Dextran hydrogels for colon-specific drug delivery. V. Degradation in human intestinal incubation models

EUROPtAN

ELSEVIER

European Journal of Pharmaceutical Sciences 3 (1995) 329-337

JOURNAL

OF

PHARMACEUTICAL SCIENCES ,

Dextran hydrogels for colon-specific drug delivery. V. Degradation in human intestinal incubation models Lene Simonsen a, Lars Hovgaard a'*, Per Br0bech Mortensen b, Helle Br0ndsted a ~Department of Pharmaceutics, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen ~, Denmark ~Department of Medicine A, Division of Gastroenterology, State University Hospital, Copenhagen, Denmark Received 18 December 1994; accepted 11 May 1995

Abstract In the present study degradation of dextran hydrogels, potential drug carriers for colon-specific drug delivery, was studied in simulated small intestinal juices as well as in a human colonic fermentation model. Dextran hydrogels were shown to be stable when incubated at 37°C with the small intestinal enzymes amyloglucosidase, invertase and pancreatin. After a 24 h incubation, less than 3.3% of free glucose was released. However, the hydrogels were still intact as measured by the dry weight remaining. The fermentation of dextran hydrogels and several mono- and polysaccharides to short-chain fatty acids (SCFA) was investigated after anaerobic incubation in a human colonic fermentation model at 37°C for 0-72 h. In addition, the dextranase activity of the incubations was determined. The amounts and ratios of S C F A formed varied considerably in relation to the type of substrate fermented (glucose, maize starch, potato starch, cellulose, soluble dextran and dextran hydrogels). Detailed S C F A analysis demonstrated that fermentable saccharides resulted in an increased S C F A production, in contrast to the metabolic inert polysaccharide, cellulose. The hydrogels were found to be completely degraded in the human colonic fermentation model. An increased crosslinking density or a decreased degree of hydration resulted in a lower degradability. The pH of the incubations were found to be inversely proportional to the SCFA production as a result of the increased acid formation.

Keywords: Dextran; Biodegradable hydrogel; Colon-specific drug delivery; Dextranase activity; Human colonic fermentation model; Short-chain fatty acid

1. Introduction

Various bacteria of the Lactobacillaceae family have been found to synthesize dextrans (Sidebotham, 1974). Dextran is a class of polysaccharides with a linear polymer backbone in which linkages are almost entirely of the 1,6-a-Dtype. Dextran derived from the bacterium Leuconostoc mesenteroides NRRL B-512 is characterized by the content of 95% 1,6-cr-oglucopyranosidic linkages and 5% 1,3-cr-D -linkages, the latter mostly consisting of 1 to 2 glucose * Corresponding author. Tel. (+45) 3537 0850; Fax (+45) 3537 1277. 0928-0987/95/$09.50 (~ 1995 Elsevier Science B.V. All rights reserved SSDI 0928-0987(95)00023-2

units (Van Cleve et al., 1956; Lindberg and Svensson, 1968). The 1,6-a-D-glucosidic linkages of dextrans are hydrolyzed by dextranases; enzymes with the systematic name: 1,6-o~-o-glucan 6-glucanohydrolases (EC 3.2.1.11). Dextranases are produced by various moulds (Hultin and Nordstr6m, 1949; Tsuchiya et al., 1952) and certain bacteria (Ingelman, 1948; Sery and Hehre, 1956; Bailey and Clarke, 1959; Janson and Porath, 1966; Sawai et al., 1974) as well as by mammalian cells (Rosenfeld and Lukomskaya, 1957; Dahlqvist, 1962). The vast majority of dextran splitters in the human colon are strictly anaerobic Gramnegative intestinal bacteria mainly of the genus

