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.

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.

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.

Comparative study of the binding of pepsin to four alkaloids by spectrofluorimetry.

The interactions between pepsin and four alkaloids, including caffeine (Caf), aminophylline (Ami), acefylline (Ace), diprophylline (Dip), were investigated by fluorescence, UV-visible absorption, resonance light scattering, synchronous fluorescence spectroscopy and 3D spectroscopy under mimic physiological conditions. The results revealed that Caf (Ami/Ace/Dip) caused the fluorescence quenching of pepsin by the formation of Caf (Ami/Ace/Dip)-pepsin complex. The binding constants and thermodynamic parameters at three different temperatures, the binding locality and the binding power were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize the complex. Results showed that aminophylline was the stronger quencher and bound to pepsin with higher affinity than other three alkaloids.

Aminophylline, administered at usual doses for rodents in pharmacological studies, induces hippocampal neuronal cell injury under low tidal volume hypoxic conditions in guinea-pigs.

OBJECTIVES: To establish whether aminophylline, administered at usual doses for rodents in pharmacological studies, induces brain injury in systemic hypoxaemia in guinea-pigs. METHODS: A hypoxaemia (partial oxygen tension of arterial blood (PaO2) = 40-60 mmHg) model was developed by low tidal volume mechanical ventilation in guinea-pigs. KEY FINDINGS: Under hypoxic conditions, aminophylline significantly increased the concentration of brain-specific creatine kinase in the serum in a dose- and time-dependent manner. A reduced number of hippocampal neuronal cells in the CA1 region, an increase in the concentration of neuron-specific enolase (NSE) in cerebrospinal fluid (CSF), an increase in lipid hydroperoxides and a decrease in the ratio of glutathione to glutathione disulfide in the brain tissues were also observed. These effects were not observed when aminophylline at the same doses was administered under normoxic conditions (PaO2 = 80-100 mmHg). There was no difference in either serum or CSF concentrations of theophylline between normoxic and hypoxic conditions. Another methylxanthine, caffeine, did not increase the concentration of NSE in CSF. CONCLUSIONS: Aminophylline potentially induces brain damage under hypoxic conditions. We suggest that aminophylline treatment has adverse effects in patients with hypoxaemia subsequent to respiratory disorders such as asthma.

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.

Use of circular permutation and end modification to position photoaffinity probes for analysis of RNA structure.

Photocrosslinking allows first-order structural analysis with relatively small amounts of biological material and can be applied in complex in vitro systems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modification of circularly permuted RNAs. Application of this technique provided a library of constraints that, together with biochemical and phylogenetic comparative data, were used to develop a structure model of the bacterial ribonuclease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted RNA transcripts can be modified with high efficiency at both the 5' and 3' termini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental conditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure.

Pharmacokinetics of theophylline metabolites in 8 Chinese patients.

AIM: To study theophylline metabolites pharmacokinetics in patients after a therapeutic dose. METHODS: Eight adult patients with mild bronchial asthma and normal liver function were infused aminophylline intravenously (6.6 mumol.kg-1). The plasma concentrations of theophylline and its 4 metabolites: 1,3-dimethyluric acid (DMUA), 3-methylxanthine (3-MX), 1-methyluric acid (MUA), and the intermediate 1-methylxanthine (1-MX) were monitored by HPLC throughout 24 h. RESULTS: The plasma concentration of DMUA was the highest one among the 4 metabolites. 3-MX showed the slowest elimination rate. The plasma concentration of 1-MX throughout a 24-h period showed that there was a picking up of 1-MX (from 0.04 mumol.L-1 to 1.05 mumol.L-1) in the next morning. CONCLUSION: The formation of DMUA was the main metabolites. During night there was an accumulation of 1-MX.

Properties controlling the diffusion and release of water-soluble solutes from poly(ethylene oxide) hydrogels: 3. Device geometry.

