Mircea-Alexandru Mateescu

N-acylated chitosan: hydrophobic matrices for controlled drug release.

N-acylation of chitosan with various fatty acid (C(6)-C(16)) chlorides increased its hydrophobic character and made important changes in its structural features. Unmodified chitosan exhibited a low degree of order (DO) and a weak tablet crushing strength. Chitosan acylated with a short chain length (C(6)) possessed similar properties, but exhibited significant swelling. Acylation with longer side chains (C(8)-C(16)) resulted in a higher DO and crushing strength but lower swelling. The best mechanical characteristics and drug release properties were found for palmitoyl chitosan (substitution degree 40-50%) tablets with 20% acetaminophen as a tracer. The high stability of these monolithic tablets appears to be due to hydrophobic interactions between side chains, as shown by a more organized structure. Infrared spectroscopy, X-ray diffractometry and proton nuclear magnetic resonance analyses of palmitoyl chitosan were consistent with a hydrophobic self-assembling model. Drug dissolution kinetics showed longer release times for higher degrees of functionalization, i.e. 30 h (for 47% substitution) and 90 h (for 69% substitution), suggesting palmitoyl chitosan excipients as interesting candidates for oral and subdermal pharmaceutical applications.

Cross-linked high amylose starch for controlled release of drugs: recent advances.

Cross-linked high amylose starches have been developed as excipients for the formulation of controlled-release solid dosage forms for the oral delivery of drugs. Advantages of this new class of excipients include cost-effectiveness, readily accessible industrial manufacturing technology, high active ingredient core loading and the possibility of achieving a quasi zero-order release for most drugs. In addition to the latter, other features distinguish cross-linked high amylose starches from other excipients used to prepare hydrophilic matrices. Among these are the absence of erosion, the limited swelling and the fact that increasing cross-linking degrees results in increased water uptake rate, drug release rate and equilibrium swelling. Thus the goal of the present study was to gain some insights into the mechanism of drug release control by matrices of cross-linked high amylose starch. Water transport kinetics and dimensional changes were studied in matrices placed in water at 37 degrees C by an image analysis technique. The results show that in the first 5 min, a gel layer is formed at the surface of the tablet, after which the gel front seems to halt its progression toward the center of the tablet. Water continues to diffuse through the front and to invade the core. As a consequence, this latter swells, with a predominance for radial swelling. Equilibrium swelling is reached over 3 days, when the water concentration in the tablet becomes homogeneous and the whole tablet gelifies. Solid-state 13C-NMR were acquired on cross-linked high amylose starch powders, tablets and hydrated tablets with varying cross-linking degrees. They show a predominance of the V-type single helix arrangement of amylose in the dry state irrespective of the cross-linking degree. Upon hydration, the homologues with a low cross-linking degrees show a transition from the V to the B-type double helix arrangement. It is therefore hypothesized that the capacity of amylose to undergo the V to B transition is an important factor in controlling water transport and drug release rate. Finally applications to different drugs are reviewed briefly. They illustrate the versatility of this technology as generic versions of zero order OROS drug (Efidac) and Fickian release conventional matrices (Voltaren SR) were developed and successfully tested in pilot clinical studies to be bioequivalent to the references. These studies further showed that cross-linked high amylose starch matrices have the lowest inter-subject variability among the systems tested and show a total absence of food effect.

Modified alginate matrices for the immobilization of bioactive agents

Bioactive agents (catalase – an enzyme, and nisin – a bacteriocin) were covalently immobilized on alginate activated with sodium periodate (oxidatively converting 2,3‐dihydroxy groups into dialdehyde residues), followed or preceded by ionotropic gelation. For the same protein coupling yield, the retained enzyme activity of the immobilized enzyme (ImE) can be markedly increased by diminishing the bead diameter, a phenomenon that illustrates the role of substrate/product diffusion through the bead gel layer. When the amount of enzyme introduced for coupling was about 15 mg/100 mg of support and the bead diameter was about 100 μm, a high retained specific activity (95–98%) was obtained. Diffusion phenomena can be markedly decreased by enzyme immobilization on the surface of microbeads (obtained by gelation of activated alginate prior to immobilization). In this case, the retained activity was approx. 75% of that of the free enzyme. A slightly higher Km value of ImE suggested that the enzyme–substrate affinity was almost maintained. The profiles of ImE activities at various pH values, at various temperatures and when undergoing proteolysis showed a overall higher stability for the immbolized than that for the free enzyme. Nisin immobilized on the microbead surface, when submitted to proteolysis, conserved its bacteriocin activity, strongly inhibiting the growth of Lactobacillus sake when subjected to an agar spot test, whereas free nisin totally lost its activity.

Novel cardiac protective effects of urea: From shark to rat

This study was carried out to investigate novel cardioprotective effects of urea and the underlying mechanisms. The cardiac functions under oxidative stress were evaluated using Langendorff perfused isolated heart. Isolated dogfish shark hearts tolerated the oxidative stress generated by electrolysis (10 mA, 1 min) of the perfusion solution (n=4), and also showed normal cardiac functions during post‐ischaemia reperfusion (n=4). The high concentration of urea (350 mM) in the heart perfusate was indispensable for maintaining the normal cardiac functions of the shark heart. Urea at 3–300 mM (n=4 for each group) protected the isolated rat heart against both electrolysis‐induced heart damage and post‐ischaemia reperfusion‐induced cardiac injury. A concentration‐dependent scavenging effect of urea (3–300 mM, n=4 for each group) against electrolysis‐induced reactive oxygen species was also demonstrated in vitro. Urea derivatives as hydroxyurea, dimethylurea, and thiourea had antioxidant cardioprotective effect against the electrolysis‐induced cardiac dysfunction of rat heart, but were not as effective as urea in suppressing the post‐ischaemia reperfusion injury. Our results suggest that urea and its derivatives are potential antioxidant cardioprotective agents against oxidative stress‐induced myocardium damage including the post‐ischaemia reperfusion‐induced injury.

