Biochem. J. (2014) 460, 309–316 (Printed in Great Britain)
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*Nitric Oxide Signalling Group, MRC Clinical Science Centre, Imperial College London, London, U.K. †Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, London, U.K. ‡Institute of Pharmaceutical Science, King’s College London, London, U.K. §School of Crystallography, Birkbeck College, University of London, London, U.K. Department of Chemistry, University College London, London, U.K.
The aim of the present study was to investigate the therapeutic effects of pharmacological inhibition of DDAH1 (dimethylarginine dimethylaminohydrolase 1), an enzyme that metabolizes endogenously produced nitric oxide synthase inhibitors, principally ADMA (asymmetric dimethylarginine). The present study employs a series of rodent models to evaluate the effectiveness a DDAH1-selective inhibitor (L-257). Short-term models involved the development of endotoxaemia using lipopolysaccharide and long-term models involved the intraperitoneal administration of faecal slurry. In order to generate the most relevant model possible, following induction of severe sepsis, animals received appropriate fluid resuscitation and in some models vasopressor therapy. The effects of L-257 on survival, haemodynamics and organ function were subsequently assessed. Survival was significantly longer in all L-257 treatment groups (P < 0.01) and no adverse effects on haemodynamics and organ function were observed following L-257 administration
to either animals with sepsis or na¨ıve animals. Haemodynamic performance was preserved and the noradrenaline dose required to maintain target blood pressure was reduced in the treated animals (P < 0.01). Animals receiving L-257 had significantly increased plasma ADMA concentrations. Plasma nitrite/nitrate was reduced as was severity of sepsis-associated renal dysfunction. The degree of tachycardia was improved as were indices of tissue and microvascular perfusion. The results of the present study show that the selective DDAH-1 inhibitor L-257 improved haemodynamics, provided catecholamine sparing and prolonged survival in experimental sepsis. Further studies will determine its potential utility in human septic shock.
INTRODUCTION
proteolysis of proteins containing methylarginine residues [14]. Its major route of clearance (>80 %) is via hydrolysis to citrulline and methylamines by DDAH. There are two isoforms of DDAH with distinct tissue expressions. DDAH1 contributes a major proportion of the overall DDAH activity in many tissues [15], whereas DDAH2 is the only isoform expressed in immune cells. We have developed a competitive inhibitor (L-257) to bind the active site of DDAH1 and achieved isoform-specific competitive inhibition [16]. L-257 has no discernible effect on cytochrome P450 enzyme activity, cellular toxicity in HepG2 cells and a range of kinases determined through diversity profiling or HERG (human ether-a-go-go-related gene) currents (Biofocus; data on file). As DDAH1 is not expressed in circulating immune cells, isoform-specific inhibition of DDAH1 avoids the detrimental effects of NOS inhibition on the innate immune response that may have contributed to the failure of isoform non-selective NOS inhibitors in clinical trials. In the present study we show that L-257 is able to improve mortality and organ function in rat models of severe sepsis by reducing systemic NO production without impairment of microvascular blood flow or immune function. Pharmacological inhibition of DDAH1 is effective even when administered after the onset of septic shock and following appropriate fluid resuscitation.
Severe sepsis remains the leading cause of morbidity and mortality in patients admitted to intensive care units [1]. This often-lethal syndrome, induced by a dysregulated systemic inflammatory response to infection, can lead to vascular endothelial dysfunction and coagulation abnormalities, bioenergetic dysfunction, circulatory shock, and multiple organ failure [2]. Overproduction of NO plays an important role in sepsis-induced hypotension and hyporeactivity to catecholamine vasopressors [3]. This occurs in large part through increased activity of NOS (NO synthase), particularly its inducible isoform (iNOS) [4], resulting in a pathological shunting of blood flow, tissue hypoperfusion and cytopathic dysoxia [5,6]. Unfortunately, the early promise shown by NOS inhibition [7] or NO scavenging [8] has not been borne out by clinical studies [8,9]. Mechanisms potentially responsible for this negative outcome include excessive vasoconstriction [10], myocardial depression [11,12] and immune suppression [13]. An alternative, and potentially safer, approach is to modulate excessive production of NO via inhibition of DDAH (dimethylarginine dimethylaminohydrolase). Inhibition of this enzyme results in accumulation of ADMA (asymmetric dimethylarginine), an endogenous inhibitor of all three isoforms of NOS. ADMA is released as a result of
Key words: drug research, infection, nitric oxide, nitric oxide synthase, shock.
Abbreviations: ADMA, asymmetric dimethylarginine; DDAH, dimethylarginine dimethylaminohydrolase; LPS, lipopolysaccharide; MAP, mean arterial pressure; L-NMMA, N G -monomethyl-L-arginine; NOS, NO synthase; SDMA, symmetric ω-N G ,N ’G -dimethylarginine. 1 To whom correspondence should be addressed (email [email protected]). 2 Joint senior authors. c The Authors Journal compilation c 2014 Biochemical Society
Biochemical Journal
Zhen WANG*†, Simon LAMBDEN*1 , Valerie TAYLOR†, Elizabeth SUJKOVIC*, Manasi NANDI‡, James TOMLINSON*, Alex DYSON†, Neil MCDONALD§, Stephen CADDICK, Mervyn SINGER†2 and James LEIPER*2
www.biochemj.org
Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock
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EXPERIMENTAL
Long-term tethered awake model of faecal peritonitis
The present study was carried out in accordance with local ethics committee and UK Home Office guidelines. Male Wistar rats (Charles River) of 280–320 g of body mass were locally housed for 1 week before the study for acclimatization, with day/night cycles and free access to food and water. L-257 was manufactured by Biofocus and stored in a freezedried form. Fresh dilution was made in distilled water before use. L-NMMA (N G -monomethyl-L-arginine) was purchased from Sigma–Aldrich.
Survival study
Short-term anaesthetized model of endotoxaemia
Full details of the instrumentation, techniques and study design are provided in the Supplementary Online Data (http://www.biochemj.org/bj/460/bj4600309add.htm). In brief, anaesthetized spontaneously breathing animals underwent instrumentation of the carotid artery, internal jugular vein and bladder to facilitate continuous monitoring of blood pressure and urine output, blood sampling, and drug and fluid administration. Transthoracic echocardiography was performed using a 14 MHz linear-array transducer and a digital ultrasound system (Vivid 7; GE Healthcare). The microcirculation of the gastrocnemius muscle was assessed using sidestream dark field imaging (MicroscanTM ; MicroVision Medical). Blood was sampled at specified time points for blood gas and laboratory analysis of biochemical and haematology indices, and plasma ADMA concentration, measured using MS as described previously [17]. After at least 30 min of stabilization post-instrumentation, baseline measurements were taken and then 40 mg of LPS (lipopolysaccharide; from Klebsiella pneumoniae; Sigma– Aldrich)/kg of body mass was infused intravenously over 30 min. Following this, a crystalloid solution (1:1 mixture of 0.9 % saline/5 % glucose) was infused at 10 ml/kg of body mass per h to prevent hypovolaemia and hypoglycaemia. This infusion rate was determined from pilot studies. MAP (mean arterial pressure) showed the characteristic early fall following injection of LPS, then transient recovery and a secondary fall at 60–90 min. When MAP fell 20 % from baseline, at approximately 60–90 min postendotoxin administration, two separate studies were performed.
Cardiorespiratory effects of L-257
Animals received an intravenous bolus dose of either 0, 3, 30 or 300 mg of L-257/kg of body mass (n = 6 per group) diluted into 1 ml of saline and given over 10 min. Another six animals (sham group) were instrumented and fluid-resuscitated in the same manner, but without injection of LPS. Animals were killed at 2 h post-administration of L-257 or the placebo, with blood and tissues sampled for subsequent analysis.
