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.

Multivalent and flexible PEG-nitrilotriacetic acid derivatives for non-covalent protein pegylation.

PURPOSE: A new approach for non-covalent protein PEGylation is translated from immobilized metal ion affinity chromatography, and based on metal coordination bonds between a chelating agent linked to PEG, nitrilotriacetic acid (NTA), and the ring nitrogen of histidines in a protein. METHODS: PEG-NTA conjugates were synthesized differing in the number of NTA units and in the polymer structure. Three derivatives were investigated in association experiments with five model proteins. The most promising complex, PEG8-(NTA)(8)-Cu(2+)-G-CSF (granulocyte colony stimulating factor), was thoroughly characterized and the pharmacokinetic profile was evaluated in rats. RESULTS: The experiments demonstrated that only PEG8-(NTA)(8), bearing eight NTA molecules on flexible PEG arms, associated strongly with those proteins having several histidines. The protein secondary structure was not affected in the complex. PEG8-(NTA)(8)-Cu(2+)-G-CSF showed a K (D) of 4.7 nM, as determined by surface plasmon resonance, but the association was not stable in vivo. CONCLUSIONS: PEG8-(NTA)(8) is the first derivative able to associate with native proteins and form soluble complexes with a nanomolar K (D). The study highlights the need of a multivalent and flexible coordination and encourages further investigations to increase the stability of PEG8-(NTA)(8) complexes in vivo either through the use of protein mutants or His-tag proteins.

Stabilization of a supplemental digestive enzyme by post-translational engineering using chemically-activated polyethylene glycol.

Many enzymes used as digestive aids exhibit, at best, moderate stability when incubated under gastrointestinal conditions. A supplemental ß-galactosidase administered orally to treat lactose intolerance was conjugated to 40 kDa, branched polyethylene glycol (PEG). PEGylation increased the enzyme's relative activity at lower pH values (2.5-4.5) and doubled enzyme stability at pH 2.5. The PEGylated enzyme retained significantly more residual activity after exposure to simulated gastric conditions (52% versus 31%), a consequence of protection from both pepsin and low pH mediated inactivation. Conjugation also provided significant protection against the proteolytic component of pancreatin. Overall, the PEGylated enzyme retained over twice the levels of residual activity recorded for non-PEGylated enzyme after exposure to complete simulated gastrointestinal conditions. PEGylation also marginally improved the enzyme's kinetic characteristics. When using its physiological substrate (lactose), K(m) values recorded were slightly decreased (from 83 to 60 µM) and k(cat)/K(m) values (M(-1) s(-1)) were increased from 100 to 147. This appears to be the first report of the use of a conjugated PEG to stabilize a digestive enzyme and the first report of the ability of conjugated PEG to stabilize a protein at low pH.

Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG.

Poly(ethylene glycol) (PEG) has been widely used to prolong the residence time of proteins in blood and to decrease their immunogenicity and antigenicity. A drawback of this polymer lies in its polydispersity that makes difficult the identification of the sites of protein modification. This is a mandatory requirement if a PEGylated protein should be approved as a drug. Here, a fast and reliable method is proposed to characterize proteins conjugated at the level of glutamine (Gln) residues using microbial transglutaminase (TGase). The novelty resides in the use of a monodisperse Boc-PEG-NH(2) for the derivatization that allows the direct identification of the sites of PEGylation by electrospray ionization mass spectrometry (ESI-MS). The procedure has been tested on three model proteins, namely, human granulocyte colony-stimulating factor, human growth hormone, and horse heart apomyoglobin. The Gln residues linked to the polymer chain were easily identified by ESI-MS and tandem MS analyses, demonstrating the advantage of using a monodisperse polymer in combination with mass spectrometry for an easy characterization of conjugated proteins. Interestingly, the PEGylation reaction led to the production only of mono- and bis-derivative products, indicating that the TGase-mediated PEGylation can be extremely selective and thus very useful for the derivatization of protein drugs.

Detection of sites of infection in mice using 99mTc-labeled PN(2)S-PEG conjugated to UBI and 99mTc-UBI: a comparative biodistribution study.

UNLABELLED: The antimicrobial peptide ubiquicidin (UBI) directly labeled with technetium-99m ((99m)Tc) has recently been shown to be specifically taken up at sites of infection; however, its chemical structure is not well defined. To address this problem, the aim of the present study was to label UBI using poly(ethyleneglycol)-N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine ligand (PEG-PN(2)S) in order to compare its ability to detect infection sites with that of (99m)Tc-UBI. METHODS: The PN(2)S-PEG-UBI conjugate was prepared and labeled with (99m)Tc, and its radiochemical purity was subsequently assessed. The stability of the conjugate to cysteine challenge and dilution with both saline solution and phosphate buffer was determined and serum stability and protein binding were also assessed. In vivo studies were carried out in healthy mice to study the biodistribution of (99m)Tc-PN(2)S-PEG-UBI and its precursor (99m)Tc-PN(2)S-PEG and in infected mice to compare the uptakes of (99m)Tc-UBI and (99m)Tc-PN(2)S-PEG-UBI at the site of infection using scintigraphic imaging and ex vivo tissue counting. RESULTS: (99m)Tc-PN(2)S-PEG-UBI was obtained with high radiochemical purity (98+/-1%) and high stability. The amphiphilic nature of the conjugate leads to a tendency to form micellar aggregates that explain the high protein binding values obtained. Biodistribution studies in mice showed low renal clearance followed by a predominant reticuloendothelial system clearance that limits its application in the abdominal area. Statistical analysis revealed no significant difference between (99m)Tc-UBI and (99m)Tc-PN(2)S-PEG-UBI uptake in infected mouse thigh, and the site of infection was clearly visualized using scintigraphic imaging. CONCLUSIONS: (99m)Tc-PN(2)S-PEG-UBI proved to be as effective as (99m)Tc-UBI in detecting sites of infection; however, the well-defined chemical structure of (99m)Tc-PN(2)S-PEG-UBI makes it a better candidate for clinical imaging of infection.

Anti-cancer PEG-enzymes: 30 years old, but still a current approach.

PEGylation (i.e. the covalent link of PEG strands) is a well known technique used to improve pharmaceutical properties of bioactive proteins and peptides. Even in cancer therapy some proteins, in particular enzymes, can find many applications, because of their antiproliferative action or ability to reduce side effects of chemotherapies, but to do so they need to be properly formulated. Unfortunately, formulation alone can not fulfil all the requirements to yield a safe and successful protein preparation for therapeutic applications. In particular, for many proteins fast clearance from the body and potential immunogenicity are severe limitations, which can not be easily overcome without taking into consideration a purposely designed drug delivery system. Among the approaches in the field of drug delivery, PEGylation has so far been the best choice for protein delivery. Here, we describe some examples of PEGylated enzymes useful in antitumoral therapies and the most recent advances in this field.

Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase.

Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the gamma-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new gamma-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH(2)) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them alpha-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor (B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.

A new PEG-beta-alanine active derivative for releasable protein conjugation.

A new PEGylating agent, PEG-betaAla-NHCO-OSu, has been studied for protein amino conjugation using human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF) as model therapeutic proteins. This new activated PEG possesses a convenient property for protein modification when compared to other activated carboxylate PEGs, namely, lower reactivity. When this polymer reacts with a protein, its features lead to fewer PEG-protein conjugate isomers because it preferentially binds the most nucleophilic and exposed amines. Furthermore, the conjugates obtained with PEG-betaAla-NHCO-OSu showed an interesting slow release of polymer chains upon incubation under physiological conditions. Further investigations determined that the PEG chains released are those coupled to histidine residues, and this finally yields less PEGylated species as well as free protein. This release allows a partial recovery of protein activity that is often remarkably and permanently reduced after stable PEGylation, and it occurs in water or blood without the involvement of enzymes. On the other hand, the rate of PEG release, tuned by the chemical structure of this new PEGylating agent, is not too high, and therefore, the achievement of a desired prolongation of protein half-life in vivo is still feasible. The pharmacokinetics of hGH-PEG6k-betaAla conjugate was compared to that of native hGH in rats and monkeys, and the blood residence times were increased by 10- and 7-fold, respectively. The conjugate potency was evaluated in hypophysectomized rats demonstrating a superior pharmacodynamic profile with respect to native hGH.

The impact of PEGylation on biological therapies.

