Safety, tolerability, pharmacokinetics, and pharmacodynamics of BI 685509, a soluble guanylyl cyclase activator, in healthy volunteers: Results from two randomized controlled trials.

This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of BI 685509 after oral single rising doses (SRDs) or multiple rising doses (MRDs) in healthy volunteers. In the SRD trial (NCT02694354; February 29, 2016), within each of the three dose groups (DGs), six subjects received BI 685509 (1.0, 2.5, or 5.0 mg) and two received placebo (N = 24). In the MRD trial (NCT03116906; April 17, 2017), within each of the five DGs, nine subjects received BI 685509 (uptitrated to 1 mg once daily [qd; DG1], 2.5 mg twice daily [DG2], 5.0 mg qd [DG3]; 3.0 mg three times daily [tid; DG4] or 4.0 mg tid [DG5]) and three received placebo, for 14-17 days (N = 60). In the SRD trial, 7/24 subjects (29.2%) had ≥ 1 adverse event (AE), most frequently orthostatic dysregulation (n = 4). In the MRD trial, 26/45 subjects (57.8%) receiving BI 685509 had ≥ 1 AE, most frequently orthostatic dysregulation and fatigue (each n = 12). Tolerance development led to a marked decrease in orthostatic dysregulation events (DG3). BI 685509 was rapidly absorbed after oral administration, and exposure increased in a dose-proportional manner after single doses. Multiple dosing resulted in near-dose-proportional increase in exposure and limited accumulation. BI 685509 pharmacokinetics appeared linear with time; steady state occurred 3-5 days after each multiple-dosing period. Increased plasma cyclic guanosine monophosphate and decreased blood pressure followed by a compensatory increase in heart rate indicated target engagement. BI 685509 was generally well tolerated; orthostatic dysregulation may be appropriately countered by careful uptitration.

Aldosterone synthase inhibitor (BI 690517) therapy for people with diabetes and albuminuric chronic kidney disease: A multicentre, randomized, double-blind, placebo-controlled, Phase I trial.

AIM: This Phase I study evaluated the safety and early efficacy of an aldosterone synthase inhibitor (BI 690517) in people with diabetes and albuminuric chronic kidney disease. METHODS: Double-blind, placebo-controlled study (NCT03165240) at 40 sites across Europe. Eligible participants [estimated glomerular filtration rate ≥20 and <75 ml/min/1.73 m2; urine albumin/creatinine ratio (UACR) ≥200 and <3500 mg/g] were randomized 6:1 to receive once-daily oral BI 690517 3, 10 or 40 mg, or eplerenone 25-50 mg, or placebo, for 28 days. The primary endpoint was the proportion of participants with drug-related adverse events (AEs). Secondary endpoints included changes from baseline in the UACR. RESULTS: Fifty-eight participants were randomized and treated from 27 November 2017 to 16 April 2020 (BI 690517: 3 mg, n = 18; 10 mg, n = 13; 40 mg, n = 14; eplerenone, n = 4; placebo, n = 9) for 28 days. Eight (13.8%) participants experienced drug-related AEs [BI 690517: 3 mg (two of 18); 10 mg (four of 13); 40 mg (two of 14)], most frequently constipation [10 mg (one of 13); 40 mg (one of 14)] and hyperkalaemia [3 mg (one of 18); 10 mg (one of 13)]. Most AEs were mild to moderate; one participant experienced severe hyperkalaemia (serum potassium 6.9 mmol/L; BI 690517 10 mg). UACR responses [≥20% decrease from baseline (first morning void urine) after 28 days] were observed for 80.0% receiving BI 690517 40 mg (eight of 10) versus 37.5% receiving placebo (three of eight). Aldosterone levels were suppressed by BI 690517, but not eplerenone or placebo. CONCLUSIONS: BI 690517 was generally well tolerated, reduced plasma aldosterone and may decrease albuminuria in participants with diabetes and albuminuric chronic kidney disease.

Safety, tolerability, pharmacodynamics and pharmacokinetics of the soluble guanylyl cyclase activator BI 685509 in patients with diabetic kidney disease: A randomized trial.

