SPINK2 Protein Expression Is an Independent Adverse Prognostic Marker in AML and Is Potentially Implicated in the Regulation of Ferroptosis and Immune Response.

There is an urgent need for the identification as well as clinicopathological and functional characterization of potent prognostic biomarkers and therapeutic targets in acute myeloid leukemia (AML). Using immunohistochemistry and next-generation sequencing, we investigated the protein expression as well as clinicopathological and prognostic associations of serine protease inhibitor Kazal type 2 (SPINK2) in AML and examined its potential biological functions. High SPINK2 protein expression was an independent adverse biomarker for survival and an indicator of elevated therapy resistance and relapse risk. SPINK2 expression was associated with AML with an NPM1 mutation and an intermediate risk by cytogenetics and European LeukemiaNet (ELN) 2022 criteria. Furthermore, SPINK2 expression could refine the ELN2022prognostic stratification. Functionally, an RNA sequencing analysis uncovered a potential link of SPINK2 with ferroptosis and immune response. SPINK2 regulated the expression of certain P53 targets and ferroptosis-related genes, including SLC7A11 and STEAP3, and affected cystine uptake, intracellular iron levels and sensitivity to erastin, a specific ferroptosis inducer. Furthermore, SPINK2 inhibition consistently increased the expression of ALCAM, an immune response enhancer and promoter of T-cell activity. Additionally, we identified a potential small-molecule inhibitor of SPINK2, which requires further characterization. In summary, high SPINK2 protein expression was a potent adverse prognostic marker in AML and might represent a druggable target.

Macrocyclic Inhibitors of HGF-Activating Serine Proteases Overcome Resistance to Receptor Tyrosine Kinase Inhibitors and Block Lung Cancer Progression.

Hepatocyte growth factor (HGF), the ligand for the MET receptor tyrosine kinase, is a tumor-promoting factor that is abundant in the tumor microenvironment. Proteolytic activation of inactive pro-HGF by one or more of the serine endopeptidases matriptase, hepsin, and HGF activator is the rate-limiting step in HGF/MET signaling. Herein, we have rationally designed a novel class of side chain cyclized macrocyclic peptide inhibitors. The new series of cyclic tripeptides has superior metabolic stability and significantly improved pharmacokinetics in mice relative to the corresponding linear peptides. We identified the lead compound VD2173 that potently inhibits matriptase and hepsin, which was tested in parallel alongside the acyclic inhibitor ZFH7116 using both in vitro and in vivo models of lung cancer. We demonstrated that both compounds block pro-HGF activation, abrogate HGF-mediated wound healing, and overcome resistance to EGFR- and MET-targeted therapy in lung cancer models. Furthermore, VD2173 inhibited HGF-dependent growth of lung cancer tumors in mice.

Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma.

Although mass spectrometry-based plasma proteomics enables sensitive and large-scale discovery and validation of biomarkers for various diseases, its integrative application to hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) is not well investigated. Therefore, we analyzed albumin- and immunoglobulin G-depleted plasma samples from 148 and 60 patients with HCC and CCA, respectively, using liquid chromatography-tandem mass spectrometry. The algorithm used to measure the content of each protein was the percentage of exponentially modified protein abundance index. From 5320 proteins assayed in plasma, 53 and 25 biomarker candidates were identified for HCC and CCA, respectively. The abundance of six and two HCC markers particularly protruded in stage II and III, respectively, whereas plasma serine protease inhibitor was the sole marker the level of which steadily decreased with CCA progression. From a prognostic facet, we showed candidate markers and their cutoff levels for evaluating probability of tumor recurrence and patient survival period. Combination Kaplan-Meier models showed that HCC stage III or IV and both the content of alpha-2-HS-glycoprotein and apolipoprotein CIII <0.2% exhibited the poorest post-surgical recurrence-free and overall survivals. Furthermore, the content of afamin ≥0.2% played a significant role on the poor prognosis in patients with CCA. Our findings, taken together, characterized novel plasma biomarker signatures in dissecting tumor stages and post-surgical outcomes of HCC and CCA.

Development and analytic validation of a sandwich ELISA for the measurement of α1-proteinase inhibitor concentrations in serum and feces of common marmosets (Callithrix jacchus).

