The Sensory Effects of l-Menthol on Human Skin

Psychophysical measurements were made of the sensory effects of l-menthol applied topically to the forearm under controlled thermal conditions. In the first experiment, subjects judged the intensity and quality of sensations produced by warming or cooling the skin in the presence of menthol or the vehicle. During cooling, menthol intensified cutaneous sensations and increased reports of burning. During warming, menthol intensified sensations transiently at low temperatures and weakened them lastingly at higher temperatures; the frequency of reports of burning varied with intensity. A second experiment tested the hypothesis that menthol would lower the threshold for warmth and raise the threshold for heat pain. No change in either threshold was observed. The primary sensory effects of l-menthol on hairy skin are therefore to heighten the perception of cooling and to attenuate the perception of moderate warming. In contrast with other common chemical irritants, menthol's pungent qualities appear to be enhanced by cooling and suppressed by warming; this suggests that its sensory irritancy may be attributable to the stimulation of a population of high-threshold cold fibers or cold-sensitive nociceptors.     

Effect of Lipophilicity and Drug Ionization on Permeation Across Porcine Sublingual Mucosa

Sublingual route is one of the oldest alternative routes studied for the administration of drugs. However, the effect of physical-chemical properties on drug permeation via this route has not been systemically investigated. The objective of this study was to determine the effect of two key physicochemical properties, lipophilicity and ionization, on the transport of drugs across porcine sublingual mucosa. A series of β-blockers were used to study the effect of lipophilicity on drug permeation across the sublingual mucosa, while nimesulide (pKa 6.5) was used as a model drug to study the effect of degree of ionization on sublingual mucosa permeation of ionized and unionized species. Permeation of β-blockers increased linearly with an increase in the lipophilicity for the range of compounds studied. The permeability of nimesulide across sublingual mucosa decreased with an increase of pH. The flux of ionized and unionized forms of nimesulide was determined to delineate the contribution of ionized and unionized species to the total flux. At low pH, the apparent flux was primarily contributed by unionized species; however, when the pH is increased beyond its pKa, the primary contributor to the apparent flux, nimesulide, is ionized species. The contribution of each species to the apparent flux was shown to be determined by the thermodynamic activity of ionized or unionized species. This study identified the roles of lipophilicity and thermodynamic activity in drug permeation across the sublingual mucosa. The findings can help guide the design of sublingual drug delivery systems with optimal pH and solubility.     

The Enhancing Effect of Ion-pairing on the Skin Permeation of Glipizide

The purpose of the present study was to investigate the permeation of glipizide (GP) and observe the effect of an interaction with amines as counter ions, including diethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, N-(2-hydroxylethyl) piperidine. Permeation experiments were performed in vitro, using rat abdominal skin as a barrier. The lipophilic donor system consisting of isopropyl myristate (IPM) and ethanol (EtOH; EI system, 8:2) produced a marked enhancement of GP flux through rat skin. All the amines investigated in this study had performed an enhancing effect on GP flux, and triethylamine had the most potent enhancing effect on GP in the vehicle IPM:EtOH = 8:2(w/w). In the presence of counter ions, the solubility of GP in the donor solution (IPM:EtOH = 8:2) was increased and the log K_o/w of GP was decreased, which may due to higher solubility of the GP in the IPM:EtOH = 8:2(w/w). ¹³C NMR spectroscopy was used to identify the ion-pairing formation between GP and the respective counter ion. It was surprising that all the four enhancers examined, such as isopropyl myristate, propylene glycol, N-methyl-2-pyrrolidone, azone, and oleic acid, had no enhancing effect on the percutaneous permeation of GP. This study showed that the formation of ion-pairs between GP and counter ions is a useful method to promote the skin permeation of GP.Key words: amine, counter ion, glipizide, ion-pair, percutaneous absorption     

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.     

