Brenda McCormack

Polysialylated insulin: synthesis, characterization and biological activity in vivo

Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96-99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12-48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60-1.74 for the 22-kDa CA and 2.37-2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.

A role for liposomes in genetic vaccination

Genetic immunization by the use of plasmid DNA encoding antigens from bacteria, viruses, protozoa and cancers has often led to protective humoral and cell-mediated immunity, and has some practical advantages over conventional vaccines. However, naked DNA vaccines can be degraded by nucleases in situ, are unable to target antigen presenting cells (APCs), and exhibit poor performance when administered by routes other than the intramuscular, all of which have reduced the value of the approach. We have been able to avoid DNA degradation and also target DNA to APCs by the use of liposomes as DNA vaccine carriers. Entrapment of plasmid DNA within the aqueous spaces of cationic liposomes is effected by a one step procedure which results in most of the DNA being incorporated into a freeze dried, ready to use preparation. Animal experiments have shown that immunization by the intramuscular or the subcutaneous route with liposome-entrapped plasmid DNA encoding the hepatitis B surface antigen leads to much greater humoral (IgG subclasses) and cell mediated (splenic IFN-gamma) immune responses than with naked DNA. In other experiments with a plasmid DNA encoding a model antigen (ovalbumin), a cytotoxic T lymphocyte (CTL) response was also observed. These results could be explained by the ability of liposomes to protect their DNA content from local nucleases and direct it to APCs in the lymph nodes draining the injected site.

Drugs-in-cyclodextrins-in liposomes: a novel concept in drug delivery

Abstract Inclusion complexes of tritiated dehydroepiandrosterone (DHEA), retinol (R) and retinoic acid (RA) were formed with 14 C-labelled 2-hydroxypropyl-β-cyclodextrin (HPβCD) or unlabelled β-cyclodextrin (βCD) polymers 2009 and 2010 (Mol. Wt 4000–4500 and 8700, respectively) at various molar ratios. Formation of inclusion complexes was confirmed by the complete or partial solubilization of the drugs used and by the simultaneous elution of drug and HPβCD radioactivities following molecular sieve chromatography of the complex solutions. Inclusion complex solutions (also containing ‘void’ cyclodextrins) were subsequently entrapped into dehydration-rehydration vesicles (DRV liposomes). Ratios of entrapment values (% of amounts used) for drugs and cyclodextrin (HPβCD) approximating unity were taken to denote entrapment that did not discriminate between complexes and void cyclodextrin. Near unity ratios and highest entrapment values (e.g., up to 32.3 ± 11.9% (DHEA) and 31.9 ± 11.8% (HPβCD) of the materials used; distearoyl phosphatidylcholine (DSPC) DRV) were achieved with liposomes made of phospholipids with a high gel liquid crystalline transition temperature ( T c ) or, when equimolar (to the phospholipid) cholesterol was also present, with all phospholipids, regardless of their T c . When DSPC liposomes (without or with equimolar cholesterol) containing drug (DHEA, R or RA) complex solutions with cyclodextrins were exposed to rat blood plasma at 37°C for up to 60 min, cyclodextrin (HPβCD) retention was nearly complete (0.7–11.9% released at 60 min). However, release of drugs was considerable with values being significantly greater for DHEA (60.2–62.0%) than for R or RA (26.6 and 26.8%, respectively). Experiments with DRV containing both car☐yfluorescein (CF) (as a marker of vesicle stability) and inclusion complex solutions revealed that entrapped cyclodextrins do not destabilize liposomes. Instead, data suggest that during or after the entrapment of complex solution into liposomes, some of the included drug is displaced from the cyclodextrin cavity by phospholipid and/or cholesterol (to a degree probably dependent on the stability constant of the complex) to end up in the lipid bilayer in a state which, on incubation, ensures rapid release into the media. Results suggest that entrapment of water-insoluble (or certain soluble drugs) in the form of cyclodextrin-inclusion complexes into the aqueous phase of liposomes may circumvent some of the problems associated with their entrapment as such.

Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics.

Abstract. Naturally occurring polymers of N-acetylneuraminic acid (polysialic acids) are biodegradable, highly hydrophilic and have no known receptors in the body. Following intravenous injection, polysialic acids exhibit long half-lives in the blood circulation and have therefore been proposed as carriers of short-lived drugs and small peptides. In addition, shorter-chain polysialic acids can be used as a means to increase the circulatory half-life of proteins and thus serve as an alternative to the nonbiodegradable monomethoxypoly(ethylene glycol). Recent work has shown that covalent coupling of a low molecular weight polysialic acid (colominic acid) to catalase and asparaginase leads to a considerable increase of enzyme stability in the presence of proteolytic enzymes or blood plasma. Comparative studies in vivo with polysialylated and intact asparaginase revealed that polysialylation significantly increases the half-life of the enzyme. The highly hydrophilic and innocuous nature of polysialic acids renders them suitable as a means to prolong the circulation of peptides and proteins.

