Fm Veronese

Polyoxazoline: Chemistry, Properties, and Applications in Drug Delivery

Polyoxazoline polymers with methyl (PMOZ), ethyl (PEOZ), and propyl (PPOZ) side chains were prepared by the living cationic polymerization method and purified by ion-exchange chromatography. The following properties of polyoxazoline (POZ) were measured: apparent hydrodynamic radius by aqueous size-exclusion chromatography, relative lipophilicity by reverse-phase chromatography, and viscosity by cone-plate viscometry. The PEOZ polymers of different molecular weights were first functionalized and then conjugated to model biomolecules such as bovine serum albumin, catalase, ribonuclease, uricase, and insulin. The conjugates of catalase, uricase, and ribonuclease were tested for in vitro activity using substrate-specific reaction methods. The conjugates of insulin were tested for glucose lowering activity by injection to naïve Sprague-Dawley rats. The conjugates of BSA were injected into New Zealand white rabbits and serum samples were collected periodically and tested for antibodies to BSA. The safety of POZ was also determined by acute and chronic dosing to rats. The results showed that linear polymers of POZ with molecular weights of 1 to 40 kDa can easily be made with polydispersity values below 1.10. Chromatography results showed that PMOZ and PEOZ have a hydrodynamic volume slightly lower than PEG; PEOZ is more lipophilic than PMOZ and PEG; and PEOZ is significantly less viscous than PEG especially at the higher molecular weights. When PEOZ was attached to the enzymes catalase, ribonuclease, and uricase, the in vitro activity of the resultant bioconjugates depended on the extent of protein modification. POZ conjugates of insulin lowered blood glucose levels for a period of 8 h when compared to 2 h for insulin alone. PEOZ, like PEG, was also able to successfully attenuate the immunogenic properties of BSA. The POZ polymers (10 and 20 kDa) are safe when administered intravenously to rats, and the maximum tolerated dose (MTD) was greater than 2 g/kg. Blood counts, serum chemistry, organ weights, and the histopathology of key organs were normal. These results conclude that POZ has the desired drug delivery properties for a new biopolymer.

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.

Influence of PEGylation on the Release of Low and High Molecular-Weight Proteins from PVA Matrices

Model proteins of therapeutic interest, ribonuclease, superoxide dismutase, catalase and albumin, extensively PEGylated with linear 5,000 Mw PEG, were entrapped in PVA hydrogels (31,000-50,000 Mw) by means of a cryogel procedure and the rate of release was evaluated. All of the PEGylated proteins were released from the gel, but at a lower rate than the unmodified proteins. The rate for both native and PEGylated species decreases as the molecular weight of the protein increased. Moreover, for both native and PEGylated proteins, the release rate was reduced when the gel was lyophilized before the release evaluation. Release was also dependent on the PVA Mw, in fact, for Mws of 124,000-186,000, permanent entrapment was achieved. This behaviour was verified with PEGylated glucose oxidase and coline oxidase which were permanently retained in high Mw PVA. This study indicates the potential for the combination of PEGylation and cryogel entrapment of enzymes for the biotechnological and therapeutic applications.

Low Molecular Weight End-Functionalized Poly(N-Vinylpyrrolidinone) for the Modification of Polypeptide Aminogroups

New monofunctionalized amphiphilic oligomers, poly (N-vinyl pyrrolidinones) with an hydroxyl end group, were prepared by radical polymerization with 2-isopropoxyethanol, fractionated by both gel-permeation chromatography and fractional precipitation. The terminal hydroxyl group oligomers was activated and reacted with the amino groups of a model peptide, and a protein. These hydroxylated oligomers were also converted to carboxylate end group which were also activated and used as protein and peptide modifying agents.

Poly(ethylene glycol)-mesalazine conjugate for colon specific delivery

Chronic inflammatory bowel diseases (IBDs) are still waiting for improved and innovative therapeutic treatments, which can overcome the limits of the current approaches. Since IBDs affect mainly the lower tract of the intestine, a localized therapy in the colon tract will avoid most of the problems caused by systemic or poor selective therapies. Particularly promising are the advance drug delivery systems that can reach specific colon delivery, thus guaranteeing active agent release only at the site of action. This approach can meet two aims at the same time, first of all the drug will not affect healthy tissue and second a lower drug dose may be used because all the administered active agent will reach the target. To obtain a specific colon delivery we exploited the azoreductase enzymes, selectively present only in colon, by inserting an azo linker between a selected drug and a macromolecular carrier. The drug employed is mesalazine, a well know and used agent against IBDs. Poly(ethylene glycol) (PEG), of different molecular weights and structures, was used as carrier. Three different conjugates were synthesized and characterized, and the most promising one, with highest drug loading thanks to the use of diamino PEG of 4 kDa, was further investigated in vitro on mouse colonic epithelial cells (CMT-9) and in vivo on model mice with induced colitis. The data presented here demonstrate that PEG conjugation of mesalazine prevents drug release and absorption in upper intestine, after oral administration of the conjugates, and that the azo linker ensures a good drug release in the colon tract. The results in vivo take into consideration mice bodyweight gain, tissue histology and interleukin-2 beta as an index of inflammation. These parameters, all together, demonstrated the conjugate effectiveness against the controls.