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Bacteroides, which produce dextranases (Hehre and Sery, 1952). The bacteroides are the numerically predominant anaerobes in the colonic region of humans. They number about 10 ~ per gram of intestinal contents and constitute approximately 30% of the total cultivable gut flora (Drasar and Hill, 1974; Salyers, 1984). The bacteroides produce both endo- and exodextranases which cleave randomly along the dextran chain and at the terminal linkages, respectively. At pH 7.0-7.5, the major effect is a rapid release of D-glucose and the enzymatic attack generally appears to be limited to the outer portions of the molecules (Sery and Hehre, 1956). Dextran has been found to be degraded in human feces due to bacterial action (~kberg, 1953). The specific enzymatic activity of dextranase has been determined in the mucosa of the human intestines (Dahlqvist, 1962), but has never been determined in human fecal samples. The enzyme activity of the small intestine is related to intracellular mammalian cell productions, whereas the main activity in the colon is associated to the surface of the cell membrane of bacteroides or extracellularly secreted by the bacteria (Salyers, 1984). The majority of the strictly anaerobic bacteria in the colon is saccharolytic. The bacteria derive their energy from the fermentation of carbohydrates, which results in the production of short chain fatty acids (SCFA) and gasses (Wolin and Miller, 1983). The composition of the human colonic flora appears to be virtually independent of diet, age and the geographic location in which a person lives (Gorbach et al., 1967; Finegold et al., 1983). Moreover, the bacterial activity of freshly defecated human feces seems to be qualitatively similar to that of the colonic flora (Drassar and Hill, 1974). Several in-vitro studies simulating colonic fermentation have used human fecal microorganisms (MacFarlane and Cummings, 1991). Comparison of SCFA molar ratios demonstrates a good in-vitro/in-vivo correlation (McBurney and Thompson, 1987). Colon-specific drug delivery is of great interest for the local treatment of colonic diseases, e.g. Crohn's disease, ulcerative colitis and cancer as well as for the oral delivery of peptides and vaccines, owing to their instability in the upper gastrointestinal tract. Colonic drug delivery has

previously been proposed by the utilization of microbial enzyme activity present in the colon (Rubinstein, 1990). Using this enzymatic activity, prodrugs have been prepared releasing the active drug after the action of microbial enzymes; e.g. dextran prodrugs were shown to release the drug specifically in the colonic region of pigs (Larsen et al., 1989). Other systems are based on matrices and coatings degradable by enzymes produced by the microflora of the colon triggering drug release. Biodegradable hydrogels based on dextran have recently been synthesized and characterized in our laboratories (Br~ndsted et. al, 1995a). These dextran hydrogels have also been shown to be fully degradable by dextranase invitro and in-vivo in rat cecum (Br~ndsted et al., 1995b). The mechanism of successfully providing colonic drug release was proposed to be a result of the complete stability of the hydrogel matrix in the stomach and small intestine followed by its disintegration in the colonic region with a subsequent release of drug. It was recently shown that drug release from dextran hydrogels was sensitive to the presence of dextranases in the release medium (Br~ndsted et al., 1995c). However, no results have been obtained on the stability of dextran hydrogels in the various regions of the human gastrointestinal tract. The aim of the present study was to investigate the stability of the dextran hydrogels in in-vitro models simulating human small intestinal- and colonic environments in order to evaluate the suitability of dextran hydrogels as carriers for colon-specific drug delivery. The degradation of dextran hydrogels was evaluated by determination of the rate and extent of polysaccharide breakdown in comparison with several chemically different polysaccharides. This was assessed by the formation of glucose and SCFA as well as by the loss in dry weight of the hydrogels in the small intestinal model and the colonic fermentation model, respectively.

2. Experimental procedures 2.1. Materials Dextrans of different molecular weights, 70000 (T-70) and 500000 (T-500) were purchased from

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Pharmacia Biosystems (Uppsala, Sweden). 1,6Hexamethylenediisocyanate (HDI), anhydrous dimethylsulfoxide (DMSO), maltose monohydrate, potassium sodium tartrate tetrahydrate, D-glucose, Merckotest glucose (GOD-PAP) diagnostic kit, SCFA and 2-ethyl butyric acid were all purchased from Merck (Darmstadt, Germany). 1,12-Dodecamethylenediisocyanate (DDI) was obtained from Aldrich Chemical Co. (St. Louis, USA), 3,5-dinitrosalicylic acid was purchased from Fluka Chemie AG (Buchs, Switzerland) and invertase (3000 EU/ml) was obtained from BDH Chemical Reagents (Dorset, UK). Potato starch, cellulose and pancreatin (amylase activity: 30.000 BPU/g) were purchased from Sigma Chemical Co. (St.Louis, USA). Amyloglucosidase solution (400 AGU/ml) was obtained from Novo Nordisk (Copenhagen, Denmark). Maize starch was kindly donated by the Department of Nutrition and Food Chemistry (Gothenburg, Sweden). Buffer substances and all other chemicals were of analytical grade. All chemicals and reagents were used as obtained.

