Ijeoma F. Uchegbu

Synthetic Anticancer Gene Medicine Exploits Intrinsic Antitumor Activity of Cationic Vector to Cure Established Tumors

The systemic delivery of genetic therapies required for the treatment of inaccessible tumors and metastases remains a challenge despite the development of various viral and synthetic vector systems. Here we show that a synthetic vector system based on polypropylenimine dendrimers has the desired properties of a systemic delivery vehicle and mediates efficient transgene expression in tumors after i.v. administration. The systemic tumor necrosis factor alpha (TNFalpha) gene therapy was efficacious in the experimental treatment of established A431 epidermoid carcinoma, C33a cervix carcinoma, and LS174T colorectal adenocarcinoma. Specifically, the systemic injection of dendrimer nanoparticles containing a TNFalpha expression plasmid regulated by telomerase gene promoters (hTR and hTERT) leads to transgene expression, regression of remote xenograft murine tumors, and long-term survival of up to 100% of the animals. Interestingly, these dendrimers and, to a lesser extent, other common polymeric transfection agents also exhibit plasmid-independent antitumor activity, ranging from pronounced growth retardation to complete tumor regression. The genetic therapy as well as treatment with dendrimer alone was well tolerated with no apparent signs of toxicity in the animals. The combination of intrinsic dendrimer activity and transcriptionally targeted TNFalpha when complexed was significantly more potent than either treatment alone or when both were administered in sequence. The combination of pharmacologically active synthetic transfection agent and transcriptionally targeted antitumor gene creates an efficacious gene medicine for the systemic treatment of experimental solid tumors.

Pharmaceutical nanotechnology: polymeric vesicles for drug and gene delivery

Improving the therapeutic index of medicines is a goal of drug delivery. Employing nanosystems that control drug biodistribution is one way of achieving therapeutic improvements, and polymeric bilayer vesicles are one such nanosystem. Polymeric vesicles, with the ability to transport drugs or genes, are prepared in one of two ways: i) the self-assembly of amphiphilic polymers and ii) the polymerisation of monomers, following self-assembly (polymerised vesicles). There are two types of self-assembling amphiphilic polymers: water-soluble polymers derivatised with hydrophobic pendant groups and amphiphilic block copolymers. Amphiphilic alkenes and alkynes are the main compounds that are used to make polymerised vesicles. This review discusses polymer architecture fundamentals that govern the self-assembly of polymers into vesicles, the fine control on vesicle size that is achievable with polymeric vesicles and the application of the vesicles to drug delivery.

The molecular shape of poly(propylenimine) dendrimer amphiphiles has a profound effect on their self assembly

The shape of dendrimer amphiphiles has an unexpected effect on their self-assembly. A series of diaminobutane poly(propylenimine) generation 3 dendrimer (DAB-dendr-(NH(2))(16)) amphiphiles has been synthesized, bearing an average of five (PD5), three (PD3) and one (PD1) palmitoyl group(s) per dendrimer molecule. Additionally DAB-dendr-(NH(2))(16) was derivatized with a layer of poly(ethylene glycol) (PEG, degree of polymerization = 12) groups and conjugated to an average of 1 palmitoyl group at the PEG end (PPD1). A final amphiphile resulted from the conjugation of DAB-dendr-(NH(2))(16) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-succinimidylpropionate (DSPE-PEG(3400)-SPA), i.e.: DPD5 (with 4 DSPE-PEG arms). The critical micellar concentration in aqueous media followed the trend: DPD5 < PD5 = PD3 < PD1 < PPD1 and amphiphiles eventually formed 10-20 nm monomolecular or multimolecular micelles and/or 200 nm spheres or tubules. Aggregation was entropy driven, as expected, for DPD5, PD5 and PD1 and enthalpy driven with the most hydrophilic compound PPD1, but was unexpectedly enthalpy driven for PD3. PD3 aggregates formed low capacity hydrophobic domains with a limited capacity for encapsulation of cyclosporine A; encapsulation levels (mole drug per mole polymer) were 0.099, 0.014, 0.099, and 0.735 for PD1, PD3, PD5, and DPD5 and, respectively. We conclude that star shaped amphiphiles such as PD3 are sterically hindered from self-assembling into high capacity hydrophobic domains in aqueous media. Amphiphile-membrane interactions were promoted by hydrophobic groups, but diminished by PEG moieties. DPD5 is the most suitable amphiphile for biomedical applications.

