Miniaturization of Drug Solubility and Dissolution Testings - Doria

Division of Pharmaceutical Technology 

Faculty of Pharmacy 

University of Helsinki 

Miniaturization of Drug Solubility and Dissolution 

Testings 

Tiina Heikkilä 

ACADEMIC DISSERTATION 

To be presented, with the permission of the Faculty of Pharmacy of the University of 

Helsinki, for public examination in auditorium 1041, Viikki Biocenter 2 (Viikinkaari 5 E), 

on 11 th June 2010, at 12 noon. 

Helsinki 2010

Supervisors: Docent Leena Peltonen 




Helsinki, Finland 

Professor Marjo Yliperttula 

Division of Biopharmacy and Pharmacokinetics 




Professor Jouni Hirvonen 





Reviewers: Professor Jukka Rantanen 

Department of Pharmaceutics and Analytical Chemistry 

Faculty of Pharmaceutical Sciences 

University of Copenhagen 

Copenhagen, Denmark 

Associate Professor Christos Reppas 

Department of Pharmaceutical Technology 


University of Athens 

Athens, Greece 

Opponent: Doctor Juha Kiesvaara 

Head of Formulation Research 

Orion Pharma R&D 

Turku, Finland 

© Tiina Heikkilä 

ISBN 978-952-10-6190-5 (paperback) 

ISBN 978-952-10-6191-2 (PDF, http://e-thesis.helsinki.fi) 

ISSN 1795-7079 

Helsinki University Print 

Helsinki, Finland, 2010

Abstract 

Solubility and drug dissolution are of crucial importance for drug formulations. Poor 

water-solubility of drug candidates is a major obstacle in drug development, since the oral 

route is the most patient convenient and cost effective way to deliver drugs. In some cases 

the low aqueous solubility may limit the bioavailability when the absorption of the drug is 

dissolution limited. About 40% of the current lead optimization compounds suffer from 

poor solubility. 

Improvements in drug solubility/dissolution testing technologies (e.g. high throughput 

screening, HTS) can enhance the possibilities of the lead compounds to success in the later 

stages of drug development process. Typically HTS protocols measure the kinetic 

solubility involving co-solvents (e.g. dimethyl sulfoxide), which might enhance the in 

vitro solubility or give erroneous results if the potential drug candidates are eliminated. 

Also, measurement of dissolution profiles is not available by the present HTS methods. 

For these reasons, there is a need for improvements in HTS solubility formats. 

Traditionally the in vitro dissolution tests are studied by pharmacopoeial methods, 

which are not utilizable in the drug discovery stage because of the large amount of 

compounds needed. However, dissolution studies at the drug discovery stage could be 

useful e.g. to classify compounds based on their dissolution rates. In addition, the initial 

steps of dissolution process might be lost by the regulatory dissolution methods. 

The aim of this thesis was to miniaturize traditional drug dissolution and solubility 

testing methods. Systematic down scaling of methods was done towards the development 

of both equilibrium and kinetic 96-well plate solubility/dissolution methods. 

Miniaturization of the regulatory dissolution methods and shake-flask solubility 

measurements on the 96-well plates was successful. 96-well plate methods for equilibrium 

drug solubilities, as well as for drug dissolution profiles as a function of time, were 

developed. The former method is the first true equilibrium solubility method, which can 

be used in the screening of drug solubility and dissolution phenomena at the early stages 

of drug development process. This method was also tested using fasted state human 

intestinal fluid as a medium for the first time. Human intestinal fluid and data obtained 

might turn out to be important for very low water-solubility compounds. Surface tension 

based microtensiometry was also presented as an alternative method for kinetic HTS of 

drug solubility properties, e.g. for classifying solubility of compounds not suitable for UVanalysis. 

Channel flow methodology was introduced enabling especially the kinetic 

follow-up at the very beginning of the dissolution process. 

This thesis provides directly applicable new miniaturized methods for the drug 

solubility and dissolution experiments. These methods enhance the throughput and 

understanding of drug solubility/dissolution phenomena and profiles in drug discovery and 

improve success in the later stages of the drug development process.

Acknowledgements 

This work was carried out at the Division of Pharmaceutical Technology, Faculty of 

Pharmacy, University of Helsinki, Finland. 

I wish to thank all the people who have been involved with this study. 

First, I would like to express my warmest gratitude to my supervisors Professor Jouni 

Hirvonen, Docent Leena Peltonen and Professor Marjo Yliperttula. In particular, I want to 

thank Leena for her constant patience and practical support. You were precent when 

needed and I felt like no stupid questions were. I wish everybody had a supervisor like 

you! Jouni and Marjo, first of all, thank you for this opportunity. Thank you also for your 

comments to all the versions of manuscripts and reports I have written, your scientifical 

guidance and providing excellent working facilities. 

The reviewers of the thesis, Professor Jukka Rantanen and Associate Professor 

Christos Reppas, are acknowledged for their careful work and valuable comments on 

impoving the manuscrpit. 

I am also grateful to all my co-authors, Professor Patrick Augustijns, Doctor Milja 

Karjalainen, Professor Kyösti Kontturi, Doctor Timo Laaksonen, Professor Frank 

Lammert, Doctor Peter Liljeroth, M.Sc. Krista Ojala, M.Sc. Kirsi Partola, M.Sc. Saila 

Taskinen and Professor Arto Urtti for their co-operation and scientific contributions. 

It has been very pleasent to work at Viikki because of all you co-workers. I felt warmly 

wellcome from the very beginning and I have got all the practical help I ever needed to 

perform this work. 

I would also like to thank my co-workers at the Lauttasaari Centralpharmacy for 

keeping me updated in practical pharmacy work. Especially, I want to thank pharmacy 

owner Liisa Heikkinen. 

In vitro in vivo relevance (IVIVRe) project in co-operation with Orion Pharma is 

acknowledged for the financial support. 

Finally, I would like to thank my most lowed ones. Especially, I would like to thank 

my Mum for introducing me the pharmacy and encouraging me to study this far, and 

Sanna, thank you for endless support. Emmi, Elsa, Petter and Alpo, thank you for just 

being there, and Timo, thank you for your love all the way. I made it! 

Espoo, May 2010 

Tiina Heikkilä

Contents 

Abstract 3 

Acknowledgements 4 

List of original publications 7 

Abbreviations 8 

1 Introduction 9 

2 Review of the literature 11 

2.1 Solubility in drug discovery and development 11 

2.1.1 Kinetic solubility 13 

2.1.1.1 Dimethyl sulfoxide (DMSO) as a co-solvent 13 

2.1.1.2 Kinetic solubility assays 14 

2.1.2 Equilibrium solubility 15 

2.2 In vitro dissolution methods for solid dosage forms 16 

2.2.1 Regulatory dissolution methods 18 

2.2.2 Initial steps of the dissolution process 19 

2.2.3 In vivo mimicking of dissolution methods 22 

2.3 In vivo dissolution 23 

2.3.1 Effect of pH 23 

2.3.2 Role of bile acids and phospholipids 24 

2.3.3 Hydrodynamics in gastrointestinal (GI) tract 25 

2.4 In vitro in vivo (IVIV) correlation 26 

2.4.1 Biopharmaceutics Classification System (BCS) 26 

2.4.2 Biopharmaceutics Drug Disposition Classification System (BDDCS) 27 

2.4.3 BCS Biowaivers 28 

3 Aims of the study 30 

4 Experimental 31 

4.1 Materials 31 

4.1.1 Chemicals (I-IV) 31 

4.1.2 Human intestinal fluid (HIF) (IV) 31 

4.2 Methods 31 

4.2.1 Preparation of tablets (I, II) 31 

4.2.2 Dissolution methods 32 

4.2.2.1 Basket method (I, II) 32

4.2.2.2 Intrinsic dissolution rate method (I, II) 33 

4.2.2.3 Channel flow method (I, II) 33 

4.2.2.4 Micro dissolution method (previously unpublished data) 33 

4.2.2.5 96-well plate method (IV) 34 

4.2.3 Solubility methods 34 

4.2.3.1. Shake-flask method (III, IV) 34 

4.2.3.2 96-well plate method (IV) 35 

4.2.3.3 Surface tension based kinetic HTS solubility method (III) 35 

4.2.4 Analyses 35 

4.2.4.1 Analyses of human intestinal fluid (HIF) (IV) 35 

4.2.4.2 Solid-state characterization (IV) 35 

5 Results and discussion 37 

5.1 Dissolution testing 37 

5.1.1 Channel flow method vs. the regulatory dissolution methods (I, II) 37 

5.1.2 Micro dissolution studies in different media (previously unpublished data) 40 

5.1.3 Dissolution profiles in 96-well plate (IV) 41 

5.1.4 Scaling down the dissolution testings (I, II, III, IV) 41 

5.2 Solubility screening 42 

5.2.1 HTS 96-well plate methods (III, IV) 42 

5.2.2 Polymorphic changes (IV) 47 

5.3 Human intestinal fluid (HIF) 49 

5.3.1 Characterization of fasted state HIF (IV) 49 

5.3.2 HIF in solubility testing (IV) 50 

6 Conclusions 52 

7 References 53

List of original publications 

This thesis is based on the following publications, which are referred to in the text by their 

respective roman numerals (I-IV): 

I Peltonen, L., Liljeroth, P., Heikkilä, T., Kontturi, K., Hirvonen, J., 2003. 

Dissolution testing of acetylsalicylic acid by a channel flow method – 

correlation to USP basket and intrinsic dissolution methods. European 

Journal of Pharmaceutical Sciences, 19, 395-401. 

II Peltonen, L., Liljeroth, P., Heikkilä, T., Kontturi, K., Hirvonen, J., 2004. A 

novel channel flow method in determination of solubility properties and 

dissolution profiles of theophylline tablets. STP Pharma Sciences, 14, 389- 

394. 

III Heikkilä, T., Peltonen, L., Taskinen, S., Laaksonen, T., Hirvonen, J., 2008. 

96-well plate surface tension measurements for fast determination of drug 

solubility. Letters in Drug Design and Discovery, 5, 471-475. 

IV Heikkilä, T., Karjalainen, M., Ojala, K., Partola, K., Lammert, F., 

Augustijns, P., Urtti, A., Yliperttula, M., Peltonen, L., Hirvonen, J., 2010. 

Equilibrium drug solubility measurements in 96-well plates. International 

Journal of Pharmaceutics, submitted. 

Reprinted with the permission from the Elsevier Ltd (I, IV), Edition de Santé (II), and 

Bentham Science Publishers Ltd (III). 

7

Abbreviations 

BCS Biopharmaceutics Classification System 

BDDCS Biopharmaceutics Drug Disposition Classification System 

CFC channel flow cell 

CMC critical micelle concentration 

CSD Cambridge Structural Database 

DMSO dimethyl sulfoxide 

DSC differential scanning calorimetry 

EMEA European Medicines Agency 

FaSSIF Fasted state simulated intestinal fluid 

FDA US Food and Drug Administration 

GI gastrointestinal 

HIF human intestinal fluid 

HPCD hydroxypropyl-β-cyclodextrin 

HPLC high pressure liquid chromatography 

HTS high throughput screening 

IDR intrinsic dissolution rate 

Int.Ph. The International Pharmacopoeia 

IR immediate release 

IUPAC The International Union of Pure and Applied Chemistry 

IVIV in vitro in vivo 

JP Japanese Pharmacopoeia 

MAD maximum absorbable dose 

MCC microcrystalline cellulose 

Ph. Eur. The European Pharmacopoeia 

rpm rotations per minute 

SDS sodium dodecyl sulphate 

USP The United States Pharmacopeia 

WHO World Health Organization 

XRPD X-ray powder diffraction 

8

1 Introduction 

Oral route is the most patient convenient and cost effective way to deliver drugs. 

