J Gastroenterol2002; 37[Suppl XIV):44-52 Journalo,f-------,

Gastroenterology

© Springer-Verlag 2002

Intestinal drug delivery systems with biodegradable micro spheres targeting mucosal immune-regulating cells for chronic inflammatory colitis

KAZUICHI OKAZAKIl, HIROSHI NAKASEl, NORIHIKO WATANABE!, YASUHIKO TABATA2, YOSHITO IKADA2,

and TSUTOMU CHIBA 1

I Division of Gastroenterology and Endoscopic Medicine, Graduate School of Kyoto University, 54 Shogoinkawara-cho, Sakyoku, Kyoto 606-8507, Japan 2Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan

We developed two kinds of delivery systems target­ing mucosal immune regulating cells with poly (D,L­lactic acid) micro spheres containing dexamethasone and dichloromethylene diphosphonate, and gelatine micro spheres containing interleukin-IO. To estimate the efficacy of these drug delivery systems, we studied the effects on experimental colitis induced by dextran sodium sulfate, 2,4,6-trinitrobenzene sulfonic acid, and interleukin-IO-deficient mice. Intestinal administration of these microspheres significantly improved colitis with decreased histological score, myeloperoxidase activity, and nitric oxide production compared with mice treated with free agents. Gene expressions of tumor necrosis factor-a, interleukin-l~, and interferon-y were down­regulated in treated animals. Serum Dx, IL-lO levels, and systemic macrophages were unchanged after treat­ment. Our findings suggest that local macrophages in the intestine play a critical role in the initiation of chronic colitis in the animal model of inflammatory bowel disease (IBD). Intestinal drug delivery systems with biodegradable microspheres targeting mucosal immune-regulating cells may become a therapeutic approach to human IBD.

Key words: IBD, drug delivery system, microsphere

Introduction

Ulcerative colitis (UC) and Crohn's disease (CD) are the two major forms of chronic human inflammatory bowel disease (IBD). Studies of immune inflammatory cells and proinflammatory cytokines in the intestine of IBD patients have suggested that IBD is caused by dysregulated immune responses to enteric antigens. 1- 5 However, multifactoral processes appear to be involved

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in the pathogenesis of human IBD. To characterize this disease, several animal models have been established. Although the etiology of this disease remains unclear, several studies have indicated that active immune regu­lating cells such as macrophages and T-cells play an important role in the pathogenesis of IBD.6-10 There­fore, the regulation of these immune cells is thought to be an important therapeutic strategy for patients with IBD. Recently, we developed some new intestinal drug delivery systems which consist of polY-D,L-lactic acid (PDLLA) micro spheres containing dexamethasone (DX-microspheres)ll or dichloromethylene dipho­sphonate,12 and gelatine micro spheres containing interleukin-lO.13 To evaluate these drug delivery sys­tems, we studied their therapeutic effects on experimen­tal colitis induced by dextran sulfate sodium (DSS),14 2,4,6-trinitrobenzene sulfonic acid (TNBS),15 and interleukin (IL)-l0+ mice. 16

Biodegradable micro spheres

Recently, we developed two kinds of biodegradable microspheres containing immune modulating agents and cytokines, namely PDLLA17,18 and gelatin micro­spheres.19 The materials used for these micro spheres are widely utilized for clinical purposes. PDLLA micro­spheres have the advantage of being able to incorporate chemical agents and DNA, whereas gelatin micro­spheres can contain proteins such as cytokines, or growth factors with active forms. We incorporated dex­amethasone phosphate (Decadrone, DX)l1 and dichlo­romethylene diphosphonate (CL2MDP) into PDLLA microcapsules (Watanabe N et al.: unpublished work) and IL-IO into gelatin microspheres.u Dx has a potent antiinflammatory and immunosuppressive action, and DL2MDP selectively induces apoptosis of macroph­ages.20,21 Each microsphere can be adjusted to an appro­priate size. PDLLA microspheres of less than 48!-Lm can

