Assessment of AAV9 distribution and transduction in rats after administration through Intrastriatal, Intracisterna magna and Lumbar Intrathecal routes.

Challenges in obtaining efficient transduction of brain and spinal cord following systemic AAV delivery have led to alternative administration routes being used in clinical trials that directly infuse the virus into the CNS. However, data comparing different direct AAV injections into the brain remain limited making it difficult to choose optimal routes. Here we tested both AAV9-egfp and AAV9-fLuc delivery via intrastriatal (IST), intracisterna magna (ICM) and lumbar intrathecal (LIT) routes in adult rats and assessed vector distribution and transduction in brain, spinal cord and peripheral tissues. We find that IST infusion leads to robust transgene expression in the striatum, thalamus and cortex with lower peripheral tissue transduction and anti-AAV9 capsid titers compared to ICM or LIT. ICM delivery provided strong GFP and luciferase expression across more brain regions than the other routes and similar expression in the spinal cord to LIT injections, which itself largely failed to transduce the rat brain. Our data highlight the strengths and weaknesses of each direct CNS delivery route which will help with future clinical targeting.

Physiologically based pharmacokinetic (PBPK) modeling of everolimus (RAD001) in rats involving non-linear tissue uptake.

Everolimus is a novel macrolide immunosuppressant developed for the prophylaxis of allogeneic renal or cardiac transplant rejection. Treatments with immunosuppressants are often associated with organ toxicity that is linked to high organ exposure. Therefore, gaining insight into the pharmacokinetics of everolimus in various organs is highly desirable especially those organs of therapeutic interest or those that pose safety concerns. The aim of this work was to characterize the disposition kinetics of everolimus in rats by physiologically based pharmacokinetic (PBPK) modeling. Blood and tissue samples were collected from male Wistar rats over 24 hr following intravenous (iv) bolus and iv infusion of 1 mg/kg and 10 mg/kg/2 hr of everolimus. Further blood samples were collected between 1 and 170 hr from a third group of rats, which received iv infusion of 1 mg/kg/2 hr of everolimus. Drug concentrations in blood and tissues were determined by a liquid chromatography reverse dilution method. Distribution of everolimus between blood fractions was determined in vitro at 37 degrees C. The results of the study demonstrated that everolimus exhibited moderate non-linear binding to red blood cells. Also, the tissue-to-blood concentration ratio decreased in all tissues as blood concentration increased. A PBPK model involving non-linear tissue binding was able to successfully describe the observed data in blood and all the organs investigated. The highest binding potential was observed in thymus, lungs, and spleen with the greatest tissue affinity observed in thymus, skin, and muscle as compared to other tissues. Everolimus exhibited a high clearance rate that was limited to the hepatic blood flow (47.2 ml/min/kg). The PBPK model was also able to predict the venous blood concentration reasonably well following oral administration. The oral bioavailability value, as estimated with the PBPK, was 12% and was similar to the value obtained by non-compartmental analysis. In conclusion, A PBPK model has been developed that successfully predicts the time course of everolimus in blood and a variety of organs. This model takes into account the non- linear binding of everolimus to red blood cells and tissues. This model may be used to predict everolimus concentration-time course in organs from other species including humans.

Physiologically based pharmacokinetic modeling of FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) in rats after oral and intravenous doses.

FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) is a new sphingosine-1-phosphate receptor agonist being developed for multiple sclerosis and prevention of solid organ transplant rejection. A physiologically based pharmacokinetic model was developed to predict the concentration of FTY720 in various organs of the body. Single oral and intravenous doses of FTY720 were administered to male Wistar rats, with blood and tissue sampling over 360 h analyzed by liquid chromatography/tandem mass spectrometry. A well stirred model (perfusion rate-limited) described FTY720 kinetics in heart, lungs, spleen, muscle, kidneys, bone, and liver, with a permeability rate-limited model being required for brain, thymus, and lymph nodes. Tissue-to-blood partition coefficients (RT) ranged from 4.69 (muscle) to 41.4 (lungs). In lymph nodes and spleen, major sites for FTY720-induced changes in sequestration of lymphocytes, RT values were 22.9 and 34.7, respectively. Permeability-surface area products for brain, thymus, and lymph nodes were 39.3, 122, and 176 ml/min. Intrinsic hepatic clearance was 23,145 l/h/kg for the free drug in blood (f(ub) 0.000333); systemic clearance was 0.748 l/h/kg and terminal half-life was 23.4 h. The fraction orally absorbed was 71%. The model characterized well FTY720 disposition for this extensive dosing and tissue collection study in the rat. On scaling the model to dogs and humans, good agreement was found between the actual and predicted blood concentration-time profiles. More importantly, brain concentrations in dogs were well predicted from those of the rat. In absolute terms, the predictions were slightly lower than observed values, just under a 1.5-fold deviation, but the model accurately predicted the terminal elimination of FTY720 from the brain.

