European Journal of Pharmaceutical Sciences 14 (2001) 115122 www.elsevier.

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A dynamic in vitro lipolysis model I. Controlling the rate of lipolysis by continuous addition of calcium
Niels Hnberg Zangenberg a , Anette Mullertz a , *, Henning Gjelstrup Kristensen a , Lars Hovgaard a ,1
a

Department of Pharmaceutics, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received 4 December 2000; received in revised form 20 April 2001; accepted 15 May 2001

Abstract Lipolysis by pancreatic lipase was investigated with the aim to establish an in vitro lipolysis model, which can be used to investigate the dissolution of poorly soluble lipophilic drug substances at controlled hydrolysis rates. The effects of three experimental parameters the concentrations of bile salts and Ca 21 and the lipase activity were investigated. The effect on the rate of hydrolysis of emulsied soybean oil was investigated in experiments in a pH-stat at pH 6.5 and 378C. The free fatty acids produced by the hydrolysis were titrated at pH 6.5. It was shown that all three investigated parameters inuence the initial rate of hydrolysis, whereas only the lipase activity and the concentration of Ca 21 affect the subsequent stages. It was also shown that the rate of lipolysis can be controlled by the rate of adding Ca 21 . Thus, it is possible to design an in vitro model using readily available and inexpensive materials in which the hydrolysis rate can be controlled by the continuous addition of Ca 21 . 2001 Elsevier Science B.V. All rights reserved.
Keywords: Dissolution; Pancreatic lipase; Rate of lipolysis; Bile salts; Poorly water-soluble drug substances

1. Introduction The bioavailability of poorly water-soluble, lipophilic drug substances belonging to Class II of the Biopharmaceutics Classication System (Amidon et al., 1995), is affected by the simultaneous administration of food (Ogunbona et al., 1985; Charman et al., 1993; Humberstone et al., 1996). In order to simulate the in vivo conditions in the fasted and fed state, complex dissolution media have been proposed to provide an in vitro test that may be correlated with in vivo data (Naylor et al., 1995; Dressman et al., 1998; Galia et al., 1998; Dressman and Reppas, 2000; Pedersen et al., 2000a). The proposed dissolution media are composed of naturally occurring surfactants and buffering agents. The solutions are prepared with bile salts (BS) (glycocholate or taurocholate), fatty acids (FA) and a phospholipid [phosphatidyl choline (PC)]. In order to ensure that solutions are standardised with regard to their solubilisation capacity, the components must be of high purity and are thus very expensive to purchase. Though the dissolution of Class II drug substances can be monitored and reproduced using such
*Corresponding author. Tel.: 145-35-306-440; fax: 145-35-306-031. E-mail address: amu@dfh.dk (A. Mullertz). 1 Present address: Galenica ApS, Hrsholm, Denmark.

media, it has been shown that the dissolution rate in aspirated gastric uids does not correlate fully with the in vitro dissolution rates in the simulated media (Pedersen et al., 2000b). Another important aspect is that the complex media simulating the fed state are static compared to in vivo conditions where the composition of uids of the small intestine is changing with the progress of lipolysis. By lipolysis, triglycerides (TG) are hydrolysed into FA, diglycerides (DG) and monoglycerides (MG). Together with BS, phospholipids and cholesterol, the lipolysis products form micellar structures and vesicles with a lipophilic core and a hydrophilic surface (Patton and Carey, 1979; Patton et al., 1985; Hernell et al., 1990). The dissolution and the increased postprandial absorption of lipophilic drug substances are usually attributed to the solubilisation capacity of these micellar structures and vesicles. There are two essential requirements to a dynamic dissolution model, which incorporates the lipolysis of lipids. First, the FA produced by the lipolysis must be removed continuously, mainly because FA, as an end product, inhibits the lipolysis of TG. Second, the rate of the lipolysis must be controllable in order to obtain reproducible results. It is important to simulate the dynamics of the luminal

0928-0987 / 01 / $ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 01 )00169-5

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uids when developing an in vitro lipolysis dissolution model. Such a model will provide information about the transport of poorly soluble drug substances into the aqueous phase by solubilisation in the micellar structures resulting from lipolysis. Similar lipolysis models have been used previously to study the enzymatic hydrolysis of emulsied lipids by pancreatic lipase (triacylglycerol lipase EC3.1.1.3.) (Patton and Carey, 1979; Reymond and Sucker, 1988; MacGregor et al., 1997). The aim of the present investigation has been to nd practical solutions both to the above-mentioned requirements, i.e., controlling the lipolysis rate and removing FA. The study is divided into two series: in the rst series, effects of three parameters likely to control the lipolysis rate are investigated. The three parameters are the concentration of BS, the concentration of Ca 21 and the lipase activity (MacGregor et al., 1997). Based on the results from the rst series, the effect of continuous addition of Ca 21 on the lipolysis rate was investigated more specically in the second series.