K. Okazaki et a1.: Intestinal drug delivery system in lED

easily be incorporated into the mucosal immune regu­lating cells of the intestine, especially membranous (M) cells and macrophages.17,18

PDLLA micro spheres containing drugs

PDLLA microspheres were synthesized by the simple polycondensation of D,L-Iactic acid at 180°C under reduced pressure without any catalyst. Dx phosphate (Decadrone) and dichloromethylene diphosphonate (CL2MDP) were kindly supplied by Banyu Pharmaceu­tical (Tokyo, Japan) and Kissei Pharmaceutical (Tokyo, Japan), respectively. These agents were prepared by the solvent-evaporation method with double emulsion, as previously described. 17 In brief, 60 fLl Dx or CL2MDP (200mg) aqueous solution (W1) was poured into 1 ml methylene chloride containing 200mg PDLLA microspheres (0), followed by emulsifying probe soni­cation to form a W1/0 emulsion. The emulsion was added to 2ml of a 1 wt% polyvinyl alcohol (PV A, weight-averaged Mr = 5400, degree of saponification 79.85 mol %) aqueous solution (W2) that had been satu­rated with methylene chloride at room temperature, and agitated by a vortex mixer to form a double emulsion. The WlIO/W2 double emulsion was stirred by an impeller (200rpm) at room temperature until the methylene chloride was completely evaporated. The micro spheres were collected by centrifugation (5000 rpm, 5min, 4°C), washed three times with cold distilled water, and finally lyophilized. After hydrolysis of the Dx or CL2MDP microspheres, the concentration of drugs was measured by high-performance liquid chromatography (HPLC) to assess the dosage incorpo­rated in the microspheres.22 The prepared Dx micro­spheres were further fractionated into different sizes by counterflow centrifugal elutriation. The size of the prepared micro spheres was assessed from photomicro­graphs according to a reference scale. We adjusted the size of the Dx micro spheres (MS-Dx) to within 4 fLm because microspheres with a diameter <4.0 fLm were phagocytosed by macrophages at the maximum level. 17

They were then collected by centrifugation, washed three times with cold distilled water, and finally lyo­philized. Lyophilized MS-CL2MDP (lOOmg), MS-Dx (9.6 X 1O-4mg Dx in 1 mg MS-Dx) were suspended in 4ml PBS before use. A similar microsphere preparation was made to obtain fluorescent (cumarine 6, laser grade; ACROS Organics, Geel, Belgium)-labeled PDLLA microspheres and 125I-Iabeled microspheres (Amersham International, Buckinghamshire, UK). The spontaneous release of the incorporated drugs from the PDLLA microspheres in vitro showed that one third of the drugs in the microspheres was released into the media within 24h, and it was all released within 48h (Fig. 1).

(%)

1 100

J 80 ! )( c - 60 c

~ :. 40

20

0 0 20

---2

40 Time (hour)

60

4S

80

Fig. 1. Spontaneous release of incorporated drugs from poly­D,L-Iactic acid (PDLLA) microspheres in vitro. First, 2.5mg dexamethasone (Dx) microspheres was incubated in O.Sml saline at 37°C, and 97% ± 3.0% of the incorporated Dx had been released 48 h after the start of incubation. Each data point represents the mean ± SE. (From ref. 11, with permission)

Selective uptake of PDLLA microspheres into macrophages and colonic lymphoid follicles

Fluorescent microscopy of the colonic mucosa showed that the microspheres were predominantly taken up into the colonic lymphoid hyperplasia of the DSS­treated mice (Fig. 2). Specifically, PDLLA microspheres containing CL2MDP (MS-CL2MDP) increased the number of dead macrophages in a dose-dependent manner. They also decreased the number of local macrophages in the Peyer's patch and lamina propria in the intestine of IL-lO-l- mice with spontaneously developed colitis. Therefore, drug-containing PDLLA microcapsules less than 4 fLm are selectively taken up into the local macrophages of the intestine.

