In vivo efficacy and safety of skin electroporation

This article reviews the studies on skin electroporation carried out in vivo in animals and emphasizes its potential therapeutic applications for transdermal and topical drug delivery. In agreement with in vitro studies, transport across skin due to high-voltage pulses in vivo was shown to increase by orders of magnitude on a timescale of minutes. Increased transdermal transport was measured by systemic blood uptake and/or pharmacological response, and demonstrated for calcein, a fluorescent tracer, fentanyl, a potent analgesic and flurbiprofen, an antiinflammatory drug. Combined electroporation with iontophoresis was shown to provide rapidly responsive transdermal transport of luteinizing hormone releasing hormone ex vivo as well. These data underline the potential of skin electroporation for improving the delivery profile of existing conventional transdermal patches, but also for replacing the injectable route.High-voltage pulses can increase drug permeation within and across skin but are also an efficient tool to permeabilize the membrane of cells of the cutaneous or subcutaneous tissue. This was shown beneficial for targeting cutaneous cells with oligonucleotides or genes and might open new opportunities for gene therapy and DNA vaccination.The safety of the application of high-voltage pulses on skin was assessed in vivo, using histological and visual scores, and bioengineering methods. While changes in skin barrier and function were observed, the irritation was mild and short-lived. Further optimization of the electrode configuration for improved targeting of the stratum corneum should still improve tolerance and levels of sensation.     

Antitumor drug delivery by tissue electroporation

Tissue electroporation has been explored to enhance the local delivery of chemotherapueutic agents to solid tumors. The technique, known as electrochemotherapy (ECT), uses high-voltage pulses to deliver drugs across cancerous tissues. ECT has been demonstrated to be an effective treatment for cutaneous malignancies. Recent studies also indicate that the applications of ECT can be extended from the treatment of cutaneous cancers to the treatment of tumors of vital organs such as brain, liver, lungs and others. This review also discusses electrogene antitumor therapy.     

电穿孔技术促进萘普生经皮渗透的研究

目的：研究电穿孔技术对小分子离子型药物经皮渗透的影响。方法：应用双室扩散池方法研究电穿孔技术对萘普生在离体大鼠腹部皮肤经皮渗透的影响。并与被动扩散和离子导入进行比较。结果：外加电脉冲（指数衰减型脉冲,脉冲幅度为４００Ｖ,电容器电容为２．２ｕＦ,脉冲率为２０ｐｕｌｓｅｓ．ｍｉｎ＾－１,脉冲宽度τ≈６．０ｍｓ）或离子导入（１ｍＡ．ｃｍ＾－２,６ｈ）时,萘普生的渗透速率和累积渗透量均大于被动扩散。外加脉     

In vivo analysis of glucocorticoid-induced reporter gene expression using gene gun DNA delivery

Glucocorticoid regulates various physiological processes via the activation and repression of gene expression. The anti-inflammatory effects and the adverse effects are believed to be dependent on the repression and the activation of genes, respectively. Reporter gene assay is a useful technique to separately evaluate these two functions and has been used for in vitro screening of novel ligands for the glucocorticoid receptor (GR). We report here the application of a reporter gene assay for the in vivo determination of the GR-mediated gene activation. A reporter plasmid containing glucocorticoid response elements was introduced to abdominal mouse skin using a gene gun. Administration of prednisolone induced the expression of the reporter gene, only when the GR expression plasmid was co-transfected with the reporter plasmid. Endogenous levels of corticosterone appeared to be negligible in this protocol. The dose response for this induction was comparable to those for the decreases in thymus weight and serum corticosterone. These results suggest that gene gun-mediated skin transfection enables the in vivo reporter gene assay and that this technique can be used to predict the potency of ligands for the GR-mediated gene activation.     

