Aminooxy functionalized oligonucleotides: preparation, on-support derivatization, and postsynthetic attachment to polymer support.

Three novel phosphoramidites, each bearing a phthaloyl-protected aminooxy tail, were prepared and applied in automated oligonucleotide synthesis. After chain assembly, the phthaloyl protection was removed with hydrazinium acetate. Normal succinyl linker turned to be stable under these conditions, and hence the support-bound oligonucleotide could be converted to a pyrene oxime conjugates by reacting with pyrene carbaldehyde or cis-retinal. Standard ammonolytic deprotection then released the deprotected conjugate in solution. Alternatively, the crude aminooxy-tethered oligonucleotide was immobilized to microscopic polymer particles by reacting the aminooxy function with the particle-bound aldehyde or epoxide groups. These immobilized oligonuceotides were shown to serve properly as probes in a mixed phase hybridization assay.

Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

Porous, uniformly sized (50 micrometer) glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six oligonucleotides from a single sample. The assay was based on categorization of the particles by two organic prompt fluorophores, viz. fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-resolved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide probes were assembled on the particles by conventional phosphoramidite strategy using a non-cleavable linker, and the category defining fluorescein and/or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hybridized to the complementary 3'-terminal sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different fluorescence intensity measurements. The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particle-bound probe was optimized to achieve maximal selectivity. Furthermore, the kinetics of hybridization and the effect of the concentration of the target oligomer on the efficiency of hybridization were evaluated. By this approach the possible presence of a three base deletion (DeltaF508), point mutation (G542X) and point deletion (1078delT) related to cystic fibrosis could unequivocally be detected from a single sample.

Influence of chinoin-170, a novel antitussive, on the mucociliary activity in respiratory airways of rats, rabbits, guinea-pigs and man.

Chinoin-170 (Ch-170; 3,7-dihydro-1,3-dimethyl-7-[(5-methyl-1,2,4- oxadiazol-3-yl)methyl]1H-purine-2,6-dione) is a new antitussive with bronchodilating activity. Its effects on the ciliary beating frequency (CBF) and mucociliary clearance were studied. In tracheal explants of rats, Ch-170 dose-dependently at concentrations 2 and 5 mg mL-1 depressed CBF by 24 and 33%, respectively. In human mucosal explants, however, no effects were seen at concentrations up to 5 mg mL-1. In anaesthetized guinea-pigs, an intravenous 50 mg kg-1 dose of Ch-170 caused no changes, and 100 mg kg-1 increased the CBF by 15%. Intravenous Ch-170 dose-dependently increased by 93 (50 mg kg-1), 179 (70 mg kg-1) and 253% (100 mg kg-1) the tracheobronchial mucociliary clearance in rabbits. The effect, studied using 99mTc-labelled red blood cells as a marker, was of similar quantity to that brought about by administering 16, 25 and 40 mg kg-1 doses of bromhexine. It is concluded that unlike many older antitussives, Ch-170 in-vitro only slightly decreases the CBF in rats and has no adverse effects on the CBF in human mucosal explants at concentrations up to 5 mg mL-1. In-vivo, Ch-170 does not significantly alter the CBF in guinea-pigs, but dose-dependently increases the mucociliary clearance in rabbits. The increase is most probably a result of changes in the production and the properties of respiratory mucus.

ATP induces respiratory ciliostimulation in rat and guinea pig in vitro and in vivo.

Adenosine triphosphate (ATP) has been shown to revitalize the disturbed nasal mucociliary function in man. We investigated the effects of ATP on the ciliary beat frequency (CBF) in animals by immersing tracheal explants from rats in various concentrations of ATP, and by infusing ATP intravenously to guinea pigs. CBF was measured with a photodetector technique from the surface of the explants or from the incised trachea. ATP (from 0.01 to 1 mg/ml) in vitro increased CBF in rat tracheal explants up to 10.5% (p less than 0.05). In vivo ATP (1 mg/kg) increased the CBF by 29% (p less than 0.01) in the guinea pig trachea. As the CBF was increased by ATP, both in vitro and in vivo, it can be suggested that the improvement in mucociliary transport by exogenous ATP as shown in previous studies is caused by the ciliostimulatory effect of ATP.

