Ken Ichi Ogawara

PEG liposomalization of paclitaxel improved its in vivo disposition and anti-tumor efficacy.

To find out potent paclitaxel (PTX) formulations for cancer chemotherapy, we formulated PTX in O/W emulsion and liposome selected as candidates of nanocarriers for PTX. Surface modification of these nanoparticles with polyethylene glycol (PEG) improved their in vivo behavior, but the effect of PEGylation on the pharmacokinetics of emulsion was not so remarkable and the release of PTX from emulsion was found to be very fast in blood circulation, indicating that emulsion would not be an adequate formulation for PTX. On the other hand, AUC of PEG liposome was 3.6 times higher than that of naked liposome after intravenous injection into normal rats due to the lower disposition into the reticuloendothelial system tissues such as liver and spleen. Since PEG liposome was able to stably encapsulate PTX in blood, AUC of PTX was also extensively enhanced after intravenous dosing of PTX-PEG liposome into normal rats. In the in vivo studies utilizing Colon-26 solid tumor-bearing mice, it was confirmed that PTX-PEG liposome delivered significantly larger amount of PTX to tumor tissue and provided more excellent anti-tumor effect than PTX-naked liposome. These results suggest that PEG liposome would serve as a potent PTX delivery vehicle for the future cancer chemotherapy.

Molecular Pathways of Endothelial Cell Activation for (Targeted) Pharmacological Intervention of Chronic Inflammatory Diseases

In chronic inflammatory conditions, endothelial cells actively recruit immune cells from the circulation into the underlying tissue and participate in angiogenesis to support the continuous demand for oxygen and nutrients. They do so in response to activation by cytokines and growth factors such as tumour necrosis factor alpha (TNFalpha), interleukin-1 (IL-1), vascular endothelial growth factor (VEGF), and fibroblast growth factors (FGFs). Receptor triggering initiates intracellular signal transduction leading to activation of nuclear factor kappaB (NFkappaB), mitogen activated protein kinase (MAPK) activity, and nitric oxide and reactive oxygen species production, among others. As a result, adhesion molecules, cytokines and chemokines, and a variety of other genes are being expressed that mediate and control the inflammatory process. In recent years, different classes of drugs have been developed that interfere with selected enzymes involved in the intracellular signalling cascades. In endothelial cell cultures, they exert potent inhibitory effects on the expression of genes, while several studies also report on in vivo effectiveness to confine the inflammatory responses. To prevent undesired toxicity and to improve drug behaviour and efficacy, drug carrier systems have been developed that selectively deliver the therapeutics into the activated endothelial cells. The above subjects are recapitulated to give an overview on the status of development of endothelial cell directed therapeutic strategies to pharmacologically interfere with chronic inflammatory diseases.

Prolongation of residence time of liposome by surface-modification with mixture of hydrophilic polymers

The objective of this study is to evaluate the biodistribution characteristics of liposomes surface-modified with the mixture of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as a drug carrier for passive targeting of drugs. The liposomes (egg phosphatidylcholine:cholesterol=55:40, molar ratio) modified with both PEG and PVA (4:1 molar ratio) (PEG4%/PVA1% liposome) provided the largest AUC, which could be attributed to the smallest hepatic clearance of the liposomes. The liver perfusion studies clearly indicated that lower hepatic disposition of PEG4%/PVA1% liposome was ascribed to the decrease in its hepatic uptake via receptor-mediated endocytosis. Furthermore, the amounts of whole serum proteins and of opsonins such as complement C3 and immunoglobulin G adsorbed on PEG4%/PVA1% liposome were significantly smaller than those on the liposome solely modified with PEG (PEG5% liposome). On the other hand, several proteins were adsorbed at larger amount on PEG4%/PVA1% liposome than PEG5% liposome, and the protein identification by LC-MS/MS suggested that some of those proteins including albumin might function as dysopsonins. The decrease in the adsorbed amount of several opsonins and the increase in the adsorbed dysopsonins would be responsible for its lower affinity to the liver and long residence in the systemic circulation of PEG4%/PVA1% liposome.

Determinants for in vivo anti-tumor effects of PEG liposomal doxorubicin: importance of vascular permeability within tumors.

