Association of inflammatory trajectory with subarachnoid hemorrhage mortality.

OBJECTIVE: White blood cells (WBC) play an important role in the inflammatory response of the body. Elevated WBC counts on admission in patients with subarachnoid hemorrhage (SAH) correlate with a poor prognosis. However, the role of longitudinal WBC trajectories based on repeated WBC measurements during hospitalization remains unclear. We explored the association between different WBC trajectory patterns and in-hospital mortality. METHODS: We analyzed a cohort of consecutive patients with SAH between 2012 and 2020. Group-based trajectory modeling (GBTM) was used to group the patients according to their white blood cell patterns over the first 4 days. Stabilized inverse probability treatment weighting (sIPTW) was used to balance baseline demographic and clinical characteristics. We analyzed the association between the WBC trajectory groups and in-hospital mortality using a Cox proportional hazards model. RESULTS: In total, 506 patients with SAH were included in this retrospective cohort. The final model identified two distinct longitudinal WBC trajectories. After adjusting for confounding factors, multivariate regression analysis suggested that an elevated longitudinal WBC trajectory increased the risk of in-hospital mortality (hazard ratio [HR], 2.476; 95% confidence interval [CI] 1.081-5.227; P = 0.024) before sIPTW, and (HR, 2.472; 95%CI 1.489-4.977; P = 0.018) after sIPTW. CONCLUSION: In patients with SAH, different clinically relevant groups could be identified using WBC trajectory analysis. The WBC count trajectory-initially elevated and then decreased- may lead to an increased risk of in-hospital mortality following SAH.

Naples Prognostic Score Predicts 6-Month Outcomes in Patients with Severe Traumatic Brain Injury: A Single-Center Retrospective Study.

AIM: To examine how Naples prognostic score (NPS) relates to 6-month outcomes in patients with severe traumatic brain injury (STBI). MATERIAL AND METHODS: We retrospectively analyzed the clinical data of 94 patients with STBI between September 2018 and September 2021. Galizia?s method was used to calculate NPS, and patients were categorized as high (NPS > 3) or low (NPS?3) NPS according to their NPS scores based on receiver operating characteristic curve analysis. In addition, the controlling nutritional status score (CONUT) and prognostic nutrition index (PNI) were calculated. Based on the modified Rankin scale (mRS), the outcome for 6-months was evaluated. The mRS score for unfavorable outcomes was ?3. RESULTS: In the univariate analyses, patients in the unfavorable group had higher NPS scores (p < 0.001). The multivariate analysis demonstrated that NPS was an independent predictor of poor outcomes after adjusting for potential confounding factors (adjusted odds ratio = 7.463, 95% confidence interval [CI]: 1.131?49.253, p < 0.05). The area under the NPS curve for predicting poor outcomes was 0.755 (95% CI: 0.655?0.837, p < 0.001), which was significantly higher than Glasgow coma score (GCS), CONUT, and PNI (NPS vs. GCS, p=0.013; NPS vs. CONUT, p=0.029; NPS vs. PNI, p=0.015). CONCLUSION: NPS can be considered to be a novel and better independent predictor of poor outcomes in patients with STBI.

Comparison of a covered stent and pipeline embolization device in intracranial aneurysm: a propensity score matching analysis.

