Bioconversion of isosorbide dinitrate by various microorganisms

Summary A total of 19 microorganisms, selected from genera of bacteria, fungi and yeasts, were screened for their ability to hydrolyse isosorbide dinitrate (ISDN) to mononitrates. Cunninghamella echinulata and Cunninghamella elegans showed rates of bioconversion of ISDN of 74% and 88% respectively, measured after 73 h. However, the two strains exhibited opposite stereoselectivity, as reflected in the ratios of isosorbide 5-mononitrate (5-ISMN) to isosorbide 2-mononitrate (2-ISMN). These were 2.57 and 0.75 for C. echinulata and C. elegans, respectively.     

High-dosage isosorbide dinitrate therapy for angina.

We investigated the possibility that high dosages (480 mg/d) of isosorbide dinitrate might reduce the frequency of angina attacks in selected patients who had not responded to low dosages of the drug (40 mg/d), and that the patients could tolerate the high levels of medication and maintain their responsiveness over the long term. In the single-blind phases of this trial 24 patients with grade 3 stable angina pectoris were given a placebo for 4 weeks and then increasing doses of isosorbide dinitrate for a further 6 weeks. The 19 patients who both responded to and tolerated high doses of the drug kept taking 480 mg/d for an average of 1 year. The average weekly rate of angina attacks fell by 74%, from 6.05 in the placebo phase to 1.6 during long-term active treatment (p less than 0.01). Nitroglycerin consumption decreased accordingly. The patients'' assessments of their levels of activity and well-being and their angina thresholds showed improvement among most of them. The trend of angina frequency was stable in 12 cases, downward in 6 and upward in only 1 case. Exercise performance as evaluated by a graded treadmill test showed a small but nonsignificant improvement of 18%. It was concluded that some patients who do not respond to the antianginal action of low-dosage isosorbide dinitrate and cannot be given beta-blockers may respond to high dosages and tolerate them for over a year. Isosorbide dinitrate may be clinically useful in patients with coronary heart disease even though their exercise performance is not significantly improved.     

Gas chromatographic determination of isosorbide dinitrate in human plasma and urine

A rapid, simple and sensitive method for the specific determination of isosorbide dinitrate concentrations down to 0.5 ng/ml in human plasma and urine is described. Following traction (with or without internal standard) of isosorbide dinitrate into toluene, the compound is determined by gas chromatography using a ⁶³Ni electron-capture detector.     

Biological denitration of propylene glycol dinitrate byBacillus sp. ATCC 51912

In previous studies, bacterial cultures were isolated that had the ability to degrade the nitrate ester glyceryl trinitrate (i.e. nitroglycerin). The goal of the present study was to examine the ability of resting cells and cell-free extracts of the isolateBacillus sp. ATCC 51912 to degrade the more recalcitrant nitrate ester propylene glycol dinitrate (PGDN). It was observed that the PGDN-denitrating activity was expressed during growth even when cells were cultured in the absence of nitrate esters. This indicates that nitrate esters are not required for expression of denitration activity. Using cell-free extracts, PGDN was observed to be sequentially denitrated to propylene glycol mononitrate (PGMN) and propylene glycol with the second denitration step proceeding more slowly than the first. Also it was observed that dialysis of the cell-free extracts did not affect denitration activity indicating that regenerable cofactors [e.g. NAD(P)H or ATP] are not required for denitration.     

Pharmacokinetic-hemodynamic studies of the enantiomers of isoidide dinitrate in conscious rats.

Our objective was to determine the pharmacokinetic properties of the D- and L-enantiomers of isoidide dinitrate (IIDN) in relation to their hemodynamic effects. Conscious male Sprague-Dawley rats were administered a bolus i.v. dose of 2 mg.kg-1 D- or IIDN and simultaneous blood samples and blood pressure recordings were taken at various times. The elimination half-life of D-IIDN was significantly shorter than that of L-IIDN (10 vs. 16 min) owing to a larger Vd area of the L-enantiomer (5.8 vs. 3.8 L.kg-1). The plasma clearance of either enantiomer was approximately 250 mL.min-1.kg-1, a value equal to plasma cardiac output. The pharmacokinetic data indicates that IIDN is distributed extensively and that significant extrahepatic biotransformation of the drug occurs. After intravenous administration of D-IIDN, there was an initial decrease in mean arterial pressure (MAP) of 29% compared with 15% for L-IIDN (p less than 0.05). For L-IIDN, the decrease in MAP was short lived (less than 2 min), while for D-IIDN, MAP remained significantly decreased for up to 60 min. The oral bioavailability of both enantiomers was low (ca. 7%). However, decreases in MAP occurred after oral administration of D-IIDN, suggesting that the mononitrate metabolite of IIDN was pharmacologically active. We conclude that, despite a faster rate of elimination from the central compartment, D-IIDN exhibits a greater vasodilator effect in the intact animal compared to L-IIDN. This is consistent with previous observations of a 10-fold greater potency of D-IIDN for relaxation of isolated vascular smooth muscle.     