PDDLA microspheres can decrease the systemic effects of agents owing to poor systemic uptake of agents

After oral administration of Dx alone (10-4mg/g), the concentration of Dx in the plasma increased markedly, and reached the maximum level after 30 min. In con­trast, no significant elevation in the plasma Dx level was observed after the administration of Dx microspheres (Fig. 3). Similarly, MS-CL2MDP treatment reduced macrophages in the Peyer's patch and lamina propria of the intestine, but not in the other peripheral lym­phoid organs such as the spleen, peritoneal cavity, and mesenteric lymph nodes. Therefore, drugs contained in PDDLA micro spheres of less than 4 fLm are not systemi-

46 K. Okazaki et al.: Intestinal drug delivery system in lED

Fig. 2a,b. Localization of PDDLA microspheres. a Fluorescence microscopy findings. The fluorescent-labeled PDLLA microspheres were predominantly present in the peripheral portion of the colonic lymphoid tissue of the dextran sulfate sodium (DSS)-treated mice 12 h after oral administration of microspheres. b H&E staining at the same section, X200. (From ref. 11, with permission)

(nglml) .. 100 c

~ I 80

i 60

'0 .. 40 i • DX-alona o Dx-mlcrospheras

E 20 2 : 0~·~~~;~5;·O~.:~ .. : .... : .... : ... ~.:: ... : .... : .... : ... ~::~::~~~ Tlma(hour)

Fig. 3. Time-course of the serum levels of dexamethasone (Dx) (ng/ml) in mice with DSS-induced colitis after the oral administration of Dx micro spheres (1O-4mg/g) or Dx alone (1O-4mg/g). Data are expressed as means ± SE (n = 3). There was no elevation of the serum level of Dx in mice treated with Dx microspheres. (From ref. 11, with permission)

cally absorbed, which results in the decrease of systemic side effects.

Effects of treatments with PDLLA microspheres on colonic inflammation in experimental coltitis models

DSS-induced colitis mice and TNBS-induced colitis rats showed multiple mucosal erosions and ulcerations with reddish and edematous mucosa. Systemic or intestinal administration of free Dx improved macroscopic and microscopic mucosal injury in both models (reddish and edematous mucosa with fewer erosions and ulcera-

tions). After oral administration of the same dose of MS-Dx, macroscopic and microscopic examinations demonstrated a remarkably improvement in mucosal injury (only mild edematous mucosa) compared with untreated and treated animals having free Dx. Mice at 4-9 weeks of age spontaneously developed mucosal inflammation in almost the entire colon, as described previously. Intestinal administraion of MS-CL2MDP drastically reduced the markedly thickened colonic wall as compared with untreated groups. Histological analy­sis in the colon of IL-lO-l- mice treated with PBS alone revealed a marked transmural infiltration of mono­nuclear cells, and mucosal ulceration with severe epi­thelial hyperplasia. In contrast, histological examination of the colon in IL-lO-l- mice treated with MS-CL2MDP showed a drastic reduction of the dense infiltration of inflammatory cells, and the disappearance of mucosal ulceration and epithelial hyperplasia. Thus, these findings demonstrated that the administration of MS­Dx or MS-CL2MDP has a potent inhibitory effect in the development of chronic colitis by targeting local macrophages without systemic effects (Fig. 4a).

DSS-induced colitis mice and TNBS-induced colitis rats showed increased MPO activity and NO production in the colonic tissue. MPO activity and NO production in the animals treated with MS-Dx were significantly lower than those treated with free Dx (Fig. 4b,c).

