polymorfi - fysikaalinenfarmasia.fi · thermal stability of lysozyme and lactase formulations ......
TRANSCRIPT
POLYMORFI 2008
ISSN: 1236-4002
1458-5820 (pdf)
2
XIX SYMPOSIUMIN OHJELMA
9.00 Ilmoittautuminen ja aamukahvi
9.30 Niina Kivikero
(FFY, Helsingin yliopisto)
Symposiumin avaus
9.45 Pekka Teppola
(VTT)
Lähi-infrapunaspektroskopian (NIRS) ja kemometrian
sovellukset lääketeollisuudessa
10.30 Pekka Keski-Rahkonen
(Kuopion yliopisto)
HPLC – principles of operation and use in pharmaceutical
analysis
11.15 Posteriesittelyt
11.45 Lounas ja posterinäyttely
13.15 Kirsi Jouppila
(Helsingin yliopisto)
Termoanalytiikka tuotekehityksen työkaluna
14.00 Kahvi
14.30 Niklas Sandler
(AstraZeneca)
In-line and off-line particle size measurement in the
pharmaceutical industry
15.15 Timo Tuomi
(Bruker)
Analysis of pharmaceutical samples with the help of FT-
IR microscopy, FT-IR imaging and micro-Raman
15.35 Mikko Tenho
(Turun yliopisto)
Novel pharmaceutical x-ray diffraction methods
15.55 Jari Leskinen
(Kuopion yliopisto)
Moisture detection of granulation processes by
electrical impedance spectroscopy (EIS)
16.15 Symposiumin päätös
16.30 Fysikaalisen farmasian yhdistyksen vuosikokous
Vaihtoehtoisena ohjelmana vapaa pääsy Heurekan
tiedenäyttelyyn (koko päivän ajan)
18.00 Illallisbuffet (Sokos Hotel Vantaa) ja parhaan posterin
palkitseminen
Fysikaalisen farmasian XIX vuosittainen symposium:
Analytical Methods in Pharmacy -
Industrial and Novel Applications
VANTAA, TIEDEKESKUS HEUREKA, 24.1.2008
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PÄÄTOIMITTAJAN PALSTA ........................................................................................................ 7
ESITYSABSTRAKTIT ..................................................................................................................... 9
HPLC – Principles of operation and use in pharmaceutical analysis ................................................ 10
Termoanalytiikka tuotekehityksen työkaluna .................................................................................... 12
In-line and off-line particle size measurement in the pharmaceutical industry ................................. 13
Analysis of pharmaceutical samples with the help of FT-IR microscopy, FT-IR imaging and micro-
Raman ................................................................................................................................................ 14
Novel pharmaceutical X-ray diffraction methods ............................................................................. 15
Moisture detection of granulation processes by electrical impedance spectroscopy (EIS) ............... 17
POSTERIABSTRAKTIT................................................................................................................ 21
New method to calculate true density and Heckel equation for pharmaceutical powders ................ 22
Controlled stabilization of traceable sol-gel silica (nano)particles via surface engineering ............. 23
Processing of powder particles by using a pulsatile application of pressurized hot steam ............... 24
Development of chitosan microparticles for mucosal vaccine delivery ............................................ 25
Compatibility screening of mesoporous silicon and silica based drug carriers with poorly soluble
drugs .................................................................................................................................................. 26
Solubility determinations of four NSAIDs - Effect of pH on total dissolved amount and dissolution
rate ..................................................................................................................................................... 27
Surface pressure measurements in interaction studies of pharmaceutical nanoparticles .................. 28
Viscosity and physical stability of amorphous citric acid and paracetamol blends upon consecutive
shearing .............................................................................................................................................. 29
Teaching of physical pharmacy in Department of Pharmaceutics at the University of Kuopio:
Cyclodextrin complexation as an example ........................................................................................ 30
Fluid bed granulations in Multipart Microscale Fluid bed powder Processor (MMFP) ................... 31
Monitoring solid state changes of theophylline-excipient binary formulations during drying ......... 32
Quantitative analysis of solid-state changes using vibrational spectroscopy and partial least squares
regression ........................................................................................................................................... 33
Failure of MTT as a toxicity testing agent for Si particles ................................................................ 34
An optical in situ dissolution rate determination method for fast dissolving drugs .......................... 35
A desiccant system to control the internal humidity of drug containers ........................................... 36
Thermal stability of lysozyme and lactase formulations ................................................................... 37
Granule water content prediction during fluidized bed granulation .................................................. 38
Raman spectroscopy as a tool for detecting isomorphic dehydration transitions .............................. 39
In situ monitoring of processing-induced phase transformations using optical microscopy ............. 40
POLYMORFI 2008
FYSIKAALISEN FARMASIAN YHDISTYKSEN JÄSENLEHTI
SISÄLLYS
5
Adsorption of two model drugs into mesoporous silicon microparticles ........................................... 41
Effect of in vitro release method on drug release from mesoporous silicon particles ....................... 42
Effect of formulation parameters and drug - polymer interactions on drug release from starch
acetate matrix tablets .......................................................................................................................... 43
Surface tension measurements as a promising method for fast solubility determinations in a 96-well
plate .................................................................................................................................................... 44
Granule size determination during fluidization in a fluidized bed granulator ................................... 45
Characterization of enzymatic degradation of oxazoline modified poly-ε-caprolactone (PCL-O) by
enzyme inhibitors and mass spectrometry ......................................................................................... 46
Tomography imaging of dissolution test ............................................................................................ 47
Preheating of granulator improved fluid bed granulation .................................................................. 48
Solid state transformation of erythromycin A dihydrate during drying monitored by near infrared
spectroscopy ....................................................................................................................................... 49
In vitro studies of mesoporous silica/silicon microparticles: Toxicity and dissolution ..................... 50
Better understanding of dissolution behavior of amorphous drugs .................................................... 51
Fundamental understanding through simulations? ............................................................................. 52
Investigations on physicochemical properties of glycerides and glyceride-based extrudates ........... 53
Preferred orientation affects the intrinsic dissolution of tolbutamide and acetylsalicylic acid
compacts ............................................................................................................................................. 54
Detection of defensins from biological matrices with HPLC-ESI-MS .............................................. 55
Quantitative analysis of natural cyclodextrins using HPLC and pulsed amperometric detection
method ................................................................................................................................................ 56
Sublingual administration of fast-dissolving perphenazine formulations .......................................... 57
Teaching of physical pharmacy in Department of Pharmaceutics at the University of Kuopio:
Emulsions as an example ................................................................................................................... 58
Crushing strength of tablets measured by FT-near infrared spectroscopy ......................................... 59
VÄITÖSKIRJOJEN TIIVISTELMÄT.......................................................................................... 61
From polymorph screening to dissolution testing – Solid phase analysis during pharmaceutical
development and manufacturing. ....................................................................................................... 62
Cyclodextrins in intraoral delivery of delta-9-tetrahydrocannabinol and cannabidiol ...................... 63
Development of LC-MS methods for quantitative and qualitative analyses of endogenous
compounds, drugs, and their metabolites to support drug discovery programs ................................. 65
Polymeric carriers in non-viral gene delivery: a study of physicochemical properties and biological
activity in human RPE cell line .......................................................................................................... 67
Significance of crystallographic properties in pharmaceutical compacts .......................................... 69
Corneal epithelial cell culture model for pharmaceutical studies ...................................................... 70
Studies on thermosensitive poly(N-vinylcaprolactam) based polymers for pharmaceutical
applications ......................................................................................................................................... 71
GRADUJEN TIIVISTELMÄT ....................................................................................................... 73
Syklodekstriinien vuorovaikutukset biologisten membraanien kanssa .............................................. 74
Jauheiden sekoittuminen, erottuminen ja jauheseosten homogeenisuuden mittaaminen .................. 75
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Development of cell-based (Caco-2) screening method for substrates of MRP2 efflux proteins ..... 76
Lääkeaineiden adsorptio muovisiin säilytysastiamateriaaleihin ja käsiteltyyn polystyreeniin ......... 77
Hydrofiiliset polymeerimatriisit / nopeasti liukenevien perfenatsiinipartikkelien stabiilisuus ja
formulointi ......................................................................................................................................... 78
Development of preformulation method for drug-containing siloxane matrices .............................. 79
Biorelevantit dissoluutiomenetelmät ................................................................................................. 80
Studies on wet granulation and roller compaction of inulin .............................................................. 81
Solid lipid dispersions as carriers for poorly water-soluble compounds ........................................... 82
II vaiheen metabolia Caco-2-soluissa ................................................................................................ 83
Farmaseuttisten binaariseosten kvantitatiivinen faasianalyysi röntgendiffraktiomenetelmin ........... 84
Kvantitatiivinen analyysi ja kidekoon määritys jauheröntgendiffraktiolla ....................................... 85
Viruksettomien geeninsiirtokompleksien biofysikaalinen karakterisointi ........................................ 86
Change of physicochemical characteristics of trimethoprim and sulfamethoxazole in a suspension
product ............................................................................................................................................... 87
Lääkemolekyylien adsorptiosta mesohuokoiseen piihin ................................................................... 88
Tabletoitavien jauheiden prosessikäyttäytyminen ja tablettirakenne ................................................ 89
Prosessiparametrien vaikutukset leijupetirakeistuksessa ................................................................... 90
Mesohuokoisten materiaalien ja lääkeainemolekyylien välisten vuorovaikutusten spesifiointi
spektroskopisin ja kalorimetrisin menetelmin ................................................................................... 91
Kitosaani ja kvatemääriset kitosaanijohdokset peptidien ja proteiinien imeytymisen edisteinä ....... 92
Askorbiinihappo-isomeerien faasidiagrammin sekä kuivattujen marjamehujen C-
vitamiinipitoisuuden määritys DSC-menetelmällä ............................................................................ 93
Screening anticancer activity: Apoptosis based methods and validation of automated SRB assay .. 94
Huokoisen piin liukeneminen simuloituihin kehonesteisiin .............................................................. 95
Pehmitevalinnan ja proteiinin kuumennuksen vaikutukset heraproteiinikalvojen mekaanisiin
ominaisuuksiin ................................................................................................................................... 96
Pintajännitykseen perustuva liukoisuuden määritys: mikrotensiometri- ja ravistelumenetelmien
tulosten vertailu ................................................................................................................................. 97
Kiinteät dispersiot veteen niukkaliukoisten lääkeaineiden formuloinnissa ....................................... 98
FYSIKAALISEN FARMASIAN AMMATTILAISET MAAILMALLA .................................. 99
SANALAATIKKO ........................................................................................................................ 105
OSALLISTUJAT ........................................................................................................................... 106
Päätoimittaja: Elina Turunen, Kuopion yliopisto
Julkaisija: Fysikaalisen farmasian yhdistys ry
www.fysikaalinenfarmasia.fi
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Tervehdys yhdistyksen jäsenet ja Fysikaalisen farmasian XIX symposiumin osallistujat!
Tämänvuotinen Fysikaalisen farmasian symposium on kerännyt ennätysmäärän ilmoittautuneita.
Symposiumin aiheena olevat analyyttiset menetelmät ovat herättäneet mielenkiintoa useilla
farmasian aloilla niin yliopistoissa, lääketeollisuudessa kuin viranomaissektorillakin. Tällä kertaa
symposiumissa esitellään sekä tuttujen analyyttisten menetelmien teollisia sovelluksia että
uudempia farmaseuttisessa tutkimuksessa käytettäviä analyysimenetelmiä.
Ensi vuonna Fysikaalisen farmasian yhdistys juhlii 20-vuotista taivaltaan juhlasymposiumin
merkeissä. Juhlavuoden symposiumin tieteellinen tarjonta liikkuu myös analyysimenetelmien
parissa: aiheena ovat lääketeollisuudessa ja -tutkimuksessa tällä hetkellä erittäin ajankohtaiset
prosessianalyyttiset tekniikat. Kuluneen toimikauden aikana Fysikaalisen farmasian yhdistys alkoi
jo valmistautua tulevaan juhlavuoteen erilaisin uudistuksin: yhdistyksen sääntöjen lisäksi
päivitettiin yhdistyksen internet-sivut. Käy tutustumassa Fysikaalisen farmasian yhdistykseen ja
lukemassa viime vuosien Polymorfi-lehtiä osoitteessa www.fysikaalinenfarmasia.fi!
Tämänvuotinen Polymorfi tarjoaa tuttuun tapaan mahdollisuuden palata myöhemminkin
symposiumissa käsiteltyihin aiheisiin luento- ja posteriabstrakteja lukemalla. Vuoden aikana
valmistuneet, fysikaalista farmasiaa sivuavat väitöskirja- ja gradutiivistelmät Helsingin, Turun ja
Kuopion yliopistoista tarjoavat katsauksen alan tämänhetkiseen tutkimukseen Suomessa. Lehden
loppuosasta löytyy myös jo lähes perinteeksi muodostunut, yleisön hartaasti odottama aivopähkinä.
Toivotamme kaikille symposiumin osallistujille mukavia ja opettavaisia hetkiä symposiumissa ja
Polymorfin parissa!
Kuopiossa 20.1.2008
Elina Turunen
PÄÄTOIMITTAJAN PALSTA
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ESITYSABSTRAKTIT
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The advent of column chromatography in the beginning of the twentieth century, followed by its
refinement into a technique know today as high performance liquid chromatography, HPLC,
proved to be one of the most significant methodological achievements in the field of
pharmaceutical analysis. The theory and foundations of the HPLC was laid in the 1960s and 1970s
and rapid spread of the technique was seen beginning in the latter decade [1]. Today it is
undoubtedly the most used method in the field of pharmaceutical and biomedical analysis, which
can be seen in the number of published methods in journals as well as in the contribution in the
compendial analysis such as in the monographs of the United States Pharmacopoeia [2].
The operating principle of a HPLC instrument is simple. It is based on the original idea of column
chromatography, where sample constituents are being separated in a particle-filled tube or column
by flushing it with suitable solvent. As the solvent flushes the column, it elutes the sample
compounds depending on their physicochemical properties. The resulting separation results from
the different affinities of the compounds to the column packing; some compounds will stay in the
column longer than the others. The elution of the compounds is controlled mostly by the properties
of the eluting solvent. There are different modes of liquid column chromatography used in HPLC,
depending on the packing material inside the column. With these modes, the compound retention
can be based on either hydrophobicity, size, ionic properties, affinity or some other phenomenon.
The most used mode is reversed phase chromatography, in which the column is filled with non-
polar species and retention is affected mostly by the Van der Waals interactions between the
analytes and column packing.
Modern HPLC instrument (Figure 1) is build around the column, comprising pump for solvent
delivery, automated injector for sample introduction and detector for monitoring the elution of
analytes from the column. The pump is capable of maintaining adjustable and stable solvent flow
rate and the injector can be programmed to perform precise and accurate sample injections
according to a pre-defined timetable. Column temperature, which plays a significant role in the
separation, is usually controllable and detection of the eluting compounds can be performed with
different types of detectors, depending on the requirements of the analysis or the physicochemical
properties of the compounds. The most used detector is based on UV absorption of the analytes as
they pass an in-line flow cell. Other types of detectors can be based on fluorescence or light
scattering as well as refractive or electrochemical properties of the chemicals. Recently, the
coupling of mass spectrometer to the HPLC instrument, a technique known as HPLC-MS, is
gained popularity as it enables highly sensitive, mass-specific detection of compounds, even from
very complex matrices, such as body fluids and tissues.
HPLC – PRINCIPLES OF OPERATION AND USE IN
PHARMACEUTICAL ANALYSIS
Pekka Keski-Rahkonen
Department of Pharmaceutical Chemistry, University of Kuopio, Kuopio, Finland
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Column Pump
Solvent
reservoir
Detector
Injector
Direction of solvent flow
Figure 1. Schematic drawing of HPLC instrument
The HPLC based methods used in the pharmaceutical research are usually divided in two groups –
pharmaceutical and biomedical analysis. The former covers the applications to the analysis of
pharmaceuticals in raw material and drug product levels, such as drug purity and stability studies
and quality assurance in the pharmaceutical industry, where the HPLC has much replaced the
previously used but poorly selective UV-VIS methods. The latter of the two groups comprises of
methods aimed at the analysis of compounds from biological matrices like in the studies on
pharmacokinetics, therapeutic monitoring and metabolic profiling. Both types of methods are based
on the same basic principles of HPLC separation, mainly the amount of sample preparation and the
effort required to produce an adequately performing method is different. Also for the method
validation, different directions and requirements are set by authorities.
References
[1] Sándor Görög, Trends in Analytical Chemistry. 26 (2007) 12.
[2] USP, United States Pharmacopoeia XXIX. USP Convention Inc., Rockville, Maryland, USA, 2006.
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Termoanalytiikkaa käytetään elintarvikkeiden ja niiden ainesosien ominaisuuksien ja fysikaalisen
tilan sekä niissä tapahtuvien muutosten tutkimukseen. Tuloksia voidaan hyödyntää esimerkiksi
prosessointi- ja säilytysolosuhteita valittaessa ja optimoitaessa sekä tuotekehityksen tukena.
Yleisimmin käytettyjä termoanalyyttisiä laitteita ovat differentiaalinen scanning-kalorimetri (DSC),
dielektrinen analysaattori (DEA), dynaamis-mekaaninen analysaattori (DMA) ja
termogravimetrinen analysaattori eli termovaaka (TGA).
Termoanalyyttisiä menetelmiä on perinteisesti käytetty rasvojen, proteiinien ja tärkkelysten
faasimuutosten ja ominaislämmön tutkimiseen. DSC:lla on tutkittu mm. rasvojen sulamis- ja
kiteytymisominaisuuksia, proteiinien denaturoitumista ja tärkkelyksen liisteröitymistä. Uudempi
termoanalyyttisten menetelmien sovellus on amorfisten materiaalien tutkimus, joka käynnistyi
1980-luvulla elintarviketutkimuksessa ja -tuotekehityksessä.
Amorfisten materiaalien karakterisoinnissa määritetään materiaalin lasisiirtymälämpötila eri
vesipitoisuuksilla, jotta saadaan selville, mikä on materiaalin fysikaalinen tila erilaisissa
säilytysolosuhteissa. Kun tunnetaan materiaalien fysikaalinen tila, materiaalille voidaan määrittää
kriittinen vesipitoisuus, joka on se vesipitoisuus, joka laskee amorfisen materiaalin
lasisiirtymälämpötilan siihen lämpötilaan, josta kulloinkin ollaan kiinnostuneita (Roos, 1993).
Säilyvyyttä tutkittaessa tämä lämpötila on useimmiten huoneen lämpötila. Useille amorfisille
hiilihydraateille on määritetty kriittinen vesipitoisuus huoneenlämmössä (Jouppila ja Roos, 2008).
Kun materiaalin vesipitoisuus on suurempi kuin kriittinen vesipitoisuus, materiaalin
lasisiirtymälämpötila on säilytyslämpötilaa matalampi, jolloin molekyylien liikkuvuus
materiaalissa lisääntyy ja materiaalissa voi tapahtua erilaisia muutoksia. Tällaisia muutoksia ovat
jauheiden tahmeutuminen ja paakkuuntuminen, amorfisten sokereiden kiteytyminen ja ei-
entsymaattinen ruskistuminen. Näistä muutoksista esimerkiksi amorfisten sokereiden kiteytymisen
ja ei-entsymaattisen ruskistumisen kinetiikkaa on tutkittu DSC:lla eri lämpötiloissa. Ei-
entsymaattista ruskistumista voidaan tutkia myös termogravimetrisesti, jolloin seurataan
reaktiotuotteena syntyvän veden sekä muiden haihtuvien komponenttien poistumista näytteestä
vakiolämpötilassa.
Termoanalyyttisen elintarviketutkimuksen haasteena on löytää uusia sovelluksia perinteisten
laitteiden sekä uusien menetelmien, kuten TOPEM-DSC:n, hyödyntämiseen.
Lähdeluettelo
Jouppila, K. & Roos, Y.H. 2008. Crystallization: measurements, data and prediction. Teoksessa: Food Properties
Handbook, 2nd
Ed. Rahman, M.S. (Ed.). In press.
Roos, Y.H. 1993. Water activity and physical state effects on amorphous food stability, J. Food Process. Preserv.
16:433–447.
TERMOANALYTIIKKA TUOTEKEHITYKSEN TYÖKALUNA
Kirsi Jouppila
Helsingin yliopisto, elintarviketeknologian laitos
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Particle size measurement of raw materials (API and excipients) and intermediate products
(granules) plays a key role in pharmaceutical development. Particle size information is essential for
decision-making in process-optimisation trials and often defines important quality attributes of raw
materials and intermediates. Particle size data often also provide indispensable information for
predictions of material behaviour in a unit operation.
For the drug substance the ICH Q6A guideline sets acceptance criteria for the particle size
distribution in terms of dissolution, solubility, bioavailability, processability, stability, appearance
and content uniformity. With regard to excipients one has to be able to relate their particle size
distributions to any functionality related characteristics. In addition to raw material sizing
intermediate particle size measurement during processing can often be advantageous enabling in-
line granule growth monitoring e.g. of wet granulation processes.
Various techniques for measuring the particle size distribution of powders exist and the choice of
method has to be made carefully based on material characteristics and type information required.
Kelly and Lerke have recently systematically covered the selection criteria of off-line particle size
analysis methods in the pharmaceutical industry and listed strengths and limitations of different
techniques [1].
This presentation will discuss the importance of particle size in pharmaceutical solid dosage form
development and will also demonstrate examples of at-line/on-line measurement e.g. in milling of
API with laser diffraction. The significant advances in imaging technologies and computing power
have had an impact on recent developments of new image analysis applications including devices
with automated sample preparation and real time process analysis systems for particle size
measurement. An image based system (QicPic, Sympatec, Germany) in granule size and shape
determination is shown and how it can support pharmaceutical development with key data. Other
techniques are also briefly covered including chord length distribution measurements. Emphasis in
the presentation will be put on the data analysis of particle size information. Multivariate
techniques provide powerful possibilities to extract relevant information from particle sizing data
measured with any given technique.
References
[1] Kelly R.N. and Lerke S.A.. Particle Size Measurement Technique Selection Within Method Development in the
Pharmaceutical Industry. American Pharmaceutical Review. Nov/Dec 2005.
IN-LINE AND OFF-LINE PARTICLE SIZE MEASUREMENT IN
THE PHARMACEUTICAL INDUSTRY
Niklas Sandler
Process Engineering & Material Science, Product Development, AstraZeneca R&D, UK
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In analytical chemistry, there is a demand to get information from smaller and smaller objects and
preferably without any, or as little as possible, sample preparation. The progress in modern FT-IR-
and Raman-technoly gives many possibilities to fulfil the demand.
In FT-IR microscopy new ATR objectives and imaging detectors give possibility for good lateral
resolution and possibilities to look at specific kinetic reactions. Micro-Raman systems give
possibilities for analysis of small objects with spatial resolution of one micron. Also confocal
analysis and fast polymorph screening is possible with modern dispersive Raman systems . FT-IR
with high throughput reader can effectively be used for fast screening purposes of synthesis
products. The modern systems are also equipped with advanced software, containing powerful
chemometric capabilities. Some Bruker Optics FT-IR systems and practical examples are
described.
ANALYSIS OF PHARMACEUTICAL SAMPLES WITH THE
HELP OF FT-IR MICROSCOPY, FT-IR IMAGING AND MICRO-
RAMAN
Matthias Boese1, Anders Nilsson
2 and Timo Tuomi
2
1 Bruker Optik GmbH, D-76275 Ettlingen, Germany,
2 Bruker Optics Scandinavia AB, SE-187 66 Täby, Sweden
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Introduction
X-ray diffraction is traditional measurement technique in the field of physical pharmacy. Through
the decades, it has been successfully applied for investigating crystal structures, crystallinity,
polymorphism and crystallite properties. Moreover, x-ray diffraction is a powerful method in
qualitative and quantitative analysis. Although the x-ray diffraction is an old technique, it still has a
great potential in the field of pharmaceutical materials research. By utilizing modern
instrumentation and innovative measurement procedures with the ever improving data analysis
methods x-ray diffraction offers a very powerful research tool for pharmaceutical physicists.
Whole Profile Fitting Methods
Various whole profile fitting procedures are increasingly applied when interpreting x-ray powder
diffractograms. These methods are based on the calculation of the ideal diffractogram of the sample
from its known crystal structure. The calculated powder pattern is then convoluted with
instrumental function, and compared with the measured diffractogram. After that, the calculated
pattern is refined to match the measured diffractogram. The refinement procedure can be used to
achieve information about the crystallite size and strain, texture and crystal structure of the sample,
for example. The whole profile fitting techniques are also very efficient in quantitative analysis.
The most widely used whole profile fitting procedure is the Rietveld method [1]. In order to
calculate the reference diffractograms, crystal information can be retrieved from different various
databases. The most used databases in the field of pharmacy are the Powder Diffraction File (PDF)
and the Cambridge Structural Database (CSD). For the crystal structures of inorganic compounds,
the Inorganic Crystal Structure Database (ICSD) is used.
Texture Measurements
The preferred orientation of crystallites, i.e. texture, often disturbs the qualitative and quantitative
determinations performed with x-ray powder diffraction [2]. Texturization may occur when the
crystallites of the powder sample are not truly randomly oriented. In the diffractograms, the effect
of texture is shown by the abnormal intensity of some diffraction maxima. Texture of
pharmaceuticals has been studied only recently, and these studies have been revealed that sample
texture will not only disturb the data analysis but also affect on the properties of pharmaceuticals.
Texturized pharmaceutical compacts have been found to have different mechanical and dissolution
properties [3,4].
Depth Profiling of Crystallographic Properties
By using so called grazing incidence diffraction (GID) geometry, the x-ray diffraction data can be
obtained as a function of depth. With GID basically all crystallographic properties can be depth
profiled. So far, GID has been utilized for studying crystal disorders (amorphicity) and phase
transitions on the surface of tablets [5,6].
NOVEL PHARMACEUTICAL X-RAY DIFFRACTION METHODS
Mikko Tenho
Laboratory of Industrial Physics, University of Turku, FI-20014 Turku, Finland
16
Pair Distribution Function in Crystallography
The traditional crystallographic tools do not work on nanoworld. However, the Pair Distribution
Function (PDF) analysis goes beyond crystallography [7]. The PDF can be obtained from x-ray
diffractogram through a Fourier transform. Once obtained, it can be used to investigate the crystal
structure of the sample. PDF analysis is also applied for determining the size of the nanoparticles.
PDF also tells whether the sample is amorphous or nanocrystalline.
References [1] Rietveld, H.M., A Profile Refinement Method for Nuclear and Magnetic Structures. J. Appl. Cryst. 2, 65-71
(1969).
[2] Stephenson, G.A. et al., Characterization of the solid state: quantitative issues, Adv. Drug Deliv. Rev. 48, 67-
90 (2001).
[3] Koivisto, M. et al., Correlation between texture and tabletting properties of some pharmaceutical tablets, Z.
Kristal. Suppl. 23, 563-568 (2006).
[4] Tenho, M. et al., Effect of texture on the intrinsic dissolution behaviour of acetylsalicylic acid and tolbutamide
compacts, J. Appl. Cryst. 40, 857-864 (2007).
[5] Koivisto, M. et al., Depth profiling of compression-induced disorders and polymorphic transition on tablet
surfaces with grazing incidence X-ray diffraction, Pharm. Res. 23, 813-820 (2006).
[6] Debnath, S. et al., Use of glancing angle x-ray powder diffractometry to depth-profile phase transformations
during dissolution of indomethacin and theophylline tablets, Pharm. Res. 21, 149-159 (2004).
[7] Billinge, S.J.L. et al., Beyond crystallography: the study of disorder, nanocrystallinity and
crystallographically challenged materials with pair distribution functions, Chem. Comm. 7, 749-760 (2004).
17
In 2004 U. S. Food and Drug Administration (FDA) announced Process Analytical Technology
concept (PAT-Framework for innovative pharmaceutical manufacturing and quality assurance)
[1]. It is described "A system for designing, analyzing, and controlling manufacturing through
timely measurements (i.e. during processing) of critical quality and performance attributes of raw
and in-process materials and processes, with the goal of ensuring final product quality." European
Union and national agencies have pronounced their keen on cheaper medicines and better care
response, too. At same time research and development of more accurate pharmaceutical molecules
and medicines is becoming more expensive. The calculated cost of a newly invented
pharmaceutical molecule has tripled. For example, the total expenses of European, American and
Japanese pharmaceutical research has doubled in ten years and in 2004 they were over 50 billion
euros [2]. As guidance for industry, PAT does not restrict the way of understanding but, finally, it
should restrict the process expenses.
Granulation is a widely used part of pharmaceutical manufacturing process. Small sized powder
particles are processed into granules by spraying a binder solution towards the fluidized particles.
In fluidized bed granulators fluidizing motion is controlled by a heated air flow through the powder
bed and is affected by numerous physical parameters. To understand the granulation process, we
should be able to measure these parameters in real-time.
Process parameters may include temperature, relative humidity of air, air flow velocity and air
pressure, but they do not provide direct information of the granule size or moisture content (Fig. 1).
However, granule size distribution can be measured by sieving of separate samples gathered from
granulator and free water content of the sample can be measured by Karl Fischer titration. These
methods do not meet the PAT ideology of "timely measurements". However, real-time moisture
estimation is done everyday in other fields of engineering. Electrical impedance measurements are
used routinely to detect unwanted water content within the structures (walls, buildings etc.).
MOISTURE DETECTION OF GRANULATION PROCESSES BY
ELECTRICAL IMPEDANCE SPECTROSCOPY (EIS)
Jari Leskinena, Mikko Hakulinen
a,b
aDepartment of Physics, P.O.Box 1627, FIN-70211 University of Kuopio
bDepartment of Pharmaceutics, University of Kuopio
18
Figure 1: Two process parameters used for monitoring of granulation process. Relative humidity
of the outlet air, RHout, can explain up to 54 % and granule bed temperature, T(mass), up to 62 %
of the variation of the moisture measurement.
We have used electrical impedance spectroscopy (EIS) to measure non-invasively water content of
fluidized bed granulation process in-line (Fig. 2). Electric impedance can be measured by from the
dielectric response of the material during small alternating current input. By measuring the
response of different frequencies of the input, spectrum can be collected. This spectral data can be
used to estimate material's moisture content.
Figure 2: Typical granulation process monitored by EIS. Moisture content measured in-line and
compared to reference method. Dotted line stands for relative granule size. Starting point of the
drying stage was in 22 minutes and optimal end point at 35 minutes. [3]
0
2
4
6
8
10
12
0 10 20 30 40
Time (min)
Mo
istu
re c
on
ten
t (%
) In-Linemeasurement
KF-titratedreference
10
20
30
40
50
60
70
80
90
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Moisture [w/w%]
T(mass) [C]
RH out [%]
19
In first experimental tests we found a good correlation (r = 0.7) between water content and EIS
method. By using density compensated algorithm during fluidization, the correlation was increased
up to r = 0.9. In industrial applications fluidization is used periodically during the granulation
because the filters in the chamber tend to clog up by fluidizing powder. By using EIS between
fluidization periods, the correlation was increased up to r = 0.98 (Fig. 3).
Figure 3: Water content of granule bed as a function of parallel capacitance. Data for parallel
capacitance was collected during granulation.
Before the whole granulation process, including wet agglomeration, granule drying and optimal
end point, can be controlled, the part processes should be understood. In our approach, we have
developed non-invasive methods to monitor granulation parameters in-line. It is shown that the
water content of the processed fluidizing bed could be determined by EIS. EIS technique has been
successfully applied to other fields of industry (e.g. wood industry [4]) and our preliminary results
shows that EIS can also offer valuable information on granulation process. The EIS technique may
not replace the traditional direct measurements in pharmaceutical manufacturing process but it
provides a good tool towards complete process understanding.
