Intravascular and endobronchial DNA delivery to murine lung tissue using a novel, nonviral vector.

Gene transfer to the lung can be achieved via either the airway or the pulmonary vasculature. We evaluated gene transfer and expression by intravascular and endobronchial routes, using DNA complexed with G9 PAMAM dendrimer or naked plasmid DNA. Intravascular tail vein delivery of dendrimer-complexed pCF1CAT plasmid resulted in high levels of transgene expression in the lung at 12 and 24 hr, followed by a second peak of expression 3 to 5 days after administration. After intravenous administration of the complexes, CAT expression was never observed in organs other than the lung. There were only minimal levels of CAT protein expressed in the lung after intravenous administration of naked plasmid DNA. Repeated intravascular doses of the dendrimer-complexed plasmid, administered four times at 4-day intervals, maintained expression at 15-25% of peak concentrations achieved after the initial dose. Endobronchial delivery of naked pCF1CAT plasmid produced significant amounts of CAT protein in the lung. Comparison of intratracheal and intranasal routes resulted in similar expression levels of CAT in the lung and trachea. However, in juxtaposition to vascular delivery, intranasal delivery of dendrimer-complexed plasmid DNA gave lower levels of CAT expression than that observed with naked plasmid DNA. In situ localization of CAT enzymatic activity suggested that vascular administration seemed to achieve expression in the lung parenchyma, mainly within the alveoli, while endobronchial administration primarily targeted bronchial epithelium. Our results show that intravenously administered G9 dendrimer is an effective vector for pulmonary gene transfer and that transgene expression can be prolonged by repeated administration of dendrimer-complexed DNA.

Effects of intravenous administration of prostacyclin on regional blood circulation in awake rats.

1. The effects of intravenous infusion of prostacyclin (PGI2, 0.1, 0.2, 0.3 and 1.0 microg kg(-1) min(-1) lasting 5 min) on regional blood flow and regional vascular resistance have been studied in awake rats using the radioactive microsphere method. 2. The control values of blood flow to the heart, kidney, small intestine, hind limb muscle, pericranial skin and brain as well as the corresponding vascular resistance were not modified by an i.v. infusion (0.1 ml min(-1)) of Tris-buffer (the vehicle of PGI2). 3. The i.v. infusion of PGI2 produced graded dose-dependent decreases of MAP (r=0.87, P<0.001; ED20=0.73 [0.13-2.55] microg kg(-1) min(-1)) as well as decreases of vascular resistance in the heart (r=0.83, P<0.001; ED30=0.17 [0.09-0.31] microg kg(-1) min(-1)), pericranial skin (r=0.88, P<0.001; ED30=0.28 [0.18-0.43] microg kg(-1) min(-1)) and small intestine (r=0.74, P<0.001; ED30=0.21 [0.11-0.39] microg kg(-1) min(-1)), which led to dose-related increases of blood flow to these territories. 4. On the contrary, PGI2 increased vascular resistance in skeletal muscle (r=0.73, P<0.001; ED30=0.20 [0.10-0.39] microg kg(-1) min(-1)) with corresponding reductions in blood flow. The low doses reduced renal blood flow but there were no significant changes during the high ones. Cerebral vessels did not dilate during any infusion of PGI2 and cerebral blood flow decreased as MAP fell (r=0.56, P<0.01). 5. We conclude that, in awake rats, the coronary vessels are extremely sensitive to the vasodilating effect of PGI2 and that the mesenteric vessels and those of the pericranial skin are very responsive too. Moreover, autoregulation is inefficient to maintain cerebral blood flow during infusion of PGI2.

Serum protein binding of lerisetron, a novel specific 5HT3 antagonist, in patients with cancer.

The aim of this study was, (1) to characterize the serum protein binding of lerisetron, a new 5-hydroxytryptamine (5-HT3) receptor antagonist under investigation as an antiemetic agent, and (2) to measure the percentage of unbound lerisetron in cancer patients. The binding parameters were determined in human serum albumin (HSA), alpha1-acid glycoprotein (AAG) and in pooled serum from six healthy volunteers. Concentrations of lerisetron ranging from 50 ng/ml to 2 microg/ml were used. The serum protein binding of 14C-lerisetron (2 microg/ml) was determined by ultrafiltration in three groups of individuals. Group I comprised healthy subjects (n = 11), group II comprised cancer patients undergoing radiotherapy (n = 9), and group III comprised cancer patients receiving chemotherapy (n = 18). The unbound concentration of lerisetron was measured in all samples by liquid scintillation counting. Concentrations of both AAG and HSA were also measured in all serum samples. The drug was extensively bound in pooled serum, involving a nonsaturated process. In HSA, lerisetron was also highly bound (4.04+/-0.8% unbound) and the protein binding was essentially unchanged within the studied concentration range of lerisetron. The extent of binding to AAG was high but significantly lower than in serum and in HSA and was also independent of lerisetron concentration. The unbound lerisetron was significantly decreased in group II cancer patients when compared with group I subjects (2.38+/-0.64% vs 3.70+/-0.70%; P < 0.001). No significant changes in lerisetron binding were observed in group III patients. HSA was diminished in both groups of patients and AAG was only significantly increased in group II. Unbound lerisetron was correlated with AAG in group II and with HSA in group III.

Effects of indomethacin and diclofenac on cerebral blood flow in hypercapnic conscious rats.

Cerebral blood flow (CBF) was measured in conscious rats treated with indomethacin, diclofenac or the vehicle for indomethacin during hypercapnia of three different degrees. Similar values were found for CBF in rats treated with diclofenac or vehicle and there was a direct correlation between CBF and the arterial blood carbon dioxide values. In contrast, indomethacin completely blocked the increase of CBF during hypercapnia. These results suggest strongly that prostaglandins are not involved in the hypercapnia-induced cerebral vasodilatation.

Distribution of cardiac output during development of two metastasizing murine tumors.

The study of cardiac output (CO) distribution to tumors and metastases is of interest for a better understanding of tumor biology and for pharmacological approaches. A radioactive microsphere method was developed to asses CO distribution in C57BL/6J mice bearing syngeneic 3LL or BALB/c mice with JWS. At the initial stages of cancer growth, the CO relative fractions per g of tissue (%CO/g) to 3LL and JWS were similar to those in surrounding tissues. In both tumors a positive, significant correlation was found between tumor weight and tumor CO fraction, but %CO/g was lower in 3LL 2 and 3 weeks after transplantation, whereas it did not change in JWS. Indeed, much larger necrotic areas developed in 3LL than in JWS. The %CO/g to the lungs increased in both models when metastases were not yet visible; subsequently, the appearance of lung nodules was accompanied by a decrease of %CO/g in JWS and a further increase in 3LL. This corresponded to the much higher ratio of metastatic to intact lung tissue in JWS than in 3LL. In fact, isolated lung metastases had a significantly lower blood supply than the surrounding tissue. This might be due to a different vascularization pattern and/or smaller amounts of vasodilator substances being produced by metastatic nodules; the latter is suggested by lower generation of prostacyclin activity in isolated lung metastases than in intact pulmonary tissue.

Cardiac output redistribution induced by noradrenaline in two murine tumor models.

The response of tumor vessels to vasoactive substances could provide useful information on experimental tumor biology. We have studied the effects of noradrenaline (20 micrograms/kg i.v.) on cardiac output (%CO) distribution in C57BL/6J mice bearing syngeneic Lewis lung carcinoma (3LL) and BALB/c mice with JW sarcoma (JWS). Mice were studied at different stages during tumor growth using microspheres labeled with 57Co (basal determination) or 58Co (after noradrenaline or saline). In control C57BL/6J mice noradrenaline induced a redistribution of CO, with an increase in the heart and brain and a decrease in the kidneys and hind limb muscle CO fractions (%CO). In 3LL-bearing mice the %CO to the tumor was not changed by noradrenaline 1 week after implantation but was significantly less after 2 and 3 weeks. %CO to the total lung tissue or to isolated metastases did not change after noradrenaline. In control BALB/c mice noradrenaline increased the %CO to the brain and decreased that to the kidneys and hind limb muscle. In JWS-bearing mice the %CO to the tumor was reduced 2 weeks after implantation, was not changed after 4 weeks and was increased after 6 weeks. These results suggest that tumor vessel reactivity to a vasoactive substance may change markedly during various phases of tumor growth and may differ in different experimental models.

Altered disposition and effect of lerisetron in rats with elevated alpha 1-acid glycoprotein levels.