BACKGROUND: The Willis covered stent (WCS) and pipeline embolization device (PED) have partly overlapping therapeutic indications. However, the differences of effect between these two treatments remain unclear. OBJECTIVE: To compare clinical outcome, angiographic outcome, and complications following treatment with a WCS versus PED. METHODS: Patients with intracranial aneurysms treated by a WCS or PED between January 2015 and December 2020 were included. The primary outcomes were complications, clinical outcome (modified Rankin Scale score >2), and angiographic outcome (incomplete aneurysm occlusion). Propensity score matching was conducted to adjust for potential confounding factors. RESULTS: A total of 94 aneurysms treated by WCS and 698 aneurysms by PED were included. Compared with the PED group, patients in the WCS group are younger, a greater number have a poor condition at admission, a larger proportion of ruptured, non-saccular, and anterior circulation aneurysms, a smaller aneurysm neck width, and less coiling assistance is required. A total of 42 (44.7%) branches were covered by WCS. After adjustment for age, sex, aneurysm type, rupture status, neck size, aneurysm location, and coiling, 50 WCS and PED pairs were examined for internal carotid artery aneurysms. No significant differences were observed in clinical (10.4% vs 2.1%, P=0.206) and angiographic outcomes (12.8% vs 18.2%, P=0.713). However, 27 branches covered by WCS, including 22 ophthalmic arteries and five posterior communicating arteries. Patients in the WCS group had a higher intraoperative complication rate than those in the PED group (28% vs 6%, P=0.008), especially in the occlusion rate of covered branches (51.9% vs 11.1%, P<0.001). CONCLUSION: The comparable clinical and angiographic outcomes of WCS or PED demonstrate the therapeutic potential of WCS as a viable alternative for aneurysms. However, the complication of occlusion of covered branches might not be negligible.

Procalcitonin/Albumin Ratio Predicts the Outcome After Severe Traumatic Brain Injury: A Propensity Score-Matched Analysis.

BACKGROUND: The procalcitonin/albumin ratio (PAR), a novel inflammation-based index, has been reported to predict the prognosis following cardiopulmonary bypass surgery and bacterial infection. However, whether PAR can predict the outcome of patients with severe traumatic brain injury (STBI) has not been fully elucidated. This study aimed to investigate the relationship between serum PAR levels and prognosis at 6 months after STBI. METHODS: We retrospectively enrolled 129 patients diagnosed with STBI and collected relevant clinical and laboratory data. Logistic regression analysis was used to estimate the association of PAR with the prognosis of STBI. The receiver operating characteristics curve was performed to examine the predictive use of PAR for prognosis. Propensity score matching (PSM) analysis was also performed to improve the reliability of the results. The primary outcome measures were expressed as a score on the modified Rankin Scale at 6 months. RESULTS: The unfavorable prognosis group had advanced age, lower Glasgow Coma Scale score, higher rate of cerebral hernia and intracranial infection, higher neutrophil/lymphocyte ratio (NLR) and C-reactive protein/albumin ratio (CAR), elevated PAR, and higher rate of pneumonia. Multivariate analysis showed that PAR (before PSM: odds ratio 3.473, 95% confidence interval 2.983-4.043, P < 0.001; after PSM: odds ratio 5.358, 95% confidence interval 3.689-6.491, P < 0.001) was independently associated with unfavorable outcome. The area under the curve of the PAR for predicting an unfavorable outcome was higher than that of the CAR and NLR. CONCLUSIONS: The PAR might be a novel independent risk factor of the outcome after STBI. Moreover, PAR was a better biomarker in predicting the outcome of patients with STBI than CAR and NLR.

Microglial pyroptosis: Therapeutic target in secondary brain injury following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a major cerebrovascular illness that causes substantial neurological sequelae and dysfunction caused by secondary brain injury (SBI), and there are no effective therapies to mitigate the disability. Microglia, the brain-resident macrophage, participates in the primary inflammatory response, and activation of microglia to an M1-like phenotype largely takes place in the acute phase following ICH. A growing body of research suggests that the pathophysiology of SBI after ICH is mediated by an inflammatory response mediated by microglial-pyroptotic inflammasomes, while inhibiting the activation of microglial pyroptosis could suppress the inflammatory cascade reaction, thus attenuating the brain injury after ICH. Pyroptosis is characterized by rapid plasma membrane disruption, followed by the release of cellular contents and pro-inflammatory mediators. In this review, we outline the molecular mechanism of microglial pyroptosis and summarize the up-to-date evidence of its involvement in the pathological process of ICH, and highlight microglial pyroptosis-targeted strategies that have the potential to cure intracerebral hemorrhage. This review contributes to a better understanding of the function of microglial pyroptosis in ICH and assesses it as a possible therapeutic target.