Nycomed Pharma (Roskilde, Denmark). The content of PC was determined as choline by using an enzymatic kit, MPR 2 manufactured by Boehringer Mannheim (Mannheim, Germany). The content of Ca 21 was determined by atomic absorption spectroscopy. Medilab A / S (Copenhagen, Denmark) performed the latter two analyses. The content of free FA in the soybean oil was determined by TLCFID as described in Zangenberg et al. (submitted).

2.2. Preparation of the emulsion for the second series

The emulsion contained 200 mg soybean oil / ml. Tween 80 and Span 80 were used as emulsiers, 1% (w / v) each. Methyl-parahydroxybenzoate, propyl-parahydroxybenzoate and a-tocopherol were used as preservatives. The emulsion was prepared using an Ultra Turrax T25 with a D25N-25Gm shaft for 5 min at 24 000 rpm at 808C. The particle size was measured in a Malvern Mastersizer (Malvern Instruments Ltd., Malvern, UK) with an RF-300 lens.

2.3. Preparation of pancreatin suspension

2. Materials and methods In order to obtain the required activity, an accurately weighed amount of pancreatin was suspended in 125.0 ml of 378C puried water and mixed thoroughly. The suspension was then centrifuged for 7 min at 4000 rpm at 378C in a Labofuge 400R, and the pH in the supernatant was adjusted to 6.5 using 1.00 M NaOH. A 100.0-ml aliquot was withdrawn and used in the model. The suspension must be freshly prepared (within 15 min) in order to avoid denaturation of the lipase.

2.1. Chemicals
Pancreatin (porcine) (P 1625), and bile extract (porcine) (B 8631) were purchased from Sigma (St. Louis, USA). Soybean oil was purchased from Unikem (Copenhagen, Denmark) and a-tocopherol from BASF AS (Asker, Norway) and both were of pharmaceutical grade. Intralipid (200 mg / ml) was manufactured by Fresenius Kabi (Copenhagen, Denmark). CaCl 2 ?2H 2 O of analytical-grade, manufactured by Merck (Darmstadt, Germany), was used. All other chemicals were of analytical-grade. The lipase activity of pancreatin was determined as described in The US Pharmacopoeial 24 (2000). Pancreatin is assumed to contain equivalent moles of colipase and lipase (Patton et al., 1978). The BS content in the bile extract was determined by using an enzymatic kit, i.e., Enzabile purchased from
Table 1 Experimental values for the rst series of lipolysis experiments a Na 1 Trizmamaleate TG b mM mM mM 150 2 15.3 Lower limit 270 1.5 4 300

2.4. Preparation of the lipolysis model 2.4.1. First series The compositions of the models are listed in Table 1. The models were prepared by mixing puried water, buffer (Trizma maleate and NaCl, pH 6.5), bile extract dissolved in puried water, Ca 21 -solution and Intralipid in a thermostated beaker (378C) and allowing the mixture to

Lipase activity BS c Ca 21 Volume (t50 min)

units / ml mM mM ml

Upper limit 1340 6.6 20

Centre point 800 4.1 12

a Standard-conditions for Na 1 , Trizma maleate and TG. Investigated intervals for lipase activity, BS and Ca 21 in the randomised, orthogonal 2 3 -central composite design with ve repetitions of the centre point. b Hernell et al. (1990); Janowitz et al. (1990). c Sjovall (1959); Hernell et al. (1990); Lindahl et al. (1997).

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equilibrate. The pH was adjusted to 6.5 with 1.00 M NaOH. The pancreatin suspension was prepared simultaneously and added to the freshly prepared substrate. At this point, the pH-stat was started.

2.4.2. Second series The lipolysis models were prepared similarly to those in the rst series, with the exception of the emulsion (prepared as described in Section 2.2) and the 0.5 M Ca 21 solution. The Ca 21 -solution was dispensed with a pump, which was started upon addition of the pancreatin suspension. In both series, a pH-stat (Titrino 718 with burette from Metrohm, Herisau, Switzerland) was used to maintain pH at 6.5. Data were collected using the software Tinet, version 2.3, also from Metrohm. The volume of the added 1.00 M NaOH reected the amount of FA generated by the lipolysis. The amount of titrated FA, as a function of time, was depicted and interpreted as a measure of the hydrolysis of TG by the lipase. 2.5. Experimental design 2.5.1. First series The purpose of the rst series was to investigate the effects of the concentration of BS, Ca 21 and lipase activity on the lipolysis rate. The experiments were designed as a randomised, orthogonal 2 3 -central composite design with ve repetitions of the centre point, in a total of 19 experiments. This design allows the investigation of a large interval for each parameter with relatively few experiments. Statgraphics 7.0 (Manugistics, Inc., Rockville, MD, USA) was used to design and statistically evaluate the results. The investigated concentrations are listed in Table 1. 2.5.2. Second series Three different rates for adding Ca 21 were investigated (see Table 2). Two other experiments were conducted with static concentrations of Ca 21 . The rst with [Ca 21 ]50.0 mM and the second with [Ca 21 ]517.4 mM. The concentration of 17.4 mM Ca 21 corresponds to the con-

centration after a 40-min experiment in which Ca 21 is added at a rate of 0.135 mmol / min. In addition, a lower (4 mM) concentration and a higher (16 mM) concentration of BS were investigated under standard conditions and with a Ca 21 -rate at 0.135 mmol / min.

3. Results

3.1. Establishing the model

The concentrations of the various model components used are listed in Tables 1 and 2. The concentrations and investigated intervals were chosen as a compromise between in vivo values as reported in the literature and ease of experimentation in the laboratory. A low Trizma maleate concentration (2 mM) was chosen in order to ensure that ionised FA were able to change the pH and is similar to that used by other authors (Patton and Carey, 1981; Larsson and Erlanson-Al bertsson, 1986; Lindstrom et al., 1988; Reymond and Sucker, 1988). The pH was set at 6.5 as a compromise between the optimum for the pancreas lipase, which is between 6 and 10 (Gargouri et al., 1989), and the measured duodenal pH, which is around 5.05.5 during a test meal (Zentler-Munro et al., 1984; Dressman et al., 1990; Carriere et al., 1993). The concentration of Ca 21 in the fasted state is 0.53 mM in the duodenum (Hofmann and Mysels, 1992; Lindahl et al., 1997), and the concentration in the fed state is highly dependent on the concentration in the food. Ca 21 is necessary for the activity of pancreatic lipase and the rate is highly dependent on the concentration. A large concentration interval (420 mM) was investigated in order to provide experimental variation. Several groups have reported the concentration of BS in the duodenum in the postprandial state. Mean values are typically between 5 and 15 mM, depending on the time after ingestion of the meal, with peak values up to 40 mM (Sjovall, 1959; Fausa, 1974; Westergaard, 1977; Rautureau et al., 1981; Hernell et al., 1990). BS concentrations in the duodenum of 37 mM during the fasted state have been reported (Sjovall, 1959; Hernell et al., 1990; Lindahl et al., 1997). An interval of 1.56.6 mM was investigated in the rst series, while concentrations ranged from 4 to 16 mM BS in the second series. Pancreatic lipase is present in the duodenum in excess. Arnesjo et al. (1969) measured an activity of 163 units / ml in aspirates from the duodenum and upper jejunum in the fed state. According to Patton (1981), the activity of 12 . 10 27 M pancreatic lipase and colipase in intestinal uids is between 150 and 300 units / ml. In order to ensure lipase activity in excess, an interval of 2701340 units / ml was investigated.

Table 2 Experimental values for the second series of lipolysis experiments Na 1 Trizma maleate TG Lipase activity BS Ca 21 Volume (t50 min) mM mM mM units / ml mM mmol Ca 21 / min ml 150 2 30.5 810 8 0.072 / 0.135 / 0.181 300

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Bile extract and pancreatin of porcine origin were chosen. According to Patton (1981), Borgstrom and Erlanson-Albertsson (1984) and Verger (1984), the replacement of human pancreatic lipase and colipase with porcine pancreatic lipase and colipase is generally accepted.

