Nitric oxide-releasing polyacrylonitrile disperses biofilms formed by wound-relevant pathogenic bacteria.

AIMS: To test the antimicrobial and antibiofilm properties of a nitric oxide (NO)-releasing polymer against wound-relevant bacterial pathogens. METHODS AND RESULTS: Using a variety of 96-well plate assay systems that include standard well plates and the minimum biofilm eradication concentration biofilm assay well plate, a NO-releasing polymer based on (poly)acrylonitrile (PAN/NO) was studied for antimicrobial and antibiofilm activity against the common wound pathogens Pseudomonas aeruginosa (PAO1), Staphylococcus aureus (Mu50) and Enterococcus faecalis (V583). The polymer was capable of dispersing single-species biofilms of Ps. aeruginosa as well as a more clinically relevant multispecies biofilm that incorporates Ps. aeruginosa along with Staph. aureus and Ent. faecalis. PAN/NO also synergistically enhanced the susceptibility of the multispecies biofilms to the common broad-spectrum antibiotic, ciprofloxacin. Multiple in vitro biocompatibility assays show that PAN/NO has limited potential for mammalian cytotoxicity. CONCLUSION: This study demonstrates the feasibility of utilizing the NO-releasing polymer, PAN/NO, to manage biofilms formed by wound-relevant pathogens, and provides proof-of-concept for use of this NO-releasing polymer platform across multiple disciplines where bacterial biofilms pose significant problems. SIGNIFICANCE AND IMPACT OF STUDY: In the clinical sector, bacterial biofilms represent a substantial treatment challenge for health care professionals and are widely recognized as a key factor in prolonging patient morbidity. This study highlights the potential role for the ubiquitous signalling molecule nitric oxide (NO) as an antibiofilm therapy.

Analysis of vasodilator responses to novel nitric oxide donors in the hindquarters vascular bed of the cat.

Controlled release of nitric oxide (NO*) may be useful in the treatment of a variety of vascular disorders. NO* donors of the diazeniumdiolate family with different rates of spontaneous NO* release have been synthesized. In the current study responses to seven diazeniumdiolate NO* donors (DEA/NO*, DETA/NO*, OXI/NO*, PIPERAZI/NO*, PROLI/NO*, SPER/NO*, and SULFI/NO*) were investigated in the hindquarters vascular bed of the cat. Intravenous injections of all NO* donors caused dose-dependent decreases in systemic arterial pressure and the rank order of potency was SNP > DEA/NO* > PIPERAZI/NO* > SPER/NO* > PROLI/NO* > OXI/NO*. Injections of all NO* donors into the hindlimb perfusion circuit caused dose-related decreases in hindquarters perfusion pressure that were similar to the order of potency in decreasing systemic arterial pressure. The rank order of the time required for the response to return to 50% of the maximal decrease in pressure (T(1/2)) and total duration of action of the NO* donors was SPER/NO* > PIPERAZI/NO* > DEA/NO* > OXI/NO* > DETA/NO* > PROLI/NO* > SULFI/NO*. After treatment with the NO* synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 mg/kg, i.v.), hindlimb vasodilator responses to the NO* donors were not significantly different, but vasodilator responses to acetylcholine were significantly reduced. After treatment with zaprinast (2 mg/kg, i.v.), a type V cyclic 3',5'-guanosine monophosphate-specific phosphodiesterase inhibitor, the duration of vasodilator responses to the NO* donors, as measured by T(1/2), was increased significantly, whereas the duration of the response to the beta2-adrenergic receptor agonist albuterol was unchanged. These data suggest that diazeniumdiolate NO* donors are endothelium-independent, directly stimulate soluble guanylate cyclase, and decrease vascular resistance by increasing cyclic 3',5'-guanosine monophosphate levels in the hindquarters vascular bed of the cat.

Reaction of nitric oxide at the beta-carbon of enamines. A new method of preparing compounds containing the diazeniumdiolate functional group.

