Detection of exhaled hydrogen sulphide gas in healthy human volunteers during intravenous administration of sodium sulphide.

INTRODUCTION: Hydrogen sulphide (H(2)S) is an endogenous gaseous signaling molecule and potential therapeutic agent. Emerging studies indicate its therapeutic potential in a variety of cardiovascular diseases and in critical illness. Augmentation of endogenous sulphide concentrations by intravenous administration of sodium sulphide can be used for the delivery of H(2)S to the tissues. In the current study, we have measured H(2)S concentrations in the exhaled breath of healthy human volunteers subjected to increasing doses sodium sulphide in a human phase I safety and tolerability study. METHODS: We have measured reactive sulphide in the blood via ex vivo derivatization of sulphide with monobromobimane to form sulphide-dibimane and blood concentrations of thiosulfate (major oxidative metabolite of sulphide) via ion chromatography. We have measured exhaled H(2)S concentrations using a custom-made device based on a sulphide gas detector (Interscan). RESULTS: Administration of IK-1001, a parenteral formulation of Na(2)S (0.005-0.20 mg kg(-1), i.v., infused over 1 min) induced an elevation of blood sulphide and thiosulfate concentrations over baseline, which was observed within the first 1-5 min following administration of IK-1001 at 0.10 mg kg(-1) dose and higher. In all subjects, basal exhaled H(2)S was observed to be higher than the ambient concentration of H(2)S gas in room air, indicative of on-going endogenous H(2)S production in human subjects. Upon intravenous administration of Na(2)S, a rapid elevation of exhaled H(2)S concentrations was observed. The amount of exhaled H(2)S rapidly decreased after discontinuation of the infusion of Na(2)S. CONCLUSION: Exhaled H(2)S represents a detectable route of elimination after parenteral administration of Na(2)S.

Washout of heme-containing proteins dramatically improves tetrazolium-based infarct staining.

INTRODUCTION: Methods to determine infarct size following ischemia-reperfusion injury include gross staining with triphenyltetrazolium chloride (TTC) and perfusion of colored dyes to demarcate the non-ischemic zone. Infarcted tissue (INF) can typically appear a mottled tan to brownish color, making a border between INF and TTC-positive tissue difficult to discern. Previous work in our lab indicated that following TTC staining, prolonged washing of thick sections dramatically sharpened this boundary. METHODS: Adult rats underwent 30 min ischemia via LAD ligation and reperfusion/recovery over 24 h. Hearts were then harvested, thick-sectioned, and stained with TTC. Stained sections were stored in PBS at 4 degrees C for up to 3 weeks. RESULTS: Histology on thin sections from infarcted hearts fixed directly after harvest revealed extensive hemorrhage within the INF. However, this hemorrhage is washed out when hearts are stored in PBS for 3 weeks. SDS-PAGE of PBS samples taken at 1, 2, and 3 weeks showed a low molecular weight band appearing over time. Peptide sequencing revealed the presence of several proteins including the heme-containing proteins (HCPs) hemoglobin, cytochrome c, and myoglobin. The loss of HCPs from thick sections to PBS corresponded with the blanching of the previously mottled INF within each section. HPLC analysis of these samples confirmed the loss of HCPs contributes to INF whitening. Further, analysis of infarct size values derived from heart slices with or without HCPs showed a significant decrease in measurement error when values were derived from slices without HCPs. DISCUSSION: These data suggest that HCPs in the heart tissue contribute to the non-uniform and discolored appearance of the INF, and that washout of these proteins produces an INF more easily distinguished from neighboring non-infarcted tissue. This method greatly reduces the error associated with infarct measurements and improves the analysis of the effects of drug treatments and other interventions designed to impact ischemia reperfusion injury.

Studying ischemia and reperfusion in isolated neonatal rat ventricular myocytes using coverslip hypoxia.

