Guinea Pig Skin Research Articles

Effect of chemical enhancers and conducting gels on iontophoretic transdermal delivery of cromolyn sodium

In vitro iontophoretic transdermal delivery (ITD) at a continuous current density of 0.1 mA/cm 2 of cromolyn sodium (CS) across hairless guinea pig skin (HGP) was studied with and without enhancers. CS was quantitated by a sensitive HPLC method. At a saturated drug concentration of CS in 80:20 mixture of ethanol:6.66 mM acetate buffer, an overall flux enhancement compared to buffer alone was observed. This enhancement was determined to be an additive effect of iontophoresis and ethanol. Chemical enhancers, such as anionic surfactants (e.g. sodium dodecyi sulfonate and sodium lauryl sulfate), inhibited the permeation of CS ions at concentration less than or equal to the critical micelle concentration. No significant change in flux ( P > 0.05) was observed when propylene glycol was added at different concentrations to yield solutions with varying dielectric constants in the aqueous donor medium. Aqueous glycerol solution was ineffective for ITD. Conducting gels of ionic polymers, polyjel-HV ® and lubrijel-MS ®, decreased the flux of CS significantly ( P < 0.05). Non-ionic polymers such as hydroxypropyl cellulose (Klucel-LF ®) and polyvinyl alcohol did not affect the flux and may be used for ITD of CS from a transdermal patch. An optimized solution formulation for CS was incorporated in a commercially available electropatch, from which delivery rates up to 46 ± 5 μg/cm 2hr −1. were achieved. The optimized formulation of CS provided about 18 fold higher flux compared to an unoptimized formulation from the electropatch. Stainless steel or Ag/AgCl electrodes showed no difference in the flux of CS from the patch. Therapeutic levels of CS in humans may be achieved by this modem non-invasive drug-delivery route.

Effect of in vivo desensitization to leukotriene B4 on eosinophil infiltration in response to C5a in guinea‐pig skin

1. The effect of in vivo desensitization to leukotriene B4 (LTB4) on eosinophil infiltration in response to recombinant C5a was examined in guinea-pig skin. 2. LTB4 (10-300 ng) and C5a (1-10 micrograms) caused a dose-dependent increase in the levels of eosinophil peroxidase activity (a measure of eosinophil infiltration) 4 h after injection into guinea-pig skin. Leukotriene B4 and C5a were approximately equipotent on a molar basis. Platelet activating factor (0.01-10 micrograms) also caused eosinophil accumulation but was much less active than LTB4 or C5a. 3. 20-Hydroxy-LTB4 caused a dose-dependent desensitization of eosinophil responses to LTB4 (ED50 = 1.6 micrograms kg-1, s.c.) and partially reduced responses to C5a. At a dose of 20-hydroxy-LTB4 (10 micrograms) which inhibited responses to LTB4 completely, responses to C5a were reduced by 56.5 +/- 1.8% (n = 5). The structurally related metabolite of 20-hydroxy-LTB4, 20-carboxy-LTB4, which does not cause desensitization to the effects of LTB4, did not inhibit eosinophil infiltration in response to C5a. 4. The LTB4 receptor antagonist, SC-41,930 (10 mg kg-1, p.o.), also inhibited eosinophil accumulation in response to C5a by 63.0 +/- 3.9% (n = 5) at a dose which inhibited responses to LTB4 by 86.5 +/- 1.9% (n = 5). 5. These data indicate that eosinophil infiltration in response to C5a may, in part, be mediated by the generation of secondary chemotactic factors such as LTB4.