3.2. Characterisation of pancreatin, bile extract, soybean oil, and emulsions

The lipase activity in pancreatin was 12 units / mg pancreatin. One unit is equivalent to one microequivalent FA hydrolysed per min. The BS content in the bile extract was determined to 0.5060.05 g BS / g bile extract. The PC-concentration was 6% (w / w). In human and porcine bile, more than 95% of the phospholipids are PC (Alvaro et al., 1986). These concentrations approximate 13 moles of BS to 1 mole of PC. Ratios found in human bile are between 3 and 4 moles of BS and 1 mole of PC (Carey and Small, 1978; Lee and Nicholls, 1986). The content of Ca 21 was determined to be less than 0.06% (w / w). No free FA were detected in the soybean oil. The particle size of Intralipid was measured with the Mastersizer S. The distribution of the particle size was unimodal and the mean size was 0.41 mm with a span value of 0.80 hspan5[d(x,0.9)2d(x,0.1)] / d(x,0.5)j. As far as the emulsion for the second series is concerned, the particle size distribution was unimodal with a mean particle size of 1.59 mm and a span value of 1.6.

3.3. First series (titration of FA)

Because of the complexity of the investigated system, it was possible to evaluate the effect of the investigated parameters qualitatively but not quantitatively. Fig. 1A and B shows some of the results from the rst series. All 19 experiments showed the same pattern: a fast initial lipolysis rate during the rst 5 min, which then levelled off to a very slow rate. Statistical evaluation showed that both the Ca 21 -concentration and lipase activity had a signicant effect on the level of lipolysis at t51, 4 and 40 min at the investigated concentrations (P,0.0001 for both parameters after all three investigated periods). Ca 21 had the greatest impact on the relative hydrolysis. The effect of the lipase activity was relatively minor. In Fig. 1A and B, three different experiments are shown at two different levels of lipase activity and the lipolysis rate changes only slightly between lipase activity levels. In contrast, a change in Ca 21 concentration from 4 to 20 mM increases the lipolysis level dramatically. The increase is apparent within a few minutes, and after 40 min at 20 mM Ca 21 at the high lipase activity, the hydrolysis of the TG into two FA and one MG is almost 100%. Statistical analysis showed that BS only had a signicant effect at t51 min (P50.0032), lipolysis increasing with BS concentration.

Fig. 1. (A and B) Comparison of the lipolysis rate in three different compositions of the lipolysis model from the rst series. The lipase activity is 270 units / ml in (A) and 1340 units / ml in (B). () 1.5 mM BS and 4 mM Ca 21 ; ( ) 6.6 mM BS and 4 mM Ca 21 ; and (- - -) 6.6 mM BS and 20 mM Ca 21 . A total of 9.2 mmol FA are equivalent to a complete hydrolysis of the TG into FA and MG.

These results indicated that it is possible to control the rate of lipolysis by continuous addition of Ca 21 , using excess lipase activity and that this can be done independent of the BS concentration.

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3.4. Second series (continuous addition of Ca 21)

Fig. 2 shows the lipolysis in ve experiments. Experiments A, B and C compare the lipolysis at three different Ca 21 -addition rates, and show that if the addition rate is increased, the rate of lipolysis increases as well. After an initial high rate, the lipolysis rate slows down and becomes almost linear after approximately 5 min for all three rates of Ca 21 -addition. In experiment E, the initial concentration of Ca 21 was 17.4 mM, corresponding to the concentration after 40 min in experiment B. The nal extent of lipolysis was the same in these two experiments. Experiment D is performed without the addition of Ca 21 as a control. In Fig. 3, the number of moles of released FA is compared to the amount of Ca 21 added after 40 min. The relationship between added Ca 21 and released FA from the experiments with 8 mM BS is linear, with a slope of 1.1 mmol FA / mmol Ca 21 . The results from similar experiments with 4 and 16 mM BS show that the amount of FA hydrolysed does not change signicantly with the concentration of BS.

Fig. 3. The amount of FA titrated as a function of added Ca 21 after 40 min at 4 mM BS (h), 8 mM BS ( ) and 16 mM BS (s) under standard conditions. The line is based on results from experiments with 8 mM BS. It has a slope of 1.1 mmol FA / mmol Ca 21 (R 2 51.00).

Fig. 4 shows the relationship between the concentration of BS and the initial rate of lipolysis measured as the amount of FA hydrolysed during the rst minute. The higher the concentration of BS, the higher the initial lipolysis rate.