References
[1] http://www.fda.gov/cder/guidance/6419fnl.htm
[2] http://www.pif.fi/tiedostot/LTK_LTkalvot2006_suomi.pdf
[3] Leskinen et al: In-Line Process Monitoring with Electrical Impedance Measurement: Preliminary Fluid Bed
Granulator Studies, 3rd International Granulation Workshop, Sheffield, UK 2007.
[4] Markku Tiitta: Non-destructive Methods for Characterization of Wood Material, PhD thesis, University of Kuopio,
Finland 2007.
EIS moisture estimation
N = 11
r = 0.9864
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
1.00E+00 1.20E+00 1.40E+00 1.60E+00 1.80E+00
Cp Ratio
Mo
istu
re w
/w%
20
21
POSTERIABSTRAKTIT
22
In this study is introduced a new mathematical formula, which can represent very accurately
densification of powder bed as a function of compression pressure. By means of the parameters of
the new formula it is possible to calculate the limit value (true density) toward which the powder
will densify as the compression pressure increases. Two medicaments: acetylsalicylic acid and
ibuprofen were direct compressed in an eccentric tablet machine. Both substances densified under
high compression pressure clearly over the true density, measured by helium densitometer. The
porosity of the powders became thus apparently negative at high pressures. Explanation to this
phenomenon is that the true density is not constant, but depends from compression pressure. In this
study we represent a method to calculate the true density as a function of compression pressure. It
is not possible to draw Heckel plot for acetylsalicylic acid and ibuprofen for a wide pressure range
if we assume a constant true density values for those materials. We represent a modified Heckel
equation were we assume that the true density is a function of compression pressure. The new
method give clearly different results for yield values to the tested materials compared to the
traditional method. This is because the shape of the Heckel plot is very sensitive to the right value
of the true density.
NEW METHOD TO CALCULATE TRUE DENSITY AND
HECKEL EQUATION FOR PHARMACEUTICAL POWDERS
Antikainen O., Pietarinen T., Yliruusi J.
University of Helsinki, Pharmaceutical Technology Division, Helsinki, Finland
23
Nanoparticles can be used in diverse biological applications, for example as fluorescent markers in
vitro and in vivo, for clinical diagnosis and drug delivery1. Silica nanoparticles are emerging as a
promising and versatile alternative to polymer-based counterparts, as they can be produced with a
tunable particle size and pore structure. Silica particles are biocompatible and chemically inert in a
wide range of conditions2. Furthermore, functionalization of the silica surface is relatively
straightforward using well-documented methods. This is an essential part of any state-of-the-art
formulation, as the surface functions serve as anchoring points for further functionalization, and
also control the extent of particle aggregation under given conditions. This is especially important
under biological conditions where the salt concentration is normally high, which puts a limit to the
possibility of utilizing electrostatic forces as the basis for dispersion stability. Here, we demonstrate
the successful use of surface modification of nonporous sol-gel derived silica particles based on
hyperbranching ―dendritic‖ surface polymerization using aziridine as the poly(ethylene imine)
precursor. The surface concentration of functional amino groups achievable is much higher than
normally obtained by surface functionalization by silanization. This leads to a marked increase in
the nanoparticles dispersion stability. The quantity, and thus also the thickness, of the PEI layer,
can be controlled simply by changing the aziridine/silica ratio in the surface functionalization step.
Furthermore, targeting of specific cells in drug delivery applications requires that additional
functions, such as specific antibodies, are introduced to the particle surface. The linking chemistry
is often relying on amine-carboxylate interactions, and thus the presence of amine or carboxylic
acid functions on the outer particle surface is of special interest. We demonstrate that the outermost
amino groups of the PEI can be ―converted‖ to carboxylic acid functions or used for conjugation
reactions as such, thus allowing full flexibility for further functionalization of the particles by
standard linking chemistry used in biochemistry. The introduced methodology also offers means
for fine-tuning of the effective surface charge of the particles at a given pH, which is one of the key
parameters for dispersion stability. The attachment of labeled fluorescent streptavidin is
demonstrated utilizing this exact linking chemistry, and imaging of the particles is demonstrated by
confocal fluorescent microscopy as well as SEM and TEM. Due to the exceptionally high affinity
of streptavidin to its water-soluble ligand biotin (vitamin H) it is easy to be bound to a wide variety
of biotin-antibody complexes enabling the targeting of nanoparticles to cell receptors of interest.
References 1 Lin, Y-S.; Tsai, C-P.; Huang, H-Y.; Kuo, C-T.; Hung, Y.; Huang, D-M.; Chen, Y-C.; Mou, C-Y. Well-Ordered
Mesoporous Silica Nanoparticles as Cell Markers Chem. Mater. 2005, 17, 4570-4573 2 Schiestel, T.; Brunner, H.; Tovar, G.E.M. Controlled Surface Functionalization of Silica Nanospheres by Covalent
Conjugation Reactions and Preparation of High Density Streptavidin Nanoparticles. J. Nanosci. Nanotech. 2004, 4,
504-511.
CONTROLLED STABILIZATION OF TRACEABLE SOL-GEL
SILICA (NANO)PARTICLES VIA SURFACE ENGINEERING
Lotta Bergmana, Jessica Rosenholm
a, Anna-Brita Öst
b,
Alain Duchanoya, Mika Lindén
a*
aDepartment of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, 20500 Turku, Finland
bDepartment of Biochemistry, Medicity, Tykistökatu 6A, 20520 Turku, Finland
*to whom correspondence should be addressed: [email protected]
24
Objective
The aim of this study was to test a novel hot steam based technique for processing powder
particles. The method is based on a pulsatile application of pressurized hot steam on to the powder
mass.
Method
Lactose monohydrate and glucose anhydrate were used as test materials and caffeine anhydrate as a
model drug in a hot steam processing. The sugars or drug powder were mixed in a fluidized bed
device and exposed to a pulsatile flow of pressurized hot steam sprayed at a constant pressure of 6
bar and at a temperature of 160°C inside the boiler (Figure 1). The batch sizes were 200-500 g and
the processing time was 10-30 min. Special interest was focused on the effect of spraying amount
of hot steam. The hot steam treated and untreated powders were evaluated with respect to yield,
moisture content, flow properties, particle surface morphology (stereomicroscope), as well as
particle size and size distribution (sieve analysis).
Results
It was observed that hot steam exposure flattened the surface of lactose and caffeine powder.
Treated glucose particles appeared to be slightly larger, but more equidimensional in size. The
disappearance of fine powder was observed in both cases. In comparison with original sugars, an
improvement of powder flow properties was evidenced. The instant changes in particle and powder
properties of drugs were obviously due to a superficial dissolution of fine species, which
recrystallized in the fractures of the bigger particles during the period between hot steam injection.
Conclusion
In conclusion, the present hot steam technique is capable of rapid influence on particle surface and
size characteristics of pharmaceutical sugars.
Figure 1. A laboratory-scale fluidized bed device equipped with a hot-steam generator: (1) Heater,
(2) Reactor chamber, (3) Temperature display, (4) Pipe for outlet air, (5) Channel blower, (6)
Frequency inverter and (7) Steam generator.
PROCESSING OF POWDER PARTICLES BY USING A
PULSATILE APPLICATION OF PRESSURIZED HOT STEAM
N. Geninaa;b
, J. Heinämäkia, P. Veski
b, J. Yliruusi
a
aDivision of Pharmaceutical Technology, Faculty of Pharmacy, P.O.Box 56, FI-00014 University of Helsinki, Finland
bDepartment of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
E-mail address: [email protected]
25
In recent decades, mucosal vaccination has received a great deal of attention as alternative for
systemic vaccines. Unlike systemic vaccination, mucosal one is non-invasive and does not require
trained personnel for administration. This would lead to lower cost and better patient compliance.
Chitosan (polysaccharide obtained by deacetylation of chitin) has been widely used as antigens
delivery systems because of its biocompatibility, biodegradability, mucoadhesiveness, low cost,
and its ability to open intercellular tight junctions. Chitosan can also be used to prepare
microparticles or nanoparticles. In the present study, biodegradable microparticles formulation for
mucosal antigen delivery was developed and evaluated based on chitosan. The microparticles were
prepared by emulsion (water/oil) and solvent evaporation technique, and Bovine Serum Albumin
(BSA) was used as a model vaccine antigen. A central composite design was used to optimize
microparticles preparation. The process parameters evaluated were stirring rate during emulsion
preparation and amount of surface active agent. Both, unloaded and loaded microspheres were
evaluated respect size, shape, antigen encapsulation efficiency and release properties. When the
amount of emulsifier agent or stirring rate increased, microparticles size was lower, being between
5 and 15 µm. Inclusion of BSA in the formulation also resulted in microparticles with uniform size
and shape and low batch-to-batch variation, similar to unload microparticles. The encapsulation
efficiency of BSA was 51.3 % ± 1.8. The protein release from microparticles occured during the
first 20 minutes. In conclusion, microparticles with appropriate characteristics were obtained to be
used as mucosal vaccine.
DEVELOPMENT OF CHITOSAN MICROPARTICLES FOR
MUCOSAL VACCINE DELIVERY
L. González1, J. Heinämäki
2, C. Peniche
3, M.A. Barrios
1, J. Yliruusi
2
1Institute of Pharmacy and Foods, University of Havana, 11400, Havana,Cuba. [email protected]
2Faculty of Pharmacy, Division of Pharmaceutical Technology,
University of Helsinki, P.O. Box 56, (Viikinkaari 5E), 00014, University of Helsinki, Finland. 3Biomaterials Center, University of Havana, 10600, Havana, Cuba.
26
User-friendly administration of poorly water soluble drug compounds over the oral route (as pills
or tablets) is an ongoing, but still largely unmet goal of the pharmaceutical industry. In recent
studies, drug delivery systems based on novel mesoporous silica/silicon materials have shown great
potential for overcoming the problems associated with the oral delivery of drugs. This is due to
their high drug load capacity, rapid drug release property as well as drug solubility and
permeability enhancing properties in the intestinal lumen models [1, 2]. These properties are
realized by housing individual drug molecules in the nanoscale (2-50 nm) pores of the solid carrier,
thereby enabling the dispersion of drug in its highly active amorphous (but still stable) form after
ingestion. The absolute in vivo safety and biocompatibility of any pharmaceutical product is of
outmost importance. Therefore, when designing drug formulations based on mesoporous materials,
the compatibility between the mesoporous solid and the applied drug has to be considered, also
taking into account the long-term stability of the product. Degradation of the loaded drug
compound can be induced by chemical reactions between the mesoporous solid, the drug and/or the
solvent used in the loading process, as well as by unfavorable treatment conditions (high
temperature etc.) of the formulation during production or storage.
In the present study, ATR-FTIR (Fig. 1), HPLC and IMC methods were used for preliminary
mapping of the reactions between several combinations of mesoporous material, solvent and poorly
water soluble model drug compounds, in order to improve the understanding of the factors
affecting drug stability and compatibility. The mesoporous materials studied were based on silicon
(AAPSi, TOPSi and TCPSi) and silica (MCM-41 and SBA-15). The compatibility of several
solvents (e.g. chloroform, acetone, acetonitrile, methanol and ethanol) was tested in combination
with various poorly water soluble model drugs (e.g. indomethacin, furosemide, griseofulvin,
beclomethasone dipropionate). The results indicate that many combinations of drug and solvent
exist that are likely to produce degraded products when applied with a mesoporous solid. Further
development of pure formulations based on mesoporous materials can be achieved by avoiding
these combinations in the processes.
1500 1400 1300 1200 1100 1000
7
6
5
4
3
2
1
1240 cm-1
Tra
nsm
itta
nce (
%)
Wavenumber (cm-1)
1. EtOH
2. EtOH/BDP
3. EtOH/BDP/AAPSi
4. EtOH/BDP/TOPSi
5. EtOH/BDP/TCPSi
6. EtOH/BDP/SBA-15
7. EtOH/BDP/MCM-41
Fig. 1. ATR-FTIR liquid phase spectras of pure
ethanol (EtOH), beclomethasone dipropionate (BDP)
dissolved in EtOH, and with the mesoporous materials
(AAPSi, TOPSi, TCPSi, SBA-15 and MCM-41).
Features indicating incompatibility were detected in
the BDP/silica spectras at 1240 cm-1
.
References
[1] Heikkilä et al., Evaluation of Mesoporous TCPSi, MCM-41, SBA-15 and TUD-1 materials as API carriers for oral
drug delivery, Drug Delivery 14 (2007), 337-347.
[2] Kaukonen et al., Enhanced in vitro permeation of furosemide loaded into thermally carbonized mesoporous silicon
(TCPSi) microparticles, Eur. J. Pharm. Biopharm. 66 (2007), 348-356.
COMPATIBILITY SCREENING OF MESOPOROUS SILICON
AND SILICA BASED DRUG CARRIERS WITH POORLY
SOLUBLE DRUGS
T. Heikkiläa, d
, J. Riikonena, E. Mäkilä
a, J. Salonen
a, J. Saarela
a, N. Kumar
b, D.Yu.
Murzinb, T. Salmi
b, H. Santos
c, L. Peltonen
c, J. Hirvonen
c, V-P. Lehto
a
a Laboratory of Industrial Physics, Department of Physics, University of Turku, FI-20014 Turku, Finland
b Laboratory of Industrial Chemistry, PCC, Åbo Akademi University, FI-20500 Turku, Finland
c Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, Finland
d Graduate School of Materials Research, Turku, Finland
27
Poor solubility is one of the most common and frustrating problems in the drug development.
Ibuprofen, ketoprofen, indomethacin and piroxicam are all classified as BCS (Biopharmaceutics
classification system) II drugs meaning that they have poor solubility. There are several compound
properties which affect the solubility. It is known for example that salts, solvates and polymorphs
of the same drug have different solubilities. It is well documented in the pharmaceutical literature
that all the model compounds in this study do exist at least two different polymorphic forms.
Changes from one polymorph to another during solubility determinations is a problematic
phenomena which is highly compound dependent. The aim of this study was to analyse the
solubility behavior of the four model compounds in different media and detect the changes on the
dissolved amount.
The solubility tests were made by the shake flask method at room temperature. As the media was
used water and aqueous buffer solutions at pH 1,2 and 6,8 (1). The assays were performed after 1h,
2h, 3h, 4h, 5h, 24h and 30h. The assays were analyzed by the UV-spectrophotometry (LKB
Biochrom, Ultrospec II).
The results showed that all the model compounds had the highest total solubility at pH 6.8. At pH
1.2 the solubility was the lowest, except for piroxicam it was the lowest in water. The highest
solubility was reached within the first 4 hours, except for piroxicam and indomethacin at pH 1.2
and 6.8 the peak value was achieved at later time-points. The colour of piroxicam was changed
during the test indicating polymorphia.
Both the circumstances of the environment (e.g. the pH of the GI tract) and the physicochemical
properties of the drug(s) affect the solubility and cause variety in the solubility results. For reliable
solubility testing it is of outmost important to perform exact physicochemical characterization of
the drug material in question.
Reference
(1) The United States Pharmacopeia and national formulary, 26th
edition (2003)
SOLUBILITY DETERMINATIONS OF FOUR NSAIDS - EFFECT
OF PH ON TOTAL DISSOLVED AMOUNT AND DISSOLUTION
RATE
Tiina Heikkiläa, Marjo Yli-Perttula
b, Arto Urtti
c, Krista Ojala
d,
Jouni Hirvonena and Leena Peltonen
a
aDivision of Pharmaceutical Technology, University of Helsinki, Finland
bDivision of Biopharmacy and Pharmacokinetics, University of Helsinki, Finland
cDDTC, Faculty of Pharmacy, University of Helsinki
, Finland
dOrion Pharma Oyj, Espoo, Finland
28
The aim of this study was to investigate the applicability of surface pressure measurements,
Langmuir-Schaefer (LS) deposition and scanning electron microscopy (SEM) in stability
evaluation of pharmaceutical nanoparticles in different environments.
Poly(lactic acid) (PLA) nanoparticles were spread on aqueous surfaces of different pH or
electrolyte concentration. The nanoparticles were compressed and the values of surface pressure (π
vs. A isotherms) were recorded and evaluated. The particle layers on aqueous subphases were
visualized by SEM after the LS deposition on silica plates.
At neutral pH, the PLA nanoparticles were electrostatically stabilized by their high surface charge
(δ-potential). When the pH was decreased, the nanoparticles started to aggregate due to the
lowering of the surface charge, which caused steepness (resistance towards compression) in the π
vs. A isotherms [1]. When the nanoparticles were prepared with a surfactant, Poloxamer 188, the
surfactant molecules located on the nanoparticle surface created a steric barrier that resisted the
compression at longer distances compared to the surfactant-free nanoparticles. This could be seen
as an earlier increase in the surface pressure during the compression. The surfactant, i.e. steric
stabilization, protected the nanoparticles against pH-induced aggregation. Increased surface charge
(δ-potential) and, thus, increased repulsion between the surfactant-free nanoparticles, induced by
electrolyte addition, caused a rise in the surface pressure values. The aggregation due to the pH
decrease resulted in percolated particle networks [2], if the surfactant was not present. With
surfactant, or if the surface charge was high enough as a result of electrolyte addition, the
formation of networks was prevented and the aggregates remained as clusters.
Surface pressure determinations together with visual observations provided useful and descriptive
information about the stability and the aggregation mechanisms of the PLA nanoparticles in
different environments.
References
[1] Hirsjärvi S., Peltonen L., Hirvonen J. (2008). Int. J. Pharm. 348, 153-160.
[2] Robinson D.J., Earnshaw J.C. (1992). Phys. Rev. A. 46, 2045-2054.
SURFACE PRESSURE MEASUREMENTS IN INTERACTION
STUDIES OF PHARMACEUTICAL NANOPARTICLES
Samuli Hirsjärvi, Leena Peltonen and Jouni Hirvonen
Division of Pharmaceutical Technology, Faculty of Pharmacy, P.O. Box 56, 00014 University of Helsinki
29
In the present literature there are only a few papers handling about viscosity of amorphous drugs
(1-2). The viscosity is one of the most important physical parameter of an amorphous material. The
viscosity of the glassy material is accounted to be approximately 1012
Pa s, but it may vary between
different systems.
In this study the main aim was to compare the effect of preparation method of amorphous sample
on viscosity and crystallisation tendency upon consecutive shearing. Amorphous samples were
produced by melting or ethanol (EtOH) evaporation method. Materials used were pure citric acid
and paracetamol (PARA) or blends of them. Viscosity measurements were measured by controlled
stress rheometer from the samples. The melt flow activation energy was determined according to
Arrhenius equation. Material parameters were determined using the Vogel-Tamman-Fulcher
equation. X-ray powder diffraction was used to confirm physical stability.
The composition of the samples affects the viscosity, the viscosity increases with increasing PARA
content. Furthermore, it was found that the melt-processed samples show slightly higher values of
moduli and viscosity than the corresponding EtOH samples having the same composition. All the
samples behave as Newtonian liquids. Upon shearing none of the EtOH samples crystallised during
the measurement, although the melt-processed samples with other compositions than 50% (w/w)
tended to crystallise. All samples can be described as "fragile" according to Angell's classification.
The main finding was that composition and processing method has an impact on physical stability
and viscosity of amorphous sample.
References
[1] B. C. Hancock, Y. Dupuis, and R. Thibert (1999). Pharm. Res. 16: 672-675.
[2] V. Andronis, and G. Zografi (1997). Pharm. Res. 14: 410-414.
VISCOSITY AND PHYSICAL STABILITY OF AMORPHOUS
CITRIC ACID AND PARACETAMOL BLENDS UPON
CONSECUTIVE SHEARING
Pekka Hoppu,a Sami Hietala,
b Staffan Schantz,
c Anne Mari Juppo
d
a Division of Pharmaceutical Technology, Faculty of Pharmacy,
Fin-00014, University of Helsinki, Finland b Laboratory of Polymer Chemistry, Department of Chemistry,
Fin-00014, University of Helsinki, Finland c AstraZeneca R&D, S-431 83, Mölndal, Sweden
d Division of Pharmaceutical Technology, Industrial Pharmacy, Faculty of Pharmacy,
Fin-00014, University of Helsinki, Finland
30
Introduction
As an example of physical pharmacy teaching in the Department of Pharmaceutics, the laboratory
work included in Advanced Physical Pharmacy course (4th year) is presented.
Materials and methods
Solubility of ibuprofen with and without 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) was
determined in acetate buffer (pH 4.5) and in phosphate buffer (pH 7.4). Complexation constant of
ibuprofen-2-HP-β-CD complex was determined from the phase-solubility diagram and by
microcalorimetry. Vacuum-dried ibuprofen-2-HP-β-CD complex and physical mixture of ibuprofen
and 2-HP-β-CD were characterized by differential scanning calorimetry (DSC) and Fourier
transform infrared spectroscopy (FT-IR). The influence of 2-HP-β-CD on the in vitro permeability
of ibuprofen was determined in phosphate buffer (pH 7.4) using poly-ε-caprolactone as a
membrane. Sink conditions were maintained and samples were analyzed with reverse phase HPLC
(isocratic conditions, acetonitrile:water 70:30 with 0.1 % TFA; 1.0 ml/min). The effect of 2-HP-β-
CD on surface tension of aqueous sodium dodecyl sulphate (SDS) solution was examined by
tensiometer.
Results and discussion
Ionization of ibuprofen (pKa 5.2) strongly affected complexation: complexation with 2-HP-β-CD
increased solubility of ibuprofen more at pH 4.5 than at pH 7.4. Solubility of ibuprofen (2.5 mM)
was increased linearly at pH 4.5 when complex formation had occurred. At pH 4.5, ibuprofen is
mainly in unionized form, thus complexation with cyclodextrin improves solubility by means of
solubilization [1-3]. Stability constant K1:1 at pH 4.5 was 9124 M-1
. ΔG values obtained from the
microcalorimetry were -20.965 kJ*mol-1
, -19.38 kJ*mol-1
and -15.50 kJ*mol-1
at pH 3.0; 5.2 and
7.4, respectively. Change in Gibbs free energy (ΔG) can be used in estimation of spontaneity of
complexation reaction [1]. Solid-state complexation was confirmed by IR and DSC. The IR spectra
of ibuprofen exhibited a peak at 1722 cm-1
. The corresponding peak was not observed in the IR
spectra of the complex that explains the complex formation. In the DSC thermogram of the
complex the melting peak of ibuprofen was disappeared. Complexation slightly decreased
permeability of ibuprofen at pH 7.4. Surface tension of aqueous SDS solution (6.02 mM) was
increased when cyclodextrin was added into solution because the complexed SDS was not able to
lower the surface tension.
Students´ feedback
According to feedback, the students were pleased with the course. The project work was interesting
and helped to understand the theory of physical pharmacy.
References
[1] Sinko P J: Martin´s Physical Pharmacy and Pharmaceutical Sciences 5th
ed, 2006
[2] Perlovich G L et al: Driving forces and the influence of the buffer composition on the complexation reaction
between ibuprofen and HPCD. Eur J Pharm Sci 20: 197-200, 2003
[3] Higuchi T, Connors K A: Phase-Solubility Techniques. Adv Anal Chem Instr 4: 117-212, 1965
TEACHING OF PHYSICAL PHARMACY IN DEPARTMENT OF
PHARMACEUTICS AT THE UNIVERSITY OF KUOPIO:
CYCLODEXTRIN COMPLEXATION AS AN EXAMPLE
Maiju Järvinen, Marko Kuosmanen, Kristiina Järvinen,
Ossi Korhonen and Tarja Toropainen
Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FI70211 Kuopio
31
Multipart Microscale Fluid bed powder Processor (MMFP) has been developed for fast
characterization of pharmaceutical materials. Miniaturized devices are advantageous especially in
formulation development studies during the expensive drug development process. The purpose of
this study was to clarify the key parameters of fluid bed granulation in microscale using
electrostatic atomization for addition of the granulation liquid.
The experiments were performed in the MMFP with a special nozzle which was connected to a
high voltage supply. In electrostatic atomization an electric field is applied at the granulation liquid
surface and strong coulombic forces disrupt the liquid surface so that droplets form. It is not
possible to use the pressurized air to generate the droplets in this device because the pressurized air
would destroy the fluidization with low air flow rates.
The model substance granulated was lactose monohydrate and granulation liquid consisted of
polyvinylpyrrolidone and water. After the granulation in MMFP, the granules were transferred for
drying to another fluid bed column due to insufficient adjustment speed of the air conditioning unit
used. The variables in this study were the binder quantity, pumping speed and atomization voltage
of the granulation liquid. The relative humidity of the inlet air was also controlled. The particle size
of the granules was determined by sieve analysis.
The preliminary results indicate that pumping speed and binder content of the granulation liquid
had the strongest positive effect on the granule size. The relative humidity of the inlet air had no
effect on granule size and the effect of atomization voltage remained unclear. In this study, only
simple linear relationships could be generated between the variables due to the large variation in
the analytical methods for small samples.
Electrostatic atomization is a good technique for generating droplets in MMFP. The granulation
process in MMFP is a delicate method and vulnerable to disturbances like cleaning of the device.
Despite the challenges, it was shown to be possible to predict the processability in fluid bed
granulation of pharmaceutical material with MMFP.
Figure 1. The nozzle
FLUID BED GRANULATIONS IN MULTIPART MICROSCALE
FLUID BED POWDER PROCESSOR (MMFP)
N. Kivikeroa, B. Ingelbeen
a, M. Murtomaa
b, O. Antikainen
c,
E. Rasanend, J-P. Mannermaa
e
a Industrial Pharmacy, University of Helsinki, Finland,
bLaboratory of Industrial Physics, University of Turku, Finland,
cDivision of Pharmaceuticla Technology, Unversity of Helsinki, Finland,
dSouth Carelian Hospital Pharmacy, Lappeenranta,Finland,
eVerman Oy, Jarvenpaa, Finland
32
Miniaturization has been an ongoing trend in pharmaceutical development for years. One of the
main reasons for miniaturization in addition to reduced cost, is the limited amount of the active
pharmaceutical ingredient in the early stages of formulation development. The purpose of this
study was to compare the Multipart Microscale Fluid bed powder Processor (MMFP) with other
drying methods using theophylline-excipient mixtures as a model material. The effect of excipient
on the solid state change and the appearance of the metastable form were also evaluated.
Theophylline anhydrate was mixed with one excipient at a time in a planetary mixer. The
excipients included the most common excipients in formulations, e.g. lactose monohydrate, starch,
low substituted hydroxypropyl cellulose and mannitol. Water was used as the granulation liquid.
The water amount for the granulations was determined beforehand. After the granulation process,
the granules were dried at fixed temperature just above the onset temperature of the dehydration in
the MMFP, tray drier and x-ray powder diffractometer (XRPD). Before, during and after the drying
procedure, the granules were examined with near infrared (NIR) and Raman spectroscopy and
multivariate analysis. The moisture content of the granules was measured with Karl-Fischer
titrimetry. Because polymorphic changes can be time-dependent, the measurements were repeated
after one hour and 24 hours in triplicate.
NIR and Raman spectroscopy combined with chemometrics were succesfully used to monitor the
solid state transitions in the MMFP. Drying method and excipient selection had an influence on the
conversion of the theophylline structure and the appearance of the metastable form. The water
content during the granulation process also significantly affected the solid state forms.
NIR and Raman spectroscopies combined with multivariate analysis are powerful tools for
monitoring solid state changes on the molecular level. MMFP is a practical tool for understanding
of solid state changes during drying.
Figure 1. The score plot of the principal component analysis of the SNV corrected NIR spectra (A)
and loadings (B) of drying process containing granules made of TP.
MONITORING SOLID STATE CHANGES OF THEOPHYLLINE-
EXCIPIENT BINARY FORMULATIONS DURING DRYING
N. Kivikeroa, C. Strachan
b, J. Aaltonen
c, S. Airaksinen
c, M. Karjalainen
c,
J. Yliruusic, A. Juppo
a
a Industrial Pharmacy, University of Helsinki, Finland,
b Drug Discovery and Development Technology Center, University of Helsinki, Finland,
c Division of Pharmaceutical Technology, University of Helsinki, Finland
33
It has been estimated that every third active pharmaceutical ingredient is capable of forming a
hydrate. Mechanical or thermal stress on a system during pharmaceutical manufacture may induce
the dehydration of hydrates and alter the physicochemical properties of a solid form. Furthermore, if
the hydrate form is present in the final product the quantification becomes an important tool in solid
state investigation.
The aim of the study was to perform quantitative analysis of the solid-state changes during
dehydration using in situ near infrared (NIR) and Raman spectroscopy combined with partial least
squares (PLS) regression modeling.
In situ NIR and Raman spectroscopy were used to monitor isothermal dehydration of piroxicam
(PRX) and carbamazepine (CBZ) hydrates at variable temperatures. Calibration models were
constructed after qualitative partial least squares discriminant analysis, and using all known forms
that occur during dehydration at these conditions. PRX monohydrate transformed directly to
anhydrous PRX form I, whereas the dehydration of CBZ dihydrate (CBZDH) included several
solid-state forms (amorphous, forms III and I). In this study, PLS regression was performed to
quantify these forms. Several pre-treatment and scaling methods were trialed. External test set
validation was utilized to determine the model quality.
The best models were obtained using standard normal variate transformation and mean-centering.
For PRX, the root mean square error of predictions (RMSEP) for the hydrate form were 2.2% and
2.9%, for NIR and Raman spectroscopy, respectively. For CBZ, RMSEP values for the hydrate
form were 3.9% and 4.0%, for NIR and Raman spectroscopy, respectively. It was demonstrated
that both NIR and Raman spectroscopy combined with PLS allowed quantitative analysis of the
dehydration. However, NIR spectroscopy models could quantify the forms in the binary system
(PRX), but were unable to quantify all the forms in the quaternary system (CBZ). Raman
spectroscopy models on the other hand could quantify all four solid-state forms that appeared upon
isothermal heating of CBZDH. This showed that Raman spectroscopy was particularly sensitive to
structural changes, whereas NIR spectroscopy was very sensitive to water of crystallization but less
sensitive to alterations in the structure.
This study demonstrates the utility of two complementary spectroscopic techniques (NIR and
Raman) combined with PLS regression to perform quantitative analysis of multiple solid-state
forms during the dehydration. This provides a basis for real time process monitoring during
pharmaceutical manufacturing.
QUANTITATIVE ANALYSIS OF SOLID-STATE CHANGES
USING VIBRATIONAL SPECTROSCOPY AND PARTIAL LEAST
SQUARES REGRESSION
Karin Kogermanna,b
, Jaakko Aaltonena, Clare J. Strachan
c, Kati Pöllänen
d, Jyrki
Heinämäkia, Jouko Yliruusi
a, Jukka Rantanen
e
aDivision of Pharmaceutical Technology, University of Helsinki, Finland
bDepartment of Pharmacy, University of Tartu, Estonia
cDrug Discovery and Development Technology Center, University of Helsinki, Finland
dDepartment of Chemical Technology, Lappeenranta University of Technology, Finland
eDepartment of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences,
University of Copenhagen, Denmark
34
Introduction
Porous Si and SiO2 microparticles (PSi) have been proposed as possible new drug carrier materials.
Before widespread use, information about their toxicology must be obtained. However, testing
them with the standard MTT indicator could be faulted. MTT testing relies on the enzymatic
reduction of MTT in living cells. Unfortunately, unprotected Si will easily form a red/ox pairs with
MTT and cause dramatic shifts in the observed toxicity. These reactions are studied here.
