Percutaneous absorption of clonazepam in rabbit

The percutaneous (p.c.) absorption of clonazepam (CZP), an antiepileptic drug, was investigated in rabbits. CZP was efficiently absorbed from a gel ointment (0.5% CZP, 1g, 9 cm2) with Azone and therapeutic plasma concentrations were maintained for 27 h. The bioavailability of CZP from the gel ointment was 47.2 +/- 3.1%, which was significantly larger than that (3.3 +/- 0.5%) after the ointment without Azone or that (12.5 +/- 3.6%) after oral administration. About a half of CZP in the ointment with Azone was absorbed during a 24 h application. The maximum and minimum plasma concentrations and the area under the plasma concentration-time curve gradually increased during repeated application of the ointment (2% CZP, 0.25 g/d, 2.25 cm2), probably due to the accumulation of drug in the skin and body. The efficient absorption and sustained plasma concentration of CZP after application suggest that a once a day p.c. administration regimen is possible by using the ointment with Azone.     

Percutaneous absorption of 1,3-dinitroglycerin and a trial of pharmacokinetic analysis

In order to estimate the pharmaceutical usefulness of 1,3-glyceryl dinitrate (1,3-GDN), an active metabolite of nitroglycerin, a trial transdermal delivery system designed to sustain a suitable plasma concentration of 1,3-GDN was produced using a porous membrane (Hipore 2100 or 4500) and it was a gel base or ethylhexyl acrylate-based adhesive (adhesive) and it was applied to rats. Additionally, for practical use of the transdermal system, a simple pharmacokinetic model to describe plasma 1,3-GDN levels after percutaneous (p.c.) application is presented. As a result, the drug was penetrated through the rat skin in vitro at a zero-order rate, although the penetration rate from the gel base was significantly greater than that from the adhesive. In vivo, the drug was rapidly absorbed through the rat skin, with a peak plasma level of 581 +/- 151 and 265 +/- 62 ng/ml for the gel ointment and adhesive systems without a porous membrane, respectively. The plasma levels after application of the systems with a membrane were relatively constant for a long time, indicating that the membranes act as a controlled-release barrier. The bioavailability of 1,3-GDN after gel base systems with and without a membrane was relatively high. The model presented was successfully able to describe the time course of plasma 1,3-GDN concentrations following p.c. application of the systems.     

External control of drug release and penetration. VI. Enhancing effect of ultrasound on the transdermal absorption of indomethacin from an ointment in rats.

The effect of an ultrasound (1 MHz) on transdermal absorption of indomethacin from an ointment was studied in rats. Ultrasound energy was supplied for between 5 and 20 min at a range of intensities (0.25, 0.5, 0.75, and 1 W cm-2), energy levels commonly used for therapeutic purposes. For evaluating skin penetration of indomethacin, the change of plasma concentration was measured. The pronounced effect of ultrasound on the transdermal absorption of indomethacin was observed at all ultrasound energy levels studied. The intensity and the time of application were found to play an important role in the transdermal phonophoretic delivery system of indomethacin; 0.75 W cm-2 appeared to be the most effective intensity in improving the transdermal absorption of indomethacin, while the 10 min ultrasound treatment was the most effective. Although the highest penetration was observed at an intensity of 0.75 W cm-2, 0.5 W cm-2 was preferred because intensities of less than 0.5 W cm-2 of ultrasound for 10 min did not result in any significant skin temperature rise nor did it have any destructive effect on rat skin. Progressively more skin damage was noted as the intensity and the time of application of ultrasound increased. When used at a proper intensity and time of application, ultrasound appears to be a safe technique for enhancing the passage of various drug molecules through human skin.     

Membrane permeation-controlled transdermal delivery system design. Influence of controlling membrane and adhesive on skin permeation of isosorbide dinitrate

A membrane permeation-controlled transdermal delivery system (MC-TDS) of isosorbide dinitrate (ISDN), a model drug, was prepared from polyvinyl alcohol aqueous gel containing the drug, a membrane consisting of ethylene-vinyl acetate copolymer membrane and acrylic adhesive (EV-a). The permeability of ISDN through the EV-a membrane was 2.5 times higher than that through excised hairless rat skin. The ratio of plasma concentration of ISDN after application of MC-TDS on stripped (damaged) skin relative to intact skin was lower than that after application of Frandol tape-S, a marketed ISDN TDS, which suggests that the EV-a membrane might work as a control membrane for overall delivery rate of ISDN to the body. When MC-TDS stored at 30 degrees C for 13.5-48h was applied to the damaged skin, however, the initial plasma concentration of ISDN was very much higher than the expected therapeutic level and was not controlled by the EV-a membrane. The initial high plasma concentration of ISDN after application of the stored MC-TDS on the damaged skin was due to migration of ISDN from the reservoir to the adhesive during storage at 30 degrees C. The migration of drugs into the adhesive might be an important problem in developing efficient MC-TDS.     

Percutaneous absorption of elcatonin and hypocalcemic effect in rat

The percutaneous absorption of elcatonin (EC), a hypocalcemic peptide, was investigated. A transdermal dosage form of EC was produced using a gel base, absorption enhancer and protease inhibitor, and applied to rats for 24 h. The combination of bile salt such as taurocholate and glycocholate, and n-octyl-beta-D-glucoside or n-octyl-beta-D-thioglucoside (OTG) exerted the potent enhancing effect on the absorption of EC, and a potent hypocalcemic effect was shown for 24 h or longer. The least level of plasma calcium was obtained 6 h or longer after application, suggesting the relatively rapid absorption of EC. The apparent bioavailability of EC in system 5 was 4.6%, this value being noteworthy in the percutaneous absorption of peptides. When the enhancing effect of taurocholate and OTG was separately measured, both agents acted additively on the absorption of EC. An EC ointment maintained the hypocalcemin effect after storage for 15 d at 40 degrees C. The transdermal dosage form has the potential to be an efficient drug delivery system for Paget's disease and osteoporosis.     

Film‐ and ointment‐based delivery systems for the transdermal delivery of TNP‐470

Pathological angiogenesis, the process of new blood vessel formation, is responsible for a broad range of neovascular‐related systemic diseases. One of the first antiangiogenic compounds tested in clinical trials against cancer was TNP‐470. Despite promising activity the injectable drug showed poor plasma stability and caused adverse side effects in high doses lead to termination of the trials. In our current work, we introduce the development of a transdermal delivery systems for controlled release of TNP‐470. Such formulation can potentially reduce toxicity due to controlled continuous dosing and improve stability by avoiding gastrointestinal first pass metabolism. Although transdermal delivery is a very challenging route for drug administration due to the low permeability of the skin, here we present a successful development of two different drug delivery systems, film and ointment for dermal application of TNP‐470. Chitosan film had high loading capacity of up to 50% w/w of TNP‐470 compared with 10% maximum loading in hydrocarbon ointment. A detailed step‐by‐step development of TNP‐470 films, from the initial solvent screening to final optimized formulation, is presented. Ex vivo skin permeation studies demonstrated a superior release of the drug from the film formulation compared with the ointment. Furthermore, histological test of the skin confirmed ointment safety showing no evidence of skin tissues damage. Our results present novel, promising, controlled release drug delivery systems with improved stability, efficacy, and safety profile of TNP‐470 via transdermal route.     

Simultaneous determination of clozapine, clozapine N-oxide, N-desmethylclozapine, risperidone, and 9-hydroxyrisperidone in plasma by high performance liquid chromatography with ultraviolet detection

A simple high performance liquid chromatography techniques with ultraviolet detection (HPLC–UV) method is described for the simultaneous determination of clozapine (CZP), clozapine N-oxide (CNO), N-desmethylclozapine (NCZ), risperidone (RSP) and 9-hydroxyrisperidone (9-OHRSP) in human plasma. After extraction process, the analytes were separated on a C₁₈ column (150 mm×3.9 mm i.d.) by the mobile phase (methanol–water–dimethylamine, 60:40:0.04 (v:v:v)). Relative recoveries of five analytes were quantitative. The precision and accuracy of intra- and interday assays were all below 8.2% for R.S.D. and 5.6% for RE, respectively. Based on 1 ml of plasma, the limits of detection were 2.0 ng/ml for CZP, 0.2 ng/ml for CZP N-oxide, 1.0 ng/ml for NCZ, 1.0 ng/ml for RSP, and 0.5 ng/ml for 9-OHRSP (S/N=3). The calibration curves were linear (r≥0.988). This method was applied to therapeutic drug monitoring of schizophrenia patients receiving CZP or RSP therapy.     

High-performance liquid chromatography determination of dipotassium clorazepate and its major metabolite nordiazepam in plasma

A rapid and sensitive high-performance liquid chromatographic method is described for the quantitative analysis of dipotassium clorazepate (CZP) and its major metabolite nordiazepam (ND) in fresh human and dog plasma. The method consists of two separate selective ND extractions from a plasma sample without and with conversion of all the CZP to ND. For quantitation, diazepam (DZP) is used as the internal standard. The chromatographic phase utilized in a reversed-phase Hibar EC-RT analytical column prepacked with LiChrosolv RP-18 with a solvent system consisting of acetonitrile-0.05 M sodium acetate buffer, pH 5.0 (45:55). The UV absorbance is monitored at 225 nm using a variable-wavelength detector. The mean assay coefficient of variation over a concentration range of 20-400 ng per ml of plasma is less than 3% for the within-day precision. Recoveries of ND, DZP and CZP (as ND) are essentially quantitative at all levels investigated. The calibration curves of ND are rectilinear (r2 = 0.99) from the lower limit of sensitivity (2 ng/ml) to at least 2000 ng/ml in plasma. Applicability of the method to CZP and ND disposition studies in the anaesthetized mongrel dog is illustrated. When the two separate selective nordiazepam extractions from plasma cannot be performed immediately after blood sampling, an extrapolation kinetic method is suggested for the estimation of CZP concentration. In all previous in vivo studies, CZP has been determined only with gas-liquid chromatographic methods.     

Improvement in bioavailability of transdermally applied flurbiprofen using tulsi (Ocimum sanctum) and turpentine oil

Penetration enhancing potential of tulsi and turpentine oil on transdermal delivery of flurbiprofen, a potent non-steroidal anti-inflammatory agent, was investigated. The transdermal permeation rate of flurbiprofen across the rat abdominal skin from binary solvent mixture composition of propylene glycol (PG):isopropyl alcohol (IPA) (30:70%, v/v) was 98.88 microg/cm(2)/h, significantly higher than other binary solvent mixtures. The corresponding steady state plasma concentration, 0.71 microg/ml, was much lower than required steady state plasma concentration of 3-5 microg/ml. Hence influence of tulsi and turpentine oil in the optimized binary solvent mixture along with the increased drug load on the flurbiprofen permeation was evaluated. The magnitude of the flux enhancement factor with turpentine oil and tulsi oil was 2.4 and 2.0 respectively at 5% (v/v) concentration beyond which there was no significant increase in the flux. Addition of 2% (w/v) hydroxypropyl methylcellulose (HPMC), as a thickening agent, resulted in desired consistency for the fabrication of patch with insignificant effect on permeation rate of flurbiprofen. The reservoir type of transdermal patch formulation, fabricated by encapsulating the flurbiprofen reservoir solution within a shallow compartment moulded from polyester backing film and microporous ethyl vinyl acetate membrane, did not modulate the skin permeation of flurbiprofen through rat skin in case of turpentine formulations whereas flux of formulations with tulsi oil was significantly altered. The influence of penetration enhancer and solvents on the anatomical structure of the rat skin was studied. Enhancement properties exhibited by turpentine oil and tulsi oil in optimized binary solvent mixture were superior as compared to solvent treated and normal control groups with negligible skin irritation. The fabricated transdermal patches were found to be stable. The bioavailability of flurbiprofen with reference to orally administered flurbiprofen in albino rats was found to increase by 2.97, 3.80 and 5.56 times with transdermal patch formulation without enhancer, tulsi and turpentine oil formulations, respectively. The results were confirmed by pharmacodynamic studies in rat edema inflammation model.     

Influence of Crosslinking Agent on the Release of Drug from the Matrix Transdermal Patches of HPMC/Eudragit RL 100 Polymer Blends

The present work was designed to develop suitable transdermal matrix patches using the polymer blends of hydroxy propyl methyl cellulose (HPMC) and Eudragit RL100 (ERL) with triethyl citrate as a plasticizer in group A and in group B, other than HPMC and ERL, crosslinking agent, succinic acid was added. A 3² full factorial design was employed for both groups. The concentration of HPMC and ERL were used as independent variables, while percentage drug release was selected as dependent variable. Physical evaluation was performed such as moisture content, moisture uptake, tensile strength, flatness and folding endurance. In vitro diffusion studies were performed using cellulose acetate membrane (pore size 0.45 μ) in a Franz's diffusion cell. The concentration of diffused drug was measured using UV-visible spectrophotometer (V-530, Jasco) at λ _max 272 nm. The experimental results shows that the transdermal drug delivery system (TDDS) containing ERL in higher proportion gives sustained the release of drug and patches containing crosslinking agent shows more release than those do not contains succinic acid.     

Pharmacokinetics of aconitine in rat skin after oral and transdermal gel administrations

The purpose of this study was to evaluate percutaneous penetration and arrhythmogenic effects of aconitine after transdermal administration, compared with the oral route. Skin penetration of aconitine was tested by a microdialysis technique in rats and in vivo recovery was determined by retrodialysis. After oral and transdermal administration of aconitine, dialysate was sampled at 20 min intervals until the end of the experiment for the determination of concentration of aconitine in skin. Blood samples were collected and analyzed using a validated HPLC‐MS/MS method. In addition, we concurrently recorded the electrocardiogram (ECG). The in vivo recovery of aconitine in the skin was calculated to be 39.59%. The C_max values for aconitine absorbed into the skin after oral and transdermal administration were 1.51 ± 0.53 and 2723.8 ± 848.8 ng/mL, respectively, and within the plasma, 215.86 ± 79.29 and 20.92 ± 3.15 ng/mL. The C_max value for the plasma concentration of aconitine after oral administration was approximately 10 times higher than with the transdermal route. For oral administration, the ECG revealed various types of arrhythmias at a period of T_max, which is normal in transdermal gel administration. These results indicate that transdermal aconitine gel is a safe formulation that can deliver the drug in sufficient amounts and safe concentrations to produce therapeutic action in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhanced transdermal delivery of indomethacin-loaded PLGA nanoparticles by iontophoresis

Nanoparticles effectively deliver therapeutic agent by penetrating into the skin. Indomethacin (IM) and coumarin-6 were loaded in PLGA nanoparticles with an average diameter of 100 nm. IM and coumarin-6 were chosen as a model drug and as a fluorescent marker, respectively. The surfaces of the nanoparticles were negatively charged. Permeability of IM-loaded PLGA nanoparticles through rat skin was studied. Higher amount of IM was delivered through skin when IM was loaded in nanoparticles than IM was free molecules. Also, iontophoresis was applied to enhance the permeability of nanoparticles. When iontophoresis with 3 V/cm was applied, permeability of IM was much higher than that obtained by simple diffusion of nanoparticles through skin. The combination of charged nanoparticle system with iontophoresis is useful for effective transdermal delivery of therapeutic agents.     

A Novel Methacryloyl Chitosan Hydrogel Microneedles Patch with Sustainable Drug Release Property for Effective Treatment of Psoriasis

Psoriasis is a chronic and recurrent skin disease that often requires long-term treatment, and topical transdermal drug delivery can reduce systemic side effects. However, it is still a challenge in efficient transdermal drug delivery for psoriasis treatment due to low penetration efficiency of most drugs and the abnormal skin conditions of psoriasis patients. Here, a safe and effective methacryloyl chitosan hydrogel microneedles (CSMA hMNs) patch is developed and served as a sustained drug release platform for the treatment of psoriasis. By systematically optimizing the CSMA preparation, CSMA hMNs with excellent morphological characteristics and strong mechanical properties (0.7 N needle⁻¹) are prepared with a concentration of only 3% (w/v) CSMA. As a proof-of-concept, methotrexate (MTX) and nicotinamide (NIC) are loaded into CSMA hMNs patch, which can produce a sustained drug release of 80% within 24 h in vitro. In vivo experiments demonstrated that the CSMA hMNs patch can effectively inhibit the skin thickening and spleen enlargement of psoriatic mice and has a good biosafety profile at sufficient therapeutic doses. This study provides a new idea for the preparation of hMN systems using modified CS or other biocompatible materials and offers an effective therapeutic option for psoriasis treatment.     

Emodin–phospholipid complex

Developing the drugs as amphiphilic lipid complexes is a potential approach for improving therapeutic efficacy of the drugs by increasing solubility, reducing drug crystallinity, modifying dissolution behavior (sustained or controlled release), and improving bioavailability. Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an anthranoid derivative, shows several biological effects like antimicrobial, antidiuretic, anti-cancerous, and potent antioxidant but due to poor solubility, the dissolution restrains its valuable importance. To overcome this limitation, the emodin–phospholipid complex was developed and investigated by thermal analysis (differential scanning calorimetry), crystallographic (X-ray diffractography), surface morphology (scanning electron microscopy), spectroscopic methods (FT-IR, ¹H-NMR), solubility, and the dissolution (in vitro drug release) study. The phospholipid complex of emodin was found, fluffy and porous with rough surface morphology in the SEM. FT-IR, ¹H-NMR, DSC, and X-RPD data confirmed the formation of the complex. The water and n-octanol solubility of emodin was improved from 2.25 to 9.97 and 53.45 to 77.62 μg/ml, respectively, in the prepared complex. The improved dissolution was shown by the phospholipid complex. Based on the results of the study, it can be concluded that the phospholipid complex may be considered as promising drug delivery system for improving the overall absorption and bioavailability of the emodin molecule.     

Development of a stick-type transdermal eyelid delivery system of ketotifen fumarate for ophthalmic diseases

A transdermal eyelid delivery system for treating ocular diseases (eye-stick) has been developed. Ketotifen fumarate (KT) was used as a model drug. An in vivo study using rabbits showed that the eye-stick device maintained a constant conjunctival concentration of the drug for an extended period of time, which was equivalent or higher than the therapeutic level following eye drop administration. Moreover, the conjunctival concentration after eye-stick application was well predicted using the physicochemical parameters, diffusion coefficient and partition coefficient, obtained from in vitro hairless mouse skin permeation experiments.     

Dual-labeled visual tracer system for topical drug delivery by nanoparticle-triggered P-glycoprotein silencing

Using nanoparticle-based drug delivery systems as enhancers is a robust strategy for transdermal delivery; however, the mechanisms by which these systems promote transdermal penetration are still unclear. Here, we fabricated a dual-labeled nano drug delivery system that allows discrete visualization of both the drug and the nanoparticle carrier. To comprehensively examine its potential mechanism, we investigated its effects on human epidermal keratinocyte HaCaT cells, including changes in cell membrane potential, intracellular Ca²⁺ concentration, and Ca²⁺-ATPase activity. P-glycoprotein (P-gp) expression in nanoparticle-treated human dermal microvascular endothelial cells was detected by western blotting and immunofluorescence. Furthermore, the transdermal absorption and biodistribution of the dual-labeled nanoparticles were deeply investigated by skin permeability study in vitro and in vivo using fluorescence microscopy and in vivo imaging, respectively. In addition to reducing membrane potential, increasing the intracellular Ca²⁺ concentration, and decreasing Ca²⁺-ATPase activity, our results indicate that the dual-labeled nanoparticles can downregulate P-gp to promote transdermal absorption. Fluorescence and in vivo imaging visually demonstrated that the nanoparticle delivery system penetrated into the dermis through the stratum corneum. All these results indicate that this dual-labeled nano delivery system provides a new method for future in-depth visual explorations of transdermal drug delivery mechanisms.     

Enhanced transdermal delivery of zidovudine in rats and human skin

Zidovudine (azidothymidine, AZT), a potent antiviral agent acting on acquired immunodeficiency syndrome virus, was examined with regard to permeation through rat and human skin. A steady state plasma concentration of AZT after transdermal application in rats estimated from both pharmacokinetics data after i.v. administration and penetration rate through excised rat skin from 10% oleic acid (OA) aqueous solution shows penetration about 85 times higher compared to that from 10% OA would be needed for therapeutic efficacy. A mixed-solvent system consisted of 5% Sefsol-318 (S-318), 10% OA, 10% N-methyl-2-pyrrolidone (MP), 20% propylene glycol (PG) and water showed promising characteristics as a vehicle in terms of permeability of AZT through excised rat skin. The maximum flux of 0.41 mumol/cm2/h was observed in excised human skin after application of a gel formulation including S-318, OA, MP and PG. The result suggests a possible use of the gel formulation to gain an effective plasma concentration in humans.     

Quantitative determination of enzalutamide,an anti‐prostate cancer drug,in rat plasma using liquid chromatography–tandem mass spectrometry,and its application to a pharmacokinetic study

This report details a method using liquid chromatography–tandem mass spectrometry (LC‐MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC‐MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 µm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple‐reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001–1 µg/mL, as well as an intra‐ and inter‐assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 μL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Formulation design of ointment base suitable for healing of lesions in treatment of bedsores

We intended to develop a desired ointment base suitable for treatment of bedsores including the proliferation of granulation and epidermis. The main bedsore bacteria detected in our hospital were S. aureus in gram-positive coccus and P. aeruginosa in gram-negative bacillus. As the macrogol ointment (MO) was found to have bactericidal effects on these bacteria, MO was adopted as the base for the objective ointment. To improve the properties of the ointment base such as regulating the humidity of the exudation and controlling the release of antibiotics formulated in the ointment, co-formulating effects of various additives to MO were evaluated. The sustained release function of the ointment base was obtained by adding hydrophilic petrolatum (HP) to MO. However, the resultant ointment was found to have a poor humidity regulating property. On the other hand, MO containing 5% of hydroxypropyl cellulose (HPC) showed both the humidity regulating and the controlled drug releasing properties. It was considered that HPC particles dispersed in the ointment could be swelled by absorbing water to form a gel network. The curd tension meter tests for the ointments prepared with the various polymers showed that the MO-HPC base, which showed the highest sustained drug releasing property, was found to have the highest hardness. This result means that HPC formulated into the base forms the most rigid gel structure to resist the erosion of the ointment and to control the drug release.     

Percutaneous absorption of bromhexine in rats

The percutaneous absorption of bromhexine (BH), an expectorant drug, through rat skin was examined in vitro and in vivo. BH in free base form penetrated better than the hydrochloride through the skin. When the in vitro penetration of BH was compared using Plastibase, macrogol and sucrose ester of fatty acid F-160 (DK-ester) formulations, the DK-ester formulation showed the best penetration of BH of the three. The addition of Azone (3%) or lauric acid (BH: lauric acid molar ratio, 1:1) considerably increased BH penetration to a relatively large penetration rate. The plasma levels of BH after in vivo application of the DK-ester formulation with Azone or lauric acid (0.6 g/3.8 cm2) were also higher than those after the formulation without an enhancer, and a constant plasma level (20-50 ng/ml) was obtained during the application for 48 h. However, the bioavailability was low, 2.5 and 2.7% respectively. When the amount of BH remaining in DK-ester ointment and the skin after an 18-h application was measured, the BH content in the ointment was 88.6 +/- 8.0% for the formulation without Azone and 93.7 +/- 6.9% for that with Azone. The low penetration and low bioavailability observed will thus be due to the high drug retention of the base.