Ultrasound-mediated transdermal in vivo transport of insulin with low-profile cymbal arrays

The purpose of this study was to demonstrate the feasibility of ultrasound (US)-mediated transdermal delivery of insulin in vivo using rats with a novel, low profile two-by-two US array based on the "cymbal" (due to its unique shape) transducer. As a practical device, the cymbal array (f = 20 kHz) was 37 x 37 x 7 mm in size, and weighed less than 22 g. A total of 20 Sprague-Dawley rats (350 to 450 g) were divided into four groups, two controls and two US exposure, with five rats in each group. The rats were anesthetized and shaved; a water-tight standoff reservoir, which held the insulin or saline, was sealed against the rat's abdomen and the US array. At the beginning of the experiment and every 30 min for 90 min, 0.3 mL of blood was collected from the jugular vein to determine the blood glucose level (mg/dL). For comparison between the rats, the change in the glucose level for each rat was normalized to a baseline (i.e., 0 mg/dL). The first control group used insulin in the reservoir with no US and the second control group had saline in the reservoir with US operating at I(SPTP) = 100 mW/cm(2) for 60 min. For the experiments, the third group employed insulin with US exposure for 60 min (I(SPTP) = 100 mW/cm(2)), whereas the last group used insulin with US operating with a 20-min exposure (I(SPTP) = 100 mW/cm(2)) to examine the effects of time on delivery. For the 60-min US exposure group, the glucose level was found to decrease from the baseline to -267.5 +/- 61.9 mg/dL in 1 h. Moreover, to study the effects of US exposure time on insulin delivery, the 20-min group had essentially the same result as the 60-min exposure at a similar intensity, which indicates that the expose time does not need to be as long for delivery.     

Glucose measurements with sensors and ultrasound

Accurate monitoring of the blood glucose level in diabetics is essential in preventing complications. Generally, conventional over-the-counter glucose meters require frequent painful finger punctures to obtain samples, which makes a noninvasive method preferable. The purpose of this study was to demonstrate that glucose levels can be measured transdermally with the combination of the low-profile cymbal array and an electrochemical glucose sensor consisting of amperometric electrodes and a novel glucose oxidase hydrogel. Interstitial fluid glucose concentrations can be determined with the electrochemical glucose sensor after the skin is made permeable to glucose by ultrasound (US) (20 kHz) with the thin (< 7 mm) and light (< 22 g) cymbal array. Using this array to deliver insulin into hyperglycemic rats, our previous experiments demonstrated that blood glucose levels would decrease 233.3 mg/dl with 5 min of US exposure. With the sensor and array, our goal was to determine the glucose levels of hyperglycemic rats noninvasively and evaluate the possible bioeffects. A total of 12 anesthetized rats were placed into two groups (US exposure group and control group) and the array (I(SPTP) = 100 mW/cm(2)) with a saline reservoir operating for 20 min was affixed to the abdomen. The array was removed and an electrochemical glucose sensor was placed on the exposed area to determine the glucose concentrations through the skin. Comparison was made using a commercial glucose meter with the blood collected from a jugular vein. The average blood glucose level determined by the sensor was 356.0 +/- 116.6 mg/dl, and the glucose level measured by the commercial glucose meter was 424.8 +/- 59.1 mg/dl. These results supported the use of this novel system consisting of the electrochemical glucose sensor and the cymbal array for glucose monitoring.     

Elucidation of the transport pathway in hairless rat skin enhanced by low-frequency sonophoresis based on the solute-water transport relationship and confocal microscopy.

