Bioadhesive N-(2-hydroxypropy1) methacrylamide copolymers for colon-specific drug delivery

Journal of Controlled Release, 28 (1994) 211-222 © 1994 Elsevier Science B.V. All rights reserved 0168-3659/94/$07.00

211

SSDI 01 68-365 9 (93 )E0089-X

COREL00905

Bioadhesive 7V-(2-hydroxypropyl)methacrylamide copolymers for colon-specific drug delivery P. Kopeckova a b , R. Rathi a , S. Takada a , B. Rihovä a , M.M. Berenson c and J. Kopecek a b Departments of*Pharmaceutics and Pharmaceutical Chemistry/CCCD and hBioengineering, andc School ofMedicine, University of Utah, Salt Lake City, UT 84112, USA (Received 4 March 1993; accepted in revised form 28 June 1993)

A^(2-Hydroxypropyl)methacrylamide (HPMA) copolymers were evaluated as colon-specific drug carriers. Their design was based on the concept of site-specific binding of carbohydrate moieties com­ plementary to colonic mucosal lectins and on the concept of site-specific drug (5-aminosalicylic acid) release by the microbial azoreductase activity present in the colon. A new 5-aminosalicylic acid-con­ taining monomer was synthesized and incorporated into the copolymer together with the fucosylamine (bioadhesive moiety)-containing comonomer by radical copolymerization. The in vitro release rate of 5-ASA from HPMA copolymers by azoreductase activity in guinea pig cecum was approx. 2.5 times lower than from a low molecular weight analog. The azoreductase activities in cecum contents of guinea pig, rat, and rabbit as well as in human feces were determined. The relative activities for ratguinea pig:human:rabbit were 100:65:50:28. Both in vitro and in vivo HPMA copolymer-containing sidechains terminated in fucosylamine showed a higher adherence to guinea pig colon when compared to HPMA copolymer without fucosylamine moieties. The incorporation of 5-ASA-containing aromatic side-chains into HPMA copolymers further increased their adherence probably by combination of nonpecific hydrophobic binding with specific recognition. Keywords: A^-(2-Hydroxypropyl)methacrylamide copolymer; Colon-specific Carbohydrate moiety; Colonic mucosal lectin; Microbial azoreductase activity Introduction Oral delivery is the most convenient and com­ monly employed route of drug delivery. How­ ever, the design of oral sustained release dosage forms is based on insufficient knowledge on the relationship between gastrointestinal (GI) phys­ iology and factors responsible for drug absorp­ tion and gastrointestinal transit time [ 1 ]. PolyCorrespondence to: P. Kopeckovä, Department of Pharma­ ceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City, UT 84112, USA

drug

delivery;

meric bioadhesives [2,3] have been proposed to control the GI residence time and attempts have been made to understand the relationship be­ tween their physicochemical properties and bioadhesion [4-7 ]. The majority of bioadhesive systems operate on physical forms of attraction. Anionic polyelectrolytes, particularly those with a high density of carboxylic residues are good bioadhesives [8]. Water soluble copolymers containing quaternary ammonium groups have been shown to alter the GI transit time in rats [8]. Oral delivery systems of the future will be

212

based on physiological mechanisms operating in the GI tract which are site-specific. An optimal case would be the combination of site-specific bioadhesion with site-specific drug release. Re­ cently, we have proposed a concept of drug de­ livery to the colon which combines both ap­ proaches [9]. Site-specific drug delivery was achieved by binding 5-aminosalicylic (5-ASA) acid to water soluble N-( 2-hydroxypropyl)methacrylamide (HPMA) copolymers via aro­ matic azo bonds [10] cleavable by the microbial azoreductase activity present only in the colon [11,12]. Site-specific bioadhesion in guinea pigs in vitro was achieved by incorporating into HPMA copolymers side-chains terminating in acylated fucosylamine, a structure complemen­ tary to lectin-like structures present in the colon [13,14]. The concept of bioadhesion was based on the observation that some bacteria, e.g., Shigella flexneri adhere to colonic cells of guinea pigs. This adherence was found to reside in the host cells and be fucose and glucose specific [15]. These results suggest that there is a potential for the synthesis of water soluble copolymers which would show a two-fold specificity: in bioadhe­ sion and drug release. In this study, a new synthetic pathway has been developed for the synthesis of bioadhesive HPMA copolymers containing 5-ASA. A new 5azolinked salicylic acid-containing monomer, (5{4- [ 2- (7V-methacryloylglycylglycyl) aminoethylcarbamoyl]phenylazo}salicylic acid) was syn­ thesized and incorporated into the HPMA copolymer together with the fucosylamine (bioad­ hesive moiety) containing comonomer by radical copolymerization. The release of 5-ASA from HPMA copolymers by azoreductase activity in the guinea pig cecum was studied and compared with the 5-ASA release from a low molecular an­ alog, (5- [ 4- (2-aminoethylcarbamoyl) phenylazo] salicylic acid), and with the cleavage of a model azocompound, methyl orange. The azo­ reductase activity in different species was com­ pared using cecum content of rats, guinea pigs, rabbits as well as human feces. In vitro bioadhe­ sion properties of radioiodinated HPMA copol­ ymers were compared with the in vivo bioadhe­

