Critical appraisal of STAT3 pattern in adult cardiomyocytes.

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.

Photocatalytic interaction of aminophylline-riboflavin leads to ROS-mediated DNA damage and cell death: A novel phototherapeutic mechanism for cancer.

The accompanied tissue devastation and systemic toxicity of chemotherapy has shifted the quest for having an effective and palliative cancer therapy towards photodynamic therapy (PDT). Riboflavin (Rf), an essential micronutrient is emerging as a potent tool of PDT, due to its excellent photosensitizing properties. It can be used as an efficient adjuvant for various anticancer drugs. The hemolytic and proteolytic effect of photoilluminated aminophylline (Am), a xanthine derivative, and Rf is well documented in literature. In this study, using human peripheral lymphocytes we have demonstrated the strong pro-oxidant effects of photocatalytic interaction between Am and Rf. The photo degradation kinetics of Am in the presence of Rf was monitored using UV spectroscopy, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. The resultant pro-oxidant action of Am was monitored through various assays like lipid peroxidation, protein carbonylation, and reactive oxygen species (ROS) generation. Furthermore, the cytotoxic potential of this system was studied using comet and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Treated lymphocytes were visualized using fluorescence and scanning electron microscopy to further validate apoptosis. ROS scavengers ameliorated the oxidative damage caused by this system suggesting pivotal role of ROS in causing apoptotic cell death. As cancer cells exhibit increased absorption of Rf as well as are very sensitive in any further ROS level increment, this putative pathway can serve as an effective anodyne phototherapeutic strategy for cancer treatment. © 2017 IUBMB Life, 69(8):611-622, 2017.

Assessing the Potency of the Novel Tocolytics 2-APB, Glycyl-H-1152, and HC-067047 in Pregnant Human Myometrium.

The intracellular signaling pathways that regulate myometrial contractions can be targeted by drugs for tocolysis. The agents, 2-APB, glycyl-H-1152, and HC-067047, have been identified as inhibitors of uterine contractility and may have tocolytic potential. However, the contraction-blocking potency of these novel tocolytics was yet to be comprehensively assessed and compared to agents that have seen greater scrutiny, such as the phosphodiesterase inhibitors, aminophylline and rolipram, or the clinically used tocolytics, nifedipine and indomethacin. We determined the IC50 concentrations (inhibit 50% of baseline contractility) for 2-APB, glycyl-H-1152, HC-067047, aminophylline, rolipram, nifedipine, and indomethacin against spontaneous ex vivo contractions in pregnant human myometrium, and then compared their tocolytic potency. Myometrial strips obtained from term, not-in-labor women, were treated with cumulative concentrations of the contraction-blocking agents. Comprehensive dose-response curves were generated. The IC50 concentrations were 53 µM for 2-APB, 18.2 µM for glycyl-H-1152, 48 µM for HC-067047, 318.5 µM for aminophylline, 4.3 µM for rolipram, 10 nM for nifedipine, and 59.5 µM for indomethacin. A single treatment with each drug at the determined IC50 concentration was confirmed to reduce contraction performance (AUC) by approximately 50%. Of the three novel tocolytics examined, glycyl-H-1152 was the most potent inhibitor. However, of all the drugs examined, the overall order of contraction-blocking potency in decreasing order was nifedipine > rolipram > glycyl-H-1152 > HC-067047 > 2-APB > indomethacin > aminophylline. These data provide greater insight into the contraction-blocking properties of some novel tocolytics, with glycyl-H-1152, in particular, emerging as a potential novel tocolytic for preventing preterm birth.

Needle-free iontophoresis-driven ß-adrenergic sweat rate test.