Investigation of the endogenous chemoattractants involved in 111In‐eosinophil accumulation in passive cutaneous anaphylactic reactions in the guinea‐pig

1. Eosinophil accumulation and plasma extravasation are features of type I allergic responses. In an attempt to characterize the mediators of these responses, we have examined the local accumulation of 111In-eosinophils and leakage of 125I-human serum albumin (125I-HSA) during passive cutaneous anaphylaxis (PCA) reactions and in response to defined inflammatory mediators in the guinea-pig. Animals were passively sensitized by intradermal injection of anti-bovine gamma globulin antibody (50 microliters, 1/50 dilution). After 20-24 h, animals were injected intravenously with 111In-eosinophils and 125I-HSA for the measurement of cell accumulation and plasma leakage, respectively. 2. When injected into sensitized sites, antigen caused a dose-related increase in the accumulation of 111In-eosinophils and plasma leakage in guinea-pig skin. Time course experiments over 24 h revealed that the maximal rate of 111In-eosinophil accumulation occurred over the first 90 min, with little accumulation at later time points. Plasma leakage was completed within the first 30 min after challenge. Responses to the mast cell degranulator, compound 48/80, exhibited very similar responses to the PCA reaction. 3. Co-injection of antigen with the PAF antagonist, WEB 2086 (10(-7) mol/site) or the 5-lipoxygenase inhibitor, PF 5901 (10(-7) mol/site) did not significantly alter the accumulation of 111In-eosinophils or plasma leakage, whereas these drug doses abolished responses to exogenous PAF (10(-9) mol/site) and arachidonic acid (AA, 3 x 10(-8) mol/site), respectively. The H1 receptor antagonist chlorpheniramine (2.5 x 10(-8) mol/site) did not reduce antigen-induced 111In-eosinophil accumulation. Drug combinations were also injected with antigen into sensitized sites, but were unable to reduce "'In-eosinophil accumulation.4. These results indicate that anaphylactic eosinophil accumulation in this model involves mediators other than histamine, PAF or lipoxygenase products. This is in contrast to plasma leakage in this reaction, which can be abolished by a combination of antagonists blocking these mediators.

Substance P-induced leukotriene B 4 release from guinea pig skin in vitro

Substance P-induced leukotriene B 4 release from guinea pig skin in vitro

Expression of fibroblast growth factor receptor 1 during wound healing in guinea pig skin

Expression of fibroblast growth factor receptor 1 during wound healing in guinea pig skin

Phospholipids (PL) in the Arthus reaction sites induced in guinea-pig skin

Phospholipids (PL) in the Arthus reaction sites induced in guinea-pig skin

Updated toxicology test methods for new industrial chemicals: Implications for regulatory acceptance of in vitro alternatives now and in the future

Toxicology studies submitted under the Notification of New Substances Regulations are conducted according to standard (‘Annex V’) methods first published in 1984. In December 1992, updated methods became available that indicate where progress has been made in developing in vitro alternatives to animal testing which are acceptable to the regulatory authority (Health and Safety Executive). Although some changes reflect concern for animal welfare (e.g. the introduction of the fixed-dose method), for most toxicological endpoints an animal test remains obligatory. The major advance in the potential use of in vitro methods is as part of a tiered approach to irritancy testing. The updated method for eye irritation, for example, allows positive results in ‘well-validated alternative studies’ to override the need for an animal test. The substance is then classified as irritant and labelled with R41. The challenge facing such methods is to achieve full validation, so that negative data will be acceptable to the regulatory authorities. There are other challenges: a validated in vitro screen for developmental toxicity is needed for new substances, as are methods for detecting respiratory sensitizers and for replacing guinea pig skin sensitization tests.

Structural basis of hairless guinea pig skin for models of cutaneous biology

Structural basis of hairless guinea pig skin for models of cutaneous biology

Optimization of iontophoretic transdermal delivery of a peptide and a non-peptide drug

