A comparative pharmacokinetic and tolerability analysis of the novel orotic acid salt form of tenofovir disoproxil and the fumaric acid salt form in healthy subjects.

A novel orotic acid salt form of tenofovir disoproxil (DA-2802) was developed and is expected to replace the fumaric acid salt form. The pharmacokinetic (PK) characteristics and tolerability profiles of DA-2802 were compared to those of tenofovir disoproxil fumarate (TDF, Viread®) in healthy subjects. A randomized, open-label, single-dose study was conducted in 36 healthy subjects using a two-treatment, two-period, and two-sequence crossover design. Subjects received a single oral dose of 319 mg DA-2802 or 300 mg TDF, during each period, with a 7-day washout. Serial blood samples were collected pre-dosing and up to 72 hours post-dosing in each period, for determination of serum tenofovir concentration, which was measured by ultra-performance liquid chromatography-tandem mass spectrometry. A non-compartmental method was used to obtain PK parameters of tenofovir. For comparison between the two tenofovir disoproxil salts, the 90% confidence intervals (90% CIs) of geometric mean ratios of DA-2802 to TDF for the maximum concentration (Cmax) and the area under the concentration-time curve to the last quantifiable concentration (AUC0-t) were determined. The tolerability profiles of tenofovir were assessed by evaluation of adverse events and vital signs, physical examination, ECG, and clinical laboratory tests. The serum tenofovir concentration-time profiles of DA-2802 or TDF were comparable in 32 subjects who completed the study. In both profiles, a two-compartmental elimination with first-order elimination kinetics in the terminal phase was reported in a few subjects, showing a secondary peak in the initial phase of elimination. The geometric mean ratio (90% CI) of DA-2802 to TDF was 0.898 (0.815-0.990) for Cmax and 0.904 (0.836-0.978) for AUC0-t. There were no clinically significant findings in the tolerability assessments. DA-2802 showed comparable PK characteristics and tolerability profiles to TDF.

Integration of genomic and metabonomic data in systems biology--are we 'there' yet?

The measurement of genes, proteins and metabolites has gained increasing acceptance as a means by which to study the response of an organism to stimuli, whether they are environmental, genetic, pharmacological, toxicological, etc. Typically referred to as genomics, proteomics, and metabonomics or metabolomics, respectively, these methods as independent entities have undoubtedly provided new biological insight that was not attainable a decade ago. Not surprisingly, scientists continue to push the boundaries to extract knowledge from data, and it is currently recognized that the full realization of these technologies is limited by a lack of tools to enable data integration. Integration of these 'omic datasets, or integromics, is desirable as it links the individual biological elements together to provide a more complete understanding of dynamic biological processes. Accordingly, in addition to developing new data analysis methods to extract further details from each of the high-content datasets individually, effort is also being expended to create or improve statistical methods, databases, annotations and pathway mapping to maximize our learning. There are several recent examples, in both mammalian and non-mammalian systems, in which genes, proteins and/or metabolites have been integrated using either biology- or data-driven strategies. Herein, key findings are reviewed, gaps in our current tools and technologies are identified and illustrated, and perspective is provided on the potential of integromics in biological research.

[The results of application of magnesium-bearing medications and different load doses of vitamins B1 and B2 in volunteers].

Presently is no consensus of opinion of the size of human optimal daily allowance as for thiamine and riboflavin, so for magnesium, what their content in polyvitamin and mineral preparations testify to. The study was conducted in 60 volunteers, who once took oral dosing of magnesium -bearing preparation "Magnerot" (500 mg of magnesium orotat converting to Mg2+ 32,8 Mg) and "Centrum" (100 mg of magnesium oxide converting to Mg2+ - 60,3 Vg) or vitamins B1 and B2 in 10, 20 and 30 mg doses. The received data testify that during the application of both of these preparations, equal concentration of this element takes place, though in case of magnesium oxide it happens later. As for the used vitamin B1 and B2 doses, it is determined that 10 mg is the optimal dose for treating their hypoavitaminosis.

Behavior of alginate gel beads containing chitosan salt prepared with water-soluble vitamins.

