Air classifier technology (ACT) in dry powder inhalation Part 4. Performance of air classifier technology in the Novolizer multi-dose dry powder inhaler

In this study, the in vitro fine particle deposition from a multi dose dry powder inhaler (Novolizer) with air classifier technology has been investigated. It is shown that different target values for the fine particle fraction (fpf<5 microm) of the same drug can be achieved in a well-controlled way. This is particularly relevant to the application of generic formulations in the inhaler. The well-controlled and predictable fpf is achieved through dispersion of different types of formulations in exactly the same classifier concept. On the other hand, it is shown that air classifier-based inhalers are less sensitive to the carrier surface and bulk properties than competitive inhalers like the Diskus. For 10 randomly selected lactose carriers for inhalation from four different suppliers, the budesonide fpf (at 4 kPa) from the Novolizer varied between 30 and 46% (of the measured dose; R.S.D.=14.2%), whereas the extremes in fpf from the Diskus dpi were 7 and 44% (R.S.D.=56.2%) for the same formulations. The fpf from a classifier-based inhaler appears to be less dependent of the amount of lactose (carrier) fines (<15 microm) in the mixture too. Classifier-based inhalers perform best with coarse carriers that have relatively wide size distributions (e.g. 50-350 microm) and surface discontinuities inside which drug particles can find shelter from press-on forces during mixing. Coarse carrier fractions have good flow properties, which increases the dose measuring accuracy and reproducibility. The fpf from the Novolizer increases with increasing pressure drop across the device. On theoretical grounds, it can be argued that this yields a more reproducible therapy, because it compensates for a shift in deposition to larger airways when the flow rate is increased. Support for this reasoning based on lung deposition modelling studies has been found in a scintigraphic study with the Novolizer. Finally, it is shown that this inhaler produces a finer aerosol than competitor devices, within the fpf<5 microm, subfractions of particles (e.g. <1, 1-2, 2-3, 3-4 and 4-5 microm) are higher.     

Studies on the effects of heterologous antisera against subcellular thymocyte fraction

Subcellular fractions of mice thymocytes were used for sensitization of rabbits. The antisera were examined for their immunosuppressive potency in vivo by allogeneic murine tumor metastases system and on skingraft survival and in vitro by leukocyte agglutination tests. The results indicated that the most potent immunosuppressive antisera was that against the second fraction (Fr. 2) of the detergent soluble endoplasmic reticulum fraction from thymocytes.     

Purine and pyrimidine salvage pathways in Leishmania donovani

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta544, 360 (1978)]. [8-¹⁴C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-¹⁴C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form xanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-¹⁴C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-¹⁴C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).     

Identification and mutagenicity of aflatoxicol-M1 produced by metabolism of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout (Salmo gairdneri) fed beta-naphthoflavone

beta-Naphthoflavone (beta NF) fed to rainbow trout (Salmo gairdneri) at 50 or 500 ppm in the diet, modified the in vitro metabolism of aflatoxin B1 (AFB1) by the postmitochondrial fraction (PMF) of the liver. Production of aflatoxicol (AFL) was significantly less in the 500 ppm beta NF-fed group (33.9 ng/mg protein) than in the control group (45.7 ng/mg protein), aflatoxin M1 production was dependent on the dose of beta NF, being greatest in the 500 ppm beta NF-fed group (48.9 ng/mg protein), intermediate in the 50 ppm beta NF-fed group (3.7 ng/mg protein), and was not detected in controls. A new trout metabolite, 4-hydroxyaflatoxicol (aflatoxicol M1, AFLM1) was also detected in small amounts from in vitro metabolism by liver PMF from beta NF-fed trout. Sufficient quantities of AFLM1 for confirmation of identity by ultraviolet spectra, mass spectra and nuclear magnetic resonance spectra were prepared by biotransformation of AFL using liver microsomes and isolation by HPLC. In a modified Ames mutagen assay with Salmonella typhimurium TA98, ALFM1 was 4.1% as mutagenic as AFB1 in a previous determination. The carcinogenicity of AFLM1 to rainbow trout is expected to be considerably less than that of AFB1.     

Para-capillary electron-dense deposits reduce glomerular filtration in patients with primary glomerular diseases

Background Electron-dense deposits are often found around glomerular capillary lumens in patients with glomerulonephritis, forming a portion of the blood-urine barrier (BUB). Methods Four hundred and four patients with primary glomerular diseases or donors for living-related kidney transplantation who underwent both percutaneous renal biopsy and renal clearance tests were included in the study. Sodium thiosulfate and paraamino hippurate double-clearance studies were performed with catheterized urinary collection. The filtration fraction (FF) was determined as follows: FF = sodium thiosulfate clearance/paraamino hippurate clearance (Cpah). Histomorphometric analyses were performed in 53 patients with overt para-capillary electron-dense deposits (PCEDD) by electron microscopic observations. Results Patients with membranous nephropathy and membranoproliferative glomerulonephritis showed significantly lower levels of FF than the donors for living-rebated kidney transplantation (normal controls). FF levels were significantly lower in patients with PCEDD than in those without (P < 0.001), while the levels of mean blood pressure and Cpah were comparable in the two groups. The PCEDD/BUB ratio demonstrated a significant negative correlation with FF (P < 0.0001; r² = 0.331). Patients with a ratio of 0.5 or more showed significantly lower FF levels than those with a ratio of 0.25 or less. Conclusions PCEDD significantly affected FF levels in patients with primary glomerular diseases. FF may not be an accurate indicator of intraglomerular blood pressure in patients with overt PCEDD.     