sion in GI tract of guinea pigs after oral and intracolonic administration. Materials and Methods Abbreviations AIBN, 2,2'-azobisisobutyronitrile; 5-ASA, 5aminosalicylic acid; DMSO, dimethyl sufoxide, ED-5-ASA, 5-[4-(2-aminoethylcarbamoyl) phenyl-azo] salicylic acid; FucN, fucosylamine; HPMA, N- (2-hydroxypropyl) methacrylamide; MA,methacryloyl; MA-Gly-Gly-FucN, TV-methacryl-oylglycylglycylfucosylamine; MA-Gly-GlyONp, 7V-methacryloylglycylglycine p-nitrophenyl ester; ONp, p-nitrophenoxy; PBS, phos­ phate-buffered saline, pH 7.4; RT, room temper­ ature; Tris, 2-amino-2-hydroxymethyl-l,3-propanediol. Chemicals 5-(4-Ethoxyphenylazo)salicylic acid (3) [11] 5- [ 4- (2-aminoethylcarbamoyl) phenylazo ] sali­ cylic acid (4) [11], N-(2-hydroxypropyl) methacrylamide (7) [16], 7V-methacryloylglycylglycine p-nitrophenyl ester (5) [17], N-methacryloylglycylglycylfucosylamine (8) [13] and N-methacryloyltyrosinamide (9) [18] were pre­ pared as previously described. Na125I was from Amersham. All other chemicals were from Sigma. Synthesis of 5-aminosalicylic acid-containing monomer 5-{ 4- [ 2- (7V-Methacryloylglycylglycyl) aminoethylcarbamoyl]phenylazo}salicylic acid (6) was prepared by aminolysis of 7V-methacryloylglycylglycine /7-nitrophenyl ester (MA-Gly-Gly-ONp, 5) with 5-[4-(2-aminoethylcarbamoyl)-pheny­ lazo] salicylic acid (4) (Scheme 1). To a solu­ tion of 2.25 g (7 mmol) MA-Gly-Gly-ONp and 2.55 g (7 mmol) hydrochloride of (4) in 12 ml DMF, triethylamine (1.6 g, 16 mmol) was added dropwise within 30 min at RT. The reaction

213 CH 3

O

ΐ"

to

Φ

NaNQ2/Ha

4c

°

_^COOH {J~OH

N II + N Ch

NH 2

1

C H 2= C

Λ

O

CQ

φ

CH 3

CO-O-CHo-CH,

2

CH 2

pH 9 -10 1 h,4°C

t ) N II

N H 2- C H 2- C H 2- N H 2 140°C; 6 h

+

«.