In this study, we examined a relationship between hydrophilic solute and water (vehicle) transports in the excised hairless rat skin in the presence of ultrasound (41 kHz, 60-300 mW/cm2) irradiation and also conducted skin surface observation using confocal microscopy. When the applied intensity was increased stepwise over the rage of 60-300 mW/cm2, the transport of tritiated water (3H2O) was increased 140-fold in an intensity-dependent manner and this returned to normal on stopping the ultrasound application. The skin permeation clearance (mul/h) of model hydrophilic solutes, calcein (MW 623) and FITC-labeled dextrans [MW 4400 (FD-4) and MW 38000 (FD-40)], across the skin under the influence of ultrasound was plotted against the corresponding 3H2O flux (microl/h) to estimate the potential contribution of convective solvent flow, induced by the ultrasound application, to the solute transport. Good correlations were observed between the 3H2O flux and solute clearances and, unexpectedly, the slope values obtained from linear regression of the plots were consistent for all solutes examined (1.04+/-0.29 for calcein, 1.07+/-0.17 for FD-4, and 1.08+/-0.23 for FD-40, respectively). Transport of intact FD-4 and FD-40 was confirmed by gel permeation chromatography. When the skin surface and deeper regions of the skin after sonophoresis of FD-40 were observed using a confocal microscope, the fluorescence of FD-40 was uniformly distributed in the area under the ultrasound horn and also evident in crack-like structures in the boundary of the horn. On the other hand, a hexagonal structure of horny cells in the stratum corneum (SC) observed by post-staining with rhodamine B was fully conserved in the area under the horn. These findings suggest that 41 kHz ultrasound can increase the transdermal transport of hydrophilic solutes by inducing convective solvent flow probably via both corneocytes and SC lipids as well as newly developed routes. Our observation also suggests that 41 kHz (low-frequency) ultrasound has the potential to deliver hydrophilic large molecules transdermally.     

Characterization of transdermal solute transport induced by low-frequency ultrasound in the hairless rat skin.

Sonophoretic drug transport with low-frequency (41-445 kHz) and low-intensity (60-240 mW/cm2) ultrasound was characterized using hydrophilic calcein and deuterium oxide (D2O) as a solvent vehicle in excised hairless rat skin. The excised skin was mounted in vertical diffusion chambers for measurement of skin resistance and sonophoretic transport of calcein and D2O. The calcein content of the skin was also measured after ultrasound application. When the stratum corneum (sc) side was exposed to ultrasound at an intensity of 60 mW/cm2 for 30 min, the calcein flux in the sc-to-dermis direction was increased by 22.3-, 6.3-, and 3.8-fold from a baseline of 0.0088+/-0.0100 nmol/(cm2 x h) at frequencies of 41, 158, and 445 kHz, respectively, without significant changes in skin resistance. The ultrasonically-enhanced fluxes returned to baseline following cessation of the ultrasound application. At 41 kHz, there was a further increase in the magnitude of enhancement and a significant decrease in skin resistance (by 50% of the baseline resistance) on increasing the intensity from 60 to 120 mW/cm2, whereas no further enhancement was observed at 158 and 445 kHz up to 240 mW/cm2. Comparison of the calcein content in the skin before, during, and after ultrasound application at 41 kHz, 120 mW/cm2, was consistent with a transient ultrasonically-induced increase in calcein flux. In the sonophoretic transport experiments at 41 kHz, 120 mW/cm2, calcein transport correlated well with D2O transport. When 41-kHz ultrasound was applied to the sc side at 120 mW/cm2, the calcein and D2O fluxes in the sc-to-dermis direction were 13.7- and 5.2-fold higher than those in the dermis-to-sc direction. Similar directionality was also observed in tape-stripped skin, suggesting possible induction of convection in the direction of sound propagation. However, dermal application under the same ultrasound conditions induced neither an increase in calcein and D2O transport nor a decrease in skin resistance. These results demonstrate that low frequency sonophoresis is a potentially useful technique for controlling transdermal drug transport. Convective solvent flow as well as structural alteration of the skin induced by ultrasound are likely to be responsible for the observed sonophoretic transport enhancement.     

Frequency and thermal effects on the enhancement of transdermal transport by sonophoresis.

The aims of this work were: (i) to examine the role of ultrasound (US) frequency and intensity on the transport of glucose and mannitol across porcine skin in vitro, (ii) to quantify the energy delivered to the skin during application of low-frequency sonophoresis, and (iii) to 'deconvolute' the thermal effect, induced by US application to the skin, to the enhanced permeability of the cutaneous barrier. Low- (20 kHz) and high-frequency (10 MHz) sonophoresis were first compared. Only low frequency US resulted in significantly increased permeation. Low-frequency, US-induced enhancement of mannitol transport was symmetric; that is, mannitol flux was the same when 'delivered' or 'extracted' from a donor solution (in both cases, the US probe was present on the surface side of the skin). Calorimetry was used to quantify the US energy delivered by the sonicator. Subsequently, the US-enhanced transdermal transport of mannitol, during which a significant (and US intensity-dependent) temperature increase occurred, was compared to that provoked, in the absence of sonophoresis, by a comparable thermal effect. Only 25% of this enhancement was attributable to the increased temperature induced by US. It follows that another mechanism, most probably cavitation, is principally responsible for the lowered skin barrier function observed.     

Effect of sonication parameters on transdermal delivery of insulin to hairless rats.

Application of low-frequency ultrasound has been shown to enhance transdermal drug transport of large molecules such as insulin. In this study, we investigated the dependence of ultrasound-induced transdermal delivery of insulin on ultrasound parameters. Insulin was delivered in vivo to hairless rats using 20 kHz ultrasound applied over a range of ultrasound intensity, application time and pulse length. Change in blood glucose levels of the animals was monitored to assess insulin transport. The results showed a threshold below which no detectable changes in blood glucose level was observed for each ultrasound parameter. Moreover, our findings indicated that sonophoretic enhancement is dependent on energy dose and length of ultrasound pulse that is consistent with a cavitation-based mechanism. The more significant effect of lowering glycemia was obtained with application of less than 15 min ultrasound and was similar to subcutaneous injection of 0.5 U of insulin. Pretreatment of hairless rat skin with ultrasound followed by application of insulin resulted in no significant modification in blood glucose level, indicating that transdermal transport of insulin mainly occurred during sonication. Sonophoresis may therefore potentially be applied for non-invasive and painless delivery of insulin in the treatment of insulin-dependent diabetes.     

Transdermal delivery of poly-l-lysine by sonomacroporation

A feasibility study of using high-amplitude ultrasound (US) to deliver large molecules transdermally was undertaken. US (20 kHz) of intensity in the range between 2 to 50 W/cm(2) was used to increase the permeability of skin in vitro to large size molecules. For example, when 20-kHz, 5% duty cycle US at the spatial average and pulse-average intensity I(SAPA) = 19 W/cm(2) was applied for 10 min and the distance between the US source and the surface of a skin specimen was 2 mm, the skin permeability was calculated to be 0.5 +/- 0.2 cm/h and 8.5 +/- 4.2 cm/h, respectively, for poly l-lysine-fluorescein isothiocyanate (FITC) (51 kDa) and octa-1-lysine-FITC (2.5 kDa). Without application of US, the skin permeability of the above-mentioned molecules would be essentially zero. A transdermal flux enhancement occurring during the process reported here was much higher than that due to sonophoresis (I(SAPA) < 2 W/cm(2)) as reported in the literature. For comparison, for example, the skin permeability for delivering erythropoeitin (48 kDa) and insulin (6 kDa) reached 9.8 x 10(-6) and 3.3 x10(-3) cm/h, respectively, by using sonophoresis for 1 h US exposure. Experimental results from transdermal flux kinetics, and confocal microscopic cross-sectional and optical images, suggested that the formation of pores in the stratum corneum, whose size varies with skin samples, may be in the range of 1 to 100 microm. The confocal images also suggest the formation of microm-size pathways in epidermis during US exposure.     

Radiofrequency-driven skin microchanneling as a new way for electrically assisted transdermal delivery of hydrophilic drugs.

The aim of this study was to increase the skin penetration of two drugs, granisetron hydrochloride and diclofenac sodium, using a microelectronic device based on an ablation of outer layers of skin using radiofrequency high-voltage currents. These radiofrequency currents created an array of microchannels across the stratum corneum deep into the epidermis. The percutaneous penetration studies were first performed in vitro using excised full thickness porcine ear skin. An array of 100 microelectrodes/cm(2) was used in these studies. The skin permeability of both molecules was significantly enhanced after pretreatment with the radiofrequency microelectrodes, as compared to the delivery through the untreated control skin. Steady state fluxes of 41.6 micro g/cm(2)/h (r=0.997) and 23.0 micro g/cm(2)/h (r=0.989) were obtained for granisetron and diclofenac, respectively. The enhanced transdermal delivery was also demonstrated in vivo in rats. It was shown that diclofenac plasma levels in the pretreated rats reached plateau levels of 1.22+/-0.32 micro g/ml after 3 h to 1.47+/-0.33 micro g/ml after 6 h, as compared to 0.16+/-0.04 micro g/ml levels obtained after 6 h in untreated rats. Similarly, application of granisetron patches (3% in crosslinked hydrogel) onto rats' abdominal skin pretreated with radiofrequency electrodes resulted in an averaged peak plasma level of 239.3+/-43.7 ng/ml after 12 h, which was about 30 times higher than the plasma levels obtained by 24-h passive diffusion of the applied drug. The results emphasize, therefore, that the new transdermal technology is suitable for therapeutic delivery of poorly penetrating molecules.     

Transdermal Delivery of Enfuvirtide in a Porcine Model Using a Low-Frequency,Low-Power Ultrasound Transducer Patch

Ultrasound-mediated transdermal delivery is a promising parenteral administration method for large-molecule or unstable medications. This study evaluated skin health and systemic delivery when administering enfuvirtide, an injectable anti-retroviral medication, over a 1-mo period in a porcine model using a low-frequency cymbal transducer. Three groups received twice-daily treatments: (i) enfuvirtide injection control (n?=?12); (ii) saline ultrasound control (n?=?6); and (iii) enfuvirtide ultrasound treatment (n?=?13). Ultrasound parameters were as follows: 30-min exposure, 90 mW/cm², 24–26 kHz and 15% duty cycle. No statistical difference in trans-epidermal water loss, a measure of skin health and function, was seen between ultrasound-treated and control skin sites for either saline (p?=?0.50) or enfuvirtide (p?=?0.29) groups. Average trough plasma concentrations of enfuvirtide were 0.6 ± 0.2 and 2.8 ± 0.8 μg/mL for ultrasound and injection, respectively. Tolerability and efficacy results indicate that chronic, low-frequency ultrasound exposure can be a practical means for transdermal delivery of medications such as enfuvirtide.     

Polymeric nanoparticles based on carboxymethyl chitosan in combination with painless microneedle therapy systems for enhancing transdermal insulin delivery

Biodegradable nanoparticles (NPs) have been frequently used as insulin transdermal delivery vehicles due to their grand bioavailability, better encapsulation, controlled release and less toxic properties. However, the skin''s barrier properties prevent insulin-loaded NP permeation at useful levels. Nowadays, microneedles have been spotlighted as novel transdermal delivery systems due to their advantages such as painlessness, efficient penetration and no hazardous residues. Herein, we introduce polymeric nanocarriers based on carboxymethyl chitosan (CMCS) for insulin delivery, combining with microneedle therapy systems, which can rapidly deliver insulin (INS) into the skin. The resulting CMCS-based nanocarriers are spherical nanoparticles with a mean size around 200 nm, which could generate supramolecular micelles to effectively encapsulate insulin (EE% = 83.78 ± 3.73%). A nanocrystalline microneedle array (6 × 6, 75/150 μm) was used to penetrate the stratum corneum (SC) for enhancing transdermal insulin delivery, while minimizing the pain sensation caused by intravenous injection. Compared with the transdermal rate of passive diffusion [2.77 ± 0.64 μg (cm⁻² h⁻¹)], the transdermal rate of the insulin-loaded NP combined with microneedle penetration shows a 4.2-fold increase [10.24 ± 1.06 μg (cm⁻² h⁻¹)] from permeation experiment in vitro. In vivo hypoglycemic experiments demonstrate the potential of using nanocarrier combination with microneedle arrays for painless insulin delivery through the skin in a clinical setting. Thus, the developed combination scheme of nanoparticles and microneedle arrays offers an effective, user-friendly, and low-toxicity option for diabetes patients requiring long-term and multiple treatments.

Biodegradable nanoparticles (NPs) have been frequently used as insulin transdermal delivery vehicles due to their grand bioavailability, better encapsulation, controlled release and less toxic properties.     

Enhancement of enzymatic fibrinolysis with 2-MHz ultrasound and microbubbles.