However, inadequate aqueous solubility and dissolution in intestines and colon limits the 

bioavailability of drugs. Nowadays even 40% of the lead optimization compounds are 

poorly water-soluble (Lipinski et al., 1997). Although the poor solubility can be 

occasionally overcome by novel formulations, e.g. oral lipid-based (Porter and Charman, 

2001; Hauss, 2007) or amorphous formulations (Kawakami 2009), or delivery systems, 

e.g. cyclodextrins (Szejtli, 1988; Brewster and Loftsson, 2007), it still increases the cost 

and time of the new drug product to enter the market. Instead, improvement of drug 

solubility/dissolution testing methods (e.g. by high throughput screening, HTS) in the drug 

discovery stage can enhance the possibilities of the lead compound to success in the later 

stages of the drug development process. 

HTS solubility methods measure the solubility of numerous compounds at small 

quantities (Pan et al., 2001; Chen et al., 2002b; Takano et al., 2006; Bard et al., 2008; Dai 

et al., 2008; Alelyunas et al., 2009). Discovery protocols typically measure the kinetic 

solubility involving small amounts of co-solvents like dimethyl sulfoxide (DMSO). The 

co-solvents might enhance the in vitro solubility due to the co-solvent effect (Chen et al., 

2002b) and the solubility in vivo might be totally different. Especially, for the poorly 

soluble drugs the solubility can increase manifold with co-solvents. Also, erroneous 

decisions can be made if wrong drug candidates are eliminated because of rapid uptake of 

water by DMSO. When the water concentration in the DMSO solution gets too high, some 

lead compounds may fall out of solution and give a false negative result (Homon and 

Nelson, 2006). For these reasons, there is a need for improvements in HTS solubility 

formats. 

Drug dissolution testing of solid dosage forms is a routine process in pharmaceutical 

industry to evaluate drug release characteristics and to predict in vivo behavior. 

Traditionally the in vitro dissolution tests are studied by pharmacopoeial methods (e.g. 

paddle and basket methods) in the product development process for quality control, e.g. to 

assess the lot-to-lot quality and to detect changes in the drug formulation or manufacturing 

process. In the development of the drug product dissolution tests have been used to study 

the excipients (Omelczuk and McGinity, 1993; Uesu et al., 2000), and also to determine 

bioequivalence. In the drug discovery process pharmacopoeial dissolution methods are not 

utilizable because of the large amount of lead compounds and/or drug candidates needed, 

however, dissolution studies at the drug discovery stage could be used to e.g. to classify 

compounds based on their dissolution rates or to show whether the dissolution has taken 

place. 

Biopharmaceutics Classification System (BCS), which divides drugs into four classes 

according to their solubility and permeability properties, correlates in vitro drug product 

dissolution and in vivo bioavailability (Amidon et al., 1995). After BCS the meaning and 

application of relevant solubility/dissolution media has become more important and a 

broad range of dissolution media have been developed, e.g. FaSSIF (fasted state simulated 

intestinal fluid) and FeSSIF (fed state simulated intestinal fluid) (Galia et al., 1998). Still, 

the most physiological medium for solubility and dissolution studies would be human 

9

intestinal fluid (HIF), because drugs are usually dissolved and absorbed in the small 

intestine. The miniaturized drug solubility and dissolution methods requested above 

should enable the use of HIF when appropiate. 

In this thesis, different approaches were studied to miniaturize solubility and 

dissolution testing methods. The in vitro in vivo (IVIV) correlation of the solubility testing 

was studied by using different buffer solutions and fasted state human intestinal fluid as 

media. The literature review focuses on the different available solubility and dissolution 

methods and on the IVIV correlation. 

10

2 Review of the literature 

2.1 Solubility in drug discovery and development 

IUPAC (The International Union of Pure and Applied Chemistry) defines solubility as 

‘the analytical composition of a mixture or solution which is saturated with one of the 

components of the mixture or solution, expressed in terms of the proportion of the 

designated component in the designated mixture or solution’ (Lorimer and Cohen-Adad, 

2003). Solubility can further be defined in a more specific manner, like unbuffered, 

buffered and intrinsic solubility. Unbuffered solubility means the solubility of a saturated 

solution of the compound at the final pH of the solution. Buffered (apparent) solubility 

refers to solubility at a given pH, measured in a defined pH-buffered system, and usually 

does not perceive the influence of salt formation with counterions of the buffering system 

on the measured solubility value. Intrinsic solubility (So) is the solubility of the neutral 

form of an ionizable compound. For neutral compounds all three definitions coincidence. 

Solubility measurements determine either kinetic or equilibrium (thermodynamic) 

solubility depending on the experimental set-up (Alsenz and Kansy, 2007). 

Compounds can be divided into highly or poorly soluble ones. Traditionally 

compounds were considered to be poorly soluble if the solubility was less than 100 μg/ml. 

In 2006 Fligge and Schuler redefined the concept of poor solubility by HTS method; a 

compound is poorly soluble when the solubility is less than 20 μg/ml, partly soluble when 

the solubility is 20-80 μg/ml and soluble when the solubility is over 80 μg/ml. According 

to Lipinski (2004), 15% of the drug-like compounds and 40% of lead optimization 

compounds were insoluble at a concentration of less than or equal to 20 μg/ml. The 

definitions of drug-likeness and lead-likeness have been discussed e.g. by Oprea et al. 

(2001), but in general it can be said that in the lead optimization process a lead compound 

is moved towards a drug candidate having drug-like properties. In the pharmacopoeias 

solubility is indicated by the descriptive terms (Table 1). 

Table 1. The descriptive terms for the solubility used in the pharmacopoeias (USP, 

2009;Ph.Eur., 2009). 

Term Parts of solvent required for 1 part of solute 

Very soluble 

Less than 1 

Freely soluble 

From 1 to 10 

Soluble 

From 10 to 30 

Sparingly soluble 

From 30 to 100 

Slightly soluble 

From 100 to 1000 

Very slightly soluble 

From 1000 to 10 000 

Practically insoluble, or insoluble 

Greater than or equal to 10 000 

11

The requirement to the drug candidate’s solubility has been studied by the concept of 

maximum absorbable dose (MAD) (Johnson and Swindell, 1996): 

MAD = S 6.5 VL k a t (1) 

The unit of MAD is milligram, the transit time, t, is 270 minutes and the estimated luminal 

volume, VL, is 250 ml. S 6.5 marks the solubility at pH 6.5 (mg/ml) and ka the 

transintestinal absorption rate constant (min -1 ). In the MAD equation the small intestine 

can be considered as a tube, which has the volume of 250 ml. The saturated drug 

suspension is set into that tube, and the timer is started. The absorption rate constant 

describes how fast the dissolved quantity of the drug ‘leaks’ out of the tube each minute. 

After 270 minutes the total amount of ‘leakage’ is the estimate of the amount of the drug 

that should be intestinally absorbed. If that estimated quantity is close to the entire drug 

dosage, the intestinal absorption should be 100%. 

Lipinski (2000) has developed further the MAD concept based on the most common 

clinical potency of drug dose, 1 mg/kg. According to Lipinski, in the case of permeability 

being the average, the equilibrium (thermodynamic) solubility should be 52 μg/ml at pH 

6.5-7. For the low permeability drugs the equilibrium solubility should be 207 μg/ml and 

for the high permeability drugs 10 μg/ml, in order to be therapeutically effective. Low 

dose drugs (0.1 mg/kg) with high permeability can be effective even at the equilibrium 

solubility of 1 μg/ml and, on the opposite cases, 10 mg/kg dose drugs having low 

permeability properties might need equilibrium solubilities of as high as 2100 μg/ml to be 

effective. The advantage of the MAD model is that it easily shows the impact that the 

required dose might have to the permeability, solubility and fraction absorbed of the dosed 

compound. 

The solubility determinations are performed several times along the drug discovery 

and development processes, only varying the focus, the amount of the drug used and also 

the level of the purity and crystallinity of the drug candidate. In the early drug discovery 

stage the goal is to rank compounds according to the solubility and to aid in the structure 

activity chemistry relationships; in the drug development stage the goal is a more accurate 

numerical solubility number (Lipinski, 2004). More precisely, during the lead 

identification the solubility assays are, e.g., used to recognize compounds with potential 

liabilities, while in the lead optimization solubility information is used to make informed 

decisions on potential development candidates. Also the equilibrium solubility is used to 

confirm earlier kinetic solubility results in the lead optimization. In the drug development 

stage the dissolution testing is an important area for solubility assays (Dokoumetzidis and 

Macheras, 2006). In the earlier drug development stages the goal is, e.g., to identify ratelimiting 

steps, while in the later stages the dissolution tests are used, e.g., in the quality 

control and bioequivalence studies. Figure 1 depicts the solubility and dissolution assays 

in the drug discovery and development processes. 

12

New 

medicines 

proposal 

DISCOVERY DEVELOPMENT 

Target 

assessment 

Lead 

identi- 

fication 

Figure 1 Solubility and dissolution determinations in the drug discovery and development 

stages. 

2.1.1 Kinetic solubility 

Lead 

optimi- 

zation 

Kinetic Equilibrium 

solubility solubility 

(HTS) (shake-flask) 

Entry into 

human 

enable 

The measurement of the kinetic (non-equilibrium) solubility usually starts from the 

dissolved compound and represents the maximum solubility of the fastest precipitating 

material, which is typically not determined. The precipitating material can be any of the 

existing different solid forms of the compound, for example, polymorphic changes can 

happen even within minutes (Aaltonen et al., 2006). Also the formation of different salts 

may be possible depending on the presence of ionic species in the medium. In practice, the 

small volume (μl) of the stock solution (most often DMSO), in which the compound is 

dissolved, is added to the aqueous solution until the solubility limit is reached. 

Experiments can be performed by adding equal volumes of DMSO stock solution to the 

aqueous medium spaced a minute apart (Lipinski et al., 1997). In an alternative 

experiment, a dilution series of DMSO stock solution is prepared into separate vials 

(Kibbey et al., 2001). The kinetic solubility is defined to be the concentration at which the 

precipitation starts. 

2.1.1.1 Dimethyl sulfoxide (DMSO) as a co-solvent 

The amount of DMSO in the aqueous solution is increased in the kinetic solubility 

experiments by every addition of the DMSO stock solution. The final DMSO 

concentration varies and concentrations 0.5% (Bard et al., 2008), 1% (Chen et al., 2002b; 

Taub et al., 2002; Bard et al., 2008), 2% (Kibbey et al., 2001; Dehring et al., 2004), 5% 

(Pan et al., 2001; Bard et al., 2008) and even up to 20% (Kramer et al., 2009) have been 

reported. DMSO might induce a higher water-solubility than the compound would have 

without the DMSO due to the supersaturation or co-solvent effects (Chen et al., 2002b; 

Loftsson and Hreinsdottir, 2006; Bard et al., 2008). The co-solvents increase the solubility 

Phase 

I 

13 

Phase 

II 

Phase 

III 

Dissolution 

(regulatory methods, intrinsic dissolution rate) 

Registration 

On 

market

y being miscible with water and acting as better solvents for the solute in question. 