The term PEGylation describes the modification of biological molecules by covalent conjugation with polyethylene glycol (PEG), a non-toxic, non-immunogenic polymer, and is used as a strategy to overcome disadvantages associated with some biopharmaceuticals. PEGylation changes the physical and chemical properties of the biomedical molecule, such as its conformation, electrostatic binding, and hydrophobicity, and results in an improvement in the pharmacokinetic behavior of the drug. In general, PEGylation improves drug solubility and decreases immunogenicity. PEGylation also increases drug stability and the retention time of the conjugates in blood, and reduces proteolysis and renal excretion, thereby allowing a reduced dosing frequency. In order to benefit from these favorable pharmacokinetic consequences, a variety of therapeutic proteins, peptides, and antibody fragments, as well as small molecule drugs, have been PEGylated. This paper reviews the chemical procedures and the conditions that have been used thus far to achieve PEGylation of biomedical molecules. It also discusses the importance of structure and size of PEGs, as well as the behavior of linear and branched PEGs. A number of properties of the PEG polymer--e.g. mass, number of linking chains, the molecular site of PEG attachment--have been shown to affect the biological activity and bioavailability of the PEGylated product. Releasable PEGs have been designed to slowly release the native protein from the conjugates into the blood, aiming at avoiding any loss of efficacy that may occur with stable covalent PEGylation. Since the first PEGylated drug was developed in the 1970s, PEGylation of therapeutic proteins has significantly improved the treatment of several chronic diseases, including hepatitis C, leukemia, severe combined immunodeficiency disease, rheumatoid arthritis, and Crohn disease. The most important PEGylated drugs, including pegademase bovine, pegaspargase, pegfilgrastim, interferons, pegvisomant, pegaptanib, certolizumab pegol, and some of the PEGylated products presently in an advanced stage of development, such as PEG-uricase and PEGylated hemoglobin, are reviewed. The adaptations and applications of PEGylation will undoubtedly prove useful for the treatment of many previously difficult-to-treat conditions.

PEGylation: Posttranslational bioengineering of protein biotherapeutics.

Polymer conjugation, especially by poly(ethylene glycol), has become a leading technology for the delivery of proteins. Nowadays, biotech drugs represent an increasing share of the new approved drugs, but their use is often prevented by drawbacks and safety concern. In particular, short in vivo half-life and immunogenicity are significant problems faced by the researchers dealing with the development of protein and peptide drugs. The chemical linking of a polymer to the protein surface has proved effective in prolonging protein blood circulation and reducing the immunogenicity by decreasing renal clearance and shielding immunogenic epitopes, respectively. So far, PEGylation has already led to nine marketed conjugates with great therapeutic success.:

Nitric oxide modulates proapoptotic and antiapoptotic properties of chemotherapy agents: the case of NO-pegylated epirubicin.

The use of the anthracycline epirubicin (EPI) is limited by the risk of a dilatory congestive heart failure that develops as a consequence of induction of a mitochondrial-dependent cardiomyocyte and endothelial cell apoptosis. Nitric oxide (NO) increases the antitumoral activity of several chemotherapics, while it provides protection against apoptosis induced by oxidative stress both in endothelial cells and cardiomyocytes. The aim of the present study was to investigate whether the addition of an NO-releasing moiety to a pegylated derivative of EPI (p-EPI-NO) confers to the drug a different cytotoxic profile against tumoral and normal cells. The cytotoxic profile of the drugs was investigated in Caco-2 cell line, in embryonic rat heart-derived myoblasts (H9c2), in adult cardiomyocytes, and in endothelial cells (HUVEC). p-EPI-NO was more efficient than EPI in inducing Caco-2 cell apoptosis, while it spared HUVEC, H9c2 cells and adult cardiomyocytes from EPI-induced toxicity. Exposure of cells to p-EPI-NO resulted in a NO-mediated inhibition of cellular respiration followed by mitochondrial membrane depolarization and cell death in Caco-2 cells but not in HUVEC and H9c2 cells in which mitochondrial membrane polarization was maintained at the expense of glycolytically generated ATP. These findings indicate that addition of an NO-releasing moiety to p-EPI increases the anti-neoplastic activity of the drug, while it reduces its cytotoxicity against nonneoplastic cells.

