Analytical method development and validation of mianserin hydrochloride and its metabolite in human plasma by LC-MS

Mianserin is a tetracyclic antidepressant drug and administered as racemate of R (-) and S (+) mianserin hydrochloride in a dose of 30-90 mg/day in divided doses. Liquid chromatography-mass spectroscopy (LC-MS) is a tool, which is widely used for determination of drug and their metabolites in biological fluids because of its high sensitivity and precision. Here we describe a liquid chromatography mass spectroscopy method for simultaneous determination of mianserin and its metabolite, N-desmethylmianserin, from human plasma using a liquid-liquid extraction with hexane:isoamylalcohol (98:2) and back extraction with 0.005 M formic acid solution. This method is specific and linear over the concentration range of 1.00-60.00 ng/ml for mianserin and 0.50-14.00 ng/ml for N-desmethylmianserin in human plasma. The lowest limits of quantification (LLQ) is 1.00 ng/ml for mianserin and 0.50 ng/ml for N-desmethylmianserin. Intraday and interday precision (%C.V.) is <10% for both mianserin and N-desmethylmianserin. The accuracy ranges from 94.44 to 112.33% for mianserin and 91.85-100.13% for N-desmethylmianserin. The stability studies showed that mianserin and N-desmethylmianserin in human plasma are stable during short-term period for sample preparation and analysis. The method was used to assay mianserin and its metabolite, N-desmethylmianserin, in human plasma samples obtained from subjects who had been given an oral tablet of 30 mg of mianserin.     

Variations in leucocyte count during menstrual cycle.

The neutrophil counts of seven women, three tkaing oral contraceptives and four nottaking them, showed cyclical variations during the menstrual cycle, most consistentlya fall in the neutrophil count at menstuation. The neutrophil count in women not taking oral contraceptives rose to a peak twice during each cycle. One womennot taking oral contraceptives was studied in detail over eight consecutive menstrualcycles. She showed two neutrophil peaks per cycle and a similiar variation in themonocyte count. The eosinophil count showed a reciprocal relation with the neutrophil count and the basophil count fell in mid-cycle. The changes in her neutrophil count seemed to follow changes in oestrogen level with a delay of one to two days. Oestrogen probably promotes release of neutrophils from the bone marrow rather than from the marginated pool.     

Vancomycin-induced neutropenia.

Although rare, neutropenia associated with long-term vancomycin therapy may occur. A 67-year-old woman with cellulitis and sepsis caused by Staphylococcus aureus was treated initially with cefazolin. Despite in-vitro susceptibility of the organism to this drug, the patient remained febrile, and therapy was changed to vancomycin. On day 17 of therapy with this medication neutropenia was noted; it progressed over the next 3 days, and therapy with the drug was stopped. A rise in the neutrophil count occurred within 5 days of discontinuation. Periodic monitoring of the leukocyte count during longterm vancomycin therapy is recommended.     

Predicting drug-induced agranulocytosis: characterizing neutrophil-generated metabolites of a model compound,DMP 406, and assessing the relevance of an in vitro apoptosis assay for identifying drugs that may cause agranulocytosis

DMP 406 is a clozapine analogue developed by Dupont-Pharma for the treatment of schizophrenia. Unfortunately it caused agranulocytosis in dogs during preclinical studies. Clozapine also causes agranulocytosis and this is believed to be due to a reactive nitrenium ion metabolite produced by neutrophils. We studied the oxidation of DMP 406 by activated neutrophils and found that the major reactive species that is produced is not a nitrenium ion but rather an imine. This metabolite is similar to the reactive metabolite that has been proposed to be responsible for mianserin-induced agranulocytosis. Therefore we also studied the oxidation of mianserin by activated neutrophils and found that, although the major species is an iminium ion, it also bears a lactam moiety in the piperazine ring resulting from further oxidation. We usually find that HOCl is a good model system for the production of reactive metabolites of drugs that are formed by activated neutrophils, but in the case of both DMP 406 and mianserin, the products produced were significantly different than those formed by activated neutrophils. In contrast, the combination of horseradish peroxidase and hydrogen peroxide (HRP/H(2)O(2)) formed a very similar pattern of products, and this system was used to produce sufficient quantities of metabolites to allow for identification. The reactive metabolites of both DMP 406 and mianserin reacted with a range of nucleophiles, but in many cases the reaction was reversible. The best nucleophile for trapping these reactive metabolites was cyanide. It has been demonstrated that the products of clozapine oxidation by HRP/H(2)O(2), presumably the nitrenium ion, induced apoptosis in neutrophils at therapeutic concentrations of clozapine. It has been suggested that this process is involved in the mechanism of clozapine-induced agranulocytosis. We tested DMP 406 and mianserin in this system to see if the ability of a reactive metabolite of a drug to cause apoptosis could predict the ability of that drug to cause agranulocytosis. We used clozapine as a positive control and we also tested olanzapine, a drug that forms a reactive metabolite similar to that of clozapine but is given at a lower dose and does not cause agranulocytosis. We found that DMP 406 did not increase apoptosis at concentrations below 50 microM, and although mianserin did increase apoptosis at 10 microM this is above the therapeutic concentration. Olanzapine caused an increase in apoptosis at the same concentration as clozapine (1 microM), but because its therapeutic concentration is lower, this concentration was above the pharmacological range. There was no increase in apoptosis with any drug in the absence of HRP/H(2)O(2). These results indicate that this assay is unable to reliably predict the ability of different types of drugs to cause agranulocytosis. This is not a surprising result given that different drugs may induce agranulocytosis by different mechanisms.     