4. Discussion

4.1. First series (titration of FA)

In this study, it is assumed that the NaOH added at pH 6.5 reects the FA released from the hydrolysis of the TG by pancreatic lipase. In one of the experiments shown in Fig. 1B (pH56.5, 6.6 mM BS, 15 mM TG, high lipase activity and 20 mM Ca 21 ), 100% FA were titrated after 40 min. One hundred percent were titrated, even though the apparent pKa value for a typical long-chain FA, oleic acid (C18:1), in pure water is about 8 (Mattson and Volpenhein, 1966; Shankland, 1970). However, the pKa value of oleic acid has been shown to be 6.4 in a system containing 0.1 M NaCl and 0.5 M Ca 21 (Benzonana and Desnuelle,

Fig. 2. The effect of three different rates of adding Ca 21 . (A) 0.072 mmol Ca 21 / min, nal concentration59.4 mM Ca 21 ( - - ); (B) 0.135 mmol Ca 21 / min, nal concentration517.4 mM Ca 21 ( - ); (C) 0.181 mmol Ca 21 / min, nal concentration523.0 mM Ca 21 ( ); (D) 0.0 mmol Ca 21 / min, nal concentration50.0 mM Ca 21 (- - -). (E) 17.4 mM Ca 21 (), the initial concentration of Ca 21 corresponds to the nal concentration in (B). In this series 18.3 mmols of FA are equivalent to a complete hydrolysis of the TG into FA and MG. The BS concentration is 8 mM in all the experiments. All experiments were conducted in duplicate.

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Fig. 4. The amount of FA titrated during the rst 1 min of the experiments with 4, 8 and 16 mM BS under standard conditions (see Table 2) and 0.135 mmol Ca 21 / min.

1968). Similar pKa values can be achieved by dissolving oleic acid in BS micelles (Hofmann, 1968; Shankland, 1970; Staggers et al., 1990). On the other hand, the addition of MG, PC and lyso-PC to BS show no synergistic effect on the pKa value (Hofmann, 1963; Shankland, 1970; Small et al., 1984). Therefore, it can be assumed that in the present system (1.56.6 mM BS and 150 mM NaCl at pH 6.5), approx. 50% of the FA from soybean oil (mainly C18:2, C18:1 and C16:0) are ionised and can be partly titrated an assumption that to some extent is conrmed by Patton and Carey (1981). By hydrolysing TG in which the FA were radioactively labelled, they investigated whether long-chain FA are titratable at pH 6 and 6.9 in the presence of 17 mM TG, 6 mM BS and 10 mM Ca 21 . They determined the released FA by both radioactivity and by titration using a pH-stat. They found that at pH 6.9, approx. 50% of the released FA are titratable and that only negligible amounts are titratable at pH 6.0. In Fig. 1B, another experiment, containing only 4 mM Ca 21 , is compared to the one mentioned above with 20 mM Ca 21 (and pH56.5, 6.6 mM BS, 15 mM TG and high lipase activity for both experiments). The experiment with 4 mM Ca 21 showed as little as 50% titrated FA, whereas the experiment with 20 mM Ca 21 showed a 100% titration of the FA. Furthermore, the additional data presented in Fig. 1A and B show an initial lipolysis rate that is dependent on both BS- and Ca 21 -concentrations, followed by a lower hydrolysis rate after 510 min at hydrolysis levels which are dependent on the amount of Ca 21 present. This strongly indicates that Ca 21 is an important determinant of the extent of hydrolysis. In vivo, solubilisation of FA in BS-phospholipid mi-

