Inhibition of antibiotic-resistant Staphylococcus aureus by the broad-spectrum dihydrofolate reductase inhibitor RAB1.

The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.

Effects of retinoids on cancerous phenotype and apoptosis in organotypic cultures of ovarian carcinoma.

BACKGROUND: Retinoic acid analogues, called retinoids, have shown promise in clinical trials in preventing breast and ovarian cancers. Classic retinoids bind to retinoic acid receptors, which regulate cell growth. Some novel retinoids, such as fenretinide, i.e., N-(4-hydroxyphenyl)retinamide (4-HPR), induce apoptosis through retinoic acid receptor-independent mechanisms; however, they appear to do so only at concentrations above those achieved in clinical chemoprevention trials. At lower concentrations (< or =1 microM), 4-HPR acts like classic retinoids, by inducing differentiation through a receptor-dependent mechanism. Our goal was to compare the effects of novel receptor-independent (apoptotic) retinoids with those of classic growth-inhibitory retinoids at clinically achievable doses on growth, differentiation, and apoptosis in ovarian tissue. METHODS: Four receptor-independent (apoptotic) and seven growth-inhibitory retinoids, including synthetic, low-toxicity compounds called heteroarotinoids, were administered at concentrations of 1 microM to organotypic cultures of ovarian primary and cancer cell lines: OVCAR-3, Caov-3, and SK-OV-3. After fixation, embedding, and sectioning, the growth fraction was quantified by measuring expression of the proliferation marker Ki-67/myb, differentiation was assessed by expression of mucin, and apoptosis was evaluated by the TUNEL assay. Spearman correlation analysis was performed on the data, and all P values were two-sided. RESULTS: All 11 retinoids reversed characteristics associated with the cancerous phenotype in all neoplastic cultures. Glandular structures were observed consistently in retinoid-treated, but not in untreated, OVCAR-3 and Caov-3 cultures. All retinoids decreased growth fractions, and some increased mucin expression. All receptor-independent retinoids and two receptor-dependent retinoids induced apoptosis, and the induction correlated significantly with increased expression of the mucin MUC1 (r =.83; P =.03). Retinoids with ester-linking groups did not induce apoptosis but decreased the growth fraction in correlation with MUC1 induction (r = -.93; P =.02). CONCLUSIONS: At clinically achievable concentrations, all retinoids tested decrease the growth fraction, induce differentiation and apoptosis. Induction of MUC1 expression is implicated in the mechanisms of action.

The acute and subchronic toxicity of BRB-I-28, a novel class Ib antiarrhythmic agent, in CD-1 mice.

The acute and subchronic toxic effects of BRB-I-28 (7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane HCl), a novel class Ib antiarrhythmic agent, were investigated in male and female mice. The estimated oral LD(50) for BRB-I-28 was 128 mg/kg (male mice) and 131 mg/kg (female mice). In subchronic oral studies, four groups of mice (15/sex/group/dose) were fed daily with diets containing BRB-I-28 for 90 consecutive days. The equivalent daily doses were approximately 0, 16, 32, 76 (male) and 0, 18, 37, 89 mg/kg (female). All mice survived. Food consumption per day was decreased, but water consumption per day was increased (in a non-dose-dependent manner). However, both mean body weight and mean body weight gain were not significantly changed as were true for hematological and clinical chemistry profiles, except for serum Na(+) concentration (male) and serum K(+) concentration in male and female mice (high dose levels). Hepatocellular necrosis occurred in male and female mice (in a dose-dependent fashion). Renal cortical vacuoles and myocardial necrosis with low numbers of lymphocytic infiltrations were present in female mice (middle and high doses). Lesions in the liver, kidney and heart were mild with (very small) changes in serum biochemical values. These data suggest that BRB-I-28 has limited toxic potential, and coupled with low proarrhythmic and other desirable cardiovascular effects, makes BRB-I-28 worthy of further development.

Preliminary acute and subchronic toxicity studies of GLG-V-13, a novel class III antiarrhythmic agent, in mice.

