Protective effect of superoxide dismutase and polyethylene glycol-linked superoxide dismutase against renal warm ischemia/reperfusion injury.

The protective effect of oxygen free radical scavenger superoxide dismutase (SOD) against the warm ischemic damage that occurs in kidneys harvested from non-heart-beating donors is controversial because of its short half-life. In this model, we compared the protective effect of SOD and two longer lasting polyethylene glycol (PEG)-linked forms of SOD in a model of renal ischemia induced by 60 min of arterial clamping in rats. Rats treated with PEG1-SOD and PEG2-SOD had a better renal function than controls, with significantly lower serum creatinine levels throughout the follow-up period and a significantly higher creatinine clearance on postoperative days 1, 2, and 4. In native SOD treated-rats, serum creatinine was lower than in controls, though not significantly so, and creatinine clearance was significantly higher on postoperative day 4. Our results indicate that the protective effect of SOD against renal warm ischemia can be enhanced by prolonging its half-life by binding the enzyme to PEG.

Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative.

Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.

PEG-Ara-C conjugates for controlled release.

The antitumour agent 1-beta-D arabinofuranosilcytosyne (Ara-C) was covalently linked to poly(ethylene glycol) (PEG) in order to improve the in vivo stability and blood residence time. Eight PEG conjugates were synthesised, with linear or branched PEG of 5000, 10000 and 20000 Da molecular weight through an amino acid spacer. Starting from mPEG-OH or HO-PEG-OH, conjugation was carried out to the one or two available hydroxyl groups at the polymer's extreme. Furthermore, to increase the drug loading of the polymer, the hydroxyl functions of PEG were functionalised with a bicarboxylic amino acid yielding a tetrafunctional derivative and, by recursive conjugation with the same bicarboxylic amino acid, products with four or eight Ara-C molecules for each PEG chain were prepared. A computer graphic investigation demonstrated that aminoadipic acid was a suitable bicarboxylic amino acid to overcome the steric hindrance between the vicinal Ara-C molecules in the dendrimeric structure. In this paper we report the optimised conditions for synthesis and purification of PEG-Ara-C products with a low amount of remaining free drug, studies toward the hydrolysis of PEG-Ara-C and the Ara-C deamination by cytidine deaminase, pharmacokinetics in mice and cytotoxicity towards HeLa human cells were also investigated. Increased stability towards degradation of the conjugated Ara-C products, in particular for the highly loaded ones, improved blood residence time in mice and a reduced cytotoxicity with respect to the free Ara-C form was demonstrated.

Distribution of catalase, ribonuclease and superoxide dismutase modified by monomethoxy (polyethylene glycol) into rat central lymph and lymphatic nodes.

The plasma-lymphatic distribution of ribonuclease (RNase), superoxide dismutase (SODase), and catalase (CTase) modified by monomethoxy (polyethylene glycol) (mPEG) was studied in rats. The lymphatic bioavailability (FL) of individual enzymes administered intravenously was determined on the basis of plasmatic and lymphatic concentration curves. It was concluded that FL values depend on enzyme-adduct molecular weight (m.w.). The highest FL value was found in mPEG-RNase (the lowest m.w.), medium value in mPEG-SODase (intermediate m.w.), and the lowest one in mPEG-CTase (the highest m.w.). The binding of these enzymes in the lymphatic tissue of iliac, intestinal, brachial and neck nodes was also proportional to their molecular weight. The lymphatic binding was dependent on the node localization, higher concentrations being found in the iliac and neck nodes in contrast to the other nodes (intestinal, brachial).

Accurate evaluation method of the polymer content in monomethoxy(polyethylene glycol) modified proteins based on amino acid analysis.

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.

Covalent modification of mushroom tyrosinase with different amphiphic polymers for pharmaceutical and biocatalysis applications.

Two different poly(ethylene glycol) derivatives (linear, mol wt 5000 and a branched form, mol wt 10000) and a new polymer (poly-[acryloylmorfoline], mol wt 5500) were covalently bound to the enzyme tyrosinase. The polymer-protein conjugates were studied with a view to their potential pharmaceutical application and to their use for the bioconversion of phenolic substrates in organic solvents. Vmax and Km for the dopa-dopaquinone conversion, thermostability, stability toward inactivation by dopa oxidation products, half-life in blood circulation, and behavior in organic solvents for the different adducts were investigated. Arrhenius plots for the dopa-dopaquinone conversion were also obtained in order to study the effects of temperature on the different enzyme forms. Covalent attachment of the polymers increased enzyme stability in aqueous solution and the solubility in organic solvents. However, organic solvent solubilization brought about loss of enzyme conformation as assessed by CD measurements, which is accompanied by a nonreversible loss of catalytic activity.

Enzyme modification by MPEG with an amino acid or peptide as spacer arms.

A method for the modification of enzymes by MPEG carrying an amino acid or peptide as a spacer arm is described and tested with aliphatic or aromatic side chains amino acids. The procedure involves MPEG activation by p-nitrophenylchloroformate for the amino acid or peptide coupling that is in turn activated for the protein binding. The advantage of the method resides in the possibility to introduce proper reporter groups between the polymer and the protein as norleucine for a direct evaluation of the bound polymer chains, tryptophan for structural studies of the polymer-protein adduct, and radioactive amino acid for pharmacokinetic investigations. The method was positively tested with arginase, ribonuclease, and superoxide dismutase as enzymes of therapeutic value.

Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase.

A single-step method of activation of monomethoxypolyethylene glycols suitable for its binding to polypeptides and proteins is proposed. Based on the reaction with 2,4,5-trichlorophenylchloroformate or p-nitrophenylchloroformate, it gives reactive PEG-phenylcarbonate derivatives. The PEG intermediate is stable on storage, the activating group is easily quantified,and the reaction with amino acid and proteins proceeds rapidly at pH near neutrality. The PEG derivatization of enzymes with this procedure is less inactivating than those previously reported. Ribonuclease and superoxide dismutase were modified and the effect of (a) bound polymer on clearance time in rats, (b) antibody recognition, and (c) on the enzymatic activity toward low and high molecular weight substrates were studied.

Evaluation of monomethoxypolyethyleneglycol derivatized superoxide dismutase in blood and evidence for its binding to blood cells.

A method to evaluate in blood free superoxide dismutase (SOD) and SOD covalently bound to monomethoxypolyethylene glycol (MPEG-5000) is reported. The method takes advantage of gel filtration and cationic exchange chromatography to remove substances present in plasma which can interfere with the SOD enzymatic assay. Using this methodology, it was demonstrated that, contrary to free SOD, MPEG-SOD, when added to blood, was not completely recovered in plasma. Binding of MPEG-SOD to blood cell components takes place involving interactions of MPEG chains with cell membranes.

Controlled release of proteins and peptides from hydrogels synthesized by gamma ray-induced polymerization.

Hydrogels prepared by radiation-induced polymerization at a low temperature have been used as carriers for the controlled release of peptides and proteins. It was found that polymerization of 2-hydroxyethyl methacrylate in the presence of poly (ethylene glycol) methyl ether (MPEG) enabled the more porous and swellable matrics to be obtained, the higher the molecular weight of MPEG. As a consequence, protein release took place at an increasing extent and, provided that MPEG molecular weight was high enough, high molecular weight proteins could also be released. Such a state of affairs was not met in the case of hydrogels based on poly (2-hydroxyethyl acrylate). SEM analysis revealed that even high molecular weight MPEG did not give rise to any porosity, even though the degree of swelling was very high. As a result, no protein release was observed. It was therefore concluded that control of hydrogel porosity for the controlled release of large proteins is of overwhelming importance.

A direct assay for evaluation of polyethylene glycol in enzyme adducts used as drugs or biocatalysts.

A specific and direct method for the evaluation of monomethoxypolyethylene glycol (MPEG) in enzyme adducts has been developed. The method is based on extensive modification by trinitrobenzensulfonic acid (TNBS) of the MPEG-enzyme, to form an acid-stable TNBS adduct with the available amino groups of lysine and alpha-amino acid. The MPEG-modified trinitrophenylated enzyme was hydrolyzed in 6N HCl and amino acid composition evaluated by a standard AA analyzer in comparison with the analysis of a MPEG enzyme sample. The number of bound MPEG polymers was calculated from the difference in amino acid composition of the two samples.

Surface modification of enzymes for therapeutic use: monomethoxypoly (ethylene glycol) derivatization of ribonuclease.

Bovine pancreatic ribonuclease A (RNase) was modified at various extent at the lysine residues by monomethoxypoly(ethylene glycol) (MPEG) activated as active ester. For pharmacokinetic experiments a radioactive adduct was also prepared with tritiated amino acid as spacer between polymer and protein. The modification reduced only slightly the RNase catalytic activity and Km towards the substrate cytidine-2',3'-cyclic monophosphate. On the other hand extensively modified MPEG-RNase samples, showed significant decrease in activity towards ribonucleic acid. The polymer modification did not change the pH activity profile, increased the stability to proteolytic digestion, while the behaviour towards denaturants and heat was not modified. The native and MPEG-RNase administered IV, IM and SC to rats, showed impressive differences in pharmacokinetics: the half-life of the modified enzyme, evaluated in blood by radioactivity, was increased of 40-50 folds with respect to the native form.

Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine).

Uricase from Bacillus fastidiosus (UC) was covalently linked to linear PEG (PEG-1) (Mw 5 kDa), branched PEG (PEG-2) (Mw 10 kDa) and to poly(N-acryloylmorpholine) (PAcM) (Mw 6 kDa). The conjugation of UC with linear PEG and PAcM was accompanied by complete loss of enzymatic activity but, if uric acid as site protecting agent was included in the reaction mixture, the conjugate protein retained enzymatic activity. On the other hand, the modification with PEG-2 gave a conjugate that also maintained enzymatic activity in the absence of any active site protection. This behaviour must be related to hindrance of the branched polymer in reaching the enzyme active site. The UC conjugates exhibited increased resistance to proteolytic digestion while minor variations in the inhibitory constant, optimal pH, heat stability, affinity for substrate, were observed. Pharmacokinetic investigations in mice demonstrated increased residence time in blood for all the conjugates as compared with native uricase. Uricase conjugated with linear PEG was longer lasting in blood UC derivative, followed by branched PEG and the PAcM conjugates. Unconjugated uricase was rapidly removed from circulation. All these data are in favour of the use of the less known amphiphilic polymer PAcM as an alternative to PEGs in modification of enzymes devised for therapeutic applications.