The diffusive release from hydrogels can be determined by both composition and geometry. This paper presents a theoretical and experimental comparison of the release characteristics of proxyphylline in water-swollen slabs, spheres, and cylinders of a urethane cross-linked poly(ethylene oxide). Contrary to general conventional wisdom it was found that practically cylinders and spheres, which have considerable potential advantages for oral delivery, can provide good 'anomalous' rates for which the 'exponent of release' into water from the dry xerogels is c. 0.8 compared with 1.0 for zero order. An exponent of 0.94 was found for release into water from 'larger' xerogel flat slabs thus confirming that these configurations can provide essentially constant delivery formulations from which the active agent cannot be 'dumped'. For up to 40% total drug release, the theoretical release profiles were essentially of identical form for all three geometries in the swollen state and, as expected in theory and practice, showed an exponent for release of close to 0.5. However, the experimental release of proxyphylline was found to be more sustained from swollen spheres of these polymers than theory would predict. The half life times for release were further extended by approximately two and a half times for the initially dry devices compared with the initially swollen ones.

Properties controlling the diffusion and release of water-soluble solutes from poly(ethylene oxide) hydrogels. 4. Extended constant rate release from partly-coated spheres.

Spheres of poly(ethylene oxide) xerogel from 4-17 mm diameter were impregnated with the drug, proxyphylline, or the herbicide, 2,4-DNa and then partly coated with a water-impermeable elastomer. When placed in water the active additive was released over periods ranging from hours to weeks at a nearly constant rate. The effect of the hydrogel sphere dimensions and the window size through the elastomer, on water uptake and consequent release of the active agent were examined. These devices superficially possess a configuration similar to some commercial osmotic devices though the active-agent release from the devices of this paper is, in fact, diffusive and not osmotically driven. They appear to provide a very versatile and simple design allowing relatively constant release of the active agent over periods of hours to months merely by changing the device geometry.

The effects of charcoal and sorbitol (alone and in combination) on plasma theophylline concentrations after a sustained-release formulation.

1. The effects of charcoal and sorbitol, alone and in combination, were investigated in eight healthy female volunteers who received 600 mg slow-release theophylline (two 300 mg capsules). 2. The area under the plasma concentration time curve to 24 h (AUC0-24) after theophylline alone was significantly greater than after both the charcoal and charcoal plus sorbitol phase. 3. Charcoal and charcoal with sorbitol also significantly reduced the maximum plasma theophylline concentration (Tmax) and time to maximum concentration (Cmax). 4. Sorbitol significantly increased Cmax and shortened Tmax. 5. Although sorbitol did not reduce the adsorptive efficacy of charcoal, its use alone may be deleterious in poisoning with sustained-release theophylline products.

Aminophylline for acute exacerbations of chronic obstructive pulmonary disease. A controlled trial.

STUDY OBJECTIVE: To determine the efficacy of intravenous aminophylline in the treatment of patients hospitalized for exacerbation of chronic obstructive pulmonary disease. DESIGN: Randomized, double-blind, placebo-controlled trial during the first 72 hours of hospitalization. PATIENTS: Thirty patients admitted from the emergency room or walk-in clinic with the primary diagnosis of an exacerbation of chronic obstructive pulmonary disease. Twenty-eight patients completed the study; 2 patients, 1 receiving placebo and 1 receiving aminophylline, were removed from the study because of respiratory failure requiring mechanical ventilation. INTERVENTIONS: PATIENTS received either intravenous aminophylline or placebo, in addition to nebulized, inhaled 0.3 mL of a 5% solution every 6 hours; methylprednisolone, 0.5 mg/kg body weight every 6 hours intravenously; ampicillin, 500 mg orally every 6 hours (tetracycline or trimethoprim-sulfamethoxazole were substituted in penicillin-allergic patients); and supplemental oxygen as needed. Aminophylline infusion rates were adjusted by an unblinded investigator to achieve theophylline levels of 72 to 83 mumol/L. Changes were also made in placebo infusion rates to maintain the double-blind design. MEASUREMENTS AND MAIN RESULTS: The forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) before and after metaproterenol inhalation were measured twice daily by a blinded investigator, who also administered a verbal dyspnea index with a scale of 1 to 10 and questioned patients regarding possible side effects of treatment (tremor, palpitations, nausea, or vomiting). Arterial blood gas measurements at 72 hours were compared with those obtained on admission. Significant improvements in FEV1 and FVC measured before and after metaproterenol treatment and in dyspnea occurred over time in both treatment groups (p less than 0.05 for all measurements). However, there were no significant differences between the placebo and aminophylline groups in any of the spirometric measurements or the dyspnea indices (p greater than 0.5 in all five analyses). The mean increases (+/- SE) in Po2 of 1.9 (+/- 0.5) kPa with placebo and 1.7 (+/- 0.7) kPa with aminophylline and the mean decreases in PCO2 of 0.5 (+/- 0.4) kPa with placebo and 1.2 (+/- 0.4) kPa with aminophylline were not significantly different (p greater than 0.6 for PO2, p greater than 0.2 for PCO2).(ABSTRACT TRUNCATED AT 400 WORDS)

