X-ray microanalysis of cryopreserved human skin to study the effect of iontophoresis on percutaneous ion transport.

PURPOSE: To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis. METHODS: Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA). The intrinsic electron absorbing characteristics of cryopreserved skin allow direct TEM examination without additional staining. XRMA can be used to obtain in a relatively nondestructive way in situ information on ion distributions across the skin. RESULTS: After passive diffusion, iodide was mainly found in the stratum corneum (SC), whereas there was little iodide in the viable epidermis. Iontophoresis up to 300 microA/cm2 did not significantly affect this distribution. With iontophoresis at 1,000 microA/cm2, the amount of iodide increased dramatically and was equally distributed over the SC and viable epidermis. The presence of iodide in the SC suggests that iodide is present inside corneocytes. CONCLUSIONS: Iontophoresis up to 300 microA/cm2 does not significantly perturb skin structures in contrast to iontophoresis at 1,000 microA/cm2. The presence of iodide inside corneocytes suggests the possibility of transcellular percutaneous iodide transport.

Visualization of percutaneous 3H-estradiol and 3H-norethindrone acetate transport across human epidermis as a function of time.

Developing transdermal therapeutic systems for estradiol and norethindrone acetate raised questions about the steroids penetration pathway across and retention in the skin. This paper describes the distribution of 3H-estradiol and 3H-norethindrone acetate in human stratum corneum after topical application to dermatomed skin in vitro. The study involved (a) permeation experiments to determine the steroid flux, (b) autoradiographical visualization of the steroid distribution in the same skin samples, and (c) a correlation between flux and skin distribution in time. On correlating the steroid flux with intraepidermal steroid distribution, it was concluded that both permeants were bound in the skin tissue. The steroids were preferentially located in or close to the intercellular lipids of the stratum corneum, indicating that both transport and binding occurred via this domain of the stratum corneum. This study demonstrated the importance of correlating drug flux with intraepidermal drug distribution as a function of time.

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum.

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.

In vivo human skin permeability enhancement by oleic acid: a laser Doppler velocimetry study.

Topical application of a skin permeation enhancer such as oleic acid (OA) can result in: (i) higher skin permeability for many exogenous substances (ii) an irritation reaction. Laser Doppler velocimetry (LDV) is one of few techniques which can assess both effects using appropriate protocols. Direct LDV measurement, measuring cutaneous blood flow, has been preferred as a tool to evaluate skin irritation. By comparing the LDV value of an irritant-treated site with an untreated site, an irritation index for the irritant can be obtained. Occlusive application of 0.16 M OA in propylene glycol (PG) for either 3 or 24 h produced irritation in form of redness and slight swelling. Using LDV, we obtained an irritation index of 2 and 4, respectively. The vehicle, PG alone, produced an index of around 1, which corresponded well to the slight to almost no irritation observed visually. The duration of the high irritation index assessed by LDV after OA-PG application is comparable to the duration of the increase in transepidermal water loss following the same application. This indicates a correlation between skin irritation and barrier perturbation caused by OA. LDV can also be used to elucidate the effect of enhancers on skin using hexyl nicotinate (HN) as a model drug, since its vasodilative effect can be clearly assessed by LDV. Pre-treatment of PG for 3 h significantly reduced the t0 and tmax of HN. No further reduction could be observed when OA was added into PG, suggesting that OA-PG is not more effective than PG alone in enhancing the permeation of HN.

Electroperturbation of human stratum corneum fine structure by high voltage pulses: a freeze-fracture electron microscopy and differential thermal analysis study.