In vitro iontophoretic transdermal delivery (ITD) of a tripeptide, enalaprilat (EP) and a non-peptide, cromolyn sodium (CS), across frozen hairless guinea pig (HGP) skin were investigated. Parameters for optimization of ITD included the influence of ionic strength (μ), buffer type and size, drug loading in the donor and the effect of pH. Drug permeation into the receptor compartment was monitored using HPLC assay methods developed for the study. An optimum μ of 6.66 mM in acetate buffer was found necessary for efficient ITD of CS. An exponential decrease in the flux of CS was observed with an increasing μ. Buffer ions larger than acetate ions inhibited the transport of CS ions. With an increase in the donor concentration of CS, a hyperbolic relationship for the increase in flux was observed. For EP, permeation was not detectable when μ was increased to greater than 31 mM in phosphate-buffered solution. With an increase in pH above the p K a1 (3.55) for EP, a linear decrease in flux was observed. Higher drug loading of EP in the donor compartment provided better permeation. Effect of freezing of HGP skin on the iontophoretic delivery of EP and CS was also evaluated. Flux values for either of the drugs studied were similar when frozen or fresh skins were used. Reversibility studies indicated that no gross current induced permeation changes occurred with the HGP skin. Passive permeation of either of the drugs investigated was negligible.

Pseudo-acylceramide with linoleic acid produces selective recovery of diminished cutaneous barrier function in essential fatty acid-deficient rats and has an inhibitory effect on epidermal hyperplasia.

Pseudo-acylceramides with different acyl properties were investigated for their capacity to restore diminished barrier function in essential fatty acid-deficient rats. Daily topical applications of synthetic pseudo-acylceramides containing ester-linked linoleic acid caused a dose-dependent, significant reduction of transepidermal water loss (TEWL). Both other pseudo-acylceramides with ester-linked oleic acid or saturated alkyl chains and ordinary ceramides exhibited a poor effect on recovery of TEWL. Furthermore, pseudoceramide containing ether-linked linoleic acid, which is biologically inactive in terms of degradation by hydrolytic enzymes, also induced a significant and similar increase in the barrier function. This restoration of barrier function by pseudo-acylceramides with linoleic acid was accompanied by suppressed DNA synthesis in the EFAD rat epidermis. In UVB-irradiated guinea pig skin, topical applications of the pseudo-acylceramides with linoleic acid immediately after the exposure significantly reduced epidermal hyperplasia, secondary to markedly diminished barrier disruption, whereas linoleic acid itself did not. A comparison of both the anti-hyperplasia and the barrier recovery effects in the series of pseudo-ceramide derivatives examined revealed that the suppressive effect on the induced epidermal hyperplasia was paralleled by the recovery of the barrier defect in EFAD rats. These findings directly suggest that acylceramide with an ester-linked linoleic acid has an essential role in the epidermal permeability barrier.

Effect of a neurokinin-1 (NK 1) receptor antagonist on oedema formation induced by tachykinins, carrageenin and an allergic response in guinea-pig skin

The effect of the neurokinin-1 (NK 1) receptor antagonist RP67580 in modulating inflammatory oedema formation has been investigated in guinea-pig skin. Oedema formation was measured over 30 min by the extravascular accumulation of intravenously-injected 125l-albumin in the anaesthetised guinea-pig. RP67580 was injected intradermally with the agents under test. Intradermal RP67580 (10 nmol/site) inhibits oedema formation induced by substance P (30 pmol) and neurokinin A (100 pmol), but not that induced by bradykinin (10–1000 pmol) or histamine (10 nmol). Substance P-induced oedema formation is similar in control (saline) and mepyramine (histamine H 1 receptor antagonist) pretreated guinea-pigs suggesting a minimal involvement of histamine in substance P induced oedema formation in guinea-pig skin. Oedema formation induced by intradermal carrageenin (0.2%) was not inhibited by RP67580 (1–10 nmol). A significant but partial inhibition of oedema formation induced in a passive cutaneous anaphylaxis (PCA) response was observed. The oedema formation in the PCA was inhibited 50% by mepyramine pretreatment but in the presence of mepyramine no further inhibition of the PCA response by RP67580 was observed.