Enhanced transdermal delivery of tetracaine by electroporation

The effect of electroporation on the transport of tetracaine through skin in vitro was studied using side-by-side compartment diffusion cells method. After achieving steady state by passive diffusion, fluxes of tetracaine achieved with passive diffusion, electroporative pulse and iontophoresis were compared. Electroporation (square-wave pulse, voltage 130 V, pulse time 0.4 s, pulse frequency 40 pulses min(-1)) or iontophoresis (0.2.mA cm(-2), lasting for 4 h) increased the transport of tetracaine through skin. The flux of tetracaine at 0.25 h after electroporation (pulse number 400) was 54.6+/-6.0 microg.cm(-2).h(-1), that after iontophoresis was 17.4+/-5.8 microg.cm(-2).h(-1) and that after passive diffusion was 8.2+/-0.5 microg.cm(-2).h(-1). In addition, the fluxes of tetracaine increased with the increasing of pulse number. From these results, it is clear that electroporation is effective in enhancing transdermal delivery of tetracaine and its function is better than iontophoresis.     

Inhibitory effect of Ca^2＋ on in vivo gene transfer by electroporation

AIM: To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice. METHODS: Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid, with or without Ca2+ and various other ions. RESULTS: For in vivo electroporation, the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression, to less than 5% of control values. Only Ca2+, not other ions, caused inhibition, and the effect was not tissue specific. More surprisingly, even when Ca2+ ions were delivered by electroporation before or after DNA administration, similar effects were still observed. CONCLUSION: The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific, ie, the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.     

In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

After a decade of clinical trials, gene therapy seems to have found its place between excessive ambitions and feasible aims, with encouraging results obtained in recent years. Intracellular delivery of genetic material is the key step in gene therapy. Optimization of delivery vectors is of major importance for turning gene therapy into a successful therapeutic method. Nonviral gene delivery relies mainly on the complexes formed from cationic liposomes (or cationic polymers) and DNA, i.e., lipoplexes (or polyplexes). Many lipoplex formulations have been studied, but in vivo activity is generally low compared to that of viral systems. This review gives a concise overview of studies on the application of cationic liposomes in vivo in animal models of diseases and in clinical studies. The transfection efficiency, the pharmacokinetic and pharmacodynamic properties of the lipid-DNA complexes, and potentially relevant applications for cationic liposomes are discussed. Furthermore, the toxicity of, and the induction of an inflammatory response in association with the administration of lipoplexes are described. Increasing understanding of lipoplex behavior and gene transfer capacities in vivo offers new possibilities to enhance their efficiency and paves the path to more extensive clinical applications in the future.     

Clinical potential of electroporation for gene therapy and DNA vaccine delivery

ABSTRACT

Introduction: Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.

Areas covered: This review explores the electroporation mechanism and the parameters affecting its efficacy. An analysis of past and current clinical trials focused on DNA electroporation is presented. The pathologies addressed, the protocol used, the treatment outcome and the tolerability are highlighted. In addition, several of the possible optimization strategies for improving patient compliance and therapeutic efficacy are discussed such as plasmid design, use of genetic adjuvants for DNA vaccines, choice of appropriate delivery site and electrodes as well as pulse parameters.

Expert opinion: The growing number of clinical trials and the results already available underline the strong potential of DNA electroporation which combines both safety and efficiency. Nevertheless, it remains critical to further increase fundamental knowledge to refine future strategies, to develop concerted and common DNA electroporation protocols and to continue exploring new electroporation-based therapeutic options.     

Mechanisms of a phosphorothioate oligonucleotide delivery by skin electroporation

Skin electroporation has great potential for topical delivery of oligonucleotides. Controled therapeutic levels of an intact phosphorothioate oligonucleotide (PS) can be reached in the viable tissue of the skin. The aim of this work was to investigate the transport mechanisms of a PS in hairless rat skin by electroporation, and hence to allow optimization of oligonucleotides (ONs) topical delivery. The pulsing condition used was five exponentially-decaying pulses of 100 V and 500 ms pulse time. The main mechanism of PS transport in the skin viable tissues during pulsing was electrophoresis. The electroosmosis contribution was negligible. Electrophoresis created within minutes a reservoir of PS in the skin viable tissues, which persisted within a therapeutic range of hours. A strong PS/stratum corneum interaction occurred.     