Full details of the instrumentation, techniques and study design are provided in the Supplementary Online Data, and have been described previously [18]. In brief, animals were instrumented under isoflurane anaesthesia with tunnelled carotid arterial and jugular venous lines. After instrumentation, animals were injected with 0.625 ml of faecal slurry/100 g of body mass intraperitoneally to induce a high mortality 48-h model of sepsis. The animals were then transferred into individual cages and their intravascular lines were then connected to a tether system to enable, on awakening, uninhibited movement and free access to food and water. To replicate a clinical scenario, no fluid was given until the MAP fell to 15–20 % below baseline. Two fluid boluses of 10 ml of hydroxyethyl starch (Voluven® ; Fresenius Kabi)/kg of body mass were then given intravenously over 10min periods until the MAP recovered and remained within 10 % of baseline. Animals (15 per group) received, in a randomized blinded manner, L-257 at a bolus dose of 0, 3, 10 or 30 mg/kg of body mass given with the second fluid challenge, followed by a continuous infusion of 0, 0.41, 1.38 or 4.1 mg/kg of body mass per h respectively, to maintain the steady-state levels. Drug was added to the resuscitation fluid described above. Thereafter animals received a 1:1 mixture of 0.9 % saline/5 % glucose infused at 10 ml/kg of body mass per h. Animals were observed for 48 h post-LPS or until premature death. Any animal appearing to be in significant discomfort was killed, as were animals whose MAP fell 0.05 compared with sham-operated animals without sepsis; Figure 2B) as was the rate of deterioration in standard base excess seen in the untreated animals which was significantly ameliorated by L-257 therapy (Figure 2C) Compared with the placebo, at 120 min post-drug bolus, animals with sepsis treated with L-257 had a higher gastrocnemius muscle perfused capillary density (with 30 mg/kg, P < 0.05; Figure 2D). In the short-term study assessing the effect of L-257 on noradrenaline requirements to maintain blood pressure (noradrenaline infusion rate 0.5–1.5 μg/kg of body mass per min), the L-257-treated animals required significantly less noradrenaline to maintain target pressures during a total of 3 h noradrenaline infusion (37.8 + − 3.2 μg compared with 47.8 + 1.3 μg; P < 0.05; Figure 2E). Significant reductions in − heart rate, urea and creatinine were also associated with supplementation of noradrenaline with L-257 (Table 2), whereas MAP was consistent in both groups (Figure 2F). In the control population of animals that underwent sham surgery and subsequent treatment with L-257, there
A DDAH1 inhibitor as a therapeutic agent in septic shock
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Figure 2 Effect of L-257 administration in the short-term endotoxaemic model of septic shock (n = 6 per group) at 2 h after bolus doses of L-257 at 0, 3 mg/kg of body mass and 30 mg/kg of body mass Data are medians and interquartile ranges. (A) An associated reduction in plasma nitrite/nitrate concentration. P < 0.05 compared with the untreated animals. (B) The administration of L-257 was associated with preservation of urine output in treated groups compared with the control, whereas untreated animals with sepsis showed a significant reduction in urine output. P < 0.05 compared with the untreated sham controls as indicated by the bracket. (C) L-257 was associated with a reduced rate of deterioration in the standard base excess in sepsis. *P < 0.05 compared with the untreated control. (D) Administration of L-257 had no negative effect on the perfused capillary density at 3 mg/kg of body mass and improved microvascular flow at 30 mg/kg of body mass following bolus administration. *P < 0.05 compared with the untreated animals as indicated by the bracket. (E) Administration of L-257 was associated with a reduction in the total amount of noradrenaline required to maintain normal MAP in the endotoxaemic model of septic shock. *P < 0.05 as indicated by the bracket. (F) Following the onset of hypotension (drop in MAP >20 % from baseline) therapy with noradrenaline alone or co-administration of L-257 with noradrenaline was undertaken to restore and maintain the MAP for the 180 min experimental period.
c The Authors Journal compilation c 2014 Biochemical Society
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Figure 3
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The effect of L-257 administration on polymicrobial peritoneal sepsis
(A) Long-term survival benefit seen with L-257 administration in a high-mortality model of polymicrobial sepsis induced by caecal ligation and puncture. P < 0.01 for all groups. (B) The effect of bolus doses of L-257 at 0, 3 mg/kg of body mass and 30 mg/kg of body mass on the MAP in the long-term study of the conscious tethered rats. The bolus L-257 was given at 16 h followed by a 10-min infusion. n = 6 in each group. (C) Change in NO production in a less-severe long-term model of polymicrobial sepsis induced by faecal slurry administration. *P < 0.05 compared with the control animals. (D) Incubation of peritoneal monocytes with an pro-inflammatory cocktail and either L-257 or L-NMMA (a global NOS inhibitor). No effect on phagocytosis of fluorescein-labelled LPS in isolated peritoneal monocytes in those cells incubated with L-257. However L-NMMA administration at a high dose resulted in significant inhibition of function. n = 3 per group. *P < 0.05.
was no difference in the MAP over the course of the experiment at any dose (Supplementary Figure 1A at http://www.biochemj.org/bj/460/bj4600309add.htm), no associated change in plasma ADMA (Supplementary Figure 1B), SDMA (Supplementary Figure 1C), arginine (Supplementary Figure 1D) or the arginine/ADMA ratio (Supplementary Figure 1E). There was no difference in cumulative urine output over the course of the experimental period in the treated animals compared with the controls (Supplementary Figure 1F).
In the second less-severe model where L-257 or placebo was commenced at 16 h after a lower-dose slurry, the MAP was better preserved at the end of the observation period (Figure 3B). Plasma nitrate/nitrite concentration was significantly lower in both treatment groups (P < 0.05; Figure 3C). The severity of tachycardia at the end of the experimental period was also reduced in both long-term treatment groups with associated trends towards improved stroke volume and renal function (Supplementary Table S3 at http://www.biochemj.org/bj/460/bj4600309add.htm).
Long-term peritonitis studies
Survival times were significantly longer (P < 0.01) in all treatment groups (18 h median) compared with the control (12 h) (Figure 3A). The decrease in the MAP was attenuated in the L257-treated animals compared with the controls with sepsis. These differences did not reach significance as increased mortality in the control group reduced the statistical power (Supplementary Table S2 at http://www.biochemj.org/bj/460/bj4600309add.htm). c The Authors Journal compilation c 2014 Biochemical Society
The effect of L-257 on peritoneal macrophage phagocytic capacity
L-257, at both 100 and 300 μM concentrations, had no effect on in vitro phagocytic function of peritoneal macrophages. However, the direct NOS inhibitor L-NMMA at concentrations similar to those achieved in clinical studies of global NOS inhibition significantly impaired phagocytic capacity (P < 0.05; Figure 3D).