Figure 1. Schematic presentation of reduction at a PSi surface.
Methods
MTT and Si/SiO2 microparticles are mixed in a range of pH solutions and the resulting formation
of purple formazan is monitored spectrometrically. The amount of MTT converted to formazan is
estimated from UV-vis spectra. Caco-2 cells were used to model the toxicity of the particles for
oral drug delivery.
Results
From the measurements, it is clear that PSi particles indeed reduce MTT directly to formazan,
causing a false non-toxic signal in cell tests. The extent of the reaction is largest for as-anodized
PSi particles and lower for thermally oxidized and carbonized PSi particles. As expected, SiO2
particles did not react with MTT, since SiO2 cannot be further oxidized and form red/ox pairs with
MTT. Comparable experiments done in a cell culturing solution, where the real toxicity test would
be carried out, showed similar trends: All Si particles were able to reduce MTT and SiO2 particles
were completely non-reactive.
It is shown that the common toxicity indicator, MTT, will fail to predict the toxicity of
formulations containing PSi particles. This is due to the spontaneous red/ox reactions where the
MTT is reduced and the PSi particle surfaces are oxidized simultaneously. It is strongly suggested
that other forms of toxicity indicators be used with any drug formulation testing involving PSi
particles.
FAILURE OF MTT AS A TOXICITY TESTING AGENT FOR SI
PARTICLES
Timo Laaksonen, Hélder Santos, Leena Peltonen, Jouni Hirvonen
Division of Pharmaceutical Technology, P.O. Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland.
E-mail: [email protected]
35
The existing in vitro drug dissolution test methods have limitations, such as long sampling times of
30-60 seconds, considering fast dissolving drugs. They also require relatively large amounts of
dissolution medium and time consuming sample preparation. There is a clear need for resource-
sparing assays of the dissolution and measurement methods that could monitor fast dissolution on-
line and would enable better simulation of the conditions e.g. in buccal cavity with smaller
dissolution medium volumes.
In this study, a method based on scattering of laser light from particles in a liquid medium is
evaluated for dissolution rate determination of two fast dissolving drugs (anhydrous caffeine and
propranolol hydrochloride). The measuring system consisted of a laser power supply, a light
detector, an oscilloscope, a magnetic stirrer and a sample vessel (Figure 1). The intensity of laser
light transmitted through the dissolution medium was recorded and displayed by the oscilloscope.
The dissolution tests were performed in 100 ml of dissolution medium varying the medium pH,
temperature and the amount of drug sample. The resulting raw data curves were fitted with Noyes-
Whitney equation and compared to the dissolution curves obtained by a traditional dissolution
method in the same conditions. The measurement system offers a fast, reproducible and accurate
on-line monitoring of fast dissolution processes with sampling interval of 0.2 s. However, the
method is not suitable for drug particles with poor wettablility. Furthermore, with this method it is
not possible to measure dissolution profiles of two or more drugs simultaneously or dissolution of
drugs formulated with excipients.
Figure 1. Assembly of the optical measuring system used for dissolution rate determination (left)
and the sample beaker showing the laser beam passing through the sample containing medium
(right).
AN OPTICAL IN SITU DISSOLUTION RATE DETERMINATION
METHOD FOR FAST DISSOLVING DRUGS
Riikka Laitinena,, Jani Lahtinen
b, Pertti Silfsten
b, Erik Vartiainen
b,
Jarkko Ketolainena
a Department of Pharmaceutics, University of Kuopio, P.O.Box 1627, FI-70211 Kuopio, Finland
b Department of Mathematics and Physics, University of Lappeenranta, P.O.Box 20, FI-53851 Lappeenranta, Finland
36
The most common way to protect moisture-sensitive pharmaceutical powders is to utilize
protective packaging. However, the most convenient package materials are all permeable to water
molecules to some extent and limited protection is normally achieved with this arrangement even
though desiccants are employed. In this study a novel system is introduced that can regulate the
internal humidity of the containers used with solid dosage forms for a desired time at a requested
level [1]. Instead of the widely used solid adsorbents the system utilizes saturated salt solutions
loaded in desiccant bags made of various polymer materials with appropriate permeation
properties. By utilizing salt solutions the size of the desiccant bag can be further reduced compared
to the conventional solid adsorbents. A wide variety of commonly used powder chambers and
desiccant bags has been tested in the study, proving the effectiveness of the introduced system
(Figure 1).
Figure 1. The mass increase (Δm) and the internal humidity (RHch) as a function of storage time at
75% RH for the polycarbonate (PC) chambers containing silicone, polyvinylchloride (PVC) or
latex desiccant bag loaded with MgCl2 solution.
Acknowledgements
The author thanks Orion Pharma Oy (Finland) for donating HDPE chambers.
Reference
[1] V-P. Lehto and I. Erling, Drug Dev. Ind. Pharm. 33:1233-1239 (2007).
A DESICCANT SYSTEM TO CONTROL THE INTERNAL
HUMIDITY OF DRUG CONTAINERS
Vesa-Pekka Lehto
Laboratory of Industrial Physics, Department of Physics, University of Turku, FI-20014 Turku, Finland
37
The objective of this study was to determine the thermal stability of model proteins lactase and
lysozyme (Lys). Proteins, that are thermally labile, were exposed to extreme temperatures, and
differences in their secondary and tertiary structure were measured. Sucrose (S), trehalose (T)
and/or mannitol (M) were added to formulations to prevent structural changes.
Protein formulations were freeze-dried. Thermal stability, such as cold and hot denaturation, glass
transition (Tg) or denaturation temperatures (Tm), of formulations was analyzed with DSC.
Corresponding heat-treatment was applied to dry (160˚C, 18 h) and reconstituted (90˚C, 18 h)
samples. FT-IR and fluorometry were used to study a protein secondary and tertiary structure,
respectively. Measurements were performed to dry, and dissolved untreated protein, to dry and
reconstituted freeze-dried formulations, and to dry and reconstituted heat-treated formulations.
The glass transition temperatures of excipient-protein formulations increased in comparison to the
Tg of pure sugars. This implies that excipients and protein have formed a continuous matrix (only
one Tg was detected). Furthermore, this indicates a higher thermal stability of formulations due to
the slower molecular movement at room temperature. Denaturation temperatures of lysozyme-
solutions were also higher than of the native (Table 1). Mannitol seemed to be the most efficient in
preventing denaturation (Tm ca. 74,5˚C) whereas Tg of trehalose formulations were the highest (ca.
129˚C). Regardless of the stabilizing excipients, the supportive methods showed alterations in the
protein structure levels. Sugar addition itself caused perturbations in the overall shape of secondary
structure, thus it is difficult to evaluate the impact of changes. Tertiary structures of formulations
were compared. Freeze-dried excipient containing formulations were more alike with the native
form than with the pure freeze-dried proteins.
In conclusion, it was observed that excipients contribute to protein stability regardless of
temperature range. Combination of trehalose and mannitol seemed to stabilize lysozyme the best.
Table 1. The effect of excipients on entalphy change (ΔH %) and denaturation temperature change
(ΔTm) of pure proteins and freeze-dried proteins-excipient formulations.
Formulation Measured
Δ H (Jg-1
)
Calculated
Δ H (Jg-1)
Δ H % (Measured Δ H/
Calculated Δ H)*100% ΔTm (°C)
Lys (native) 3,2 3,2 - -
Lys 3,04 3,2 95 - 0,23
MLys 0,79 0,8 98,8 3,11
SLys 1,37 1,6 85,6 1,95
TLys 1,27 1,6 79,4 1,99
MSLys 0,51 0,64 79,7 3,25
MTLys 0,53 0,64 82,8 3,39
THERMAL STABILITY OF LYSOZYME AND LACTASE
FORMULATIONS
Katri M. Levonen, A. Penttonen, J. Ketolainen and O. Korhonen
Department of Pharmaceutics, University of Kuopio, Finland
38
The fluidized bed granulation is a complex and multivariate process in nature like any other
process1 and it must be handled in a multivariate way. Until recently granulation has been followed
by recording separately few process parameters, e.g. in - and outlet air humidity and temperature2.
Separate parameters give information of the process based on the earlier experiments using the
same process conditions, and the end-point of the granulation could be estimated using the
experience gathered from these process parameters and conditions which have led to a desired end
product. However, separate process variables do not give a comprehensive picture of the
granulation process and such operating methods, which are ultimately based on users experience,
do not fit well into the high-quality product manufacturing proposed in FDA's PAT initiative. Thus,
developing more sophisticated controlling and monitoring systems for fluidized bed granulation is
needed.
Binder content of the fluidized bed affects the granule growth rate, granule size and eventually,
yield. An even distribution of the aquous binder produces narrow granule size distribution, while
over wetting the mass can lead to a bed collapse. Thus, the granulation process could be followed
by tracing the water content of the fluidized mass. A common technique for water content
measurement is Karl Fischer titration, but it requires the removal of the sample from the process
line, and obtaining statistically reliable results could be cumbersome. Near-infrared spectroscopy is
a capable tool in determining water content during granulation3, but collecting representative
spectra can be problematic due to the probe or window contamination. Passive acoustic emission is
a technology, which have been used in a wide variety of chemical engineering processes4. As the
moisture content changes during granulation, so does the elastic properties of the material,
affecting the acoustic emissions caused by particle impact. Also, since the water content of the
mass depends heavily on the process parameters, taking account of the parameters is important for
the water content prediction.
Our study aimed to develop a model for real-time quantification of the water content of the powder
mass during the granulation. For the model, we combined the knowledge from the process
parameters and acouctic emission using PLS regression. As a result the water content of the
granule mass can be followed using acoustic emission and process parameters. The relative
humidity of ambient air is crucial for determining the water content of granules and it is of
importance to be able to stabilize its effect in the model. The data analysis were carried out using
PLS_toolbox5 version 4.0. More detailed results will be provided in the poster
GRANULE WATER CONTENT PREDICTION DURING
FLUIDIZED BED GRANULATION
Sanni Materoa, Sami Poutiainen
b, Jari Leskinen
c, Mikko Hakulinen
b, Maija Lahtela-
Kakkonena, Antti Poso
a,Jarkko Ketolainen
b, Reijo Lappalainen
c, Kristiina Järvinen
b
aDepartment of Pharmaceutical Chemistry
bDepartment of Pharmaceutics
cDepartment of Physics
University of Kuopio, Kuopio, Finland, FIN-70211
39
Dehydration of a hydrate is a common solid-state reaction that can be facilitated by pharmaceutical
unit operations. The solid-state changes resulting from dehydration are reflected as altered physical
and chemical properties of pharmaceuticals, with potential effects on their processability,
dissolution rate and bioavailability, and stability. The ability to detect these changes as well as the
knowledge of dehydration mechanisms are, therefore, prerequisites to controlling the
manufacturing process and storage conditions of a drug product. Lattice channel hydrates, which
are capable of forming isomorphic dehydrates, comprise a challenging class of hydrates from
analytical standpoint and hence the development of analytical tools to ensure the appropriate solid
state of the pharmaceuticals is clearly requested.
The purpose of this study was twofold: (1) to investigate the potential of Raman spectroscopy to
detect isomorphic dehydration transition and (2) to obtain molecular level insight into dehydration
mechanism of a model drug, erythromycin A dihydrate. Based on the multi-technical approach
(variable-temperature X-ray powder diffraction, differential scanning calorimetry,
thermogravimetry and hot-stage Raman spectroscopy), solid-state dehydration of the model drug
was verified. Furthermore, it was demonstrated that, for any isomorphic dehydration transition,
Raman spectroscopy provides a powerful tool to detect the transition. Finally, the experimental
data were combined with molecular modeling tools in order to postulate the mechanism involved in
the solid-state dehydration of erythromycin A dihydrate.
RAMAN SPECTROSCOPY AS A TOOL FOR DETECTING
ISOMORPHIC DEHYDRATION TRANSITIONS
Inna Miroshnyka, Sabiruddin Mirza
a, Jukka Rantanen
a,b, Jyrki Heinämäki
a and
Jouko Yliruusia,b
a Division of Pharmaceutical Technology, University of Helsinki, Finland
b Department of Pharmaceutics and Analytical Chemistry,
The Danish University of Pharmaceutical Sciences, Denmark
40
Pharmaceutical unit operations are well known to facilitate phase transformations of active
ingredients and/or excipients. Since the quality and performance of a pharmaceutical solid
formulation depend on solid state of the drug and excipients, a thorough investigation of these
transformations is required. The aim of this study was on the physical phenomena taking place
during (1) recrystallization of anhydrous baclofen from water and (2) formulation of erythromycin
dihydrate solid dispersion with polyethylene glycol (PEG) 6000 by melting method.
Optical microscopy was utilized for in situ monitoring of phase transitions during these processes.
X-ray powder diffraction (XRPD) and Raman spectroscopy were used as complementary analytical
techniques to verify solid-state changes.
The process visualization using optical microscopy revealed that both model drugs undergo a
solvent-mediated phase transformation that involves dissolution of the initial phase and formation
of a new solid phase with characteristic morphology. The phase transitions (specifically, baclofen
anhydrate baclofen monohydrate and erythromycin dihydrate erythromycin dehydrate
erythromycin anhydrate) were further verified by XRPD and Raman spectroscopy.
In conclusion, the present study demonstrates that optical microscopy is a fast tool in obtaining an
insight into the physical phenomena taking place during processing of pharmaceuticals, which
enables better understanding and control of the process.
IN SITU MONITORING OF PROCESSING-INDUCED PHASE
TRANSFORMATIONS USING OPTICAL MICROSCOPY
Sabiruddin Mirzaa, Inna Miroshnyk
a, Jyrki Heinämäki
a, Jukka Rantanen
a,b,
Jouko Yliruusia,b
a Division of Pharmaceutical Technology, University of Helsinki, Finland
b Department of Pharmaceutics and Analytical Chemistry,
The Danish University of Pharmaceutical Sciences, Denmark
41
Figure 1 Ibuprofen adsorption
isotherms into TCPSi and TOPSi
from chloroform solution
One of the most promising applications of mesoporous silicon (PSi) is in the field of drug delivery. The
mesoscale dimensions induce solid-state restructuring of the drug adsorbed inside the pores into a more
disordered state, which has been shown to improve drug dissolution and permeation.
However, in order to benefit from the improved pharmacokinetic properties of the drug, a suitable and
easily controlled method for loading the drug inside the pores has to be chosen. The most common choice is
adsorption from solution, which provides several controllable parameters, such as solution concentration,
the choice of solvent and temperature. The physicochemical properties of the drug molecule and the
influence of the particle surface chemistry also have a considerable effect to the outcome of the adsorption.
As the improvement of the drug properties requires the drug molecules to reside inside the pores, the amount
of drug molecules that crystallize on the outer surface of the porous material has to be minimized, in order to
avoid erratic and unpredictable behavior of the delivery method.
The present study outlines the effects of the solvent and solution concentration on the adsorption of the
drug into PSi. Adsorption isotherms with two BCS-II class model drugs, ibuprofen and griseofulvin, were
determined with various organic solvents. The effect of PSi surface chemistry was obtained by stabilizing
the porous silicon by thermal carbonization (TCPSi) and thermal oxidation (TOPSi). Employing
thermoanalytical methods in the drug load characterization, the separation of the actual drug load inside the
pores from the crystallized surface fraction of the drug was possible. Interactions between the drugs and the
solvents were characterized with FTIR.
The results obtained indicate, that beside the solution concentration, drug-solvent and drug-surface
interactions have significant effect on the adsorption process. For ibuprofen, adsorption from less polar
solvents provides an isotherm where the initial slope of the isotherm is steep, and high drug loads are
achievable with low relative concentration. However as can be seen from Fig. 1, at higher concentrations the
quickly increasing surface fraction blocks the pore entrances
resulting in diminishing of the actual drug load. In contrast,
reaching the maximum drug load required slightly higher
concentrations of the loading solution when highly polar alcohols
were used. However, the surface fraction was negligible compared
to the less polar solvent providing a Langmuir-type isotherm with a
clear plateau. In the case of ibuprofen, the choice of solvent does
not affect the actual drug load, as much as it does the surface
fraction. This can be partly explained by differing tendencies of the
solvents to cleave the ibuprofen dimeric structure, which can be
observed in the solution FTIR spectra. Thus, in some cases
crystallization on the outer surfaces becomes less favorable.
Adsorption of griseofulvin was considerably lower, as the
maximum drug loads obtained were less than half compared to
ibuprofen. Also notable was the complete lack of surface
crystallization. Other difference was the greater affinity of
griseofulvin towards carbonized (TCPSi) surface, which appears to be consistent with the behavior related
to oxide surfaces, as griseofulvin payloads in mesoporous silica (SiO2) have also remained quite low in
similar loading tests. Adsorption was also more dependent on the solvent properties. The highest drug loads
were obtained with solvate-forming solvents, emphasizing the important role of drug-solvent interactions.
Through concentration and solvent selection, the adsorption can be optimized to provide maximum drug
load inside the pores without significant amount of crystallized drug on the outer surfaces. The extent and
nature of interactions has decisive effect on the outcome of the adsorption, however the outcome seems to
be situation-specific, making them either beneficial or detrimental.
ADSORPTION OF TWO MODEL DRUGS INTO MESOPOROUS
SILICON MICROPARTICLES
Ermei Mäkilä, Joakim Riikonen, Teemu Heikkilä, Jarno Salonen,
and Vesa-Pekka Lehto
Laboratory of Industrial Physics, Department of Physics, University of Turku, FI-20014, Turku, Finland
0 200 400 600 800 10000
10
20
30
40
50
60
Dru
g load [w
-%]
Concentration [mg/ml]
Drug inside pores (TCPSi)
Total drug load (TCPSi)
Drug inside pores (TOPSi)
Total drug load (TOPSi)
Ibuprofen-chloroform solution
42
Different methods have been employed to investigate in vitro release from microparticles but so
far, no standard method has been estabilished.1 The aim of this study was to evaluate the effect of
two in vitro release methods, centrifuge and cell culture inserts in 6-well plates, on the drug release
profiles of mesoporous silicon microparticles.
Model drugs were ibuprofen and antipyrine that were loaded into the thermally carbonized porous
silicon (TCPSi) microparticles (<38 µm) as described earlier.2 The loading degrees were
determined by thermogravimetry3 and they were 57.9 % w/w and 48.6 % w/w for antipyrine and
ibuprofen, respectively. In the centrifuge method, 1 mg loaded particles or drug powder was
suspended in 1.5 ml of phosphate buffered saline (PBS) at pH 7.4 and test tubes were placed in the
water bath shaker (+37 ˚C and 120 rpm). Samples were centrifuged for 2 minutes (13 000 rpm,
17 000 g) at the pre-determined time intervals, followed by the supernatant collection and
replacement by fresh PBS. When using 6-well plates, 2 mg particles, 1 mg unloaded drug or 0.67
mg/ml drug solution was placed in the donor compartment. Volumes of the donor and acceptor
compartments were 1.5 ml and 2.75 ml PBS, respectively, and they were separated by polyester
membrane (pore size 0.4 µm, membrane area 4.7 cm2 and 4x10
6 pores/cm
2). 6-well plates were
placed on the orbital shaker (+37 ˚C and 130 rpm) during the study. Drug concentrations were
analysed with UV-spectrophotometer.
The release rates of ibuprofen and antipyrine were fast when the centrifuge method was used but
when employing 6-well plates, the release seemed to be considerably slower (Fig. 1, shows
ibuprofen data). Despite of method or drug, the release profiles of loaded drugs were similar to the
release profiles of the corresponding unloaded drug or drug solution. The polyester membrane
creates a diffusion barrier, thus delaying the mass transfer from the donor to the acceptor
compartment. Therefore, this method does not only describe the drug release from the particles but
also the drug diffusion through the membrane. In contrast, drug can be released freely to the buffer
in the centrifuge method. In conclusion, this study shows that different in vitro methods can result
in dissimilar release profiles.
Acknowledgements
The authors thank Timo Korjamo for
assistance and advice. The financial
support from the Finnish Academy is
acknowledged (PEPBI consortium #
117906).
References
[1] D’Souza and Deluca. Pharm. Res. 23
(2006) 460-474
[2] Salonen J, Laitinen L, Kaukonen A.M,
Tuura J, Björqvist M, Heikkilä T, Vähä-
Heikkilä K, Hirvonen J and Lehto V-P. J.
Control. Release 108 (2005) 362-374
[3] Lehto V-P, Vähä-Heikkilä K, Paski J
and Salonen J. J. Therm. Anal. Calorim.
80 (2005) 393-397
EFFECT OF IN VITRO RELEASE METHOD ON DRUG
RELEASE FROM MESOPOROUS SILICON PARTICLES
Juha Mönkärea, Joakim Riikonen
b, Elina Rauma
a, Mika Pulkkinen
a Jarno Salonen
b,
Vesa-Pekka Lehtob and Kristiina Järvinen
a
a Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland
b Laboratory of Industrial Physics, Department of Physics, University of Turku, FI-20014 Turku, Finland
Figure 1. Ibuprofen release from TCPSi microparticles:
centrifuge and 6-well plate method.
0
10
20
30
40
50
60
70
80
90
100
0 60 120 180 240 300 360 420 480
Time (min)
Rele
ased
dru
g (
%)
Ibuprofen pow der
(Centrifuge)
TCPSi + Ibuprofen
(Centrifuge)
Ibuprofen solution (6-w ell
plate)
Ibuprofen pow der (6-w ell
plate)
TCPSi + ibuprofen (6-w ell
plate)
43
Reservoir and matrix type tablets are the most commonly used orally administrated sustained
release preparations. In particular matrix tablets, which are produced by direct compressing using
hydrophobic polymer, are sufficiently fast and easy to manufacture. The functionality and the
safety, i.e. drug release properties, of the product can be determined by in vitro dissolution tests. In
order to achieve desired in vitro drug release properties for drug compound in question different
formulations can be utilized. However, due to large variation of drug compounds, it is not always a
simple task to achieve well working formulation.
Starch acetates (SA) are modified starches produced by acetylating native starch. The modification
converts starch into a more hydrophobic derivative, as the average degree of substitution (DS)
increases from 0 up to 3.0. Starch acetate is a suitable excipient for controlling drug release from
tablets when DS is greater than 2.1. Although starch acetate is not anymore a novel biopolymer,
studies focused on interactions between drug compound and polymer, which affect in vitro drug
release, have not been performed. Therefore, it should be examined, whether there are interactions
between drug and starch acetate and how they affect the drug release. Furthermore, it should be
determined, which are the most important formulation parameters, e.g. porosity or structure of the
tablet, which have the greatest impact as the formulation for desired drug release profile is
searched.
In this study six different drug compounds, allopurinol, acyclovir, metronidazole, paracetamol,
salicylamide and theophylline, were used in different formulations with starch acetate as a filler
binder in order to explain the interactions and to specify their impact. The drug compounds were
fully characterized in order to achieve an adequate overview of their properties. Furthermore, nine
different formulations were occupied for one drug compound in order to determine how different
formulation parameters affect the drug release and which of the parameters are the most important.
Due to the complex nature of the setup, i.e. large number of formulations and properties of drug
compounds, multivariate data analysis, namely partial least squares projections to latent structures
(PLS), was utilized.
Results indicate that formulation parameters describing the structure of the matrix, such as
porosity, compaction force and the particle size fraction of the starch acetate, have the strongest
impact on drug release. The importance of drug property based variables is not as high as
formulation parameters, but they can not be overlooked. The importance of water solubility and
solubility rate of the compound are obvious, but there seems to be other significant parameters,
which describes the hydrophobic and hydrophilic regions of the molecule, that affect the drug
release. This can be seen especially with salicylamide: compound seems to have a strong and
sufficiently great hydrophobic region that interacts with starch acetate and impairs the drug release.
EFFECT OF FORMULATION PARAMETERS AND DRUG -
POLYMER INTERACTIONS ON DRUG RELEASE FROM
STARCH ACETATE MATRIX TABLETS
Jari Pajandera, Maria Laamanen
a, Eeva-Leena Ryynänen
a, Ian Grimsey
b,
Ossi Korhonena, Bert van Veen
c, and Jarkko Ketolainen
a
aDepartment of Pharmaceutics, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland
bSchool of Life Sciences, University of Bradford, Bradford, United Kingdom
cOrion, Finland
44
The purpose of this study was to test the feasibility of microtensiometric measurements as a fast
(HTS) analytical tool for solubility determinations of poorly soluble drug substances.
Solubility properties of a poorly soluble model drug, ibuprofen, were measured in different media
both with microtensiometry (Delta-8 multichannel microtensiometer, Kibron Inc., Finland) and
traditional shake flask -method. Microtensiometric measurements were performed in a 96-well
plate system, and the sample volume was 50 μl. The system contains eight probes that are used to
measure the surface activity/surface pressure properties of the solution/suspension tested. The
measurement is based on Du Nouy ring method.
According to the results, microtensiometric measurements could be a feasible new predictive tool
in solubility measurements. Changes in the values of surface tension were caused by precipitated
drug material in the well plate. These changes were repeatably seen from the surface tension vs.
ibuprofen concentration curves. Due to the small sample volume, the accuracy of the results was
not as good as with shake flask -method, but the results correlated clearly with the traditional
method. Easily volatile liquids may also cause problems due to the small sample volume.
Microtensiometric measurements provide a very promising method for fast and predictive
solubility screening. At present, accuracy of the method may not necessarily be high enough for
determinations of absolute solubility values, but for comparative and classifying solubility
screening it fits nicely.
SURFACE TENSION MEASUREMENTS AS A PROMISING
METHOD FOR FAST SOLUBILITY DETERMINATIONS IN A 96-
WELL PLATE
Leena Peltonen, Saila Taskinen, Jouni Hirvonen
Division of Pharmaceutical Technology, Faculty of Pharmacy,
P.O. Box 56, 00014 University of Helsinki, Finland
E-mail: [email protected]
45
.
Building quality into pharmaceutical systems has been a big issue in recent years, notably after the
launching of the FDA's PAT initiative. As stated, quality should be a build-in feature of the end-
product, resulting from the understanding and controlling of the process. Fluidized bed granulation
is a common size-enlargement process in pharmaceutical industry, in which a fine powder is
agglomerated using liquid binder to give larger granules. However, fluidized bed granulation is a
complex process in nature, and is hard to control due to the strong interactions between different
process variables. Since the granule size distribution is one of the main characteristics of the
evolution of the granulation process, there is a need to design process controlling methods which
aim to characterize the size distribution. Several applications of image analysis have tackled this
problem in recent years.1,2
During granulation the size and the pore saturation of the granules alter due to coalescence and
consolidation. They have an effect on the elastic properties of the granules, which in turn affects
the acoustic emissions caused by the granule impacts. Our previous results showed the potential of
the passive acoustic emission technique in monitoring the fluidized bed granulation process3, and
the extension of this technique as a process control method seems prospective. In this study, the
applicability of the acoustic emission technique in characterizing granule size during fluidization
was examined. Several granulations were carried out in a custom made top-spray granulation
chamber. The granular end-products were sieved in eight fractions. The acoustic emissions were
collected by fluidizing each size fraction separately at different bulk volumes and fluidization gas
flow rates. Several models were developed from the emission data using multivariate data analysis.
The results showed that acoustic emission technique can be used in determining granule size during
fluidization. As expected, the fluidization gas flow rate as well as the amount of fluidized material
both have significant effect on the acoustic emission. These effects between divergent granule size
fractions must be taken into a carefull consideration when developing model for the granule size
determination. The data analysis was carried out using SIMCA-P5 version 11. More detailed results
will be provided in the poster.
Acknowledgment
We thank The Finnish Funding Agency for Technology and Innovation (TEKES) and all collaborating companies in
PATKIVA-project for supporting the work financially.
References 1 Watano S et al. Powder Technol. . 1995, 83, 55-60
2 Laitinen N. et al. Chemom. Intell. Lab. Syst. 2002, 62, 47-60
3 Poutiainen, Leskinen, Matero, Hakulinen, Lappalainen, Lahtela-Kakkonen, Närvänen, Järvinen, Ketolainen. The 5th
International Symposium on Solid Oral Dosage Forms Stockholm Sweden 2007 4 Soft Independent Modelling of Class Analogies, versio SIMCA-P 11, Umetrics,
http://www.umetrics.com/default.asp/pagename/software_simcap/c/3%5B16 [17 August 2007]
GRANULE SIZE DETERMINATION DURING FLUIDIZATION
IN A FLUIDIZED BED GRANULATOR
Sami Poutiainena, Sanni Matero
b, Laura Piispanen
a, Jari Leskinen
c, Mikko
Hakulinena, Reijo Lappalainen
c, Kristiina Järvinen
a, Jarkko Ketolainen
a
aDepartment of Pharmaceutics
bDepartment of Pharmaceutical Chemistry
cDepartment of Physics
University of Kuopio, Kuopio, Finland, FIN-70211
46
Our previous results have shown that new 2,2’-bis(2-oxazoline)-linked poly-ε-caprolactone (PCL-
O) degrades by enzymatic surface erosion, and the erosion rate can be controlled by the PCL block
length [1,2]. The aim of the study was to evaluate the responsible enzymes for enzymatic
degradation of PCL-O and characterize the enzymatic degradation products by mass spectrometry.
The enzymatic degradation and erosion (weight loss) of solvent cast PCL-O films were studied in
the presence of pancreatin (1%), with and without enzyme inhibitors. To identify the
biodegradation products, a new liquid chromatography mass spectrometry method was developed.
The chromatographic separation was performed by Xterra reversed-phase column (2.1 x 150 mm,
3.5 µm, C8). Gradient method (5 mM ammonium acetate and 2-90 % ACN in 37 min, 200 µl/min)
was used for separation. Measurements were performed with a LTQ quadrupole ion trap mass
spectrometer equipped with an ESI ion source. Before analyzing, the samples were purified by
size-exclusion filter (Mw 30000) coupled with centrifuge.
The presence of the lipase inhibitors, paraoxon-ethyl and tetrahydrolipstatin delayed the weight
loss of the PCL-O films. This indicates that lipase was mainly responsible for the enzymatic
erosion of the PCL-O films. LC-ESI-MS/MS method was successfully used to characterize
enzymatic degradation products of PCL-O polymer. The enzymatic degradation of PCL-O
produced a complex mixture, however, main biodegradation products (about 20) were effectively
separated within 30 min (Fig. 1). Results suggest that oxazoline link remained intact in the
degradation products, which is confirming that lipase was mainly responsible for the enzymatic
degradation of PCL-O as it is primarily cleaving ester bonds.
References
[1] Tarvainen T, Malin M, Suutari T, Pöllänen M, Tuominen J, Seppälä J, Järvinen K. J. Control. Rel. 2003; 86: 213
[2] Pulkkinen M, Malin M, Tarvainen T. Saarimäki T, Seppälä J, Järvinen K. Eur. J. Pharm. Sci. 2007; 31: 119
CHARACTERIZATION OF ENZYMATIC DEGRADATION OF
OXAZOLINE MODIFIED POLY-Ε-CAPROLACTONE (PCL-O)
BY ENZYME INHIBITORS AND MASS SPECTROMETRY
Mika Pulkkinena, Joni J. Palmgrén
b, Seppo Auriola
b, Minna Malin
c,
Jukka Seppäläc, Kristiina Järvinen
a
a Department of Pharmaceutics,
b Department of Pharmaceutical Chemistry,
University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio, Finland c Department of Chemical Technology, Laboratory of Polymer Technology,
Helsinki University of Technology, Helsinki, Finland
47
Traditionally drug concentration in dissolution testing is monitored by taking a set of samples with
a sample cannula. The collected samples are analyzed e.g. with a spectrophotometer. This method
is therefore intrusive and offline. The purpose of this study was to develop and validate a
tomography based method for dissolution testing. Tomography techniques are nonintrusive and
usually online.
