Tomoyuki Furubayashi

Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells

The effects of various experimental conditions on in vitro drug permeability to Caco-2 monolayers were investigated to determine the optimized conditions for the prediction of intestinal drug absorption. Concerning the pH of the transport medium in the Caco-2 study, two different pH values, 6.0 and 7.4, were tested for the apical medium with the pH of the basolateral medium fixed to 7.4. The change in the apical pH showed pronounced effects on the permeability of both passively and actively transported drugs. It was found that the transport study under the condition of an apical pH value of 6.0 showed a better prediction of in vivo drug absorption in human. The appropriate conditions for determining the permeability of poorly soluble drugs were also examined. First, the effects of bile acids, surfactant and some agents used for solubilizing drugs on the permeability and transepithelial electrical resistance (TEER) of Caco-2 monolayers were investigated. Taurocholic and cholic acid showed no effects on the permeability of 3H-Dexamethasone (DEX) and TEER at 10 mM concentration, suggesting the possibility of use in the Caco-2 study. Polyethyleneglycol-400 and dimethylsulfoxide reduced the permeability of DEX concentration dependently, whereas ethanol induced no significant changes in the permeability. Furthermore, it was demonstrated that the addition of plasma protein (bovine serum albumin) to the basolateral medium apparently facilitated the transport of poorly soluble drugs with high lipophilicity across Caco-2 monolayers. These findings clearly suggest the importance of considering the physiological conditions of in vivo drug absorption in optimizing the in vitro experimental conditions for transport study using Caco-2 cells, in order to obtain a satisfactory in vitro-in vivo correlation.

Transnasal Delivery of Methotrexate to Brain Tumors in Rats: A New Strategy for Brain Tumor Chemotherapy

Brain tumors are one of the most lethal and difficult to treat. Their treatment is limited by the inadequate delivery of antitumor drugs to the tumor. In order to overcome this limitation, the possibility of the nose-brain direct transport pathway was evaluated using methotrexate (MTX) as a model antitumor agent. The direct transport of nasal MTX to the cerebrospinal fluid (CSF) was examined by comparing the concentration of MTX in the plasma and the CSF after intraperitoneal (IP) and intranasal (IN) administrations. The brain uptake of MTX was evaluated based on a multiple-time/graphical analysis by measuring the concentration of MTX in the plasma and in the brain. The feasibility of nasal chemotherapy was examined by three nasal dosings of MTX to tumor-bearing rats in vivo at two day intervals with peritoneal application as a positive control. MTX showed a significant inhibitory effect on the in vitro growth of 9L glioma cells with 50% growth inhibitory concentration at 7.99 ng/mL. The pharmacokinetic studies clarified the significant direct transport of MTX from nasal cavity both to the CSF and to the brain. Nasal chemotherapy with MTX significantly reduced the tumor weight as compared to nontreatment control and IP group. The strategy to utilize the nose-brain direct transport can be applicable to a new therapeutic system not only for brain tumors but also for other central nervous system disorders such as neurodegenerative diseases.

The transnasal delivery of 5-fluorouracil to the rat brain is enhanced by acetazolamide (the inhibitor of the secretion of cerebrospinal fluid)

The purpose of the research is to evaluate the effect of acetazolamide (AZA), an inhibitor of the secretion of cerebrospinal fluid (CSF), on the direct drug transport from the nasal cavity to the CSF and the brain uptake of a model drug, 5-fluorouracil (5FU). 5FU was infused intravenously or perfused nasally in the presence and absence of intravenously administered AZA. Concentrations of 5FU in plasma, CSF and the cerebral cortex were measured. The AUC and the concentration of 5FU in the brain were used to calculate the apparent brain uptake clearance (CL(up)) of 5FU, which is an index of drug delivery to the brain under the two experimental conditions. Intravenous AZA markedly increased the concentration of 5FU in the CSF and brain following the nasal perfusion of 5FU, although the plasma concentrations of 5FU were similar with intravenous infusion and nasal perfusions of 5FU. CL(up) of 5FU after the nasal perfusion with AZA was significantly increased by 104% and 46% as compared to intravenous infusion and nasal perfusion without AZA, respectively. AZA enhanced the 5FU delivery to the brain through a nose-to-brain pathway by increasing the concentration of the nasally applied drug in the CSF.

