Effects of ABCB1 3435C>T genotype on serum levels of cortisol and aldosterone in women with normal menstrual cycles.

ABCB1, also known as MDR1/P-glycoprotein, can transport cortisol and aldosterone. We examined the effects of ABCB1 polymorphisms on serum levels of cortisol and aldosterone among different phases of the normal menstrual cycle in 51 non-pregnant healthy Japanese female volunteers (22 +/- 1 years old). The menstrual cycle was divided into three phases: premenstrual phase (14 days preceding the onset of menstruation, N = 22; menstrual phase, N = 11, and postmenstrual phase, N = 18). ABCB1 -129T>C, 1236C>T, 2677G>A/T, and 3435C>T genotypes were determined. Serum levels of cortisol, aldosterone, estradiol, progesterone, and testosterone were measured. The serum levels of estradiol in the pre- and post-menstrual phases and of progesterone in the premenstrual phase were significantly increased when compared to their serum levels in the menstrual phase (P < 0.005). In the postmenstrual phase, the mean serum cortisol level in subjects with the 3435CT and 3435TT genotype was 7.6 +/- 3.4 microg/dL (mean +/- SD, N = 7), which was significantly lower than in women with the 3435CC genotype (9.9 +/- 1.8 microg/dL, N = 11) (P = 0.037). The opposite effect was observed in the serum aldosterone level during the postmenstrual phase (97.2 +/- 23.4 and 141.2 +/- 48.5 pg/mL for 3435CC and 3435CT + 3435TT, respectively; P = 0.041). These findings suggest that ABCB1 3435C>T genotype can influence serum levels of cortisol and aldosterone during the postmenstrual phase of the normal menstrual cycle.

S-2474, a novel nonsteroidal anti-inflammatory drug, rescues cortical neurons from human group IIA secretory phospholipase A(2)-induced apoptosis.

The elevated level of group IIA secretory phospholipase A(2) (sPLA(2)-IIA) activity contributes to neurodegeneration in the cerebral cortex after ischemia. The up-regulation of cyclooxygenase-2 (COX-2) is also relevant to cerebral ischemia in humans. Studies of ischemia with COX-2 inhibitors suggest a clinical benefit. In the present study, we investigated effects of S-2474 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons, which was established as an in vitro model of brain ischemia. S-2474 is a novel nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 and contains the di-tert-butylphenol antioxidant moiety. S-2474 significantly prevented neurons from undergoing sPLA(2)-IIA-induced cell death. S-2474 completely ameliorated sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. sPLA(2) also generated neurotoxic prostaglandin D(2) (PGD(2)) and free radicals from neurons before cell death. S-2474 significantly inhibited the sPLA(2)-IIA-induced generation of PGD(2). The present cortical cultures contained few non-neuronal cells, indicating that S-2474 affected neuronal survival directly, but not indirectly via non-neuronal cells. The inhibitory effect of S-2474 on COX-2 might contribute to its neuroprotective effect. In conclusion, S-2474 exhibits neuroprotective effects against sPLA(2)-IIA. Furthermore, the present study suggests that S-2474 may possess therapeutic potential for stroke via ameliorating neurodegeneration.

Gas6 rescues cortical neurons from amyloid beta protein-induced apoptosis.

Gas6, a product of the growth-arrest-specific gene 6, protects neurons from serum deprivation-induced apoptosis. Neuronal apoptosis is also caused by amyloid beta protein (Abeta), whose accumulation in the brain is a characteristic feature of Alzheimer's disease. Abeta induces Ca(2+) influx via L-type voltage-dependent calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on Abeta-induced cell death in primary cultures of rat cortical neurons. Abeta caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from Abeta-induced cell death. Gas6 ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, Abeta increased influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the Abeta-induced Ca(2+) influx. The inhibitor of L-VSCCs also suppressed Abeta-induced neuronal cell death. The present cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from Abeta-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.

