Comparison of lung deposition of amikacin by intrapulmonary percussive ventilation and jet nebulization by urinary monitoring.

The intrapulmonary percussive ventilation (IPV), frequently coupled with a nebulizer, is increasingly used as a physiotherapy technique; however, its physiologic and clinical values have been poorly studied. The aim of this study was to compare lung deposition of amikacin by the nebulizer of the IPV device (Percussionaire; Percussionaire Corporation; Sandpoint, ID) and that of standard jet nebulization (SST; SideStream; Medic-Aid; West Sussex, UK). Amikacin was nebulized with both devices in a group of five healthy subjects during spontaneous breathing. The deposition of amikacin was measured by urinary monitoring. Drug output of both devices was measured. Respiratory frequency (RF) was significantly lower when comparing the IPV device with SST (8.2 +/- 1.6 breaths/min vs. 12.6 +/- 2.5 breaths/min, p < 0.05). The total daily amount of amikacin excreted in the urine was significantly lower with IPV than with SST (0.8% initial dose vs. 5.6% initial dose, p < 0.001). Elimination halflife was identical with both devices. Drug output was lower with IPV than with SST. The amount of amikacin delivered to the lung is sixfold lower with IPV than with SST, although a lower respiratory frequency was adopted by the subjects with the IPV. Therefore, the IPV seems unfavorable for the nebulization of antibiotics.

Enzymic N-demethylation reaction catalysed by red blood cell cytosol.

Red blood cell cytosol promotes enzymic N-demethylation reactions which display typical Michaelis-Menten kinetics with respect to N-methylaniline as substrate. The demethylase activity is linked with hemoglobin (Hb) and is enhanced in the presence of NADH and the NADH-methemoglobin reductase system. It has been adduced that Hb in its oxygenated form is involved in the reaction.

Dopamine receptor-modulated [35S]GTPgammaS binding in striatum of 6-hydroxydopamine-lesioned rats.

The role of dopamine receptor-G protein coupling in the development of striatal dopamine receptor supersensitivity was studied in rats with a 6-hydroxydopamine (6-OHDA)-induced unilateral lesion of the nigrostriatal pathway. This coupling was assessed by the measurement of dopamine agonist-induced guanosine 5'-O-(gamma[35S]thio)triphosphate ([35S]GTP-gammaS) binding in striatal membranes, at different periods of time (1-5 weeks) following the microinjection of the neurotoxin. From the first to the fifth week following the lesion, basal and dopamine-stimulated [35S]GTPgammaS-specific binding were found to be enhanced in the denervated striata as compared to their control counterpart. D2 dopamine receptors were clearly demonstrated to be involved in this supersensitivity, as assessed by measuring N-propylnorapomorphine (NPA)-, quinpirole- and bromocriptine-induced [35S]GTPgammaS-specific binding. The involvement of D1 dopamine receptors was indirectly studied by the combination of dopamine with a saturating concentration of the selective and potent D2 antagonist domperidone. In these conditions, the remaining response to dopamine was also found to be significantly increased following the lesion. These results are consistent with the hypothesis that, in addition to D2 dopamine receptor upregulation, modulation of dopamine receptor-G protein interaction is involved in the hypersensitivity accompanying striatal dopamine depletion.

Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats.

The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Ki's for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).

Evaluation of the formalin test to assess the analgesic activity of diflunisal in the rat.

The formalin test was evaluated to assess the analgesic activity of diflunisal in the rat. Fifty microliters of a 5% formalin solution was injected into the hindpaw of rats and two distinct nociceptive behaviors, i.e. flinching/shaking and licking/biting of the injected paw, were recorded over 120 min. The effect of factors such as age of the animal, time of injection (morning vs. afternoon), site of injection (right vs. left hind paw) were evaluated. Both nociceptive behaviors exhibited a biphasic time course. Rats weighing 210-220 grams showed a more intense response compared to older rats weighing 240-250 or 270-280 grams. The nociceptive behavior response was affected by the time of formalin injection and was more pronounced in the morning. Diflunisal (100 mg/kg, i.v. infusion over 3 min) caused a significant delay in the flinching/shaking response vs. time curve, whereas the licking/biting response was significantly inhibited. When carried out under carefully controlled conditions, the formalin test may be useful to study the analgesic effect of diflunisal in the rat. It seems to be less sensitive, however, than other commonly used nociceptive tests.

Phenyl-substituted normethadones: synthesis and pharmacology.

Phenyl-substituted normethadone derivatives were synthesized and their affinity (IC50) for opioid receptors was determined by displacement of the specific binding sites of [3H]sufentanyl on rat brain preparations. Substitution resulted in a decrease of affinity in-vitro. These results suggest that normethadone-like compounds may interact with the P subsite of the mu-opioid receptor and that the P subsite has a well-defined cavity shape of stringent dimensions.

The use of a non-linear regression approach for the analysis of the ouabain-K+ interaction with (Na+ + K+)-ATPase from guinea pig and rat hearts.

1. The interaction between ouabain and K+ and their effects on (Na+ + K+)-ATPase activity were studied using microsomes from guinea pig and rat heart. 2. Microsomes were incubated in the presence of various concentrations of K+ and ouabain and ATPase activity was estimated by measuring the inorganic phosphate liberated. The experimental data were analyzed statistically by micro-computer, using a non-linear regression program based on the steepest descent technique. 3. The experimental data were best fitted by a model which assumes that ouabain acts like a mixed inhibitor with respect to the apparently cooperative K+ activation of (Na+ + K+)-ATPase. This quantitative approach provided estimates (with approximate standard deviations) of all the parameters involved in the model. 4. The inhibition constant for the uncompetitive term of the effect was 7- to 9-fold higher than the inhibition constant for the competitive term for both the guinea pig and rat heart preparations. 5. The present results indicate that graphical analyses are helpful for illustrative purposes but suggest that a computerized, non-linear regression program simultaneously analyzing all the non-linearized data should be used to quantify the complex kinetic parameters and to discriminate objectively among possible models.

Diagnostic specificities and sensitivities of anti dsDNA, anti membrane DNA and anti nucleosomes autoantibodies.

The aim of this study is to evaluate, from 369 routine sera of SLE and control patients, the worth of anti double stranded nuclear DNA, anti nucleosomes autoantibodies and anti membrane DNA for the diagnosis of SLE. Cell membrane associated DNA (mDNA) has been described on B lymphocytes and monocytes, but not on T cells. Antibodies to mDNA were identified by an indirect immunofluorescence assay using a B cell line fixed but not permeabilised. At a 1:40 serum dilution, anti mDNA is almost associated with the diagnosis of systemic lupus erythematosus (SLE). Anti mDNA were shown to be different in specificity as compared with anti double stranded nuclear DNA. We compare its characteristics as diagnostic procedure to the conventional anti dsDNA antibody detection and to the recently introduced anti nucleosome antibody test documented as associated with SLE. It appears that the best sensitivity (0.65) and specificity (0.98) is given by the anti mDNA test.

[Enrichment of cytochrome P-450 in human liver microsomes by the action of proteases]. / Enrichissement en cytochrome P450 de microsomes de foie humain par l'action de la protéase.

Human liver microsomes have been partially enriched in cytochrome P450 using a simple and rapid method. Microsomes were digested with protease XXVII (40 micrograms/nmole cyt. P450), then incubated with CHAPS, a zwitterionic detergent (25 mg/nmole cyt. P450) in combination with 0.07% protamine sulfate and the solubilized cyt. P450 was separated by ultracentrifugation. By this method, about 35% of total microsomal protein was solubilized with more than 80% of cyt. P450. This technique increases the specific activity of cyt. P450 by three fold.

[Conditions for analysis of cytochrome p-450 activity in human liver microsomes]. / Conditions de dosage de l'activité des CYT-P450 des microsomes de foie humain.

Multiples forms of CYT-P450 have been isolated from human liver microsomes. The distribution of CYT-P450 could be correlated with pathologic and influence the individual's response to therapeutic drugs and susceptibility to the toxic and cancerogenic effects of environmental pollutants. The aim of the this work is to determine the amounts of CYT-P450's in small samples from human liver such as biopsies and eventually to correlate them with pathology. For these reasons, tests providing informations about the distribution of CYT-P450 in individual subjects are very important. In this study we have reviewed the method for measuring the activity of CYT-P450 and the dosage of Benzopyrene hydroxylase. We modified some of these methods for the study of CYT-P450 from human liver microsomes.

[Purification of cytochrome P-450 and NADPH cytochrome p-450 reductase from human liver]. / Purification des cytochromes-P450 et de la NADPH cytochrome-P450 réductase de foie humain.

Two methods for the purification of cytochromes-P450 from microsomes of human liver are described. Method A: Cyt-P450 were solubilized from microsomes using a non ionic detergent, the Lubrol. The Cyt-P450 were purified by affinity, hydrophobicity followed by ion-exchange chromatography on DEAE-5PW column (HPLC) with an overall yield of 18% and a specific activity of 10 nmole/mg of protein. The recovery of NADPH Cyt-P450 reductase by method A (affinity) is about 60% with a specific activity of 16.2 U.I./mg of protein. Method B: Cyt-P450 were solubilized from microsomes using a zwitterionic detergent, the CHAPS. Cyt-P450 were filtered and separated by chromatofocusing on Mono-P column (HPLC). By this method it was possible to increase strongly the specific activity keeping a yield of 50% of Cyt-P450. Also it was possible to apply this method to small samples of human liver like biopsies (0.5 to 2.5 g).