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the local buildup of metabolic byproducts that occur in ischemic myocardium in vivo. Coverslip hypoxia (CSH) is a recently described method for studying hypoxia and ischemia derived from the byproducts and metabolites of contractile ventricular myocytes. Hence, this method is dependent on the purity and contractile activity of the isolated myocytes. We describe herein methods for isolating neonatal rat ventricular myocytes with these characteristics, as well as means for performing CSH, identifying viable and compromised myocytes after coverslipping, and tracking pH changes during CSH.

Defining the minimally effective dose and schedule for parenteral hydrogen sulfide: long-term benefits in a rat model of hindlimb ischemia.

BACKGROUND: Peripheral arterial disease (PAD) affects millions of Americans and leads to critical limb ischemia (CLI) in the most severe cases. Investigators have demonstrated the utility of hydrogen sulfide for restoring perfusion in rodent models of chronic ischemia. We sought to determine the minimum effective dose (MED) of sulfide necessary to restore perfusion in the rat hindlimb, to assess the persistence of limb perfusion after cessation of treatment, and to compare perfusion measurements between laser doppler and ultrasound methods. METHODS: In 3 separate experiments, sodium sulfide (1.0, 0.5, or 0.25 mg/kg twice daily for 14 days, 0.25 mg/kg twice daily for 7 days, 0.5 mg/kg once daily for 7 days, or 0.25 mg/kg twice daily for 3 days) or vehicle was administered after left femoral artery ligation and transection. Hindlimb perfusion was assessed by laser doppler flowmetry and contrast enhanced ultrasound over the duration of each study, and cellular proliferation and vascular density were assessed by immunohistochemical means in the initial experiment. RESULTS: Intravenous sodium sulfide at 0.25, 0.5, or 1.0 mg/kg twice daily for 2 weeks significantly enhanced the recovery of blood flow to the ischemic hindlimb by 7 days. The enhancement of blood flow with 1.0 mg/kg dosing was coincident with an increase in cellular proliferation and vascular density in the ischemic tissue. In a final experiment, i.v. administration of sodium sulfide at 0.5 mg/kg once daily for 7 days or 0.25 mg/kg twice daily for 7 days significantly elevated blood flow and skeletal muscle perfusion in the ischemic hindlimb, whereas 0.25 mg/kg twice daily for 3 days had no effect. This enhancement of blood flow appeared long lived, as blood flow remained elevated 3 weeks after cessation of treatment. CONCLUSIONS: These data, together with other published observations, demonstrate the efficacy of hydrogen sulfide in restoring perfusion to chronically ischemic tissue and establish a minimum efficacious dose in the rat hindlimb model.

A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2)S) is a labile, endogenous metabolite of cysteine, with multiple biological roles. The development of sulphide-based therapies for human diseases will benefit from a reliable method of quantifying H(2)S in blood and tissues. EXPERIMENTAL APPROACH: Concentrations of reactive sulphide in saline and freshly drawn whole blood were quantified by reaction with the thio-specific derivatization agent monobromobimane, followed by reversed-phase fluorescence HPLC and/or mass spectrometry. In pharmacokinetic studies, male rats were exposed either to intravenous infusions of sodium sulphide or to H(2)S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H(2)S/HS(-) were concomitantly measured using an amperometric sensor. KEY RESULTS: Monobromobimane was found to rapidly and quantitatively derivatize sulphide in saline or whole blood to yield the stable small molecule sulphide dibimane. Extraction and quantification of this bis-bimane derivative were validated via reversed-phase HPLC separation coupled to fluorescence detection, and also by mass spectrometry. Baseline levels of sulphide in blood were in the range of 0.4-0.9 microM. Intravenous administration of sodium sulphide solution (2-20 mg x kg(-1) x h(-1)) or inhalation of H(2)S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg x kg(-1) x h(-1) of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H(2)S gas inhalation. CONCLUSIONS AND IMPLICATIONS: The monobromobimane derivatization method is a sensitive and reliable means to measure reactive sulphide species in whole blood. Using this method, we have established a bioequivalence between infused sodium sulphide and inhaled H(2)S gas.