Optimisation of Synthetic Vector Systems for Cancer Gene Therapy – The Role of the Excess of Cationic Dendrimer Under Physiological Conditions

We have previously demonstrated in a therapeutic study that a single systemic course of DAB-Am16 dendriplexes loaded with plasmid expressing TNFα over a period of time of 10 days led to a regression of 100% of tumours and to long term cures of up to 80% of animals. However, the formulation had a relatively low colloidal stability requiring administration soon after nanoparticle preparation. Similar to other cationic polyplex and dendrimer DNA delivery systems, DAB-AM16 dendrimer formulations contained a substantial proportion of free polymer; this free polymer is present independently of the specific polymer:DNA ratio and increases with increasing proportion of polymer (N:P charge ratio) in the formulation. It has previously been shown for this and other systems that the excess of polymer plays a role in promoting the transfection efficiency of synthetic vectors. This has been linked to effects of the polymer on the efficiency of intracellular processing, e.g. endosomal release. However, the free polymer may have additional effects that are relevant to the efficiency of the formulation. This study therefore considered the effect of free dendrimer on the colloidal stability of the complexes, the interaction of the complex with the formulation medium, and with biological components, i.e. electrolytes and serum proteins after administration. Analysis of the total potential of interaction shows that, even at high N:P ratios, the excess of free dendrimer in the medium is not enough to induce the aggregation of the formulation due to depletion forces. This finding is unusual and can be attributed to the particularly low Mw of these dendrimers (1.6 kDa). On the other hand, formulations are highly sensitive to the strength of the dendrimer:DNA interactions. These can be controlled by the degree of protonation (α) of the dendrimer which is strongly dependent on bulk pH. Modulation of the protonation level to α≥0.4 allows reproducible production of colloidally stable particles. Finally, we have demonstrated that electrolytes and proteins present in physiological media play a crucial role to favour the efficiency of these synthetic vectors reducing the toxicity associated with their cationic groups.

Amphotericin B Formulations – The Possibility of Generic Competition

From the 1950s, Amphotericin B (AmB) in the form of Fungizone® was considered the “gold standard” in anti- fungal therapy. This was due to its broad-spectrum of activity. In the 1990s, Fungizone® was relegated to a second line treatment option after the commercialization of lipid-based formulations (AmBisome®, Abelcet® and Amphocil®). These new medicines have similar efficacies but more favourable safety profiles. These formulations will be coming off patent over the coming months and so there is an opportunity for generics manufacturers to enter the arena. However, the lack of clear regulatory guidance on how to perform bioequivalence studies with liposomal drugs makes it difficult to ensure that generic parenteral formulations present quality, efficacy and safety profiles similar to the innovator product. To date, AmB therapy relies mostly on parenteral administration although infusion-related side effects and nephrotoxicity can lead to treatment being halted. Non-parenteral AmB administration would potentially lead to safer treatments and expand the benefits of antifungal and antileishmanial therapy with this drug worldwide. Despite real scientific advances in the area over the last ten years, very few delivery systems have progressed from proof-of-concept to clinical trials as oral, topical or pulmonary AmB medicines. Cyclodextrins and nanoparticle based formulations (self-emulsifying systems, cochleates and liposomes) are the most promising delivery systems to date.

Nanostructures Overcoming the Blood-Brain Barrier : Physiological Considerations and Mechanistic Issues

Neurological diseases, such as cancers, neurodegenerative conditions, infections, pain and psychiatric disorders, are a leading cause of disability, morbidity and mortality. At any time 1.5 billion people worldwide are suffering from some form of central nervous system disorder, with this number pre...

Chapter 7.3:Drug Delivery Strategies: Nanostructures for Improved Brain Delivery

The effective nature of the blood-brain-barrier (BBB) frequently precludes pharmacotherapy or requires the use of invasive procedures to bypass the BBB and deliver the drugs directly into the cerebrospinal fluid or brain parenchyma. While this method may be acceptable for the treatment of acute, lif...