Different effects of serotonin antagonists on 3H-mianserin and 3H-ketanserin recognition sites

In minces prepared from the frontal cortex of rats treated with ketanserin (10 mg/kg i.p.) or mianserin (5 mg/kg i.p.) twice daily for 21 days, the Vmax of the adenylate cyclase stimulated by NE (100 microM) is attenuated, suggesting that ketanserin and mianserin share with a number of antidepressants the ability to attenuate the adenylate cyclase stimulation by NE. Ketanserin, given with the above mentioned dose schedule for 7 consecutive days, reduced the Bmax of 5HT2 recognition sites but failed to change either the Bmax or the apparent Kd of H-mianserin binding. A significant decrease in the Bmax of 5HT2 binding sites is elicited also by a single injection of mianserin (1). This drug also down-regulates its own binding when given twice daily for 3 weeks. From this and other information (2,3), it is concluded that ketanserin and mianserin bind to distinct recognition sites. The possibility that 5HT2 and mianserin recognition sites are functionally related and that serotonergic synapses are modulated by multiple chemical signals might be considered.     

Putative serotonin reuptake inhibitor-induced spawning and parturition in freshwater bivalves is inhibited by mammalian 5-HT2 receptor antagonists

Selective serotonin reuptake inhibitors (SSRIs) can mimic the physiological actions of serotonin, and in bivalve molluscs they induce zebra mussel spawning and fingernail clam parturition. We have elucidated further the pharmacology of SSRI-induced spawning and part-urition by blocking these reproductive processes with two mammalian 5-HT(2) receptor antagonists, cyproheptadine and mianserin. These two antagonists were potent inhibitors of both spawning and parturition induced by the SSRIs fluvoxamine, fluoxetine, and zimelidine. In zebra mussels, both cyproheptadine and mianserin significantly blocked spawning induced by fluvoxamine and by zimelidine. In the fingernail clams Sphaerium spp., both cyproheptadine and mianserin blocked fluvoxamine-induced parturition. A possible mechanism of action for SSRI-induced spawning and parturition in bivalves is suggested.     

Simplified method for determination of the tetracyclic antidepressant mianserin in human plasma using gas chromatography with nitrogen detection

A simplified gas chromatographic method for determination of the antidepressant drug mianserin in human plasma is described. Application of a nitrogen-sensitive detector reduces the assay procedure to extraction, concentration and gas chromatographic determination. The method is suitable to determine mianserin in human plasma at the 1 ng/mol level on a routine basis. At the 20 ng/ml level the deviation of the mean from the true value and the relative standard deviation amount to 1.0% and 6.8%, respectively.     