The acute and subchronic toxic effects of GLG-V-13 (3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nona ne dihydroperchlorate, CAS 155029-33-7), a novel class III with some class Ib antiarrhythmic activity, were investigated in mice. The estimated LD50 for GLG-V-13 given orally were 419 mg/kg for male mice and 383 mg/kg for female mice, respectively. The acute toxic signs appeared to be of the central nervous system in origin. Four groups of mice (15 per sex, group and dose) were fed daily with diets containing GLG-V-13 for 90 consecutive days. The equivalent daily doses were 0, 22, 50 and 121 mg/kg/day and 0, 27, 60 and 136 mg/kg/day for male and female mice, respectively. All of the mice survived. Food consumption was decreased. However, mean body weight and body weight gain were not significantly changed. Gross pathological changes, especially in the lungs and liver, were found in the middle and high dose groups. Consistent increased mean corpuscular hemoglobin concentration and decreased mean corpuscular hemoglobin were observed in all dose groups. Hepatocellular necrosis was found in both male and female mice treated with the drug and was dose-dependent. Marked vacuolation of the X zone in the adrenal gland with mild to moderate deposition of ceroid pigments (brown degeneration) was observed in female mice. Lesions in the kidneys and adrenal glands may be a possible reason for changes in serum sodium and potassium ions concentrations leading to an increase in water intake. A significant reduction in cholesterol in the high dose group may be a favorable pharmacological effect of GLG-V-13. The data from the 90-day subchronic toxicity studies indicate that GLG-V-13 appears to have limited systemic toxicity potential.

Effect of GLG-V-13, a class III antiarrhythmic agent, on potassium currents in rabbit ventricular myocytes.

The effects of a new Class III antiarrhythmic drug, GLG-V-13, on the 4-aminopyridine sensitive transient outward current, on the inward rectifier potassium current, on the ATP sensitive potassium current and on the rapid and slow components of the delayed rectifier potassium current were studied in single rabbit ventricular myocytes using the whole-cell voltage-clamp technique. GLG-V-13 blocked the rapid component of the delayed rectifier potassium current in a dose-dependent manner, with an estimated EC50 value of 0.36 microM. At high concentration, the slow component of the delayed rectifier potassium current was also depressed by the drug (40% effect at 10 microM concentration). The transient outward current, the inward rectifier potassium current and the ATP sensitive potassium current were not influenced by GLG-V-13, even at 10 microM concentration. Thus, GLG-V-13 blocks predominantly the rapid component of the delayed rectifier potassium current which may play a significant role in the prolongation of repolarization by the drug in ventricular tissue.

Heteroarotinoids inhibit head and neck cancer cell lines in vitro and in vivo through both RAR and RXR retinoic acid receptors.

A class of less toxic retinoids, called heteroarotinoids, was evaluated for their molecular mechanism of growth inhibition of two head and neck squamous cell carcinoma (HNSCC) cell lines SCC-2 and SCC-38. A series of 14 heteroarotinoids were screened for growth inhibition activity in vitro. The two most active compounds, one that contained an oxygen heteroatom (6) and the other a sulfur heteroatom (16), were evaluated in a xenograph model of tumor establishment in nude mice. Five days after subcutaneous injection of 10(7) SCC-38 cells, groups of 5 nu/nu mice were gavaged daily (5 days/week for 4 weeks) with 20 mg/kg/day of all-trans-retinoic acid (t-RA, 1), 10 mg/kg/day of 6, 10 mg/kg/day of 16, or sesame oil. After a few days, the dose of t-RA (1) was decreased to 10 mg/kg/day to alleviate the side effects of eczema and bone fracture. No significant toxic effects were observed in the heteroarotinoid groups. All three retinoids caused a statistically significant reduction in tumor size as determined by the Student t-test (P < 0. 05). Complete tumor regression was noted in 3 of 5 mice treated with t-RA (1), 4 of 5 mice treated with 16, 1 of 5 mice treated with 6, and 1 of 5 mice treated with sesame oil. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine that the expression levels of RARalpha, RXRalpha, and RXRbeta were similar in the two cell lines, while RARbeta expression was higher in SCC-2 over SCC-38, and RARgamma expression was higher in SCC-38 over SCC-2. Receptor cotransfection assays in CV-1 cells demonstrated that 16 was a potent activator of both RAR and RXR receptors, while 6 was selective for the RXR receptors. Transient cotransfection assays in CV-1 cells using an AP-1 responsive reporter plasmid demonstrated that t-RA (1), 6, and 16 each inhibited AP-1-driven transcription in this cell line. In conclusion, the growth inhibition activity of the RXR-selective 6 and the more potent growth inhibition activity of the RAR/RXR pan-agonist 16 implicate both RARs and RXRs in the molecular mechanism of retinoid growth inhibition. Moreover, the chemoprevention activity and the lack of toxicity of heteroarotinoids demonstrate their clinical potential in head and neck cancer chemoprevention.