Differential biotransformation of the enantiomers of isoidide dinitrate in isolated rat aorta

Previous studies have demonstrated that the D-enantiomer of isoidide dinitrate (IIDN) is 10-fold more potent than the L-enantiomer for relaxation and cyclic GMP accumulation in isolated rat aorta. To test whether preferential biotransformation of D-IIDN to a species that activates guanylate cyclase is the basis for this observed enantioselectivity, paired segments of rat aorta were exposed to D- and L-IIDN and the tissue accumulation of the parent compound and the formation of their respective metabolites (D- and L-isoidide mononitrate, IIMN) were determined. The extent of relaxation of rat aorta following exposure to 2 microM D-IIDN was greater than that by L-IIDN over a 5-minute time course, and this was associated with a higher rate of D-IIDN biotransformation to D-IIMN at all time points. In addition, the rate of D-IIDN biotransformation was greater than that of L-IIDN at most IIDN concentrations tested. By contrast, the amount of D- and L-IIDN in the tissue was the same at all time points and concentrations tested, indicating that selective uptake of D-IIDN into blood vessels did not occur. When tissues were made tolerant to organic nitrate-induced relaxation by treatment with a high concentration of glyceryl trinitrate, the biotransformation of both D- and L-IIDN was attenuated. This suggests that mechanism-based biotransformation may be affected during tolerance development. Furthermore, the association of preferential D-IIDN biotransformation with its greater potency for vasodilation and cyclic GMP accumulation suggests than an enantioselective site for biotransformation is an important component of organic nitrate-induced vasodilation.     

Isosorbide dinitrate bioconversion by Beauveria strains: implication of glutathione transferase levels

Summary The level of glutathione S-transferase (GSH0ST) activity was determined in growing cultures and in washed resting cells of Beauveria strains with and without addition of isosorbide dinitrate (ISDN), by following the reaction with o-dinitrobenzene (o-DNB). The level of GSH-ST varied according to the pH changes of the medium and decreased during culture. The enzymatic activity measured with o-DNB did not correlate with ISDN bioconversion carried out either with B. sulfurescens or B. tenella. Immediately after starting incubation of the resting cells with ISDN, the level of GSH-ST activity initially increased, but declined afterwards, whereas the bioconversion process continued and reached 500 mg/l isosorbide 5-mononitrate. When 1-chloro-2,4-dinitrobenzene was used as a substrate for the evaluation of GSH-ST activity using B. tenella, a conjugation product having a UV absorption at 410 nm was formed.     

The effects of isosorbide dinitrate on methemoglobin reductase enzyme activity and antioxidant states

Isosorbide dinitrate (ISDN) has been used in the treatment of ischaemic cardiovascular diseases for many years. ISDN is the most popular nitric oxide donor and causes methemoglobinemia as an important side-effect. The purpose of this study was to examine antioxidant states and methemoglobin reductase activity after giving ISDN and ISDN plus vitamin E. Rats were divided into three groups according to the treatment: control group, ISDN group and ISDN plus vit. E group. We measured reduced glutathione in blood (GSH), plasma malondialdehyde (MDA), erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and NADH-dependent methemoglobin reductase activities. In the ISDN group, plasma MDA levels were significantly high compared to the control and ISDN + vit. E groups (p < 0.001). In the ISDN and ISDN + vit. E groups, blood GSH levels were higher than those of the control group (p < 0.05). Changes of SOD and GPx activities were not significant. In the ISDN and ISDN + vit. E groups the erythrocyte catalase and NADH-dependent methemoglobin reductase activities were significantly higher than that in the control group (p < 0.001). We conclude that oxidant drugs such as ISDN need to be carefully used because of lipid peroxidation and methemoglobinemia. These findings support the notion that vitamine E protects tissues against oxidative stress.     