A DDAH1 inhibitor as a therapeutic agent in septic shock DISCUSSION
Cardiovascular collapse is a cardinal feature of septic shock in which excessive NO accumulation causes profound vasodilatation, impaired vasoreactivity to exogenous catecholamines [19– 21] and myocardial depression. L-257 acts via inhibition of DDAH1 that, in turn, results in elevated intracellular levels of the endogenous NOS inhibitor ADMA. We hypothesized on the basis of our previous work [15,16] and have confirmed in the present study that L-257 causes a significant reduction in plasma nitrate/nitrite levels in sepsis, with a consequent improvement in cardiovascular function and indices of tissue perfusion with associated preservation of organ function and increased survival. Previous laboratory studies of novel therapeutic interventions in septic shock have often given treatment before, at or shortly after the induction of sepsis [22–24]. The models employed in the present study allow significant systemic compromise to occur, followed by appropriate fluid resuscitation and then vasopressor support before drug administration. This more closely replicates the pattern of therapy that might be employed in the clinical environment and resulted in a significant prolongation of survival time associated with improved macro- and micro-circulatory perfusion and the preservation of organ function without apparent toxicity at any dose, albeit in short experimental time courses. In clinical practice, catecholamine therapy is associated with a variety of complications [19] and, although not formally tested in prospective randomized trials, there are strong suggestions that catecholamine use is associated with excess mortality [25]. Agents that provide a catecholamine-sparing effect may prove advantageous, provided their safety profile is superior. Direct NOS inhibition and NO scavenging in sepsis provides short-term improvement in blood pressure [26], although at the expense of decreased cardiac output and worse survival rates [8,9]. Although an excess of NO production probably contributes to mortality in critically ill patients, some NO is essential to maintain coronary blood flow [27] and myocardial contractility [28], to decrease platelet adhesiveness [29], and to bolster innate immune defences, exerting both direct antimicrobial properties [30,31] and immunomodulatory effects [13]. DDAH1 is found throughout the systemic vasculature and may be localized within vessels to endothelial cells [32]. It is minimally expressed in the heart [33] and, therefore, L-257 is unlikely to impair NO-driven autoregulation of cardiac function in sepsis. Likewise, we show that L-257 has no direct effect on macrophage phagocytic ability, unlike the direct NOS inhibitor L-NMMA that compromised macrophage function. This ex vivo preservation of macrophage function is likely to be replicated in vivo as DDAH1 it is not expressed in immune cells [33]. Previous studies have also shown that elevated circulating ADMA has no effect on leucocyte number in rat models [34]. Given the absence of DDAH1 in immune cells and the specificity of L-257 for DDAH1, an exhaustive examination of the effects of the effect of L-257 on immune function was beyond the scope of the present study. The present study employs the Griess assay as an index of NO flux. Since NO production cannot be measured reliably during in vivo experiments, changes in the plasma nitrate/nitrite level measured using this technique have been widely employed as a measure of NO production. It should be noted that under normal circumstances nitrate levels may be altered by things other than changes in NO level such as diet. In our short-term experiments animals did not receive any enteral feeding and were treated with matched doses of crystalloid. In the long-term studies, there were no changes in diet during the experiment and so changes in exogenous nitrate uptake are unlikely to confound the results.
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Therapeutic doses of L-257 in our models increased plasma ADMA levels. However the absolute rise in plasma ADMA, although reflecting increased intracellular levels, is not sufficient to result in the systemic NOS inhibition that results from pharmacological doses of direct inhibitors. Although comparable fold elevations in plasma ADMA are associated with severity of illness and organ dysfunction in adults with severe sepsis or septic shock [35,36], it is unknown whether this is epiphenomenal, reflecting chronic cardiovascular dysfunction or causative. Of relevance, much higher plasma levels must be achieved in human volunteers to produce cardiovascular effects with exogenous administration of ADMA [37]. Secondary inhibition of nonDDAH1-expressing cells by treatment-induced increases in the circulating ADMA level is therefore unlikely. In summary, the present study demonstrates that L-257, a specific DDAH-1 inhibitor, may offer a novel, and seemingly safe, therapeutic option for septic shock. Additional studies are warranted to explore this drug further.
AUTHOR CONTRIBUTION Zhen Wang, Valerie Taylor, James Tomlinson, Alex Dyson and Elizabeth Sujkovic contributed to performing the experiments and collection of the data. Zhen Wang, Simon Lambden, James Leiper, Stephen Caddick, Neil McDonald, Mervyn Singer and Manasi Nandi contributed to conception of the study, and analysis and interpretation of data. James Leiper, Simon Lambden and Mervyn Singer wrote the paper.
FUNDING This work was supported a Wellcome Trust Seeding Drug Discovery Grant [to J.L. (Principal Investigator), M.S., S.C. and N.M.] and the British Heart Foundation [grant numbers PG/02/165/14797 and RG/02/005 (to J.L.)]. This work was undertaken at UCLH (University College London Hospitals)/UCL (University College London) who received a proportion of funding from the UK Department of Health’s NIHR (National Institute of Health Research) Biomedical Research Centres funding scheme. J.L. is an MRC (Medical Research Council) research programme leader. M.S. is an NIHR senior investigator.
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c The Authors Journal compilation c 2014 Biochemical Society
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Biochem. J. (2014) 460, 309–316 (Printed in Great Britain)
doi:10.1042/BJ20131666
SUPPLEMENTARY ONLINE DATA
Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock Zhen WANG*†, Simon LAMBDEN*1 , Valerie TAYLOR†, Elizabeth SUJKOVIC*, Manasi NANDI‡, James TOMLINSON*, Alex DYSON†, Neil MCDONALD§, Stephen CADDICK, Mervyn SINGER†2 and James LEIPER*2 *Nitric Oxide Signalling Group, MRC Clinical Science Centre, Imperial College London, London, U.K. †Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, London, U.K. ‡Institute of Pharmaceutical Science, King’s College London, London, U.K. §School of Crystallography, Birkbeck College, University of London, London, U.K. Department of Chemistry, University College London, London, U.K.
EXPERIMENTAL Short-term model of endoxtoxaemia
While spontaneously breathing under 2 % isoflurane anaesthesia, the animals were placed on to a heating mat to maintain their core temperature 37 ◦ C, and then underwent cannulation of their right internal jugular vein and left carotid artery using PVC tubing (external diameter 0.96 mm; SteriHealth). The MAP was continually monitored and recorded on to a laptop computer using a precalibrated PowerLab system (Powerlab 16/30; ADInstruments). Urine drainage was facilitated via PVC tubing sited in the bladder lumen under direct vision. Vetergesic (buprenorphine hydrochloride; Alstoe Veterinary) was injected subcutaneously at 40 mg/kg of body mass. After instrumentation, the inhaled isoflurane concentration was dropped to 1.5 % and the animals allowed to stabilize for 15–30 min.
consecutive cycles from the start of each Doppler trace. Cardiac output was calculated as the product of stroke volume and heart rate. Long-term tethered awake model of faecal peritonitis
Briefly, under 2 % isoflurane anaesthesia, spontaneously breathing animals had their right internal jugular vein and left carotid artery cannulated using a PVC tubing catheter (external diameter 0.96 mm). The catheters were tunnelled subcutaneously to the nape of the neck and mounted on to a swivel/tether system that enabled unimpaired movement around the cage after recovery from anaesthesia. The MAP was monitored and recorded continuously using a precalibrated PowerLab system (ADInstruments). Blood sampling
Assessment of microcirulatory flow
In this short-term endotoxaemic model, blood flow within the microvasculature of the gastrocnemius muscle was visualized and recorded using SDF (sidestream dark-field) imaging (Microvision Medical BV) [1] at baseline and at 2 h post-bolus administration of L-257 or placebo. The device probe was gently placed on to the surface area of the muscle without causing a pressure artefact. Five video images, with recording time of 20 s each, were captured from different sites in each animal. Analysis of microcirculatory flow was made offline in a blinded way based on a scoring system developed by De Backer et al. [2].
Blood (1 ml) was anti-coagulated with EDTA (Sigma–Aldrich) for haematology analyses. A further 1 ml of blood was added to sodium citrate for subsequent coagulation assays. The remainder was allowed to clot for 20 min and then centrifuged at 3000 g for 10 min at 4 ◦ C. Serum was collected for biochemistry assays and other analyses. Measurements of arginine, ADMA and SDMA
Methylarginines were measured in serum and tissue lysates in the liver and kidney using LC–MS/MS as described previously [4].
Transthoracic echocardiography
Measurement of serum NOx (nitrate/nitrate)
Transthoracic echocardiography was performed at baseline and at the end of the experiment to assess cardiac function. In brief, using a Vivid 7 DimensionTM device (GE Healthcare) with a 14 MHz probe recording at a depth of 2 cm, aortic blood flow velocities were determined before the bifurcation of the right carotid artery using pulsed-wave Doppler; the direction of blood flow was confirmed by colour Doppler imaging. Stroke volume was determined as the product of the VTI (velocity time integral) and vessel cross-sectional area [π×(0.5×diameter)2 ]. Previous studies in rats of this age showed the aortic diameter to be 0.26 cm [3], thus a cross-sectional area of π×0.132 cm2 was assumed. Heart rate was determined by measuring the time between six
A modified Griess assay was used to determine NOx by first enzymatically converting nitrate into nitrite by the addition of NADPH-dependent nitrate reductase (Roche). Nitrite was then determined colorimetrically at 540 nM using a plate reader (V max Kinetic microplate reader; Molecular Devices). Standard curves for nitrite and nitrate enabled the calculation of total serum NOx concentration and the percentage nitrate conversion.
