Few bicyclic acetals at reducing end of low-molecular-weight heparins: might they restrict specification of pharmacopoeia?

The alkaline hydrolysis of heparin benzyl ester originates enoxaparin. The depolymerization by beta-elimination is the primary effect of reaction; but side reactions can happen and the bicyclic acetal at the reducing end of glucosamine N,6-disulphate, called 1,6-anhydro ring, is a product of a side reaction. The amount of this predictable moiety of enoxaparin can be controlled to a lowest extent (6%) and to extent higher than 40% by modulating the alkalinity and duration of the reaction of hydrolysis. With the exclusion of the beta-elimination effects and of these non significative side reactions, the chemical structure of the parent heparin is entirely maintained in enoxaparin as it results by the same profiles of constituent disaccharides. The content of 1,6-anhydro rings is assessed by a not yet validated NMR method. The chains of enoxaparin bearing, at their reducing end, 1,6-anhydro rings could be regarded as Related Substances of enoxaparin. If present, even in an amount less than, or equal to, 30% of chains, these "related substances" affect neither activities nor safety of enoxaparin.

Semisynthetic chondroitins as chiral buffer additives in capillary electrophoresis.

Chemically oversulfated galactosaminoglycans with potential as therapeutic agents (inhibitors of human leukocyte elastase) were tested as chiral selectors in capillary electrophoresis of basic racemates. The high anionic character of these compounds provides them with anodic mobility in acidic buffer; using uncoated capillaries, the enantioresolution of racemic basic drugs was obtained at pH 2.5. Dimethindene, chloroquine and chlorpheniramine were enantioresolved applying negative voltage (-15 kV) while the other analytes (propranolol, pindolol, tetrahydrozoline and cloperastine) exhibited catodic migration. The addition of organic solvents to the running buffer was evaluated in order to increase the resolution; methanol provides the best results and in general, baseline separation of the analytes was reached. The studied oversulfated mucopolysaccharide, shows the same ionic character of heparin but presents different stereochemistry and sites of sulfation. A comparison with heparin, used in the same acidic conditions, may underline the role of ionic, spatial and steric features of glycosaminoglycans in the enantiorecognition.

Fractions of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped viruses: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus.

A series of chemically oversulphated galactosaminoglycans (SO3H:COOH ratio > or = 2) were tested in vitro as antiviral agents against human immunodeficiency virus type 1 (HIV-1), the aetiological agent of AIDS, and against herpes simplex virus type 1 and human cytomegalovirus, two agents responsible for opportunistic infections in HIV-infected people. The oversulphated derivatives displayed an increase in activity ranging from one to four orders of magnitude against the three viruses, as compared to the natural parent compounds (SO3H:COOH, ratio approx. 1). The antiviral activity of these polyanions appears to be favoured by a high degree of sulphation and a high molecular mass. An oversulphated dermatan, with a SO3H:COOH ratio of 2.86 and molecular mass of 23.2 kDa, was the most potent anti-HIV-1 compound (EC50 0.04 microgram/ml). A second oversulphated dermatan, with a SO3H:COOH ratio of 2.40 and molecular mass of 25 kDa, displayed the highest activity against HSV-1 (EC50 0.01 microgram/ml). An oversulphated chondroitin, with a SO3H:COOH ratio of 2.80 and molecular mass of 17.3 kDa, was the strongest anti-HCMV agent (EC50 0.4 microgram/ml). In view of the absence of the side-effects typical of heparin-like compounds, a combination of these derivatives could have therapeutic potential.

Dermatan sulfate as useful chiral selector in capillary electrophoresis.