OBJECTIVE To develop and validate a sandwich ELISA for the measurement of α1-proteinase inhibitor (α1-PI) concentrations in serum and fecal samples obtained from common marmosets (Callithrix jacchus). SAMPLE Leftover serum (n = 42) and fecal (23) samples submitted for diagnostic testing; paired serum and fecal samples obtained from 30 common marmosets at 2 research colonies. PROCEDURES A sandwich ELISA was developed and analytically validated by determining the lower limit of detection, linearity, accuracy, precision, and reproducibility. Reference intervals for α1-PI concentrations in serum and feces of common marmosets were calculated. RESULTS The standard curve was generated for concentrations between 1 and 100 ng/mL. Mean ± SD observed-to-expected ratio for serial dilutions of serum and fecal samples was 117.1 ± 5.6% (range, 112.2% to 123.0%) and 106.1 ± 19.7% (range, 82.6% to 130.2%), respectively. Mean observed-to-expected ratio for spiking recovery of serum and fecal samples was 102.9 ± 12.1% (range, 86.8% to 115.8%) and 97.9 ± 19.0% (range, 83.0% to 125.1%), respectively. Reference interval for serum concentrations of α1-PI was 1,254 to 1,813 µg/mL, for 3-day mean fecal concentrations was 11.5 to 42.2 µg/g of feces, and for 3-day maximum fecal concentrations was 13.2 to 51.2 µg/g of feces. CONCLUSIONS AND CLINICAL RELEVANCE The ELISA was linear, accurate, precise, and reproducible for quantification of α1-PI concentrations in serum and feces of common marmosets. However, the ELISA had limited linearity and accuracy for spiking recovery of fecal samples.

Plasminogen activator inhibitor-1 activity and long-term outcome in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention: a prospective cohort study.

AIM: To determine the relationship between plasminogen activator inhibitor-1 (PAI-1) activity rise during the first 24 hours of ST-elevation myocardial infarction (STEMI) treatment and death after 5 years. METHODS: From May 1, 2009 to March 23, 2010, 87 STEMI patients treated with primary percutaneous coronary intervention (PCI) at the Sestre Milosrdnice University Hospital Center were consecutively enrolled in prospective single-center cohort study. PAI-1 activity was determined on admission and 24 hours later. The primary end-point was death after 5 years. The predictive value of PAI-1 activity variables as biomarkers of death was assessed using receiver operating characteristic (ROC) curve, independent predictors of death were assessed using multivariate Cox regression, and covariates independently related to higher PAI-1 activity rise were assessed using linear regression. RESULTS: Two patients died during the hospital treatment and 11 during the follow-up. PAI-1 activity rise had the largest area under curve (0.748) for predicting death rate (optimal cut-off point 3.7 U/mL, sensitivity 53.8%, specificity 90.5%). Patients with PAI-1 activity rise higher than 3.7 U/mL had significantly higher mortality (P<0.001). Kaplan-Meier survival curve diverged within the first year after STEMI. Independent predictors of death were PAI-1 rise and final Thrombolysis in Myocardial Infarction flow. PAI-1 activity rise was independently related to heart failure, thrombus aspiration, and body weight. CONCLUSION: PAI-1 activity rise higher than 3.7 U/mL is associated with higher 5-year death rate in STEMI patients treated with primary PCI.

A long-acting PAI-1 inhibitor reduces thrombus formation.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.

Kallistatin levels in HIV-infected patients and effects of statin therapy.

BACKGROUND: Kallistatin, a serine proteinase inhibitor, has vasodilatory and anti-inflammatory properties and is increased in other inflammatory conditions. We measured kallistatin in HIV for the first time, examined its relationship with inflammation, and determined if statin therapy affected levels. METHODS: Kallistatin levels were measured in subjects from a randomized, double-blinded, placebo-controlled trial. RESULTS: One hundred and thirty-five HIV-infected subjects were included. Kallistatin levels were 28.4 µg/mL at baseline and not affected by rosuvastatin. Levels were correlated with high-sensitivity C-reactive protein (hsCRP), interleukin-6, fibrinogen and insulin resistance. CONCLUSIONS: Kallistatin levels were correlated with some markers of systemic inflammation and should be further explored in the HIV population.