Preparation and properties of monomethoxy poly(ethylene glycol) doxorubicin conjugates linked by an amino acid or a peptide as spacer.

Polymeric doxorubicin prodrugs were prepared linking monomethoxy poly(ethylene glycol), 5000 D molecular weight, to the doxorubicin amino group, using an amino acid or a peptide as a spacer arm. As spacers glycine, L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine were used. The conjugates showed enhanced stability to alkaline degradation compared to the free doxorubicin. Towards Ehrlich solid tumor in mice the glycin spaced derivative was devoid of activity, whereas the phenylalanine and tryptophan derivatives were 20% and 16% active and the tripeptide one 50% active with respect to free doxorubicin. On the other hand the derivatization was accompanied by a great decrease of toxicity in mice with respect to the free drug. Doxorubicin was not released from conjugates by chymotrypsin incubation or in plasma.

In vitro and in vivo behaviour of narciclasine released from matrices based on poly (2-hydroxyethyl methacrylate).

Narciclasine (1,2,3,7-tetrahydroxy-8,9-methylendioxy-1,2,3,4-tetrahydrophena ntridone) is a natural substance with strong antimitotic effects on cells and potential antitumor activity. Its release form a hydrogel matrix was studied with the purpose of avoiding the concentration spikes of the parenteral administration. The matrix prepared by gamma ray polymerization of a mixture of 2-hydroxyethyl methacrylate (85%) and trimethylolpropane trimethacrylate (15%) was found to release narciclasine for several days, according to a diffusion controlled mechanism. In agreement with its antimitotic effect, narciclasine inhibited the growth rate of healthy mice, when the drug-loaded matrix was introduced subcutaneously. Antitumor effect was observed in an experimental model of Erlich ascitic tumor when low amounts of tumor cells were inoculated. No effect was observed at high concentrations of inoculum or towards solid tumors (Sarcoma 180). This behaviour was related to the rapid clearance of narciclasine from the body which prevented the reaching of sufficient therapeutical concentrations. A pharmacokinetic investigation carried out by an original method of assay demonstrated that narciclasine was accumulated in significant amounts in the kidney only and eliminated in urine with a half time of less than 20 min.

Pegylated enzyme entrapped in poly(vinyl alcohol) hydrogel for biocatalytic application.

A procedure for enzyme entrapment into matrices suitable for biocatalytic applications is reported. The method, which takes advantage of the stable formation of polyvinyl alcohol (PVA) hydrogels by freezing and thawing PVA aqueous solutions, was assayed using lipase as model enzyme. The leakage of lipase was minimised by using high molecular weight PVA and by previous conjugation of the enzyme to PEG. The immobilised PEG enzyme maintained its catalytic activity in organic solvents also, thus allowing enzymatic activity towards water insoluble substrates. The activity was largely increased reducing the diffusional constrain by cutting the matrices into slices of micron size. Matrix-entrapped lipase-PEG, when used in the hydrolysis of acetoxycoumarins, showed a conversion rate of about 10 times lower than the enzyme-PEG in the free form, and maintained regioselectivity when a diacetylated product was used as substrate.

Evaluation of proteolytic enzymes in effervescent tablets.

A procedure for the evaluation of proteolytic enzymes in effervescent tablets is reported. A proper chromatographic step followed by enzymatic activity assay allows to overcome the interferences due to the high salt content of such formulations as well as to chromophores eventually present in the tablet components.

[The modification of Cu, Zn-superoxide dismutase by monomethoxypolyethylene glycol improves the indices of the experimental therapy of the ischemic myocardium in rats]. / Modifikatsiia Cu, Zn-superoksiddismutazy monometoksipoliétilenglikolem uluchshaet pokazateli ékpserimental'noi terapii ishemizirovannogo miokarda krys.

Mice were used to make a comparative study of the biological distribution of intravenous preparations of native and monomethoxypolyethylene glycol-modified superoxide dismutase isolated from bovine liver, as well as native and aldehyde dextran. The study demonstrated that the biodistribution of the native enzymes from various sources was, however, equal, but in the mouse liver there was a higher accumulation of SOD isolated from the rat liver. AD-SOD was found to have a longer half-life in the blood and in the liver of mice, in particular, while MPEG-SOD showed 10, 15, and 16 times longer in the lungs, blood and heart of the animals examined, respectively. The elevated accumulation of MPEG-SOD in some organs was used for their treatment, particularly for experimental therapy of rat myocardial ischemia. A rat model of ischemia demonstrated that the intravenous bolus administration of MPEG-SOD reduced the size of a myocardial necrotic area by 40% as compared to a 13% decrease when the other compounds were assayed. The findings suggest that the MPEG-SOD preparation is promising for decreasing reperfusion injuries of the cardiovascular system and the lungs.