Comparison of the percutaneous absorption of hydrophilic and lipophilic compounds in shed snake skin and human skin

The in vitro transdermal permeation of eight hydrophilic drugs (antipyrine, L-dopa, dopamine hydrochloride, diclofenac sodium, 5-fluorouracil, isoprenaline hydrochloride, nicorandil and morphine hydrochloride) and eight lipophilic drugs (aminopyrine, cyclobarbital, ibuprofen, indomethacin, isosorbide dinitrate, flurbiprofen, ketoprofen and lignocaine) was determined using shed snake skin of Elaphae obsoleta and human skin. The permeation parameters and physiological characteristics of the skin, e.g. the water and lipid content, and the thickness of shed snake skin and human skin were evaluated and compared. In shed snake skin, the permeability coefficients (P) of lipophilic drugs were in the same range as those through the human skin (0.9 to 1.8-times); whereas those of hydrophilic drugs were remarkably lower (3.3 to 6.1-times). The thickness and lipid content of shed snake skin and human stratum corneum were not significantly different (P > 0.05), whereas the water content of shed snake skin was significantly lower than that of human stratum corneum (P < 0.05). The lower permeability of shed snake skin for hydrophilic compounds might be caused by the lower porosity of skin strata. The results suggested a potential use of shed snake skin as barrier membrane for lipophilic compounds percutaneous absorption studies in vitro.     

Evaluation of simultaneous permeation and metabolism of methyl nicotinate in human, snake, and shed snake skin

The transdermal permeation and metabolic characteristics of methyl nicotinate (MN) in stratum corneum and split-thickness human skin and three species of shed snake and snake skin (Elaphae obsoleta, Naja kaouthia, and Python molurus bivittatus) were evaluated. In vitro skin transport using excised skin and hydrolysis experiments using skin homogenate were carried out. The flux of MN, a metabolite, nicotinic acid (NA), and the total (MN+NA), as well as kinetic parameters (V(max) and K(m)) for hydrolysis of MN were determined and compared among various skin types. The total flux from MN-saturated solution through human skin was not significantly different from that through snake and shed snake skin of Elaphae obsoleta, Naja kaouthia but was significantly higher than that through snake and shed snake skin of Naja kaouthia (p < 0.05). A great difference in skin esterase activity was observed between human and snake in both snake skin and shed snake skin of all species. In all skins except the stratum corneum of human skin, NA flux increased with an increase in MN donor concentration and reached a plateau, suggesting that metabolic saturation was taking place in the skin. NA flux at the plateau and MN donor concentrations at which the NA flux reached a plateau also varied by species. These findings indicated that the discrepancy in transdermal profiles of MN among skins tested was predominantly due to the difference in the esterase activity in the skin.     

Comparison of skin transport and metabolism of ethyl nicotinate in various species.

The skin transport and metabolism characteristics of ethyl nicotinate (EN) in rabbit, rat, guinea-pig, pig, shed snake skin and human were compared. In vitro skin transport using excised skin and hydrolysis experiments using skin homogenate were carried out. Flux of EN, a metabolite, nicotinic acid (NA), and the total (EN + NA), as well as kinetic parameters (V(max) and K(m)) for hydrolysis of EN were determined and compared among various species. The enzymatic conversion of EN to NA was observed for all skin permeation experiments. Total flux from EN-saturated solution between rabbit, rat, guinea-pig and human was significantly different (P < 0.05). A great difference between species was observed in skin esterase activity. The NA/total flux ratio of human was significantly lower than that of rabbit, rat or guinea-pig but lower than that of shed snake skin (P < 0.05). There is no significant difference in skin permeation and metabolism between human and pig (P > 0.05). Total flux increased linearly with an increase in EN donor concentration for all species. For pig, shed snake skin and human, NA flux increased with an increase in EN donor concentration and reached a plateau, suggesting the metabolic saturation was taking place in the skin. NA flux at plateau and EN donor concentration in which the NA flux reached a plateau were also affected by species difference. These findings indicated that the discrepancy in transdermal profiles of EN among species tested was predominantly due to the difference in the esterase activity in the skin.     

Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption

For the study of in-vitro skin penetration of candidate drugs, excised animal skin is frequently used as a replacement for human skin. Reconstructed human skin or epidermis equivalents have been proposed as alternatives. We compared the penetration properties of human, pig and rat skin with the Graftskin LSE (living skin equivalent) and the Skinethic HRE (human reconstructed epidermis) models using four topical dermatological drugs (salicylic acid, hydrocortisone, clotrimazole and terbinafine) with widely varying polarity. In agreement with published data, pig skin appeared as the most suitable model for human skin: the fluxes through the skin and concentrations in the skin were of the same order of magnitude for both tissues, with differences of at most two- or fourfold, respectively. Graftskin LSE provided an adequate barrier to salicylic acid, but was very permeable for the more hydrophobic compounds (e.g. about 900-fold higher flux and 50-fold higher skin concentrations of clotrimazole as compared to human skin), even more than rat skin. In the case of the Skinethic HRE, we found similar concentrations of salicylic acid as in human skin and an approximately sevenfold higher flux. In contrast, the permeation of hydrophobic compounds through the epidermal layer was vastly higher than through split-thickness human skin (up to a factor of about 800). To conclude, currently available reconstituted skin models cannot be regarded as generally useful for in-vitro penetration studies.     

Evaluation of Simultaneous Permeation and Metabolism of Methyl Nicotinate in Human,Snake, and Shed Snake Skin

The transdermal permeation and metabolic characteristics of methyl nicotinate (MN) in stratum corneum and split-thickness human skin and three species of shed snake and snake skin (Elaphae obsoleta, Naja kaouthia, and Python molurus bivittatus) were evaluated. In vitro skin transport using excised skin and hydrolysis experiments using skin homogenate were carried out. The flux of MN, a metabolite, nicotinic acid (NA), and the total (MN+NA), as well as kinetic parameters (V_max and K_m) for hydrolysis of MN were determined and compared among various skin types. The total flux from MN-saturated solution through human skin was not significantly different from that through snake and shed snake skin of Elaphae obsoleta, Naja kaouthia but was significantly higher than that through snake and shed snake skin of Naja kaouthia (p < 0.05). A great difference in skin esterase activity was observed between human and snake in both snake skin and shed snake skin of all species. In all skins except the stratum corneum of human skin, NA flux increased with an increase in MN donor concentration and reached a plateau, suggesting that metabolic saturation was taking place in the skin. NA flux at the plateau and MN donor concentrations at which the NA flux reached a plateau also varied by species. These findings indicated that the discrepancy in transdermal profiles of MN among skins tested was predominantly due to the difference in the esterase activity in the skin.     

Development of a polymerase chain reaction to distinguish monocellate cobra (Naja khouthia) bites from other common Thai snake species, using both venom extracts and bite-site swabs.

A PCR technique was used in this study to identify and distinguish monocellate cobra snake bites using snake venoms and swab specimens from snake bite-sites in mice from bites by other common Thai snakes. The sequences of nucleotide primers were selected for the cobrotoxin-encoding gene from the Chinese cobra (Naja atra) since the sequences of monocellate cobra (Naja kaouthia) venom are still unknown. However, the 113-bp fragment of cDNA of the cobrotoxin-encoding gene was detected in the monocellate cobra venom using RT-PCR. This gene was not found in the venoms of Ophiophagus hannah (king cobra), Bungarus fasciatus (banded krait), Daboia russelii siamensis (Siamese Russell's Viper, and Calloselasma rhodostoma (Malayan pit viper). Moreover, direct PCR could detect a 665-bp fragment of the cobrotoxin-encoding gene in the monocellate cobra venom but not the other snake venoms. Likewise, this gene was only observed in swab specimens from cobra snake bite-sites in mice. This is the first report demonstrating the ability of PCR to detect the cobrotoxin-encoding gene from snake venoms and swab specimens. Further studies are required for identification of this and other snakes from the bite-sites on human skin.     

Comparison of different membranes with cultures of keratinocytes from man for percutaneous absorption of nitroglycerine.

The permeability barrier function of cell-culture membranes to the permeation of nitroglycerine was evaluated to find an alternative to skin from man for ex-vivo skin-permeation tests. The membranes were prepared, under submerged conditions, by inducing the growth of keratinocytes, from different donors, on a film of esterified jaluronic acid for different times (10, 20 and 30 days). Their permeability barrier functions were compared with those of some of the most widely used artificial membranes, silicone rubber (Silastic), cellulosic material (Cuprophan, Millipore HAWP), polysulphone membrane (Supor) and polytetrafluoroethylene membrane (TF-PTFE), and with those of biological membranes such as fresh and frozen skin, stratum corneum and epidermis from man, and hairless mouse skin. For each membrane the permeation profile was obtained and the flux was calculated. The permeation profiles for nitroglycerine were similar and linear in the first 2-3 h for all the synthetic membranes tested except TF-PTFE. For this membrane the profile was linear throughout the period considered and the amount permeating in 24 h (1603 microg cm(-2)) was significantly lower than those obtained for the other artificial membranes (between 1926 and 2508 microg cm(-2)). The amounts permeating through all the biological membranes in 24 h were in the range 520 to 781 microg cm(-2), except those for the keratinocyte-culture membranes, which were in the range 1730 to 2553 microg cm(-2). Prolonging the growth period of cultured keratinocytes did not affect nitroglycerine permeation. The findings suggest that these keratinocyte-culture membranes have some advantages--good reproducibility if obtained from the same donor; many membranes can be obtained from the same donor; the preparation is simple; they can be handled more easily than traditional cell-culture membranes; and they afford constant penetration rates for a longer period than synthetic membranes. The membranes could be used for preliminary in-vitro permeation studies.     

An approach to reduce the number of skin samples in testing the transdermal permeation of drugs

Although glyceryl trinitrate (GT) is a drug that easily permeates through skin, the variations in its transepidermal fluxes were high. The arithmetic mean of the GT flux (n = 31 skin samples from different individuals) was 16.5 micrograms cm-2 h-1 with a standard deviation of 42%. The extreme values were 4.1 and 36.9 micrograms cm-2 h-1, i.e. they differed by a factor of 9. Wide variations were also found for ephedrine, frusemide, caffeine, ethacrynic and benzoic acids and especially trospium chloride. All these fluxes were determined in an in-vitro permeation model at 32 degrees C using human epidermis. With the aim of standardizing epidermal preparations on their permeability, the extent to which the in-vitro GT fluxes through a human epidermal preparation correlate with those of other compounds was evaluated. The resulting standardization procedure consisted of two interactive parts: the correlation of the flux of a test-substance with that of GT using epidermal samples from three donors and estimating the minimum, mean and maximum flux of the test compound and quantitation of the transepidermal permeation of the test compound with those standardized epidermal preparations by calculating the GT standard coefficient defined by the slope of the line derived from the relation between GT flux units and the corresponding flux from the test compound.     

In-vitro and in-vivo evaluation of oligoethylene esters as dermal prodrugs of 18beta-glycyrrhetic acid

Novel polyoxyethylene esters of 18 beta-glycyrrhetic acid (GA) were synthesized and evaluated as potential dermal prodrugs. The permeation of these prodrugs (1a-e) was studied in-vitro, using excised human skin membranes (SCE; stratum corneum/epidermis) mounted in Franz type cells, and in-vivo, evaluating the ability of these compounds to inhibit methyl nicotinate (MN)-induced skin erythema in healthy human subjects. All the esters synthesized showed a good water stability, while the enzymatic hydrolysis rate was significantly affected by the length of the polyoxyethylenic chain used as promoiety. In in-vitro percutaneous absorption studies, only esters 1b and 1c (respectively triethylen- and tetraethylenglycol derivatives) showed an increased flux through SCE membranes compared with GA. Furthermore, we observed an appreciable and sustained in-vivo topical anti-inflammatory activity of esters 1b and 1c compared with the parent drug.     

An investigation of the chick chorioallantoic membrane as an alternative model to various biological tissues for permeation studies

Objectives The chick chorioallantoic membrane (CAM) was explored as a biological membrane for use in the study of drug permeation with a Franz diffusion cell. Methods The CAM was removed from fertilized chicken eggs of embryo age 9–18 days. The permeation profiles of nicotine through the fresh CAM were first obtained with a Franz diffusion cell. The permeation profiles of nicotine through frozen CAM, snake skin, pig skin, pig retina and pig buccal mucosa were also determined and compared with those of the fresh CAM. Key findings The permeability coefficient of the CAM varied with its age. The CAM at embryo age 13 was the most robust, showing the lowest standard error in permeability. It was thus chosen for comparative studies with snake skin, pig skin, retina and buccal mucosa. The CAM was found to be most similar to the buccal mucosa in terms of permeation profile and permeability coefficient values. Frozen CAM was also found to have a higher permeability coefficient than fresh CAM. The enhanced permeability was attributed to freezing, which affected the integrity of the CAM structure. Conclusions From the findings, CAM shows potential as an alternative to the pig buccal mucosa as an in‐vitro buccal model. The robustness of the CAM for drug permeation studies is affected by its age.     

Preparation of curcuminoid niosomes for enhancement of skin permeation

Curcuminoids (curcumin, desmethoxycurcumin, and bisdesmethoxycurcumin) are major bioactive substances found in turmeric (Curcuma longa L.) extracts and possess antioxidant, anti-inflammatory, antimicrobial and anticancer properties. In this study, curcuminoid niosomes prepared with a series of Span non-ionic surfactants were developed to enhance the skin permeation of curcuminoids. Formulations were evaluated based on aggregation of niosomes, curcuminoid loading, % entrapment efficiency and in vitro permeation of curcuminoids through shed snake skin. Optimal formulations of curcuminoid niosomes including sorbitan monooleate, cholesterol, and Solulan C-24 at a mole ratio of 47.5:47.5:5 were obtained. Up to 11 micromoles of curcuminoids could be loaded in the niosome with a % entrapment efficiency of 83%. About 90% of the niosomes had a diameter of 12.25 +/- 5.00 microm. The niosomes significantly enhanced permeation of curcuminoids compared with a methanolic solution of curcuminoids: 4% of entrapped curcuminoids traversed the shed snake skin, whereas permeation from the methanolic solution was undetectable. The fluxes of curcumin, desmethoxycurcumin, and bisdesmethoxycurcumin were 1.117, 0.263, and 0.057 microg/(cm2h), respectively, consistent with the relative hydrophobicity of curcumin > desmethoxycurcumin > bisdesmethoxycurcumin. In conclusion, our data show that curcuminoids can be successfully formulated as niosomes and that such formulations have improved properties for transdermal delivery.     

Transdermal Delivery of Narcotic Analgesics: pH,Anatomical, and Subject Influences on Cutaneous Permeability of Fentanyl and Sufentanil

The permeation of fentanyl and sufentanil through cadaver skin membranes was investigated using in vitro diffusion cell techniques. Neither drug influenced the permeation of the other when they were concurrently applied to the skin membrane. With respect to transdermal delivery, short diffusion lag times of less than 0.5 hr were observed for each compound. Their permeation rates through heat-isolated epidermis and dermatomed (200- to 250-µm) skin sections were essentially the same. However, when the stratum corneum was removed by tape stripping, the respective permeability coefficients were increased >30-fold, establishing the stratum corneum as the principal barrier to their skin permeation. Permeation was also studied as a function of pH. From pH 4 to pH 8 the permeability coefficients of both fentanyl and sufentanil, calculated from the total solution concentration regardless of ionization, increased exponentially. The free base is thus responsible for the relatively facile skin permeation of these drugs. Factoring of the independent permeability coefficients of the ionized and free-base forms was possible, with the latter being over two log orders larger than seen for the protonated species. Permeability coefficients of fentanyl and sufentanil through skin sections obtained from different cadavers varied four- to fivefold. Neither gender nor age was a flux-determining variable for either drug. The permeability coefficients of the drugs through skin sites as diverse as the sole of the foot, chest, thigh, and abdomen were remarkably similar. Their fluxes were sufficient for transdermal administration.     

磺胺嘧啶银霜治疗中华眼镜蛇咬伤坏死的价值探讨

【摘要】 目的:探讨中华眼镜蛇咬伤致局部皮肤软组织肿胀坏死合理有效的治疗方法。方法:回顾性分析我院2003年6月至2009年6月间收治中华眼镜蛇咬伤中毒67例患者的临床资料,并将67例患者分为三组,其中磺胺嘧啶银霜组22例、40%硫酸镁甘油组26例以及季德胜蛇药片组19例,分别外敷治疗局部皮肤软组织肿胀坏死,以消肿时间和愈合时间为观察对象进行统计学分析。结果:磺胺嘧啶银霜组、40%硫酸镁甘油组与季德胜蛇药片组的平均消肿时间分别为（8.82±1.92）天、（10.35±2.10）天和（11.42±2.61）天,三组间差异有统计学意义(F=7.305, p <0.05);组间两两比较发现磺胺嘧啶银霜组与40%硫酸镁甘油组、季德胜蛇药片组间差异均有统计学意义(p <0.05),而40%硫酸镁甘油组与季德胜蛇药片组间差异无统计学意义(p ＞0.05)。磺胺嘧啶银霜组、40%硫酸镁甘油组与季德胜蛇药片组的平均愈合时间分别为（17.45±6.67）天、（19.92±7.63）天和（20.74±8.50）天,三组间差异无统计学意义(F=1.077, p ＞0.05)。结论: 磺胺嘧啶银霜治疗中华眼镜蛇咬伤所致的局部皮肤软组织肿胀坏死在消肿方面较40%硫酸镁甘油与季德胜蛇药片占优,而在愈合时间上三种方法无优劣。     

A Novel in Vitro Percutaneous Penetration Model: Evaluation of Barrier Properties with P-Aminobenzoic Acid and Two of Its Derivatives

Purpose The objective of this study was to evaluate the utility of a stratum corneum substitute (SCS) as a novel in vitro percutaneous penetration model. The SCS consists of synthetic stratum corneum (SC) lipids (cholesterol, free fatty acids, and specific ceramides) applied onto a porous substrate. The composition, organization, and orientation of lipids in the SCS bear high resemblance to that of the intercellular barrier lipids in SC. Methods The barrier integrity of the SCS was evaluated by means of passive diffusion studies, using three model compounds with different lipophilicities. The effects of lipid layer thickness, permeant lipophilicity, and altered lipid composition on the barrier properties were investigated, using isolated human SC as a control sample. Results For all three model compounds, the permeability characteristics of the SCS with a 12-μm-thick lipid layer closely resemble those of human SC. Modification of the lipid composition, generating an SCS that lacks the characteristic long periodicity phase as present in SC, was accompanied by a 2-fold increased permeability. Conclusions The SCS offers an attractive tool to predict solute permeation through human skin. Moreover, as its lipid composition can be modified, they may also serve as a suitable screening model for diseased skin.     

Sandwich enzyme-linked immunosorbent assay for Taiwan cobra venom

Poisonous snake bite victims usually have difficulty identifying the species, and clinical manifestations alone are not reliable because of overlapping symptoms. Thus, it is important to develop a quick and reliable mean of identifying the snake responsible. We describe the development of a sandwich-ELISA method for detection of venom in biological samples and apply it to a case of snakebite to confirm the clinical diagnosis. The sandwich-ELISA takes 6 h to complete. Cobra venom antigen gave positive absorbance at about 500 pg/ml. Good linearity with R2 values over 0.99 were observed in dilution series of 1:100 ng/mL of cobra venom in calf serum and human urine. A snakebite initially thought to be Trimeresurus mucrosquamatus was proven cobra with a serum venom level up to 288 ngmL 3 h after envenoming. Sandwich-ELISA provides a rapid and accurate method for clinical identification and evaluation of toxic antigens circulating in individuals bitten by the Taiwan cobra snake.     

Enhanced Topical and Transdermal Delivery of Antineoplastic and Antiviral Acyclic Nucleoside Phosphonate cPr-PMEDAP

Purpose

Acyclic nucleoside phosphonates possess unique antiviral and antineoplastic activities; however, their polar phosphonate moiety is associated with low ability to cross biological membranes. We explored the potential of transdermal and topical delivery of 2,6-diaminopurine derivative cPr-PMEDAP.

Methods

In vitro diffusion of cPr-PMEDAP was investigated using formulations at different pH and concentration and with permeation enhancer through porcine and human skin.

Results

Ability of 0.1?C5% cPr-PMEDAP to cross human skin barrier was very low with flux values ~40 ng/cm²/h, the majority of compound found in the stratum corneum. The highest permeation rates were found at pH 6; increased donor concentration had no influence. The permeation enhancer dodecyl 6-dimethylaminohexanoate (DDAK, 1%) increased flux of cPr-PMEDAP (up to 61 times) and its concentration in nucleated epidermis (up to ~0.5 mg of cPr-PMEDAP/g of the tissue). No deamination of cPr-PMEDAP into PMEG occurred during permeation studies, but N-dealkylation into PMEDAP mediated by skin microflora was observed.

Conclusions

Transdermal or topical application of cPr-PMEDAP enabled by the permeation enhancer DDAK may provide an attractive alternative route of administration of this potent antitumor and antiviral compound.     

Ester prodrugs of morphine improve transdermal drug delivery: a mechanistic study

Two alkyl esters of morphine, morphine propionate (MPR) and morphine enanthate (MEN), were synthesized as potential prodrugs for transdermal delivery. The ester prodrugs could enhance transdermal morphine delivery. The mechanisms of this enhancing effect were elucidated in this study. Both prodrugs were more lipophilic than their parent drug as evaluated by the skin/vehicle partition coefficient (log P) and capacity factor (log K'). The in-vitro skin permeation of morphine and its prodrugs from pH 6 buffer was in the order of MEN > MPR > morphine. MPR and MEN respectively enhanced the transdermal delivery of morphine by 2- and 5-fold. A contrary result was observed when using sesame oil as the vehicle. The prodrugs were stable against chemical hydrolysis in an aqueous solution, but were readily hydrolysed to the parent drug when exposed to skin homogenate and esterase. Approximately 98% MPR and approximately 75% MEN were converted to morphine in an in-vitro permeation experiment. The viable epidermis/dermis contributed to a significant resistance to the permeation of ester prodrugs. According to the data of skin permeation across ethanol-, alpha-terpineol-, and oleic acid-pretreated skin, MEN was predominantly transported via lipid bilayer lamellae in the stratum corneum. The intercellular pathway was not important for either morphine or MPR permeation.     

In vitro permeation of progesterone from a gel through the shed skin of three different snake species

The in vitro diffusion of progesterone from a gel formulation using the European Pharmacopoeia method for transdermal dosage forms is described. The membranes used were the dorsal and ventral portions of the shed skin of three different species of snake. Considerable differences are apparent between the dorsal and ventral sites and between the different species of snake. The dorsal area shows better permeability for progesterone and the permeability order for the different species is python>cobra>viper. These differences may be due to the thickness of the skin and the hinge:scale ratio. The results indicate that shed snake skin is not a model membrane for human skin.     

Skin permeation of nickel,cobalt and chromium salts in ex vivo human skin,visualized using mass spectrometry imaging

Skin permeation and distribution of three of the most common skin sensitizers was investigated using a previously developed animal-free exposure method combined with imaging mass spectrometry. Nickel, cobalt, and chromium (III) salts were dissolved in a buffer and exposed to human skin ex vivo, to be analyzed using time of flight secondary ion mass spectrometry (ToF-SIMS). Our findings demonstrate that metal haptens mainly accumulated in the stratum corneum, however all three metal sensitizers could also be detected in the epidermis. Cobalt and chromium (III) species penetrated into the epidermis to a larger extent than nickel species. The degree of penetration into the epidermis is suggested to be affected by the sensitization potency of the metal salts, as well as their speciation, i.e. the amount of the respective metal present in the solution as bioaccessible and solubilised ions. Our method provided permeation profiles in human skin for known sensitizers, on a level of detail that is not possible to achieve by other means. The findings show that the permeation profiles are different, despite these sensitizers being all metal ions and common causes of contact allergy. Studying skin uptake by only considering penetration through the skin might therefore not give accurate results.     

Effectiveness of Thai cobra (Naja kaouthia) antivenom against sea snake (Lapemis hardwickii) venom: verification by affinity purified F(AB')2 fragments

Commercial Thai cobra (Naja kaouthia) antivenom was found to be effective in neutralizing sea snake (Lapemis hardwickii) venom. Neurotoxin specific F(ab')2 fragments obtained from the antivenom by chromatography using Thai cobra neurotoxin or sea snake venom affinity columns were able to neutralize both venoms.