CO

CO NH

CH 2

CH 2

CH 2

CH-OH

CO

CH 3

L

CH 2 = C CO NH CH-CO-NH2

to

CH 2

6

v ^ 2 NH

CH 2

CH 2

CO

CH 2 1 NH

T

OH

2

CO

NH 2

CH 2

Φ

AH

N

C H 2= C - C O - f N H - C H 2- C O ) - N H

CH 2 I CH 2 I NH

CH 2

CO

T

T

c

NH

NH

r^j

CH 2 =

CO 1 NH

z

OH

1

CH 3

CH 3

CH 3 CH 2 == C

+

io

y"3

N

CH 2=C -co 4 N H -CH 2-CO )^Ο — £ } - N

N II N OH

5

DMSO; RT

OH N II

AIBN, 50°C, 24 h acetone/DMSO

N

S^COOH 4 OH

6 Scheme 1. Synthesis of 5-aminosalicylic acid-containing monomers.

mixture was stirred overnight at RT. DMF was evaporated under vacuum and the residual red oil was acidified with 30 ml of diluted hydro­ chloric acid (1:1). The crude product precipi­ tated as a yellow powder. The product was recrystallized from EtOH/H 2 0 (2:1), m.p. 239°C (decomp.), 72% yield, e = 2.75xl0 4 1 mol" 1 cm - 1 (362 nm, 50% EtOH/H 2 0, measured after 24 h in the dark). TLC in EtOH, one spot, R{: 0.5, (original compound (4), free base, Rf: 0.06). Anal. calc. for C 24 H 26 N 6 0 7 (510.5): Calculated: C 56.46%, H 5.13%, N 16.46%; found: C 56.30%, H5.17%,N16.34%. Synthesis of 7V-(2-hydroxypropyl) methacrylamide copolymers HPMA copolymers (lOa-d) were synthesized as described previously [17] by radical copolymerization in acetone containing 20%-vol. of DMSO (to dissolve the monomer mixture) at 50 °C for 24 h using AIBN as the initiator (Scheme 2). The ratio of monomers to initiator and solvent was 12.5:0.6:86.9 wt.%. The com­ position of the monomer mixture was manipu­ lated to afford a variety of HPMA copolymer

CH 3

I co

CH 2 - C —

CH 3 CH 2 - C

-

CH 2 - C -

x

NH

CH - C O - N H 2 I CH 2

CH 2 CH-OH I CH 3

Φ 109-d

Scheme 2. Synthesis of copolymers.

compositions (Table 1). After polymerization the copolymers 10a, 10c, and lOd were filtered off (from the solution of 10b acetone was evap­ orated before precipitation) and reprecipitated from a DMSO solution into an excess of acetone. The higher molecular weight fractions (less than 10%) were removed by size exclusion chromatography on a preparative Superose 6 column (HR 16/50, Pharmacia FPLC chromatography system). After dialysis and freeze drying the yield of polymers was 50-56%. The weight- and num­ ber average molecular weights Mw and Mn were estimated using a Superose 6 (HR 10/30) col­ umn, (buffer 0.05 M Tris + 0.5 M NaCl, pH 8.0) calibrated with poly (HPMA) fractions. The content of azo comonomer (6) was deter­ mined spectrophotometrically using € = 2.75x 104 1 mol" 1 cm" 1 (362 nm, 50% EtOH/H 2 0; measured after 24 h in the dark). The content of 9 was determined spectrophotometrically (for

214 TABLE 1 Characterization of HPMA copolymers Copolymer

10a 10b 10c lOd

mol %

Mw

y

z

(FucN)

(5-AS A)

w (Tyr)

31.6 30.9 0 0

10.0 0 10.9 0

-1 -1 -1 -1

Mw/Mn>

58700 63800 43800b 73800

1.74 1.78 1.60 1.82

aPolydispersity

b InTris

of copolymer. buffer (0.05 M tris + 0.5 M NaCl, pH 8.0)/acetonitrile (70:30; v/v).

copolymers 10b and lOd) using e = 1 . 7 x l 0 3 1 mol - 1 c m - 1 (278 nm, water). It was assumed that the content of 9 was comparable in copoly­ mers 10a and 10c. The content of fucose-containing comonomer (8) was determined by the following two methods. (a) Anion exchange chromatography. The copol­ ymers were hydrolysed 6 h in 6 N HC1 at 100°C. The hydrolysate was analysed on a Dionex Carbopac PA-1 column using pulsed amperometric detection and 15 mM NaOH as eluent [13]. (b) Colorimetric L-cysteine sulphuric acid method. The L-cysteine sulphuric acid assay [19] was used with a slight modification. 0.5 ml copoly­ mer solution in water (containing approx. 0.15 //mol fucosylamine) was heated with sulphuric acid (86%) for 15 min at 100°C. After cooling to RT, 50 μ\ of aqueous L-cysteine hydrochloride (3 wt.%) was added and the mixture incubated for 15 h. The difference of absorbancies at 396 and 426 nm was determined and compared with standard curves obtained using MA-Gly-GlyFucN (8) or €-aminocaproylfucosylamine. The relative difference in values between the anion exchange chromatography method and the col­ orimetric method was less than 5%. Radioiodination of polymers The HPMA copolymers were radioiodinated using the modified chloramine-T method [20].

To a solution of 10 mg polymer in 400 μΐ PBS, 0.3 mCi of Na125I was added followed by 0.4 mg chloramine-T in 100 μΐ PBS. The reaction mix­ ture was stirred 30 min at RT. Nonreacted io­ dine was removed by three consecutive separa­ tions on Sephadex G-25 PD-10 columns. To calculate the specific activity, the copolymer concentration was determined spectrophotometrically using aromatic azo bond or tyrosine absorbancy in separate experiments with cold copolymers under the same experimental condi­ tions (Table 2). To verify that the radiolabel was not on the (cleavable) 5-ASA residue, the aromatic azo bond was chemically reduced and the radioac­ tive profile of the reaction mixture determined by size exclusion chromatography. To a mixture of cold (5 mg in 500 μΐ PBS) and hot (5 μΐ, 0.5 μα 125Ι) copolymer 10a, a solution of 500 μΐ of fresh 0.2 N sodium hydrosulfite in PBS was TABLE 2 Characterization of 125l-labelled HPMA copolymers Copolymer Radioactivity added per 10 mg copolymer (kcpmXlO- 5 ) 4.81 4.29 3.97 4.77

Labelling efficiency3

(%)

Specific activity (//Ci/mg)

46 52 57 48

22 20 21 23

a% of radioactivity bound to the copolymer (based on total radioactivity added to the reaction mixture).

215

added. The sample lost color immediately indi­ cating reduction of the azo bonds. The mixture was applied on a Sephadex G-25 PD-10 column and 16 fractions (1 ml) were collected and counted for radioactivity. No radioactivity was found in the low molecular weight fractions. In a separate experiment with cold copolymer 10a, the released 5-ASA was spectrophotometrically de­ tected in low molecular weight fractions after diazotization (NaN0 2 /HCl) and subsequent coupling with sodium salicylate. Biological samples for azoreductase activity measurements Cecum contents were isolated from SpragueDawley male rats (150-200 g), Hartley strain male guinea pigs (200-250 g), and New Zealand white rabbits (2.5-3 kg). Human feces were col­ lected from two healthy individuals on a normal Western diet. The fresh contents of cecum or hu­ man feces were suspended in a 0.1 M potassium phosphate buffer pH 7.4, previously bubbled with nitrogen, to yield 10 wt.% suspension. The sus­ pensions were kept frozen until use at — 20 °C. In some experiments, as indicated, the suspen­ sions were used after 14 h preincubation in 0.1 M phosphate buffer containing a-D-glucose (1.5 mg/ml) or immediately after preparation. Degradation by cecum contents/feces The suspension of cecum contents/feces (2.5 ml) was mixed under nitrogen atmosphere with solutions (total volume 7.5 ml) of azo substrate and α-D-glucose in 0.1 M potassium phosphate buffer, pH 7.4. The final concentrations were: 0.025 g/ml cecum content/feces, 0.1 mM azo substrate and 1.5 mg/ml α-D-glucose. The reac­ tion mixtures were incubated under anaerobic conditions at 37°C in a shaking water bath. At time intervals indicated, 1 ml samples were withdrawn under nitrogen. To stop the reaction (and to shift the absorption profile), 20 μΐ of cone. HC1 was added to methyl orange samples, and 20 /il 10 N NaOH to polymeric substrates or ED-5-ASA. After centrifugation for 20 min at 3000 xg the supernatant was diluted 1:1 with

water and the difference in absorbancies at 540 and 600 nm (methyl orange), or 480 and 560 nm (polymeric substrates or ED-5-ASA) were determined. The degradation was expressed either as a percent of the original azo substrate degraded at a particular time interval or as a deg­ radation rate in μτηοΐ azo bonds degraded per g of cecum/feces per hour. The degradation rates were calculated from the slope of the linear por­ tion of the degradation vs. time curves (omitting the lag time). The results represent the mean ( ± S.D.) of three experiments. Bioadhesion of polymers in vitro The bioadhesion experiments were performed as described previously [13,14]. Briefly, radiolabelled copolymers were incubated with ev­ erted sacs isolated from guinea pig small intes­ tine and colon in preoxidized MEM (minimum esential medium) containing 5% FCS (fetal calf serum) for 30 min. at 37°C. Each segment (one fourth of small intestine or one third of colon) was incubated in 10 ml of incubation media con­ taining 2 //Ci of 125I labelled HPMA copolymer. After washing, the radioactivity of the segments was determined using a Packard gamma counter. Results were expressed as percentage of radio­ activity bound per g of tissue, and represent the mean ( ± S.D.) of at least five experimental values. Gastrointestinal distribution of polymers in vivo Oral administration Hartley strain male guinea pigs (200-250 g) were fasted for 18 h prior to the study, but were given water ad libitum. One ml of saline solution containing a mixture of hot (2 //Ci per animal) and cold polymer (20 mg/kg body weight) was administered via a flexible catheter into the stomach of animals under light ether anesthesia. After polymer administration the animals were kept in separate cages and given 20 g of standard diet in one portion. After 24 h the animals were given an ether overdose and the stomach, small intestine, cecum, and colon were excised. The content of stomach was suspended in 50 ml PBS, the content of cecum in 200 ml of PBS and ali-