Synthesis, structure-activity relationships, and RARgamma-ligand interactions of nitrogen heteroarotinoids.

Three heteroarotinoids containing a nitrogen atom in the first ring and a C-O linking group between the two aryl rings were synthesized and evaluated for RAR and RXR retinoid receptor transactivation, tumor cell growth inhibition, and transglutaminase (TGase) induction. Ethyl 4-(N,4,4-trimethyl-1,2,3,4-tetrahydroquinolinyl)benzoate (1) contained an N-CH(3) group and activated all retinoid receptors except for RARgamma. Inceasing the hydrophobicity around the rings with analogues ethyl 4-(N,4,4,7-tetramethyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (2) [7-methyl group added] and ethyl 4-(4,4-dimethyl-N-isopropyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (3) [NCH(CH(3))(2) group at C-4] increased the potency and specificity for RARalpha, RARbeta, and RXRalpha, compared to 1, but had little effect on RXRbeta and RXRgamma activation. Although 1 and 3 were unable to activate RARgamma, 2 did activate this receptor with efficacy and high potency equal to that of 9-cis-retinoic acid (9-c-RA). All three heteroarotinoids exhibited 5-8-fold greater specificities for RARbeta over RARalpha. In addition, esters 1-3 inhibited the growth of two cell lines each derived from cervix, vulvar, ovarian, and head/neck tumors with similar efficiencies to that of 9-c-RA through a mechanism independent of apoptosis. The vulvar cell lines were the most sensitive, and the ovarian lines were the least sensitive. Ester 2 was similar to 1 and 3 except that 2 was a much more potent growth inhibitor of the two vulvar cell lines, which is consistent with strong RARgamma activation by 2 (but not by 1 and 3) and the high levels of RARgamma expression in skin. All three heteroarotinoids induced production of TGase, a marker of retinoid activity in human erythroleukemic cells. Esters 2 and 3 were the more potent TGase activators than 1, in agreement with the stronger activation of the RAR receptors by 2 and 3. The biological activities of these agents, and the RARgamma potency of 2 in particular, demonstrate the promise of these compounds as pharmaceutics for cancer and skin disorders.

Synthesis and characterization of heteroarotinoids demonstrate structure specificity relationships.

Heteroarotinoids are synthetic retinoids derived from trans-retinoic acid and the arotinoid structures and include a heteroatom in a five- or six-membered cyclic ring. This is the first systematic study of influences of the heteroatom, ring size, number of aryl groups, and terminal side chain on retinoid receptor specificity. Two new heteroarotinoids were synthesized and characterized. Although all heteroarotinoids activated RAR receptors, two dominant associations between structure and specificity were identified across all compounds. The six-membered ring conferred increased RARbeta specificity over the five-membered ring. The sulfur atom conferred greater specificity for RARgamma than the oxygen atom. RARalpha specificity was attenuated by a combination of influences from the heteroatom and aryl groups. In summary, the heteroatom and cyclic ring size exerted dominant effects, while the number of aryl rings and terminal side chain had attenuating effects on retinoid receptor specificity of heteroarotinoids.

Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity.

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-dependent alterations in growth rate in response to treatment with trans-retinoic acid. Activation of nuclear retinoic acid receptors was determined in a reporter cell line established from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated in a statistically significant manner between assays. RARE transactivation activity in CC-B cells correlated with induction of TGase in GMO6141A (R = 0.96) and with a decrease in the growth rate of CC-1 cells (R = -0.90). The ability of the selected heteroarotinoids to induce differentiation, inhibit proliferation, and activate nuclear receptors demonstrates the chemotherapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylchroman-6-yl)-2, 4,6-heptatrienoic acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05 mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid] as reference controls. The target organs affected in the mice by the three heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively, which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these heteroarotinoids over the parent compound. The biological activity and reduced toxicity of these heteroartinoids demonstrate the potential efficacy as anticancer agents.

Class III electrophysiologic actions of imidazole-substituted diheterabicyclononanes in canine myocardium.