Activation of RKIP Binding ASC Attenuates Neuronal Pyroptosis and Brain Injury via Caspase-1/GSDMD Signaling Pathway After Intracerebral Hemorrhage in Mice.

Pyroptosis has been proven to be responsible for secondary brain injury after intracerebral hemorrhage (ICH). A recent study reported that Raf kinase inhibitor protein (RKIP) inhibited assembly and activation of inflammasome in macrophages. Our present study aimed to investigate the effects of RKIP on inflammasome-mediated neuronal pyroptosis and underlying neuroprotective mechanisms in experimental ICH. Here, we showed that RKIP expression was decreased both in cerebrospinal fluid (CSF) samples from patients with ICH and in the peri-hematoma tissues after experimental ICH. In mouse ICH model, activation of RKIP remarkably improved neurological deficits, reduced brain water content and BBB disruption, and promoted hematoma absorption at 24 h after ICH, as well as alleviated neuronal degeneration, reduced membrane pore formation, and downregulated pyroptotic molecules NLRP3, caspase-1 P20, GSDMD-N, and mature IL-1ß. Besides, RKIP activation decreased the number of caspase-1 P20-positive neurons after ICH. However, RKIP inhibitor reserved the neuroprotective effects of RKIP at 24 h following ICH. Moreover, RKIP could bind with ASC, then interrupt the assembly of NLRP3 inflammasome. Mechanistically, inhibiting the caspase-1 by VX-765 attenuated brain injury and suppressed neuronal pyroptosis after RKIP inhibitor-pretreated ICH. In conclusion, our findings indicated that activation of RKIP could attenuate neuronal pyroptosis and brain injury after ICH, to some extent, through ASC/Caspase-1/GSDMD pathway. Thus, RKIP may be a potential target to attenuate brain injury via its anti-pyroptosis effect after ICH.

Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage.

Neuroinflammation has been proven to exert an important effect on brain injury after intracerebral hemorrhage (ICH). Previous studies reported that Didymin possessed anti-inflammatory properties after acute hepatic injury, hyperglycemia-induced endothelial dysfunction, and death. However, the role of Didymin in microglial pyroptosis and neuroinflammation after ICH is unclear. The current study aimed to investigate the effect of Didymin on neuroinflammation mediated by microglial pyroptosis in mouse models of ICH and shed some light on the underlying mechanisms. In this study, we observed that Didymin treatment remarkably improved neurobehavioral performance and decreased BBB disruption and brain water content. Microglial activation and neutrophil infiltration in the peri-hematoma tissue after ICH were strikingly mitigated by Didymin as well. At the molecular level, administration of Didymin significantly unregulated the expression of Rkip and downregulated the expression of pyroptotic molecules and inflammatory cytokines such as Nlrp3 inflammasome, GSDMD, caspase-1, and mature IL-1ß, TNF-α, and MPO after ICH. Besides, Didymin treatment decreased the number of Caspase-1-positive microglia and GSDMD-positive microglia after ICH. Inversely, Locostatin, an Rkip-specific inhibitor, significantly abolished the anti-pyroptosis and anti-neuroinflammation effects of Didymin. Moreover, Rkip binding with Asc could interrupt the activation and assembly of the inflammasome. Mechanistically, inhibition of Caspase-1 by VX-765 attenuated brain injury and suppressed microglial pyroptosis and neuroinflammation by downregulation of GSDMD, mature IL-1ß, TNF-α, and MPO based on Locostatin-treated ICH. Taken together, Didymin alleviated microglial pyroptosis and neuroinflammation, at least in part through the Asc/Caspase-1/GSDMD pathway via upregulating Rkip expression after ICH. Therefore, Didymin may be a potential agent to attenuate neuroinflammation via its anti-pyroptosis effect after ICH.

Systemic immune-inflammation index predicts the outcome after aneurysmal subarachnoid hemorrhage.

Systemic inflammatory response is closely related to the pathogenesis and prognosis in critical patients. Recently, systemic immune-inflammation index (SII), an indicator of systemic inflammatory response, was proved to predict the outcome in cancerous and non-cancerous diseases. The aim of this study is to investigate the association between SII on admission and 6-month outcome in patients with aneurysmal subarachnoid hemorrhage (aSAH). The clinical data and prognosis of 76 patients with aSAH were analyzed. The 6-month outcome was assessed by the modified Rankin scale(mRS). The unfavorable outcome was defined as mRS score ≥ 3. In addition, multivariate analysis was conducted to investigate factors independently associated with the favorable outcome. Receiver operating characteristic (ROC) curve analysis was undertaken to identify the best cut-off value of SII for the discriminate between favorable and unfavorable outcome in these patients. Thirty-six patients (47.4%) in our study had an unfavorable outcome (mRS ≥ 3) at 6 months, and twenty-four (66.7%) of them were in the high-SII group. A significantly higher SII on admission was observed in patients with unfavorable functional outcome at 6 months. Binary logistic regression analysis showed that there was an independent association between SII on admission and 6-month clinical outcome (adjusted OR = 4.499, 95%CI: 1.242-16.295, P < 0.05). The AUC of the SII for predicting unfavorable outcome was 0.692 (95% CI: 0.571-0.814, P < 0.05). Systemic immune-inflammation index (SII) could be a novel independent prognostic factor for aSAH patients at the early stage of the disease.

Co-administration of dual-targeting nanoparticles with penetration enhancement peptide for antiglioblastoma therapy.

Chemotherapy is an indispensable auxiliary treatment for glioma but highly limited by the existence of both blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). The dysfunctional brain tumor blood vessels and high interstitial pressure in glioma also greatly hindered the accumulation and deep penetration of chemotherapeutics into the glioma. Lactoferrin (Lf), with its receptor overexpressed on both the brain endothelial cells and glioma cells, was here functionalized to the surface of poly(ethylene glycol)-poly(lactic acid) nanoparticles to mediate BBB/BBTB and glioma cell dual targeting. tLyP-1, a tumor-homing peptide, which contains a C-end Rule sequence that can mediate tissue penetration through the neuropilin-1-dependent internalization pathway, was coadministrated with Lf-functionalized nanoparticles (Lf-NP) to enhance its accumulation and deep penetration into the glioma parenchyma. Enhanced cellular association in both BCEC and C6 cells, increased cytotoxicity of the loaded paclitaxel, and deep penetration in the 3D glioma spheroids was achieved by Lf-NP. Following coadministration with tLyP-1, the functionalized nanoparticles obtained improved tumor targeting, glioma vascular extravasation, and antiglioma efficacy. The findings here suggested that the strategy by coadministrating BBB/BBTB and glioma cells dual-targeting nanocarriers with a tumor penetration enhancement peptide represent a promising platform for antiglioma drug delivery.

[Research of bornrol promote drugs through blood-brain barrier].

Malignant tumor, epilepsy, dementia, cerebral ischemia and other brain diseases have very high rates of disability and mortality. Currently, many drugs are developed to treat such diseases and the effect is obviously. But they can not achieve the purpose to control these diseases because many of the drugs can not pass through the blood-brain barrier (BBB). Therefore, the treatment is not good. Borneol as the represent of the aromatic resuscitation medicine, it has strong fat-soluble active ingredients, small molecular weight, volatile and through the BBB quickly. It can also promote other therapeutic drugs through the BBB. It has two-ways regulations on BBB permeability and the damage of brain tissue is small, this have important theoretical significances and application values.

Analytical characterization of an orally-delivered peptide pharmaceutical product.