Epidermal lipids and the natural history of hydrofluoric acid (HF) injury

To explain several fortuitous observations, we hypothesized that there is a naturally occurring lipid ‘barrier’ to HF injury in guinea-pig skin and sought to characterize both the barrier and its role in the natural history of such injuries. Under anaesthesia, the dorsal trunk skin of groups of guinea-pigs was gently clipped of hair, washed with chloroform, soap and water, acetone or nothing (controls), and examined histologically for the presence of neutral lipid. Thereafter, in animal groups similarly washed, 1.5 in × 1.5 in (38 mm × 38 mm) areas were exposed to 40 per cent HF for up to 50 min and: (a) mean percentages of exposed areas with gross necrosis 5 days postinjury plotted on dose—response curves; or (b) less than 4 h after exposure to HF, intra-aortic India ink was injected and skin specimens examined to discern depth of ischaemia and necrosis. In contrast to controls, washing reduced neutral lipid in epidermis and significantly (at P < 0.001) increased susceptibility to injury by HF. With very rare (but interesting) exceptions, HF injury was found to be full thickness in depth with ischaemia and coagulative necrosis. In this study, development of guinea-pig skin necrosis due to HF was typically an ‘all-or-nothing’ ‘barrier-penetration’ phenomenon relating as much to the integrity of an epidermal lipid barrier as to the duration and intensity of noxious exposure.

Evaluation of a cultured skin equivalent as a model membrane for iontophoretic transport

Recently, cultured skin equivalents of human origin have become available. These materials, alson known as living skin equivalents (LSEs), are made up of human cells and matrix equivalents normally present in skin. LSEs closely resemble human skin, consisting of a dermis and a stratified epidermis with a well-differentiated stratum corneum (Ponec et al., Toxicity screening of N-alkylazacycloheptan-2-one derivatives in cultured human skin cells: structure-toxicity relationships. J. Pharm. Sci., 78 (1989) 738–741; Ponec et al., Nitroglycerin and sucrose permeability as quality markers for reconstructed human epidermis Skin Pharmacol., 3 (1990) 126–135) but lack the structural appendages found in human skin (e.g., hair follicles, sweat glands). This paper will discuss a preliminary evaluation of a cultured skin equivalent as a model membrane for in vitro screening experiments of iontophoretic candidates. Transport rates of pindolol hydrochloride, synthetic salmon calcitonin and benzyl alcohol are compared across both guinea pig skin (a common in vitro model membrane) and the cultured skin equivalent. Preliminary results with a cultured skin model indicate that this material may be useful as a model membrane for in vitro experiments. The results obtained using the LSE correlate well with those obtained with guinea pig skin in vitro. The iontophoretic flux of pindolol through both membranes was indistinguishable from one another. Similar results are also reported for synthetic salmon calcitonin and benzyl alcohol.

Action spectrum for bergamot-oil phototoxicity measured by sunburn cell counting.

The present study investigated the phototoxic effect of bergamot oil and its photosensitive component, bergapten, on sunburn cell (SBC) production in guinea pig skin. The back skin was pretreated with bergamot oil or bergapten and exposed to monochromatic light under various conditions. After irradiation, skin specimens were excised, and histological sections were prepared. The number of sunburn cells in the interfollicular epidermis was counted. The SBC formation by bergamot oil or bergapten plus UVB radiation was the same as that without pretreatment with any photosensitizer. In contrast, a significant number of SBCs were induced by bergamot oil or bergapten plus UVA radiation, but no SBCs were found after the treatment with UVA alone. The result indicates that bergamot oil or bergapten was photosensitized by UVA irradiation. The SBCs were linearly increased in a UV-dose dependent manner. On the basis of the regression lines, an action spectrum and spectral peak for the photosensitizers plus UVA were obtained. The action spectrum for bergamot oil- and bergapten-induced SBC formation was in the ranges of 325-365 nm and 325-350 nm, and their spectral peaks were at 335-345 nm and 335-350 nm, respectively. The data are in good accordance with those estimated from skin erythema reactions. Therefore, counting SBCs is a very useful parameter for quantitative evaluation of phototoxicity.

