Upconversion Nanoparticles Decorated with Polysialic Acid for Solid Tumors Visualization In Vivo.

Upconversion nanoparticles (UCNPs) are a promising nanoplatform for bioreagent formation for in vivo imaging, which emit UV and blue light under the action of near-infrared radiation, providing deep tissue penetration and maintaining a high signal-to-noise ratio. In the case of solid tumor visualization, the UCNP surface functionalization is required to ensure a long circulation time, biocompatibility, and non-toxicity. The effective UCNP accumulation in the solid tumors is determined by the disturbed architecture of the vascular network and lymphatic drainage. This work demonstrates an approach to the UCNP biofunctionalization with endogenous polysialic acid for in vivo bioreagent formation. Bioreagents possess a low level of nonspecific protein adsorption and macrophage uptake, which allow the prolongation of the circulation time in the bloodstream up to 3 h. This leads to an intense photoluminescent signal in the tumor.

Clinical Application of Polysialylated Deoxyribonuclease and Erythropoietin.

BACKGROUND: While protein therapeutics are invaluable in managing numerous diseases, many require frequent injections to maintain therapeutically effective concentrations, due to their short half-life in circulation. PolyXen™, a platform and patented technology employing biodegradable, non-immunogenic and hydrophilic Polysialic Acids (PSA) for drug delivery, is being utilized to overcome such limitations, thereby potentially enabling the clinical utility of a broad range of protein therapeutics. Here, we report the recent progress on two development candidates, polysialylated deoxyribonuclease I (PSA-DNase) and polysialylated erythropoietin (PSA-EPO). METHODS AND RESULTS: Chemical polysialylation of DNase I (DNase) using PSA with different chain length at various conjugation sites led to improved stability against proteases and thermal stress, and slightly reduced enzymatic activity. Polysialylation of EPO resulted in retention of protein structure and PSA-EPO remained biologically active. PSA-EPO had a significantly prolonged circulating half-life (e.g. t1/2 of PSA-EPO = ~400 h in patients after subcutaneous administration, aimed for once monthly administration, vs. t1/2 of EPO = ~22 h; administered twice or thrice weekly), and retained in vivo efficacy. CONCLUSION: This approach has been clinically validated in phase I (in healthy volunteers) and II studies of PSA-EPO [for managing anemia in patients with chronic kidney disease (CKD)].

Hydrophobically modified polysaccharide-based on polysialic acid nanoparticles as carriers for anticancer drugs.

This study presented the development of hydrophobically modified polysialic acid (HPSA) nanoparticles, a novel anticancer drug nanocarrier that increases therapeutic efficacy without causing nonspecific toxicity towards normal cells. HPSA nanoparticles were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling between N-deacetylated polysialic acid (PSA) and 5ß-cholanic acid. The physicochemical characteristics of HPSA nanoparticles (zeta-potential, morphology and size) were measured, and in vitro cytotoxicity and cellular uptake of PSA and HPSA nanoparticles were tested in A549 cells. In vivo cancer targeting of HPSA nanoparticles was evaluated by labeling PSA and HPSA nanoparticles with Cy5.5, a near-infrared fluorescent dye, for imaging. HPSA nanoparticles showed improved cancer-targeting ability compared with PSA. Doxorubicin-loaded HPSA (DOX-HPSA) nanoparticles were prepared using a simple dialysis method. An analysis of the in vitro drug-release profile and drug-delivery behavior showed that DOX was effectively released from DOX-HPSA nanoparticles. In vivo cancer therapy with DOX-HPSA nanoparticles in mice showed antitumor effects that resembled those of free DOX. Moreover, DOX-HPSA nanoparticles had low toxicity toward other organs, reflecting their tumor-targeting property. Hence, HPSA nanoparticles are considered a potential nanocarrier for anticancer agents.

Sialic acid acquisition in bacteria-one substrate, many transporters.

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, ß-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

Assessing the oral bioavailability of difluorosialic acid prodrugs, potent viral neuraminidase inhibitors, using a snapshot PK screening assay.