Dermatan sulfate (DS), a complex, polydispersed, sulfate polysaccharide was investigated as a useful chiral selector in capillary electrophoresis for the enantioresolution of a variety of drugs. Analysis was carried out in a fused-silica capillary column of 48.5 cm length (40 cm to detector window) x 50 microns I.D., and the separation buffer consisted of citric acid-Tris containing DS; the applied voltage was 15 kV and the detection wavelength was 220 nm. The effects of buffer pH, the dermatan concentration and run temperature on the enantioseparation and migration were examined. The method was applied to the enantioresolution of a representative set of twenty basic drugs. At all pH values used (3.0, 4.5 and 6.5) the addition of DS resulted in an increased migration time due to analyte-DS interaction. Using DS concentration of 2% (w/W), at pH 4.5, enantiomeric separations could be obtained for more than 50% of the examined drugs; resorcinic drugs; resorcinic moiety was found to be a very favourable structural feature for obtaining high enantioresolution values.

Heterogeneity of unfractionated heparins studied in connection with species, source, and production processes.

The heterogeneity of unfractionated heparins (Hep) can be correlated to the species and organs of origin and to the process of production. Heparins, extracted by different, validated processes from different organs and/or tissues (mucosa, thymus, pancreas, placenta, lung, intestine) or mammals (pig, beef, sheep, man) and other vertebrates (chicken), have been examined by HPLC analysis of heparinase digests. By analysis of disaccharides many observations have been made. Porcine mucosa heparin (pm-Hep) was always found to contain higher amounts of the disaccharides delta UA-GlcNS,6S and delta UA-2S-GlcNS,6S, than did bovine mucosa heparin (bm-Hep), whereas bm-Hep always showed higher amounts of the sequence IdoA(2OSO3)-GlcNSO3 than did pm-Hep. These findings mean that the last step of the biosynthesis, the 6-O-sulfation of glucosamine-N-sulfate (GlcNSO3), is accomplished; in bm-Hep, to a lesser extent than in pm-Hep. The 6-O-sulfated molar fractions of pig mucosa, chicken intestine, beef pancreas, beef placenta, and beef lung heparins were higher than the corresponding molar fractions of beef mucosa and beef thymus Heps. Also the manufacturing processes can partially rearrange the heparin structure. Even 6-O-sulfation enrichment (by chromatographic purification) or base-catalyzed displacement of sulfate groups from IdoA2SO3 occurred. The resulting anticoagulant activity roughly correlated with the percentage of trisulfated disaccharide and the 6-O-sulfated molar fraction. The heparin from human placenta was similar to pm-Hep. The observed species- and organ-dependent structural characteristics support the suggestion by Nader and Dietrich (in Heparin, Chemical and Biological Properties, Lane DA, U Lindahl (Eds). Arnold, London, 1989, p 81) on the antipathogenic role of heparin. The 6-O-sulfation of glucosamine, present in higher amounts in organs that function as barriers against many foreign bodies, like lung, placenta, intestine of chicken and pig, may play an important role in this antipathogenic action of Hep.

Active site for heparin cofactor II in low molecular mass dermatan sulfate. Contribution to the antithrombotic activity of fractions with high affinity for heparin cofactor II.

Dermatan sulfate (DS) is currently under clinical investigation as new antithrombotic agent. Unlike heparin, DS does not act through Antithrombin III (ATIII) but primarily through thrombin on Heparin Cofactor II (HCII). HCII is activated by the oversulfated sequence (IdoA2SO3-GalNAc4SO3)4 or by both the sequences (IdoA2SO3-GalNAc4SO3)n and (IdoA-GalNAc-4,6SO3)n, [n > or = 2]. A Low Molecular Mass Dermatan Sulfate (LMM-DS), endowed with a bioavailability three-four times higher than DS, by subcutaneous route, was obtained by chemical depolymerization of DS. The LMM-DS was fractionated by anion exchange and size exclusion chromatography. Fractions with high and low charge densities, high and low molecular masses, and high (2.66) and low (0.07) potencies on HCII were isolated. A relationship between the in vitro HCII-mediated inhibition of thrombin and the chain length of DS fractions containing oversulfated sequences was found [by a multiple regression test]. The in vivo activity increased until it reached a plateau. The important influence on the HCII activity of natural IdoA-GalNAc-4,6SO3 disaccharide was confirmed by investigation on oversulfated DS obtained by a limited and selective chemical 6-O-sulfation in GalNAc4SO3 units of DS.