celles together with MG is believed to be the most important way of removing the FA from the oilwater interface. In vitro, the formation of Ca 21 -soaps from Ca 21 and palmitate was found to take place at concentrations relevant to the present experiments (Lichtenberg et al., 1988), and the precipitation of Ca(FA) 2 is believed to be an important reaction in the presence of Ca 21 (Patton et al., 1985; Hernell et al., 1990). By precipitating FA with Ca 21 , FA are removed from the interface of the emulsion. This prevents the inhibition of pancreatic lipase by FA, which is explained as an effect of product accumulation at the interface (Brockerhoff, 1968; Scow et al., 1979), making the substrate inaccessible to the enzyme (Brockerhoff and Jensen, 1974). This effect is observed in vitro when a substantial amount of substrate is hydrolysed in the absence of FA-acceptors like Ca 21 and BS (Wieloch et al., 1982). Therefore, to allow the lipolysis to continue unimpeded, the FA must be removed either with Ca 21 or BS. Scow (1988) investigated the effect of Ca 21 on lipolysis in a study where he also included BS (Scow, 1988). He found that 3.3 mM Ca 21 in the medium affected the hydrolysis but not the release of lipolytic products from oil droplets during lipolysis. By contrast, 17 mM sodium taurodeoxycholate affected the release but not the hydrolysis by the lipase. In the same study, morphological studies were performed which showed that in the medium containing sodium taurodeoxycholate but no Ca 21 the surface of the oil droplets was smooth except for constantly moving stubby projections that seemed to melt into the aqueous medium. In the medium with Ca 21 but no BS, the droplets at rst became rufed and then crusted showing structures similar to those observed by Patton and Carey (1979) in analogous experiments. Thus, in the medium with BS, the lipolytic products were released into the medium in mixed BS micelles, whereas the droplets in the medium with Ca 21 were enveloped in crystalline Ca 21 soaps. The formation of Ca 21 -soaps draws the equilibrium towards the ionised FA (le Chateliers principle) and makes the FA titratable. The intensive stirring removes the crystalline shell from the oil droplets and allows the lipolysis to continue unimpeded at the surface of the emulsion droplet.

4.2. Second series (continuous addition of Ca 21)

The results from the rst series showed that the continuous addition of Ca 21 provides a way of controlling the lipolysis rate. The curves in Fig. 2 show an increased lipolysis rate when the rate of Ca 21 -addition is increased. This is both in accordance with the results from the rst series and with the work of MacGregor et al. (1997). They increased the concentration of Ca 21 from 5 to 40 mM and found the factor to be 2.6 for the extent of lipolysis after 30 min using 8 mM sodium taurocholate, 1.5 mM PC and 150 mM NaCl at pH 6.5. After an initial burst of hydrolysis, the rate slows down

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and becomes constant. In Fig. 3, the amount of hydrolysed FA after 40 min is plotted against the amount of added Ca 21 over the 40 min at the four different rates. The slope of this curve is expected to approximate 2 mmol FA / mmol Ca 21 , if all Ca 21 precipitates with two FA. The observed slope is only 1.1, indicating that Ca 21 must be in excess to keep the lipolysis rate constant. The initial extent of the hydrolysis in the rst 23 min, as shown by the curves in Fig. 2, can be explained in terms of the solubilisation of the lipolysis products in the BS micelles, whereby one mole of BS can solubilise approximately 1 mole of lipolytic product (Scow, 1988; Hernell et al., 1990). In the current experiments (Fig. 2), 2.4 mmols of BS are present. The lipolytic products in the initial period are primarily FA and DG in the absence of Ca 21 (Desnuelle et al., 1948; Constantin et al., 1960), and once 2.4 mmols of FA are hydrolysed, the hydrolysis rate starts to level off. This indicates that the initial lipolysis rate is inuenced by the concentration of BS. In Fig. 4, the initial lipolysis rate as a function of BS concentration is shown. If the BS concentration is increased, the initial rate increases as well. This nding agrees with the results from the rst series that the initial rate is inuenced by the BS concentration. In Fig. 3, the results from the experiments with 4 or 16 mM BS are also plotted. The amount of hydrolysed FA after 40 min does not change signicantly with the concentration of BS, which was also found to be the case in the rst series.

References
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5. Conclusions In the presence of BS, Ca 21 and physiological concentrations of NaCl, FA are titratable with NaOH at pH 6.5, even though the apparent pKa for long-chain FA is 6.5, presumably because the proposed formation of Ca 21 -soaps pushes the equilibrium towards the ionised FA (le Chateliers principle). In the system with continuous addition of Ca 21 , the initial hydrolysis rate is inuenced by the BS-concentration, and after the initial stage the rate levels off and becomes dependent on the rate of Ca 21 addition. Thus, using physiological concentrations of BS and NaCl and readily available and easy to prepare lipase sources and substrates, a reproducible model for controlled lipolysis has been established. This model should prove useful for the study of dissolution of poorly soluble drug substances and lipophilic formulations under biorelevant conditions in vitro.

Acknowledgements Dumex-Alpharma A / S, Novo Nordisk A / S and Nycomed Amersham A / S supported this project nancially.

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