Tadatoshi Tanino

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.

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.

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.

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

Organic anion transporting polypeptide 2-mediated uptake of paclitaxel and 2'-ethylcarbonate-linked paclitaxel in freshly isolated rat hepatocytes.

Objectives The P‐glycoprotein (P‐gp) efflux pump plays an important role in paclitaxel detoxification. However, hepatic uptake of paclitaxel mediated by a solute‐linked carrier transporter family is still poorly understood in animals and humans. Freshly isolated hepatocyte suspensions are a well established in‐vitro model for studying drug transport and xenobiotic metabolism. Therefore, the hepatic uptake of paclitaxel and its P‐gp‐insensitive prodrug, 2′‐ethylcarbonate‐linked paclitaxel (TAX‐2′‐Et), has been characterized using freshly isolated and pregnenolone‐16‐α‐carbonitrile (PCN)‐treated hepatocytes in rats.

Enhancement of oral bioavailability of phenytoin by esterification, and in vitro hydrolytic characteristics of prodrugs

To improve the oral absorbability of phenytoin (DPH), prodrugs of DPH with a small acyl substituent, N-carboethoxy- and N-carboisopropoxy-DPH (PT-1 and PT-2, respectively), were synthesized and bioavailabilities of them were evaluated after oral administration in rats, compared to that of DPH dosed. The prodrugs were rapidly hydrolyzed in the intestinal fluid, intestinal mucosa, liver homogenates and plasma of rats, the plasma giving the highest hydrolytic activity. Two different eliminations of DPH, slow and rapid, were observed after intravenous and oral administrations of prodrugs. The bioavailabilities of DPH after oral administration at a dose of 25 mg/kg of PT-1 and PT-2 (DPH equivalent), increased to approximately 8.5- and 6.0-fold for PT-1 and PT-2 (rapid elimination group) or 3.0- and 3.0-fold (slow elimination group), respectively, compared to those after dosing of DPH. The plasma levels of DPH converted from PT-2 dosed were lower, but more sustained in slow elimination groups than those from PT-1. The normalized AUC values after oral dosing of prodrugs at a dose of 50 mg/kg were increased dramatically, compared to those at a dose of 25 mg/kg, suggesting non-linear clearance at a high dose. In order to clarify the mechanism for preponderance of intestinal absorption of the prodrugs, concentrations of parent drug and prodrug were measured in intestinal mucosa after a single oral dosing of 50 mg/kg (DPH equivalent). Upon the administration of PT-1 and PT-2, greater amounts of DPH, in comparison with those after dosing of DPH, and small amounts of intact prodrugs were detected in the duodenal and jejunal mucosa. These data indicated that these prodrugs was subjected to the extensive intestinal absorption compared to DPH, giving comparatively high plasma levels. Therefore, PT-1 and PT-2 will be useful prodrugs as an orally applicable form. In particular, PT-2 seems to serve as a benign prodrug with the intention of improving the absorption of DPH.

Effect of High Glucose Levels on Amyloid β Production in Retinas of Spontaneous Diabetes Mellitus Otsuka Long-Evans Tokushima Fatty Rats

The accumulation of amyloid β(1-42) peptide (Aβ(1-42)) in retina is implicated in the development of retinal ganglion cell apoptosis and diabetic retinopathy. In this study we demonstrate that spontaneous diabetes mellitus Otsuka Long-Evans Tokushima Fatty (OLETF) rats can be used as an animal model in studies to identify the expression of Aβ in diabetic retinas. In addition, we investigated the relation between glucose level and Aβ production in the retinas of OLETF rats. In the retinas of Long-Evans Tokushima Otsuka (LETO) rats used as normal controls and OLETF rats, no expression of neprilysin (NEP), which degrades Aβ, was detected, and the expression levels of genes associated with Aβ production (amyloid precursor protein, β site APP cleaving enzyme, and presenilin) and Aβ(1-42) levels in the retinas of 60-week-old OLETF rats with diabetes mellitus were significantly higher than in 60-week-old LETO rat retinas. Furthermore, the increase in the expression levels of genes associated with Aβ production was enhanced by administration of glucose (3.0 g/kg; OGT test), and close relations between the retinal Aβ(1-42) level and plasma blood glucose and HbA1c were observed. In conclusion, we have found that Aβ accumulates easily in the retinas of LETO and OLETF rats due to the absence of NEP. In addition, we determined that the accumulation of Aβ(1-42) in the retinas of OLETF rats is promoted by high plasma glucose levels. Therefore OLETF rats may be a suitable model for studies to identify the expression of Aβ in diabetic retinas.