Effects of S-2474, a novel nonsteroidal anti-inflammatory drug, on amyloid beta protein-induced neuronal cell death.

1. The accumulation of amyloid beta protein (Abeta) in the brain is a characteristic feature of Alzheimer's disease (AD). Clinical trials of AD patients with nonsteroidal anti-inflammatory drugs (NSAIDs) indicate a clinical benefit. NSAIDs are presumed to act by suppressing inhibiting chronic inflammation in the brain of AD patients. 2. In the present study, we investigated effects of S-2474 on Abeta-induced cell death in primary cultures of rat cortical neurons. 3. S-2474 is a novel NSAID, which inhibits cyclo-oxygenase-2 (COX-2) and contains the di-tert-butylphenol antioxidant moiety. S-2474 significantly prevented neurons from Abeta(25 - 35)- and Abeta(1 - 40)-induced cell death. S-2474 ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely. 4. Prior to cell death, Abeta(25 - 35) generated prostaglandin D(2) (PGD(2)) and free radicals from neurons. PGD(2) is a product of cyclo-oxygenase (COX), and caused neuronal cell death. 5. S-2474 significantly inhibited the Abeta(25 - 35)-induced generation of PGD(2) and free radicals. 6. The present cortical cultures contained little non-neuronal cells, indicating that S-2474 affected neuronal survival directly, but not indirectly via non-neuronal cells. Both an inhibitory effect of COX-2 and an antioxidant effect might contribute to the neuroprotective effects of S-2474. 7. In conclusion, S-2474 exhibits protective effects against neurotoxicity of Abeta. Furthermore, the present study suggests that S-2474 may possess therapeutic potential for AD via ameliorating degeneration in neurons as well as suppressing chronic inflammation in non-neuronal cells.

Controlled release and ocular absorption of tilisolol utilizing ophthalmic insert-incorporated lipophilic prodrugs.

To control ocular drug delivery, the O-butyryl ester prodrug of tilisolol (BUTL) and the O-palmitoyl ester prodrug of tilisolol (PalTL) were incorporated into an ophthalmic insert. The released TL from BUTL inserts and PalTL inserts in pH 7.4 phosphate-buffered saline until 5 h were approximately 25% and 3% of that from TL inserts, respectively. In addition, BUTL was also released from BUTL inserts. However, PalTL was not released from the PalTL insert. The release of drugs from TL inserts and BUTL inserts was little affected by the addition of bovine serum albumin (BSA) in pH 7.4 phosphate-buffered saline. In contrast, the release of drugs from PalTL inserts were enhanced by the addition of BSA. After application of TL, BUTL, and PalTL inserts to the rabbit eye, the aqueous humor concentration of TL was prolonged compared with TL instillation, and the plasma concentration of TL was much lower than that of TL instillation. The ratios of the area under the TL concentration-time curve (AUC) in the aqueous humor to AUC in the plasma (AUC(aqueous)/AUC(plasma)) after application of BUTL until 8 h were 3.1-fold and 3.8-fold higher than those of the TL insert and PalTL insert, respectively.

Effect of viscous additives on drug absorption from the liver surface in rats using phenol red as a model.

The purpose of this study is to obtain information that can be used to improve controlled release and residence time of drugs on the liver surface. Using carboxymethylcellulose sodium salt (CMC-Na) and polyvinyl alcohol (PVA), we examined the effect of viscous formulations on the absorption of phenol red as a model. In the presence of 3% CMC-Na or 15% PVA, the maximum plasma concentration of phenol red decreased after application to the rat liver surface using a cylindrical glass cell. The absorption ratios in 6 h calculated from the remaining amount of phenol red in the glass cell were 68.6, 60.5 and 48.7% (control: 73.1%) in the presence of 1 or 3% CMC-Na and 15% PVA, respectively. As a result of the reduction in the absorption ratio, the amount of phenol red excreted into the bile and urine in 6 h was decreased by the addition of the viscous additives. The decrease in absorption rate was characterized by a pharmacokinetic analysis of the plasma concentration profile. The change in absorption rate differed between the viscous additives, reflecting the result of the in vitro release experiment. Accordingly, the possibility that the drug absorption rate from the liver surface can be altered by viscous additives was suggested to have a promising prospect for therapeutic use.