AIMS: Albuminuria is associated with abnormalities in the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate pathway. We assessed safety and efficacy of the NO-independent sGC activator BI 685509 in patients with diabetic kidney disease and albuminuria. MATERIALS AND METHODS: In this Phase Ib trial (NCT03165227), we randomized patients with type 1 or 2 diabetes, estimated glomerular filtration rate (eGFR) 20-75 mL/min/1.73 m2 and urinary albumin:creatinine ratio (UACR) 200-3500 mg/g to oral BI 685509 (1 mg three times daily, n = 20; 3 mg once daily, n = 19; 3 mg three times daily, n = 20, after final titration) or placebo (n = 15) for 28 days. Changes from baseline in UACR in first morning void (UACRFMV ) and 10-hour (UACR10h ) urine (3 mg once daily/three times daily only) were assessed. RESULTS: Baseline median eGFR and UACR were 47.0 mL/min/1.73 m2 and 641.5 mg/g, respectively. Twelve patients had drug-related adverse events (AEs; 16.2%: BI 685509, n = 9; placebo, n = 3), most frequently hypotension (4.1%: BI 685509, n = 2; placebo, n = 1) and diarrhoea (2.7%: BI 685509, n = 2; placebo, n = 0). Four patients experienced AEs leading to study discontinuation (5.4%: BI 685509, n = 3; placebo, n = 1). Placebo-corrected mean UACRFMV decreased from baseline in the 3-mg once-daily (28.8%, P = 0.23) and three-times-daily groups (10.2%, P = 0.71) and increased in the 1-mg three-times-daily group (6.6%, P = 0.82); changes were not significant. UACR10h decreased by 35.3% (3 mg once daily, P = 0.34) and 56.7% (3 mg three times daily, P = 0.09); ≥50.0% of patients (UACR10h 3 mg once daily/three times daily) responded (≥20% UACR decrease from baseline). CONCLUSIONS: BI 685509 was generally well tolerated. Effects on UACR lowering merit further investigation.

Perspectives on a Way Forward to Implementation of Precision Medicine in Patients With Diabetic Kidney Disease; Results of a Stakeholder Consensus-Building Meeting.

Aim: This study aimed to identify from different stakeholders the benefits and obstacles of implementing precision medicine in diabetic kidney disease (DKD) and to build consensus about a way forward in order to treat, prevent, or even reverse this disease. Methods: As part of an ongoing effort of moving implementation of precision medicine in DKD forward, a two-day consensus-building meeting was organized with different stakeholders involved in drug development and patient care in DKD, including patients, patient representatives, pharmaceutical industry, regulatory agencies representatives, health technology assessors, healthcare professionals, basic scientists, and clinical academic researchers. The meeting consisted of plenary presentations and discussions, and small group break-out sessions. Discussion topics were based on a symposium, focus groups and literature search. Benefits, obstacles and potential solutions toward implementing precision medicine were discussed. Results from the break-out sessions were presented in plenary and formed the basis of a broad consensus discussion to reach final conclusions. Throughout the meeting, participants answered several statement and open-ended questions on their mobile device, using a real-time online survey tool. Answers to the statement questions were analyzed descriptively. Results of the open-ended survey questions, the break-out sessions and the consensus discussion were analyzed qualitatively. Results and conclusion: Seventy-one participants from 26 countries attended the consensus-building meeting in Amsterdam, April 2019. During the opening plenary on the first day, the participants agreed with the statement that precision medicine is the way forward in DKD (n = 57, median 90, IQR [75-100]). Lack of efficient tools for implementation in practice and generating robust data were identified as significant obstacles. The identified benefits, e.g., improvement of the benefit-risk ratio of treatment, offer substantive incentives to find solutions for the identified obstacles. Earlier and increased multi-stakeholder collaboration and specific training may provide solutions to alter clinical and regulatory guidelines that lie at the basis of both obstacles and solutions. At the end of the second day, the opinion of the participants toward precision medicine in DKD was somewhat more nuanced (n = 45, median 83, IQR [70-92]) and they concluded that precision medicine is an important way forward in improving the treatment of patients with DKD.