216

quots of 2 ml were taken for counting. The small intestine and colon were divided into small (approx. 2 cm) segments and the whole pieces (tis­ sue and content) were counted. The radioactiv­ ities of stomach content, cecum content, small intestine, colon, and feces (collected thoughout the experiment) were determined. The results were expressed as percentage of radioactivity re­ covered (83 ±9.5% of administered dose). Intracolonic administration Hartley strain male guinea pigs (200-250 g) were fasted for 18 h before study, but had free access to water. Under ether anesthesia, the abdomen was exposed through a midline inci­ sion and the ileocecal junction identified. Two hundred and fifty μΐ of a saline solution contain­ ing a mixture of hot (1 /zCi) and cold polymer (10 mg/kg body weight) was injected into the colon 5 cm distal to the ileocecal junction. The injection site was securely closed and the abdo­ men was closed with a doubled suture. After 24 h the animals were sacrificed by an ether over­ dose and colon and cecum removed. The colon was divided into three equal parts (starting from ascending colon segments were designated as co­ lon 1, colon 2, colon 3) and the radioactivities of colon segments, cecum and feces (collected throughout the experiment) were determined. The results are expressed as the percent of radio­ activity recovered in colon and feces. The total recoveries were 78 ± 14%. Results and Discussion The design of polymeric drug delivery systems for colon-specific delivery has recently attracted considerable interest [21]. The enzymatic activ­ ity of colonic bacteria appears to be the target of the tailor-made synthesis of low molecular weight as well as polymeric prodrugs. Dexamethasoneand prednisolone-/?-D-glucosides were evaluated as prodrugs capable of releasing the active agent in rat [22] and guinea pig colon [23,24]. This approach has been shown to be effective in the experimental treatment of inflammatory bowel disease [23] and morphine-dependent consti­

pation [25]. The dextranase activity in the co­ lon was utilized for the release of naproxen from dextran carriers [26]. Chondroitin sulfate [27] and pectin [28] were also evaluated as colonspecific drug carriers. The potential of the azoreductase activity for colon-specific drug delivery was also evaluated. Two prodrugs, i.e., 5-[4-(2-pyridylaminosulfonyl)phenylazo] salicylic acid [29] and 5,5'azodisalicylic acid [30] are used in the clinic. The use of water-soluble copolymers for the co­ lon-specific oral delivery of 5-ASA has been pro­ posed by Brown et al. [10], crosslinked (branched) copolymers for the colonic delivery of proteins were introduced by Saffran et al. [31]. Based on the reports [15,32,33] that in­ testinal cells produce lectin-like mucosal adhesins, which in the presence of calcium cause fucose-sensitive adherence of certain invasive enteropathogens, bioadhesive HPMA copoly­ mers containing 5-ASA were synthesized and their bioadhesion [8,13,14] and drug release [11,12] were evaluated in vitro. Synthesis To achieve successful biorecognition of syn­ thetic macromolecules in living systems a careful control of chemical and physical properties of synthesized macromolecules is necessary. We have developed a new pathway for the synthesis of bioadhesive colon-specific HPMA copoly­ mers that permits the incorporation of variable amounts of drug (5-ASA) and bioadhesive ligand (FucN) into the copolymer structure and also provides a means to control the molecular weight of synthesized copolymers. By choosing the comonomer composition, solution proper­ ties in the physiological environment may be partially regulated. Our original method [12] was the synthesis of a polymer precursor by copolymerization of HPMA and 7V-methacryloylglycylglycine p-nitrophenyl ester, followed by binding to this pre­ cursor, by consecutive aminolysis, of /?-aminoethyl /?-aminobenzamide and aminosugars (e.g., FucN). The aromatic amino groups present in the side-chains of HPMA copolymers were dia-

