Guinea Pig Skin Research Articles

In vitro transdermal permeation of fenoterol hydrobromide

The aim of this study was to determine if transdermal penetration of fenoterol, a β-agonist drug, could be enhanced and controlled by formulation modification and formulation of transdermal patches. Pre-formulation studies were performed to determine the feasibility of a transdermal dosage form of fenoterol. Penetration of fenoterol was determined using the hairless guinea pig skin with unjacketed Franz diffusion cell. Transdermal patches were formulated using drug in-adhesive technique. Several enhancers were investigated for fenoterol skin penetration. Transcutol–oleic acid co-solvent gives the highest drug flux among all tested liquid formulations. Pretreatment of the skin with oleic acid 2h before patch application significantly increases drug diffusion. Cis-oleic acid gives best results compared to oleic acid. Azone derivative (1-dodecyl-2-pyrrolidinone) gives the highest drug diffusion amongst all tested enhancers. Results of this study show the feasibility of using fenoterol formulated in transdermal delivery system in the treatment of chronic asthma to improve patient compliance, bioavailability and reduce the inter-subject variability.

Microemulsions as vehicles for topical administration of voriconazole: formulation and in vitro evaluation

This work was undertaken to investigate microemulsion (ME) as a topical delivery system for the poorly water-soluble voriconazole. Different ME components were selected for the preparation of plain ME systems with suitable rheological properties for topical use. Two permeation enhancers were incorporated, namely sodium deoxycholate or oleic acid. Drug-loaded MEs were evaluated for their physical appearance, pH, rheological properties and in vitro permeation studies using guinea pig skin. MEs based on polyoxyethylene(10)oleyl ether (Brij 97) as the surfactant showed pseudoplastic flow with thixotropic behavior and were loaded with voriconazole. Jojoba oil-based MEs successfully prolonged voriconazole release up to 4 h. No significant changes in physical or rheological properties were recorded on storage for 12 months at ambient conditions. The presence of permeation enhancers favored transdermal rather than dermal delivery. Sodium deoxycholate was more effective than oleic acid for enhancing the voriconazole permeation. Voriconazole-loaded MEs, with and without enhancers, showed significantly better antifungal activity against Candida albicans than voriconazole supersaturated solution. In conclusion, the studied ME formulae could be promising vehicles for topical delivery of voriconazole.

Disruption of tyrosinase glycosylation by N-acetylglucosamine and its depigmenting effects in guinea pig skin and in human skin

Disruption of tyrosinase glycosylation by N-acetylglucosamine and its depigmenting effects in guinea pig skin and in human skin

Evaluation of a novel non-penetrating electrode for use in DNA vaccination.

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination.

UVB-induced suppression of resistance to Mycobacterium tuberculosisin guinea pigs after BCG vaccination: cellular and molecular changes in the skin (99.19)

Abstract Previously, we reported that ultraviolet-B radiation (UVR) suppressed BCG vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs as it induced immuno-suppression and increased pathogenesis. In this study, we investigated the cellular and molecular changes within the guinea pig epidermis.The dorsal skin of guinea pigs was exposed to 5.6 kJ/m2 UVR and the epidermal cells were harvested 24 h later. Although UVR did not induce any gross morphological changes in the epidermis, a significant increase in the angular nucleated keratinocytes and a reduction in the number of cells with high nuclear to cytoplasmic ratio and binucleated cells were observed. Flow cytometry studies indicated that the proportions of CD3+ and CD4+ T cells increased in the epidermis while spleen showed a decrease in the CD3+, CD4+, CD8+, CD25+, MHC class II+ and CD14+ cells. Upregulation of IL-10, IL-4, IL-12p40, TGF-β, IL-1β and INOS mRNA expression was observed in the UV-irradiated epidermal cells. In addition, IL-4, IL-12p40, IFN-γ, and IL-1β mRNAs were increased in the spleen cells after UVR. Furthermore, culture supernatants from in vitro UV-irradiated keratinocytes reduced the MHC class II expression in the peritoneal cells and reduced the spleen cell proliferation to ConA. Future studies will address the identity of the suppressive factors induced by UVR and the systemic effect of culture supernatants from UV-irradiated epidermal cells on M. bovis BCG or M. tuberculosis infection.