The characterization of orally-delivered peptide pharmaceuticals presents several challenges to analytical methods in comparison to characterization of conventional small-molecule drugs. These challenges include the analysis and characterization of difficult-to-separate impurities, secondary structure, the amorphous solid-state form, and the integrity of enteric-coated drug delivery systems. This work presents the multidisciplinary analytical characterization of a parathyroid hormone (PTH) peptide active pharmaceutical ingredient (API) and an oral formulation of this API within enteric-coated sucrose spheres. The analysis of impurities and degradation products in API and formulated drug product was facilitated by the development of an ultrahigh-performance liquid chromatography (UHPLC) method for analysis by high-resolution mass spectrometry (MS). The use of UHPLC allowed for additional resolution needed to detect impurities and degradation products of interest. The secondary structure was probed using a combination of solution-state NMR, infrared, and circular dichroism spectroscopic methods. Solid-state NMR is used to detect amorphous API in a nondestructive manner directly within the coated sucrose sphere formulation. Fluorescence and Raman microscopy were used in conjunction with Raman mapping to show enteric coating integrity and observe the distribution of API beneath the enteric-coating on the sucrose spheres. The methods are combined in a multidisciplinary approach to characterize the quality of the enteric-coated peptide product.

Eliminating pharmaceutical impurities: Recent advances in detection techniques.

The elimination of organic impurities to produce highly pure drug substances is an important goal of process chemistry. For the detection of general impurities, hyphenated techniques (eg, liquid chromatography-mass spectrometry [LC-MS]) play a critical role in rapid structural identification (qualitative detection) and in understanding the mechanisms of formation of the impurities, enabling informed decisions to control and eliminate the impurities resulting from the chemical process where possible. Concern regarding genotoxic impurities (GTIs), which must typically be controlled at low parts-per-million limits, continues to increase, and significant advances have been achieved in recent years for the selective and sensitive quantitation (quantitative detection) of such impurities. Conventional detection techniques, such as ultraviolet (UV) detection, are often inadequate for the detection of potentially minute quantities of GTIs; therefore, various advanced MS-based detection strategies, either stand-alone or in conjunction with chemical approaches, are playing an increasing role in this field. The primary aim of this review is to highlight recent advances in qualitative and quantitative detection of impurities at trace levels, with a particular focus on GTIs.

Enhancing the detection sensitivity of trace analysis of pharmaceutical genotoxic impurities by chemical derivatization and coordination ion spray-mass spectrometry.

Many pharmaceutical genotoxic impurities are neutral molecules. Trace level analysis of these neutral analytes is hampered by their poor ionization efficiency in mass spectrometry (MS). Two analytical approaches including chemical derivatization and coordination ion spray-MS were developed to enhance neutral analyte detection sensitivity. The chemical derivatization approach converts analytes into highly ionizable or permanently charged derivatives, which become readily detectable by MS. The coordination ion spray-MS method, on the other hand, improves ionization by forming neutral-ion adducts with metal ions such as Na(+), K(+), or NH(4)(+) which are introduced into the electrospray ionization source. Both approaches have been proven to be able to enhance the detection sensitivity of neutral pharmaceuticals dramatically. This article demonstrates the successful applications of the two approaches in the analysis of four pharmaceutical genotoxic impurities identified in a single drug development program, of which two are non-volatile alkyl chlorides and the other two are epoxides.

Matrix deactivation: A general approach to improve stability of unstable and reactive pharmaceutical genotoxic impurities for trace analysis.