To elucidate the determinants of the in vivo anti-tumor efficacy of polyethylene glycol (PEG)-modified liposomal doxorubicin (DOX), we examined its anti-tumor effect against three different tumor cell lines (Lewis lung cancer (LLC), Colon-26 (C26) and B16BL6 melanoma (B16)) in vitro and in vivo. In vitro, LLC was the most sensitive tumor to DOX and liposomal DOX based on the MTT assay. However, the strongest in vivo anti-tumor effect was observed in the C26 tumor-bearing mice. The in vivo accumulation of radiolabelled PEG liposome in the C26 tumor after intravenous injection was significantly larger than in other tumors. The extent of vascularity assessed by immunohistochemical staining of CD31 was not directly related with the tumor accumulation of PEG liposome. On the other hand, Evans blue extravasation and secretion of VEGF in C26 tumors were higher than in LLC tumors, clearly demonstrating that the vasculature permeability was higher within C26 tumors. These results indicated that the vascular permeability within the tumor substantially affects the tumor accumulation of PEG liposome and may be one of the important determinants in the in vivo anti-tumor efficacy of PEG liposomal DOX.

In vivo anti-tumor effect of PEG liposomal doxorubicin (DOX) in DOX-resistant tumor-bearing mice: Involvement of cytotoxic effect on vascular endothelial cells.

We evaluated the in vivo anti-tumor effect of polyethylene glycol-modified liposomal doxorubicin (PEG liposomal DOX) in the DOX-resistant Colon-26 cancer cells (C26/DOX)-bearing mice model. IC(50) value of DOX to C26/DOX in vitro (40.0 microM) was about 250 times higher than that to control C26 (C26/control) (0.15 microM). However, in vivo anti-tumor effect of PEG liposomal DOX was similar in both C26/control- and C26/DOX-bearing mice, suggesting that the in vivo anti-tumor effect of PEG liposomal DOX was not directly reflecting the sensitivity of these tumor cells to DOX. IC(50) value (0.10 microM) of DOX to HUVEC, a model vascular endothelial cell, was similar to that of C26/control. Double immunohistochemical staining of vascular endothelial cells and apoptotic cells within the tumor tissue after intravenous administration of PEG liposomal DOX showed that the extent of co-localization of apoptotic cells with endothelial cells was significantly higher for C26/DOX tumors (60%) than C26/control ones (20%), suggesting that the apoptosis is caused preferentially for vascular endothelial cells in C26/DOX tumor. From these results, it was suggested that the cytotoxic effect of DOX on vascular endothelial cells in the tumor would be involved in the in vivo anti-tumor effect of PEG liposomal DOX in C26/DOX-bearing mice.

Estimation of intradermal disposition kinetics of drugs: II. Factors determining penetration of drugs from viable skin to muscular layer

To develop a more efficient transdermal delivery system, it is very important to regulate the intradermal disposition of drugs after topical application. We tried to elucidate the factors determining the intradermal disposition kinetics, especially drug penetration from the viable skin to the muscular layer, mainly based on the six-compartment model, including the contralateral skin and muscle for ten model drugs with different physicochemical characteristics. In vivo transdermal absorption study was performed for six model drugs using the stripped-skin rats. The fitting analyses by the six-compartment model gave the theoretical curves describing the observed data very well and the reasonable pharmacokinetic parameters, showing the pharmacokinetic model should be useful for the estimation of the intradermal disposition kinetics of drugs applied topically again. The simulation study using the pharmacokinetic parameters obtained above could show the relative contribution of the direct penetration and the distribution from the systemic circulation to the muscular distribution of drugs. The largest contribution of direct penetration was observed for antipyrine (90.8%) and the smallest was for felbinac (43.3%). Among the pharmacokinetic parameters obtained above, the clearance from the viable skin to the muscle (CL(vs-m)) was found to be significantly correlated with the unbound fraction of drugs in the viable skin (fu(vs)). Although the clearance from the viable skin to the plasma (CL(vs-p)) also tended to increase as fu(vs) increased, the ratio of CL(vs-m) to CL(vs-p) was significantly correlated with fu(vs), meaning that the larger amount of unbound drug in the viable skin significantly contributes to the direct penetration into the muscle more than to the systemic absorption. On the other hand, k(direct) values obtained in in vitro penetration study-the penetration rate constant of drugs from the viable skin to the muscular layer-were found to be correlated with CL(vs-m) values for seven model drugs. Therefore, adding the in vitro experiments for the other three model drugs, the multiple linear regression analysis of k(direct) was performed for ten model drugs in terms of fu(vs), logarithm of the partition coefficient (Log P) and molecular weight. The results clearly showed the largest and significant contribution of fu(vs) to the direct penetration of drugs from the viable skin to the muscular layer, indicating that a drug with the higher value of fu(vs) in the viable skin can penetrate more into the muscular layer.

