- Email: [email protected]
Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis
YCLIM-07450; No. of pages: 11; 4C: Clinical Immunology (2015) xx, xxx–xxx
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
4Q2
Ke Ren, Hongjiang Yuan, Yijia Zhang, Xin Wei, Dong Wang ⁎
5
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
O
R O
P
2
F
3
Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis
1Q1
7
Received 20 December 2014; accepted with revision 30 March 2015
8
13 14 15 16 17
E
R
R
18
Abstract A macromolecular prodrug (P-Dex) of dexamethasone (Dex) was developed to improve the treatment of inflammatory bowel disease (IBD). Colonic inflammation was induced by feeding mice with dextran sulfate sodium. Mice were treated with daily i.p. injection of free Dex or single i.v. injection of P-Dex, PBS or free polymer. Both P-Dex and free Dex could lower disease activity index and histology scores when compared to the controls. A single injection of P-Dex with 1/4 equivalent Dex dose had a better therapeutic effect than daily free Dex treatment. Mechanism study found that P-Dex could target the inflamed colon, and be retained by epithelial cells and local inflammatory infiltrates, suggesting that the improved efficacy of P-Dex may be attributed to its inflammation targeting, subcellular processing and activation. Collectively, these data support our hypothesis that the development of macromolecular prodrug of glucocorticoid may have the potential to improve the clinical management of IBD. © 2015 Elsevier Inc. All rights reserved.
Inflammatory bowel diseases; Dextran sulfate sodium; Dexamethasone; ELVIS; HPMA copolymer; Prodrug; Inflammation targeting
T
12
KEYWORDS
C
11
E
Q310
D
6
31 33
C
34
1. Introduction
36
Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder in the gastrointestinal tract. It is comprised of two types of chronic intestinal disorders: Crohn's disease and ulcerative colitis. There are approximately 1.4 million IBD patients in the United States and
39 40
U
38
N
35
37
20 21 22 23 24 25 26 27 28 29 30
O
32
19
⁎ Corresponding author at: University of Nebraska Medical Center, 986025 Nebraska Medical Center, COP 3026, Omaha, Nebraska 68198-6025, USA. Fax: + 1 402 559 9543. E-mail address: [email protected] (D. Wang).
its peak onset is in persons 15 to 30 years of age [1,2]. The annual total estimated expenditures for the treatment of IBD in US alone is $2.8 billion, making it one of the most expensive gastrointestinal tract disorders to treat [3,4]. The common symptoms of IBD include diarrhea, rectal bleeding, abdominal pain, fever and weight loss. In addition to these painful and often debilitating symptoms, individuals with IBD may experience other complications including bowel obstruction, ulcers, and malnutrition. The pathogenesis of IBD remains elusive and multifactorial. Accumulating evidences suggest that IBD may result from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host [5,6].
http://dx.doi.org/10.1016/j.clim.2015.03.027 1521-6616/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
41 42 43 44 45 46 47 48 49 50 51 52 53
2
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111
Macromolecular prodrug of dexamethasone (P-Dex) was synthesized by conjugating Dex to N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer via an acid labile hydrazone bond as previously described [14]. The weight average molecular weight was determined to be 3.68 × 104 with a PDI of 1.36 using an ÄKTA FPLC system (GE HealthCare, Piscataway, NJ) equipped with UV and RI detectors. After being fully hydrolyzed by HCl (0.1 M), the Dex content in P-Dex was determined to be 140 mg/g of polymer using high performance liquid chromatography. Alexa Flour® 488 labeled P-Dex (P-Dex-Alexa) and IRDye® 800 CW labeled P-Dex (P-Dex-IRDye) were obtained via polymer analogous reaction between APMA-containing P-Dex and NHS esters of the dyes. The Alexa Flour® 488 and IRDye® 800 CW contents in the conjugate were determined as 2.0 × 10−5 mol/g and 1.1 × 10− 5 mol/g, respectively, using a Lambda 10 UV/vis spectrometer.
2.3. Histological evaluation
141
After fixation, the colons were paraffin embedded and sectioned (5 μm thickness). The sections were stained with hematoxylin and eosin (H&E) for light microscopy (Olympus BX51 microscopy, Olympus, Japan) observation and graded for inflammation, extent of inflammation, regeneration, crypt damage and percent involvement by two investigators blind to the group design. The histological grading criteria were shown in Table 1. The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement to yield the final score (0–12 for inflammation and extent of inflammation, 0–16 for regeneration and crypt damage) [22,23].
142
2.4. Optical imaging analysis of the targeting of P-Dex to intestinal inflammation
154
Twenty mice were used for the optical imaging study. They were evenly divided into two groups: the health control group with normal water intake and the DSS group with 3% DSS in drinking water. On day 6, mice were given with P-Dex-IRDye (0.3 mg/mice, IRDye content 1.1 × 10− 5 mol/g) by i.v. injection. They were sacrificed one day or three days post injection and the isolated major organs were imagined using Pearl® Impulse small animal imaging system (LI-COR, Lincoln, NE) to evaluate the biodistribution of the IRDyelabeled P-Dex prodrug.
156
F
66
C
65
E
64
R
63
R
62
N C O
61
U
60
114
O
2.1. Synthesis and characterization of HPMA copolymer conjugates
59
Forty-two male Swiss Webster mice were randomly assigned into six groups. Group 1 was designated as health control group with normal water intake and no treatment. Groups 2–6 were given water containing 3% (w/v) dextran sulfate sodium salt (DSS, Mw 40,000; Alfa Aesar, Ward Hill, MA) for 10 days. Starting from the 6th day, the animals were given the following treatments: group 2, PBS (daily i.v. injection, five injections in total); group 3, HPMA homopolymer without Dex (PHPMA, the amount of polymer used was equivalent to that in P-Dex 2.5 mg/kg group, one i.v. injection on the 6th day); group 4, P-Dex (equivalent Dex dose = 2.5 mg/kg, one i.v. injection on the 6th day); group 5, P-Dex (equivalent Dex dose = 1.25 mg/kg, one i.v. injection on the 6th day); group 6, free Dex (total dose = 5 mg/kg, daily i.v. injections for 5 days). Body weight, stool consistency and hemoccult positivity were examined daily for all the animals. The disease activity index (DAI) scores, an indicator of the severity of colitis, were determined based on a standard scoring system as previously described in literatures [20,21]. The DAI scoring was performed using a 0–3 scale, where for diarrheal score, 0 = normal stool, 1 = mild soft stool, 2 = very soft stool, 3 = watery stool; for blood score, 0 = normal colored stool, 1 = brown stool, 2 = reddish stool, 3 = bloody stool. The mice were euthanized on the 10th day. The colons were removed and fixed with 4% paraformaldehyde in PBS.
R O
93
58
112
P
2. Material and methods
57
2.2. Establishment of a DSS induced colitis model and treatment
D
92
56
T
91
Immunosuppressant, anti-inflammatory and steroid-based medications are the typical treatment options for IBD [7,8]. They are used to control flare ups, maintain remission, prevent repeated attacks and help the colon and intestinal tract heal [9]. Glucocorticoids (GC) are highly effective in reducing inflammation and are widely used to induce remission in patients with active IBD [10,11]. Although the benefits of GC treatment has been widely proven in treating the disease, prolonged or high-dose GC therapy, however, is associated with significant side effects, such as hypertension, osteoporosis, immunodeficiency and adrenal gland atrophy [12,13]. In order to further potentiate the therapeutic efficacy and dampen the off-target toxicities associated with GC, we have developed a macromolecular prodrug of dexamethasone (Dex) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer (P-Dex) [14]. The prodrug P-Dex has been found to passively target and be retained at the sites of inflammation due to its extravasation through leaky vasculature and inflammatory cell-mediated sequestration (ELVIS) mechanism, providing sustained inflammation amelioration (N 1 month/injection). This observation has been validated in multiple inflammatory disease animal models, including inflammatory arthritis, lupus nephritis and aseptic implant loosening [15–17]. In addition to its superior therapeutic efficacy, it was also found that the prodrug could effectively avert the typical side effects associated with GC treatment [17,18]. As a chronic inflammatory disorder, IBD shares the general features of inflammation, such as angiogenesis and inflammatory cell infiltration, which constitute the pathological components of ELVIS mechanism [19]. Therefore, we hypothesized that the successful application of P-Dex in treatment of these inflammatory conditions may be extrapolated to IBD. Given the fact that GCs are still one of the first line treatments for IBD and they are often given in the hospital setting as i.v. infusion, the validation of the P-Dex' superior efficacy and safety in IBD models will be clinically relevant and highly translational.
55
E
54
K. Ren et al.
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
113
115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
143 144 145 146 147 148 149 150 151 152 153
155
157 158 159 160 161 162 163 164 165
Potential of P-Dex to improve the clinical management of IBD
Inflammation
0 1 2 3 0 1 2 3 0
t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25
Extent
Regeneration
Crypt damage
Percent involvement
1 2 3 4 0 1 2 3 4 1 2 3 4
None Slight Moderate Severe None Mucosa Mucosa and submucosa Transmural Complete regeneration or normal tissue Almost complete regeneration Regeneration with crypt depletion Surface epithelium not intact No tissue repair None Basal 1/3 damage Basal 2/3 damage Only surface epithelium lost Entire crypt and epithelium lost 1–25% 26–50% 51–75% 76–100%
F
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12
O
Score Histological features
colons were collected at 24 h post injection. They were fixed, paraffin embedded and sectioned (5 μm thickness). The slides obtained were first incubated with 10% goat serum (Sigma-Aldrich) for 30 min at room temperature. After addition of the primary antibodies [rat anti-mouse F4/ 80 (Invitrogen, Camarillo, CA), rat anti-mouse EpCAM (Ebioscience, San Diego, CA), rat anti-mouse Ly-6G (Gr-1) (EBioscience), rat anti-mouse Ly-6B (7/4) (AbD Serotec), Armenian hamster anti-mouse CD11c (AbD Serotec) and rabbit anti-mouse prolyl-4-hydroxylase (P4HB, Abcam, Cambridge, MA)], the sections were incubated at room temperature for 1 h in a humidified chamber. After washing with PBS, diluted secondary antibodies [Alexa Flour® 647 labeled goat anti-rat IgG (Invitrogen, Carlsbad, CA), Alexa Flour® 647 labeled goat anti-Armenian hamster IgG (Jackson ImmunoResearch, West Grove, PA) or Alexa Flour® 647 labeled donkey anti-rabbit IgG (Invitrogen)] were added and incubated for another 30 min at room temperature in darkness. In control experiments, primary antibodies were replaced by corresponding isotype controls [purified rabbit IgG (Sigma-Aldrich), purified rat IgG (Sigma-Aldrich) and purified hamster IgG (Jackson ImmunoResearch)], and the samples were processed similarly as described above. The tissue sections were then evaluated with a Nikon Swept Field confocal microscope (Nikon Instruments Inc., Melville, NY).