Joint Chromatographic Purification of Bovine Serum Ceruloplasmin and Amineoxidase

A purification procedure leading to a joint separation of two serum copper-enzymes: ceruloplasmin (EC 1.16.3.1) and amineoxidase (EC 1.4.3.6), is described. Both enzymes are obtained in electrophoretically homogeneous form and their specific activities are higher than those obtained by previously described purification techniques. Two common steps: precipitation of bovine plasma proteins with ammonium sulphate (at 35% and 55% saturation) followed by column chromatography on AE-Agarose (obtained by treatment of agarose beads with 1-chloro-2-ethylamine), lead to an electrophoretically homogeneous ceruloplasmin. At the same time, the ceruloplasmin-free protein preparation eluted in a first peak, following further Q-Sepharose and Con A-Sepharose chromatography, leads to purified bovine serum amine oxidase (BSAO) with an improved yield. The emphasis was given to a mutual improving effect as a consequence of the integration of the two enzymes purification procedures.

NMR imaging of chitosan and carboxymethyl starch tablets: swelling and hydration of the polyelectrolyte complex.

The hydration and swelling properties of the tablets made of chitosan, carboxymethyl starch, and a polyelectrolyte complex of these two polysaccharides have been studied by NMR imaging. We studied the effect of pH and ionic strength on the swelling of the tablets and on the diffusion of fluid into the tablets in water and simulated physiological fluids. The pH value of the fluids exerts a more significant effect than their ionic strengths on the swelling of the tablets. The tablets are compared also with those made of cross-linked high amylose starch. The formation of complex helps to keep the integrity of the tablets in various media and render a slow and restricted swelling similar to that of the tablets of the cross-linked high amylase starch, which is significantly lower than the swelling of chitosan and of carboxymethyl starch. The capacities to modulate the release rate of drugs in different media are discussed by comparing the matrices and evaluating the preparation process of the complex. A sustained release of less soluble drugs such as aspirin in gastrointestinal fluids can be provided by the complex, due to the ionic interaction and hydrogen bonding between the drug and the biopolymer complex.

l-Tyrosine prevents aggregation of therapeutic proteins by γ-irradiation

The present paper describes a protective role of l‐tyrosine against aggregation of caeruloplasmin and haemoglobin therapeutic proteins during their sterilization by γ‐irradiation in the aqueous phase. Irradiation of proteins, known to induce their degradation in the presence of oxygen, generates aggregation under oxygen‐free conditions. It was found that l‐tyrosine present during irradiation in deoxygenated media prevents protein aggregation even at high doses (10 kGy), as asserted by SDS/PAGE and high‐performance size‐exclusion chromatography. The protective role of l‐tyrosine, allowing the γ‐irradiation treatment of therapeutic proteins in solution without conformational alteration, is probably exerted by scavenging oxygen radicals produced by irradiation‐induced water radiolysis. It was also found that haemoglobin had a greater stability than caeruloplasmin under γ‐irradiation treatment.

Deglycosylated ceruloplasmin maintains its enzymatic, antioxidant, cardioprotective, and neuronoprotective properties

Ceruloplasmin (CP), an important serum antioxidant, is a blue copper glycoprotein with ferroxidase and oxidase activities. Among other physiological actions, plasma CP was shown to protect isolated rat hearts and cultured P19 neurons exposed to oxidative stress conditions, raising the possibility of using this protein in the treatment of cardiac and neuronal diseases related to oxidative damage. However, since therapeutic applications of CP must be compatible with restrictions in the administration of blood derivatives to humans, there is a need to produce the protein by genetic engineering. To help in the choice of adequate expression systems, we undertook this study to determine if the carbohydrate moiety on the protein is essential for its functions. CP was completely deglycosylated using N-glycosidase F under nondenaturing conditions. Deglycosylated CP was found to retain most of the conformational, antioxidant, and enzymatic properties of the native protein in vitro. Moreover, both forms of the protein had similar cardioprotective and neuronoprotective effects against oxidative stress as evaluated with isolated rat hearts undergoing ischemia-reperfusion and with cultured P19 neurons exposed to xanthine-xanthine oxidase. The data thus indicate that the carbohydrate moiety of CP is not essential for its enzymatic and protective actions. Accordingly, even the use of expression systems that do not glycosylate mammalian proteins could provide a recombinant CP that retains its therapeutic potential.

Peculiar effects of temperature and polyvinylalcohol on the activity of bovine serum amine oxidase.

An inflexion point of enzyme activity at 38 - 42 degrees C of the bovine serum amineoxidase was found. This result, associated with non-strict Arrhenius curves and slightly different activation energies in various temperature intervals, suggests some conformational transitions at the mentioned temperatures. The high molecular weight polyvinylalcohol (100,000 Da) generated an activatory effect and a sigmoidal (non-Michaelis) curve of the dependence of the activity on the substrate concentrations, while the low molecular weight polyvinylalcohol (20,000 Da) does not produce this effect. The different ratio of the two types of polyvinylalcohol/enzyme monomer sizes is considered to be responsible for these different effects on the enzyme kinetics.

The effects of nitric oxide-oxidase and putative glutathione-peroxidase activities of ceruloplasmin on the viability of cardiomyocytes exposed to hydrogen peroxide.

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia-reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H(2)O(2)), the former activity by consuming H(2)O(2) and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO(+) species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H(2)O(2). In contrast to human CP, bovine CP consumed H(2)O(2) in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H(2)O(2) could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H(2)O(2)-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CPs putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue.