Nobuyasu Mizuno

Phosphatidylcholine-coated silica as a useful stationary phase for high-performance liquid chromatographic determination of partition coefficients between octanol and water

Phospholipid-coated silica gel is a useful stationary phase to determine the partition coefficient between octanol and water, Poct, by high-performance liquid chromatography (HPLC). A column of silica gel coated with dipalmitoyl phosphatidylcholine is easy to prepare, and is stable over a long period. With this column and an aqueous mobile phase containing acetonitrile, a good single correlation was observed between the log of the capacity factor, k′, and log Poct, for a wide variety of compounds. Thus, this chromatographic system, which overcomes the limitations of the standard shake-flask method and the HPLC method on an octadecyl silica column, is very useful for determining log Poct.

Characterization of the influence of polyol fatty acid esters on the permeation of diclofenac through rat skin.

In this study, we investigated the effect of sefsols on the skin to clarify the mechanism of the sefsol enhancement effect. In vitro percutaneous absorption experiments were performed using Franz diffusion cells. Removal of the stratum corneum and delipidization of the skin increased the permeation of diclofenac from aqueous suspension, with the enhancement effects being similar for both treatments. Further enhancement effects of diclofenac permeation by sefsol through the stripped and delipidized skin were not observed. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was employed to investigate the biophysical changes in the stratum corneum lipids by sefsols. One of the sefsols, propylene glycol mono caprylate (S-218), induced higher and broader absorbance shifts in both asymmetric and symmetric C-H bond stretching regions. However, no significant differences were observed among the sefsols with respect to peak heights and areas for both absorbances when compared with H2O treatment. These results suggest that sefsol may change the lipid-chain fluidity of the stratum corneum without lipid extraction. The accumulated amounts of diclofenac in the skin significantly increased in the presence of sefsol. Also, the amounts of diclofenac in the skin increased with the amount of sefsol in the skin. This sefsol enhancement effect was reversed at 12 h after treatment. Thus, enhancement of the diclofenac flux by sefsols is reversible and may be due to a change in the lipid-chain fluidity of the stratum corneum and improvement in drug partitioning to the stratum corneum.

Effect of hydrogen bonding on the high-performance liquid chromatographic behaviour of organic compounds: relationship between capacity factors and partition coefficients

Abstract Capacity factors ( k ′) of various compounds were determined by high-performance liquid chromatography on ODS (octadecylsilica) and gly-CPG (glyceryl-coated controlled-pore glass) with a mobile phase consisting of various proportions of methanol and water, and the correlation of the k ′ values with the P oct values (partition coefficients between octanol and water) of these compounds was examined. On both columns, log k ′ increased with increase in log P oct . However, introduction of a hydrogen bonding term was necessary to obtain a single correlation between log k ′ and log P oct for a wide variety of compounds. On both columns, the effect of hydrogen bonding on k ′ became smaller with a decrease in the proportion of methanol in the mobile phase, but in different manners on the two columns: among compounds with the same log P oct values, those with the ability to form hydrogen bonds had less affinity than those with no ability to form hydrogen bonds on ODS, and vice versa on gly-CPG.

Effect of Fatty Acid Diesters on Permeation of Anti-Inflammatory Drugs Through Rat Skin

ABSTRACT Four fatty acid diesters (diethyl succinate, diethyl adipate, diethyl sebacate, and diisopropyl adipate) were used to study their enhancement effect on the permeation of four non-steroidal anti-inflammatory drugs (NSAIDs: ketoprofen, indomethacin, diclofenac sodium, and ibuprofen) through rat abdominal skin. With the diester pretreatment, drug permeation increased and the lag times decreased. No relationship was observed between the solubilities of the drugs in the diesters and the diester enhancement effects. The enhancement effect decreased with an increase of the drug lipophilicity, but increased with an increase of the lipophilic index of the diester up to about 3.5, after which the enhancement effect decreased or remained constant. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was employed to investigate the biophysical changes in the stratum corneum lipids caused by the diesters. The FTIR results showed that treatment of the skin with diesters did not produce a blue shift in the asymmetric and symmetric C–H stretching peak positions. However, all of the above diesters showed a decrease in peak heights and areas for both asymmetric and symmetric C–H stretching absorbances in comparison with water treatment. These results suggested that the diesters were more effective for enhancing the penetration of hydrophilic drugs than lipophilic drugs, and the enhancing effect of lipophilic diesters was more effective than that of hydrophilic diesters. The enhancement effects of diesters may be due to their causing lipid extraction in the skin.

Percutaneous Permeation of Basic Compounds Through Shed Snake Skin as a Model Membrane

Abstract— Relationships between the in‐vitro permeability of basic compounds through shed snake skin as a suitable model membrane for human stratum corneum and their physicochemical properties were investigated. Compounds with low pKa values were selected to compare the permeabilities of non‐ionized forms of the compounds. Steady‐state penetration was achieved immediately without a lag time for all compounds. Flux rate and permeability coefficient were calculated from the steady‐state penetration data and relationships between these parameters and the physicochemical properties were investigated. The results showed that permeability may be controlled by the lipophilicity and the molecular size of the compounds. Equations were developed to predict the permeability from the molecular weights and the partition coefficients of basic compounds.

Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors

Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma.

