Ronald R. Burnette

Evidence that oleic acid exists in a separate phase within stratum corneum lipids

Oleic acid is known to be a penetration enhancer for polar to moderately polar molecules. A mechanism related to lipid phase separation has been previously proposed by this laboratory to explain the increases in skin transport. In the studies presented here, Fourier transform infrared spectroscopy (FT-IR) was utilized to investigate whether or not oleic acid exists in a separate phase within stratum corneum (SC) lipids. Per-deuterated oleic acid was employed allowing the conformational phase behavior of the exogenously added fatty acid and the endogenous SC lipids to be monitored independently of each other. The results indicated that oleic acid exerts a significant effect on the SC lipids, lowering the lipid transition temperature (Tm) in addition to increasing the conformational freedom or flexibility of the endogenous lipid alkyl chains above their Tm. At temperatures lower than Tm, however, oleic acid did not significantly change the chain disorder of the SC lipids. Similar results were obtained with lipids isolated from the SC by chloroform:methanol extraction. Oleic acid, itself, was almost fully disordered at temperatures both above and below the endogenous lipid Tm in the intact SC and extracted lipid samples. This finding suggested that oleic acid does exist as a liquid within the SC lipids. The coexistence of fluid oleic acid and ordered SC lipids, at physiological temperatures, is consistent with the previously proposed phase-separation transport mechanism for enhanced diffusion. In this mechanism, the enhanced transport of polar molecules across the SC can be explained by the formation of permeable interfacial defects within the SC lipid bilayers which effectively decrease either the diffusional path length or the resistance, without necessarily invoking the formation of frank pores.

A comparison of pulsed and continuous current iontophoresis

Na+ flux resulting from pulsed constant currents (50 μA amplitude with respect to virtual ground, 50% duty cycle at 10 Hz, 100 Hz, 1 kHz, and 10 kHz), with and without the presence of the analog switch (to obtain enhanced skin depolarization), and from constant voltage pulses (2 V amplitude with respect to ground, 50% duty cycle at 10 kHz, 100 kHz, and 1 MHz resulting in average currents which were < 150 μA) was measured and compared with the Na + flux obtained from an equivalent continuous current. All studies were performed with excised full thickness nude mouse skin which was bathed in a solution containing 25 mM HEPES and 0.133 M NaCl, maintained at pH 7.4 and at 37° C. The pulsed (constant current and constant voltage) iontophoretic Na + flux obtained without enhanced skin depolarization was shown to be equal to the Na+ flux obtained from an equivalent continuous current (P < 0.05). Enhanced depolarization of the skin during pulsed constant current iontophoresis resulted in a statistically significant (P < 0.05) decrease in Na+ flux relative to an equivalent continuous current only at 10 kHz. In addition, the application of all constant pulsed current protocols resulted in a decrease in the skins low frequency impedance, a decrease which was greater than that obtained by skin hydration alone (P < 0.05). Finally, the voltage waveforms obtained across the skin during pulsed constant current iontophoresis could be experimentally and mathematically modelled by assuming the skin is a parallel resistor-capacitor combination.

Characterization of the effects of amphotericin B on ion channels in MDCK cells using the patch-clamp technique

Cultured Madin-Darby Canine Kidney cells were used as a model to study the mechanism of nephrotoxicity of amphotericin B using the patch-clamp technique. At the whole-cell level, amphotericin B altered potassium conductances in two types of these cells categorized on the basis of whole-cell potassium currents. The first cell type, classified as Type I, exhibited no significant whole-cell potassium currents. The second type, Type II, exhibited depolarization-induced outward potassium currents that rundown over time. In both of these subpopulations, exposure to amphotericin B at a concentration of 68 nM for a prolonged period of time (approximately 30-45 min) led to an increased whole-cell potassium conductance. In Type I cells, it increased by a factor of 16 and in Type II cells, by a factor of 3.5. Furthermore, the potassium currents observed in Type I cells following amphotericin B treatment bore no resemblance to currents through pores formed by amphotericin B in artificial membranes. At the single-channel level, incubation with amphotericin B led to a significantly higher potassium channel activity in both inside-out and outside-out patches. Kinetic studies in inside-out patches revealed that the increases in channel activity were associated with a decrease in the mean closed time and an overall increase in the mean open time. In summary, our data suggest that the direct toxicity of amphotericin B is primarily related to its ability to disturb normal ion channel functioning rather than to formation of pores in cell membranes.

Plasma Clearance and Half-Life of Prostaglandin F2alpha: A Comparison Between Mares and Heifers

ABSTRACT Horses are about five times more sensitive to the luteolytic effect of prostaglandin F2alpha (PGF) than cattle, as indicated by a recommended clinical dose of 5 mg in horses and 25 mg in cattle. Novel evaluations of the PGF plasma disappearance curves were made in mares and in heifers, and the two species were compared. Mares and heifers (n = 5) of similar body weight were injected (Min 0) intravenously with PGF (5 mg per animal). Blood was sampled every 10 sec until Min 3, every 30 sec until Min 5, every 10 min until Min 60, and every 30 min until Min 240. The mean PGF concentration was greater (P < 0.05) in mares than in heifers at Min 1 through Min 60 and at Mins 180 and 240. The mean time to maximum PGF concentration was not different between mares (42.0 ± 8.6 sec) and heifers (35.0 ± 2.9 sec). The apparent plasma clearance, distribution half-life, elimination half-life, and maximum plasma PGF concentration were 3.3 ± 0.5 L h−1 kg−1, 94.2 ± 15.9 sec, 25.9 ± 5.0 min, and 249.1 ± 36.8 ng/ml, respectively, in mares and 15.4 ± 2.3 L h−1 kg−1, 29.2 ± 3.9 sec, 9.0 ± 0.9 min, and 51.4 ± 22.6 ng/ml, respectively, in heifers. Plasma clearance was about five times less (P < 0.0005), maximum plasma PGF concentration was five times greater (P < 0.002), and the distribution half-life and elimination half-life were about three times longer (P < 0.005) in mares than in heifers. The fivefold greater plasma clearance of PGF in heifers than in mares corresponds to the recommended fivefold greater clinical dose of PGF in cattle and supported the hypothesis that the metabolic clearance of PGF is slower in mares than heifers.

Evaluation of Mucosal Damage and Recovery in the Gastrointestinal Tract of Rats by a Penetration Enhancer

PurposeTo evaluate absorption barrier recovery in the gastrointestinal tract after treatment with a penetration enhancer by using a poorly absorbed marker and correlate results with morphological recovery.MethodsOral gavage of sodium dodecyl sulfate (SDS) was given to Wistar rats. Phenol red (PR) was given at different time points following administration of SDS. Blood samples were obtained from the jugular vein. Pharmacokinetic analysis was performed on the in vivo data using WinNonlin and MATLAB®5 software. The pharmacokinetic parameters of PR were compared to the negative control to measure functional recovery. The intestinal tissues were observed using light and transmission electron microscopy.ResultsAbsorption was highest when PR was co-administered with SDS. Cmax, AUC and Ka decreased and Tmax and MAT increased as the recovery period (time between administration of SDS and PR) increased. The pharmacokinetic parameters approached the negative control profile in one hour after treatment with 1% SDS. Microscopy results showed recovery of paracellular and transcellular barrier at this time.ConclusionsAbsorption barrier recovery could be measured using a poorly absorbed marker. Functional recovery showed a good correlation with morphological recovery. The local effects of SDS were found to be temporary and reversible.

The effect of amphotericin B on the K-channel activity of MDCK cells.

By using the whole-cell patch technique, it is shown that the total outward current is increased, as a function of time, after the addition of amphotericin B to the bathing solution. The whole-cell current is shown to be primarily a K-channel current by the blockage of this current upon application of TEA to the bathing solution. Single K-channel studies, using the outside-out patch-clamp technique, reveal that the single K-channel opening probability increases by a factor of six after the addition of amphotericin B. In addition, single K-channel voltage dependent studies, using the inside-out patch-clamp technique, demonstrate that this increase in opening probability is due to an increase in the amplitude of Po(v). In contrast to the present belief that amphotericin B simply creates pores in a cells membrane, these results suggest that amphotericin B can also influence the function of the cells K-channel proteins.

Statistical Properties of Thermodynamic Quantities for Cyclodextrin Complex Formation

Literature values of DeltaG degrees (change in Gibbs free energy), DeltaH degrees (change in enthalpy), and TDeltaS degrees (temperature times change in entropy) for 1:1 complex formation by alpha-, beta-, and gamma-cyclodextrins constitute normally distributed populations with the following statistical parameters (all energy quantities in kcal mol(-1); n is the number of data points; mu is the population mean; sigma is the standard deviation): for alpha-cyclodextrin, n = 512, micro(DeltaG) = -2.85, sigma(DeltaG) = 1.23, micro(DeltaH) = -4.77, sigma(DeltaH) = 2.98, micro(TDeltaS) = -1.96, and sigma(TDeltaS) = 2.72; for beta-cyclodextrin, n = 415, micro(DeltaG) = -3.67, sigma(DeltaG) = 1. 37, micro(DeltaH) = -4.24, sigma(DeltaH) = 2.89, micro(DeltaS) = -0. 56, and sigma(TDeltaS) = 2.63; for gamma-cyclodextrin, n = 42, micro(DeltaG) = -3.71, sigma(DeltaG) = 1.19, micro(DeltaH) = -3.10, sigma(DeltaH) = 3.39, micro(TDeltaS) = +0.69, and sigma(TDeltaS) = 3. 29. The temperature is 298.15 K. The mean DeltaG degrees values correspond to binding constants of 123, 490, and 525 M(-1) for alpha-, beta-, and gamma-cyclodextrins, respectively.

A Monte-Carlo model for the passive diffusion of drugs through the stratum corneum

Abstract The diffusion of solute molecules through the stratum corneum has been described using a Monte-Carlo random walk model. The results obtained are in agreement with the solutions obtained from Ficks first and second laws. The method is computationally simple to apply, can handle time variation of the stratum corneums thickness and diffusion coefficient, and provides physical insight into the process of diffusion on a molecular level.

Dosimetric effectiveness of targeted radionuclide therapy based on a pharmacokinetic landscape.

Assessment of targeted radionuclide therapy (TRT) agent effectiveness based on its pharmacokinetic (PK) properties could provide means to expedited agent development or its rejection. A broad PK model that predicts the relative effectiveness of TRT agents based on the relationship between their normal body (k(12), k(21)) and tumor (k(34), k(43)) PK parameters has been developed. A classic two-compartment open model decoupled from a tumor was used to represent the body. Analytically solved differential equations were used to develop a relationship that predicts TRT effectiveness. Various PK scenarios were created by pairing normal body PK parameters of 38 pharmaceuticals found in the literature with estimated tumor PK parameters. Each PK scenario resulted in a maximum permissible injected activity that limited the whole-body dose to 2 Gy and yielded a maximum delivered tumor dose. The model suggests that a k(34):k(43) ratio greater than 5 and a k(12):k(21) ratio less than 1 is effective at delivering doses that ensure sufficient solid tumor control. It was also shown that there is no direct relationship between tumor dose and acid dissociation constant (pK(a)), lipophilicity (log P), and fraction unbound (fu), which are important physicochemical properties. This study suggests that although effective TRT may be difficult to achieve for solid tumors, good TRT agents must have extremely desirable normal body PKs in conjunction with very high tumor retention. The developed PK TRT model could serve as a tool to compare the relative dosimetric effectiveness of existing TRT agents and novel TRT agents early in the developmental phase to potentially reject those that possess unfavorable PKs.

Lipid microencapsulation of hemoglobin

The idea of microencapsulating a solution of hemoglobin (Hb) to make a prototypal artificial red blood cell (RBC) has been around for decades. T.M.S. Chang discussed the possibility in the late 50s and early 60s. The ideal properties and characteristics of a transfusable suspension of microencapsulated Hb have been specified. Progress has been limited by three major obstacles: (i) selecting an acceptable microencapsulation material; (ii) developing a microencapsulation process that yields the desired size range, but avoids denaturation of Hb; and (iii) encapsulating sufficient Hb while maintaining an acceptable final viscosity. We have made considerable progress in all three fronts. The resulting microcapsules are called Neohemocytes.