The gene expressions of TNF-a, IL-l~,and IFN-y were all up-regulated in the colons of DSS-induced coli­tis mice and TNBS-induced colitis rats compared with those from normal animals. The expressions of each

K. Okazaki et a1.: Intestinal drug delivery system in lED

(a) 20

!! § .. E 10 8

N.S r-,

(X10-2 U/g tissue) (b) 8

7

6

5

4

3

2

0

(C) (1lM) 80

70

60 c 0 50 :.= u

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20

10

0

N.S r-,

A B

N.S

A B

N.S r-1

C o

N.S ,---,

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E normal

E normal

Fig. 4a-c. Effects of treatments with dexamethasone micro spheres (MS-Dx) on the colonic inflammation of DSS­induced colitis. a Colonic histological scores, b mycloperoxi­dase (MPO) activity, and c nitric oxide (NO) production in colonic tissue. Data are means::':: SE (n = 7 in each group). Group A, no medication; group B, PDLLA microspheres (0.1 mgg-1 day-I) alone; group C, Dx (l0-4 mgg-1 day-I) alone; group D, PDLLA microspheres (0.lmgg-1 day-l) + Dx (10-4 mgg-Iday-l) (i.e., the mixture ofDx and microspheres); group E, Dx microspheres (0.1 mgg-Iday-I). *p < 0.05 com­pared with groups A and B. #p < 0.05 compared with groups C and D. N.S = not significant. (From ref. 11, with permission)

TNF-a. 354bp ~

IL-1 ~ 563bp ~

IFN-y 365bp ~

G3PDH 983bp ~

ABC D E

~ ... ' ",,',

)~~L'~· rtii1i~l~ _

, _ -.!"~:T

47

N

Fig. 5. Gene expression of proinflammatory cytokines deter­mined by reverse transcriptase - polymerase chain reaction (RT -PCR) in the colonic tissues of mice with or without Dx treatment. PCR products were separated on 2% agarose gels and stained with ethidium bromide. The mRNA expressions ofTNF-a, IFN-y, and IL-1~ were upregulated in groups A and B. There were weaker expressions of these cytokines in groups C and D as compared with those in groups A and B. There was no mRNA cytokine in group E. Lanes are as marked: lanes A-E, groups A-E; lane N, normal mice. DNA sizes are indicated on the left-hand side of the gels. TNF, tumor necrosis factor; fL, interleukin; fFN, interferon. (From ref. 11, with permission)

cytokine in animals treated with free Dx were weaker than those in untreated animals. The expression of each cytokine was undetectable in animals treated with MS­Dx (Fig. 5). Taken together, intestinal administration of MS-Dx or MS-CL2MDP has a potent inhibitory effect on the development of chronic colitis by targeting local macrophages without systemic effects.

Gelatine microspheres containing IL-IO

Preparation of gelatin microspheres containing lL-10

Gelatin microspheres were prepared as reported pre vi­ously.19 Briefly, 10ml of an aqueous solution of acidic gelatin (10 wt% preheated to 40°C) was added drop­wise into 375ml olive oil while stirring at 420rpm at 40°C for 10min to yield a water-in-oil emulsion. The

48

emulsion temperature was lowered to lSoC, followed by further stirring for 30min to allow for natural gelation of the gelatin aqueous solution. Acetone (lOOml) was added to the emulsion, and stirring was continued for 1 h. The resulting microspheres were washed three times with acetone, recovered by centrifugation (SOOOrpm, 4°C, Smin), passed through sieves with dif­ferent apertures for size fractionation, and air-dried. The average size of the microspheres was adjusted to be less than 12/.1m. Gelatin aqueous solution (O.2ml; 10 wt%) and olive oil (S ml) were agitated with a vortex mixer for 1 min, and then sonicated at 3.0 W Icm2 for variable time periods. The prepared emulsion was cooled, washed with acetone by centrifugation, and air­dried. The non-cross-linked and dried gelatin micro­spheres (2S mg) were placed in S ml of 0.1 wt% Tween 80 aqueous solution containing glutaraldehyde (40 /.1g1 ml) and stirred at 4°C for lSh to facilitate cross-linking. Following collection by centrifugation (SOOOrpm, 4°C, Smin), the microspheres were agitated in Sml of 10mM aqueous glycine solution at 37°C for 1 h to block the residual aldehyde groups on unreacted glutaraldehyde. The resulting microspheres were washed three times with double-distilled water by centrifugation, and freeze-dried. The incorporation of rmIL-lO into ge­latine microsphere (GM) was performed by allowing the freeze-dried micro spheres to swell in an aqueous solution of the protein. Briefly, 10/.11 IL-10 solu­tion (S /.1g/ml) was dropped onto 2.S mg freeze-dried glutaraldehyde-crosslinked microspheres, and allowed to sit at 37°C for 1 h. Both 125I-labeled and unlabeled GM were prepared in the same way.