Transdermal delivery of nalbuphine and its prodrugs by electroporation.

The aim of this study was to assess the effects of electroporation on transdermal permeation of nalbuphine (NA) and its prodrugs. The permeation characteristics were investigated under various electrical factors and skin barriers to elucidate the mechanisms involved in transdermal delivery of NA and its prodrugs by skin electroporation. The in vitro permeation studies were performed using side-by-side diffusion cells. The various electrical factors investigated were pulse voltage, pulse duration and pulse number; the different skin barriers studied were intact hairless mouse skin, stratum corneum (SC)-stripped skin, delipid skin as well as furry Wistar rat skin. The prodrugs were fully converted to parent drug after skin permeation. Application of electroporation significantly enhanced transdermal permeation of NA and its prodrugs. The enhancement ratios were highest for NA and the four prodrugs showed the similar permeability after electroporation. The permeation amounts of NA and its prodrugs may be increased by application of higher pulse voltage, pulse duration as well as pulse number. Various kinetics and mechanisms were observed for the permeation of the hydrophilic NA and lipophilic nalbuphine enanthate through different skin barriers by applying electroporation. This study demonstrated that electroporation may enhance and control transdermal permeation of NA and its prodrugs. The results also indicated that the physicochemical properties of prodrug had significant effects on kinetics as well as mechanisms of transdermal permeation by electroporation.     

A practical assessment of transdermal drug delivery by skin electroporation

Transdermal drug delivery has many potential advantages, but the skin's poorly-permeable stratum corneum blocks delivery of most drugs at therapeutic levels. Short high-voltage pulses have been used to electroporate the skin's lipid bilayer barriers and thereby deliver compounds at rates increased by as much as four orders of magnitude. Evidence that the observed flux enhancement is due to physical alteration of the skin by electroporation, as opposed to only providing an iontophoretic driving force, is supported by a number of different transport, electrical and microscopy studies. Practical applications of electroporation's unique effects on skin are motivated by large flux increases for many different compounds, rapidly responsive delivery profiles, and efficient use of skin area and electrical charge. Greater enhancement can be achieved by combining skin electroporation with iontophoresis, ultrasound, and macromolecules. Sensation due to electroporation can be avoided by using appropriate electrical protocols and electrode design. To develop skin electroporation as a successful transdermal drug delivery technology, the strong set of existing in vitro mechanistic studies must be supplemented with studies addressing in vivo/clinical issues and device design.     

Lipid and electroosmosis enhanced transdermal delivery of insulin by electroporation

Transdermal transport of insulin and extraction of interstitial glucose under anodal iontophoresis (electroosmosis) following electroporation in the presence of 1,2-dimyristoylphophatidylserine (DMPS) was studied. An earlier study showed that DMPS increased the transport of insulin across porcine epidermis under electroporation by approximately fourfold. It was suggested that DMPS increased the lifetime of electropores in the epidermis resulting in an enhanced transport of permeants. When electroosmosis was applied across the epidermis following electroporation with DMPS, the enhancement of insulin transport was approximately 18-fold over electroporation alone. When the same strategy was applied to extract interstitial glucose, the enhancement was approximately 23-fold over electroporation alone. Real-time transdermal insulin transport kinetics was measured using FITC-labeled insulin and a custom-made vertical diffusion apparatus that had a fluorescence cuvette as the receiver compartment. Insulin transport by electroporation alone showed a nonlinear kinetics that is most likely due to the resealing of the electropores with time. The transport kinetics when electroporation was carried out in the presence of DMPS was more linear, confirming earlier studies that suggested the DMPS stabilizes transport paths formed by electroporation. The data suggests that in vivo, noninvasive insulin delivery to therapeutic levels and glucose extraction may be achieved by combining electroporation with anionic lipids and electroosmosis.     