The electrophysiologic effects of the imidazole-substituted diheterabicyclo[3.3.1]nonane compounds GLG-V-13 and KMC-IV-84 were evaluated in canine ventricular tissues using intracellular and extracellular recordings. The drugs produced a concentration-dependent prolongation of action potential duration at 90% of repolarization in Purkinje (338 +/- 26 to 611 +/- 43 msec, 10 mg/l GLG-V-13; 328 +/- 17 to 468 +/- 18 msec, 10 mg/l KMC-IV-84), in right ventricular subendocardium (260 +/- 18 to 335 +/- 18 msec, 10 mg/l GLG-V-13; 221 +/- 9 to 264 +/- 13 msec, 10 mg/l KMC-IV-84) and in left ventricular epicardium (195 +/- 13 to 256 +/- 18 msec, 10 mg/l GLG-V-13; 203 +/- 11 to 273 +/- 26 msec, 10 mg/l KMC-IV-84) without altering resting membrane potential, action potential amplitude, overshoot potential, Vmax, conduction velocity or Purkinje fiber automaticity. Prolongation of the effective refractory period was proportional to the change in action potential duration at 90% of repolarization. Prolongation of action potential duration at 90% of repolarization was maximal at paced cycle lengths exceeding 1000 msec and was minimal at a paced cycle length of 250 msec (Purkinje: 266 +/- 20 vs. 6 +/- 8 msec, GLG-V-13; 178 +/- 12 vs. 10 +/- 10 msec, KMC-IV-84. Right ventricular subendocardium: 70 +/- 12 vs. 10 +/- 2 msec, GLG-V-13; 60 +/- 8 vs. 19 +/- 6 msec. Left ventricular epicardium: 67 +/- 13 vs. 10 +/- 5 msec, GLG-V-13; 68 +/- 12 vs. 16 +/- 8 msec, KMC-IV-84). An increase in K+(o) to 12 mM reduced action potential prolongation by GLG-V-13 and KMC-IV-84 in left ventricular epicardium. The results demonstrate selective class III electrophysiologic properties for imidazole-substituted diheterabicyclo[3.3.1]nonane compounds.

Electrophysiological and inotropic characterization of a novel class III antiarrhythmic agent, GLG-V-13, in the mammalian heart.

GLG-V-13, a novel 3,7-diheterabicyclo(3.3.1)nonane, was examined both in vivo and in vitro to characterize its electrophysiological, hemodynamic, and inotropic properties. In anesthetized guinea pigs, GLG-V-13 [0.5-500 micrograms/kg intravenously (i.v.), n = 6] lengthened the epicardial monophasic action potential (MAP) duration, the atrioventricular (AV) conduction time and the RR interval in a dose-dependent manner. At the highest dose, these variables were increased by 30, 13, and 23%, respectively. No significant effects were noted on QRS duration or blood pressure (BP). In rabbit atrial and papillary muscle preparations, GLG-V-13 (0.32-3.2 mg/L) did not exert a negative inotropic action and in isolated rabbit cardiomyocytes the agent blocked the rapidly activating delayed rectifier K+ current (IKr, EC50 = 48 micrograms/L). In 10 intact anesthetized mongrel dogs, the left ventricular (LV) endocardial MAP was measured during atrial pacing before and after administration of GLG-V-13 (3 and 6 mg/kg i.v.). As compared with the drug-free state, the agent induced a significant prolongation of the MAP at all pacing frequencies (2.0-4.5 Hz). In 15 anesthetized dogs studied 1-4 days after two-stage ligation of the left anterior descending coronary artery (LAD), the antiarrhythmic/proarrhythmic potential of GLG-V-13 was compared with that of lidocaine. ECG, His bundle, LV (IZepi), and composite and normal zone composite electrograms were recorded. Programmed electrical stimulation (PES) and burst pacing (4.0-7.0 Hz) were delivered to the right ventricular outflow tract. In the drug-free state, sustained monomorphic ventricular tachycardia (SMVT) was inducible in 6 dogs (6 of 15). After lidocaine, SMVT was induced in 7 other dogs (13 of 15). GLG-V-13 prevented induction of SMVT in 5 of 6 dogs; a proarrhythmic action was noted in 1 dog only. GLG-V-13 slowed the heart rate (HR), increased the AH and the HV intervals, prolonged the paced (2.5 Hz) QT interval, and increased the ventricular effective refractory period (VERP). These effects were associated with 2:1 block of late potentials in the IZepi electrograms, a phenomenon also observed during rapid atrial pacing (2.5-3.5 Hz), suggestive of a marked prolongation of refractoriness in the ischemically damaged myocardium. In light of the recent Cardiac Arrhythmia Suppression Trial (CAST) study, the antiarrhythmic efficacy, together with the low proarrhythmic potential and lack of cardiodepressant properties of GLG-V-13, may merit further investigation of this novel class III antiarrhythmic agent.

