Factorial analysis of the influence of dissolution medium on drug release from carrageenan-diltiazem complexes

This research studied the influence of buffer composition, pH, and ionic strength on the release of diltiazem hydrochloride from a complex of the drug with lambda carrageenan. Two viscosity grades of carrageenan were also compared. A factorial analysis was used to evaluate the influence of individual variables and their interactions. Both the complex solubility, measured as the drug concentration in equilibrium with the solid complex, and the drug release rate from constant surface area were considered. The increase of ionic strength significantly increased complex solubility in all the buffer systems. A significant effect of polymer grade on complex solubility was evidenced only in phosphate buffer with a pH of 6.8, indicating lower solubility of the complex when higher polymer molecular weight was involved. In most cases, drug release rate decreased when high polymer grade was involved in the complex. Ionic strength did not always have a significant effect on drug release rate and was quantitatively less important than for solubility lonic strength especially affected the drug release profiles. At higher ionic strength drug release was no longer constant, but decreased with time, probably because of lower polymer solubility.     

Follow-up of drug permeation through excised human skin with confocal Raman microspectroscopy

Skin is a multilayered organ which covers and protects the surface of human body by providing a barrier function against exogenous agents. Meanwhile, the efficacy of several topically applicated drugs is directly related to their penetration through the skin barrier. Several techniques are commonly used to evaluate the rate, the speed and the depth of penetration of these drugs, but few of them can provide real-time results. Therefore, the use of nondestructive and structurally informative techniques permits a real breakthrough in the investigations on skin penetration at a microscopic scale. Confocal Raman microspectroscopy is a nondestructive and rapid technique which allows information to be obtained from deep layers under the skin surface, giving the possibility of a real-time tracking of the drug in the skin layers. The specific Raman signature of the drug enables its identification in the skin. In this study, we try to follow the penetration of Metronidazole, a drug produced by Galderma as a therapeutic agent for Rosacea treatment, through the skin. The first step was the spectral characterization of Metronidazole in the skin. Then micro-axial profiles were conducted to follow the penetration of the drug in the superficial layers, on excised human skin specimens. For more accurate information, transverse sections were cut from the skin and spectral images were conducted, giving information down to several millimeters deep. Moreover, the collected spectra permit us to follow the structural modifications, induced by the Metronidazole on the skin, by studying the changes in the spectral signature of the skin constituents. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

The effect of the lysosomotropic drug chloroquine on the binding of N-acetyl-beta-D-glucosaminidase to mannose-6-phosphate recognizing receptors of rat liver

The binding of N-acetyl-beta-D-glucosaminidase to rat liver receptors was studied in the presence of chloroquine. The association rate constant was not affected in the presence of the drug, while the dissociation rate constant and consequently the equilibrium dissociation binding constant significatively decreased. This results may explain effects of chloroquine on lysosomal enzyme transport found in cultured cells by other authors.     

Inhibition of nicotinic acetylcholine receptor by philanthotoxin-343: kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The mechanism of inhibition of a nicotinic acetylcholine receptor (nAChR) in BC(3)H1 muscle cells by philanthotoxin-343 (PhTX-343), a synthetic analogue of philanthotoxin-433, a wasp toxin, was investigated using a laser-pulse photolysis technique with microsecond time resolution and in a carbamoylcholine concentration range of 20-100 microM and PhTX-343 concentration range of 0-200 microM. The rate constant for nAChR channel opening determined by the chemical kinetic techniques decreased with increasing PhTX-343 concentration, whereas there was no significant effect on the rate constant for channel closing. The resulting decrease in the channel-opening equilibrium constant accounted quantitatively for the inhibition of the receptor by the toxin. Single-channel current measurements suggest an additional step in which the open channel:inhibitor complex isomerizes to a nonconducting receptor form. Cell-flow experiments with a time resolution of 10 ms indicate that this isomerization step is only important at very high inhibitor concentrations. The inhibitor binds to the open-channel receptor form, with an affinity that is at least 5 times smaller than that for the closed-channel form. This indicates that receptor inhibition mainly involves the binding of PhTX-343 to the closed-channel form of the receptor. PhTX-343, and an analogue of this polyamine, had no effect when applied to the inside of the cell membrane. However, there was significant inhibition of the nAChR when these compounds were applied to the outside of the cell membrane, indicating an extracellular site for inhibition. Furthermore, increasing the transmembrane potential results in a decrease in the ability of PhTX-343 to inhibit the receptor. This observation is related to the voltage dependence of the effect of PhTX-343 on the rate constant for nAChR channel opening with increasing transmembrane voltage (-60 to 50 mV). This suggests that the voltage dependence of inhibition mainly reflects the effect of transmembrane voltage on the rate constant of channel opening and, therefore, the channel-opening equilibrium constant. PhTX-343 competes with cocaine and procaine for its binding site. The finding that this toxin, which binds to a common inhibitory site with compounds such as cocaine, exerts its effect by decreasing the channel-opening equilibrium constant suggests an approach for the development of therapeutic agents. A compound that binds to this regulatory site but does not affect the channel-opening equilibrium constant may be developed. Such a compound can displace an abused drug such as cocaine and thereby alleviate the toxic effect of this compound on the organism.     

