Taro Ogiso

Transfollicular Drug Delivery: Penetration of Drugs Through Human Scalp Skin and Comparison of Penetration Between Scalp and Abdominal Skins In Vitro

In order to quantitatively investigate the importance of transfollicular pathway for drug delivery, drug penetration through human scalp skin was investigated using liquid formulations containing lipophilic and hydrophilic drugs in vitro. The penetration pathway for drugs through the scalp skin was examined using fluorescent probes. Additionally, the drug penetration through the scalp skin was compared with that via human abdominal skin to clarify the usefulness of intrafollicular delivery. Lipophilic melatonin (MT) and ketoprofen (KP) showed high permeabilities through the scalp skin, although the flux of KP was much higher. Absorption enhancers, N -methyl-2-pyrrolidone and isopropylmyristate, only slightly increased the fluxes. Hydrophilic fluorouracil (5FU) and acyclovir (ACV) penetrated through the scalp skin with relatively large fluxes. However, there was large variability in the fluxes of these drugs across scalp skin from different sources. When the relationship between the flux and hair follicle density was estimated, there was good correlation between the two (r =0.651 for MT and r =0.666 for ACV, P <0.05) . The histologic examination of the scalp skin, following application of the formulation with nile red or sodium fluorescein, indicated that the probes permeated into the junction of the internal and external root sheath of follicles and diffused into the dermis via the outer root sheath at the initial times. The penetration of nile red, a lipophilic probe, via the stratum corneum of scalp skin was later than that via the follicles. The permeation of MT and 5FU through the scalp skin was much higher than that via the abdominal skin, being 27 and 48 times as high as the abdominal skin, respectively. These results indicate that the drug delivery through the scalp skin will offer an available delivery means for drugs, particularly for hydrophilic drugs.

Fluidity of human erythrocyte membrane and effect of chlorpromazine on fluidity and phase separation of membrane

The fluidity of human erythrocyte membrane, and the effect of chlorpromazine at prelytic and lytic concentrations on the fluidity have been studied by using three kinds of fatty acid spin labels and measuring the temperature dependence of Mg2+-ATPase activity. The Arrhenius plot of the apparent rotational correlation time, tau c, for probes I(12,3) and I(5,10) showed an abrupt discontinuity at about 30 degrees C, and the plot for I(1,14) at 25 degrees C, indicating that a large difference in the fluidity exists between the interior and the outer surface of the lipid bilayer. The portions of the fatty acid chain near the ten carbon bond lengths removed from the bilayer surface became more fluid by chlorpromazine treatment; there was a decrease in the break point to around 26 degrees C following treatment with 0.6 or 1 mM of the drug. Two breaks at 21 and 30 degrees C in the Arrhenius plot of the Mg2+-ATPase activity were observed in normal erythrocyte membrane. The activation energy of the Mg2+-ATPase reaction has the values of 3.0 and 22.1 kcal/mol above the upper break and below the lower break, respectively. The drug exposure induced only a slight shift in the break temperatures, while the treatment significantly enhanced the associated activation energies of the reaction. These results suggest that the boundary phospholipids of the Mg2+-ATPase in the membrane are probably more rigid than the bulk lipids.

Effect of Positively and Negatively Charged Liposomes on Skin Permeation of Drugs

To clarify the effect of the surface charge of liposomes on percutaneous absorption, the permeation of liposomal drugs through rat skin was investigated in vitro and in vivo. Liposomes were prepared using egg yolk lecithin (EPC, phase transition temperature, -15 to -17°C), cholesterol and dicetylphosphate (DP) or stearylamine (SA) (10:1:1, mol/mol). Also examined was the penetration behavior of positively and negatively charged liposomes, using a fluorescent probe (Nile Red). The in vitro penetration rate of melatonin (MT) entrapped in negatively charged liposomes was higher than that of positively charged ones (p<0.05). When the percutaneous absorption of ethosuximide (ES) encapsulated was estimated in vivo, the absorption of ES from negatively charged liposomes was slightly higher than that from positively charged liposomes. Additionally, the absorption of ES from both types of liposomes was superior to that from the lipid mixtures consisting of the same composition as the vesicles. The percutaneous absorption of betahistine (BH) from a gel formulation containing negatively charged liposomes of BH was much more than that from the formulation with positively charged ones, with 2-fold higher AUC (p<0.05). Histological studies revealed that the negatively charged liposomes diffused to the dermis and the lower portion of hair follicles through the stratum corneum and the follicles much faster than the positive vesicles at the intitial time stage after application. Thus, the rapid penetration of negatively charged liposomes would contribute to the increased permeation of drugs through the skin.

Polar solute transport across the pigmented rabbit conjunctiva: size dependence and the influence of 8-bromo cyclic adenosine monophosphate.