BACKGROUND: Microbubbles used for echo-contrast agents accelerate enzymatic fibrinolysis of clots exposed to low-frequency ultrasound (US). It is not known whether microbubbles are also effective in enhancing high-frequency US-driven enzymatic fibrinolysis. METHODS AND RESULTS: Calibrated whole blood clots were exposed to US, or US and galactose-based microbubbles (Levovist), with or without recombinant tissue plasminogen activator (rt-PA) in an in-vitro flow system. We used low-intensity, 2-MHz, pulsed wave US. Relative weight reduction of clot +/- SD was 30.7 +/- 9.5% after exposure to microbubbles, rt-PA and US, 13.1 +/- 2.6% after exposure to rt-PA and US, 10.9 +/- 3.6% after exposure to microbubbles and US, and 6.1 +/- 1.9% after exposure to US alone. anova demonstrated a significant effect of rt-PA (P =0.001), microbubbles (P = 0.012), and interaction of both (P = 0.022). CONCLUSIONS: The application of galactose-based microbubbles (Levovist) strongly accelerates lysis of clots exposed to 2 MHz, low-intensity US in vitro both with and without rt-PA. The findings suggest a synergy between microbubbles and rt-PA. These methods routinely used for transcranial diagnostic applications have the potential to improve the efficacy of intravenous rt-PA in acute ischemic stroke.     

Reverse iontophoresis for non-invasive transdermal monitoring

Iontophoresis is the application of a small electric current to enhance the transport of both charged and polar, neutral compounds across the skin. Manipulation of either the total charge delivered and/or certain electrode formulation parameters allows control of electromigration and electroosmosis, the two principal mechanisms of transdermal iontophoresis. While the approach has been mainly used for transdermal drug delivery, 'reverse iontophoresis', by which substances are extracted to the skin surface, has recently been the subject of considerable effort. Glucose monitoring has been extensively studied and other applications, including therapeutic drug monitoring, are contributing to the development of the technique. An internal standard calibration procedure may ultimately render this novel monitoring technique completely non-invasive.     

Ultrasound nucleolysis: an in vitro study

Thermal intradiscal therapy for chronic low back pain, using a catheter inserted into the intervertebral disc, is becoming more popular in the treatment of low back pain. The aim of this study was to investigate the possibility of heating the nucleus pulposus of the intervertebral disc with high-intensity focused ultrasound (US) or HIFU. Two specific situations were considered, invasive transducers that would be in contact with the annulus fibrosus of the disc, and noninvasive transducers that could be used externally. Theoretical simulations were performed to find the optimal parameters of US transducers and then experimental studies were done using transducers made to these specifications. These experiments confirmed that it was possible to heat the discs with HIFU. Two orthogonal transducers resulted in a superior temperature distribution than using just one transducer. It is, therefore, feasible to consider thermal treatment of the nucleus pulposus of the disc using noninvasive US.     

Transendothelial insulin transport is not saturable in vivo. No evidence for a receptor-mediated process.

In vitro, insulin transport across endothelial cells has been reported to be saturable, suggesting that the transport process is receptor mediated. In the present study, the transport of insulin across capillary endothelial cells was investigated in vivo. Euglycemic glucose clamps were performed in anesthetized dogs (n = 16) in which insulin was infused to achieve concentrations in the physiological range (1.0 mU/kg per min + 5 mU/kg priming bolus; n = 8) or pharmacologic range (18 mU/kg per min + 325 mU/kg priming bolus; n = 8). Insulin concentrations were measured in plasma and hindlimb lymph derived from interstitial fluid (ISF) surrounding muscle. Basal plasma insulin concentrations were twice the basal ISF insulin concentrations and were not different between the physiologic and pharmacologic infusion groups (plasma/ISF ratio 2.05 +/- 0.22 vs 2.05 +/- 0.23; p = 0.0003). The plasma/ISF gradient was, however, significantly reduced at steady-state pharmacologic insulin concentrations (1.37 +/- 0.25 vs 1.98 +/- 0.21; P = 0.0003). The reduced gradient is opposite to that expected if transendothelial insulin transport were saturable. Insulin transport into muscle ISF tended to increase with pharmacologic compared with physiologic changes in insulin concentration (41% increase; 1.37 +/- 0.18 10(-2) to 1.93 +/- 0.24 10(-2) min-1; P = 0.088), while at the same time insulin clearance out of the muscle ISF compartment was unaltered (2.53 +/- 0.26 10(-2) vs 2.34 +/- 0.28 10(-2) min-1; P = 0.62). Thus, the reduced plasma/ISF gradient at pharmacologic insulin was due to enhanced transendothelial insulin transport rather than changes in ISF insulin clearance. We conclude that insulin transport is not saturable in vivo and thus not receptor mediated. The increase in transport efficiency with saturating insulin is likely due to an increase in diffusionary capacity resulting from capillary dilation or recruitment.     

