided fingerprints of rat metabolomic changes during obesity and an overview of multiple metabolic pathways during the progression of obesity involving glucose metabolism, lipid metabolism, and amino acid metabolism.     

An integrated metabonomic and proteomic study on Kidney-Yin Deficiency Syndrome patients with diabetes mellitus in China

Aim:

To investigate specific changes in metabolites and proteins of Kidney-Yin Deficiency Syndrome (KYDS) patients with diabetes mellitus (DM) in China.

Methods:

KYDS (n=29) and non-KYDS (n=23) patients with DM were recruited for this study. The KYDS was diagnosed by two senior TCM clinicians separately. The metabonomic and proteomic profiles of the patients were assessed using a metabonomic strategy based on NMR with multivariate analysis and a proteomic strategy based on MALDI-TOF-MS, respectively.

Results:

Eighteen upregulated peptides and thirty downregulated peptides were observed in the plasma of the KYDS patients. Comparing the proteomic profiles of the KYDS and non-KYDS groups, however, no significantly differentially expressed peptides were found. At the same time, major metabolic alterations were found to distinguish the two groups, including eight significantly changed metabolites (creatinine, citrate, TMAO, phenylalanine, tyrosine, alanine, glycine and taurine). The levels of creatinine, citrate, TMAO, phenylalanine and tyrosine were decreased, whereas the levels of alanine, glycine and taurine were increased in the KYDS patients. These biochemical changes were found to be associated with alterations in amino acid metabolism, energy metabolism and gut microflora.

Conclusion:

The identification of distinct expression profiles of metabolites and signaling pathways in KYDS patients with DM suggests that there are indeed molecular signatures underlying the principles of ''Syndrome Differentiation'' in traditional Chinese medicine.     

4epatic cytochrome P450s play a major role in monocrotaline-induced renal toxicity in mice

Aim：Monocrotaline （MCT） in plants of the genus Crotalaria induces significant toxicity in multiple organs including the liver, lung and kidney. Metabolic activation of MCT is required for MCT-induced toxicity. In this study, we attempted to determine whether the toxicity of MCT in kidney was a consequence of the metabolic activation of MCT in the liver. Methods： Liver-specific cytochrome P450 reductase-null （Null） mice, wild-type （WT） mice and CYP3A inhibitor ketoconazole-pretreated WT （KET-WT） mice were examined. The mice were injected with MCT （300, 400, or 500 mg/kg, ip）, and hepatotoxicity and nephrotoxic- ity were examined 24 h after MCT treatment. The levels of MCT and its metabolites in the blood, liver, lung, kidney and bile were deter- mined using LC-MS analysis. Results： Treatment of WT mice with MCT increased the serum levels of alanine aminotransferase, hyaluronic acid, urea nitrogen and creatinine in a dose-dependent manner. Histological examination revealed that MCT （500 mg/kg） caused severe liver injury and mod- erate kidney injury. In contrast, these pathological abnormalities were absent in Null and KET-WT mice. After injection of MCT （400 and 500 mg/kg）, the plasma, liver, kidney and lung of WT mice had significantly lower MCT levels and much higher N-oxide metabolites contents in compared with those of Null and KET-WT mice. Furthermore, WT mice had considerably higher levels of tissue-bound pyr roles and bile GSH-conjugated MCT metabolites compared with Null and KET-WT mice. Conclusion： Cytochrome P450s in mouse liver play a major role in the metabolic activation of MCT and thus contribute to MCT-induced renal toxicity.     

Cordyceps sinensis protects against liver and heart injuries in a rat model of chronic kidney disease： a metabolomic analysis

Aim： To test the hypothesis that the traditional Chinese medicine Cordyceps sinensis could improve the metabolic function of extrarenal organs to achieve its anti-chronic kidney disease （CKD） effects. Methods： Male SD rats were divided into CKD rats （with 5/6-nephrectomy）, CKD rats treated with Cordyceps sinensis （4 mg.kg1.d-1, pc）, and sham-operated rats. After an 8-week treatment, metabolites were extracted from the hearts and livers of the rats, and then subjected to 1H-NMR-based metabolomic analysis. Results： Oxidative stress, energy metabolism, amino acid and protein metabolism and choline metabolism were considered as links between CKD and extrarenal organ dysfunction. Within the experimental period of 8 weeks, the metabolic disorders in the liver were more pronounced than in the heart, suggesting that CKD-related extrarenal organ dysfunctions occurred sequentially rather than simultaneously. Oral administration of Cordyceps sinensis exerted statistically significant rescue effects on the liver and heart by reversely regulating levels of those metabolites that are typically perturbed in CKD. Conclusion： Oral administration of Cordyceps sinensis significantly attenuates the liver and heart injuries in CKD rats. The 1H NMR- based metabolomic approach has provided a systematic view for understanding of CKD and the drug treatment, which can also be used to elucidate the mechanisms of action of other traditional Chinese medicines.     