The reaction of nitric oxide (NO) with enamines has been investigated. Unlike previously reported reactions of NO as a free radical with alkenes, the electrophilic addition of NO to the beta-carbon of enamines results in the formation of compounds containing the diazeniumdiolate functional group (-[N(O)NO](-)). This reaction between NO and enamines has been shown to be quite general and a variety of enamine-derived diazeniumdiolates have been isolated and characterized. While enamines derived from aldehydes and ketones whose structures allow for sequential multiple electrophilic additions tended to undergo overreaction leading to unstable products, it has been shown that this complication may be overcome by suitable choice of reaction solvent. The products obtained may exist as zwitterionic iminium salts or as neutral species depending upon the structure of the parent enamine. The diazeniumdiolate derived from 1-(N-morpholino)cyclohexene is unique among the new compounds in that it spontaneously releases NO upon dissolution in buffered aqueous solution at pH 7.4 and 37 degrees C. While the total quantity of NO released by this material (ca. 7% of the theoretical 2 moles) is apparently limited by a competing reaction in which it hydrolyzes to an alpha-diazeniumdiolated carbonyl compound and the parent amine, this feature may prove to be of great value in the development of multiaction pharmaceuticals based upon this new type of NO-releasing compound. Reports of enzymatic (oxidative) release of NO from previously known carbon-bound diazeniumdiolates also suggest that analogues of these compounds may be useful as pharmaceutical agents. This new method of introducing the relatively rarely studied diazeniumdiolate functional group into organic compounds should lead to further research into its chemical and biological properties.

Conversion of proteins to diazeniumdiolate-based nitric oxide donors.

Michael reaction of the methoxymethyl-protected monodiazeniumdiolate of piperazine (MOM-PIPERAZI/NO) with 4-maleimidobutyric acid followed by its conversion to the N-hydroxy-succinimido ester produces a reagent capable of transferring the nitric oxide (NO)-donating diazeniumdiolate group to the terminal amines of the lysine residues contained in proteins. The reagent has been used to produce diazeniumdiolated bovine serum albumin (D-BSA) and diazeniumdiolated human serum albumin (D-HSA) containing 22 and 19 modified lysyl groups, respectively. Upon dissolution in pH 7.4 phosphate buffer at 37 degrees C, these albumin derivatives gradually released all of their contained NO (approximately 40 mol/mol of protein) with initial rates of about 30-40 pmol/min/mg and half-lives on the order of 3 weeks. This methodology is now available for use in exploiting the unique specific metabolic interactions of proteins to target NO therapy to specific physiological processes in vivo.

Induction of penile erection by intracavernosal and transurethral administration of novel nitric oxide donors in the cat.

PURPOSE: The effects of novel nitric oxide (NO) donors administered intracavernosally and transurethrally on erectile function in the anesthetized cat were evaluated. MATERIALS AND METHODS: In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal and transurethral injections of novel NO donors (MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZI/NO and PROLI/NO). All parameters were measured after administration of NO donors intracavernosally via a 30-gauge needle and urethrally via a Jelco i.v. catheter in a volume of 200 microliters. Systemic arterial pressure was also assessed in these experiments. All NO donors were compared with a triple-drug control combination comprised of papaverine (1.65 mg.), prostaglandin E1 (0.5 microgram.), and phentolamine (25 micrograms.). RESULTS: MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZI/NO and PROLI/NO induced dose dependent increases in intracavernosal pressure and penile length (p < 0.05) when administered intracavernosally. The increases in cavernosal pressure and penile length were comparable to those observed with the triple-drug control combination. The maximum increase in cavernosal pressure in response to PROLI/NO and PAPA/NO was associated with no significant change in systemic arterial pressure. Transurethral administration of PROLI/NO and PIPERAZI/NO induced dose-dependent increases in cavernosal pressure and penile length (p < 0.05). The response was similar to that of the triple-drug control combination, except that transurethral PROLI/NO and PIPERAZI/NO had no significant effect on systemic blood pressure. CONCLUSIONS: NO donors caused dose-dependent increases in cavernosal pressure when administered intracavernosally and transurethrally. These data suggest further exploration of the use of NO donors for the treatment of erectile dysfunction.

Pharmacokinetics and consistency of pericardial delivery directed to coronary arteries: direct comparison with endoluminal delivery.