O/W lipid emulsions for parenteral drug delivery. III. Lipophilicity necessary for incorporation in oil particles even after intravenous injection.

The potential usefulness of oil-in-water (O/W) lipid emulsions as injectable drug delivery systems was examined. Plasma concentrations of oil particles after intravenous injection of a standard lipid emulsion composed of soybean oil and egg yolk phosphatides were monitored based on the plasma concentrations of phospholipids and triglycerides, and the light scattering intensity of the plasma. Their time profiles were similar to each other, and the oil particle size decreased time-dependently. Pretreatment with dextran sulfate, a known reticuloendothelial system (RES) suppressor, resulted in marked reduction of the plasma clearance of the oil particles and of the time-dependent alteration of oil particle size, suggesting that oil particles were trapped by RES. The lipophilicity of the drug needed for its incorporation in the oil particles even after intravenous injection was found to be clog P > 8, where clog P is the calculated logarithm of the partition coefficient between n-octanol and water. In the case of sudan II (clog P = 5.4), the release from the oil particles was very quick after intravenous injection, resulting in slight alteration in biodistribution when compared with its micellar solution. In contrast, menatetrenone (clog P = 9.5) was selectively delivered to the liver, lungs and spleen, being consistent with the oil particles taken up by RES.

Effect of composition on biological fate of oil particles after intravenous injection of O/W lipid emulsions.

Plasma concentrations of oil particles after intravenous injection of oil-in-water (O/W) lipid emulsions were monitored based on the plasma concentration of phospholipids (PL) and triglycerides (TG), and the light scattering intensity (LSI) of plasma. Previously, we found that their time profiles after injection of the standard O/W lipid emulsion composed of soybean oil (SO) and egg yolk phosphatides (EYP) were similar and suggested that the oil particles with diameter of about 200 nm were entrapped by reticuloendothelial system (RES). Herein, in order to develop a delivery system to avoid the RES uptake by using the lipid emulsions, biological fate of lipid emulsions with oil particles of various sizes or those emulsified by surfactants with polyoxyethylene segments were subjected to the investigations. Lipid emulsions with oil particles of various sizes (about 150-550 nm) were prepared by altering EYP content. The oil particles were stable in plasma in vitro, but oil particle size decreased time-dependently after intravenous injection. Plasma clearance of oil particles depended on their initial size and was decreased by pretreatment with dextran sulfate 500 (DS500), a known RES suppressor. These results suggested that oil particles are still entrapped by RES, even for small-sized oil particles (about 150 nm). Lipid emulsion with small-sized oil particles was also prepared using medium chain triglycerides. The oil particles were stable in vitro, but the time profiles of plasma concentrations of PL and TG, and LSI of plasma were different, and oil particle size decreased time-dependently after intravenous injection. Plasma clearance of the oil particles also depended on their initial size and was decreased by DS500, suggesting that in vivo instability could be due to RES-mediated processes. Artificial surfactants with polyoxyethylene segments, HCO-60 (HCO60) and polysorbate 80 (PS80), were used for RES avoidance. HCO60 resulted in drastic reduction of the plasma clearance of the oil particles for both lipid emulsions composed of soybean oil and medium chain triglycerides. The time-dependent decrease of oil particle size after intravenous injection was marginal. In contrast, PS80 could not prolong the circulation time of the oil particles, and their size decreased time-dependently after intravenous injection.

O/W lipid emulsions for parenteral drug delivery. II. Effect of composition on pharmacokinetics of incorporated drug.