An integrated pathway interaction network for the combination of four effective compounds from ShengMai preparations in the treatment of cardio-cerebral ischemic diseases

Aim:

SMXZF (a combination of ginsenoside Rb1, ginsenoside Rg1, schizandrin and DT-13) derived from Chinese traditional medicine formula ShengMai preparations) is capable of alleviating cerebral ischemia-reperfusion injury in mice. In this study we used network pharmacology approach to explore the mechanisms of SMXZF in the treatment of cardio-cerebral ischemic diseases.

Methods:

Based upon the chemical predictors, such as chemical structure, pharmacological information and systems biology functional data analysis, a target-pathway interaction network was constructed to identify potential pathways and targets of SMXZF in the treatment of cardio-cerebral ischemia. Furthermore, the most related pathways were verified in TNF-α-treated human vascular endothelial EA.hy926 cells and H2O2-treated rat PC12 cells.

Results:

Three signaling pathways including the NF-κB pathway, oxidative stress pathway and cytokine network pathway were demonstrated to be the main signaling pathways. The results from the gene ontology analysis were in accordance with these signaling pathways. The target proteins were found to be associated with other diseases such as vision, renal and metabolic diseases, although they exerted therapeutic actions on cardio-cerebral ischemic diseases. Furthermore, SMXZF not only dose-dependently inhibited the phosphorylation of NF-κB, p50, p65 and IKKα/β in TNF-α-treated EA.hy926 cells, but also regulated the Nrf2/HO-1 pathway in H2O2-treated PC12 cells.

Conclusion:

NF-κB signaling pathway, oxidative stress pathway and cytokine network pathway are mainly responsible for the therapeutic actions of SMXZF against cardio-cerebral ischemic diseases.     

Excretion of [3H]triptolide and its metabolites in rats after oral administration

Aim： To investigate the routes of elimination and excretion for triptolide recovered in rats.
Methods： After a single oral administration of [3H]triptolide （0.8 mg/kg, 100 μCi/kg） in Sprague Dawley rats, urine and fecal samples were collected for 168 h. To study biliary excretion, bile samples were collected for 24 h through bile duct cannulation. Radioactivity was measured using a liquid scintillation analyzer, and excretion pathway analysis was performed using an HPLC/on-line radioactivity detector.

Results： The total radioactivity recovered from the urine and feces of rats without bile duct ligation ranged from 86.6%–89.1%. Most of the radioactivity （68.6%–72.0%） was recovered in the feces within 72 h after oral administration, while the radioactivity recovered in the urine and bile was 17.1%–18.0% and 39.0%–39.4%, respectively. The HPLC/on-line radiochromatographic analysis revealed that most of the drug-related radioactivity was in the form of metabolites. In addition, significant gender differences in the quantity of these metabolites were found： monohydroxytriptolide sulfates were the major metabolites detected in the urine, feces, and bile of female rats, while only traces of these metabolites were found in male rats.

Conclusion： Radiolabeled triptolide is mainly secreted in bile and eliminated in feces. The absorbed radioactivity is primarily eliminated in the form of metabolites, and significant gender differences are observed in the quantity of recovered metabolites, which are likely caused by the gender-specific expression of sulfotransferases.     

β-Naphthoflavone protects mice from aristolochic acid-l-induced acute kidney injury in a CYPIA dependent mechanism

Aim:

The role of CYP1A in the protection of aristolochic acid (AA)I-induced nephrotoxicity has been suggested. In the present study we investigated the effects of β-naphthoflavone (BNF), a non-carcinogen CYP1A inducer, on AAI-induced kidney injury.

Methods:

Mice were pretreated with 80 mg/kg BNF by daily intraperitoneal injection (ip) for 3 days followed by a single ip of 10 mg/kg AAI. AAI and its major metabolites in blood, liver and kidney, the expression of CYP1A1 and CYP1A2 in microsomes of liver and kidney, as well as the nephrotoxicity were evaluated.