Demostración del papel de la estimulación de receptores purinérgicos en el efecto electrofisiológico de los compuestos adenilicos en el hombre / The electrophysiological actions of ampare medrate through purinergic receptor stimulation

Para estudiar el mecanismos de acción del AMP en el nodo A-V, se estudiaron 10 pacientes con taquicardia supraventricular por re-entrada (TVS) que incluía el nodo AV en su circuito.. El promedio de edad fue 32.8 + ou - 11.1 años. La taquicardia se indujo por estimulación auricular programada y se realizaron registros electrofisiológicos intracavitarios en todos los casos. En la primera etapa la administración de AMP suprimió la arrtmia en todos los casos en un tiempo promedio de 15.2 + ou - 2.6 seg., con una dosis de 0.095 + ou - 0.037mg/kg. En la segunda etapa la administración de atropina (0.04mg/kg.) no impidió la supresión de la arritmia. La dosis de AMP fue 0.122 + ou - 0.045mg/kg (P3NDS) y el tiempo promedio 16.2 + ou - 2.2 (p=NDS). En la tercera etapa: la administración de AMP no interrumpió la arritmia inducida bajo el efecto de atropina y aminofilina (4mg/kg) en 8 de los pacientes. En uno de los restantes la dosis necesarias fue 0.250mg/kg., y en el otro fue 0.075mg/kg. No hubo efectos colaterales graves. Se demuestra que la acción del AMP está mediada por estimulación agonista de receptores purinérgicos en el nodo AV

Effect of organic vehicles on the pharmacokinetics of aminophylline administered intravenously to goats.

Aminophylline dissolved in water, propylene glycol, or dimethyl sulfoxide was administered intravenously to goats in a randomized cross-over experiment. Model-dependent and model-independent pharmacokinetic parameters for theophylline were compared on the basis of the solvent used in the dosage form administered. No difference was found in any pharmacokinetic parameter. Thus, we found no evidence for the possibility that the organic solvents studied would confound pharmacokinetic investigations of theophylline and similar lipophilic drugs.

Theophylline disposition in tetraplegic man.

We studied the time-course of serum theophylline concentration after a 30-minute intravenous infusion of aminophylline (6 mg/kg) in 18 traumatic tetraplegic (nine smoking, nine nonsmoking) and in six control (nonsmoking) subjects. All study subjects were healthy men with normal renal function. Pharmacokinetic parameters were calculated from a linear regression analysis of the terminal log-linear portion of the log serum theophylline concentration-time curves. The terminal elimination kinetics of theophylline in this study were best characterized by first order elimination from a one compartment-open model. A statistically significant difference (P less than .05) was demonstrated between the means and variances of theophylline clearance (Cl ml/kg/min) and hours of half-life (t1/2) for control subjects and smoking tetraplegics. Total body clearance of theophylline was greater in all tetraplegic subjects, the greatest difference in total clearance of theophylline adjusted to body weight being observed between tetraplegic smokers and nonsmoking controls with an intact central nervous system (P less than .05). Theophylline volume of distribution (Vdarea) did not differ significantly from Vdarea in able-bodied subjects. Tetraplegic individuals have multiple disorders and marked pharmacokinetic variation, which might be expected to make serum concentration and toxicity of theophylline unpredictable using population averages.

Aminophylline toxicity.

Aminophylline therapy has undergone change in the past decade. With the changes in usage and dosage forms, the frequency of toxicity in the pediatric population, especially in adolescents, has increased dramatically. Two distinct patterns, chronic and acute, have been recognized and treatment methods for both are changing. Table 4 summarizes the emerging state-of-the-art therapy for aminophylline toxicity. Judging from the activity seen in the literature, investigation into aminophylline toxicity will continue to be a priority. We will see a greater understanding of the disease process and a refining of the therapeutic process. The ultimate goal is the elimination of mortality and the minimization of morbidity from aminophylline toxicity.