NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

How to achieve higher repeatability and reproducibility in capillary electrophoresis.

The influence of experimental parameters on precision of migration times and accuracy of integration has been studied. The repeatability of migration times strongly depends on the proper selection of the rinse steps between each run of a CE system. The rinse steps have to be optimized additionally with each separation system. This is especially important for systems providing an anodic electroosmotic flow. The errors introduced by the integration software were studied by transferring the same data sets to different commercial available integration softwares. A strategy for the transfer of raw data files between several softwares is described. By using systems with identical peak areas or with identical peak heights it could be shown that the newly introduced softwares can cope better with the leading or tailing peaks. The RSD of quantitation strongly depends on the signal-to-noise ratio. At S/N ratios larger than 35 no differences between the various softwares studied could be observed. At lower S/N ratios the newly released softwares are to be preferred.

Simultaneous determination of alkali, alkaline earth and transition metal ions by capillary electrophoresis with indirect UV detection.

Determination of metal ions in aqueous samples using capillary electrophoresis can be accomplished with indirect UV detection. For optimal determination of alkali, alkaline earth, and transition metal ions, several electrolyte systems were studied. Detection at 214 nm was performed with a background electrolyte containing reagents with inherent absorbance in the UV range: imidazole, 4-methylbenzylamine and 4-aminopyridine. Glycolic acid and alpha-hydroxyisobutyric acid were used as complexing reagents. A mixture of 16 metal ions was successfully separated. The detection limits were between 92 ppb for Ca and 454 ppb for Cu with hydrostatic injection. All peaks were completely resolved and well separated. A separation efficiency of about 650,000 theoretical plates per meter was achieved for the Mg ion. The described methods can be used successfully in routine analysis of real samples. One of the methods was applied to an environmental water sample from the Georgian river Kasretula.

Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide: quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabelling.

7-(2-Hydroxypropyl)guanine (7-HPG) constitutes the major adduct from alkylation of DNA by the genotoxic carcinogen, propylene oxide. The levels of 7-HPG in DNA of various organs provides a relevant measure of tissue dose. 7-Alkylguanines can induce mutation through abasic sites formed from spontaneous depurination of the adduct. In the current study the formation of 7-HPG was investigated in male Fisher 344 rats exposed to 500 ppm of propylene oxide by inhalation for 6 h/day, 5 days/week, for up to 20 days. 7-HPG was analyzed using the 32P-postlabelling assay with anion-exchange cartridges for adduct enrichment. In animals sacrificed directly following 20 days of exposure, the adduct level was highest in the respiratory nasal epithelium (98.1 adducts per 10(6) nucleotides), followed by olfactory nasal epithelium (58.5), lung (16.3), lymphocytes (9.92), spleen (9.26), liver (4.64), and testis (2.95). The nasal cavity is the major target for tumor induction in the rat following inhalation. This finding is consistent with the major difference in adduct levels observed in nasal epithelium compared to other tissues. In rats sacrificed 3 days after cessation of exposure, the levels of 7-HPG in the aforementioned tissues had, on the average, decreased by about one-quarter of their initial concentrations. This degree of loss closely corresponds to the spontaneous rate of depurination for this adduct (t 1/2 = 120 h), and suggests a low efficiency of repair for 7-HPG in the rat. The postlabelling assay used had a detection limit of one to two adducts per 10(8) nucleotides, i.e. it is likely that this adduct could be analyzed in nasal tissues of rats exposed to less than 1 ppm of propylene oxide.

First-pass metabolism of 1,3-butadiene in once-through perfused livers of rats and mice.

First-pass metabolism of 1,3-butadiene (BD) leading to 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), 3-butene-1,2-diol (B-diol), 3,4-epoxy-1,2-butanediol (EBD) and crotonaldehyde (CA) was studied quantitatively in the once-through BD perfused liver of mouse and rat by means of an all-glass gas-tight perfusion system. Metabolites were analyzed using gas chromatography equipped with mass selective detection. The perfusate consisted of Krebs-Henseleit buffer (pH 7.4) containing bovine erythrocytes (40%v/v) and BD. The perfusion flow rates through the livers were 3-4 ml/min (mouse) and 17-20 ml/min (rat). The BD concentrations in the liver perfusates were 330 nmol/ml (mouse) and 240 nmol/ml (rat) being high enough to reach almost saturation of BD metabolism. The mean rates of BD transformation were about 0.014 and 0.055 mmol/h per liver of a mouse and a rat, respectively, being similar to the values expected from in-vivo measurements. There were marked species differences in the formation of BD metabolites. In the effluent of mouse livers, all three epoxides (EB: 9.4 nmol/ml; DEB: 0.06 nmol/ml; EBD: 0.07 nmol/ml) and B-diol (8.2 nmol/ml) were detected. In the perfusate leaving naïve rat livers, only EB and B-diol were found. In that of rat liver, EB concentration was 8.5 times smaller than in that of mouse liver, whereas B-diol concentrations were similar in the effluent liver perfusate of both species. CA was below the limit of its detection (60 nmol/l) in the liver perfusate of mice and of naïve rats. Of BD metabolized, the sum of the metabolites investigated in the effluent amounted to only 30% (mouse) and 20% (rat). In first experiments with rat liver, glutathione (GSH) was depleted by pretreating the animals with diethylmaleate. With the exception of EBD (not quantifiable due to an interfering peak), all other metabolites including CA were found in the effluent perfusate summing up to about 70 and 100% of BD metabolized, which indicates the quantitative importance of the GSH dependent metabolism. In summary, the results demonstrate the relevance of an intrahepatic first-pass metabolism for metabolic intermediates of BD, which undergo further transformation immediately after their production in the liver before leaving this organ. Hitherto, the occurrence of this first-pass metabolism was only hypothesized. The findings will help to explain the drastic species difference between mice and rats in the carcinogenic potency of BD.

