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Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

For cystic fibrosis (CF) gene therapy using an aerosolized adenovirus expressing the CFTR gene, optimization of the inhalation conditions is a prerequisite to obtain sufficient amount of CFTR protein expression in the target areas of the respiratory tract. For such a purpose, in vivo radioisotopic imaging of the radiolabeled virus is a unique strategy for a quantitative assessment of the actual deposition. In the present study, an adenovirus CFTR (AdCFTR) was labeled with 99m Technetium gamma emitting isotope in such conditions that its bioactivity was preserved. The 99mTc-AdCFTR aerosol was characterized using both laser diffraction and cascade impaction for sizing with further determination of nebulized and inhalable fractions. After administration to baboons, scintigraphic quantitation of the regional lung distribution was performed and the actual dose deposited in the target area was estimated and expressed as an equivalent viral titer. Since a virus scintigraphy is not realistic in a hospital setting, we have developed an approach using 99mTc-DTPA (diethylene triamino pentaacetic acid) that could be used to predict the virus deposition. Indeed, similarities observed between 99mTc-DTPA and 99mTc-adenovirus aerosol imaging patterns validates the use of the 99mTc-DTPA scintigraphy that we propose as a pretherapeutic test for each patient prior to gene transfer.

A surgical procedure was evaluated to allow bile collection from the freely moving male Sprague-Dawley rats for the assessment of drug biliary excretion during regulatory safety studies. A catheter was implanted into the bile duct to divert the bile flow via an exteriorized loop. Following recovery from the surgery and verification of normal hepatic function, the exteriorized catheter was sectioned to allow collection of the bile and replacement with a commercial bile salt solution. Approximately 80% of the catheterized animals (10 females and 10 males) had normal serum liver enzyme levels 2 days after surgery. Then, the effect of acute or repeated administrations of the immunosuppressant tacrolimus on the biliary excretion of 14C diazepam was studied to validate the technique. A first group of 12 rats received an intravenous injection of 10 mg/kg 14C-diazepam and the total and sequential amounts of diazepam excreted in the bile were measured over 72 h. Biliary excretion accounted for 80% of diazepam elimination. These rats were then given an oral administration of 3 mg/kg tacrolimus on days 7 and 8 followed by the same intravenous dose of 14C-diazepam. Another group of 10 catheterized rats was given 21 daily oral doses of 3 mg/kg tacrolimus followed by a single intravenous administration of 14C-diazepam. No significant changes in diazepam biliary excretion were observed following either acute or repeated administration of tacrolimus. This study demonstrates the feasibility of drug biliary excretion investigations under Good Laboratory Practices conditions as a complement to regulatory acute or repeated dose safety studies.

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