Assessment of dermal glyceryl trinitrate and isosorbide dinitrate for patients with angina pectoris.

Dermal nitrate preparations are claimed to be useful in the treatment of angina, as their slow absorption by-passing the liver leads to a sustained action. Ten patients with angina were exercised on a treadmill after dermal application of 16.64 mg glyceryl trinitrate or 100 mg isosorbide dinitrate or placebo. Exercise duration was significantly increased at one and three hours for both nitrate preparations but not at six hours after application. The calculated workload achieved was significantly increased (p less than 0.01) at one and three hours for both preparations and at six hours (p less than 0.05) for isosorbide dinitrate. Headaches were common with glyceryl trinitrate cream. The dermal nitrate preparations studied had a duration of antianginal action similar to that of oral nitrate tablets. Aside from their value when the oral route cannot be used or absorption may be delayed, dermal nitrate preparations have no advantage over oral preparations for angina pectoris.     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes

Since nitric oxide (NO) in many cells is involved in energy metabolism, the aim of this study was to evaluate the role of isosorbide dinitrate (ISDN), a NO donor, in energy metabolism of rat reticulocytes, particularly due to their high content of hemoglobin--an effective scavenger of NO. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated in the absence (control) or in the presence of different concentrations of ISDN. ISDN decreased total and coupled oxygen consumption (p<0.05) while increased uncoupled oxygen consumption (p<0.05) in a dose- and time-dependent manner. This was followed by enhancement of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.05). Levels of all glycolytic intermediates in the presence of ISDN indicate only stimulation of pyruvate kinase activity. ISDN did not alter the concentration of ATP, while increased ADP and AMP levels (p>0.05). In rat reticulocytes under steady-state conditions, 95.4% of overall energy was produced by oxidative phosphorylation but only 4.6% by glycolysis. Due to a reduced coupled oxygen consumption in the presence of ISDN, ATP production via oxidative phosphorylation was significantly diminished. A simultaneous increase of glycolytic ATP production is not enough to ensure constant ATP production. The calculated mean ATP turnover time was prolonged by 199% in the presence of 1.5 mmol/l ISDN. In conclusion, ISDN a) inhibited total and coupled respiration but enhanced uncoupled respiration, b) stimulated glycolysis, c) decreased ATP production and d) prolonged ATP turnover time in rat reticulocytes. These effects were mediated by NO as the effector molecule.     

Effect of isosorbide dinitrate,verapamil, and labetalol on portal pressure in cirrhosis.

The effects on portal pressure of the vasodilatory drugs isosorbide dinitrate and verapamil and of an alpha and beta blocking agent, labetalol, were assessed in 21 patients with cirrhosis and portal hypertension. The wedged hepatic venous pressure gradient (wedged minus free hepatic venous pressures) was used as an index of portal pressure and was not significantly changed by treatment with labetalol (n = 5) but was significantly decreased by verapamil (n = 6; p less than 0.05) and isosorbide dinitrate (n = 10; p less than 0.01). Long term administration of isosorbide dinitrate also had a significant effect (p less than 0.01).     

Solid-phase extraction of isosorbide dinitrate and two of its metabolites from plasma for gas chromatographic analysis

A rapid, accurate and selective method for the determination of isosorbide dinitrate and its 2- and 5-isosorbide mononitrate metabolites in 1.0 ml of human plasma has been developed. Before chromatographic quantitation by gas-liquid chromatography with electron-capture detection, the compounds are subjected to solid-phase extraction, using ENVI 18 cartridges (Supelco). The intra-day and inter-day coefficients of variation are less than 10%, except the inter-day coefficient of variation for the assay of 5-isosorbide dinitrate which is less than 15%. Limits of quantitation are 10, 10 and 20 ng/ml for isosorbide dinitrate, 2-isosorbide mononitrate and 5-isosorbide mononitrate, respectively. Recoveries are in excess of 90% for isosorbide dinitrate and 70% for its two metabolites.     