BACKGROUND AND HYPOTHESIS: Pharmacologic modulation of the contents of the pericardial space has been shown to influence the response of coronary arteries to balloon injury. Endoluminal (EL) local delivery of various drugs into coronaries has been found to be limited by short residence time, as well as by highly variable deposited agent concentration. We hypothesized that compounds placed into the pericardial space (P) would penetrate into coronary tissue with greater consistency than seen after EL delivery and provide for prolonged coronary exposure to agents. METHODS AND RESULTS: 125I-labeled basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), albumin, or 131I-labeled diazeniumdiolated albumin (NONO-albumin) were delivered as model/therapeutic proteins into the porcine pericardial space (n = 15 pigs) or into coronaries using an EL delivery catheter (n = 48 arteries). In subjects receiving 125I-labeled proteins, the delivery target or mid-regions of the left anterior descending (LAD) and left circumflex (LCx) arteries were harvested at 1 h or 24 h for gamma-counting and autoradiography, and fractional intramural delivery (FID) or retention measured as percent agent in 100 mg artery/agent in infusate for both time points. In the animals receiving 131I-labeled NONO-albumin, serial gamma imaging was employed to evaluate the rate of redistribution in individual animals following either pericardial or endoluminal delivery. At 1 h, FID values ranged from 0.00064 to 0.0052% for P delivery (median 0.0022%), and from 0.00021 to 6.7 for EL delivery (median 0.27%). At 24 h, FID values ranged from 0.00011 to 0.003 for P delivery (median 0.0013), and from 0.0002 to 1.4 for EL delivery. The estimated T1/2 for bFGF redistribution from the vascular tissue was 22 h (P) and 7 h (EL), respectively, while the directly determined T1/2 values for NONO-albumin redistribution from the delivery region were 22.2 h (P) and 2.5 h (EL). CONCLUSIONS: These data show that pericardial fluid contents can access coronary arteries with intramural concentrations which typically vary by 10-15-fold, while EL delivery results in a remarkably wide intramural concentration range with up to 33,000-fold variability. The apparent redistribution rate is more rapid following EL delivery, possibly due to sustained diffusive tissue loading from the pericardial space. Pericardial delivery appears to offer substantial advantages over EL administration with respect to residence time and reproducibility.

Nitric oxide-releasing polymers containing the [N(O)NO]- group.

Ions of structure X[N(O)NO]- display broad-spectrum pharmacological activity that correlates with the rate and extent of their spontaneous, first-order decomposition to nitric oxide when dissolved. We report incorporation of this functional group into polymeric matrices that can be used for altering the time course of nitric oxide release and/or targeting it to tissues with which the polymers are in physical contact. Structural types prepared include those in which the [N(O)NO]- group is attached to heteroatoms in low molecular weight species that are noncovalently distributed throughout the polymeric matrix, in groupings pendant to the polymer backbone, and in the polymer backbone itself. They range in physical form from films that can be coated onto other surfaces to microspheres, gels, powders, and moldable resins. Chemiluminescence measurements confirm that polymers to which the [N(O)NO]- group is attached can serve as localized sources of nitric oxide, with one prototype providing sustained NO release for 5 weeks in pH 7.4 buffer at 37 degrees C. The latter composition, a cross-linked poly-(ethylenimine) that had been exposed to NO, inhibited the in vitro proliferation of rat aorta smooth muscle cells when added as a powder to the culture medium and showed potent antiplatelet activity when coated on a normally thrombogenic vascular graft situated in an arteriovenous shunt in a baboon's circulatory system. The results suggest that polymers containing the [N(O)NO]- functional group may hold considerable promise for a variety of biomedical applications in which local delivery of NO is desired.

Nitric oxide/nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release.

Cell-mediated antitumor effects have, in part, been attributed to the production of NO. Compounds which generate NO might, therefore, be useful in attenuating the growth of tumor cells. Six nitric oxide/nucleophile adducts that release NO spontaneously in solution were tested for their effectiveness in inhibiting DNA synthesis in A375 human melanoma cells. The complexes of NO with spermine, 3-(n-propylamino)propylamine (PAPA/NO), and diethylamine reduced thymidine incorporation by 50% at concentrations of 24, 44, and 128 microM, respectively. The degree of inhibition was, in general, related to the rate and extent of NO release in solution. A melanoma cell clone sensitive to interleukin 1-mediated cytostasis (A375-C6) was no more sensitive to PAPA/NO than a clone resistant to interleukin 1 (A375-C5), suggesting that the differing inhibitory effects of interleukin 1 in the two A375 cell clones are not due to a differential sensitivity to nitric oxide. Oxymyoglobin (125 microM), a known scavenger of NO, restored the ability of A375-C6 cells to incorporate thymidine in the presence of up to 200 microM PAPA/NO. When PAPA/NO was added to a solution of oxymyoglobin, nitrosylmyoglobin was formed, indicating that the protective effect of myoglobin was due to scavenging of NO. The results are consistent with a nitric oxide-mediated mechanism for NO/nucleophile cytostasis and suggest that such compounds may be useful as tools for investigating the role of reactive nitrogen intermediates in cytostasis and cytotoxicity.

Tumor-promoting and hepatocarcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in DBA/2NCr and C57BL/6NCr mice and an apparent promoting effect on nasal cavity tumors but not on hepatocellular tumors in F344/NCr rats initiated with N-nitrosodiethylamine.

