Robert George Whittaker

Delivery of nucleic acids

Nucleic acids have revolutionized biomedical research and have become indispensable research tools. In pharmaceutical development, nucleic acids are at present mostly used as diagnostic tools and for target validation (1– 3). Applications of microarrays and PCR, treatment with antisense oligonucleotides or small interfering RNA and breeding of knock-in/knock-out-models can be used for accurate diagnosis and biomarker detection, can improve insight into disease processes and can pinpoint pathways where treatments may interfere. Although the application of nucleic acids as therapeutics promises to be even more exciting, their role as clinically applied drugs is still modest. At present, two nucleic acid-based drugs (Vitravenei and Macugeni) are on the market (4). Both drugs are oligonucleotides. Macugeni is an extracellularly acting aptamer that functions as a growth factor decoy and Vitravenei is an intracellularly acting antisense molecule that inhibits a viral gene. Both oligonucleotides contain chemically modified backbones and are injected at the site of the pathology in the vitreous of the eye. This exemplifies the difficulties associated with the use of nucleic acids for therapeutic intervention, both regarding their physicochemical as well as their biological properties. The physicochemical properties of nucleic acids, with molecular weights ranging from 7 kDa for antisense oligonucleotides to over 1 MDa for plasmid DNA, and strong negative charge do not favor membrane passage. Only one class of nucleic acids, aptamers, can act extracellularly, which circumvents the need for cell membrane translocation. Conversely, all other classes need to interact with intracellular targets to be active. The problem is most prominent for plasmid DNA, which has the largest size of all proposed nucleic acid therapeutics and also needs to arrive inside the cell nucleus to be effective. Nuclear localization would in principle require passage through the nuclear pore for which the DNA-molecule is too large (5). These qualities at least partly explain why the marketed drugs are an aptamer and an antisense oligonucleotide. The biological properties also do not support their application as therapeutics. Nucleic acids are susceptible to the action of nucleases. Therefore the two marked oligonucleotides bear chemically modified backbones. In addition, nucleic acids are rapidly cleared from the body, either via glomerular filtration by the kidneys and excretion into the urine or by (scavenger) receptor uptake and intracellular degradation. Therefore, local injection at the site of the pathology is the preferred administration route for the clinically applied oligonucleotides. Despite these difficulties, nucleic acids still capture the mind of many pharmaceutical scientists as possible therapeutics. One of the most appealing properties is that a change in a disease target would in principle only require a change in the nucleic acid sequence to obtain a new drug. As the physicochemical properties like size and charge of the molecules remain the same, the same principles can be applied during the drug formulation steps for this new sequence. After successful formulation of the first nucleic acid drug it can be expected that subsequent formulations will follow more easily. In contrast, for small molecular weight drugs, lead compound identification requires high throughput screening, and drug formulation is dependent on the physicochemical and biological characteristics of the compound. Nevertheless, the difficult biopharmaceutical characteristics of nucleic acids put a lot of demands on the delivery systems that should compensate for these qualities by increasing stability against the action of nucleases, reducing excretion and uptake by non-target tissues and promoting target tissue interaction, target cell association, membrane translocation, and correct intracellular trafficking (6). The articles in this theme issue address this difficult drug formulation process. The group of Klibanov approached the problem of identifying suitable vectors for plasmid DNA delivery using a high-throughput-synthesis coupled to combinatorial chemistry approach. Their study is based on the cationic polymer poly(ethylene imine) (PEI). Experimental observations of their group and others indicate that PEI molecular weight is positively correlated with degree of transfection but also with severity of toxicity (7, 8). These observations provided the input for synthesizing small molecular weight PEI-derivatives that were cross-linked with oligo-acrylate esters. As many of the factors that contribute to degree of transfection and toxicity as well as the relative contribution of each factor to the overall transfection efficiency are unknown, the highthroughput synthesis approach likely provides a higher chance of finding successful polymers. Indeed, their results show that superior PEI-derivatives could be identified as compared to the presently used Fgolden standard_ 22 kD PEI both with respect to degree of transfection as well as toxicity both in vitro and in vivo. Most cationic polymers exhibit a molecular weight distribution. De Wolf et al. investigated the effects of fractionation of the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) into four different molecular weight fractions on in vitro/in vivo transfection of plasmid DNA and polymer-DNA-complex-

Substrate preference profiles of proteases released by allergenic pollens.

Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation.