PURPOSE: To examine the effect of changes in plasma alpha1-acid glycoprotein (AAG) levels on the pharmacokinetics (PK) and pharmacodynamics (PD) of lerisetron, a novel serotonin 5-HT3 receptor antagonist, in the rat. METHODS: After subcutaneous administration of turpentine oil, AAG was significantly elevated compared with controls. The PK of unchanged lerisetron (UL; high-performance liquid chromatography with radioactivity monitoring) and total lerisetron (TL; unchanged + changed, scintillation counting) was characterized post intravenous (i.v.) 14C lerisetron (50 microg/kg) in control and turpentine oil pretreated rats. The PK (0-180 min) was described by a two-compartmental model. Protein binding of lerisetron in vitro was measured using an ultrafiltration technique. The effect of lerisetron (5 microg/kg, i.v.) over 180 min was measured in anesthetized rats (control and pretreated) with the Bezold-Jarisch reflex (inhibition of bradycardia after 16 microg/kg serotonin i.v.) as the endpoint. PD parameters were estimated by sigmoid Emax models. RESULTS: The unbound fraction was significantly diminished in pretreated rats (mean +/- SEM) (6.60 +/- 1.23% vs. control 14.4 +/- 1.40%, P < 0.05). Volume of distribution (V) and clearance for UL and TL were significantly decreased when compared to the controls (P < 0.0001 for UL and P < 0.05 for TL). Plasma clearance based on unbound concentration for UL did not differ between groups but the unbound V and steady-state unbound V remained decreased (P < 0.05 and P < 0.0001). Pretreated rats showed a significantly diminished drug effect: the area under the E-t curve over 180 min was (mean +/- SEM) 5,189 +/- 657.7 in control animals vs. 3,486 +/- 464.4 in the pretreated group (P < 0.05). The EC50 (concentration at half maximum effect) for UL and TL were increased in pretreated rats and were not compensated when the unbound concentration was used. CONCLUSIONS: An increase in AAG causes alterations in the PK and PD of lerisetron, and because this is not compensated with the unbound concentration, we suggest that mechanisms not linked to protein binding may be involved.

Effects of aspirin and indomethacin on cerebral circulation in the conscious rat: evidence for a physiological role of endogenous prostaglandins.

The purpose of this work was to evaluate the effects of equipotent doses of two different inhibitors of cyclo-oxygenase, indomethacin and aspirin, on cerebral blood flow and cerebral vascular resistances in the conscious undisturbed rat, using the reference sample radioactive microsphere method. We found that both, aspirin (50 mg/kg) and indomethacin (5 mg/kg) at 3, 15 and 60 min after their intravenous administration, increased cerebral vascular resistances and decreased cerebral blood flow to a similar extent. Both drugs completely abolished the hypotensive effect of 5 mg/kg i.v. arachidonic acid and they did not change arterial PO2, PCO2 or pH values. We conclude that the pharmacological inhibition of cyclooxygenase in the conscious undisturbed rat leads to a cerebral vasoconstriction and consequently to a decrease in cerebral blood flow. Our results evidence that prostaglandins are a physiological factor that actively contributes to the maintenance of cerebral circulation.

The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo.

Gene transfer in the lung holds promise for the treatment of diseases such as pulmonary fibrosis, cystic fibrosis and asthma. Pulmonary surfactant has been reported to enhance expression from endobronchial, adenovirus-mediated gene transfer in experimental animals. This study examines the effect of exogenous synthetic surfactant (Exosurf) on gene expression from naked plasmid DNA administered endobronchially to adult mice. Transfection efficiency was evaluated by quantifying the expression of chloramphenicol acetyltransferase (CAT) and luciferase (Luc) genes in the lung. Endobronchial administration of either CAT or Luc expression plasmid DNA resulted in detectable concentrations of each reporter protein. CAT expression from plasmid DNA was monitored after endobronchial administration with the maximal expression observed at 3-5 days after administration and decreasing for 5 days thereafter. When DNA was delivered in a 50% suspension of Exosurf, the expression of either CAT or Luc was significantly reduced by 89.6 +/- 1.4% and 82.7 +/- 10.5%, respectively. The decrease in Luc expression was closely correlated (r = 0.99, P < 0.001) to log concentration of surfactant in the plasmid buffer solution (IC50 = 8.6%). CAT expression was not altered when surfactant was administered either 2 h before or after plasmid DNA instillation. Examination of the components of Exosurf revealed that two compounds, DPPC and tyloxapol, showed inhibitory effects on CAT expression. However, the inhibition caused by Exosurf appeared greater than that of either component. Our results suggest that the lung surfactant is a barrier to transfection of the endobronchial airway and may be partly responsible for the low expression of exogenous DNA in vivo in the bronchial tree.