R O
Parameters
Histological grading criteria.
P
Table 1
D
t1:1 t1:2 t1:3
3
171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
2.6. Immunohistochemistry analysis of P-Dex' retention at intestinal inflammation Ten mice were either fed with normal water or 3% DSS. P-Dex-Alexa (5.0 mg/mice) was given to mice by tail vein injection on day 6. The animals were sacrificed and the
196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
For subcellular localization studies, mouse macrophage cell line (J774, ATCC, VA) and human epithelial cell line (Caco-2, ATCC, Manassas, VA) were cultured on 13 mm diameter coverslips (Thermo Fisher scientific, Waltham, MA) placed on tissue culture Petri dishes (Becton, Dickinson and Company, Franklin Lakes, NJ). DMEM medium (ATCC) supplemented with 10% fetal bovine serum (VWR, Radnor, PA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) was used. The cells were allowed to adhere for 24 h and then activated with lipopolysaccharide (LPS, 1 mg/mL) for 24 h. After activation, cells were treated with Alexa Flour® 488-labeled P-Dex (1 mg/mL) in the presence of LPS (500 ng/mL) for 24 h. Cells were fixed with 4% paraformaldehyde in PBS, rinsed and then lysed with 0.1% Triton X-100. After addition of the primary antibodies [rat anti-mouse LAMP-1 (EBioscience) for J774 cells, rabbit anti-human LAMP-1 (Santa Cruz, Dallas, Texas) for Caco-2 cells], the slides were incubated at 4 °C overnight in a humidified chamber. The next day, after washing, diluted secondary antibodies [Alexa Flour® 647 labeled goat anti-rat IgG (Invitrogen) or Alexa Flour® 647 labeled goat anti-rabbit IgG (Santa Cruz)] were added and incubated for 60 min at room temperature in darkness. After rinsing, cells were stained with DAPI, fixed, mounted and observed using confocal microscope. In control experiments, primary antibodies were replaced with corresponding isotype controls [purified rabbit IgG (Sigma-Aldrich), purified rat IgG (Sigma-Aldrich)]. The samples were processed similarly as described above.
222
2.8. Statistical analysis
250
Comparisons were made by one-way ANOVA followed by a post hoc Tukey’s test for multiple comparisons using SPSS
251
T
C
E
170
R
169
Thirty mice were either fed with normal water or 3% DSS. Alexa Flour® 488-labeled P-Dex (P-Dex-Alexa, 5.0 mg/mice) was given to mice by tail vein injection on day 6. At necropsy (24 h post injection), the colons were isolated and minced aseptically. The tissues were further digested with collagenase type II (1 mg/mL, Sigma-Aldrich, St. Louis, MO) at 37 °C for 30 min. After passing through a 70 μm cell strainer, a single cell suspension was obtained. The red blood cells were removed by ACK lysing buffer (Quality Biological, Gaithersburg, MD). For fluorescence-activated cell sorting (FACS) evaluation of F4/80, EpCAM, CD11c, Ly-6G (Gr-1) and Ly-6B (7/4) positive cells, the samples were incubated with antibodies [Allophycocyanin (APC)-labeled rat anti-mouse F4/80 (AbD Serotec, Raleigh, NC), APC-labeled hamster anti-mouse CD11c (BD Pharmingen, San Jose, CA), Phycoerythrin (PE) labeled rat anti-mouse EpCAM (Ebioscience, San Diego, CA), PE-labeled rat anti-mouse Ly-6B (7/4) (AbD Serotec) and PE-labeled rat anti-mouse Ly-6G (Gr-1) (Ebioscience)] for 30 min on ice. Isotype-matched APC-labeled hamster IgG1 (BD Biosciences, San Jose, CA), APC-labeled rat IgG2a (Ebioscience), APC-labeled rat IgG2b (BD Pharmingen) and purified rabbit IgG (Ebioscience) were used as negative controls. After the final wash, the cells were analyzed with FACSCalibur flow cytometer (BD, Franklin Lakes, NJ).
R
168
2.5. Analysis of P-Dex' retention mechanism at intestinal inflammation using flow cytometry
N C O
167
U
166
E
2.7. Cells culture study
Q5
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
252
4
K. Ren et al. 256
3.1. Disease activity evaluation
257
The clinical symptoms of rectal bleeding and loose stools were observed on day 5 for mice treated with DSS. As shown in Fig. 1, DAI was 0 in the health control group on day 10, indicating no disease symptom (diarrhea and blood) were observed. In contrast, the DAI scores were increased in all DSS treated groups. There were significantly lower DAI scores in both P-Dex treated groups when compared with PBS and PHPMA treated groups. High dose (2.5 mg/kg) P-Dex treatment had a significantly better therapeutic effect than the free Dex treated group (5 mg/kg) (P b 0.05). There was ~ 3% weight increase for health control animals at day 10. In contrast, weight losses were observed in all the DSS treated groups. The mice treated with free Dex (5 mg/kg) had the most weight loss, with ~9% on the 10th day. Animal treated with PBS and PHPMA had the least weight loss (~5%). The weight losses of P-Dex treated groups were between PBS/PHPMA and free Dex groups.
258
R O
O
F
3. Results
P
Figure 1 The DAI score of each group on day 10. The DAI in PBS and PHPMA groups were significantly higher than the DAI in both P-Dex groups. P-Dex high dose group has a significantly lower DAI than free Dex group. *, P b 0.05.
R
E
C
T
E
The severity of colonic inflammation and the effects of drugs on the recovery from DSS induced colitis were evaluated by examining H&E stained colon sections. Normal colonic tissue
R
255
software. Data were expressed as means ± standard deviation. A value of P b 0.05 was considered to be statistically significant.
N C O
254
U
253
D
3.2. Histology evaluation
Figure 2 Representative colon H&E staining images. Group A represented health control mice with normal water uptake. Mice in B–F groups were fed with 3% DSS in drinking water. B: PBS treated group; C: PHPMA treated group; D: P-Dex (0.5 mg/kg) treated group; E: P-Dex (0.25 mg/kg) treated group; F: free Dex (1 mg/kg) treated group. Crypt damages and inflammatory cell infiltration were found in DSS treated groups (B–F), especially in PBS (B) and PHPMA (C) treated groups. Both P-Dex groups (D&E) exhibited relatively better crypt structure and less inflammation when compared with the other groups (B&C&F). Bars = 100 um. Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274
275 276 277 278
Potential of P-Dex to improve the clinical management of IBD
The optical imaging results are shown in Fig. 4. One day after injection, there were signals in all the major organs in both groups, especially in the liver and kidneys. In addition, obvious accumulation of P-Dex-IRDye signal in the inflamed
292 293 294 295 296 297 298
302 303 304
314
Around 8% of total cells isolated from the colon 24 h after systemic administration of P-Dex-Alexa tested positive of the prodrug. The P-Dex-Alexa positive cells were further analyzed. Representative data from fluorescence-activated cell sorting (FACS) analysis were shown in Fig. 5. The histogram plots showed the intensity of staining with the specific antibodies designated on the x-axis (purple fills) with isotype control antibodies (black lines) on the same plots. The percentages represented the amount of antibody positive cells among P-Dex-Alexa positive cells: (A) ~ 76% P-Dex-Alexa positive cells were EPCam (epithelial cell marker) positive; (B) ~ 3% P-Dex-Alexa positive cells were 7/4 (neutrophil marker) positive; (C) ~ 5% P-Dex-Alexa positive cells were Ly-6G (neutrophil and monocyte marker) positive; (D) ~ 7% P-Dex-Alexa positive cells were F4/80 (macrophage marker) positive; b 1% P-Dex-Alexa positive
315
D
291
3.4. Flow cytometry analysis
T
290
C
289
E
288
R
287
R
286
N C O
285
U
284
F
301
283
O
3.3. Optical imaging
282
305
R O
300
281
colon was observed in DSS fed group. Three days after injection, there was almost no signal in the colon in health group while the strong signal in the inflamed colon in DSS group remained. In addition, compared with healthy small intestine, there was obvious accumulation of P-Dex-IRDye in the inflamed colon in DSS group. The sustained presence of near infrared signal in the colon in DSS group after P-Dex-IRDye injection validates the passive targeting and retention of P-Dex prodrug at the inflammatory lesions.
P
299
architecture is shown in Fig. 2A. Severe erosion, crypt abscesses and inflammatory cells infiltration were observed in mice with DSS water uptake and PBS treatment (Fig. 2B). The inflammation was mostly confined to the mucosa, and in some areas edema of the submucosa was observed. PHPMA treated group (Fig. 2C) showed no histological differences when compared to PBS group, while there were visible reductions in crypt damage and inflammatory cell infiltration in both P-Dex treated groups (Figs. 2D&E). There were obvious distorted crypt architecture and infiltration of inflammatory cells into the mucosa in free Dex treated group (Fig. 2F). The histology damage scores of the H&E slides were shown in Fig. 3 based on the grading criteria in Table 1. Oral administration of DSS for 10 days induced colitis. There were high scores for all the parameters in animals treated with PBS and PHPMA. After treating with P-Dex and free Dex, inflammation and crypt damages were significantly reduced when compared with PBS and PHPMA treatment. Both P-Dex groups (1.25 mg/kg and 2.5 mg/kg) had significantly lower scores than the free Dex (5 mg/kg) treated group.