The tumor-promoting and carcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in the liver and in other organs were quantified and compared to those of phenobarbital (PB) in two inbred strains of mice (C57BL/6NCr, DBA/2NCr) and in F344/NCr rats initiated at 5 weeks of age with N-nitrosodiethylamine (NDEA; 90 mg/kg in mice, 75 mg/kg in rats). Two weeks later animals were placed on a regimen of TCPOBOP once every 2 weeks (administered i.p. or i.g.) or on a diet containing 500 p.p.m. PB as a positive control for the duration of the experiment. Mice were administered TCPOBOP (3.0 mg/kg/dose) for 30 weeks followed by control diet, while rats were given the TCPOBOP regimen (3.0 or 30 mg/kg/dose) for the full 78 weeks of the experiment. TCPOBOP was a complete carcinogen and an extremely potent promoter in both strains of mice, particularly the DBA strain in which NDEA followed by TCPOBOP (i.p.) resulted in death of all the animals within 30 weeks from multiple hepatocellular tumors. TCPOBOP alone induced 100% tumor incidence in DBA mice within 60 weeks. In addition, in both strains of mice, a high proportion of those animals with liver tumors had metastases to the lungs. In contrast, TCPOBOP was ineffective as a liver tumor promoter in F344 rats at even 10 times the dose administered to mice. Interestingly however, TCPOBOP, when given subsequent to NDEA, caused a significant increase in nasal cavity tumors in F344 rats. PB was an effective liver tumor promoter in male DBA mice and male F344 rats, but was relatively ineffective as a promoter in C57 mice. When tumor-promoting activity and induction of cytochrome P450 IIB1 were compared, good agreement between these two parameters was observed. PB was an effective inducer of P450 IIB1 in the rats and in both strains of mice and a potent liver tumor promoter in both DBA mice and F344 rats, whereas TCPOBOP was a potent inducer and tumor promoter in both strains of mice but was negligibly effective as either an inducer or a promoter in F344 rats at even 10-fold higher dosage.

Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects.

Selected nucleophile/nitric oxide adducts [compounds which contain the anionic moiety, XN(O-)N = O] were studied for their ability to release nitric oxide spontaneously in aqueous solution and for possible vasoactivity. The diversity of structures chosen included those in which the nucleophile residue, X, was that of a secondary amine [Et2N, as in [Et2NN(N = O)O]Na, 1], a primary amine [iPrHN, as in [iPrHNN(N = O)O]Na, 2], a polyamine, spermine [as in the zwitterion H2N(CH2)3NH2+(CH2)4N[N(N = O)O-](CH2)3NH2, 3], oxide [as in Na[ON(N = O)O]Na, 4], and sulfite [as in NH4[O3SN(N = O)O]NH4, 5]. The rate constants (k) for decomposition in pH 7.4 phosphate buffer at 37 degrees C, as measured by following loss of chromophore at 230-260 nm, were as follows: 1, 5.4 x 10(-3) s-1; 2, 5.1 x 10(-3) s-1; 3, 0.30 x 10(-3) s-1; 4, 5.0 x 10(-3) s-1; and 5, 1.7 x 10(-3) s-1. The corresponding extents of nitric oxide release (ENO) were 1.5, 0.73, 1.9, 0.54, and 0.001 mol/mol of starting material consumed, respectively, as determined from the integrated chemiluminescence response. Vasodilatory activities expressed as the concentrations required to induce 50% relaxation in norepinephrine-constricted aortic rings bathed in pH 7.4 buffer at 37 degrees C (EC50) were as follows: 1, 0.19 microM; 2, 0.45 microM; 3, 6.2 microM; 4, 0.59 microM; and 5, 62 microM. Vasorelaxant potency (expressed as 1/EC50) was strongly correlated with the quantity of .NO calculated from the physicochemical data to be released in the interval required to achieve maximum relaxation at the EC50 doses (r = 0.995). This suggests that such nucleophile/.NO adducts might generally be useful as vehicles for the nonenzymatic generation of nitric oxide, in predictable amounts and at predictable rates, for biological purposes. The particular significance for possible drug design is underscored in the very favorable potency comparison between several of these agents and the established nitrovasodilators sodium nitroprusside and glyceryl trinitrate (EC50 values of 2.0 and greater than 10 microM, respectively) in parallel aortic ring tests.

Stereoselectivity in the microsomal conversion of N-nitrosodimethylamine to formaldehyde.