The amniotic band syndrome as a cause of anencephaly

Summary Extremely severe gliosis develops at the end stage of infantile neuronal ceroid-lipofuscinosis (INCL), a fatal encephalopathy characterized by accumulation of autofluorescent storage material in the brain and other tissues followed by a terminal subtotal neuronal and myelin loss. A major fraction of highly enriched intermediate filaments was obtained with a density gradient centrifugation method from INCL brain tissue, whereas the storage material represented only a minor fraction. SDS-polyacrylamide gel electrophoresis of the filament fraction showed a major protein with molecular weight of 51 kD and three to four polypeptides of 40–48 kD identified as glial fibrillary acidic protein (GFAP) and its degradation products by the immunoblotting technique with monoclonal antibodies against GFAP. Immunization experiments with the isolated INCL glial filament fraction produced antibodies reacting only with GFAP but not with other types of intermediate filament proteins, furthermore indicating a high content of GFAP in the isolated fraction. No significant amounts of vimentin or other types of intermediate filament proteins could be detected. These results document the extremely high content of glial filaments at the terminal stage of INCL and suggest that INCL brain may serve as a good human model for studies on the composition of glial filaments in vivo and on the pathogenesis of gliosis.Supported by grants from the Research Department of Rinnekoti Foundation, Finska Läkaresällskapet, the Sigrid Juselius Foundation and the Finnish Medical Research Council     

The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae

Preliminary studies had indicated the inadequacy of the wheat germ and rabbit reticulocyte cell-free translation systems for the in vitro translation of mRNA isolated from Plasmodium lophurae. To identify the factors which are important for the efficient translation of parasite proteins, an homologous system was established using polysomes, the pH 5 fraction, and tRNA prepared from P. lophurae. For comparison, the same components were isolated from the host duck reticulocytes and tested. The effect of each of these factors was evaluated by analysis of the translation products and by comparison with products synthesized in vivo. The results indicated that P. lophurae tRNA had a marked stimulatory effect on the synthesis of parasite proteins while it inhibited the synthesis of host proteins. Duck reticulocyte tRNA could not be used as a substitute for the parasite tRNA. Based on these findings, a commercially available rabbit reticulocyte system was supplemented with P. lophurae tRNA, which markedly increased the efficiency of translation of P. lophurae proteins by this system.     

Mutagenic activation of 2,4-diaminoanisole and 2-aminofluorene in vitro by liver and kidney fractions from aromatic hydrocarbon responsive and nonresponsive mice.

The mutagenicity of the two carcinogenic arylamines 2,4-diaminoanisole (2,4-DAA) and 2-aminofluorene (AF) was compared using liver and kidney fractions from two aromatic hydrocarbon (3-methylcholanthrene, MC) responsive and two nonresponsive mouse strains. MC pretreatment of mice caused an increase in 2,4-DAA mutagenicity with liver fractions from all four strains; however, much higher increases were seen in the two responsive than in the two nonresponsive strains. Kidney fractions had very low basal 2,4-DAA mutagenic activity. MC treatment led to 14–27-fold increase in 2,4-DAA mutagenicity in the responsive C57BL/6/BOM (B6) strain, but not in any of the other strains. AF mutagenicity was increased with liver fractions from all four mouse strains, but to the greatest extent in the B6 mice. AF showed high basal mutagenic activity with kidney fractions from all four strains, but MC treatment did not cause any increase in AF mutagenicity in any of the strains. Thus, there was a clear difference in the pattern of metabolic activation of the two arylamines 2,4-DAA and AF by liver and kidney fractions in mice, both with respect to constitutive activities and to the response to aromatic hydrocarbons.     

Tc-99m-IDA gallbladder kinetics and response to CCK in chronic cholecystitis

The cholecystographic pattern and the contractile response of the gallbladder (GB) to cholecystokinin (CCK) were studied in 101 consecutive patients with uncomplicated chronic cholecystitis confirmed by pathology. Sequential GB images were obtained after administration of 5 mCi ^99mTc-Disofenin and the ejection fraction was determined following a 15 min infusion of CCK. Sixteen of 101 (16%) GB failed to visualize upto 4 h; of the remaining patients, 3/85 (4%) showed delayed visualization beyond 1 h, and 82/85 visualized within 1 h. The mean ejection fraction (EF) in 67 patients was 56.9%±27.5% compared to 74.8%±19.8% in a normal control group of 27 subjects (P0.005). However, there was a large overlap as 76% of chronic cholecystitis patients had EF values falling within the full normal range. GB disease could be identified with confidence when the EF was less than 35%, i.e. below the 2 standard deviation range of normal. On the basis of radionuclide kinetic studies alone, the majority of patients with chronic cholecystitis cannot be distinguished from normal.