Dabigatran treatment simulation in patients undergoing maintenance haemodialysis.

Patients with atrial fibrillation requiring maintenance haemodialysis are at increased risk of ischaemic stroke and bleeds. Currently, vitamin K antagonists such as warfarin are predominantly used in these patients as limited data are available on the use of non-vitamin K oral anticoagulants, including dabigatran etexilate (dabigatran). Dabigatran is approximately 85 % renally eliminated, thus, its half-life is prolonged in renal impairment. This study simulated the doseexposure relationship of dabigatran in patients undergoing haemodialysis. Dabigatran exposure was modelled at once- and twice-daily doses of 75 mg, 110 mg and 150 mg and at variations in non-renal clearance and dialysis settings. Resultant dose exposure (area under the curve [AUC]) was compared with values simulated from typical patients in the RE-LY® trial (based on a previously characterised pharmacometric model). In this simulation, all twice-daily dosages resulted in exposures above those simulated from typical RE-LY patients (1.5- to 3.3-fold increase in AUC) and thus may not be optimal for use in haemodialysis patients. However, dabigatran doses of 75 mg or 110 mg once daily produced exposures comparable to those simulated in typical RE-LY patients (-13.3 and +4.4 %, respectively). Of patient and dialysis variables, non-renal clearance had the highest impact on exposure (≤ 30.8 % change). These data could potentially inform dose selection in patients undergoing maintenance haemodialysis and the findings warrant investigation in future clinical trials.

Symmetric dimethylarginine predicts all-cause mortality following ischemic stroke.

OBJECTIVE: Methylarginines have been shown to interfere with nitric oxide (NO) formation by inhibiting NO synthase (asymmetric dimethylarginine, ADMA, and monomethylarginine, NMMA) and the cellular l-arginine uptake system (ADMA, NMMA and symmetric dimethylarginine, SDMA), thereby causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in diverse populations. METHODS: We investigated whether methylarginines are predictors of mortality in 394 patients after acute ischemic stroke during 7.4 years of follow-up. RESULTS: Patients who died (N=231) were older and more frequently had one of the traditional risk factors for stroke (previous stroke/TIA, atrial fibrillation, prevalent ischemic heart disease, peripheral vascular disease, each p<0.05). ADMA (0.52 micromol/l vs. 0.50 micromol/l, p=0.015) and SDMA (0.56 micromol/l vs. 0.43 micromol/l, p<0.001) were higher in patients who died. In multivariable-adjusted hazard models, SDMA but not ADMA or NMMA was an independent predictor of all-cause mortality after stroke (SDMA, hazard ratio 2.41 (1.55-3.72), p<0.001; ADMA, hazard ratio 1.43 (0.99-2.07), p=0.06). SDMA was significantly associated with atrial fibrillation (0.55 micromol/l vs. 0.50 micromol/l, p=0.03) but there was no significant interaction between SDMA and AF in relation to mortality (p=0.81). SDMA remained significantly associated with mortality after adjusting for eGFR and also additionally adjusting for C-reactive protein, albumin, beta-thromboglobulin, and von Willebrand factor. CONCLUSION: Our study demonstrates that SDMA is an independent predictor of total mortality after acute stroke irrespective of renal function. SDMA is associated with atrial fibrillation, endothelial and platelet activation, and may therefore play a previously unknown role in the pathophysiology of stroke.

Association of plasma ADMA levels with MRI markers of vascular brain injury: Framingham offspring study.

BACKGROUND AND PURPOSE: Asymmetrical dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase, is a marker of endothelial dysfunction. Elevated circulating ADMA concentrations have been associated with systemic and carotid atherosclerosis, an elevated risk of developing stroke, and magnetic resonance imaging white-matter hyperintensities (WMHs). The relation of plasma ADMA to subclinical vascular brain injury has not been previously studied in a middle-aged, community-based sample. METHODS: In 2013 stroke-free Framingham offspring (mean+/-SD age, 58+/-9.5 years; 53% women), we related baseline plasma ADMA levels (1995-1998) to subsequent brain magnetic resonance imaging measures (1999-2004) of subclinical vascular injury: presence of silent brain infarcts (SBIs) and large white-matter hyperintensity volumes (LWMHs; defined as >1 SD above the age-specific mean). RESULTS: Prevalences of SBIs and LWMHs were 10.7% and 12.6%, respectively. In multivariable analyses adjusting for age, sex and traditional stroke risk factors, higher ADMA levels were associated with an increased risk of prevalent SBIs (odds ratio [OR] per 1-SD increase in ADMA=1.16; 95% CI, 1.01 to 1.33; P=0.04). We observed that participants in the upper 3 age-specific quartiles (Qs) of plasma ADMA values had an increased prevalence of SBIs (OR for Q2-Q4 vs Q1=1.43; 95% CI, 1.00 to 2.04; P<0.05). The prevalence of SBIs in Q1and Q2-Q4 was 8.3% and 11.6%, respectively. The prevalence of LWMHs did not differ according to ADMA concentrations. CONCLUSIONS: Higher plasma ADMA values were associated with an increased prevalence of SBIs, after adjustment for traditional stroke risk factors. Thus, ADMA may be a potentially useful new biomarker of subclinical vascular brain injury, which is an important correlate of vascular cognitive impairment and risk of stroke.

Asymmetric dimethylarginine (ADMA) as a prospective marker of cardiovascular disease and mortality--an update on patient populations with a wide range of cardiovascular risk.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthases. By inhibiting NO formation, ADMA causes endothelial dysfunction, vasoconstriction, elevation of blood pressure, and aggravation of experimental atherosclerosis. Cross-sectional studies in humans have revealed that ADMA plasma concentration is elevated in numerous populations with vascular diseases or at high cardiovascular risk. However, the potential causal relationship between elevated ADMA and cardiovascular events and mortality in humans can only be revealed in prospective clinical studies. This review gives an overview of currently available data from prospective clinical studies in which ADMA has been measured in populations at high, intermediate, or low global vascular risk. Although the analytical methods used to quantify ADMA varied and statistical approaches to assess the association of ADMA with risk differed, these data reveal that ADMA is significantly associated with an increased risk of incident cardiovascular events and total mortality in subjects at a broad range of global risk. Hazard ratios were mostly in a range comparable to that of traditional cardiovascular risk markers even after multivariable adjustments, suggesting that ADMA may be suitable as a diagnostic marker for risk assessment, and that the biochemical pathways that regulate ADMA may be promising therapeutic approaches to treat cardiovascular disease in the future.

Association of the endogenous nitric oxide synthase inhibitor ADMA with carotid artery intimal media thickness in the Framingham Heart Study offspring cohort.

BACKGROUND AND PURPOSE: Higher plasma concentrations of the endogenous nitric oxides synthase inhibitor asymmetrical dimethylarginine (ADMA) are associated with increased risk of cardiovascular and cerebrovascular events and death, presumably by promoting endothelial dysfunction and subclinical atherosclerosis. We hypothesized that plasma ADMA concentrations are positively related to common carotid artery intimal-media thickness (CCA-IMT) and to internal carotid (ICA)/bulb IMT. METHODS: We investigated the cross-sectional relations of plasma ADMA with CCA-IMT and ICA/bulb IMT in 2958 Framingham Heart Study participants (mean age, 58 years; 55% women). RESULTS: In unadjusted analyses, ADMA was positively related to both CCA-IMT (beta per SD increment, 0.012; P<0.001) and ICA/bulb IMT (beta per SD increment, 0.059; P<0.001). In multivariable analyses (adjusting for age, sex, systolic blood pressure, antihypertensive treatment, smoking status, diabetes, BMI, total-to-HDL cholesterol ratio, log C-reactive protein, and serum creatinine), plasma ADMA was not associated with CCA-IMT (P=0.991), but remained significantly and positively related to ICA/bulb IMT (beta per SD increment, 0.0246; P=0.002). CONCLUSIONS: In our large community-based sample, we observed that higher plasma ADMA concentrations were associated with greater ICA/bulb IMT, but not with CCA-IMT. These data are consistent with the notion that ADMA promotes subclinical atherosclerosis in a site-specific manner, with a greater proatherogenic influence at known vulnerable sites in the arterial tree.

L-Arginine enhances the triglyceride-lowering effect of simvastatin in patients with elevated plasma triglycerides.

We recently noticed a possible triglyceride-lowering effect during dietary supplementation with L-arginine. The major limitation of prior studies on L-arginine, however, was that triglyceride levels were not the primary end point, and patients were not necessarily hypertriglyceridemic. Therefore, we conducted a 2-arm, randomized, double-blind study in 33 hypertriglyceridemic patients to investigate the hypothesis that oral L-arginine may lower serum triglyceride levels in hypertriglyceridemic patients on and off statins. The study consisted of a 6-week run-in phase, 6 weeks of treatment with L-arginine (n = 22, 1.5 g bid) or placebo (n = 11), and a 6-week extension period where simvastatin (20 mg qd) was added. All patients received dietary advice during each study visit. Routine and lipid laboratory parameters were determined in the local routine clinical laboratory. Treatment with L-arginine alone had no effects on serum lipids compared to placebo. The combination of L-arginine with simvastatin led to a significantly stronger reduction in triglycerides compared to placebo plus simvastatin (-140.5 +/- 149.2 mg/dL vs -56.1 +/- 85.0 mg/dL; P = .048). In addition, we found simvastatin-induced increases in aspartate transaminase and fibrinogen to be attenuated by L-arginine as compared to placebo. We conclude from our data that L-arginine enhances the effects of simvastatin on lipid metabolism, but it has no triglyceride-lowering effects when given alone.

Asymmetric dimethylarginine reference intervals determined with liquid chromatography-tandem mass spectrometry: results from the Framingham offspring cohort.

BACKGROUND: Accumulating evidence links higher circulating asymmetric dimethylarginine (ADMA) to greater risk of cardiovascular disease (CVD). Relatively small differences in ADMA concentrations between healthy individuals and those with disease underscore the need to formulate reference intervals that may aid risk stratification of individuals. METHODS: We formulated reference intervals for plasma ADMA concentrations using a community-based reference sample from the Framingham Offspring Study consisting of 1126 nonsmoking individuals [mean (SD) age 56 (9) years; 60% women] who were free of clinical CVD, hypertension, diabetes, and obesity and who attended a routine examination at which ADMA was assayed. ADMA concentrations were determined using a validated tandem mass spectrometry-liquid chromatography assay. RESULTS: In the study sample, the mean ADMA concentration was 0.52 (0.11) micromol/L, and the reference limits were 0.311 and 0.732 (2.5th and 97.5th percentile). The sex-specific reference limits were 0.310 and 0.745 in men and 0.313 and 0.721 micromol/L in women. In multivariable regression analysis, ADMA plasma concentrations were positively correlated with age and total plasma homocysteine (both P < 0.001). CONCLUSIONS: Reference limits calculated for circulating ADMA in our large community-based healthy reference sample confirm the previous observation of a relatively narrow distribution of concentrations. This suggests a tight physiological control of ADMA plasma concentrations, presumably by dimethylarginine dimethylaminohydrolase (DDAH) metabolism of ADMA.

Plasma asymmetric dimethylarginine and incidence of cardiovascular disease and death in the community.