Detection of exhaled hydrogen sulphide gas in rats exposed to intravenous sodium sulphide.

BACKGROUND AND PURPOSE: Sodium sulphide (Na(2)S) disassociates to sodium (Na(+)) hydrosulphide, anion (HS(-)) and hydrogen sulphide (H(2)S) in aqueous solutions. Here we have established and characterized a method to detect H(2)S gas in the exhaled breath of rats. EXPERIMENTAL APPROACH: Male rats were anaesthetized with ketamine and xylazine, instrumented with intravenous (i.v.) jugular vein catheters, and a tube inserted into the trachea was connected to a pneumotach connected to a H(2)S gas detector. Sodium sulphide, cysteine or the natural polysulphide compound diallyl disulphide were infused intravenously while the airway was monitored for exhaled H(2)S real time. KEY RESULTS: Exhaled sulphide concentration was calculated to be in the range of 0.4-11 ppm in response to i.v. infusion rates ranging between 0.3 and 1.1 mg x kg(-1) x min(-1). When nitric oxide synthesis was inhibited with N(omega)-nitro-L-arginine methyl ester the amount of H(2)S exhaled during i.v. infusions of sodium sulphide was significantly increased compared with that obtained with the vehicle control. An increase in circulating nitric oxide using DETA NONOate [3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene] did not alter the levels of exhaled H(2)S during an i.v. infusion of sodium sulphide. An i.v. bolus of L-cysteine, 1 g.kg(-1), and an i.v. infusion of the garlic derived natural compound diallyl disulphide, 1.8 mg x kg(-1) x min(-1), also caused exhalation of H(2)S gas. CONCLUSIONS AND IMPLICATIONS: This method has shown that significant amounts of H(2)S are exhaled in rats during sodium sulphide infusions, and the amount exhaled can be modulated by various pharmacological interventions.

Differentially regulated functional gene clusters identified during ischemia and reperfusion in isolated cardiac myocytes using coverslip hypoxia.

INTRODUCTION: Coverslip hypoxia (CSH) is a recently described method for producing rapid and severe ischemia derived from the metabolic activity of synchronously contracting isolated neonatal rat ventricular myocytes (NRVMs). While the effect of acute ischemia produced by CSH is documented, the contribution of reperfusion to cell viability has not been fully studied. METHODS: We therefore used fluorescence microscopy and expression profiling by microarray to determine the morphological and genetic effects in NRVMs of both the ischemic and reperfusion events of CSH. RESULTS: Fluorescence microscopy studies in coverslipped NRVMs showed cell death at 1 h as previously reported. Matched samples coverslipped for up to 2 h and then reperfused 18 h showed myocyte recovery prior to but not beyond 1 h upon post-staining, suggesting a limited window of recovery. Expression profiling of more than 30,000 genes using total RNA collected from NRVMs subjected to varying periods of ischemia and reperfusion revealed 103 genes regulated at least 2-fold at p<10(-7). These genes fall into discrete functional groups including apoptosis, metabolism, and hypoxia/acidosis. The regulation of a subset of genes from these groups was confirmed by RT-PCR. Interestingly, the hypoxia/acidosis gene BNip3 (a Bcl-2 family member implicated in hypoxia/acidosis-associated cell death) was upregulated early during ischemia and persisted throughout reperfusion. In addition, other hypoxia/acidosis genes such as heme oxygenase 1, pyruvate dehydrogenase kinase 1, prolyl hydroxylases, and hypoxia-inducible protein 2 were upregulated. DISCUSSION: These data suggest that the ischemic and reperfusion events created by CSH induce gene regulation within distinct functional groups related to in vivo ischemia.

Alfimeprase: a novel recombinant direct-acting fibrinolytic.