The disposition and intestinal absorption of zinc in rats

The variety of physiologic and biologic functions of zinc is expected to enable the development of zinc-related medicines. In this study, the distribution of endogenous zinc, the disposition after intravenous injection, and the intestinal absorption of zinc were investigated in vivo using rats from the viewpoints of pharmaceutical science and pharmacokinetics. High levels of endogenous zinc were observed in bone, testis, and liver. RT-PCR analysis on the mRNA of metallothionein in tissues clarified that it is significantly correlated with the distribution of zinc, suggesting that zinc is accumulated in tissues as a complex with MT. Following intravenous injection, uptake of zinc was high in liver, spleen, pancreas, kidney, and intestine. Fractional absorptions of zinc after oral administration to fasted rats were greater than those to fed rats, suggesting that some factors in diet inhibit the absorption of zinc. In fasted rats, fractional absorption was slightly decreased in high-dose group, suggesting the involvement of carrier-mediated transport. Study utilizing an in situ closed-loop method also indicated saturable intestinal absorption of zinc. These findings will further the research and development of zinc-related medicines by providing basic and important information on zinc.

Nasal drug absorption from powder formulations: The effect of three types of hydroxypropyl cellulose (HPC)

&NA; Despite the numerous advantages of powder formulations, few studies have described their nasal drug absorption. The first aim of this study was to compare the drug absorption from powder formulation with that from a liquid formulation in rats. Since pharmaceutical excipients are usually added to most powder formulations, the second aim of the study was to investigate the effect of hydroxypropyl cellulose (HPC) on nasal drug absorption from the powder. Three types of HPC with different polymerization degrees were used: HPC(SL), HPC(M), and HPC(H). The model drugs were warfarin (BCS Class I), piroxicam (BCS Class II), and sumatriptan (BCS Class III). The absorption of these model drugs in the powder form was higher than that from the solution. All HPCs failed to enhance warfarin absorption, while the piroxicam absorption was enhanced only by HPC(M). Sumatriptan absorption was not enhanced by HPC(SL), but by HPC(M) and HPC(H). The differences in nasal absorption of the three model drugs promoted by HPCs depend on the permeability and solubility of the drug. Moreover, the nasal retention of different formulations was increased by HPCs. Because HPCs showed no toxic effect on the nasal epithelium. These findings indicate that powder formulations supplemented with HPC are a valuable and promising approach to increase the nasal absorption of highly soluble and poorly permeable drugs. Graphical abstract The differences in nasal absorption of the three model drugs promoted by various types of hydroxypropyl cellulose (HPCs) depend on the permeability and solubility of the drug. Figure. No caption available.

Nasal Absorption of Macromolecules from Powder Formulations and Effects of Sodium Carboxymethyl Cellulose on Their Absorption.

The nasal absorption of macromolecules from powder formulations and the effect of sodium carboxymethyl cellulose (CMC-Na) as a pharmaceutical excipient on their absorption were studied. Model macromolecules were fluorescein isothiocyanate-labeled dextran (average molecular weight of 4.4kDa, FD4) and insulin. The plasma concentration of FD4 after application of the powder containing 50% starch (control) was higher than that after application of the solution, and the absorption from 50% starch powder was enhanced by the substitution of starch with CMC-Na. The fractional absorption of FD4 after administration of the CMC-Na powder formulation was 30% and 40% higher than that after administration from the solution and the starch powder, respectively. The nasal absorption of insulin from the powder and the effect of CMC-Na were similar with those of FD4. The effective absorption of FD4 and insulin after application of powder with CMC-Na could be due to the increase in the nasal residence of FD4 and insulin. No damage in the nasal mucosa or dysfunction of the mucociliary clearance was observed after application of the drug powder and CMC-Na. The present findings indicate that nasal delivery of powder formulations with the addition of CMC-Na as an excipient is a promising approach for improving the nasal absorption of macromolecules.

Delivery of Oxytocin to the Brain for the Treatment of Autism Spectrum Disorder by Nasal Application

Oxytocin (OXT) is a cyclic nonapeptide, two amino acids of which are cysteine, forming an intramolecular disulfide bond. OXT is produced in the hypothalamus and is secreted into the bloodstream from the posterior pituitary. As recent studies have suggested that OXT is a neurotransmitter exhibiting central effects important for social deficits, it has drawn much attention as a drug candidate for the treatment of autism. Although human-stage clinical trials of the nasal spray of OXT for the treatment of autism have already begun, few studies have examined the pharmacokinetics and brain distribution of OXT after nasal application. The aim of this study is to evaluate the disposition, nasal absorption, and therapeutic potential of OXT after nasal administration. The pharmacokinetics of OXT after intravenous bolus injection to rats followed a two-compartment model, with a rapid initial half-life of 3 min. The nasal bioavailability of OXT was approximately 2%. The brain concentration of OXT after nasal application was much higher than that after intravenous application, despite much lower concentrations in the plasma. More than 95% of OXT in the brain was directly transported from the nasal cavity. The in vivo stress-relief effect by OXT was observed only after intranasal administration. These results indicate that pharmacologically active OXT was effectively delivered to the brain after intranasal administration. In conclusion, the nasal cavity is a promising route for the efficient delivery of OXT to the brain.

