Development of an Oral Drug Delivery System Targeting Immune-Regulating Cells in Experimental Inflammatory Bowel Disease: A New Therapeutic Strategy1

Several studies have indicated the involvement of macrophages and dendritic cells in active inflammatory bowel disease (IBD). Manipulation of these cells is considered a very important therapeutic strategy for patients with IBD. We evaluated the effect of a new drug delivery system targeting microfold cells and macrophages with poly(DL-lactic acid) microspheres containing dexamethasone (Dx). Colitis was induced in BALB/c mice by 5% dextran sodium sulfate. Dx microspheres (n 5 10) and only Dx (n 5 10) were orally administered to dextran sodium sulfate-treated mice. Thereafter, serum levels and tissue distributions of Dx were investigated. To estimate the efficacy of this drug delivery system, we measured the histological score, myeloperoxidase activity and nitric oxide production, and gene expressions of tumor necrosis factor-a, interleukin1b, and interferon-g in the colonic tissue. Serum Dx levels were not increased after oral administration of Dx microspheres. The tissue distribution of microspheres containing I-labeled Dx in inflamed colon was significantly higher than in other organs. The histological score, myeloperoxidase activity, and nitric oxide production of the group treated with Dx microspheres were significantly lower than of those treated with Dx alone. Gene expressions of tumor necrosis factor-a, interleukin-1b, and interferon-g were down-regulated in mice treated with Dx microspheres. Microspheres containing glucocorticoids such as Dx, which target microfold cells and macrophages, can facilitate mucosal repair in experimental colitis and could be an ideal agent for treatment of human IBD. Many patients with ulcerative colitis (UC) have been successfully treated with steroidal drugs and immunosuppressants. Among these drugs, glucocorticosteroid is known to be very effective in the treatment of UC (Hanauer and Kirsner, 1988). However, because of various systemic side effects, the administration of glucocorticosteroids by oral and i.v. routes is often restricted to patients with severe or acute UC (Elson, 1988). Therefore, to circumvent such side effects, topical rectal administration of glucocorticosteroid (Patterson, 1958; Lee et al., 1980) and its alternative regimens (Swartz and Dluhy, 1978) have been used for UC patients. Recently, newer corticosteroids, such as budesonide, have been widely used because of their low systemic availability (Keller et al., 1997; Friend, 1998). However, some patients are still resistant to these treatments (Elson, 1988; Keller et al., 1997). It is well known that macrophages and dendritic cells play important roles in the regulation of immunoresponses in the gastrointestinal tract as antigen-presenting cells (Wilders et al., 1984; Allison et al., 1988; Seldenrijk et al., 1989). Microfold (M) cells, which exist in the follicle-associated epithelium overlying the lymphoid follicles of Peyer’s patches, take up various macromolecules, bacteria, viruses, and protozoa and transport them to the follicular areas for uptake by macrophages (Owen et al., 1981; Inman and Cantey, 1983; Wolf et al., 1983; Wassef et al., 1989; Amerongen et al., 1994; Owen, 1997). The existence of colonic mucosal lymphoid organs with M cells was reported, and antigen can be taken up in these organs as in Peyer’s patch in the small intestine (Perry and Sharp, 1988; Owen et al., 1991). Several studies have indicated involvement of macrophages and dendritic cells in active inflammatory bowel disease (IBD) (Wilders et al., 1984; Allison et al., 1988; Seldenrijk et al., 1989). Moreover, CD4 Received for publication June 10, 1999. 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Culture and Science of Japan (09670543, 11670495, and 10470134), a Grant-in-Aid for Research for the Future Program from the Japan Society for the Promotion of Science (JSPS-RFTF 97100201), and Supporting in Research Funds from the Japanese Foundation for Research and Promotion of Endoscopy (JFE-1997). ABBREVIATIONS: UC, ulcerative colitis; IBD, inflammatory bowel disease; M cells, microfold cells; PDLLA, poly(DL-lactic acid); Dx, dexamethasone; DSS, dextran sodium sulfate; MPO, myeloperoxidase; NO, nitric oxide; RT-PCR, reverse-transcription polymerase chain reaction; TNF-a, tumor necrosis factor-a; IL-1b, interleukin-1b; IFN-g, interferon-g. 0022-3565/00/2921-0015$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 292, No. 1 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 292:15–21, 2000 15 at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from T cells also have the important role of pathophysiology of IBD (Okazaki et al., 1993; Probert et al., 1996). Therefore, the regulation of these immune cells is thought to be an important therapeutic strategy for patients with