Trace analysis of unstable and reactive pharmaceutical genotoxic impurities (GTIs) is a challenging task in pharmaceutical analysis. Many method issues such as insufficient sensitivity, poor precision, and unusual (too high/low) spiking recovery are often directly related to analytes' instability. We report herein a matrix deactivation approach that chemically stabilizes these analytes for analytical method development. In contrast to the conventional chemical derivatization approach where the analytes are transformed into stable detectable species, the matrix deactivation approach chemically deactivates the hypothetical reactive species in the sample matrix. The matrix deactivation approach was developed on the premise that the instability of certain analytes at trace level is caused by reactions between the analytes and low level reactive species in the sample matrix. Thus, quenching the reactivity of the reactive species would be a key to stabilizing the unstable and reactive analytes. For example, electrophilic alkylators could be destabilized by nucleophiles or bases through either nucleophilic substitution or elimination reactions. One way to mask those reactive species is via protonation by adding acids to the diluent. Alternatively, one can use nucleophile scavengers to deplete reactive unknown species in the sample matrix completely, in analogy to the use of antioxidants and metal chelators to prevent oxidation in the analysis of compounds liable to oxidation. This paper reports the application of the matrix deactivation to the analyses of unstable and reactive pharmaceutical genotoxic impurities. Some of the methods have been used to support development of manufacturing processes for drug substances and a recent regulatory filing.

Recent advances in trace analysis of pharmaceutical genotoxic impurities.

Genotoxic impurities (GTIs) in pharmaceuticals at trace levels are of increasing concerns to both pharmaceutical industries and regulatory agencies due to their potentials for human carcinogenesis. Determination of these impurities at ppm levels requires highly sensitive analytical methodologies, which poses tremendous challenges on analytical communities in pharmaceutical R&D. Practical guidance with respect to the analytical determination of diverse classes of GTIs is currently lacking in the literature. This article provides an industrial perspective with regard to the analysis of various structural classes of GTIs that are commonly encountered during chemical development. The recent literatures will be reviewed, and several practical approaches for enhancing analyte detectability developed in recent years will be highlighted. As such, this article is organized into the following main sections: (1) trace analysis toolbox including sample introduction, separation, and detection techniques, as well as several 'general' approaches for enhancing detectability; (2) method development: chemical structure and property-based approaches; (3) method validation considerations; and (4) testing and control strategies in process chemistry. The general approaches for enhancing detection sensitivity to be discussed include chemical derivatization, 'matrix deactivation', and 'coordination ion spray-mass spectrometry'. Leveraging the use of these general approaches in method development greatly facilitates the analysis of poorly detectable or unstable/reactive GTIs. It is the authors' intent to provide a contemporary perspective on method development and validation that can guide analytical scientists in the pharmaceutical industries.

A generic approach for the determination of trace hydrazine in drug substances using in situ derivatization-headspace GC-MS.

In situ derivatization-headspace GC-MS methodology has been developed for the determination of hydrazine in drug substance at low ppm levels. This general method uses acetone or acetone-d(6) as the derivatization reagent. The resulting acetone azine or acetone azine-d(12) can then be analyzed by headspace GC-MS. The method gives excellent sensitivity with a limit of quantitation (LOQ) as low as 0.1ppm when the API (active pharmaceutical ingredient) samples are prepared at 10mg per headspace injection vial. The spike recoveries of hydrazine at the 1ppm level were between 79% and 117% in various APIs tested. The precisions (%RSD) of six preparations of the hydrazine standards at the concentration of 1ppm level were typically between 2.7 and 5.6%. A linear range of concentrations from 0.1 to 10ppm has been demonstrated with R(2)> or =0.999. This general method has been tested in a number of API matrices and successfully applied to the determination of hydrazine in support of API batch releases and process chemistry at GlaxoSmithKline.

A practical derivatization LC/MS approach for determination of trace level alkyl sulfonates and dialkyl sulfates genotoxic impurities in drug substances.

