Oddone Schiavon

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 monomethoxy-polyethylene glycols suitable for its binding to polypeptides and proteins is proposed. Based on the reaction with 2,4,5-trichloro-phenylchloroformate orp-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 super-oxide 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.

Polyethylene glycol-superoxide dismutase, a conjugate in search of exploitation

Without a doubt PEG-SOD has been the enzyme most studied in PEGylation. One can say that it represents the preferred model to assess chemistries for PEG activation, analytical procedures suitable for conjugate characterization, the influence of PEG size in conjugate removal from circulation and elimination of immunogenicity and antigenicity, and the effect of route of administration. The effect of PEG conjugation was studied in vitro and in vivo models in comparison with the free enzyme and the following conclusions may be drawn: (1) At the blood vessel level, PEG-SOD has been shown to provide a greater resistance to oxidant stress, to improve endothelium relaxation and inhibit lipid oxidation. (2) In the heart, PEG-SOD proved to be at least as effective as native SOD in treatment of reperfusion-induced arrhythmias and myocardial ischemia. (3) In the lung, PEG-SOD appeared to be able to reduce oxygen toxicity and E. coli-induced lung injury, but not in the treatment of lung physiopathology associated with endotoxin-induced acute respiratory failure and in the reduction of asbestos-induced cell damage. (4) On cerebral ischemia/reperfusion injuries the effect of PEG-SOD was uncertain, also due to the difficulty of cerebral cell penetration. (5) In kidney and liver ischemia both enzyme forms were found to ameliorate reperfusion damage. In view of so much positive research on PEG-SOD, it is surprising that no approved application in human therapy has been established and approved.

Branched and Linear Poly(Ethylene Glycol): Influence of the Polymer Structure on Enzymological, Pharmacokinetic, and Immunological Properties of Protein Conjugates

Linear and branched poly(ethylene glycol)s, with similar molecular weights, were conjugated with uricase and asparaginase, and an investigation of enzymological, immunological, and pharmacokinetic properties of the conjugates was carried out. It was found that the steric hindrance of the branched polymer has a relevant role in determining the biological properties of the conjugates. Conjugations with branched polymers inactivate the enzyme less than the linear ones. Compared to the native and the linear polymer conjugate counterparts the branched polymer derivatives: (1) are more stable to proteolysis by elastase, pronase, and trypsin, (2) stay longer in the blood with increased systemic availability after intravenous administration in mice, and (3) give rise to lower levels of antinative enzyme antibodies after immunization. These data are consistent with a greater surface area of protein covered by the branched PEG.

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.

Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly( ethylene glycol)

A comparative pre formulation study of native asparaginase and conjugated with linear monomethoxypoly(ethylene glycol) 5000 (mPEG) or branched monomethoxypoly(ethylene glycol) (mPEG2) is reported. The first polymer, mPEG, was obtained with norleucine as spacer between polymer chain and protein, while mPEG2 was obtained by linking of mPEG 5000 Mw to the α- and ϵ-lysine amino groups. The comparison regarded the enzymatic activity, stability, pharmacokinetic and immunological properties of the different enzyme forms. The enzyme modified by the branched polymer showed increased in vitro activity and proteolytic resistance, improved stability towards temperature and pH variations and enhanced half life. Furthermore, the conjugation with mPEG largely reduced recognition by polyclonal antibodies raised against native asparaginase, the effect being more marked in the mPEG2 conjugate. All these data are in favour of the use of asparaginase modified by the mPEG2 in the treatment of patients with tumour responding to this enzyme, in particular in case of intolerance to native asparaginase.

PREPARATION, PHYSICO-CHEMICAL AND PHARMACOKINETIC CHARACTERIZATION OF MONOMETHOXYPOLY(ETHYLENE GLYCOL)- DERIVATIZED SUPEROXIDE DISMUTASE

Abstract The surface modification of enzymes by polymers has recently been proposed in order to improve their use in therapy. It is found that the derivatization with monomethoxypoly(ethylene glycol) (MPEG) of Superoxide dismutase (SOD), an enzyme now used in inflammatory diseases, gives a heterogenous product. It is demonstrated that this heterogeneity depends on the presence of bifunctional PEG coming from non-methoxylated molecules. Covalent binding of PEG is accompanied by some loss in enzymatic activity, which has been demonstrated to be due to the decrease in the affinity of the Superoxide ion at the SOD surface, while the active site structure at the metals is not modified. The MPEG bound to the SOD surface modifies certain physico-chemical properties of the enzyme, such as solvent solubility, metal binding and structural stability. The pharmacokinetic parameters of the enzyme, which were evaluated in rats following i.V., i.m., i.p. and s.c. administration, has been found to be greatly modified by the polymer binding.

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 byp-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.

PEG-epirubicin Conjugates with High Drug Loading

PEG is used as a polymeric carrier for low molecular weight drugs, but limitations arise from the fact that only one or two hydroxyl residues are on each polymer. Therefore, the synthesis of dendrimeric structures, based on amino adipic acid or beta-glutamic acid, as a branching molecule, built on a PEG diol of Mw 10,000Da was investigated. The large polycyclic drug epirubicin molecule was chosen as a model to investigate the influence of structure branching and drug steric hindrance during coupling reactions. Several derivatives with increased numbers of drug molecules linked to each PEG chain were synthesized and their physical, chemical and biological properties were studied. The use of specific amino bicarboxylic acids (amino adipic acid or -glutamic acid), as the branching moiety for the dendrimer synthesis, allowed linking the hindered molecule epirubicin to multibranched PEG. Most drug loaded conjugates only dissolve in water following dissolution in DMSO. This solubility problem was solved by adding a hydrophilic peptide linker between the drug and the polymer. The conjugates, synthesized in good yield and purity, showed better stability than free epirubicin in different pH buffers and in plasma as well as prolonged residence time in blood. Dynamic light scattering investigation showed that these products have a high tendency to aggregate forming stable micelles.

Relevance of folic acid/polymer ratio in targeted PEG-epirubicin conjugates

A series of PEG-epirubicin conjugates with different folic acid contents per polymer chain was synthesized in order to study the influence of polymer/targeting moiety ratio on selective cytotoxicity, cellular uptake and intracellular localization. Analogous carboxyl-terminated conjugates without folic acid were studied as control. The heterobifunctional HO-PEG-COOH was used as polymeric carrier, allowing the synthesis of conjugates with a good control over the chemical structure and the drug/polymer and polymer/targeting residue ratios. A dendron structure was synthesized at one end of the PEG chain with the aim to increase the number of folic acid molecules. L-2-aminoadipic acid was used as branching unit. The conjugates showed high stability under several physiological conditions. Biological evaluation was carried out in A549, HeLa and KB-3-1 human cell lines, as these cells have different levels of folate receptor (FR) expression. In particular A549 cells are FR negative (FR-), HeLa cells are FR positive (FR+) and KB-3-1 cells over-express FR (FR++). It was clearly shown that the biological activity of the conjugates was influenced by the presence and the number of folic acid molecules per polymer chain and by the level of FR expression on cell surface. Conjugates conformation in solution was also studied, as differences in size might well affect cell internalization. In the cell viability assay, conjugates without folic acid were unexpectedly more cytotoxic than the targeted conjugates, but their IC(50) values were similar in the three cell lines. Differently, the anti-proliferative activity of targeted derivatives markedly increased going from FR(-) to FR(++) cells. FACS and confocal microscopy studies showed greater cellular internalization with the targeted conjugates than with their non-targeted analogues; more importantly, this relationship is clearly dependent on folic acid content in the conjugates and FR expression level in the cell line used.

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 nor-leucine (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.