DMSO is an efficient solvent for poorly water-soluble compounds because of its 

amphipathic structure with highly polar domain and two apolar groups enabling it to mix 

both hydrophilic (with water) and hydrophobic (organic compounds) environments 

(Santos et al., 2003). Supersaturated solutions are solutions, which have higher solute 

concentration than in the equilibrium state. The equilibrium is established between the 

solute in the solution and the excess of (undissolved) compound. DMSO enhances the 

solubility in a compound specific way (Chen et al., 2002b; Taub et al., 2002). Taub et al. 

(2002) investigated the possibilities of replacing DMSO by another co-solvent, for 

example ethanol or HPCD (hydroxypropyl-β-cyclodextrin), but the effect of enhanced 

solubility remained. According to Alsenz and Kansy (2007), the DMSO concentration 

should be kept as low as possible (1%) to reduce the potential co-solvent effect and to 

achieve better correlation with the equilibrium solubility methods. The final co-solvent 

and the amount of it should always be reported, although the solubility results obtained 

from different kinetic methods are typically not comparable to each other and the kinetic 

solubility values are not even expected to be reproducible between different kinetic 

methods (Stuart and Box, 2005). Comparison of the results is complicated because of the 

different DMSO concentrations at the end-point (Chen et al., 2002b). Di and Kerns (2006) 

have reviewed the strategies for optimizing bioassays with DMSO, including 

improvements in storage and handling. Serial dilutions in DMSO are recommended, and 

these dilutions should be added directly to the assay media. Assays should also run at the 

lowest useful concentrations. 

DMSO, the current industry standard solvent for screening purposes, has some very 

problematic features, like it suffers from the rapid uptake of water; if the water content 

gets too high in the DMSO solutions, some compounds may fall out of solution and give a 

false negative result (Homon and Nelson, 2006). DMSO is hygroscopic and can absorb 

water up to 10% of its original volume (Di and Kerns, 2006), which also causes reduced 

solubility with the crystallization in the freeze-thaw cycles. DMSO has also several 

systemic adverse effects, e.g. nausea, vomiting, diarrhea, anaphylactic reactions, 

bradycardia and heart block are reported (O’Donnell et al., 1981; Davis et al., 1990; 

Rapoport et al., 1991; Stroncek et al., 1991; Styler et al., 1992). Besides, even 20% of the 

compounds in the commercial libraries are poorly soluble in DMSO according to Balakin 

et al., 2004. 

2.1.1.2 Kinetic solubility assays 

In general, kinetic solubility values are measured in the drug discovery process to screen 

the lead compounds. A high-throughput turbidimetric method for kinetic solubility assay, 

in which the detection was based on nephelometry (Davis and Parke, 1942), was 

introduced by Lipinski et al. (1997). Detection is based on the intensity of the scattered 

light as a function of concentration. In general, kinetic solubility measurements are fast 

and suitable for the HTS processes handling more than 100.000 samples per day and cover 

14

the required concentration range in assays used in early discovery (Homon and Nelson, 

2006). More details about the methods are presented in the Table 2. 

Table 2. Kinetic solubility in the drug discovery stage. Precipitation quantified by 

nephelometry or UV-absorbance. 

Sensitiveness 

Sample volume 

DMSO conc. 

Advantages 

Nephelometry a-d Diode array UV a UV plate reader d-g 

20-54 μM 

200-300 μl 

0.67-5% 

Colored compounds do 

not interfere, 

Shaking or 

ultrasonication is not 

required. 

5-65 μg/ml 

2.5 ml 

0.67% 

Accurate determination based 

on a calibration curve. 

0.5-1 μM 

200 μl 

0.5–5% 

Able to detect impurities 

or low UV absorption. 

Disadvantages Small undissolved Colored compounds miscalled Compounds should 

particles cause as insoluble. 

have sufficient 

misinterpretations; 

absorbance in the 

High/low 

region > 230 nm, 

concentrations 

Absorption of residues 

can be problematic. 

or other materials at the 

same wavelength as 

drug compounds. 

a b c d 

Lipinski et al., 1997; Bevan and Lloyd, 2000; Dehring et al., 2004; Hoelke et al., 2009; 

e f g 

Pan et al., 2001; Chen et al., 2002b; Bard et al., 2008 

2.1.2 Equilibrium solubility 

The equilibrium (thermodynamic) solubility test is performed by dispensing a surplus of 

solid compound into a liquid medium. After reaching the equilibrium state between the 

excess of undissolved compound and saturated solution, the saturation solubility can be 

defined. To confirm that equilibrium state has been reached, the solubility has to be 

constant for several consecutive time-points, which takes several hours or days, even 

months to reach. Usually equilibrium solubility is studied in the later stages of drug 

15

discovery process and it is regarded as the golden standard for development needs (Alsenz 

and Kansy, 2007). 

A classical equilibrium solubility assay is the shake-flask method. In this method an 

excess of solid drug is dispensed into aqueous solution, after which the container is shaken 

at certain speed and temperature. Samples are collected regularly, usually during 24-48 

hours, to make certain that the equilibrium plateau has been reached. The shake-flask 

method has not been officially defined in pharmacopoeias, so several protocols exist 

(Avdeef et al., 2000; Blasko et al., 2001; Kerns, 2001; Bergström et al., 2002; Lipinski et 

al., 2003; Chen and Venkatesh, 2004; Glomme et al., 2005; Avdeef, 2007; Zhou et al., 

2007) (Table 3). 

Table 3. Different protocols to perform shake-flask solubility measurements (N.A. not 

available in the article). 

Blasko et al., 2001 

Kerns, 2001 

Bergström et al., 2002 

Chen and Venkatesh, 2004 

Glomme et al., 2005 

Avdeef, 2007 

Zhou et al., 2007 

Medium 

volume (ml) 

5.0 

0.45 

0.05-1.0 

1.0 

2.0 

2.0-20.0 

1.0-5.0 

Shaking 

(rpm) 

N.A. 

N.A. 

300 

N.A. 

450 

N.A. 

600 

16 

Residue removal Quantification 

filtration 

filtration 

centrifugation 

filtration 

filtration 

filtration or 


filtration or 


2.2 In vitro dissolution methods for solid dosage forms 

Dissolution (dissolution rate) can be expressed by the Noyes-Whitney equation: 

HPLC 

HPLC 

HPLC-UV/fluorescence 

HPLC-UV 

HPLC-UV 

HPLC-UV 

HPLC-UV 

(LC-MS) 

dm DA 

= (Cs – Cb). (2) 

dt h 

The rate of mass transfer of solute molecules or ions through a static diffusion layer, 

dm/dt, is directly proportional to the area available for molecular or ionic migration, A, the 

concentration difference, Cs-Cb, and D, the diffusion coefficient of the solute (usually 

m 2 /s) and is inversely proportional to the thickness of the boundary layer, h. Cs is the

saturation solubility and Cb is the concentration of drug at a particular time in the bulk 

dissolution medium (Noyes and Whitney, 1897). If the Cb does not exceed 10% of the Cs, 

the dissolution is said to occur under sink conditions. In the table 4 are the factors 

affecting the dissolution rate. 

Table 4. Factors affecting the dissolution rate. 

Term Factor Examples 

A Particle size and shape 

- Aggregation 

Dokoumetzidis and Macheras, 

2006 

- Crystal shape affected by crystallization process 

Fini et al., 1995 

Cs 

Cb 

h 

D 

Solid-state form 

- Change in the solid form properties 

Complex formation 

Salts 

- common ion-effect 

Solubilizing agents 

Temperature 

Dissolution medium 

- e.g. pH (ionizable compounds), co-solvents 

Volume of medium 

Processes that remove dissolved solute from the medium 

- e.g. adsorption 

Thickness of boundary layer 

- affected by agitation 

Diffusion coefficient of solute in the dissolution medium 

- affected by the viscosity of medium 

17 

Pudipeddi and Serajuddin, 2005 

Brewster and Loftsson, 2007 

Serajuddin, 2007 

Bakatselou et al., 1991 

Hill and Petrucci, 1999 

Taub et al., 2002 

Dressman et al., 1998 

Lindenberg et al., 2005 

Alsenz et al., 2007 

Dressman et al., 1998

The importance of dissolution to the drug absorption and bioavailability is emphasized in 

the Biopharmaceutics Classification System (see also later chapters) by Dn, dissolution 

number (Amidon et al., 1995): 

Dn = 

t 

t 

res 

Diss 

where tres is the mean intestinal residence time (ca. 180 min, which differs from MAD 

model time) and tDiss is the time required for a drug particle to dissolve. From the above 

equation it is obvious that the higher the Dn, the better the drug absorption, and the 

maximal drug absorption is expected when Dn>1. In the case of Dn

Table 5. The official dissolution testing conditions. 

Medium 

Volume (ml) 

pH of buffers 

Temperature ( o C) 

Enzymes 

Agitation (rpm) 

Basket method 

Paddle method 

a 

FDA, 1997a 

Ph. Eur. USP a JP 

900 

1.0-7.5 

37±0.5 

case-by-case 

100 

50 

500, 900, 1000 

1.2-6.8 

37±0.5 

case-by-case 

50-100 

50-75 

2.2.2 Initial steps of the dissolution process 

19 

900 

1.2-6.8 

37±0.5 

case-by-case 

The main difference between the stirring methods described above and intrinsic 

dissolution method (IDR) (Ph. Eur., 2009; USP, 2009) or channel flow cell technique 

(Compton et al., 1988; Brown et al., 1993; Compton et al., 1993) is that in the latter 

methods only one tablet side is exposed to the dissolution medium (Figure 2). 

100 

50

Figure 2 Schematic illustrations and liquid flows of a) Intrinsic dissolution apparatus and b) 

Channel flow cell method. 

Intrinsic dissolution rate (IDR) 

The IDR method is based on the idea that the dissolution environment (surface area, 

temperature, pH, stirring speed and ionic strength) of pure drug substances is kept constant 

during the whole measurement (Nicklasson et al., 1981; Nicklasson et al., 1985). The fluid 

flow has been studied (Mauger, 1996; Mauger et al., 2003) and the IDR method has also 

been successfully miniaturized requiring only 5 mg of pure drug (Berger et al., 2007; 

Persson et al., 2008). As an advantage the compression pressure, dissolution medium 

volume or die position have no significant effect on IDR (Yu et al., 2004). The IDR of the 

pure drug is an important physicochemical property, which is useful, e.g., when predicting 

20

problems related to the absorption of the drugs. The evaluation of the IDR of pure drug 

substances demonstrates chemical purity and equivalence. 

The intrinsic rate (j) is expressed in terms of dissolved mass of compound per time per 

exposed area (mg min -1 cm -2 ): 

V dc 

j = , (4) 

A dt 

where V is volume, A is area of the drug disk, c is concentration and t is time. IDR can 

also be calculated by Levich equation (Yu et al., 2004): 

2 / 3 

D 

j = 0.62 ( 1/ 

6 

v 

1/ 

2 

) Cs, (5) 

where Cs is saturated solubility or concentration, D is the diffusion coefficient, ω is the 

angular velocity of the rotating disk and v is the kinematic viscosity of the dissolution 

medium. It is assumed in this model that equilibrium is predominant at the interface of 

solid and liquid, and the liquid at the interface is the same as the liquid in bulk dissolution. 

