Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl ester in mononuclear cells by high-performance liquid chromatography with fluorescence detection.

Oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and anti-inflammatory effects. This paper describes a rapid, selective, and sensitive HPLC method with fluorescence detection for determination of 4'-geranyloxyferulic acid (GOFA) and its conjugate with l-nitroarginine methyl ester (GOFA-L-NAME) in mononuclear cells. Analytes were extracted from cells using methanol and eluted on a GraceSmart RP18 analytical column (250×4.6mm i.d., 5µm particle size) kept at 25°C. A mixture of formic acid 1% in water (A) and methanol (B) were used as mobile phase, at a flow-rate of 1.2mL/min in gradient elution. A fluorescence detector (excitation/emission wavelength of 319/398nm for GOFA and GOFA-L-NAME), was used for the two analytes. Calibration curves of GOFA and GOFA-L-NAME were linear over the concentration range of 1.0-50µg/mL, with correlation coefficients (r2)≥0.9995. Intra- and inter-assay precision do not exceed 6.8%. The accuracy was from 94% to 105% for quality control samples (2.0, 25.0 and 40µg/mL). The mean (RSD%) extraction recoveries (n=5) for GOFA and GOFA-L-NAME from spiked cells at 2.0, 25.0 and 40.0µg/mL were 92.4±1.5%, 94.7±0.9% and 93.8±1.1%, for GOFA and 95.3±1.2%, 94.8±1.0% and 93.9±1.3%, for GOFA-L-NAME. The limits of detection and quantification were 0.3µg/mL and 1.0µg/mL for GOFA and GOFA-L-NAME. This method was successfully applied to measure GOFA and GOFA-L-NAME concentrations in a mononuclear cells.

A newly synthesized compound, 4'-geranyloxyferulic acid-N(omega)-nitro-L-arginine methyl ester suppresses inflammation-associated colorectal carcinogenesis in male mice.

We previously reported the cancer chemopreventive activity of 4'-geranyloxyferulic acid (GOFA, Miyamoto et al., Nutr Cancer 2008; 60:675-84) and a ß-cyclodextrin inclusion compound of GOFA (Tanaka et al., Int J Cancer 2010; 126:830-40) in colitis-related colorectal carcinogenesis. In our study, the chemopreventive effects of a newly synthesized GOFA-containing compound, GOFA-N(omega)-nitro-L-arginine methyl ester (L-NAME), which inhibits inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX) enzymes, were investigated using a colitis-associated mouse colorectal carcinogenesis model with azoxymethane (AOM) and dextran sodium sulfate (DSS). The dietary administration of GOFA-L-NAME after the AOM and DSS treatments significantly reduced the multiplicity of adenocarcinomas (inhibition rates: 100 ppm, 84%, p < 0.001; 500 ppm, 94%, p < 0.001) compared with the AOM + DSS group. Dietary GOFA-L-NAME significantly decreased the proliferation (p < 0.001) and increased the apoptosis (p < 0.001) of colonic adenocarcinoma cells. A subsequent short-term experiment revealed that dietary GOFA-L-NAME decreased the mRNA expression of inflammatory enzymes, such as iNOS and COX-2, and proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6 and macrophage inflammatory protein (MIP)-2 in the colonic mucosa of mice that received 1.5% DSS in their drinking water for 7 days. Our findings indicate that GOFA-L-NAME is able to inhibit colitis-associated colon carcinogenesis by modulating inflammation, proliferation, apoptosis and the expression of proinflammatory cytokines in mice.

17ß-estradiol-linked nitro-L-arginine as simultaneous inducer of apoptosis in melanoma and tumor-angiogenic vascular endothelial cells.

Aggressive melanoma is commonly associated with rapid angiogenic growth in tumor mass, tumor cells acquiring apoptosis resistance, inhibition of cellular differentiation etc. Designing a single anticancer molecule which will target all these factors simultaneously is challenging. In the pretext of inciting anticancer effect through inhibiting nitric oxide synthase (NOS) via estrogen receptors (ER) in ER-expressing skin cancer cells, we developed an estrogen-linked L-nitro-arginine molecule (ESAr) for inciting anticancer effect in melanoma cells. ESAr showed specific anticancer effect through diminishing aggressiveness and metastatic behavior in melanoma cells and tumor. In comparison, ESAr showed significantly higher antiproliferative effect than parent molecule L-nitroarginine methyl ester (L-NAME, a NOS inhibitor) through induction of prominent apoptosis in melanoma cells. ESAr-pretreated aggressive melanoma cells could not form tumor possibly because of transformation/differentiation into epithelial-type cells. Furthermore, its antiangiogenic effect was demonstrated through ESAr-induced antiproliferation in HUVEC cells and apoptosis-induction in tumor-associated vascular endothelial cells, thereby significantly restricting severe growth in melanoma tumor. The targeting moiety, estrogen, at the therapeutic concentration of ESAr has apparently no effect in tumor-growth reduction. Albeit, no specific NOS-inhibition was observed, but ESAr could simultaneously induce these three cancer-specific antiaggressiveness factors, which the parent molecule could not induce. Our data rationalize and establish a new use of estrogen as a ligand for potentially targeting multiple cellular factors for treating aggressive cancers.