IBD. Considerable attention has been paid to the use of polymer microspheres for the sustained release of various drugs and the targeting of therapeutic or diagnostic agents to their site of action (Tabata and Ikada, 1990a). The use of biodegradable microspheres is particularly preferable from the perspective of avoiding the accumulation of foreign materials in the body (Tabata and Ikada, 1988). It was reported that biodegradable poly(DL-lactic acid) (PDLLA) microspheres can be efficiently taken up by macrophages and M cells (Tabata et al., 1996). Polymer microspheres worked effectively as carriers for biological response modifiers that activate macrophages (Tabata et al., 1989; Tabata and Ikada, 1990a,b,c). Judging from these results, the direct uptake of anti-inflammatory agents by macrophages, achieved with the use of microspheres, appears to have a superior immunosuppressive effect and to be more useful for the treatment of patients with IBD. We have successfully incorporated dexamethasone (Dx) into microspheres via a solvent double-emulsion method and developed a new therapy with PDLLA microspheres containing Dx (Dx microspheres), which directly target macrophages and M cells. The objectives of this study were to evaluate the pharmacokinetics of Dx microspheres in mice and to examine the therapeutic effects of this drug in treating experimental colitis in mice. Materials and Methods Animals. Female BALB/c mice weighing 20 to 25 g (Japan SLC Inc., Shizuoka, Japan) were used for the experiments. They had access to food and water ad libitum. For induction of colitis, mice (n 5 10/group) received three cycles of treatment with dextran sulfate sodium (DSS) (5000 Mr, 40 kDa; Sigma Chemical Co., St. Louis, MO). Each cycle consisted of 5% DSS in drinking water for 7 days, followed by a 7-day period with normal drinking water (Okayasu et al., 1990). Preparation of Dx Microspheres. PDLLA microspheres were synthesized by the simple polycondensation of DL-lactic acid at 180°C under reduced pressure without any catalyst. Dx phosphate (Decadrone) was kindly supplied from Banyu Pharmaceutical Co. (Tokyo, Japan). Dx microspheres were prepared by the solvent-evaporation method with double emulsion, as previously described (Tabata et al., 1996). In brief, 60 ml of a Decadrone aqueous solution (W1) was poured into 1 ml of methylene choride containing 200 mg of PDLLA microspheres (O), followed by emulsifying probe sonication to form a W1/O emulsion. The emulsion was added to 2 ml of a 1 wt% polyvinyl alcohol (PVA; weight-averaged Mr 5 5400; degree of saponification, 79.85 mol%) aqueous solution (W2) that had been saturated with methylene choride at room temperature and agitated by a vortex mixer to form a double emulsion. The W1/O/W2 double emulsion was stirred by an impeller (200 rpm) at room temperature until the methylene choride was completely evaporated. The microspheres were collected by centrifugation (5000 rpm, 5 min, 4°C), washed three times with cold distilled water, and finally lyophilized. After hydrolysis of Dx microspheres, the concentration of Dx was measured by HPLC to assess the dosage incorporated in the microspheres (Haeberlin et al., 1993). As a result, 9.6 3 10 mg of Dx could be incorporated into 1 mg of PDLLA microspheres. The prepared Dx microspheres were further fractionated into different sizes by counterflow centrifugal elutriation. The size of the prepared Dx microspheres was assessed with microscopic photographs according to a reference scale. We adjusted the size of the Dx microspheres to within 4 mm because the microspheres with diameters ,4.0 mm were phagocytosed by macrophages at the maximum level (Tabata and Ikada, 1990a). A similar microsphere preparation was performed to obtain fluorescent (cumarine 6, laser grade; ACR # OS, Organics, Geel, Belgium)-labeled PDLLA microspheres and I-labeled Dx microspheres (Amersham International, Buckinghamshire, UK). In Vitro Release of Dx from PDLLA Microspheres. First, 2.5 mg of PDLLA microspheres containing Dx was suspended in 0.5 ml of normal saline and incubated at 37°C in a shaking bath. After centrifugation, this suspension was serially sampled. The Dx concentration of the sample solution was assessed by HPLC as previously described (Haeberlin et al., 1993). Tissue Distribution of PDLLA Microspheres in Mice with DSS-Induced Colitis. For the determination of the systemic distribution of PDLLA microspheres, microspheres containing I-labeled Dx (0.1 mg/g) were orally administered once to normal mice and mice with DSS-induced colitis. The radioactivity of each organ was determined at 6, 12, and 24 h and 3 and 7 days after administration (n 5 3 at each time point for each treatment) by gamma counter (ARC300; Aloka Co., Tokyo, Japan). Other mice with DSS-induced colitis were orally administered, via gastric intubation, 2.5 mg of PDLLA microspheres containing