Preparation and Properties of Monomethoxypoly(Ethylene Glycol)-Modified Enzymes for Therapeutic Applications

The therapeutic value of enzymes as drugs would be considerably increased if drawbacks such as immunogenicity and antigenicity, rapid clearance from circulation, difficulty in targeting, instability, and inadequate supply were overcome. Genetic engineering seems to be promising in obtaining large amounts of useful enzymes, although doubts exist concerning the correct folding of expressed proteins.1 Nevertheless, the disadvantages of limited tissue distribution and rapid clearance from circulation of enzymes remain a major problem. Moreover, genetic methods cannot be used for producing enzymes carrying post-transcriptional modifications, but with this aim the so-called transgenic animal technology appears to be quite promising. For these reasons, alternative strategies are being actively investigated in several laboratories. These strategies include surface modification of the enzymes by chemical modification or compartmentalization of the enzyme onto complex structures which isolate it from body cells, tissues, and proteolytic enzymes.23

A Comparative Study of Enzymatic, Structural, and Pharmacokinetic Properties of Superoxide Dismutase Isolated from Two Sources and Modified by Monomethoxypolyethylene Glycol Using Different Methods of Couplinga

The surface modification of therapeutically useful enzymes by covalent linkage of nontoxic, nonimmunogenic, and biocompatible polymers is a technique now widely investigated to overcome several disadvantages of the use of enzymes in their native forms. These disadvantages often include rapid clearance from circulation, immunogenicity and antigenicity, degradation by proteolytic enzymes, and finally low stability in handling and conservation.’ Engineered forms, although very promising, do not solve all of these problems because they may still be rapidly cleared; moreover, evidences for immunogenicity of human enzymes cloned in bacterial culture have been reported.2 Among the polymers so far proposed in surface protection of enzymes are dextran, homologous albumin, polyvinylpyrrolidone, ficoll, eparin, and monomethoxypolyethylene glycol (MPEG).’.3 The last is actually one of the best-investigated polymers in several laboratories studying different enzymes and different techniques of activation for binding to pr~teins.~-’So far, a comparative investigation about the influence of the coupling method and the source of the enzymes on the properties of MPEG enzyme adducts is missing. Only recently, a study on the interference of coupling on alkaline phosphatase was reported.’ This paper reports the modification of superoxide dismutase (SOD), an enzyme that presents many potential therapeutical applications, obtained from bovine erythrocytes or from yeast. The reason of the decrease of activity after polymer modification is investigated; furthermore, structural, functional, and pharmacokinetic properties of

Hydrogels obtained by radiation-induced polymerization as delivery systems for peptide and protein drugs

Abstract The controlled release of peptides and proteins from hydrogels obtained by radiation-induced polymerization of 2-hydroxyethyl methacrylate at a low temperature was studied. It was found that the extent of release progressively decreased as protein molecular weight increased until no further release occurred above a critical value of the latter. However, an increasing rate of protein release was found if the polymerization was carried out in the presence of poly(ethylene glycol), PEG. Moreover, only with high molecular weight PEGs were large proteins released. The release data as a function of swellability and porosity of polymer matrices were discussed.

Poly (acryloyl-l-proline methyl ester) hydrogels obtained by radiation polymerization for the controlled release of drugs

Abstract Thermosensitive hydrogels were obtained by radiation-induced polymerization of acryloyl- l -proline methyl ester in the presence of a crosslinking agent. The measurements of equilibrium water content in the temperature range between 0° and 60°C showed that the samples swelled at low temperatures while they shrank at high temperatures. These hydrogels were used as drug delivery systems for the controlled release of insulin. In vivo studies carried out on diabetic rats ascertained a significant reduction in the hyperglycemic level in the blood which continued for about 2 months.

Anti-inflammatory activity of monomethoxypolyethylene glycol superoxide dismutase on adjuvant arthritis in rats

In previous studies we observed an enhanced anti-inflammatory activity of MPEG-SOD derivatives in acute inflammation in rat. To assess the activity in chronic inflammation we tested the compound with longer half-life (MPEG-SOD 18) in complete adjuvant arthritis in rat. According to the prophylactic schedule of treatment (i.m. administration at alternative days of 10 mg/kg from day 3 to day 21), the MPEG-SOD derivative reduced arthritic lesions in a significant way (P less than 0.01 at 14th, 21st, 28th day). Indomethacin, administered i.m. daily at the dose of 1.5 mg/kg according to the same schedule, significantly inhibited adjuvant arthritis each time it was considered (% of inhibition are 66.5% at 14th day, 58.3% at 21st day and 50.8% at 28th day). Native SOD and inactivated enzyme, administered from day 3 to day 21 did not show any anti-inflammatory properties. According to the therapeutic schedule of treatment (from day 14 to day 28), neither MPEG-SOD nor native SOD showed antiarthritic activity.