Results:

BNF pretreatment prevented AAI-induced renal damage by facilitating the disposal of AAI in liver. BNF pretreatment induced the expression of CYP1A1 in both liver and kidney; but the induction of CYP1A2 was only observed in liver.

Conclusion:

BNF prevents AAI-induced kidney toxicity primarily through CYP1A induction.     

Anti-pruritic effect of baicalin and its metabolites, baicalein and oroxylin A, in mice Anti-tumorPharmacology

Aim:

To explore whether intestinal microflora plays a role in anti-pruritic activity of baicalin, a main constituent of the rhizome of Scutellaria baicalensis (SB).

Methods:

Baicalin was anaerobically incubated with human fecal microflora, and its metabolites, baicalein and oroxylin A, were isolated. The inhibitory effect of baicalin and its metabolites was accessed in histamine- or compound 48/80-induced scratching behavior in mice.

Results:

Baicalin was metabolized to baicalein and oroxylin A, with metabolic activities of 40.2±26.2 and 1.2±1.1 nmol·h⁻¹·mg⁻¹ wet weight of human fecal microflora, respectively. Baicalin (20, 50 mg/kg) showed more potent inhibitory effect on histamine-induced scratching behavior when orally administered than intraperitoneally. In contrast, baicalein and oroxylin A had more potent inhibitory effect when the intraperitoneally administered. The anti-scratching behavior activity of oral baicalin and its metabolites was in proportion to their inhibition on histamine-induced increase of vascular permeability with oroxylin A more potent than baicalein and baicalin. In Magnus test using guinea pig ileum, oroxylin A is more potent than baicalein and baicalin in inhibition of histamine-induced contraction. The anti-scratching behavioral effect of oral baicalin was significantly reduced when oral antibiotics were simultaneously administered, whereas the effect of baicalein and oroxylin A were not affected.

Conclusion:

Oral baicalin may be metabolized by intestinal microflora into baicalein and oroxylin A, which ameliorate pruritic reactions through anti-histamine action.     

Combined prostaglandin E1 and lithium exert potent neuroprotection in a rat model of cerebral ischemia

Aim:

To examine the effects of a mixed formulation composed of prostaglandin E1 and lithium (PGE1+Li mixture) on brain damage after cerebral ischemia. The effects of the mixture on protein expression of heat shock proteins (HSPs), p53, and Bcl-2 were also determined.

Methods:

Brain ischemia was induced with a permanent middle cerebral artery occlusion (pMCAO) in rats. Rats were treated with a single intravenous administration of PGE1, lithium or a PGE1+Li mixture immediately after the ischemic insult. The infarct volume and motor behavior deficits were analyzed 24 h after the ischemic insult. The protein levels of HSP70, glucose-regulated protein 78 (GRP78), HSP60, Bcl-2, and p53 in the striatum of the ipsilateral hemisphere were examined using immunoblotting.

Results:

The mixture (PGE1 22.6 nmol/kg+Li 0.5 mmol/kg) reduced infarct volume and neurological deficits induced by focal cerebral ischemia. Moreover, the mixture had a greater neuroprotective effect against cerebral ischemia compared with PGE1 or lithium alone. The mixture was effective even if it was administered 3 h after ischemia. PGE1+Li also significantly upregulated cytoprotective HSP70, GRP78, HSP60, and Bcl-2 protein levels, while decreasing p53 expression.

Conclusion:

These results demonstrated a PGE1+Li mixture with a therapeutic window of up to 3 h for clinical treatment of cerebral ischemia. The PGE1+Li mixture potentially exerts a protective effect after stroke through the induction of HSPs and Bcl-2 proteins.     

The edaravone and 3-n-butylphthalide ring-opening derivative 10b effectively attenuates cerebral ischemia injury in rats

Aim:

Compound 10b is a hybrid molecule of edaravone and a ring-opening derivative of 3-n-butylphthalide (NBP). The aim of this study was to examine the effects of compound 10b on brain damage in rats after focal cerebral ischemia.

Methods:

SD rats were subjected to 2-h-middle cerebral artery occlusion (MCAO). At the onset of reperfusion, the rats were orally treated with NBP (60 mg/kg), edaravone (3 mg/kg), NBP (60 mg/kg)+edaravone (3 mg/kg), or compound 10b (70, 140 mg/kg). The infarct volume, motor behavior deficits, brain water content, histopathological alterations, and activity of GSH, SOD, and MDA were analyzed 24 h after reperfusion. The levels of relevant proteins in the ipsilateral striatum were examined using immunoblotting.