Difluorosialic acids (DFSAs) are potent inhibitors of viral neuraminidase that demonstrate activity against oseltamivir- and zanamivir-resistant strains of influenza. Unfortunately, low oral bioavailability precludes their development as second generation neuraminidase inhibitors for treating influenza as this leaves them unsuitable for use in an oral formulation. Herein is described the preparation of a series of DFSA prodrugs designed to increase oral bioavailability. These prodrugs were evaluated using a snapshot PK screen and stability tests, with successful candidates being further assessed with a full pharmacokinetic workup. These new prodrugs increased oral bioavailability by up to three times that seen for the parent DFSAs.

Modulating antibody pharmacokinetics using hydrophilic polymers.

INTRODUCTION: The use of hydrophilic polymers as a substitute for the Fc-domain in immuno- or non-immuno-based binding proteins is accelerating. Chemical PEGylation has led the way and is still the most advanced and clinically-approved approach. Hydrophilic polymers act by maintaining a flexible conformation and hydrogen bonding to a network of water molecules to acquire a larger hydrodynamic volume and apparent mass than their actual molecular mass suggest. The benefits are increased blood half-life and bioavailability, stability and reduced immunogenicity. In the case of PEG, there is also evidence of enhanced targeting and reduced side effects, but drawbacks include the fact that PEG is non-biodegradable. AREAS COVERED: This report reviews the state of the art for antibody PEGylation in terms of approaches and effects. Additionally, non-biological (such as N-(2-hydroxypropyl)methacrylamide) and potentially superior biological alternatives (such as polysialylation) are described, ending with recombinant approaches (such as hydrophilic peptides and glyco-engineering), which promise to circumvent the need for chemical modification altogether. EXPERT OPINION: The emergence of many small, antibody fragment-like mimics will drive the need for such technologies, and PEGylation is still the choice polymer due to its established use and track record. However, there will be a place for many alternative technologies if they can match the pharmacokinetics of PEG-conjugates and bring addition beneficial features such as easier production.

Localization and imaging of sialylated glycosphingolipids in brain tissue sections by MALDI mass spectrometry.

In this study, we describe a simple and efficient method for mapping the distribution and localization of all sialylated sphingoglycolipids present in coronal mouse brain sections using a conventional axial matrix-assisted laser desorption/ionization time of flight. A single scan of a histological tissue section gives a complete profile of ganglioside species without derivatization or labeling. We have developed and tested a new matrix preparation (2,6-dihydroxyacetophenone [DHA]/ammonium sulfate/heptafluorobutyric acid [HFBA]) to maximize the detection of all ganglioside species; the ammonium sulfate limits the formation of salt adducts, while the addition of HFBA increases the stability of DHA in a vacuum, thus facilitating imaging applications. Our results, in both extracted samples and whole tissue sections using negative ion reflectron and linear modes, show differences in localization in several brain regions depending on the sialic acids and the ceramide-associated core gangliosides.

Low molecular weight antagonists of the myelin-associated glycoprotein: synthesis, docking, and biological evaluation.

The injured adult mammalian central nervous system is an inhibitory environment for axon regeneration due to specific inhibitors, among them the myelin-associated glycoprotein (MAG), a member of the Siglec family (sialic-acid binding immunoglobulin-like lectin). In earlier studies, we identified the lead structure 5, which shows a 250-fold improved in vitro affinity for MAG compared to the tetrasaccharide binding epitope of GQ1balpha (1), the best physiological MAG ligand described so far. By modifying the 2- and 5-position, the affinity of 5 could be further improved to the nanomolar range (-->19a). Docking studies to a homology model of MAG allowed the rationalization of the experimental binding properties. Finally, pharmacokinetic parameters (stability in the cerebrospinal fluid, logD and permeation through the BBB) indicate the drug-like properties of the high-affinity antagonist 19a.

Synthesis and structural characterization of sialic acid-glutamic acid hybrid foldamers as conformational surrogates of alpha-2,8-linked polysialic acid.

Surface expression of alpha-(2,8)-linked polymers of sialic acid in adult tissues has been correlated with metastasis of several human cancers. One approach to chemotherapeutic intervention against the spread of these cancers involves the development of immunogenic molecules that elicit an antibody response against alpha-(2,8)-linked polysialic acids. Naturally occurring polysialic acids are not viable candidates because they are present during embryonic development and are recognized as self by the immune system. These natural polymers also have poor pharmacokinetic properties because they are readily degraded by neuraminidase enzymes. We have been interested in developing structural surrogates of polysialic acids in an effort to overcome these limitations. Reported herein are microwave-assisted solid-phase peptide syntheses and structural characterization studies of a series of alpha/delta hybrid peptides derived from Fmoc-Neu2en and Fmoc-Glu(OtBu)-OH. Conformational experiments including circular dichroism, NH/ND exchange, and ROESY in aqueous solution were performed to study the secondary structures of these hybrid foldamers. ROESY data were analyzed with the assistance of XPLOR-NIH that was modified to include parameter and topology files to accommodate unnatural amino acids and the delta amide linkages. The results indicate that stable secondary structure is dependent upon both the amino acid sequence and the configuration of Glu. The most stable foldamer was composed of a total of 6 residues beginning with L-Glu at the carboxy terminus and alternating Neu2en and L-Glu residues. In water, this foldamer adopts a right-handed helical conformation with 3.7 residues per turn, 7.4 A pitch, 5.8 A diameter, and a length of 18.5 A, which is stabilized by both classical C=O...H-N backbone interactions and by pyranose ring O and L-Glu HN H-bonding. These structural features orient the L-Glu carboxylates along the helical backbone with a periodicity that matches the carboxylate positions along the reported G2(+) left-handed helix of alpha-(2,8)-polysialic acid. However, the charge density of the foldamer is one-half that of the natural polymer. These findings provide a fundamental understanding of the factors that influence stable secondary structure in hybrid Neu2en/Glu systems, and the tools we have developed establish a viable platform for the rational design of alpha-(2,8)-polysialic acid surrogates.

Modulation of antibody pharmacokinetics by chemical polysialylation.

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.

Zanamivir: an influenza virus neuraminidase inhibitor.

Zanamivir is the first of two registered neuraminidase inhibitors for the treatment and prophylaxis of influenza. Relenza, an orally inhaled powder form of zanamivir, is currently approved in 19 countries for treatment, and in two for prophylaxis. Relenza reduces the time to alleviation of symptoms by 1 to 2 days in the influenza-positive population, if taken within 48 h of symptom onset, and in prophylaxis in family settings, it confers an 80% reduction in the odds of contracting influenza. The resistance profile of zanamivir is encouraging in the sense that there are still no reports of patients on acute therapy shedding drug-resistant virus. However, patient uptake of the inhaled drug has been insufficient to conclude that drug resistance will not be an issue in the future. All zanamivir-resistant variants selected in the laboratory so far have diminished viability.

Dimeric zanamivir conjugates with various linking groups are potent, long-lasting inhibitors of influenza neuraminidase including H5N1 avian influenza.

The synthesis, antiviral and pharmacokinetic properties of zanamivir (ZMV) dimers 8 and 13 are described. The compounds are highly potent neuraminidase (NA) inhibitors which, along with dimer 3, are being investigated as potential second generation inhaled therapies both for the treatment of influenza and for prophylactic use. They show outstanding activity in a 1 week mouse influenza prophylaxis assay, and compared with ZMV, high concentrations of 8 and 13 are found in rat lung tissue after 1 week. Retention of compounds in rat lung tissue correlated both with molecular weight (excluding 3 and 15) and with a capacity factor K' derived from immobilized artificial membrane (IAM) chromatography (including 3 and 15). Pharmacokinetic parameters for 3, 8 and 13 in rats show the compounds have short to moderate plasma half-lives, low clearances and low volumes of distribution. Dimer 3 shows NA inhibitory activity against N1 viruses including the recent highly pathogenic H5N1 A/Chicken/Vietnam/8/2004. In plaque reduction assays, 3, 8 and 13 show good to outstanding potency against a panel of nine flu A and B virus strains. Consistent with its shorter and more rigid linking group, dimer 8 has been successfully crystallized.

Distribution of sialic acids in the milk of spanish mothers of full term infants during lactation.

OBJECTIVES: The protective effect of human milk against infection is well known. Several non-immunologic components, including complex carbohydrates, have been described. The present study was undertaken to determine the sialic acid distribution in different milk fractions (complex carbohydrates). METHODS: Milk samples from 12 Spanish women at three different lactational stages (colostrum, transitional milk and mature milk) were analyzed. Total and glycoprotein-bound, oligosaccharide-bound, casein-bound, and lipid-bound sialic acids were determined. RESULTS: Sialic acids from human milk are mainly bound to oligosaccharides and only a small amount is present bound to glycoproteins or in the free form. All the fractions analyzed showed a similar trend: sialic acids decrease rapidly along lactation. Casein-bound sialic acid does not follow this trend. We detected the presence of an O-acetylated species of N-acetylneuraminic acid. CONCLUSIONS: In human milk from Spanish women we observed slightly different values than those previously reported. This could be a result of population differences but nutritional or methodological aspects can not be discarded.

[Neuraminidase inhibitor, anti-influenzal agent--mechanism of action, and how to use clinically].

Neuraminidase inhibitor has changed the treatment of influenza dramatically. The drug is effective for both influenza A and B whereas amantadine, another anti-influenzal agent, inhibits influenza A virus infection but not B. Neuraminidase inhibitor blocks the process of release of influenza virus from infected cells and inhibit the virus transmission to the neighboring cells. Neuraminidase inhibitor improves the symptoms of influenza and shortens the duration of illness. It is emphasized that this drug should be given within forty-eight hours after the onset, and it must be not noted that the possibility of the drug resistant virus although it is observed infrequently. Prophylactic use of the drug is also discussed.

Zanamivir in the treatment of influenza.

Influenza is a common illness, affecting many people every winter, with a considerable impact on mortality, hospital admissions, healthcare utilisation and sickness absence from work and school. Influenza management is currently focused on annual vaccinations for those in certain risk groups. Risk is determined by age and chronic illness, particularly diabetes, chronic respiratory and cardiac disease, and persons immunocompromised from disease or concomitant therapy. Amantadine (and in some countries, rimantadine is available but has not been widely used, because it is only effective against influenza A infections. The use of amantadines for treatment has been associated with the rapid emergence of resistant viruses capable of transmission, compromising its potential as a prophylactic, as well as a treatment. Side effects are well recognised and are a particular problem in the most vulnerable elderly populations, where dose restriction is necessary and prior knowledge of creatinine clearance desirable. The potential market for a new influenza treatment is large and the potential role of neuraminidase inhibitors in addressing this market has been covered in several review articles [1-4]. This review reports on the introduction of zanamivir (Relenza) to the market with particular reference to experience in the UK.

Synthesis and anti-influenza evaluation of orally active bicyclic ether derivatives related to zanamivir.

We synthesized bicyclic ether sialidase inhibitors such as tetrahydro-furan-2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl derivatives related to zanamivir. These compounds substituted by diol at the C-3' and C-4' positions resulted in the retention of low nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. Compound 11a in particular showed comparable efficacy in vivo relative to that of oseltamivir phosphate.

The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics.

Erwinia carotovora L-asparaginase was conjugated via the epsilon-amino groups of its lysine residues with colominic acid (CA) (polysialic acid) of average molecular mass of 10 kDa by reductive amination in the presence of NaCNBH3. Polysialylation using 50-, 100- and 250-fold molar excess CA relative to the enzyme led to an increasing proportion of the enzyme's in-amino groups (5.8, 7.6 and 11.3%, respectively) being conjugated to CA. Polysialylated and native (intact) asparaginase were used to immunize mice intravenously. Results (total IgG immune responses) indicate that all preparations elicited antibody production against the enzyme moiety but not against the CA of the conjugates. Moreover, antibody titres appeared highest for the native enzyme and were generally reduced as the degree of polysialylation increased. In other experiments mice pre-immunized with native or polysialylated asparaginase, with anti-asparaginase antibodies in their blood, were injected intravenously with the corresponding enzyme preparations. Results revealed that polysialylation reduces the antigenicity of asparaginase thus leading to circulatory half-lives (t 1/2 beta) that were 3-4-fold greater than that of the native enzyme, and similar to those observed in naive, non-immunized mice. Our data suggest that polysialylation of therapeutic enzymes and other proteins may be useful in maintaining their pharmacokinetics in individuals with antibodies to the therapeutic proteins as a result of chronic treatment.