Alginate gel beads were prepared which contained weak acid salts of chitosan (Alg-CS) and water-soluble vitamins (e.g. ascorbic acid (AS)) and the behavior of the beads, uptake of bile acids was investigated in vitro. The Alg-CS beads rapidly took up bile acid and this phenomenon was observed for both hydrogel beads and dried beads. About 120 micromol of taurocholic acid was taken up into Alg-CS (1 g) prepared with orotic acid. Dried Alg-CS is the granule which can be made easily, and keeps the ability of CS salt, and all elements can be taken as a food. Therefore, Alg-CS could serve as a useful dietary agent for the prevention of hyperlipidemia.

Plasma concentrations and renal clearance of orotic acid in argininosuccinic acid synthetase deficiency.

Argininosuccinic acid synthetase deficiency (ASD) is a rare disorder of urea cycle metabolism, with pronounced citrullinemia and orotic aciduria being characteristic biochemical features. To further investigate the role of plasma orotic acid and its possible use for monitoring the metabolic status in ASD, we determined plasma orotic acid, amino acid, and ammonium levels in plasma samples collected over a period of 3 years from a patient who is now 8 years of age. Orotic acid plasma concentrations varied widely from less than 1 micromol/l to more than 60 micromol/l. The renal clearance of orotic acid was eightfold the glomerular filtration rate, thus supporting an active mechanism underlying the excretion of this pyrimidine. Data obtained during a metabolic crisis yielded a statistically significant linear correlation of orotic acid plasma levels with those of glutamine and ammonium, which are generally accepted for assessment of the successful treatment of this disorder. Our data revealed no advantage of plasma orotic acid concentrations over the established amino acids (glutamine and arginine) and ammonium for determining acute treatment responses. Since several effects of high levels of orotic acid have been described in mammals, further research is necessary to assess a possible contribution of orotic acid to the pathogenesis of ASD and the use of plasma orotic acid levels in the long-term monitoring of these patients.

Diffusion enhancement of drugs by loaded nanoparticles in vitro.

1. Dalargin, a Leu-enkephaline analogue, does normally not pass the blood-brain barrier (BBB). When it was adsorbed onto the surface of polybutylcyanoacrylate, nanoparticles dalargin can cross the BBB and induce central analgesic effects after intravenously as well as after oral application. 2. The mechanisms of this effect are unknown. Therefore, the authors evaluated whether neuronal transport was involved in this effect. In hippocampal synaptosomes and in tissue slices in vitro the active neuronal uptake and diffusion processes were determined by use of labelled D-aspartate as a marker of the aspartate/glutamate transporter and orotic acid as marker of diffusion. 3. Transporter-mediated uptake into hippocampal tissue preparations was not altered in comparison to control whereas diffusion processes were enhanced. These data indicate that the nanoparticles can modify neuronal uptake mechanisms.

Antimalarial activity of a combination of 5-fluoroorotate and uridine in mice.

Malarial parasites, in contrast to mammalian cells, utilize orotic acid more efficiently than uracil or uridine. Recently, chloroquine-susceptible and chloroquine-resistant clones of Plasmodium falciparum were shown to be inhibited by 5-fluoroorotate, with a 50% inhibitory concentration of 6 nM in vitro. Mammalian cells were far less sensitive to 5-fluoroorotate, particularly in the presence of uridine. In this report, the antimalarial activity of 5-fluoroorotate was tested in vivo. Initially, levels of 5-fluoroorotate in plasma were determined in Swiss mice injected intraperitoneally with radioactive 5-fluoroorotate. On the basis of the pharmacokinetics profile, mice infected with Plasmodium yoelii were treated with 5-fluoroorotate at a dose of 0.2 or 5 mg/kg (body weight) every 4 h for 3 days. At the nontoxic dose of 0.2 mg/kg, the reduction in parasitemia was followed by a temporary resurgence of parasitemia. This second wave of parasitemia cleared without additional 5-fluoroorotate treatment. To radically eliminate P. yoelii from mice and avoid the second wave of parasitemia, a higher dose of 5 mg of 5-fluoroorotate per kg had to be used. In the absence of uridine, repeated doses of 5 mg/kg were toxic to mice, as judged by weight loss, diarrhea, decreased numbers of leukocytes, and increased mortality. However, in the presence of uridine, repeated doses of 5 mg/kg could be used for antimalarial chemotherapy without obvious toxicity. Mice cured with 5-fluoroorotate and uridine were immune to subsequent challenge with a potentially lethal inoculum of P. yoelii.