2.2. Hydrogels Hydrogels based on dextran T-70 were prepared as described by BrOndsted et al. (1995a). Dextran was dissolved in anhydrous DMSO, and HDI was added as the crosslinking agent. The solution was transferred to Teflon coated aluminum blocks for fabrication of films. The reaction took place at 70°C for 24 h. In order to investigate the effect of crosslinking density and equilibrium degree of swelling on degradability, gels of two crosslinking densities were synthesized. Table 1 gives the compositions of the hydrogels used. 2.3. Degradation in simulated small intestinal juices

The equilibrium swollen dextran hydrogels and soluble dextran (T-70) were incubated with an enzyme mixture representing small intestinal enzymes as described by Englyst et al. (1992). The enzyme mixture was prepared as follows. First, 3 g of pancreatin was suspended in 20 ml distilled water and stirred magnetically for 10 min. The

Table 1 Chemical composition of dextran hydrogels Sample #

Dextran (MW)

Crosslinker a (% mol)

DMSO (.% v/v)

1

70 000

4.0

85

2

70 000

5.9

85

a 1,6-Hexamethylenediisocyanate was used as crosslinking agent.

suspension was then centrifuged for 10 min at 1500g and 13.5 ml of the cloudy supernatant was mixed with 1.5 ml of an aqueous solution of amyloglucosidase (140 A G U / m l ) and 1 ml of invertase. The swollen hydrogels and dextran T70 (60 mg of dry weight) in 0.5 ml enzyme mixture and 2 ml of 0.1 M sodium acetate buffer pH 5.2 were incubated at 37°C for 24 h. All experiments were done in triplicate. The released glucose was analyzed using the glucose oxidase kit. The absorbency of the standards and the samples was measured at 510 nm on a LKB Ultra Spech 4050 UV-spectrophotometer (UK) against reagent blanks. The percentage of degraded polysaccharide was defined using glucose as a standard. Degradation of the hydrogels was further evaluated by measuring the dry weights remaining in relation to the initial dry weights.

2.4. Degradation in a human colonic fermentation model Incubation of samples. Feces from two healthy female volunteers (38 and 40 years old) was used. No dietary restrictions or recommendations were given. Antibiotics were not administered in a period of at least 2 weeks before sampling. Fecal samples were collected and homogenized in an Ultra-Turrax (Janke and Kunkel, Germany) in 5 times their weight of an 0.125 M aqueous sodium bicarbonate solution of pH 7.8 at anaerobic nitrogen conditions, creating 16.6% fecal homogenates. This was done within 30 min after defecation. Different substrates were selected for incubation in addition to dextran T-70 and the hydrogel samples: glucose as a rapidly fermentable monosaccharide, maize starch as a rapidly fermentable polysaccharide, potato starch as a fer-

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mentable polysaccharide, and cellulose as a resistant polysaccharide. The hydrogels were cut as 6.6 mm diameter discs, 1-2 mm in height, from the equilibrium swollen state in aqueous sodium bicarbonate solution of pH 7.8 and dried. The substrates (60 mg dry weight) were placed in nitrogen filled vials as dry powders and swollen hydrogels, respectively. The incubations were started by addition of 9 ml fecal homogenate, to give final substrate concentrations of 6.66 mg/ml. Dispersion was performed by shaking for 1 min. The vials were closed with screw caps after additional filling with nitrogen. Incubations were started within 1 h after defecation. As controls, aliquots of fecal homogenates were incubated without substrates. The samples were incubated for 0, 6, 24, 48 and 72 h at 37°C while continuously shaking. The fermentation was terminated by freezing of the vials, which were kept closed during the procedure, and stored at - 18°C until analysis. Degradation was evaluated by determination of the SCFA production. Furthermore, degradation of hydrogels was assessed by measuring the dry weight in relation to the initial dry weight.