Histaminase PEGylation: preparation and characterization of a new bioconjugate for therapeutic application.

Copper amine oxidase catalyses the oxidative deamination of primary amino groups of several biogenic amines, one of which is histamine, the principal chemical mediator of the first phase of allergic reactions. Looking forward to a possible future therapeutic application of this enzyme in the field of histamine-mediated afflictions, we developed a simple method for the purification of a histaminase from grass pea shoots, a source particularly enriched with the enzyme. Furthermore, in order to improve its therapeutic potential, in particular to reduce the high impurity due to its heterologous source, we conjugated the protein with poly(ethylene glycol) and tested the molecular, immunogenic and pharmacokinetic properties of the native and modified forms. The PEGylated enzyme showed molecular and enzymatic properties similar to those of the unmodified one, but the PEGylation extended the permanence of the injected drug in the body and eliminated its high immunogenic behaviour. The considerable ease of native histaminase production as well as the improved properties after PEGylation, make this engineered plant enzyme a suitable drug candidate for alternative treatment of histamine-mediated affections.

PEGylation, successful approach to drug delivery.

PEGylation defines the modification of a protein, peptide or non-peptide molecule by the linking of one or more polyethylene glycol (PEG) chains. This polymer is non-toxic, non-immunogenic, non-antigenic, highly soluble in water and FDA approved. The PEG-drug conjugates have several advantages: a prolonged residence in body, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity. Thanks to these favorable properties, PEGylation now plays an important role in drug delivery, enhancing the potentials of peptides and proteins as therapeutic agents.

PEG-metronidazole conjugates: synthesis, in vitro and in vivo properties.

Metronidazole (MTZ), a drug used for the treatment of protozoal infections caused by protozoa and anaerobic microorganisms, was conjugated to linear or branched poly(ethylene glycol) of 5,000, 10,000 and 20,000 Da. An ester linkage between polymer and drug was used in the coupling to yield a polymeric prodrug. The modification allowed overcoming the known MTZ solubility problem leading us to obtain a bioconjugate more suitable for parental administration. The conjugates of various molecular weight polymers have been tested in vitro toward chemical degradation and digestive enzymes. It was found that molecular weight and shape of PEG is critical for the prodrugs stability. Good resistance in the stomach acidic media was found and a slow release of the drug in the large intestinal fluid may take place. In vivo studies carried out following i.v. or s.c. administration to mice revealed improved pharmacokinetics properties upon conjugation.

Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates.

Peptide and protein PEGylation is usually undertaken to improve the biopharmaceutical properties of these drugs and, to date, several examples of conjugates with long permanence in the body as well as with localization ability in disease sites have been reported. Although a number of studies on the in vivo behavior and fate of conjugates have been performed, forecasting their pharmacokinetics is a difficult task since the pharmacokinetic profile is determined by a number of parameters which include physiological and anatomical aspects of the recipient and physico-chemical properties of the derivative. The most relevant perturbations of the protein molecule following PEG conjugation are: size enlargement, protein surface and glycosylation function masking, charge modification, and epitope shielding. In particular, size enlargement slows down kidney ultrafiltration and promotes the accumulation into permeable tissues by the passive enhanced permeation and retention mechanism. Charge and glycosylation function masking is revealed predominantly in reduced phagocytosis by the RES and liver cells. Protein shielding reduces proteolysis and immune system recognition, which are important routes of elimination. The specific effect of PEGylation on protein physico-chemical and biological properties is strictly determined by protein and polymer properties as well as by the adopted PEGylation strategy. Relevant parameters to be considered in protein-polymer conjugates are: protein structure, molecular weight and composition, polymer molecular weight and shape, number of linked polymer chains and conjugation chemistry. The examples reported in this review show that general considerations could be useful in developing a target product, although significant deviations from the expected results can not be excluded.

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.

Synthesis, characterization, and preliminary in vivo tests of new poly(ethylene glycol) conjugates of the antitumor agent 10-amino-7-ethylcamptothecin.