Application of high voltage pulses (HVP) to the skin has been shown to promote the transdermal drug delivery by a mechanism involving skin electroporation. The aim of this study was to detect potential changes in lipid phase and ultrastructure induced in human stratum corneum by various HVP protocols, using differential thermal analysis and freeze-fracture electron microscopy. Due to the time involved between the moment the electric field is switched off and the analysis, only "secondary" phenomena rather than primary events could be observed. A decrease in enthalpies for the phase transitions observed at 70 degrees C and 85 degrees C was detected by differential thermal analysis after HVP treatment. No changes in transition temperature could be seen. The freeze-fracture electron microscopy study revealed a dramatic perturbation of the lamellar ordering of the intercellular lipid after application of HVP. Most of the planes displayed rough surfaces. The lipid lamellae exhibited rounded off steps or a vanished stepwise order. There was no evidence for perturbation of the corneocytes content. In conclusion, the freeze-fracture electron microscopy and differential thermal analysis studies suggest that HVP application induces a general perturbation of the stratum corneum lipid ultrastructure.

Mechanistic and quantitative prediction of aminopeptidase activity in stripped human skin based on the HaCaT cell sheet model.

HaCaT cell culture sheets were recently demonstrated to be a useful tool to study epidermal metabolism. Here we report on a mechanistic and quantitative correlation between the kinetics of aminopeptidase-based cleavage of L-Ala-4-methoxy-2-naphthylamide (Ala-MNA) in HaCaT sheets versus stripped human skin. Fresh human skin (breast or abdominal) was obtained from cosmetic surgery, tape-stripped, and dermatomed. HaCaT sheets were cultured on porous membranes. Diffusion and concurrent metabolism were studied under reflection and permeation conditions. Numerical simulations of simultaneous diffusion and saturable Michaelis-Menten metabolism were based on a physical model and a fixed set of independently obtained parameters (diffusion coefficient D, distance x, partition coefficient P, Michaelis constant Km, maximum metabolic rate Vmax). Under reflection conditions, cleavage of Ala-MNA in HaCaT sheets was very close to stripped skin. In contrast, in permeation studies substrate only permeated through HaCaT whereas passage through stripped skin led to full cleavage of Ala-MNA to MNA. All experimental data were in reasonable to excellent agreement with numerically generated data. Differences between HaCaT and stripped skin could be quantitatively and mechanistically explained by the thickness of the metabolically active layer, i.e., approximately 10 microm in HaCaT and approximately 40 microm in stripped skin. Full cleavage of permeating Ala-MNA in stripped skin was predicted to occur within the upper approximately 20 microm of viable epidermis. Thus epidermal aminopeptidase activity may act as an efficient metabolic barrier to fully block the permeation of aminopeptidase labile xenobiotics. Within the settings of this study the kinetics of metabolism in the viable epidermis of skin is predictable from HaCaT sheets.

Validation and testing of a new iontophoretic continuous flow through transport cell.

A new continuous flow through transport cell is presented. The design is based on a minimisation of contributions of the cell conformation and experimental protocol to the overall transport kinetics. The system is validated by measuring the washout period of methylene blue and apomorphine. Preliminary results on the iontophoretic transport of apomorphine across human stratum corneum are presented. These data are used to calculate the deviation of the measured apparent flux from the calculated intrinsic flux across the membrane. The steady state iontophoretic flux was 90 +/- 6 nmol cm-2 h-1 when a 15 mM apomorphine solution was applied in the anodal chamber at a current density of 500 microA cm2. At any point in time the contribution of the cell to the transport rate was < or = 3%. The supporting membrane does not contribute significantly to the overall transport rates. Sink conditions are guaranteed at a flow rate > or = 6.5 ml h-1. For this new transport cell it can be concluded that the apparent flux that was measured, at validated experimental conditions, is a reliable estimate for the intrinsic flux.

Pharmacokinetics, enantiomer interconversion, and metabolism of R-apomorphine in patients with idiopathic Parkinson's disease.