217

zotized and salicylic acid coupled via aromatic azobonds. However, during this procedure sev­ eral side-reactions may occur: attachment of sal­ icylic acid in the 3-position, formation of nonreactive trans-diazotate, formation of arylcarbonium ions and their reaction with prevalent nucleophiles (such as hydroxide and chloride) as well as formation of triazine crosslinks [10]. To avoid these side-reactions we have synthe­ sized a 5-ASA derivative, ED-5-ASA (4). By binding of (4) to polymeric precursor, side re­ actions can be avoided [11]. Still this procedure was not optimal, since only up to 15 mol% of re­ active side-chains terminating in p-nitrophenyl ester groups could be incorporated into the copolymer since the p-nitrophenyl ester-containing comonomer is a chain-transfer agent in radical polymerization. Consequently, the content of drug and bioadhesive moieties in the copolymer was limited. To increase the amount of FucN units in the copolymer, a polymerizable deriva­ tive of FucN, N-methacryloylglycylglycylfucosylamine (8), was synthesized [13]. Here we re­ port on the synthesis of a polymerizable derivative of 5-ASA, 5-{4-[2-(7V-methacryloylglycylglycyl) aminoethyl-carbamoyl ] phenylazo} salicylic acid (6), and its incorporation into HP MA copolymers. Theoretically, any combination of comonomers (6,7,8 and 9) may be used for the copolymer synthesis. However, to provide good water solu­ bility, the ratio of FucN: 5-ASA in the copolymer should be > 1:1 for higher contents (> 20 mol.%) of the hydrophobic comonomer (6). Four HPMA copolymers (lOa-d) with different con­ tents of comonomers were prepared (Table 1). All copolymers contain a small amount ( « 1 mol.%) of tyrosinamide-containing monomer units to permit radioiodination (Table 2). The analysis of radioiodinated copolymers has shown that only the tyrosine units were labelled whereas the salicylic acid moieties were not. The hot and cold copolymer 10a was cleaved simultaneously by a chemical reducing agent (Na 2 S 2 0 4 ) and by rat cecum contents. Released 5-ASA was de­ tected spectrophotometrically in both proce­ dures, however, the radioactivity was associated

only with high molecular weight fractions after separation of the reaction mixtures on a Sephadex G-25 column (results not shown). Degradation by colonic azoreductase activity Methyl orange was used as a marker of enzy­ matic activity of all preparations to ensure the reproducibility of results. No exogenous elec­ tron-carriers were added. The degradation rate of copolymers 10a, 10c, ED-5-ASA (4), and methyl orange were compared in fresh guinea pig cecum contents (Fig. 1). After a short lag time (<0.5 h), the degradation of methyl orange in fresh cecum was complete in 2 h, ED-5-ASA in 2.5 h and approx. 50% of the both copolymers was degraded within 6 h (Fig. 1). The calculated degradation rates are in Table 3. These results indicate that the accessibility of the reduction sites is hindered for polymeric substrates com­ pared to substrates of low molecular weight. It is generally accepted that the degradation mecha­ nism of aromatic azo bonds by azoreductase ac­ tivity in colon is a non-enzymatic reduction of azo bonds by reduced flavins which act as an electron shuttle between NAD(P)H-dependent flavoprotein (enzyme) and the substrate [34]. The observed difference in the rate of cleavage of low molecular weight and polymeric sub­ strates may probably be explained by the solu­ tion properties of the latter. The copolymers may

#

OH

0

—

1

5 time, h

1

10

Fig. 1. Anaerobic degradation of low molecular and poly­ meric azo compounds by fresh guinea pig cecum contents at 37°C. Methyl orange, ■; ED-5-ASA (4), · ; copolymer 10a, O; copolymer 10c, D.

218 TABLE 3 Degradation rates of low molecular weight and polymeric azocompounds by guinea pig cecum contents* Azo substrate

Degradation rateb (μπιοΐ/g/h) Fresh

Methyl orange ED-5-ASA (4) Copolymer 10a Copolymer 10c

100

Frozen

14 h preincubation

2.0±0.3 1.310.3 1.4 10.32 n.d. 1.2 10.26 1.6±0.4 0.35±0.12 n.d. 0.4810.14 0.3010.18 n.d. 0.4410.15

degradation by guinea pig cecum content suspension (0.025 g/ml), fresh (used immediately after isolation), or stored fro­ zen and melted before experiment, or preincubated in glucosecontaining buffer. Concentration of azo substrate 0.1 mM. ^ h e values are maximal degradation rates. The values were calculated from the linear part of degradation profile omitting the lag time. They are expressed in μτηοΐ azo bonds degraded per gram of wet cecum content/feces per hour and are the mean of two or three experiments 1 S.D. n.d., not determined.

form aggregates in solution [ 35 ] with the hydrophobic side-chains in the core of the micelle, thus hindering contact between the mediators and the azo bond to be cleaved. In all cases studied, it appeared that the azoreduction proceeded via the hydrazo intermediate to the formation of aro­ matic amines. The same was observed during the cleavage of hydrogels containing aromatic azo bonds in crosslinks [ 9 ] contrary to the results of Kimura et al. [36] who observed, during the cleavage of azo-containing polyurethanes, only reduction to hydrazo groups.