Despite the high antitumor activity of camptothecins, few derivatives have been developed and tested for human treatment of solid tumors, due to unpredictable toxicity mainly connected to their poor water solubility. We report the conjugation of the antitumor agent 10-amino-7-hydroxy camptothecin (SN-392) to linear or branched poly(ethylene glycol)s (PEGs) of different loading capacity through a tri- or tetrapeptide spacer selectively cleaved by lysosomal enzymes (cathepsins). A synthetic strategy based on the chemoselective acylation of the aromatic amino group in the presence of the unprotected C20 tertiary alcohol allowed high overall yields. Two conjugates demonstrated good stability at physiological pH and in mouse plasma (nonspecific proteases) but slowly released the drug payload in the presence of the lysosomal enzyme cathepsin B1. Compound 3, selected for in vivo experiments, was very active against P388, P388/ADM leukaemia, and Meth A fibrosarcoma cell lines, scoring T/C% values comparable with the camptothecin derivative CPT-11. Pharmacokinetic studies indicated that 3 acts as a reservoir of 10-amino-7-ethylcamptothecin, as the mean residence time (MRT) is about 3-fold higher than that of the free drug.

Poly(ethylene glycol)-avidin bioconjugates: suitable candidates for tumor pretargeting.

Avidin-poly(ethylene glycol) (PEG) conjugates were obtained by derivatization of about 10% of the protein amino groups (four amino groups per protein molecule) with linear 5 kDa PEG or branched 10 or 20 kDa PEGs. Circular dichroism analysis showed that the polymer conjugation neither altered the protein structure nor the environment of the aromatic amino acids which are present at the level of the biotin binding site. Spectroscopic studies were carried out to evaluate the biotin recognition activity of the conjugates either in terms of number of biotin binding sites or avidin/biotin affinity. Avidin-PEG 5 kDa and avidin-PEG 10 kDa displayed over 90% of the native protein biological activity while a reduction in the recognition of biotinylated antibodies of about 25% was found with PEG 20 kDa. In vivo studies demonstrated that the protein immunogenicity was in the order: wild type avidin>avidin-PEG 5 kDa>avidin-PEG 10 kDa>avidin-PEG 20 kDa. By intravenous injection into mice bearing a solid tumor, all conjugates displayed prolonged permanence in the circulation with respect to the native protein. The area under the curve values of avidin-PEG 5 kDa, avidin-PEG 10 kDa and avidin-PEG 20 kDa were about 3-, 7- and 30-times higher than the wild type avidin with reduced accumulation in kidneys and liver. Interestingly, all conjugates accumulated significantly in the tumor mass. In particular, in the case of avidin-PEG 20 kDa, 8% of the injected dose (ID)/g of tissue accumulated in the tumor after 5 h from the administration and over 6% of the ID/g was maintained throughout 72 h.

Conjugates of peptides and proteins to polyethylene glycols.

This chapter provides a critical overview of the technology presently available in the field of protein PEGylation. The chemistry of the polymer and of its reactive derivatives is discussed and presented together with several protocols used to obtain PEG-protein conjugates. The coupling protocols are critically discussed on the basis of the properties of the protein to be modified and those desired for the final product. Methods for product purification and characterization are also provided. The overall information provided will guide the reader toward all of the critical steps involved in the preparation of PEG-protein adducts.

Synthesis, characterization and preliminary cytotoxicity assays of poly(ethylene glycol)-malonato-Pt-DACH conjugates.

Oxalate 1,2-diaminocyclohexane platinum (oxaliplatin(R)), a successfully employed platinum compound belonging to the family of Pt-DACH complexes, has been conjugated to different molecular weight poly(ethylene glycols) (PEG) by means of peptide spacers and a malonic acid bidentate residue. Tri- and tetrapeptidic substrates of lysosomal enzymes were used in order to increase the release of Pt-DACH complex inside the cell following endocytosis and enzymatic degradation of the peptide spacer. Other aminoacids (e.g. norleucine) have been also employed. 1H-NMR of some conjugates was performed as characterisation of the product, while 195Pt-NMR analysis was carried out to detect the rearrangement of the platinum complex from the Pt(O,O) to the Pt(O,N) form. The compound PEG(5000)-Nle-malonato-Pt-DACH (4) has been tested against L1210-implanted mice and showed and appreciable increase in cytotoxicity as compared to the reference standard Cl(2)PtDACH.