1 2
In vitro phagocytosis assay
Phagocytic function was evaluated using a commercial assay kit (Vybrant Phagocytosis Assay Kit; Molecular Probes). Peritoneal
To whom correspondence should be addressed (email [email protected]). Joint senior authors. c The Authors Journal compilation c 2014 Biochemical Society
Z. Wang and others Table S1
Biochemistry, haematology and coagulation variables obtained in a short-term study of organ function in a mouse model of endotoxaemia
n = 6 per group. *P < 0.05 compared with the control. ALT, alanine transaminase; APTT, activated partial thromboplastin time; Hb, haemoglobin; Plt, platelet count; PT, prothrombin time; WBC, white cell count. Groups with sepsis Variables
No L-257
3 mg of L-257/kg of body mass
30 mg of L-257/kg of body mass
Sham-operated group
Urea (mmol/l) Creatinine (μM) ALT (units/l) WBC (109 /l) Plt (109 /l) Hb (g/dl) PT (s) APTT (s)
16.62 + − 1.542 76.00 + − 17.0 230 + − 83.8 2.6 + − 0.75 204 + − 59.1 13 + − 0.91 15.50 + − 1.7 39.3 + − 5.0
12.24 + − 2.421 33.2 + − 5.6* 68.1 + − 9.1 1.3 + − 0.26 269 + − 46.7 12.6 + − 0.21 15.25 + − 1.6 37.3 + − 11.3
13.92 + − 1.094 42.80 + − 4.6 181 + − 99.7 1.3 + − 0.15 301 + − 42.5 11.7 + − 0.42 14.60 + − 0.4 3 3.8 + − 3.0
8.7 + − 3.6 26.6 + − 8.6 40.8 + − 5.9 4.5 + − 2.4 701 + − 97 11.8 + − 0.4 11.5 + − 0.8 22 + − 2.8
Table S2 Mean arterial pressure values and survivor numbers at sequential time points in control mice and animals treated with three different regimes of L-257 therapy in a long-term model of peritonitis Numbers in parentheses indicate the number of surviving animals at the corresponding time points.
Table S3
Groups
Baseline
Pre-treatment
12 h
18 h
24 h
Control 3 mg of L-257/kg of body mass 10 mg of L-257/kg of body mass 30 mg of L-257/kg of body mass
112 + − 2 (15) 112 + − 4 (15) 119 + − 2 (15) 113 + − 3 (15)
104 + − 3 (15) 107 + − 3 (15) 109 + − 3 (15) 107 + − 3 (15)
103 + − 6 (8) 98 + − 5 (10) 97 + − 9 (12) 91 + − 4 (12)
105 + − 4 (3) 98 + − 9 (7) 105 + − 5 (5) 93 + − 7 (7)
91 + − 3 (2) 87 + − 6 (4) 109 + − 4 (2) 95 + − 8 (4)
The effect of L-257 administration in a long-term model of sepsis induced by intraperitoneal faecal slurry administration
Echocardiographic examination and organ function assessment after 22 h on conscious tethered rats. n = 6 in each group. *P < 0.05 compared with the control. ALT, alanine transaminase; APTT, activated partial thromboplastin time; Hb, haemoglobin; Plt, platelet count; PT, prothrombin time; U, blood urea. Groups with sepsis Variables
No L-257
3 mg of L-257/kg of body mass
30 mg of L-257/kg of body mass
Sham-operated
Stroke volume (ml) Heart rate (b.p.m.) CO (ml/min) U (mM) Creatinine (μM) ALT (U/L) Leucocytes (109 /l) Plt (109 /l) Hb (g/dl) PT (s) APTT(s)
0.15 + − 0.01 550 + − 10.35 84.1 + − 2.5 9.2 + − 2.6 37.83 + − 6.82 80.20 + − 29.2 2.36 + − 0.45 156 + − 35.48 12.97 + − 0.2801 11.00 + − 0.3162 28.83 + − 2.738
0.19 + − 0.01* 501 + − 14.47* 95.5 + − 4.9 9.4 + − 1.6 40.67 + − 6.54 57.50 + − 9.26 2.88 + − 0.53 140 + − 22.98 13.67 + − 0.8586 10.80 + − 0.2000 30.80 + − 2.200
0.17 + − 0.01 504 + − 10.29* 87.4 + − 3.6 8.4 + − 0.8 33.80 + − 7.34 48.60 + − 8.17 3.17 + − 0.52 142 + − 28.54 13.50 + − 0.3592 10.60 + − 0.2449 36.80 + − 5.472
0.30 + − 0.03 413 + − 39 122 + −2 8.0 + − 1.6 25 + −3 38 + −7 5.5 + − 0.7 701 + − 91 11.1 + − 0.5 9+ − 0.5 20 + − 0.8
macrophages, isolated from peritoneal washouts of C57/BL6 mice, were incubated overnight at a cell density of 106 in a 96well plate and then stimulated by a cytokine cocktail consisting of 10 ng/ml TNFα (tumour necrosis factor α), 100 units/ml IFNγ (interferon γ ) and 5 μg/ml LPS (from Klebsiella pneumoniae; Sigma–Aldrich). Cells were allocated into five experimental groups in triplicate as: (i) control, and in the presence of (ii) 100 μM L-257, (iii) 300 μM L-257, (iv) 30 μM L-NMMA, and
c The Authors Journal compilation c 2014 Biochemical Society
(v) 100 μM L-NMMA. Three blank wells that were loaded with only culture medium were used as negative controls. The media was removed 4 h later, then fluorescein-labelled E. coli was added and the procedure performed as per the manufacturer’s instructions (Sigma–Aldrich). The wells were measured for fluorescence on a plate reader at 480 nm excitation and 520 nm emission (Fluoroskan Ascent FL; LabSystems), and results expressed as the fluorescence intensity.
A DDAH1 inhibitor as a therapeutic agent in septic shock
Figure S1 Impact of L-257 administration on na¨ıve animals (n = 6 per group) either untreated or 2 h after bolus doses of L-257 at 3 mg/kg of body mass, 30 mg/kg of body mass or 300 mg/kg of body mass Data are presented as the median and interquartile range. (A) The impact of the three dose regimens on the MAP over the 2 h following bolus dose administration. (B) Administration of L-257 does not increase plasma ADMA concentration in healthy animals. (C–E) L-257 administration has no impact on SDMA level (C), arginine level (D) or arginine/ADMA ratio (E). (F) Urine output is not affected by L-257 administration at any of the test doses.
c The Authors Journal compilation c 2014 Biochemical Society
Z. Wang and others REFERENCES 1 Caplin, B., Wang, Z., Slaviero, A., Tomlinson, J., Dowsett, L., Delahaye, M. and Salama, A. (2012) Alanine-glyoxylate aminotransferase-2 metabolizes endogenous methylarginines, regulates no, and controls blood pressure. Arterioscler. Thromb. Vasc. Biol. 32, 2892–2900 CrossRef PubMed
Received 19 December 2013/18 February 2014; accepted 10 March 2014 Published as BJ Immediate Publication 10 March 2014, doi:10.1042/BJ20131666
c The Authors Journal compilation c 2014 Biochemical Society
2 De Backer, D., Hollenberg, S., Boerma, C., Goedhart, P., Buchele, G., Ospina-Tascon, G., Dobbe, I. and Ince, C. (2007) How to evaluate the microcirculation: report of a round table conference. Crit. Care 11, R101 CrossRef PubMed 3 Spronk, P., Ince, C., Gardien, M., Mathura, K., Oudemansvan Straaten, H. and Zandstra, D. (2002) Nitroglycerin in septic shock after intravascular volume resuscitation. Lancet 360, 1395–1396 CrossRef PubMed 4 Slama, M., Susic, D., Varagic, J., Ahn, J. and Frohlich, E. D. (2003) Echocardiographic measurement of cardiac output in rats. Am. J. Physiol. Heart Circ. Physiol. 284, H691–H697 PubMed
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*Nitric Oxide Signalling Group, MRC Clinical Science Centre, Imperial College London, London, U.K. †Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, London, U.K. ‡Institute of Pharmaceutical Science, King’s College London, London, U.K. §School of Crystallography, Birkbeck College, University of London, London, U.K. Department of Chemistry, University College London, London, U.K.