Short-chain fatty acid analysis.

Fecal suspensions were pretreated by steam distillation as described by Zijlstra et al. (1977) before gasliquid chromatographic analysis of SCFA. A 0.5 /zl distilled sample was automatically injected splitless into a Hewlett-Packard 5890A gas chromatograph equipped with a widebore, 530 /xm internal diameter, 30 m long HP-FFAP crosslinked fused silica capillary column with a film thickness of 1 /zm (Hewlett-Packard, Palo Alto, USA). Carrier and make-up gas was helium with flow rates of 8 and 20 ml/min, respectively. Injection and flame ionisation detector temperatures were 200°C. The oven temperature was 115°C for 2 min before being raised 5°C/min to 150°C. SCFA concentrations were calculated in relation to those of standards from the areas of gas-liquid chromatographic peaks by a HP-3396A integrator connected on-line to the gas chromatograph. 2-Ethylbutyrate was used as the internal standard. Acetate showed a retention time of 2.8 min, propionate 3.7 min, isobutyrate 4.0 min, butyrate 4.9 min, isovalerate 5.5 min, valerate

6.6 min, hexanoate 8.4 min and 2-ethylbutyrate appeared after 6.9 min.

Determination of dextranase activity in human fecal incubations. The dextranase activity was determined by measuring the production of reducing sugars (Tsuchiya et al., 1952). The analytical method was modified from Somogyi (1945). A color reagent was prepared by moistening 5 g 3,5-dinitrosalicylic acid with double distilled water and adding 100 ml aqueous 2 N N a O H and 250 ml double distilled water to complete dissolution. Finally, 150 g potassium sodium tartrate tetrahydrate was dissolved in the reagent solution and the volume was adjusted to 500 ml. In a 40°C water bath 2 ml aqueous solution of dextran T-500 (25 mg/ml) in 0.1 M acetate buffer of pH 5.4 was preheated. Then 1 ml of human fecal homogenate ( - 1 Dextranase Unit (DU)) was added and the samples were kept anaerobic in a nitrogen atmosphere at 40°C. After additional 20 rain 3 ml color reagent was added. A control was prepared by addition of the color reagent prior to the incubation. The vials were boiled for 5 min and cooled in ice for 10 rain. The color complex formed was measured at 540 nm on a Shimadzu UV-160A UV-spectrophotometer (Japan). The dextranase activity was defined using maltose as a standard. One dextranase activity unit, 1 DU, was determined as the amount of enzyme that degrades dextran to reducing sugar equivalent to 1 mg maltose per h. The pH of the incubations was determined by use of a Radiometer PHM 83 Autocal pH-meter (Copenhagen, Denmark).

3. Results and discussion

3.1. Stability in simulated small intestinal juices Dextran T-70 and dextran hydrogels were studied in simulated small intestinal juices by incubation at 37°C with the enzymes representing those of the human small intestine; amyloglucosidase, invertase and pancreatin. During the 24 h incubation, dextran T-70 and the dextran hydrogels were found to be stable. Less than 3.3% of free glucose was released (gel sample

L. Simonsen et al. / European Journal Of Pharmaceutical Sciences 3 (1995) 329-337

#1: 1.9%-_-0.9, gel sample #2: 2 . 4 % - 1.9 and dextran T-70: 3.3% +-2.6) determined by the glucose-oxidase method (see Table 1 for composition of hydrogels). This was further confirmed by measuring the loss in dry weight of the hydrogel samples where 100.3%-+ 0.0 of gel sample #1 and 100.2%-+ 0.1 of gel sample #2 was recovered. Obviously, the hydrogels were not degraded since no reduction in dry weight was found. Therefore, the amounts of free glucose measured by the glucose oxidase method should not erroneously be taken as the result of a degradation but more as an indication of the high sensitivity of the test. Correlating with these results, the lack of degradation of dextran prodrugs was shown in the small intestine of pigs (Larsen et al. 1989).