Nasal Delivery of P-gp Substrates to the Brain through the Nose­Brain Pathway

The objective of this study was to evaluate in rats the potential utility of the nasal route to enhance central nervous system (CNS) delivery of drugs recognized by P-glycoprotein (P-gp). Well-known P-gp substrates verapamil and talinolol were perfused nasally or infused intravenously, and when plasma concentrations following intravenous infusion and nasal perfusion showed similar profiles. The concentration of verapamil in the brain after nasal perfusion was twice that after intravenous infusion. Although talinolol in the brain and the cerebrospinal fluid after i.v. infusion were below the detection limit, it was detected after nasal perfusion. When rats were treated with cyclosporin A, brain concentrations of verapamil after both administration modes were increased significantly, while those of talinolol were not significantly changed. Since the permeability of talinolol is low, talinolol in the brain which was transported directly from the nasal cavity has little chance of transport by P-gp localized in the apical membrane of cerebral microvessel endothelial cells. The potential for drug delivery utilizing the nose-CNS route was confirmed for P-gp substrates. The advantage of nasal delivery over i.v. delivery of talinolol to the brain was more significant than that of verapamil, suggesting that nasal administration is more useful strategy for the brain delivery of low-permeability P-gp substrates than the use of P-gp inhibitors.

Importance of the Direct Contact of Amorphous Solid Particles with the Surface of Monolayers for the Transepithelial Permeation of Curcumin

The amorphization has been generally known to improve the absorption and permeation of poorly water-soluble drugs through the enhancement of the solubility. The present study focused on the direct contact of amorphous solid particles with the surface of the membrane using curcumin as a model for water-insoluble drugs. Amorphous nanoparticles of curcumin (ANC) were prepared with antisolvent crystallization method using a microreactor. The solubility of curcumin from ANC was two orders of magnitude higher than that of crystalline curcumin (CC). However, the permeation of curcumin from the saturated solution of ANC was negligible. The transepithelial permeation of curcumin from ANC suspension was significantly increased as compared to CC suspension, while the permeation was unlikely correlated with the solubility, and the increase in the permeation was dependent on the total concentration of curcumin in ANC suspension. The absorptive transport of curcumin (from apical to basal, A to B) from ANC suspension was much higher than the secretory transport (from basal to apical, B to A). In vitro transport of curcumin through air-interface monolayers is large from ANC but negligible from CC particles. These findings suggest that the direct contact of ANC with the absorptive membrane can play an important role in the transport of curcumin from ANC suspension. The results of the study suggest that amorphous particles may be directly involved in the transepithlial permeation of curcumin.

The Enhancement of Nasal Drug Absorption From Powder Formulations by the Addition of Sodium Carboxymethyl Cellulose

For nasal drug absorption, powder formulations can be expected to provide many advantages. The first aim of this study was to examine drug absorption following nasal administration of powder formulations in rats. Pharmaceutical excipients are typically added to most powder formulations. The second aim was to investigate the change in nasal drug absorption of powder formulations in the presence of sodium carboxymethyl cellulose (CMC-Na). Model drugs used were norfloxacin (NFX), warfarin (WF), and piroxicam (PXC). The absorption from bulk powders is different from that of solutions. The absorption of PXC and WF from powder formulations was enhanced compared to those of the other solutions, while that of NFX, which has a low solubility, was decreased, suggesting that the nasal absorption of many drugs, except poorly soluble drugs, is enhanced when they are administered as powder formulations. CMC-Na enhanced the absorption of NFX and PXC. The presence of CMC-Na slightly decreased the absorption of WF. In vitro transepithelial transport from the powder formulation was not affected by the presence of CMC-Na. Furthermore, the nasal retention of the powder formulation was significantly increased in the presence of CMC-Na. In conclusion, the nasal absorption of many drugs, except those that are poorly soluble, can be increased by administering them as a powder formulation and the nasal absorption of the formulation is enhanced further in the presence of CMC-Na.