Propylene oxide: mutagenesis, carcinogenesis and molecular dose.

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO-DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC-HRMS) analysis. The second method utilized 32P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4 +/- 80.1 (respiratory), 396.8 +/- 53.1 (olfactory) and 34.6 +/- 3.0 (liver) pmol adduct/mumol guanine using GC-HRMS. Lower values, 592.7 +/- 53.3, 296.5 +/- 32.6 and 23.2 +/- 0.6 pmol adduct/mumol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by 32P-postlabeling yielded the following values: 445.7 +/- 8.0 (respiratory), 301.6 +/- 49.2 (olfactory) and 20.6 +/- 1.8 (liver) pmol adduct/mumol guanine in DNA of rats killed immediately after exposure cessation and 327.1 +/- 21.7 (respiratory), 185.3 +/- 29.2 (olfactory) and 15.7 +/- 0.9 (liver) pmol adduct/mumol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.

[Consciousness disorders in focal lesions of the right and left cerebral hemispheres]. / Rasstroistva soznaniia pri ochagovom porazhenii pravogo i levogo polusharii mozga

In the light of modern concepts of functional asymmetry in the human brain, the authors analyse paroxysmal conditions of changed consciousness which occurred in 160 patients with lesions of the hemispheres due to brain tumors (75 patients) and in epilepsy (85 patients). It is shown that certain forms of changed consciousness are characteristic of disorders of the right and left hemispheres in right-handedness. This clinical fact is taken as a basis for the assumption that in the formation of consciousness both hemispheres in different ways play a certain role and they provide different "formating" kinds of consciousness: the right hemisphere--a direct perception or a sensory perception of the environment and itself, the left--process of abstract perception based on speech, symbols, semantics.

[Dynamics of diencephalic-hypothalamic disorders after the removal of basal meningiomas]. / Dinamika diéntsefal'no-gipotalamicheskikh narushenii posle udaleniia bazal'nykh meningiom.

Comprehensive dynamic examination of 27 patients after removal of basal meningiomas revealed some characteristic changes in the results of REG and EEG and in the neurological status. It was also found that the dynamics of the data studied differed with the course of the postoperative period. Some EEG and REG data of favourable prognostic importance were noted. The study of these data promotes objectivization of the course of the postoperative period and is thus also important in the choice of rational therapy in the postoperative period.

[Status of biogenic amine metabolism in tumors of chiasm-sellar localization]. / Sostoianie obmena biogennykh aminov pri opukholiakh khiazmal'no-selliarnoi lokalizatsii.

Histamine, serotonin, and catecholamine metabolism was studied in 47 patients with tumors of diencephalic localization. It was shown that the degree of involvement of the hypothalamus in the tumor determined the different character of metabolic changes of biogenous amines. Three stages of biogenous amine metabolism were distinguished: activation, compensation, and exhaustion. The degree of metabolic changes manifestation before the operation determined to a great measure the organism's reaction to it. The work substantiates the necessity for taking into consideration the metabolism of biogenous amines before undertaking surgery.

[Experience with transcranial surgery of hypophyseal adenomas]. / Opyt transkranial'nykh operatsii pri adenomakh gipofiza.

The article generalizes experience in intracranial operations on 453 patients performed at the Institute of Neurosurgery, AMS, USSR in 1983-1987. The tumors had a pronounced extrasellar growth in most cases. The surgical approaches and the peculiarities of surgical techniques in removal of pituitary adenomas are described. The importance of microsurgical techniques which raise the radical character of the surgical interventions and improve their results is discussed.

[Transnasosphenoidal microsurgery of hypophyseal adenomas]. / Transnazosfenoidal'naia mikrokhirurgiia adenom gipofiza.

The results of surgical treatment of 550 patients through a transnasosphenoidal approach are analysed. The results of management of endosellar and endoextrasellar adenomas are discussed. Various groups of patients are set apart according to the hormonal activity of the tumor. The indications and contraindications for operations through a transnasosphenoidal approach are specified beforehand. The complications in transnasal operations are described and mortality is analysed.