3.2. Human colonic fermentation Degradation. The degradability of the hydrogels was studied in a human colonic fermentation model modified from Mortensen et al. (1991). The hydrogels and several mono- and polysaccharides were incubated in 16.6% human fecal homogenates for 0-72 h. Previously, it was found that fecal homogenates diluted to 10-20% were optimal for fermentation studies of at least 6 h of incubation (Mortensen et al., 1991). Table 2 gives the total and specific SCFA production after fermentation of the chemically different substrates; glucose, maize starch, potato starch, cellulose, dextran T-70 and dextran hydrogels. As seen, a substantial production of SCFA was found in the control incubations, which were homogenates not given any substrate. Table 2 further lists the measured pH of the incubations and the dry weights of the recovered hydrogel samples. The fermentation which resulted in increased SCFA concentrations was associated with a decrease in pH, as seen in Table 2. The decrease in pH was explained by the formation of the weak organic acids (SCFA) due to the fermented saccharides. The pH-values of the various incubations were found to depend on the addition of substrate. The higher SCFA concentrations found after incubation and fermentation of dextran hydrogels caused a decrease in pH, in

333

contrast to what was found in the control incubation. Cummings et al. (1987) found pH and SCFA concentrations to be inversely related in the human large intestine; the SCFA concentrations being high in the cecum, due to bacterial fermentation, resulting in low pH. Metabolic inert substrates, such as cellulose, has previously been found not to alter pH (Mortensen et al., 1991).

Dextranase activity.

The dextranase activity of the fecal incubations was found to be as follows after 0, 6, 24, 48 and 72 h of incubation (results given as D U per g of hydrated feces): control incubation (15, 20, 31, 55), hydrogel #1 (15, 30, 36, 51) and hydrogel #2 (15, 16, 27, 56). The freshly defecated stools were found to contain approximately 15 D U per g of hydrated feces. The dextranase activity of human fecal samples has not previously been reported. However, Dahlqvist (1962) found that the mucosa of the human small and large intestine possessed considerably lower dextranase activity. Increased incubation time was found to be associated with an increased dextranase activity. The dextranase activity was determined to be similar for the dextran hydrogel incubations to that of the control incubation. However, determination of dextranase activity involves detection of the formation of maltose, which might have an erroneously low concentration due to the further fermentation of sugars to SCFA. This is in full accordance with a report by Pettipher and Latham (1979). It has previously been stated that most colonic microbial enzymes are inducible (Englyst et al., 1987; Salayers and Leedle, 1983). If colonic bacteria in growth were supplied various polysaccharides as carbon source they rapidly developed the ability to degrade the compound in question.

Substrate specificity.

The production of total SCFA varied considerably in relation to the type of substrate fermented, as illustrated in Fig. 1. The shown SCFA concentrations are the SCFA values subtracted the basal SCFA concentration found in the control incubations, to illustrate the specific pattern of the SCFA production from each substrate. Glucose, maize starch, soluble

L. Simonsen et al. / European Journal o f Pharmaceutical Sciences 3 (1995) 329-337

334

Table 2 Production of short-chain fatty acids (SCFA) after incubation of glucose, maize starch, potato starch, cellulose, soluble dextran a n d d e x t r a n h y d r o g e l s in a h u m a n c o l o n i c f e r m e n t a t i o n m o d e l (in a d d i t i o n , p H o f t h e i n c u b a t i o n s a n d t h e r e m a i n i n g p e r c e n t dry weight of the hydrogels) Substrate (6.6 mg/ml)

Time (h)

pH

Dry weight ~ (%)

Total SCFAb (mmol/l)

Specific production of SCFAc (mmol/I) Acetate

Propionate

Butyrate

iC4 + C5 - 6 J

Control e

0 6 24 48 72

-

-

8.5 18.8 34.1 38.8 48.5

5.8 (68%) 11.6(62%) 20.7(61%) 22.9(59%) 29.0(60%)

1.0 (12%) 2.9(15%) 5.2(15%) 6.2(16%) 8.0(16%)