The potential usefulness of O/W lipid emulsions as injectable drug delivery systems for lipophilic drugs was examined using a model lipophilic drug, sudan II (clogP = 5.4) in the normal rats. The standard lipid emulsion composed of soybean oil and egg yolk phosphatides increased the blood concentration of sudan II after i.v. injection when compared with its solubilized solution by plasma. However, it was still lower than that of the oil particles, and the distribution of sudan II to liver, lungs, adipose tissue, heart, and muscle was not altered, and only that to brain and kidneys was decreased. Herein, the effect of extensive alterations in the lipid emulsion composition on the blood concentration and organ distribution of sudan II was examined in comparison with the standard formulation. Addition of cholesterol, use of pure egg yolk phosphatidylcholine, use of phospholipids with saturated alkyl chain, use of saturated long chain triglycerides, and use of saturated medium chain triglycerides were tested. The oil particles of all tested lipid emulsions were still located in plasma space, and use of saturated medium chain triglycerides was the most effective way to increase blood concentration of sudan II, resulting in higher distribution to liver, lungs, spleen, and brain. This was caused by the increase of the steady-state partition of sudan II to the oil particles, and not by alteration of their organ distribution clearance.

Enhancement of transport of D-melphalan analogue by conjugation with L-glutamate across bovine brain microvessel endothelial cell monolayers.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37 degrees C to 4 degrees C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Effect of injection volume on the pharmacokinetics of oil particles and incorporated menatetrenone after intravenous injection as O/W lipid emulsions in rats.

Oil-in-water lipid emulsions are promising drug carriers for lipophilic drugs, however, the pharmacokinetics after entering the circulation should be clarified at clinical injection volume in order to utilize them in a clinical situation. In the present study, the standard lipid emulsions, consisting of soybean oil, egg yolk phosphatides and menatetrenone with diameters of about 150 nm, were prepared using a microfluidizer system. The pharmacokinetics of menatetrenone and the oil particles after intravenous injection as standard lipid emulsions at various injection volumes, from the clinical injection volume (0.1 ml/kg) to the experimental injection volume (3.0 ml/kg), were examined in rats. The plasma concentrations of menatetrenone and the oil particles were similar after administration, showing that menatetrenone was not released even after entering the circulation. Menatetrenone was delivered to the liver and spleen at the clinical injection volume, and more menatetrenone was delivered to the liver at clinical injection volume compared with the experimental volume. Moreover, additional information on injection volume-dependency was also obtained from these findings. These results at various injection volumes suggested that the standard lipid emulsions can be utilized as a useful drug delivery system at the clinical injection volume, especially for liver and spleen targeting.

Blood flow rate in normal and tumor-bearing rats in conscious state, under urethane anesthesia, and during systemic hypothermia.

The blood flow rates of 14 tissues in the body were determined by microsphere method using normal and tumor-bearing rats kept conscious or under urethane anesthesia. The effects on the blood flow rate in the tissues were assessed for multimodal therapy, systemic hypothermia for ischemic brain injury, and local hyperthermia and angiotensin II-induced hypertensive chemotherapy for cancer. Urethane anesthesia showed no effect on cardiac output, while there was a tendency of decrease of blood flow rate and % of cardiac output in each tissue other than muscle tissue, in which they increased as a counterbalance, in normal and tumor-bearing rats. Systemic hypothermia gave results similar to those of urethane anesthesia in normal rats, but for tumor-bearing rats, it decreased cardiac output, and consequently the blood flow rate in most tissues. Brain blood flow rate was about half of that in the conscious rats. Local hyperthermia also decreased the cardiac output and blood flow rate in each tissue, including the tumor tissue. Angiotensin II-induced hypertension showed no effect on cardiac output, had various effects on blood flow rate in each tissue, and led to no increase in the tumor blood flow rate. Simulations based on the physiological pharmacokinetic modeling suggested that intramuscular injection of a lung-specific derivative of ceftazidime would provide the ideal biodistribution to ensure its optimal therapeutic efficacy during systemic hypothermia. This methodology, namely the pharmacokinetic simulation based on the physiological values of the body, will provide a useful piece of information on drug delivery systems under various conditions.