The proposed method is based on electrical impedance tomography (EIT). In EIT, an array of metal
electrodes is attached on the boundary of the dissolution vessel and a set of alternating electric
currents is injected through these electrodes and the resulting voltages are measured. By using
sophisticated mathematical algorithms, an approximation for the spatial/temporal distribution of the
electrical conductivity within the vessel can be estimated based on the known currents and
measured voltages. Drug concentration distribution can be estimated if relationship between
electrical conductivity and concentration is known or empirically determined.
Method was tested with a cylindrical testing tank and a USP II dissolution testing vessel without a
rotating paddle. To get clear changes in electrical properties sodium chloride tablets were used as
sample drugs and water was used as dissolution medium. It was found that EIT is a suitable method
for imaging of tablet dissolution. Two-dimensional conductivity distribution was calculated inside
the cylindrical tank and changes in conductivity due to dissolution were noticed. Also dissolution
induced changes in a voltage across the USP II vessel were observed.
In the future the proposed method will be tested with more realistic drugs and dissolution mediums.
Also the USP defined rotating paddle will be included. Aim is to estimate three-dimensional
concentration distribution during USP II dissolution testing.
Acknowledgements
Authors wish to thank The Finnish Funding Agency for Technology and Innovation (TEKES) and the collaborating
companies in PATKIVA project for financial support.
TOMOGRAPHY IMAGING OF DISSOLUTION TEST
Ville Rimpiläinena, Lasse Heikkinen
a, Anssi Lehikoinen
a, Arto Voutilainen
a,
Marko Kuosmanenb, Kristiina Järvinen
b, Jarkko Ketolainen
b
aDepartment of Physics, University of Kuopio, P.O.B. 1627, FI-70211 Kuopio, Finland
bDepartment of Pharmaceutics, University of Kuopio, P.O.B. 1627, FI-70211 Kuopio, Finland
48
The aims of this study were to optimize the whole fluid bed granulation process of ibuprofen
formulation and also to optimize the particle size distribution of final granules. If the particle size
of granules is small, the granules also dry faster. The materials used in the fluid bed granulations
(Glatt WSG 5, Germany) were ibuprofen, alpha-lactose monohydrate and polyvinylpyrrolidone
(PVP). The variables of experiments were the preheating of granulator before fluid bed granulation
and the humidity of process air (inlet air) during fluid bed granulation. Humidity of inlet air was
either the ambient inlet air (i.e. ~20-30% RH) or wet inlet air (~75-95% RH). Granule particle size
was determined by sieve analysis and laser diffraction method. Ibuprofen has low melting point
(~70 °C) and therefore high drying temperatures in granulation process were impossible to use.
Ibuprofen is a hydrophobic compound and its wettability is poor. This resulted to the large particle
size distribution of final granules especially when the moisture content of the inlet air was high
(> 70% RH). The preheating of granulator proved to be useful technique to minimize the variability
in particle sizes of different granule batches especially at high humidity of the inlet air. The size
distribution of final granules was uniform and narrow when preheating technique was used. This
narrow distribution is beneficial when granules will be used at subsequent processes, like tabletting
and mixing. Preheating of the granulator decreased also the amount of oversize granules
(sized > 3200μm) at high humidity. The preheating technique produced more optimal granules in
spite of granulation conditions.
PREHEATING OF GRANULATOR IMPROVED FLUID BED
GRANULATION
Räikkönen Heikkia, Lipsanen Tanja
b, Airaksinen Sari
a, Yliruusi Jouko
a
aUniversity of Helsinki, Helsinki, Finland;
bOrion Pharma, Espoo, Finland
49
Solid-state transformations have caused problems during manufacturing of many active
pharmaceutical ingredients (APIs). One example for a drug that changes its solid state during
processing is the antibiotic erythromycin (EM). Erythromycin dihydrate (EM.DH) incorporates two
easily removable water molecules in its lattice channels. The lost of the two incorporated water
molecules in erythromycin dihydrate results in the hygroscopic and isomorphic erythromycin
dehydrate (EM.DD). The aim of the present study was to detect the conditions under which this
transformation occurs.
The pellets, contained 50% (w/w) erythromycin dihydrate and 50% (w/w) microcrystalline
cellulose, were produced by extrusion-spheronisation. The pellets were dried with a microscale
fluid bed device at 30°C, 45°C and 60°C. An in-line NIR was applied to monitor the fluid bed
drying process. Principle component analysis was used to characterise changes in solid-state
properties. Phase purity of the pellets was analysed by X-ray powder diffraction (XRPD).
At a drying temperature of 30°C no changes occurred for both drying techniques. For the drying
process in the oven tray drier at 60°C and the fluid bed drier at 45°C transformation to
erythromycin dehydrate was only detectable for one batch. For both batches dried in the fluid bed
at 60°C dehydration was detectable by in-line NIR and XRPD. During fluid bed drying
transformations to the isomorphic dehydrate form of erythromycin dihydrate are induced.
Erythromycin dehydrate was observed at the moisture content of 1.4% [w/w] while at 1.8% [w/w]
neither XRPD nor NIR were able to detect dehydration of the hydrate form. Transformation to
erythromycin dehydrate is strongly depended on the moisture content of the pellet formulation.
Figure 1:
XRPD pattern of single
crystal data (NAFTAV),
experimental reference
substances and
Figure 2:
Unit cell of erythromycin
dihydrate
Figure 3:
Scores plot of the first two principle components of
the near infrared data (SNV corrected, Savitzky
Golay first derivative smoothing, mean centred)
Reference
Römer M et al. Eur. J Pharm Biopharm 2007 Aug; 67(1):246-52.
SOLID STATE TRANSFORMATION OF ERYTHROMYCIN A
DIHYDRATE DURING DRYING MONITORED BY NEAR
INFRARED SPECTROSCOPY
Meike Römera, Jyrki Heinämäki
a, Inna Miroshnyk
a, Niina Kivikero
a, Niklas Sandler
b,
Jukka Rantanenc, Jouko Yliruusi
a
a Division of Pharmaceutical Technology, Faculty of Pharmacy, Helsinki, Finland
b AstraZeneca Process Pharmaceutical & Analytical Research & Development, Macclesfield, United Kingdom
c Department of Pharmaceutics and Analytical Chemistry, Copenhagen, Denmark
50
The convenient and preferred route for the administration of drug compounds is via oral delivery.
However, many existing and new drug molecules have low oral bioavailability due to the low
solubility of the drug in the intestinal lumen and/or poor permeability across the intestinal
membranes.
Recent in vitro studies showed a clear improvement in the dissolution profiles of poorly dissolving
drug compounds when using mesoporous silicon (PSi) and silica microparticles [1–3].
Interestingly, it was recently reported that PSi microparticles also have a permeability enhancing
effect on furosemide across Caco-2 cell monolayers [4]. Furthermore, it was shown that
mesoporous carriers can also be used to reduce the pH dependence of ibuprofen dissolution [2].
Literature data on the in vitro quantification of cellular toxicity of mesoporous PSi microparticles is
scarce, because many of the traditional assays used for the determination of cellular toxicity, such
as MTT or LDH assays fail to correctly predict the effect of the PSi particles in cellular toxicity
due to the redox reactions between those chemicals and PSi surfaces [5].
In the first part of this study the dissolution profiles of PSi and silica (SiO2) microparticles loaded
with poorly water-soluble model drugs (BCS classes II and IV) were investigated, and in the
second part cell toxicity of the PSi particles using Caco-2 cell cultures was studied.
Acknowledgements
Financial support from the Academy of Finland (HUMALA-project number 211048) and ComPSi-project number
122314, is gratefully acknowledged.
References
[1] T. Salonen, et al., J. Control. Rel. 108 (2005) 362–374.
[2] T. Heikkilä, et al., Drug Deliv. 14 (2007) 337–347.
[3] V. Ambrogi, et al, Eur. J. Pharm. Sci. 32 (2007) 216–222.
[4] A. M. Kaukonen, et al., Eur. J. Pharm. Biopharm. 66 (2007) 348–356.
[5] T. Laaksonen, et al., Chem Res. Toxicol. 20 (2007) 1913–1918.
IN VITRO STUDIES OF MESOPOROUS SILICA/SILICON
MICROPARTICLES: TOXICITY AND DISSOLUTION
H.A. Santos1*, T. Heikkilä
2, J. Riikonen
2, T. Lautenschlager
1, L. Peltonen
1,
J. Salonen2, N. Kumar
3, D.Y. Murzin
3, T. Salmi
3, M. Kemell
4, M. Ritala
4,
V.-P. Lehto2, J. Hirvonen
1
1Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland;
2Laboratory of Industrial Physics, Department of Physics, University of Turku, FI-20014 Turku, Finland;
3Laboratory of Industrial Chemistry, Process Chemistry Centre, Åbo Akademi University, FI-20500 Turku, Finland; 4Laboratory of Inorganic Chemistry, Department of Chemistry, University of Helsinki, FI-00014 Helsinki, Finland
*email: [email protected]
51
Purpose
Formulating a drug in the amorphous form is commonly suggested as a solution to enhance the
solubility of poorly water soluble drugs. However, in order for the dissolution rate of the active
pharmaceutical ingredient to improve, it has to remain amorphous during the dissolution in the
body. The aim of this study was to use in situ Raman spectroscopy to monitor solid-state transitions
during dissolution testing of a model drug indomethacin.
Methods
Amorphous indomethacin was prepared by cooling the melt. The dissolution tests of the
amorphous samples and the crystalline α- and γ-polymorphs were carried out in a channel flow
intrinsic dissolution test apparatus with a sight window for Raman probe. Two different dissolution
media were used: water-ethanol mixture 50% (m/m) and phosphate buffer (pH 7.2). A partial least
squares discriminant analysis (PLS-DA) of the Raman spectra was used to gather qualitative
information about the solid-state transitions. X-ray powder diffraction (XRPD) was used to analyze
the solid state of the samples before and after the dissolution testing.
Results
As expected, the dissolution rate of amorphous indomethacin was several-fold higher than from the
crystalline counterparts. However in both dissolution media, the dissolution rate of indomethacin
from the amorphous sample slowed down significantly during the duration of the experiments.
Based on the Raman spectra, crystals of α-polymorph were formed on the surface of the amorphous
sample at that time. PLS-DA analysis showed that the amorphous indomethacin converted directly
to the α-form. Thus, the transition did not go through the γ-polymorph.
Conclusions
In situ measurement of solid-state transition using Raman spectroscopy offers a valuable tool to get
a better insight into the phenomena that occur during the dissolution of an amorphous drug. PLS-
DA enables the monitoring of multiple solid-state forms that might appear during dissolution.
BETTER UNDERSTANDING OF DISSOLUTION BEHAVIOR OF
AMORPHOUS DRUGS
M. Savolainena, A Heinz
a, b, c, J. Aaltonen
a, L. Peltonen
a, C. Strachan
b, J. Yliruusi
a
aDivision of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, Finland
bDrug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, Finland
cSchool of Pharmacy, University of Otago, New Zealand
52
Pharmaceutical powder and granule systems are complex, but they are so important in formulation
and process research that more attention should be paid on their theoretical understanding. In many
cases direct measurements and observations from powder systems are limited. In such cases
computer simulations, which are especially designed for pharmaceutical systems can increase our
understanding of pharmaceutical powders. The applicability of such simulations is very versatile
ranging from basic educational needs to model powder flow to fundamental understanding of
complex nanotechnological structures of new drug delivery systems.
The purpose of this study is to develop new simulation software based on Newtonian mechanics.
Particle properties taken into account at this stage include size, density, friction coefficient,
elasticity of collisions and stiffness. Similar properties of container were also considered, and
conservation of energy throughout the simulation is tracked. Effects of different parameters could
be studied by changing one parameter at a time. To ensure that given parameters are realistic,
comparison with measurements is required. For example, when particle size gets smaller, electric
forces start to contribute significantly to particle arrangement. Comparison helps to find the
minimum particle size in which it is not necessary to take other interactions into account. Our
simulations have so far been performed in normal table-top PC-computer. Systems containing up to
few thousand particles can be modeled in reasonable time with such hardware. Further studies aim
to simulation of segregation, mixing and compaction phenomena. Through these simulations it
would be possible to understand which physical properties are essential in powder flow, mixing
and packing.
FUNDAMENTAL UNDERSTANDING THROUGH
SIMULATIONS?
Simo Siiriä, Jouko Yliruusi
Division of Pharmaceutical Technology, University of Helsinki.
53
Purpose
To characterize the solid-state structure of different triglycerides and a partial glyceride in
powdered form and as extrudates and investigate their complex behaviour during processing and
storage.
Methods
Different types of powdered glycerides (Dynasan, Imwitor) and their mixtures were extruded
below their melting ranges. Extrusion was also performed with theophylline anhydrate (50%) as a
model drug. The physical structure of powders and extrudates was examined using differential
scanning calorimetry (DSC), x-ray powder diffraction (XRPD), vibrational spectroscopy (ATR-IR,
Raman, NIR) and Raman microscopy.
Results
Examination of the pure triglycerides (Dynasan) revealed a chain length dependant behaviour of
the fatty acids. Powders were in the stable -form before extrusion. Dynasan 112 and Dynasan 116
also stayed in the -form after extrusion. Dynasan 118 was partially changed to the -form after
extrusion depending on the processing temperature, and the amount of -form remained stable
during storage in ambient conditions for 3 months. Extrusion of the partial glyceride Imwitor
showed no solid-state changes. The addition of the model drug theophylline anhydrate showed no
change in crystal structure before and after extrusion and even after storage no transformations
could be noticed. Spatial analysis with Raman microscopy showed that the crystalline drug
particles are randomly distributed within the lipid matrix. There was no evidence of interaction
during extrusion between triglycerides and theophylline. Although the crystal structure appeared
unchanged thermoanalysis results suggest a limited interaction of theophylline with the partial
glyceride Imwitor during extrusion. Mixture of Dynasan 118 and Imwitor resulted in interactions
of the glycerides during processing.
Conclusions
The solid-state behaviour of glycerides and their extrudates during extrusion and storage strongly
depends on the glyceride composition. Triglycerides show no interaction with the model drug,
whereas the partial glyceride appears to interact with the drug. In the mixture partial glyceride and
triglyceride interact. The combination of XRPD, DSC and vibrational spectroscopy including
Raman microscopy gives new insight into the solid- state properties of glycerides and glyceride-
based extrudates.
INVESTIGATIONS ON PHYSICOCHEMICAL PROPERTIES OF
GLYCERIDES AND GLYCERIDE-BASED EXTRUDATES
Maike Stiersa,b,c
, Clare J. Strachanb, Peter Kleinebudde
a
aInstitute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University Duesseldorf, Germany
bDrug Discovery and Development Technology Center (DDTC), University of Helsinki, Finland
cDivision of Pharmaceutical Technology, University of Helsinki, Finland
54
Preferred orientation of crystallites, i.e. texture, often occurs when the particles of the sample
powder are needle or plate-like. Assuming that different crystal planes possess different energetics,
and therefore also different dissolution properties, there could be differences in the intrinsic
dissolution rates of the compacted samples having different type of texture. The purpose of this
study was to clarify the effects of preferred orientation in tolbutamide and acetylsalicylic acid
compacts on the intrinsic dissolution.
In order to obtain particles with different habits, various crystallization methods were used. The
preferred orientation of the compacts was measured with several different methods using both
ordinary and texture x-ray diffraction goniometer. The channel flow technique was used to
determine the intrinsic dissolution rate of the samples. Moreover, the crystal plane attachment
energies were calculated theoretically.
All the crystallographic parameters of the samples except texture were comparable. Acetylsalicylic
acid samples had different type of texture whereas the texture of the tolbutamide compacts was of
similar nature and only the amount of preferred orientation varied between the samples. The results
of the intrinsic dissolution studies indicated that the most texturized compacts had a clearly lower
intrinsic dissolution rate than the compacts with a lower degree of preferred orientation (Fig. 1.).
The calculated energy values were in good agreement with the results of the texture and intrinsic
dissolution determinations. Moreover, it was found that by using the results of the texture
measurements the habit of the compacted powder could be predicted (Fig. 2.).
Based on the present study in the case of acetylsalicylic acid and tolbutamide, the preferred
orientation should be taken into account when interpreting the results of the intrinsic dissolution
studies. Moreover, there could be a possibility to modify the dissolution behavior of
pharmaceuticals by modifying their texture.
Fig. 1. Channel flow intrinsic dissolution
profiles and the intrinsic dissolution rates of
the acetylsalicylic acid samples. Error bars
indicate the standard deviation of the results.
Fig 2. Predicted crystal habits of the acetylsalicylic acid samples
compared with the SEM micrographs of the powders.
PREFERRED ORIENTATION AFFECTS THE INTRINSIC
DISSOLUTION OF TOLBUTAMIDE AND ACETYLSALICYLIC
ACID COMPACTS
Mikko Tenhoa,c
, Jaakko Aaltonenb, Paula Lehto
c, Leena Peltonen
b
and Vesa-Pekka Lehtoa
a Laboratory of Industrial Physics, University of Turku, FI-20014 Turku, Finland
b Division of Pharmaceutical Technology, University of Helsinki, FI-00014 Helsinki, Finland
c Orion Pharma, PO Box 65, FI-02101 Espoo, Finland
55
Defensins are cationic, 3-6 kDa molecular weight peptides that are found in most human tissues
and fluids. They are part of the innate immunity system and have direct antibacterial, antifungal
and antiviral properties and can indirectly enhance the immune system. There is a lot of research of
the properties of defensins and several methods to measure the activity of the genes, but there is
very little development of qualitative and quantitative methods for the peptides in different
biological fluids. In this study a pre-treatment and detection method was developed for different
biological samples: human saliva and pancreatic juice and rat ileum. The pre-treatment of a
biological sample consists of gel permeation chromatography and reversed phase chromatography.
Detection was done with mass spectrometry.
Samples were treated with 30% formic acid to precipitate large proteins. The first separation was
done with gel permeation chromatography: TSKgel 4000 and 2000 in a series, isocratic 20%
acetonitrile +0,1% trifluoroacetic acid. The fractions acquired were separated again with RP-
HPLC, using a silica C4 column with 300Å pore size. LTQ linear ion trap mass spectrometer was
used to detect the peptides.
Different columns were tested for RP-HPLC, and C4 with 300Å pore size was found to be the best.
The reason for this is, that there's a significant carryover of peptides in columns with larger side
chains and smaller pore sizes. This is probably due to the smaller active surface of the column,
which can strongly bind the cationic peptides.
Human neutrophil defensins (HNP) nos. 1, 2, and 3 were detected in human saliva. HNPs 1, 2, 3
and 4 were detected in human pancreatic juice. Rat neutrophil defensins 2 and 4 were detected in a
sample of rat intestine (ileum). Next target will be colostrum, which contains a lot of antibacterial
components. Also a quantitation method is being developed. This enables the studying of the effect
nutrition or illnesses to the level of defensins in humans and animals.
DETECTION OF DEFENSINS FROM BIOLOGICAL MATRICES
WITH HPLC-ESI-MS
Jussi Tervonen, Ilpo Jääskeläinen and Seppo Auriola
University of Kuopio, Department of pharmaceutical chemistry, Kuopio, Finland
56
Purpose
The aim of the present study was to develop simple and quantitative methods for analysis of natural
cyclodextrins (α-CD, β-CD and γ-CD) in aqueous sample matrices from the Calu-3 cell permeation
studies of CDs.
Methods
As a pretreatment method, C-18 solid phase extraction was used to remove the interfering
compounds (i.e. glucose and inorganic salts) of the sample matrix. An aliquot of 450 µl of the
sample was washed with 1.0 ml of water and the analytes were eluted with 2.0 ml of
methanol:water (75:25). The samples were analyzed with HPLC using a mobile phase of
acetonitrile:water (4:96 V/V for α-CD or 7:93 V/V for β-CD and γ-CD, respectively) with a flow
rate 0.65 ml/min. Zorbax SB-Aq column was used for α-CD analyses while Zorbax SB-Phenyl
column was used for β-CD and γ-CD analyses. A pulsed amperometric detector (PAD) with a gold-
plated surface electrode was used for the detection of CDs and therefore, the pH of the mobile
phase was alkalised (pH>12) by infusing 0.5 M NaOH (0.65 ml/min) via a post-column t-piece.
The CD analysis methods were studied for specificity, linearity, accuracy, precision and
repeatability at the concentration ranges of 4-975 µg/ml (α-CD), 4-1150 µg/ml (β-CD) and 4-1300
µg/ml (γ-CD). Accuracy and precision were determined at three concentration levels (n = 3 at each
concentration level) and the repeatability was evaluated on three separate days (n = 3 /day).
Results The sample pretreatment recovery of the CDs was > 90% (while that of glucose was < 0.5 %). All
the CDs were eluted within 4 minutes with a peak symmetry of 0.9 and with a sufficient resolution
to glucose (2.9, 3.2 and 2.7 for α-CD, β-CD and γ-CD, respectively). The methods showed good
linearity (r ≥ 0.999) and accurary (90-107 %). The RSD values indicated an acceptable intra-day
precision (RSD< 11 %) and inter-day repeatability (RSD < 15 %). The limits of quantitation for α-
CD, β-CD and γ-CD were 0.78, 0.46 and 0.52 µg/ml (RSD < 20 %, n = 4-5), respectively.
Conclusions
In conclusion, rapid and sensitive HPLC-PAD methods have been developed for quantitative
analysis of natural CDs in aqueous matrices containing glucose and inorganic salts. The present
method has been applied to analysis of samples from cell permeation studies (involving the
pretreatment step) and dissolution studies (without the pretreatment step).
QUANTITATIVE ANALYSIS OF NATURAL CYCLODEXTRINS
USING HPLC AND PULSED AMPEROMETRIC DETECTION
METHOD
Tarja Toropainena,b
, Marko Lehtonena, Pekka Jarho
a, Pekka Keski-Rahkonen
a,
Heli Raatikainena and Tomi Järvinen
a
a Department of Pharmaceutical Chemistry, University of Kuopio, Finland
b Department of Pharmaceutics, University of Kuopio, Finland
57
Perphenazine (PPZ) is a drug with relatively poor oral bioavailability due to first-pass metabolism.
The first-pass metabolism can possibly be avoided using sublingual administration route. However,
the sublingual absorption of PPZ may be limited due to its poor aqueous solubility. Two
formulation approaches were investigated in order to improve the aqueous solubility and
bioavailability of PPZ in sublingual delivery.
The effects of cyclodextrin complexation and solid dispersion formation on the aqueous solubility
and dissolution rate of PPZ were investigated. Solid formulations were prepared by spray-drying or
freeze-drying. The absorption of PPZ (1 mg/kg) was studied in rabbits after sublingual
administration of solid PPZ/cyclodextrin complex, PPZ/polymer solid dispersion and plain
micronized PPZ, and after oral administration of aqueous PPZ solution.
The aqueous solubility and dissolution rate of PPZ were increased by both formulation techniques.
The AUC(0-360min) values (mean±SD, n=3-5) after sublingual administration of
PPZ/cyclodextrin complex, PPZ/polymer solid dispersion and plain PPZ were 5530±1730,
7070±1120 and 8650±3590 ng min ml-1
, respectively. After oral administration of PPZ solution,
the AUC(0-360min) value was 1090±877 ng min ml-1
.
In conclusion, the absorption of PPZ could be enhanced using the sublingual administration route.
However, cyclodextrin complexation and solid dispersion formation did not improve the sublingual
absorption of PPZ when compared to plain PPZ, possibly due to lower wettability and larger bulk
volume of these formulations.
SUBLINGUAL ADMINISTRATION OF FAST-DISSOLVING
PERPHENAZINE FORMULATIONS
Elina Turunen1, Janne Mannila
1, Riikka Laitinen
2, Kristiina Järvinen
2,
Tomi Järvinen1, Jarkko Ketolainen
2, Pekka Jarho
1
1Department of Pharmaceutical Chemistry
2Department of Pharmaceutics
University of Kuopio, P.O.Box 1627, 70211 Kuopio, Finland
58
Courses
Topics related to physical pharmacy are mainly taught in the following courses:
Physical Pharmacy (1st year)
Preparation of Dosage Forms (1st year)
Pharmaceutical Physical Chemistry I (2nd
year)
Pharmaceutical Physical Chemistry II (3rd
year)
Advanced Preparation of Dosage Forms (3rd
year)
Advanced Physical Pharmacy (4th
year)
As an example, Formulation of an Emulsion in the course of Advanced Preparation of Dosage
Forms is presented.
Emulsions
An emulsion consists of two immiscible liquid phases, usually water and oil phase. In oil -in-water
-emulsion small oil droplets are dispersed throughout water phase and in water-in-oil -emulsion the
situation is vice versa. The phase that forms droplets is called disperse phase and the other phase is
called continuous phase. The third component necessary to stabilize the emulsion is emulsifying
agent. The function of the emulsifying agents is to form an interfacial film around the dispersed
droplets i.e. to stabilize the emulsion. However emulsions typically are thermodynamically
unstable: flocculation, phase-inversion or creaming may occur. (1)
Formulation of Emulsion
In the course the students have to formulate an emulsion using given materials. They have to plan a
composition for oil-in-water, water-in-oil -emulsion or an oral emulsion. Each emulsion contains
water and oil phases and two emulsifying agents (one in oil phase and one in water phase). They
use the HLB-values of oil phase ingredients to calculate the required hydrophile-lipohile balance
value, RHLB, for the formulation and the amounts of two emulsifying agents (2). The students
prepare the emulsion according the basic principles that includes that both phases are liquids with
same temperature when the phases are combined.
Characterisation of Emulsion
The students have to determinate the emulsion type of the emulsion they formulated. They use
three methods: 1. a miscibility method, 2. a staining method and 3. a conductivity method.
1: The emulsions are mixed either with water or oil. 2: An oil-in-water -emulsion dyes evenly
when using of a water soluble methyl blue solution and water-in oil -emulsion is respectively
evenly coloured with an oil soluble Sudan solution. 3. Oil-in-water emulsion conducts electricity.
The students follow during one week the physical stability of their emulsions.
Students' Feedback
In conclusion: "This is a great lab work"
References
1. Aulton ME: Aulton's Pharmaceutics The design and manufacture of medicines, 3rd
ed. Churchill Livingstone,
Elsevier 2007, pp 70-97
2. Sinko PJ: Martin's Physical Pharmacy and Pharmaceutical Sciences, 5th
ed. Lippincott Williams & Wilkins 2006, p
449
TEACHING OF PHYSICAL PHARMACY IN DEPARTMENT OF
PHARMACEUTICS AT THE UNIVERSITY OF KUOPIO:
EMULSIONS AS AN EXAMPLE
Jaana Veki, Outi Raunio, Kristiina Järvinen, Päivi Harjunen
University of Kuopio, PO Box 1627, 70211 Kuopio
59
The purpose of the study was to find correlation between crushing strengths of theophylline tablets
and spectra measured by FT-NIR spectroscopy. Theophylline was granulated with fluid bed
granulator. The composition of the granules included theophylline anhydrate and
polyvinylpyrrolidone (PVP, Kollidon K25) as a binder. The granules were mixed with 0.5 % (w/w)
magnesium stearate for 3 minutes in Turbula mixer with mixing speed of 46 rpm. The tablets were
compressed with single punch eccentric tableting machine with rotating speed of 36 rpm using a
feed shoe. The tablet weight was 250 mg. The tablets were compressed so that the crushing
strengths of the tablets were 30, 60, 80, 100 and 125 N. The crushing strengths were measured with
indirect diametral compression apparatus. Three parallel tablets from each crushing strength group
were measured using FT-NIR spectrometry. The distance between tablet and fiber-optic probe was
adjusted by measuring trough the bottom of a sapphire glass vial. The gained spectra were
normalized at the wavelength of 1054 nm. The Partial Least Squares (PLS) method was used for
modeling the FT-NIRS data with SIMCA-P software (Umetrics, Umeå. Sweden). The range of
whole spectra (1000-2500 nm) was used in the modeling. The correlation between crushing
strength and FT-NIR spectra was observed. Both the R2 and Q
2 value were above 0.9 which means
that the correlation for these theophylline tablets was very good.
CRUSHING STRENGTH OF TABLETS MEASURED BY FT-
NEAR INFRARED SPECTROSCOPY
Satu Virtanen, Osmo Antikainen, Heikki Räikkönen,
Sari Airaksinen, Jouko Yliruusi
Division of Pharmaceutical Technology, 00014 University of Helsinki, P.O Box 56, Helsinki, Finland
60
61
VÄITÖSKIRJOJEN TIIVISTELMÄT
62
Solid materials can exist in different physical structures without a change in chemical composition.
This phenomenon, known as polymorphism, has several implications on pharmaceutical
development and manufacturing. Various solid forms of a drug can possess different physical and
chemical properties, which may affect processing characteristics and stability, as well as the
performance of a drug in the human body. Therefore, knowledge and control of the solid forms is
fundamental to maintain safety and high quality of pharmaceuticals. During manufacture, harsh
conditions can give rise to unexpected solid phase transformations and therefore change the
behavior of the drug. Traditionally, pharmaceutical production has relied on time-consuming off-
line analysis of production batches and finished products. This has led to poor understanding of
processes and drug products. Therefore, new powerful methods that enable real time monitoring of
pharmaceuticals during manufacturing processes are greatly needed.
The aim of this thesis was to apply spectroscopic techniques to solid phase analysis within different
stages of drug development and manufacturing, and thus, provide a molecular level insight into the
behavior of active pharmaceutical ingredients (APIs) during processing. Applications to polymorph
screening and different unit operations were developed and studied. A new approach to dissolution
testing, which involves simultaneous measurement of drug concentration in the dissolution medium
and in-situ solid phase analysis of the dissolving sample, was introduced and studied. Solid phase
analysis was successfully performed during different stages, enabling a molecular level insight into
the occurring phenomena. Near-infrared (NIR) spectroscopy was utilized in screening of
polymorphs and processing-induced transformations (PITs). Polymorph screening was also studied
with NIR and Raman spectroscopy in tandem. Quantitative solid phase analysis during fluidized
bed drying was performed with in-line NIR and Raman spectroscopy and partial least squares
(PLS) regression, and different dehydration mechanisms were studied using in-situ spectroscopy
and partial least squares discriminant analysis (PLS-DA). In situ solid phase analysis with Raman
spectroscopy during dissolution testing enabled analysis of dissolution as a whole, and provided a
scientific explanation for changes in the dissolution rate. It was concluded that the methods applied
and studied provide better process understanding and knowledge of the drug products, and
therefore, a way to achieve better quality.
FROM POLYMORPH SCREENING TO DISSOLUTION TESTING
– SOLID PHASE ANALYSIS DURING PHARMACEUTICAL
DEVELOPMENT AND MANUFACTURING.
Jaakko Aaltonen
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki, 9/2007, 41 pp.
63
Modern drug development programmes can identify many highly potent drug candidates. However,
many promising drugs end up discarded due to their unfavourable pharmacokinetic properties such
as inadequate bioavailability. Poor bioavailability may result from high first-pass metabolism
and/or instability of the drug in the GI-tract.
Intraoral drug delivery can be utilized for the avoidance of the conditions in the GI-tract and to
circumvent first-pass metabolism. However, in order to gain access to the systemic circulation
through the oral mucosa, the drug molecule must first dissolve into the saliva. Due to the small
volume of saliva, dissolution enhancers must be applied in cases of poorly soluble drugs.