Drugs-in-cyclodextrins-in-liposomes: an approach to controlling the fate of water insoluble drugs in vivo

Distearoyl phosphatidycholine liposomes containing entrapped complexes of 14 C-labelled hydroxypropyl-8-cyclodextrin (HPβ-CD) with 3 H-labelled dehydroepiandrosterone (DHEA), retinol (RET) and dexamethasone (DEX) were prepared and incubated with rat blood plasma at 37°C. The rate of drug dissocation and release in the media was minimal for DEX/HPβ-CD (11%), modest for RET/HPβ-CD (23%) and considerable for DHEA/HPβ-CD (56%, 60 min). However, the HPβ-CD moiety of each of the complexes was retained by liposomes quantitatively. Intravenous injection of free complexes into rats led to their rapid clearance from the circulation with up to 94% of HPβ-CD recovered in 24 h urine together with lesser and variable amounts of drug (up to 46% of the dose. DHEA < RET < DEX). A proportion of the drugs (up to 25% of the dose), but very little HPβ-CD, was removed by the liver where drugs were catabolised rapidly, presumably following complex dissociation in the blood and drug transport to the tissue via plasma proteins. After injection of complexes entrapped in liposomes, these were found to alter the pharmacokinetics of the complexes with only 6-13% of HPβ-CD and a moderate proportion of drugs (up to 26% of the dose) recovered in 24 h urine. Much of the HPβ-CD moiety was recovered in the liver (up to 83%) and spleen (up to 13% of the dose) 30 min after injection, together with a variable proportion of drugs (DHEA < RET < DEX). In longer term (up to 24 days) experiments with liposome-entrapped complexes, there was removal of HPβ-CD from the tissues albeit at a very slow rate. Moreover, the metabolism of individual drugs. both the liver and spleen following vesicle disintegration appeared to depend directly on the rate of complex dissociation. Administration of drug/cyclodextrin inclusion complexes via liposomes could serve as a means to control the action of a wide range of therapeutic agents.

Comparative studies of the fate of free and liposome-entrapped hydroxypropyl-β-cyclodextrin/drug complexes after intravenous injection into rats: implications in drug delivery

Inclusion complexes of 14C-labelled hydroxypropyl-beta-cyclodextrin (HP beta-CD) with tritiated dehydroepiandrosterone (DHEA), retinol (RET) and dexamethasone (DEX) were entrapped in the aqueous phase of liposomes composed of distearoyl phosphatidylcholine. Incubation of liposome-entrapped complexes in the presence of rat blood plasma at 37 degrees C resulted in complex dissociation and drug leakage in the media at a rate which was minimal for DEX/HP beta-CD (11%), modest for RET/HP beta-CD (23%) and considerable for DHEA/HP beta-CD (56%; 60 min). In contrast, the HP beta-CD moiety of the complexes was retained by liposomes quantitatively. Free complexes injected intravenously into rats were cleared rapidly from the circulation with most (up to 94%) of the HP beta-CD moiety recovered in 24 urine together with lesser and variable amounts of drug (up to 46% of the dose) in a pattern (DHEA < RET < DEX) that was consistent with the extent of drug association with HP beta-CD in the presence of plasma. A significant proportion of the drugs (up to 25% of the dose)(but very little HP beta-CD) was removed by the liver where drugs were catabolised rapidly, probably following complex dissociation and transport to the tissue via plasma proteins. Injection of entrapped complexes revealed that liposomes alter their pharmacokinetics with only 6-13% of HP beta-CD and a moderate proportion of drugs (up to 26% of the dose) recovered in the urine. Much of the HP beta-CD moiety of the liposomal complexes was recovered in the liver (up to 83%) and spleen (up to 13% of the dose) by 30 min after injection, together with a variable proportion of drugs in a pattern (DHEA < RET < DEX) which reflected that of complex dissociation in vitro. Longer term experiments where animals injected with liposome-entrapped complexes were killed at time intervals (up to 24 days), revealed that HP beta-CD is eliminated from the tissues, albeit at a very slow rate. Moreover, the metabolism of individual drugs in the tissues following vesicle disintegration, appeared to depend on the rate of complex dissociation with DEX, for instance, reaching values of 4-6% (liver) only after 14 days. It is concluded that administration of drug/cyclodextrin inclusion complexes via liposomes could serve as a means to control the action of a wide range of therapeutic agents.

The ‘Co-Delivery’ Approach to Liposomal Vaccines: Application to the Development of influenza-A and hepatitis-B Vaccine Candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination ‘co-delivery’ and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell – a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.