In vitro release of recombinant (rm) IL-IO from GM-IL-IO

The in vitro release test of rm1L-10 from the microspheres was conducted at 37°C. GM containing 1251-labeled rmlL-lO was immersed with shaking in col­lagenase solution (1 ml; 0.8mg/ml) prepared from phos­phate buffered saline (PBS). After centrifugation, the radioactivity of 1251 -labeled rm1L-10 in the supernatants was determined using a gamma counter (ARC-300, Aloka, Tokyo, Japan). rm1L-10 was released from GM­IL-lO in a time-dependent manner: 38% ± S% (2h), 66% ± 4% (6h), 83% ± 3% (12h), and 90% ± S% (48h) (Fig. 6).

Retention ratio of IL-IO in the colon and the blood distribution of IL-IO

1251-labeled rmlL-lO (O.OS /.1g) or GM containing 1251_ labeled rmIL-lO (2.S mg GM which contained O.OS /.1g IL-10) was suspended in 200/.11 PBS, allowing the solu­tion to reach the entire colon. The in vivo retention ratio of rm1L-10 (percentage of 1251-labeled rmIL-10 re-

K. Okazaki et al.: Intestinal drug delivery system in lED

(%) 100

6~ 1> .I.

80 6/ oQ) m 11 m oQ)

1S 1 ... 60 -c 8 ...

J oQ) a.

40

1 20

o 10 20 30 40 50 Time (hr)

Fig.6. In vitro release of recombinant (rm) IL-IO from gelatin microspheres. Gelatin microspheres (2.5mg) containing 1251_ labeled rmlL-lO was immersed under shaking in 1 ml phos­phate buffered saline (PBS) with collagenase (O.8mg/ml). Each point represents the mean ± SE of the percentage of the radioactivity released (n = 3). (From ref. 28, with permission)

maining in the colon) and the blood distribution of rm1L-10 (percentage of 125I-labeled rmIL-10 remaining in the blood) were determined using a gamma counter at lS, 30, and 4Smin, and 1, 12,24, and 48h by mea­suring the radioactivity of the removed colon and blood.

The retention ratios of rm1L-10 remaining in the colon were measured at lS, 30, and 4Smin, and 1,6,12, 24, and 48h after rectal administration of GM contain­ing 125I-labeled IL-10 or 125I-labeled IL-10 alone (Fig. 7). The retention ratios of rmIL-lO in the colon of GM IL-10-treated normal mice at 1,6, and 12h after admin­istration were significantly higher than those of rmlL-10-treated normal mice. Furthermore, the retention ratios of rm1L-10 in the colon of GM IL-10-treated IL-10-1- mice at 6, 12, and 24h were significantly higher than those of GM IL-10-treated normal mice.

The blood distribution of 1251-labeled rmlL-lO after rectal administration of 125I-labeled IL-10 alone was detected at lS, 30, and 4S min, but was not detectable at 60min. In contrast, the blood distribution of 1251-labeled rm1L-10 after rectal administration of GM containing 1251-labeled rmIL-lO was detectable from 30min to 24h (Fig. 8). The blood distributions of IL-10 in GM IL-10-treated IL-lO-I- mice throughout the experiment were significantly higher than those of IL-10-treated normal mice.