Analysis of heparin origin by HPLC quantitation of disaccharide components.

A simple method was developed to analyze by high performance liquid chromatography unsaturated disaccharide isomers, derived from heparin by enzymatic digestion. This method was successfully exploited in the investigation of heparin origin. The percent amount of 6-sulphated disaccharides in the heparin extracted from porcine mucosa was found to be higher than that contained in the heparin extracted from bovine mucosa. The differences in the contents of the disaccharides obtained by enzymatic beta-elimination cleavage of heparin were confirmed by 13C-NMR measures of heparin in toto. The processes for extracting and purifying heparin may, however, modify the sulphation pattern of heparin. The structure of the latter seems to depend on the species owing to the specificity of the biosynthesis.

Inhibition of human leukocyte elastase by chemically and naturally oversulfated galactosaminoglycans.

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

Quantitation of dermatan sulfate active site for heparin cofactor II by 1H nuclear magnetic resonance spectroscopy.

The sequence (IdoA2SO3-GalNAc4SO3)n contributes to the HCII-mediated inhibition of thrombin by dermatan sulfate (DS). This sequence clearly results from the 13C NMR spectrum and can be quantified by the signal C1-H of IdoA2SO3 in the 1H NMR spectrum. A linear correlation has been found between the content in the disulfated disaccharide delta Di-2,4diS obtained by enzymatic demolition with ABC lyase, the percentage content in IdoA2SO3 quantified by 1H NMR, and the HCII-mediated activity of dermatan sulfates from beef mucosa and pig skin. DSs have been obtained also from pig mucosa and contain an amount, not negligible, of delta Di-4, 6diS. This disulfate disaccharide contributes to the activity expressed by the IdoA2SO3-GalNAc4SO3 sequence. The analytical techniques HPLC and 1H NMR, applied to the currently performed analyses of DS, are described and discussed.

Relative influence of different disulphate disaccharide clusters on the HCII-mediated inhibition of thrombin by dermatan sulphates of different origins.

Besides the major monosulphated disaccharide sequences (IdoA-GalNAc4SO3), dermatan sulphates (DS) contain the oversulphated sequences (IdoA2SO3-GalNAc4SO3) and (IdoA-GalNAc4, 6SO3), the concentration of which is correlated with the HCII-mediated inhibition of thrombin by DS. The effect of the chemical removal of the sulphate groups on the HCII-mediated activity was studied. The base-catalyzed sulphate group displacement from IdoA2SO3 residues, leading to formation of the epoxyde aGulA, is a 1st order reaction. When the content of sequences (IdoA-GalNAc4, 6SO3) is higher than that of sequences (IdoA2SO3-GalNAc4SO3), removal of the sulphate groups from Ido2SO3 reduces the HCII activity less than when the latter sequences prevail. (IdoA-GalNAc4, 6SO3) cooperates with (IdoA2SO3-GalNAc4SO3) in the activation of the HCII. Also the removal of 6-SO3- groups from GalNAc4, 6SO3, in absence of IdoA2SO3-GalNAc4SO3, considerably reduces the activity. A low molecular mass natural fraction rich in IdoA2SO3 as well as glucuronic acid, having higher electrophoretic mobility than the higher molecular mass DS which contains less glucuronic acid, is remarkably active.

Structure and contribution to the heparin cofactor II-mediated inhibition of thrombin of naturally oversulphated sequences of dermatan sulphate.