Pharmacokinetic and pharmacodynamic modelling of the novel human granulocyte colony-stimulating factor derivative Maxy-G34 and pegfilgrastim in rats

Objectives:  This study aims to compare pharmacokinetics and pharmacodynamics of pegfilgrastim, a pharmaceutical recombinant human granulocyte colony-stimulating factor (rhG-CSF), with that of a newly developed reagent, Maxy-G34. This comparison was performed using rat experiments and biomathematical modelling of granulopoiesis.
Methods:  Healthy rats and those with cyclophosphamide-induced neutropenia were treated with either pegfilgrastim or Maxy-G34 under various schedules. Time courses of absolute neutrophil count (ANC) and G-CSF serum level were measured and we constructed a combined pharmacokinetic/pharmacodynamic model of both drugs. Neutropenic episodes were assessed by experimental data and model simulations.
Results:  Both Pegfilgrastim and Maxy-G34 showed strong dose-dependent efficacy in reducing neutropenic episodes. However, time courses of ANC and G-CSF serum levels were markedly different. The biomathematical model showed good agreement with these data. We estimated that differences between the two drugs could be explained by lower bioavailability and reduced elimination of Maxy-G34. Based on the data and model interpolations, we estimated that Maxy-G34 is superior in reducing neutropenic episodes. Also, we predicted that G-CSF administration 48 h after cyclophosphamide would be superior to its administration after 2 or 24 h, for both derivatives.
Conclusion:  Maxy-G34 is a highly potent drug for stimulation of neutrophil production in rats. By our modelling approach, we quantified differences between Maxy-G34 and pegfilgrastim, related to pharmacokinetic parameters. Model simulations can be used to estimate optimal dosing and timing options in the present preclinical rat model.     

Influence of somatostatin on peripheral leucocyte count and granulation tissue in man and rats.

Subcutaneous injection of synthetic protamin-zink-somatostatin completely prevents endotoxin-induced leucocytosis in normal rats. Piromen-induced elevated stab neutrophil, neutrophil and monocyte counts remain within the normal range during somatostatin administration. There is an inhibiting effect of synthetic protamin-zink-somatostatin on the wet weights of granulation tissue of cotton pellet granulomata, too. Incorporation of 35S-sulfate in sulfated mucopolysaccharides of granulation tissue in cotton pellet granulomata is not inhibited. Intravenous administration of synthetic linear somatostatin decreases stab neutrophil and neutrophil blood count in patients with acute bacterial leucocytosis. After the termination of somatostatin infusion a rebound phenomenon occurs. In healthy subjects lymphocyte count increases during somatostatin infusion. This effect can not be demonstrated in patients with bacterial leucocytosis.     

Effect of methiothepin on imipramine- or mianserin-induced subsensitivity of serotonergic receptors

Effect of methiothepin on imipramine- or mianserin-induced subsensitivity of serotonergic receptors was examined in rat brain. Treatment with either imipramine plus methiothepin or mianserin plus methiothepin for 4 days resulted in a significant decrease in [³H] 5-hydroxytryptamine ([³H] 5-HT) binding in synaptic membranes. The binding was not significantly decreased after treatment for this period of time with either methiothepin, imipramine or mianserin alone. It is suggested that the elevated intrasynaptic 5-HT levels could contribute to potentiation effect of methiothepin on imipramine- or mianserin-induced down regulation of serotonergic receptors.     

Letter: Limited licensure.

We examined the relationship of leukocyte colony-stimulating activity (CSA) in vitro to neutrophil count in vivo. Using a standard two-layer system, cultures of 10⁶ leukocytes were assayed for their ability to stimulate colony formation by human bone marrow colony-forming cells. The total leukocyte CSA per ml (TLCSA) of blood varied directly with the blood neutrophil count in a group of patients with a wide range in blood neutrophil count, and in two patients recovering from neutropenia in whom serial observations were made. In the latter two patients the rise in TLCSA did not antedate the rise in blood neutrophil count, suggesting that blood leukocyte colony-stimulating factor (CSF) per se probably has little biologic significance. However, release into the circulation of cells which generate CSF could be an important way of controlling the amount of CSF acting within the marrow. In one patient the CSA of dialyzed serum increased after the rise in TLCSA, while undialyzed serum contained no CSA.     