cumarin 6 in 0.3 ml of PBS for the investigation of sites at which the microspheres were taken up in the colon. The mice were sacrificed by cervical dislocation at 12 h after administration of the microspheres. The excised colons were immediately mounted in optimal cutting temperature (OCT) freezing compound (4583; Miles Inc., Elkhart, IN) and frozen in liquid nitrogen. They were cut into 4to 6-mm serial sections, which were then viewed by confocal fluorescent microscopy. Blood Distributions of Dx in Mice with DSS-Induced Colitis Treated with Dx Microspheres or Dx Alone. In the amount 0.1 mg, Dx microspheres contain about 10 mg of Dx. Therefore, Dx microspheres (0.1 mg z g z day) or Dx (10 mg z g z day) in 0.3 ml of PBS was administered by gastric tube to mice with DSSinduced colitis. The mice were anesthetized with ether at 0.5, 1, 1.5, 2, 2.5, 3, and 4 h after drug administration (n 5 3 at each time point for each treatment), and 1 to 1.5 ml of blood was then taken before sacrificing the mice. The plasma was separated from the blood by centrifugation and stored at 4°C until assayed. Treatments. Fifty mice with DSS-induced colitis were divided into five groups (10 mice in each; groups A–E) and treated immediately after the above-described three cycles of DSS administration as follows: A, no medication; B, PDLLA microspheres (0.1 mg z g z day) alone; C, Dx (10 mg z g z day) alone; D, PDLLA microspheres (0.1 mg z g z day) 1 Dx (10 mg z g z day) (the mixture of Dx and microspheres); E, Dx microspheres (0.1 mg z g z day). Mice were sacrificed by cervical dislocation after a 2-week administration of these treatments. The colonic tissues were processed according to the procedures described below. Determination of the Histological Score of Colitis. The colon was removed, washed with PBS, and turned inside out by cutting longitudinally. The colon was then fixed in 10% formalin in PBS overnight. Tissues from the distal third of the colon were fixed in 3.3% buffered formaldehyde and stained with H&E. Histological analysis was performed in a blind fashion. Microscopically, mucosal damage was quantitated by the scoring system of Kojouharoff et al. (1997). The colitis score of each mouse represented the sum of the different histological subscores. Assessment of Colonic Myeloperoxidase (MPO) Activity. MPO activity was measured according to the method of Bradley et al. (1982). In brief, tissues from the midcolon, were homogenized with a Polytron homogenizer (Brinkman Instruments, Rexdale, Ontario, Canada) three times in hexadecyltrimethylammonium bromide buffer. The homogenate was centrifuged, and MPO activity in the supernatants was measured. One unit of MPO activity was defined as the amount required to degrade 1 mM hydrogen peroxide in 1 min at 25°C. Assessment of Nitric Oxide (NO) Production. Tissues from the proximal third of the colon were homogenized for 15 s in HEPES buffer solution (40 mM, pH 7.4) containing sucrose (320 mM) (Bough16 Nakase et al. Vol. 292 at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from ton-Smith et al., 1993; Denenberg et al., 1995). The combined concentration of nitrite and nitrate and the degradation products of NO in supernatants (10,000g for 20 min at 4°C), were determined by Griess reaction after nitrate reduction, as previously described (Salzman et al., 1995). Total nitrite/nitrate production is described in the text as NO production. Measurement of Cytokine mRNA Expression in Colonic Tissue. Samples of colonic tissue for mRNA isolation were removed from the distal third of the colon. Total RNA was isolated with the guanidium isothiocyanate method (Khan and Collins, 1994). The concentration of RNA was determined by absorbance at 260 nm in relation to that at 280 nm. The RNA was stored at 270°C until used for reverse-transcription polymerase chain reaction (RT-PCR). One microliter of RT product was added to 1 mM of each primer and a solution of 1 U of Taq DNA polymerase (Takara Biochemicals, Ohtsu, Japan) in a final volume of 20 ml. The mixture underwent PCR amplification for 35 cycles (1 min at 94°C, 1 min at 52°C, and 20 s at 20°C). Negative controls (cDNA-free solution) were included in each reaction. The sequences of primers for the cytokine genes are as follows: Tumor necrosis factor (TNF)-a forward, 59-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-39 TNF-a reverse, 59-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-39 Interleukin (IL)-1b forward, 59-ATGGCAACTGTTCCTGAACTCAACT-39 IL-1b reverse, 59-CAGGACAGGTATAGATTCTTTCCTTT-39 interferon (IFN)-g forward, 59-TGCATCTTGGCTTTGCAGCTCTTCCTCATGGC-39 IFN-g reverse, 59-TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-39 Statistics. Student’s t test and the Mann-Whitney test were used where appropriate for statistical analysis. The data were presented as means 6 S.E. A two-tailed P value of ,.05 was considered significant.