Dermatan sulphate (DS) obtained from bovine and pig mucosa and pig skin, and charge-enriched fractions of a selected DS preparation, were characterized in terms of charge density, M(r) and disaccharide composition of chondroitin ABC lyase digests, and by 13C-n.m.r. spectroscopy. Besides the major IdoA-GalNAc4SO3 sequences, all DS preparations contain about 10% disulphated disaccharide sequences (mostly IdoA2SO3-GalNAc4SO3, with minor amounts of IdoA-GalNAc4,6SO3). DS fragments (prepared by radical-catalysed depolymerization of DS and retaining the internal structure of the parent polysaccharide) as well as Smith degraded fragments [SD-DS, obtained by controlled degradation of periodate-oxidized and borohydride-reduced DS (RO-DS)] with the general structure GalNAc4SO3(IdoA2SO3-GalNAc4SO3)n-R (where R is the remnant of a glycol-split uronic acid, and n = 2-3 and 3-4) were characterized by one- and two-dimensional 1H-n.m.r., 13C-n.m.r. and disaccharide composition analysis. In accordance with previous findings [Maimone and Tollefsen (1990) J. Biol. Chem. 265, 18263-18271], only fragments with n > or = 3 significantly enhance the heparin cofactor II-mediated inhibition of thrombin. In natural DS preparations and their fractions, this activity (as well as the antithrombotic activity in an animal model) appears to require IdoA2SO3-containing sequences. The heparin cofactor II activity of DS, RO-DS and SD-DS fragments decreases with decreasing M(r). However, RO-DS fragments are more active than DS fragments of similar M(r), probably because of the extra flexibility endowed by glycol-split IdoA residues.

Effect of heparin, dermatan sulfate, and related oligo-derivatives on human polymorphonuclear leukocyte functions.

The in vitro effect of unfractionated heparin and dermatan sulfate, as well as oligo-heparin and oligo-dermatan sulfate, on human PMN function was investigated. Superoxide anion generation in fMLP-stimulated PMN was dose-dependently reduced by heparin and oligo-heparin, while DS and oligo-DS lacked inhibitory activity. FMLP-stimulated PMN adhesion to endothelial cells was reduced to a similar extent by both heparin and oligo-heparin, but not by DS and oligo-DS. On the other hand, none of the compounds affected the adhesion of unstimulated PMN to either IL-1- or PMA-activated endothelial cells. Heparin and oligo-heparin also inhibited the homotypic aggregation of fMLP-stimulated PMN. As reported, coincubation of platelets with fMLP-stimulated PMN resulted in platelet activation, a process mainly mediated by the PMN-derived serine protease cathepsin G. Both heparin and DS, as well as their oligo-derivatives, reduced platelet activation induced by either fMLP-stimulated PMN or purified leukocytic cathepsin G. Finally, besides cathepsin G, also the activity of beta-glucuronidase and lysozyme released by stimulated PMN were reduced by heparin, oligo-heparin and DS. These data support the hypothesis that heparin and other GAGs may exert an antiinflammatory role.

Different pharmacokinetics, antithrombotic activity and bleeding effects of heparin and two new fragments administered in rat by subcutaneous route.

Native heparin (CAS 9005-49-6) and its two new fragments, low molecular weight heparin (LMW-H, 5 kDa) and oligo-heparin (oligo-H, 2 kDa) obtained by radical degradation were characterized as to their physicochemical properties. Heparin fragments differ from unfractionated heparin only in molecular weight. The pharmacokinetics and some pharmacological effects, bleeding and antithrombotic activity, of the three different molecular weight heparins were investigated. The plasma concentrations were determined by an amidolytic method which measures inhibiting effect on factor Xa. The blood levels of each substance were derived from their in vitro calibration curves. The examination of the pharmacokinetics parameters allowed to evaluate the differences in the bioavailability, absorption rate and elimination mechanisms between the three different heparins. The bioavailability, the absorption rate and the distribution of the molecules of heparins in biological compartments depend on the molecular weight. LMW-H and oligo-H exhibit greater antithrombotic activity than unfractionated heparin when administered subcutaneously. The pharmacokinetic behaviour of oligo-H considerably differs from that of unfractionated heparin and LMW-H. This new drug is able to bind cells and plasma proteins differently from heparin and LMW-H. The capacity of oligo-H to bind smooth muscle cells and to interact with myosin is discussed in relation to the bleeding effect.

Low molecular weight heparins (5 kDa) and oligoheparins (2 kDa) produced by gel permeation enrichment or radical process: comparison of structures and physicochemical and biological properties.