In vivo nose-to-brain delivery of the hydrophilic antiviral ribavirin by microparticle agglomerates

Nasal administration has been proposed as a potential approach for the delivery of drugs to the central nervous system. Ribavirin (RBV), an antiviral drug potentially useful to treat viral infections both in humans and animals, has been previously demonstrated to attain several brain compartments after nasal administration. Here, a powder formulation in the form of agglomerates comprising micronized RBV and spray-dried microparticles containing excipients with potential absorption enhancing properties, i.e. mannitol, chitosan, and α-cyclodextrin, was developed for nasal insufflation. The agglomerates were characterized for particle size, agglomeration yield, and ex vivo RBV permeation across rabbit nasal mucosa as well as delivery from an animal dry powder insufflator device. Interestingly, permeation enhancers such as chitosan and mannitol showed a lower amount of RBV permeating across the excised nasal tissue, whereas α-cyclodextrin proved to outperform the other formulations and to match the highly soluble micronized RBV powder taken as a reference. In vivo nasal administration to rats of the agglomerates containing α-cyclodextrin showed an overall higher accumulation of RBV in all the brain compartments analyzed as compared with the micronized RBV administered as such without excipient microparticles. Hence, powder agglomerates are a valuable approach to obtain a nasal formulation potentially attaining nose-to-brain delivery of drugs with minimal processing of the APIs and improvement of the technological and biopharmaceutical properties of micronized API and excipients, as they combine optimal flow properties for handling and dosing, suitable particle size for nasal deposition, high surface area for drug dissolution, and penetration enhancing properties from excipients such as cyclodextrins.     

Skin electroporation for transdermal and topical delivery

Electroporation is the transitory structural perturbation of lipid bilayer membranes due to the application of high voltage pulses. Its application to the skin has been shown to increase transdermal drug delivery by several orders of magnitude. Moreover, electroporation, used alone or in combination with other enhancement methods, expands the range of drugs (small to macromolecules, lipophilic or hydrophilic, charged or neutral molecules) which can be delivered transdermally. Molecular transport through transiently permeabilized skin by electroporation results mainly from enhanced diffusion and electrophoresis. The efficacy of transport depends on the electrical parameters and the physicochemical properties of drugs. The in vivo application of high voltage pulses is well tolerated but muscle contractions are usually induced. The electrode and patch design is an important issue to reduce the discomfort of the electrical treatment in humans.     

Novel polyethylenimine-derived nanoparticles for in vivo gene delivery

Introduction: Branched and linear polyethylenimines (PEIs) are cationic polymers that have been used to deliver nucleic acids both in vitro and in vivo. Owing to the high cationic charge, the branched polymers exhibit high transfection efficiency, and particularly PEI of molecular weight 25 kDa is considered as a gold standard in gene delivery. These polymers have been extensively studied and modified with different ligands so as to achieve the targeted delivery.

Areas covered: The application of PEI in vivo promises to take the polymer-based vector to the next level wherein it can undergo clinical trials and subsequently could be used for delivery of therapeutics in humans. This review focuses on the various recent developments that have been made in the field of PEI-based delivery vectors for delivery of therapeutics in vivo.

Expert opinion: The efficacy of PEI-based delivery vectors in vivo is significantly high and animal studies demonstrate that such systems have a potential in humans. However, we feel that though PEI is a promising vector, further studies involving PEI in animal models are needed so as to get a detailed toxicity profile of these vectors. Also, it is imperative that the vector reaches the specific organ causing little or no undesirable effects to other organs.     