100

100

100 Human 501

Comparison of azoreductase activity in different species An important consideration in the develop­ ment of colon-specific polymeric 5-ASA-containing drugs is the azoreductase activity in dif­ ferent species including man. We have compared the azoreductase activity in rat, rabbit, guinea pig and man. Human feces were used in this study as the composition of the bacterial flora of feces re­ sembles that of the large intestine and it is as­ sumed that freshly passed feces can be consid­ ered as representative of the large gut flora [ 37 ]. A model azo compound, methyl orange, was used

Fig. 2. Azoreductase activity in different species. Methyl or­ ange was anaerobically cleaved at 37°C by cecum content from rat, guinea pig and rabbit, and by human feces. Cecal/ fecal suspensions were kept frozen until the experiment and used after thawing without preincubation. Incubation under aerobic conditions (dashed line) is also shown. Each curve represents the cecal activity from one animal. Human fecal activity was determined on two samples, each of them twice. No. 1, D B ; N o . 2 , 0 · .

219 TABLE 4 Azoreductase activity in different speciesa Species

Source

Azoreductase activity0 (//mol/g/h)

Rat Guinea Pig Rabbit Human

Cecum content Cecum content Cecum content Feces

2.0 ± 0.2 1.3 ±0.3 0.57 ± 0.2 1.0 ± 0.3

aMethyl orange (0.1 mM) was used as a substrate. Azoreductase activity was determined without preincubation using cecum/feces samples (0.025 g/ml) stored frozen before experiment. bActivity expressed as degradation rate in μπιοΐ azo bonds degraded per gram of wet cecum content/feces per hour. The values represent maximal rates and were calculated from the linear part of degradation profile omitting the lag time (Fig. 2). The values are the mean of three or four experiments ± S.D.

time, h Fig. 3. Effect of preincubation on azoreductase activity (full lines). Methyl orange was incubated with cecal/fecal suspen­ sions which were stored frozen and used after thawing fol­ lowed by 14 h preincubation in a-D-glucose (1.5 mg/ml) containing 0.1 M phosphate buffer. Control experiments without preincubation are also shown (dashed lines). Cecum contents of: rat, Δ; guinea pig, O; rabbit, D; human feces, X.

to determine the azoreductase activity in differ­ ent species since its spectrophotometric profile did not interfere with the background of the in­ cubation mixture as it was the case with 5-ASAcontaining copolymers (particularly when incu­ bated with human feces). Thawed cecum contents/feces (stored frozen) were used without preincubation. In all cases a time lag (3 to 6 h)

was observed (Fig. 2) followed by fast degrada­ tion. The slope of the dependence of azo bond concentration vs. time was used for calculation of degradation rates (Table 4). The fastest deg­ radation was observed with rat cecum, the activ­ ity of guinea pig cecum was very close to human feces and rabbit cecum showed the lowest activ­ ity from all preparations evaluated. A similar ra­ tio of azoreductase activities between rat and guinea pig was found by Rowland et al. [ 38 ]. Some cofactors, important for the cleavage process can be be lost during the isolation pro­ cedure and/or storage in the frozen state [ 34 ]. Consequently, after thawing a time lag in the degradation process was observed (Fig. 2). Deg­ radation experiments were also performed with cecum contents/feces after preincubation (14 h) in an α-D-glucose-containing phosphate buffer (Fig. 3). During preincubation the regeneration of cofactors and cell growth take place and con­ tribute to the increased activity of the prepara­ tion. All preincubated preparations cleaved the methyl orange with a minimal time lag (Fig. 3). It appears that the guinea pig cecum contents possess the closest activity to man when compar­ ing the species studied. Consequently, guinea pig is a suitable animal model for the study of both, degradation and bioadhesion. Bioadhesion of HPMA copolymers to everted intestinal sacs The binding of HPMA copolymers containing bioadhesive fucosylamine moieties to everted sacs and enterocytes isolated from guinea pig small intestine and colon were evaluated previ­ ously [13,14]. Binding of FucN-containing HPMA copolymer to everted sacs isolated from colon was substantially higher when compared to small intestine. The higher the fucosylamine content, the higher the binding. The fact that the binding of fucosylamine-containing HPMA co­ polymers was partially inhibited by free fucose, and to a lesser extent, by free glucose [14] indi­ cated that lectin-like structures may be present in the colon of guinea pigs in accordance with results of Ashkenazi [ 33 ]. Incorporation of hydrophobic 5-ASA-contain-