O/W lipid emulsions for parenteral drug delivery. IV. Changes in the pharmacokinetics and pharmacodynamics of a highly lipophilic drug, menatetrenone.

The pharmacokinetics and pharmacodynamics of antihemorrhagic vitamin, menatetrenone after intravenous injection as the lipid emulsion, were compared to those as the micellar solutions. Menatetrenone was selectively delivered to the liver, lungs and spleen and retained in them. Hepatic and splenetic concentration at 6 h (C6h) increased 21.6- and 27.1-fold, respectively, and the area under the tissue concentration-time curve up to 6 h (AUC(0-6h)) were 2.3- and 11.4-fold, respectively, when compared with its micellar solution. Antihemorrhagic effect of menatetrenone was assessed using warfarin-induced hypoprothrombinemic rats. The lipid emulsion of menatetrenone decreased the prothrombin time at 6h after intravenous injection more effectively than micellar solution. The dose response curves indicated that the efficacy of the lipid emulsion was 2.4-2.9 times that of a micellar solution, and this was correlated with AUC(0-6h) rather than C6h. The plasma level of clotting factor VII and the hepatic level of descarboxyprothrombin were also recovered more effectively, while no significant differences were noted between the two formulations for the plasma level of factor II or descarboxyprothrombin at the dose levels examined. Although selective delivery of menatetrenone in the liver by the lipid emulsion was due to phagocytosis by non-parenchymal cells, menatetrenone in the whole liver appeared to contribute to recovery from hypoprothrombinemia.

Derivatives of melphalan designed to enhance drug accumulation in cancer cells.

The objective of this study was to develop chemical strategies to improve the uptake and accumulation of melphalan (L-Mel and D-Mel), a cytotoxic agent, into cancer cells. Dipeptides synthesized from L- (or D-) Mel and L-glutamic acid (L-Glu) or L-valine (L-Val) and their methyl or ethyl esters (all compounds were trifluoroacetic acid salts) were evaluated for cytotoxicity and cellular uptake using Caco-2 cells, a human colon carcinoma cell line, and RT-2 cells, a rat brain glioma cell line. Treatment of Caco-2 cells with L-Mel or D-Mel (0.5 mg/ml equivalent of melphalan) for 48 h resulted in approximately 50% cell survival. Treatment of the Caco-2 cells with dipeptide derivatives of L-Mel (or D-Mel) (11c-d, 12c-d and 13) caused similar cytotoxicity effects (approximately 50-70% of cell survival). When the cytotoxicities of the esters of L-Mel, D-Mel and their dipeptide derivatives (11a-b, 12a-b and 14) in Caco-2 cells were determined, less than 10% cell survival was observed. Similar results were observed in RT-2 cells. When the cellular uptake properties of these compounds were determined in Caco-2 cell monolayers, L-Glu-L-Mel (12c), L-Glu-D-Mel (12d), and L-Mel-L-Glu (11c) generated slightly lower intracellular levels of L-Mel or D-Mel than when the cell monolayer was treated with the amino acids (L-Mel or D-Mel). In Caco-2 cells treated with 11c, 12c or 12d, low levels of the dipeptides were also detected. Caco-2 cell monolayers treated with D-Mel-L-Glu (11d) or D-Mel-L-Val (13) showed very low levels of the amino acids (L-Mel or D-Mel), but generally higher levels of the dipeptides. In contrast to the amino acids (L-Mel, D-Mel) or the dipeptide derivatives (11c-d, 12c-d and 13), the ester derivatives of the amino acids [L-Mel(OEt), D-Mel(OEt)] or the dipeptides (11a-b, 12a-b and 14) produced 5-20 times higher intracellular concentrations of potentially cytotoxic metabolites (e.g., L-Mel, D-Mel, Mel-containing dipeptides or Mel-containing dipeptide monoesters). L-Mel(OEt), D-Mel(OEt), L-Glu(OEt)-L-Mel(OEt) (12a), L-Glu(OEt)-D-Mel(OEt) (12b), and L-Mel-L-Glu(OEt)2 (11a) accumulated mainly as either L-Mel or D-Mel, and the percentages of L-Mel or D-Mel were 99%, 99%, 90%, 75% and 98% of the total intracellular concentration of potentially cytotoxic agents, respectively. D-Mel-L-Glu(OEt)2 (11b) accumulated as its monoester (> 95%) and D-Mel-L-Val(OMe) (14) accumulated as its dipeptide metabolite (> 98%). Inclusion of Gly-Pro, carnosine, L-Phe or L-Glu did not inhibit uptake of the dipeptide derivatives of L-Mel (or D-Mel) or their esters. These results suggest that the cellular uptake of the dipeptide derivatives of melphalan and their esters is probably via passive diffusion rather than being facilitated by an amino acid transporter or a di/tripeptide transporter. The higher intracellular levels of cytotoxic agents generated from the ester derivatives of the amino acids and the dipeptides are probably due to their higher lipophilicity and the overall neutral charge of the esters and subsequent intracellular formation of the more polar amino acids (L- or D-Mel) and/or Mel-containing dipeptides. Finally, these studies suggest that dipeptides of D-Mel [11b, 11d, 13] have inherent cytotoxicity properties.