Surface hydrophobicity of particles is not necessarily the most important determinant in their in vivo disposition after intravenous administration in rats

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) and lecithin-coated MS-50 (LMS-50) after intravenous administration to rats was characterized. While a rapid elimination from the systemic circulation was observed for MS-50, much more prolonged circulating property was observed for LMS-50. In addition, this in vivo disposition property of LMS-50 was suggested to be ascribed to its lower affinity to the liver, which is the determining organ of the in vivo disposition of MS-50. The evaluation of surface hydrophobicity of MS-50 and LMS-50 in buffer solution revealed that the surface of MS-50 is more hydrophobic than that of LMS-50. However, LMS-50 was oppositely found to be more hydrophobic than that of MS-50 in rat serum. The profiles of serum proteins associated with MS-50 and LMS-50 were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the amounts of some adsorbed proteins are greatly different between MS-50 and LMS-50. From these findings, it was suggested that the substantial difference in the in vivo disposition between MS-50 and LMS-50 would not be attributed to the difference in their surface hydrophobicity in the blood, but the difference in the type of serum proteins associated with them.

Appearance of double peaks in plasma concentration-time profile after oral administration depends on gastric emptying profile and weight function.

PurposeMechanism for double-peak occurrence in plasma concentration profile after oral administration of drugs is controversial, although irregular gastric emptying would be an important factor. The objective of this study was to assess the effect of gastric emptying and a weight function, i.e. pharmacokinetics after reaching the systemic circulation, on the double-peak appearance in plasma concentration profiles.Materials and MethodsAlprazolam, which generates irregular gastric emptying, was orally co-administered with theophylline to rats, and the plasma concentration profiles or absorption rates were compared between the two drugs. Both drugs are highly absorbable, but alprazolam is rapidly eliminated from plasma, while the elimination of theophylline is very slow.ResultsOral administration of alprazolam generated the irregular gastric emptying profiles, resulting in multiple peaks in the absorption rate profiles of both drugs. The double peaks in the absorption rate profiles led to the double peaks in plasma concentration profiles for alprazolam, but not necessarily for theophylline. Simulation study clearly indicated that the slower elimination from plasma made the first peak less recognizable.ConclusionsThe irregular gastric emptying could be a main reason for the double peaks in plasma concentration profiles. However, the frequency of double-peak occurrence depends on the weight function, particularly the elimination rate, of each drug.

Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver

Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.

Evaluation of absorption kinetics of orally administered theophylline in rats based on gastrointestinal transit monitoring by gamma scintigraphy

The gastrointestinal (GI) transit and absorption of orally administered theophylline, a highly absorbable drug without presystemic elimination, were investigated under fasted and fed conditions using three rats in a crossover study. To evaluate the GI transit rate for each segment in vivo, a noninvasive technique, gamma scintigraphy, was employed using a nonabsorbable compound, (99m)Tc-labeled diethylenetriamine pentaacetic acid (DTPA). Using a gamma scintigraphic technique it is possible to simultaneously evaluate the GI transit and absorption of orally administered drug in the same individual. Theophylline was simultaneously administered along with [(99m)Tc]DTPA to animals in the fasted and fed states. Each GI transit pattern, simulated using the GI transit-kinetic model with a lag time factor, was well fitted to the experimental data. Gastric emptying rate varied in each study, even under the same experimental condition. The GI transit pattern for each segment was highly variable, especially in animals in the fed state. This inconsistency in transit pattern was mainly due to the variability in gastric emptying, which was much slower in animals in the fed compared with the fasted state. However, in spite of a large variability of GI transit kinetics, the plasma concentration-time curves of theophylline were well predicted by the GI transit-absorption model using the individual GI transit parameters obtained in the study. The absorption rate of theophylline was considerably reduced in animals in the fed state, because of the reduction of gastric emptying rate. Analysis using GI transit-absorption model and gamma scintigraphic technique made it possible to estimate the variable absorption kinetics regulated by GI transit with huge variability.