Determination of the permeability of biological membranes to various chemical markers, including anti-HIV drugs

[摘要] ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds areproduced by parallel synthesis and combinatorial chemistry. The pharmaceuticalindustry therefore requires rapid and accurate methods to screen new drugs leads formembrane permeability potential in the early stages of drug discovery. Around 50 %of all investigational new drugs fail in pre-clinical and clinical phases of developmentdue to inadequate absorption/permeation, distribution, metabolism, excretion and/orunacceptable toxicity. This may be decreased by applying in vitro screening methodsearly in the discovery process. Reliable in vitro models can be applied to determinepermeation of the test compounds, which will help avoid the wasting of valuableresources for the development of drugs that are destined to fail in preclinical andclinical phases due to insufficient permeability properties. It is important to decide asearly as possible on the most promising compound and physical formulation for theintended route of administration.With awareness of the increasing importance of in vitro models in the investigationsof the permeability properties of drug compounds, this research project wasspecifically devoted to determine the suitability of our in vitro model to evaluate andpredict drug permeability. A continuous flow-through diffusion system was employedto evaluate the permeability of nine different compounds/drugs with differentchemical properties, across three biological membranes. The biological membraneschosen for the present study were human vaginal mucosa, human skin tissue andhuman small intestine mucosa. The continuous flow-through diffusion system wasfurthermore utilised to investigate the effects of de-epithelialisation of mucosalsurfaces, chemical enhancers, temperature, permeant concentration and formulationon the permeability of the test compounds/drugs. The in vitro permeabilityinformation and data from the flow-through diffusion model were compared to in vitroand in vivo literature studies and drug profile. An in vitro model that is able to reliablypredict in vivo data will shorten the drug development period, economise resourcesand may potentially lead to improved product quality.In this thesis research results are reported on the permeability of the mentionedbiological membranes to the various chemical markers, including anti-HIV (humanimmunodeficiency virus) drugs. The permeability studies will be discussed in threesections: vaginal mucosa, skin tissue, small intestine mucosa.The results of the vaginal permeability studies showed that the three peptides (MEA-5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higherflux rate than MEA-5, commensurate with its smaller molecular size (weight). Thesurfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreasedthe lag time of the peptide. Removal of the vaginal epithelium increased the fluxrates of the peptides across the mucosa and may have implications for a more rapiduptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCsof the overall mean flux values of MDY-19 and PCI increased with concentrationaccording to well-established diffusion theory.The experiments on the permeability of different terbinafine hydrochlorideformulations through human skin demonstrated that the terbinafine hydrochlorideformulations used in this study, readily diffused into the skin tissue. However, no fluxvalues for any of the terbinafine hydrochloride formulations through the skin into theacceptor fluid were found. The mean terbinafine concentrations in the skin after 24 hexposure to the three commercial, terbinafine hydrochloride formulations were 3.589,1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skinexposed to the 10 mg/ml PBS/Methanol solution was higher than those from thethree commercial formulations.The results of the temperature study demonstrated that an increase of 5 ºC caused asignificant increase in flux values of tritiated water across skin. The flux values fortritiated water across skin at 37 ºC were on average double those at a temperature of32 ºC.The permeability of excised human small intestine mucosa to different oral dosagedrugs was investigated over a 24 h period. The four drugs selected were zidovudine,propranolol hydrochloride, didanosine and enalapril maleate. They were selected asrepresentative model compounds of drug classes 1 (high solubility, high permeability)and 3 (high solubility, low permeability) according to the BiopharmaceuticsClassification System. The flux rates of the four chosen test drugs were influencedby the length of the experiment. Between the time periods 2-4 h and 4-6 h,zidovudine's mean flux values across small intestine tissue were respectively 1.8 and2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril.Propranolol's mean flux values were respectively 1.2 and 1.4 times higher thandidanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods.Between both the time periods 2-4 and 4-6 h AZT's mean flux values were 1.4 timeshigher than propranolol and didanosine's mean flux values were respectively 1.3 and1.1 times higher than enalapril during the mentioned time periods. Class 1 drugsshowed a significantly higher flux rate across the jejunal mucosa compared to theclass 3 drugs and these results are in line with their Biopharmaceutics ClassificationSystem classification. The in vitro model has proved to be reliable to predictpermeability of class 1 and 3 drugs and also showed correlation with human in vivodata.It seems that the in vitro flow-through diffusion model used in the present study havethe potential to overcome some of the problems and limitations demonstrated byother in vitro techniques and may potentially serve as a future tool for pharmaceuticalcompanies to predict the diffusion characteristics of new drugs and differentformulations, across different biological membranes. Furthermore, it may serve as aprospective method for assessing the bioequivalence of alternative (generic) vehiclesor formulations containing the same drug/compound.

[发布机构] Stellenbosch University