Kinetics of the daunomycin--DNA interaction

The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the two slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by two sequential isomerization steps. The six rate constants for this mechanism were extracted from our data by relaxation analysis. The values determined for the six rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using first sodium dodecyl sulfate to dissociate bound drug and second pseudo-first-order conditions to study the fast bimolecular step, provide independent verification of three of the six rate constants. The temperature dependence of four of the six rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. We speculate that the three steps in the proposed mechanism may correspond to a rapid "outside" binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.     

In vivo measurement and mapping of skin redox stress induced by ultraviolet light exposure

Exposure of skin to UV light presents a potent oxidative stress and this could alter the skin redox state. In this context, we evaluated the ability of electron paramagnetic resonance (EPR) imaging to provide noninvasive in vivo mapping of the redox status of the skin of living rats. The redox status was measured using a topically applied nitroxyl spin probe, (15)N-PDT. The nitroxyl intensity profile obtained across the skin layers showed that the concentration of the probe was higher in the epidermis and lower in the dermis and hypodermis. Skin permeability and reduction metabolism were evaluated in the skin exposed to UVB (312 nm) radiation. Exposure of skin to UVB decreased the overall reduction rate constant of the nitroxyl probe to 25 +/- 6% of the value obtained in the untreated skin. EPR imaging data showed that after the UVB treatment, the reduction rate constant decreased to 41 +/- 1% in epidermis, 28 +/- 1% in dermis, and 21 +/- 8% in hypodermis layers. The data suggested that UVB decreased the overall reducing capability of the skin with a larger decrease in the dermis and hypodermis. In summary, in vivo EPR imaging measurements showed significant alterations in the redox state of the skin exposed to UV light.     

Comparison of skin permeability of drugs in mice and human cadaver skin

In vitro percutaneous absorption of four antihypertensive drugs were carried out across the mice and human cavader skin in order to compare their skin permeability. An interesting trend was noticed in these experiments. Poorly water soluble drug prazosin hydrochloride showed 13 times enhanced flux in the mice skin whereas the steady-state flux of the water soluble drug propranolol hydrochloride was almost same in both human cadaver and mice skin. The permeation rate of prazosin hydrochloride and propranolol hydrochloride through the human cadaver skin fluctuated widely over time, but in mice skin, distinct trends were noticed. The study indicates that the overall permeation rate in mice skin is higher than that in the cadaver skin and the meeting of the target-flux in mice skin does not guarantee its good permeability in human skin.     

Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase.

The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.     

Kinetics of 9-aminoacridine block of single Na channels

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.     

Evaluation of a simple carrier molecule to enhance drug penetration of dermal layers by utilizing multivariate methods, structure property correlations, and continuous system modeling

Nicotinic acid is shown to be comparable to dihydropyridine in its capacity to facilitate penetration of an attached antibacterial drug through dermal layers. Antibacterial drugs examined with nicotinic acid or dihydropyridine carriers were beta-lactam antibiotics: methicillin, oxacillin, benzylpenicillin, penicillin F, penicillin dihydro F, propicillin, carbenicillin, penicillin K, penicillin X, and ampicillin. An oxymethyl (-O-CH2-) group is inserted as the linker between the antibiotic and the carrier group. Structure Property Correlations and multivariate methods such as regression analysis, cluster analysis, principal component analysis, discriminate analysis, self-organizing tree algorithm, and factor analysis clearly showed that nicotinic acid performs as an effective carrier drug and is comparable to dihydropyridine. The skin penetration constant Kp was calculated for all 10 antibiotics having either dihydropyridine or nicotinic acid as carrier, and was found to have a mean of 5.13E-05 cm/hour and 1.83E-05 cm/hour, respectively. The standard deviation for each group showed the numerical values overlap as did the 90% confidence interval for each group. A hierarchical tree organization of skin shows the overlapped dermal layers as they exist in normal skin and for the model utilized in this work. A Deming-Regression analysis also shows the nicotinic acid and dihydropyridine structures to have similar and correlated water solubility. Plotting Kp of the dihydropyridine structures as independent variable versus Kp of the nicotinic acid structures show good correlation (Pearson correlation r = 0.6606) and no significant departure from linearity. Connected box plots showed the majority of Kp values for each group of modified antibiotics to exist in a tight cluster. Polar graph of the Log Kow values showed the two groups of modified antibiotics to be correlated and numerically adjacent in trend. ChemSketch property calculations and modeling demonstrates the affects of structural oxygens, nitrogens, carbonyl groups, amide groups, and aromatic rings that are important in understanding the pervasiveness through dermal layers. Continuous model analysis by acslXtreme is utilized and demonstrates three models of the dispersion of drugs through dermal layers based on diffusivity constant (D), Log Kp from Log Kow and formula weight, and Kp as a function of time.     

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.     

The in vivo and in vitro interactions of elastic and rigid vesicles with human skin

Elastic vesicles are the most novel development in vesicular systems design for dermal and transdermal drug delivery. However, interactions between these vesicles and human skin are not yet fully understood. In this study, the in vivo and in vitro interactions between elastic-, rigid vesicles and micelles with human skin were investigated. Vesicle and micelle solutions were applied onto human skin in vitro and in vivo. Subsequently, a series of tape strippings were performed, which were visualised by freeze fracture electron microscopy (FFEM). The results showed no ultrastructural changes in skin treated with rigid vesicles. Skin treated with elastic vesicles, however, showed a fast partitioning of intact vesicles into the deeper layers of the stratum corneum (SC), where they accumulated in channel-like regions. Only little vesicle material was found in the deepest layers of the SC, suggesting that the partitioning of intact vesicles from the SC into the viable epidermis is unlikely to happen. Treatment with micelles resulted in rough, irregular fracture planes. Similar results were obtained in vitro and in vivo, indicating an excellent in vitro/in vivo correlation. These results support the hypothesis that elastic vesicles have superior characteristics to rigid vesicles for the interaction with human skin. Elastic vesicles and micelles demonstrated very different interactions with human skin and hence probably also have different mechanisms of action for the enhancement of drug transport.     

Theoretical analysis of the effect of the transbilayer movement of phospholipid molecules on the dynamic behavior of a microtube pulled out of an aspirated vesicle

Observations over extended times of a lipid microtube (tether) formed from a lecithin vesicle have shown that under constant external loads the tether exhibits a continuous slow growth. It is considered that this growth is a consequence of the net transbilayer movement of phospholipid molecules in a direction which relieves the membrane strain resulting from the elastic deformation of the vesicle. The elastic deformation mode responsible for this effect is identified as the relative expansion of the two membrane layers reflecting the non-local contribution to membrane bending. An equation for the consequent rate of transbilayer movement of phospholipid molecules is derived. The dynamic behavior of the system is modeled by including frictional contributions due to interlayer slip and Stokes drag on the glass bead used to form the tether. The general numerical solution reveals a complex dependence of the tether growth rate on the system parameters and a continuous increase in the rate of tether growth at long times. Closed form expressions approximating the system behavior are derived and the conditions under which they can be applied are specified. Modeling the mechanically-driven lipid transport as a simple, stochastic, thermal process, allows the rate of lipid translocation to be related to the equilibrium transbilayer exchange rate of phospholipid molecules. Consideration of experimental results shows that the time constant for mechanically-driven translocation is shorter than the time for diffusion-driven translocation by approximately two orders of magnitude, indicating that lipid translocation is not a simple diffusive process. Received: 29 November 1996 / Revised version: 1 December 1997 / Accepted: 9 January 1998     

Relaxation methods for the determination of drug receptor affinities

According to the occupation theory of drug receptor interaction, the response is a functionf of the number of receptors occupied by drug molecules. Considerable controversy exists regarding assumptions about this function. Without knowledge of the nature of the function, it is not possible to determine directly the rate constants, and hence the affinity constant, in the reaction between the receptor and an agonist drug. Instead, indirect determinations involving the use of antagonists have been employed, limiting the determination of affinity to those agents for which specific antagonists exist. The present paper discusses a method for the direct determination of affinity of an agonist drug. It is a “relaxation method,” i.e., the equilibrium is perturbed and the kinetics of the restoration process are studied. Assuming only thatf is non-decreasing and approximately linear over a limited domain of concentrations, it is shown that the change in response obeys first order kinetics, permitting a determination of the rate constants from the time course of the restoration process.     

No dynamic effector responses to fast changes of core temperature at constant skin temperature

Experiments in conscious goats were done to see whether heat production and respiratory evaporative heat loss show dynamic responses to changing core temperature at constant skin temperature. Core temperature was altered by external heat exchangers acting on blood temperature, while skin temperature was maintained constant by immersing the animals up to the neck in a rapidly circulating water bath. Core temperature was altered at various rates up to 0.9 degrees C/min. Step deviations of core temperature from control values were always followed by a positive time derivative of effector response, but never by a negative time derivative during sustained displacement of core temperature. Ramp experiments showed that the slopes at which heat production or heat loss rose with core temperature deviating from its control level grew smaller at higher rates of change of core temperature. It is concluded that neither heat production nor respiratory evaporative heat loss respond to the rate of change of core temperature. At constant skin temperature, thermoregulatory effector responses appear to be proportional to the degree to which core temperature deviates from its set level.     

Drug release properties of polyethylene-glycol-treated ciprofloxacin-Indion 234 complexes

The polyethylene glycol (PEG) treatment of ciprofloxacin-Indion 234 complex was aimed to retard rapid ion exchange drug release at gastric pH. Ciprofloxacin loading on Indion 234 was performed in a batch process, and the amount of K⁺ in Indion 234 displaced by drug with time was studied as equilibrium constant K_DM. Drug-resin complex (DRC) was treated with aqueous PEG solution (0.5%–2% wt/vol) of different molecular weights (MWs) for 2 to 30 minutes. The PEG-treated ciprofloxacin-Indion 234 complex was evaluated for particle size, water absorption time, and drug release at gastric pH. During drug loading on Indion 234, the equilibrium constant (K_DM) increased rapidly up to 20 minutes with efficient drug loading. Increased time of immersion of the drug resinate in PEG solutions significantly retained higher size particles upon dehydration. The larger DRC particles showed longer water absorption times owing to compromised hydrating power. The untreated DRC showed insignificant drug release in deionized water; while at gastric pH, ciprofloxacin release was complete in 90 minutes. A trend of increased residual particle size, proportionate increase in water absorption time, and hence the retardation of release with time of immersion was evident in PEG-treated DRC. The time of immersion of DRC in PEG-treated DRC. The time of immersion of DRC in PEG solution had predominant release retardant effect, while the effect of molecular weight of PEG was insignificant. Thus, PEG treatment of DRC successfully retards ciprofloxacin ion exchange release in acidic pH.     

Liquid Crystalline Systems for Transdermal Delivery of Celecoxib: In Vitro Drug Release and Skin Permeation Studies

Liquid crystalline systems of monoolein/water could be a promising approach for the delivery of celecoxib (CXB) to the skin because these systems can sustain drug release, improve drug penetration into the skin layers and minimize side effects. This study evaluated the potential of these systems for the delivery of CXB into the skin based on in vitro drug release and skin permeation studies. The amount of CXB that permeated into and/or was retained in the skin was assayed using an HPLC method. Polarizing light microscopy studies showed that liquid crystalline systems of monoolein/water were formed in the presence of CXB, without any changes in the mesophases. The liquid crystalline systems decreased drug release when compared to control solution. Drug release was independent of the initial water content of the systems and CXB was released from cubic phase systems, irrespective of the initial water content. The systems released the CXB following zero-order release kinetics. In vitro drug permeation studies showed that cubic phase systems allowed drug permeation and retention in the skin layers. Cubic phase systems of monoolein/water may be promising vehicles for the delivery of CXB in/through the skin because it improved CXB skin permeation compared with the control solution.KEY WORDS: celecoxib, drug delivery systems, liquid crystalline system, monoolein, skin permeation     

药物经皮转运通道的网络热力学模型

阐述药物在经皮转运过程中皮肤通道导纳特性,为经皮给药技术提供理论和方法。用药物经皮转运通道的网络热力学模型建立药物浓度与渗透通量的定量关系,提出皮肤通道导纳由激活因子和失活因子控制的理论。以替硝唑为模式药物,进行被动扩散(对照组)和电脉冲增强扩散(实验组)的离体漏槽实验,结果显示皮肤通道导纳的理论线与实验值拟合较好。理论线计算的激活因子初值m0、激活因子时间常数Tm、失活因子初值‰、失活因子时间常数Th对分析皮肤通道特性有重要价值。     

Studies in formulation and pharmacotechnical evaluation of controlled release transdermal delivery system of bupropion

The objective of the present study was to design and evaluate unilaminate transdermal adhesive matrix systems capable of diffusing bupropion base at a constant rate over an extended period of time as an alternative route of administration. Unilaminate transdermal adhesive matrices have been fabricated with different concentrations of Eudragit E as the adhesive and rate-controlling polymer. The in vitro release and epidermal flux through human cadaver skin were studied. The release of drug from the matrices obeyed zero order release kinetics (r ²=0.9810 to 0.9960). The delivery rate of bupropion ranged from 10.5 mg to 31.4 mg per day from a 3.14 cm² area of matrix. The relation between concentration of bupropion base in matrix and epidermal flux, concentration of drug in matrix, and epidermal adsorption of bupropion during diffusion follow hyperbolic fashion. Triethylcitrate (TEC) and dibutylphthalate (DBP) have no influence on the diffusion of bupropion through human cadaver skin when used as plasticizers. Incorporation of succinic acid in the adhesive matrix retarded diffusion due to the formation of rigid cross linking of the polymer, while propylene glycol and myristic acid, alone or in combination, significantly enhanced the flux of bupropion through human cadaver skin.