Effect of a blocker of nitric oxide production on albumin excretion by rat kidney.

Experiments on Wistar rats showed that single intraperitoneal injection nonselective NO-synthase inhibitor L-NAME in a dose of 50 mg/kg was followed by transient proteinuria and albuminuria. This effect was not reproduced by injection of ODQ, an inhibitor of intracellular effects of NO, and arginine, but D-NAME, an optical isomer of L-NAME not blocking NO-synthase, produced similar, though less pronounced effect. The degree of proteinuria and albuminuria increased in combined treatment with nitroarginine methyl esters and 1-deamino-arginine vasotocin or arginine vasopressin. Proteinuria during treatment with arginine derivatives attests to not only their effect on the charge of the filtration membrane, but also the participation of NO-dependent processes in the regulation of ultrafiltration in renal glomeruli.

Alterations in dendritic morphology of hippocampal neurons in adult rats after neonatal administration of N-omega-nitro-L-arginine.

The dendritic length and dendritic-spine density of the pyramidal neurons of the prefrontal cortex and the CA1 hippocampus of rats using the nonselective nitric oxide synthase inhibitor N-omega-nitro-L-arginine (L-NNA) at different postnatal day (P) periods of the brain development (P1-P3, P4-P6, and P7-P9) were assessed using Golgi-Cox staining after puberty (P60). At P4-P6, the L-NNA treatment produced a significant decrease of the dendritic length and dendritic-spine density of the pyramidal cells of the CA1 hippocampus. In addition, the dendritic length of the pyramidal neurons of the CA1 hippocampus decreased because of the L-NNA treatment at P1-P3. These data suggest that during a specific step in the development of the brain, the nitric oxide levels may play a critical role in the morphological modifications of the pyramidal neurons of the CA1 hippocampus at postpubertal age.

Probing heme coordination states of inducible nitric oxide synthase with a ReI(imidazole-alkyl-nitroarginine) sensitizer-wire.

Mammalian inducible nitric oxide synthase (iNOS) catalyzes the production of l-citrulline and nitric oxide (NO) from L-arginine and O2. The Soret peak in the spectrum of the iNOS heme domain (iNOSoxy) shifts from 423 to 390 nm upon addition of a sensitizer-wire, [ReI-imidazole-(CH2)8-nitroarginine]+, or [ReC8argNO2]+, owing to partial displacement of the water ligand in the active site. From analysis of competitive binding experiments with imidazole, the dissociation constant (Kd) for [ReC8argNO2]+-iNOSoxy was determined to be 3.0+/-0.1 microM, confirming that the sensitizer-wire binds with higher affinity than both L-arginine (Kd=22+/-5 microM) and imidazole (Kd=14+/-3 microM). Laser excitation (355 nm) of [ReC8argNO2]+-iNOSoxy triggers electron transfer to the active site of the enzyme, producing a ferroheme in less than approximately 1 micros.

Structure-based design and synthesis of N(omega)-nitro-L-arginine-containing peptidomimetics as selective inhibitors of neuronal nitric oxide synthase. Displacement of the heme structural water.

The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N'-nitroguanidine (1) and 4-N-(Nomega-nitro-l-argininyl)-trans-4-amino-l-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). On the basis of this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3-5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 5 and 6). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.

Inhibitory effects of benzoate on chiral inversion and clearance of N(G)-nitro-arginine in conscious rats.

N(G)-nitro-arginine (NNA) is known to exhibit stereoselective pharmacokinetics in which N(G)-nitro-d-arginine (d-NNA) has a faster clearance rate than N(G)-nitro-l-arginine (l-NNA) in anesthetized rats, and d-NNA undergoes unidirectional chiral inversion. It was postulated that chiral inversion of d-NNA was performed in a two-step pathway by d-amino acid oxidase (DAAO) followed by an unidentified transaminase. Such chiral inversion contributes (at least partially) to the pharmacokinetic stereoselectivity of NNA. This study used the selective inhibitor of DAAO, sodium benzoate, to test the above hypothesis. An i.v. bolus injection of d-NNA (32 mg/kg) and l-NNA (16 mg/kg) in conscious rats exhibited biphasic disposition with different pharmacokinetic parameters in a stereospecific manner (approximately 5-10-fold differences). Unidirectional chiral inversion of d-NNA but not l-NNA was found from these animals. In addition to its similar inhibitory effects on the d-NNA conversion and DAAO activity in kidney homogenates, sodium benzoate completely blocked chiral inversion of d-NNA and led to a smaller stereospecific difference, reflected by a nearly 50% reduction of d-NNA clearance and a 2-fold increase in t(1/2) and area under the curve of d-NNA in benzoate-pretreated rats. The results suggest that DAAO plays an essential role in chiral inversion of d-NNA and chiral inversion contributes mostly to the pharmacokinetic stereospecificity of NNA.