0.8 (9%) 2.1(11%) 3.8(11%) 4.2(11%) 4.7(10%)

0.9 (11%) 2.3(12%) 4.4(13%) 5.5(14%) 6.7(14%)

Glucose f

0 6 24 48 72

-

-

0 40.7 41.1 43.2 45.2

0 28 (69%) 26.1(64%) 27.4 (63%) 27.8(62%)

0.1 3.6 (9%) 4.9(12%) 5.2 (12%) 5.6(12%)

0.1 8.4 (21%) 9.5(23%) 9.9 (23%) 11.0(24%)

0.1 0.6 (1%) 0.6(1%) 0.7 (2%) 0.9(2%)

Maize starch f

0 6 24 48 72

-

-

3.4 42.9 47.1 51.6 43.2

0.9 29.4(69%) 30.6 (65%) 32.4(63%) 26.3(61%)

1.1 3.9(9%) 5.3 (11%) 6.2(I2%) 5.2(12%)

1.0 8.9(21%) 10.3 (22%) 11.7(23%) 11.0(25%)

0.4 0.6(1%) 0.8 (2%) 1.3(3%) 0.5(1%)

Potato starch t

0 6 24 48 72

-

-

0.5 1.4 15.9 31.3 25.2

0 0.7(50%) 8.1(51%) 16.7 (53%) 12.8 (51%)

0.2 0.2(14%) 0(0%) 1.0 (3%) 0.4 (2%)

0.3 0.2(14%) 5.4(34%) 10.0 (32%) 9.2 (37%)

0.2 0.1 (7%) 2.6(16%) 3.5 (11%) 2.9 (12%)

Cellulose t

0 6 24 48 72

-

-

0.6 2.9 0.4 0 0.8

Soluble dextran f

-

0 6 24 48 72

0 1.9 0.5 0.2 0.9

0. I 0.3 0 0 0

0.3 0.3 0 0 0.4

0.2 0.2 0.1 0 0.1

-

0.6---0.8 23.5-+3.0 45.9-+7.5 51.8-+1.1 51.5-+2.3

0_+0.7 19.3-+2.3(82%) 26.1_+4.9(57%) 28.4_+0.7(55%) 27.7-+0.4(54%)

0.2-+0.1 2.9-+0.5(12%) 18-+2.0(39%) 20.0_+0.5(39%) 19.7-+1.7(38%)

0.1 -+0.1 1.4-+0.3(6%) 2.5-+0.(5%) 3.7-+0.3(7%) 4.4-+0.3(9%)

0.1 -+0.1 0-+0.2(0%) 0_+0.3(0%) 0±0.4(0%) 0-+0.6(0%)

Hydrogel #1 f

0 6 24 48 72

8.4-+0.3 7.9-+0.1 7.8-+0.2 7.7-+0.1 7.3---0.1

100-+ 0.0 I00-+ 0.0 100_+ 0.0 24.2_+7.8 0.0-+ 0.0

1.0-+0.2 3.1-+4.7 2.3_+2.2 35.7-+2.3 45.1+-4.9

0.6-+0,0 2.4+__3.4(77%) 1.1_+1.5 23.2_+3.1(65%) 24.9-+3.2(55%)

0.1 -+ 0.0 0.3_+0.5(10%) 1.7-+0.5 11.2_+1.5(31%) 18.2-+1.4(40%)

0.1 -+0.0 0.2_+0.4(6%) 0-+0.2 2.2-+0.2(6%) 2.8-+0.2(6%)

0.1-+0.2 0.2-+0.4(6%) 0-+0.5 0-+0.6(0%) 0_+0.8(0%)

Hydrogel #2 ~

0 6 24 48 72

8.1-+0.0 8.0-+0.5 8.1_+0.2 7.9-+0.0 7.2-+0.2

100_+ 0.0 100-+ 0.0 100-+ 0.0 59.2-+11.5 0.0 -+ 0.0

0.9---1.0 1.1-+1.2 0_+1.9 13.0-+8.7 31.8-+5.5

0.2-+0.6 0.7-+1.0 0_+0.9 8.5-+6.1(65%) 19.0-+2.3(60%)