The possibility that N-nitrosodimethylamine (NDMA) might be metabolized preferentially at either the syn (relative to the nitroso oxygen) or the anti methyl group has been examined by comparing the rates of formaldehyde production when unlabeled NDMA, its fully deuteriated analogue (NDMA-d6), and (Z)- or (E)-N-nitrosomethyl(methyl-d3)amine (NDMA-d3) were incubated in turn at concentrations of 0-2.4 mM with acetone-induced rat liver microsomes. The Km values for the conversion of (Z)- and (E)-NDMA-d3 to formaldehyde were identical to each other within experimental error (32 +/- 2 and 35 +/- 1 microM, respectively) but different from those for NDMA (24 +/- 6 microM) and NDMA-d6 (116 +/- 3 microM); similar Vmax values were observed for the four isotopic variants [7.5-8.1 nmol/(mg of protein.min)]. The observed similarity of kinetic parameters for (Z)- and (E)-NDMA-d3 suggested that the isotopic composition of the methyl group is an energetically more important determinant of its rate of oxidation at the NDMA demethylase active site than is its orientation relative to the nitroso oxygen atom. The absence of syn vs anti stereospecificity was confirmed via product isolation studies, in which the formaldehyde generated from each of the four isotopomers was trapped as the dimedone adduct and assayed for deuterium content by mass spectrometry; again, a strong preference for metabolism at CH3 vs CD3 regardless of stereochemistry was observed, though the data on CH2O generation suggested that there may be a slight net excess of anti attack. The results indicate that the microsomal enzymes employed display little regioselectivity in metabolizing the syn vs anti methyl groups of NDMA.

Deuterium isotope effect on the toxicokinetics of monomethylamine in the rat.

The single-dose toxicokinetics of monomethylamine has been characterized in the rat by HPLC assay of serial blood samples. Biphasic first-order elimination was observed following an iv bolus dose of 19 mumol/kg with a terminal half-life of 19.1 +/- 1.3 min (mean +/- SE, N = 4). The apparent steady state volume of distribution, systemic blood clearance, and renal blood clearance were 1.21 +/- 0.09 liter/kg, 53.4 +/- 3.5 ml/min/kg, and 5.72 +/- 0.53 ml/min/kg, respectively. The administration of an intragastric dose permitted the calculation of the systemic bioavailability of monomethylamine as 69 +/- 3%. Duplicate experiments using the structural analogue with deuterium atoms substituted for hydrogens on the methyl group revealed a much slower elimination of the compound, although ultimately, 5 times as much was excreted unchanged in the urine. Isotope effects calculated as the ratios of terminal half-life, systemic blood clearance, and systemic bioavailability were 1.9, 2.2, and 1.8, respectively.

Sex differences in the single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine in the rat.

The single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine (NMHA) has been characterized in 8-week-old Fischer 344 rats by analysis using high-performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to male rats revealed biphasic first-order elimination with a terminal half-life of 37.4 +/- 1.7 min for unchanged NMHA and 101 +/- 6 min for total radioactivity, and extensive conversion to polar metabolites was seen in the high-performance liquid chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMHA were 13.1 +/- 0.9 ml/min/kg, and 685 +/- 31 ml/kg, respectively. Renal blood clearance and intrinsic hepatic clearance were estimated to be 0.805 +/- 0.024 and 16.7 +/- 2.1 ml/min/kg, respectively. A similar dose given to female rats yielded a terminal half-life for NMHA of 27.2 +/- 1.2 min, a steady-state volume of distribution of 652 +/- 23 ml/kg, and systemic blood, renal blood, and intrinsic hepatic clearances of 16.9 +/- 1.3, 1.45 +/- 0.14, and 22.5 +/- 0.3 ml/min/kg, respectively. The sex differences in terminal half-life and systemic blood, renal blood, and intrinsic hepatic clearances were significant at the P less than 0.05 level. Larger doses given by gavage, which appeared to be completely absorbed from the gut, indicated systemic bioavailabilities for unchanged NMHA of 78 +/- 10% and 69 +/- 1% for male and female rats, respectively. Binding of NMHA to plasma proteins was found to be negligible. Taken together the data allow for the conclusion that the observed sex differences in toxicokinetic parameters are due to differences in the intrinsic hepatic clearance of the compound. This difference in the ability of the liver to metabolize NMHA in vivo correlates with and may contribute to the greater susceptibility of female rats to hepatocarcinogenesis and of male rats to development of tumors in the nasal epithelium following oral exposure to NMHA.

Deuterium isotope effect on denitrosation and demethylation of N-nitrosodimethylamine by rat liver microsomes.

In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([2H6]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The Vmax calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the Vmax for either pathway, but it did increase the Km for demethylation from 0.06 to 0.3 mM. The Km for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-15N-labeled NDMA with [2H6]NDMA and measuring the methyl[15N]amine:[2H3]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the Km values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [VmaxH/VmaxD approximately 1 and (Vmax/Km)H/(Vmax/Km)D approximately 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an alpha-nitrosamino radical which either combines with a hydroxyl radical to form the alpha-hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.