Effect of Experimental Temperature on the Permeation of Model Diffusants Across Porcine Buccal Mucosa

The influence of experimental temperature on the permeability of model diffusants across porcine buccal mucosa was investigated in vitro. The permeability increased significantly as the experimental temperature was increased in increments of approximately 7°C. It was observed that the apparent permeability and temperature were related by an exponential relationship that conformed to the Arrhenius equation. Diffusants with higher lipophilicities—buspirone and bupivacaine—had lower activation energies for diffusion when compared to hydrophilic diffusants—antipyrine and caffeine. The activation energy for diffusion of the model diffusants decreased linearly with increasing distribution coefficients across porcine buccal mucosa. The results suggested that the buccal mucosa acts as a stronger barrier to the diffusion of hydrophilic diffusants than the lipophilic ones. The log-linear relationship between permeability and temperature indicates that temperature should be carefully controlled in diffusion experiments. These results also point to the possibility of developing heat-generating buccal delivery devices, especially for hydrophobic diffusants.     

Archaea on Human Skin

The recent era of exploring the human microbiome has provided valuable information on microbial inhabitants, beneficials and pathogens. Screening efforts based on DNA sequencing identified thousands of bacterial lineages associated with human skin but provided only incomplete and crude information on Archaea. Here, we report for the first time the quantification and visualization of Archaea from human skin. Based on 16 S rRNA gene copies Archaea comprised up to 4.2% of the prokaryotic skin microbiome. Most of the gene signatures analyzed belonged to the Thaumarchaeota, a group of Archaea we also found in hospitals and clean room facilities. The metabolic potential for ammonia oxidation of the skin-associated Archaea was supported by the successful detection of thaumarchaeal amoA genes in human skin samples. However, the activity and possible interaction with human epithelial cells of these associated Archaea remains an open question. Nevertheless, in this study we provide evidence that Archaea are part of the human skin microbiome and discuss their potential for ammonia turnover on human skin.     

Effect of Formulation Components on the In Vitro Permeation of Microemulsion Drug Delivery System of Fluconazole

The purpose of this study was to evaluate the effect of formulation components on the in vitro skin permeation of microemulsion drug delivery system containing fluconazole (FLZ). Lauryl alcohol (LA) was screened as the oil phase of microemulsions. The pseudo-ternary phase diagrams for microemulsion regions were constructed using LA as the oil, Labrasol (Lab) as the surfactant and ethanol (EtOH) as the cosurfactant. The formulation which showed a highest permeation rate of 47.15 ± 1.12 μg cm⁻² h⁻¹ and appropriate physicochemical properties was optimized as containing 2% FLZ, 10% LA, 20% Lab/EtOH (1:1), and 68% double-distilled water (w/w). The efficiency of microemulsion formulation in the topical delivery of FLZ was dependent upon the contents of water and LA as well as Lab/EtOH mixing ratio. It was concluded that the percutaneous absorption of FLZ from microemulsions was enhanced with increasing the LA and water contents, and with decreasing the Lab/EtOH ratio in the formulation. Candida albicans was used as a model fungus to evaluate the antifungal activity of the best formula achieved, which showed the widest zone of inhibition as compared to FLZ reference. The studied microemulsion formulation showed a good stability for a period of 3 months. These results indicate that the studied microemulsion formulation might be a promising vehicle for topical delivery of FLZ.     

Genistein对卵巢癌铂类耐药细胞株CP70增殖凋亡的影响及与细胞内ROS的相关性

目的:探讨Genistein对卵巢癌铂类耐药细胞CP70增殖、凋亡的影响及与细胞内活性氧水平的关系。方法:采用MTT法检测Genistein对CP70细胞增殖的影响;流式细胞仪分析不同药物处理后对细胞凋亡的影响,线粒体膜电位及细胞内ROS水平的变化情况。结果:Genistein对CP70细胞增殖表现出剂量和时间依赖性的抑制作用,并能诱导其凋亡;Genistein作用于CP70细胞后,可使其线粒体膜电位降低,并引发了细胞内ROS水平的显著升高;ROS抑制剂NAC预处理CP70细胞后,有效抑制了ROS的产生,并降低了细胞凋亡率,与未加NAC组相比差异有显著性(P0.05)。结论:Genistein能抑制铂类耐药卵巢癌细胞CP70的增殖,并促进其凋亡,这与细胞内ROS水平的升高有关,可能是Genistein抗肿瘤诱导细胞凋亡的机制之一。     