Since the intrinsic dissolution is primarily a rate phenomenon, it is expected to correlate 

more closely with in vivo drug dissolution dynamics than solubility. Still, the correlation 

between the solubility and intrinsic dissolution rate of a drug compound has been found 

both in water or aqueous buffer solutions (Nicklasson et al., 1981; Gu et al., 1987; 

Nicklasson et al., 1988) and water-ethanol binary solvent systems (Nicklasson and Brodin, 

1984). Also the correlation between the IDR and BCS solubility classification has been 

found by Yu et al. (2002). 

Channel flow cell (CFC) method 

The hydrodynamics (laminar flow) in the channel flow cell (CFC) is well defined 

(Compton et al., 1988; Brown et al., 1993; Compton et al., 1993), which enables 

acquisition of quantitative information of the dissolution kinetics and mechanism, even at 

early stages of the process, and modelling. This information is often lost in the traditional 

dissolution methods. In addition, accurate modelling of the mass transfer in the CFC 

enables comparisons of different dissolution mechanisms and experimental results. The 

flow rate in the channel presents an additional experimental parameter that can be used to 

tune the rate of mass transfer in the flowing aqueous phase. 

When the dissolution experiments involve pure compounds, the model consists of a 

convective mass transfer in the medium coupled to a dissolution reaction at the tablet 

surface with an equilibrium constant, K (Singh and Dutt, 1985). Theoretical expression to 

the concentration, c b , as a function of time: 

21

c b mA 

(t) = K (1 – exp (- t)) (6) 

V 

where m is the mass transfer coefficient, A is the exposed area of the tablet and V is the 

volume of the dissolution medium and t is time. 

In the CFC the tablet is located in the cavity so that the disintegration of the tablet is 

hindered when, e.g., the properties of disintegration excipients do not affect the 

dissolution as in the traditional methods. In the case of tablet formulations, it is not 

expected that the mass transfer coefficient would be equal for all tablets and only a 

function of the flow rate. With tablet formulations including excipients, diffusion and 

dissolution inside the tablet and the sink conditions were included in the model (Higuchi, 

1961): 

2 b 

b 

V c ( t) 

V V 2K 

K − c ( t) 

t + + ( + ) ln ( ) = 0 (7) 

2 o 

2 o 

D A c mA D A c K 

T 

T 

where DT is the diffusion coefficient of the drug inside the tablet and c 0 the concentration 

of the drug in the dry tablet. 

The combination of dissolution testing and solid-state analysis has been proposed to be 

called physico-relevant dissolution testing (Aaltonen and Rades, 2009). Recently, the 

channel flow method has been utilized, e.g., to study the solvent-mediated solid-state 

transformation during dissolution (Aaltonen et al., 2006; Lehto et al., 2008), the solid-state 

behavior during dissolution of amorphous drugs (Savolainen et al., 2009) and the effect of 

texture on the intrinsic dissolution behavior (Tenho et al., 2007). 

2.2.3 In vivo mimicking of dissolution methods 

Alternative methods have been developed to complete the lacks of the regulatory methods. 

In general, the official basket and paddle dissolution techniques suffer from high 

variability in results (Achanta et al., 1995; Qureshi and McGilvery, 1999). Further, the 

basket method suffers, e.g., from the hydrodynamic ‘dead zone’ under the basket, 

blockings in the mesh by drug or excipient and hindered visual examination of the 

disintegration process (Banaker, 1992). The paddle method, for its part, suffers above all 

from the coning effect (Beckett et al., 1996). 

Several unofficial in vitro dissolution methods exist (Pernarowski et al., 1968; 

Shepherd et al., 1972; Shah et al., 1973; Cakiryildis et al., 1975), some of them modified 

from the regulatory methods (Qureshi and Shabnam, 2003; Röst and Quist, 2003). The 

new methods, e.g. the dissolution stress-test device, are more complicated (Garbacz et al., 

2008; Garbacz et al., 2009). This method mimics the discontinuous movements of the 

dosage form and breaks of it from the intestinal fluid in the GI tract. Even more 

complicated release and solubility testing methods are the physiologically based TIM-1 

22

(stomach and small intestine) and TIM-2 systems (large-intestine) (Havenaar and 

Menikus, 1994). For example the TIM-1 has four compartments simulating stomach, 

duodenum, jejunum and ileum. The TIM devices mimic, e.g., the peristaltic movements, 

GI transition times, pH of GI tract and secretion of bile, gastric acids and enzymes. Results 

with the TIM-1 system compared to the in vivo data have been similar (Blanquet et al., 

2004; Souliman et al., 2006). Still, the new dissolution devices need a large volume of 

medium, which enables their use only in the later stages of drug development process. 

2.3 In vivo dissolution 

The most common and patient convenient way to deliver drugs is the oral route. After oral 

administration, the physiology of the GI tract affects the dissolution and absorption of the 

drug. The GI tract is divided into three major anatomical regions (the stomach, the small 

intestine and the colon), from which the small intestine is the most important site for drug 

absorption. The length of the small intestine is about 3.3 meters (Ho et al., 1983) and the 

area available for absorption is about 200 m 2 (Ashford, 2007). The large area of small 

intestine is possible because of the folds of Keckring, the villi and the microvilli. The 

absorption sites of drugs in the small intestine differ from each other. Highly permeable 

drugs absorb mainly at the tips of the villi and the low permeability drugs may diffuse 

down the length of the villi to be absorbed over a larger surface area (Artursson et al., 

2001). The main anatomical barrier for the drug absorption is the enterocytes, which 

apical side constitutes of densely packed microvilli and the lateral membranes connected 

through tight junctions. The absorption routes from the intestinal epithelium are based on 

paracellular passive diffusion, transcellular pathway (passive diffusion, carrier-mediated, 

receptor-mediated) or carrier-mediated efflux pathways (Back and Rogers, 1987; Tsuji 

and Tamai, 1996; Stenberg et al., 2000; Artursson et al., 2001; Martinez and Amidon, 

2002). 

Before possible absorption the drug needs to be released and dissolved from the solid 

dosage form to the GI fluids. The pH, composition, volume and hydrodynamics of the 

contents in the lumen are the main factors in the dissolution of drugs in the GI tract 

(Dressman et al., 1998). 

2.3.1 Effect of pH 

The pH in the GI tract varies depending on the anatomical region and the nutrition state. 

Stomach has an acidic pH in the fasted state, median values are found between 1.7 and 2.4 

with high interindividual variations. After meal the gastric median pH increases up to 6.4- 

6.7 depending on the type and volume of the meal. The pH of stomach is returned acidic 

2-4 hours after the meal (Dressman et al., 1990; Kalantzi et al., 2006a). Intestinal pH is 

higher than the gastric pH. In the fasting state the median pH is about 6.1-7.2 (Lindahl et 

al., 1997; Kalantzi et al., 2006a; Moreno et al., 2006; Brouwers et al., 2006; Clarysse et 

al., 2009b) and in the fed state 6.1-6.6 (Dressman et al., 1990; Kalantzi et al., 2006a; 

23

Clarysse et al., 2009b), with less interindividual variation compared to the gastric pH 

(Table 6). The pH has been reported to be similar in the jejunum (median 7.0) and 

duodenum (median 6.5) in the fasted state (Moreno et al., 2006). The pH in the colon 

depends on the region. In the caecum the pH is around 5.6-6.6, increasing up to 7-7.5 in 

the distal parts of the colon (Fallingborg et al., 1989). Especially, for ionizable compounds 

the pH of the GI tract is important; Ratio of unionized and ionized forms depends on pH. 

Usually the ionized form has higher solubility than unionized form. For example weak 

acids has slow solubility and dissolution in stomach, whereas they can achieve high 

solubility and dissolution in intestine. On the contrary, for poorly soluble compounds the 

pH of GI is not the rate-limiting step for dissolution, but in the GI tract is not enough fluid 

available or the entire dose of the drug dissolves too slowly (Dressman et al., 1998). 

2.3.2 Role of bile acids and phospholipids 

Bile is secreted to the small intestine. The secretion of bile is at maximum about 30 

minutes after ingestion of a meal and the gallbladder is emptied up to 73% (Mathus- 

Vliegen et al., 2004). The major components of bile are the bile acids, phospholipids, 

cholesterol and free fatty acids. The bile salt concentration compared to the phospholipid 

concentration has been reported to be 2-3:1 in the fed state and 9:1 in the fasted state 

(Persson et al., 2005; Kalantzi et al., 2006b). Bile has an important role in improving 

solubility and dissolution of poorly water-soluble drugs. Especially, bioavailability of 

poorly soluble drugs increases manifold when administered in fed state or with lipid-based 

formulations (Persson et al., 2005; Sunesen et al., 2005; Kalantzi et al., 2006b). The effect 

could result from decreased first pass metabolism, lymphatic uptake (Porter et al., 1996), 

inhibition of efflux transporters (Wu et al., 2005) or increased solubility (Persson et al., 

2005; Kalantzi et al., 2006b) and dissolution. Bile acids enhance solubility due to the 

wetting (Bakatselou et al., 1991) and/or solubilization effect (Bakatselou et al., 1991; 

Charman et al., 1993) which has been demonstrated e.g. with steroidal compounds 

(Mithani et al., 1995). 

For the prediction of in vivo dissolution and solubility, simulated intestinal fluids, like 

FaSSIF and FeSSIF, have been developed (Galia et al., 1998). Jantratid et al. (2008) 

proposed further a set of media to represent various regions of the gastrointestinal tract: 

fasted state simulated gastric fluid (FaSSGF) (Vertzoni et al., 2005), fed state simulated 

gastric fluid (FeSSGF), fasted and fed state simulated upper small intestine fluids 

(FeSSIF-V2, FaSSIF-V2) and various media for conditions that represent digestion. 

Simulated media contain e.g. lecithin and sodium taurocholate to represent the 

phospholipids and the bile salts. 

24

Table 6. The compositions of fasted and fed state HIF. 

Bile Acids/salts (mM) 

Taurocholic acid (%) 

Glycocholate (%) 

Glycochenodeoxycholate (%) 

Glycodeoxycholate (%) 

Taurochenodeoxycholate (%) 

Taurodeoxycholate (%) 

Phospholipids (mM) 

Lyso-phosphatidylcholine (%) 

Phosphatidylcholine (%) 

Parameters 

Osmolality (mOsm/kg) 

pH 

Buffer capacity (mmol/l pH -1 ) 

Fasted HIF Fed HIF 

2.0-3.5 a-e 

22.0 a , 44.95±18.17 d 

25.0 a , 17.21±6.37 d 

18.0 a , 13.77±9.59 d 

1.0 a , 10.40±7.32 d 

20.0 a , 7.89±2.16 d 

1.0 a , 4.71±2.47 d 

0.2–0.6 a,i 

99.0 a 

1.0 a 

200–271 b-d 

6.6-7.5 a-f 

2.4-2.8 a-e 

25 

8.0-15.0 a,g,i 

18.0 a 

25.0 a 

20.0 a 

13.0 a 

15.0 a 

9.0 a 

0.07-4.8 a,g,i 

87.0 a 

13.0 a 

287 k 

5.1-6.1 f,j,k 

13.2-14.6 a 

a Persson et al., 2005; b Clarysse et al., 2009b; c Lindahl et al., 1997; d Moreno et al., 2006; 

e Brouwers et al., 2006; f Kalantzi et al., 2009a; g Mansbach et al., 1975; h Sheng et al., 2009; 

i Armand et al., 1996; j Clarysse et al., 2009a; k Dressman et al., 1998 

2.3.3 Hydrodynamics in gastrointestinal (GI) tract 

In the fasted state GI tract has only little or no motoric activity. Still, the ‘’house-keeping’’ 

waves empty the stomach regularly about every two hours from the stomach to the small 

intestine. In the fed state mixing is more active and dissolution is expected to be more 

efficient. The gastric emptying time is higher after meal ingestion or after large liquid 

volume compared to the fasted state and the lipid contents of the meal increases the transit 

time further (Sunesen et al., 2005). For small intestine the transit time varies from three to 

four hours and stays the same whether the drug is taken with meal or not (Sunesen et al., 

2005). Instead, the flow velocity varies in different parts of the small intestine e.g. being 

higher in the ileum as in the jejunum (Ho et al., 1983). The volume of stomach and 

intestine in fasted state is 20-30 ml and 120-350 ml (Dressman et al., 1998). Under fed 

conditions the volume might be as high as 1500 ml in the upper intestine.