280
E
279
5
Figure 3 Histology grading of colitis according to Table 1. A: Histologic colitis score (the sum of the scores in B, C and D); B: inflammation score; C: extent of inflammation score; and D: crypt damage/regeneration score. Both P-Dex treated groups showed significantly lower score than PBS, PHPMA and free Dex treated groups. *, P b 0.05. Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
306 307 308 309 310 311 312 313
316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
K. Ren et al.
E
D
P
R O
O
F
6
cells were CD11c (dendritic cell marker) positive (data not shown).
E
332
R
331
C
T
Figure 4 The distribution of P-Dex-IRDye in major organs by optical imaging. P-Dex-IRDye was given via tail vein injection on day 6 after DSS treatment. The mice were sacrificed 1 day or 3 days after the administration of the optical imaging conjugates. Compared to the health group, there were obvious accumulation and retention of the P-Dex-IRDye signals in the inflamed colons in DSS group.
3.5. Immunohistochemistry analysis
334
348
Immunohistochemical staining with specific cell markers was performed to identify the cell phenotypes within the inflamed colon that were responsible for the cellular sequestration and retention of P-Dex. As shown in Fig. 6, numerous P-Dex-Alexa positive cells (green fluorescence) were observed in the colon. Antibody stained positive cells were shown in red. Blue color from DAPI indicated cell nucleus. Co-localization with Alexa Flour® 647 labeled cell type markers indicated that among the cells that had sequestered P-Dex-Alexa, the majority were EPCam (epithelial cell marker), F4/80 (macrophage marker), Ly-6B (7/4, neutrophil marker) and Ly-6G (Gr-1, neutrophil and monocyte marker) positive cells. Very weak co-localization was observed for CD11c (dendritic cell marker) positive cells (data not shown).
349
3.6. Cell culture study
350
From the data shown in Sections 3.4 and 3.5, it was obvious that the epithelial cells and macrophages were the major cell types responsible for the sequestration of P-Dex as evaluated by flow cytometry and immunohistochemistry.
337 338 339 340 341 342 343 344 345 346 347
351 352 353
N C O
336
U
335
R
333
The internalization and subcellular trafficking of P-DexAlexa of these cells were further validated in Caco-2 epithelial cells and J774 macrophages cultures. Both Caco-2 and J774 cells internalized P-Dex-Alexa as shown in Fig. 7. The internalized conjugate co-localized with the lysosome marker LAMP-1 in both cell lines, suggesting that P-Dex-Alexa was processed by an endocytic pathway that resulted in sequestration in the lysosomal compartment, where P-Dex would be activated in the acidic environment, leading to the gradual release of Dex and sustained amelioration of inflammation [24,25].
354
4. Discussion
365
Clinically recommended treatments for IBD include 5-aminosalicylates, GC, immunosuppressive agents, biologic agents etc. GC is one of the most commonly used therapies for treating IBD. Although the exact anti-inflammatory and immunosuppressive mechanism of GC are still unknown, studies have proven that majority of the effects of GC are mediated through the binding of the glucocorticoid receptor as a homodimer to specific DNA sequences (GC response elements). The transcription of inflammatory proteins by NF-κB and activator protein 1 are blocked and the expression of anti-inflammatory proteins such as IκB, annexin I, and MAPK phosphatase I are induced [26,27], resulting in a variety of anti-inflammatory effects such as inhibition of the
366
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
355 356 357 358 359 360 361 362 363 364
367 368 369 370 371 372 373 374 375 376 377 378
7
T
E
D
P
R O
O
F
Potential of P-Dex to improve the clinical management of IBD
382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402
R
381
recruitment and proliferation of lymphocytes, monocytes and macrophages, migration of neutrophils to sites of inflammation, and decreased production of inflammatory mediators including cytokines, leukotrienes, and prostaglandins [28–30]. The therapeutic efficacy of a drug depends on its intrinsic specificity for the molecular target and its concentration at the target site. Though GC is a class of very potent anti-inflammatory drugs, the toxicity induced by universal distribution of GC in off-target tissue after systemic administration limit the long-term use of GC. The developing of targeted delivery system may partially address the problems related to the inability of controlling the in vivo drug concentration at the intended site of action and off-target sites. Due to the leaky vasculature at the inflammatory site [19], P-Dex would passively target to these lesions. Compared with other normal tissues, optical imaging showed obvious near infrared signal in the inflamed colon in DSS fed mice. The data indicate the preferential accumulation of the prodrug to the inflamed tissue. The prodrug system alternates the tissue distribution of the parent drug by increasing the drug concentration at the intended disease site. Compared with free drug without targeting ability, more prodrug would distribute to the
N C O
380
U
379
R
E
C
Figure 5 Representative data from fluorescence-activated cell sorting analysis of cells isolated from colon. A: EPCam; B: 7/4; C: Ly-6G; and D: Ly-6B. The histogram plots show the intensity of staining with the specific antibodies (purple fills) with isotype control antibodies (black lines) on the same plots. EPCam (epithelial cell marker) positive cells were the major cell type responsible for the retention of P-Dex in the inflammatory site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
inflammatory tissue. The prodrug has superior therapeutic efficacy to the free drug even at one-fourth of the equivalent dose. In our previous studies, we have proved that prodrug could effectively avert the typical side effects (e.g. osteopenia) associated with GC treatment in two other inflammatory disease models after long-term treatment [17,18]. In the present study, P-Dex demonstrated greater therapeutic benefits even at reduced dosing level in DSS induced colitis animal model. The preferential accumulation of P-Dex to the inflammatory lesion may significantly decrease the side effect of Dex. Future studies using chronic IBD models are necessary to establish the long-term safety profile of P-Dex in management of IBD. Optical imaging also showed the strong signal in the inflamed colon in DSS group remained three days after prodrug administration, while there was almost no signal in colon in health mice. The sustained presence of the near infrared signal in colon in the DSS group after P-Dex-IRDye injection validates the retention of prodrug at the inflammatory lesion. FACS and immunohistochemical staining of the inflamed colon tissues showed epithelial cells and macrophages were the major cell types responsible for the internalization and local retention of the macromolecular prodrug. Cell culture study utilizing Alexa Flour® 488
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426
K. Ren et al.
C
T
E
D
P
R O
O
F
8
U
N C O
R
R
E
Figure 6 Representative confocal images of anti-EpCAM (A), anti-7/4 (B), anti-Ly-6G (Gr-1, Gr1) (C) and anti-F4/80 (D) antibody stained sections of the colon from DSS administrated mice. Each panel was composed of four subimages: antibody red staining, P-Dex-Alexa green fluorescence, DAPI blue staining and the colocalization of the three. The colocalization of red and green color in both panels yielded a yellow color, which confirmed the internalization of the HPMA copolymer conjugate by EpCAM, F4/80, Ly-6G (Gr-1, Gr1) or 7/4 positive cells at the sites of inflammation. Bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Figure 7 Representative confocal fluorescence microscopy of J774 and Caco-2 cells stained with LAMP-1 antibody (red, lysosome marker), and P-Dex-Alexa conjugate (green). The nucleus was stained with DAPI (blue). Co-localization (yellow) of P-Dex-Alexa conjugate and lysosome marker was observed, indicating the internalization of HPMA copolymer into the lysosome department of cells. Bars = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
Potential of P-Dex to improve the clinical management of IBD
440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488
5. Conclusions
524
We have evaluated a macromolecular prodrug of Dexamethasone based on HPMA copolymers in DSS-induced mouse colitis model. The prodrug has superior therapeutic efficacy compared to the free drug even at one-fourth of the equivalent dose. Mechanistic studies indicate that the passive targeting and cell-mediated local sequestration and retention by epithelial cells and inflammatory cells likely contribute to its superior and sustained therapeutic efficacy. The reduced Dex dose may have the benefit of improving long-term safety profile of the drug in the clinical management of IBD. Given the fact that GCs are still one of the first line treatments for IBD, and the toxicity of GCs is one of the major causes of iatrogenic illness associated with chronic inflammatory disease, the validation of the P-Dex prodrug's superior efficacy and its potential for improved safety are highly desirable for clinical management of IBD patients.
525
Conflict of interest statement
542
The authors declare that there are no conflicts of interest.
543
F
439
O
438
R O
437
P
436
D
435
489
E
434
[40]. It is hypothesized that the exposure of the luminal content to the underlying mucosal immune system due to increased permeability leads to an aberrant autoimmune response [41]. In addition, by forming nano-lipocomplexes with medium-chain-length fatty acid in colon, DSS activates intestinal inflammatory signaling pathway [41]. Cooper et al. investigated the clinical and histopathological of DSS induced acute colitis in Swiss-Webster mice [31]. They observed crypt damage without any inflammatory cell infiltration at the early stage of experiment. Inflammation became statistically significant after erosion appeared, which was on day 4. Therefore, our treatments were started on day 5 when there are inflammatory cell infiltration in the mucosa and submucosa. Although DSS induced colitis model represents many pathological features of IBD, the inflammation is generally limited to colon. Therefore, DSS induced colitis model is interpreting as a model for ulcerative colitis. In addition, DSS is directly toxic to epithelial cells of the basal crypts and affects the integrity of the mucosal barrier. T-cell and B-cell deficient Rag1−/− or C.B-17scid mice also developed severe colitis, indicating the adaptive immune system does not play a major role (at least in the acute phase) in DSS induced colitis mice model [38,42]. Due to the intrinsic limitations of DSS induced colitis model, efficacy and safety of P-Dex prodrug will be further investigated in other IBD models, including TNBS induced colitis model, T cell transfer model of colitis and interleukin-10 deficient mouse model [38,43,44]. As these animal models could recapitulate certain aspect of the IBD pathology, further evaluation of P-Dex in these models would confirm the translational potential of P-Dex for clinical management of IBD. Different from the DSS induced acute colitis, evaluation of P-Dex in a chronic IBD model would also permit the proper evaluation of P-Dex' potential in reducing GC side effects.