The aim of the present study was to investigate the therapeutic effects of pharmacological inhibition of DDAH1 (dimethylarginine dimethylaminohydrolase 1), an enzyme that metabolizes endogenously produced nitric oxide synthase inhibitors, principally ADMA (asymmetric dimethylarginine). The present study employs a series of rodent models to evaluate the effectiveness a DDAH1-selective inhibitor (L-257). Short-term models involved the development of endotoxaemia using lipopolysaccharide and long-term models involved the intraperitoneal administration of faecal slurry. In order to generate the most relevant model possible, following induction of severe sepsis, animals received appropriate fluid resuscitation and in some models vasopressor therapy. The effects of L-257 on survival, haemodynamics and organ function were subsequently assessed. Survival was significantly longer in all L-257 treatment groups (P < 0.01) and no adverse effects on haemodynamics and organ function were observed following L-257 administration
to either animals with sepsis or na¨ıve animals. Haemodynamic performance was preserved and the noradrenaline dose required to maintain target blood pressure was reduced in the treated animals (P < 0.01). Animals receiving L-257 had significantly increased plasma ADMA concentrations. Plasma nitrite/nitrate was reduced as was severity of sepsis-associated renal dysfunction. The degree of tachycardia was improved as were indices of tissue and microvascular perfusion. The results of the present study show that the selective DDAH-1 inhibitor L-257 improved haemodynamics, provided catecholamine sparing and prolonged survival in experimental sepsis. Further studies will determine its potential utility in human septic shock.
INTRODUCTION
proteolysis of proteins containing methylarginine residues [14]. Its major route of clearance (>80 %) is via hydrolysis to citrulline and methylamines by DDAH. There are two isoforms of DDAH with distinct tissue expressions. DDAH1 contributes a major proportion of the overall DDAH activity in many tissues [15], whereas DDAH2 is the only isoform expressed in immune cells. We have developed a competitive inhibitor (L-257) to bind the active site of DDAH1 and achieved isoform-specific competitive inhibition [16]. L-257 has no discernible effect on cytochrome P450 enzyme activity, cellular toxicity in HepG2 cells and a range of kinases determined through diversity profiling or HERG (human ether-a-go-go-related gene) currents (Biofocus; data on file). As DDAH1 is not expressed in circulating immune cells, isoform-specific inhibition of DDAH1 avoids the detrimental effects of NOS inhibition on the innate immune response that may have contributed to the failure of isoform non-selective NOS inhibitors in clinical trials. In the present study we show that L-257 is able to improve mortality and organ function in rat models of severe sepsis by reducing systemic NO production without impairment of microvascular blood flow or immune function. Pharmacological inhibition of DDAH1 is effective even when administered after the onset of septic shock and following appropriate fluid resuscitation.
Severe sepsis remains the leading cause of morbidity and mortality in patients admitted to intensive care units [1]. This often-lethal syndrome, induced by a dysregulated systemic inflammatory response to infection, can lead to vascular endothelial dysfunction and coagulation abnormalities, bioenergetic dysfunction, circulatory shock, and multiple organ failure [2]. Overproduction of NO plays an important role in sepsis-induced hypotension and hyporeactivity to catecholamine vasopressors [3]. This occurs in large part through increased activity of NOS (NO synthase), particularly its inducible isoform (iNOS) [4], resulting in a pathological shunting of blood flow, tissue hypoperfusion and cytopathic dysoxia [5,6]. Unfortunately, the early promise shown by NOS inhibition [7] or NO scavenging [8] has not been borne out by clinical studies [8,9]. Mechanisms potentially responsible for this negative outcome include excessive vasoconstriction [10], myocardial depression [11,12] and immune suppression [13]. An alternative, and potentially safer, approach is to modulate excessive production of NO via inhibition of DDAH (dimethylarginine dimethylaminohydrolase). Inhibition of this enzyme results in accumulation of ADMA (asymmetric dimethylarginine), an endogenous inhibitor of all three isoforms of NOS. ADMA is released as a result of
Key words: drug research, infection, nitric oxide, nitric oxide synthase, shock.
Abbreviations: ADMA, asymmetric dimethylarginine; DDAH, dimethylarginine dimethylaminohydrolase; LPS, lipopolysaccharide; MAP, mean arterial pressure; L-NMMA, N G -monomethyl-L-arginine; NOS, NO synthase; SDMA, symmetric ω-N G ,N ’G -dimethylarginine. 1 To whom correspondence should be addressed (email [email protected]). 2 Joint senior authors. c The Authors Journal compilation c 2014 Biochemical Society
Biochemical Journal
Zhen WANG*†, Simon LAMBDEN*1 , Valerie TAYLOR†, Elizabeth SUJKOVIC*, Manasi NANDI‡, James TOMLINSON*, Alex DYSON†, Neil MCDONALD§, Stephen CADDICK, Mervyn SINGER†2 and James LEIPER*2
www.biochemj.org
Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock
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Z. Wang and others
EXPERIMENTAL
Long-term tethered awake model of faecal peritonitis
The present study was carried out in accordance with local ethics committee and UK Home Office guidelines. Male Wistar rats (Charles River) of 280–320 g of body mass were locally housed for 1 week before the study for acclimatization, with day/night cycles and free access to food and water. L-257 was manufactured by Biofocus and stored in a freezedried form. Fresh dilution was made in distilled water before use. L-NMMA (N G -monomethyl-L-arginine) was purchased from Sigma–Aldrich.
Survival study
Short-term anaesthetized model of endotoxaemia
Full details of the instrumentation, techniques and study design are provided in the Supplementary Online Data (http://www.biochemj.org/bj/460/bj4600309add.htm). In brief, anaesthetized spontaneously breathing animals underwent instrumentation of the carotid artery, internal jugular vein and bladder to facilitate continuous monitoring of blood pressure and urine output, blood sampling, and drug and fluid administration. Transthoracic echocardiography was performed using a 14 MHz linear-array transducer and a digital ultrasound system (Vivid 7; GE Healthcare). The microcirculation of the gastrocnemius muscle was assessed using sidestream dark field imaging (MicroscanTM ; MicroVision Medical). Blood was sampled at specified time points for blood gas and laboratory analysis of biochemical and haematology indices, and plasma ADMA concentration, measured using MS as described previously [17]. After at least 30 min of stabilization post-instrumentation, baseline measurements were taken and then 40 mg of LPS (lipopolysaccharide; from Klebsiella pneumoniae; Sigma– Aldrich)/kg of body mass was infused intravenously over 30 min. Following this, a crystalloid solution (1:1 mixture of 0.9 % saline/5 % glucose) was infused at 10 ml/kg of body mass per h to prevent hypovolaemia and hypoglycaemia. This infusion rate was determined from pilot studies. MAP (mean arterial pressure) showed the characteristic early fall following injection of LPS, then transient recovery and a secondary fall at 60–90 min. When MAP fell 20 % from baseline, at approximately 60–90 min postendotoxin administration, two separate studies were performed.