Derivatization LC/MS methodology has been developed for the determination of a group of commonly encountered alkyl esters of sulfonates or sulfates in drug substances at low ppm levels. This general method uses trimethylamine as the derivatizing reagent for ethyl/propyl/isopropyl esters and triethylamine for methyl esters. The resulting quaternary ammonium derivatization products are highly polar (ionic) and can be retained by a hydrophilic interaction liquid chromatography (HILIC) column and readily separated from the main interfering active pharmaceutical ingredient (API) peak that is usually present at very high concentration. The method gives excellent sensitivity for all the alkyl esters at typical target analyte level of 1-2 ppm when the API samples were prepared at 5mg/mL. The recoveries at 1-2 ppm were generally above 85% for all the alkyl esters in the various APIs tested. The injection precisions of the lowest concentration standards were excellent with R.S.D.=0.4-4%. A linear range for concentrations from 0.2 to 20 ppm has been established with R(2)>or=0.99. This general method has been tested in a number of API matrices and used successfully for determination of alkyl sulfonates or dialkyl sulfates in support of API batch releases at GlaxoSmithKline.

Fragmentation of aromatic sulfonamides in electrospray ionization mass spectrometry: elimination of SO(2) via rearrangement.

Arylsulfonamides are attractive pharmacophores for drug candidates. Fragmentation behaviors of selected aromatic sulfonamides were investigated using electrospray ionization mass spectrometry in the positive ion mode. Some of the sulfonamides afforded unique loss of 64 (loss of SO(2)) ions upon collision-induced dissociation followed by intramolecular rearrangements in the gas phase. This SO(2) elimination-rearrangement pathway leading to the generation of [M + H - SO(2)](+) ions appeared to be susceptible to substitutions on the aromatic (Ar) ring that would affect the Ar--sulfur bond strength and the stability of the partially positive charge developed at the ipso position upon bond dissociation. Electron withdrawing groups such as chlorine attached to the aromatic ring at ortho position seem to promote the SO(2) extrusion. Although this fragmentation pathway in atmospheric pressure ionization MS is less predictable than in electron impact MS, it is a frequently encountered reaction. The absence of this fragmentation pathway in some of the arylsulfonamides indicates that other factors such as nucleophilicity of the nitrogen may also play a role in the process. With respect to the site of attachment of the migrating NR'R'', ipso-substitution on the aromatic ring is evident since this fragmentation mechanism is operative in the fully ortho-substituted arylsulfonamides.

Platelet CD36 links hyperlipidemia, oxidant stress and a prothrombotic phenotype.

Dyslipidemia is associated with a prothrombotic phenotype; however, the mechanisms responsible for enhanced platelet reactivity remain unclear. Proatherosclerotic lipid abnormalities are associated with both enhanced oxidant stress and the generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet glycoprotein. Using multiple mouse in vivo thrombosis models, we now demonstrate that genetic deletion of Cd36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and the accompanying prothrombotic phenotype. Structurally defined oxidized choline glycerophospholipids that serve as high-affinity ligands for CD36 were at markedly increased levels in the plasma of hyperlipidemic mice and in the plasma of humans with low HDL levels, were able to bind platelets via CD36 and, at pathophysiological levels, promoted platelet activation via CD36. Thus, interactions of platelet CD36 with specific endogenous oxidized lipids play a crucial role in the well-known clinical associations between dyslipidemia, oxidant stress and a prothrombotic phenotype.

On-line H/D exchange LC-MS strategy for structural elucidation of pharmaceutical impurities.

Structural elucidation of pharmaceutical impurities in drug substances and drug products is an important task in pharmaceutical analysis in various phases of drug development. Liquid chromatography-mass spectrometry (LC-MS) technologies play a key role in this task owing to their general attributes of superior selectivity, sensitivity and speed. Full scan and product ion scan analysis, providing molecular weight information and fragmentation data, respectively, offer rich structural information and allow proposal of candidate structures rather quickly. However, these proposed structures often lack certainty especially when dealing with structural isomers. On-line hydrogen/deuterium (H/D) exchange by LC-MS using D2O as the mobile phase component is a powerful tool for identifying active hydrogen atoms, thus constituting a simple strategy for distinguishing between isomeric structures which are sometimes difficult by product ion spectral data or accurate mass data. This review describes the typical experimental setup we use routinely in the laboratories for performing H/D exchange LC-MS experiments in conjunction with representative applications of the strategy in structural elucidation of pharmaceutical impurities.