Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are
produced by parallel synthesis and combinatorial chemistry. The pharmaceutical
industry therefore requires rapid and accurate methods to screen new drugs leads for
membrane permeability potential in the early stages of drug discovery. Around 50 %
of all investigational new drugs fail in pre-clinical and clinical phases of development
due to inadequate absorption/permeation, distribution, metabolism, excretion and/or
unacceptable toxicity. This may be decreased by applying in vitro screening methods
early in the discovery process. Reliable in vitro models can be applied to determine
permeation of the test compounds, which will help avoid the wasting of valuable
resources for the development of drugs that are destined to fail in preclinical and
clinical phases due to insufficient permeability properties. It is important to decide as
early as possible on the most promising compound and physical formulation for the
intended route of administration.
With awareness of the increasing importance of in vitro models in the investigations
of the permeability properties of drug compounds, this research project was
specifically devoted to determine the suitability of our in vitro model to evaluate and
predict drug permeability. A continuous flow-through diffusion system was employed
to evaluate the permeability of nine different compounds/drugs with different
chemical properties, across three biological membranes. The biological membranes
chosen for the present study were human vaginal mucosa, human skin tissue and
human small intestine mucosa. The continuous flow-through diffusion system was
furthermore utilised to investigate the effects of de-epithelialisation of mucosal
surfaces, chemical enhancers, temperature, permeant concentration and formulation
on the permeability of the test compounds/drugs. The in vitro permeability
information and data from the flow-through diffusion model were compared to in vitro
and in vivo literature studies and drug profile. An in vitro model that is able to reliably
predict in vivo data will shorten the drug development period, economise resources
and may potentially lead to improved product quality.
In this thesis research results are reported on the permeability of the mentioned
biological membranes to the various chemical markers, including anti-HIV (human
immunodeficiency virus) drugs. The permeability studies will be discussed in three
sections: 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 higher
flux rate than MEA-5, commensurate with its smaller molecular size (weight). The
surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased
the lag time of the peptide. Removal of the vaginal epithelium increased the flux
rates of the peptides across the mucosa and may have implications for a more rapid
uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19
and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs
of the overall mean flux values of MDY-19 and PCI increased with concentration
according to well-established diffusion theory.
The experiments on the permeability of different terbinafine hydrochloride
formulations through human skin demonstrated that the terbinafine hydrochloride
formulations used in this study, readily diffused into the skin tissue. However, no flux
values for any of the terbinafine hydrochloride formulations through the skin into the
acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h
exposure to the three commercial, terbinafine hydrochloride formulations were 3.589,
1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin
exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the
three commercial formulations.
The results of the temperature study demonstrated that an increase of 5 ºC caused a
significant increase in flux values of tritiated water across skin. The flux values for
tritiated water across skin at 37 ºC were on average double those at a temperature of
32 ºC.
The permeability of excised human small intestine mucosa to different oral dosage
drugs was investigated over a 24 h period. The four drugs selected were zidovudine,
propranolol hydrochloride, didanosine and enalapril maleate. They were selected as
representative model compounds of drug classes 1 (high solubility, high permeability)
and 3 (high solubility, low permeability) according to the Biopharmaceutics
Classification System. The flux rates of the four chosen test drugs were influenced
by 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 and
2.0 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 than
didanosine 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 times
higher than propranolol and didanosine’s mean flux values were respectively 1.3 and
1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs
showed a significantly higher flux rate across the jejunal mucosa compared to the
class 3 drugs and these results are in line with their Biopharmaceutics Classification
System classification. The in vitro model has proved to be reliable to predict
permeability of class 1 and 3 drugs and also showed correlation with human in vivo
data.
It seems that the in vitro flow-through diffusion model used in the present study have
the potential to overcome some of the problems and limitations demonstrated by
other in vitro techniques and may potentially serve as a future tool for pharmaceutical
companies to predict the diffusion characteristics of new drugs and different
formulations, across different biological membranes. Furthermore, it may serve as a
prospective method for assessing the bioequivalence of alternative (generic) vehicles
or formulations containing the same drug/compound. / AFRIKAANSE OPSOMMING: As gevolg van moderne hoë spoed tegnologie kan groot hoeveelhede middels
vervaardig word deur ooreenkomende sintese en kombinasieleer chemie. Die
farmaseutiese industrie benodig dus vinnige en akkurate metodes om nuwe
geneesmiddels te evalueer t.o.v. membraan deurlaatbaarheid. Hierdie evaluasie
moet verkieslik so vroeg moontlik in die geneesmiddel se ontwikkelingsproses
geskied. Ongeveer 50 % van alle potensiële geneesmiddels misluk in pre-kliniese en
kliniese fases van geneesmiddelontwikkeling. Die mislukte pogings kan toegskryf
word aan onvoldoende absorbsie/deurlaatbaarheid, distribusie, metabolisme,
ekskresie en/of onaanvaarbare middel toksisiteit. Dit is daarom belangrik om so
vroeg moontlik in die geneesmiddelontwikkelingsproses te besluit op die mees
belowende middel, asook die geskikte formulasie vir die spesifieke roete van
toediening van die middel. Die farmaseutiese industrie benodig tans in vitro modelle
met die potensiaal om die deurlaatbaarheid van geneesmiddels te bepaal en te
voorspel. Betroubare in vitro modelle kan aangewend word om die deurlaatbaarheid
van potensiële geneesmiddels te toets. Sodoende sal die onnodige uitgawes op die
ontwikkkeling van geneesmiddels wat in elk geval later gaan faal in pre-kliniese en
kliniese fases van geneesmiddelproewe a.g.v. deurlaatbaarheidseienskappe, vermy
word.
Hierdie navorsingsprojek was dus spesifiek onderneem om die waarde en
toepaslikheid van ‘n in vitro deurlopende-vloei perfusie model te ondersoek. Die
model se potensiaal om geneesmiddels se deurlaatbaarheid en absorpsie te
voorspel was geëvalueer. Die deurlopende-vloei perfusie apparaat was gebruik om
die deurlaatbaarheidsvloede van drie verskillende biologiese membrane t.o.v. nege
chemiese stowwe (MEA-5, MDY-19, PCI, terbinafien hidrochloried, getritieerde
water, zidovudien, propranolol, hidrochloried, didanosien, enalapril maleaat) te
bepaal. Die drie biologiese membrane wat gebruik was, was vaginale weefsel, vel
en klein intestinale weefsel. Al drie weefsel tipes was van menslike oorsprong. Die
deurlopende-vloei perfusie apparaat was ook gebruik om die effek wat verwydering
van die mukosa se epiteellaag op deurlaatbaarheidsvloede het, te ondersoek.
Verder was navorsing gedoen op die effek van temperatuur en die konsentrasie en
formulasie van die toetsmiddels op hulle diffusie vloedwaardes. Daar was ook gekyk
na die invloed van ander chemiese stowwe op die toetsmiddels se diffusie
vloedwaardes. Die in vitro deurlaatbaarheidsinformasie en -gegewens was vergelyk
met ander in vitro en in vivo literatuurstudies en geneesmiddel databasisse. ‘n In
vitro model wat in staat is om in vivo resultate betroubaar te voorspel, het die
potensiaal om die tyd wat dit neem om geneesmiddels te ontwikkel, te verkort,
finansiële uitgawes te besnoei en om geneesmiddelkwaliteit te verseker.
In die tesis word dan die resultate gerapporteer van die deurlaatbaarheidsvloede van
die verskillende tipes weefsel ten op sigte van verskeie chemiese stowwe,
insluitende anti-MIV (menslike immuniteitsgebreksvirus) middels. Die
deurlaatbaarheidstudies word bespreek in drie afdelings: vaginale mukosa, vel en
klein intestinale mukosa.
Die resultate van die deurlaatbaarheidstudies op die vaginale weefsel dui daarop dat
die drie peptiede (MEA-5, MDY-19 and PCI) die vaginale mukosa goed penetreer.
Soos verwag, het MDY-19 hoër diffusie vloedwaardes as MEA-5 gehad. Dit kan
toegeskryf word aan MDY-19 se kleiner molekulere grootte (gewig). Surfaktant het
die diffusie vloedwaardes van MDY-19 1.3 keer vergroot en het ook die tyd na vaste
vlak verminder. Die verwydering van die vaginale epiteel het die diffusie
vloedwaardes van die peptiede verhoog en mag dus dui op die vinniger opname van
peptiede en moontlike ander mikrobisiede in vivo, wanneer die belyning van die
epiteel onderbreek. Die deurlaatbaarheid van 1 mM MDY-19 en PCI by 37 °C was
satisties beduidend (p<0.05) hoer as teem 20 °C. Die area onder die kurwe (AOK)
van die gemiddelde vloedwaardes van MDY-19 en PCI by 37 °C, het toegeneem met
‘n toename in die konsentrasie van hierdie peptiede. Die toename vloedwaardes
ondersteun dus die alombekende diffusie teorie.