OBJECTIVES: Two CFTR-dependent ß-adrenergic sweat rate tests applying intradermal drug injections were reported to better define diagnosis and efficacy of CFTR-directed therapies. The aim of this work was to develop and test a needle-free image-based test and to provide an accurate analysis of the responses. METHODS: The modified method was conducted by applying two successive iontophoresis sessions using the Macroduct device. Efficiency of drug delivery was tested by evaporimetry. Cholinergically stimulated sweating was evoked by pilocarpine iontophoresis. ß-adrenergically stimulated sweating was obtained by iontophoresis of isoproterenol and aminophylline in the presence of atropine and ascorbic acid. A nonlinear mixed-effects (NLME) approach was applied to model volumes of sweat and subject-specific effects displaying inter- and intra-subject variability. RESULTS: Iontophoresis provided successful transdermal delivery of all drugs, including almost neutral isoproterenol and aminophylline. Pilocarpine was used at a concentration ∼130-times lower than that used in the classical Gibson and Cooke sweat test. Addition of ascorbic acid lowered the pH of the solution, made it stable, prevented isoproterenol degradation and promoted drug iontophoresis. Maximal secretory capacity and kinetic rate of ß-adrenergic responses were blunted in CF. A cutoff of 5.2 minutes for ET50, the time to reach the half maximal secretion, discriminated CF from controls with a 100% sensitivity and specificity. Heterozygous showed an apparently reduced kinetic rate and a preserved secretory capacity. CONCLUSION: We tested a safe, well-tolerated needle-free image-based sweat test potentially applicable in children. Modelling responses by NLME allowed evaluating metrics of CFTR-dependent effects reflecting secretory capacity and kinetic rate.

Plasma protein binding monitoring of therapeutic drugs in patients using single set of hollow fiber centrifugal ultrafiltration.

AIM: Plasma protein binding (PPB), as a significant influenced factor of pharmacokinetic and pharmacodynamic properties of a medicine, is a suitable index for therapeutic drug monitoring (TDM) strategies. A suitable measurement technique of PPB of patients is in urgent need and attracts many analysts' attention. Results & methodology: In this study, a novel method was proposed to analyze free drug concentration and total drug concentration (Ct) successively in one unit with a sample. All RSDs were less than 3%. The absolute recovery of Ct ranged from 98.1 to 101.2%. DISCUSSION & CONCLUSION: It is extremely valuable to consider PPB as an important index for TDM, perfecting information of medication, reflecting the disease condition more comprehensively, providing assistance for doctors to adjust the dose regimen. The proposed technique, convenience, accuracy and without the influence of plasma condition, provides a feasible method to monitor PPB of various patients, facilitating the popularization of monitoring PPB in TDM.

Increased myocardial N-myristoyltransferase activity in rotenone model of Parkinsonism.

There is widespread brain pathology in Parkinson's disease (PD), with the primary pathology in the substantia nigra. Oxidative stress is believed to play a role in cell death in PD. Rotenone is a mitochondrial toxin which can produce Parkinson syndrome (PS) in rats. Myristoyl-CoA:protein N-myristoyltransferase (NMT), which catalyzes the co-translational transfer of myristate from myristoyl-CoA to the amino-terminal glycine residue of selected polypeptides, is increased in the myocardium of ischemia-reperfusion rat model myocardium. Animals received rotoneone (n=10) or placebo vehicle (n=6) via Alzet osmotic pumps. Mean cardiac muscle NMT activity of placebo treated (control) rats was 0.608+/-0.366 units/mg protein. Rats with mild or no detectable PS features on rotenone showed slight (mean 0.853+/-0.192) but insignificantly increased activity. Rats that had moderately severe PS features had higher level of NMT activity (mean 1.223+/-0.057), which was borderline significant compared to controls (P=0.066). Rats with severe PS features had the highest NMT activity (1.353+/-0.128) which was significantly greater compared to controls (P=0.003) and to the rats that had equivocal or no motor slowing (P=0.005). Our data show cardiac metabolic dysfunction in a rotenone rat model of PS. The severity of this change correlates with the severity of motor manifestations. Further studies of NMT activity in human PD cases and patients with cardiomyopathy of unknown cause may provide valuable information in these disorders.

Theophylline disposition in obesity.