A process of chemical depolymerization of heparin initiated by free radicals has been developed. The process follows reaction kinetics of apparent first order, and it is possible to obtain heparins with different molecular weights. Low and very low molecular weight heparins (LMWHs, approximately 5 kDa, and Oligo-H, approximately 2 kDa) have been prepared by enriching fractions derived from natural heparin by gel permeation. The LMWHs produced by a radical reaction have been characterized by their physicochemical properties, patterns of constitutive disaccharides, NMR spectra, and biological activities in comparison with those of the LMWHs produced by an enrichment process. The process of free radical degradation is shown to produce heparins with a desired molecular weight (from 1 to greater than 10 kDa) without changes in their primary structure and in their physicochemical properties and with the in vitro biological activities expected for LMWHs. Furthermore, the limits of enzymatic cleavage of heparins with different molecular weights by heparinases and the possibility of obtaining a precise pattern of constitutive disaccharides are discussed.

Structural features of dermatan sulfates and their relationship to anticoagulant and antithrombotic activities.

Dermatan sulfate is a polydisperse, microheterogeneous sufated copolymer of N-acetyl-D-galactopyranose and idopyranosyluronic acid that is currently under clinical investigation as a new antithrombotic agent. The structure and activity of two pairs of dermatan sulfates, isolated from bovine and porcine mucosa, were studied. One dermatan sulfate from each species demonstrated high in vivo antithrombotic activity in the rat vena cava assay. The in vitro anticoagulant activity of each dermatan sulfate was determined using activated partial thromboplastin time (APTT), thrombin time (TT) (5 units), calcium thrombin time (CaTT) (5 units), Heptest, anti-factor Xa and anti-factor IIa antithrombin assays and heparin cofactor II amidolytic assays. The coagulation-based assays gave the best correlation to in vivo antithrombotic activity. The physical and chemical properties of each dermatan sulfate were determined using 1H-NMR and 13C-NMR spectroscopy, molecular weight determination, potentiometric titration, chemical degradative analysis, chondroitin lyase degradative analysis and oligosaccharide mapping. These analyses indicated that the major difference between dermatan sulfates from a particular species having high and low in vivo antithrombotic activity was their iduronic acid content. The relation between increased iduronic acid content and increased in vivo antithrombotic activity may be the result of the conformational flexibility of this residue.

Quinolonecarboxylic acids. 2. Synthesis and antibacterial evaluation of 7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzothiazine-6-carboxylic acids.

A series of pyridobenzothiazine acid derivatives was synthesized and their in vitro antibacterial activity was evaluated. The 1,4-benzothiazine intermediates, which by Gould-Jacobs quinoline synthesis produced pyridobenzothiazine acids, were prepared by hydrolytic basic cleavage of substituted 2-aminobenzothiazoles and successive cyclocondensation with 1-bromo-2-chloroethane or alternatively with monochloroacetic acid, hence reduction by LiAlH4. The pyridobenzothiazine acids 10c, 30, and 31 show potent antibacterial activities against Gram-positive and Gram-negative pathogens. Structure-activity relationships are discussed. The compound 9-fluoro-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-d e] [1,4]benzothiazine-6-carboxylic acid (31) (MF-934) has been found to possess, together with the antibacterial activity, a weak acute toxicity and interesting pharmacokinetic characteristics in several animal species (rat, dog, monkey, man).

Synthesis and beta-adrenergic blocking activity of oxipropanolamines of 3,4-dihydro-3-oxo-2H(1,4)benzothiazine.

The synthesis of a series of oxypropanolamines of 3,4-dihydro-3-oxo-2H(1,4)benzothiazine is reported. Some of these compounds proved more potent than propranolol and carteolol as beta-adrenergic blocking agents in in vitro tests. The 8-(3-tert-butylamino-2-hydroxy)propoxy-3,4-dihydro-3-oxo-2H(1,4) benzothiazine fumarate (XVI a), which gave better results, confirmed its remarkable activity in in vivo tests.