Transdermal delivery using surface electrodes in porcine skin.

The main barrier to cutaneous or transcutaneous drug and gene delivery is the impermeability of the stratum corneum (SC), the outermost layer of the skin (1). If the integrity of the SC is disrupted, the barrier to molecular transit may be greatly reduced. Cutaneous absorption can be increased by removal of the SC by tape-stripping or dermabrasion, by vehicle (solvent-carrier) optimization, or by the use of penetration enhancers like DMSO (dimethylsulfoxide), oleic acid, and alcohols (2,3). An electric field can also be used to enhance delivery. Disruption of the SC can be achieved by electroporation, which is the creation of penetration sites by an electric pulse. Ions and molecules move through induced gaps of the SC by diffusion and electromotive or electroosmotic transport (4-6). Electroporation differs from iontophoresis, in which there is an increased migration of ions or charged molecules through the skin when an electrical potential gradient is applied. The primary transdermal route for iontophoresis seems to be appendageal or intercellular through preexisting pathways (5,7), or as a result of low-voltage (<5 V) induced permeabilization of appendageal bilayers (8). A third form of electroenhanced drug delivery, electrochemotherapy (9), refers to localized delivery of electric pulses across a tumor following systemic or intratumor drug administration, and usually does not involve cutaneous or transcutaneous delivery.

Increased DNA synthesis in INIT/10T1/2 cells after exposure to a 60 Hz magnetic field: a magnetic-field or a thermal effect?

This study was designed to test the hypothesis that a 0.1-0.8-mT 60 Hz magnetic field may act as a promoter of carcinogenesis. C3H 10T1/2 mouse fibroblasts initiated with the carcinogen methylcholanthrene (INIT/10T1/2 cells) were used; in these cells, expression of the carcinogenic phenotype is suppressed indefinitely by the presence of retinyl acetate in the culture medium. After withdrawal of retinyl acetate, expression of the carcinogenic phenotype may be observed as the loss of contact inhibition. Cells grown without retinyl acetate were exposed to 0.1-0.8-mT (rms) 60 Hz magnetic fields or to sham fields. Eight days after exposure, magnetic-field and sham-exposed cells showed the same levels of incorporation of [3H]thymidine, and both had counts significantly higher than those of unexposed cells. The rate of incorporation of [3H]thymidine was very sensitive to small (0.1-0.8 degrees C) and transient (60 min) increases in incubation temperature during the first few days of withdrawal of retinyl acetate. Exposure of Jurkat (human acute T-cell lymphoma) and GH3 (rat pituitary tumor) cells to magnetic fields and sham conditions yielded similar results. INIT/10T1/2 cells cultured in the presence of retinyl acetate showed no effect of exposure conditions. Both magnetic-field and sham exposures caused a slight increase in temperature within the exposure zone in the incubator. Thus the differences between rates of incorporation of [3H]thymidine in magnetic field-exposed, sham-exposed and unexposed cells seem to be attributable at least in part to a slight elevation in temperature during exposure. Since some cells appear to be extremely sensitive to small increases in temperature, measurements of magnetic-field effects must be made and interpreted with caution.

A pulsed electric field enhances cutaneous delivery of methylene blue in excised full-thickness porcine skin.

We used electric pulses to permeabilize porcine stratum corneum and demonstrate enhanced epidermal transport of methylene blue, a water-soluble cationic dye. Electrodes were placed on the outer surface of excised full-thickness porcine skin, and methylene blue was applied to the skin beneath the positive electrode; 1 ms pulses of up to 240 V were delivered at frequencies of 20-100 Hz for up to 30 min. The amount of dye in a skin sample was determined from absorbance spectra of dissolved punch biopsy sections. Penetration depth and concentration of the dye were measured with light and fluorescence microscopy of cryosections. At an electric exposure dose VT (applied voltage x frequency x pulse width x treatment duration) of about 4700 Vs, there is a threshold for efficient drug delivery. Increasing the applied voltage or field application time resulted in increased dye penetration. Transport induced by electric pulses was more than an order of magnitude greater than that seen following iontophoresis. We believe that the enhanced cutaneous delivery of methylene blue is due to a combination of de novo permeabilization of the stratum corneum by electric pulses, passive diffusion through the permeabilization sites, and electrophoretic and electroosmotic transport by the electric pulses. Pulsed electric fields may have important applications for drug delivery in a variety of fields where topical drug delivery is a goal.

The effect of tacrolimus (FK506) on intestinal barrier function and cellular energy production in humans.

BACKGROUND & AIMS: The maintenance of the intestinal mucosal barrier may be energy dependent. Tacrolimus is a potent immunosuppressive drug that decreases mitochondrial adenosine triphosphate production and increases intestinal permeability in animals. METHODS: Twelve liver graft recipients receiving tacrolimus, 9 healthy volunteers, and 5 liver graft recipients not receiving immunosuppression underwent a combined absorption-permeability-mitochondrial function test using 5 g lactulose, 1 g L-rhamnose, 0.5 g D-xylose, 0.2 g 3-O-methyl-D-glucose, 1 mg/kg 2-keto[1-13C]isocaproic acid ([13C]KICA), and 20 mg/kg L-leucine. The respiratory quotient and resting energy expenditure were measured by indirect calorimetry. Tacrolimus pharmacokinetic profiles and levels of endotoxin and IgM and IgG endotoxin core antibodies were determined. RESULTS: Tacrolimus inhibited the decarboxylation of [13C]KICA, the resting energy expenditure, and the respiratory quotient in an exposure-dependent manner, suggesting an inhibition of mitochondrial respiration. Tacrolimus inhibited intestinal absorptive capacity in an exposure-dependent manner. Tacrolimus-treated patients had an increased intestinal permeability and significantly higher endotoxin levels compared with healthy volunteers. CONCLUSIONS: Tacrolimus inhibits cellular energy production in humans at clinically relevant doses. This is associated with an increased intestinal permeability, endotoxemia, and an impaired intestinal absorptive capacity.

Absence of 60-Hz, 0.1-mT magnetic field-induced changes in oncogene transcription rates or levels in CEM-CM3 cells.

Our objective was to assess the reproducibility of the 60-Hz magnetic field-induced, time-dependent transcription changes of c-fos, c-jun and c-myc oncogenes in CEM-CM3 cells reported by Phillips et al. (Biochim. Biophys. Acta, 1132 (1992) 140-144). Cells were exposed to a 60-Hz magnetic field (MF) at 0.1 mT (rms), generated by a pair of Helmholtz coils energized in a reinforcing (MF) mode, or to a null magnetic field when the coils were energized in a bucking (sham) mode. After MF or sham exposure for 15, 30, 60 or 120 min, nuclei and cytoplasmic RNA were extracted. Transcription rates were measured by a nuclear run-on assay, and values were normalized against either their zero-time exposure values, or against those of the c-G3PDH (housekeeping) gene at the same time points. There was no significant difference, at P=0.05, detected between MF and either sham-exposed or control cells at any time point. Transcript levels of the oncogenes were measured by Northern analysis and normalized as above. No significant difference (P=0.05) in transcript levels between MF and either sham-exposed or control cells was detected.

Photosensitization of murine tumor, vasculature and skin by 5-aminolevulinic acid-induced porphyrin.

The effects of topical and systemic administration of 5-aminolevulinic acid (ALA) were examined in several murine tumor systems with regard to porphyrin accumulation kinetics in tumor, skin and blood, vascular and tumor cell photosensitization and tumor response after light exposure. Marked, transient increases in porphyrin levels were observed in tumor and skin after systemic and topical ALA. Rapid, transient, dose-dependent porphyrin increases were also observed in blood; these were pronounced after systemic ALA injection and mild after topical application. They were highest within 1 h after ALA injection, thereafter declining rapidly. This matched the clearing kinetics of injected exogenous protoporphyrin IX (PpIX). Initially, vascular photosensitivity changed inversely to blood porphyrin levels, increasing gradually up to 5 h post-ALA, as porphyrin was clearing from the bloodstream. This pattern was again matched by injected, exogenous PpIX. After therapeutic tumor treatment vascular disruption of the tumor bed, while observed, was incomplete, especially at the tumor base. Minimal direct tumor cell kill was found at low photodynamic therapy (PDT) doses (250 mg/kg ALA, 135 J/cm2 light). Significant, but limited (< 1 log) direct photodynamic tumor cell kill was obtained when the PDT dose was raised to 500 mg/kg systemic ALA, followed 3 h later by 270 J/cm2, a dose that was however toxic to the animals. The further reduction of clonogenic tumor cells over 24 h following treatment was moderate and probably limited by the incomplete disruption of the vasculature. Tumor responses were highest when light treatment was carried out at the time of highest tumor porphyrin content rather than at the time of highest vascular photosensitivity. Tumor destruction did not reach the tumor base, regardless of treatment conditions.

Damage induced in episomal EBV DNA in Raji cells by antitumor drugs as measured by pulsed field gel electrophoresis.

The studies described below were carried out to analyze the damage induced by DNA active drugs to episomal (Epstein-Barr virus, EBV) DNA in the Raji Burkitt's lymphoma cell line. This work: (i) applies pulsed-field gel electrophoresis (PFGE) techniques to quantify DNA damage on a large (approximately 180 kbp), circular target, (ii) investigates the DNA strand-scission behavior of different classes of drugs on the EBV episome, and (iii) compares EBV episomal damage to that generated in genomic DNA in the Raji cell line. Cells were treated with ionizing radiation to induce random strand scission, and the migration of topological forms of EBV was measured using PFGE. DNA damage induced in the episome by DNA active drugs was then assayed. Three drugs, acting by different types of DNA interactive mechanisms, were used: bleomycin, an intercalative DNA strand-scission agent; and amsacrine (mAMSA) and teniposide (VM26), intercalative and nonintercalative topoisomerase II active drugs, respectively. Rad equivalency of damage was determined by comparing the drug-induced change in percentage of Forms I and III to that generated by ionizing radiation. Additionally, single- and double-strand scission induced in genomic (total cellular) DNA by X-rays, bleomycin, amsacrine, and teniposide were assayed by high-sensitivity alkaline and neutral filter elution techniques. We demonstrate that pulsed-field gel electrophoresis is a useful technique for measuring form conversion in large episomal DNA. While all three drugs effect both episomal and genomic DNA strand scission, bleomycin appears to preferentially damage the EBV episome. The topoisomerase II active drugs mAMSA and VM26 show no evidence of episome-directed damage in this system and, in fact, damage genomic DNA at somewhat higher rates.