T
433
C
432
E
431
R
430
R
429
labeled P-Dex and immunohistochemical staining with the lysosomal marker LAMP-1 confirmed the co-localization of the prodrug in a lysosomal compartment, which is in consistent with internalization via an endocytic pathway. After internalized by the cells, water-soluble P-Dex is restricted by the lysosomal membrane from escaping the endosome/lysosome compartments. P-Dex is exposed to an acidic environment (pH ~ 5.5) within these subcellular vesicles. Since Dex is conjugated to the HPMA copolymer via an acid-labile hydrazone bond, P-Dex is subject to gradual hydrolysis and subsequent release of the active drug [15,24]. The intracellular prodrug activation and the presence of free Dex was confirmed by the capacity of the internalized P-Dex to inhibit the release of TNF-α and IL-6 from LPS-treated macrophages [24]. Therefore, we speculate that the sustained therapeutic effect observed in the P-Dex treatment is due to the prolonged retention of P-Dex within local inflammatory cells and their gradual low pH-triggered activation within lysosomes, followed by the release of free Dex into the cytosol and extracellular space. The sequestration of the P-Dex by local activated epithelial cells and inflammatory cells and the sustained activation/release of Dex from these cells explain why one single injection could achieve a sustained therapeutic effect. Body weight is an important parameter monitoring disease activity in mice [31,32]. Weight loss was observed in all the DSS treated groups. The animals treated with free Dex and P-Dex had more weight losses than mice in PBS group. As weight change is an important indicator of severity of colitis [31,32], Dex treatments seem to exacerbate the disease. We monitored the body weight of health animals with normal water intake. Interestingly, for health mice with daily free Dex injection (5 mg/kg total dosage) for five days, there were also gradual body weight losses. At the end of the 5th day, ~ 4% weight losses were observed. This result suggests that Dex itself could induce body weight loss, which is in agreement with literature report that GC would inhibit young animals' growth [33–35]. Therefore, though the animal body weight change is a frequently used parameter for the evaluation of IBD progression and IBD treatment efficacy, it was not used in the present study due to the intrinsic weight loss side effect of Dex treatment. The reduced weight losses in P-Dex treated groups when compared with free Dex groups might be considered as a sign of decreased side effects. It may also be explained by the decreased Dex dosage. Many chemicals are used to induce colitis in animal models for IBD research, including DSS, 2,4,6-trinitrobenzenesulfonic acid (TNBS), oxazolone and acetic acid [36–38]. Among them, DSS induced colitis model is widely used for the purpose of evaluating novel therapeutic agents. It is cost-efficiency and highly reproducible. Feeding mice for several days with DSS polymers in the drinking water induce an inflammatory response in the colon that mimics human ulcerative colitis. It is characterized by bloody diarrhea, inflammatory cells infiltration, body weight loss and elevated colonic ROS productions [36,39,40]. DSS is toxic to gut epithelial cells of the basal crypts and therefore affects the integrity of the mucosal barrier [38]. In DSS-induced murine colitis model, decreased expression of tight junction proteins and increased intestinal permeability were observed in the inflamed mucosa
N C O
428
U
427
9
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523
526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541
10
K. Ren et al.
547 Q4
This study was supported in part by a UNMC graduate student fellowship (KR), a China Scholar Council scholarship (KR) and the UNMC College of Pharmacy.
548
References
549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606
[1] P.L. Lakatos, Recent trends in the epidemiology of inflammatory bowel diseases: up or down? World J. Gastroenterol. 12 (38) (2006) 6102–6108. [2] J. Cosnes, C. Gower-Rousseau, P. Seksik, A. Cortot, Epidemiology and natural history of inflammatory bowel diseases, Gastroenterology 140 (6) (2011) 1785–1794. [3] R.B. Sartor, Mechanisms of disease: pathogenesis of Crohn's disease and ulcerative colitis, Nat. Clin. Pract. Gastroenterol. Hepatol. 3 (7) (2006) 390–407. [4] C. Fiocchi, Inflammatory bowel disease: evolutionary concepts in biology, epidemiology, mechanisms and therapy, Curr. Opin. Gastroenterol. 29 (4) (2013) 347–349. [5] C. Abraham, J.H. Cho, Inflammatory bowel disease, N. Engl. J. Med. 361 (21) (2009) 2066–2078. [6] D.K. Podolsky, Inflammatory bowel disease, N. Engl. J. Med. 347 (6) (2002) 417–429. [7] S. Melgar, F. Shanahan, Inflammatory bowel disease—from mechanisms to treatment strategies, Autoimmunity 43 (7) (2010) 463–477. [8] G.R. Lichtenstein, M.T. Abreu, R. Cohen, W. Tremaine, American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease, Gastroenterology 130 (3) (2006) 940–987. [9] A.A. Sohrabpour, R. Malekzadeh, A. Keshavarzian, Current therapeutic approaches in inflammatory bowel disease, Curr. Pharm. Des. 16 (33) (2010) 3668–3683. [10] J.A. Katz, Treatment of inflammatory bowel disease with corticosteroids, Gastroenterol. Clin. N. Am. 33 (2) (2004) 171–189 (vii). [11] E.I. Benchimol, C.H. Seow, A.H. Steinhart, A.M. Griffiths, Traditional corticosteroids for induction of remission in Crohn's disease, Cochrane Database Syst. Rev. (2) (2008) CD006792. [12] S. Moghadam-Kia, V.P. Werth, Prevention and treatment of systemic glucocorticoid side effects, Int. J. Dermatol. 49 (3) (2010) 239–248. [13] H. Schacke, W.D. Docke, K. Asadullah, Mechanisms involved in the side effects of glucocorticoids, Pharmacol. Ther. 96 (1) (2002) 23–43. [14] X.M. Liu, L.D. Quan, J. Tian, Y. Alnouti, K. Fu, G.M. Thiele, et al., Synthesis and evaluation of a well-defined HPMA copolymer–dexamethasone conjugate for effective treatment of rheumatoid arthritis, Pharm. Res. 25 (12) (2008) 2910–2919. [15] D. Wang, S.C. Miller, X.M. Liu, B. Anderson, X.S. Wang, S.R. Goldring, Novel dexamethasone–HPMA copolymer conjugate and its potential application in treatment of rheumatoid arthritis, Arthritis Res. Ther. 9 (1) (2007) R2. [16] K. Ren, P.E. Purdue, L. Burton, L.D. Quan, E.V. Fehringer, G.M. Thiele, et al., Early detection and treatment of wear particleinduced inflammation and bone loss in a mouse calvarial osteolysis model using HPMA copolymer conjugates, Mol. Pharm. 8 (4) (2011) 1043–1051. [17] F. Yuan, R.K. Nelson, D.E. Tabor, Y. Zhang, M.P. Akhter, K.A. Gould, et al., Dexamethasone prodrug treatment prevents nephritis in lupus-prone (NZB × NZW)F1 mice without causing systemic side effects, Arthritis Rheum. 64 (12) (2012) 4029–4039. [18] K. Ren, A. Dusad, F. Yuan, H. Yuan, P.E. Purdue, E.V. Fehringer, et al., Macromolecular prodrug of dexamethasone
[20]
[21]
[22]
O
[23]
[24]
D
[25]
E
[26]
[27]
T
C
E
R
R
N C O
U
546
[19]
F
545
prevents particle-induced peri-implant osteolysis with reduced systemic side effects, J. Control. Release 175C (2013) 1–9. P.M. Henson, Dampening inflammation, Nat. Immunol. 6 (12) (2005) 1179–1181. Y. Nishiyama, T. Kataoka, K. Yamato, T. Taguchi, K. Yamaoka, Suppression of dextran sulfate sodium-induced colitis in mice by radon inhalation, Mediat. Inflamm. 2012 (2012) 239617. K. Tanaka, T. Namba, Y. Arai, M. Fujimoto, H. Adachi, G. Sobue, et al., Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis, J. Biol. Chem. 282 (32) (2007) 23240–23252. C.J. Kim, J. Kovacs-Nolan, C. Yang, T. Archbold, M.Z. Fan, Y. Mine, L-cysteine supplementation attenuates local inflammation and restores gut homeostasis in a porcine model of colitis, Biochim. Biophys. Acta 1790 (10) (2009) 1161–1169. L.A. Dieleman, M.J. Palmen, H. Akol, E. Bloemena, A.S. Pena, S.G. Meuwissen, et al., Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines, Clin. Exp. Immunol. 114 (3) (1998) 385–391. L.D. Quan, P.E. Purdue, X.M. Liu, M.D. Boska, S.M. Lele, G.M. Thiele, et al., Development of a macromolecular prodrug for the treatment of inflammatory arthritis: mechanisms involved in arthrotropism and sustained therapeutic efficacy, Arthritis Res. Ther. 12 (5) (2010) R170. K. Ren, A. Dusad, F. Yuan, H. Yuan, P.E. Purdue, E.V. Fehringer, et al., Macromolecular prodrug of dexamethasone prevents particle-induced peri-implant osteolysis with reduced systemic side effects, J. Control. Release 175 (2014) 1–9. R.I. Scheinman, P.C. Cogswell, A.K. Lofquist, A.S. Baldwin Jr., Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids, Science 270 (5234) (1995) 283–286. Y. Tao, C. Williams-Skipp, R.I. Scheinman, Mapping of glucocorticoid receptor DNA binding domain surfaces contributing to transrepression of NF-kappa B and induction of apoptosis, J. Biol. Chem. 276 (4) (2001) 2329–2332. P.J. Barnes, Glucocorticosteroids: current and future directions, Br. J. Pharmacol. 163 (1) (2011) 29–43. T. Rhen, J.A. Cidlowski, Antiinflammatory action of glucocorticoids—new mechanisms for old drugs, N. Engl. J. Med. 353 (16) (2005) 1711–1723. M.J.R. Nikhil Singh, M. Jane Tucker, Mechanisms of glucocorticoid-mediated antiinflammatory and immunosuppressive action, Paediatr. Perinat. Drug Ther. 6 (2) (2004) 107–115. H.S. Cooper, S.N. Murthy, R.S. Shah, D.J. Sedergran, Clinicopathologic study of dextran sulfate sodium experimental murine colitis, Lab. Invest. 69 (2) (1993) 238–249. O.H. Kang, D.K. Kim, Y.A. Choi, H.J. Park, J. Tae, C.S. Kang, et al., Suppressive effect of non-anaphylactogenic anti-IgE antibody on the development of dextran sulfate sodiuminduced colitis, Int. J. Mol. Med. 18 (5) (2006) 893–899. M. Silbermann, U. Kleinhaus, E. Livne, T. Kedar, Retardation of bone growth in triamcinolone-treated mice, J. Anat. 121 (Pt 3) (1976) 515–535. G. Ortoft, H. Gronbaek, H. Oxlund, Growth hormone administration can improve growth in glucocorticoid-injected rats without affecting the lymphocytopenic effect of the glucocorticoid, Growth Horm. IGF Res. 8 (3) (1998) 251–264. D.B. Allen, M. Mullen, B. Mullen, A meta-analysis of the effect of oral and inhaled corticosteroids on growth, J. Allergy Clin. Immunol. 93 (6) (1994) 967–976. S. Wirtz, C. Neufert, B. Weigmann, M.F. Neurath, Chemically induced mouse models of intestinal inflammation, Nat. Protoc. 2 (3) (2007) 541–546. M. Kawada, A. Arihiro, E. Mizoguchi, Insights from advances in research of chemically induced experimental models of human inflammatory bowel disease, World J. Gastroenterol. 13 (42) (2007) 5581–5593.