Biotransformation of glyceryl trinitrate and isosorbide dinitrate in vascular smooth muscle made tolerant to organic nitrates

It has been proposed that organic nitrates are prodrugs and biotransformation to a pharmacologically active metabolite (i.e., nitric oxide) must occur before the onset of vasodilation. If this postulated mechanism is correct, tolerance to organic nitrate-induced vasodilation might involve decreased biotransformation of organic nitrates by vascular smooth muscle. In this study, biotransformation of isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) was estimated by measuring isosorbide mononitrate (ISMN) and glyceryl dinitrate (GDN), respectively, rather than the nitrate anion, because of a more sensitive method for measurement of ISMN and GDN. To test this hypothesis, isolated rabbit aortic strips (RAS) were made tolerant in vitro by incubation with 500 microM GTN or ISDN for 1 h. After a washout period and submaximal contraction with phenylephrine, the tissues were incubated with either 2.0 microM [14C]ISDN or 0.5 microM [14C]GTN for 2 min. ISDN- or GTN-induced relaxation of RAS was monitored and tissue parent drug and metabolite contents were determined by thin-layer chromatography and liquid scintillation spectrometry. ISDN- and GTN-induced relaxation of RAS and the metabolite concentrations were significantly less for both GTN- and ISDN-tolerant tissue compared with nontolerant tissue. These results are consistent with the hypothesis that organic nitrate biotransformation is required for organic nitrate-induced vasodilation.     

Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites

A sensitive, specific capillary gas chromatographic—electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs>76% and for GTN and the GDNs>95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1,3-GDN maximum concentrations (C_max) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the C_max ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN C_max ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.     

Crystal structure of the cyclomaltohexaose (alpha-cyclodextrin) complex with isosorbide dinitrate. Guest-modulated channel-type structure

The crystal structure of the 2:1 complex of cyclomaltohexaose (alpha-cyclodextrin, alpha-CD) with isosorbide dinitrate was determined by single-crystal X-ray analysis. In the crystal with the space group C2, two cyclomaltohexaose molecules form a head-to-head dimer with the secondary hydroxy-group sides facing each other. The dimer unit is stacked along the crystallographic c-axis to form a channel-type structure. The isosorbide dinitrate molecule is encapsulated in the cylindrical cavity of the cyclomaltohexaose dimer. The dimeric structure exhibits pseudo twofold symmetry, and the guest molecule is disordered on the local symmetry axis. The isosorbide moiety is located at the center of the dimer cavity, and the nitrate groups penetrate into the cyclomaltohexaose rings. The guest molecule modulates the dimer structure to attain the most stable accommodation into the cavity. The cyclomaltohexaose molecules are laterally shifted away from each other to create the cavity fitted to the shape of the guest molecule. As the result, the intermolecular hydrogen bonds between secondary hydroxy-groups are not fully formed, but the dimeric structure is stabilized by the interaction with the guest molecule.     

Crystal and molecular structure of tris(propane-1,3-diamine)nickel(II) dinitrate complex

The title compound, [Ni(1,3-pn)₃](NO₃)₂, crystallizes in the orthorhombic space group Pbca with eight formula units in a cell of dimensions a = 17.146(8), b = 14.364(5) and c = 15.054(7). The structure was solved by the heavy-atom method and refined by least-squares calculations to R = 0.053 for 1439 counter data. It consists of discrete, slightly distorted octahedral [Ni(1,3-pn)₃]²⁺ cations and NO₃^? anions. One of the three six-membered chelate rings show a pronounced flattening unusual chair conformation. Magnetic and spectroscopic data agree to a lower stability of six-membered chelate rings, compared to five-membered chelate ones.     

Patterns of isosorbide dinitrate and glyceryl trinitrate metabolites formed by selected segments of the rabbit gastrointestinal tract