Die transdermale diffusie eksperimente van verskillende terbinafien formulasies het
getoon dat terbinafien geredelik vrygestel word vanuit hierdie formulasies na die vel.
Geen terbinafien vloedwaardes, van enige van die formulasies, was egter gevind in
die ontvangselle van die deurlopende-vloei perfusie apparaat nie. Die gemiddelde
terbinafien konsentrasies in die vel na 24 h se blootstelling aan drie kommersiële
terbinafien hidrochloried formulasies was onderskeidelik 3.589, 1.590 en 4.219
μg/ml. Die gemiddelde terbinafien konsentrasie in die vel wat aan 10 mg/ml
PBS/metanol blootgestel was, was hoër as die konsentrasies in die vel wat aan die
drie kommersiële formulasies blootgestel was.
Die resultate van die temperatuurstudie op vel het aangetoon dat ‘n temperatuur
toename van 5 ºC ‘n statisties beduidende toename in vloedwaardes van
getritieerde water oor vel veroorsaak. Die vloedwaardes van die getritieerde water
oor vel teen ‘n temperatuur van 37 ºC was gemiddeld dubbeld so veel as teen 32 ºC.
Die deurlaatbaarheidsvloede van klein intestinale mukosa ten opsigte van
verskillende geneesmiddels (wat oraal toegedien word) was ondersoek gedurende ‘n
24 h eksperiment. Die vier geneesmiddels wat gebruik was, was zidovudine,
propranolol hidrochloried, didanosien en enalapril maleaat. Hierdie geneesmiddels is
verteenwoordigers van die Biofarmaseutiese Klassifikasie Sisteem se klas 1 (hoë
oplosbaarheid, hoë deurlaatbaarheid) en klas 3 (hoë oplosbaarheid, lae
deurlaatbaarheid) geneesmiddels. Die vloedwaardes van die vier geneesmiddels het
gewissel na aanleiding van die tydsverloop in die eksperiment. Zidovudien se
gemiddelde vloedwaardes tussen 2-4 en 4-6 h was onderskeidelik 1.8 en 2.0 keer
hoër as didanosien se gemiddelde vloedwaardes vir hierdie tyd periodes en
onderskeidelik 2.3 en 2.2 keer hoër as enalapril se gemiddelde vloedwaardes.
Tydens hierdie selfde periodes was propranolol se gemiddelde vloedwaardes 1.2 en
1.4 keer hoër as didanosien en vir beide periods 1.6 keer hoër as enalapril se
gemiddelde vloedwaardes. Gedurende beide genoemde tyd periodes was
zidovudien se gemiddelde vloedwaardes 1.4 keer hoer as propranolol en didanosien
se gemiddelde vloedwaardes was onderskeidelik 1.3 en 1.1 keer hoër as enalapril
tydens 2-4 en 4-6 h. Die klas 1 geneesmiddels het statisties beduidende hoër
vloedwaardes gehad as die klas 3 geneesmiddels. Hierdie resultate stem ooreen
met die geneesmiddels se Biofarmaseutiese Klassifikasie Sisteem klassifikasie. Dit
wil dus voorkom asof die in vitro model wat gebruik was in die studie, gebruik kan
word om die deurlaatbaarheidsvloede van klas 1 en 3 te voorspel. Die resultate van
hierdie studie stem ooreen met ander in vivo studies.
Dit wil voorkom asof die in vitro deurlopende-vloei perfusie apparaat die potensiaal
het om sommige van die probleme en tekortkominge van ander in vitro modelle te
oorkom en dat dit moontlik die potensiaal het om die diffusie-eienskappe van nuwe
geneesmiddels en verskillende formulasies oor verskillende biologiese membrane te
voorspel. Die model kan verder moontlik dien as ‘n potensiële toestel om
biogelykbaarheid van alternatiewe (generiese) formulasies, wat dieselfde
geneesmiddel/chemiese stof bevat, te bepaal.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1289 |
| Date | 12 1900 |
| Creators | Pretorius, Erina |
| Contributors | Bouic, P.J.D., University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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