Saghir Akhtar

Biosynthetic polyhydroxyalkanoates and their potential in drug delivery

Abstract Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polyesters produced as energy storage materials by many bacteria. The most common PHA, poly(3-hydroxybutyrate) (PHB), can be produced in high yield by fermentation of a variety of bacterial strains. PHB is a isotactic semi-crystalline polyester with great potential as a biodegradable commodity; it has useful physico-mechanical properties and appears to be biocompatible. Hydrolytic degradation occurs by surface erosion which makes it an attractive material for controlled release applications. The homopolymer PHB has a relatively high melting point and crystallizes rapidly, making entrapment of drug technically difficult. The related copolymers with 3-hydroxyvalerate, P(HB-HV)s, have similar semi-crystalline properties though their slower rates of crystallization result in matrices with different properties; this merits further investigation. Release of low molecular weight drugs from PHB and P(HB-HV) matrices tends to proceed by penetration of water and pore formation, at least above loadings of approximately 5% drug. Release from such matrices is predominantly independent of polymer erosion; though at lower loadings it is possible to trap drug more effectively. PHB and P(HB-HV) matrices lose mass very slowly when compared to bulk-degrading poly(lactide-glycolide) systems. Therefore the applications of these materials in drug delivery are likely to depend on the formulation of suitable blends with other biocompatible polymers. Porosity, erosion rate and hence drug release rate can be controlled by blending techniques. At a more fundamental level there is considerable potential for design and bioengineering of other PHAs for applications in drug delivery. For example, medium chain PHAs are rubbery materials with low melting points which may be much more suitable as matrices for applications in drug delivery.

Cellular uptake and intracellular fate of antisense oligonucleotides

Antisense oligonucleotides with sequences complementary to a given genetic target can enter cells in sufficient quantities to selectively inhibit gene expression. Thus, they have a potential therapeutic use in preventing undesirable gene expression in diseases such as cancer and AIDS. However, it is remarkable that these molecules, which have high molecular weights and are often charged, gain entry to cells at all. In this article, we review the possible mechanisms by which oligonucleotides enter cells and their subsequent intracellular fates. We also discuss current approaches for improving cellular uptake and delivery of antisense nucleic acids to their intended targets.

The delivery of antisense therapeutics

Antisense oligonucleotides, ribozymes and DNAzymes have emerged as novel, highly selective inhibitors or modulators of gene expression. Indeed, their use in the treatment of diseases arising from genetic abnormalities has become a real possibility over the past few years. The first antisense drug molecule is now available for clinical use in Europe and USA. However, their successful application in the clinic will require improvements in cellular targeting and intracellular delivery. This review aims to look at recent advances in the in vitro and in vivo delivery of antisense oligodeoxynucleotides and ribozymes.

In vivo studies with antisense oligonucleotides

The ability of short, synthetic, single- stranded DNA or RNA oligonu- cleotides to interdict individual gene expression in a sequence-specific manner is the basis for antisense oligonucleotide-based therapies (for recent reviews see Refs 1,2). Unlike traditional drugs which typically interact with proteins, these oligonu- cleotide molecules are designed to selectively bind, by Watson-Crick base pairing, to complementary sequences in the target messenger RNA to interfere with and/or pre- vent formation of the gene product. In principle, antisense oligonu- cleotides can bind to and inhibit the translation of a given messenger RNA by several putative mecha- nisms including simply blocking ribosomal reading or by activating RNaseH, an endogenous enzyme which selectively degrades the message. The fact that very specific Watson-Crick binding of oligo- nucleotides to target sequences may be possible suggests that antisense- based therapies have the potential to be orders of magnitude more specific than conventional therapies. Antisense therapeutics, therefore, represents an exciting new tech- nology for manipulating gene expression in the treatment of human viral diseases such as AIDS, inflammatory disorders and cancers. The purpose of this article is to review the current literature on in vioo pre-clinical and clinical studies with a view to assessing the extent to which antisense oligonucleotides have ‘lived up’ to their initial thera- peutic potential and expectations. Pre-clinical efficacy studies The fact that antisense oligonu- cleotides may be useful therapeutic agents, particularly as antiviral agents, was demonstrated almost a decade ag+. However, it was quickly realized that unmodified phosphodiester oligonucleotides were rapidly degraded in biological fluid&-7 and in viao following intra- venous (i.v.) administration”. Hence, for therapeutic purposes biologically stable analogues would be desirable. The most widely studied of these analogues at present are the phosphorothioate oligonucleotides (S-oligos) where one of the non- bridging oxygens in the phospho- diester backbone is replaced with a sulphur. The initial ‘proof-of concept’ dis- coveries were made with such first- generation oligonucleotides in cell culture (for review see Ref. 9) but rapid development to in viva models followed. Table 1 (see Refs 10-23) highlights some of the in viva studies that have formed the basis for evalu- ating oligonucleotide-based thera- pies in clinical trials (see below). These and the pharmacokinetic dis- tribution studies (Table 2; see Refs 24-36) suggest that the organs of the reticuloendothelial system (RES) such as liver, spleen, kidney and lungs may be good sites for oligonucleotide efficacy as much of the oligonucleotide administered, by almost any route, appears to end up in these tissues (see below). For example, the study of Dean and McKay17 reported a dose-dependent, antisense-mediated downregulation (up to 90%) of pro- tein kinase C-a mRNA in the livers of hairless mice following intraperi- toneal (i.p.) administration. In another study, an oligonucleotide complementary to angiotensinogen that was administered to the liver via the portal vein showed a measurable reduction in angiotensinogen levels in plasma and a lowering of mean blood pressure for three to five days20. However, preferential accu- mulation of oligonucleotides within RES organs may also precipitate adverse effects (see below).

The cellular delivery of antisense oligonucleotides and ribozymes

The design and development of antisense oligonucleotides and ribozymes for the treatment of diseases arising from genetic abnormalities has become a real possibility over the past few years. Improvements in oligonucleotide chemistry have led to the synthesis of nucleic acids that are relatively stable in the biological milieu. However, advances in cellular targeting and intracellular delivery will probably lead to more widespread clinical applications. This review looks at recent advances in the in vitro and in vivo delivery of antisense oligodeoxynucleotides and ribozymes.

Antisense oligonucleotide delivery to cultured macrophages is improved by incorporation into sustained-release biodegradable polymer microspheres

Macrophages are important reservoirs of infection including the human immunodeficiency virus (HIV) virus and strategies which target therapeutic agents to these cells appear worthwhile. In this study, we have evaluated the use of biodegradable poly(lactide-co-glycolide) (P(LA-GA)) microspheres for the improved delivery of anti-HIV oligonucleotides (ODNs) to macrophage cells in culture. Phosphodiester (PO) or phosphorothioate (PS) sequences, including those antisense to the tat gene in HIV, were incorporated into biodegradable P(LA-GA) microspheres (1–2 μm) using a double-emulsion solvent evaporation procedure. For a given polymer molecular weight and ODN chemistry, entrapment efficiencies and in vitro release rates were dependent on microsphere size. Smaller microspheres (1–2 μm) released 70% of the entrapped ODN within 4 days compared with 40 days for larger microspheres (10–20 μm), suggesting that this delivery system also offers the potential for sustained release of ODNs. The cellular association of ODNs entrapped within small microspheres was improved 10-fold in murine macrophages compared with free ODNs. Uptake was enhanced when macrophages were activated with interferon-γ (INF-γ) and lipopolysaccharide (LPS) treatment but decreased significantly in the presence of metabolic and phagocytosis inhibitors. Fluorescence microscopy studies with macrophages showed that a more diffuse subcellular distribution of ODNs was observed when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. These results indicate that polymer microspheres represent an attractive strategy for the improved cellular delivery of ODNs.

Biodegradable poly(L-lactic acid) matrices for the sustained delivery of antisense oligonucleotides

Abstract Antisense oligonucleotides have emerged as exciting novel therapeutic agents which can inhibit gene expression in a sequence-specific manner and are currently undergoing clinical trials evaluation in the treatment of cancer and viral diseases. The therapeutic application of these molecules is limited by their poor biological stability and rapid in vivo elimination kinetics which necessitates frequent administration of oligonucleotides for sustained efficacy. The potential use of a sustained-release biodegradable delivery system, that would protect oligonucleotides from degradation by nucleases whilst delivering the nucleic acid in a controlled or sustained manner to the site of action, may circumvent these problems. In this study, we report on the biological stability, hybridization potential and in vitro release kinetics of antisense phosphodiester (D-oligos) and phosphorothioate (S-oligos) oligonucleotides entrapped within biodegradable poly( l -lactic acid) (PLA) matrices. The in vitro release profiles of 5′-end radiolabelled oligonucleotides entrapped within solvent-cast polymer film matrices (100±10 μ m thickness) were monitored over a period of at least 28 days in either serum, citrate buffer (pH 5.5) or phosphate buffer (pH 7.4). The release profiles over 28 days suggested that the entrapped oligonucleotide was released biphasically from the polymer films, characterized by a rapid burst release during the first 48 h followed by a more sustained release. Oligonucleotide release from PLA matrices was dependent on oligomer chemistry (S-oligos were released more slowly than D-oligos) and on oligomer length (a 20-mer S-oligo was released more slowly than a 7-mer). During the release experiments, little or no degradation of the polymer matrices was observed by SEM or by DSC methods. Oligonucleotide release could be described by the mathematical model for drug release from a solution within a monolithic polymer slab. In all cases, the polymer-entrapped oligonucleotides were resistant to degradation from serum nucleases over the entire study period whereas free phosphodiester oligonucleotides were completely degraded within 1 h. Gel mobility shift analyses and duplex melting point determinations suggested that the hybridization capability of antisense oligonucleotides released from the polylactide matrices was unaffected by the solvent-casting procedure for preparing sustained release polymer devices. These results suggest that biodegradable polymer matrices may be suitable delivery systems for the sustained administration of antisense oligonucleotides.