Time course of lung ischemia-reperfusion-induced ICAM-1 expression and its role in ischemia-reperfusion lung injury.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression is an important mechanism underlying ischemia-reperfusion (I/R) induced neutrophil activation and tissue injury in other organs. However, I/R of the lungs has not been shown to upregulate ICAM-1 expression. We determined the time course profile of lung I/R-induced ICAM-1 expression and assessed the role of ICAM-1 in mediating neutrophil sequestration, transmigration, and I/R injury in the isolated blood-perfused rat lungs. I/R had a biphasic effect on ICAM-1 expression, an early downregulation and a late-phase upregulation. Superoxide dismutase and neutrophil depletion prevented the early ICAM-1 downregulation. The late-phase ICAM-1 upregulation coincided with the I/R-induced increase in pulmonary microvascular leakage index. ICAM-1 monoclonal antibody (MAb) reversed the I/R-induced increase in pulmonary microvascular leakage index, with control antibody being ineffective. Neither I/R nor ICAM-1 MAb affected lung MPO activity and circulating neutrophil count. Lung I/R significantly increased bronchoalveolar lavage fluid neutrophil count and the GSSG-to-(GSSG+GSH) ratio. ICAM-1 MAb blocked the I/R-induced increase in GSSG-to-(GSSG+GSH) ratio but had no effect on bronchoalveolar lavage fluid neutrophil count. Our results demonstrated that lung I/R up- and downregulates ICAM-1 expression depending on the duration of reperfusion. ICAM-1 upregulation is an important mechanism of I/R-induced pulmonary endothelial injury.     

Seasonal changeability in the leukocytic composition of blood in dogs]

The leukocyte blood composition was studied for seven years in dogs kept outdoors. The lymphocyte count proved to be higher from May to September than from November to March. Variations in the neutrophil and monocyte count were subject to reverse regularity. Changes in the total leukocyte count failed to coincide with the seasonal periodicity.     

Sputum mediator profiling and relationship to airway wall geometry imaging in severe asthma

Background

Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts.

Methods

In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses.

Results

Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing.

Conclusions

We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.     

Metabolism of Some “Second”– and “Fourth”–Generation Antidepressants: Iprindole, Viloxazine, Bupropion, Mianserin, Maprotiline, Trazodone, Nefazodone, and Venlafaxine

1. This review summarizes the major known aspects of the metabolism of second-generation (iprindole, viloxazine, bupropion, mianserin, maprotiline, and trazodone) and fourth-generation (nefazodone and venlafaxine) antidepressants.2. Discussions about specific enzymes involved and about possible pharmacokinetic drug–drug interactions, particularly as they relate to cytochrome P450 enzymes, are provided.     

Elimination of HIV in South Africa through Expanded Access to Antiretroviral Therapy: A Model Comparison Study

Background

Expanded access to antiretroviral therapy (ART) using universal test and treat (UTT) has been suggested as a strategy to eliminate HIV in South Africa within 7 y based on an influential mathematical modeling study. However, the underlying deterministic model was criticized widely, and other modeling studies did not always confirm the study''s finding. The objective of our study is to better understand the implications of different model structures and assumptions, so as to arrive at the best possible predictions of the long-term impact of UTT and the possibility of elimination of HIV.

Methods and Findings

We developed nine structurally different mathematical models of the South African HIV epidemic in a stepwise approach of increasing complexity and realism. The simplest model resembles the initial deterministic model, while the most comprehensive model is the stochastic microsimulation model STDSIM, which includes sexual networks and HIV stages with different degrees of infectiousness. We defined UTT as annual screening and immediate ART for all HIV-infected adults, starting at 13% in January 2012 and scaled up to 90% coverage by January 2019. All models predict elimination, yet those that capture more processes underlying the HIV transmission dynamics predict elimination at a later point in time, after 20 to 25 y. Importantly, the most comprehensive model predicts that the current strategy of ART at CD4 count ≤350 cells/µl will also lead to elimination, albeit 10 y later compared to UTT. Still, UTT remains cost-effective, as many additional life-years would be saved. The study''s major limitations are that elimination was defined as incidence below 1/1,000 person-years rather than 0% prevalence, and drug resistance was not modeled.

Conclusions

Our results confirm previous predictions that the HIV epidemic in South Africa can be eliminated through universal testing and immediate treatment at 90% coverage. However, more realistic models show that elimination is likely to occur at a much later point in time than the initial model suggested. Also, UTT is a cost-effective intervention, but less cost-effective than previously predicted because the current South African ART treatment policy alone could already drive HIV into elimination. Please see later in the article for the Editors'' Summary     

Treatment Effect and Drug-Resistant Mutations in Chinese AIDS Patients Switching to Second-Line Antiretroviral Therapy