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, induces endothelial dysfunction. Although elevated ADMA has been associated with an increased risk of cardiovascular disease events and death in referral samples, the prognostic significance of ADMA in the community has not been adequately evaluated. METHODS AND RESULTS: We related plasma ADMA, l-arginine (Arg), and the ratio of Arg to ADMA to the incidence of cardiovascular disease (fatal or nonfatal myocardial infarction, coronary insufficiency, angina pectoris, stroke or transient ischemic attack, intermittent claudication, or heart failure) and death in 3320 Framingham Offspring Study participants (1769 women; mean age, 59 years). Over a follow-up period of 10.9 years, 281 individuals of 2956 free of cardiovascular disease at baseline developed incident cardiovascular disease, and 285 participants died. In multivariable models adjusting for established risk factors and other biomarkers (B-type natriuretic peptide, renin, homocysteine, urine albumin excretion, and C-reactive protein), ADMA and the ratio of Arg to ADMA were significantly associated with all-cause mortality (adjusted-hazard ratio [HR] per 1-SD increment, 1.21; 95% CI, 1.07 to 1.37; and HR per 1-SD increment, 0.80; 95% CI, 0.69 to 0.93, respectively), whereas Arg was not (HR per 1-SD increment, 0.89; 95% CI, 0.77 to 1.02). We noted effect modification by diabetes status; ADMA was associated with death in individuals without diabetes (adjusted HR per 1-SD increment, 1.30; 95% CI, 1.13 to 1.50) but not in individuals with diabetes (adjusted HR per 1-SD increment, 0.85; 95% CI, 0.62 to 1.16). ADMA, Arg, and the ratio of Arg to ADMA were not associated with cardiovascular disease incidence (P>0.10). CONCLUSIONS: In our large community-based sample, ADMA was significantly associated with all-cause mortality, particularly in nondiabetic individuals.

Plasma asymmetric dimethylarginine, L-arginine and left ventricular structure and function in a community-based sample.

OBJECTIVE: Increasing evidence indicates that cardiac structure and function are modulated by the nitric oxide (NO) system. Elevated plasma concentrations of asymmetric dimethylarginine (ADMA; a competitive inhibitor of NO synthase) have been reported in patients with end-stage renal disease. It is unclear if circulating ADMA and L-arginine levels are related to cardiac structure and function in the general population. METHODS: We related plasma ADMA and L-arginine (the amino acid precursor of NO) to echocardiographic left ventricular (LV) mass, left atrial (LA) size and fractional shortening (FS) using multivariable linear regression analyses in 1919 Framingham Offspring Study participants (mean age 57 years, 58% women). RESULTS: Overall, neither ADMA or L-arginine, nor their ratio was associated with LV mass, LA size and FS in multivariable models (p>0.10 for all). However, we observed effect modification by obesity of the relations of ADMA and LA size (p for interaction p=0.04): ADMA was positively related to LA size in obese individuals (adjusted-p=0.0004 for trend across ADMA quartiles) but not in non-obese people. CONCLUSION: In our large community-based sample, plasma ADMA and l-arginine concentrations were not related to cardiac structure or function. The observation of positive relations of LA size and ADMA in obese individuals warrants confirmation.

Asymmetric dimethylarginine predicts outcome and time of stay in hospital in patients attending an internal medicine emergency room.

INTRODUCTION: For patients attending the emergency room (ER) valid diagnostic criteria which identify patients at risk for an adverse outcome are needed. We investigated the predictive value of asymmetric dimethylarginine (ADMA) in unselected patients attending an internal medicine ER regarding outcome of the patients and duration of stay in the hospital. PATIENTS AND METHODS: Patients (n=417) attending the ER were classified according to their primary diagnosis. Routine laboratory tests were performed and ADMA was determined. Patients were followed for a primary endpoint of in hospital death and complicated outcome. RESULTS: ADMA levels were highest in patients with a cancer-related diagnosis (0.76 (0.63-0.93) micromol/L) and in patients with a cardiovascular diagnosis (0.69 (0.60-0.80) micromol/L; p<0.001). Overall, we found increasing proportions of patients experiencing the primary end point over the quartiles of ADMA (4.6%, 8.2%, 9.6%, and 15.8%; p=0.007). ADMA had the highest predictive value for the primary endpoint in patients with cardiovascular disease (odds ratio 19.4; p=0.029). In a Cox proportional hazard model ADMA was an independent predictor of the length of hospitalization (hazard ratio 2.0 (95% CI: 1.3-3.3); p=0.006) in the entire cohort. CONCLUSION: We conclude that ADMA independently predicts future complications and hospitalization in patients attending an ER.