Enzymatic stability of 2′‐ethylcarbonate‐linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapy

In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2′‐ethylcarbonate‐linked paclitaxel (TAX‐2′‐Et) in the sera of several different species including humans. TAX‐2′‐Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX‐2′‐Et hydrolysis, a CES isolated from rabbit liver (Ra‐CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX‐2′‐Et with a half‐life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16 h−1) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p‐nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX‐2′‐Et was 5.1 × 10−2 and 0.15 h−1, respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX‐2′‐Et. The amount of TAX produced accounted for only 8–11% of the decrease in TAX‐2′‐Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5 h incubation with human or rabbit serum. Ra‐CES enzyme converted TAX‐2′‐Et to TAX with Vmax and Km of 74.7±13.8 nmol/min/mg protein and 8.8±2.8 µM, respectively. These results indicate that TAX‐2′‐Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species‐dependent hydrolysis of TAX‐2′‐Et. Although human serum allows the slow release of TAX, TAX‐2′‐Et is expected to reduce the side‐effects of TAX. The Ra‐CES enzyme is capable of hydrolysing TAX‐2′‐Et, which may be beneficial for the development of a TAX‐2′‐Et/enzyme therapy strategy for ovarian cancer. Copyright

Percutaneous absorption of terodiline and the membrane-controlled transdermal therapeutic system

Abstract In an attempt to prepare a transdermal dosage form of terodiline which is extensively metabolized in man, the absorption of terodiline, an anticholinergic and calcium antagonist, through rat skin was estimated in vitro and in vivo. Terodiline penetrated rapidly through the skin from the gel formulation without enhancers (penetration rate, 167.6 μg/h per cm2). Laurocapram, a potent enhancer, did not enhance the absorption in vitro. When the gel formulation (0.9 g) was applied to rat abdominal skin (6 cm2), high plasma concentrations (Cmax, 8.8±3.4 μg/ml) of terodiline were observed for 24 h, thereby resulting in a high bioavailability equivalent to that after intravenous injection. The application of a transdermal therapeutic system, prepared using the gel formulation and a microporous membrane, gave a constant plasma level of terodiline, 250–820 ng/ml, for about 48 h, indicating that the membrane-controlled systems may be an efficient drug delivery system for treatment of urinary urge incontinence.

Enhanced Production of Nitric Oxide Leads to ATP Collapse in the Retinas of Otsuka Long-Evans Tokushima Fatty Rats, a Model of Human Diabetes

Abstract Purpose: We determined nitric oxide (NO) production via inducible NO synthase (iNOS) by hyperglycemia using the retina of Otsuka Long-Evans Tokushima Fatty rats (OLETF rats), and investigated the relationship between ATP contents and NO production in the retinas of OLETF rats. Methods: Long-Evans Tokushima Otsuka rats (LETO rats, normal rats) and OLETF rats (model rat for diabetes mellitus) aged 60 weeks of age were used. Plasma glucose (Glu) levels were determined using an Accutrend GCT System, and NO levels were measured by the microdialysis method as nitrite (). Cytochrome c oxidase (CCO) activity was measured using a Mitochondrial Isolation Kit and Cytochrome c Oxidase Assay Kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200. Results: levels in the retinas of OLETF rats were significantly higher than in LETO rats, and the levels in the retinas of 60-week-old OLETF rats increased with increasing Glu. CCO activity in the retinas of OLETF rats showed no significant difference from that in LETO rats; however, ATP levels in the retinas of OLETF rats were significantly lower than those in LETO rats. The oral administration of aminoguanidine or disulfiram, an iNOS inhibitor, attenuated the decrease in ATP levels in the retinas of 60-week-old OELTF rats. Conclusion: The present study demonstrates that NO production via iNOS in the retinas of 60-week-old OLETF rats is caused by hyperglycemia, and that NO causes a decrease in ATP contents in the retinas of 60-week-old OELTF rats. It is possible that the low ATP contents caused by NO may affect the normal functioning of the retina in OLETF rats.