Inhibitory Effect of Rose Hip (Rosa caninaL.) on Melanogenesis in Mouse Melanoma Cells and on Pigmentation in Brown Guinea Pigs

The compounds present in rose hips exerting an inhibitory action against melanogenesis in B16 mouse melanoma cells were investigated by dividing an aqueous extract of rose hips (RE) into four fractions. The 50% ethanol eluate from a DIAION HP-20 column significantly reduced the production of melanin and was mainly composed of procyanidin glycosides. We also found that this 50% ethanol eluate reduced the intracellular tyrosinase activity and also had a direct inhibitory effect on tyrosinase obtained as a protein mixture from the melanoma cell lysate. We also investigated the effect of orally administering RE on skin pigmentation in brown guinea pigs, and found that the pigmentation was inhibited together with the tyrosinase activity in the skin. These data collectively suggest that proanthocyanidins from RE inhibited melanogenesis in mouse melanoma cells and guinea pig skin, and could be useful as a skin-whitening agent when taken orally.

Influence of MSCs injection on the expressions of SOD, MDA on photoaging skin in guinea pigs

Objective To observe the expressions of SOD, MDA on photoaging skin when it was repaired by bone marrow mesenchymal stem cells (MSCs) . Methods MSCs were isolated and cultured according to density gradient centrifugation. The model of photoaging skin was prepared in guinea pigs by exposure to UVB lamp. The animals were divided into the normal group, model group and treatment group. The expressions of SOD and MDA were detected in skin tissue. Results The expression of MDA in treatment group was significantly decreased compared with that of the model ( P ＜ 0. 01 ), but higher than that in the normal group. The expression of SOD in treatment group was significantly higher than that of model group ( P ＜ 0. 01 ), but lower than that of normal group. Conclusions MSCs can remove the harmful substances such as oxygen free radicals and keep the antioxidant balance of the system. MSCs play the role on the treatment of skin photoaging. Key words: MSCs; Skin photoaging; Repair

Development and <i>In vitro</i> Evaluation of Betahistine Adhesive-Type Transdermal Delivery System

Purpose: To develop a transdermal betahistine (BTH) delivery system using different pressure sensitive adhesives (PSAs) including acrylics, polyisobutylene and styrenic rubber solution.Methods: Formulations were prepared by solvent casting and adhesive transfer method. PSAs - acrylate vinylacetate (AVA), hydrophilic acrylate (HA), acrylic non-curing (ANC), polyisobutylene (PIB), and tackified styrenic rubber solution (TSR) - were evaluated for their suitability in terms of miscibility, maximum drug loading, effect on tack property and in vitro permeation through excised guinea pig skin. Furthermore, one of the PSAs was tested in relation to effect of penetration enhancers on tack property, in vitro permeation, in vivo patch adhesion performance and stability.Results: Only formulations prepared with AVA and HA were stable. Increased drug loading in these PSAs significantly reduced tack. In vitro permeation data across guinea pig skin demonstrated that BTH flux from from the formulation containing HA (F1) was significantly (p < 0.001) higher than that containing AVA (F2). Formulations containing 2 % enhancer showed good tack. Specifically, the formulation containing 2 % oleic acid as enhancer not only showed the highest permeation but also good tack property, non-irritancy for up to 36 h and stability under accelerated conditions.Conclusion: The formulation containing HA as the PSA and 2 % oleic acid as enhancer demonstrated a good potential for further development to an adhesive-type transdermal delivery system for BTH.Keywords: Meniere’s syndrome, Transdermal delivery, Betahistine, Pressure-sensitive adhesives, Penetration enhancersTropical Journal of Pharmaceutical Research December 2010; 9 (6): 516-524

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Characterization of Transcutaneous Protein Delivery by a Hydrogel Patch in Animal, Human, and Tissue-Engineered Skin Models