Asymmetrical dimethylarginine is increased in plasma and decreased in cerebrospinal fluid of patients with Alzheimer's disease.

BACKGROUND: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase and may alter NO production during pathological conditions. Concerning Alzheimer's disease (AD), there are reports on altered cerebral NO metabolism, but only few studies on ADMA concentrations in plasma and cerebrospinal fluid (CSF). METHODS: We assessed plasma ADMA in 80 AD patients and 80 age- and gender-matched controls and CSF ADMA in a subgroup of 53 AD patients and 20 controls. RESULTS: ADMA plasma concentrations were increased, while CSF ADMA concentrations were decreased in AD patients. There was a significant association between decreasing CSF ADMA levels and the severity of cognitive impairment. CONCLUSION: Elevated ADMA in plasma might be a contributing factor for AD through alterations of NO metabolism, for example decreased cerebral microperfusion, while decreased levels of CSF ADMA might lead to a cerebral increase of NO, peroxynitrite production and oxidative protein damage. Our study reveals different mechanisms of plasma and CSF ADMA regulation, both potentially contributing to AD pathology.

Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism.

AIMS: Oral L-arginine supplementation has been used in several studies to improve endothelium-dependent, nitric oxide (NO)-mediated vasodilation. L-Arginine treatment is hampered by extensive presystemic elimination due to intestinal arginase activity. In contrast, L-citrulline is readily absorbed and at least in part converted to L-arginine. The aim of our study was to assess this metabolic conversion and its subsequent pharmacodynamic effects. METHODS: In a double-blind, randomized, placebo-controlled cross-over study, 20 healthy volunteers received six different dosing regimes of placebo, citrulline, and arginine. Pharmacokinetic parameters (C(max), T(max), C(min), AUC) were calculated after 1 week of oral supplementation. The ratio of plasma L-arginine over asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase (arginine/ADMA ratio), urinary cyclic guanosine monophosphate (cGMP) and nitrate excretion rates, and flow-mediated vasodilation (FMD) was measured to assess pharmacodynamic effects. RESULTS: L-Citrulline dose-dependently increased AUC and C(max) of plasma L-arginine concentration more effectively than L-arginine (P < 0.01). The highest dose of citrulline (3 g bid) increased the C(min) of plasma L-arginine and improved the L-arginine/ADMA ratio from 186 +/- 8 (baseline) to 278 +/- 14 [P < 0.01, 95% confidence interval (CI) 66, 121]. Moreover, urinary nitrate and cGMP were increased from 92 +/- 10 to 125 +/- 15 micromol mmol(-1) creatinine (P = 0.01, 95% CI 8, 58) and from 38 +/- 3.3 to 50 +/- 6.7 nmol mmol(-1) creatinine (P = 0.04, 95% CI 0.4, 24), respectively. No treatment improved FMD over baseline. However, pooled analysis of all FMD data revealed a correlation between the increase of arginine/ADMA ratio and improvement of FMD. CONCLUSION: Our data show for the first time that oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling in a dose-dependent manner.

A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue.

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the hydrolysis of asymmetric dimethylarginine (ADMA) to L-citrulline and dimethylamine. DDAH is currently investigated as a promising target for therapeutic interventions, as ADMA has been found to be elevated in cardiovascular disease. In many tissues continuous endogenous formation of ADMA and L-citrulline poses considerable limitations to the presently used assays for DDAH activity, which are commonly based on the measurement of ADMA or L-citrulline. We therefore developed a stable-isotope-based assay suitable for 96-well plates to determine DDAH activity. Using deuterium-labeled ADMA ([(2)H(6)]-ADMA) as substrate and double stable-isotope labeled ADMA ([(13)C(5)-(2)H(6)]-ADMA) as internal standard we were able to simultaneously determine formation and metabolism of ADMA in renal and liver tissue of mice by LC-tandem MS. Endogenous formation of ADMA could largely be abolished by addition of protease inhibitors, while metabolism of [(2)H(6)]-ADMA was not significantly altered. The intra-assay coefficient of variation for the determination of endogenous ADMA and [(2)H(6)]-ADMA was 2.4% and 4.8% in renal and liver tissue, respectively. The inter-assay coefficient of variation for DDAH activity based on degradation of [(2)H(6)]-ADMA determined in separate samples from the same organs was determined to be 8.9% and 10% for mouse kidney and liver, respectively. The present DDAH activity assay allows for the first time to simultaneously determine DDAH activity and endogenous formation of ADMA, SDMA, and L-arginine in tissue.