R O
Acknowledgments
P
544
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
Potential of P-Dex to improve the clinical management of IBD
chain-length fatty acids in the colon, PLoS One 7 (3) (2012) e32084. [42] L.A. Dieleman, B.U. Ridwan, G.S. Tennyson, K.W. Beagley, R.P. Bucy, C.O. Elson, Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice, Gastroenterology 107 (6) (1994) 1643–1652. [43] S.Y. Oh, K.A. Cho, J.L. Kang, K.H. Kim, S.Y. Woo, Comparison of experimental mouse models of inflammatory bowel disease, Int. J. Mol. Med. 33 (2) (2014) 333–340. [44] F.R. Byrne, J.L. Viney, Mouse models of inflammatory bowel disease, Curr. Opin. Drug Discov. Dev. 9 (2) (2006) 207–217.
N C O
R
R
E
C
T
E
D
P
R O
O
F
[38] S. Wirtz, M.F. Neurath, Mouse models of inflammatory bowel disease, Adv. Drug Deliv. Rev. 59 (11) (2007) 1073–1083. [39] I. Okayasu, S. Hatakeyama, M. Yamada, T. Ohkusa, Y. Inagaki, R. Nakaya, A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice, Gastroenterology 98 (3) (1990) 694–702. [40] M. Mahler, I.J. Bristol, E.H. Leiter, A.E. Workman, E.H. Birkenmeier, C.O. Elson, et al., Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis, Am. J. Physiol. 274 (3 Pt 1) (1998) G544–G551. [41] H. Laroui, S.A. Ingersoll, H.C. Liu, M.T. Baker, S. Ayyadurai, M.A. Charania, et al., Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-
U
675 676 677 678 679 680 681 682 683 684 685 686 687 700
11
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
688 689 690 691 692 693 694 695 696 697 698 699
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
4Q2
Ke Ren, Hongjiang Yuan, Yijia Zhang, Xin Wei, Dong Wang ⁎
5
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
O
R O
P
2
F
3
Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis
1Q1
7
Received 20 December 2014; accepted with revision 30 March 2015
8
13 14 15 16 17
E
R
R
18
Abstract A macromolecular prodrug (P-Dex) of dexamethasone (Dex) was developed to improve the treatment of inflammatory bowel disease (IBD). Colonic inflammation was induced by feeding mice with dextran sulfate sodium. Mice were treated with daily i.p. injection of free Dex or single i.v. injection of P-Dex, PBS or free polymer. Both P-Dex and free Dex could lower disease activity index and histology scores when compared to the controls. A single injection of P-Dex with 1/4 equivalent Dex dose had a better therapeutic effect than daily free Dex treatment. Mechanism study found that P-Dex could target the inflamed colon, and be retained by epithelial cells and local inflammatory infiltrates, suggesting that the improved efficacy of P-Dex may be attributed to its inflammation targeting, subcellular processing and activation. Collectively, these data support our hypothesis that the development of macromolecular prodrug of glucocorticoid may have the potential to improve the clinical management of IBD. © 2015 Elsevier Inc. All rights reserved.
Inflammatory bowel diseases; Dextran sulfate sodium; Dexamethasone; ELVIS; HPMA copolymer; Prodrug; Inflammation targeting
T
12
KEYWORDS
C
11
E
Q310
D
6
31 33
C
34
1. Introduction
36
Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder in the gastrointestinal tract. It is comprised of two types of chronic intestinal disorders: Crohn's disease and ulcerative colitis. There are approximately 1.4 million IBD patients in the United States and
39 40
U
38
N
35
37
20 21 22 23 24 25 26 27 28 29 30
O
32
19
⁎ Corresponding author at: University of Nebraska Medical Center, 986025 Nebraska Medical Center, COP 3026, Omaha, Nebraska 68198-6025, USA. Fax: + 1 402 559 9543. E-mail address: [email protected] (D. Wang).
its peak onset is in persons 15 to 30 years of age [1,2]. The annual total estimated expenditures for the treatment of IBD in US alone is $2.8 billion, making it one of the most expensive gastrointestinal tract disorders to treat [3,4]. The common symptoms of IBD include diarrhea, rectal bleeding, abdominal pain, fever and weight loss. In addition to these painful and often debilitating symptoms, individuals with IBD may experience other complications including bowel obstruction, ulcers, and malnutrition. The pathogenesis of IBD remains elusive and multifactorial. Accumulating evidences suggest that IBD may result from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host [5,6].
http://dx.doi.org/10.1016/j.clim.2015.03.027 1521-6616/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
41 42 43 44 45 46 47 48 49 50 51 52 53
2
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111
Macromolecular prodrug of dexamethasone (P-Dex) was synthesized by conjugating Dex to N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer via an acid labile hydrazone bond as previously described [14]. The weight average molecular weight was determined to be 3.68 × 104 with a PDI of 1.36 using an ÄKTA FPLC system (GE HealthCare, Piscataway, NJ) equipped with UV and RI detectors. After being fully hydrolyzed by HCl (0.1 M), the Dex content in P-Dex was determined to be 140 mg/g of polymer using high performance liquid chromatography. Alexa Flour® 488 labeled P-Dex (P-Dex-Alexa) and IRDye® 800 CW labeled P-Dex (P-Dex-IRDye) were obtained via polymer analogous reaction between APMA-containing P-Dex and NHS esters of the dyes. The Alexa Flour® 488 and IRDye® 800 CW contents in the conjugate were determined as 2.0 × 10−5 mol/g and 1.1 × 10− 5 mol/g, respectively, using a Lambda 10 UV/vis spectrometer.
2.3. Histological evaluation
141
After fixation, the colons were paraffin embedded and sectioned (5 μm thickness). The sections were stained with hematoxylin and eosin (H&E) for light microscopy (Olympus BX51 microscopy, Olympus, Japan) observation and graded for inflammation, extent of inflammation, regeneration, crypt damage and percent involvement by two investigators blind to the group design. The histological grading criteria were shown in Table 1. The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement to yield the final score (0–12 for inflammation and extent of inflammation, 0–16 for regeneration and crypt damage) [22,23].
142
2.4. Optical imaging analysis of the targeting of P-Dex to intestinal inflammation
154
Twenty mice were used for the optical imaging study. They were evenly divided into two groups: the health control group with normal water intake and the DSS group with 3% DSS in drinking water. On day 6, mice were given with P-Dex-IRDye (0.3 mg/mice, IRDye content 1.1 × 10− 5 mol/g) by i.v. injection. They were sacrificed one day or three days post injection and the isolated major organs were imagined using Pearl® Impulse small animal imaging system (LI-COR, Lincoln, NE) to evaluate the biodistribution of the IRDyelabeled P-Dex prodrug.
156
F
66
C
65
E
64
R
63
R
62
N C O
61
U
60
114
O
2.1. Synthesis and characterization of HPMA copolymer conjugates
59
Forty-two male Swiss Webster mice were randomly assigned into six groups. Group 1 was designated as health control group with normal water intake and no treatment. Groups 2–6 were given water containing 3% (w/v) dextran sulfate sodium salt (DSS, Mw 40,000; Alfa Aesar, Ward Hill, MA) for 10 days. Starting from the 6th day, the animals were given the following treatments: group 2, PBS (daily i.v. injection, five injections in total); group 3, HPMA homopolymer without Dex (PHPMA, the amount of polymer used was equivalent to that in P-Dex 2.5 mg/kg group, one i.v. injection on the 6th day); group 4, P-Dex (equivalent Dex dose = 2.5 mg/kg, one i.v. injection on the 6th day); group 5, P-Dex (equivalent Dex dose = 1.25 mg/kg, one i.v. injection on the 6th day); group 6, free Dex (total dose = 5 mg/kg, daily i.v. injections for 5 days). Body weight, stool consistency and hemoccult positivity were examined daily for all the animals. The disease activity index (DAI) scores, an indicator of the severity of colitis, were determined based on a standard scoring system as previously described in literatures [20,21]. The DAI scoring was performed using a 0–3 scale, where for diarrheal score, 0 = normal stool, 1 = mild soft stool, 2 = very soft stool, 3 = watery stool; for blood score, 0 = normal colored stool, 1 = brown stool, 2 = reddish stool, 3 = bloody stool. The mice were euthanized on the 10th day. The colons were removed and fixed with 4% paraformaldehyde in PBS.
R O
93
58
112
P
2. Material and methods
57
2.2. Establishment of a DSS induced colitis model and treatment
D
92
56
T
91
Immunosuppressant, anti-inflammatory and steroid-based medications are the typical treatment options for IBD [7,8]. They are used to control flare ups, maintain remission, prevent repeated attacks and help the colon and intestinal tract heal [9]. Glucocorticoids (GC) are highly effective in reducing inflammation and are widely used to induce remission in patients with active IBD [10,11]. Although the benefits of GC treatment has been widely proven in treating the disease, prolonged or high-dose GC therapy, however, is associated with significant side effects, such as hypertension, osteoporosis, immunodeficiency and adrenal gland atrophy [12,13]. In order to further potentiate the therapeutic efficacy and dampen the off-target toxicities associated with GC, we have developed a macromolecular prodrug of dexamethasone (Dex) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer (P-Dex) [14]. The prodrug P-Dex has been found to passively target and be retained at the sites of inflammation due to its extravasation through leaky vasculature and inflammatory cell-mediated sequestration (ELVIS) mechanism, providing sustained inflammation amelioration (N 1 month/injection). This observation has been validated in multiple inflammatory disease animal models, including inflammatory arthritis, lupus nephritis and aseptic implant loosening [15–17]. In addition to its superior therapeutic efficacy, it was also found that the prodrug could effectively avert the typical side effects associated with GC treatment [17,18]. As a chronic inflammatory disorder, IBD shares the general features of inflammation, such as angiogenesis and inflammatory cell infiltration, which constitute the pathological components of ELVIS mechanism [19]. Therefore, we hypothesized that the successful application of P-Dex in treatment of these inflammatory conditions may be extrapolated to IBD. Given the fact that GCs are still one of the first line treatments for IBD and they are often given in the hospital setting as i.v. infusion, the validation of the P-Dex' superior efficacy and safety in IBD models will be clinically relevant and highly translational.
