Inhibitory effect of prostaglandin E1 on laurate-induced peripheral vascular occlusive sequelae in rabbits: optimized topical formulation with beta-cyclodextrin derivative and penetration enhancer HPE-101.

Prostaglandin E1 (PGE1) and its inclusion complexes with beta-cyclodextrin (beta-CyD) and O-carboxymethyl-O-ethyl-beta-cyclodextrin (CME-beta-CyD) were made as topical preparations. The PGE1 preparations, when applied with a penetration enhancer, 1-[2-(decylthio)ethyl]azacyclopentane-2-one (HPE-101), markedly increased the regional blood flow in the ear of rabbits and were longer acting than when administered by the intravenous route. Topical application of the PGE1 preparations significantly protected rabbits against laurate-induced peripheral vascular occlusive sequelae; the protective potency increased in the order of PGE1 alone = beta-CyD complex < CME-beta-CyD complex preparation. The PGE1 preparations elicited skin reactions such as erythema and oedema depending on their vasodilating actions. These reactions disappeared gradually after removal of the preparations, and hence may not be serious obstacles for their safe use. These results suggest that combinations of CME-beta-CyD and HPE-101 work synergistically to facilitate the entry of PGE1 into the skin, and consequently enhance the therapeutic potential of PGE1 in the topical preparation tested.     

Combination effects of O-carboxymethyl-O-ethyl-β-cyclodextrin and penetration enhancer HPE-101 on transdermal delivery of prostaglandin E1 in hairless mice

An inclusion complex of prostaglandin E₁ (PGE₁) with β-cyclodextrin (β-Cyd) or O-carboxymethyl-O-ethyl-β-cyclodextrin (CME-β-CyD) was made as topical preparations in a fatty alcohol/propylene glycol ointment base. When the PGE₁ preparations were applied onto the skin of hairless mice, the vasodilating effect of the PGE₁-CME-β-CyD complex supplemented with a penetration enhancer, 1-[2-(decylthio)ethyl] azacyclopentane-2-one (HPE-101) was approximately 100 times that of the PGE₁ alone and approximately 10 times that of PGE₁ with HPE-101 or the PGE₁-β-CyD complex with HPE-101. The combination of CME-β-CyD and HPE-101 enhanced the percutaneous penetration of PGE₁ in a synergistic manner; CME-β-CyD assisted the release of HPE-101 from the ointment base and its entry into the skin which may facilitate the percutaneous penetration of PGE₁. Furthermore, this combination suppressed the bioconversion of PGE₁ to give less pharmacologically active metabolites during the passage through the skin, a situation delivering intact PGE₁ more effectively to the site of action. The present data suggest that the combination of CME-β-CyD and HPE-101 is particularly useful for improving topical bioavailability of PGE₁.     

Effect of admixture of commercially available corticosteroid ointments and/or creams on vasoconstrictor activity

A commonly used admixture of commercially available ointments and/or creams was selected from the prescribed sheets in our hospital, and questionnaire to dermatologists. To assess the relationship between permeability of corticosteroid through murine skin and clinical effects in human, we attempted to investigate the vasoconstrictor activity of these admixtures of topical corticosteroid by double-blind controlled study. Test samples were occluded at random on the back of 20 healthy volunteers for 4 hours. The vasoconstrictor activity of corticosteroid creams (Lidomex) alone was significantly large as compared with that of ointments alone. The vasoconstrictor activity of corticosteroid in the admixture of Lidomex ointment and urea ointments or heparinoid ointment was 1.5-2 fold significantly larger than that from ointments alone. The extent of the stability of the emulsion after mixing was related to the vasoconstrictor activity. These experiments demonstrated a close relationship between the vasoconstrictor activity of human skin and permeability of hairless mice skin. These results suggested that the vasoconstrictor activity of topical corticosteroids mixed with commercially available ointments and/or creams depends upon their physicochemical characteristics.     

Effects of HPE-101, a skin penetration enhancer, on human erythrocyte membranes

The primary aim of this study was to investigate the skin permeation-enhancing mechanism of HPE-101 using erythrocyte ghost cells prepared from human whole blood as a biomembrane model. The extent of hemolysis of erythrocytes induced by HPE-101 was measured using a spectrophotometer at 540nm. The effect of HPE-101 on lipid fluidity was examined by observing the change of intramolecular excimer formation and fluorescence polarization using an intramolecular probe (1,3-bis(pyrene) propane) and a lipid probe (1,6-diphenyl 1,3,5-hexatriene), respectively. Hemolysis of erythrocytes was observed at 0.01mM and completed at 1.0mM of HPE-101. The fluorescence polarization of the ghost membrane decreased with the addition of HPE-101, whereas the intramolecular excimer formation increased. HPE-101 thus enhanced the rotational mobility and the lateral diffusion, thereby decreasing the microviscosity of ghost membranes, implying that HPE-101 increases the lipid fluidity of ghost membranes. Therefore, HPE-101 seems to cause an increase in fluidity of the lipid bilayers in the stratum corneum of the skin, resulting in the reduction of diffusion resistance.     

Enhanced Bioavailability of Subcutaneously Injected Insulin by Pretreatment with Ointment Containing Protease Inhibitors

The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycemic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.     

Evaluation of In-vivo Transdermal Absorption of Cyclosporin with Absorption Enhancer Using Intradermal Microdialysis in Rats

The purpose of this study was to evaluate the effect of absorption enhancer on in-vivo transdermal absorption of cyclosporin using intradermal microdialysis in rats. Cyclosporin oily solutions (0.5, 2, 8% w/v) were prepared from Sandimmun (10% w/v oily oral preparation of cyclosporin) by diluting with olive oil. 1-[2-(Decylthio)ethyl]azacyclopentan-2-one (HPE-101) and glycerin were added to the cyclosporin formulation as an absorption enhancer at various concentrations between 1 and 20%. These formulations were applied to the shaved abdomen of rats treated with intradermal microdialysis at a flow rate of 2.5 μL min^?1 for 6 h. Cyclosporin was immediately detected and attained a plateau in the dermal dialysate after topical application of cyclosporin oily solution alone. Cyclosporin levels in the dialysate increased with increasing cyclosporin concentrations in the formulation from 0.5 to 8% (w/v). HPE-101 did not influence cyclosporin absorption at concentrations less than 6% (w/v). Addition of 10% (w/v) HPE-101 significantly enhanced an apparent absorption rate of cyclosporin by 4.9 times. However, 20% (w/v) HPE-101 did not show the enhancing activity. On the other hand, addition of glycerin at concentrations of 6, 10, and 20% (v/v) significantly enhanced an apparent absorption rate of cyclosporin by 3.0, 64, and 6.9 times, respectively. The time lag for cyclosporin absorption was less than 0.21 h in all tested cases. This microdialysis study shows that glycerin is a suitable enhancer for improving the in-vivo cyclosporin absorption from the skin.     

Study of tolnaftate release from fatty acids containing ointment and penetration into human skin ex vivo

Five fatty acids (oleic, linoleic, myristic, lauric and capric) were incorporated in 10% (w/w) into ointment formulation and their influence on lipophilic model drug tolnaftate release in vitro and enhancing effect on tolnaftate penetration into epidermis and dermis of human skin ex vivo were investigated. The prepared ointments were tested for homogeneity, pH and theological properties. In vitro release studies and ex vivo skin penetration experiments were carried out using Hanson and Bronaugh-type flow-through diffusion cells, respectively. Tolnaftate cumulative amount liberated from semisolids was assayed using UV-Vis spectrophotometer. After in vitro skin penetration studies, appropriately extracted human skin layers were analyzed for tolnaftate content using a validated HPLC method. Statistical analysis revealed that release rate of tolnaftate from control ointment and ointments with fatty acids was not significantly different and only 7.34-8.98% of drug was liberated into an acceptor medium after 6 h. Tolnaftate amount penetrating into 1 cm2 of epidermis from ointments containing oleic, linoleic, myristic and lauric acids was significantly greater (p < 0.05) than from the control ointment. Penetration enhancing ratios for these fatty acids for tolnaftate penetration into epidermis ranged from 1.48 to 1.75. In conclusion, fatty acids did not increase the liberation of tolnaftate from ointment formulation, but demonstrated their enhancing effect on tolnaftate penetration into human epidermis in vitro. Results from in vitro release experiments do not suit for prediction of the situation in the skin in vitro, if chemical penetration enhancers are incorporated into the ointment formulation.     

A study on flow properties of semisolid dosage forms

There are a wide variety of semi-solid ointments used for healing the skin diseases, whose therapeutic and skin penetration abilities may greatly differ from one another depending on the compositions of ointment vehicles. A computer optimization technique was applied to obtain the optimum formula of o/w type ointment giving the in vitro maximum absorption rate through hairless rat skin membrane. Some of the formulations were selected to find out a relationship between skin penetration of ointment and its rheological characteristics. The experimental value of absorption rate obtained from the ointment by optimum formula agreed well with the theoretical value obtained from a polynomial regression analysis. Three kinds of ointments selected among 15 formulations were obtained with a concentric cylinder type rheometer (Model; Rheolab SM-HM Physica, Germany) at 20, 30, 40 and 50°C for rheograms of rhelolgical properties of o/w type ointments. As the temperature was raised, all products showed a decrease in both shear stress and yield values. The higher skin penetration, the lower shear stress showed.     

Biopharmaceutics: Effect of Ointment Bases on Topical and Transdermal Delivery of Salicylic Acid in Rats: Evaluation by Skin Microdialysis

Microdialysis has been used to determine the concentration of salicylic acid in skin tissue and plasma periodically for 4 h to evaluate the effect of ointment bases on topical and transdermal delivery of salicylic acid. The ointment bases examined were solbase (water-soluble), poloid and white petrolatum (oleaginous), hydrophilic poloid (water in oil (w/o) type emulsion lacking water) and absorptive ointment (w/o-type emulsion containing water). The ointments (0.1 g) containing 25 μmol salicylic acid were applied for 2 h to the surface of rat skin (1 cm²) with (intact) or without the stratum corneum. For intact skin, the extent of topical delivery from different ointments, evaluated by the area under the concentration-time curve (AUC) of salicylic acid in the skin tissue (AUCskin), increased in the order solbase. white petrolatum, poloid, hydrophilic poloid. absorptive ointment. The ratio of AUC_skin (topical delivery) to the AUC of salicylic acid in plasma (AUC_plasma, transdermal delivery) varied remarkably among the different bases, the greatest ratio being observed for absorptive ointment. When the ointments were applied to skin surface without stratum corneum, AUC_skin for solbase was much higher (about 45 times that for intact skin), whereas only a small (two-fold) increase was observed for poloid and hydrophilic poloid and the increase was negligible for white petrolatum and absorptive ointment. For skin without the stratum corneum, the ratio AUC_skin/AUC_plasma for the different ointments was comparable, although the magnitudes of AUC_skin and AUC_plasma still varied substantially. The variance of AUC values arises as a result of the different rates of release of salicylic acid from the bases. These results indicate that: the topical and transdermal delivery of salicylic acid in intact skin varies substantially among different ointment bases, and the greatest topical delivery is observed for absorptive ointment; use of absorptive ointment increases the retention of salicylic acid in the stratum corneum; and the stratum corneum functions strongly as a penetration barrier for solbase, moderately for poloid and hydrophilic poloid, and less for absorptive ointment and white petrolatum.     

Investigation of the absorption of hypericin into the skin of hairless mice

The skin absorption of hypericin was evaluated in hairless mice to develop an optimised hypericin topical formulation that could be used in the clinical study of psoriasis. Hypericin (0.01-1.0%) in Beeler basis, polyethylene glycol ointment, carbopol gel, cetomacrogol cream, petrolatum or emulsifying ointment, with and without skin-absorption enhancers (isopropylidene glycerol and diethylene glycol monoethyl ether), was tested in-vivo on hairless mice skin. Using a skin-stripping technique and the intrinsic fluorescence of hypericin under standardised UV365 irradiation, it was demonstrated that the absorption of hypericin very much depended on the vehicle used. The concentrations of hypericin in the skin were then estimated by HPLC analysis. For this purpose, two vehicles were employed, with which hypericin penetrated the skin of hairless mice well (emulsifying ointment with isopropylidene glycerol) or very poorly (polyethylene glycol ointment). In the case of emulsifying ointment with isopropylidene glycerol (0.05% hypericin), a substantial concentration of hypericin (8.6+/-3.2 microg g(-1)) (mean +/- s.d., n = 5) was found in the skin. With polyethylene glycol ointment, however, only a limited hypericin skin concentration (0.38+/-0-34 microg g(-1), n = 5) was achieved. These results show that emulsifying ointment with polyethylene glycol holds promise as an effective topical vehicle for the treatment of skin diseases, such as psoriasis, with hypericin.     

Further Evaluation of a New Penetration Enhancer,HPE-101

Abstract— The penetration enhancer, 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101), significantly enhanced the excretion of topically applied [¹⁴C]indomethacin when dissolved in dipropylene glycol, triethylene glycol, diethylene glycol, 1,3-butylene glycol, trimethylene glycol, glycerin, water, silicone or triethanolamine, but not when dissolved in ethanol, isopropyl alcohol, oleyl alcohol, olive oil, peppermint oil, isopropyl myristate or hexylene glycol. HPE-101 significantly enhanced the excretion of [¹⁴C]indomethacin, [¹⁴C]nicotinic acid, [¹⁴C]5-fluorouracil, [³H]oestradiol and [³H]triamcinolone acetonide, but not that of [³H]testosterone. HPE-101 also significantly enhanced the excretion of [¹⁴C]indomethacin applied to intact skin of rabbit, guinea-pig and rat, and to tape-stripped skin of guinea-pig, but did not enhance the excretion of [¹⁴C]indomethacin applied to tape-stripped skin of rat or rabbit.     

Application of in Vivo Microdialysis to Transdermal Absorption of Methotrexate in Rats

Microdialysis was applied to determine the in vivo transdermal absorption of methotrexate (MTX) in rats with or without a new penetration enhancer, l-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101). A solution composed of 2.5 mM MTX and 3% (w/v) HPE-101 was applied to the shaved abdomen, in which a semipermeable membrane cannula of 10-mm length was inserted intracutaneously with the use of an L-shaped needle. Intradermal microdialysis was performed at a flow rate of 1.0 µL/min for 12 hr. The concentration of MTX in the dialysate was measured by fluorescence polarization immunoassay (FPIA). HPE-101 (3%, w/v) significantly increased the dermal MTX concentration from 0.06±0.04 µM in the control to 56±26 µM in the dialysate from 8 to 12 hr. HPE-101 at concentrations of 0.75, 1.5, 2.25, and 3% (w/v) enhanced the total recovery of MTX in dermal dialysate from 0 to 10 hr by approximately 5, 18, 42, and 500 times compared with the control, respectively. The microdialysis system is useful for assessing in vivo transdermal drug absorption.     

A comparison of sodium fusidate ointment and mupirocin ointment in superficial skin sepsis

Sodium fusidate ointment and mupirocin ointment were compared in 354 patients with superficial skin sepsis. The ointments were applied 3-times daily, or once daily when covered by a dressing, and the response assessed after 6 to 8 days. Both preparations proved effective clinically with 86% of patients responding. There was no difference between the two preparations in cases of primary infection (85% to both ointments), including a sub-group with impetigo (sodium fusidate 88% and mupirocin 84%), or secondary infection (sodium fusidate 81% and mupirocin 89%). Sodium fusidate ointment (98%) was significantly better (p less than 0.05) than mupirocin (82%) in patients with other superficial infections. Both ointments were equally effective in cases where Gram-positive, Gram-negative or mixed Gram-positive/Gram-negative bacteria were isolated. Adverse effects were reported in 1.0% of patients using sodium fusidate ointment and in 7.4% of patients using mupirocin ointment. The majority of complaints concerned the greasiness of mupirocin ointment.     

Evaluation of the permeability of corticosteroid in hairless mouse and hairless micropig skin from admixture of commercially available corticosteroid ointments and/or creams

Yucatan hairless micropig (YHMP) skin has been shown to have histology and physiologic properties similar to human skin. To assess the relationship between the permeability of corticosteroid ointments and five types of commonly used admixtures of corticosteroid through hairless mice (HM) or YHMP skin and the clinical effects in humans, we conduct by in vitro experiments using HM and YHMP skin. The permeability of corticosteroid in admixtures with urea or heparinoid ointments across HM or YHMP skin was 1.5-4-fold greater than that of corticosteroid ointments alone. HM skin was found to have faster permeability than YHMP skin, but otherwise was similar to YHMP skin. These experiments demonstrated a close relationship between the permeability of HM or YHMP skin and vasoconstrictor activity in humans. These results suggest that the in vitro permeability of corticosteroid measurements across HM skin could be a useful, rapid, and easy method for assessing the vasoconstrictor activity of topical corticosteroids and the admixtures of commercially available ointments and/or creams in humans.     

Analysis of in vitro skin permeation of 22-oxacalcitriol from ointments based on a two- or three-layer diffusion model considering diffusivity in a vehicle

In the present study, in vitro rat skin permeation of 22-oxacalcitriol (OCT) from ointments having differing compositions was determined and discussed based on a diffusion model. Diffusion coefficients of OCT in two ointments, one containing 3% (w/w) medium chain triglyceride (MCT) (3MO) and the other 30% (w/w) MCT (30MO), were determined using a modified membraneless method resulting in values of 0.89x10(-4) and 1.87x10(-4) cm2/h, respectively. At 24 h after application with 3MO, 7% of the applied OCT dose permeated through full-thickness skin and 22% remained in the ointment, whereas with 30MO, 2% of the applied dose permeated through full-thickness skin and 65% remained in the ointment. The diffusion coefficient of OCT in 3MO was lower than 30MO but the cumulative amount of OCT permeated was higher. From analysis of skin permeation of OCT based on a diffusion model considering diffusivity in an ointment, the partition coefficient of OCT from the ointment to stratum corneum (K(SC/V)) was calculated to be five-fold higher with 3MO than with 30MO. Our simulation study based on a diffusion model suggests that the diffusion coefficients of OCT in both ointments were high enough to have no affect on the skin permeation of OCT in the present case and that the difference in the skin permeations of the ointments was mainly caused by a difference in K(SC/V).     

Specific augmentation of plantar skin blood flow by lipo-PGE1 assessed in tetrodotoxin- and N(G)-nitro-L-arginine-treated rats

1Vasodilating effects of prostaglandin E1 incorporated in lipid microspheres (lipo-PGE1) were compared with those of prostaglandin E1 (PGE1) or its cyclodextrin clathrated preparation (PGE1-CD) on plantar skin blood flow in rats treated with tetrodotoxin and N(G)-nitro-L-arginine (L-NNA). Tetrodotoxin (50 microg/kg, i.v.) could totally inhibit the pressor response to electrical stimulation of the spinal cord, and the reflex tachycardia due to the depressor response to acetylcholine. Furthermore, L-NNA (30 mg/kg, i.v.) was used to counteract the lowering of the systemic blood pressure and peripheral vascular tone by elimination of sympathetic nerve activity, and to maintain the arterial blood pressure at the control level. Lipo-PGE1 increased plantar skin blood flow 4 to 6 times more potently than PGE1-CD or PGE1 in the treated rats. Furthermore, lipo-PGE1 increased plantar skin blood flow about 3 times more selectively than PGE1-CD. We also assessed several vasodilators, including terbutaline, nitroprusside, nicardipine, and papaverine in tetrodotoxin- and L-NNA-treated rats. However, none of them could selectively increase plantar blood flow despite the prominent depressor responses achieved. These results suggest that PGE1 preparations, especially lipo-PGE1 could potently and selectively increase plantar skin blood flow in rats treated with tetrodotoxin and L-NNA.     

Enhancing effect of hydroxypropyl-beta-cyclodextrin on cutaneous penetration and activation of ethyl 4-biphenylyl acetate in hairless mouse skin.

The effect of hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) on the cutaneous penetration and activation of ethyl 4-biphenylyl acetate (EBA), a prodrug of non-steroidal anti-inflammatory drug 4-biphenylylacetic acid (BPAA), from hydrophilic ointment was investigated, using hairless mouse skin in vitro. When the hydrophilic ointment containing a complex of EBA with HP-beta-CyD was applied to the full-thickness skin, HP-beta-CyD facilitated the penetration of EBA into the skin, the conversion of EBA to BPAA in the epidermis and the transfer of BPAA to the receptor phase. Under the present condition, pre- and post-application of the ointment containing HP-beta-CyD onto the skin did not affect the cutaneous penetration of EBA and its activation. When the ointment containing the EBA:HP-beta-CyD complex was applied to the skin, the flux of BPAA through the tape-stripped skin was greater than that through the full-thickness skin, while the activation of the prodrug in the skin was slowed down by the tape-stripping. When propylene glycol was used as a vehicle, HP-beta-CyD no longer enhanced the cutaneous permeation of BPAA through the full-thickness skin. These results suggest that the enhancing effect of HP-beta-CyD on the cutaneous penetration of EBA would be ascribable largely to an increase in effective concentration of EBA in the ointment. Furthermore, the slow diffusion of EBA solubilized in HP-beta-CyD through the stratum corneum, together with the vehicle effect, could make the prodrug more susceptible to the metabolic process that is active in the epidermis, eventually leading to the facilitated activation of the prodrug.     

Comparison of the antiphlogistic effect of mucopolysaccharide-polysulfate ointments with heparin-containing ointments in the UV erythema test

It could be demonstrated by means of the UV erythema model that four different ointment preparations containing mucopolysaccharide polysulfate (MPS) have a marked antiinflammatory effect and inhibit the formation of erythemas to a large extent. In this trial arrangement the different preparations containing MPS were significantly superior to altogether eight commercial heparin ointments, which were tested, too. With an inhibition of 69% of the erythema induced by UV rays, Hirudoid 40000 ointment is the most effective of all preparations tested.     

Enhanced penetration of mitomycin C through hairless mouse and rat skin by enhancers with terpene moieties

The effects of four new percutaneous absorption enhancers containing an azacyclo ring and terpene chain (1-geranylazacycloheptan-2-one (GAH), 1-farnesylazacycloheptan-2-one (FAH), 1-geranylazacyclopentan-2,5-dione (GAPD), and 1-farnesylazacyclopentan-2-one (FAP] and 1-dodecylazacycloheptan-2-one (Azone) on the percutaneous penetration of mitomycin C (MMC) through hairless mouse and rat skin in-vitro has been investigated. GAH, FAH, FAP and Azone enhanced MMC penetration by 20 to 60 times that of the control (ethanol). During the early part of the experiments, when the sink condition was maintained, FAH was the most effective for hairless mouse skin, whereas Azone showed the highest effect in the rat skin. The enhancing effect of GAPD was only about half that of the other enhancers, suggesting the importance of the polar group of the ring moiety in these compounds. The penetration of MMC through rat skin was also increased by pretreatment with these compounds, suggesting that the enhancers had a direct effect on the skin.     

Development of a topical ointment of betamethasone dipropionate loaded nanostructured lipid carrier

The purpose of this study was to design an innovative topical ointment containing betamethasone dipropionate loaded nanostructured lipid carrier (BD-NLC) for the treatment of atopic dermatitis (AD). BD-loaded NLC was produced with precirol ATO 5 and oleic oil (OA) by melt emulsification method. Effects of surfactant concentration, amount of solid lipid and liquid lipid on skin retention and skin penetration were investigated by in vitro percutaneous permeation experiment. The optimized BD-NLC showed a homogeneous particle size of 169.1 nm (with PI = 0.195), negatively charged surface (−23.4 mV) and high encapsulation efficiency (85%). Particle morphology assessed by TEM revealed a spherical shape. In vitro skin permeation study was carried out to investigate the percutaneous behaviors of W/O ointment with BD-NLC and Carbopol emulgel ointment with BD-NLC. W/O ointment with BD-NLC showed high skin retention (35.43 µg/g) and low penetration (0.87 µg/ml). In vitro drug release studies were carried out to demonstrate the drug releasing properties of the two ointments. W/O ointment with BD-NLC showed an advantage for skin retention as it was better for drug release. The tissue distribution test suggested that BD distribution was skin > muscle > blood. Self-made topical ointment in mice showed no skin irritation. The animal experiments indicated that BD-loaded NLC ointment was effective and safe for topical use.