In vivo iontophoretic delivery and pharmacokinetics of salmon calcitonin

In vivo iontophoretic delivery of salmon calcitonin (SCT) in hairless rats using a self-contained wearable and disposable iontophoretic patch was investigated. Iontophoretic patches with built-in proprietary Zn/AgCl electrodes were used. SCT was formulated in citrate buffer (50mM, pH 4.0) to impart a positive charge for anodal iontophoresis. SCT was delivered intravenously to determine primary pharmacokinetic parameters. Pharmacokinetics of iontophoretic delivery of SCT was compared with subcutaneous route of administration. Blood samples were collected through tail vein and analyzed for serum SCT and calcium levels. Pharmacokinetic parameters were calculated by non-compartmental analysis. An average current of 0.43+/-0.01 mA was maintained during patch application. Iontophoretic patches delivered SCT at an average infusion rate of 177.9+/-58.7 ng/(min kg) and an average steady state concentration of 7.58+/-1.35 ng/ml was achieved. There was no difference between the calcium lowering effect of iontophoretic patch and subcutaneous injection (p>0.05). Clearance and half-life of SCT after IV administration were found to be 16.8+/-0.9 ml/(min kg) and 33.5+/-3.3 min, respectively. The iontophoretic delivery of SCT was well defined by a one-compartment model with zero-order infusion. Iontophoretic patch delivered therapeutically relevant concentrations of SCT in hairless rats and delivery was comparable to conventional routes.     

Gene expression and antitumor effect following imelectroporation delivery of human interferon α2 gene

AIM: To investigate the gene expression and antitumor effect following im electroporation delivery of human interferon alpha 2 (hIFN-alpha 2) gene. METHODS: The pcD2/hIFN-alpha 2 was injected into the middle of the quadriceps muscle of female BALB/c mice or the leukemia-bearing female BALB/c nude mice, and then electroporation was given to the injection site. Optimal electrical parameters and the efficiency of gene transfer was studied with hIFN-alpha 2 ELISA kit. The HL-60 tumor model in BALB/c nude mice was used to investigate therapeutic effects of im electroporation delivery of pcD2/hIFN-alpha 2. RESULTS: The optimal conditions for the electric pulses were as follows: voltage at 200 V/cm; pulse duration at 40 ms per pulse; number of pulse at 6 pulses and frequency at 1 Hz. Under optimal conditions, the serum hIFN-alpha 2 levels in electroporation group (160 microg/L+/-31 microg/L) were 45-fold higher than those of nonelectroporation group (3.6 microg/L+/-1.6 microg/L, P<0.01). The growth of leukemia was inhibited more obviously and the survival time of the leukemia-bearing nude mice was prolonged after im electroporation delivery of pcD2/hIFN-alpha 2 100 microg or 200 microg. CONCLUSION: Electroporation was an efficient method for the delivery of plasmid DNA and im electroporation delivery of pcD2/hIFN-alpha 2 was effective in treating leukemia.     

Bleomycin--electrical pulse delivery: electroporation therapy-bleomycin--Genetronics; MedPulser-bleomycin--Genetronics

