Ultrasound-assisted nonviral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion.

Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial using Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system used in this pioneering trial preclude its advancement to a Phase II trial, and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted nonviral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Swine irradiated unilaterally with a 10-Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 h and 1 week post treatment (1.62±0.48 ml and 1.87±0.45 ml) to preinjury levels (1.34±0.14 ml) in a manner comparable to Adenoviral delivery (2.32±0.6 ml and 1.33±0.97 ml). UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model, and it is a candidate for advancement to a Phase I human clinical trial.

An AAV2/5 vector enhances safety of gene transfer to the mouse salivary gland.

This study was designed to improve AAV-mediated gene transfer to the murine submandibular salivary glands. Our first aim was to utilize AAV pseudotype vectors, containing the genetic elements of the canonical AAV2, packaged within capsids of AAV serotypes 5, 8, and 9. Having determined that this pseudotyping increased the efficiency of gene transfer to the glands by several orders of magnitude, we next asked whether we could reduce the gene transfer inoculum of the pseudotype while still achieving gene transfer comparable with that achieved with high-dose AAV2. Having achieved gene transfer comparable with that of AAV2 using a pseudotype vector (AAV2/5) at a 100-fold lower dose, our final objective was to evaluate the implications of this lower dose on two pre-clinical parameters of vector safety. To evaluate systemic toxicity, we measured AAV vector sequestration in the liver using qPCR, and found that the 100-fold lower dose reduced the vector recovered from the liver by 300-fold. To evaluate salivary gland function, we undertook whole-proteome profiling of salivary gland lysates two weeks after vector administration and found that high-dose (5 × 109) AAV altered the expression level of ~32% of the entire salivary gland proteome, and that the lower dose (5 × 107) reduced this effect to ~7%.

Ultrasound-assisted non-viral gene transfer to the salivary glands.

We report a non-viral gene transfer method using ultrasound induced microbubble destruction to allow the uptake of plasmid gene transfer vectors to the cells of the mouse salivary gland. The Luciferase (Luc) reporter gene, driven by a cytomegalovirus (CMV) promoter, was delivered unilaterally to the submandibular salivary gland via retroductal cannulation and Luc expression was monitored with in vivo imaging. The CMV-Luc plasmid was delivered to the salivary gland in a carrier solution containing microbubbles composed of lipid-encased perfluoropropane gas, with two different concentrations of microbubbles used (100 and 15% volume/volume). An Adenoviral (Ad) vector using an identical CMV-Luc expression cassette was used as a positive control at two different dosages. Whereas ultrasound-assisted gene transfer (UAGT) with 100% microbubbles was weak and rapidly extinguished, UAGT with the 15% microbubble solution was robust and stable for 28 days. UAGT seems to be a practicable and promising method for non-viral gene delivery to the salivary glands.

Pharmacokinetics of ethylene glycol. III. Plasma disposition and metabolic fate after single increasing intravenous, peroral, or percutaneous doses in the male Sprague-Dawley rat.

1. The pharmacokinetic fate of [1,2-14C]-ethylene glycol (EG) was evaluated in the male Sprague-Dawley rat in order to characterize its overall uptake and elimination. Doses of 10 and 1000 mg/kg were administered by the intravenous (i.v.), peroral (p.o.), or percutaneous (p.c.) route; additional doses of 400, 600 and 800 mg/kg were evaluated by the p.o route. 2. Baseline data obtained by the i.v. route for bioavailability comparisons showed that while plasma radioactivity concentrations declined in a biexponential manner with t1/2 beta of 26-37 h, the disappearance of unmetabolized EG from the plasma was quite rapid (t1/2 beta of 0.8-1.2 h). Peroral doses were rapidly and almost completely absorbed, showing t1/2 abs in the order of minutes, and a bioavailable fraction for unmetabolized EG of 92-100%. Conversely, EG applied to rat skin was slowly and rather poorly absorbed, showing t1/2 abs which were an order of magnitude longer than for comparable p.o. and i.v. doses, and a bioavailability of approximately 22%. 3. The major route of elimination for the 10 mg/kg dose by any route was by metabolism to 14CO2 and exhalation, while urinary elimination of 14C was the secondary excretion pathway. 4. Plasma clearance of 14C was linear with increases in p.o. doses over the 400-800 mg/kg range, with AUC proportional to dose for these and the 10 mg/kg p.o. dose levels. However, a dose-dependent shift in excretion routes was observed following the p.o. 1000 mg/kg dose, with urine becoming the major excretion route, and similar capacity limited pharmacokinetics were observed for the i.v. 1000 mg/kg dose. Plasma pharmacokinetic data for unchanged EG after i.v. and p.o. doses demonstrated an apparent first-order kinetic behaviour between the 10 and 1000 mg/kg dose levels for the disappearance of EG. 5. Following both i.v. and p.o doses, dose-independent relationships were seen in the values obtained for the area under the plasma curve (AUC infinity), the total clearance of EG (CltotalEG), mean residence time (MRT infinity), apparent volume of distribution at steady state (Vdss), the terminal half-life (t1/2 beta) and the renal and metabolic clearance values. However, this dose-linear plasma time course was not apparent from the dose-dependent excretion profiles for these two exposure routes. 6. Increases in urinary 14C-glycolate were also observed when the i.v. or p.o. doses were increased from 10 to 1000 mg EG/kg, indicating that metabolism of EG makes a substantial contribution to AUC infinity in the beta disposition phase of the plasma curves for this high dose. Oxalate, a metabolite found in man after EG exposure, was detected at very low levels after both the 10 and 1000 mg/kg dose levels and by either i.v or p.o. routes. 7. Thus, EG given by three different routes demonstrated apparent first-order pharmacokinetic behaviour for disposition in and the elimination from plasma in the male rat, but dose-dependent changes occurred for the elimination of metabolites in urine and as 14CO2 after single i.v. and p.o. doses, but not for the p.c. routes.