Kinetics of glucose disposal in whole body and across the forearm in man.

We reevaluated the concept that the in vivo glucose disposal rate in man is determined by the activity of the glucose transport system. Rates of glucose disposal were determined in whole body and across forearm at four insulin levels (approximately 9, approximately 50, approximately 160, and approximately 1700 microU/ml) and at each insulin level at four glucose levels (approximately 90, approximately 160, approximately 250, and approximately 400 mg/dl). At the lowest insulin level, the Michaelis constants (Ks:s) for glucose disposal in whole body (8.7 +/- 1.1 mM) and across forearm (7.4 +/- 1.4) mM) were compatible with a Ks determined in vitro for the transport system. At higher insulin levels, the apparent Ks increased significantly in whole body (16.2-37.7 mM) and across forearm (20.7-31.2 mM). We interpret the apparent increase of Ks by insulin to reflect a shift in the rate-limiting step from glucose transport to some step beyond transport.     

Bubble growth within the skin by rectified diffusion might play a significant role in sonophoresis.

Low frequency ultrasound has successfully been used for enhancing transdermal transport of a variety of different molecules. This phenomenon is referred to as sonophoresis. Several attempts have been made to investigate the enhancing mechanism in order to modulate the overall process. In this study we assess whether rectified diffusion is a process that occurs within the skin, which could eventually lead to channeling and thereby to transdermal sonophoresis. The model presented in this paper is based on the following postulate: gas bubbles are randomly distributed within the lipid bilayers of the stratum corneum (SC). As the skin is subjected to ultrasound, gas bubbles grow by rectified diffusion. During this period, bubbles may merge with the outer or inner boundaries of the SC, or merge with neighboring bubbles. Eventually, channels are created, allowing drugs to easily penetrate through the most significant barrier to transdermal delivery, the SC. As a result, transdermal transport rate is enhanced. In this work, a mathematical model has been formulated, in which permeability enhancement of the SC is linked to channels, possibly created by means of rectified diffusion. Sonophoresis may result from various mechanisms that act in synergy. The present model predicts that rectified diffusion might be one of the factors that lead to sonophoresis during ultrasound treatment.     

Identification of penetration enhancers for testosterone transdermal delivery from spray formulations.

The goal of this study was to identify a suitable penetration enhancer-containing formulation for the transdermal delivery of testosterone from a spray. The first step involved in vitro measurement of drug flux from a 1:1 ethanol/water saturated solution across hairless rat skin, which had been pre-treated with a series of penetration enhancers. Isopropyl myristate (IPM) was found to be the most efficient excipient, increasing testosterone transport by more than a factor of 5. The enhancing ability of IPM was also apparent when the drug was formulated in 3:1 ethanol/propylene glycol, a more compatible vehicle for use in a spray. IPM was then incorporated into this formulation directly (as opposed to being used to pre-treat the skin) over a range of concentrations from 10-25% v/v, and testosterone transport was evaluated when delivered from either a solution, or from a mechanical spray, or from an aerosol (which also contained 50% v/v propellant). At the highest level of enhancer, the flux was improved 2.5-fold from both the spray and the aerosol, relative to a control. However, these formulations were far from optimally conceived, in that the amount of drug which eventually contacted the skin represented only approximately 10% of the pulverized quantity from the spray, and approximately 40% of that from the aerosol. Repeated application, especially from the aerosol, improved matters somewhat, but further work is clearly required before the concept can be developed for practical application.     