The development of a simple, easy-to-use, and noninvasive vaccination system is in high demand. For transcutaneous immunization (TCI), we previously reported that a hydrogel patch was an effective TCI device that accelerates antigen penetration through the stratum corneum in mouse and rat models. The present study was performed to characterize the transcutaneous protein delivery induced by the hydrogel patch in mouse, guinea pig, LWD pig, human, or tissue-engineered skin models, and to assess the activity of proteins delivered into the skin. The hydrogel patch promoted protein penetration through the stratum corneum in all skin models, indicating that our original hydrogel patch might have practical application for use in humans. In addition, proteins delivered into the skin by the hydrogel patch retained their activity, suggesting that the hydrogel patch is applicable for the delivery of therapies for other diseases as well. On the basis of these results, translational research in human is now in progress.

Fermentative Production of Harpin Protein by a Recombinant &lt;i&gt;E. coli&lt;/i&gt; Strain and its Toxicity Determination

Harpin protein was produced by a recombinant E. coli BL21(DE3)/pET30a(+)hrpNEcc in a 1-ton fermentor, and acute oral toxicity test in rats and guinea pig skin sensitization test for Harpin protein were carried out in order to determine its toxicity. The experimental results show that the protein production is up to 4.78 g/L after 16-18 h of fermentation, and the production cost of this protein is 1.75 yuan/g. Harpin protein produced by the recombinant E. coli strain is non-toxic (LD50&gt;5000mg/kg) and has a low allergenicity (rate of sensitization is 0%). The results obtained in this study lay the foundation of the industrial manufacturing and the application on agriculture for Harpin protein.

Final Report of the Safety Assessment of Kojic Acid as Used in Cosmetics

Kojic acid functions as an antioxidant in cosmetic products. Kojic acid was not a toxicant in acute, chronic, reproductive, and genotoxicity studies. While some animal data suggested tumor promotion and weak carcinogenicity, kojic acid is slowly absorbed into the circulation from human skin and likely would not reach the threshold at which these effects were seen. The available human sensitization data supported the safety of kojic acid at a use concentration of 2% in leave-on cosmetics. Kojic acid depigmented black guinea pig skin at a concentration of 4%, but this effect was not seen at 1%. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that the 2 end points of concern, dermal sensitization and skin lightening, would not be seen at use concentrations below 1%; therefore, this ingredient is safe for use in cosmetic products up to that level.

Inhibitory Effects of Neo Muscat Grape Vine Extracts on Melanin Biosynthesis

Neo Muscat grape vine extracts significantly inhibited melanin production in melanocyte stimulating hormone-stimulated B16 cells. They also exhibited tyrosinase inhibitory activity and reduced the levels of intra-cellular tyrosinase. Moreover, when the extracts were treated with UV B-induced hyper-pigmented brown guinea pig skin, depigmenting activity was observed, but with no visible edema. These results indicate that the vine extracts of Neo Muscat grapes might prove useful as a skin depigmenting agent.

Inhibitory effect of N-adamantyl-3,4-dihydroxybenzamide on melanogenesis in melan-a cells and brown guinea pigs

To find novel depigmenting agents, a new synthetic compound, N-adamantyl-3,4-dihydroxybenzamide (NADB) was produced and the effects on melanogenesis were investigated. Our results showed that NADB reduced melanin synthesis in a dose-dependent manner in melan-a cells. Tyrosinase activity was also reduced by NADB treatment in melan-a cells. However, NADB did not inhibit tyrosinase activity directly in a cell-free system. Treatment of melan-a cells with NADB caused a marked decrease in protein and mRNA levels of tyrosinase along with tyrosinase-related protein 1 and dopachrome tautomerase. To determine whether NADB reduces skin pigmentation, the dorsal skin of brown guinea pigs was shaved and irradiated with UV for 3 weeks using a solar simulator. Then NADB (2 or 1% in propylene glycol:ethanol:water = 5:3:2) was applied topically twice daily for 4 weeks. Visual inspection and Fontana-Masson staining both demonstrated that NADB resulted in lower skin pigmentation and total epidermal melanin in comparison to vehicle-treated areas. These findings suggest that NADB is useful in the treatment of hyperpigmentation.

Long-term persistence after acute Q fever of non-infective Coxiella burnetii cell components, including antigens

Previous studies of inciting factors for a prolonged post-infection fatigue syndrome after Q fever (variously termed QFS or Q fever associated CFS/ME in the literature) showed that after the acute infection a high proportion of asymptomatic and QFS patients had Q fever antibody and also low levels in PBMC and bone marrow of Coxiella burnetii (C.b.) DNA with PCR assays directed against three different target sequences in different parts of the coxiella genome. Attempts to isolate a strain of C.b. in A/J mice, and cell culture from PCR positive PBMC and bone marrow were consistently negative. The detailed composition of the persisting coxiella residues remains to be defined. To retest and provide detailed results on selected PCR positive samples from the Birmingham Q fever outbreak patients tested by a highly sensitive method to detect viable organisms and to determine the nature of the residual coxiella cell components. Laboratory case study. NOD/SCID mice were inoculated with samples from the 1989 Q fever outbreak in Birmingham and followed for evidence of infection and the presence of coxiella DNA and specific antigens in spleen and liver macrophages. A significant, unexpected finding of specific antigen was followed by assessment of its ability to provoke production of inflammatory and non-inflammatory cytokines in mice, in THP-1 human macrophage cell cultures and to induce inflammatory lesions in the skin of guinea pigs hyperimmunized against Q fever vaccine. Culture of samples from 10 Birmingham Q fever patients in NOD/SCID mice, 12 years from infection did not yield viable Coxiella burnetii, as shown earlier. However complexes of material with coxiella antigens were found in mouse spleens in all cases but in significantly greater amounts in samples from those with post Q fever fatigue syndrome. The antigenic complexes [now designated 'immunomodulatory complexes' (IMC)] were shown to stimulate cytokine release in the mice and in the THP-1 macrophages and to provoke an inflammatory reaction on intradermal injection into the skin of Q fever hyperimmunized guinea pigs. The study identifies a non-infective complex of C.b. antigens able to survive in the host and provoke aberrant humoral and cell medicated immunity responses - a possible pathogenic link between initial infection and a subsequent long-term post Q fever fatigue syndrome.

Hexavalent chromium induced ROS formation, Akt, NF-κB, and MAPK activation, and TNF-α and IL-1α production in keratinocytes

In certain cell types, it has been found that, hexavalent chromium could increase ROS formation, activate cell signaling and stimulate the release of cytokines. But, in keratinocytes, these effects have not yet fully been demonstrated. Our aim is to observe the above effects of hexavalent chromium on keratinocytes. By utilizing HaCaT cells and the skin of albino guinea pigs, we showed that hexavalent chromium could increase ROS formation, activate the Akt, NF-kB, and MAPK pathways as well as increase the production of cytokines, including TNF-α and IL-1α. The release of these cytokines from keratinocytes is considered to be a key participant in the pathogenesis of contact hypersensitivity. Among cement workers, chromium hypersensitivity is an important occupational skin disease issue. Therefore, the observations of our study help us better understand the role of hexavalent chromium on the development of chromium hypersensitivity, which might provide clues for clinicians in the development of chemopreventative agents for the prevention of chromium hypersensitivity among cement workers.

Transdermal Delivery of Naltrexol and Skin Permeability Lifetime after Microneedle Treatment in Hairless Guinea Pigs

Controlled-release delivery of 6-beta-naltrexol (NTXOL), the major active metabolite of naltrexone, via a transdermal patch is desirable for treatment of alcoholism. Unfortunately, NTXOL does not diffuse across skin at a therapeutic rate. Therefore, the focus of this study was to evaluate microneedle (MN) skin permeation enhancement of NTXOL's hydrochloride salt in hairless guinea pigs. Specifically, these studies were designed to determine the lifetime of MN-created aqueous pore pathways. MN pore lifetime was estimated by pharmacokinetic evaluation, transepidermal water loss (TEWL) and visualization of MN-treated skin pore diameters using light microscopy. A 3.6-fold enhancement in steady-state plasma concentration was observed in vivo with MN treated skin with NTXOL.HCl, as compared to NTXOL base. TEWL measurements and microscopic evaluation of stained MN-treated guinea pig skin indicated the presence of pores, suggesting a feasible nonlipid bilayer pathway for enhanced transdermal delivery. Overall, MN-assisted transdermal delivery appears viable for at least 48 h after MN-application.

Tranexamic acid can treat ultraviolet radiation-induced pigmentation in guinea pigs

Exposure of human skin to ultraviolet (UV) radiation causes erythema and pigmentation. Guinea pigs are recognized as having pigmentation similar to that of humans, after exposure to UV radiation. Recently, the curative effect of oral treatment of tranexamic acid in skin pigmentation was confirmed by many doctors in China. Our aim was to investigate the effects of tranexamic acid on pigmentation in the skin of guinea pigs exposed to UV irradiation and its possible mechanism. Guinea pigs were exposed to UVB radiation (60 min per day) for 30 days, skin pigmentation was clearly observed after that. 30 days later, tranexamic acid (5 mg/mL) was intradermally injected into the exposed regions every day after radiation to prevent or inhibit the pigmentation process. After 30 days the skin was removed and stained by HMB45 immunohistochemistry and the Masson Fontana-Ponceau method. HMB45 positive expression melanocytes in the basal layer of UV-exposed epidermis showed no significant differences in the regions to which tranexamic acid solutions had been applied compared with the control. But the melanin content was significantly reduced. We concluded that tranexamic acid has no effect on the number of melanocytes, it is likely that it affects the function of melanocytes, to inhibit the melanin expression in order to lighten moth patches.

Inhibitory effects of 5-chloroacetyl-2-piperidino-1,3-selenazole, a novel selenium-containing compound, on skin melanin biosynthesis

Increased production and accumulation of melanin leads to many hyperpigmentation disorders such as melasma, freckles and geriatric pigment spots. Thus, there is a need for the development of depigmenting agents. Based on our previous reports, selenium derivatives as anti-melanogenic lead compounds could be very important. The aim of this study was to investigate the depigmenting effect of novel selenium-containing compounds. The inhibitory effects of 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), a novel selenium-containing compound, on melanogenesis were investigated in B16F10 melanoma cells and cultured brownish guinea pig skin tissue with alpha-melanocyte-stimulating hormone stimulation. We found that CS1 inhibited melanin production in B16F10 cells by suppressing tyrosinase activity and its protein expression. In addition, Western blotting analysis revealed that CS1 suppressed the expression of tyrosinase-related protein (TRP)-1 and TRP-2. Therefore, the depigmenting effect of CS1 might have been due to inhibition of tyrosinase activity and expression of melanogenic enzymes. Furthermore, CS1 had inhibitory effects on melanin biosynthesis of primary cultured skin of brownish guinea pig. The results suggested that CS1 could be a useful candidate for the treatment of skin hyperpigmentation.

Morphological effect of lipid carriers on permeation of lidocaine hydrochloride through lipid membranes

We have studied how the transdermal delivery of lidocaine hydrochloride (LHC) is affected by the morphology of lipid carriers, liposomes and micelles, having the same lipid composition of 1-stearoyl-sn-glycero-3-phosphocholine (LPC) and cholesteryl hemisuccinate (CHEMS). In vitro drug permeation study, carried out on guinea pig skin, has revealed that the liposomes made of LPC and CHEMS significantly enhance the permeation rate of entrapped LHC; by contrast, the mixed micelles with the same composition decrease the degree of delivering co-existing LHC. Basically, we have also investigated the release kinetics of LHC through the cellulose membrane and found that both liposomes and micelles have a similar releasing profile. To experimentally demonstrate this unique behavior, we have observed the fluidity of stratum corneum liposomal membranes in the presence of either our liposomes or micelles. From this study, we have found that LPC/CHEMS liposomes fluidize the lipid membrane of stratum corneum lipids; however, lipid micelles rather make the membrane rigid. These findings highlight that controlling the morphology of drug carriers provides us with a means to modulate the permeability of encapsulated drug molecules.