Conjugation with L-Glutamate for in vivo brain drug delivery.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

Ocular absorption behavior of palmitoyl tilisolol, an amphiphilic prodrug of tilisolol, for ocular drug delivery.

The objective of this study was to examine the ocular absorption behavior of an amphiphilic prodrug after instillation onto the cornea of rabbits. A micellar solution of O-palmitoyl tilisolol (PalTL), an amphiphilic prodrug, was prepared. After instillation of tilisolol (TL) and PalTL, the drug concentrations in the tear fluid, cornea, aqueous humor, iris-ciliary body, vitreous body, and blood were measured. In addition, in situ ocular absorption behavior was also evaluated. After instillation of TL, the concentration of TL in the tear fluid quickly decreased. After instillation of PalTL, prolonged retention and high concentrations of PalTL in tear fluid and the cornea were observed. In addition, more prolonged retention of the TL concentration after instillation of PalTL than after instillation of TL was observed in the cornea, aqueous humor, and iris-ciliary body. In situ experiments demonstrated that PalTL was mainly absorbed by the corneal route and the improvement effects of PalTL under in vivo conditions was due to an enhanced transit time of PalTL in ocular tissues. PalTL, an amphiphilic prodrug, exhibited increased retention in the precorneal area compared with the parent drug, TL, resulted in improved ocular absorption of the parent drug.

Sustained ocular delivery of tilisolol to rabbits after topical administration or intravitreal injection of lipophilic prodrug incorporated in liposomes.

To improve the retention time of tilisolol in the precorneal area or vitreous body, we prepared liposomes incorporating the O-palmitoyl prodrug of tilisolol. O-Palmitoyl tilisolol was completely incorporated in the liposomes. After topical administration of O-palmitoyl tilisolol liposomes to the rabbit eye, O-palmitoyl tilisolol rapidly disappeared from the tear fluid. The inclusion of 2% carmellose sodium slightly prolonged the retention of O-palmitoyl tilisolol in the tear fluid. After intravitreal injection of O-palmitoyl tilisolol liposomes, there was a relatively prolonged retention of O-palmitoyl tilisolol in the vitreous body. At 24 and 48 h after intravitreal injection of O-palmitoyl tilisolol liposomes, the tilisolol concentration in the vitreous body was significantly higher compared with the concentration after intravitreal injection of tilisolol liposomes.