Cardiorespiratory effects of L-257
Animals received an intravenous bolus dose of either 0, 3, 30 or 300 mg of L-257/kg of body mass (n = 6 per group) diluted into 1 ml of saline and given over 10 min. Another six animals (sham group) were instrumented and fluid-resuscitated in the same manner, but without injection of LPS. Animals were killed at 2 h post-administration of L-257 or the placebo, with blood and tissues sampled for subsequent analysis.
Full details of the instrumentation, techniques and study design are provided in the Supplementary Online Data, and have been described previously [18]. In brief, animals were instrumented under isoflurane anaesthesia with tunnelled carotid arterial and jugular venous lines. After instrumentation, animals were injected with 0.625 ml of faecal slurry/100 g of body mass intraperitoneally to induce a high mortality 48-h model of sepsis. The animals were then transferred into individual cages and their intravascular lines were then connected to a tether system to enable, on awakening, uninhibited movement and free access to food and water. To replicate a clinical scenario, no fluid was given until the MAP fell to 15–20 % below baseline. Two fluid boluses of 10 ml of hydroxyethyl starch (Voluven® ; Fresenius Kabi)/kg of body mass were then given intravenously over 10min periods until the MAP recovered and remained within 10 % of baseline. Animals (15 per group) received, in a randomized blinded manner, L-257 at a bolus dose of 0, 3, 10 or 30 mg/kg of body mass given with the second fluid challenge, followed by a continuous infusion of 0, 0.41, 1.38 or 4.1 mg/kg of body mass per h respectively, to maintain the steady-state levels. Drug was added to the resuscitation fluid described above. Thereafter animals received a 1:1 mixture of 0.9 % saline/5 % glucose infused at 10 ml/kg of body mass per h. Animals were observed for 48 h post-LPS or until premature death. Any animal appearing to be in significant discomfort was killed, as were animals whose MAP fell 0.05 compared with sham-operated animals without sepsis; Figure 2B) as was the rate of deterioration in standard base excess seen in the untreated animals which was significantly ameliorated by L-257 therapy (Figure 2C) Compared with the placebo, at 120 min post-drug bolus, animals with sepsis treated with L-257 had a higher gastrocnemius muscle perfused capillary density (with 30 mg/kg, P < 0.05; Figure 2D). In the short-term study assessing the effect of L-257 on noradrenaline requirements to maintain blood pressure (noradrenaline infusion rate 0.5–1.5 μg/kg of body mass per min), the L-257-treated animals required significantly less noradrenaline to maintain target pressures during a total of 3 h noradrenaline infusion (37.8 + − 3.2 μg compared with 47.8 + 1.3 μg; P < 0.05; Figure 2E). Significant reductions in − heart rate, urea and creatinine were also associated with supplementation of noradrenaline with L-257 (Table 2), whereas MAP was consistent in both groups (Figure 2F). In the control population of animals that underwent sham surgery and subsequent treatment with L-257, there
A DDAH1 inhibitor as a therapeutic agent in septic shock
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Figure 2 Effect of L-257 administration in the short-term endotoxaemic model of septic shock (n = 6 per group) at 2 h after bolus doses of L-257 at 0, 3 mg/kg of body mass and 30 mg/kg of body mass Data are medians and interquartile ranges. (A) An associated reduction in plasma nitrite/nitrate concentration. P < 0.05 compared with the untreated animals. (B) The administration of L-257 was associated with preservation of urine output in treated groups compared with the control, whereas untreated animals with sepsis showed a significant reduction in urine output. P < 0.05 compared with the untreated sham controls as indicated by the bracket. (C) L-257 was associated with a reduced rate of deterioration in the standard base excess in sepsis. *P < 0.05 compared with the untreated control. (D) Administration of L-257 had no negative effect on the perfused capillary density at 3 mg/kg of body mass and improved microvascular flow at 30 mg/kg of body mass following bolus administration. *P < 0.05 compared with the untreated animals as indicated by the bracket. (E) Administration of L-257 was associated with a reduction in the total amount of noradrenaline required to maintain normal MAP in the endotoxaemic model of septic shock. *P < 0.05 as indicated by the bracket. (F) Following the onset of hypotension (drop in MAP >20 % from baseline) therapy with noradrenaline alone or co-administration of L-257 with noradrenaline was undertaken to restore and maintain the MAP for the 180 min experimental period.
c The Authors Journal compilation c 2014 Biochemical Society
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Figure 3
Z. Wang and others
The effect of L-257 administration on polymicrobial peritoneal sepsis
(A) Long-term survival benefit seen with L-257 administration in a high-mortality model of polymicrobial sepsis induced by caecal ligation and puncture. P < 0.01 for all groups. (B) The effect of bolus doses of L-257 at 0, 3 mg/kg of body mass and 30 mg/kg of body mass on the MAP in the long-term study of the conscious tethered rats. The bolus L-257 was given at 16 h followed by a 10-min infusion. n = 6 in each group. (C) Change in NO production in a less-severe long-term model of polymicrobial sepsis induced by faecal slurry administration. *P < 0.05 compared with the control animals. (D) Incubation of peritoneal monocytes with an pro-inflammatory cocktail and either L-257 or L-NMMA (a global NOS inhibitor). No effect on phagocytosis of fluorescein-labelled LPS in isolated peritoneal monocytes in those cells incubated with L-257. However L-NMMA administration at a high dose resulted in significant inhibition of function. n = 3 per group. *P < 0.05.
was no difference in the MAP over the course of the experiment at any dose (Supplementary Figure 1A at http://www.biochemj.org/bj/460/bj4600309add.htm), no associated change in plasma ADMA (Supplementary Figure 1B), SDMA (Supplementary Figure 1C), arginine (Supplementary Figure 1D) or the arginine/ADMA ratio (Supplementary Figure 1E). There was no difference in cumulative urine output over the course of the experimental period in the treated animals compared with the controls (Supplementary Figure 1F).
In the second less-severe model where L-257 or placebo was commenced at 16 h after a lower-dose slurry, the MAP was better preserved at the end of the observation period (Figure 3B). Plasma nitrate/nitrite concentration was significantly lower in both treatment groups (P < 0.05; Figure 3C). The severity of tachycardia at the end of the experimental period was also reduced in both long-term treatment groups with associated trends towards improved stroke volume and renal function (Supplementary Table S3 at http://www.biochemj.org/bj/460/bj4600309add.htm).
Long-term peritonitis studies
Survival times were significantly longer (P < 0.01) in all treatment groups (18 h median) compared with the control (12 h) (Figure 3A). The decrease in the MAP was attenuated in the L257-treated animals compared with the controls with sepsis. These differences did not reach significance as increased mortality in the control group reduced the statistical power (Supplementary Table S2 at http://www.biochemj.org/bj/460/bj4600309add.htm). c The Authors Journal compilation c 2014 Biochemical Society
The effect of L-257 on peritoneal macrophage phagocytic capacity
L-257, at both 100 and 300 μM concentrations, had no effect on in vitro phagocytic function of peritoneal macrophages. However, the direct NOS inhibitor L-NMMA at concentrations similar to those achieved in clinical studies of global NOS inhibition significantly impaired phagocytic capacity (P < 0.05; Figure 3D).