Homogenates of selected segments of the rabbit gastrointestinal tract (GIT) were studied for their ability to biotransform isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) to their mono- and di-nitrate metabolites, respectively. In addition, preferential formation of certain metabolites was investigated by examination of the patterns of metabolites formed by the various homogenates. After a 30-min incubation of ISDN with GIT homogenates (pH 7.4, 37 degrees C), the percent disappearance of ISDN and the ratio of isosorbide-2-mononitrate (2-ISMN) to isosorbide-5-mononitrate (5-ISMN) were as follows: stomach, 32%, 0.8; duodenum, 65%, 0.1; jejunum, 59%, 0.2; ileum, 38% , 1.2; cecum, 33%, 2.7; and colon, 32%, 3.4. After a 5-min incubation of GTN with GIT homogenates, the percent disappearance of GTN and the ratio of glyceryl-1,3-dinitrate (1,3-GDN) to glyceryl-1,2-dinitrate (1,2-GDN) were as follows: duodenum, 54%, 0.65; ileum, 73%, 0.68; and colon, 61%, 0.17. Incubation of 2 x 10(-7) M ISDN with mucosal and muscularis homogenates of duodenum, jejunum, and ileum resulted in significant losses of ISDN with an equimolar formation of the mononitrate metabolites. Most of the metabolic activity for ISDN resided in the mucosal layer of each section. The ratio of 2-ISMN to 5-ISMN varied in each section (stomach to colon) and cross section (mucosal versus muscularis) of the GIT. We conclude that the metabolism of ISDN and GTN by the GIT may contribute to the high clearance of these organic nitrates, and the low oral bioavailability of ISDN. Also, multiple mechanisms appear to be involved in the biotransformation of ISDN and GTN in the rabbit GIT.     

Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography-mass spectrometry with electrospray ionization

We have developed a liquid chromatographic-mass spectrometric method for the simultaneous determination of nitroglycerin (NTG) and its active metabolites, glyceryl 1,2-dinitrate (1,2-GDN) and glyceryl 1,3-dinitrate (1,3-GDN), for metabolism studies in cell cultures. 1,2,4-Butanetriol-1,4-dinitrate was chosen as an internal standard. Using a linear gradient of water/methanol containing 0.025 mM NH(4)Cl, the compounds were eluted within 12.5 min on an Allure Aqueous C(18) column (100 mm x 2.1 mm). Detection and quantification was achieved with multiple reaction monitoring in the negative ion mode. Intra- and inter-day variabilities for simultaneous determination of the three nitrates were below 10 and 18%, respectively, over a range of NTG and GDN concentrations of 0.5-15 ng/ml. The lower limit of quantification was found to be about 0.01 ng on column. Application of this method was illustrated through in vitro metabolism studies of NTG in culture media bathing LLC-PK1 cells and human vascular smooth muscle cells (HA-VSMC) at 37 degrees C. The degradation half-life of NTG was found to be 4.5 +/- 0.4 h and 39.2 +/- 0.02 h, respectively, for LLC-PK1 cells versus HA-VSMC. At 5 h, the 1,2-GDN versus 1,3-GDN metabolite distribution ratio in the bathing medium was found to be 1.5 +/- 0.1 and 0.2 +/- 0.02 for LLC-PK1 and HA-VSMC cells, respectively. With this method, the degradation half-life of NTG in rat plasma at 37 degrees C was shown to be 26.8 +/- 1.8 min, consistent with previous values obtained using gas chromatography.     

Sex- and species-related differences in the biotransformation of isosorbide dinitrate by various tissues of the rabbit and rat

The biotransformation of isosorbide dinitrate (ISDN) by various tissues of the rabbit and rat was examined. Incubation of 2 X 10(-7) M ISDN at 37 degrees C with tissue homogenates of liver, lung, kidney, intestine, skeletal muscle, aorta, and erythrocytes from the rabbit and rat resulted in a significant disappearance of ISDN after a 30-min incubation (also, 5-min incubation for liver). The disappearance of ISDN in each tissue homogenate was accompanied by an equimolar production of the mononitrate metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN), with the exception of liver homogenates where the loss of ISDN could not be accounted for by mononitrate formation. The relative rate of ISDN disappearance in various tissue homogenates was for the male rabbit, liver greater than lung approximately intestine greater than kidney greater than erythrocytes approximately skeletal muscle approximately aorta; for the female rabbit, liver greater than kidney approximately lung approximately intestine greater than erythrocytes approximately skeletal muscle approximately aorta; and for the male rat, liver greater than intestine greater than erythrocytes greater than skeletal muscle greater than lung approximately kidney. A sex difference in the percent disappearance of ISDN was observed in homogenates of lung and intestine from male and female rabbits. In addition, a sex difference in the ratio of metabolite (2-ISMN/5-ISMN) formed by denitration of ISDN was seen in homogenates of lung, skeletal muscle, and erythrocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)