K. Okazaki et al.: Intestinal drug delivery system in lED

(%)

100 -- GM-IL.-IO+IL· 1O·/-mice

__ GM-ll_-IO+I101·mol micc

80 ~ IL.-l 0

0 .~

60 .,

6 .i§ 4)

~ 40

20

o o 10 20 30 40 50

Time (hr)

Fig.7. In vivo retention ratio of IL-10 in the colon after rectal administration of gelatin microspheres containing 1251-labeled IL-10 or 1251-labeled IL-lO alone. Each point represents the mean ± SE of the percentage of the radioactivity remaining in the colonic tissue (n = 3). #p < 0.05 compared with IL-10-treated normal mice. *p < 0.01 compared with GM-IL-10-treated normal mice. (From ref. 28, with permission)

(%)

0 .10

6 0 .08

's .. .. .c

:~ 0, 06

'0 '0 0 0 ,0. 0 :c

0.0 2

..

10

hour 20

--*- G~I. fI~ IO+IL. IO·/-mi<c

--*- G M-IL-IO+f1o r-nlfll m ke

-0- IL·IO

30 50

Fig. 8. Blood distribution of IL-lO after rectal administration of gelatin microspheres containing 1251-1abeled IL-10 or 1251_ labeled IL-lO alone. Each point represents the mean ± SE of the percentage of the radioactivity remaining in the blood (n = 3). #p < 0.05 compared with IL-lO-treated normal mice. *p < 0.05 compared with GM-IL-10-treated normal mice. (From ref. 28, with permission)

Effects of treatment with gelatin microspheres on colonic inflammation in experimental coltitis models

Macroscopic examination of the colon from nontreated, GM-alone, and rmIL-lO-alone animals revealed a marked thickening of the colonic walL In contrast, mac­roscopic examination of GM IL-IO-treated animals re­vealed no thickening of the colonic wall. There was no difference in the GM IL-IO treatment effect in terms of the site within the colon. Histologic findings revealed

49

~ 4

8 (1'1

"" .s, 3

..s S 2 .lQ ::

1

o

Fig. 9. Histological findings of the effects of GM-IL-10 and IL-lO on the colonic mucosa of IL-lO-l- mice with various treatments. Histological colon scores. A score of 4 represents maximal injury, and score 0 no injury. Data are means ± SE (n = 5 for each group). Group A, nontreated IL-10-1- mice; group B, GM-treated IL-10-1- mice; group C, IL-10-treated IL-lO-l- mice; group D, GM IL-lO-treated IL-lO-l- mice. *p < 0.01 vs groups A, B, and C. (From ref. 28, with permission)

epithelial hyperplasia, mucosal ulceration, and a re­markable infiltration of mononuclear cells in the colons of nontreated, GM-alone, rmIL-lO-alone animals. In contrast, histologic findings in GM IL-IO-treated ani­mals were almost normal except for low levels of infil­trating monocytes. The histological score for the GM IL-IO group was significantly lower than that for nontreated groups, GM-alone groups, and rmIL-lO­alone groups. There were no significant differences in the histological scores among nontreated, GM-alone, and rmIL-lO-alone groups (Fig. 9). The jejunum and the ileum were histologically normal in all groups.

mRNA expression of Mac-I-positive cells from the lamina propria of the colon

The reverse transcriptase-polymerase chain reaction (RT-PCR) results showed that mRNA expressions of both IL-12 p40 and IL-12 p35 were up-regulated in Mac-I-positive cells in the colon of nontreated, GM­alone, and rmIL-lO-alone groups. However, transcript levels of these cytokines in the GM IL-IO group were significantly lower than those in nontreated, GM-alone, and rmIL-lO-alone groups (Fig. 10).

Expression of CD40 on Mac-I-positive cells from the lamina propria of the colon and spleen

Compared with untreated IL-lO-I- mice, CD40 expres­sion on Mac-I-positive cells in both the lamina propria and the spleen were markedly decreased in the GM IL-IO group, but not in the rmIL-lO-alone group (Fig. 11).

50 K. Okazaki et al.: Intestinal drug delivery system in IBD

#

0 .8 #

#

c ... U t'Il C ~ ... ...... u 0 t'Il

I

~ <lQ. ...... N If"l .... M

I a. ....J N ....