渗透促进剂对紫草素透过大鼠腹部皮肤的作用

研究不同浓度的丙二醇、氮酮和油酸对紫草素透过大鼠腹部皮肤的影响．实验结果表明,不同浓度的丙二醇对紫草素经皮渗透均有促进渗透作用,丙二醇浓度为10％时促渗效果最佳,油酸也具有促进紫草素的经皮渗透作用,但是在本实验中氮酮抑制紫草素的经皮渗透。     

N-乙酰葡萄糖胺对人皮肤成纤维细胞透明质酸合成的影响

目的:研究外源性N-乙酰葡萄糖胺(NAG)对人皮肤成纤维细胞(HSF)透明质酸(HA)合成的影响,探索其作为护肤品成分的可能性。方法:分别用MTT、ELISA、改良Schales法检测不同NAG浓度下HSF的增殖、HA合成和分泌、对NAG的吸收率,RT-PCR测定NAG对HSF细胞透明质酸合成酶(HAS)3种同工酶HAS1、HAS2和HAS3 mRNA的影响。结果:MTT和改良Schales法结果显示,随NAG浓度升高,HSF细胞的增殖受抑制程度增加,对NAG的吸收率降低;ELISA结果表明,HSF细胞HA的合成对NAG呈剂量依赖性促进,5mmol/L浓度最佳;RT-PCR结果显示,HAS3 mRNA的表达在NAG浓度为3mmol/L和5mmol/L出现上调。结论:NAG可促进HSF细胞HA的合成和分泌,可能机制是NAG作为原料或者通过上调HAS3 mRNA表达水平来增加HAS活性从而提高HA的合成,提示NAG作为护肤品成分的可能。     

Liposomes as Carriers for Verbascoside: Stability and Skin Permeation Studies

In this study the influence of liposomal incorporation on both the stability and the in vitro (trans) dermal delivery of verbascoside was evaluated. The effect of drug entrapment into vesicles on its radical scavenging activity was also studied. Liposomes were obtained from soy phosphatidylcholine and cholesterol according to the film hydration method. Stability of verbascoside-loaded vesicles was studied over 6 months. Results showed that verbascoside can be incorporated in liposomes (E%?=?57–66%), preventing its degradation. Stability studies (dynamic lager light scattering [DLLS] measurements and transmission electron microscopy [TEM] visualization) pointed out that vesicles were stable for 90 days and neither verbascoside leakage nor vesicle size alteration occurred during this period. The effects of vesicular incorporation on verbascoside diffusion through skin were investigated in vitro using newborn pig skin. Results showed that liposomes promoted drug accumulation into the stratum corneum but they did not give rise to any significant transdermal verbascoside delivery. Finally, results obtained from a 1, 1-diphenyl-2-pierylhydrazyl (DPPH) radical assay demonstrated that liposomes did not interfere with the radical scavenging activity of verbascoside.     

Effects of Niacin Restriction on Sirtuin and PARP Responses to Photodamage in Human Skin

Sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), NAD(+)-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3), actinic keratoses (SIRTs 2, 3, 5, 6, and 7) and squamous cell carcinoma (SIRTs 1-7). Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD(+), the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention.     

Factors Affecting Enhanced Permeation of Amphotericin B Across Cell Membranes and Safety of Formulation

The aim of this study was to determine amphotericin B (AmB) permeation across lipid bilayer membranes mounted on Transwell® and to observe the phagocytosis of the AmB and the AmB-lipid formulations by alveolar macrophage (AM) cell lines using a fluorescence microscope. The lipid bilayer membranes were prepared from phospholipid and ergosterol as well as phospholipid and cholesterol in a ratio (67:33 mol%). AmB-lipid formulations were prepared from AmB incorporated with four lipid derivatives during a lyophilization process. In vitro cytotoxicity studies were carried out on kidney cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide production by AMs exposed to these AmB-lipid formulations were determined by the Griess reaction. Phagocytosis of the AmB-lipid formulations was carried out using AM cells. The lipid bilayer membranes and AmB-lipid formulations were successfully prepared. In vitro cytotoxicity results showed less toxicity to kidney cells than pure AmB, and a 1,000-fold less production of nitric oxide by NR8383 cell lines was obtained when compared to lipopolysaccharide. Permeation results were two- to fivefold higher than for pure AmB in the ergosterol containing lipid bilayer and two- to fourfold higher than AmB in the cholesterol containing compositions, both of which were enough to kill the fungi according to their MICs and MFCs. AM phagocytosed the AmB-lipid formulations. We suggest that these products especially the AmB-sodium deoxycholate sulfate are potential candidates for targeting AM cells for the treatment of invasive pulmonary aspergillosis.     