The pharmacokinetics and metabolism of R-apomorphine were determined in 10 patients with idiopathic Parkinson's disease after intravenous infusion of 30 micrograms.kg-1 in 15 min. Specifically, emphasis was on enantiomeric interconversion into S-apomorphine and on the formation of apocodeine and isoapocodeine, since these metabolites may interfere with the pharmacodynamics of R-apomorphine. The pharmacokinetics of R-apomorphine in plasma were determined using an enantioselective high-performance liquid chromatography assay. In most patients, the plasma concentration versus time profile was characterized by a biexponential function. The values of relevant pharmacokinetic parameters were as follows: clearance 40 +/- 15 ml.min-1.kg-1, volume of distribution at steady state 1.6 +/- 0.5 l.kg-1, and terminal half-life 41 +/- 13 min. No measurable concentrations of S-apomorphine were detected in plasma, indicating that enantiomeric interconversion does not occur in vivo. Furthermore, no measurable concentrations of the methylated metabolites apocodeine and isoapocodeine could be detected in plasma. The metabolism of apomorphine was characterized on basis of the excretion of unchanged R-apomorphine, S-apomorphine, apocodeine, isoapocodeine, and their respective sulfate and glucuronide conjugates in urine. The total excretion of unconjugated S-apomorphine, apocodeine, and isoapocodeine was less than 0.1% of the administered dose. The total excretion of unchanged apomorphine, apomorphine sulfate, and apomorphine glucuronide amounted to 0.3 +/- 0.4%, 3.8 +/- 1% and 6.0 +/- 2.2% of the administered dose, respectively. The findings of this study show that on intravenous administration, S-apomorphine and the metabolites apocodeine and isoapocodeine are unlikely to interfere with the pharmacologic actions of R-apomorphine in patients with idiopathic Parkinson's disease. Furthermore, no pharmacokinetic interaction between R-apomorphine and catechol-O-methyl transferase inhibitors is expected.

Stepwise intravenous infusion of apomorphine to determine the therapeutic window in patients with Parkinson's disease.

A new experimental strategy was applied to determine the concentration-effect relation and the therapeutic window of apomorphine in individual patients with Parkinson's disease. Apomorphine was administered by a stepwise intravenous infusion. The infusion rate was increased by 10 micrograms/kg/h every 20 minutes, up to 100 micrograms/kg/h or less when adverse effects occurred. Thereafter, the infusion rate was decreased in a stepwise fashion until zero. Plasma apomorphine concentrations were measured every 20 minutes. Clinical efficacy (tapping score and tremor), dyskinesia, and adverse effects were monitored at the same time. The mean clearance of apomorphine was 4.5 L/min (2.2 to 6.6 L/min). Of the 10 patients, 8 responded to apomorphine. The effects were quantal rather than continuous. Within each patient, the concentrations at onset and offset of effect generally were similar. Significant interpatient variability was observed with respect to minimal concentration for each of the effects. Clinical efficacy occurred at a mean minimal effective concentration (MEC) of 4.7 ng/mL (range 1.4 to 10.7 ng/mL). Dyskinesia was observed at a mean concentration of 8.5 ng/mL (range 2.7 to 20 ng/mL). This value was not significantly different from the MEC. The mean minimal toxic concentration was 16.7 ng/mL (8.5 to 24.5 ng/mL) and was significantly different from the mean MEC. In conclusion, the stepwise increase and decrease of the intravenous infusion rate is a suitable tool for the establishment of the concentration-effect relation of apomorphine in individual patients. The finding of a narrow therapeutic window, in which the onset concentrations vary from patient to patient, underlines the need for accurate and individualized dosing.

In vivo human skin barrier modulation by topical application of fatty acids.

The in vivo effects of fatty acids on skin barrier function were assessed by measuring: (i) transepidermal water loss (TEWL), (ii) diffusion lag times for hexyl nicotinate (HN), and (iii) irritant skin response using laser Doppler velocimetry (LDV) in combination with visual scoring. Two classes of fatty acids have been investigated: straight-chain saturated fatty acids (SFA), having 6-12 carbon atoms, and unsaturated fatty acids (UFA): oleic, linoleic, alpha-linolenic and arachidonic acids. It has been reported that these acids can enhance the permeation of various compounds across the skin. After topical and occlusive application as a solution in propylene glycol (PG) for 3 h on the volar arm of human subjects, SFA only caused a slight irritation and increase in TEWL. The diffusion lag times of HN were reduced by the application SFA to the same extent as and not more than by the application of the pure solvent PG. In contrast, the application of UFA caused a significant increase in TEWL and LDV (irritation) responses. The TEWL values after oleic acid application were higher than those observed for the other three acids, while the irritation potential of arachidonic acid was the highest among UFA. As with SFA, sites treated with UFA did not show significantly different lag times of HN diffusion from PC-treated sites. The data suggest that the degree of irritation and the degree of barrier modulation for fatty acids are not necessarily correlated.