Effects of phosphodiesterase isoenzyme inhibitors on cutaneous inflammation in the guinea‐pig

1. Inflammation is central to the pathophysiology of asthma. The recent findings that different inflammatory cells may express different phosphodiesterase (PDE) isoenzymes have centered attention on inhibitors of these isoenzymes as new drugs for the treatment of asthma. In this study, we investigated the effect of different PDE isoenzyme inhibitors on the accumulation of 111In-labelled eosinophils and local oedema formation at sites of allergic- and mediator-induced inflammation in guinea-pig skin. 2. Systemic treatment with SK&F 94120, a type III PDE inhibitor, or zaprinast, a type V PDE inhibitor, had no effect on the 111In-eosinophil accumulation and oedema formation induced by i.d. injection of zymosan-activated plasma (ZAP), PAF, histamine or in a passive cutaneous anaphylaxis (PCA) reaction. 3. Systemic treatment with rolipram, a type IV PDE inhibitor, effectively inhibited 111In-eosinophil accumulation induced by ZAP, PAF, histamine and in a PCA reaction. However, oedema formation measured in the same sites was not affected. Systemic administration of higher doses of theophylline produced similar results. In contrast, 111In-neutrophil accumulation induced by ZAP or in a PCA reaction was not altered by systemic treatment with rolipram. 4. Locally-injected rolipram had little effect on 111In-eosinophil accumulation and oedema formation induced by histamine, PAF and in a PCA reaction. 5. These data show that systemic, but not local, treatment with rolipram effectively inhibits allergic- and mediator-induced 111In-eosinophil accumulation but not oedema formation or 111In-neutrophil accumulation. This, taken together with the potent inhibitory effects of PDE type IV inhibitors on eosinophil function in vitro, suggest that this class of drugs may be beneficial in disease states such as asthma where eosinophils are thought to play a major pathophysiological role.

Structure-activity relationships for contact allergenic potential of gamma,gamma-dimethyl-gamma-butyrolactone derivatives. 1. Synthesis and electrophilic reactivity studies of alpha-(omega-substituted-alkyl)-gamma,gamma-dimethyl-gamma-butyrolacton es and correlation of skin sensitization potential and cross-sensitization patterns with structure.

A series of alpha-(X-substituted-methyl)-gamma,gamma-dimethyl-gamma-butyrolactones (series 1), a series of alpha-(2-X-substituted-ethyl)-gamma,gamma-dimethyl-gamma-butyrolactones (series 2), where X is a leaving group, and the compound alpha-(3-bromopropyl)-gamma,gamma-butyrolactone (3) were synthesized. Their reactions as electrophiles toward n-butylamine, used as a model for nucleophilic groups on skin proteins whose in vivo chemical modification leads to skin sensitization, were investigated. The compounds of series 1 were shown to react via a two-stage elimination--Michael addition sequence whereby the elements of HX are eliminated to form alpha-methylene-gamma,gamma-dimethyl-gamma-butyrolactone, which reacts more slowly with n-butylamine to give alpha-[(N-butylamino)methyl]-gamma,gamma-dimethyl-gamma-butyrolactone. The compounds of series 2 and compound 3 were shown to react with n-butylamine via a single-stage substitution reaction to give respectively alpha-[2-(N-butylamino)ethyl]- and alpha-[3-(N-butylamino)propyl]-gamma,gamma-dimethyl-gamma-butyrolactones . Rate constants for these reactions have been determined, and it is found that substitution reactions of series 2 and compound 3 are slower than Michael addition of alpha-methylene-gamma,gamma-dimethyl-gamma-butyrolactone, which in turn is slower than the elimination reactions of series 1. The results of guinea pig skin sensitization tests on these compounds were found to be consistent with the above findings in that the compounds of series 1 were found to be in general much stronger sensitizers than those of series 2 and compound 3. The results of cross-challenge tests indicate that sensitizing compounds from series 2 were cross-reactive with both series 1 and compound 3 but that compound 3 is only weakly cross-reactive with series 1. These observations indicated that for these compounds a difference of two carbon atoms between the determinant groups transferred to protein had a markedly greater effect than a difference of one carbon atom on antigenic specificity.