Enhancement of Galantamine HBr Skin Permeation Using Sonophoresis and Limonene-Containing PEGylated Liposomes

This study aimed to investigate the effect of low-frequency sonophoresis (SN) and limonene-containing PEGylated liposomes (PL) on the transdermal delivery of galantamine HBr (GLT). To evaluate the skin penetration mechanism, confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) were employed. The application of SN led to more GLT penetration into and through the skin than GLT solution alone. The liposomes also improved GLT permeation, and 2% limonene-containing PL (PL-LI2%) exhibited the highest GLT permeation, followed by PL-LI1%, PL-LI0.1%, and PL. The CLSM images of PL-LI2% resulted in the highest fluorescence intensity of fluorescent hydrophilic molecules in the deep skin layer, and the rhodamine PE-labeled liposome membrane was distributed in the intercellular region of the stratum corneum (SC). PL-LI2% induced significant changes in intercellular lipids in the SC, whereas SN had no effect on intercellular lipids of the SC. DSC thermograms showed that the greatest decrease in the lipid transition temperature occurred in PL-LI2%-treated SC. SN might improve drug permeation through an intracellular pathway, while limonene-containing liposomes play an important role in delivering GLT through an intercellular pathway by increasing the fluidity of intercellular lipids in the SC. Moreover, a small vesicle size and high membrane fluidity might enhance the transportation of intact vesicles through the skin.     

Effects of an Antibacterial Soap on the Ecology of Aerobic Bacterial Flora of Human Skin

The effects of ad lib use of an antibacterial soap containing 1.0% trichlorocarbanilide and 0.5% trifluoromethyldichlorocarbanilide on the bacterial flora of six skin sites of 132 subjects were measured by comparison with the flora of 93 control subjects who avoided the use of topical antibacterials. Each subject was examined once. The test soap produced significant reductions in geometric mean counts of the total aerobic flora on the back, chest, forearm, calf, and foot; counts were also reduced in the axilla, but not to a significant extent. The overall reduction by the test soap on all sites was 62% (P < 0.001). Neither age nor sex influenced the effect of the soap on the flora. The antibacterial soap also reduced the prevalence of Staphylococcus aureus on the skin, mostly by virtually eliminating it from areas other than the axilla. Partial inhibition of the gram-positive flora was not accompanied by an increase in gram-negative species. The latter were found principally in the axilla; Klebsiella pneumoniae and Enterobacter aerogenes were the species most frequently found.     

Skin Innervation and Its Effects on the Epidermis

Sensory innervation of the skin subserves protective sensations for the body to prevent thermal and noxious injuries. Neurophysiologically, they belong to the categories of A and C fibers, usually with caliber less than one µm in diameter. Morphological demonstration of the terminals of these nerves in the epidermis has been recognized recently by sensitive immunocytochemistry and an axonal marker, the protein gene product 9.5 (PGP). PGP is a ubiquitin C-terminal hydrolase, which is abundantly present in the nervous system, and particularly enriched in the unmyelinated nerves. Sensory nerves positive for PGP arise from the dorsal root ganglion, pass through the dermis, parallel the epidermis-dermis border, penetrate the basement membrane, move vertically and upwards in the epidermis with tortuous course and knobby appearance, and finally terminate at the granular layers of the epidermis. In rodents, denervation of the skin results in degeneration of epidermal nerves within 48 h of nerve transection, and thinning of the epidermis. In humans, application of this technique to evaluate disorders of the peripheral nervous system makes study of the degeneration of sensory nerve terminals possible. Patients with sensory neuropathy had fewer epidermal nerves than normal subjects, consistent with the notion of distal axonopathy. This approach has the potential to evaluate human sensory neuropathy in temporal and spatial domains. In addition, the influences of epidermal denervation open a new field to explore the interactions between sensory nerves and keratinocytes.     