A DDAH1 inhibitor as a therapeutic agent in septic shock DISCUSSION
Cardiovascular collapse is a cardinal feature of septic shock in which excessive NO accumulation causes profound vasodilatation, impaired vasoreactivity to exogenous catecholamines [19– 21] and myocardial depression. L-257 acts via inhibition of DDAH1 that, in turn, results in elevated intracellular levels of the endogenous NOS inhibitor ADMA. We hypothesized on the basis of our previous work [15,16] and have confirmed in the present study that L-257 causes a significant reduction in plasma nitrate/nitrite levels in sepsis, with a consequent improvement in cardiovascular function and indices of tissue perfusion with associated preservation of organ function and increased survival. Previous laboratory studies of novel therapeutic interventions in septic shock have often given treatment before, at or shortly after the induction of sepsis [22–24]. The models employed in the present study allow significant systemic compromise to occur, followed by appropriate fluid resuscitation and then vasopressor support before drug administration. This more closely replicates the pattern of therapy that might be employed in the clinical environment and resulted in a significant prolongation of survival time associated with improved macro- and micro-circulatory perfusion and the preservation of organ function without apparent toxicity at any dose, albeit in short experimental time courses. In clinical practice, catecholamine therapy is associated with a variety of complications [19] and, although not formally tested in prospective randomized trials, there are strong suggestions that catecholamine use is associated with excess mortality [25]. Agents that provide a catecholamine-sparing effect may prove advantageous, provided their safety profile is superior. Direct NOS inhibition and NO scavenging in sepsis provides short-term improvement in blood pressure [26], although at the expense of decreased cardiac output and worse survival rates [8,9]. Although an excess of NO production probably contributes to mortality in critically ill patients, some NO is essential to maintain coronary blood flow [27] and myocardial contractility [28], to decrease platelet adhesiveness [29], and to bolster innate immune defences, exerting both direct antimicrobial properties [30,31] and immunomodulatory effects [13]. DDAH1 is found throughout the systemic vasculature and may be localized within vessels to endothelial cells [32]. It is minimally expressed in the heart [33] and, therefore, L-257 is unlikely to impair NO-driven autoregulation of cardiac function in sepsis. Likewise, we show that L-257 has no direct effect on macrophage phagocytic ability, unlike the direct NOS inhibitor L-NMMA that compromised macrophage function. This ex vivo preservation of macrophage function is likely to be replicated in vivo as DDAH1 it is not expressed in immune cells [33]. Previous studies have also shown that elevated circulating ADMA has no effect on leucocyte number in rat models [34]. Given the absence of DDAH1 in immune cells and the specificity of L-257 for DDAH1, an exhaustive examination of the effects of the effect of L-257 on immune function was beyond the scope of the present study. The present study employs the Griess assay as an index of NO flux. Since NO production cannot be measured reliably during in vivo experiments, changes in the plasma nitrate/nitrite level measured using this technique have been widely employed as a measure of NO production. It should be noted that under normal circumstances nitrate levels may be altered by things other than changes in NO level such as diet. In our short-term experiments animals did not receive any enteral feeding and were treated with matched doses of crystalloid. In the long-term studies, there were no changes in diet during the experiment and so changes in exogenous nitrate uptake are unlikely to confound the results.
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Therapeutic doses of L-257 in our models increased plasma ADMA levels. However the absolute rise in plasma ADMA, although reflecting increased intracellular levels, is not sufficient to result in the systemic NOS inhibition that results from pharmacological doses of direct inhibitors. Although comparable fold elevations in plasma ADMA are associated with severity of illness and organ dysfunction in adults with severe sepsis or septic shock [35,36], it is unknown whether this is epiphenomenal, reflecting chronic cardiovascular dysfunction or causative. Of relevance, much higher plasma levels must be achieved in human volunteers to produce cardiovascular effects with exogenous administration of ADMA [37]. Secondary inhibition of nonDDAH1-expressing cells by treatment-induced increases in the circulating ADMA level is therefore unlikely. In summary, the present study demonstrates that L-257, a specific DDAH-1 inhibitor, may offer a novel, and seemingly safe, therapeutic option for septic shock. Additional studies are warranted to explore this drug further.
AUTHOR CONTRIBUTION Zhen Wang, Valerie Taylor, James Tomlinson, Alex Dyson and Elizabeth Sujkovic contributed to performing the experiments and collection of the data. Zhen Wang, Simon Lambden, James Leiper, Stephen Caddick, Neil McDonald, Mervyn Singer and Manasi Nandi contributed to conception of the study, and analysis and interpretation of data. James Leiper, Simon Lambden and Mervyn Singer wrote the paper.
FUNDING This work was supported a Wellcome Trust Seeding Drug Discovery Grant [to J.L. (Principal Investigator), M.S., S.C. and N.M.] and the British Heart Foundation [grant numbers PG/02/165/14797 and RG/02/005 (to J.L.)]. This work was undertaken at UCLH (University College London Hospitals)/UCL (University College London) who received a proportion of funding from the UK Department of Health’s NIHR (National Institute of Health Research) Biomedical Research Centres funding scheme. J.L. is an MRC (Medical Research Council) research programme leader. M.S. is an NIHR senior investigator.
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Biochem. J. (2014) 460, 309–316 (Printed in Great Britain)
doi:10.1042/BJ20131666
SUPPLEMENTARY ONLINE DATA
Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock Zhen WANG*†, Simon LAMBDEN*1 , Valerie TAYLOR†, Elizabeth SUJKOVIC*, Manasi NANDI‡, James TOMLINSON*, Alex DYSON†, Neil MCDONALD§, Stephen CADDICK, Mervyn SINGER†2 and James LEIPER*2 *Nitric Oxide Signalling Group, MRC Clinical Science Centre, Imperial College London, London, U.K. †Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, London, U.K. ‡Institute of Pharmaceutical Science, King’s College London, London, U.K. §School of Crystallography, Birkbeck College, University of London, London, U.K. Department of Chemistry, University College London, London, U.K.
EXPERIMENTAL Short-term model of endoxtoxaemia
While spontaneously breathing under 2 % isoflurane anaesthesia, the animals were placed on to a heating mat to maintain their core temperature 37 ◦ C, and then underwent cannulation of their right internal jugular vein and left carotid artery using PVC tubing (external diameter 0.96 mm; SteriHealth). The MAP was continually monitored and recorded on to a laptop computer using a precalibrated PowerLab system (Powerlab 16/30; ADInstruments). Urine drainage was facilitated via PVC tubing sited in the bladder lumen under direct vision. Vetergesic (buprenorphine hydrochloride; Alstoe Veterinary) was injected subcutaneously at 40 mg/kg of body mass. After instrumentation, the inhaled isoflurane concentration was dropped to 1.5 % and the animals allowed to stabilize for 15–30 min.
consecutive cycles from the start of each Doppler trace. Cardiac output was calculated as the product of stroke volume and heart rate. Long-term tethered awake model of faecal peritonitis
Briefly, under 2 % isoflurane anaesthesia, spontaneously breathing animals had their right internal jugular vein and left carotid artery cannulated using a PVC tubing catheter (external diameter 0.96 mm). The catheters were tunnelled subcutaneously to the nape of the neck and mounted on to a swivel/tether system that enabled unimpaired movement around the cage after recovery from anaesthesia. The MAP was monitored and recorded continuously using a precalibrated PowerLab system (ADInstruments). Blood sampling
Assessment of microcirulatory flow
In this short-term endotoxaemic model, blood flow within the microvasculature of the gastrocnemius muscle was visualized and recorded using SDF (sidestream dark-field) imaging (Microvision Medical BV) [1] at baseline and at 2 h post-bolus administration of L-257 or placebo. The device probe was gently placed on to the surface area of the muscle without causing a pressure artefact. Five video images, with recording time of 20 s each, were captured from different sites in each animal. Analysis of microcirculatory flow was made offline in a blinded way based on a scoring system developed by De Backer et al. [2].
Blood (1 ml) was anti-coagulated with EDTA (Sigma–Aldrich) for haematology analyses. A further 1 ml of blood was added to sodium citrate for subsequent coagulation assays. The remainder was allowed to clot for 20 min and then centrifuged at 3000 g for 10 min at 4 ◦ C. Serum was collected for biochemistry assays and other analyses. Measurements of arginine, ADMA and SDMA
Methylarginines were measured in serum and tissue lysates in the liver and kidney using LC–MS/MS as described previously [4].