[Study of nomifensine in the treatment of depressive states (author's transl)]. / Etude de la nomifensine dans le traitement des états dépressifs.

The nomifensine antidepressant activity, tolerance and rapidity of action have been studied by 95 psychiatrists in 498 patients during a 4 week open treatment. A 12 items evaluation scale allowed the estimation of the efficacy of the drug in depressed patients distributed to 4 classes of diagnosis: reactional, neurotic, endogenous and other type of depression. Side effects and rapidity of action of the treatment have been recorded during the whole treatment. Nomifensine's antidepressant activity was already observed after one week of treatment: the global scores were significantly diminished at this time in the 4 types of depressive patients. Items such as loss of interest, depressive mood and idea of suicide have been strongly ameliorated and have put in evidence the therapeutic profile and the security of use of this drug The significant and rapid decrease of severity of the symptoms as well as the excellent tolerance of the drug support the psychiatrists' judgement of satisfaction and confirm nomifensine as a major antidepressant, well tolerated and with a rapid onset of action.

Dietary chicory inulin increases whole-body bone mineral density in growing male rats.

Chicory inulin is a natural linear fructan that is not digested in the upper part of the gastrointestinal tract but is fermented in the cecocolon. It enhances calcium absorption in rats and improves femur and tibia mineral contents in gastrectomized or ovariectomized rats. We studied the effect of inulin (0, 5 and 10 g/100 g diet) on whole-body bone mineral content (WBBMC), whole-body bone area (WBBA) and whole-body bone mineral density (WBBMD) in live, growing male rats fed diets containing 0.2, 0.5 or 1 g Ca/100 g. Three experiments, each corresponding to one of the different dietary Ca concentrations, were performed using male Wistar rats (n = 108; 4 wk old). WBBMC was measured by dual-energy X-ray absorptiometry every 4 wk up to wk 22. Inulin increased WBBMC (P < 0.05) and WBBMD (P < 0.001) significantly but not WBBA at all ages and all dietary calcium concentrations. This is the first report to demonstrate that chicory inulin not only increases calcium absorption but also increases mineral parameters in whole-body bones.

Concentration-dependent plasma protein binding of flurbiprofen in the rat: an in vivo microdialysis study.

PURPOSE: The in vivo plasma protein binding and pharmacokinetics of flurbiprofen were studied in awake, unrestrained rats using intravenous microdialysis sampling. METHODS: Flurbiprofen (20 mg/kg) was administered i.v. to 2 groups of 6 rats: in both groups sampling was carried out by microdialysis, but in the second group an additional 10 blood samples were withdrawn via a jugular cannula. In vitro and ex vivo (following i.v. administration of flurbiprofen 20 mg/kg to another group of 13 rats) plasma protein binding of the drug was determined by equilibrium dialysis. RESULTS: The area under the unbound plasma concentration-time profile of flurbiprofen (AUCu), determined by microdialysis sampling was somewhat smaller (-19%, p = 0.666) in the rats undergoing simultaneous serial blood sampling (2.21 +/- 0.36 micrograms.h/ml) as compared to the rats undergoing microdialysis sampling only (2.73 +/- 0.60 micrograms.h/ml). Comparison of total and unbound concentrations of flurbiprofen showed an in vivo plasma binding varying between 99.5% at low and 98.0% at high total flurbiprofen plasma concentrations. Plasma binding of flurbiprofen determined in vitro over the same concentration range was higher (99.5-99.9%) but also concentration-dependent. Plasma binding of flurbiprofen determined ex vivo, on the other hand, corresponded well with the in vivo binding. CONCLUSIONS: Monitoring the fraction of drug unbound in blood of an individual rat throughout a pharmacokinetic experiment has now become possible by using simultaneous sampling of blood and intravenous microdialysates.

A new method for the purification of cytochrome-P450 from human liver microsomes.

A procedure for the solubilization and purification of cytochrome-P450 (cyt-P450) from human liver microsomes is described. Successive treatment of microsomes with protease XXVII and 3-(3-cholamidopropyl)dimethylammoniopropanesulphonic acid gave a solubilized cyt-P450 in more than 80% yield and with a three-fold increase in specific activity. With this treatment it was possible to eliminate 80% of cytochrome-b5 and 75% of NADPH cyt-P450 reductase. The solubilized cyt-P450 was filtered on a Sephacryl-200 column and then subjected to high performance liquid chromatography with a Mono-P column (chromatofocussing). The recovery of separated cyt-P450 was about 50% with a specific activity of 11.5 nmol cyt-P450/mg protein. Also with this technique it was possible to determine the isoelectric points of cyt-P450. These results allowed us to confirm the usefulness of our method, for the study the cyt-P450 from surgical biopsies.