Regulating the Skin Permeation Rate of Escitalopram by Ion-pair Formation with Organic Acids

In order to regulate the skin permeation rate (flux) of escitalopram (ESP), ion-pair strategy was used in our work. Five organic acids with different physicochemical properties, benzoic acid (BA), ibuprofen (IB), salicylic acid (SA), benzenesulfonic acid (BSA), and p-aminobenzoic acid (PABA), were employed as counter-ions to regulate the permeation rate of ESP across the rabbit abdominal skin in vitro. The interaction between ESP and organic acids was characterized by FTIR and ¹³C NMR spectroscopy. Results showed that all organic acids investigated in this study performed a controlling effect on ESP flux. To further analyze the factors concerned with the permeation capability of ESP-acid complex, a multiple linear regression model was used. It is concluded that the steady-state flux (J) of ESP-acid complexes had a positive correlation with log K _o/w (the n-octanol/water partition coefficient of ion-pair complex) and pK _a (the acidity of organic acid counter-ion), but a negative correlation with MW (the molecular weight of ion-pair complex). The logK _o/w of ion-pair complex is the primary one in all the factors that influence the skin permeation rate of ESP. The results demonstrated that organic acid with appropriate physicochemical properties can be considered as suitable candidate for the transdermal drug delivery of escitalopram.     

Positive Promoting Effects of Smilax China Flower Absolute on the Wound Healing/Skin Barrier Repair-Related Responses of HaCaT Human Skin Keratinocytes

Smilax china (SC) has pharmacological effects including anti-inflammatory activity, but its effects on skin wound healing and skin barrier function have not been investigated. Here, we investigated the effects of absolute extracted from SC flowers (SCF) on skin wound healing-linked responses and functional skin barrier proteins using human epidermal keratinocytes (HaCaT cells). SCF absolute contained 20 components and was non-toxic to HaCaT cells. The absolute increased the proliferation, migration, and sprout outgrowth of HaCaT cells, and enhanced the activations of serine/threonine-specific protein kinase and extracellular signal-regulated kinase1/2. In addition, it increased the syntheses of type I and IV collagens and the expressions of skin barrier proteins (filaggrin and loricrin). These results indicate SCF absolute may has positive effects on skin wound healing by accelerating keratinocyte migration and proliferation activities and collagen synthesis, and on skin barrier function by upregulating barrier proteins in keratinocytes. We suggest SCF absolute to be considered as a potential means of promoting skin wound and barrier repair.     

In Vitro Percutaneous Permeation and Skin Accumulation of Finasteride Using Vesicular Ethosomal Carriers

In order to develop a novel transdermal drug delivery system that facilitates the skin permeation of finasteride encapsulated in novel lipid-based vesicular carriers (ethosomes)finasteride ethosomes were constructed and the morphological characteristics were studied by transmission electron microscopy. The particle size, zeta potential and the entrapment capacity of ethosome were also determined. In contrast to liposomes ethosomes were of more condensed vesicular structure and they were found to be oppositely charged. Ethosomes were found to be more efficient delivery carriers with high encapsulation capacities. In vitro percutaneous permeation experiments demonstrated that the permeation of finasteride through human cadaver skin was significantly increased when ethosomes were used. The finasteride transdermal fluxes from ethosomes containing formulation (1.34 ± 0.11 μg/cm²/h) were 7.4, 3.2 and 2.6 times higher than that of finasteride from aqueous solution, conventional liposomes and hydroethanolic solution respectively (P < 0.01).Furthermore, ethosomes produced a significant (P < 0.01) finasteride accumulation in the skin, especially in deeper layers, for instance in dermis it reached to 18.2 ± 1.8 μg/cm². In contrast, the accumulation of finasteride in the dermis was only 2.8 ± 1.3 μg/cm² with liposome formulation. The study demonstrated that ethosomes are promising vesicular carriers for enhancing percutaneous absorption of finasteride.