Entrapment of plasmid DNA vaccines into liposomes by dehydration/rehydration

Intramuscular injection of naked plasmid DNA is known (1-3) to elicit humoral and cell-mediated immune responses against the encoded antigen. It is thought (2,3) that immunity follows DNA uptake by muscle cells, leading to the expression and extracellular release of the antigen which is then taken up by antigen presenting cells (APC). In addition, it is feasible that some of the injected DNA is taken up directly by APC. Disadvantages (1-3) of naked DNA vaccination include: uptake of DNA by only a minor fraction of muscle cells, exposure of DNA to deoxyribonuclease in the interstitial fluid thus necessitating the use of relatively large quantities of DNA, and, in some cases, injection into regenerating muscle in order to enhance immunity. We have recently proposed (1,4) that DNA immunization via liposomes (phospholipid vesicles) could circumvent the need of muscle involvement and instead facilitate (5) uptake of DNA by APC infiltrating the site of injection or in the lymphatics, at the same time protecting DNA from nuclease attack (6). Moreover, transfection of APC with liposomal DNA could be promoted by the judicial choice of vesicle surface charge, size and lipid composition, or by the co-entrapment, together with DNA, of plasmids expressing appropriate cytokines (e.g., interleukin 2), or immunostimulatory sequences.

Polysialic Acids: Potential for Long Circulating Drug, Protein, Liposome and Other Microparticle Constructs

Optimal use of drugs often requires their extended presence within the vascular system or in extravascular areas (Gregoriadis et al, 1994). For instance, some antibiotics and cytostatics and a variety of peptides and proteins including hormones, cytokines, enzymes, antibodies and haemoglobin (as a blood surrogate) are excreted or removed from the circulation by tissues rapidly and before therapeutic concentrations in target areas can be achieved. Such drugs could be more effective, less toxic and also used in smaller quantities if their presence in the blood circulation (and hence interaction with corresponding receptors or substrates intravascularly or extravascularly) could be prolonged (Lee et al., 1995). In the same way, prolonged circulation of drug delivery systems such as liposomes (Gregoriadis, 1995), other colloidal systems (Davis et al., 1984) and polymers (Domb et al., 1997) would facilitate targeting of drugs to cells other than those (e.g. the reticuloendothelial system; RES) by which many of these systems are normally intercepted (Gregoriadis, 1995; Lee et al., 1995)

Polysialic Acids: In Vivo Properties and Possible Uses in Drug Delivery

Alongside the development of new drugs and improving the efficacy of established drugs is the need to design ever more effective carrier systems to enhance pharmacological drug action. Conventional use of promising compounds can have many shortcomings in vivo, such as failure to reach target tissues — thus giving rise to undesirable side-effects, inability to penetrate intracellular sites, reduced residence time within the vascular system and extravascular areas (Florence and Gregoriadis, 1991) all pre-empting their therapeutic purpose. During the last two decades a variety of delivery systems have been investigated in an attempt to circumvent these problems and have had variable success in directing drugs to tissues in need of treatment and enabling their release there. Such carriers are antibodies, including monoclonal (Nelson, 1991), ligands designed to bind specifically to cell surface receptors and antigens, polymers with distinct attached ligands, and paniculate systems such as liposomes and microspheres. More recent interest has focused on attempts to prolong the presence within vascular and extravascular areas of many established drugs such as antibiotics and cytostatics, as well as a vast range of peptides and proteins which include hormones, cytokines, enzymes and antibodies, all of which are known to be excreted or otherwise removed rapidly, rendering them pharmacologically valueless, unless given in unacceptably toxic amounts. Extending the residence time of liposomes (Gregoriadis, 1988) and other colloidal systems (Roerdink and Kroon, 1989) would also increase their flexibility allowing perhaps contact with tissues other than those of the reticuloendothelial system (RES) by which they are mainly intercepted. (Gregoriadis, 1988; Roerdink and Kroon, 1989). This has been done successfully by coating liposomes and other particles with low molecular weight (750–5000) monomethoxypoly(ethyleneglycol)(mPEG) (Blume and Cevc, 1990; Klibanov et al, 1990; Senior et al, 1991; Papahadjopoulos et al, 1991) or poloxamers (Moghimi et al, 1991; Jamshaid et al, 1988). mPEG seems to act by forming a shell of mPEG molecules (Nucci et al, 1991) around the surface of proteins and particles which sterically interferes with factors in the biological milieu responsible for their removal. However, low molecular weight mPEG, which is known (Nucci et al, 1991) to be excreted rapidly by the kidneys, is of little benefit in extending the half-lives in the circulation of many useful small peptides and conventional drugs.