AbstractPurpose. To characterize the conjunctival permeability to polar solutes ranging from 182 to 167,000 daltons in molecular weight (m.w.). Methods. Solute transport across the excised pigmented rabbit conjunctiva with a baseline transepithelial electrical resistance (TEER) of 1,285 ± 46 ohm·cm2 was evaluated in the modified Ussing chamber under open-circuit conditions. The model solutes were mannitol (m.w. 182), 6-carboxyfluorescein (m.w. 376), and fluorescein isothiocyanate-labeled dextrans (FD4, m.w. 4,400—FD150, m.w. 167,000). Results. For a given solute, the apparent permeability coefficient (Papp) was independent of solute concentration and direction of transport. As expected, the Papp decreased with solute size, from 27.7 × 10−8 cm/sec for mannitol to 0.31 × 10−8 cm/sec for FD150. When the experimental temperature was lowered from 37°C to 4°C, Papp decreased by ~50% for FD4 through FD40 and by >80% for both FD70 and FD150. Equivalent pore analysis, assuming restricted solute diffusion via cylindrical, water-filled pores across the isolated tissue, revealed a radius of 5.5 nm at a pore density of 1.9 × 108 pores per cm2. The addition of 1 mM 8-bromo cyclic adenosine monophosphate (8-BrcAMP), known to stimulate Cl− secretion and decrease TEER, to the mucosal side of the conjunctiva increased the transport of mannitol, FD4, and FD40 by 28%, while not affecting FD150 transport. Conclusions. Our findings suggest that polar solutes up to FD40 traverse the conjunctival epithelial barrier primarily by restricted diffusion through equivalent pores of 5.5 nm radius and that solute movement is affected by reduction of TEER. On the other hand, polar solutes of the FD70 or larger may cross the barrier primarily via non-diffusional pathways such as non-specific endocytosis.

Effect of temperature on percutaneous absorption of terodiline, and relationship between penetration and fluidity of the stratum corneum lipids

Abstract The effect of temperature on the skin permeation of terodiline (TD) hydrochloride and the free base form was examined. The in vitro penetration experiment at 25–50°C was carried out using the full-thickness skin (FS) and the stratum corneum (SC) sheet of Wistar rat. The fluidity of the stratum corneum lipids was measured by ESR. The relationship between the flux and the phase state of the SC lipids was evaluated based on the data obtained. The effect of heating on the in vivo percutaneous absorption was also estimated. Increasing temperatures resulted in increased penetration of both hydrochloride and free base forms. A significant difference in the penetration rates through the FS was not observed between the hydrochloride and free base forms, whereas the fluxes of the free base form through the SC sheet were slightly higher than those of the hydrochloride form at each temperature. The Arrhenius plots of permeability coefficient ( K p ) yielded straight lines for both FS and SC sheet. The activation energies (15.5 and 20.0 kJ/mol), calculated from the slope of curves, for the SC sheet were smaller than those (45.7 and 39.3 kJ/mol) for the FS. The spin label mobility of the SC lipids, measured by ESR, was increased with rising temperatures. The plots of apparent rotational correlation time ( τ c ) versus temperature represented the temperature dependence, with abrupt breaks at 41.3±0.7°C and 68.1±1.4°C ( n =4), suggesting the phase transition of the lipids. When 1/ τ c was plotted against the K p for the free base form at four temperatures, straight lines were obtained for both skins ( r 2 =0.842 and 0.938). This indicates that the penetration of TD free base through the skin was dependent on the temperatures of the SC lipids and the drug penetrated via the lipoidal pathway within the SC. A notable result was not obtained from heating the transdermal system using a heat patch, because of the lesser efficiency of the patch.

Studies on dextranase. Purification of dextranase from Penicillium funiculosum and its enzymatic properties.

Dextranase (α-1,6-glucan 6-glucanohydrolase,, EC 3.2.1.11) from Penicillium funiculosum was purified by Bio Gel P-60 gel filtration, CM-cellulose and DEAE-cellulose chromatography. The enzyme preparation was further fractionated into two fractions, dextranase I (pI 3.98) and dextranse II (pI 4.19), by isoelectrofocusing. The purified dextranases I and II were both electrophoretically homogeneous. A molecular weight of 44 000 for both enzymes was obtained by gel filtration. The enzymes were most active at pH 6.0 and stable over a pH range from 5.0 to 7.5 at 37 °C for 60 min. They were activated by Co2+, Mn2+ and Cu2+, and inactivated by Ag+, Hg2+, N-bromosuccinimide and iodine. Dextranase II hydrolyzed preferentially a series of dextrans with α-1,6, linkages.

