Topical delivery of diclofenac into and across equine skin from a novel liquid diclofenac epolamine formulation.

The aim was to investigate diclofenac delivery into and across equine skin in vitro using Franz diffusion cells from a novel diclofenac epolamine (DIC-EP; 1.3%) formulation and to compare the results to those of Surpass® (1% diclofenac sodium liposomal cream) and a 1% aqueous solution of diclofenac sodium. Skin was harvested from the lower legs of Freiberger geldings immediately after slaughter and sliced to a thickness of ~2 mm. Skin samples were divided into two groups [Group 1: 1 year old (n = 2) and Group 2: 6-8 years old (n = 3)]. Cumulative permeation of diclofenac in Groups 1 and 2 after 24 h using diclofenac sodium solution was 1.91 ± 0.27 and 1.76 ± 0.34 µg/cm2 , respectively. The values for Surpass® and DIC-EP were 3.2 ± 0.8/3.3 ± 0.7 µg/cm2 and 230 ± 59/89.2 ± 32.5 µg/cm2 , respectively. Thus, diclofenac permeation from DIC-EP was significantly greater and appeared to show an age-dependent effect. Mathematical modelling showed that the DIC-EP formulation significantly increased diclofenac partitioning into the skin and a linear correlation was observed between steady-state flux and the partition parameter. Greater skin deposition of diclofenac was also observed with DIC-EP. These preliminary results suggest that the DIC-EP formulation may be effective in treating inflammatory conditions in horses.

Semi-synthetic heparinoids.

New chemical-enzymatic technology based on the modification of the bacterial polysaccharide K5 from Escherichia coli leads to the synthesis of a number of heparin/heparan sulfate-like molecules with different biological activities. With this technology, two families of sulfated compounds were synthesized, which differ in their uronic acid content. The first group contains only glucuronic acid, whereas the second group contains about 50% iduronic acid following epimerization by immobilized recombinant C5 epimerase. This has led to the development of various anticoagulant and nonanticoagulant K5 derivatives endowed with different - and sometimes highly specific - antitumor, antiviral, and/or anti-inflammatory activities.

Structural requirements for duodenal permeability of heparin-diamine complexes.

We have compared the physico-chemical behaviors alone and in the presence of a synthetic bilayer membrane, in aqueous solution and the bioavailability after intraduodenal administration to rabbits, of the two heparin diamine salts ITF-300 and ITF-331 with those of the heparin-amine salt ITF-1175. The three salts have similar structures but different characteristics of compounds tend to form aggregates in solution, but at different critical concentrations. The compounds induce fusion of single-walled vesicles of a synthetic peptide lipid into multi-walled lamellae. The minimal concentrations of the compounds required for the formation of such lamellae differ. This behavior in solution explains the differences in absorption in the animal model. This makes it possible to correlate enhanced heparin bioavailability with the structural nature of the diamine counter-ions used to prepare heparin salts.

Anti-HIV type 1 properties of chemically modified heparins with diminished anticoagulant activity.

Several groups have reported that sulfated polysaccharides are potent and selective in vitro inhibitors of human immunodeficiency virus type 1 (HIV-1); however, their therapeutic application is limited by their anticoagulant activity. In view of possible improvements in therapeutic potential, a number of heparin derivatives with reduced anticoagulant activity were studied for their inhibitory activity of an HIV-dependent syncytium formation assay, in comparison with standard anionic polysaccharides, such as sodium heparin, dextran sulfate, and heparin sulfate. The chemical modifications introduced in the heparin molecule included succinylation of desulfated N groups (Suc-H), exhaustive periodate oxidation and reduction (RO-H), and controlled nitrous acid degradation (LMW-H). The most pronounced anti-HIV activity was observed with RO-H, Suc30-H (standard heparin, 30% succinylated), and Suc100-LMW-H (low molecular weight heparin, 100% succinylated); the latter retained only 5% of the anticoagulant activity of standard heparin, whereas RO-H and Suc30-H retained approximately 35% of the anticoagulant activity of standard heparin. A safety ratio (arbitrary units of anti-HIV activity per anticoagulant international unit) was calculated: by this parameter, RO-H, Suc30-H, and Suc100-LMW-H were, respectively, 48-, 3.6-, and 1644-fold more safe than standard heparin.

Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5.

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

Sulfated K5 Escherichia coli polysaccharide derivatives inhibit human immunodeficiency type-1 (HIV-1) infection: candidate microbicides to prevent sexual HIV transmission.

The ideal microbicide must fulfill a number of criteria including a broad and potent activity against transmission of HIV and other sexually transmitted agents in the absence of toxicity and inflammation. We have described that derivatives of K5 polysaccharide from Escherichia coli inhibit HIV entry in target cells. K5 derivatives have a structure that resembles that of heparin, but they are devoid of the anticoagulant activity typical of heparin. Moreover, in contrast to heparin, they inhibit a broad spectrum of HIV-1 laboratory-adapted and primary isolates that use either CCR5 or CXCR4 or both coreceptors in terms of their infection and replication in primary CD4+ lymphocytes and monocytes-derived macrophages (MDM). Therefore, these compounds could be developed as candidate microbicides for preventing sexual HIV transmission, a predominant modality of HIV spreading in both the developed and underdeveloped world.

Detection of the low molecular weight heparin component of ITF 1300 in urines after intranasal administration to dogs.

GAGs were purified from urine of dogs after intranasal administration of 40 mg/kg ITF 1300. The electrophoretic patterns of urine GAGs in acidic buffer showed the presence of heparin together with chondroitins, heparan sulfate, and hyaluronic acid. The heparin present in urines was purified using chondroitinase ABC, and its purity was tested by electrophoresis in acidic buffer. The sample obtained was characterized by 13C-NMR, showing the same characteristic signals of the heparin starting material.

Heparin by alternative routes of administration.

In a study investigating the bioavailability of heparin administered by different routes, we also compared its bioavailability in the dog after oral administration of two enteric-coated formulations, one containing a heparin preparation (ITF-300) and the other sodium heparin. After administration of the formulation containing sodium heparin, there was no heparin in plasma, but when the formulation containing ITF-300 was given, plasma heparin levels were detectable.

Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor.

Heparins from bovine mucosa and lung, and chemically modified heparins were assayed for their capacity to: (i) protect human recombinant basic fibroblast growth factor (bFGF) from tryptic cleavage; (ii) prevent 125I-bFGF binding to heparan sulphate proteoglycans present in the extracellular matrix and on the cell surface of fetal bovine aortic endothelial GM 7373 cell cultures; (iii) affect 125I-bFGF binding to high-affinity tyrosine kinase FGF receptors present on the cell membrane of GM 7373 cells; (iv) inhibit the mitogenic activity exerted by bFGF in the same cells. The results demonstrate that the potency shown by mucosal heparins in the different assays is a direct function of size, very-low-molecular-mass heparin (2.0 kDa) being significantly less effective on a molar basis than unfractionated heparin (13.6 kDa). Increased flexibility of the backbone structure, as observed in reduced/oxidized heparins of different size, does not affect the capacity of the polysaccharide to interact with bFGF. In contrast, selective 2-O-desulphation, but not 6-O-desulphation, drastically reduced the capacity of heparin to protect bFGF from proteolytic cleavage, to affect its interaction with low- and high-affinity sites, and to inhibit its mitogenic activity. Two preparations of bovine lung heparin, differing in molecular mass, were as effective as mucosal heparin in the bFGF-tryptic-digestion assay and the endothelial-cell proteoglycan-binding assay, but they were highly inefficient at inhibiting the capacity of bFGF to interact with its tyrosine kinase receptors. Bovine lung heparins were also less effective than mucosal heparin as bFGF antagonists in GM 7373-cell-proliferation assays. N-Desulphated/N-acetylated bovine lung heparin retained only a significant capacity to protect bFGF from tryptic cleavage. The results demonstrate that different chemical features of the heparin molecule, including decrease in molecular mass, selective desulphation, disaccharide composition and clustering, affect differently the capacity of the glycosaminoglycan to interact with bFGF and to influence its biological behaviour in different assays in vitro and in endothelial cell cultures. Our findings should aid the design of synthetic oligosaccharides aimed at improving the bioavailability of bFGF when administered in vivo as a therapeutic agent.

