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The chemical modification of synthetic oligonucleotides has recently been investigated to improve their pharmacological utilization. In addition to chemical alterations of the backbone and of the heterocyclic bases, their conjugation with amphiphylic moieties, such as the polyethylene glycol has been proposed. The large scale production of these molecules as demanded for commercial purposes is hampered by the heterogeneity of the solid-phase processes and by the low reactivity of high-molecular weight PEGs in solution. A new synthetic procedure based on the recently developed liquid-phase method (HELP), has been set up to overcome these limitations.

In the attempt of prolonging the effect of drugs, a new branched, high-molecular weight multimeric poly(ethylene glycol) (MultiPEG), synthesized with a simple assembling procedure that devised the introduction of functional groups with divergent and selective reactivity, was employed as drug carrier. In particular, the attention was focused on the study of theophylline (THEO) and THEO-MultiPEG conjugates pharmacokinetic after oral administration in rabbit. Pharmacokinetic behavior was studied according to an ad hoc developed mathematical model accounting for THEO-MultiPEG in vivo absorption and decomposition into drug (THEO) and carrier (MultiPEG). The branched high-molecular weight MultiPEG proved to be a reliable drug delivery system able to prolong theophylline staying in the blood after oral administration of a THEO-MultiPEG solution. The analysis of experimental data by means of the developed mathematical model revealed that the prolongation of THEO effect was essentially due to the low THEO-MultiPEG permeability in comparison to that of pure THEO., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The design of new photosensitizers with enhanced phototoxicity and pharmacokinetic properties remains a central challenge for cancer photodynamic therapy (PDT). In this study, Pheophorbide a (Pba) has been pegylated to methoxypolyethylene glycol (mPE G-Pba) to produce a soluble photosensitizer that exhibits a higher tissue distribution than free Pba. In vitro studies have shown that mPE G-Pba promotes a fairly strong photosensitizing effect in cancer cells, as previously observed for the unpegylated molecule. mPE G-Pba targets the mitochondria where, following photoactivation, ROS are produced which cause a cellular injury by lipid peroxidation. The effect of pegylation on the photosensitizer biodistribution has been examined in different selected organs of female mice, at different time points after intraperitoneal administration of the drug (50 μmol/Kg body weight). Other than free Pba, which showed a low tissue accumulation, mPE G-Pba has been detected in significant amounts (8 to 16 μg/ml) in liver, spleen, duodenum and kidney and, 3-5 hours after intraperitoneal injection, in moderate amounts (3 to 8 μg/ml) in brain and lung. In vivo optical imaging performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Our results indicate that although pegylation somewhat decreases the phototoxicity, it significantly increases the drug solubility and tissue distribution and tumor uptake of mPE G-Pba, making the conjugate an interesting photosensitizer for PDT.

Taking advantage of the peculiar features of poly(ethylene glycol), a wide range of applications in the field of drug delivery has been devised. In these last years, a series of patents have been applied on the use of this polymer in delivering of biopolymers, as peptides, proteins and nucleic acid derivatives, as well as of low molecular weight drugs. Moreover, several related strategies have been outlined for the modification of biocompatible materials employed in the field of drug delivery. In this review we focused our attention on the patents appeared in the last years; the description is not exhaustive but the reported citations have been selected as examples of the aforementioned applications.

The successful conjugation of active theophylline molecules to two new multifunctional high-molecular weight poly(ethylene glycol) derivatives (MultiPEG) and their pharmacokinetic evaluations are reported. The drug loading was increased up to six times in comparison with commercial PEG of the same molecular weight. A clear increase of the time of persistence within the body and a concomitant improvement of the overall pharmacokinetic properties of those prodrugs were also observed. These studies sustain the use of these new PEG-based polymeric supports as a valuable alternative for an effective drug delivery system.

The conjugation of a bioactive, fluorescent PNA sequence to high-molecular weight poly(ethylene glycol) (PEG) is described and the properties of the PEG-PNA conjugate are evaluated.

A new process for the preparation of large amounts of thioated oligonucleotides in a quasi-classical solution condition is described. This method takes advantage of the use of polyethylene glycol as a soluble, inert support during synthesis. The easy purification of intermediates from a moderate excess of reagents allows very high coupling yields and, consequently, efficient production of the long oligonucleotide sequences required for pharmacological applications. The reaction of properly protected and activated oligonucleotides with high-molecular-weight polyethylene glycols allows a convenient procedure for the postsynthetic conjugation of those biopolymers in solution. The oligonucleotides are modified at the 5' terminus using a liquid-phase procedure with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. The use of N, N'-disuccinimidyl carbonate for the activation of the terminal OH groups of the PEG was preferred.

The Ki-ras gene is frequently mutated and/or overexpressed in human cancer. Since it is suspected to play a key role in the pathogenesis of many tumors, there is interest to search for strategies aiming at the specific inhibition of this oncogene. In this paper, we investigated the capacity of a 20 mer G-rich oligonucleotide (ODN20) conjugated to high molecular weight monomethoxy polyethylene glycol (MPEG) to inhibit the expression of the Ki-ras gene and the proliferation of pancreatic cancer cells. The conjugate, MPEG ODN20, was designed to form a triplex with a critical pur/pyr sequence located in the promoter of the Ki-ras gene. To make the conjugate resistant to endogenous and exogenous nucleases, five phosphorothioate linkages were introduced in its backbone. Confocal microscopy and FACS experiments showed that MPEG ODN20 had a higher capacity to penetrate the cell membranes and accumulate in the nucleus of Panc-1 cells than ODN20. Incubation of Panc-1 cells with MPEG ODN20 reduced specifically the levels of Ki-ras mRNA and RAS protein p21RAS. A single-dose administration of MPEG ODN20 was sufficient to inhibit cell proliferation by about 50% compared with control. By contrast, the antiproliferative activity of the unconjugated ODN20 analog was found to be not significant. Band-shift and footprinting experiments showed that MPEG ODN20 formed a weak triplex (Kd approximately 1.5 microM at 37 degrees C, 50 mM Tris-acetate, pH 7.4, 10 mM NaCl, 10 mM MgCl2, 5 mM spermidine) with the Ki-ras pyr/pur motif, suggesting that its bioactivity can hardly be mediated by a triplex-based mechanism. Here, we provide evidence that, in vitro, ODN20 and MPEG ODN20 competitively inhibit the binding to the Ki-ras pur/pyr motif of a nuclear protein, suggesting that the activity of MPEG ODN20 occurs with an aptameric mechanism. The biological implications of this study are discussed.

The reaction of oligonucleotides with high molecular weight monomethoxy poly(ethylene glycol)s (MPEGs) has been tested to set up a convenient procedure for the postsynthetic conjugation in solution of biopolymers. A first oligonucleotide was previously modified in 5', using a liquid-phase procedure, with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. Two procedures commonly utilized for the activation of the terminal OH groups of the MPEG were evaluated, that is, the reaction with pNO(2)-phenyl chloroformate and with N,N'-disuccinimidyl carbonate. Both water as well as organic solution conditions were employed and compared. In a second test, a 3'-amino modified, commercial 20-mer was also conjugated in a microscale condition to verify the effect of size and concentration of MPEG on the postsynthetic conjugation of these biopolymers under troublesome synthetic conditions.

The synthesis of a peptide-PEG-oligonucleotide chimera is compared when starting from the peptide or from the oligonucleotide sequence.

Triplex-forming oligonucleotides are able to modulate gene expression by site-specific binding to genomic DNA. Their use as therapeutic agents is limited by inefficient cellular uptake, scarce nuclear internalization, and oligonucleotide self-aggregation. In this study, we demonstrate that a 13-mer AG motif oligonucleotide covalently linked to a high-molecular mass (9000 Da) polyethylene glycol (PEG ODN(13)) exhibits uptake and biological properties that are superior to those of the nonconjugated isosequence analogue (free ODN(13)). Band-shift and footprinting experiments showed that PEG ODN(13) forms a stable triple helix (apparent K(d) between 10(-6) and 10(-7) M in 50 mM Tris-acetate, 10 mM MgCl(2), pH 7.4, 37 degrees C) with a natural polypurine-polypyrimidine target located in the 5' flanking region of the human bcr/abl oncogene. Confocal laser microscopy performed on unfixed live cells stained with hexidium iodide as well as on glass-fixed cells stained with propidium iodide showed that fluorescein-labeled PEG ODN(13) is far more efficiently taken up and internalized in the nucleus by K562 and HeLa cells than the nonconjugated free ODN(13). It was found that PEG ODN(13) specifically downregulated the transcription of bcr/abl mRNA at 65 +/- 5% with respect to control and inhibited cell growth by 32 +/- 3% in a 3 day liquid culture assay. Moreover, PEG ODN(13) was more resistant against S1 and fetal bovine serum nucleases than free ODN(13), and less inclined to self-associate into multistrand structures in solution. Taken together, these results provide useful elements for designing artificial transcription repressors with enhanced potency in vivo.

Long "all-purine" oligonucleotides, up to the 20mer, known to be active as antigene effectors, conjugated to high molecular weight monomethoxy poly(ethylene glycol)s (MPEG)s, were successfully synthesized. Through a liquid-phase, MPEG-supported process, both natural and chimeric sequences containing selected phosphorothioate backbone modifications were obtained, purified, and characterized. To follow their cellular trafficking, a fluorescent probe was linked by soluble supported organic reactions to the 5'-terminus, and the efficiency of the different synthetic procedures for the introduction of a fluorescein moiety was compared. The usefulness of the fluorescent marker was estimated by laser confocal microscopy that ascertains that the MPEG-conjugation enhances the oligonucleotide capacity to cross the cellular membranes and to be accumulated inside the nuclei.

The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.

A new amphiphilic, high-molecular weight poly (N-acryloylmorpholine) (PAcM) polymer has been used to be linked to oligonucleotide chains through a liquid-phase stepwise synthesis. This new conjugate has been investigated for its melting property, nuclease stability and capacity to elicit RNase H activity. Its antisense activity against an HIV-1 target has been also evaluated.

Two complete series of N-protected, monodispersed oligopeptide esters to the pentamer level from 1-aminocyclododecane-1-carboxylic acid (Ac(12)c), an alpha-amino acid conformationally constrained through C(alpha)(i) <--> C(alpha)(i) cyclization, and either L-Ala or Aib residues, along with the N-protected Ac(12)c homopeptide alkylamide series from monomer to trimer, have been synthesized by solution methods and fully characterized. The solution-preferred conformations of these peptides have been assessed by Fourier transform ir absorption and (1)H-nmr techniques. Moreover, the molecular structures of one derivative (Z-Ac(12)c-OH) and three peptides [the tripeptide ester Z-L-Ala-Ac(12)c-L-Ala-OMe, the tripeptide alkylamide Z-(Ac(12)c)(3)-NHiPr, and the tetrapeptide ester Z-(Aib)(2)-Ac(12)c-Aib-OtBu (Aib, alpha-aminoisobutyric acid)] have been determined in the crystal state by x-ray diffraction. The results obtained point to the conclusion that beta-bends and 3(10)-helices are preferentially adopted by peptides based on Ac(12)c, the largest cycloaliphatic C-disubstituted glycine known. A comparison with the structural tendencies extracted from published works on peptides from Aib, the prototype of C-disubstituted glycines, and the other extensively studied members of the class of 1-aminocycloalkane-1-carboxylic acids (Ac(n) c, with n = 3-9), is made and the implications for the use of the Ac(12)c residue in the Ac(n) c scan approach of conformationally restricted analogues of bioactive peptides are briefly discussed., (Copyright 2000 John Wiley & Sons, Inc.)

Two conjugates of an anti-HIV oligonucleotide (ODN) with different high molecular weight monomethoxy polyethylene glycols (MPEGs) have been tested for their activity as substrate towards RNase H. The MPEG does not impede the formation of the regular hybrid duplex with the target RNA sequence as pointed out by the persistence of the RNase H activity; thus, these derivatives stimulate the hydrolysis of RNA by the enzyme at the same site and with the same extent of cleavage as the native sequence.

An anti-HIV 12mer oligonucleotide (ODN) conjugated to two different high molecular weight monomethoxy polyethylene glycols (MPEGs) has been tested for its antisense activity. The capacity of these conjugates to protect the MT-4 cells against HIV infection has been compared to the unmodified, native ODN, and the effect of the different structures of the supporting polymer has been discussed. It was found that only the ODN conjugated to the linear MPEG shows an anti-HIV activity in the investigated conditions. The same 12mer, when conjugated to a branched (MPEG)2, is fully inactive, as well as the native, unmodified ODN.

The use of synthetic oligonucleotides as possible drugs for human therapy is usually hampered by their low in vivo stability and capacity to achieve high concentrations at their cellular targets. To overcome this, it has been suggested that they be modified chemically. Among the various options, conjugation with short- and long-chain polyethylene glycols (PEGs) has several advantages: PEG is nontoxic and very soluble, reduces immunogenic reactions, and increases the stability of the conjugated molecules. PEG is generally joined to oligonucleotides while bound to the insoluble solid-phase supports, after their synthesis, which does not allow for their being easy scaled up. The use of the liquid-phase approach as an alternative synthetic process, utilizing the PEG polymer both as a soluble, inert synthetic support and a stabilizing agent of the oligonucleotide, is proposed. A new protocol has been set up, characterized by a stable phosphate bond between the support and the oligonucleotide. This method has been tested on a 12mer previously investigated as an antisense agent against HIV. The PEG-conjugated 12mer was efficiently synthesized and purified. It was found that the high-molecular mass PEG chain results in enzymatic stability and does not interfere with the formation of the duplex with its nucleic acid target.

A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C alpha,alpha-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac9c) and two Ala/Ac9c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives mCIAc-Ac9c-OH and Z-Ac9c-OtBu, the dipeptide pBrBz-(Ac9c)2-OtBu, the tetrapeptide Z-(Ac9c)4-OtBu, and the pentapeptide Z-(Ac9c)5-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac9c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac9c residue is a strong beta-turn and helix former. A comparison with the structural propensity of alpha-aminoisobutyric acid, the prototype of C alpha,alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3-8) is made and the implications for the use of the Ac9c residue in conformationally constrained analogues of bioactive peptides are briefly examined.

A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined.

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C (alpha,alpha)-disubstituted glycine 1-aminocyclooctane-1-carboxylic acid (Ac8c), and two Ala/Ac8c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and 1H-NMR. The molecular structures of the amino acid derivative Z-Ac8c-OH, the dipeptide pBrBz-(Ac8c)2-OH and the tripeptide pBrBz-(Ac8c)3-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac8c-NHMe. Taken together, the results obtained strongly support the view that the Ac8c residue is an effective beta-turn and helix former. A comparison is also made with the conformational preferences of alpha-aminoisobutyric acid, the prototype of C (alpha,alpha)-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc with n = 3, 5-7) investigated so far. The implications for the use of the Ac8c residue in peptide conformational design are considered.

A N alpha-blocked, Aib-rich octapeptide methylamide containing two N omega-benzoylated L-Lys residues at positions 3 and 6 was synthesized by solution methods and fully characterized. A solution and crystal-state conformational analysis, performed by using FT-IR, 1H NMR, CD, and X-ray diffraction techniques, showed that the peptide is folded into a regular, right-handed 3(10)-helix stabilized by seven consecutive N-H...O=C intramolecular H-bonds of the beta-turn III type. The two benzamidobutyl L-Lys side chains, located on the same side of the helix after one complete turn, generate a cleft the minimal width of which was found to be 3.47 A.