Results:

Administration of compound 10b (70 or 140 mg/kg) significantly reduced the infarct volume and neurological deficits in MCAO rats. The neuroprotective effects of compound 10b were more pronounced compared to NBP, edaravone or NBP+edaravone. Furthermore, compound 10b significantly upregulated the protein levels of the cytoprotective molecules Bcl-2, HO-1, Nrf2, Trx, P-NF-κB p65, and IκB-α, while decreasing the expression of Bax, caspase 3, caspase 9, Txnip, NF-κB p65, and P-IκB-α.

Conclusion:

Oral administration of compound 10b effectively attenuates rat cerebral ischemia injury.     

Phenotype-dependent alteration of pathways and networks reveals a pure synergistic mechanism for compounds treating mouse cerebral ischemia

Aim:

Our previous studies have showed that ursodeoxycholic acid (UA) and jasminoidin (JA) effectively reduce cerebral infarct volume in mice. In this study we explored the pure synergistic mechanism of these compounds in treatment of mouse cerebral ischemia, which was defined as synergistic actions specific for phenotype variations after excluding interference from ineffective compounds.

Methods:

Mice with focal cerebral ischemia were treated with UA, JA or a combination JA and UA (JU). Concha margaritifera (CM) was taken as ineffective compound. Cerebral infarct volume of the mice was determined, and the hippocampi were taken for microarray analysis. Particular signaling pathways and biological functions were enriched based on differentially expressed genes, and corresponding networks were constructed through Ingenuity Pathway Analysis.

Results:

In phenotype analysis, UA, JA, and JU significantly reduced the ischemic infarct volume with JU being superior to UA or JA alone, while CM was ineffective. As a result, 4 pathways enriched in CM were excluded. Core pathways in the phenotype-positive groups (UA or JA) were involved in neuronal homeostasis and neuropathology. JU-contributing pathways included all UA-contributing and the majority (71.7%) of JA-contributing pathways, and 10 new core pathways whose effects included inflammatory immunity, apoptosis and nervous system development. The functions of JU group included all functions of JA group, the majority (93.1%) of UA-contributing functions, and 3 new core functions, which focused on physiological system development and function.

Conclusion:

The pure synergism between UA and JA underlies 10 new core pathways and 3 new core functions, which are involved in inflammation, immune responses, apoptosis and nervous system development.     

Neural Correlates of Suicidal Ideation and Its Reduction in Depression

Background:

The neural correlates of suicidal ideation and its reduction after treatment are unknown. We hypothesized that increased regional cerebral glucose metabolism in the infralimbic cortex (Brodmann area 25), amygdala, and subgenual anterior cingulate cortex would be associated with suicidal ideation and its reduction after ketamine infusion.

Methods:

Medication-free patients (n=19) with treatment-resistant major depressive disorder underwent positron emission tomography imaging at baseline and 230 minutes after an open-label ketamine infusion (0.5mg/kg for 40 minutes).

Results:

Baseline suicidal ideation and regional cerebral glucose metabolism in the infralimbic cortex were significantly correlated (r=.59, P=.007); but not overall mood scores (r=−.07, P=.79). Reductions in suicidal ideation after ketamine infusion were correlated with decreased regional cerebral glucose metabolism in the infralimbic cortex (r=.54, P=.02). Metabolism in other areas of interest was not significantly correlated with suicidal ideation or depression.

Conclusion:

The infralimbic cortex may be implicated in suicidal ideation.     

Pharmacokinetics,metabolism and bioavailability of the triazole antifungal agent voriconazole in relation to CYP2C19 genotype

AIMS

The aim was to determine the pharmacokinetics of voriconazole after a single oral dose in comparison with intravenous (i.v.) administration in healthy individuals stratified according to the cytochrome P450 (CYP) 2C19 genotype. In addition, the possible metabolic pathways and their modulation according to CYP2C19 genotype were investigated after oral and i.v. administration of voriconazole.

METHODS

In a single-centre, open-label, two-period crossover study 20 participants received single doses of 400 mg voriconazole orally and 400 mg voriconazole intravenously in randomized order. Blood and urine samples were collected up to 96 h post dose and the voriconazole and three major metabolites were quantified by high-performance liquid chromatography coupled to mass spectroscopy.