Pharmacokinetics of oral doses of telmisartan and nisoldipine, given alone and in combination, in patients with essential hypertension.

This randomized, single-blind, parallel-group study was performed to assess pharmacokinetic interactions potentially occurring during concomitant use of telmisartan and nisoldipine. Patients with essential hypertension (n = 37) were treated with once-daily doses of telmisartan, nisoldipine, or their combination for 6 weeks. The regimen was started at low dose with an increase of dosage after 3 weeks of treatment. AUC(ss) (132%; P < .01) of telmisartan applied in doses of 80 mg was significantly higher after concomitant application with nisoldipine (10 mg), whereas CL/f(ss) (-54%; P < .05) and Vz/f(ss) (-72%; P < .05) were significantly lower. Regarding pharmacokinetic parameters of nisoldipine, significant differences between treatment groups were not detected. In conclusion, the results of this study strongly suggest that concomitant treatment with nisoldipine enhances telmisartan bioavailability in hypertensive individuals. Larger crossover trials will have to establish these observations and investigate whether interaction of both drugs affects telmisartan efficacy and tolerability in clinical use.

Asymmetric dimethylarginine, smoking, and risk of coronary heart disease in apparently healthy men: prospective analysis from the population-based Monitoring of Trends and Determinants in Cardiovascular Disease/Kooperative Gesundheitsforschung in der Region Augsburg study and experimental data.

BACKGROUND: An increased plasma concentration of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) predicts adverse clinical outcome in patients with coronary heart disease. We investigated the association between plasma concentrations of ADMA and risk in initially healthy smoking and nonsmoking men. METHODS: Participants for this nested case-control study came from the population-based Monitoring of Trends and Determinants in Cardiovascular Disease/Kooperative Gesundheitsforschung in der Region Augsburg study. ADMA was measured by liquid chromatography-tandem mass spectrometry in 88 men with incident coronary events (fatal and nonfatal myocardial infarction and sudden cardiac death) and 254 age-matched controls, with a median (interquartile range) follow-up of 6.2 (3.3-7.9) years. RESULTS: After adjustment for potential confounders, the relative risk for a future coronary event was 2.00 [95% confidence interval (CI) 1.27-3.16; P = 0.003] for smokers compared with nonsmokers and 1.35 (95% CI 0.78-2.33; P = 0.282) for the top vs the bottom tertile of the ADMA distribution. In cases and controls, lower ADMA plasma concentrations were observed in smokers. Analysis of ADMA-associated risk in smokers and nonsmokers separately revealed substantial differences: the adjusted relative risk for future coronary events (top vs bottom tertile of the ADMA distribution) was 0.48 (95% CI 0.16-1.46; P = 0.198) in smokers and 2.40 (95% CI 1.14-5.08; P = 0.021) in nonsmokers. Exposure of human endothelium-derived EAhy 926 cells to tobacco smoke enhanced expression of the ADMA metabolizing enzyme dimethylarginine dimethylaminohydrolase 2 and reduced ADMA concentration. CONCLUSIONS: In apparently healthy men, increased ADMA predicts the risk for coronary events in nonsmokers, but not in smokers. This may be explained in part by an alteration of ADMA metabolism by tobacco smoke.

High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma.

The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. L-Arginine is converted to NO and L-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-mul aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74+/-19 (SD), 0.46+/-0.09, and 0.37+/-0.07 microM in healthy humans (n=85), respectively, and 44+/-14, 0.72+/-0.23, and 0.19+/-0.06 microM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166+/-50 and 85+/-22, respectively.