[Catamnestic evaluation of the results of surgical and combined treatment of pituitary tumors]. / Katamnesticheskaia otsenka rezul'tatov khirurgicheskogo i kombinirovannogo lecheniia opukholei gipofiza.

The work deals with the study of the results of surgical and combined treatment of 455 patients with adenoma of the hypophysis who were operated on between 1971 and 1977. the catamnestic follow-up periods ranged from 2 to 7 years and more. Microsurgical techniques were used in operations on 146 patients, 309 patients underwent operation by the traditional method. Among 455 patients, 190 were treated by surgery alone, whereas 265 patients received combined treatment (surgical intervention and radiotherapy, mostly in the postoperative period). Study of the results of catamnestic investigation showed that with the use of microsurgical techniques the intervention was more radical and tumors with pronounced extrasellar growth could be removed. The current diagnostic and surgical means as well as combined therapy (surgery combined with radiotherapy) do not solve the problem of the prevention of recurrent tumors of the hypophysis. Recurrences were observed in 22.1% of cases after surgical intervention and in 11.1-15.18% of cases after combined treatment. Combined therapy produces poorer results from the standpoint of correction of the visual and hormonal disorders. The existence, of a considerable number of recurrences makes necessary exact identification of the histobiological and hormonal nature of the whole group of adenomas because the classification accepted today does not meet the essence of the clinical problems encountered in the treatment of these tumors. An exact classification of adenomas of the hypophysis according to their spread is necessary.

[The effects of halothane and enflurane on uterine activity during childbirth (author's transl)]. / Halothan, Enfluran und ihr Einfluss auf die Uterusaktivität am Geburtstermin.

The effects of equipotent low concentrations of halothane and enflurane on uterine contractions during labour were studied by means of transcervical toko-ergometry. Administration of halothane was followed by a highly significant reduction of uterine activity to 48% +/- 14 of its original value. Ethrane in equipotent concentration of 1.0% reduced uterine activity in 5 women to only 83.7% +/- 29 (not significant). Five other women were also tested with ethrane in 1.5% concentrations: uterine activity was reduced to 79% +/- 24 of its original value (not significant). The findings indicate that low concentrations of ethrane relax uterine contractions less than do equipotent concentrations of halothane. The clinical applications of the observations are discussed.

[Current problems in the surgical management of parasellar meningiomas]. / Aktuelle Probleme der chirurgischen Behandlung von parasellären Meningiomen.

In the period from 1967 to 1972, 302 patients suffering from basal parasellar meningiomas were operated on at the Burdenko Institute of Moscow. Further, an analysis of the results obtained in the surgical treatment of 43 patients is given who were operated on in the last two years by employing the microsurgical technique. The considerable advantages of the use of the operating microscope and the microtechnique are shown. The mortality rate was reduced to one half and the radicality increased from 50 to 80--85 per cent. All Tuberculum sellae meningiomas could be removed totally. Also in the presence of bad general conditions of the patients no contraindications were seen but the very last chances were used. Postoperative supervision consisted in measuring and checking the cerebrospinal pressure, intracerebral pressure, interstitial fluid pressure, local and regional blood flow, pCO2 and pO2, electrolyte balance, osmolarity and pH. In this way, low pressure conditions could frequently also be determined. The influence of an early diagnosis on the prognosis of the disease is specially pointed out.

Kinetics of propylene oxide metabolism in microsomes and cytosol of different organs from mouse, rat, and humans.

Kinetics of the metabolic inactivation of 1,2-epoxypropane (propylene oxide; PO) catalyzed by glutathione S-transferase (GST) and by epoxide hydrolase (EH) were investigated at 37 degrees C in cytosol and microsomes of liver and lung of B6C3F1 mice, F344 rats, and humans and of respiratory and olfactory nasal mucosa of F344 rats. In all of these tissues, GST and EH activities were detected. GST activity for PO was found in cytosolic fractions exclusively. EH activity for PO could be determined only in microsomes, with the exception of human livers where some cytosolic activity also occurred, representing 1-3% of the corresponding GST activity. For GST, the ratio of the maximum metabolic rate (V(max)) to the apparent Michaelis constant (K(m)) could be quantified for all tissues. In liver and lung, these ratios ranged from 12 (human liver) to 106 microl/min/mg protein (mouse lung). Corresponding values for EH ranged from 4.4 (mouse liver) to 46 (human lung). The lowest V(max) value for EH was found in mouse lung (7.1 nmol/min/mg protein); the highest was found in human liver (80 nmol/min/mg protein). K(m) values for EH-mediated PO hydrolysis in liver and lung ranged from 0.83 (human lung) to 3.7 mmol/L (mouse liver). With respect to liver and lung, the highest V(max)/K(m) ratios were obtained for GST in mouse and for EH in human tissues. GST activities were higher in lung than in liver of mouse and human and were alike in both rat tissues. Species-specific EH activities in lung were similar to those in liver. In rat nasal mucosa, GST and EH activities were much higher than in rat liver.