Validation of freeze-drying to visualize percutaneous 3H-estradiol transport: the influence of skin hydration on the efficacy of the method.

A study was made of the validity of freeze-drying to visualize the distribution of 3H-estradiol in human stratum corneum after topical application of a dry dose, a patch or a buffer solution. Each of these donor formulations was applied to human dermatomed skin for 24 h using Franz permeation cells. Subsequently, small pieces of skin were subjected to cryofixation, freeze-drying, osmium tetroxide vapor fixation, Spurr resin embedding and electron microscopic autoradiography. Stratum corneum from dry dose and patch application experiments was well preserved by freeze-drying, allowing an accurate localization of 3H-estradiol. In contrast, stratum corneum from buffer solution experiments suffered from cryofixation artifacts due to excessive hydration of the skin. The corresponding autoradiographs showed strong redistribution of 3H-estradiol. Thus, the visualization method under investigation has its limitations regarding the hydration level of the skin.

Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy.

The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination with potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6). Instead of fixing skin with either OsO4 or RuO4, these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K4Fe(CN)6 and K3Fe(CN)6 in combination with RuO4 were examined. Furthermore, we compared two dehydration solvents, methanol and acetone. The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO4 and RuO4 with K3Fe(CN)6, i.e. with Fe in its highest oxidation state (Fe3+). No differences were observed between dehydration in methanol and acetone.

Electroperturbation of the human skin barrier in vitro: II. Effects on stratum corneum lipid ordering and ultrastructure.

In transdermal iontophoresis, drugs can be driven across the skin by electrorepulsion, but their transport can also be enhanced by electrical perturbation of the skin barrier. Our objective was to study perturbing effects of electrical current on human stratum corneum lipid fine structure combining techniques including freeze-fracture electron microscopy. Human stratum corneum was subjected to pulsed constant currents, varying from 0.013-13 mA.cm-2. The voltage across the stratum corneum was high-frequency-sampled and s.c. impedence values derived from it. Upon termination of the current, skin samples were rapidly frozen and processed for freeze-fracture electron microscopy or subjected to X-ray diffraction analysis. Initially a rapid decrease of the resistance and, overall, a rapid increase of the capacitances was observed; generally, these effects became more pronounced with increasing current density. Wide- and small-angle X-ray diffractograms of human stratum corneum exposed for 1 h to the highest current indicated a disordering of both the lateral packaging arrangement and long-range lamellar stacking of the intercellular lipids of stratum corneum. Furthermore, an increase in the stratum corneum hydration level as a result of electrical current application was observed. On electron micrographs of freeze-fracture replicas of human stratum corneum, exposed for 1 h to current densities between 0.013 and 13 mA.cm-2, perturbations of the intercellular lipid structure were observed in accordance with the results of X-ray diffraction; these perturbations aggravated with increasing current density. Together, the data suggest that both the lateral and the longitudinal disordering of the intercellular lipids observed with X-ray diffraction may be responsible for the appearance of perturbed structures observed with freeze-fracture electron microscopy. The lipid disordering may be due to polarization of the lipid head groups induced by the electrical field, followed by mutual repulsion.

In vitro human skin barrier modulation by fatty acids: skin permeation and thermal analysis studies.