Cyclodextrins (CDs) are known to solubilize lipophilic compounds since they can for water soluble
inclusion complexes with lipophilic drug molecules and/or the lipophilic parts of larger
compounds. However, few questions remain to be answered before one can recommend using CDs
in intraoral formulations, e.g. what is the effect of CDs on formulation bulk mass and what is the
rate at which the drug is released from CD.
In the present study, two cannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD),
were used as model compounds. Cannabinoids such as THC and CBD originate from Cannabis
sativa and have been used for thousands of years for medicinal purposes. Two of the main
cannabinoids in marijuana, THC and CBD have been recognized as being useful in the treatment of
various medical conditions such as nausea, AIDS associated wasting, anorexia, multiple sclerosis,
pain and glaucoma. The per oral use of THC and CBD is, however, limited by their substantial
first-pass metabolism and low aqueous solubility. The intraoral drug delivery route in combination
with CD technology may be useful when administering compounds such as cannabinoids.
The general aim of the present study was to improve intraoral absorption of THC and CBD by
using CDs. The detailed aims were: (1) to develop GC-MS method for the analysis of THC and
CBD from rabbit plasma, (2) to develop an in vivo rabbit model for the evaluation of intraoral
cannabinoid/CD formulations, (3) to increase aqueous solubilities and dissolution rates of THC and
CBD by using CD, (4) to efficiently prepare CD-complexes of THC and CBD and (5) to study the
sublingual absorption of THC and CBD after intraoral administration of cannabinoid/CD
complexes in vivo in rabbits.
A sensitive and accurate GC-MS method was developed for monitoring the two cannabinoids THC
and CBD from rabbit plasma: the method was validated over the range of 0.3–530 ng/ml for THC
and 0.2–450 ng/ml for CBD. The aqueous solubilities of THC and CBD were markedly improved
using CD-derivatives. For example, RM-β-CD (10%; w/v) improved the aqueous solubilities of
THC and CBD by 10000 and 60000-fold, respectively. With β-CD, THC and CBD formed
complexes with limited solubilities, enabling the use of the precipitation complexation method.
Based on the solubility studies, RM-β-CD and β-CD were selected for the preparation of solid THC
and CBD complexes. The amounts of RM-β-CD complexes corresponding to 1 mg of THC and
CBD were 26 and 12 mg, respectively. With β-CD, the corresponding values for THC and CBD
were 125 and 8 mg. The dissolution studies showed that the dissolution rates of THC and CBD
were significantly (p < 0.05) improved by CD-complexation. The in vivo absorption studies were
performed by administering cannabinoid/CD formulations or plain cannabinoids either under the
tongue, per orally via a catheter or intravenously. The absolute bioavailabilities of THC after
intraoral administration of THC/CD complexes were increased by 9-fold (RM-β-CD) and by 12-
fold (β-CD), when compared to per oral absorption of ethanolic THC. The absorption of CBD after
CYCLODEXTRINS IN INTRAORAL DELIVERY OF DELTA-9-
TETRAHYDROCANNABINOL AND CANNABIDIOL
Janne Mannila
Kuopio University Publications A. Pharmaceutical Sciences 101. 2007. 90 p.
64
intraoral administration of a solid CBD/β-CD complex was comparable to intraoral absorption of
ethanolic CBD. The bioavailability of CBD after per oral administration of ethanolic CBD was
markedly lower than that after intraoral administration. The critical factors in the absorption of
THC and CBD from intraoral CD-formulations were assumed to be the rate of dissolution and the
rate of release of cannabinoids from the CD complex.
The results indicate that CD-complexation in combination with intraoral delivery can be utilized in
the development of novel cannabinoid delivery systems.
65
Drug discovery and development is a long and expensive process. In order to decrease drug
development cycle time and costs pharmaceutical industry is searching methods to ease the
decision making and speed up the development processes.
Introduction of atmospheric pressure ionization (API) tehcniques allowed direct coupling of liquid
chromatography (LC) to mass spectrometer (MS). Over the past decade LC-MS has emerged as
preferred analytical tool for many pharmaceutical and bioanalytical applications because of its
sensitivity, selectivity and ease of automation.
The general aim of the present study was to develop LC-MS methods for identification and
quantitation of synthesis products, endogenous compounds, drugs and their metabolites to support
drug development programs. The specific aims of the study were (a) to develop method for
identification of trinucleotide synthesis products, (b) to develop method for quantitation of
endogenous acyl-CoA compounds, and (c) to develop methods for quantitation and identification
of drugs and their metabolites.
Trimeric nucleotide building blocks have been found to be valuable in synthesis of randomized
oligonucleotides for creating peptide libraries to support drug discovery programs. In this study a
LC-MS method for quality control of protected trinucleotides was developed.
The characterization of metabolism profile of a drug candidate is essential to evaluate its efficacy
and toxicity.The metabolism data needs to be available as soon as possible in order to support drug
development process. In this study methods for metabolite identification were developed. New
techniques for sample collection, sample handling and mass spectrometric detection were
implemented. This study also presents a strategy for drug level determination and detection of
metabolites using dried blood spots for sample collection. In addition, new scan modes of hybrid
quadrupole linear ion trap mass spectrometer were evaluated. Furthermore, an approach to
metabolite identification that leverages the fast scanning and high mass accuracy of hybrid
quadrupole time-of-flight mass spectrometry was developed.
The usefulness of a high-field asymmetric waveform ion mobility spectrometer (FAIMS) for
quantitation of low level prostaglandins was evaluated. FAIMS combined with LC-MS was found
to be able to separate the analytes from co-eluting compounds in biological samples. Furthermore,
separation of interconverting anomers was achieved.
DEVELOPMENT OF LC-MS METHODS FOR QUANTITATIVE
AND QUALITATIVE ANALYSES OF ENDOGENOUS
COMPOUNDS, DRUGS, AND THEIR METABOLITES TO
SUPPORT DRUG DISCOVERY PROGRAMS
Timo Mauriala
Kuopio University Publications A. Pharmaceutical Sciences 103. 2007. 126 p.
66
The ability to deliver the drug to the patient in a safe, efficacious and cost-effective manner
depends largely on the physicochemical properties of the active pharmaceutical ingredient (API) in
the solid state. In this context, crystallization is of critical importance in pharmaceutical industry, as
it defines physical and powder properties of crystalline APIs. An improved knowledge of the
various aspects of crystallization process is therefore needed. The overall goal of this thesis was to
gain better understanding of the relationships between crystallization, solid-state form and
properties of pharmaceutical solids with a focus on a crystal engineering approach to design
technological properties of APIs. Specifically, solid-state properties of the crystalline forms of the
model APIs, erythromycin A and baclofen, and the influence of solvent on their crystallization
behavior were investigated. In addition, the physical phenomena associated with wet granulation
and hot-melting processing of the model APIs were examined at the molecular level. Finally, the
effect of crystal habit modification of a model API on its tabletting properties was evaluated.
The thesis enabled the understanding of the relationship between the crystalline forms of the model
APIs, which is of practical importance for solid-state control during processing and storage.
Moreover, a new crystalline form, baclofen monohydrate, was discovered and characterized. Upon
polymorph screening, erythromycin A demonstrated high solvate-forming propensity thus
emphasizing the need for careful control of the solvent effects during formulation. The solvent
compositions that yield the desirable crystalline form of erythromycin A were defined.
Furthermore, new examples on solvent-mediated phase transformations taking place during wet
granulation of baclofen and hot-melt processing of erythromycin A dihydrate with PEG 6000 are
reported. Since solvent-mediated phase transformations involve the crystallization of a stable phase
and hence affect the dissolution kinetics and possibly absorption of the API these transformations
must be well documented. Finally, a controlled-crystallization method utilizing HPMC as a crystal
habit modifier was developed for erythromycin A dihydrate. The crystals with modified habit were
shown to posses improved compaction properties as compared with those of unmodified crystals.
This result supports the idea of morphological crystal engineering as a tool for designing
technological properties of APIs and is of utmost practical interest.
CRYSTALLIZATION AS A TOOL FOR CONTROLLING AND
DESIGNING PROPERTIES OF PHARMACEUTICAL SOLIDS
Sabiruddin Mirza
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki, 25/2007, 64 pp.
67
Gene therapy is a promising new tool to treat some diseases that currently are incurable such as,
genetic disorders, cancer diseases and some retinal diseases, but it has still not become an
established practice in medicine mainly because of either insufficient efficacy or safety problems.
The basic idea in gene therapy is straightforward: the failure to produce some protein coded by a
defective gene is overcome by delivering a new intact gene into the nucleus of the cells. Since
naked DNA as such is not usually efficiently internalized by cells, a carrier system is needed for
gene delivery. The first systems were based on modified viruses. However, the safety issues of
viral gene delivery systems generated a new research field, non-viral gene delivery systems. A
battery of different kinds of alternatives has been generated but none have achieved ultimate
success: non-viral gene delivery systems are still less effective than viral systems.
The objective of the present study was to develop new kinds of gene carriers and to study their
suitability for gene delivery purposes, and also to study cellular mechanisms/properties involved in
gene delivery. Systematic physicochemical and biological characterization of plasmid DNA
(pDNA) carriers and mechanistic studies can help in designing new more efficient non-viral
carriers. We investigated the role of structural properties (shape, PEGylation, molecular weight
(MW)) of poly-L-lysine (PLL) gene carriers, and the influence of biological processes and
properties (cell cycle, intracellular kinetics, glycosaminoglycans, (GAGs)) on polymeric DNA
delivery into a cultured human retinal pigment epithelium (hRPE) cell line, D407. This is important
cell line, since RPE maintains the function of photoreceptors and eyesight, and therefore, is a
potential target for gene delivery. Physicochemical and biological structure-property relationships
of PLLs (3–20 kDa) exhibited no clear correlations between the tested physicochemical properties
(condensation, relaxation, zeta-potential, size and shape of the polyplexes) and biological activities
(cell uptake, transgene expression and cytotoxicity of the polyplexes). Most PLLs (20 kDa)
condense DNA (linear, grafted, branched > dendritic) and condensation is not decreased if the
polyethylene glycol (PEG) content is about 60 % or less (fraction of MW). PEGylated PLLs (20
kDa) form sterically stabilized toroidal or rod-like complexes with diameters of 27–123 nm, but
they are not totally protected from interacting with polyanionic chondroitin sulphate. Further
studies with two carriers with different gene delivery properties, PLL 200 kDa and PEI 25 kDa
(polyethyleneimine), demonstrated a relationship between cell cycle phase (G1, S, G2, M) and
transfection efficiency. The transgene expression of the polyplexes is influenced by cellular uptake
and transcription, and both processes are cell cycle-dependent. Cellular uptake of the polyplexes
was at its highest during the S phase (80–90 %) and lowest during the G1 phase (5–30 %). PEI 25
kDa was a more efficient as a transfection agent than PLL 200 kDa. Furthermore, all promoters
(CMV, SV40, tk, PDE-β) and reporter genes (β-galactosidase, luciferase) showed dependence on
the cell cycle. However, as expected, only a small fraction of the pDNA was found in the nucleus,
partly carrier-bound, but having been accumulated in a cell cycle-dependent manner. Interestingly,
the gene expression after PEI25 kDa mediated transfection was 1–2 orders of magnitude higher
than after PLL mediated delivery even though the gene transfer into the nuclei was approximately
the same. This indicates that there is higher transcriptional efficacy after PEI transfection. Finally,
since it is possible that interactions with endogenous polyanionic GAGs could interfere with
cellular uptake or transgene expression, the GAG profile of synchronized D407 cells was
determined. However, we found that the GAG contents alone do not explain the transfection
efficiencies, since major changes occur simultaneously in the general rate of fluid-phase
endocytosis, and nuclear access of the endocytosed pDNAs.
POLYMERIC CARRIERS IN NON-VIRAL GENE DELIVERY: A
STUDY OF PHYSICOCHEMICAL PROPERTIES AND
BIOLOGICAL ACTIVITY IN HUMAN RPE CELL LINE
Marjo Männistö
Kuopio University Publications A. Pharmaceutical Sciences 102. 2007. 65 p.
68
In conclusion, the present study indicates that the physicochemical properties of PLL polyplexes
can differ to some extent without this having any great impact on biological activity. Also, a
knowledge of cell cycle-dependent variation in gene transfer can hep promote targeting in gene
therapy into uncontrollably dividing cells in diseases such as proliferative vitreoretinopathy (PVR)
and retinal generations, or different types of cancer diseases. Nonetheless even greater
understanding of the intracellular kinetics of polyplexes and underlying molecular mechanisms of
the diseases is needed before of non-viral gene therapy can become a clinical reality.
69
This doctoral thesis discusses the significance of crystallographic properties in pharmaceutical
compacts. The term crystallographic properties here stands for crystallinity, polymorphism,
crystallite size and defects, and texture. The main emphasis of this study is on the formation,
characterization and effects of preferred orientation of crystallites, i.e. texture. The measurements
were mainly made using different x-ray diffraction techniques.
The main objective of this study was to clarify the effects of texture and other crystallographic
properties on the dissolution of pharmaceutical compacts. Since the texture of pharmaceuticals has
not been a subject of wide research prior to this study, another important goal of this study was to
study the texturization of pharmaceuticals and the parameters affecting it.
According to the results, most crystalline pharmaceutical powders will texturize in compression.
The major properties, which affect the degree of texture, were found to be the tabletting behaviour
(described by the yield pressure) of the material, and the size and habit of the sample crystallites.
Plastic materials were found to texturize strongly in compression, whereas substances with elastic
tabletting behaviour did not notably texturize. Strong texture is likely to be found in samples with
large needle or plate-like particles. The compaction pressure and time were not found to have a
major effect on texturization. Moreover, based on the results, the texture of pharmaceutical
compacts is more likely to be unhomogenous.
By performing some theoretical calculations and combining them with the texture measurement
results, it was found that energetic properties of the surface of highly texturized compact differ
from the properties of the sample with less preferred orientation. This was shown in the dissolution
studies indicating that the compacts with less texture had a higher intrinsic dissolution rate than the
samples with a higher degree of preferred orientation. Therefore, when performing dissolution
studies from samples with strong texture, the results should be analyzed and interpreted with great
care. Since the nature of texture of the samples could be modified by modifying the habit of the
particles to be compacted, there is a possibility to modify the dissolution properties of
pharmaceuticals by modifying their texture.
The classical properties affecting the dissolution behaviour of pharmaceuticals are polymorphism
and amorphicity. In the present study, several methods for characterization of amorphicity were
investigated. Amongst others, x-ray powder diffraction proved to be a good method for studying
crystallinity. Moreover, by utilizing x-ray diffraction, it is also possible to get information about the
polymorphic purity and crystallite properties of the sample. In this study, the grazing incidence x-
ray diffraction method was used to study these effects as a function of distance from the sample
surface. The results indicated that the compaction process induced disorders (e.g. amorphicity and
small crystallite size) on the surface of the compacts. These disorders were dependent on the
compaction pressure, and diminished as a function of depth.
Although x-ray diffraction is an old measurement technique, it still has great potential to become a
more effective characterizing method in the field of pharmaceutical materials research. By utilizing
the modern measurement methods, such as texture goniometry and grazing incidence diffraction,
its potential increases even further. Combining modern instrumentation with the ever improving
data analysis methods, including whole profile fitting methods, x-ray diffraction offers a powerful
research tool for pharmaceutical physics.
SIGNIFICANCE OF CRYSTALLOGRAPHIC PROPERTIES IN
PHARMACEUTICAL COMPACTS
Mikko Tenho
Annales Universitatis Turkuensis A I 375, University of Turku, 2007, 108 p.
70
In general, drug absorption into the eye from eye drops is limited. Only 17% of the dose
eventually reaches aqueous humor since corneal epithelium effectively limits drug delivery into the
eye. Gene therapy offers new therapeutic possibilities in ophthalmology, but delivery is an
important issue in this case. Ocular drug delivery experiments require sacrification of at least five
animals at each time point in the drug concentration profile. Improved corneal cell culture model
would therefore be useful in ocular drug and gene delivery experiments, and might reduce the need
for animal experiments.
The aim of the study was to develop a cell culture model of corneal epithelium for pharmaceutical
studies. The cell culture model was tested as a tool in drug and gene delivery experiments.
Immortalised human corneal epithelial cells (HCE) were grown on collagen or laminin covered
permeable support filters with or without feeder fibroblasts. After airlift the cells stratify and
differentiate forming epithelium approximately seven cell layers thick with flattened superficial
cells, tight junctions and microvilli. In the optimised cell model the penetration of β-blockers
increased with lipophilicity following an almost similar sigmoidal relationship with that of excised
rabbit cornea. Paracellular permeability in the HCE-model was generally found to be slightly
higher than in the excised rabbit cornea. The HCE-model has larger paracellular pores at lower
density than the excised cornea, but overall paracellular space was fairly similar. The HCE-model
has esterase activity.
Rabbit corneal epithelium in vivo was transfected using non-viral liposomes (1,2-dioleoyl-3-
trimethylammonium-propane; DOTAP and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DOPE) to secrete transgene product (SEAP; a secreted form of human placental alkaline
phosphatase) into the tear fluid and aqueous humor. Furthermore, suitability of the differentiated
corneal epithelial cell culture for transfection studies was evaluated with DOTAP/DOPE and
cationic polymer (polyethyleneimine; PEI). The transfection levels decreased with the increased
differentiation of HCE cells. PEI was particularly effective in transfecting the dividing cells but
ineffective in the differentiated cells. DOTAP/DOPE also showed high activity in differentiated
cell cultures. Significant SEAP expression was seen for at least three days after in vivo transfection
in the tear fluid and aqueous humor. Rates of SEAP secretion to the basolateral side of
differentiated HCE-cells and into the aqueous humor in vivo were in the same range showing the
predictive applicability of the cell model.
In conclusion, the morphology, physical barrier and permeability properties demonstrate that the
HCE-model closely resembles those of the excised rabbit cornea. Corneal epithelium can be
transfected topically to reach prolonged protein secretion into the tear fluid and aqueous humor,
and the levels of this protein secretion can be predicted correctly with the cell culture model.
CORNEAL EPITHELIAL CELL CULTURE MODEL FOR
PHARMACEUTICAL STUDIES
Elisa Toropainen
Kuopio University Publications A. Pharmaceutical Sciences 100. 2007. 81 p.
71
Polymers as drug carriers provide benefits and means for controlled drug release applications, like
sustained circulation time or cellular targeting of a drug. At all stages of the drug delivery process
the polymeric carrier system has to be safe and biocompatible and, at the same time, maintain or
improve the efficacy of the medical treatment. Several new implications for the polymeric drug
delivery devices have been proposed; among others are the polymers, which are responsive to
temperature changes. These materials require a signal, i.e. critical temperature, which triggers their
phase transition, when water-soluble and hydrophilic polymers become hydrophobic and collapse.
The drug release from these polymers can then be regulated in response to the changes of
temperature.
In this study, the properties and behaviour of a thermosensitive polymer, poly(N-
vinylcaprolactam), PVCL, was evaluated. The phase transition temperature of this polymer is near
the physiological temperature, thus it can be considered as a potential candidate for pharmaceutical
use. The toxicity of PVCL was evaluated by two colorimetric methods using in vitro cell cultures.
The results did not show evidence of cellular toxicity: the cell membranes were found to remain
intact and the cells viable. Fluorescently labelled model particles with thermosensitive PVCL
coating were used in cellular interaction studies. Enhanced cellular attachment was achieved with
PVCL-shell around the particles. This was found to be due to the polymer’s ability to bind to the
cellular surfaces. Poly(N-isopropylacrylamide), PNIPAM, another thermosensitive polymer with
similar characteristics, was compared to PVCL with respect to cellular interactions. It was found
out that the attachment of PNIPAM coated particles to the cellular surfaces was inhibited,
presumably by steric repulsion created by the PNIPAM-chains. Release profiles of different model
drugs from the PVCL hydrogels were estimated, and the effect of physical cross-linking to the
loading and release of drugs was further evaluated. Hydrogen bonding and hydrophobic
interactions were found to be strongly involved in the loading and release of the drugs and the
inhibitory effect of the increasing temperature on drug release was clearly demonstrated. Also,
physico-chemical properties of the drugs and the surrounding environment affected the loading and
release. By physical crosslinking, stable thermosensitive hydrogel particles could be obtained by
creating a tight net, which affected also the drug release. As hydrophilic poly(ethylene oxide),
PEO, is known to improve circulation time and to increase the biocompatibility of polymeric
systems, the in vitro cellular studies were performed also with the PEO-macromonomer grafted to
the PVCL polymer or to the fluorescent model particles. It was concluded that the cellular
interactions of PVCL were diminished when grafted with the PEO-macromonomer, presumably
again by the creation of steric repulsion. The release of drugs from the physically cross-linked
hydrogels containing PEO-grafts was also inhibited because of the ability of PEO to interact with
the cross-linking agent by forming tight hydrogen bonds.
STUDIES ON THERMOSENSITIVE POLY(N-
VINYLCAPROLACTAM) BASED POLYMERS FOR
PHARMACEUTICAL APPLICATIONS
Henna Vihola
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki, 20/2007, 53 pp.
72
73
GRADUJEN TIIVISTELMÄT
74
Syklodekstriinit (CD) ovat rengasrakenteisia sokereita, jotka koostuvat kuudesta (α-CD),
seitsemästä (β-CD) tai kahdeksasta (γ-CD) glukoosiyksiköstä. Renkaan sisäpuolelle jää lipofiilinen
onkalo, jonka sisälle hydrofobiset vierasaineet hakeutuvat muodostaen inkluusiokompleksin.
Syklodekstriinin ulko-osa on hydrofiilinen, ja siksi syklodekstriini on hyvin vesiliukoinen. Tämän
takia syklodekstriinin ja vierasaineen muodostama inkluusiokompleksi liukenee myös hyvin
veteen.
Syklodekstriinejä käytetään farmasiassa apuaineina lipofiilisten lääkeaineiden annostelemisessa.
Elimistöön joutuessaan syklodekstriinit kuitenkin pystyvät reagoimaan myös elimistön omien
lipofiilisten rakenteiden kanssa. Yksi tärkeimmistä lipofiilisista rakenteista on solukalvo, jonka
oikea toiminta on tärkeä solulle ja sitä kautta myös kudoksen ja koko elimistön toiminnalle.
Syklodekstriinit pystyvät liuottamaan solukalvosta erilaisia lipidejä. Sen seurauksena solukalvon
kestävyys kärsii ja samalla myös joidenkin proteiinien toiminta häiriintyy.
Vaikka syklodekstriineillä on suoria vaikutuksia solukalvon toimintaan, syklodekstriinilääkintä on
kuitenkin turvallista. Sopivia syklodekstriinejä mahdollisimman matalilla pitoisuuksilla
käyttämällä saavutetaan syklodekstriinilääkinnän edut samalla haittoja välttäen.
Kokeellisessa osassa tutkittiin hydroksipropyyli-β-syklodekstriini/hydrokortisonikompleksin (HP-
β-CD/HC) aggregaatiota Loftssonin ym. artikkelin pohjalta. Artikkelin mukaan toistettiin
faasiliukoisuus- ja permeaatiokokeet, ja lisäksi liuosten partikkelikokojakauma määritettiin DLS-
laitteella. Artikkelissa todettiin hydrokortisonin permeaation hidastuvan puoliläpäisevän
dialyysikalvon läpi syklodekstriinipitoisuutta kohotettaessa. Hidastumisen selitettiin johtuvan
aggregaattien syntymisestä. Artikkelin mukaisia kokeita toistettaessa faasiliukoisuuskokeiden
tulokset olivat verrattavissa artikkelin tuloksiin. Myös hydrokortisonin permeaatio hidastui
artikkelin mukaisesti, mutta koejärjestelyssä huomattiin muutamia virheitä, jotka korjattiin.
Korjauksien jälkeenkin hydrokortisonin permeaatio hidastui artikkelin tulosten kaltaisesti
syklodekstriinipitoisuutta kasvatettaessa, eikä se noudattanut faasiliukoisuuskuvaajan lineaarista
muotoa. Hidastuminen voi johtua joko aggregoitumisesta tai syklodekstriiniliuoksen kasvavasta
viskositeetista. DLS-laitteen herkkyys ei riittänyt pienen partikkelikoon määrittämiseen.
Suoritettujen kokeiden perusteella ei kuitenkaan pystytä luotettavasti sanomaan syntyykö
syklodekstriiniaggregaatteja kokeen aikana.
SYKLODEKSTRIINIEN VUOROVAIKUTUKSET BIOLOGISTEN
MEMBRAANIEN KANSSA
Hanne Ahtiainen
Farmaseuttisen kemian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
75
Kiinteät aineet sekoittuvat pääasiassa kolmella eri mekanismilla: konvektiolla, diffuusiolla ja
leikkausvoimilla (shear). Sekoittuminen on usein näiden kolmen mekanismin yhdistelmä
tasapainossa segregaation kanssa.
Segregaatio on ilmiö, joka on läsnä kaikissa partikkelisysteemeissä. Segregaatio on termi kiinteiden
aineiden erottumiselle. Segregaatio johtuu partikkelien fysikaalisten ominaisuuksien
eroavaisuuksista. Näitä ominaisuuksia ovat muun muassa koko, muoto, pinnan ominaisuudet ja
kitka. Partikkelien välinen ilmakin voi aiheuttaa segregaatiota. Segregaatio vaikuttaa suuresti
partikkelien sekoittumiseen ja sekoitinvalintaan. Sekoitinvalintaan vaikuttavia muita tekijöitä ovat
esimerkiksi jauheseoksen koheesiivisuus ja valumisominaisuudet.
Jauheseosten homogeenisuuden määrittämisen on sanottu olevan enemmän taidetta kuin tiedettä.
Teollisuudessa käytetyt nykymenetelmät ovat lähinnä invasiivinen näytteenotto ja off-line -
analyysi. Prosessianalyyttiset tekniikat tarjoavat mahdollisuuden non-invasiiviseen, non-
destruktiiviseen ja nopeaan in-line - analyysiin. Tämä edesauttaa prosessin tehokkuutta sekä
parantaa prosessien yleistä ymmärtämistä. Kaksi tavallisinta prosessianalyyttisten tekniikoiden
mittausmenetelmää ovat lähialueen infrapuna- ja Raman-spektrofotometria.
Työn kokeellisessa osassa kehitettiin menetelmä jolla on mahdollista seurata yksittäisen
kohdehelmen liikkeitä 2000-4000 vertikaalisesti ravistellun lasihelmen systeemissä. Ensimmäisen
lähestymistavan lähtökohta oli päällystää kohdehelmi fosforoivalla maalilla ja kuvata systeemi
pimeässä kahdesta kulmasta, jotta saataisiin määritettyä tarkka kolmiulotteinen sijainti
kohdehelmelle. Kohdehelmen oli tarkoitus näkyä kuvissa pimeässä hohtavana alueena.
Ensimmäinen lähestymistapa ei ollut menestyksekäs johtuen lasihelmen maalaamisen validoinnin
vaikeudesta ja riittämättömästä fosforöinnista ja valokuvauskalustosta.
Toisen lähestymistavan perusta oli systeemin voimakas valaiseminen ja lasihelmen päällystäminen
valoa läpäisevällä värillä. Valokuvaaminen tehtiin samaan tapaan kuin ensimmäisessä
lähestymistavassa, muttei pimeässä. Valokuvamateriaali analysoitiin käyttäen MatLab-
tietokoneohjelmaa. Ohjelma laski kohdehelmen tarkat koordinaatit jokaisesta kuvasta 66
mikrometrin tarkkuudella. Menetelmä osoittautui toimivaksi tietyin rajoittein. Edelleen kehiteltynä
menetelmä voisi tarjota tietoa, mikä edesauttaisi segregaation yleistä ymmärrystä ilmiönä. Saadusta
informaatiosta voisi myös olla hyötyä parannettaessa olemassa olevia partikkelisysteemien
tietokonemallinnusmenetelmiä.
JAUHEIDEN SEKOITTUMINEN, EROTTUMINEN JA
JAUHESEOSTEN HOMOGEENISUUDEN MITTAAMINEN
Henrik Ehlers
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
76
Drugs and nutrients are absorbed across intestinal epithelium via one or more routes. The transport
may occur by passive transcellular or paracellular mechanism, active carrier mediated route or by
transcytosis. A variety of transporters are expressed in the small intestine. They mediate the influx
and efflux of endogenous and exogenous compounds. Together with metabolizing enzymes efflux
transporters limit drag absorption and bioavailability of orally administrated drugs by transporting
compounds back to lumen br via circulation to excretive organs. Efflux proteins are also present in
many other tissues, like liver, kidney and brain where they control the fate of drag by affecting
absorption, distribution and elimination processes. In cancer cells efflux proteins located in cell
membranes limit the absorption of anticancer drugs and facilitate resistance against anticancer agents.
Endogenous or exogenous compounds may regulate the function and activity of efflux proteins.
The main physiological role of MRP2 is to limit the absorption of xenobiotics in the intestine, secrete
compounds into bile and facilitate the excretion by transporting xenobiotics to liver and kidney.
MRP2 localizes to the apical membrane of enterocytes and polarized cells. MRP2 can transport a broad
range of endo- and exogenous compounds.
Screening of compounds that bind to efflux proteins is beneficial during drag discovery and
development as efflux proteins can limit the oral bioavailability and lead to drag interactions. Caco-2
cells can be used to predict the intestinal drag absorption via different routes and as a screening method
for substrates of efflux proteins.
The aim of this experimental Pro gradu was to develop a screening method for drags that bind to MRP2
efflux protein using Caco-2 based screening method. The screening method was based on measuring
the fluorescense caused by a marker transported by active efflux proteins while the cells are exposed to
drags. A non-fluorescent promoiety of the marker was applied to passively permeate thröugh cell
membranes and hydrolyze to fluorescent marker by intracellular esterases. The effect of compounds
on the fluorescense of marker and hydrolysis to the fluorescent form was determined before cell
experiments. The specificity and sensitivity of screening method was improved by minimizing the
background fluorescence. The results were compared to the known MRP2 inhibitor, probenecid. The
binding of the drag to the MRP2 efflux protein decreased the transport of the fluorescent marker and
the detected fluorescence. The method underestimated the activity of efflux proteins for poorlj
permeable drags. Compounds with high permeability prevented the efflux of fluorescenl marker by
binding to MRP2-transporter.
DEVELOPMENT OF CELL-BASED (CACO-2) SCREENING
METHOD FOR SUBSTRATES OF MRP2 EFFLUX PROTEINS
Jenni Hannukainen
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
77
KIRJALLISUUSKATSAUS
Adsorptiolla tarkoitetaan atomin tai molekyylin kiinnittymistä kiinteän aineen pintaan ja se voi
aiheuttaa hävikkiä lääkevalmisteissa ja virhettä laboratoriotyöskentelyssä. Lääkeaineen ja astian
välisen interaktion todennäköisyys on suurin liuosmaisilla lääkemuodoilla. Molekyylit ja atomit
voivat kiinnittyä kahdella tapaa kiinteille pinnoille, fysikaalisesti (van der Waalsin voima) ja
kemiallisesti (kovalenttinen- tai ionisidos). Liuosadsorptio on yleisimmin yksikerroksinen ja siten
luonteeltaan kyllästyvä. Muovisten lääkeastioiden tulee olla mahdollisimman inerttejä
soveltuakseen lääkkeiden säilöntään. Lääkeaineilla on havaittu hävikkiä pääasiassa PVC-muoviin,
jossa adsorptiolla on kuitenkin vain pienehkö osuus kokonaishävikeissä. Lääkeaineiden adsorptiota
säilytysastioihin tutkitaan perinteisesti määrittämällä pitoisuuden erotus näytteen ja alkuperäisen
konsentraation välillä. Lääkeainesorptionäytteitä on analysoitu yleisimmin UV-spektrofotometrillä.