Elastase inhibitor AZD9668 treatment prevented progression of experimental abdominal aortic aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a particular form of arterial disease characterized by the dilation of the aortic wall and the presence of an intraluminal thrombus linked to a high proteolytic activity. The aim of this study was to investigate the effect of an elastase inhibitor (AZD9668 from AstraZeneca) on aneurysm progression. METHODS: For this purpose, we have used a rat model of elastase perfusion followed by repeated injection of Porphyromonas gingivalis (Pg), a weak periodontal pathogen recently reported to enhance AAA thrombus formation. Pg (1.10(7) colony-forming units) was injected through the jugular vein once a week for 4 weeks, and AZD9668, incorporated in the food, was delivered concomitantly. RESULTS: Our results show a beneficial effect of AZD9668 treatment on AAA progression and composition. The increased AAA diameter induced by Pg was significantly reduced by AZD9668 treatment. Histologic analyses allowed us to observe the persistence of a neutrophil-rich luminal thrombus associated with calcifications in Pg-injected rats and the presence of a healing process in the Pg/AZD9668-treated group. The enhanced concentrations of markers of neutrophil activation, cell-free DNA, and myeloperoxidase and elastase activity in Pg-injected rats were significantly reduced both in the conditioned medium of AAA tissue samples and in plasma of rats injected with Pg and treated with AZD9668. CONCLUSIONS: AZD9668 treatment could therefore constitute a new therapeutic tool for treatment of AAA.

Discovery of potential serum protein biomarkers for lymph node metastasis in oral cancer.

BACKGROUND: The purpose of our study was to identify serum protein biomarkers for node-positive oral squamous cell carcinoma (OSCC). Biomarkers indicating lymph node metastasis provides a valuable classification methodology to optimize treatment plans for patients with OSCC. METHODS: Quantitative serum proteomic analysis of OSCCs with either node-positive or node-negative disease was performed with tandem mass spectrometry and isobaric tagging for relative and absolute quantitation (iTRAQ). Immunoassays were used to validate a panel of candidate protein biomarkers and receiver operating characteristic (ROC) analysis was used to evaluate the performance of the candidate biomarkers. RESULTS: A total of 282 serum proteins were quantified between node-positive and node-negative OSCCs with the proteomic approach. Four candidate biomarkers, gelsolin, fibronectin, angiotensinogen, and haptoglobin, were validated in an independent group of patients with node-positive or node-negative OSCC. The best candidate biomarker, gelsolin, yielded a ROC value of 89% for node-positive OSCC, although the sample size for validation is relatively small. Fibronectin, gelsolin, and angiotensinogen were also found to be differentially expressed between cancer cell lines of node-positive and node-negative cancer origin. CONCLUSION: Our studies suggest that testing of serum protein biomarkers might help detect lymph node metastasis of oral cancer. Because of limited sample size in our studies, long-term longitudinal studies with large populations of individuals with oral cancer are needed to validate these potential biomarkers.

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay.

BACKGROUND: Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. METHODOLOGY/PRINCIPAL FINDINGS: Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman's rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). CONCLUSIONS/SIGNIFICANCE: Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system.

Association of inflammatory and hemostatic markers with stroke and thromboembolic events in atrial fibrillation: a systematic review and meta-analysis.

BACKGROUND: Atrial fibrillation (AF) increases the risk of stroke and thromboembolic events. Recently, biomarkers have been proposed as a practical tool to predict adverse outcomes in patients with AF. The prognostic value of inflammatory and hemostatic markers in AF has been widely studied; however, the results of previous studies have been inconclusive. METHODS: We conducted a systematic review and meta-analysis to evaluate the association of inflammatory and hemostatic markers with stroke and thromboembolic events in patients with AF. RESULTS: A total of 27 studies including 22,176 participants met our inclusion criteria for the systematic review. Our meta-analysis determined that elevated circulating plasminogen activator inhibitor-1 (PAI-1) and thrombin-antithrombin (TAT) were significantly associated with increased risk of stroke in patients with AF (standardized mean difference [SMD], 0.89; 95% confidence interval [CI], 0.20-1.59 and 1.43; 95% CI, 0.40-2.47, respectively). Higher levels of D-dimer were associated with increased subsequent thromboembolic event risk with a pooled hazard ratio of 2.90 (95% CI, 1.22-6.90) for cohort studies and an SMD of 0.93 (95% CI, 0.36-1.50) for case-control studies. There was also very limited evidence indicating that other biomarkers-such as interleukin-6, von Willebrand factor, P-selectin, and mean platelet volume-could predict adverse outcomes in AF. CONCLUSIONS: In conclusion, increased circulating PAI-1 and TAT levels were significantly associated with subsequent stroke in patients with AF, and high levels of D-dimer were associated with thromboembolic events in AF. Further epidemiologic studies are needed to accumulate more evidence on the prognostic role of inflammatory and hemostatic markers in AF.