Development of a Sustained-Release Biodegradable Polymer Delivery System for Site-Specific Delivery of Oligonucleotides: Characterization of P(LA-GA) Copolymer Microspheres In Vitro

Development of a Sustained-Release Biodegradable Polymer Delivery System for Site-Specific Delivery of Oligonucleotides: Characterization of P(LA-GA) Copolymer Microspheres In Vitro Antisense oligodeoxynucleotides (ODNs) can selectively inhibit individual gene expression provided they gain access to and remain stable at the target site for a sufficient period of time. Biodegradable sustained-release delivery systems may facilitate site-specific delivery and also prevent degradation of ODNs by nucleases whilst delivering the nucleic acid in a controlled manner to the desired site of action. In this study, we have characterized biodegradable poly (lactide-co-glycolide) (P(LA-GA)) 50:50 microspheres for the potential delivery of antisense oligonucleotides in vivo. Phosphodiester (PO) oligonucleotides complementary to either c-myc proto-oncogene or the tat gene in HIV-RNA were adequately incorporated within P(LA-GA) microspheres with entrapment efficiencies up to 60% depending on particles size. In vitro release profiles of antisense nucleic acids from 10-20 microm size microspheres over 56 days in physiological buffer were triphasic. Profiles were characterised by an initial burst effect during the first 48 hours (phase 1) of release followed by a more sustained release (phase 2) with an additional increased release (phase 3) being observed after 25 days which corresponded with bulk degradation of the copolymer matrix. The release profiles were influenced by microsphere size, copolymer molecular weight, ODN loading, ODN length and by the pH of release medium used. The serum stability of PO ODNs was significantly improved when entrapped within P(LA-GA) microspheres and the hybridization capability, as assessed by duplex melting (Tm) measurements, of released ODN was not impaired by the double-emulsion microsphere fabrication procedure used. Thus, P(LA-GA) microspheres appear to be promising candidates for improving site-specific delivery profiles for ODNs and are worthy of further evaluation in vivo.

ANTISENSE TECHNOLOGY : SELECTION AND DELIVERY OF OPTIMALLY ACTING ANTISENSE OLIGONUCLEOTIDES

AbstractAntisense technology has increasingly become a reality. Its objective is simple: to inhibit gene expression in a highly sequence-specific and selective manner. The technology has come to encompass three distinct strategies: antigene and antisense oligonucleotides (ODNs) and ribozymes. The antigene strategy relies on short, synthetic ODNs to form triple helical structures with duplex DNA to interfere with gene expression at the level of transcription, whereas antisense ODNs and ribozymes are designed to interfere with gene expression at the level of single stranded RNA (pre-mRNA or mRNA). Antisense ODNs simply rely on the fidelity of Watson:Crick base pairing interactions to hybridize (bind) to the target RNA and inhibit translation by several putative mechanisms including steric hindrance of ribosomal read-through or, depending on the ODN chemistry, by activation of RNaseH to selectively destroy the RNA component of the duplex. In contrast, ribozymes are RNA-based “antisense” oligonucleotides whi...

Angiotensin‐(1‐7) inhibits epidermal growth factor receptor transactivation via a Mas receptor‐dependent pathway

BACKGROUND AND PURPOSE The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes‐induced vascular dysfunction. Angiotensin‐(1‐7) [Ang‐(1‐7)] via its Mas receptor can prevent the development of hyperglycaemia‐induced cardiovascular complications. Here, we investigated whether Ang‐(1‐7) can inhibit hyperglycaemia‐induced EGF receptor transactivation and its classical signalling via ERK1/2 and p38 MAPK in vivo and in vitro.