Effect of instillation method on the absorption of phenolsulphonphthalein as a model drug from the liver and small intestinal serosal surface in rats.

We have examined the effect of the instillation method on the absorption of a drug from the liver and the small intestinal serosal surface in rats. We performed continuous microinstillation via an infusion pump and bolus instillation via a syringe, using phenolsulphonphthalein (phenol red) as the model drug. After continuous microinstillation of phenolsulphonphthalein 2.35 mg in 235 microL for 5 min on the liver and small intestinal serosal surface in rats, the AUC (area under the curve) of the plasma concentration profile up to 60 min was significantly higher compared with bolus instillation. A similar trend was observed after continuous microinstillation of phenolsulphonphthalein 2.35 mg in 117.5 microL for 2.5 min. The calculated absorption rate constants (Ka) after continuous microinstillation of phenolsulphonphthalein based on a two-compartment model with first-order absorption were higher than those after bolus instillation on the liver and small intestinal serosal surface at either instillation concentration. Moreover, Ka was increased after continuous microinstillation of 2.35 mg in 117.5 microL at either instillation site. Instillation of phenolsulphonphthalein on the liver surface resulted in a 1.2- to 2.3-fold higher Ka compared with the small intestinal serosal surface. This tendency was marked after continuous microinstillation of 2.35 mg in 117.5 microL. In conclusion, absorption could be enhanced by instilling a small amount of drug solution on the liver surface gradually and continuously, suggesting a promising approach for instillation site-selective drug delivery in the peritoneal cavity.

O/W lipid emulsions for parenteral drug delivery. I. Pharmacokinetics of the oil particles and incorporated sudan II.

The potential usefulness of oil in water (O/W) lipid emulsions as parenteral drug delivery system for lipophilic drugs was examined in tumor-bearing rats. A model lipophilic drug, sudan II (PCoct = 226000), was formulated in five lipid emulsions consisting of soybean oil and various surfactants. Compared with HCO-60 micellar and plasma solutions of sudan II, the blood concentration of sudan II was markedly elevated by administration as a lipid emulsion. However, the distribution of sudan II to the liver, lungs, spleen, and adipose tissue was not altered, and that to the brain, heart, kidneys, muscle, and tumor was slightly decreased. To understand these results, pharmacokinetic analysis was performed using a newly derived compartmental model, and moreover, the organ distribution clearance was analyzed. It was suggested that the oil particles deliver the incorporated drug selectively to the liver, lungs, and spleen, and the speed of delivery could be surpressed by using HCO-60. However, in the case of sudan II, its rapid release from the oil particles after i.v. injection prevented a drastic alteration in the distribution of sudan II. The simulation studies suggested that a considerable decrease in the release rate or an increase in partition coefficient (experimentally more than 10(8) would be required for delivery.

Identification of N-acetyltransferase 2 and CYP2C19 genotypes for hair, buccal cell swabs, or fingernails compared with blood.

Genotyping of polymorphic drug metabolizing enzymes may be useful to estimate the blood concentration, efficacy, and toxicity of drugs before administration. Blood samples are most generally used for genotyping; however, sampling is invasive and complicated by handling and transport. Therefore, the authors developed genotyping methods using nonblood specimens, and then each genotype was compared with that from blood. Healthy Japanese volunteers provided hairs (n = 50), buccal cell swabs (n = 50), and fingernails (n = 30) for N-acetyltransferase 2 and CYP2C19 genotyping. Recovery of genomic DNA from each nonblood specimen was lower than that from 0.5 mL blood. Using a modification of the DNA extraction and polymerase chain reaction amplification method, genotypes were diagnosed without failure, even for those with very low levels of DNA. Both genotypes from these specimens completely matched the genotypes from the blood of the same subject. These nonblood specimens can be convenient, accessible, and economical alternatives to blood as a source of DNA for genotyping.