2.4 In vitro in vivo (IVIV) correlation 

IVIV correlation has been defined as ‘a predictive mathematical model describing the 

relationship between in vitro property of a dosage form and relevant in vivo response, for 

example plasma drug concentration or amount of drug absorbed’ (FDA, 1997b). IVIV 

correlation serves as a surrogate for in vivo data and is also used to validate the dissolution 

methods and in quality control. Three main levels of the IVIV correlation have been 

determined: level A, level B and level C (FDA, 1997b). The level A correlation represents 

the linear relationship between in vitro dissolution rate and in vivo input curves of the drug 

from the dosage form. Level A correlation is the most informative and recommended, if 

possible. Table 7 compares the release testing in vitro and in vivo. Medium, volume, 

hydrodynamics, temperature, location of the formulation and amount of the drug are 

considered. 

Table 7. Differences between the in vitro and in vivo release testing of immediate release 

drug products. 

Parameter IN VIVO IN VITRO 

Medium GI fluids 

Water, 

Buffer solutions, 

Simulated media 

Volume 250 ml (fasted state) 

Depending on the device 

(interindividual variation) (from μl:s to1000 ml) 

Hydrodynamics GI motility Mechanical agitation, 

Depending on the device 

Temperature Body temperature Depending on the test 

circumstances 

Location of the formulation Variation with time Depending on the device 

Amount of the drug Decreases as it is absorbed Constant in closed systems, 

(e.g. open system) 

Decreases in open systems 

2.4.1 Biopharmaceutics Classification System (BCS) 

The meaning of BCS is to correlate in vitro drug product dissolution and in vivo 

bioavailability (Amidon et al., 1995). In practice, BCS divides drugs into four classes 

according to their solubility and permeability properties. The drugs are classified as highly 

soluble if the highest orally administered single dose can be completely dissolved in 250 

ml of water, independently of the pH value at the range of 1.0–7.5 at 37 o C. Further, the 

drug is considered to be highly permeable when 90% or more of the orally administered 

dose is absorbed in the small intestine. The ideal drug would be class I drug having high 

26

solubility and permeability. The dissolution test for these drugs is only to verify that the 

drug is rapidly released from the dosage form. For the BCS class I drugs the IVIV 

correlation exist if dissolution rate is slower than gastric emptying rate. For BCS class II 

drugs the drug absorption is expected to be dissolution limited and IVIV correlation 

should exist if the dose is not very high. For BCS class III drugs limited or no IVIV 

correlation is expected because permeability is the rate limiting step. These drugs dissolve 

rapidly, while the slow absorption restricts bioavailability during the passage in the 

absorption site in the GI tract. The BCS class IV drugs are often not very suitable for oral 

administration at all, so limited or no IVIV correlation is expected. 

Fleischer et al. (1999) noticed that food (high-fat meals) effects to the extent of 

bioavailability could generally be predicted based on the BCS. For example, high-fat 

meals have no significant effect on the extent of availability for class I drugs, although 

food may delay the stomach emptying time. Class II drugs would benefit from high-fat 

meals, e.g., because of additional solubilization and inhibition of efflux transporters in the 

lumen. High-fat meal effects would be decreased if the class II drugs were formulated 

towards having better solubility. 

2.4.2 Biopharmaceutics Drug Disposition Classification System (BDDCS) 

In 2005 Wu and Benet represented a modified version of BCS called Biopharmaceutics 

Drug Disposition Classification System (BDDCS) (Table 8), which is used in the 

prediction of overall drug disposition, including the rates of elimination routes and the 

effects of efflux and absorptive transporters on oral drug absorption. Accordingly, the 

extent of drug metabolism would be a better predictor than the 90% absorption 

characteristic. It was noticed that BCS class I and II compounds are eliminated primarily 

via metabolism, while class III and IV compounds end up unchanged into the urine and 

bile; if the major route of elimination for a drug is metabolism, then the drug exhibits the 

properties of high permeability compounds, and if the major route of elimination is renal 

and biliary excretion of unchanged drug, the drug is classified as low permeability 

compound. Using the extent of metabolism instead of permeability (BCS) changed the 

classification of 10 drugs, changed 11 multiple classified drugs to one class, and added 38 

drugs to the totally 168 drugs’ BDDCS list. The BDDCS predicted 93% of the drugs’ 

permeabilities correctly related to major route of elimination (Benet et al., 2008). 

27

Table 8. The Biopharmaceutics Drug Disposition Classification System by Wu and Benet 

(2005). The major route of elimination (metabolized vs. unchanged) serves as the 

permeability criteria. 

Class I 

High Solubility 

Extensive Metabolism 

Class III 

High Solubility 

Poor Metabolism 

28 

Class II 

Poor Solubility 

Extensive Metabolism 

Class IV 

Poor Solubility 

Poor Metabolism 

The BDDCS can also predict the effects of transporters. According to the Wu and Benet 

(2005), the transporter effects are minimal for class I drugs; efflux transporter effects 

predominate for the class II drugs and transporter-enzyme interplay in the intestines is 

important for the CYP3A substrates and phase 2 conjugation enzymes; absorptive 

transporter effects predominate for the class III drugs and; absorptive or efflux transporter 

effects could be important for the class IV drugs. 

2.4.3 BCS Biowaivers 

BCS is utilized for the so-called biowaivers, which means that in vivo bioavailability 

and/or bioequivalence studies may be waived. In that case a dissolution test could be 

adopted as the surrogate basis for the decision as to whether two pharmaceutical products 

are equivalent. According to the regulatory authorities (e.g. FDA, 2000; EMEA, 2002), 

only highly soluble and highly permeable BCS class I drugs can have a biowaiver status in 

IR solid oral-dosage forms. Furthermore, biowaiver products may not contain excipients 

that could influence the absorption of a drug compound, drug compound can not have a 

narrow therapeutic index, and the product can not be designed to be absorbed from the 

oral cavity. 

Yu et al. (2002) have proposed to extend the biowaiver status to BCS class II and III 

drugs. They criticized the regulatory limits of solubility and permeability being too strict. 

The changes they proposed were to narrow the required solubility pH range to the 1.0-6.8 

and to reduce the high permeability requirement from 90% to 85%. Also the boundaries 

for solubility of acidic BCS class II drugs have been shown to be too restrictive by 

Yazdanian et al. (2004). Other studies (Cheng et al., 2004; Kortejärvi et al., 2005; 

Kortejärvi et al., 2007; Kortejärvi et al., 2010) and reports (WHO, 2006) have been 

published recommending a biowaiver status for BCS class III drugs. Benet et al. (2008) 

recommended the regulatory authorities to add the extent of drug metabolism (i.e. 90%

metabolized) as an alternative method for the extent of drug absorption (i.e. 90% 

absorbed) in defining class I drugs suitable for biowaivers. This would increase the 

number of class I drugs eligible for biowaivers. 

29

3 Aims of the study 

The overall aim of the study was to develop predictive, reliable and rapid kinetic and 

equilibrium solubility and dissolution screening methods, which do not require large 

amounts of compounds and would (ultimately) correlate well with in vivo results. Several 

solubility and dissolution testing methods were developed and characterized by using 

different volumes of aqueous buffer solutions and fasted state HIF as media. The specific 

aims of the study were: 

1. To construct and characterize the channel flow method and to compare it with 

the regulatory USP basket and intrinsic dissolution methods (I, II). 

2. To miniaturize dissolution testing stepwise from the regulatory dissolution 

methods and shake-flask solubility measurements via micro dissolution method 

to the 96-well plates (micro dissolution testing is previously unpublished data). 

3. To develop 96-well plate HTS methodology to determine both the equilibrium 

solubility and dissolution rate while avoiding the co-solvent(s) related 

problems. The model compounds were initially in solid form as in the 

traditional equilibrium solubility method in shake-flasks (IV). 

4. To develop kinetic HTS drug solubility testing method suitable for compounds 

having problems with UV absorption. The method is based on compounds’ 

surface tension measurements by a novel microtensiometer (III). 

5. To compare the aqueous equilibrium solubility/dissolution results in the 96well 

plate method with corresponding testings in fasted state HIF medium (IV). 

30

4 Experimental 

4.1 Materials 

4.1.1 Chemicals (I-IV) 

The model compounds and references to the corresponding publications are summarized 

in Table 9. The model compounds were from Orion Pharma (Finland) except for 

antipyrine (Aldrich Chemical Company Inc., USA), diclofenac sodium (Sigma-Aldrich 

Chemie GmbH, Germany), indomethacin (Fluka Biochemika, Italy), piroxicam (Hawkins 

Inc., USA) and theophylline anhydrous (Helm AG, Germany). As tablet excipients were 

used microcrystalline cellulose (MCC) (Avicel PH 102, FMC, Ireland) (I, II), lactose 

(monohydr., 80 M, DMV International, The Netherlands) (I, II), magnesium stearate 

(University Pharmacy, Finland) (II) and talc (Pharmia Oy, Finland) (II). All the other 

laboratory chemicals were of analytical grade. 

4.1.2 Human intestinal fluid (HIF) (IV) 

The HIF was collected from five volunteers after an overnight fast by introducing one 

double-lumen catheter (Salem Sump Tube 14 Ch, external diameter 4.7 mm, Sherwood 

Medical, Petit Rechain, Belgium) in the duodenum (D2/D3) via the mouth. Sampling of 

HIF was continued for 3 hours every 15 minutes. HIF was stored at -30 o C or colder and 

before solubility studies the individual HIF samples were pooled together. 

4.2 Methods 

4.2.1 Preparation of tablets (I, II) 

Tabletting masses of 200 g (I) and 150 g (II) were prepared. The powders were mixed for 

15 min in turbula mixer (Turbula T2C, Willy A. Bachofen AG Machinenfabrik, 

Switzerland). Tablets were directly compressed with a single punch tabletting machine 

(Korsch EK-0, Erweka Apparatebau, Germany) using a flat punch with a diameter of 9 

mm. The target weights of tablets were 250 mg. 

31

Table 9. The model compounds. 