Theophylline disposition was examined in 14 obese subjects and 57 normal subjects. A single oral dose of aminophylline solution was given and serum and saliva samples were collected over several hours and assayed by high-pressure liquid chromatography. The apparent volume of distribution (Vd) and body clearance (ClB) were analyzed for total body weight (TBW) and ideal body weight (IBW). The Vd averaged 0.482 (SD = 0.084) L/kg TBW in normals vs 0.382 (0.069) L/kg TBW and 0.77 (0.189) L/kg IBW in obese subjects. The ClB averaged 63.0 (28.5) ml/hr/kg IBW in normals compared to 32.8 (11.1) ml/hr/kg TBW and 64.1 (20.8) ml/hr/kg IBW in obese subjects. Similar Vd values between the two groups when TBW is used indicates that loading dose is best calculated based on TBW. Similar ClB based on IBW in normal and obese subjects indicates that IBW should be used to calculate maintenance doses for theophylline. Mean half-lives were longer in obese subjects than in normals, 8.6 (2.0) and 6.0 (2.1) hr, respectively, suggesting that obese patients may need less frequent dosing.

Enhanced biotransformation of theophylline in marihuana and tobacco smokers.

Single oral doses of theophylline were administered to 7 chronic marihuana smokers, to 7 chronic users of both marihuana and tobacco, and to 43 appropriate control subjects. Theophylline was cleared from the blood more rapidly in both marihuana and tobacco smokers with a mean increase in total clearance from 52 ml/kg/hr in nonsmokers to 74 ml/kg/hr in subjects who smoked either material alone. There was an additive increase in clearance to 93 ml/kg/hr in those who smoked both substances. Concern over enhanced metabolism of other drugs is probably warranted in tobacco and cannabis smokers.

Kinetics of theophylline transfer to breast milk.

Investigation of three nursIng women given theophylline intravenously defined the kinetics of theophylline transfer into breast milk. In each subject, R was constant, with no significant delay between the attainment of peak plasma and peak milk concentrations. The amount of theophylline eliminated into milk was equal to the product of R, milk volume, and the simultaneous maternal plasma concentration. The data indicate that theophylline cumulation to toxic concentrations should not occur in most breast-fed infants of asthmatic women treated with appropriate doses of theophylline.

Effects of three antibiotics on theophylline kinetics.

Nine healthy subjects received single doses of intravenous aminophylline (5 mg/kg) on four occasions separated by at least 1 wk. One trial was a control; the other three were performed during administration of therapeutic doses (250 mg every 6 hr) of tetracycline, erythromycin, and cephalexin. Theophylline concentrations in multiple plasma samples drawn during 24 hr after each dose were determined by high-pressure liquid chromatography. Mean kinetic variables for theophylline during control, tetracycline, erythromycin, and cephalexin trials, respectively, were: volume of distribution, 0.57, 0.61, 0.63, and 0.62 l/kg; elimination half-life (t1/2), 7.9, 9.2, 10.3, and 8.6 hr; total clearance, 0.90, 0.84, 0.78, and 0.89 ml/min/kg. In no case did differences between treatment conditions approach significance. Thus there is no consistent evidence of a kinetic interaction between theophylline and three commonly prescribed antibiotics.

Kinetics of intravenous theophylline.

The pharmacokinetics of theophylline was studied in 6 normal, nonsmoking, adult male volunteers. A constant-rate intravenous infusion of 3.84 to 4.98 mg/kg of theophylline (as the ethylenediamine salt, aminophylline) was administered over 40 min. Serum theophylline concentrations were measured for 24 hr by means of a gas chromatographic method specific for theophylline. Within 30 min of an average intravenous dose of 4.4 mg/kg of theophylline, serum levels reached 10 microgram/ml. The highest serum level at the end of the infusion was 17 microgram/ml. The serum concentration-time data were fitted to a two-compartment open model and yielded a mean serum half-life (t1/2) of 11.02 hr, a value longer than those previously reported. Our results indicated that after the original loading dose of 4.4 mg/kg was infused for 40min, an immediate infusion rate of 1.40 mg/kg/hr (1.65 mg/kg/hr aminophylline) would be necessary to maintain a serum level of 10 microgram/ml.

Enhancement of theophylline clearance by oral activated charcoal.