Improved pharmacokinetic and biodistribution properties of the selective urokinase inhibitor PAI-2 (SerpinB2) by site-specific PEGylation: implications for drug delivery.

PURPOSE: Overexpression of the serine protease urokinase (uPA) is recognised as an important biomarker of metastatic disease and a druggable anticancer target. Plasminogen activator inhibitor type-2 (PAI-2/SerpinB2) is a specific uPA inhibitor with proven potential for use in targeted therapy. However, PAI-2 is rapidly cleared via the renal system which impairs tumor uptake and efficacy. Here we aimed to improve the pharmacological properties of PAI-2 by site-specific PEGylation. METHODS: Several cysteine to serine substitution mutants were generated for PEGylation with PEG-maleimide (size range 12-30 kDa) and the physico-chemical and biochemical properties of the PEG-PAI-2 conjugates characterised. Radiolabeled proteins were used for evaluation of blood clearance and tissue uptake profiles in an orthotopic breast tumor xenograft mouse model. RESULTS: PEGylation of the PAI-2(C161S) mutant gave a predominant mono-PEGylated-PAI-2 product (~90%) with full uPA inhibitory activity, despite a significant increase in hydrodynamic radius. Compared to un-PEGylated protein the plasma half-life and AUC for PEG20-PAI-2(C161S) were significantly increased. This translated to a 10-fold increase in tumor retention after 24 h compared to PAI-2(C161S), an effect not seen in non-target organs. CONCLUSIONS: Our data underscores the potential for PEG20-PAI-2(C161S) drug conjugates to be further developed as anti-uPA targeted therapeutics with enhanced tumor retention.

Low levels of circulating alpha-1 antitrypsin are associated with spontaneous abortions.

OBJECTIVE: Alpha-1 antitrypsin (AAT), a circulating anti-inflammatory molecule, rises four- to sixfold during acute phase responses and during pregnancy. AAT deficiency is linked with various pregnancy complications. The aim of this study is to determine plasma concentrations and activity of AAT and serum cytokine levels in blood samples from women undergoing spontaneous abortions as compared with elective abortions. METHODS: A prospective case-control study consisted of patients with sporadic abortions (n = 15), recurrent spontaneous abortions (n = 14) and healthy pregnancies going through elective terminations (n = 11). Circulating AAT and cytokine levels were determined before dilatation and curettage. RESULTS: AAT levels were lower in both recurrent and sporadic spontaneous abortion groups compared with healthy pregnancies (1.421 ± 0.08, 1.569 ± 0.14 and 3.224 ± 0.45 mg/ml, respectively, p < 0.001). Reduced AAT levels correlated with elevated proinflammatory cytokines. CONCLUSIONS: AAT levels in patients with either sporadic or recurrent spontaneous abortions were lower than normal pregnancies, and were associated with an inflammatory profile. Future studies should examine larger cohort groups, effects of earlier time-points and the influence of antithrombotic therapy in such patients who are diagnosed with relatively low levels of circulating AAT, in an effort to improve pregnancy outcomes.

Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

Alpha-1-antitrypsin acts as a preeclampsia-related protein: a proteomic study.