Genetronics Biomedical is using its electroporation therapy technology to deliver bleomycin to tumour cells for the treatment of cancer. Genetronics have developed the MedPulser Electroporation Therapy System, which consists of an electrical pulse generator and disposable electrode applicators. The MedPulser system enables the delivery of large molecules into cells by briefly applying an electric field to the cell. This causes a transient permeability in the cell's outer membrane characterised by the appearance of pores across the membrane. After the field is discontinued, the pores close, trapping the therapeutic molecules inside the target cells. Genetronics is using the MedPulser System in conjunction with bleomycin, an antineoplastic antibiotic that binds to DNA causing strand scissions. Genetronics is seeking a licensing partner for the use of electroporation for the delivery of drugs in chemotherapy. In 1998, Genetronics entered a licensing and development agreement with Ethicon for electroporation and electrofusion. Under the terms of this agreement, Ethicon was to develop and clinically test the Genetronics electroporation delivery system and conduct all regulatory activities throughout the world except Canada. Ethicon would also market the products once regulatory approval has been obtained and Genetronics was to receive a percentage of the net sales and as license fees. However, in July 2000, Ethicon exercised its rights to terminate the agreement without cause. All rights were returned to Genetronics in January 2001. In 1997, Genetronics entered an agreement with Abbott Laboratories for the manufacture of bleomycin for use in the US in its MedPulsar system after regulatory approval had been granted for its use in the treatment of solid tumours. In a separate supply agreement, Faulding Inc. has agreed to manufacture bleomycin for Genetronic for use in Canada after regulatory approval had been granted. The MedPulsar Electroporation Therapy System with bleomycin is currently in phase III pivotal studies in the US as a treatment for recurrent and second primary squamous cell carcinomas of the head and neck. Genetronics received approval for the Electroporation Therapy system as a device in March 1999 when it achieved CE Mark certification. In February 2004, Genetronics announced that it had completed a Special Protocol Assessment review process with the US FDA for two new trials that will compare bleomycin electroporation therapy to surgery. The primary endpoint will be tissue and function preservation rather than survival. One proposal is for recurrent head and neck cancer, and the other is for disfiguring cutaneous cancer. Three Institutional Review Boards in the US have approved the two protocols and Genetronics has initiated enrollment. In June 2004, Genetronics was granted fast-track status for its MedPulsar Electroporation Therapy System clinical development programme for patients with head and neck cancer. Shifting from a primary endpoint of survival to a quality-of-life outcome will enable those clinical trials to be carried out faster with less cost and with a higher likelihood of success. As a result, Genetronic's phase III trials focussing on survival as a primary endpoint have been discontinued. This includes a phase III trial for late-stage, recurrent head and neck cancer in combination with the normal standard of treatment compared with normal standard of treatment alone. Interim results from this trial had suggested bleomycin electroporation therapy demonstrated local tumour control and preservation of organ function, as well as non-inferiority when compared with surgery. This trial was initiated in May 2002. In March 2004, Genetronics initiated a post-European regulatory approval clinical study in patients with primary or recurrent squamous cell carcinoma of the head and neck (SCCHN). This study aims to enroll approximately 100 patients at 12-15 hospitals located in the UK, Germany, Italy, France, Austria and other western European countries. The study is designed to support the commercialisation of the MedPulser Electroporation System in the EU. Prior clinical trials established the safety and performance of the MedPulser System for the treatment of SCCHN, leading to approval for sale in the EU based on achieving the CE Mark. This study will document the clinical and pharmacoeconomic benefit in support of reimbursement approval throughout Western Europe, establish centres of excellence to facilitate early sales, create a reference and customer base for a projected European commercial launch in 2005, and generate safety and efficacy data to support marketing applications in the US. The bleomycin delivery system has completed phase IIB trials in the US, Canada and Europe in patients with squamous cell carcinoma of the head and neck who have failed conventional therapies. Phase II data were submitted to the FDA in the first quarter of 2002 and a phase III trial was launched in May 2002. The therapy is also being used in France in patients with cancers of the head and neck, liver (metastatic) and melanoma. A review of the data from these phase II trials was completed in April 2001. In June 2004, Genetronics was granted two US patents. US patent 6,748,265 covers its trans-surface drug and gene delivery technology and provides additional proprietary rights for an apparatus and method to deliver genes, drugs and other molecules through tissue surfaces. The second US patent, 6,746,441, pertains to the field of ex vivo therapies and covers the introduction of molecules into cells by electroporation, either in a continuous-flow or batch mode, with a variable electric field orientation. In July 2004, Genetronics received a US patent (no. 6,763,264) covering methods for the in vivo delivery of a recombinant expression vector (DNA) or a pharmaceutical agent into tissue cells, and a method for the therapeutic application of electroporation to a patient to introduce macromolecules.