Tumor Necrosis Factor α-Gene Therapy for an Established Murine Melanoma Using RGB (Arg-Gly-Asp) Fiber-mutant Adenovirus Vectors

Although adenovirus vectors (Ad) provide high‐level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie‐adenovirus receptor, a primitive Ad‐receptor, was very low in murine and human melanoma cells. We also found that fiber‐mutant Ad containing the Arg‐Gly‐Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting αv‐integrins. In addition, intratumoral injection of RGD fiber‐mutant Ad containing the tumor necrosis factor α gene (AdRGD‐TNFα) revealed dramatic anti‐tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad‐RGD‐TNFα required one‐tenth the dosage of Ad‐TNFα to induce an equal therapeutic effect. These results suggest that αv‐integrin‐targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy.

Correlation between dissolution rate and bioavailability of different commercial mefenamic acid capsules

A dissolution test was performed with five brands of 250 mg mefenamic acid capsule products available on the market. Three of them, the fast dissolving A and the slow dissolving D and E were subjected to a bioavailability study using a commercially available suspension as the reference. The products were administered orally in a cross-over design to 6 healthy men, and then parameters for the bioavailability were calculated from the plasma concentration-time curve. Analysis of variance indicated several significant differences among the products with respect to Cmax, Tmax and AUC. The relative availabilities of A, D and E were 86, 81 and 28%, respectively, with the AUC value (0-7 h) for the suspension as 100%. No correlation was observed between the in vitro dissolution rate of the drug from the capsules and the in vivo data, because the dispersing behavior of the capsule exerted a marked influence on its in vitro dissolution rate. To eliminate the influence of the capsule disintegrating process, a dissolution test was done on the contents of the capsules. A good correlation was found between the bioavailability and the dissolution rate of the drug from the capsule contents. Product E with the lowest bioavailability was passed through a 200-mesh sieve, placed in a new capsule, and tested for its bioavailabilky in humans. The AUC value was greater than that of the original product and the bioavailability was about equal to that of the suspension. The in vitro dissolution rate of the drug from the pulverized product E was also markedly increased.

An investigation of adverse effects caused by the injection of high-dose TNFα-expressing adenovirus vector into established murine melanoma

We previously reported that RGD fiber-mutant adenovirus vector carrying human TNFα cDNA (AdRGD-TNFα) could more effectively induce mouse B16 BL6 melanoma regression than conventional Ad-TNFα on intratumoral injection at less than 109 vector particles (VP). Although mice treated with either Ad type at 1010 VP showed remarkable tumor regression due to hemolytic necrosis, severe adverse effects including extreme reduction in body weight were also induced by Ad treatment. Here, we attempted to elucidate the cause of the adverse effects to optimize the application of AdRGD-TNFα. More than 99% of systemically administered Ad accumulated in the liver, and the rate of Ad leakage into systemic circulation from the B16 BL6 tumors injected with AdRGD or conventional Ad at 1010 VP was about 1% of the administered VP. Although the leaked Ad did not directly induce hepatotoxicity or body weight reduction, excessive TNFα produced in the tumors leaked into the blood at high concentrations and caused systemic inflammation, tissue denaturation, and body weight reduction in mice injected intratumorally with AdRGD-TNFα or Ad-TNFα at 1010 VP. Our results demonstrated that an exact AdRGD-TNFα dosage must be determined to prevent TNFα leakage from tumors into systemic circulation, thereby enabling safe application of AdRGD-TNFα to clinical melanoma gene therapy in the future.

Effect of age on the gastrointestinal absorption of acyclovir in rats.

Abstract— Drug elimination from the body after intravenous administration of acyclovir (20 mg kg−1) was delayed in 1‐week‐old rats but the pharmacokinetic data for 2–5‐week‐old rats were the same as those for 8‐week‐old rats. The areas under the plasma concentration‐time curves at 0–∞ h (AUC) after oral administration of acyclovir (20 mg kg−1) decreased with increasing age. The absolute bioavailabilities for 1‐, 2·5‐, 3‐ and 8‐week‐old rats were 77·59, 51·52, 14·61 and 7·30%, respectively. The gastrointestinal absorption of poorly absorbed acyclovir was good for rats younger than 2·5 weeks but dropped abruptly between 2·5 and 3 weeks of age. The intestinal membrane permeability of acyclovir was studied using the everted sac method. The rate of transfer of an initial concentration of 10 μm acyclovir from the mucosal to the serosal side was constant until 60 min in rats of different ages while the rate in 2–5‐week‐old rats was significantly greater than that in 3‐, 4‐ and 8‐week‐old rats. Abrupt in‐vivo and in‐vitro changes were observed in the experimental results between 2·5‐ and 3‐week‐old rats; this period coincided with the weaning period of the rat. The membrane transport mechanism of acyclovir in 2·5‐ and 8‐week‐old rats was also studied. Cumulative transferred amounts of acyclovir were linear (r = 0·99) over the range 5 μm–1 min and dose‐independent. The influence of metabolic inhibitors (sodium azide, 2,4‐dinitrophenol, ouabain), purine and pyrimidine analogues (2‐deoxyguanosine, guanine, adenine, uridine) and temperature on the permeation of acyclovir was studied. The permeation of acyclovir was inhibited only by 2‐deoxyguanosine and guanine in 2·5‐week‐old rats. These results suggest that throughout the maturation period, the gastrointestinal absorption mechanism of acyclovir is predominantly via passive diffusion with little or no active or facilitated transport. The abrupt change that occurs during the weaning period is attributable not to facilitated transport but to a change in the factors regarding passive transport.