AIMS: To screen the preeclampsia-related protein by proteomics. METHODS: Proteomics was performed to identify differential protein expression profiles between normal full-term pregnancy, early-onset severe preeclampsia (ES-PE) or late-onset severe preeclampsia (LS-PE; n = 10 per group). Real-time quantitative PCR and immunohistochemistry were conducted to confirm the expression of α(1)-antitrypsin (α(1)-AT) in the decidual tissues of different subjects. ELISA was employed to detect the α(1)-AT content in the peripheral blood of 90 women (n = 30 per group). RESULTS: We successfully constructed two-dimensional electrophoresis maps of decidual tissues, and a total of 20 differentially expressed proteins were identified. The α(1)-AT expression was different among the three groups. The normal full-term pregnancy women expressed the most α(1)-AT, and the LS-PE women expressed the least amount of α(1)-AT. The difference in the α(1)-AT expression was consistent with the proteomics data. The peripheral α(1)-AT content was the highest in the normal full-term pregnancy group (1.85 ± 0.15 g/l), moderate in the ES-PE group (0.77 ± 0.14 g/l) and lowest in the LS-PE group (0.42 ± 0.07 g/l; p < 0.05). CONCLUSION: Using 2D PAGE, we identified twenty proteins with significantly altered expression in PE. These differentially expressed proteins include prevention protein, in which α(1)-AT is downregulated in PE.

Effects of rosiglitazone on the cardiovascular profile in postmenopausal women without diabetes mellitus: interplay of thiazolidinediones and hormone therapy.

OBJECTIVE: Thiazolidinediones have antiatherothrombotic effects on persons with diabetes. Hormone therapy among postmenopausal women has both positive and negative cardiovascular effects. However, the effects of rosiglitazone with or without concurrent long-term hormone therapy on the cardiovascular profile of nondiabetic postmenopausal women are unknown. METHODS: Thirty-eight nondiabetic postmenopausal women were enrolled in this double-blind and placebo-controlled study. Eighteen participants received 4 mg rosiglitazone, and 20 participants took placebo daily for 12 weeks. Global endothelial function and plasma biomarkers were measured. RESULTS: Baseline characteristics and parameters were similar between the groups. Rosiglitazone, but not placebo, significantly reduced leukocyte count and plasma levels of matrix metalloproteinase-9 and inhibited the elevation of plasma levels of plasminogen activator inhibitor-1 and tissue plasminogen activator (P < 0.05 for all). Most of the favorable effects provided by rosiglitazone were still present in participants with concurrent hormone therapy. Increased body weight and waist size as well as elevation of the plasma levels of total and low-density lipoprotein cholesterol were noted after rosiglitazone treatment among participants without concurrent hormone therapy. No significant change in the global endothelial function occurred in response to treatment in either group. CONCLUSIONS: Rosiglitazone treatment provided both protective and harmful cardiovascular effects in nondiabetic postmenopausal women. Concurrent hormone therapy resulted in the maintenance of the major beneficial effects while neutralizing the unfavorable effects of rosiglitazone.

α1-Antitrypsin low-density-lipoprotein serves as a marker of smoking-specific oxidative stress.

AIM: While smoking cessation (SC) leads to a reduction of cardiovascular events, atherogenic biomarkers specifically connected with cigarette smoking and SC are unknown. Circulating levels of oxidatively modified low-density-lipoprotein (LDL) are associated with a high risk of cardiovascular diseases. Recently, two novel, oxidatively modified LDL markers, serum amyloid A-LDL (SAA-LDL) and α1-antitrypsin-LDL (AT-LDL), were identified; however, the significance of SAA-LDL and AT-LDL as cardiovascular risk markers is unknown. METHODS: We carried out a cross-sectional study involving 243 patients, and determined serum levels of SAA-LDL and AT-LDL. RESULTS: Both serum levels of SAA-LDL and AT-LDL were significantly increased in current compared to non-current smokers. Stepwise regression analysis revealed that the current smoking status and duration of smoking were strong independent determinants of the AT-LDL level. In contrast, high-sensitivity C-reactive protein was the strongest determinant of the SAA-LDL level. In multiple logistic regression analysis, the current smoking status was most closely associated with the AT-LDL level. Successful SC employing a 12-week program significantly increased the body mass index and serum levels of obesity-related markers. Notably, successful SC significantly decreased levels of AT-LDL, but not those of SAA-LDL. CONCLUSIONS: The present study provides the first evidence for the distinct characteristics of two novel, oxidatively modified LDL markers, SAA-LDL and AT-LDL. In contrast to SAA-LDL, an inflammatory marker, AT-LDL serves as a marker of smoking-specific oxidative stress. These findings warrant further investigations to clarify if AT-LDL provides a key link between smoking and cardiovascular diseases.