PURPOSE: This study aims to elucidate the skin permeation enhancement and the skin perturbation effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA). METHODS: The skin permeation enhancement effects were studied using human stratum comeum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. The perturbation effects were studied using differential thermal analysis (DTA) on SC after pretreatment with FA/PG. RESULTS: SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). Nonanoic and decanoic acids exert barely noticeable effects on the thermal behaviour of SC, suggesting that they easily mix with the skin lipids. All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. DTA revealed that the cis-9- and 13-isomers form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70 degrees/80 degrees C. PUFA--linoleic (LA), alpha-linolenic (ALA) and arachidonic acids--enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. LA and ALA form separate domains but do not completely suppress the SC lipid transitions at 70 degrees/80 degrees C. Increase in the enthalpy changes of 70 degrees/80 degrees transitions linearly correlates to the decrease in the permeability coefficients, suggesting that an increased perturbation of the skin lipids not necessarily has to yield an increased PABA permeation. CONCLUSIONS: The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids.

Localization of aminopeptidase activity in freshly excised human skin: direct visualization by confocal laser scanning microscopy.

The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue. To separate the kinetics of diffusion and degradation of Leu2-R110, a lateral application mode was introduced: the probe was applied at the cutting plane of a mechanical cross-section of the sample, and optical cross-sections were made parallel to the cutting plane of the mechanical section. By this means, simultaneous and equal access of the substrate to the various strata and domains of the skin was achieved. The observations revealed that the fluorescence, i.e., aminopeptidase activity, was evenly distributed throughout the viable part of the epidermis, with enhanced fluorescence ("hot spots") in the upper layers of the stratum granulosum, while dermis and stratum corneum showed considerably less aminopeptidase activity. Independent studies with hair follicles (obtained from trypsin-separated stratum corneum) also showed aminopeptidase activity, mostly at the root sheath. Because of the advantage of direct visualization and localization of enzymatic activity in intact tissue, the lateral application mode of substrate administration in combination with CLSM may be beneficial to further elucidate the location and intensity of metabolic activity in other living tissues as well.

An in vivo-In vitro study of the use of a human skin equivalent for irritancy screening of fatty acids.

A human skin equivalent (HSE) consisting of reconstructed epidermis on a fibroblast-populated collagen gel was evaluated as a model for irritancy screening. The irritancy potential of a series of saturated and unsaturated fatty acids was investigated in vivo under short-term exposure conditions using transepidermal water loss (TEWL), laser Doppler velocimetry (LDV) and the penetration of hexyl nicotinate as parameters. The effects of the fatty acids in vitro were studied after topical application on HSE using changes in epidermal morphology, changes in interleukin (IL)-1alpha and interleukin-8 mRNA expression and protein levels, and alterations in activity of plasminogen activators as endpoints. The unsaturated fatty acids increased both TEWL and LDV and elevated IL-1alpha and IL-8 mRNA levels, whereas their effects on protein levels were minimal. In contrast, the saturated fatty acids were not very effective in vivo but induced an increase in IL-1alpha protein levels. The type of fatty acid determines not only the way and the extent of skin barrier modulation, but also the pattern of cell mediator production and release. This study stresses the neccessity of investigating multiple endpoints for the characterization of a test compound, in particular when studying mild and moderate irritants.

Iontophoretic delivery of apomorphine. I: In vitro optimization and validation.

PURPOSE: To investigate the feasibility of transdermal iontophoretic delivery of apomorphine in patients with Parkinson's disease, transdermal transport rates were optimized and validated across human stratum corneum and freshly dermatomed human skin in vitro. METHODS: In all experiments R-apomorphine hydrochloride was applied in the anodal compartment. The effect on the flux of the following parameters was studied, using a flow through transport cell current density, pH, concentration, ionic strength, osmolarity, buffer strength, temperature and skin type. RESULTS: Transdermal transport of apomorphine was directly controlled by the presence or absence of current. Passive delivery was minimal and no depot effect was observed. A linear relationship was found between current density and steady-state flux. At room temperature the lag time was 30 to 40 minutes. A maximal steady-state flux was obtained when the donor concentration approached maximum solubility. By increasing the temperature of the acceptor chamber to 37 degrees C the steady-state flux was increased by a factor of 2.3 and the lag time decreased to +/- 3 minutes. No effect of osmolarity and buffer strength and only a small effect of ionic strength and pH on the transport rate were observed. The flux through dermatomed human skin was decreased compared to stratum corneum. This effect was shown not to be caused by skin metabolism. CONCLUSIONS: The results obtained in vitro indicate that the iontophoretic delivery of apomorphine can be controlled and manipulated accurately by the applied current. The in vitro flux furthermore depends on the donor composition, temperature and skin type. Under optimized conditions, transport rates resulting in therapeutically effective plasma concentrations are feasible, assuming a one to one in vitro/in vivo correlation.