A randomized crossover trial of the effect of oral activated charcoal on the kinetics of intravenous theophylline was carried out in six normal male subjects. After intravenous aminophylline (6 mg/kg), subjects received water or water with activated charcoal (140 gm) in divided doses over 12 hr. Serum theophylline concentrations were measured from 0 to 24 hr after the aminophylline infusion. Treatment with activated charcoal decreased the serum t 1/2 from 6.4 +/- 1.2 to 3.3 +/- 0.4 (SEM) hr and the serum AUC from 78 +/- 14 to 42 +/- 4 mg . hr/l. Percent decrease in AUC after treatment with charcoal correlated positively with the endogenous theophylline serum t 1/2 (r = 0.94). These results suggest that oral activated charcoal (1) enhanced the total body clearance of theophylline and (2) may be efficacious in the treatment of theophylline poisoning, especially in patients with prolonged serum t 1/2s of theophylline.

Development of a radioimmunoassay for theophylline. Application to studies in premature infants.

The use of theophylline in premature infants has necessitated the development of an assay applicable to samples under 0.1 ml. To accomplish this we have developed a radioimmunoassay for theophylline. The antiserum to theophylline has been characterized and found to be highly specific for theophylline and sensitive to 2 ng. The affinity of our antibody and the titer developed in the goat allowed the assay to be performed routinely with the use of a plasma sample size of only 2 to 5 micronl. Theophylline levels of 6 to 20 microng/ml were found 2 hr after maintenance doses of aminophylline, 4 mg/kg per rectum in 12 premature infants. Terminal serum half-lives ranged from 12 to 54 hr.

Model for theophylline overdose treatment with oral activated charcoal.

The effect of repeated oral doses of activated charcoal on theophylline kinetics was studied in six subjects with hepatic cirrhosis and five patients with moderate theophylline poisoning to determine whether an activated charcoal regimen would be a useful strategy in patients with theophylline poisoning who did not require hemoperfusion. Six subjects with cirrhosis were injected IV with 6 mg/kg aminophylline followed by either water or water with activated charcoal (140 gm) in divided doses over 12 hr. In these subjects, treatment with activated charcoal decreased the mean (+/- SE) serum theophylline t1/2 from 12.7 +/- 4.0 hr to 4.0 +/- 0.7 hr. Subjects with the longest control t1/2s demonstrated the greatest charcoal effect. We developed a mathematical model that predicts that treatment with repeated oral doses of activated charcoal would result in an average serum theophylline t1/2 of 7.1 hr or less even if the subject's endogenous theophylline t1/2 is very long. In a pilot study of five patients with moderate theophylline poisoning, treatment with repeated oral doses of activated charcoal was well tolerated and led to a mean (+/- SE) t1/2 that was shorter than expected (4.9 +/- 0.8 hr, range 3.1 to 7.1 hr). We conclude that repeated oral doses of activated charcoal are relatively more effective in decreasing the serum theophylline t1/2 in persons with long endogenous t1/2s and that this may be useful for certain patients with mild or moderate theophylline poisoning.

Metabolic responses to plasma concentrations of theophylline.

We investigated the effect of intravenous infusions of aminophylline on plasma glucose, insulin (IRI), glucagon (IRG), growth hormone (HGH), cortisol, and free fatty acid (FFA) levels in healthy young subjects. Six received an intravenous loading dose of aminophylline (6.0 mg/kg over 20 min) followed by a maintenance dose (0.9 mg/kg/hr) for 100 min. Another 7 subjects initially received smaller loading (3.0 mg/kg) and maintenance (0.45 mg/kg/hr) doses, and after 60 min they received a second loading dose (3.0 mg/kg) followed by a larger maintenance dose (0.9 mg/kg/hr) over 120 min. In these fasting volunteers, infusion of aminophylline, which produced theophylline levels in the usual therapeutic range (10 to 20 microgram/ml) caused small increases in plasma glucose levels without changing IRI, IRG, HGH, or cortisol. There were rapid, pronounced, and prolonged rises in FFA associated with the aminophylline infusion. Increases in FFA paralleled the rise in theophylline levels. It is concluded that routine therapeutic doses of theophylline, i.e., doses that achieve serum levels normally encountered in treatment for bronchial asthma, cause a marked rise in FFA and a slight rise in glucose (8 +/- 3 mg/dl) without changing levels of IRI, IRG, HGH, or cortisol.

Pharmacokinetics of theophylline in children with asthma.