55
E
54
K. Ren et al.
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
113
115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
143 144 145 146 147 148 149 150 151 152 153
155
157 158 159 160 161 162 163 164 165
Potential of P-Dex to improve the clinical management of IBD
Inflammation
0 1 2 3 0 1 2 3 0
t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25
Extent
Regeneration
Crypt damage
Percent involvement
1 2 3 4 0 1 2 3 4 1 2 3 4
None Slight Moderate Severe None Mucosa Mucosa and submucosa Transmural Complete regeneration or normal tissue Almost complete regeneration Regeneration with crypt depletion Surface epithelium not intact No tissue repair None Basal 1/3 damage Basal 2/3 damage Only surface epithelium lost Entire crypt and epithelium lost 1–25% 26–50% 51–75% 76–100%
F
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12
O
Score Histological features
colons were collected at 24 h post injection. They were fixed, paraffin embedded and sectioned (5 μm thickness). The slides obtained were first incubated with 10% goat serum (Sigma-Aldrich) for 30 min at room temperature. After addition of the primary antibodies [rat anti-mouse F4/ 80 (Invitrogen, Camarillo, CA), rat anti-mouse EpCAM (Ebioscience, San Diego, CA), rat anti-mouse Ly-6G (Gr-1) (EBioscience), rat anti-mouse Ly-6B (7/4) (AbD Serotec), Armenian hamster anti-mouse CD11c (AbD Serotec) and rabbit anti-mouse prolyl-4-hydroxylase (P4HB, Abcam, Cambridge, MA)], the sections were incubated at room temperature for 1 h in a humidified chamber. After washing with PBS, diluted secondary antibodies [Alexa Flour® 647 labeled goat anti-rat IgG (Invitrogen, Carlsbad, CA), Alexa Flour® 647 labeled goat anti-Armenian hamster IgG (Jackson ImmunoResearch, West Grove, PA) or Alexa Flour® 647 labeled donkey anti-rabbit IgG (Invitrogen)] were added and incubated for another 30 min at room temperature in darkness. In control experiments, primary antibodies were replaced by corresponding isotype controls [purified rabbit IgG (Sigma-Aldrich), purified rat IgG (Sigma-Aldrich) and purified hamster IgG (Jackson ImmunoResearch)], and the samples were processed similarly as described above. The tissue sections were then evaluated with a Nikon Swept Field confocal microscope (Nikon Instruments Inc., Melville, NY).
R O
Parameters
Histological grading criteria.
P
Table 1
D
t1:1 t1:2 t1:3
3
171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
2.6. Immunohistochemistry analysis of P-Dex' retention at intestinal inflammation Ten mice were either fed with normal water or 3% DSS. P-Dex-Alexa (5.0 mg/mice) was given to mice by tail vein injection on day 6. The animals were sacrificed and the
196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
For subcellular localization studies, mouse macrophage cell line (J774, ATCC, VA) and human epithelial cell line (Caco-2, ATCC, Manassas, VA) were cultured on 13 mm diameter coverslips (Thermo Fisher scientific, Waltham, MA) placed on tissue culture Petri dishes (Becton, Dickinson and Company, Franklin Lakes, NJ). DMEM medium (ATCC) supplemented with 10% fetal bovine serum (VWR, Radnor, PA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) was used. The cells were allowed to adhere for 24 h and then activated with lipopolysaccharide (LPS, 1 mg/mL) for 24 h. After activation, cells were treated with Alexa Flour® 488-labeled P-Dex (1 mg/mL) in the presence of LPS (500 ng/mL) for 24 h. Cells were fixed with 4% paraformaldehyde in PBS, rinsed and then lysed with 0.1% Triton X-100. After addition of the primary antibodies [rat anti-mouse LAMP-1 (EBioscience) for J774 cells, rabbit anti-human LAMP-1 (Santa Cruz, Dallas, Texas) for Caco-2 cells], the slides were incubated at 4 °C overnight in a humidified chamber. The next day, after washing, diluted secondary antibodies [Alexa Flour® 647 labeled goat anti-rat IgG (Invitrogen) or Alexa Flour® 647 labeled goat anti-rabbit IgG (Santa Cruz)] were added and incubated for 60 min at room temperature in darkness. After rinsing, cells were stained with DAPI, fixed, mounted and observed using confocal microscope. In control experiments, primary antibodies were replaced with corresponding isotype controls [purified rabbit IgG (Sigma-Aldrich), purified rat IgG (Sigma-Aldrich)]. The samples were processed similarly as described above.
222
2.8. Statistical analysis
250
Comparisons were made by one-way ANOVA followed by a post hoc Tukey’s test for multiple comparisons using SPSS
251
T
C
E
170
R
169
Thirty mice were either fed with normal water or 3% DSS. Alexa Flour® 488-labeled P-Dex (P-Dex-Alexa, 5.0 mg/mice) was given to mice by tail vein injection on day 6. At necropsy (24 h post injection), the colons were isolated and minced aseptically. The tissues were further digested with collagenase type II (1 mg/mL, Sigma-Aldrich, St. Louis, MO) at 37 °C for 30 min. After passing through a 70 μm cell strainer, a single cell suspension was obtained. The red blood cells were removed by ACK lysing buffer (Quality Biological, Gaithersburg, MD). For fluorescence-activated cell sorting (FACS) evaluation of F4/80, EpCAM, CD11c, Ly-6G (Gr-1) and Ly-6B (7/4) positive cells, the samples were incubated with antibodies [Allophycocyanin (APC)-labeled rat anti-mouse F4/80 (AbD Serotec, Raleigh, NC), APC-labeled hamster anti-mouse CD11c (BD Pharmingen, San Jose, CA), Phycoerythrin (PE) labeled rat anti-mouse EpCAM (Ebioscience, San Diego, CA), PE-labeled rat anti-mouse Ly-6B (7/4) (AbD Serotec) and PE-labeled rat anti-mouse Ly-6G (Gr-1) (Ebioscience)] for 30 min on ice. Isotype-matched APC-labeled hamster IgG1 (BD Biosciences, San Jose, CA), APC-labeled rat IgG2a (Ebioscience), APC-labeled rat IgG2b (BD Pharmingen) and purified rabbit IgG (Ebioscience) were used as negative controls. After the final wash, the cells were analyzed with FACSCalibur flow cytometer (BD, Franklin Lakes, NJ).
R
168
2.5. Analysis of P-Dex' retention mechanism at intestinal inflammation using flow cytometry
N C O
167
U
166
E
2.7. Cells culture study
Q5
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
252
4
K. Ren et al. 256
3.1. Disease activity evaluation
257
The clinical symptoms of rectal bleeding and loose stools were observed on day 5 for mice treated with DSS. As shown in Fig. 1, DAI was 0 in the health control group on day 10, indicating no disease symptom (diarrhea and blood) were observed. In contrast, the DAI scores were increased in all DSS treated groups. There were significantly lower DAI scores in both P-Dex treated groups when compared with PBS and PHPMA treated groups. High dose (2.5 mg/kg) P-Dex treatment had a significantly better therapeutic effect than the free Dex treated group (5 mg/kg) (P b 0.05). There was ~ 3% weight increase for health control animals at day 10. In contrast, weight losses were observed in all the DSS treated groups. The mice treated with free Dex (5 mg/kg) had the most weight loss, with ~9% on the 10th day. Animal treated with PBS and PHPMA had the least weight loss (~5%). The weight losses of P-Dex treated groups were between PBS/PHPMA and free Dex groups.
258
R O
O
F
3. Results
P
Figure 1 The DAI score of each group on day 10. The DAI in PBS and PHPMA groups were significantly higher than the DAI in both P-Dex groups. P-Dex high dose group has a significantly lower DAI than free Dex group. *, P b 0.05.
R
E
C
T
E
The severity of colonic inflammation and the effects of drugs on the recovery from DSS induced colitis were evaluated by examining H&E stained colon sections. Normal colonic tissue
R
255
software. Data were expressed as means ± standard deviation. A value of P b 0.05 was considered to be statistically significant.
N C O
254
U
253
D
3.2. Histology evaluation
Figure 2 Representative colon H&E staining images. Group A represented health control mice with normal water uptake. Mice in B–F groups were fed with 3% DSS in drinking water. B: PBS treated group; C: PHPMA treated group; D: P-Dex (0.5 mg/kg) treated group; E: P-Dex (0.25 mg/kg) treated group; F: free Dex (1 mg/kg) treated group. Crypt damages and inflammatory cell infiltration were found in DSS treated groups (B–F), especially in PBS (B) and PHPMA (C) treated groups. Both P-Dex groups (D&E) exhibited relatively better crypt structure and less inflammation when compared with the other groups (B&C&F). Bars = 100 um. Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274
275 276 277 278
Potential of P-Dex to improve the clinical management of IBD
The optical imaging results are shown in Fig. 4. One day after injection, there were signals in all the major organs in both groups, especially in the liver and kidneys. In addition, obvious accumulation of P-Dex-IRDye signal in the inflamed
292 293 294 295 296 297 298
302 303 304
314
Around 8% of total cells isolated from the colon 24 h after systemic administration of P-Dex-Alexa tested positive of the prodrug. The P-Dex-Alexa positive cells were further analyzed. Representative data from fluorescence-activated cell sorting (FACS) analysis were shown in Fig. 5. The histogram plots showed the intensity of staining with the specific antibodies designated on the x-axis (purple fills) with isotype control antibodies (black lines) on the same plots. The percentages represented the amount of antibody positive cells among P-Dex-Alexa positive cells: (A) ~ 76% P-Dex-Alexa positive cells were EPCam (epithelial cell marker) positive; (B) ~ 3% P-Dex-Alexa positive cells were 7/4 (neutrophil marker) positive; (C) ~ 5% P-Dex-Alexa positive cells were Ly-6G (neutrophil and monocyte marker) positive; (D) ~ 7% P-Dex-Alexa positive cells were F4/80 (macrophage marker) positive; b 1% P-Dex-Alexa positive
315
D
291
3.4. Flow cytometry analysis
T
290
C
289
E
288
R
287
R
286
N C O
285
U
284
F
301
283
O
3.3. Optical imaging
282
305
R O
300
281
colon was observed in DSS fed group. Three days after injection, there was almost no signal in the colon in health group while the strong signal in the inflamed colon in DSS group remained. In addition, compared with healthy small intestine, there was obvious accumulation of P-Dex-IRDye in the inflamed colon in DSS group. The sustained presence of near infrared signal in the colon in DSS group after P-Dex-IRDye injection validates the passive targeting and retention of P-Dex prodrug at the inflammatory lesions.