0.4-+0.2 0.1-+0.2 0.1-+0.4 4.0-+2.2(31%) 11.3-+2.8(26%)

0.2-+0.1 0.1-+0.1 0+-0.3 0.8_+0.5(6%) 2.4-+2.8(26%)

0.2-+0.2 0.0-+0.4 0.1-+0.1 0-+1.0(0%) 0-+ 1.2(0%)

a Dry weight in relation to initial dry weight. b Values are averages (n = 2), for the dextran and dextran hydrogel incubations values are m e a n s -+s.d. (n = 3). c Percentage of total S C F A in parenthesis for total SCFA values > 2 . 9 m m o l / l and incubation time different from 0 h. a iC4 + C5 - 6, the production of isobutyrate, valerate, isovalerate and hexanoate. e Incubations without any substrate. r Values for S C F A are corrected by subtraction of values for S C F A in control incubations.

L. Simonsen et al. / European Journal of Pharmaceutical Sciences 3 (1995) 329-337

z

50 ¸

,...,

40

=

.

m o b

'~

mm~h

•.~

3o-

.<

20-

~T~

"d

17 72h

lo-

t..., o

-

~

~

=

,~

~

Fig. 1. Degradation of dextran hydrogels and various monoand polysaccharides in a human colonic fermentation model. Time-dependence of total short-chain fatty acids (SCFA) production (control incubation values are subtracted) on the chemical composition of substrates at 37°C (see Table 1 for composition of hydrogels). Values for dextran and dextran hydrogel incubations are averages---s.d. (n = 3), values for other saccharide incubations are averages (n = 2).

dextran and hydrogel sample #1 were completely f e r m e n t e d to SCFA, whereas potato starch and hydrogel sample # 2 were only partially ferm e n t e d . Glucose and maize starch were more rapidly f e r m e n t e d than the other substrates, noticed by the high S C F A production after 6 h of incubation. Glucose, maize starch, potato starch and soluble dextran yielded 45.2 mmol/1, 43.2 mmol/1, 25.3 mmol/1 and 51.5 mmol/1 of total SCFA, respectively. Cellulose was hardly ferm e n t e d at all. These levels of total SCFA are in full agreement with the levels found in-vivo in h u m a n s on E u r o p e a n diet, where a daily intake of 50-60 g of fermentable carbohydrate yields a production of 500-600 m m o l total SCFA per day (Bergman, 1990). It has previously been shown that between 35-59% of the carbon in saccharides will end up as SCFA, the theoretical m a x i m u m being 70% (MacFarlane and Cummings, 1991). In accordance with our results it was shown earlier that glucose and starch were completely fermentable to S C F A and that cellulose was resistant to degradation (Englyst et al.,

335

1987; Mortensen et al., 1991). The hydrogel samples # 1 and # 2 were degraded to 45.1 m m o l / 1 and 31.9 m m o l / I of SCFA, respectively. This indicates that crosslinking of the hydrogels caused a decreased SCFA production in comparison with the soluble dextran and that the more crosslinked hydrogel (gel sample # 2 ) was less fermented, although all gels were completely degraded as measured by the dry weight remaining. It is reasonable to conclude that the slower degradation rate of the crosslinked dextran hydrogels is due to steric reasons, since it might be more difficult for the dextranase macromolecule to diffuse into the hydrogel matrix (Brondsted et al., 1995b). Furthermore, the chemical crosslinks of the hydrogels are enzymatically stable. Therefore, the glucose units attached hereto will not undergo complete fermentation and contribute to the SCFA production. Hence, lower degree of fermentation of the hydrogels based on m o r e crosslinked dextran was fully expected to result in lower production of total SCFA.