Kuitenkin UV-menetelmillä on omat rajoituksensa herkkyyden ja selektiivisyyden suhteen. HPLC-
MS/MS-laitteisto soveltuu havaitsemaan pieniäkin yhdistemääriä heterogeenisistä seoksista ja
soveltuu siten hyvin myös lääkeainehävikin tutkimiseen.
KOKEELLINEN OSA
In vitro soluläpäisykokeissa käytetty kuoppalevymateriaali, käsitelty polystyreeni, muodostaa
pintaominaisuuksiensa perusteella mahdollisen virhelähteen lääkeainehävikille. Työssä kehitettiin
ja validoidoitiin tarkka ja luotettava HPLC-MS/MS-analyysimenetelmä adsorptionäytteiden
analysoimiseen ja suunniteltiin koejärjestelyt materiaaliadsorptiokokeille. Erilaisten lääkeaineiden
materiaalihävikkiä käsitellyssä polystyreenimateriaalissa verrattiin lasiin ja polypropyleeniin
puhdistetussa vedessä ja puskuriliuoksessa. Lasiin ja polypropyleeniin ei havaittu merkittävää
hävikkiä yhdelläkään tutkituista lääkeaineista. Erivarauksellisista lääkeaineista vain positiivisesti
varautuneilla lipofiilisillä emäslääkeaineilla havaittiin huomattavaa hävikkiä vedessä käsiteltyyn
polystyreeniin. Merkittävimmät hävikit havaittiin 4,5 tunnin kuluttua metoprololilla (64,7 ± 6,8 %),
medetomidiinilla (38,4 ± 9,1 %, propranololilla (31,9 ± 6,7 %) ja midatsolaamilla (23,5 ± 6,1 %).
Puskuriliuoksessa adsorptiohävikkiä ei havaittu, syynä tähän pidettiin puskuriliuoksen kumoavaa
vaikutusta materiaalin pintavaraukseen. Käytettäessä lääkeaineilla suurempia konsentraatioita
havaittu suhteellinen hävikki väheni, joka viittaa käsitellyn polystyreenin omaavan vain rajatun
määrän interaktiopaikkoja. In vitro olosuhteissa (suuret konsentraatiot, puskuriliuos)
lääkeaineadsorptio soluläpäisykokeissa käytettyyn polystyreenimateriaaliin on merkityksetöntä.
LÄÄKEAINEIDEN ADSORPTIO MUOVISIIN
SÄILYTYSASTIAMATERIAALEIHIN JA KÄSITELTYYN
POLYSTYREENIIN
Anssi Hassinen
Farmaseuttisen kemian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
78
Säädellysti lääkeainetta vapauttavilla hydrofiilisillä polymeerimatriisivalmisteilla voidaan
saavuttaa lääkeaineen tasainen pitoisuus plasmassa. Muita hydrofiilisten polymeerimatriisien etuja
on yksinkertainen ja taloudellinen valmistusteknologia. Hydrofiiliset selluloosajohdokset ovat
yleisimmin käytettyjä polymeerejä matriiseissa. Ulkopuolisen nesteen tunkeutuessa hydrofiiliseen
polymeerimatriisiin, matriisi turpoaa ja sen ympärille muodostuu geelikerros. Muodostunut
geelikerros hidastaa lääkeaineen vapautumista ulos matriisista. Hydrofiilisistä
polymeerimatriiseista lääkeaineet vapautuvat diffundoituen geelikerroksen läpi ja/tai erodoitumalla
matriisin ulkoreunalta. Geelikerroksen paksuuden pysyessä vakiona lääkeaine voi vapautua
noudattaen 0. kertaluvun kinetiikkaa. Geelikerroksen paksuus, stabiilisuus ja geelikerroksessa
tapahtuvat muutokset vaikuttavat merkittävästi lääkeaineen vapautumiseen. Geelikerroksessa
tapahtuvia muutoksia lääkeaineen vapautumisen aikana on tutkittu yleisimmin tekstuurianalyysillä
ja kuva-analyysillä.
Kokeellisessa osassa tutkittiin kylmäkuivattujen kiinteiden dispersioiden stabiilisuuttaja niitä
sisältävien tablettiformulaatioiden ominaisuuksia. Dispersiot sisälsivät eri suhteissa perfenatsiinia
ja polyetyleeniglykolia tai perfenatsiinia ja polyvinyylipyrrolidonia. Dispersioita tutkittiin
isotermisellä mikrokalorimetrillä (IMC), differentiaalisella pyyhkäisykalorimetrillä (DSC) ja
materiaalin hygroskooppisuutta mittaavalla HMA-laitteella. Stabiilein dispersio tutkimusten
mukaan oli 1/5 perfenatsiini/PEG. Tästä dispersiosta ja vastaavasta PVP-dispersiosta valmistettiin
tablettiformulaatioita, jotka suunniteltiin suussa hajoaviksi tableteiksi. Tableteissa käytettiin
täyteaineena mannitolia ja hajotusaineina krospovidonia ja kalsiumsilikaattia. Hajotusaineet ja
niiden määrät vaihtelivat formulaatioissa suhteessa toisiinsa. Tabletteja puristettiin kahdella eri
puristusvoimalla. Tableteista tutkittiin hajoamisaika, murtolujuus ja perfenatsiinin vapautuminen.
Lisäksi verrattiin differentiaaliselta pyyhkäisykalorimetriltä saatuja tablettimassan ja tablettien
termogrammeja. Tableteista tehtiin myös säilyvyysseurantaa säilyttämällä niitä 60 % suhteellisessa
ilmankosteudessa yksi kuukausi ja tutkimalla ne sen jälkeen samoilla menetelmillä kuin
vastavalmistetut tabletit. Suussa hajoavien tablettien tulee hajota nopeasti, mutta toisaalta niiden
tulee olla riittävän lujia kestääkseen valmistuksen, pakkauksen ja käsittelyn. Tällaiset ominaisuudet
todettiin olevan tableteilla, jotka sisälsivät stabiileinta dispersiota ja krospovidonia.
.
HYDROFIILISET POLYMEERIMATRIISIT / NOPEASTI
LIUKENEVIEN PERFENATSIINIPARTIKKELIEN
STABIILISUUS JA FORMULOINTI
Kristiina Heikkilä
Farmasian teknologian ja biofarmasian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
79
Tässä tutkimuksessa kehitettiin preformulointimenetelmä lääkeainetta sisältäville siloksaani-
matriiseille. Arvioimalla useita menetelmiä, pyyhkäisykalorimetria (DSC), dynaaminen mekaaninen
analyysi (DMA) ja reometria valittiin sopivimmiksi menetelmiksi. Näitä käytettiin tutkimaan
peroksidi- ja platinakatalysoituja siloksaanimatriiseja, jotka sisälsivät joko levonorgestreelia,
estradioli hemihydraattia, traneksaamihappoa tai mefenaamihappoa. DSC:llä analysoitavat
ominaisuudet olivat yksittäisten aineiden yhteensopivuus sekä siloksaanimatriisien
ristiinsitomisentalpia. Reometriaa käytettiin selvittämään silloittamattomien näytteiden G’/G"
risteämisfrekvenssi ja moduli, geeliytymispisteen lämpötila ja moduli, sekä silloittumisnopeus.
Silloitettujen näytteiden lasipistettä, sekä niiden elastista modulia eri lämpötiloissa selvitettiin DMA:n
avulla.
DSC:llä nähdyt yhteensopimattomuuden korreloivat varsin hyvin näytteiden mekaanisten
ominaisuuksien kanssa. Heikompiin mekaanisiin ominaisuuksiin johtavat yhteen-
sopimattomuudet havaittiin aina lääkeaineen ja reaktiokatalyytin välillä. Tästä syystä DSC-analyysit
voitaisiin keskittää koskemaan ainoastaan lääkeaineita ja reaktiokatalyyttejä tutkittaessa suurta
lääkeainejoukkoa. Elastomeerien silloittumisentalpiat eivät korreloineet niin selvästi lopputuotteiden
mekaanisten ominaisuuksien välillä, joten pelkästään näillä tutkimuksilla ei saada tarpeeksi tietoa
lopputuotteen ominaisuuksista. Reometrialla huomattiin, että kaikki lääkeiaineet nostivat
elastomeerien mekaanista lujuutta. Lääkeainetta sisältävien näytteiden G’/G" risteämisarvot tai
geeliytymispisteen arvot eivät poikenneet suuresti toisistaan, silloittumisnopeuksien välillä oli selviä
eroja. Tämä saattaa kuitenkin johtua suuresta hajonnasta silloittamattomien ja silloitettujen näytteiden
G'-arvojen välillä. DMA:lla havaittiin selviä eroja silloitettujen näytteiden E'-arvojen välillä, joita ei
muilla menetelmillä havaittu. Näin ollen lopputuotteen elastisuutta voitiin arvioida tehokkaasti.
Lasipisteen ilmenemislämpötilalla ei kuitenkaan havaittu olevan selkeää yhteyttä käytetyn lääkeaineen
kanssa. Lisäksi näytteet, joilla ei havaittu silloittumista reometrin avulla, eivät soveltuneet
analysoitaviksi DMA:lla. Käytetty preformulaatiomenetelmä pystyi selkeästi tekemään eron
yhteensopivien ja yhteensopimattomien siloksaaninäytteiden välille.
Käytetyt kolme menetelmää tukivat toisiaan, ja kaikki tuottivat tietoa mitä muut menetelmät eivät
tuottaneet. Tästä syystä kaikkia kolme menetelmää tulisi käyttää myös tulevissa siloksaanimatriisien
preformulaatiokokeissa. DSC:llä tulisi tutkia lääkeaineiden ja katalyyttien yhteensopivuutta,
reometrillä tulisi tutkia silloittumisen nopeutta ja lämpötilaa, ja DMA:ta voisi käyttää tutkimaan niiden
näytteiden mekaanisia ominaisuuksia, jotka ovat silloittuneet reometritutkimuksissa. Lisäksi, mikäli
DSC:llä havaittaisiin termodynaaminen reaktio siloksaanien ekstruusiolämpötiloissa, XRPD:tä
voitaisiin käyttää tutkimaan muutoksia näytteiden morfologiassa. Tämä menetelmä ei keskittynyt
analysoimaan lääkeaineen vapautumista matriiseista, joten tulevissa tutkimuksissa tulisi paneutua
näihin ominaisuuksiin.
DEVELOPMENT OF PREFORMULATION METHOD FOR
DRUG-CONTAINING SILOXANE MATRICES
Ville Petteri Heljo
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
80
Suun kautta nautittavan lääkeaineen päätyminen valmisteesta elimistöön vaatii monia tapahtumia.
Lääkeaineen on mm. liuettava ruoansulatuskanavan nesteisiin, ennen kuin se pystyy imeytymään.
Keskeisiä tekijöitä lääkeaineiden liukenemiselle ruoansulatuskanavassa ovat mm. sisällön pH,
pintajännitys, solubilisoivien sappisuolamisellien määrä ja muu nautittu ravinto. Joissakin
tapauksissa lääkeaineen dissoluutiota ruoansulatuskanavassa voidaan arvioida in vitro -kokeiden
avulla, mitä voidaan käyttää hyödyksi mm. tuotekehitystarkoituksessa.
In vitro -dissoluutiota tutkitaan erilaisilla väliaineilla ja laitteistoilla. In vivo/in vitro -korrelaation
saavuttamiseksi on kehitetty biorelevantteja väliaineita, jotka sisältävät ruoansulatuskanavassa
esiintyviä pinta-aktiivisia aineita. Ne ovat kuitenkin kalliita ja analytiikaltaan haastavia, ja eivät
siksi sovi lääketeollisuuden rutiinikäyttöön.
Tutkimuksen tavoitteena oli kehittää synteettisistä surfaktanteista uusia dissoluutioväliaineita ja
verrata niillä tehtyjen dissoluutioajojen tuloksia aikaisemmin käytettyihin väliaineisiin.
Väliaineiden ominaisuuksia tutkittiin mittaamalla niistä pintajännitys, lääkeaineen liukoisuus ja
tutkimalla dissoluutiota. Pintajännitys on ruoansulatuskanavan nesteissä alhainen, mikä nopeuttaa
kostumista ja dissoluutiota. Lääkeaineiden tasapainoliukoisuus tutkittuihin väliaineisiin
määritettiin. Dissoluutiota tutkittiin läpivirtausdissoluutiolaitteella, jossa lääkeaine liukenee
valmisteesta väliaineeseen avoimessa systeemissa.
Lääkeaineen luonne määrää sen käyttäytymisen; ionittuvan lääkeaineen dissoluutiota ohjaa lähinnä
väliaineen pH, ionittumattoman dissoluutiossa keskeinen tekijä on pinta-aktiivisten aineiden määrä.
Malliaineiksi valittiin ionittuva ibuprofeeni, lipofiilinen spironolaktoni ja erittäin lipofiilinen
danatsoli. Tutkimus tuki käsitystä, että tasapainoliukoisuuden dynamiikka on hyvin erilainen
verrattuna dissoluutiotapahtumaan. Siksi lääkeaineen tasapainoliukoisuus ei anna suoraa viitettä
lääkeaineen dissoluutiokäyttäytymisestä, vaikka väliaine olisi sama.
Danatsolille onnistuttiin löytämään dissoluutioväliaine, jossa dissoluutio vastasi aiemmin käytettyä
sappisuolaliuosta. Danatsolin keskeisin dissoluutiomekanismi on solubilisaatio, ja pinta-aktiivisen
aineen määrällä havaittiin olevan suora vaikutus dissoluutioon. Ionittuvana lääkeaineena
ibuprofeeni käyttäytyi pikemminkin väliaineen pH:n kuin pinta-aktiivisen aineen määrän
mukaisesti. Spironolaktonin käyttäytyminen vaihteli erilaisissa liuoksissa epäennustettavasti.
Spironolaktonille voisi kuitenkin olla mahdollista löytää pinta-aktiivinen aine, jossa dissoluutio on
ennustettavampi. Tutkituista surfaktanteista tällainen voisi olla natriumlauryylisulfaatti.
BIORELEVANTIT DISSOLUUTIOMENETELMÄT
Liisa Itkonen
Farmasian teknologian ja biofarmasian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
81
Inulin is physiologically inert, non-toxic long-chain fructose and interesting for its potential use in
pharmaceutical applications e.g. as a filler-binder in tablets. The purpose of the study was to
evaluate the suitability of inulin in wet granulation and roller compaction processes. The main
process parameters affecting the properties of the granules were determined. The usability of the
inulin granules produced by these two granulation methods was investigated in tablet compaction.
Three inulin qualities were characterised to find out the most suitable inulin quality for wet
granulation. Frutafit® TEX! was the quality with the best wet mass consistency measured on mixer
torque rheometer and a laboratory-scale high-shear granulation and therefore it was chosen for further
granulation experiments. Frutafit® TEX! had the highest degree of polymerization and was less
sticky on nature that the other two qualities as water was added to the granulation process.
Scanning electrone microscope pictures showed that Frutafit® TEX! consisted of spherical and
hollow particles.
Full factorial experimental design for three variables at two levels was performed both for
granulation on a laboratory-scale high-shear granulator and a roller compactor to investigate their
effects on the properties of the granules. Frutafit® TEX! was granulated in a laboratory-scale high-
shear granulator using water as granulation liquid. Process variables were water amount, impeller
speed and wet massing time. The granules were tray-dried and milled. Roller compaction was
performed after blending inulin with lubricant, sodium stearyl fumarate. Process variables were
pressure, roll speed and screw feeder speed. Roller compacted flakes were milled.
Granules produced on both of the methods were characterized by performing a sieve analysis, bulk
and tap density measurements and tablet compaction experiments on all the batches. Porosity
measurement was performed on a mercury porosimeter of the extreme points in regard to porosity.
Scanning electron microscope pictures were taken of the extreme and the center points.
The properties of the dry granulated granules were found to be mostly affected by pressure. The
granules produced at high pressures had the highest bulk densities and the lowest tablet hardness
values. The properties of the wet granulated batches were mostly dependent on the amount of water
added into the granulation process although no strong correlations were found.
It was found that Frutafit® TEX! was not a suitable excipient to be used as a single excipient in
we1 granulation with water as granulation liquid because of its equipment stressing sticky nature, the
low water absorption capacity and the poor liquid distribution abilities. Frutafit® TEX! was well
processable on roller compaction due to its good flowability and binding properties. However
there was a remarkable loss of flowability and compactability of the granules in comparison with the
original powder as the spherical, hollow structure of the original particles was altered by the roller
compaction process.
STUDIES ON WET GRANULATION AND ROLLER
COMPACTION OF INULIN
Henna Juvonen
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
82
Nowadays an increasing amount of the drug candidates are lipophilic and poorly water-soluble,
which is problematic, because the drug has to be dissolved before it can be absorbed. Fatty food or
lipid based formulations are known to increase the bioavailability of many poorly water-soluble drugs.
For several different reasons there are, however, not many oral lipid based formulations on the market.
This Master´s thesis deals with solid lipid based dispersions as carriers for poorly water-soluble
compounds. The literature review covers different types of lipid formulations, methods for
analysing the solid state, the use of triglycerides as excipients and polymorphism. The emphasis
is on the production and analysis of solid dispersions. The physical characteristics of the carrier and
the drug plays an important role in the functionality of the lipid based solid dispersion. The aim of
the experimental part was to study the phase behaviour of three model lipids (trimyristin, tripalmitin
and tristearin) and a hydrophobic model drug (phytosterol). The effect of the chosen lipid, drug
content and production method on the physical state of the dispersion, for example crystallinity and
polymorphism, was studied. The triglycerides were known to exist in several different polymorphs, of
which the α-, β'- and β -forms are the most important. β-sitosterol, the main component of
phytosterol, was known to exist as an anhydrate, hemihydrate and monohydrate. The water content of
these three forms is different. Solid lipid based dispersions were prepared at several different mixture
ratios by crystallizing the components from the melt or from solution (antisolvent method). Physical
mixtures were prepared for comparison by grinding the components in a mortar. Ali dispersions were
analysed by differential scanning calorimeter (DSC) and X-ray powder diffractometer (XRPD). Some
of the dispersions were also analysed by Raman and Infrared spectroscopy.
The triglycerides and phytosterol crystallized separately at ali tested compositions. On the DSC scan
the melting of both components was seen separately and the X-ray diffraction patterns consisted of
peaks originating from the pure components. The components showed no or insignificant solid solubility,
vvhich could be because of the sizes of the molecules. The results obtained with the different methods
were mostly consistent, which shows that DSC, XRPD, Raman and IR-spectroscopy are feasible
methods for the analysis of solid lipid based dispersions. The research provided preliminary
information about possible production methods for solid lipid based dispersions in the laboratory scale
and problems associated with the methods.
SOLID LIPID DISPERSIONS AS CARRIERS FOR POORLY
WATER-SOLUBLE COMPOUNDS
Johanna Laiho
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
83
Peroraalisesti annosteltujen lääkeaineiden pääasiallinen imeytymispaikka on ohutsuoli, jonka
enterosyyteissä on sekä endogeenisten yhdisteiden että lääkeaineiden ja muiden elimistölle
vieraiden aineiden metaboliaan osallistuvia entsyymejä. Näin ollen lääkeaine altistuu ensikierron
metabolialle jo imeytymisvaiheessa. Lääkeainemetabolialla tarkoitetaan kaikkia niitä kemiallisia
reaktioita, jotka lääkeaine käy läpi elimistössä ennen erittymistään. Hyvin hydrofiilinen tai
hydrofobinen lääkeaine voi erittyä elimistöstä myös sellaisenaan. Metaboliareaktiot voidaan jakaa I
ja II vaiheen reaktioihin. II vaiheen reaktioita ovat erilaiset konjugaatioreaktiot, kuten
glukuronidaatio, sulfonaatio, asetylaatio, metylaatio ja glutationikonjugaatio. Lääkeaineelle
tapahtuva metabolia vaikuttaa usein sen farmakokinetiikkaan ja sillä voi olla vaikutusta myös
lääkeaineen farmakologiseen tehoon ja toksisuuteen, minkä vuoksi lääkeaineen metaboliareittien
tunteminen on tärkeää. Lisäksi metaboliareittien tunteminen auttaa välttämään lääkeaineiden
yhteisvaikutuksia, jotka usein aiheutuvat metaboliaentsyymien induktiosta tai inhibitiosta.
Caco-2-solulinja on laajasti hyväksytty in vitro -malli ohutsuolessa tapahtuvan imeytymisen
ennustamiseen ja sen hyödyntämistä ohutsuolessa tapahtuvan ensikierron metabolian tutkimisessa
selvittävät tällä hetkellä useat tutkimusryhmät. Caco-2-solut ovat peräisin ihmisen paksusuolen
syöpäkasvaimesta ja ne erilaistuvat spontaanisti ohutsuolen enterosyyttejä muistuttavaksi
soluiksi kasvatusjakson aikana.
Työn tarkoituksena oli selvittää Caco-2-solulinjan käyttökelpoisuutta II vaiheen metabolian in vitro
tutkimisessa. Kirjallisuuskatsauksessa keskityttiin tutkimuksiin, joissa on selvitetty II vaiheen
konjugaatioentsyymien ekspressiota ja aktiivisuutta Caco-2-soluissa. Lisäksi selvitettiin kuinka
hyvin Caco-2-solulinja vastaa II vaiheen metabolian osalta in vivo olosuhteita eli metaboliaa
ohutsuolessa. Kokeellisessa osassa Caco-2-solujen soveltuvuutta II vaiheen metabolian tutkimiseen
selvitettiin entsyymiaktiivisuus- ja kulkeutumiskokeilla. Entsyymiaktiivisuuskokeiden tulokset
viittasivat erilaistuneiden Caco-2-solujen sisältävän enemmän metaboloivia entsyymejä kuin
erilaistumattomat Caco-2-solut. Kulkeutumiskokeissa muodostui glukuronidi- ja
sulfaattikonjugaatteja, mikä osoitti Caco-2-soluissa olevan aktiivisia UGT- ja SULT-isoformeja.
Muodostuneiden glukuronidi- ja sulfaattikonjugaattien erittymiseen Caco-2-soluista osallistuivat
sekä apikaali- että basolateraalikalvolla sijaitsevat MRP-effluksiproteiinit.
Caco-2-solut vaikuttavat kehityskelpoiselta menetelmältä ohutsuolessa tapahtuvan II vaiheen
metabolian ennustamiseen. Menetelmän käyttökelpoisuutta rajoittaa Caco-2-solujen
karakterisoinnin keskeneräisyys, erityisesti eri UGT-isoformien ekspression osalta, sekä Caco-2-
solujen heterogeenisyyden aiheuttama vaihtelu tuloksissa laboratorioiden sisällä ja eri laboratorioiden
kesken.
II VAIHEEN METABOLIA CACO-2-SOLUISSA
Laura Laine
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
84
Tutkielmassa tarkastellaan erilaisten binääriseosten kvantitatiiviseen faasianalyysin kehitettyjen
röntgendiffraktiomenetelmien luotettavuutta, ja toisaalta pyritään määrittämään kulloisenkin
seostyypin kannalta mielekkäin ja tarkoituksenmukaisin datankäsittelymenetelmä. Seostyypit olivat
saman yhdisteen kahden polymorfin, polymorfin ja amorfisen muodon, kahden kiteisen yhdisteen
sekä polymorfin ja pseudopolymorfin väliset seokset.
Tutkielmassa tutkittiin karbamatsepiinin III-polymorfin faasiosuuksia erilaisissa binääriseoksissa
röntgendiffraktiomenetelmin. Kvantitaviisen faasianalyysin tekemiseen käytetyt röntgendiffraktio-
menetelmät olivat suoran vertailun, Wakelinin, sisäisen standardin ja Rietveldin menetelmät.
Tutkittavat yhdisteet olivat karbamatsepiinin III-polymorfin binääriseoksia karbamatsepiinin I-
polymorfin, amorfisen muodon, karbamatsepiinin dihydraatin seka kalsiumvetyfosfaattidihydraatin
kanssa.
Suoritettujen analyysien perusteella päädyttiin suosittelemaan tiettyjä menetelmiä eri
binääriseosten faasianalyysiin. Yleisesti kahden kiteisen aineen seoksissa hyödyllisimmat
menetelmät ovat Rietveldin menetelmä tai suoran vertailun menetelmä. Jos seoksen muodostavilla
yhdisteillä on sama lineaarinen absorptiokerroin, saadaan Wakelinin menetelmällä myöskin hyviä
tuloksia, tämän tutkielman perusteella parempia kuin suoran vertailun menetelmällä. Yhdisteen
kiteisyysastetta määritettäessä suoran vertailun menetelmä on parempi kuin Wakelinin menetelmä,
ja menetelmänä varsin luotettava, kunhan kiteisyysaste on kohtuullinen. Polymorfin ja
pseudopolymorfin välisten binääriseosten tarkastelussa lienee syytä huomioida tekstuurin vaikutus.
Tässä tutkielmassa sitä ei tehty.
FARMASEUTTISTEN BINAARISEOSTEN KVANTITATIIVINEN
FAASIANALYYSI RÖNTGENDIFFRAKTIOMENETELMIN
Kimmo Lehtinen
Fysiikan laitos, Turun yliopisto
85
Röntgendiffraktiolla voidaan faasiseoksista identifioida sen sisältämät faasit ja määrittää eri faasien
suhteellisten massojen suuruudet. Teofylliini- ja tolbutamidi-lääkeaineiden tableteista tutkittiin
dehydraatiokinetiikkaa sekä polymorfisten muotojen faasimuutoksia massaosuuksineen yhteistyönä
Kuopion yliopiston kanssa. Katalyyteistä Pt/Al203 ja Ni/SiO2 mitattiin, yhteistyönä Åbo
Akademin kanssa, diffraktogrammien piikin profiilista platina- ja nikkelifaasin keskimääräisiä
kidekokoja.
Tolbutamidin polymorfisia muotoja identifioitiin jauheesta ja tabletista. Tavoitteena oli selvittää,
muuttuuko tolbutamidin IV-muoto tabletissa puristuksen aikana eri polymorfiseksi muodoksi.
Tolbutamidi-mittaukset todensivat kylmäkuivausmetodilla valmistetun tolbutamidi IV-muodon
jauheen muuttuneen II-muodoksi. Tolbutamidin IV-muodosta puristetuissa tableteissa havaittiin II-
muodon lisaksi pieni määrä III-muotoa. On mahdollista, että III-muotoa on syntynyt puristuksen
aikana.
Teofylliinin metastabiilimuodon jauheen faasimuutosta tutkittaessa havaittiin anhydraatin II-
muodon määrän kasvavan koko 22 % RH:n säilytyksen ajan. Metastabiilin III-muodon määrä
väheni koko säilyttämisen ajan. Muodon III nopeamman vähenemisen aikana monohydraatin
massaosuus kasvoi 23 %:sta 41 %:iin. Tämä kasvu todistanee, että III-muoto voi muuttua II-
muodon lisaksi myös monohydraatin suuntaan.
Teofylliinin röntgendiffraktiomittauksissa tutkittiin myös teofylliinin neljästä eri muodosta eri
puristuspaineissa valmistettuja tabletteja. Selvitettävänä oli, kuinka stabiileja neljästä eri
teofylliinimuodosta valmistetut puristeet ovat ja miten mahdolliset transitiot etenevät, kun tabletteja
säilytetään kuivassa ympäristössä. Teofylliinin monohydraattitableteissa havaittiin suuntaus, että
korkeammilla puristuspaineilla valmistetuissa tableteissa faasimuutos kohti II-muotoa oli edennyt
pidemmälle kuin alhaisemmilla puristuspaineilla. Teofylliinin anhydraatin III-muoto todettiin
erittäin epästabiiliksi vallitsevissa olosuhteissa. Metastabiilin III-muodon tableteista havaittiin, että
III-muodon pitoisuus väheni ja II-muodon pitoisuus kasvoi puristuspaineen kasvaessa. Voidaan
olettaa, että paineen nostaminen nopeuttaa huomattavasti transitiota muoto III -> muoto II.
Teofylliinin anhydraatin I-muodon tableteissa havaittiin I-muodon hidas transitio kohti II-muotoa.
Muodon II havaittiin olevan stabiili vallitsevissa olosuhteissa.
Katalyytin Pt/Al2O3 mittaussarjassa tutkittiin kahdella eri tavalla valmistettua katalyyttiä.
Analyysituloksena havaittiin platinan keskimääräisen kidekoon kasvavan platinan massaosuuden
mukana. Kidekoko oli suurempi H2PtCl6(4H20)-liuoksella valmistetussa katalyytissä kuin
Pt(NO3)2-liuoksella valmistetussa. Katalyytissä Ni/Si02 nikkelin keskimääräisen kidekoon
havaittiin kasvavan nikkelin massaosuuden mukana.
KVANTITATIIVINEN ANALYYSI JA KIDEKOON MÄÄRITYS
JAUHERÖNTGENDIFFRAKTIOLLA
Tero Lehto
Fysiikan laitos, Turun yliopisto
86
Geeniterapia on lupaava tulevaisuuden hoitomuoto, joka mahdollistaisi lukuisien sairauksien
hoidon ja ennaltaehkäisyn. Sen edut nykyisiin hoitomuotoihin verrattuna ovat kiistattomat, mutta
ongelmana on edelleen se, ettei riittävän tehokasta ja turvallista geeninsiirtomenetelmää ole
löydetty. Tämän vuoksi myöskään formulointiin ei ole toistaiseksi kiinnitetty juuri huomiota.
Geeninsiirtoon käytettävistä menetelmistä virusperäiset geeninkantajat ovat jo teholtaan varsin
hyviä, mutta niiden ongelmana on turvallisuus. Viruksettomat kantaja/DNA-kompleksit puolestaan
kärsivät heikosta biologisesta tehosta.
Viruksettomien geeninsiirtokompleksien biofysikaalisella karakterisoinnilla pyritään selvittämään
kompleksien biologisen aktiivisuuden taustalla olevat fysikaaliset ominaisuudet. Korrelaatioiden
löytäminen helpottaisi osaltaan riittävän tehokkaan viruksettoman kantaja/DNA-kompleksin
kehitystyötä. Mahdollisia tutkimusmenetelmiä on useita. Yhdistämällä mikroskopisia,
spektrometrisia, kalorimetrisia ja valonsirontaan perustuvia menetelmiä, pyritään saamaan tietoa
siitä, miten esimerkiksi kompleksien koko, pintavaraus, rakenne tai lämpöstabiilius, vaikuttavat
niiden biologiseen aktiivisuuteen. Korrelaatioiden löytäminen on vielä toistaiseksi ollut vaikeaa,
mutta eri menetelmillä on jo nyt saatu paljon hyödyllistä tietoa kompleksien fysikaalisista
ominaisuuksista.
Tutkielmaan kuuluneessa erikoistyössä selvitettiin apu aineiden vaikutuksia viruksettomien
geeninsiirtokompleksien biologiseen aktiivisuuteen in vitro. Apuaineiden vaikutusta geeninsiirron
tehokkuuteen tutkittiin kahdella eri geeninkantajalla.. Lisäksi selvitettiin apuaineiden pitoisuuden
merkitystä ja seurattiin eri formulaatioiden biologisen tehon säilyvyyttä ajan funktiona.
Erikoistyöhön sisällytettiin lisäksi biofysikaalinen tutkimus, jonka tarkoituksena oli selvittää
formulaatioiden ominaisuuksien merkitystä niiden lämpöstabiiliudelle ja sitä kautta biologiselle
aktiivisuudelle in vitro. Tutkimus toteutettiin differentiaalisella pyyhkäisykalorimetrialla.