Model compound 

Molecular 

structure 

BCS 

class 

32 

pKa 

Acidic 

/basic 

Paper 

Acetylsalicylic acid C16H12O6 I 3.48 A I 

Antipyrine C11H12N2O I 0.7 B IV 

Atenolol C14H22N2O3 III 9.16 B IV 

Caffeine C8H10N4O2 I 0.73 B IV 

Carbamazepine C15H12N2O II 13.94 A IV 

Diclofenac sodium C14H10Cl2NNaO2 II 4.18 A IV 

Furosemide C12H11ClN2O5S IV 3.04 A IV 

Hydrochlorothiazide C7H8ClN3O4S2 III 8.95 A IV 

Ibuprofen C13H18O2 II 4.41 A III, IV 

Indomethacin C19H16ClNO4 II 3.93 A III, IV 

Ketoprofen C16H14O3 II 4.23 A IV 

Metformin HCl C4H12ClN5 III 13.86 B IV 

Metoprolol tartrate C15H25NO3 I 9.17 B IV 

Naproxen C14H14O3 II 4.84 A IV 

Phenytoin sodium C15H11N2NaO2 II 8.33 A IV 

Piroxicam C15H13N3O4S II 3.80,4.50, 

13.33 

A/B IV 

Propranolol HCl C16H22ClNO2 I 9.14 B IV 

Ranitidine HCl C13H23ClN4O3S III 8.40 B IV 

Rifampicin C43H58N4O12 II 9.92 A IV 

Spironolactone C24H32O4S IV N.A. N.A. IV 

Theophylline 

anhydrous 

C7H8N4O2 II 8.60,1.70 A/B II 

Trimethoprim C14H18N4O3 II 7.2 B IV 

4.2.2 Dissolution methods 

4.2.2.1 Basket method (I, II) 

USP basket method (Sotax AT 7, Sotax, Switzerland) was used as a reference dissolution 

method (USP XXVI, 2003). The rotational speed was 50 rpm, the volume of medium was

900 ml and the temperature was 23 o C. The samples were analyzed by a spectrophotometer 

(Perkin-Elmer Lambda 2, Germany). 

4.2.2.2 Intrinsic dissolution rate method (I, II) 

The intrinsic dissolution test was done according to the USP XXVI (2003), which is based 

on Wood’s rotating disk method (Wood, 1965). The model compounds (100 mg) were 

pressed with a hydraulic pressure of 1500 kg (Pye Unicam, UK) and rotated at 50 rpm in 

900 ml of the medium at the temperature of 23 o C. (Sotax AT 7, Sotax, Germany). The 

samples were conducted into a spectrophotometer (Perkin-Elmer Lambda 2, Germany) for 

analyses. 

4.2.2.3 Channel flow method (I, II) 

In the channel flow system only one tablet surface was in contact with the media (Figure 

3). The tablet was located at the centre of the channel flow cell in order to avoid the edgeeffects 

(Compton et al., 1988). The flow rate of the aqueous phase was controlled by a 

peristaltic pump (Ismatec IPN-12, Switzerland) and temperature (23 o C) by a thermostatted 

bath. The channel flow cell was coupled to a UV-VIS spectrophotometer (HP8541A, 

Hewlett-Packard, USA). 

Figure 3 a) The channel flow cell and b) the channel flow test apparatus. Modified from 

publications I and II 

. 

4.2.2.4 Micro dissolution method (previously unpublished data) 

By this method the solubility of selected model compounds (ibuprofen, ketoprofen, 

indomethacin and piroxicam) was determined in 0.1 M hydrochloric acid solution in pH 

1.2 and 6.8 buffer solutions and in biorelevant medium, FaSSIF. Excess of drug 

33

compound was added into 2 ml flat-bottomed test tubes of the micro dissolution apparatus 

μDISS ® (pION, USA). Dissolution medium was added into the test tubes and the solution 

was stirred at 180 rpm using magnet stirrers. The temperature of the test solutions was 

maintained at 37 o C with the aid of a water bath surrounding the test tube block. The test 

solutions were kept under these circumstances for 3 hours after which the solutions were 

filtered using preheated Millipore PVDF 0.45 μm filters. The solutions were then left at 

37 o C for 24 hours and the concentration of the solutions were measured by detecting the 

UV absorbance of the solutions in situ. 

Quantification of the sample solutions was determined against a calibration curve with 

four to five dilutions of standard stock solution. The concentrations of the solutions were 

determined at the absorption maximum wavelength. Further, to assure the buffer capacity 

of the solutions, pH of the sample solutions was measured after the solubility runs. 

4.2.2.5 96-well plate method (IV) 

Model drugs were first dissolved into a presolvent (methanol or water), and the presolvent 

solutions were then pipetted into 96-well plates (96F untreated 260895, Nunc, Denmark), 

after which the presolvent was evaporated in order to avoid co-solvent effects. After 

evaporation, 250 μl of the medium was added to the wells and the 96-well plates were 

shaken orbitally at 600 rpm (Heidolph titramax 100, Germany). Samples were withdrawn 

after 30 min, 1, 2, 5, 24 and 30 hours and pipetted into UV 96-well plates (UV FB 

microtiter plates 8404, Thermo Fisher Oy, USA) for analyses with UV plate reader 

λ=230-400 nm (Varioskan, Thermo Electron corporation, Finland). The tests were 

performed at 23 o C. 

4.2.3 Solubility methods 

4.2.3.1. Shake-flask method (III, IV) 

Shake-flask method was used as a reference method in the solubility tests (Lipinski, 

2003). The model compounds were weighted into 25 ml (III) or 40 ml (IV) glass vials, 

which were mixed by magnetic stirrer at 100 rpm at 23 o C during the tests. Samples were 

taken after 1, 2, 3, 4, 5 and 24, even after 30 hours for some compounds, until the 

equilibrium plateau was reached. Samples were filtered (0.45 μm, Minisart, Sartorius, 

UK) before analyses by UV-spectrophotometry (Pharmacia LKB, Ultrospec, Sweden). 

34

4.2.3.2 96-well plate method (IV) 

The method was described in chapter 4.2.2.5. 

4.2.3.3 Surface tension based kinetic HTS solubility method (III) 

The model drugs were first dissolved into a presolvent (methanol, ethanol or DMSO). 12- 

22 samples were prepared so that the first one had a drug concentration of ca. 100 mg/ml, 

and each subsequent sample was diluted to half the concentration of the previous one. 

Final sample was always a blank sample with no drug. Then, the presolvent solutions were 

added to the aqueous medium in the 96-well plate by a volume of 50 μl. Surface tension 

based measurements were performed with recently introduced Delta-8 multichannel 

microtensiometer (Kibron Inc., Finland). 

4.2.4 Analyses 

4.2.4.1 Analyses of human intestinal fluid (HIF) (IV) 

Bile salts were measured by GC-MS-selected ion monitoring analysis developed by 

Lütjohann et al. (2004). Free bile acids were extracted with diethylether and 

trimethylsilylated after alkaline hydrolysis and acidification. The chromatographic 

separation was performed with a DB-XLB capillary column in a constant flow mode of 

0.8 ml/min with a final temperature of 290 o C having helium as a carrier gas. 

Total phospholipid levels were determinated enzymatically according to Gurantz et al. 

(1981), using phospholipase D and choline oxidase. The reagent kit was from Wako 

Chemicals (Neuss, Germany). 

4.2.4.2 Solid-state characterization (IV) 

X-ray powder diffraction (XRPD) was used to study the solid state forms of the model 

compounds and to confirm possible (pseudo)polymorphic changes. Measurements were 

done by an X-ray powder diffraction theta-theta diffractometer (Bruker AXS D8 advance, 

Bruker AXS GmbH, Germany). The XRPD experiments were performed in symmetrical 

reflection mode with CuKα radiation (1.54 Å) using Göbel Mirror bent gradient multilayer 

optics. The scattered intensities were measured with a scintillation counter. The angular 

range was from 5 o to 30 o with the steps of 0.1 o and the measuring time was 20 s/step. 

The reference codes to identify experimental polymorphs were from CSD database 

(Cambridge Structural Database, The Cambridge Crystallographic Data Centre, UK) 

35

(Allen, 2002). The reference crystal structure codes were for diclofenac form I (P21/c) 

SIKLIH02 (Castellari and Ottani, 1997), diclofenac form II (C2/c) SIKLIH05 (Muangsin 

et al., 2004), diclofenac form III (Pcan) SIKLIH04 (Jaiboon et al., 2001), indomethacin αform 

INDMET2 (Chen et al., 2002a), indomethacin γ-form INDMET (Kistenmacher and 

Marsh, 1972), piroxicam monohydrate CIDYAP0 (Bordner, 1984), piroxicam α-form 

BIYSEH06 (Vrecer et al., 2003), piroxicam β-form BIYSEH (Kojic-Prodic et al., 1982) 

and spironolactone polymorph 1 from acetone ATPRCL10 (Dideborg and Dupont, 1972). 

Differential scanning calorimetry (DSC) was used to further confirm the XRPD 

results. The thermal analyses were performed with a Mettler DSC 823e (Mettler-Toledo 

AG, Switzerland) and analyzed by STAR e software (Mettler-Toledo AG, Switzerland). 

Samples were weighted (4-5 mg) to the aluminium pans and crimped by aluminium caps 

with a pinhole. The samples were heated at the rate of 10 o C/min from 25 o C to the 10 o C 

above the melting point of each compound. The measurements were performed under N2 

atmosphere and the calibration was performed with indium. 

36

5 Results and discussion 

5.1 Dissolution testing 

5.1.1 Channel flow method vs. the regulatory dissolution methods (I, II) 

Dissolution profiles of pure acetylsalicylic acid and theophylline from the channel flow 

method correlated well with the intrinsic dissolution profiles. In both methods the 

dissolution rate of acetylsalicylic acid at the time-point 25-min was around 5-10 mg/cm 2 at 

pH 1.2 and around 20-25 mg/cm 2 at pH 6.8 (I). The dissolution rates of theophylline at the 

time-point 10-min were around 11-12 mg/cm 2 at pH 1.2 and 10 mg/cm 2 at pH 6.8 by both 

the methods (II). 

The solubility of acetylsalicylic acid tablets (I) containing microcrystalline cellulose 

(MCC) (compositions A and C) was slightly increased by the lowering pH in the channel 

flow method opposite to the basket method. This could be due to the MCC swelling 

properties being in more important role in the channel flow method compared to the 

solubility of acetylsalicylic acid. Swelling of MCC affects the flow and the mass transfer 

characteristics hindering MCC’s disintegrating effect of the tablet in the CFC. The 

dissolution results with tablets containing only lactose as an excipient correlated well 

between the methods and the rank order of the dissolution profiles with all tablet batches 

(A-C) was similar by both methods (Figure 4). 

37

(a) 

(c) 

pH 1.2 pH 1.2 

pH 6.8 

Figure 4 Channel flow dissolution of acetylsalicylic acid tablet formulations A-C at pH 1.2 (a) 

and at pH 6.8 (c) (Flow rate was ca. 190 ml/h). Dissolution of acetylsalicylic acid 

with the USP basket method at pH 1.2 (b) and at pH 6.8 (d). Modified from paper I. 

The tablet compositions were more complicated with theophylline (II) containing four 

excipients (MCC, lactose, talc, magnesium stearate). The amounts of MCC and lactose 

varied between the tablet batches (A-D). The drug release rate measured by the channel 

flow method was decreased as the amount of MCC was increased. The USP basket 

method gave different result; the slowest drug release was from the tablets having the 

highest amount of lactose (A) or MCC (D). The difference can be explained by the 

different dissolution set-ups. In the channel flow method the position of the tablet hinders 

the disintegrating effect of MCC and even the formulation C (MCC 56%) remained as a 

coherent matrix. From the basket method the tablet formulation C disintegrated 

immediately. Also with theophylline the two methods lead to similar conclusions (Figure 

5). 