This study aimed to investigate treatment effect, drug resistance changes, and their influencing factors in Chinese AIDS patients after switching to second-line antiretroviral therapy, and thus provide important information for the scale-up of second-line antiretroviral treatment in China. In Weishi county of Henan province, where second-line antiretroviral therapy was introduced early in China, 195 AIDS patients were enrolled, of which 127 patients met the switching criterion and 68 patients volunteered to switch drugs without meeting the switching criterion. CD4 cell count, viral load and in-house PCR genotyping for drug resistance were measured for all 195 subjects before drug switch, as well as 6 and 12 months after drug switch. Extensive secondary mutations to the protease inhibitor were observed, which suggested that long-term drug resistance surveillance is necessary for patients switching to second-line antiretroviral therapy. Multidrug resistance and cross-resistance were extensive in Chinese patients that experienced first-line treatment failure. Patients need timely CD4 count, viral load, and drug resistance monitoring in order to switch to second-line therapy under conditions of relatively good immunity and low viral duplication levels.     

Mianserin-Induced Down-Regulation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Stably Expressed in the Human Neuroblastoma Cell Line SH-SY5Y

Abstract: We have assessed the ability of the serotonergic antagonist mianserin to modulate the number and functional activity of human 5-hydroxytryptamine_2A (5-HT_2A) and 5-HT_2C receptors stably expressed in the human neuroblastoma cell line SH-SY5Y. Incubation of cells expressing the 5-HT_2A receptor with mianserin (100 n M ) for 24 h caused a significant decrease (48%) in the binding capacity of [³H]ketanserin. This receptor down-regulation was associated with a corresponding decrease in the maximal production of inositol phosphates induced by 5-HT but not by carbachol. Exposure of cells expressing the 5-HT_2C receptor to mianserin (100 n M ) for 72 h but not for 24 h similarly resulted in a significant reduction (44%) in [³H]mesulergine binding. Corresponding analysis of inositol phosphate production by 5-HT at the 5-HT_2C receptor after incubation with mianserin showed no change in maximal response after 24 h. No change in the binding capacity of either radioligand was seen after incubation with mianserin for 1 h. A decrease in the binding affinity of both radioligands was also observed after mianserin treatment, but this decrease was similar after 1 h of incubation to that seen after 24 or 72 h, and was probably due to the retention of mianserin within the tissue. We conclude that antagonist down-regulation is evident at human 5-HT_2A and 5-HT_2C receptors stably expressed in a human neuroblastoma cell line and is probably mediated by a direct action of mianserin at the receptor.     

Pharmacokinetic aspects of tert-butylaminoethyl disulfide, an experimental drug against schistosomiasis in mice

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethyl disulfide was performed after administration of two different single doses (35 and 300 mg/kg) of either the cold or labelled drug. Plasma or blood samples were treated with dithiothreitol, perchloric acid, and, after filtration, submitted to further purification with anionic resin. In the final step, the drug was retained on a cationic resin column, eluted with NaCl 1M and detected according to the method of Ellman (1958). Alternatively, radioactive drug was detected by liquid scintillation counting. The results corresponding to the smaller dose of total drug suggested a pharmacokinetic behavior related to a one open compartment model with the following parameters: area under the intravenous curve (AUCi.v.): 671 +/- 14; AUCoral: 150 +/- 40 micrograms.min.ml-1; elimination rate constant: 0.071 min-1; biological half life: 9.8 min; distribution volume: 0.74 ml/g. For the higher dose, the results seemed to obey a more complex undetermined model. Combining the results, the occurrence of a dose-dependent pharmacokinetic behavior is suggested, the drug being rapidly absorbed and rapidly eliminated; the elimination process being related mainly to metabolization. The drug seems to be more toxic when administered I.V. because by this route it escapes first pass metabolism, while being quickly distributed to tissues. The maximum tolerated blood level seems to be around 16 micrograms/ml.     

Unusual acute effects of antidepressants and neuroleptics on S2-serotonergic receptors

The antidepressants mianserin and amoxapine, and the neuroleptic loxapine caused significant decreases in the number of rat frontal cortex S2-serotonergic receptors after a single acute injection. The affinity of serotonin for this site was also decreased after acute mianserin. Daily injections of loxapine and amoxapine for 2, 7 or 28 days resulted in decreased receptor density but no change in Kd. Down-regulation of S2 sites by mianserin was not dependent on endogenous serotonin stores or occupation of the S2 recognition site since chronic PCPA or acute ketanserin preadministration did not affect the mianserin-induced decreases. The results suggest that mianserin may be acting on other sites which it does not share in common with other S2-antagonists such as ketanserin.