Transthoracic echocardiography
Measurement of serum NOx (nitrate/nitrate)
Transthoracic echocardiography was performed at baseline and at the end of the experiment to assess cardiac function. In brief, using a Vivid 7 DimensionTM device (GE Healthcare) with a 14 MHz probe recording at a depth of 2 cm, aortic blood flow velocities were determined before the bifurcation of the right carotid artery using pulsed-wave Doppler; the direction of blood flow was confirmed by colour Doppler imaging. Stroke volume was determined as the product of the VTI (velocity time integral) and vessel cross-sectional area [π×(0.5×diameter)2 ]. Previous studies in rats of this age showed the aortic diameter to be 0.26 cm [3], thus a cross-sectional area of π×0.132 cm2 was assumed. Heart rate was determined by measuring the time between six
A modified Griess assay was used to determine NOx by first enzymatically converting nitrate into nitrite by the addition of NADPH-dependent nitrate reductase (Roche). Nitrite was then determined colorimetrically at 540 nM using a plate reader (V max Kinetic microplate reader; Molecular Devices). Standard curves for nitrite and nitrate enabled the calculation of total serum NOx concentration and the percentage nitrate conversion.
1 2
In vitro phagocytosis assay
Phagocytic function was evaluated using a commercial assay kit (Vybrant Phagocytosis Assay Kit; Molecular Probes). Peritoneal
To whom correspondence should be addressed (email [email protected]). Joint senior authors. c The Authors Journal compilation c 2014 Biochemical Society
Z. Wang and others Table S1
Biochemistry, haematology and coagulation variables obtained in a short-term study of organ function in a mouse model of endotoxaemia
n = 6 per group. *P < 0.05 compared with the control. ALT, alanine transaminase; APTT, activated partial thromboplastin time; Hb, haemoglobin; Plt, platelet count; PT, prothrombin time; WBC, white cell count. Groups with sepsis Variables
No L-257
3 mg of L-257/kg of body mass
30 mg of L-257/kg of body mass
Sham-operated group
Urea (mmol/l) Creatinine (μM) ALT (units/l) WBC (109 /l) Plt (109 /l) Hb (g/dl) PT (s) APTT (s)
16.62 + − 1.542 76.00 + − 17.0 230 + − 83.8 2.6 + − 0.75 204 + − 59.1 13 + − 0.91 15.50 + − 1.7 39.3 + − 5.0
12.24 + − 2.421 33.2 + − 5.6* 68.1 + − 9.1 1.3 + − 0.26 269 + − 46.7 12.6 + − 0.21 15.25 + − 1.6 37.3 + − 11.3
13.92 + − 1.094 42.80 + − 4.6 181 + − 99.7 1.3 + − 0.15 301 + − 42.5 11.7 + − 0.42 14.60 + − 0.4 3 3.8 + − 3.0
8.7 + − 3.6 26.6 + − 8.6 40.8 + − 5.9 4.5 + − 2.4 701 + − 97 11.8 + − 0.4 11.5 + − 0.8 22 + − 2.8
Table S2 Mean arterial pressure values and survivor numbers at sequential time points in control mice and animals treated with three different regimes of L-257 therapy in a long-term model of peritonitis Numbers in parentheses indicate the number of surviving animals at the corresponding time points.
Table S3
Groups
Baseline
Pre-treatment
12 h
18 h
24 h
Control 3 mg of L-257/kg of body mass 10 mg of L-257/kg of body mass 30 mg of L-257/kg of body mass
112 + − 2 (15) 112 + − 4 (15) 119 + − 2 (15) 113 + − 3 (15)
104 + − 3 (15) 107 + − 3 (15) 109 + − 3 (15) 107 + − 3 (15)
103 + − 6 (8) 98 + − 5 (10) 97 + − 9 (12) 91 + − 4 (12)
105 + − 4 (3) 98 + − 9 (7) 105 + − 5 (5) 93 + − 7 (7)
91 + − 3 (2) 87 + − 6 (4) 109 + − 4 (2) 95 + − 8 (4)
The effect of L-257 administration in a long-term model of sepsis induced by intraperitoneal faecal slurry administration
Echocardiographic examination and organ function assessment after 22 h on conscious tethered rats. n = 6 in each group. *P < 0.05 compared with the control. ALT, alanine transaminase; APTT, activated partial thromboplastin time; Hb, haemoglobin; Plt, platelet count; PT, prothrombin time; U, blood urea. Groups with sepsis Variables
No L-257
3 mg of L-257/kg of body mass
30 mg of L-257/kg of body mass
Sham-operated
Stroke volume (ml) Heart rate (b.p.m.) CO (ml/min) U (mM) Creatinine (μM) ALT (U/L) Leucocytes (109 /l) Plt (109 /l) Hb (g/dl) PT (s) APTT(s)
0.15 + − 0.01 550 + − 10.35 84.1 + − 2.5 9.2 + − 2.6 37.83 + − 6.82 80.20 + − 29.2 2.36 + − 0.45 156 + − 35.48 12.97 + − 0.2801 11.00 + − 0.3162 28.83 + − 2.738
0.19 + − 0.01* 501 + − 14.47* 95.5 + − 4.9 9.4 + − 1.6 40.67 + − 6.54 57.50 + − 9.26 2.88 + − 0.53 140 + − 22.98 13.67 + − 0.8586 10.80 + − 0.2000 30.80 + − 2.200
0.17 + − 0.01 504 + − 10.29* 87.4 + − 3.6 8.4 + − 0.8 33.80 + − 7.34 48.60 + − 8.17 3.17 + − 0.52 142 + − 28.54 13.50 + − 0.3592 10.60 + − 0.2449 36.80 + − 5.472
0.30 + − 0.03 413 + − 39 122 + −2 8.0 + − 1.6 25 + −3 38 + −7 5.5 + − 0.7 701 + − 91 11.1 + − 0.5 9+ − 0.5 20 + − 0.8
macrophages, isolated from peritoneal washouts of C57/BL6 mice, were incubated overnight at a cell density of 106 in a 96well plate and then stimulated by a cytokine cocktail consisting of 10 ng/ml TNFα (tumour necrosis factor α), 100 units/ml IFNγ (interferon γ ) and 5 μg/ml LPS (from Klebsiella pneumoniae; Sigma–Aldrich). Cells were allocated into five experimental groups in triplicate as: (i) control, and in the presence of (ii) 100 μM L-257, (iii) 300 μM L-257, (iv) 30 μM L-NMMA, and
c The Authors Journal compilation c 2014 Biochemical Society
(v) 100 μM L-NMMA. Three blank wells that were loaded with only culture medium were used as negative controls. The media was removed 4 h later, then fluorescein-labelled E. coli was added and the procedure performed as per the manufacturer’s instructions (Sigma–Aldrich). The wells were measured for fluorescence on a plate reader at 480 nm excitation and 520 nm emission (Fluoroskan Ascent FL; LabSystems), and results expressed as the fluorescence intensity.
A DDAH1 inhibitor as a therapeutic agent in septic shock
Figure S1 Impact of L-257 administration on na¨ıve animals (n = 6 per group) either untreated or 2 h after bolus doses of L-257 at 3 mg/kg of body mass, 30 mg/kg of body mass or 300 mg/kg of body mass Data are presented as the median and interquartile range. (A) The impact of the three dose regimens on the MAP over the 2 h following bolus dose administration. (B) Administration of L-257 does not increase plasma ADMA concentration in healthy animals. (C–E) L-257 administration has no impact on SDMA level (C), arginine level (D) or arginine/ADMA ratio (E). (F) Urine output is not affected by L-257 administration at any of the test doses.
c The Authors Journal compilation c 2014 Biochemical Society
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Received 19 December 2013/18 February 2014; accepted 10 March 2014 Published as BJ Immediate Publication 10 March 2014, doi:10.1042/BJ20131666
c The Authors Journal compilation c 2014 Biochemical Society
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