Effect of codeine on rat and guinea pig tracheal ciliary beat frequency.

Codeine, the basic antitussive drug, has generally been thought to impair mucociliary function. Using the photodetection method the effect of codeine on mucociliary function was studied in rat after local and systemic administration and in guinea pig after systemic administration. In vitro rat tracheal ciliary beat frequency (CBF) was measured up to 40 min. There was 24.6 +/- 2.5% and 26.6 +/- 1.6% decrease in CBF after 1 mg/ml and after 10 mg/ml codeine solutions, respectively. In vivo codeine was administered in single i.v. doses of 10 and 15 mg/kg. No statistically significant differences were found in CBF responses, although there was a CBF decreasing tendency after the 15 mg/kg dose. The total follow-up time was 120 min. In guinea pig, 10 days s.c. codeine administration in daily doses of 3, 10 and 30 mg/kg had no effect on CBF. Although codeine was used in much higher concentrations than tissues concentrations after therapeutic doses of codeine, the effects on CBF were minimal. So it can be concluded that the generally accepted ciliostatic effect of codeine is overestimated.

The effect of ATP on the ciliary activity of normal and pathological human respiratory mucosa in vitro.

The effect of adenosine triphosphate (ATP) on the airway ciliary beating frequency (CBF) of mucosal explants excised from human maxillary sinuses was studied by measuring CBF photoelectrically before and after immersion of the explants in a solution containing ATP. In samples from 64 patients with chronic sinusitis the CBF was not significantly different from the CBF in mucosal specimens from healthy tissue of 22 patients without infection. During 15 min immersion, ATP (1 mg/ml) slightly (by 5%, p less than 0.05 in healthy tissue; by 2.7%, p less than 0.01, in tissue from sinusitis patients) increased the CBF. The effect of 10 mg/ml concentration was more pronounced (19.6%). It is concluded that the impairment in ciliary function caused by chronic sinusitis is reversible when the mucosal endothelium is cleansed of the infected mucus, and that the ciliostimulatory action of ATP seen in animals is also present in human respiratory mucosa.

The effects of vadocaine, dextromethorphan, diphenhydramine and hydroxyzine on the ciliary beat frequency in rats in vitro.

Mucociliary function is a major cleansing mechanism of the respiratory tract. Many drugs used in the treatment of respiratory diseases impair the ciliary beat frequency (CBF) of mucous membrane. Our aim was to study by means of a photoelectric technique, the effects of two antitussives--dextromethorphan and vadocaine--and two antihistamines--hydroxyzine and diphenhydramine--on the rat tracheal CBF in vitro. The CBF was measured from tracheal explants immersed in drug solutions. Dextromethorphan (1.0 mg/ml and 10.0 mg/ml) caused 16.9-20.8% decrease in the CBF during the 40 min. measurement period. Vadocaine (0.1 mg/ml and 0.5 mg/ml) decreased the CBF by 6.9%. Higher vadocaine concentrations caused a dose-dependent inhibitory effect so that mucociliary function stopped totally within 20 min. with 5.0 mg/ml vadocaine solution. Both diphenhydramine and hydroxyzine totally stopped the ciliary activity during 20 min. with concentrations of 2.5 mg/ml and 1.0 mg/ml. respectively. Locke-Ringer solution used as a control did not cause any change in the CBF. These results suggest that the antihistamines diphenhydramine and hydroxyzine are more ciliostatic than the antitussives dextromethorphan and vadocaine on the rat tracheal cilia in vitro. The results suggest further in vivo studies. The used photoelectric detection method proved to be suitable for evaluating drug effects on the CBF of respiratory mucosa.

Treatment of hypertension in the elderly with labetalol.

The response of elderly hypertensive patients to labetalol, possessing alpha- and beta-blocking properties, was studied. The dosage was first titrated out and assessed in a double-blind cross-over trial. Altogether 24 patients were treated for one year. The dosage established during the titration period maintained its efficacy. In most cases 200 mg labetalol daily gave satisfactory result. The side-effects were few. The only reason for discontinuance of the drug was gastrointestinal discomfort. Because of the low dosage, the typical side-effects of beta-blockade were lacking and alpha-blockade seemed to be well tolerated. The normal precautions in the treatment of elderly hypertensive patients should be observed.