Painless electroporation with a new needle-free microelectrode array to enhance transdermal drug delivery

A microelectrode array was designed to minimize the pain sensation of electroporation for enhancing transdermal drug delivery. The influence of the size of the electrode–skin contact area and of the distance between electrodes on the pain sensation was tested on human volunteers. The pain level decreased with the dimension of electrode–skin contact area and with inter-electrode distance. When both reached about 0.5 mm, the pain level was not perceptible even at the threshold of transdermal electroporation level of sixty electric pulses at 150 V, 1 ms at 1–10 Hz. An array of 11 × 11 alternately connected electrodes with 0.6 × 0.6 mm dimension was fabricated. The electric thresholds for effective drug delivery, using toluidine blue O as a marker on mouse skin, was found to be the same for microelectrode arrays as for larger electrodes and wider inter-electrode distances. In vivo transdermal electroporation using microelectrode array with 180 pulses of 150 V, 0.2 ms at 1 Hz, followed by 30 min methotrexate (MTX) occlusion increased more than 4 fold the systemic MTX level in mice. The results demonstrated the potential of painless delivery of significant amounts of chemotherapeutic agents through skin with the new electrode arrays in a clinical setting.     

The effect of electroporation on iontophoretic transdermal delivery of calcium regulating hormones.

Electrically-assisted delivery by iontophoresis and/or electroporation was used in vitro to deliver the calcium regulating hormones, salmon calcitonin (sCT) and parathyroid hormone (1-34) (PTH) through human epidermis. Such delivery could be useful for chronic treatment of post-menopausal osteoporosis and other clinical indications as a superior alternative to parenteral delivery. sCT (50 microg/ml) or PTH (1-34) (100 microg/ml) formulation was prepared in citrate buffer (pH 4.0 or 5.0, respectively). Epidermis separated from human cadaver skin was used. Iontophoresis was applied using a constant current power source and electroporation with an exponential pulse generator. Silver/silver chloride electrodes were used. A combination of electroporation and iontophoresis resulted in higher transdermal permeation than either one technique alone. Electroporation also shortened the lag time of iontophoretic transdermal delivery of salmon calcitonin. Pulsing at lower voltages followed by iontophoresis did not result in increased transport (over iontophoresis alone), perhaps because the transdermal voltage was very low. The transdermal transport of salmon calcitonin by pulsing with 15 pulses (1 ppm) of 500 V (200 ms) followed by iontophoresis led to a quick input and high flux. The average transdermal voltage was only about 50 V for a 500 V study.     

Evidence for entry of plasma insulin into cerebrospinal fluid through an intermediate compartment in dogs. Quantitative aspects and implications for transport.

To study the route by which plasma insulin enters cerebrospinal fluid (CSF), the kinetics of uptake from plasma into cisternal CSF of both insulin and [14C]inulin were analyzed during intravenous infusion in anesthetized dogs. Four different mathematical models were used: three based on a two-compartment system (transport directly across the blood-CSF barrier by nonsaturable, saturable, or a combination of both mechanisms) and a fourth based on three compartments (uptake via an intermediate compartment). The kinetics of CSF uptake of [14C]inulin infused according to an "impulse" protocol were accurately accounted for only by the nonsaturable two-compartment model (determination coefficient [R2] = 0.879 +/- 0.044; mean +/- SEM; n = 5), consistent with uptake via diffusion across the blood-CSF barrier. When the same infusion protocol and model were used to analyze the kinetics of insulin uptake, the data fit (R2 = 0.671 +/- 0.037; n = 10) was significantly worse than that obtained with [14C]inulin (P = 0.02). Addition of a saturable component of uptake to the two-compartment model improved this fit, but was clearly inadequate for a subset of insulin infusion studies. In contrast, the three-compartment model accurately accounted for CSF insulin uptake in each study, regardless of infusion protocol (impulse infusion R2 = 0.947 +/- 0.026; n = 10; P less than 0.0001 vs. each two-compartment model; sustained infusion R2 = 0.981 +/- 0.003; n = 5). Thus, a model in which insulin passes through an intermediate compartment en route from plasma to CSF, as a part of a specialized transport system for the delivery of insulin to the brain, best accounts for the dynamics of this uptake process. This intermediate compartment could reside within the blood-CSF barrier or it may represent brain interstitial fluid, if CNS insulin uptake occurs preferentially across the blood-brain barrier.