Erikoistyön tulokset osoittavat, että apuaineilla voi olla suuri merkitys kantajan geenin siirron
tehokkuudelle lyhytkestoisesti. Apuaineet eivät kuitenkaan riitä ratkaisuksi suspensiomuotoisten
formulaatioiden stabiilius- ja säilyvyysongelmiin.
VIRUKSETTOMIEN GEENINSIIRTOKOMPLEKSIEN
BIOFYSIKAALINEN KARAKTERISOINTI
Anna Lukkarinen
Farmasian teknologian ja biofarmasian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
87
The goal of this study was to find out the physicochemical characteristics of Trimethoprim and
Sulfamethoxazole. This research is important because former studies have made it clear that these drugs
have weaker and variable bioavailability when treating infants and small children with suspensions.
The aim of this study was find out in vitro characteristics of these drugs as pure substances and
compare these results with results of suspended drugs. After this study it could be possible to develop a
product which has a repeatable and reliable absorptive amount.
There might be many reasons why Trimethoprim and Sulfamethoxazole have diminished availability.
One reason might be that former pharmacokinetic studies have been made in developing countries,
where malnourished children are not rare. Malnutrition might incur pathological changes and change
the absorbed amounts of drugs. Another reason could be normal physiological changes in the body
during the first months of life. It's also possible that some changes happen in the physicochemical
characteristics of drugs during storage in product or in the gastrointestinal tract.
Theoretical part goes into special features of a child's gastrointestinal tract and is about normal in
the development of a child's gastrointestinal tract. There are discussed about some theoretical
physicochemical characteristics of Trimethoprim and Sulfamethoxazole. The final part of work is
about methods used to analyze physicochemical characteristics of drugs. First in the practical part of
the research was to find out the behaviour of pure and separated or mixed Trimethoprim and
Sulfamethoxazole. Many methods were used to analyze these drugs, for example XRPD and FTIR.
The aim was to find out something about crystal structure and solubility's in different pH-values, like
hydrate or polymorphic forms.
In this study it was found out that the crystal structure of Trimethoprim was without any crystal
water and there were no molecular changes in suspended Trimethoprim. The crystal structure of
Sulfamethoxazole was as dry substance in polymorphic form I. The same form was also analyzed
when it was suspended in citrate buffered solution, but there was strong evidence that some part of
Sulfamethoxazole turned into semihydrate form. In Trimethoprim and Sulfamethoxazole mixtures (1:5)
there was no evidence of semihydrate nor complex forming. pH-solubility profile of Trimethoprim
studies exposed that its solubility increases surprisingly close to pH five. There was no explanation to
clarify this phenomenon. pH-solubility profile of Sulfamethoxazole was like it should be according to
theory of ionization. The solubility increases when pH increases. Solubility studies didn't expose any
findings that could support complex forming in mixtures.
According to previous studies and the results of the present study it seems that failure of bioavailability
of Trimethoprim and Sulfamethoxazole exists because physicochemical changes of substances
happen later in the gastrointestinal tract. Another possibility is that malnutrition is incurred
pathological changes. In this study behaviours of these medicines in the circumstance of the
gastrointestinal tract were not studied.
CHANGE OF PHYSICOCHEMICAL CHARACTERISTICS OF
TRIMETHOPRIM AND SULFAMETHOXAZOLE IN A
SUSPENSION PRODUCT
Iiris Mainio
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
88
Eräs huokoisen piin (PSi) uusimmista sovelluksista on lääkeaineen kantajamateriaalina
toimiminen. Huokosrakenteen tuoma suuri ominaispinta-ala ja huokostilavuus mahdollistavat
erilaisten lääkemolekyylien adsorption huokosiin. Adsorption tapahtuessa halkaisijaltaan vain
muutamia kertoja lääkemolekyyliä suurempiin huokosiin, jää lääkeaine epäjärjestyneeseen tilaan,
jonka on havaittu parantavan lääkeaineen vesiliukoisuutta sekä permeaatiota.
Edellytys toimivalle lääkeannostelumenetelmälle on lääkeaineella täytetyn materiaalin
tasalaatuisuus lääkeaineen latausasteen ja vapautumisnopeuden suhteen. Pyrittäessä
mahdollisimman korkeaan latausasteeseen voi huokosten ulkopuolelle jäädä huomattava määrä
lääkeainetta, joka kiteytyy pinnalle. Niukkaliukoisten lääkeaineiden tapauksessa huokosten
ulkopuolelle kiteytynyt osuus tekee lääkeaineen vapautumisesta ennakoimatonta.
Tutkimuksen tarkoituksena oli selvittää voidaanko lääkeaineen latausastetta ja pinnalle kiteytyvää
osuutta hallita adsorptioon vaikuttavien parametrien kautta. Lääkeaineen latausmenetelmäksi
valittiin adsorptio liuoksesta, sillä tämä mahdollisti eri parametrien helpon kontrolloinnin.
Adsorboituneen lääkeaineen määrä selvitettiin termogravimetrisesti, josta voitiin erottaa
differentiaalista pyyhkäisykalorimetria käyttäen pinnalle kiteytynyt osuus. Tutkimuksessa
mallilääkeaineina käytettiin niukkaliukoisia ibuprofeenia ja griseofulviinia, joita ladattiin termisesti
karbidoituun (TCPSi) ja termisesti oksidoituun (TOPSi) meso-huokoiseen piihin. Molemmille
mesohuokoisen piin pintakäsittelyille määritettiin lääkeaineen adsorptioisotermit eri liuottimia
käyttäen. Mahdollisia vuorovaikutuksia lääakeaineen ja liuottimen välillä tutkittiin FTIR-
spektrometrillä.
Ibuprofeenilla havaittiin, että liuoksen konsentraatiota säätelemällä oli mahdollista saavuttaa
korkea latausaste, huokosten ulkopuolelle kiteytyneen osuuden silti jäädessä mitättömäksi.
Ibuprofeenilla havaittiin myös liuottimen ja lääkeaineen välisten vuorovaikutusten roolin olevan
merkittävä suurilla konsentraatioilla. Erityisesti TOPSi tehosti ibuprofeenin adsorptiota johtaen
suureen pinnalle kiteytyneeseen osuuteen. Seurauksena korkeasta pintaosuudesta havaittiin
huokosten osittainen tukkeutuminen, mikä heikensi adsorptiota huokosiin. Griseofulviinin ei
puolestaan havaittu kiteytyvän huokosten ulkopuolelle adsorptiossa käyteillä liuottimilla. Korkein
latausaste saavutettiin poikkeuksetta suurimmalla liuoksen konsentraatiolla.
Jatkossa tutkimusta tulisi laajentaa ottamaan huomioon entistä tarkemmin eri vuorovaikutuksia ja
niiden merkitystä latausasteelle. Erittäin tärkeää olisi selvittää myös huokosissa olevan lääkeaineen
vakaus ja säilytysolosuhteiden vaikutus vakauteen.
LÄÄKEMOLEKYYLIEN ADSORPTIOSTA MESOHUOKOISEEN
PIIHIN
Ermei Mäkilä
Fysiikan laitos, Turun yliopisto
89
Tablettirakenne syntyy jauhepartikkelien muodostaessa välilleen sidoksia pysyvien
muodonmuutosten keinoin. Muodostuvaan rakenteeseen vaikuttavat jauheen ominaisuuksista
partikkelikoko ja -muoto sekä jauheen todellinen tiheys. Jauheen käyttäytymistä tabletoinnin
aikana voidaan tutkia tabletointiominaisuuksien avulla, jotka jaetaan tabletoitavuuteen,
puristuvuuteen ja puristettavuuteen. Myös erilaisten puristusyhtälöiden avulla saadaan arvokasta
tietoa jauheen käyttäytymisestä puristusprosessin aikana. Tablettirakennetta voidaan kuvantaa
esimerkiksi pyyhkäisyelektronimikroskopian tai tietokoneavusteisen röntgensädekerroskuvauksen
avulla, jotka antavat visuaalista käsitystä muodostuneesta tablettirakenteesta.
Kokeellisen osan tavoitteena oli tutkia kahdesta eri lääke aineesta ja tärkkelysasetaatista
muodostuvien matriisitablettien rakennetta ja tablettien puristuksessa käytettyjen jauheiden
tabletointiominaisuuksia. Tabletoitavia materiaaleja analysoitiin tabletoitavuuden, puristuvuuden,
puristettavuuden ja modifioidun Heckelin yhtälön avulla. Tablettien kuvantamiseen käytettiin sekä
pyyhkäisyelektronimikroskopiaa että tietokoneavusteista röntgensädekerroskuvausta.
Pyyhkäisyelektronimikroskopian avulla pystyttiin tutkimaan aineiden pintarakennetta, kun taas
tietokoneavusteisella röntgensädekerroskuvauksella saatiin parempi käsitys tablettien
sisärakenteesta ja tiheyseroista tabletin sisällä.
Tutkimusten perusteella puristuksessa käytetyistä aineista ja seossuhteista riippuen tabletteihin
muodostuvat erilaiset tablettirakenteet. Modifioidun Heckelin yhtälön avulla tutkittiin jauheiden
todellista tiheyttä ja muodonmuutosominaisuuksia. Tällöin tiheyden arvot kuitenkin vaihtelivat
verrattuna heliumpyknometrillä määriteltyihin arvoihin, eikä tulosten oikeellisuutta voitu varmistaa
ilman lisätutkimuksia. Jauheiden muodonmuutosominaisuudet olivat kirjallisuuden mukaisia
lukuun ottamatta atenololia sisältäviä jauheita, joiden osalta tulosten varmistamiseen tarvitaan
lisätutkimuksia.
TABLETOITAVIEN JAUHEIDEN
PROSESSIKÄYTTÄYTYMINEN JA TABLETTIRAKENNE
Suvi Nurro
Farmasian teknologian ja biofarmasian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
90
Leijupetirakeistuksessa rakeistusneste sumutetaan pisaroina ilmavirran avulla leijuvien
rakeistettavien hiukkasten päälle. Rakeistuksella parannetaan lähtöaineiden käsiteltävyyttä,
valuvuutta ja tabletoitavuutta. Rakeisiin saadaan haluttuja ominaisuuksia muokkaamalla
rakeistusprosessia. Rakeistusprosessiin kuuluu prosessiparametrien lisäksi materiaaliparametrit ja
laiteparametrit. Rakeiden laatuun voidaan vaikuttaa erityisesti säätämällä prosessiparametreja.
Prosessiparametrit voidaan jakaa rakeistusnesteen lisäämiseen ja leijuttavan ilman ominaisuuksiin
liittyviin parametreihin. Koska useat prosessiparametrit vaikuttavat yhtä aikaa rakeiden
ominaisuuksiin, yksittäisten parametrien vaikutusten erittely on haastavaa. Rakeistuksen
suunnittelussa pystytään hyödyntämään rakeiden ominaisuuksiin vaikuttavia tärkeimpiä
parametreja, kun kaikki prosessiin vaikuttavat parametrit ja niiden yhteisvaikutukset on analysoitu.
Prosessiparametrien tunteminen on edellytys prosessin muokkaamiselle, hallinnalle ja
automatisoinnille.
Kokeellinen osa kuului suurempaan tutkimuskokonaisuuteen, jonka tarkoituksena on kehittää
leijupetirakeistuksen prosessinaikaista seurantaa. Prosessia voi seurata esimerkiksi niin sanotun
prosessi-ikkunan avulla. Prosessi-ikkuna mahdollistaa rakeistuksen ja raekoon seurannan ja
tulkinnan akustisella emissiolla häiritsemättä prosessia sekä rakeistuksen optimoinnin ja hallinnan
prosessiparametrien avulla. Menetelmän tulee olla reaaliaikainen, jotta lopputuote vastaa asetettuja
laatuvaatimuksia. Tässä työssä selvitettiin akustisen emission yhteys rakeiden kokoon
analysoimalla tehdyt määritykset monimuuttuja-analyysin avulla. Monimuuttuja-analyysillä
määrityksistä voitiin löytää oleellisin tieto. Pääkomponenttianalyysia (principal component
analysis, PCA) käytettiin raekokofraktioiden visualisointiin ja erikokoisten rakeiden
mallintamiseen ja rakeiden kokoa ennustettiin PLS:llä (partial least squares, osittainen pienimmän
neliösumman regressioanalyysi), jota voidaan pitää PCA:n regressiolaajennoksena. Lisäksi työssä
tutkittiin rakeistusnesteen lisäämiseen liittyvien prosessiparametrien vaikutuksia rakeiden saantoon
sekä rakeiden kokojakaumaa ja kosteuspitoisuutta. Tulokset ovat samansuuntaisia kuin muissakin
tutkimuksissa saadut, eli rakeistettavan massan kosteuden lisääminen kasvattaa raekokoa.
PROSESSIPARAMETRIEN VAIKUTUKSET
LEIJUPETIRAKEISTUKSESSA
Laura Piispanen
Farmasian teknologian ja biofarmasian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
91
Mesohuokoisten materiaalien soveltuvuutta lääketeollisuuden tarpeisiin tutkitaan parhaillaan
maailmanlaajuisesti. Tutkimuksissa on muun muassa havaittu, että monilla niukkaliukoisilla
lääkeaineilla liukoisuus on merkittävästi parantunut huokoiseen materiaaliin lataamisen jälkeen.
Myös lääkeaineiden vapautumiskäyttäytymiseen ja stabiilisuuteen pystytään vaikuttamaan
lataamalla niitä huokoisiin materiaaleihin. Yhtenä tavoitteena laajoissa tutkimuksissa olisi saada
kehitettyä annostelumenetelmä, joka vapauttaisi lääkeainetta tasaiseen tahtiin ja myös mahdollisesti
vähentäisi lääkkeenottokertoja pitkävaikutteisuutensa ansiosta.
Tutkimuksen päätarkoituksena oli mesohuokoisten materiaalien ja lääkeainemolekyylien välisten
vuorovaikutusten spesifioiminen. Päämittalaitteena oli FT-IR-spektroskooppi, jonka antamia
mittaustuloksia varmennettiin IMC:lla. Tutkimuksen toisena tarkoituksena oli selvittää, miten
hyvin FT-IR-spektroskopia yhdessä isotermisen mikrokalorimetrian kanssa toimi
tutkimusmenetelmänä tämäntyyppisessä tutkimuksessa. Tutkielman viisi ensimmäistä lukua
keskittyvät mittalaitteiden ja vuorovaikutusten teorioihin sekä aineiden karakterisointiin. Kuudes
luku käsittelee mittaustuloksia ja viimeinen, eli seitsemäs luku pitää sisällään tutkimuksen
päätulokset ja johtopäätökset.
Molemmilla mittalaitteilla mitattaessa havaittiin vuorovaikutuksia eri yhdisteiden välillä. FT-IR- ja
IMC-mittaukset eivät kuitenkaan tukeneet toisiaan kovinkaan hyvin, sillä usein FT-IR:llä havaittua
vuorovaikutusta ei havaittu IMC:llä tai toisinpäin. Pääsyy ristiriitaisiin tuloksiin oli
todennäköisimmin se, että FT-IR ja IMC keräsivät tietoa näytteestä pääasiallisesti eri ajankohtina.
FT-IR mittasi näytettä lähes välittömästi näytteen lisäämishetkestä ensimmäisen tunnin ajan, kun
IMC alkoi kerätä realistista tietoa tutkittavasta näytteestä vasta noin tunnin kuluttua näytteen
lisäämisestä. Lisäksi FT-IR havaitsee ainoastaan kemialliset vuorovaikutukset, kun IMC havaitsee
sekä kemialliset että fysikaaliset vuorovaikutukset.
Tutkimuksen päätulokset olivat, että mesohuokoisten materiaalien ja lääkeaineiden välillä
löydettiin lukuisia vuorovaikutuksia. FT-IR ja IMC eivät kuitenkaan soveltuneet hyvin yhdessä
käytettäessä vuorovaikutusten spesifioimiseen. Parhaiten huokoisten materiaalien lataamiseen
lääkeaineilla soveltuivat ne yhdistekombinaatiot, joilla ei (adsorptioita lukuunottamatta) esiintynyt
vuorovaikutuksia.
MESOHUOKOISTEN MATERIAALIEN JA
LÄÄKEAINEMOLEKYYLIEN VÄLISTEN
VUOROVAIKUTUSTEN SPESIFIOINTI SPEKTROSKOPISIN JA
KALORIMETRISIN MENETELMIN
Jukka Saarela
Fysiikan laitos, Turun yliopisto
92
Proteiini- ja peptidilääkkeitä on perinteisesti annettu injektiona, koska imeytyminen muiden
antoreittien kautta, kuten esimerkiksi oraalisesti tai nasaalisesti, on hyvin vähäistä. Viimeksi
mainittujen antoreittien käyttö, jossa imeytyminen tapahtuu epiteelipinnalla, vaikeutuu erilaisten
esteiden takia. Imeytymisen esteet kuten entsymaattinen hajoaminen, lääkkeiden pääsyä
epiteelipinnalle hidastava limakalvo ja hydrofobinen solukalvo pienentävät proteiinien ja peptidien
biologista hyötyosuutta. Vaikka proteiinit ja peptidit välttäisivät entsymaattista hajoamista ja
saavuttaisivat epiteelipinnan, on solukalvo silti suurin este näille suurikokoiselle ja usein hyvin
vesiliukoisille molekyyleille. Tämän esteen ohittamiseksi on ehdotettu erilaisia imeytymisen
edisteitä, joista eniten on tutkittu kelatoivia aineita, rasvahappoja ja pinta-aktiivisia aineita. Vaikka
nämä edisteet ovat parantaneet proteiinien ja peptidien hyötyosuutta, niiden toksisuudesta
imeyttävään solupintaan nähden ei ole yksiselitteistä näyttöä.
Vaihtoehdoksi edellä mainituille edisteille on ehdotettu biopolymeerien käyttöä. Eräs niistä on
kitosaani. Tällä luonnon polymeerillä on paljon hyödyllisiä ominaisuuksia farmaseuttisen
lääkekehityksen kannalta. Kitosaani on biohajoava, ei-toksinen ja hyvin siedetty polymeeri. Sen on
todettu edistävän proteiinien ja peptidien parasellulaarista imeytymistä sekä in vitro - että in vivo -
kokeissa. Tämän lisäksi kitosaanilla ei ole havaittu toksisia sivuvaikutuksia imeyttävään
solupintaan. Kitosaanin imeytymisen ediste -vaikutus on erilainen verrattuna aikaisempiin
proteiinien ja peptidien imeytymisessä käytettyihin edisteisiin. Kuitenkin kitosaanin käyttöä
imeytymisen edisteenä rajoittaa kitosaanin huono vesiliukoisuus fysiologisessa pH:ssa. Kitosaani
liukenee vain happamiin vesiliuoksiin sen emäksisen aminoryhmän protonoituessa. Kitosaanin
synteettisellä muokkauksella on saatu kvatemäärisiä ammoniumjohdoksia, joilla on kitosaania
parempi vesiliukoisuus. Näilläjohdoksilla on kitosaanin tavoin kyky edistää proteiinienja peptidien
imeytymistä. Kvatemäärisistä ammoniumjohdoksista trimetyylikitosaania (TMC) on tutkittu eniten.
TMC:n valmistusmenetelmä ja -olosuhteet vaikuttavat sen imeytymisen ediste -ominaisuuksiin.
Imeytymistutkimuksissa kvatemääriset kitosaanijohdokset ovat osoittautuneet turvallisiksi
imeytymisen edisteiksi.
KITOSAANI JA KVATEMÄÄRISET KITOSAANIJOHDOKSET
PEPTIDIEN JA PROTEIINIEN IMEYTYMISEN EDISTEINÄ
Rustam Safin
Lääkeainekemian koulutusohjelma, Luonnontieteiden ja ympäristötieteiden tiedekunta, Kuopion yliopisto
93
C- vitamiini eli askorbiinihappo on luonnossa esiintyvä ihmiselle hyvin hyödyllinen antioksidantti,
jolla on runsaasti parantavia ominaisuuksia. Aikuisen ihmisen suositeltava päivittäinen C-
vitamiiniannos on n. 60 mg. C- vitamiinin puute saattaa aiheuttaa keripukkia.
Askorbiinihapolla on yhteensa neljä stereoisomeeriä, joista ainoastaan L-muodolla on keripukkia
ehkäisevä vitamiiniaktiivisuus. Askorbiinihapon diastereomeeriä, Disoaskorbiinihappoa, lisätään
kuitenkin ruokiin sen antioksidanttisten ominaisuuksien vuoksi. C-vitamiiniaktiivisuudesta sillä on
vain 5 % L-muodon aktiivisuudesta.
Differentiaalinen pyyhkäisykalorimetria on tekniikka, jonka avulla pystytään helposti havaitsemaan
aineessa tapahtuvat faasimuutokset. Tässä työssä differentiaalista pyyhkäisykalorimetria hyväksi
käyttäen määritettiin C- vitamiinin L- ja D-muotojen sulamislämpötila faasidiagrammi. Perkin
Elmerin tehokompensoivan DSC 7 laitteiston avulla mitattiin yhteensa 18 näytettä, jotka kattoivat
tasaisesti koko konsentraatio välin 0 - 100 % L-muotoa, lämpötilavälillä (125 - 225) C /
(130 - 205) C.
Sulamislämpötilafaasidiagrammin avulla määritettiin askorbiinihapon L- ja D-muodon
muodostavan eutektisen konsentraation seossuhteella 39,4 % L-muotoa. Eutektinen sulaminen
tapahtuu lämpötilassa Te = 155,08 C.
Kuivattujen tyrni- ja mustaherukka mehujen askorbiinihapon L- ja D-muodon osuuksia pyrittiin
määrittämään kuivattujen mehujen sulamiskäyrien avulla. Sulamiskäyristä ei kuitenkaan pystytty
havaitsemaan minkään yksittäisen aineen sulamista, joten myöskään askorbiinihapon
esiintymisestä kuivatuissa mehuissa ei voida sanoa mitään.
ASKORBIINIHAPPO-ISOMEERIEN FAASIDIAGRAMMIN SEKÄ
KUIVATTUJEN MARJAMEHUJEN C-VITAMIINIPITOISUUDEN
MÄÄRITYS DSC-MENETELMÄLLÄ
Tuomas Salonen
Fysiikan laitos, Turun yliopisto
94
The SRB assay demonstrated to have a good linearity. It seems to be sensitive enough to detect
both the lowest and the highest cell concentrations and the antiproliferative action of the
compounds tested. The SRB assay is easy to perform and it is cheap. It requires several steps but
they can be easily automated as we have shown. Indeed the SRB assay allows the evaluation of the
total amount of protein that can be easily related to the proliferation ability of the cells and to the
growth inhibition effect of the drugs.
The Caco-2 cells are part of a very resistant cell lines due to their ability to synthesize the P-gP
efflux pump. The future project is to validate the SRB assay against others cell lines that are more
sensitive to anticancer treatment like one of the leukemia cells lines and one of the prostate cancer
cell lines. The idea is to validate the SRB assay against a panel of cell lines, even if small. This
could allow the use of the assay in the screening of a library of new compounds with supposed
anticancer activities.
SCREENING ANTICANCER ACTIVITY: APOPTOSIS BASED
METHODS AND VALIDATION OF AUTOMATED SRB ASSAY
Cristina Sempio
Università degli Studi di Pavia, Facoltà di Farmacia, Dipartimento di Chimica Farmaceutica, Italy
95
Tutkielmassa on perehdytty huokoisen piin (PSi) käyttäytymiseen kolmessa simuloidussa
kehonesteessä. Työn teoriaosuudessa on käsitelty PSi:tä, sen ominaisuuksia, valmistusta,
bioyhteensopivuutta ja teollisia sovelluksia. Tutkimuksen pääasiallisena tavoitteena oli selvittää
liukeneeko eritavoin pintakäsitellyt PSi:t simuloituihin kehonesteisiin. Lisäksi tutkimuksessa
testattiin uutta menetelmää PSi:n liukenemisen seuraamiseen piihapon avulla ja laskettiin
näytteiden oksidoitumisen aktivaatioenergiat Arrheniuksen yhtälön avulla.
Tutkimuksessa käytettiin Fourier-muunnos infrapuna (FTIR) spektrometriä (Perkin Elmer), jolla
mitatut spektrit analysoitiin. Lisäksi käytettiin differentiaalista pyyhkäisykalorimetria (DSC)
(Perkin Elmer), jolla pyrittiin selvittämään voitaisiinko mittauksilla saada nopeasti tietoa näytteiden
liukenevuuskäyttäytymisestä ja oksidoitumisesta. Käytetyt liuokset olivat simuloitu kudosneste
(SBF), simuloitu aivoneste (CSF) ja simuloitu suolineste (SIF). Liukoisuusmittaukset tehtiin
pitämällä PSi partikkeleja eri liuoksissa, kahdessa eri lämpötilassa (37C ja 80C) ja tarkkailemalla
niiden massojen muutoksia eri aikavälein. Useimmista partikkeleista oli kaksi eri kokoluokkaa:
<38 um ja 38 - 75 um. Tutkimuksessa käytettiin sekä käsittelemättömiä että käsiteltyjä PSi
näytteitä. Käsitellyt piinäytteet olivat termisesti oksidoituja, termisesti karbidoituja (sekä
oksikarbidoitu että hydrokarbidoitu) ja viinihappohappokäsitelty (TAPSi).
Partikkelien kokoluokalla tai lämpötilalla ei havaittu olevan suurta merkitystä naytteiden
massanmuutoskäyrien muotoihin. Sen sijaan lämpötilan kohotessa useimpien näytteiden
oksidoituminen ja liukenemisaika nopeutui. PSi:n liukenemisen seuraaminen piihapon avulla
epäonnistui, sillä mitattavaa määrää happoa ei näyttäisi muodostuvan. DSC-mittauksilla sen sijaan
on mahdollista saada tietoa naytteiden oksidoitumisesta, mutta ei liukenemisesta.
HUOKOISEN PIIN LIUKENEMINEN SIMULOITUIHIN
KEHONESTEISIIN
Mauri Sinkkonen
Fysiikan laitos, Turun yliopisto
96
Hera on meijeriteollisuuden sivutuote, joka - huolimatta heraproteiinien monipuolisista
ominaisuuksista ja ravintoarvosta - jää suurelta osin ilman jatkojalostusta. Viime vuosina onkin
alettu etsiä heraproteiineille uusia käyttösovelluksia. Heraproteiinikalvojen tutkimus on tapahtunut
pääasiassa elintarviketutkimuksen parissa osana ns. syötävien kalvojen tutkimusta.
Proteiinirakenteisten lääkeaineiden kehitystyön edetessä lisääntyy myös tarve kehittää näihin
yhteensopivia päällystysmateriaaleja. Lehmänmaidosta saatu hera sisältää 4—7 g/1 proteiineja.
Heraproteiinit ovat ryhmä keskenään melko erityyppisiä, globulaarisia proteiineja, joita ovat β -
laktoglobuliini, α-laktalbumiini, seerumin albumiini, immunoglobuliinit, sekä eräät pienemmän
molekyylikoon omaavat peptidit, β -laktoglobuliini on tärkein heraproteiini, noin 50 %
heraproteiinista on β-laktoglobuliinia. Heraproteiinien etuihin voidaan lukea vesiliukoisuus,
heraproteiini-konsentraatin hyvä saatavuus ja edullinen hinta, sekä mahdollisuus muokata kalvon
ominaisuuksia denaturoimalla heraproteiineja.
Erikoistyön kokeellisen osuuden tarkoituksena oli tutkia erilaisten, osin uusien
pehmitevalintojen vaikutusta heraproteiinikalvojen mekaanisiin ominaisuuksiin valamalla valmistettuja
vapaita kalvoja käyttäen. Kalvojen mekaanisista ominaisuuksista tutkittiin vetolujuutta ja venyvyyttä.
Käytetty kalvonmuodostaja oli heraproteiini-konsentraatti, joka sisälsi 77 % heraproteiineja, käytetyt
pehmitteet olivat glyseroli, akaasiahunaja ja fruktoosin ja glukoosin yhdistelmä. Kalvoja valmistettiin
sekä kuumennetusta, että kuumentamattomasta heraproteiiniliuoksesta. Lisäksi tutkittiin suppeammin
kalvoliuoksen pH:n muuttamisen vaikutusta kalvojen mekaanisiin ominaisuuksiin, kalvojen
vesihöyryn läpäisevyyttä sekä kalvojen dispergoitumista veteen.
Heraproteiinikal vojen venyvyydet lisääntyivät ja vetolujuudet pienenivät, kun pehmitteen määrää
lisättiin. Vetolujuudet olivat pääsääntöisesti korkeampia akaasiahunajaa tai monosakkarideja, kuin
glyserolia käytettäessä. Vetolujuudet olivat suurimmillaan, kun käytettiin kuumennettua
heraproteiinia. Toisaalta proteiinin käyttö kuumentamattomana mahdollistaa laajemman vaihteluvälin
kalvoliuoksen proteiinipitoisuudessa sekä pH:ssa. Heraproteiinikalvojen vetolujuuteen voitiin
vaikuttaa säätämällä kalvoliuoksen pH:ta. Tutkittujen kalvojen vesihöyrynläpäisevyys oli
suurinta, kun pehmitteenä käytettiin glyserolia. Heraproteiinikalvojen dispergoituvuutta veteen
voitiin säädellä sekoittamalla kuumennettua ja kuumentamatonta proteiinia sopivassa suhteessa.
Heraproteiinin hydrofiilisyys antaa sille hyvän vesiliukoisuuden, mutta lisää toisaalta
kalvojen vesihöyrynläpäisevyyttä ja tekee ne herkäksi ympäristön kosteuden vaikutukselle.
Sakkaridien käyttö heraproteiinikalvojen pehmitteenä näyttäisi tarjoavan hyvän vaihtoehdon
yleisemmin käytetylle pehmitteelle, glyserolille.
PEHMITEVALINNAN JA PROTEIININ KUUMENNUKSEN
VAIKUTUKSET HERAPROTEIINIKALVOJEN MEKAANISIIN
OMINAISUUKSIIN
Jussi Soininen
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
97
Työn kirjallisessa osassa käsitellään lääkeaineiden liukoisuuden ja liukoisuuteen vaikuttavien
tekijöiden merkitystä farmasiassa. Suuri osa lääkevalmisteista annostellaan elimistöön kiinteässä
olomuodossa. Lääkeaineen on ensin vapauduttava valmisteesta ja sen jälkeen liuettava elimistön
nesteisiin, ennen kuin se voi imeytä biologisten kalvojen läpi ja kulkeutua vaikutuspaikkaansa.
Liukoisuudella on yhdessä permeaatio-ominaisuuksien kanssa suuri vaikutus lääkeaineen
hyötyosuuteen elimistössä. Liukoisuuden selvittäminen lääkekehityksen alkuvaiheessa on tärkeää,
jotta aikaa ei tuhlata ei-lääkkeenomaisten yhdisteiden tutkimiseen.
Monet erilaiset fysikaalis-kemialliset tekijät vaikuttavat lääkeaineiden liukoisuusominaisuuksiin.