Product specificity. The amounts and proportions of SCFA formed during the fermentation of the various substrates in the fecal h o m o g e n a t e s were also found to differ with regard to the variety of SCFA produced, as shown in Table 2. The breakdown of glucose, maize starch and potato starch was characterized by the production of large amounts of butyrate. In contrast, comparatively less butyrate and more propionate were formed during the incubation of the soluble dextran and the dextran hydrogels. The production ratios of acetate: propionate: butyrate: and the remaining SCFA (isobutyrate, valerate, isovalerate and hexanoate) after 72 h of fermentation in the human colonic fermentation model, were for glucose: (62:12:24:2), maize starch: (61:12:25:1), potato starch: (51:2:37:12), soluble dextran: (54:38:9:0), hydrogel sample #1: (55:40:6:0) and for hydrogel sample #2: (60:26:8:0). The variety of S C F A p r o d u c e d after fermentation of a saccharide is d e p e n d e n t on a n u m b e r of variables; e.g. pH, type and a m o u n t of substrate together with rate and extent of degradation (Nordgaard et al., 1995). Correlating with our results Englyst et al. (1987) found starch to give large quantities of butyrate. Similarly it

336

L. Simonsen et al. / European Journal o f Pharmaceutical Sciences 3 (1995) 329-337

was observed that fermentation of glucose resuited in large amounts of butyrate (Mortensen et al., 1991). In our experimental design all substrates were exposed to the same fecal homogenates under identical conditions. We propose that the reason for the high levels of propionate produced in the fermentation of dextran can be explained by the events in the fermentation process. The microbes responsible for the degradation of dextran to fermentable glucose are probably the bacteroides (Hehre and Sery, 1952). As the bacteroides have been shown to be the main producers of propionate (Macy and Probst, 1979), large quantities of propionate are to be expected. In Fig. 2 the degradation of the dextran hydrogels as a function of incubation time is shown. The change in dry weight with the accompanied changes in SCFA concentration were followed. The relationship was found to be inversely proportional; as the total SCFA concentration increased, percentage of dry weight remaining decreased. In other words, as the hydrogels were degraded SCFA were formed. Hydrogel sample #1 was found to be degraded faster and fermented to a greater extent than gel sample #2. After 48 h incubation of gel sample #1 24% of the dry weight remained and 36 mmol/1 of total SCFA was formed, in contrast to gel sample #2 where 52% of the dry weight remained and only

.~

100

50

80

40

60

30

4o

20

20

10

0

,.. 0

10

20

30

40

50

60

70

~'~ m

0 80

Time (h) Fig. 2. Degradation of dextran hydrogels in a human colonic fermentation model (see Table 1 for composition of hydrogels). Degradation evaluated as the remaining dry weight of the hydrogels in percent of initial dry weight ( 0 , gel sample #1; I , gel sample #2) and the production of total short chain fatty acids (SCFA) (control incubation values are subtracted) (O, gel sample #1; O, gel sample #2) as a function of incubation time at 37°C. The shown values are

averages -+s.d. (n = 3).

13 mmol/! of SCFA was produced. Despite the fact that the more crosslinked hydrogel was less fermented to SCFA after 72 h of incubation it was, however, completely degraded. As mentioned earlier, the hydrogels were not expected to be completely fermented to SCFA due to the chemical crosslinks. This degradation pattern supports our previous findings (BrOndsted et al., 1995b). It was proposed that the degradation of dextran hydrogels by dextranase was controlled by surface erosion. The fact that the production of SCFA is proportional to the weight loss of the hydrogels leave us to believe that this hypothesis is valid for degradation in the human colonic fermentation model.

4. C o n c l u s i o n s

In conclusion it has been shown that the dextran hydrogels were completely degraded in a human colonic fermentation model and at the same time stable in a mixture of model enzymes for the human small intestine. The degradation time in the human colonic fermentation model was, however, fairly long; between 48 and 72 h. This time period could be shortened by changing the structure or the thickness of the hydrogel (Br0ndsted et al., 1995b); e.g. by preparing a thin gel layer as a coating of a tablet. Experiments to optimize a drug device are presently being carried out in our laboratories. The dextran hydrogels appear to be promising as drug carriers for colon-specific drug delivery.

Acknowledgements

This work was partly supported by the Danish Medical Research Council with a scholarship to Ms. Lene Simonsen. The authors are indebted to Ms. Jette Christiansen for her qualified technical assistance.

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