RESULTS

Absolute oral bioavailability of voriconazole was 82.6% (74.1, 91.0). It ranged from 94.4% (78.8, 109.9) in CYP2C19 poor metabolizers to 75.2% (62.9, 87.4) in extensive metabolizers. In contrast to voriconazole and its N-oxide, the plasma concentrations of both hydroxylated metabolites showed a large second peak after 24 h. Independent of the route of administration, voriconazole partial metabolic hydroxylation after i.v. administration was eightfold higher compared with N-oxidation [48.8 ml min⁻¹ (30.5, 67.1) vs. 6.1 ml min⁻¹ (4.1, 8.0)]. The formation of the metabolites was related to CYP2C19 activity.

CONCLUSIONS

Independent of the route of administration, voriconazole exposure was three times higher in CYP2C19 poor metabolizers compared with extensive metabolizers. Voriconazole has a high bioavailability with no large differences between the CYP2C19 genotypes. The hydroxylation pathway of voriconazole elimination exceeded the N-oxidation, both influenced by the CYP2C19 genotype.     

Evaluation of a New Vancomycin Dosing Protocol in Morbidly Obese Patients

Background:

Optimal dosing of vancomycin in morbidly obese patients (>100 kg and at least 140% of their ideal body weight) has not been determined. Conventional dosing strategies have led to the observation of supratherapeutic trough concentrations (>20 mcg/mL).

Objective:

To evaluate the effectiveness of a new vancomycin dosing protocol in morbidly obese patients in achieving therapeutic trough concentrations between 10 and 20 mcg/mL and to determine patient-specific factors influencing the trough concentration attained.

Methodology:

A single-center, retrospective chart review included morbidly obese adult patients with a pharmacy-to-dose vancomycin consult and at least 1 trough concentration obtained at steady state. Patients were excluded if they had a creatinine clearance (CrCl) less than 35 mL/min or unstable renal function, were not dosed according to the revised protocol, or received vancomycin prior to initiation of the protocol.

Results:

Of the 48 patients included, 17 (35.4%) achieved a therapeutic vancomycin trough concentration. Subtherapeutic concentrations (<10 mcg/mL) were observed in 27 patients (56.3%) and supratherapeutic concentrations were observed in 4 (8.3%) patients. Age less than 45 years and CrCl greater than 100 mL/min were associated with subtherapeutic trough concentrations.

Conclusion:

This study demonstrates that the revised vancomycin dosing protocol led to the attainment of therapeutic trough concentrations in 35.4% of patients. The majority had subtherapeutic concentrations, which increases the risk of treatment failures and resistance. Further study is needed to determine the optimal dosing strategy in this patient population.     

Isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP and 10-hydroxy-NBP,across the rat blood-brain barrier

Aim:

To investigate the mechanisms underlying the isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP (3-OH-NBP) and 10-hydroxy-NBP (10-OH-NBP), across the blood brain barrier (BBB).

Methods:

After oral administration of NBP (20 mg/kg) to rats, the pharmacokinetics of two major hydroxylated metabolites, 3-OH-NBP and 10-OH-NBP, in plasma and brains were investigated. Plasma and brain protein binding of 3-OH-NBP and 10-OH-NBP was also assessed. To evaluate the influences of major efflux transporters, rats were pretreated with the P-gp inhibitor tariquidar (10 mg/kg, iv) and BCRP inhibitor pantoprazole (40 mg/kg, iv), then received 3-OH-NBP (12 mg/kg, iv) or 10-OH-NBP (3 mg/kg, iv). The metabolic profile of NBP was investigated in rat brain homogenate.

Results:

After NBP administration, the plasma exposure of 3-OH-NBP was 4.64 times that of 10-OH-NBP, whereas the brain exposure of 3-OH-NBP was only 11.8% of 10-OH-NBP. In the rat plasma, 60%±5.2% of 10-OH-NBP was unbound to proteins versus only 22%±2.3% of 3-OH-NBP being unbound, whereas in the rat brain, free fractions of 3-OH-NBP and 10-OH-NBP were 100%±9.7% and 49.9%±14.1%, respectively. In the rats pretreated with tariquidar and pantoprazole, the unbound partition coefficient K_p,uu of 3-OH-NBP was significantly increased, while that of 10-OH-NBP showed a slight but not statistically significant increase. Incubation of rat brain homogenate with NBP yielded 3-OH-NBP but not 10-OH-NBP.

Conclusion:

The isomer-selective distribution of 10-OH-NBP and 3-OH-NBP across the BBB of rats is mainly attributed to the differences in plasma and brain protein binding and the efflux transport of 3-OH-NBP. The abundant 10-OH-NBP is not generated in rat brains.     

Compatibility of Vancomycin and Oxacillin During Simulated Y-Site Delivery

Background:

Vancomycin and oxacillin may be used together as empiric coverage in patients with proven or suspected Staphylococcus aureus infections. Though vancomycin hydrochloride 20 mg/mL and oxacillin sodium 160 mg/mL are reported to be compatible via Y-site delivery, Y-site compatibility of commonly used concentrations, vancomycin 10 mg/mL and oxacillin 20 mg/mL, has not yet been reported.

Objective:

To determine the Y-site compatibility of vancomycin 10 mg/mL and oxacillin 20 mg/mL.

Methods:

One vancomycin hydrochloride 1 g vial was reconstituted with 10 mL sterile water for injection (SWFI) and diluted with 90 mL 5% dextrose in water (D5W) in an evacuated intravenous (IV) bag. One oxacillin sodium 2 g vial was reconstituted with 11.5 mL sterile water for injection and diluted with 88 mL sterile water for injection in an evacuated IV bag. Three mL of each vancomycin and oxacillin were mixed in 4 test tubes to simulate Y-site delivery. Spectrometry, pH evaluation, and visual examination were performed for each test tube immediately following mixing and at 30 minutes, 1 hour, and 2 hours after mixing.

Results:

Upon visual examination with multiple backgrounds, a white precipitant was immediately evident in the test tubes with vancomycin and oxacillin combined. Spectrometry results strongly supported evidence of precipitation throughout the duration of the experiment.

Conclusions:

Vancomycin 10 mg/mL and oxacillin 20 mg/mL were determined to be physically incompatible for Y-site delivery in this study, despite prior evidence that the 2 medications in different concentrations were suitable for Y-site co-administration.     

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

Aim:

To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).

Methods:

CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.

Results:

CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min⁻¹·kg⁻¹, which was comparable to the mean human hepatic blood flow rate (20.7 mL·min⁻¹·kg⁻¹). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.

Conclusion:

CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.     

Fungal transformation of androsta-1,4-diene-3,17-dione by Aspergillus brasiliensis

Background

The biotransformation of steroids by fungal biocatalysts has been recognized for many years. There are numerous fungi of the genus Aspergillus which have been shown to transform different steroid substances. The possibility of using filamentous fungi Aspergillus brasiliensis cells in the biotransformation of androsta-1,4-diene-3,17-dione, was evaluated.

Methods

The fungal strain was inoculated into the transformation medium which supplemented with androstadienedione as a substrate and fermentation continued for 5 days. The metabolites were extracted and isolated by thin layer chromatography. The structures of these metabolites were elucidated using ¹H-NMR, broadband decoupled ¹³C-NMR, EI Mass and IR spectroscopies.

Results

The fermentation yielded one reduced product: 17β-hydroxyandrost-1,4-dien-3-one and two hydroxylated metabolites: 11α-hydroxyandrost-1,4-diene-3,17-dione and 12β-hydroxyandrost-1,4-diene-3,17-dione.

Conclusions

The results obtained in this study show that A. brasiliendsis could be considered as a biocatalyst for producing important derivatives from androstadienedione.     

The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism

Aim:

To evaluate the effects of the fibrinolytic enzyme FII_a from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models.

Methods:

Both rabbit and dog APT models were used. For the rabbit APT model, the thrombi weight before and after administration was measured. Central venous pressure (CVP) and mean arterial pressure (MAP) were measured before and 15, 30, 60, and 120 min after the injection of the blood clot. Partial thromboplastin time (APTT), prothrombin time (PT), platelet count, and fibrinogen concentration were measured using auto analyzers. Plasminogen activity was measured based on chromogenic substrates. In the dog APT model, pulmonary blood flow was recorded using pulmonary angiography.

Results:

Intravenous administration of FIIa (0.1–5.0 mg/kg) improved the APT-induced hemodynamic derangements and reduced thrombi weight. The angiography evidence also showed that the pulmonary emboli had almost disappeared after FII_a infusion. FII_a (0.1, 0.5, or 1.0 mg/kg) did not impair the coagulation pathways, although very high doses of FII_a (5.0 mg/kg) could stimulate the production of plasminogen and result in impairment of the pathways.

Conclusion:

FII_a could effectively protect against APT via degradation of thrombi with less activation of plasminogen, and may provide a novel fibrinolytic enzyme for targeting the main pathological processes of the disease.