ABSTRACT
Though differing only slightly in their degrees of sulfation, heparin preparations from pig mucosa and those from beef mucosa have consistently different 13C- and 1H-NMR spectra, which provide useful fingerprints for distinguishing the two types of heparin. Integrated areas of NMR signals associated with minor, undersulfated sequences (assigned by comparison with mono-dimensional spectra of selectively desulfated heparins and by analysis of two-dimensional spectra of heparins prepared from pig and beef mucosa) permit quantitation of differences in sulfation patterns. Undersulfation of pig mucosal heparins at position 6 of the hexosamine units, determined by 13C-NMR and expressed as percent glucosamines nonsulfated at C6 referred to total glucosamines, is substantially lower for pig mucosal heparins than for beef mucosal heparins (16.9-21.7% vs 36.7-40.7%; average values: 18.6% vs 40.3%). By contrast, undersulfation at position 2 of the iduronic acid units, determined by 1H-NMR and expressed as percent nonsulfated iduronic acid referred to total (sulfated + nonsulfated) iduronic acid is significantly higher for pig mucosal preparations (9.6-13.5% vs 2.1-2.7%; average values: 12.7% vs 2.3%). Pig mucosal heparins also have a significantly higher content of 3-O-sulfated glucosamine units, which are markers for the active site of heparin for antithrombin-III.
Subject(s)
Heparin/chemistry , Intestinal Mucosa/chemistry , Sulfates/chemistry , Animals , Carbohydrate Sequence , Cattle , Glucosamine/chemistry , Glucosamine/metabolism , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Species Specificity , SwineABSTRACT
In the present study, the pharmacokinetics of extractive GAGs used as therapeutic agents have been studied after intravenous and oral administration on volunteers. The use of native or deuterium-labeled compounds, followed by HPLC/MS detection, allowed the quantitation of exogenous heparin and DS as major disaccharide fragments, obtained either by enzymatic or chemical depolymerization. In particular the high level of labeling reached in DS allowed its differentiation from structurally related endogenous species. The estimated plasmatic bioavailability was about 18% for DS. Notwithstanding the impossibility of evaluating the same parameters for heparin species, due to the interferences of endogenous GAGs, the results obtained provided clear evidence of oral availability of heparin and DS through detection and quantitation of structures specifically related to these GAGs. Due to the selectivity of the lyases used, the enzymatic degradation specifically allowed the detection of both DS and heparin species still retaining the original sulfation pattern. Additionally, the chemical degradation could detect the main metabolites of the drugs, consisting of partially to totally desulfated GAGs showing a more or less marked reduction in their molecular weight.
Subject(s)
Dermatan Sulfate/blood , Glycosaminoglycans/pharmacokinetics , Intestinal Absorption/physiology , Administration, Oral , Chromatography, High Pressure Liquid , Cross-Over Studies , Deuterium , Humans , Injections, Intravenous , Mass Spectrometry , Reference ValuesABSTRACT
The binding of single-stranded polydeoxyribonucleotides to adenosine A1 and A2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [3H]CHA (N6-cyclohexyl-adenosine) and [3H]NECA (5'-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. Ki values of 371 +/- 68 and 688 +/- 115 micrograms/ml (mean +/- S.E.M. of 4-5 replications) were computed for adenosine A1 and A2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K(+)-contracted guinea-pig trachealis muscle (IC50 = 4001 micrograms/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A1/A2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A1/A2 receptor blocker 8-phenyltheophylline, but not by the selective A1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.
Subject(s)
Fibrinolytic Agents/pharmacology , Polydeoxyribonucleotides/pharmacology , Receptors, Purinergic P1/drug effects , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/pharmacology , Adenosine-5'-(N-ethylcarboxamide) , Animals , Binding, Competitive , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Fibrinolytic Agents/metabolism , Guinea Pigs , Male , Polydeoxyribonucleotides/metabolism , Purinergic P1 Receptor Antagonists , Radioligand Assay , Rats , Receptors, Purinergic P1/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolismABSTRACT
We describe an HPLC method for the determination of whole polydeoxyribonucleotides in animal plasma. This method was compared to a colorimetric method, which evaluates the sugar moiety of polydeoxyribonucleotides, and to an agarose gel electrophoresis method, which evaluates the whole polydeoxyribonucleotides as does the HPLC method, and was found to give results very close to those obtained with these two other methods. A pharmacokinetic study of the antithrombotic, profibrinolytic, polydeoxyribonucleotidic drug defibrotide was carried out by evaluating the plasma drug levels by these three methods. The pharmacokinetic parameters calculated from the data are very similar.