Novel 3,7-diheterabicyclo[3.3.1]nonanes that possess predominant class III antiarrhythmic activity in 1-4 day post infarction dog models: X-ray diffraction analysis of 3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nona ne dihydroperchlorate.

Several 3,7-diheterabicyclo[3.3.1]nonanes (DHBCNs) were prepared and screened in the Harris dog model for their ability to abolish pace-induced and sustained ventricular tachycardia (SVT) or prevent induction of ventricular tachycardia. In addition, an electrophysiological examination was made in the infarcted hearts of each animal to determine if more than one class activity was present. The examples exhibited predominately class III antiarrhythmic activity via a prolongation of the ventricular effective refractory period (VERP) in the models, although there may well be an underlying class Ib action present as exemplified by the ability of several of the agents to slow conduction in the myocardial infarcted dog hearts. 3-[4-(1H-Imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nonan e dihydroperchlorate displayed powerful class III activity in the model systems while several other DHBCNs exhibited various degrees of class III action. An X-ray diffraction analysis revealed that this compound has a 3,7-diazabicyclo[3.3.1]nonane bicyclic unit in a chair-chair conformation.

7-Benzyl-3-thia-7-azabicyclo[3.3.1]nonane 3-oxide.

The molecular geometry of a major metabolite, C14H19NOS, of a potent anti-arrhythmic drug, 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane (BRB-I-28), has been determined by X-ray diffraction. The 3,7-dihetero bicyclic system of the sulfoxide molecule adopts a chair--chair conformation like that of the HCLO4 salt of the BRB-I-28 molecule. The S...N contact distance of 2.863(2)A in the present molecule is significantly shorter than that found in the crystal structure of its precursor [3.038(4)A]. The overall molecule possesses pseudo-mirror symmetry.

The cardiac electrophysiological effects of sparteine and its analogue BRB-I-28 in the rat.

This study compares the cardiovascular and antiarrhythmic effects of sparteine and a 3,7-diheterobicyclo[3.3.1]nonane analogue of sparteine, BRB-I-28, in pentobarbitone-anaesthetized rats subjected to left-ventricle electrical stimulation and occlusion of the left anterior descending coronary artery. Sparteine and BRB-I-28 produced a dose-dependent reduction in heart rate and blood pressure over the dose range 1-64 mumol/kg/min. As well, the P-R and Q-aT intervals of the electrocardiogram (ECG) were prolonged. The thresholds for induction of premature beats and ventricular fibrillation were dose-dependently increased and both drugs increased refractoriness. While sparteine and BRB-I-28 (at 16 and 64 mumol/kg/min, respectively) did not change the incidence of premature beats or ventricular tachycardia with coronary occlusion, both drugs equally reduced the incidence of ventricular fibrillation. We characterized the actions of sparteine and BRB-I-28 on cardiac Na+, transient outward and sustained outward plateau K+ currents of rat myocytes using the whole-cell patch-clamp. Sparteine and BRB-I-28 produced a concentration-dependent reduction in Na+ current with EC50 values of 110 and 230 microM, respectively. Both drugs produced hyperpolarizing shifts of 8 and 11 mV, respectively, for Na+ channel inactivation while neither produced a change in channel activation. Both drugs produced a concentration-dependent block of the sustained plateau K+ current and increased the rate of decay of the transient outward K+ current. Thus, sparteine and BRB-I-28 possess Na+ and K+ channel blocking properties which may account for their antiarrhythmic actions against electrical and ischaemic arrhythmias.

Relationships among petroleum refining, water and sediment contamination, and fish health.