(b) 

(d) 

38 

B 

B A 

A 

C 

A C 

pH 6.8

(a) 

(c) 

pH 1.2 

pH 6.8 

(d) 

(b) 

Figure 5 Channel flow dissolution of theophylline from tablet formulations A-D at pH 1.2 (a) 

and at pH 6.8 (c) (Flow rate was ca. 190 ml/h). Dissolution of theophylline with the 

USP basket method at pH 1.2 (b) and at pH 6.8 (d) (A , B , C, D). Modified 

from paper II. 

The main difference between the channel flow and USP basket methods is that in the 

channel flow method only one tablet surface is exposed to the dissolution medium and, 

thus, the disintegration of the tablet is hindered. This enables collection of accurate 

information from the initial steps of the dissolution process. The intrinsic dissolution 

method is based on the idea of keeping the dissolution area of pure compound constant 

(Nicklasson et al., 1981; Nicklasson and Brodin, 1984) and achieving the dissolution by 

rotating or shear-like motion in the medium, which differs from the laminar flow in the 

channel flow method (Compton et al., 1988). Also the mass transfer can be followed with 

the channel flow method, which allows comparison between the different dissolution 

mechanisms and the experimental results. Channel flow cell method is also used in the 

modelling because of the well-defined laminar flow in the CFC (Aaltonen et al., 2006; 

Lehto et al., 2008). 

39 

pH 1.2 

pH 6.8

5.1.2 Micro dissolution studies in different media (previously unpublished 

data) 

Micro dissolution studies were performed in Orion Pharma with four model drugs 

(ibuprofen, indomethacin, ketoprofen and piroxicam) in three different media (aqueous 

solution pH 1.2, buffer solution pH 6.8 and FaSSIF) at +37 o C. Table 10 presents results 

comparing equilibrium solubilities with micro dissolution, shake-flask and equilibrium 96well 

plate methods. The latter were both performed at room temperature (23±1 o C). 

Table 10. Equilibrium solubilities (mg/ml) of four model compounds in different media 

(SF=Shake-flask, MD=Micro dissolution and HTS=equilibrium 96-well plate 

method, n=2). 

Compound 

Ibuprofen 

Indomethacin 

Ketoprofen 

Piroxicam 

SF 

pH 1.2 

0.07 

0.004 

0.08 

0.06 

MD 

pH 1.2 

0.05 

0.001 

0.13 

0.19 

SF 

pH 6.8 

2.9 

0.4 

5.5 

0.2 

40 

MD 

pH 6.8 

3.5 

0.6 

5.4 

0.6 

MD 

FaSSIF 

pH 6.5 

3.3 

0.6 

5.6 

0.4 

HTS 

HIF 

pH 6.2 

2.0 

2.4 

3.2 

0.8 

As expected, the equilibrium drug solubilities by the micro dissolution and shake-flask 

methods were higher at pH 6.8 than at pH 1.2 (pKa values of the model compounds are 

mostly around 4). The micro dissolution solubilities in the pH 6.8 buffer solution and 

FaSSIF (pH 6.5) correlated well suggesting that the buffer might be an adequate medium 

at this solubility scale for the model compounds, although FaSSIF contains taurocholic 

acid and lecithin. Instead, the solubility results in HIF (pH 6.2) were somewhat lower for 

ibuprofen and ketoprofen and somewhat higher for piroxicam and indomethacin compared 

to the aqueous buffer pH 6.8 or FaSSIF pH 6.5. The compositions of fasted state HIF and 

FaSSIF are more complicated than the composition of aqueous buffer at pH 6.8 containing 

e.g. bile acids, which enhance solubility due to the solubilization effect (Bakatselou et al., 

1991; Charman et al., 1993) and/or wetting (Bakatselou et al., 1991). Here, the dissolution 

method (micro dissolution vs. 96-well plate) has important role to give similar results. 

According to our study, the medium (aqueous buffer, FaSSIF, HIF) has minor role. The 

use of HIF (and FaSSIF) in the solubility studies may have more pronounced importance 

for very low solubility compounds at the discovery stage, although the more simple, fast 

and economic methods/techniques are preferred also there.

5.1.3 Dissolution profiles in 96-well plate (IV) 

Besides the equilibrium solubility determinations, the 96-well plate method can be used to 

study the dissolution rates of drugs as a function of time (IV). Figure 6 distinguishes 

nicely the dissolution profiles of ibuprofen and indomethacin regardless the shaking 

speeds in question. At the moment the method is useful for the classification of dissolution 

rates because of rather long centrifugation times before the sampling. By shortening the 

centrifugation time, more detailed dissolution studies are possible for more rapidly 

dissolving materials. 

Concentration (mg/ml) 

Figure 6 Aqueous dissolution profiles of ibuprofen and indomethacin at different shaking 

speeds (300, 600 and 1200 rpm) (n=3). Modified from paper IV. 

5.1.4 Scaling down the dissolution testings (I, II, III, IV) 

The scaling down of the dissolution testing from the traditional basket method (e.g. USP, 

2009; Ph. Eur., 2009) via micro dissolution method to the 96-well plates was successful. 

Main focus in the dissolution testing has been in the quality control and the several stages 

of drug development (FDA, 1997a). The large amount of drug compound needed has often 

limited the use of dissolution testing at the drug discovery stage, which the new methods 

now enable. 

Alongside with the regulatory dissolution methods, the small scale dissolution methods 

can be used to study the results, e.g., at the initial states (0-5 min) of the dissolution 

process. Dissolution profiles from the early stage of drug discovery process can be used to 

improve the knowledge on lead candidates. Further, while more complicated, hard to get 

and expensive media, e.g. HIF, can be utilized with these small scale methods, if needed. 

41

5.2 Solubility screening 

5.2.1 HTS 96-well plate methods (III, IV) 

Both kinetic (III) and equilibrium (IV) 96-well plate methods were developed in this thesis 

work. The fact that amphiphilic compounds decrease the surface tension (γ) of water 

linearly within a concentration range limited by air/water partitioning at the lower 

concentration end, and solubility/critical micelle concentration (CMC) at the higher end, 

was utilized in the development of the kinetic method. The solubility value can be 

determined from these isotherms, if the compound is more likely to precipitate than to 

form micelles. The phenomenon is described by the classical Gibbs adsorption isotherm: 

Γ = − 

1 ∂γ 

RT ∂ ln c 

where Γ is surface excess, R is gas constant, T is temperature and c is concentration. 

The measured Gibbs isotherm for ibuprofen is shown in Figure 7. The solubility of 

ibuprofen can be obtained from the focal point between the sloping part and the level part 

of the isotherm. After this point, solid and solvated phases are in equilibrium and the 

amount of the free compound is constant in the solution. At higher concentrations, all 

material exceeding the solubility limit will be in the solid phase, which has no effect on 

the surface tension. Figure 7 also shows the effect of pH on the isotherm of ibuprofen. The 

solubility of ibuprofen is lower at pH 1.2 (0.19 mg/ml) than in pure water (0.23 mg/ml). 

This is seen from the shift of the adsorption curve. 

42 

(8)

Figure 7 Gibbs isotherm for ibuprofen in aqueous solution. Surface tension is reduced with 

increasing ibuprofen concentration until a solubility limit is reached. By lowering the 

pH decreases the solubility of ibuprofen, which is seen from the transition point at 

lower concentration (n=12, methanol was used as a presolvent). Modified from paper 

III. 

The slope of the linear part just before the transition point is inversely proportional to the 

cross-sectional area of the compound at the interface (Suomalainen et al., 2004) (Figure 

7). With ibuprofen the molecular area differed between the two pHs; the absolute value of 

the slope for ibuprofen at pH 6.8 was 43.0 corresponding the cross-sectional area per 

molecule 0.0096 Å 2 , compared to the absolute value of the slope (17.9) and cross-sectional 

area (0.023 Å 2 ) at pH 1.2. With indomethacin the slopes and cross-sectional areas were 

similar in both media. These differencies may be due to the different interactions between 

solvent and solute molecule. 

A linear dependency was found between the solubilities in the microtensiometer and 

shake-flask measurements for ibuprofen (Figure 8). Best fit of the linear curve gave a 

slope of 1.16 with an intercept at around 180 μg/ml. Differences in the solubility between 

the samples were clearly detected, and the correlation was at a reasonably good level for 

fast screening purposes and, thus, clear distinction between low and high solubility 

environments is possible. The indomethacin results did not fall on the same fitted line as 

the ibuprofen results, which is likely due to the differences in the precipitation rates of the 

two compounds. Still, also with indomethacin it was possible to distinguish between the 

high and low solubility conditions. Not at all unexpectedly, for both the model compounds 

the solubilities measured by the surface tension method were consistently higher than 

those from the (equilibrium) shake-flask method − due to the co-solvent effect (Fligge and 

Schuler, 2006). This is common feature to all HTS kinetic methods (Pan et al., 2001; Chen 

et al., 2002b; Bard et al., 2008) (Figures 8 and 9). 

43

Figure 8 Measured ibuprofen solubilities with the novel surface tension method (y-axis) as a 

function of the solubilities from the shake-flask measurements (x-axis). Modified from 

paper III. 

Several variables were tested in order to study the effects of different solvent systems on 

the reliability and applicability of the microtensiometry measurements. Studied media 

environments included, e.g., electrolytes, and surfactants. NaCl and CaCl2 (0.9% and 2%) 

had only limited effects on the solubility of ibuprofen. Surfactants, sodium dodecyl 

sulphate (SDS) and Tween 80, increased the solubility slightly below their CMC values, 

but the increase was much more evident above the CMC values. The pH effect on the 

acidic model compounds was as expected; the higher the pH, the higher the solubility. 

Most importantly, the presence of other materials in the medium did not disturb the 

determinations. 

44

Figure 9 Drug solubilities by the surface tension (black, n=8-12) and shake-flask (grey, n=2) 

measurements; effect of the dissolution media. Modified from paper III. 

Most of the HTS methods measure the kinetic solubility when the lead compound is at 

dissolved state in the beginning of the measurement and the co-solvent(s) may 

overestimate the solubility (Chen et al., 2002b). The HTS equilibrium solubility method 

was developed to avoid that, so in the starting point the compound was in the solid form. 

The results from the equilibrium 96-well plate correlated well (R 2 =0.931) with the values 

from the shake-flask method over five orders of magnitude in aqueous solubility with 

twenty model drugs (Figure 10) (IV). The relative deviation of the 96-well plate results 

was always less than 7-fold. Relative overestimation by the 96-well plate method was the 

most significant for compounds with solubility values lower than 10 μg/ml (circled area in 

Figure 10). Still, the 96-well plate method gave reliable solubility profiling: 98% of the 

tests were within the predictive confidence intervals of 95%. 

45

Figure 10 The solubilities from shake-flask (y-axis) and equilibrium HTS methods (x-axis). 

Modified from paper IV. 

Despite the medium, the methods gave similar values. Even 10.000-fold changes in 

solubility were detected depending on the medium, but the correlation between the 

equilibrium HTS and shake-flask methods remained good. For example, the solubility of 

diclofenac sodium at pH 1.2 was 0.002 mg/ml and at pH 6.8 15.3 mg/ml using the HTS 

method; the corresponding values obtained by the shake-flask method were 0.003 mg/ml 

and 24.0 mg/ml, respectively. The solubilities were determined accurately over a wide 

solubility scale (0.002–169.2 mg/ml), which is a true advantage of the 96-well plate 

method. Compounds with vastly different solubilities can be determined with the method 

at small drug quantities and utilize method in the early drug discovery stage when only 

small quantities of compounds are available. Equilibrium 96-well plate method from 

Alelyunas et al. (2009) included HPLC, which might slow down the process and, 

traditionally, the equilibrium solubility has not been measured until the early phase of 

drug development stage (Lipinski, 2003). 