I .....J

Group D Group E

Fig. 10. Effect of treatments with gelatin micospheres containing IL-lO (GM IL-IO) on the transcript levels of both IL12 p40 and IL12 p35 in Mac-I-positive cells. The amounts of the cytokine transcripts are expressed as relative concentrations of ~-actin (~­actin = 1). Results are means::':: SE. Group A, nontreated IL-lO+ mice; group B, GM-treated IL-lO-'- mice; group C, IL-IO­treated IL-IO+ mice; group D, GM IL-lO-treated IL-IO+ mice; group E, normal. #p < 0.01 vs groups A, B, and C. (From ref. 28, with permission)

S lamina propria

S spleen

a s

i 3

~ <> e-

m .... G)

~ <>

.c <>

E ~

~ <>

Z <>

'i) 0

'0 ' 10 ' 10 • 10 ' 10' 10 ' 10' 10 '

CD40

Fig.n. Flow cytometric analysis of CD40 expression on Mac-I-positive cells in the lamina propria and spleen of IL-IO-'- mice after treatment with GM-IL-lO and IL-lO. CD40 expression on Mac-I-positive cells was up regulated in IL-lO+ mice in both the lamina propria and the spleen (black line). CD40 expression was downregulated in the GM-IL-lO-treated group (red line), but not in the IL-IO-treated group (blue line) in both the lamina propria and spleen. (From ref. 28, with permission)

Discussion

Several animal models of intestinal inflammation have been described.I 4--16,23-27 Among them, hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol­induced colitis has been well standardized.ls The TNBS­induced colitis model resembles human CD in terms of

its histopathological features and T -helper 1 profile of cytokines, including interferon (IFN)-g, whereas DSS­induced colitis resembles human UC.l4,28-30

The regulation of macrophages which present anti­gens for activating T cells and produce various cyto­kines is a key issue in the treatment of IBD. The results of our study clearly demonstrate that microspheres

K. Okazaki et al.: Intestinal drug delivery system in IBD

containing dexamethasone (MS-DX) and dichloro­methylene diphosphonate (MS-DL2MDP), which tar­get macrophages or M cells, have novel therapeutic effects in several types of colitis model. Confocal laser microscopic examination of colonic tissue confirmed that the fluorescence-labeled microspheres were taken up into the colonic lymphoid tissue and Peyer's patches.ls Moreover, analysis of the tissue distribu­tion of microspheres containing 125I-labeled drugs re­vealed that the microspheres were predominantly distributed in the inflamed colon. These results suggest that the PDLLA micro spheres have been taken up pre­dominantly by the inflamed colonic lymphoid tissue, which is thought to be the initiation site of immune responses. Several reasons may be considered for the difference in distribution of PDLLA-microspheres in the inflamed and the normal colon. First, there are more activated macrophages in the inflamed colon than in the normal colon. In the inflamed colon, PDLLA­microspheres may be coated with various proteins such as immunoglobulins, and complement components, which are produced by colonic inflammation. These coating proteins may render the microspheres more recognizable, so that they are phagocytosed by macrophagesP

Our findings showed that the elimination of intestinal macrophages by intestinal administration of MS­DL2MDP is sufficient to suppress the development of chronic colitis in IL-lO- l - mice. These results suggest that local macrophages in the intestine play a critical role in the initiation of chronic colitis in IL-IO-I- mice. Previous studies have shown that the intraveous admin­istration of liposomes containing CL2MDP can selec­tively deplete circulating monocytes and can prevent autoimmune diabetes and experimental autoimmune encephalomyelitis (EAE). In NOD mice, a depletion of monocytes reduces Thl immune response by decreasing the expression of macrophage-derived IL-12.21 In con­trast' the depletion of circulating monocytes in EAE produces an imbalance in matrix ' metalloproteinase and/or chemokine production, resulting in a reduced invasion of autoreactive T cells and secondary glial recruitment that ordinarily leads to demyelinating pa­thology. Therefore, the elimination of intestinal macro­phages by MS-CL2MDP may reduce IL-12 production or other chemokine secretion, which can increase the migration of Thl-type CD4+ T cells and monocytes from the circulation. The pharmacokinetics of Dx con­tained in MS-Dx, and the lack of effect of MS-CL2MDP on macrophages of the systemic lymphoid organs, sug­gested that oral or intestinal administration of PDDLA­microspheres resulted in very little absorbance of drugs, which resulted in fewer systemic effects. It is considered that microspheres cannot be absorbed by enterocytes because of their size.1s