The pharmacokinetics of theophylline following intravenous injection of aminophylline, 4 mg/kg of body weight, were determined in 30 children with asthma and in 6 normal adult volunteers. The average total clearance of theophylline was 87 ml/hr/kg in the children and 57 ml/hr/kg in the adults. The biologic half-life of theophylline in the children ranged from 1.42 to 7.85 hours, reflecting mainly pronounced interindividual differences in the elimination rate constant of the drug. There was no significant difference between the children and adults with respect to the distribution rate constants and apparent volumes of distribution of theophylline, but the elimination rate constant of the drug was considerably higher in children than in adults. Thus, children eliminate theophylline more rapidly on the average than do adults and also show pronounced interindividual differences in the elimination of the drug. Compared to adults, children tend to require relatively larger amounts of theophylline per day and the doses may have to be given at shorter intervals of time.

Inhibition of hepatic microsomal drug metabolism by the immunosuppressive agent cyclosporin A.

Cyclosporin A (CsA), an orally active immunosuppressive agent, was shown to inhibit cytochrome P-450 dependent biotransformation of drugs in the mouse. It competitively inhibited the hydroxylation of benzo[a]pyrene and the N-demethylation of aminopyrine in hepatic microsomes with Ki values of 93 and 1540 microM respectively. This selective inhibition for benzo[a]pyrene hydroxylase by CsA was substantiated in vivo by selective inhibition of total body clearance of theophylline, but not of antipyrine. CsA was itself N-demethylated by hepatic microsomes with a Km of 808 microM. CsA interacted directly with cytochrome P-450, causing a reverse type I spectral change in hepatic microsomes. No metabolic intermediate complexes could be demonstrated. These results suggest that CsA has the potential to cause drug interactions involving inhibition of drug biotransformation, particularly of drugs that are metabolised by the same types of cytochrome P-450 which oxidise benzo[a]pyrene and theophylline.

Kinetics of theophylline; variability and effect of arterial pH in chronic obstructive lung disease.

The pharmacokinetic behavior of theophylline was determined in 12 patients during an acute exacerbation of their chronic obstructive pulmonary disease. A 5.6 mg/kg loading dose of aminophylilne was administered, followed three hours later by a 0.9 mg/kg/hr continuous infusion. The loading dose increased the serum theophylline level an average of only 5.77 microgram/ml. After the loading dose, only five patients had levels greater than 10 microgram/ml. Mean initial drug clearance was 0.77 L/kg/hr, half-life 9.1 hr, and apparent volume of drug distribution .887 L/kg. Wide inter- and intrapatient pharmacokinetic variability was observed. The variability of drug distribution was inversely correlated with the arterial pH. These patients with chronic obstructive pulmonary disease appeared to require more theophylline when acidemic than when alkalemic to achieve similar serum theophylline concentrations.

Pharmacokinetics of theophylline in infancy.

Theophylline clearance rates and half-life values were measured in 15 infants aged three to 23 months, after infusion of aminophylline by the intravenous route for at least 24 hours. The mean clearance rate was 1.07 +/- 0.55 ml/min-kg, which is comparable with values obtained by others in older children. There was some correlation of clearance rates with age. The mean half-life was 4.4 +/- 2.2 hours. There was a tenfold variability in half-life, suggesting that individualization of theophylline dose is especially important in infants if undertreatment and toxicity are to be avoided.

Temporal variations in trough serum theophylline concentrations at steady state.

Temporal variations in serum theophylline concentrations were observed in 14 healthy volunteers receiving multiple doses of theophylline. After repeated oral doses (6.9--18.2 mg/kg/day) of theophylline as either a nonalcoholic aminophylline solution or a controlled-release capsule, trough theophylline levels at steady state were significantly higher (p less than 0.05) in the morning than in the afternoon or evening. With the solution, the mean (+/-SE) trough serum level at 7 am was 11.1 +/- 0.9 micrograms/ml, and at 1 pm it was 9.6 +/- 0.8 micrograms/ml. With the capsule, the mean (+/-SE) trough serum level at 8 am was 13.8 +/- 0.9 micrograms/ml, and at 8 pm it was 10.7 +/- 0.9 micrograms/ml. Temporal variations in serum theophylline concentrations have not been reported previously and may be important in therapeutic monitoring.