Alfimeprase is a recombinant, direct-acting fibrinolytic zinc metalloprotease. Alfimeprase has direct proteolytic activity primarily against the fibrin(ogen) Aalpha chain. Alfimeprase is covalently bound and neutralised by serum alpha(2)-macroglobulin, a prevalent mammalian protease inhibitor. Preclinical pharmacology studies have shown that fibrinolysis with alfimeprase is up to sixfold more rapid than with select plasminogen activators, such as tissue-type plasminogen activator and urokinase. Alfimeprase directly delivered to a site of thrombosis has the potential to be a fast and effective fibrinolytic, which does not generate the systemic lytic state seen with plasminogen activators that is associated with major bleeding, including intracerebral haemorrhage. Phase I and II studies in individuals with arterial or venous thrombotic events indicate that alfimeprase is active and generally well tolerated.

Non-clinical and clinical characterization of a novel acting thrombolytic: alfimeprase.

Alfimeprase (ALF) is a recombinant, truncated form of fibrolase, a directly fibrinolytic zinc metalloproteinase that was first isolated from the venom of the Southern copperhead snake (Agkistrodon contortrix contortrix). ALF has direct proteolytic activity against the fibrin(ogen) Aalpha chain. ALF can be covalently bound and neutralized by serum alpha2-macroglobulin, a prevalent mammalian protease inhibitor. Preclinical pharmacology studies have shown that thrombolysis with ALF is up to 6-times more rapid than with select plasminogen activators. Additional studies suggest that intra-thrombus ALF has the potential to be a fast and effective thrombolytic without generation of a systemic lytic state. Investigations of phases 1 and 2 indicate that ALF is active and generally well tolerated. This paper reviews the biochemical characteristics of ALF and a review of the preliminary clinical experience in subjects with acute peripheral arterial occlusion and in those with central venous access device occlusion.

A phase I trial of alfimeprase for peripheral arterial thrombolysis.

PURPOSE: To evaluate the safety profile, pharmacokinetics, and thrombolytic activity of alfimeprase, a novel direct-acting thrombolytic agent, in patients with chronic peripheral arterial occlusion (PAO). MATERIALS AND METHODS: In this multicenter, open-label, single-dose, dose-escalation study, 20 patients with worsening symptoms of lower extremity ischemia within 6 months of enrollment were treated with alfimeprase in five escalating dose cohorts (0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, and 0.5 mg/kg) by means of intraarterial and sometimes intrathrombic pulsed infusion. The primary endpoint was safety assessed by adverse event rates. Additional safety assessments included vital sign monitoring, serum chemistry testing, hematologic testing, and coagulation testing for 28 days after the procedure, as well as alpha2-macroglobulin and antialfimeprase antibody testing for as long as 3 months after treatment. Pharmacokinetic parameters were evaluated with use of an assay that measures free and alpha2-macroglobulin-bound (ie, total) alfimeprase. RESULTS: No patient experienced a hemorrhagic adverse event. Mean plasminogen and fibrinogen concentrations were not substantially altered by treatment. Three transient treatment-related adverse events were reported in the same patient: one incidence each of increased blood fibrinogen level, skin rash, and headache. All three adverse events were graded as mild. The pharmacokinetic profile of alfimeprase suggested that the half-life for total alfimeprase ranges from 11 to 54 minutes (median, 25 min) in patients with PAO. The serum alpha2-macroglobulin concentrations decreased transiently in a dose response-like manner between 12 and 24 hours and returned to within normal limits approximately 14 days after alfimeprase exposure. CONCLUSIONS: Alfimeprase in doses as high as 0.5 mg/kg was generally well-tolerated in patients with chronic PAO. No bleeding complications were noted. The stable fibrinogen concentrations suggest that the activity of alfimeprase may be limited to the target thrombus. Alfimeprase holds the potential to achieve dissolution of thrombus with a diminished risk of hemorrhage.

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro.

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.