P
299
architecture is shown in Fig. 2A. Severe erosion, crypt abscesses and inflammatory cells infiltration were observed in mice with DSS water uptake and PBS treatment (Fig. 2B). The inflammation was mostly confined to the mucosa, and in some areas edema of the submucosa was observed. PHPMA treated group (Fig. 2C) showed no histological differences when compared to PBS group, while there were visible reductions in crypt damage and inflammatory cell infiltration in both P-Dex treated groups (Figs. 2D&E). There were obvious distorted crypt architecture and infiltration of inflammatory cells into the mucosa in free Dex treated group (Fig. 2F). The histology damage scores of the H&E slides were shown in Fig. 3 based on the grading criteria in Table 1. Oral administration of DSS for 10 days induced colitis. There were high scores for all the parameters in animals treated with PBS and PHPMA. After treating with P-Dex and free Dex, inflammation and crypt damages were significantly reduced when compared with PBS and PHPMA treatment. Both P-Dex groups (1.25 mg/kg and 2.5 mg/kg) had significantly lower scores than the free Dex (5 mg/kg) treated group.
280
E
279
5
Figure 3 Histology grading of colitis according to Table 1. A: Histologic colitis score (the sum of the scores in B, C and D); B: inflammation score; C: extent of inflammation score; and D: crypt damage/regeneration score. Both P-Dex treated groups showed significantly lower score than PBS, PHPMA and free Dex treated groups. *, P b 0.05. Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
306 307 308 309 310 311 312 313
316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
K. Ren et al.
E
D
P
R O
O
F
6
cells were CD11c (dendritic cell marker) positive (data not shown).
E
332
R
331
C
T
Figure 4 The distribution of P-Dex-IRDye in major organs by optical imaging. P-Dex-IRDye was given via tail vein injection on day 6 after DSS treatment. The mice were sacrificed 1 day or 3 days after the administration of the optical imaging conjugates. Compared to the health group, there were obvious accumulation and retention of the P-Dex-IRDye signals in the inflamed colons in DSS group.
3.5. Immunohistochemistry analysis
334
348
Immunohistochemical staining with specific cell markers was performed to identify the cell phenotypes within the inflamed colon that were responsible for the cellular sequestration and retention of P-Dex. As shown in Fig. 6, numerous P-Dex-Alexa positive cells (green fluorescence) were observed in the colon. Antibody stained positive cells were shown in red. Blue color from DAPI indicated cell nucleus. Co-localization with Alexa Flour® 647 labeled cell type markers indicated that among the cells that had sequestered P-Dex-Alexa, the majority were EPCam (epithelial cell marker), F4/80 (macrophage marker), Ly-6B (7/4, neutrophil marker) and Ly-6G (Gr-1, neutrophil and monocyte marker) positive cells. Very weak co-localization was observed for CD11c (dendritic cell marker) positive cells (data not shown).
349
3.6. Cell culture study
350
From the data shown in Sections 3.4 and 3.5, it was obvious that the epithelial cells and macrophages were the major cell types responsible for the sequestration of P-Dex as evaluated by flow cytometry and immunohistochemistry.
337 338 339 340 341 342 343 344 345 346 347
351 352 353
N C O
336
U
335
R
333
The internalization and subcellular trafficking of P-DexAlexa of these cells were further validated in Caco-2 epithelial cells and J774 macrophages cultures. Both Caco-2 and J774 cells internalized P-Dex-Alexa as shown in Fig. 7. The internalized conjugate co-localized with the lysosome marker LAMP-1 in both cell lines, suggesting that P-Dex-Alexa was processed by an endocytic pathway that resulted in sequestration in the lysosomal compartment, where P-Dex would be activated in the acidic environment, leading to the gradual release of Dex and sustained amelioration of inflammation [24,25].
354
4. Discussion
365
Clinically recommended treatments for IBD include 5-aminosalicylates, GC, immunosuppressive agents, biologic agents etc. GC is one of the most commonly used therapies for treating IBD. Although the exact anti-inflammatory and immunosuppressive mechanism of GC are still unknown, studies have proven that majority of the effects of GC are mediated through the binding of the glucocorticoid receptor as a homodimer to specific DNA sequences (GC response elements). The transcription of inflammatory proteins by NF-κB and activator protein 1 are blocked and the expression of anti-inflammatory proteins such as IκB, annexin I, and MAPK phosphatase I are induced [26,27], resulting in a variety of anti-inflammatory effects such as inhibition of the
366
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
355 356 357 358 359 360 361 362 363 364
367 368 369 370 371 372 373 374 375 376 377 378
7
T
E
D
P
R O
O
F
Potential of P-Dex to improve the clinical management of IBD
382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402
R
381
recruitment and proliferation of lymphocytes, monocytes and macrophages, migration of neutrophils to sites of inflammation, and decreased production of inflammatory mediators including cytokines, leukotrienes, and prostaglandins [28–30]. The therapeutic efficacy of a drug depends on its intrinsic specificity for the molecular target and its concentration at the target site. Though GC is a class of very potent anti-inflammatory drugs, the toxicity induced by universal distribution of GC in off-target tissue after systemic administration limit the long-term use of GC. The developing of targeted delivery system may partially address the problems related to the inability of controlling the in vivo drug concentration at the intended site of action and off-target sites. Due to the leaky vasculature at the inflammatory site [19], P-Dex would passively target to these lesions. Compared with other normal tissues, optical imaging showed obvious near infrared signal in the inflamed colon in DSS fed mice. The data indicate the preferential accumulation of the prodrug to the inflamed tissue. The prodrug system alternates the tissue distribution of the parent drug by increasing the drug concentration at the intended disease site. Compared with free drug without targeting ability, more prodrug would distribute to the
N C O
380
U
379
R
E
C
Figure 5 Representative data from fluorescence-activated cell sorting analysis of cells isolated from colon. A: EPCam; B: 7/4; C: Ly-6G; and D: Ly-6B. The histogram plots show the intensity of staining with the specific antibodies (purple fills) with isotype control antibodies (black lines) on the same plots. EPCam (epithelial cell marker) positive cells were the major cell type responsible for the retention of P-Dex in the inflammatory site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
inflammatory tissue. The prodrug has superior therapeutic efficacy to the free drug even at one-fourth of the equivalent dose. In our previous studies, we have proved that prodrug could effectively avert the typical side effects (e.g. osteopenia) associated with GC treatment in two other inflammatory disease models after long-term treatment [17,18]. In the present study, P-Dex demonstrated greater therapeutic benefits even at reduced dosing level in DSS induced colitis animal model. The preferential accumulation of P-Dex to the inflammatory lesion may significantly decrease the side effect of Dex. Future studies using chronic IBD models are necessary to establish the long-term safety profile of P-Dex in management of IBD. Optical imaging also showed the strong signal in the inflamed colon in DSS group remained three days after prodrug administration, while there was almost no signal in colon in health mice. The sustained presence of the near infrared signal in colon in the DSS group after P-Dex-IRDye injection validates the retention of prodrug at the inflammatory lesion. FACS and immunohistochemical staining of the inflamed colon tissues showed epithelial cells and macrophages were the major cell types responsible for the internalization and local retention of the macromolecular prodrug. Cell culture study utilizing Alexa Flour® 488
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426
K. Ren et al.
C
T
E
D
P
R O
O
F
8
U
N C O
R
R
E
Figure 6 Representative confocal images of anti-EpCAM (A), anti-7/4 (B), anti-Ly-6G (Gr-1, Gr1) (C) and anti-F4/80 (D) antibody stained sections of the colon from DSS administrated mice. Each panel was composed of four subimages: antibody red staining, P-Dex-Alexa green fluorescence, DAPI blue staining and the colocalization of the three. The colocalization of red and green color in both panels yielded a yellow color, which confirmed the internalization of the HPMA copolymer conjugate by EpCAM, F4/80, Ly-6G (Gr-1, Gr1) or 7/4 positive cells at the sites of inflammation. Bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Figure 7 Representative confocal fluorescence microscopy of J774 and Caco-2 cells stained with LAMP-1 antibody (red, lysosome marker), and P-Dex-Alexa conjugate (green). The nucleus was stained with DAPI (blue). Co-localization (yellow) of P-Dex-Alexa conjugate and lysosome marker was observed, indicating the internalization of HPMA copolymer into the lysosome department of cells. Bars = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
Potential of P-Dex to improve the clinical management of IBD
440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488
5. Conclusions
524
We have evaluated a macromolecular prodrug of Dexamethasone based on HPMA copolymers in DSS-induced mouse colitis model. The prodrug has superior therapeutic efficacy compared to the free drug even at one-fourth of the equivalent dose. Mechanistic studies indicate that the passive targeting and cell-mediated local sequestration and retention by epithelial cells and inflammatory cells likely contribute to its superior and sustained therapeutic efficacy. The reduced Dex dose may have the benefit of improving long-term safety profile of the drug in the clinical management of IBD. Given the fact that GCs are still one of the first line treatments for IBD, and the toxicity of GCs is one of the major causes of iatrogenic illness associated with chronic inflammatory disease, the validation of the P-Dex prodrug's superior efficacy and its potential for improved safety are highly desirable for clinical management of IBD patients.