An alternative study of the skin irritant effect of an homologous series of surfactants

An attempt has been made to differentiate between in vivo and in vitro skin reactions to a homologous series of surfactants (sodium alkyl sulfate, R-OSO 3 Na) and to determine the usefulness of percutaneous absorption in vitro as an alternative test system. Sodium alkyl sulfate showed considerable biological activity by virtue of its polar head groups. The length of the lipophilic chain in the surfactants was an important factor in their overall activity. The following in vivo tests were performed: a primary skin irritation test in guinea pigs, a primary eye irritation test in rabbits and a closed patch test in humans. Peak skin irritation occurred with C 10-C 16 sodium alkyl sulfate, which had lipophilic groups of different alkyl chain lengths. Cell injury was also evaluated by the neutral red dye uptake assay in rabbit corneal (RC) cells. C 4 and C 6 compounds had no effect, while maximal effects occurred with C 18. Protein denaturation and haemolysis occurred with C 10-C 16 compounds. In the percutaneous absorption test in guinea pig skin, permeation was low for the C 18 compound and high for the C 4 compound. The results with the C 18 compound suggest that differences between cell injury and skin irritation result from skin permeation. Although the C 18 compound caused cell injury, membrane destruction and protein denaturation were more severely with the C 10-C 16 compounds, owing to their strong haemolytic and protein-denaturation action.

52 First experiences to compare classic LDR and PDR irradiation with help of the animal model guinea pig skin

52 First experiences to compare classic LDR and PDR irradiation with help of the animal model guinea pig skin

Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation.

Eosinophil accumulation is a prominent feature of allergic inflammatory reactions, such as those occurring in the lung of the allergic asthmatic, but the endogenous chemoattractants involved have not been identified. We have investigated this in an established model of allergic inflammation, using in vivo systems both to generate and assay relevant activity. Bronchoalveolar lavage (BAL) fluid was taken from sensitized guinea pigs at intervals after aerosol challenge with ovalbumin. BAL fluid was injected intradermally in unsensitized assay guinea pigs and the accumulation of intravenously injected 111In-eosinophils was measured. Activity was detected at 30 min after allergen challenge, peaking from 3 to 6 h and declining to low levels by 24 h. 3-h BAL fluid was purified using high performance liquid chromatography techniques in conjunction with the skin assay. Microsequencing revealed a novel protein from the C-C branch of the platelet factor 4 superfamily of chemotactic cytokines. The protein, "eotaxin," exhibits homology of 53% with human MCP-1, 44% with guinea pig MCP-1, 31% with human MIP-1 alpha, and 26% with human RANTES. Laser desorption time of flight mass analysis gave four different signals (8.15, 8.38, 8.81, and 9.03 kD), probably reflecting differential O-glycosylation. Eotaxin was highly potent, inducing substantial 111In-eosinophil accumulation at a 1-2 pmol dose in the skin, but did not induce significant 111In-neutrophil accumulation. Eotaxin was a potent stimulator of both guinea pig and human eosinophils in vitro. Human recombinant RANTES, MIP-1 alpha, and MCP-1 were all inactive in inducing 111In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with RANTES on guinea pig eosinophils. This is the first description of a potent eosinophil chemoattractant cytokine generated in vivo and suggests the possibility that similar molecules may be important in the human asthmatic lung.

Chrysosporium tropicum as a probable cause of mycosis of poultry in India.

Chrysosporium tropicum was isolated from comb lesions in two different breeds of chickens in India and subcultures were shown to be pathogenic when inoculated onto prepared skin of guinea pigs. This report provides additional evidence to consider Ch. tropicum as a pathogenic fungus and a probable cause of a dermatomycosis in chickens.