Water, sediment, and fish were sampled from three streams that were receiving or had received effluents from oil refineries. Water and sediment samples were analyzed by gas chromatography/mass spectrometry. Each stream contained aromatic carbons including substituted benzenes and naphthalenes, which are related to oil refinery operations. Fish were identified, counted, and examined for external lesions. Lengths and weights were recorded for older bullhead catfish, and their livers were examined histologically. Differences were seen in the diversity and abundance of fish among the upstream, impacted (effluent-receiving), and downstream stations. In one stream, differences in liver pathology were observed between reference bullhead, collected from an upstream station, and those collected at impacted stations with more than 50% of the bullheads taken from impacted stations having some sort of pathological change, including one with a liver clear-cell focus, which is considered a preneoplastic lesion in rodents. These data suggest a correlation between contamination of water and sediments with aromatic hydrocarbons, presumably from refinery effluents, and compromised fish health.

Pigmented subcutaneous spindle cell tumors in native gizzard shad (Dorosoma cepedianum).

An epizootic of pigmented subcutaneous spindle cell tumors affected nearly 25% of the adult gizzard shad (Dorosoma cepedianum) sampled from Lake of the Arbuckles in central Oklahoma over a 2 year period. Grossly, the tumors were primarily distributed over the head, trunk and fins as superficial raised masses that were almost always darkly pigmented. Histologically, they were located in the dermis, had a variable amount of connective tissue, and consisted of cells in a variety of forms and arrangements. Most tumors were composed of fusiform or spindle cells arranged in wavy bundles, whirling patterns or interwoven fascicles. Pigmentation was attributed to large dense deposits of melanin or to scattered individual melanin-containing cells. Immunohistochemical detection of proliferating cell nuclear antigen revealed a high proliferative activity in the spindle cells. Electron microscopy showed that the tumors were composed of several cell types, including host reactive cells, melanocytes in stages of maturity, and fibroblast-like cells. Tumor cells had neither cell-to-cell junctions nor an external lamina. Although the cell of origin of the tumors was not identified, evidence points toward melanocytes or, possibly, nerve sheath cells. However, an origin from fibroblasts or some other poorly differentiated cell cannot be ruled out. The etiology of the tumors was not determined. Fractionation of lake water and sediment samples followed by GC-MS analysis revealed no carcinogenic compounds. A retroviral etiology is unlikely because assays for reverse transcriptase in tumor homogenates were negative, and no evidence of viral particles was found in specimens examined by electron microscopy.

Pharmacokinetics and plasma protein binding of the new potent class III antiarrhythmic agent 3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3, 7-diazabicyclo[3.3.1]nonane dihydroperchlorate.

GLG-V-13 (3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo [3.3.1] nonane dihydroperchlorate, CAS 155029-33-7) has been shown to be a potent class III antiarrhythmic agent. The oral and intravenous pharmacokinetics and plasma protein binding of GLG-V-13 in dogs and in rabbits have now been investigated. Plasma GLG-V-13 concentration-time profiles, following an i.v. bolus dose of 6 mg/kg, were fitted to a 2-compartment model. The volume of distribution at steady state (Vd(ss)), the total systemic (ClB), and the elimination half-life (t1/2 beta) were 4.441 l/kg, 1.113 l/h/kg, and 2.485 h in dogs and 3.723 l/kg, 1.548 l/h/kg, and 1.401 h in rabbits. Following i.v. dosing, approximately 9.38% of the parent compound was excreted in dogs urine (0-72 h). Changes in plasma GLG-V-13 concentrations, after oral administration of GLG-V-13 (6 mg/kg), were best described by the 1-compartment pharmacokinetic model. The tmax and Cmax were 1.69 h, 0.54 mg/l in dogs and 1.44 h, 0.35 mg/l in rabbits. On oral administration, GLG-V-13 was moderately eliminated (t1/2kel' 1.867 h-1 in dogs and 3.961 h-1 in rabbits, respectively). Oral bioavailability was estimated to be 53.2% +/- 11.3% in dogs and 66.7% +/- 7.7% in rabbits. About 8.74% of the oral dose (6 mg/kg) was excreted via the dog urine (0-72 h). In vitro binding of GLG-V-13 to dog plasma protein was 29.4 +/- 9.90% (from 0.5 to 4 mg/l). Ex vivo binding of GLG-V-13 to dog plasma protein was 10.4 +/- 7.20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism and metabolite pharmacokinetics of BRB-I-28, a class Ib antiarrhythmic agent.