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action.

N(alpha)-vanillyl-N(omega)-nitroarginine (N - 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N - 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide 'NO' production induced by calcium ionophore in NG 108-15 cells. N - 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy (-10.2 kcal/mol) obtained from docking N - 1 to nNOS supported the additional mode of action of N - 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N - 1 at 75 micromol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.

Studies on the synthesis of N(G)-nitro-L-arginine derivatives and their effects on septic shock.

(HCl x N(G)-NO2-Arg)2Lys-OCH3, (HCl x N(G)-NO2-Arg)2Lys-OH, [(HCl x N(G)-NO2-Arg)2Lys]2Lys-OCH3, and [(HCl x N(G)-NO2-Arg)2Lys]2Lys-OH were synthesized by use of a solution method. Their effect on septic shock was studied in vivo. The results indicate that increasing the number of N(G)-NO2-Arg residues in a molecule may be useful to improve the response to septic shock.

N(omega)-Nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide (NO). Recently, we reported the dipeptide methyl ester, D-Phe-D-Arg(NO)()2-OMe (19), as a modest inhibitor of nNOS (K(i) = 2 microM), but with selectivity over iNOS as high as 1800-fold (Silverman, R. B.; Huang, H.; Marletta, M. A.; Martasek, P. J. Med. Chem. 1997, 40, 2813-2817). Here a library of 152 dipeptide amides containing nitroarginine and amino acids other than Phe are synthesized and screened for activity. Excellent inhibitory potency and selectivity for nNOS over eNOS and iNOS is achieved with the dipeptide amides containing a basic amine side chain (20-24), which indicates a possible electrostatic (or hydrogen bonding) interaction at the enzyme active site. The most potent nNOS inhibitor among these compounds is L-Arg(NO)()2-L-Dbu-NH(2) (23) (K(i) = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with a 192-fold selectivity over iNOS. These compounds do not exhibit time-dependent inhibition. The order and the chirality of the amino acids in the dipeptide amides have profound influences on the inhibitory potency as well as on the isoform selectivity. These dipeptide amide inhibitors open the door to the design of potent and highly selective inhibitors of nNOS.

The in vivo unidirectional conversion of nitro-D-arginine to nitro-L-arginine.

We recently reported that nitric oxide synthase in the brain can be inhibited not only by nitro-L-arginine (L-NA) but also by its D-enantiomer nitro-D-arginine (D-NA). In the present study, we found that D-NA, when tested in vitro, was 400 times less potent than L-NA. However, when D-NA was injected in vivo, its L-enantiomer, L-NA, was found to rapidly appear in plasma samples (approximately 1 min), rose to a maximum concentration at 30 min (approximately 40% conversion), and remained at this plateau for about 5 h. This was consistent with the changes in blood pressure. There was no conversion of L- to D-NA. The results suggested that D-NA has very weak biological actions by itself, but when administered in vivo, D-NA can be converted to L-NA.

Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). To define the mechanisms by which GAPDH is regulated by hypoxia, EC were exposed to cobalt, other transition metals, carbon monoxide (CO), deferoxamine, or cycloheximide in the presence or absence of hypoxia for 24 h, and GAPDH protein and mRNA levels were measured. GAPDH was induced in cells by the transition metals cobalt, nickel, and manganese and by deferoxamine, and GAPDH mRNA induction by hypoxia was blocked by cycloheximide. GAPDH induction by hypoxia, unlike that of other hypoxia-regulated genes, was not inhibited by CO or by 4,6-dioxoheptanoic acid, an inhibitor of heme synthesis. GAPDH induction was not altered by mediators of protein phosphorylation, a calcium channel blocker, a calcium ionophore, or alterations in redox state. GAPDH induction by hypoxia or transitional metals was partially blocked by sodium nitroprusside but was not altered by the inhibitor of nitric oxide synthase N omega-nitro-L-arginine. These findings suggest that GAPDH induction by hypoxia in EC occurs via mechanisms other than those involved in other hypoxia-responsive systems.