Phase transitions of rat stratum corneum lipids by an electron paramagnetic resonance study and relationship of phase states to drug penetration

In order to relate barrier function to stratum corneum structure and the thermal transitions of corneum lipids, samples from hairless rat skin were investigated by using ESR and drug penetration techniques. The phase transition of stratum corneum lipids was estimated using a deeper probe (16-doxyl-stearic acid) inserted in the lipid bilayers and measuring the rotational correlation time, tau(c). Results of ESR study showed that stratum corneum lipids underwent thermal transitions at 39.3 +/- 1.6 degrees C and 63.6 +/- 2.6 degrees C roughly similar to the data obtained by differential scanning calorimetry measurements. Cholesterol oxidase treatment decreased the fluidity of the lipids at lower temperatures. The treatment of stratum corneum with laurocapram (1%) and isopropyl myristate (IPM, 2%) little changed both phase transition temperatures, although the treatment highly increased the molecular motion of the lipids. The flux (J(s)) of lipophilic drugs (beta-estradiol, indomethacin and betahistine) through the skin was enhanced with increasing temperatures, with an increase in the diffusion constant within skin and a decrease in the lag time. There was a good relationship between log J(s) or log permeability coefficient (K(p)) and 1/tau(c) in the temperature range of 45 to 64 degrees C. The calculated activation energy (delta E) for diffusion of these drugs across skin was 17-40 kcal/mol. Judging from our data, stratum corneum lipids of rat probably exist as the gel, crystalline state below 39 degrees C, the mesomorphic state between 39 and 64 degrees C and the fluid, liquid-crystalline state at temperatures of 64 degrees C or above. These results are in line with the permeability of these lipophilic drugs through the intercellular lipids disordered is highly increased.

Studies on trypsin inhibitors in sweet potato I. Purification and some properties

Abstract Three different trypsin inhibitors I, II and III were isolated from a sweet potato, i.e. Ipomoea batatas LMA var. edulis Makino ( Okinawa Kokei No. 14), by column chromatography on Sephadex G-50, DEAE- and CM-cellulose. The finally purified preparations of Inhibitors II and III were found to be homogeneous by both choromatographic and electrophoretic analysis. These inhibitors showed strong and stoichiometric inhibition of trypsin whereas they showed only weak inhibition on other proteinases, such as plasmin and kallikrein, and no effect with α-chymotrypsin and pepsin. The molecular weight of Inhibitors II and III were calculated to be 23 000 and 24 000, respectively, by gel filtration on Sephadex G-50. Both inhibitors were fairly stable over a pH range from 2–11 at 37 °C, and thermostable. Some heavy metal ions, Mn 2+ , Fe 3+ , Cu 2+ and Hg 2+ , interfered with the inhibitor activity. By chemical modification of arginyl residues in the inhibitors with 1,2-cyclohexanedione, these inhibitors were shown to be arginine inhibitors.

Mechanism for enhancement effect of lipid disperse system on percutaneous absorption: Part II

Abstract To further clarify the mechanism involved in the enhancement effect of lipid disperse systems (LDS) on percutaneous absorption, the effect of particle size of LDSs on percutaneous absorption of betahistine (BH), the comparison of the enhancement effect of LDS with the lipid mixtures or the plain LDS, the effect of pretreatment of skin with gel formulation on penetration of LDS-BH and the fluidising effect of LDSs on the stratum corneum (SC) lipids were estimated using Wistar and hairless rats. No major differences in BH absorption were seen between the gel formulations containing LDS with three different particle size (128±4, 336±15, 596±37 nm), prepared using egg phosphatidylcholine (EPC), cholesterol and dicetylphosphate. The percutaneous absorbability of BH from the formulations containing the lipid mixtures or plain LDS did not reach to the extent from EPC–LDS formulation. Following pretreatment with gel formulation containing enhancer ( d -limonene or n-octyl-β- d -thioglucoside), BH absorption significantly decreased at the initial stage after application compared with that from LDS formulation, suggesting the additive enhancement effect of LDS and enhancer on the absorption. The treatment of the SC of hairless rat with LDSs significantly decreased the rotational correlation time (τc) and shifted downwards the slope of curves (τc versus temperature) at temperatures ranging from 25 to 60°C, compared with that of untreated SC. However, the significant differences in the fluidising effect between LDSs with different particle size were not observed

Simultaneous measurement of nicotinic acid and its major metabolite, nicotinuric acid in urine using high-performance liquid chromatography: application of solid—liquid extraction

The concentrations of nicotinic acid (NiAc) and nicotinuric acid (NiUAc), a major metabolite of NiAc, were simultaneously determined in urine using solid-phase extraction (cation-exchange extraction) and reversed-phase high-performance liquid chromatography with ultraviolet detection. The intra- and inter-day precision studies showed good reproducibilities: the coefficients of variations were less than 8.1% for NiAc and 8.8% for NiUAc. The calibration curves were linear (r2 > 0.9934) in the concentration range 10-1000 micrograms/ml. The removal of endogenous interferences in urine by solid-phase extraction presented here is superior to the pretreatment protocols reported previously by other workers. The method was used in a preliminary pharmacokinetic study in rats after intravenous administration of NiAc (5 and 15 mg/kg).