Metabolization of a retention-improving dosage of methylglucamine orotate in rat brain.

The present study was carried out to investigate the time course of metabolization in brain and the influence on RNA synthesis of a retention-improving dosage of methylglucamine orotate after intracerebroventricular application. As determined by the HPLC technique, 73% of acid-soluble radioactivity were recovered in unmetabolized orotic acid 30 min after injection of 1 mumole methylglucamine [6-14C]orotate. Two hours later the amount of radioactivity found in this compound was negligible. Analysis of the sequence of labelling of uridine compounds revealed uridine to be the metabolite exhibiting the highest radioactivity at 30 min, whereas UMP- and UDP-sugars attained their maximum 90 min after injection of the precursor. Incorporation of [3H] guanosine into brain RNA was not altered by intraventricular application of 1 mumole methylglucamine orotate as compared to methylglucamine chloride-treated controls. The results are interpreted in the light of behavioural findings in which the pyrimidine nucleotide precursor at the dosage used improved the retention performance of an acquired behaviour in the rat.

RNA labelling with 3H-orotic acid and 3H-fluorouracil of rat liver tumour following transient hepatic arterial ischaemia.

Recent studies have demonstrated that transient (1 h) hepatic arterial ischaemia is followed by increased incorporation of labelled thymidine into tumour DNA 24 h later. The present study aimed at investigating if and when there is a corresponding increase in incorporation of labelled 5-fluorouracil (5-FU) or orotic acid into tumour tissue. Incorporation was studied at 0, 6, 24 and 48 h after discontinuation of transient hepatic arterial ischaemia in Wistar-Furth rats having liver tumour. The transient ischaemia was followed by increased RNA and RNA/DNA ratios in normal liver but not in tumour. Incorporation of orotic acid into RNA and DNA was unchanged in tumour but increased at 0 and 6 h in normal liver. Incorporation of 5-FU decreased with time in liver and tumour DNA and in tumour RNA. It is suggested that 5-FU has an optimal effect on tumour tissue when infused immediately after transient hepatic arterial ischaemia.

The permeability properties of septate junctions in Malpighian tubules of Rhodnius.

This paper describes the structural characteristics and permeability properties of the smooth septate junctions between the upper Malpighian tubule cells of a blood-sucking bug, Rhodnius prolixus. The permeability of the paracellular route was tested only for solutes that could be demonstrated not to cross the epithelium via the cellular route. The intercellular clefts were readily permeated by sucrose, inulin and polyethylene glycol (PEG), showing a higher permeability to molecules of smaller radius (PEG versus sucrose). Negatively charged molecules permeated the clefts more readily than positively charged ones. The effects of pH, urea and luminal flow rate on permeability were studied. The results are discussed in relation to the physiological tightness of the Malpighian tubules to certain solutes and to its function as an excretory epithelium.

Flow of protein and ribonucleic acid in peripheral nerve.

Radioactively labelled leucine and orotic acid were injected into the ventral horn of the lumbar region of the spinal cord. The outflow of labelled products into the sciatic nerve was studied. Leucine is rapidly incorporated into protein and to a lesser extent into lipid. The labelled protein is transported down the nerve, apparently by axoplasmic flow. This labelled protein was present in all sub-cellular fractions of nerve although the soluble fraction had the highest specific activity. Orotic acid is converted to a number of nucleotide derivatives and RNA. Both the nucleotides and RNA move down the sciatic nerve, but the pattern of movement is diffuse whereas protein appears to move as a discrete band. The results suggest that two systems may be operating, one in which part of the RNA is transported by axoplasmic flow along the nerve and the other involving a synthesis of RNA in the nerve from precursors which flow down the nerve. This labelled RNA was membrane bound and on sedimentation analysis, proved to be predominantly of ribosomal type.