Iontophoretic delivery of apomorphine. II: An in vivo study in patients with Parkinson's disease.

PURPOSE: Transdermal transport rates of the dopamine agonist R-apomorphine were determined in patients with idiopathic Parkinson's disease (IPD). Apomorphine was applied by iontophoresis at two current densities. METHODS: In ten patients apomorphine was applied passively for one hour. Thereafter, in the first five patients, a current density of 250 microA.cm-2 was applied for one hour and a current density of 375 microA.cm-2 in the second group. The individual pharmacokinetic parameters were obtained separately following a 15-minute zero-order intravenous infusion of 30 micrograms.kg-1. Skin resistance was measured during current delivery. Current-induced irritation was measured by Laser Doppler Flowmetry (LDF). The pharmacodynamics were quantified by a unilateral tapping score. Qualitative clinical improvements (decreased tremor, rigidity or cramp) were also recorded. RESULTS: In all patients increasing plasma concentrations of R-apomorphine were found during the interval of current application. The maximum concentrations that were attained were related to the applied current density: 1.3 +/- 0.6 ng.ml-1 at 250 microA.cm-2 and 2.5 +/- 0.7 ng.ml-1 at 375 microA.cm-2. When the current was switched off all concentrations returned to baseline values in about 90 minutes. By mathematical deconvolution of the profiles it was shown that steady-state fluxes were reached within the one-hour interval of current driven transport Steady-state fluxes were calculated to be 69 +/- 30 nmol.cm-2.h-1 at 250 microA.cm-2 and 114 +/- 34 nmol.cm-2.h-1 at 375 microA.cm-2. Individual drug input rates were inversely related to the overall resistance. Significantly elevated LDF values were found after patch removal, indicating mild current induced erythema. Only subtherapeutic plasma concentrations were obtained in all patients except for one. CONCLUSIONS: The results show that current-dependent delivery of apomorphine is possible in vivo at acceptable levels of skin irritation. Excellent correlation was found between the calculated in vivo transport rates and the rates that were previously obtained in vitro.

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease.

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 microm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min(-1) the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml(-1) for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml(-1) for plasma samples, and <4% in the concentration range of 40-400 ng ml(-1) for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 microm C18 column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min(-1) the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml(-1) for apomorphine and 2.5 ng ml(-1) for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml(-1) for plasma samples and 7% in the concentration range of 50-500 ng ml(-1) for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with beta-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 microg kg(-1) for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.

In vivo buccal delivery of the peptide drug buserelin with glycodeoxycholate as an absorption enhancer in pigs.

PURPOSE: To study the potential of buccal delivery of the peptide drug in pigs. METHODS: Intravenous administration and buccal delivery without and with 10 mM sodium glycodeoxycholate (GDC) as absorption enhancer were investigated as a randomised cross-over study in six pigs. The buccal delivery device consisted of an application chamber with a solution of buserelin and was attached to the buccal mucosa for 4 hours using an adhesive patch. RESULTS: Buccal administration of buserelin resulted in rapidly reached steady state plasma levels. The absolute bioavailability of the peptide after buccal delivery for 4 hours could be increased from 1.0 +/- 0.3 to 5.3 +/- 1.1% (mean +/- SD.) by co-administration of 10 mM GDC (0.45% w/v)). CONCLUSIONS: The results of this study demonstrate that buccal administration with the use of absorption enhancers is a useful approach for the delivery of peptide drugs such as buserelin.