525
Conflict of interest statement
542
The authors declare that there are no conflicts of interest.
543
F
439
O
438
R O
437
P
436
D
435
489
E
434
[40]. It is hypothesized that the exposure of the luminal content to the underlying mucosal immune system due to increased permeability leads to an aberrant autoimmune response [41]. In addition, by forming nano-lipocomplexes with medium-chain-length fatty acid in colon, DSS activates intestinal inflammatory signaling pathway [41]. Cooper et al. investigated the clinical and histopathological of DSS induced acute colitis in Swiss-Webster mice [31]. They observed crypt damage without any inflammatory cell infiltration at the early stage of experiment. Inflammation became statistically significant after erosion appeared, which was on day 4. Therefore, our treatments were started on day 5 when there are inflammatory cell infiltration in the mucosa and submucosa. Although DSS induced colitis model represents many pathological features of IBD, the inflammation is generally limited to colon. Therefore, DSS induced colitis model is interpreting as a model for ulcerative colitis. In addition, DSS is directly toxic to epithelial cells of the basal crypts and affects the integrity of the mucosal barrier. T-cell and B-cell deficient Rag1−/− or C.B-17scid mice also developed severe colitis, indicating the adaptive immune system does not play a major role (at least in the acute phase) in DSS induced colitis mice model [38,42]. Due to the intrinsic limitations of DSS induced colitis model, efficacy and safety of P-Dex prodrug will be further investigated in other IBD models, including TNBS induced colitis model, T cell transfer model of colitis and interleukin-10 deficient mouse model [38,43,44]. As these animal models could recapitulate certain aspect of the IBD pathology, further evaluation of P-Dex in these models would confirm the translational potential of P-Dex for clinical management of IBD. Different from the DSS induced acute colitis, evaluation of P-Dex in a chronic IBD model would also permit the proper evaluation of P-Dex' potential in reducing GC side effects.
T
433
C
432
E
431
R
430
R
429
labeled P-Dex and immunohistochemical staining with the lysosomal marker LAMP-1 confirmed the co-localization of the prodrug in a lysosomal compartment, which is in consistent with internalization via an endocytic pathway. After internalized by the cells, water-soluble P-Dex is restricted by the lysosomal membrane from escaping the endosome/lysosome compartments. P-Dex is exposed to an acidic environment (pH ~ 5.5) within these subcellular vesicles. Since Dex is conjugated to the HPMA copolymer via an acid-labile hydrazone bond, P-Dex is subject to gradual hydrolysis and subsequent release of the active drug [15,24]. The intracellular prodrug activation and the presence of free Dex was confirmed by the capacity of the internalized P-Dex to inhibit the release of TNF-α and IL-6 from LPS-treated macrophages [24]. Therefore, we speculate that the sustained therapeutic effect observed in the P-Dex treatment is due to the prolonged retention of P-Dex within local inflammatory cells and their gradual low pH-triggered activation within lysosomes, followed by the release of free Dex into the cytosol and extracellular space. The sequestration of the P-Dex by local activated epithelial cells and inflammatory cells and the sustained activation/release of Dex from these cells explain why one single injection could achieve a sustained therapeutic effect. Body weight is an important parameter monitoring disease activity in mice [31,32]. Weight loss was observed in all the DSS treated groups. The animals treated with free Dex and P-Dex had more weight losses than mice in PBS group. As weight change is an important indicator of severity of colitis [31,32], Dex treatments seem to exacerbate the disease. We monitored the body weight of health animals with normal water intake. Interestingly, for health mice with daily free Dex injection (5 mg/kg total dosage) for five days, there were also gradual body weight losses. At the end of the 5th day, ~ 4% weight losses were observed. This result suggests that Dex itself could induce body weight loss, which is in agreement with literature report that GC would inhibit young animals' growth [33–35]. Therefore, though the animal body weight change is a frequently used parameter for the evaluation of IBD progression and IBD treatment efficacy, it was not used in the present study due to the intrinsic weight loss side effect of Dex treatment. The reduced weight losses in P-Dex treated groups when compared with free Dex groups might be considered as a sign of decreased side effects. It may also be explained by the decreased Dex dosage. Many chemicals are used to induce colitis in animal models for IBD research, including DSS, 2,4,6-trinitrobenzenesulfonic acid (TNBS), oxazolone and acetic acid [36–38]. Among them, DSS induced colitis model is widely used for the purpose of evaluating novel therapeutic agents. It is cost-efficiency and highly reproducible. Feeding mice for several days with DSS polymers in the drinking water induce an inflammatory response in the colon that mimics human ulcerative colitis. It is characterized by bloody diarrhea, inflammatory cells infiltration, body weight loss and elevated colonic ROS productions [36,39,40]. DSS is toxic to gut epithelial cells of the basal crypts and therefore affects the integrity of the mucosal barrier [38]. In DSS-induced murine colitis model, decreased expression of tight junction proteins and increased intestinal permeability were observed in the inflamed mucosa
N C O
428
U
427
9
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523
526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541
10
K. Ren et al.
547 Q4
This study was supported in part by a UNMC graduate student fellowship (KR), a China Scholar Council scholarship (KR) and the UNMC College of Pharmacy.
548
References
549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606
[1] P.L. Lakatos, Recent trends in the epidemiology of inflammatory bowel diseases: up or down? World J. Gastroenterol. 12 (38) (2006) 6102–6108. [2] J. Cosnes, C. Gower-Rousseau, P. Seksik, A. Cortot, Epidemiology and natural history of inflammatory bowel diseases, Gastroenterology 140 (6) (2011) 1785–1794. [3] R.B. Sartor, Mechanisms of disease: pathogenesis of Crohn's disease and ulcerative colitis, Nat. Clin. Pract. Gastroenterol. Hepatol. 3 (7) (2006) 390–407. [4] C. Fiocchi, Inflammatory bowel disease: evolutionary concepts in biology, epidemiology, mechanisms and therapy, Curr. Opin. Gastroenterol. 29 (4) (2013) 347–349. [5] C. Abraham, J.H. Cho, Inflammatory bowel disease, N. Engl. J. Med. 361 (21) (2009) 2066–2078. [6] D.K. Podolsky, Inflammatory bowel disease, N. Engl. J. Med. 347 (6) (2002) 417–429. [7] S. Melgar, F. Shanahan, Inflammatory bowel disease—from mechanisms to treatment strategies, Autoimmunity 43 (7) (2010) 463–477. [8] G.R. Lichtenstein, M.T. Abreu, R. Cohen, W. Tremaine, American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease, Gastroenterology 130 (3) (2006) 940–987. [9] A.A. Sohrabpour, R. Malekzadeh, A. Keshavarzian, Current therapeutic approaches in inflammatory bowel disease, Curr. Pharm. Des. 16 (33) (2010) 3668–3683. [10] J.A. Katz, Treatment of inflammatory bowel disease with corticosteroids, Gastroenterol. Clin. N. Am. 33 (2) (2004) 171–189 (vii). [11] E.I. Benchimol, C.H. Seow, A.H. Steinhart, A.M. Griffiths, Traditional corticosteroids for induction of remission in Crohn's disease, Cochrane Database Syst. Rev. (2) (2008) CD006792. [12] S. Moghadam-Kia, V.P. Werth, Prevention and treatment of systemic glucocorticoid side effects, Int. J. Dermatol. 49 (3) (2010) 239–248. [13] H. Schacke, W.D. Docke, K. Asadullah, Mechanisms involved in the side effects of glucocorticoids, Pharmacol. Ther. 96 (1) (2002) 23–43. [14] X.M. Liu, L.D. Quan, J. Tian, Y. Alnouti, K. Fu, G.M. Thiele, et al., Synthesis and evaluation of a well-defined HPMA copolymer–dexamethasone conjugate for effective treatment of rheumatoid arthritis, Pharm. Res. 25 (12) (2008) 2910–2919. [15] D. Wang, S.C. Miller, X.M. Liu, B. Anderson, X.S. Wang, S.R. Goldring, Novel dexamethasone–HPMA copolymer conjugate and its potential application in treatment of rheumatoid arthritis, Arthritis Res. Ther. 9 (1) (2007) R2. [16] K. Ren, P.E. Purdue, L. Burton, L.D. Quan, E.V. Fehringer, G.M. Thiele, et al., Early detection and treatment of wear particleinduced inflammation and bone loss in a mouse calvarial osteolysis model using HPMA copolymer conjugates, Mol. Pharm. 8 (4) (2011) 1043–1051. [17] F. Yuan, R.K. Nelson, D.E. Tabor, Y. Zhang, M.P. Akhter, K.A. Gould, et al., Dexamethasone prodrug treatment prevents nephritis in lupus-prone (NZB × NZW)F1 mice without causing systemic side effects, Arthritis Rheum. 64 (12) (2012) 4029–4039. [18] K. Ren, A. Dusad, F. Yuan, H. Yuan, P.E. Purdue, E.V. Fehringer, et al., Macromolecular prodrug of dexamethasone
[20]
[21]
[22]
O
[23]
[24]
D
[25]
E
[26]
[27]
T
C
E
R
R
N C O
U
546
[19]
F
545
prevents particle-induced peri-implant osteolysis with reduced systemic side effects, J. Control. Release 175C (2013) 1–9. P.M. Henson, Dampening inflammation, Nat. Immunol. 6 (12) (2005) 1179–1181. Y. Nishiyama, T. Kataoka, K. Yamato, T. Taguchi, K. Yamaoka, Suppression of dextran sulfate sodium-induced colitis in mice by radon inhalation, Mediat. Inflamm. 2012 (2012) 239617. K. Tanaka, T. Namba, Y. Arai, M. Fujimoto, H. Adachi, G. Sobue, et al., Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis, J. Biol. Chem. 282 (32) (2007) 23240–23252. C.J. Kim, J. Kovacs-Nolan, C. Yang, T. Archbold, M.Z. Fan, Y. Mine, L-cysteine supplementation attenuates local inflammation and restores gut homeostasis in a porcine model of colitis, Biochim. Biophys. Acta 1790 (10) (2009) 1161–1169. L.A. Dieleman, M.J. Palmen, H. