Assessment of cutaneous drug delivery using microdialysis

During the last decade microdialysis has been successfully applied to assess cutaneous drug delivery of numerous substances, indicating the large potential for bioequivalence/bioavailability evaluation of topical formulations. The technique has been shown to be minimally invasive and supply pharmacokinetic information directly in the target organ for cutaneous drug delivery with high temporal resolution without further intervention with the tissue after implantation. However, there are a few challenges that need to be addressed before microdialysis can be regarded as a generally applicable routine technique for cutaneous drug delivery assessments. Firstly, the technique is currently not suitable for sampling of highly lipophilic compounds and, secondly, more studies are desirable for elucidation of the variables associated with the technique to increase reproducibility. The present literature indicates that the condition of the skin at the individual assessment sites is the main variable, but also variables associated with relative recovery, differentiation between the pharmacokinetic parameters (i.e., lag time, distribution, absorption and elimination rate) can influences the reproducibility of the technique. Furthermore, it has been indicated that cutaneous microdialysis in rats may be useful for prediction of dermal pharmacokinetic properties of novel drugs/topical formulations in man.     

In vivo study of percutaneous absorption of 4-chloroaniline using microdialysis in the rat

The present work was undertaken to study the percutaneous absorption of 4-chloroaniline (parachloroaniline, PCPA, CAS 106-47-8), a chemical intermediate in pesticide manufacture, using in vivo microdialysis in the rat. The results of the microdialysis study showed that PCPA was able to cross the skin (Tmax = 3 h and area under the curve (AUC) = 332.1 ng.h/ml) and rapidly enter the systemic circulation (Tmax = 3.3 +/- 0.6 h). It could be also shown that PCPA was partly metabolised during its percutaneous absorption. The analysis of cutaneous dialysate samples using gas chromatography/mass spectrometry (GC-MS) showed that the metabolite was 4-chloracetanilide. Taken together, the data obtained showed that contact with the skin is a danger for exposed persons.     

Nasal administration of ondansetron using a novel microspheres delivery system Part II: Ex vivo and in vivo studies

Gellan gum-based mucoadhesive microspheres of ondansetron hydrochloride for intranasal systemic delivery were prepared to avoid first pass effect, an alternative route of administration to injection and to enhance systemic bioavailability of ondansetron hydrochloride. The microspheres were prepared using spray method. The evaluation results of microspheres were reported in our previous study. The aim of this work was to study the in vivo performance of mucoadhesive microspheres in comparison with oral and intravenous preparations of ondansetron hydrochloride. The nasal delivery system gave increased AUC_0-240 and C_max as compared to those of oral delivery. In conclusion, the gellan gum-based microsphere formulation of ondansetron hydrochloride with mucoadhesive properties with increased permeation rate is promising for prolonging nasal residence time and thereby nasal absorption.     

Ex vivo absorption of promestriene from oil-in-water emulsion into infant foreskin

Hypospadias is a birth defect in which the urinary tract opening is not at the tip of the penis. Hypospadias surgery is frequently complicated by healing deficiencies. Topical treatments with oestrogens were reported to improve healing. In the present study, ex vivo percutaneous absorption of promestriene, a synthetic oestrogen resulting of the double esterification of estradiol was conducted as a pre-requisite for further clinical trial in infants. Penetration of promestriene into infant foreskin treated with commercial oil in water emulsion (10 μg mg⁻¹) for 24 h was characterized showing controlled release properties enabling epidermal concentration more than six times higher than dermal concentration (4.13 ± 2.46 mg g⁻¹ versus 0.62 ± 0.84 mg g⁻¹, respectively). Furthermore, apparent promestriene fluxes into and through the skin (i.e., 1.5 μg cm⁻² h⁻¹ and < 0.89 μg cm⁻² h⁻¹, respectively) were calculated from (i) drug amount retained into epidermis and dermis, or (ii) the limit of detection into the receptor fluid. In conclusion, less than 2% of initial dose were absorbed within 24 h which compared well with others steroids applied topically in colloidal systems.     

Decylglucoside-based microemulsions for cutaneous localization of lycopene and ascorbic acid

Cutaneous delivery of combinations of antioxidants offers the possibility of enhanced protection against UV-radiation. In this study, we investigated the potential of sugar-based microemulsions containing monoglycerides to promote simultaneous cutaneous delivery of lycopene and ascorbic acid, and increase tissue antioxidant activity. Lycopene and ascorbic acid were incorporated (0.04% and 0.2% (w/w), respectively) in decylglucoside-based microemulsions containing isopropyl myristate mixed with monocaprylin (ME-MC), monolaurin (ME-ML) or monoolein (ME-MO) as oil phase. The microemulsions increased lycopene delivery into porcine ear skin by 3.3- to 8-fold compared to a drug solution. The effect of microemulsions on ascorbic acid cutaneous delivery was more modest (1.5-3-fold), and associated with an approximately 2-fold increase in transdermal delivery. According to their penetration-enhancing ability, the microemulsions were ranked ME-MC>ME-MO>ME-ML. This superiority of ME-MC coincided with a stronger effect in decreasing skin electrical resistance. After 18 h of treatment, the viability of bioengineered skin treated with ME-MC was 2.2-times higher compared to Triton-X100 (moderate irritant), demonstrating that ME-MC is less cytotoxic. Skin treatment with ME-MC containing both antioxidants increased the tissue antioxidant activity by 10.2-fold, but no synergism between the antioxidants was observed.     

Ex vivo and in vivo diffusion of ropivacaine through spinal meninges: influence of absorption enhancers

Following epidural administration, cerebrospinal fluid bioavailability of local anesthetics is low, one major limiting factor being diffusion across the arachnoid mater barrier. The aim of this study was to evaluate the influence of absorption enhancers on the meningeal permeability of epidurally administered ropivacaine. Five enhancers known for their ability to increase drug permeability via transcellular and/or paracellular pathways, i.e. palmitoyl carnitine, ethylenediaminetetraacetic acid, sodium caprate, dodecylphosphocholine and pentylglycerol, were tested ex vivo on fresh specimen of meninges removed from cervical to lumbar level of rabbit spine following laminectomy and placed in diffusion chambers. Among them, sodium caprate lead to the best permeability improvement for both marker and drug (440% and 112% for mannitol and ropivacaine, respectively) and was therefore selected for in vivo study in a sheep model using microdialysis technique to evaluate epidural and intrathecal ropivacaine concentrations following epidural administration. Resulting dialysate and plasma concentrations were used to calculate pharmacokinetic parameters. Following sodium caprate pre-treatment, ropivacaine intrathecal maximal concentration (Cmax) was 1.6 times higher (78 ± 16 μg ml(-1) vs 129 ± 26 μg ml(-1), p<0.05) but the influence of the absorption enhancer was only effective the first 30 min following ropivacaine injection, as seen with the significantly increase of intrathecal AUC(0-30 min) (1629 ± 437 μg min ml(-1) vs 2477 ± 559 μg min ml(-1), p<0.05) resulting in a bioavailable fraction 130% higher 30 min after ropivavaine administration. Co-administration of local anesthetics with sodium caprate seems to allow a transient and reversible improvement of transmeningeal passage into intrathecal space.     

反向透析法用于体内经皮吸收微透析探针回收率的测定

目的考察反向透析法用于体内经皮吸收过程中微透析探针回收率校正的可行性.方法在体外试验中用零净流量法测定探针的回收率和传递率,在体内试验中用反向透析法测定探针的回收率;以小猪为试验动物,观察模型药雷公藤内酯醇软膏的体内经皮吸收过程.结果体外试验中,微透析探针的回收率为(64.29±2.34)%,回收率和传递率一致;体内经皮微透析实验中探针的回收率在实验前为(51.47±3.51)%,实验结束后为(51.96±4.97)%.雷公藤内酯醇软膏经皮吸收后,药物在皮内浓度很快达到稳态,去除药物后皮内药物浓度则迅速下降.结论反向透析法测定的探针回收率在实验过程中稳定,可以用于测定体内经皮吸收微透析探针回收率.     

New microencapsulation system for ascorbic acid using pea protein concentrate as coat protector

Microencapsulation is essential to preserve biological activity of ascorbic acid (AA) and pea protein has not been used as a carrier in such processes. This work aimed to produce microparticles by a spray-drying process using pea protein (PPC) as wall material of AA and evaluate the retention of the core by HPLC, overall morphology SEM, size distribution by light scattering and release kinetics. Carboxymethylcellulose (CMC) and blends with maltodextrin (M) were produced for comparative analyses. The yields were compatible with the applied technology and the retention was above 84% for all materials. The PPC microparticles presented irregular and rough surfaces, CMC produced a regular and smooth surface and agglomeration was more intense in microparticles with M. Mean particle diameters were all below 8?µm. The microparticle release rates were lower than those with free AA, being best correlated to the Higuchi kinetic model. These results support the utilization of PPC for microencapsulation of AA.     

New microencapsulation system for ascorbic acid using pea protein concentrate as coat protector

Microencapsulation is essential to preserve biological activity of ascorbic acid (AA) and pea protein has not been used as a carrier in such processes. This work aimed to produce microparticles by a spray-drying process using pea protein (PPC) as wall material of AA and evaluate the retention of the core by HPLC, overall morphology SEM, size distribution by light scattering and release kinetics. Carboxymethylcellulose (CMC) and blends with maltodextrin (M) were produced for comparative analyses. The yields were compatible with the applied technology and the retention was above 84% for all materials. The PPC microparticles presented irregular and rough surfaces, CMC produced a regular and smooth surface and agglomeration was more intense in microparticles with M. Mean particle diameters were all below 8 microm. The microparticle release rates were lower than those with free AA, being best correlated to the Higuchi kinetic model. These results support the utilization of PPC for microencapsulation of AA.     

Study of the extent of ocular absorption of acetazolamide from a developed niosomal formulation, by microdialysis sampling of aqueous humor

Acetazolamide, a carbonic anhydrase inhibitor is used orally (no topical formulation being available in the market) for the reduction of intraocular pressure (IOP) in patients suffering from glaucoma. Two major reasons responsible for the failure to develop a topically effective formulation of acetazolamide are its low solubility (0.7mg/ml) and its low permeability coefficient. It is generally recognized that topical acetazolamide formulation possessing efficacy similar to that achieved upon oral administration would be a significant advancement in the treatment of glaucoma. In order to enhance the bioavailability of acetazolamide by topical route and to improve the corneal permeability of the drug, the niosomes of acetazolamide were prepared (by reverse phase evaporation method) and coated with Carbopol for the latter's bioadhesive effect. The pharmacodynamic studies showed 33% fall in IOP with the developed formulation, and the effect was sustained for 6h after instillation. The effect compared well with a four times higher concentration of dorzolamide (Dorzox, a topical CAI available in the market. In the present study, the aqueous humor disposition of the drug from the developed bioadhesive coated niosomal formulation (ACZREVbio) is compared with the aqueous suspension of the drug (containing 1% (w/v) Tween 80 as a dispersing agent) at similar concentrations. The concentration of acetazolamide absorbed in the aqueous humor at various times from the control suspension and from ACZREVbio was determined by microdialysis in male albino rabbits. Microdialysis provides a complete concentration versus time profile and hence is an important advance to the regional sampling of tissues. The peak concentration of drug absorbed in the aqueous humor from the ACZREVbio formulation (14.94microg/ml) was almost two times of that obtained with the equivalent amount of acetazolamide control suspension (6.93microg). The results show a significant broadening of peak from 80 to 120min with the concentration of more than 13microg being maintained at these times, for the bioadhesive coated niosomal formulation (ACZREVbio). An important observation was the fact that a high drug concentration of 12.02microg reached immediately, i.e., 20min after instillation of ACZREVbio indicating a high penetration being achieved, while a meagre concentration of only 3.53microg is obtained at 60min after instillation of the control suspension. The aqueous humor disposition indicates peaks and troughs in drug concentration which may be related to the decrease in aqueous humor formation, such that the drug concentration/volume increased at these points.     

Effective intestinal absorption of insulin in diabetic rats using a new formulation approach

Insulin injected intra-jejunally together with the non-ionic surfactant cetomacrogol was effective in streptozocin-induced diabetes in the rat, as measured by the hypoglycaemic effect. The reduction in blood sugar was maximal at about 2 h after administration but continued at a high level for the 4 h of the experiment. No hypoglycaemic effect was observed in controls injected with insulin or saline alone. Intestinal absorption of insulin has thus been effected by the addition of cetomacrogol, which appears to enhance membrane-permeability to insulin rather than to function as a protective agent preventing insulin degradation, as in liposome-encapsulation. In support of this, a significant hypoglycaemic action was still obtained when the insulin injection was given half-hour after that of the cetomacrogol, both intra-jejunally. Furthermore, oral administration of the surfactant followed by intra-jejunal injection of the insulin also gave a hypoglycaemic effect. The use of this agent to enhance insulin absorption offers the possibility of a new approach to oral insulin therapy.     

Differentiated in vivo skin penetration of salicylic compounds in hairless rats measured by cutaneous microdialysis.

The purpose was to investigate the in vivo skin penetration of four 14C-salicylic compounds using microdialysis and to relate dermal concentrations to structural features. Furthermore, to compare two in vivo retrodialysis recovery methods for estimation of true unbound extracellular concentrations. Microdialysis probes were inserted in the dermis of hairless rats. Equimolal 14C-salicylic formulations were applied topically and dialysate sampled consecutively for 4h. True extracellular concentrations were estimated by retrodialysis by drug method (the 14C-salicylic compounds themselves) and by retrodialysis by calibrator method (3H-salicylic acid as internal standard). Probe depth was measured by ultrasound scanning. High dermal concentrations were found after application of 14C-salicylamide (low protein-binding) and the lipophilic ester 14C-butyl salicylate, which was completely hydrolysed to 14C-salicylic acid during skin diffusion. Protein binding and dissociation may explain the lower dermal concentrations of 14C-salicylic acid and 14C-diethylamine salicylate, respectively. Probe depth did not significantly influence dialysate concentrations. The two in vivo recovery correction methods did not reduce the variation in concentration-time curves. In conclusion, differentiated penetration kinetics was found ranking: 14C-salicylamide >/= 14C-butyl salicylate > 14C-salicylic acid > 14C-diethylamine salicylate. Dermal concentrations were related to structural features of the model compounds. The two correction methods performed alike; however, the calibrator method has the advantage of serving as a quality control during experiments.     

Intestinal absorption of oxalate in scorbutic and ascorbic acid supplemented guinea pigs

Radiolabelled U-14C oxalic acid uptake was measured in the intestine of scorbutic and ascorbic acid (AA) supplemented guinea pigs. The feeding of vitamin C deficient diet to the animals for 26 days resulted in a significant fall in the ascorbic acid levels in the various tissues studied. Supplementation of vitamin C (10, 25 or 50 mg per 200 g body weight) increased ascorbic acid levels of spleen, adrenals, liver and leucocytes. The intestinal uptake of oxalate follows a passive diffusion mechanism in normally fed guinea pigs. The oxalate uptake rate was significantly increased (p less than 0.001) in the vitamin C administered group. Vitamin C depletion significantly decreased the oxalate uptake rate as compared to control animals. The changes observed in the uptake rate appear to be related with the chemical aberrations produced in the brush border membranes.     

Determination of ascorbic acid and isoascorbic acid by capillary zone electrophoresis: Application to fruit juices and to a pharmaceutical formulation

Capillary zone electrophoresis was applied to the determination of ascorbic and isoascorbic acid, analysing the various parameters of influence such as the separation voltage, the buffer pH and concentration, the type of separation capillary or the loading conditions. Both analytes could be adequately determined within 5 min. The proposed method uses a 20 cm x 25 microns i.d. coated column, 0.1 M phosphate buffer pH 5.0, 8 kV separation voltage and light absorption detection at 265 nm. Linear calibration curves were obtained in the 0-1 mg mi-1 range, with detection limits of 0.5 micrograms ml-1. This method proved to be very rapid, simple and practical for the qualitative and quantitative determination of ascorbic acid in lemon and orange juices, as well as in a commercially available pharmaceutical formulation.     

In vivo antineoplastic activity of ascorbic acid for human mammary tumor

The effect of ascorbic acid on the growth of human mammary tumor xenografts was investigated using the 6-day subrenal capsule assay method. The results showed that ascorbic acid (1 or 5 g/liter) administered in the drinking water significantly inhibited the growth of tumor fragments implanted beneath the renal capsule of immunocompetent mice. The results agree with other work carried out in animal experiments with animal tumors. Administration of ascorbic acid in the mouse diet did not affect the growth of the human mammary tumor fragments within the 6-day experimental period. Tumor growth was inhibited when mice were fed a diet containing ascorbic acid (50g/kg diet) together with cupric sulfate (18 or 90 mg/liter) in the drinking water. The results support the hypothesis that certain oxidation and degradation products of ascorbic acid are active antineoplastic agents for the human mammary carcinoma studied.     

The use of a nonlinear absorption model in the study of ascorbic acid bioavailability in man

A two-compartment disposition model of ascorbic acid (AA) pharmacokinetics with saturable and time-constrained intestinal absorption was developed. The model was fitted to pharmacokinetic data obtained after oral administration to nine healthy volunteers of two effervescent dosage forms differing in AA content: Celaskon 60 mg (CK60) and Celaskon 500 mg (CK500). It was demonstrated that in the case of CK500 less than 30% of the dose was absorbed as compared with CK60. Parameters of the AA nonlinear absorption kinetics were assessed by simultaneous fitting of mean concentration—time data for both doses and placebo. The relatively short duration of absorption found (3.2 h) can explain the failure of past attempts to increase the AA bioavailability using sustained-release dosage forms. Model simulation showed that the ingestion of 60 mg with 3–4 h intervals is optimal for maximal bioavailability of AA.     

Assessment of in vitro and in vivo recovery of gallamine using microdialysis

The application of microdialysis technique for the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of probe performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of the neuromuscular blocker, gallamine. In vitro recovery of gallamine from the microdialysis probe when different perfusion flow rates were employed was evaluated leading to selection of a flow rate of 2 microl/min with 15-min sampling intervals for the subsequent studies. In vitro recovery of gallamine from the microdialysis probe was independent of concentration, stable over an 8-h period and reproducible. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of gallamine from rat muscle tissue. The in vivo recovery was stable over a 5.5-h sampling period. In vitro performance of the probe subsequent to the in vivo study remained stable supporting reusage of the probe. These data highlight the importance of a systematic examination of microdialysis probe validation.     

Evaluation of microdialysis sampling of aqueous humor for in vivo models of ocular absorption and disposition

The dynamics of β-adrenergic-associated reductions in aqueous humor production for treatment of elevated intraocular pressure are not well understood. In particular, the relationship between ocular pharmacokinetics and pharmacodynamics has yet to be established. This study was undertaken to develop a procedure for examining the ocular absorption and disposition of topically administered ophthalmic β-adrenergic antagonists in individual animals. Dogs were anesthetized with isoflurane and a microdialysis probe was implanted in the anterior chamber of one eye and perfused with 0.9% saline at a rate of 2 μl min⁻¹. ³H-propranolol was administered by intracameral injection or topically. Each dog received intracameral and topical propranolol, in alternate eyes on separate days, in a randomized cross-over fashion. Microdialysis probe effluent was collected every 5 min for ≥2.5 h; concentrations of propranolol were determined by liquid scintillation spectroscopy and were corrected for probe recovery of the substrate as determined by in vivo retrodialysis (∼46%) to estimate aqueous humor concentrations. In separate experiments in rabbits, microdialysis probes were implanted in each eye. ³H-propranolol was administered topically to one eye; the contralateral eye received intracameral ³H-propranolol. Model-independent pharmacokinetic parameters for each treatment phase were calculated. The mean±S.D. times to peak concentration of propranolol in aqueous humor were 86.6±47.6 min in the dog and 54.1±20.4 min in the rabbit. The terminal rate constant was 0.0189±0.00429 min⁻¹ in the dog vs. 0.00983±0.00546 min⁻¹ in the rabbit. Intraocular tissue availability of propranolol differed markedly between the dog (n=3) and rabbit (n=3) (∼0.056 in the dog vs. ∼0.55 in the rabbit). These results demonstrate the utility of microdialysis sampling for examination of ocular pharmacokinetics.