220

TABLE 5 In vitro bioadhesion of 125l-labelled HPMA copolymers to everted sacs of guinea pig small intestine and colon3 Copolymer

10a 10bc 10c PolyHPMAc

% radioactivity bound/g tissue15 Small intestine

Colon

0.947 ±0.240 0.652±0.305 0.757±0.084 0.088 ±0.029

2.665±0.342 2.358±0.911 1.317 ±0.049 0.152 ±0.064

aSegments

were incubated for 30 min with 2 //Ci of labelled copolymer. bThe values represent the mean ± S.D. of at least five experiments. cData from Ref. 14.

ing side-chains into HPMA copolymers in­ creases their binding to guinea pig everted sacs isolated from small intestine and colon (Table 5; compare copolymers 10a vs. 10b, and 10c vs. poly (HPMA). Most probably the mechanism of bioadhesion of copolymer 10a is a combination of non-specific hydrophobic binding with spe­ cific biorecognition, whereas the adherence of copolymer 10c may be due solely to hydrophobic interactions. Bioadhesion of HPMA copolymers in vivo The results of gastrointestinal distribution of copolymers 10a-10d after oral administration are shown on Fig. 4. The animals were fasted 18 h before the experiment but received 20 g of stan­ dard diet after administration of the sample. This protocol was adapted when preliminary experi­ ments showed that fasted animals retained 4060% of the copolymers in the cecum. The pres­ ent protocol decreased this value to 20-40%. The results indicate the higher binding of the fucosylamine-containing copolymers (10a, 10b) when compared to copolymers without fucosylamine (10c, lOd, respectively). The observed adher­ ence of HPMA copolymers, i.e., 10a > 10b>10c>10d has the same structure-adher­ ence relationship as observed in in vitro everted sac experiments (Table 5). The potential of hy­ drophobic aromatic azo side-chains to increase

Stomach

SI

Cecum

Colon

Feces

Fig. 4. Gastrointestinal distribution of copolymers 24 h after oral administration. See Materials and Methods for experi­ mental details. The results represent the mean (± SE) of five experimental values. Significantly different from control (lOd) by Student's Mest (*P<0.05). Copolymers: 10a, ■; 10b, $ ; 10c, El;10d, D.

the adherence both in vitro and in vivo indicates the cooperativity of hydrophobic binding with specific biorecognition. These results are in ac­ cordance with our preliminary in vivo data ob­ tained with HPMA copolymers containing low amounts of side-chains terminated in 5-ASA and/or FucN [39]. It is also interesting to note that the HPMA copolymers, as expected, are not transported across the gastrointestinal tract. After oral administration no radioactivity in blood was detected. A small amount of radioactivity (<0.1°/o of administered dose) was detected in the urine. However, the analysis on a Sephadex G-25 column has shown that the radioactivity was associated with the low molecular weight fractions, i.e., it was most probably free iodine (results not shown). Results on the distribution of copolymers 10ad after intracolonic administration to fasted guinea pigs (Fig. 5) show a different pattern when compared to oral administration (Fig. 4). The copolymers 10a and 10c containing aro­ matic azo side-chains show an increased adher­ ence to colonic segments indicating that non­ specific hydrophobic interactions predominate over specific recognition. Several factors may contribute to the observed differences in adher­ ence after oral or intracolonic administration: (a) the higher local concentration of copolymers after intracolonic administration may contribute to higher adherence of hydrophobic aromatic azo

221

■ P,

01

3

0

4 5

6 Colon 1

Colon 2

Colon 3

Feces

Fig. 5. Distribution of copolymers 24 h after intracolonic administration. The colon was divided into three parts of equal length. Starting from ascending colon segments were designated as colon 1, colon 2, and colon 3. The results rep­ resent the mean ( ± SE) of six or seven experimental values. Significantly different from control (lOd) by Student's /-test (*P<0.05). Copolymers: 10a, ■; 10b, W\ 10c, 1 ; lOd, D.

side-chain-containing copolymers; (b) after oral administration a substantial part of aromatic azo side-chains is cleaved by the azoreductase activ­ ity in the cecum resulting in copolymers contain­ ing side-chains terminated in aromatic amine. Consequently, during transit the structure of the copolymers is modified to a structure possessing a lower adherence to the colon (S. Takada, P. Kopeckovä and J. Kopecek, unpublished re­ sults) when compared to copolymers with noncleaved side-chains. Experiments with tailormade HPMA copolymers having structures sim­ ilar to those formed after cleavage are necessary to verify this hypothesis.

7 8

9

10

11

12

13

Acknowledgement The research was supported in part by NIH grant DK 39544. References 1

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