The metabolism of BRB-I-28 (7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane), a novel class Ib antiarrhythmic agent, was characterized in vivo in dogs and rats and in vitro with rat liver microsomal preparations containing a NADPH-generating system. In dogs, rats and the in vitro hepatic microsomal oxidation system, BRB-I-28 was extensively metabolized to form 7-benzyl-3-thia-7-azabicyclo [3.3.1]nonane-3-oxide (I), a major metabolite. The metabolite I was produced via S-oxidation, presumably by the hepatic P-450 system. Formation of minor metabolite, 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane (II) via the oxidation of the benzylic site was also identified in rats. Following intravenous and oral administration of BRB-I-28 to dogs, the plasma concentration of major metabolite I could be test described by a 1-compartmental model. The plasma AUC of metabolite I was 20% (i.v.) and 179.4% (oral) of that of the parent BRB-I-28, respectively, suggesting that BRB-I-28 was metabolized significantly by the first pass effect following oral administration. Extensive metabolism of BRB-I-28 to form metabolites I and II, which have demonstrated much lower antiarrhythmic activities, further supports previously observed pharmacodynamic and pharmacokinetic findings.

Electrophysiological characterization of a novel class III antiarrhythmic agent, GLG-V-13 in the mammalian heart.

GLG-V-13, a novel 3,7-diheterabicyclo[3.3.1]nonane, was examined both in vivo and in vitro in order to characterize its electrophysiological, haemodynamic and inotropic properties. Left ventricular epicardial monophasic action potential (MAP), surface ECG and mean arterial blood pressure (MBP) were recorded in six pentobarbital-anaesthetized, artificially ventilated and thoracotomized guinea-pigs. When studied in an intravenous dose interval ranging between 0.5 micrograms/kg and 500 micrograms/kg, GLG-V-13 dose-dependently lengthened the MAP duration (p < 0.05 at doses above 5 micrograms/kg), the atrioventricular conduction time (p < 0.05 at doses above 1 microgram/kg) and the RR interval (p < 0.05 at doses above 25 micrograms/kg). At the highest dose (500 micrograms/kg) these variables were increased by 30%, 13% and 23%, respectively. Only minor effects were noted on intraventricular conduction time (QRS interval) and MBP. In rabbit atrial and papillary muscle preparations, GLG-V-13 (0.32 to 3.2 mg/l) did not exert negative inotropic action. In 10 intact anaesthetized mongrel dogs, left ventricular endocardial MAP at 90% repolarization (MAP90) was measured during atrial pacing before and after administration of GLG-V-13, 3 and 6 mg/kg i.v., respectively.

Effects of novel antiarrhythmic agents, BRB-I-28 and its derivatives, on the heart mitochondrial respiratory chain and sarcoplasmic reticulum Ca(2+)-ATPase.

The effects of BRB-I-28 and its derivatives (GLG-V-13, SAZ-VII-22 and SAZ-VII-23), a novel group of antiarrhythmic agents, were investigated on the rat heart mitochondrial respiratory chain. The results indicate that BRB-I-28 and its derivatives have concentration-dependent inhibitory effects on NADH oxidase and NADH-CoQ reductase (complex I), but they have no significant effects on succinate oxidase, succinate dehydrogenase (complex II), CoQ-cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and NADH-K3Fe(CN)6 reductase. The site of inhibition of BRB-I-28 and its derivatives on the respiratory chain was localized between flavoprotein n (FPn) and CoQ, which is similar to the effect of rotenone and several other antiarrhythmic drugs such as amiodarone, propranolol, etc. BRB-I-28 and its derivatives also have significant inhibitory effects on mitochondrial ATPase activity as reported for other antiarrhythmic drugs such as amiodarone, propranolol, quinidine, and lidocaine. However, BRB-I-28 and its derivatives have no direct effects on sarcoplasmic reticulum Ca(2+)-ATPase activity. The inhibitory effects of BRB-I-28 and its derivatives on mitochondrial oxidative phosphorylation may result in the depletion of ATP. This effect, in combination with their effects on Na+,K(+)-ATPase, could possibly produce an increase in Ca2+ concentration in cytosol. This may be another mechanism by which these DHBCN derivatives produce an increase in systemic arterial blood pressure and contractile force of isolated cardiac muscle. On the other hand, inhibition on mitochondrial respiration may account for some of the potential toxic effects of these diheterabicyclo[3.3.1]nonane derivatives.