Resumen de Estudio de la absorción transdérmica de fármacos para la migraña = Study of the transdermal absorption of drugs for migraine
María Aracely Calatayud Pascual
This doctoral thesis is part of a wider research project to develop transdermal therapeutic systems for the treatment of migraine. Migraine is a transient chronic disorder characterized by recurrent episodes of headache. It is of particular relevance given the frequency with which it occurs and the negative impact it has on the quality of life of sufferers. In recent years the prevalence of neurological disease has greatly increased; it is now estimated to affect one in five people in the world and 12.6% of the Spanish population. Thus, there is a need to develop effective pharmaceutical approaches that include drugs to prevent episodes (beta-blockers) or to treat symptoms (e.g. triptans), and which can be administered transdermally, as this route has proved to be more convenient and better tolerated by patients. However, human skin constitutes a highly efficient barrier that limits or even blocks the transport of drugs, thus restricting the transdermal delivery of therapeutic agents. Different strategies have been employed to enhance the passage of drugs across the skin and thus achieve a therapeutic blood concentration, including the use of chemical substances as absorption enhancers and application of iontophoresis. The main objectives of the present work have differed depending upon whether the focus of study was the transdermal absorption through porcine skin of the beta-blockers, nadolol and propranolol hydrochloride, or of almotriptan. On the one hand, the aim was to characterize and compare the in vitro transdermal absorption of nadolol and propranolol hydrochloride and to study the effect of chemical enhancers and iontophoresis on this absorption. In addition, given that the majority of enhnacing techniques affect the barrier function of the skin, both drugs were quantified in the stratum corneum and their dermatopharmacokinetics parameters determined to describe the rate and extent of delivery across the skin. In order to characterize the transdermal absorption of both beta-blockers, percutaneous diffusion experiments were performed. Two simple and rapid High Performance Liquid Chromatographic (HPLC) methods with ultraviolet detection were chosen to determine the in vitro transdermal absorption of nadolol and propranolol hydrochloride (chapter III). Both methods were validated for specificity, linearity, precision, accuracy, limit of detection, limit of quantification and robustness. Separation was carried out on a 250 mm Kromasil® C18 column at room temperature. The detector wavelength was fixed at 269 nm for nadolol and 291 nm for propranolol hydrochloride and the response was linear for all the concentrations evaluated (range of 0.17 to 167 µM in the case of nadolol and 0.11 to 113 µM in the case of propranolol hydrochloride). The maximum relative error and relative standard deviation detected were 5.19% and 6.08% respectively for nadolol and 3.57% and 7.28% respectively for propranolol hydrochloride. The limits of detection and quantification were 0.058 and 0.038 µM for nadolol and 0.171 and 0.115 µM for propranolol hydrochloride, respectively. The results of robustness were highly satisfactory. Once both analytical methods had been validated and shown to be adequate, the transdermal absorption of nadolol and propranolol hydrochloride was characterized and the effects of the application of chemical enhancers and iontophoresis on this absorption was investigated by means of in vitro permeation studies (chapter IV). As expected, the passive transdermal flux of the more lipophilic drug (propranolol) was higher than that of nadolol (48.9 ± 1.3 ¿g/cm2h vs. 17.1 ± 0.7 ¿g/cm2h). Pretreatment of porcine skin with ethanol (vehicle), Azone®, R-(+)-limonene, caproleic acid and oleic acid (at 5% in ethanol, w/w) and the application of two current densities (0.25 and 0.50 mA/cm2) produced varying effects depending on the drug studied. In the case of nadolol, treatment of the skin with Azone® and oleic acid produced an increase in the flux compared with the control; however, in vitro data suggested that only Azone® produced a significant increase, with a value 7.18-fold that of the control (p<0.01). Of all the enhancers tested, R-(+)-limonene was incapable enhancing the absorption of nadolol. Although some of the chemical substances assayed increased the absorption of nadolol, the most significant increment in the transdermal transport of nadolol was clearly produced by iontophoresis, specifically that of 0.50 mA/cm2, which produced transdermal flux values of 216 ± 11 ¿g/cm2h for nadolol (14.8-fold that of passive diffusion). On the other hand, the effect of chemical enhancers and iontophoresis on the absorption of propranolol hydrochloride across the porcine skin proved to be more moderate. All the strategies slightly increased the transdermal absorption of this agent; Azone® and oleic acid produced 1.74 and 1.39-fold increments with respect to control values, while R-(+)-limonene and iontophoresis at a current density of 0.50 mA/cm2, proved to be the most effective of the techniques, resulting in values of 106 ± 2 ¿g/cm2h and 108 ± 10 ¿g/cm2h, respectively, which represents a 1.88-fold increase with respect to the control. Therefore, the application of iontophoresis did not produce the same effect on both drugs. When the fluxes obtained in the first 8 hours with current densities of 0.25 and 0.50 mA/cm2 were compared, statistically significant differences were detected between the two drugs: 120 ± 21 ¿g/cm2h (nadolol) and 61.6 ± 7.1 ¿g/cm2h (propranolol hydrochloride) vs. 216 ± 11 ¿g/cm2h (nadolol) and 108 ± 10 ¿g/cm2h (propranolol) (p<0.05). These results provide clear evidence of the modulating influence of current density on the transdermal transport of a drug through the skin and of the enhancing effect of this physical strategy in increasing the flux of the more hydrophilic of the two drugs assayed (nadolol). After evaluating the transdermal absorption of both beta-blockers through porcine skin, the in vitro distribution profile of the drugs in stratum corneum were compared by calculating dermatopharmacokinetic parameters. The objective of these experiments was to determine the effect of the various strategies tested. The results were used to deduce the stratum corneum-vehicle partition coefficient (K) and its diffusion kinetic (D/H2), as well as the amount of drug present in the outer layer of the skin (chapter V). When 0.50 mA/cm2 iontophoresis was applied, the total amount values of nadolol in the stratum corneum was 9.61 ± 1.28 mg/cm3 (almost 3-fold that observed after passive diffusion). Moreover, the deduced values of K and D/H2 obtained with oleic acid and Azone® were statistically different from those obtained under other conditions (p<0.05). The partitioning parameter of propranolol hydrochloride by passive diffusion was high 4.37 ± 0.62, though, oleic acid produced the highest value of K and the greatest amount of drug in the stratum corneum (p<0.05), values that were similar to those achieved with 0.50 mA/cm2. When the accumulated amounts of each beta-blocker in the donor compartments were compared they were found to be lower in the case of nadolol. It was thus determined that the higher amount of nadolol in the stratum corneum was a result of permeation of the drug, especially when iontophoresis had been applied. Based on promising results obtained with sumatriptan in previous studies by our research group, the second main objective of this project was to investigate whether or not almotriptan can be administered transdermally, and to evaluate the feasibility of using iontophoresis in order to enhance its transdermal passage (chapter VI). Transdermal flux of almotriptan through porcine skin was determined in different conditions: (a) passive diffusion (control) and (b) application of iontophoresis (0.25 and 0.50 mA/cm2) controlling the effect of ionic strength on the almotriptan transport. The transdermal flux of almotriptan malate over 8 hours was 0.64 ± 0.11 µg/cm2h (control). Application of iontophoresis produced a statistically significant increment of the transdermal transport of almotriptan malate (p<0.01). When 0.25 mA/cm2 iontophoresis was applied, transdermal flux values of 115 ± 17 µg/cm2h were obtained, and when the current was doubled the flux increased to 264 ± 24 µg/cm2h, which was 411-fold that obtained with the control, thus indicating the efficacy of iontophoresis in enhancing the transdermal absorption of almotriptan malate. From the experimental results we can highlight the following conclusions: 1. Two new methods of High Performance Liquid Chromatographic with ultraviolet detection have been developed and validated for the rapid and precise determination of nadolol and propranolol hydrochloride in samples obtained from transdermal diffusion experiments. 2. The effect of the chemical substances assayed as transdermal absorption enhancers varies according to the beta-blocker in question. Of the substances studies, Azone® and R-(+)-limonene are the most powerful enhancers of nadolol and propranolol hydrochloride, respectively. The differences observed between the transdermal flux and permeation parameters of the two beta-blockers demonstrate the importance of the lipophilicity of both the drug and the chemical enhancer in the transdermal absorption. 3. Iontophoresis is the most effective of the strategies for increasing the transdermal absorption of the beta-blockers studied here. However, this technique does not produce the same effect on both drugs. The application of a current density of 0.50 mA/cm2 enhances the flux of the more hydrophilic drug to values that are 14.8-fold those achieved with passive diffusion of nadolol. 4. The results of the dermatopharmacokinetic study, in which tape-stripping was used, allow the penetration of nadolol and propranolol hydrochloride through the stratum corneum and the pharmacokinetic parameters of this diffusion to be characterized. This research shows that shorter duration studies can predict the behavior that will be seen during longer periods of application. The low permeability of the stratum corneum is evident, thus confirming its role as a barrier with a high resistance and capacity for blocking the passage of molecules. 5. Almotriptan malate is characterized by a low transdermal absorption that is increased significantly by iontophoresis. The application of a current density of 0.50 mA/cm2 is the most effective strategy for enhancing the transdermal transport of a drug among those assessed, resulting in values 411-fold those obtained in controls. 6. Our results for the iontophoretic delivery of nadolol and almotriptan across porcine skin support the viability of the transdermal administration of these drugs. In this way, iontophoresis would seem to be a promising alternative to oral administration of pharmacological therapy for the prevention and acute treatment of migraine.