Also the kinetic HTS method (microtensiometry) is suitable for drug solubility 

screening purposes at the drug discovery stage. Although the method has few limitations, 

e.g. it might be difficult to distinguish between the precipitation and micelle formation 

behavior or some very soluble compounds might not sufficiently partition to the water/air 

interface, it has several benefits compared to other kinetic HTS methods. The method does 

not require standard curves or filtrations, solid residues do not disturb the determinations, 

large concentration scales are measurable and low UV absorption of poorly soluble drugs 

does not matter (Bevan and Lloyd, 2000; Pan et al., 2001). It can be stated that the method 

is very well utilizable for poorly soluble drugs, which partition sufficiently to the water/air 

interface. Kinetic HTS method was also used to measure the solubility of ibuprofen as a 

46

function of time. Samples were remeasured after 5 hours, in which time the solubility 

values did not change too much indicating that overestimation was more due to the cosolvent 

effect than supersaturation. The dissolution of ibuprofen was fast and the 

dissolution profile as a function of time just ascertained that dissolution had taken place. It 

is compound specific, how fast the dissolution takes place and the equilibrium solubility is 

reached. 

Further, ibuprofen and indomethacin results from both the 96-well plate methods were 

compared to each other at aqueous media pH 1.2 and 6.8. In both cases the 96-well plate 

methods gave maximum 7-fold higher solubility values compared to the shake-flask 

method. Only when using ethanol or methanol as presolvents the kinetic solubility values 

were more than 7-fold higher than the shake-flask solubility values (Figure 8). The 

overestimation by the HTS 96-well plate methods compared to the shake-flask method 

could be due to the different scales more than due to the partitioning during the kinetic and 

equilibrium solubility testings. 

5.2.2 Polymorphic changes (IV) 

During the shake-flask solubility tests, changes in the solid-state form of piroxicam were 

recognized; the solubility of piroxicam was first increased in the aqueous solutions, after 

which it was decreased to the equilibrium level. Also the color of the solid residue was 

changed during the test from white (anhydrate) to yellow (monohydrate) indicating the 

change in the physical state of the material. The changes were confirmed by XRPD and 

DSC techniques. Piroxicam raw material contains α- and β-forms (both anhydrates), but 

crystalline solid residues of piroxicam monohydrate and β-form (anhydrate) were formed 

in the solubility test at 5h time-point (Kojic-Prodic et al., 1982; Bordner, 1984; Vrecer et 

al., 2003) (Figure 11). For the other model compounds, no polymorphic changes were 

noticed during the shake-flask determinations. 

47

Figure 11 XRPD diffraction patterns of piroxicam in the shake-flask measurements. The 

diffractograms represent the solid residue recrystallized from methanol (starting 

material in HTS), raw material (starting material in shake-flask), pure piroxicam 

monohydrate and anhydrated α- and β-forms. 

In the equilibrium HTS method (pseudo)polymorphic changes occurred with four model 

drugs; piroxicam, indomethacin, diclofenac sodium and spironolactone (Table 11) (Figure 

11). From XRPD and DSC results it was obvious that the starting materials in the shakeflask 

and HTS methods were not of the same form. Also spironolactone is known to 

crystallize from different solvents (e.g. methanol, acetonitrile, ethanol, ethyl acetate and 

benzene) in different polymorphic forms (El-dalsh et al., 1983; Beckstead et al., 1993). 

During the HTS solubility tests no polymorphic changes were, however, observed. 

Although some materials had (pseudo)polymorphic changes during the solubility testing, 

these changes were not problematic for the interpretation of the results, because both the 

shake-flask and 96-well plate methods do measure the equilibrium solubility and the 

possible transformations are easily observed. Still, possible polymorphic changes of drug 

candidates may have an influence on the solubility results in solubility screening and, later 

on, on the drug development (Bauer et al., 2001). 

48

Table 11. Starting and raw material results from the XRPD (polymorphic form) and DSC 

(melting temperatures) measurements (n=3). Modified from paper IV. 

Compound XRPD DSC Reported melting 

temperature 

Diclofenac sodium 

raw material (Shake-flask) 

starting material (HTS) 

Indomethacin 



Piroxicam 



I, III 

II, III 

γ 

α, γ 

α, β 

monohydrate 

49 

288.3 o C 

285.5 o C 

160.0 o C 

154.0 o C, 160.5 o C 

202.4 o C 

201.5 o C 

a Llinas et al., 2007; b Chen et al., 2002a; c Vrecer et al., 2003 

5.3 Human intestinal fluid (HIF) 

5.3.1 Characterization of fasted state HIF (IV) 

II=180.5 o C a 

γ=160-161 o C b 

α=152-154 o C b 

α=202.6 o C c 

β=199.7 o C c 

monohydr.=202.0 o C c 

HIF was collected from five healthy volunteers’ duodenum. Inter- and intraindividual 

variations were observed in the study (Tables 12 and 13), like in the earlier studies 

(Lindahl et al., 1997; Clarysse et al., 2009b). The mean pH value measured from the 

pooled HIF was 6.24 compared to values from 6.6 to 7.5 and the mean osmolality was 205 

mOsmkg -1 compared to values of 200–271 mOsmkg -1 (Table 6). 

The mean of the total bile salt concentration in fasted HIF was 3.63 mM, which is in 

line with the earlier literature values from 2.0 to 3.5 mM (Table 6). Instead, the 

phospholipid concentration (1.8 mM, median 1.1 mM) was higher than in the earlier 

studies (0.2–0.6 mM) (Table 6) because of one sample, which was very concentrated. The 

samples vary between individuals and individual concentrations of up to 2.7 mM have 

been found in fasted upper small intestine earlier (Clarysse et al., 2009b). The mean 

cholesterol level of HIF was 1.8 mM, which was close to the level found by Mansbach et 

al. (1975) (1.5 mM).

Table 12. Summary of volunteers’ (V1-5) characteristics and human intestinal fluid 

collection from duodenum. 

Volunteer Gender Age BMI 

(kg/m 2 Volume pH Osmolality 

) (ml) 

(mOsm/kg) 

V1 Male 25 21.7 70 6.62 176 

V2 Female 39 20.8 100 5.19 219 

V3 Male 37 23.1 100 6.60 209 

V4 Female 24 21.6 40 6.27 213 

V5 Female 31 20.9 35 6.51 207 

Mean 

- 29 21.6 69 

6.24 205 

±s.d. 

8 

0.9 31 

0.60 17 

Table 13. Composition of fasted HIF from five healthy volunteers (V1-5). In the table 

DC=deoxycholate, C=cholate, CDC=chenodeoxycholate and 

UDC=ursodeoxycholate 

Bile 

salts (mM) 

Phospholipids 

(mM) 

Cholesterol 

(mM) 

50 

DC 

(%) 

C 

(%) 

CDC 

(%) 

V1 2.45 1.35 1.48 29.5 42.5 23.8 4.2 

V2 2.62 1.11 1.68 24.6 56.7 18.1 0.6 

V3 4.54 2.18 2.27 3.4 64.6 25.3 6.7 

V4 1.65 0.99 1.71 0.8 57.7 35.4 6.1 

V5 8.60 4.69 1.48 7.1 66.6 25.6 0.7 

5.3.2 HIF in solubility testing (IV) 

UDC 

(%) 

Equilibrium HTS solubility tests were performed using the fasted state HIF (pH 6.24) for 

13 model drugs and the results were compared to the HTS solubility results in the aqueous 

buffer solution at pH 6.8 (Figure 12) – resulting in a fair correlation (R 2 =0.630). More 

importantly, the absolute values of compound solubility differed only modestly (less than 

6.9-fold in all cases), even though 1000-fold range of solubilities were studied. Slightly 

increased solubility values in HIF compared to the buffer solution pH 6.8 were seen for 

piroxicam, naproxen, rifampicin and diclofenac sodium. Vice versa, the solubilities in HIF

compared to pH 6.8 buffer solution were decreased for ibuprofen and ketoprofen. No 

major differences were seen between the solubility in buffer solution pH 6.8 and HIF 

suggesting that buffer is an adequate medium when studying dissolution and solubility 

with compounds in the 100 μg/ml–100mg/ml solubility range. However, we can not rule 

out the possibility that more significant solubility differences would be seen for the 

compounds with solubility below 100 μg/ml. For example, preliminary solubility test of 

beclomethasone dipropionate, showed 150 times higher solubility in HIF (0.6 μg/ml) than 

in the buffer at pH 6.8 (0.004 μg/ml). Obviously, more detailed studies are needed to 

determine the limits and utilization potential of HIF in drug solubility and dissolution 

testing, especially in the case of the compounds with very low solubility. It is worth 

noting, however, that the simple buffer is useful medium over surprisingly wide solubility 

range that extends at least to 10 2 μg/ml. 

HIF was, found to be a suitable solubility/dissolution medium for the 96-well plate 

testings. The small volumes make it possible to utilize the HIF also as a medium in drug 

discovery process, which may have an even more pronounced importance for very low 

solubility compounds. HIF is the most relevant solubility medium, and in this system it 

can be used instead of the buffers or simulated intestinal fluids, if needed. 

Figure 12 The solubilities in HIF (y-axis) and buffer solution pH 6.8 (x-axis). Modified from 

paper IV. 

51

6 Conclusions 

The main conclusions drawn based on the results of this study are: 

1. Channel flow method was introduced to the dissolution testing. The method is 

applicable on drug dissolution testing and enables especially kinetic follow-up 

at the very beginning of the dissolution process. The results from the channel 

flow method and the intrinsic dissolution method correlated well with each 

other. Later the channel flow method has been further developed to study the 

solvent-mediated solid-state transformation during dissolution (Aaltonen et al., 

2006; Lehto et al., 2008). 

2. A micro dissolution method was used to determine equilibrium solubilities of 

model compounds with smaller volumes than by the shake-flask method. The 

solubility results from the micro dissolution were reliable indicating utilization 

potential of the micro dissolution method instead of shake-flask method. 

Miniaturization was successful to the 96-well plates. 

3. A 96-well plate method was developed for measuring the equilibrium drug 

solubilities and dissolution profiles as a function of time in microlitre scale. 

The method is useful for screening purposes at early stages of drug 

development. Also polymorphic changes of compounds can be studied. The 

methodology developed is the first true equilibrium solubility measuring HTS 

system utilizing 96-well plates, meaning that in the beginning the compound 

was in solid form. 

4. A novel surface tension based microtensiometric method was presented for 

kinetic HTS of drug solubility properties. The method was tested by 

determining physiologically relevant parameters on the solubility of model 

compounds. The method is well suited for classifying purposes and solubility 

screening tests. 

5. HIF from duodenum was used as a medium for the first time in 96-well plate 

equilibrium solubility testings. Microliter volumes make it possible to utilize 

HIF, the most relevant solubility medium, in dissolution testing at drug 

discovery stage. This may be even more important in the future with very low 

solubility compounds. Still, the aqueous buffer solutions are the first choice 

due to good prediction capacity, low costs, ease of access and good correlation 

with model drugs. 

52

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