51

For the intestinal administration of bioactive pro­teins , we developed gelatin microspheres containing IL-10 (GM-IL-IO).28 Our study clearly demonstrated that intestinal administration of GM-IL-IO inhibits colonic mucosal inflammation in IL-IO-I- mice more efficiently than treatment with IL-lO alone. Analysis of the reten­tion ratio of 125I-labeled IL-IO in the colon indicated that IL-IO remained in the colon of GM-IL-IO-treated mice longer than in IL-IO-treated mice. Thus, IL-IO appears to be released from GM-IL-IO gradually and continu­ously in the colonic mucosa, resulting in the prolonged availability of IL-lO to the colon. Moreover, the reten­tion ratio of IL-lO after the administration of GM-IL-IO in IL-IO-I- mice was greater at each time point than the corresponding values in normal mice.

As for the distribution of IL-IO in the blood, there was a longer period of distribution in the GM-IL-lO­treated group than in the IL-IO-treated group. This re­sult is considered to be due to a difference in the colonic retention ratios of IL-IO and GM-IL-lO, and reflects the local sustained release of GM-IL-IO in the colon. The ratio of distribution in the blood is extremely low (less than 0.07% of the 125I-labeled rmIL-IO administered) compared with that of the intestinal distribution, and therefore it is unlikely to induce systemic side effects. Thus, rectal administration of GM-IL-IO might be an ideal cytokine delivery system for treating lED.

The results of both macroscopic and histological studies revealed that GM-IL-IO has more potent inhibi­tory effects on colitis than IL-IO alone, and therefore they suggest that GM-IL-IO not only prevents but also improves colonic inflammation.

IL-12 is a cytokine involved in Thl T-cell differentia­tion, which promotes the production of IFN-y.29 Sys­temic administration of monoclonal antibodies against IL-12leads an improvement in colitis in mice by elimina­tion of the Thl T cells through induction of Fas­mediated apoptosis.30,31 These data suggest that IL-12 has a key role in the development of Thl-dominant colitis.

In monocytes/macrophages, the interaction of CD40 with CDI54 results in the production of inflammatory cytokines, and many fewer monocytes suffering from apoptosis.32 Several clinical studies have indicated that the CD40-CD154 interaction is involved in the patho­genesis of IBD.33-35 In the present study, the expression of CD40 on Mac-I-positive cells in both the intestinal lamina propria and the spleen were downregulated more prominently in GM-IL-IO-treated mice than in IL-IO-treated mice. These results emphasize the impor­tance of locally sustained release of IL-IO from GM­IL-IO for blocking CD40-CD154 pathways both sys­temically and locally. IL-lO-related side effects in lED patients can then be greatly reduced.

In conclusion, we developed two kinds of biodegrad­able microspheres made of polY-D,L, lactic acid, and

52

gelatin. The first has the advantage of being able to incorporate chemical agents or DNAs, and the last can incorporate bioactive proteins such as cytokines or growth factors. Our studies have clearly demonstrated the favorable therapeutic effects of microspheres con­taining Dx or CL2MDP in experimental models of IBD by targeting intestinal macrophages. Drugs contained in PDLLA microspheres are absorbed in very small quan­tities, which results in fewer systemic side effects. GM­IL-lO may be more suitable for clinical application than other delivery systems currently available for human IBD.

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