Valtaosa lääkeaineista on heikkoja elektrolyyttejä, jotka voivat ionisoitua elimistön
vesiympäristössä. Ionisoitunut muoto liukenee yleensä huomattavasti ionisoitumatonta muotoa
paremmin, ionisoitumisasteeseen vaikuttaa ympäristön pH. Lääkeaineen korkea lipofiilisyys
heikentää tavallisesti liukoisuutta. Toisaalta imeytymisominaisuudet paranevat, jolloin
niukkaliukoisuus ei välttämättä rajoita tai estä yhdisteen käyttöä vaikuttavana lääkkeenä. Kiinteän
aineen liukoisuutta parantavia tekijöitä suuren ionisaatioasteen ja matalan lipofiilisyyden lisäksi
ovat pieni molekyylikoko, kyky muodostaa vetysidoksia liuottimen kanssa, matala sulamispiste,
amorfisuus, pieni partikkelikoko sekä tavallisesti lämpötilan kasvattaminen. On kuitenkin
muistettava, että liukoisuusominaisuuksien parantaminen johtaa usein imeytymisominaisuuksien
heikentymiseen. Liukoisuuden parantaminen kaikin keinoin ei ole tarkoituksenmukaista, vaan
pyrkimys tasapainon löytämiseen näiden ominaisuuksien välille.
Liukoisuuden määrittämiseksi on kehitetty useita erilaisia menetelmiä. Ne ovat kuitenkin
huomattavasti hitaampia kuin yhdisteiden biologisen aktiivisuuden selvittämismenetelmät.
Liukoisuuden määrittäminen on lääkekehitystä hidastava vaihe, joten uusia ja nopeita menetelmiä
kaivataan.
Työn kokeellisen osan tarkoituksena oli kehittää pintajännityksen mittaamiseen perustuva
liukoisuuden määritysmenetelmä. Se perustuu ilmiöön, jossa näyteliuosten pintajännitys alkaa
pitoisuuden kasvaessa laskea lähestyttäessä liuotetun yhdisteen liukoisuusrajaa. Liukoisuusrajan
ylittymisen jälkeen pintajännitys pysyy muuttumattomana, vaikka pitoisuutta kasvatettaisiin
edelleen. Työssä liukoisuus määritettiin pisteestä, jossa pintajännityksen pieneneminen lakkaa ja
tasaantuminen alkaa. Tuloksia verrattiin perinteisellä liukoisuuden määritysmenetelmällä,
ravistelumenetelmällä, määritettyihin tuloksiin. Tutkimusaineena käytettiin ibuprofeenia, jonka
liukoisuutta tutkittiin erilaisissa olosuhteissa. pH:n vaikutusta tutkittiin pH:ssa 1,2, 7,0 ja 9,0.
Liukoisuutta tutkittiin myös erilaisten pinta-aktiivisten aineiden ja suolojen vesiliuoksissa sekä
pelkässä vedessä. Pintajännitysmenetelmällä määritetyt liukoisuudet olivat kaksi - kolme kertaa
suurempia kuin samoissa olosuhteissa ravistelumenetelmällä määritetyt liukoisuudet. Menetelmällä
voitiin määrittää siis vain suuntaa antava liukoisuus. Se voisi soveltua lääkkeiden aikaisen
kehittämisvaiheen liukoisuuden määritysmenetelmäksi, sillä menetelmä on nopea eikä tarkkaa
liukoisuusarvoa vielä siinä vaiheessa tarvita. Kokeissa tutkittiin lisäksi erilaisten esiliuottimien
vaikutusta liukoisuustuloksiin. Dimetyylisulfoksidi osoittautui jatkotutkimusten kannalta
lupaavimmaksi esiliuottimeksi.
PINTAJÄNNITYKSEEN PERUSTUVA LIUKOISUUDEN
MÄÄRITYS: MIKROTENSIOMETRI- JA
RAVISTELUMENETELMIEN TULOSTEN VERTAILU
Saila Taskinen
Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto
98
Useiden lipofiilisten lääkeaineiden liukenemisnopeuden on havaittu parantuvan, kun lääkeaine on
dispergoitu vesiliukoiseen kantajaan. Kiinteässä dispersiossa lääkeaine ja kantaja voivat säilyttää
omat kiderakenteesta tai ne voivat muodostaa yhtenäisen kiderakenteen eli molekulaarisen
dispersion (kiinteä liuos). Lääkeaineen nopeampi liukeneminen kiinteästä dispersiosta on
mahdollista, koska kiinteissä dispersioissa lääkeaine on hienojakoisina partikkeleina ja
mahdollisesti amorfisessa muodossa. Lisäksi kantaja voi lisätä lääkeaineen liukoisuutta ja parantaa
sen kostumista. Useimmiten kiinteissä dispersioissa kantajina on käytetty hydrofiilisiä polymeerejä
kuten polyvinyylipyrrolidonia (PVP), polyetyleeniglykolia (PEG), hydroksipropyylimetyyli-
selluloosaa (HPMC) tai pinta-aktiivisia aineita kuten polysorbaatti 80:a, Vitamiini E TPGS:ä ja
Gelucirea. Kiinteitä dispersioita on alunperin valmistettu sulatus-tai haihdutusmenetelmillä, mutta
viime aikoina niiden valmistukseen on pyritty käyttämään paremmin teolliseen mittakaavaan
sovellettavia menetelmiä kuten ylikriittistä nesteuuttoteknologiaa tai kuumasulapuristusta.
Kiinteiden dispersioiden liukoisuusnopeutta lisäävästä vaikutuksesta huolimatta kaupallisia kiinteä
dispersio sovellutuksia on markkinoilla vain vähän. Se johtuu muun muuassa lääkeaineen huonosta
stabiilisuudesta kiinteästä dispersiossa, jossa lääkeaine on useimmiten ainakin osittain amorfisessa
muodossa.
Kokeellisen osan tarkoituksena oli parantaa veteen heikosti liukenevan perfenatsiinin
liukenemisnopeutta muodostamalla siitä ja polyvinyylipyrrolidonista (PVP) tai
polyetyleeniglykolista (PEG) kiinteitä dispersioita. Partikkelit valmistettiin sumu (PVP).:tai
kylmäkuivaamalla (PVP ja PEG) kolmella eri seossuhteella (5: 1, 1:5 ja 1:20). Partikkeleille tehtiin
tyypillisimmät karakterisointitutkimukset; FT - IR, SEM -kuvaus, XRPD-mittaukset, säilyvyyskoe
rasitusolosuhteissa (45°C, RH 75 %) sekä liukoisuuskokeet pH:ssa 6,8. Perfenatsiinin
liukeneminen nopeutui formulaatiosta riippuen noin 1O-35-kertaiseksi käsittelemättömään
perfenatsiiniin verrattuna. Perfenatsiini oli partikkeleissa amorfisessa muodossa ja sen
partikkelikoko pieneni huomattavasti kiinteässä dispersiossa. Tosin useimmat kiinteä dispersio
formulaatiot eivät kestäneet 30 päivän säilyvyyskokeita, koska perfenatsiini oli kiteytynyt
partikkeleissa eri polymorfiseen muotoon.
KIINTEÄT DISPERSIOT VETEEN NIUKKALIUKOISTEN
LÄÄKEAINEIDEN FORMULOINNISSA
Kaisa Toukola
Farmasian teknologian ja biofarmasian laitos, Farmaseuttinen tiedekunta, Kuopion yliopisto
99
Matkailu avartaa – työelämässäkin. Post doc -tutkijana Uudessa-Seelannissa työskentelevä
Jaakko Aaltonen sekä lääketeollisuudessa Iso-Britanniassa ja Ruotsissa työskentelevät Niklas
Sandler ja Pirjo Luukkonen kertovat Polymorfin lukijoille työskentelystään ulkomailla ja
antavat vinkkejä ulkomailla työskentelyä harkitseville.
Jaakko Aaltonen
Koulutus:
FaT, Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto, 2007
Proviisori, Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto, 2003
Nykyinen työ:
Post doctoral fellow, School of Pharmacy, University of Otago, Dunedin, Uusi-Seelanti
Mitä työnkuvaasi ulkomailla sisältyy?
Työskentelen professori Thomas Radesin johtamassa kiinteän tilan tutkimusryhmässä.
Työnkuvaani kuuluu oman tutkimusprojektini lisäksi tohtoriopiskelijoiden ohjausryhmiin
kuuluminen. Tällä hetkellä olen mukana kolmessa väitöskirjaprojektissa, jotka kaikki liittyvät
enemmän tai vähemmän amorfisiin kiinteisiin lääkeaineisiin. Väitöskirjaprojekteista kaksi on vielä
alkutekijöissään, joten minulla on ollut tilaisuus päästä vaikuttamaan niiden (tulevaan)
sisältöönkin. Kolmas projekti alkaa olla jo loppusuoralla, joten sen ohjauksessa minun roolini on
enemmän painottunut tulosten käsittelyn ja raportoinnin tukemiseen.
Miten päädyit työskentelemään ulkomaille? Mitä kautta paikka avautui?
Ulkomaille työskentelemään lähtö on kiinnostanut minua farmasian opintojen alusta lähtien, mutta
kuukauden vierailua Kööpenhaminan yliopistoon lukuun ottamatta en ole aikaisemmin saanut
toimeksi lähteä ulkomaille opiskelemaan. Väitöskirjatyön loppupuolella aloin kartoittaa minulle
parhaiten soveltuvia ulkomaisia tutkimusyksiköitä. Muitakin kiinnostavia vaihtoehtoja oli, enkä
tehnyt valintaa täysin ammatillisin perustein – Uusi-Seelanti asuinpaikkana vaikutti huomattavasti
mielenkiintoisemmalta kuin esimerkiksi USA:n Keskilänsi.
Helsingin yliopiston farmasian teknologian osastolla tein tiivistä yhteistyötä Otagon yliopistosta
valmistuneen Clare Strachanin kanssa. Häneltä sain paljon tietoja nykyisestä kotiseudusta ja
työpaikasta. Teknon osastolla ja täkäläisellä tutkimusryhmällä on viime vuosina ollut tiivistä
tutkijanvaihtoa: FFY:n jäsenistä Niklas Sandler ja Marja Savolainen olivat jo käyneet täällä
tutkijavaihdossa ennen minua, ja myös Otagon yliopiston tutkijoita on käynyt Viikissä.
Oletko ollut tyytyväinen oloosi ulkomailla? Miten arkielämä, harrastukset ym. ovat sujuneet
ulkomailla?
Uuteen-Seelantiin asettuminen on sujunut oikeastaan helpommin kuin osasin kuvitella. Dunedinin
kaupunki on mukavan kokoinen kaupunki (n. 120 000 asukasta). Kaupungissa on paljon
opiskelijoita ympäri maailman joihin paikalliset ovat tottuneet, joten ulkomaalaisen on helppo
asioida täällä. Kansainvälisyyden ja suuren opiskelijamäärän vuoksi kaupungissa on kokoonsa
FYSIKAALISEN FARMASIAN AMMATTILAISET
MAAILMALLA
100
nähden hämmästyttävän hyvä ravintola- ja kahvilatarjonta, joten lounaspaikoista ei ole pulaa.
Harrastusmahdollisuuksista sen verran, että Uudessa-Seelannissa voi tehdä kaikkea ulkoiluun
liittyvää, ja viikonloput ovatkin kuluneet kotimaan matkailua ja erilaista ulkoilua harrastaessa.
Miten työskentely ja työkulttuuri ulkomaisessa työpaikassasi eroaa Suomessa työskentelystä?
Työskentely ei hirveästi eroa suomalaisesta muuten kuin että ihmiset luonnollisesti puhuvat
enemmän toistensa kanssa. Tämä heijastuu myös palavereihin ja seminaareihin, joissa keskustelu
on runsaampaa ja värikkäämpää kuin yleensä Suomessa. Tutkimusaiheet ja -työ luonnollisesti ovat
melko samanlaisia kuin muuallakin, mutta on hyvä nähdä, miten asioita tehdään muualla.
Laboratoriot ja laitekanta eivät täällä ole ihan yhtä korkeatasoiset kuin Viikissä.
Miten työpaikka on auttanut sopeutumisessa ja käytännön järjestelyissä?
Työpaikka tarjoaa työpisteen ja laboratoriot käyttööni. Työhön perehdytys, kuten oikeastaan kaikki
muukin tämän lisäksi, on ollut oman aktiivisuuden varassa. Rahoitukseni tulee Suomesta, joten
työpaikan panos paperiasioihin ja muuhun byrokratiaan on ollut kutsukirjeen lähettäminen.
Asunnon löytäminen ei ollut ihan helppoa, mutta sopiva kämppä löytyi lopulta reilun viikon
etsimisen jälkeen. Suomalaiset asuinkelpoisuusstandardit täyttävien kalustettujen perheasuntojen
tarjonta ei täällä ole järin suurta.
Millaisia vinkkejä ja neuvoja antaisit ulkomailla työskentelystä haaveileville tai sitä jo
suunnitteleville alan ihmisille?
Kannattaa varata aikaa asioiden järjestelyyn, nimittäin viisumien ja apurahojen hankkiminen on
aikaavievää puuhaa. Suosittelen myös tutustumaan kohteeseen etukäteen, ettei tule kovin pahoja
pettymyksiä paikan päällä. Kun nämä asiat ovat järjestyksessä, ei muuta kuin avoimin mielin
matkaan vaan. Tärkeintä on lähteminen, sen jälkeen asiat yleensä etenevät omalla painollaan.
Terveisiä kaikille FyFan symppariin osallistujille, antoisaa seminaaripäivää ja vielä
antoisampaa iltaa!
Niklas Sandler
Koulutus:
Dosentti, Farmasian teknologia, Helsingin yliopisto, 2007
FaT, Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto, 2003
Proviisori, Farmasian teknologian osasto, Farmasian tiedekunta, Helsingin yliopisto, 1998
Nykyinen työ:
Senior Scientist, AstraZeneca, Iso-Britannia
Kerro hieman itsestäsi Polymorfin lukijoille.
Nimeni on Niklas Sandler ja työnantajani kesästä 2006 asti on ollut AstraZeneca. Toimin
senioritutkijan roolissa AstraZenecan tuotekehityksessä ja tarkemmin sen prosessikehitysosaston
materiaalitukimusyksikössä. Valmistuin Helsingin yliopistosta proviisoriksi vuonna 1998 ja
farmasian tohtoriksi vuonna 2003 pääaineenani farmasian teknologia. Minut nimitettiin farmasian
teknologian dosentiksi Helsingin yliopistoon vuonna 2007. Väitöskirjani käsitteli pääasiassa uusien
kuva-analyysitekniikoiden ja monimuuttuja-analyysin hyödyntämistä partikkelikoon mittaamisessa
ja partikkelikoon monitoroinnissa kostearakeistusprosessin aikana. Väitöksen jälkeen vuonna 2005
101
matkasin post doc -tutkijaksi Uuteen-Seelantiin (University of Otago), jolloin tutkimukseni
painopistealueina olivat polymorfiset faasimuutokset ja niiden tutkiminen erityisesti Raman-
spektroskopian avulla.
Mitä työnkuvaasi ulkomailla sisältyy?
Kuulun materiaalitutkimusryhmään (Material Science), jonka tehtävänä on tukea uusien lääkkeiden
kehitysprojekteja lääkeaineisiin ja apuaineiden ominaisuuksiin liittyvällä asiantuntemuksella. Olen
itse vastuussa myöhäisessä vaiheessa (faasi 3) olevan syöpälääkevalmisteen prosessikehitysosaston
projektia tukevien toimintojen koordinoinnista ja lääkeaineen sekä apuaineiden materiaalitutkimus-
strategiasta. Minun tulee varmistua siitä, että me hallitsemme projektissa käytettävien materiaalien
fysikaaliset ominaisuudet ja niiden vaikutukset lääkevalmisteen prosessointiin ja laatuun.
Osallistuin hiljattain kyseisen tuotteen teknologiansiirtoon tuotantomittakaavaan (technological
transfer ja scale-up) tehtaaseemme Väli-Amerikassa. Työ on monipuolista ja eri osaajien kanssa
työskentelyä, ja projektityön lisäksi työnkuvaan kuuluu myös yrityksen sisäisiin strategisiin
teknologian kehitysohjelmiin osallistuminen.
Miten päädyit työskentelemään ulkomaille?
Suunnitelmissa oli koko ajan, että jossain vaiheessa olisi mukavaa ja kiinnostavaa muuttaa
muutamaksi vuodeksi työskentelemään ulkomaille. Noin vuosi väitökseni jälkeen oli sopiva aika
aloittaa tutkimusrahoituksen järjestelyt, ja lopulta päädyimme perheeni kanssa ensin Uuteen-
Seelantiin Otagon yliopiston School of Pharmacyn Solid State -tutkimusryhmään. Vaikka
viihdyimme Uudessa-Seelannissa erinomaisesti, teollisuusura kiinnosti. Hain avoinna olevia
paikkoja lääketeollisuudesta Englannista, ja minut kutsuttiin haastatteluun AstraZenecalle. Minut
valittiin työhön ja muutimme Manchesterin lähelle syksyllä 2006.
Oletko ollut tyytyväinen oloosi ulkomailla? Miten arkielämä, harrastukset ym. ovat sujuneet
ulkomailla?
Ensimmäinen muutto Uuteen-Seelantiin oli erittäin helppo ja elämä leppoisaa mahtavissa
maisemissa ja rennossa ilmapiirissä. Akateemisen post doc -työn vapaus ja uudenlainen vaihe
elämässämme teki ensi vaiheista ulkomailla todella nautittavia. Englantiin muutto oli yllättäen
vaikeampi kuin luulin. Ihmisten ja autojen paljous ja yhteiskunnan sosiaalinen jakautuminen olivat
joitakin asioita, jotka olivat jonkinlainen kulttuurisokki, ja asettuminen kesti kauemmin kuin luulin.
Harrastuksina ovat pääasiassa 1, 3,5 ja 5-vuotiaat lapset, joten vapaa-ajanongelmia ei ole ollut.
AstraZeneca on järjestänyt työntekijöilleen erittäin hyvät urheilumahdollisuudet: mm. kuntosaleja
ja palloilulajeja on helppo harrastaa töiden jälkeen tai lounastauolla.
Miten työskentely ja työkulttuuri ulkomaisessa työpaikassasi eroaa Suomessa työskentelystä?
En ole huomannut selkeitä eroja työkulttuurissa sinänsä suomalaiseen verrattuna. Töistä sovitaan
selkeästi ja ihmiset ovat ahkeria ja aikaansaavia. On kuitenkin ollut mielenkiintoista työskennellä
yrityksessä, jossa teemme monikansallista yhteistyötä Englannin, USA:n, ja Ruotsin tuotekehitys-
organisaatioiden välillä. Eri alueiden työskentelytavoissa havaitsee usein erilaisuuksia.
Miten työpaikka on auttanut sopeutumisessa ja käytännön järjestelyissä?
Koska siirryin Englantiin yrityksen palvelukseen, paperiasiat, pankkitilin avaamiset ja asunnon
löytäminen oli osaltani hoidettu varsin jouhevasti. Näistä pienimmistä käytännön asioista syntyy
useimmiten suurimmat kultturishokin ainekset, kun asiat eivät toimi, kuten on tottunut: esimerkiksi
jos puhelinasentajaa saa odottaa kuukauden, jotta saa internet -yhteyden avattua, tai jos yrittää etsiä
asuntoa, jossa ei olisi kokolattiamattoa kylpyhuoneessa…
102
Millaisia vinkkejä ja neuvoja antaisit ulkomailla työskentelystä haaveileville tai sitä jo
suunnitteleville alan ihmisille?
Jos asia on vakavasti mielessä, kannattaa lähteä yrittämään, muuten se voi jäädä kokonaan
tekemättä ja se voi harmittaa loppuelämän. Vaikka perheen kanssa lähteminen voi tuntua
hankalalta, niin vaikka se on kieltämättä haastavampaa, me olemme selvinneet mainiosti ensin
kahden ja nyt kolmen lapsen kanssa maailmalla.
Pirjo Luukkonen
Koulutus:
Dosentti, Farmasian teknologia, Helsingin yliopisto, 2005
FaT, Farmasian teknologian osasto, Farmasian TDK, Helsingin yliopisto, 2001
Nykyinen työ:
Associate Principal Scientist, AstraZeneca, Mölndal, Ruotsi
Kerro hieman itsestäsi Polymorfin lukijoille.
Olen peruskoulutukseltani proviisori, väittelin farmasian teknologiasta Helsingissä 2001 ja sain
dosentuurin Helsingin yliopistossa 2005. Työskentelin yliopistolla kokonaiset 9 vuotta, joista
väitöskirjaa tein aktiivisesti reilut neljä vuotta. Tein osan kokeellisesta osuudesta Kööpenhaminassa
Torben Schæferin ohjauksessa ja osan Lontoossa Mike Newtonin ohjauksessa. Muutama viikko
väitöksen jälkeen aloitin työskentelyn AstraZenecan farmaseuttisessa tuotekehityksessä
Mölndalissa (lähellä Göteborgia).
Mitä työnkuvaasi ulkomailla sisältyy?
Työskentelen Material Science –ryhmässä, ja tittelini on tällä hetkellä Associate Principal Scientist
Powder Processing -alueella. Vastuualueenani on Ruotsin kolme tuotekehityssitea; Mölndal, Lund
ja Södertälje. Työnkuvaan kuuluu tuotekehitysprojektien auttaminen oikean prosessin ja
apuaineiden valitsemisessa sekä vaikuttaminen siihen, että projektit työskentelevät Quality by
Design -mallin mukaisesti. Työskentelen paljon myös raaka-ainevariaation, prosessiaikaisten
mittausten sekä väli- ja lopputuotteiden karakterisoinnin parissa. Toimin myös globaalin tutkimus-
projektin vetäjänä sekä kuuluun AstraZenecan sisäisiin verkostoihin.
Miten päädyit työskentelemään ulkomaille? Mitä kautta paikka avautui?
Minut rekrytoi professori Anne Juppo, joka silloin työskenteli ryhmäpäällikkönä AstraZenecalla.
En ollut kuvitellut muuttavani Ruotsiin, sillä osasin ruotsia kovin huonosti, mutta kun tilaisuus
tarjoitui, en voinut jättää sitä kokeilematta.
Oletko ollut tyytyväinen oloosi ulkomailla? Miten arkielämä, harrastukset ym. ovat sujuneet
ulkomailla?
Olen ollut tyytyväinen tekemääni päätökseen lähteä ulkomaille. Tietenkään elämä ei aina ole ollut
ruusuilla tanssimista, mutta harvoinpa se on. Pidän itseäni suhteellisen sosiaalisena ihmisenä, joten
ennen muuttoa en hetkeäkään epäillyt, etten saisi Göteborgista uusia ystäviä. Mutta uusien
ystävyyssuhteiden solmiminen ei ollut niin helppoa kuin kuvittelin.
103
Miten työskentely ja työkulttuuri ulkomaisessa työpaikassasi eroaa Suomessa työskentelystä?
Työkulttuuria täällä ja Suomessa on vaikea verrata, sillä Suomessa olen työskennellyt vain
apteekissa ja yliopistolla. Ruotsalaiset tykkäävät pitää kokouksia, ja niinpä minunkin työajastani
menee suurin osa kokouksissa istumiseen. Kahvitauot eli fikapaus ovat myös hyvin tärkeässä
asemassa ruotsalaisessa työkulttuurissa.
Mikä ulkomailla työskentelyssä on ollut haastavinta, mikä parasta tai palkitsevinta?
Haastavinta aluksi oli ruotsin kieli. Vaikka AstraZeneca on kansainvälinen yritys, jonka virallinen
kieli on englanti, kaikki Ruotsin sisäinen toiminta tapahtuu ruotsiksi. Parasta työssäni on se, että
saan tehdä juuri sellaista työtä, johon olen saanut koulutuksen. Työn tekee erittäin
mielenkiintoiseksi työskentely monien eri ammattiryhmien välillä.
Miten työpaikka on auttanut sopeutumisessa ja käytännön järjestelyissä?
AstraZeneca maksoi muuton ja järjesti vuokra-asunnon, jossa sain asua ensimmäiset kolme vuotta.
Samoin sain apua alussa paperiasioiden hoitamiseen.
Millaisia vinkkejä ja neuvoja antaisit ulkomailla työskentelystä haaveileville tai sitä jo
suunnitteleville alan ihmisille?
Minun neuvoni on lähteä rohkeasti ulkomaille. Kukaan ei ole seppä syntyessään, mutta työpaikka
ja siellä hankittu kokemus opettavat sen, mitä peruskoulutuksessa kenties on jäänyt paitsi.
Paikallinen kieli kannattaa toki opiskella ja aloittaa sen käyttö mahdollisimman nopeasti, se auttaa
huomattavasti myös sosiaalista sopeutumista.
SIVUN 105 SANALAATIKON OIKEAT VASTAUKSET
1. Polymorfi 2. Pintajännitys 3. Formulaatio 4. Raman 5. Amorfinen 6. Rakeistus 7. Monohydraatti 8. Leijupeti
9. Triglyseridi 10. Adsorptio 11. Kemometria 12. Kromatografia 13. Offline 14. Ohutsuoli 15. Katalyytti
16. Segregaatio 17. Diffraktogrammi 18. Infrapuna 19. Dispersio 20. Interaktio 21. Pehmite 22. Faasi 23. PAT 24. Miselli
104
105
Etsi sanalaatikosta alla olevien vihjeiden perusteella 24 fysikaaliseen farmasiaan liittyvää sanaa.
Sanat voivat sijaita vaaka- tai pystysuorassa. Sanat voivat olla kirjoitettuna myös oikealta
vasemmalle ja alhaalta ylös.
A K E J I M M A R G O T K A R F F I D O
J A S P N A M O R F I N E N H I R P T F
I T L V K I F E T O H U T S U O L I Y F
K A P C B N A M A R N G P K N L K C A L
K L S O T T G O L M I S E L L I S T G I
I Y M I N E S N Y U W L H S A V G R Y N
S Y K T G R K O D L E I J U P E T I V E
Y T Ä A P A R H U A G P E B A N E G L O
T T R A A K H Y R A K E I S T U S L D J
I I H G I T E D S T A I S I S N Ä Y S H
N A Ö E R I B R U I D D L N N Y V S O N
N E H R T O S A R O G T L F P I T E Ä E
Ä S I G E O L A D I S P E R S I O R W T
J T S E M V C T A J L F H A R E C I E I
A M A S O A G T I T O I T P R O S D A M
T P A N M S O I H R U P L U L G G I J H
N V F C E Ä T N J U S B I N A S Ö B S E
I Ö J E K R O M A T O G R A F I A K L P
P O L Y M O R F I R P V S M L N A V Y T
1. Kidemuoto
2. Johtuu nesteen molekyylien välisestä
koheesiosta
3. Tuotekehityksen tulos
4. Sirontaa hyödyntävä spektroskopian laji
5. Järjestäytymätön kiinteä aine
6. Mm. valuvuutta ja puristuvuutta parantava
farmaseuttinen prosessi
7. Sisältää yhden kideveden
8. Rakeistuksessa käytettävä laite
9. Muodostuu glyserolista ja kolmesta
rasvahappoketjusta
10. Pintaan tarttuminen
11. Tilastomenetelmiä ja tietotekniikkaa
hyödyntävä kemian tieteenhaara
12. Aineiden erottelumenetelmä
13. Onlinen vastakohta
14. Lääkeaineiden yleisin imeytymispaikka
15. Nopeuttaa kemiallista reaktiota
16. Seoksen komponenttien erottuminen
17. XRD-laitteesta saatava kuvaaja
18. Säteily, jonka aallonpituus on välillä
750 nm–1 mm
19. Voi olla molekulaarinen, kolloidaalinen
tai karkea
20. Vuorovaikutus
21. Parantaa kalvopäällysteen elastisuutta
22. Olomuotoalue
23. Valmistusprosesseihin liittyvä
ajankohtainen lyhenne
24. Surfaktanttimonomeereistä koostuva
pallomainen rakenne
SANALAATIKKO
106
Osmo Antikainen Helsingin yliopisto
Seppo Auriola Kuopion yliopisto
Lotta Bergman Åbo Akademi
Henrik Ehlers Helsingin yliopisto
Natalja Genina Helsingin yliopisto
Noora Gisselberg Orion Pharma
Lisette Gonzáles Helsingin yliopisto
Maria Hagman Lääkelaitos
Maija-Riitta Halonen Orion Pharma
Teemu Heikkilä Turun yliopisto
Tiina Heikkilä Helsingin yliopisto
Petteri Heljo Helsingin yliopisto
Pekka Hoppu Helsingin yliopisto
Samuli Hirsjärvi Helsingin yliopisto
Minna Holopainen Orion Pharma
Johanna Husman-Piirainen Orion Pharma
Kirsi Jouppila Helsingin yliopisto
Anne Juppo Helsingin yliopisto
Kristiina Järvinen Kuopion yliopisto
Maiju Järvinen Kuopion yliopisto
Marko Kaija Orion Pharma
Tarja Kankkunen Lääkelaitos
Esko Karkkonen Malvern
Alma Kartal Helsingin yliopisto
Pekka Keski-Rahkonen Kuopion yliopisto
Jarkko Ketolainen Kuopion yliopisto
Niina Kivikero Helsingin yliopisto
Karin Kogermann Helsingin yliopisto
Ossi Korhonen Kuopion yliopisto
Karin Krogars Lääkelaitos
Marko Kuosmanen Kuopion yliopisto
Timo Laaksonen Helsingin yliopisto
Riikka Laitinen Kuopion yliopisto
Eija Lauronen Orion Pharma
Vesa-Pekka Lehto Turun yliopisto
Johanna Lehtonen Orion Pharma
Tuula Leskelä Orion Pharma
Jari Leskinen Kuopion yliopisto
Katri Levonen Kuopion yliopisto
Tarja Limnell Orion Pharma
Juha Lintunen Orion Pharma
Tanja Lipsanen Orion Pharma
Anchang Liu Helsingin yliopisto
Tinna Lücke Orion Pharma
Petteri Lyytinen Orion Pharma
Saara Mahlamäki Orion Pharma
Janne Marvola Helsingin yliopisto
Fysikaalisen farmasian XIX symposium
OSALLISTUJAT
107
Tuuli Marvola Helsingin yliopisto
Inna Miroshnyk Helsingin yliopisto
Sabiruddin Mirza Helsingin yliopisto
Matti Murtomaa Turun yliopisto
Mika Myrskyranta Orion Pharma
Ermei Mäkilä Turun yliopisto
Juha Mönkäre Kuopion yliopisto
Tero Närvänen Orion Pharma
Satu Paalanen Orion Pharma
Jari Pajander Kuopion yliopisto
Leena Peltonen Helsingin yliopisto
Sami Poutiainen Kuopion yliopisto
Heli Puintila Orion Pharma
Mika Pulkkinen Kuopion yliopisto
Anu Pulliainen Lääkelaitos
Joakim Riikonen Turun yliopisto
Ville Rimpiläinen Kuopion yliopisto
Heikki Räikkönen Helsingin yliopisto
Meike Römer Helsingin yliopisto
Kirsti Saarnivaara Orion Pharma
Henri Salokangas Orion Pharma
Minna Salonen Fennolab
Niklas Sandler AstraZeneca
Hélder Santos Helsingin yliopisto
Terhi Santtila Lääkelaitos
Marja Savolainen Helsingin yliopisto
Kari Seppälä Orion Pharma
Simo Siiriä Helsingin yliopisto
Heidi Sjögren Orion Pharma
Maike Stiers Helsingin yliopisto
Clare Strachan Helsingin yliopisto
Marjo Taive Orion Pharma
Veli Pekka Tanninen Orion Pharma
Mikko Tenho Turun yliopisto
Pekka Teppola VTT
Jussi Tervonen Kuopion yliopisto
Saara Tiittanen Orion Pharma
Tarja Toropainen Kuopion yliopisto
Timo Tuomi Bruker
Elina Turunen Kuopion yliopisto
Kari Vahervuo Orion Pharma
Bert van Veen Orion Pharma
Jaana Veki Kuopion yliopisto
Tetta Venäläinen Kuopion yliopisto
Henna Vihola Vitabalans Oy
Satu Virtanen Helsingin yliopisto
108