Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies.

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (D,L-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5'-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

Antitumor activity of erlotinib (OSI-774, Tarceva) alone or in combination in human non-small cell lung cancer tumor xenograft models.

Our objective was the preclinical assessment of the pharmacokinetics, monotherapy and combined antitumor activity of the epidermal growth factor receptor (HER1/EGFR) tyrosine kinase inhibitor erlotinib in athymic nude mice bearing non-small cell lung cancer (NSCLC) xenograft models. Immunohistochemistry determined the HER1/EGFR status of the NSCLC tumor models. Pharmacokinetic studies assessed plasma drug concentrations of erlotinib in tumor- and non-tumor-bearing athymic nude mice. These were followed by maximum tolerated dose (MTD) studies for erlotinib and each chemotherapy. Erlotinib was then assessed alone and in combination with these chemotherapies in the NSCLC xenograft models. Complete necropsies were performed on most of the animals in each study to further assess antitumor or toxic effects. Erlotinib monotherapy dose-dependently inhibited tumor growth in the H460a tumor model, correlating with circulating levels of drug. There was antitumor activity at the MTD with each agent tested in both the H460a and A549 tumor models (erlotinib 100 mg/kg: 71 and 93% tumor growth inhibition; gemcitabine 120 mg/kg: 93 and 75% tumor growth inhibition; cisplatin 6 mg/kg: 81 and 88% tumor growth inhibition). When each compound was given at a fraction of the MTD, tumor growth inhibition was suboptimal. Combinations of gemcitabine or cisplatin with erlotinib were assessed at 25% of the MTD to determine efficacy. In both NSCLC models, doses of gemcitabine (30 mg/kg) or cisplatin (1.5 mg/kg) with erlotinib (25 mg/kg) at 25% of the MTD were well tolerated. For the slow growing A549 tumor, there was significant tumor growth inhibition in the gemcitabine/erlotinib and cisplatin/erlotinib combinations (above 100 and 98%, respectively), with partial regressions. For the faster growing H460a tumor, there was significant but less remarkable tumor growth inhibition in these same combinations (86 and 53% respectively). These results show that in NSCLC xenograft tumors with similar levels of EGFR expression, the antitumor activity of erlotinib is robust both as monotherapy and in combination with chemotherapies.

Evaluation of rat intestinal absorption data and correlation with human intestinal absorption.

The absorption of 111 drug and drug-like compounds was evaluated from 111 references based on the ratio of urinary excretion of drugs following oral and intravenous administration to intact rats and biliary excretion of bile duct-cannulated rats. Ninety-eight drug compounds for which both human and rat absorption data were available were selected for correlation analysis between the human and rat absorption. The result shows that the extent of absorption in these two species is similar. For 94% of the drugs the absorption difference between humans and rats is less than 20% and for 98% of drugs the difference is less than 30%. There is only one drug for which human absorption is significantly different from rat absorption. The standard deviation is 11% between human and rat absorption. The linear relationship between human and rat absorption forced through the origin, as determined by least squares regression, is %Absorption (human)=0.997%Absorption (rat) (n=98, SD=11). It is suggested that the absorption in rats could be used as an alternative method to human absorption in pre-clinical oral absorption studies.

Rate-limited steps of human oral absorption and QSAR studies.

PURPOSE: To classify the dissolution and diffusion rate-limited drugs and establish quantitative relationships between absorption and molecular descriptors. METHODS: Absorption consists of kinetic transit processes in which dissolution, diffusion, or perfusion processes can become the rate-limited step. The absorption data of 238 drugs have been classified into either dissolution or diffusion rate-limited based on an equilibrium method developed from solubility, dose, and percentage of absorption. A nonlinear absorption model derived from first-order kinetics has been developed to identify the relationship between percentage of drug absorption and molecular descriptors. RESULTS: Regression analysis was performed between percentage of absorption and molecular descriptors. The descriptors used were ClogP, molecular polar surface area, the number of hydrogen-bonding acceptors and donors, and Abraham descriptors. Good relationships were found between absorption and Abraham descriptors or ClogP. CONCLUSIONS: The absorption models can predict the following three BCS (Biopharmaceutics Classification Scheme) classes of compounds: class I, high solubility and high permeability; class III, high solubility and low permeability; class IV, low solubility and low permeability. The absorption models overpredict the absorption of class II, low solubility and high permeability compounds because dissolution is the rate-limited step of absorption.

On the mechanism of human intestinal absorption.

In order to investigate whether the main step in intestinal absorption in humans is dominated by partition or by diffusion, we have transformed % human intestinal absorption into a first-order rate constant, and have regressed the latter, as logk, against our solvation parameters. The obtained regression coefficients are compared with those for diffusion and partition processes. The coefficients in the logk equation are completely different to those for water/solvent partitions, but are very similar to those for processes (not involving transport through membranes) in which diffusion is the major step. It is suggested that the main step in the absorption process is diffusion through a stagnant mucus layer, together with transfer across the mucusmid R:membrane interface. It is further shown that for strong Bronsted acids and bases, the rate constant for absorption of ionic species is close to that for absorption of the corresponding neutral species, so that to a first approximation the % intestinal absorption can be calculated from properties of the neutral species.