On the prediction of the human response: a recycled mechanistic pharmacokinetic/pharmacodynamic approach.

Although it is routine to predict the blood or plasma pharmacokinetics of compounds for man based upon preclinical studies, the real value of such predictions only comes when linked to drug effects. In the first example, the immunomodulator, FTY720, the first sphingosine-1-phosphate receptor agonist, stimulates the sequestration of lymphocytes into lymph nodes thus removing cells from blood circulation. A prior physiology-based pharmacokinetic model fitted the concentration-time course of FTY720 in rats. This was connected to an indirect response model of the lymphocyte system to characterise the cell trafficking effects. The IC(50) of FTY720 was different in the rat compared with the monkey; man was assumed to be similar to the monkey. The systemic lymphocyte half-lives were also different between species. To make predictions of the pharmacodynamic behaviour for man, two elements are required, i) systemic exposure, in this case from an upscaled physiology based model, and ii) an estimate of lymphocyte turnover in man, gained from the literature from other drug treatments. Predictions compared well with clinical results. The second example is the monoclonal antibody Xolair, designed to bind immunoglobulin E for atopic diseases. A mechanism based two-site binding model described the kinetics of both Xolair and endogenous IgE. This model has been reused for other monoclonal antibodies designed to bind fluid-phase ligands. Sensitivity analysis shows that if differences across species in the kinetics of the endogenous system are not accounted for, then pharmacokinetic/pharmacodynamic models may give misleading predictions of the time course and extent of the response.

Pharmacokinetics and cell trafficking dynamics of 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720) in cynomolgus monkeys after single oral and intravenous doses.

The pharmacokinetics and cell trafficking dynamics of 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720), a novel immunosuppressive agent, were examined in cynomolgus monkeys (three males and three females). After single doses of 0.1 mg/kg p.o. or i.v. bolus and 1 mg/kg p.o. were administered to the animals, the concentrations of FTY720, and the numbers of lymphocytes, CD20+CD2-B cells, and CD2+CD20-T cells in blood were measured over 23 days. A linear three-compartment model characterized the time course of FTY720 concentrations with a terminal half-life of about 31 h, clearance of about 0.53 l/h/kg, and bioavailability of about 38%. The dynamic responses were not area under the curve (or dose) proportional for either males or females. An indirect response model with a distribution pool captured the cell trafficking data for all doses for each cell type, where initial blood counts (R(0)) were about 7650, 2100, and 5250 cells/microl; maximum fractional inhibition (I(max)) about 0.88, 0.85, and 0.91; influx (k(in)) about 6014, 1312, and 5662 cells/microl/h; efflux (k(out)) about 0.798, 0.555, and 1.08 h(-1); intercompartmental k(cp) about 0.134, 0.192, and 0.082 h(-1); and intercompartmental k(pc) rate constants about 3.9 x 10(-4), and 0.016 and 8.9 x 10(-6) h(-1) for lymphocytes, B cells, and T cells, respectively. The inhibition concentration IC(50) was about 0.48 microg/l for all cells, which was remarkably low. The apparent distribution volumes of peripheral pool (V(p)) were markedly larger than blood volume (V(b)) for all cells. The I(max) for cell trafficking was achieved at doses smaller than that producing graft protection, indicating stronger central than peripheral effects of this drug. The profound cell trafficking effects of FTY720 can be readily captured and interpreted with an extended indirect response model.