The arginine mimicking ß-amino acid ß³hPhe(3-H2N-CH2) as S1 ligand in cyclotheonamide-based ß-tryptase inhibitors.

ß-Tryptase, a mast-cell specific serine protease with trypsin-like activity, has emerged in the last years as a promising novel therapeutic target in the field of allergic inflammation. Recently, we have developed a potent and selective ß-tryptase inhibitor based on the natural product cyclotheonamide E4 by implementing a basic P3 residue that addresses the determinants of the extended substrate specificity of ß-tryptase. To further improve the affinity/selectivity profile of this lead structure, we have now investigated ß-homo-3-aminomethylphenylalanine as S1 ligand. In contrast to the corresponding ß-homo amino acids derived from lysine or arginine, we demonstrate that this particular basic ß-homo amino acid is a privileged S1 ligand for the development of ß-tryptase inhibitors. Besides affinity, selectivity and reduced basicity, these novel cyclotheonamide E4 analogs show excellent stability in human plasma and serum.

Brain pharmacokinetics of two prolyl oligopeptidase inhibitors, JTP-4819 and KYP-2047, in the rat.

Prolyl oligopeptidase (PREP) inhibitors are potential drug candidates for the treatment of neurological disorders, but little is known about their ability to cross the blood-brain barrier and to reach the target site. This study characterizes brain pharmacokinetics of two potent PREP inhibitors, JTP-4819 and KYP-2047. Firstly, the in vitro permeability (P(app) ) of JTP-4819 and KYP-2047 through a bovine brain microvessel endothelial cell monolayer was assessed. Then, the in vivo brain/blood ratio was determined for the total brain and plasma concentrations and also for the unbound extracellular drug concentrations after a single dose (50 µmol/kg i.p.). KYP-2047 had a significantly higher P(app) than JTP-4819. In vivo, KYP-2047 had higher total and unbound brain/blood ratios. KYP-2047 was equally distributed between the cortex, hippocampus and striatum. In the case of JTP-4819, the unbound brain extracellular concentrations could not be readily predicted from the unbound blood levels, probably because of its poor membrane penetration properties. KYP-2047 displayed a better ability to reach the intracellularly located brain PREP, and it inhibited this enzyme more effectively than JTP-4819 after an equimolar single dose. In conclusion, KYP-2047 showed better brain penetration characteristics than JTP-4819 both in vitro and in vivo. KYP-2047 is a brain-penetrating, potent and long-acting PREP inhibitor; thus, it represents a convenient pharmacological tool for assessing the potential of PREP as a drug target.

Population pharmacokinetic analysis and dosing regimen optimization of aprotinin in neonates and young infants undergoing cardiopulmonary bypass.

The objective of this study was to determine an optimal dosing regimen for maintaining the therapeutic target range of aprotinin in neonates and young infants during cardiopulmonary bypass (CPB). A total of 27 patients scheduled for open heart surgery were enrolled. Aprotinin was administered a 25 000 KIU (kallikrein inhibition unit)/kg bolus before operation, a 35 000 KIU/kg for CPB circuit priming, and a 12 500 KIU/kg/hour continuous infusion intra- and immediate postoperative period. Blood samples were obtained at 12 time points per patient. Population pharmacokinetic modeling and Monte-Carlo simulations were used to optimize the aprotinin dosing regimen. No mortality or aprotinin-related complication was encountered. A CPB adjusted 2-compartment model best fit the data. Clearance was 687 mL/hour during CPB and 350 mL/hour pre- and post-CPB, and corresponding volumes of distribution were 1577 mL and 1352 mL, respectively. The simulations conducted showed that more than twice the dose administered in this study is required to maintain the target concentration of aprotinin. The pharmacokinetics of aprotinin appears to be affected more sensitively by CPB in neonates and young infants than in adults. Therefore, dosage adjustment considering these pharmacokinetic differences and the influence of CPB is needed in neonates and young infants.