Characterization of insulin autoantibodies in a patient with autoimmune hypoglycemia.

A 60-year-old man referred because of hypoglycemic bouts was found to have insulin autoantibodies. Total plasma insulin was as high as 1.44 nmol/l. Both plasma free insulin and C-peptide were in the normal range. The indirect immunofluorescence technique showed positivity for antinuclear antibodies. The T-lymphocyte populations in the peripheral blood were normal. When serum binding capacity for pork insulin was measured, antibodies binding pork insulin were not detected. The patient's serum bound 125I-insulin. The binding protein was identified to be an immunoglobulin G. The kinetics of dissociation, studied by the Scatchard analysis of the autoantibody, showed a curvilinear plot, which was analyzed in two components. Cold human insulin was able to compete with 125I-insulin for the antibody binding site (I.C.50 = 1.35 nmol/ml). These antibodies were apparently not associated with antibodies directed against the insulin receptor.

Serum autoantibodies and thyroid lymphocytic infiltration in endemic goitre.

Patients with thyroid diseases from areas of endemic goitre in Northern Italy were examined for thyroid antibodies by passive haemoagglutination. Of 40 schoolboys with goitre only one had thyroid antibodies (AT) in the blood. Among 182 adults with grade 1-2 goitre, examined within an area of low endemia, the frequency of AT was 7%, not significantly different from that found in the 286 persons with thyroid 'O' living in the same area. Among 181 adults with grade 2-3 goitre, examined within an area of serious endemia, the frequency of AT was 16%; the percentage went up to 24% in 144 patients operated on for goitre. AT frequency was 36% in 87 patients with toxic goitre, more than 70% in 97 patients with Graves' disease and 17% in 60 patients with cancer of the thyroid. AT occurred three times more frequently in women than in men. Microsomal antibodies were more frequently detected than anti-thyroglobulin antibodies: the opposite was true in thyroid cancer. Lymphocytic infiltration (IL) of the gland was observed in 45% of the 464 simple goitres and in 52% of the 60 cancers of the thyroid: it was more frequent and intense in women. Among the 144 patients operated on for goitre the frequency and the titre of AT progressed in parallel with the intensity of the lymphocytic infiltration. Patients with a greater lymphocytic infiltration and higher AT had a higher TSH. Multinodular non-toxic goitre and autoimmune lymphocytic thyroiditis can be present in the same thyroid gland and the clinical expression will depend on which condition predominates.

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence. Chemical and 13C nuclear-magnetic-resonance studies.

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].

Distinct role of 2-O-, N-, and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1.

Interaction of basic fibroblast growth factor (bFGF) with heparan sulfate proteoglycans (HSPGs) plays an important role in the binding of bFGF to its tyrosine kinase receptor (FGFR). The molecular bases of this interaction were investigated by evaluating the capacity of conventional and selectively desulfated heparins i) to affect the binding of bFGF to FGFR and HSPGs of NIH 3T3 cells transfected with FGFR-1/flg cDNA, ii) to facilitate the interaction of bFGF with a recombinant soluble form of the extracellular domain of FGFR-1/flg (xcFGFR-1), and iii) to protect xcFGFR-1 from tryptic cleavage. 6-O-desulfated (6-O-DS) heparin, but not 2-O-desulfated (2-O-DS) and N-desulfated/N-acetylated (N-DS/N-Ac) heparins, retains the capacity to bind bFGF, as assessed by its ability to inhibit bFGF-binding to cell-associated FGFR-1 and HSPGs. On the other hand, at variance with conventional heparin, 2-O-DS, N-DS/N-Ac, and 6-O-DS heparins are all ineffective in potentiating the binding of bFGF to xcFGFR-1 and protecting xcFGFR-1 from tryptic cleavage. The data indicate that 6-O-sulfate groups are not essential for the interaction of heparin with bFGF but are involved in the interaction with xcFGFR-1. Our findings support the hypothesis that HSPGs modulate the binding of bFGF to FGFR through the formation of a ternary complex in which the glycosaminoglycan chains interact with bFGF via 2-O- and N-sulfate groups and with FGFR also via 6-O-sulfate groups.