A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Tris and the ready production of drug-fatty acyl conjugates

The technology described in this article utilises the common laboratory reagent tris to readily produce drug–fatty acid conjugates. Tris has structural similarities to glycerol, allowing the coupling of one to three fatty acyl groups giving compounds that mimic mono‐, di‐, and triglycerides. The symmetrical carbon atom of tris avoids structural isomerisation and enables large‐scale production without the isolation problems generally associated with glycerol derivatives. The amine group provides an ideal attachment site for drugs. Tris is readily available, inexpensive, and approved for pharmaceutical use. We prepared representative fatty acid conjugates of an analgesic (e.g., morphine), nonsteroidal anti‐inflammatory drug (e.g., indomethacin), antiviral drug (e.g., AZT), and antineoplastics (e.g., methotrexate and chlorambucil). Biological activities of these conjugates demonstrated altered properties, such as cellular uptake, delivery profiles, skin retention, and toxicity. These changes may improve the therapeutic use of some compounds. Drug Dev. Res. 46:302–308, 1999.

Tris lipidation: a chemically flexible technology for modifying the delivery of drugs and genes.

1. One of the major challenges in the development of pharmaceuticals is their formulation with other materials to give them the desired bioavailability profile when administered into the body.

The use of Tris-lipidation to modify drug cytotoxicity in multidrug resistant cells expressing P-glycoprotein or MRP1.

Increasing the lipophilicity is a strategy often used to improve a compounds cellular uptake and retention but this may also convert it into a substrate for an ATP‐dependent transporter such as P‐glycoprotein or the multidrug resistance‐associated protein (MRP1), which are involved in cellular efflux of drugs. Tris‐Lipidation of compounds is a convenient way of modifying drug lipophilicity and generating an array of derivatives with diverse properties. To determine the effect of Tris‐Lipidation on a drugs cytoxicity in multidrug resistant cells, various glycyl‐Tris‐mono‐ (GTP1), di‐ (GTP2) and tri‐palmitate (GTP3) derivatives were prepared of the cancer chemotherapeutic drugs chlorambucil and methotrexate, and of the anti‐HIV drug AZT. The cytotoxicity of these derivatives and their parent compounds was determined in the CEM/VLB100 cells with increased P‐glycoprotein expression, the CEM/E1000 cells that overexpress MRP1 and the parent, drug‐sensitive CCRF‐CEM cells. Increasing the lipophilicity of AZT increased its cytotoxicity in the sensitive CCRF‐CEM parental cell line while decreased cytotoxicity was observed for the methotrexate derivatives. For the chlorambucil derivatives, both increased (GTP1) and decreased (GTP2) cytotoxicity occurred in the CCRF‐CEM cells. With the exception of AZT‐GTP1, all GTP1 and GTP2 derivatives of chlorambucil, methotrexate and AZT had decreased cytotoxicity in the P‐glycoprotein‐expressing CEM/VLB100 cells while chlorambucil‐GTP1, methotrexate‐GTP2 and methotrexate‐GTP3 were the only compounds with decreased cytotoxicity in the MRP1‐overexpressing CEM/E1000 cells. The number of palmitate residues, the position of derivatisation and the type of linkage all may affect the P‐glycoprotein and MRP1 substrate properties. Tris‐Lipidation may therefore provide a useful way of manipulating the pharmacokinetic properties of drugs.

Tris Lipidation--A Novel Drug Delivery System that Alters Biodistribution

Tris-lipidation uses Tris to produce drug-fatty acyl conjugates. Radiolabelled Tris-fatty acyl conjugates of methotrexate (MTX) were examined in biodistribution studies in BALB/c mice. Following delivery via a variety of routes, the Tris-lipidated compounds demonstrated features in common with other colloid drug delivery systems. Tissues of the reticuloendothelial system localised the drug following intravenous administration, and the compounds showed prolongation at the site of injection into muscle or fatty tissue, subcutaneously or when inhaled. These findings indicate that the Tris-lipidation platform could be classed as an alternative colloid drug delivery system.

Rapid purification of α-lytic protease and its mutants from small cultures of recombinant Escherichia coli

A single-step procedure is described for purifying α-lytic protease and its mutants on a small scale (approx. 5mg) from cell-free supernatants of recombinant E. coli. Although free of non-enzyme protein, these preparations often contain fragments that arise from enzymatic self-digestion. Two strong cation exchange matrices are shown to vary greatly in their ability to separate such degradation products from intact enzyme.

Over-expression of α-lytic protease and its mutants by recombinantEscherichia coli

SummaryWe report simple fermentation conditions for high-level extracellular production of α-lytic protease and its mutants by recombinantE. coli. Although transcription was controlled by thelac promoter, addition of the inducer isopropyl-ß-D-thiogalactopyranoside (IPTG) was generally found to reduce the expression of active protease. We also describe the effects of other factors on the levels of enzyme found in the culture medium.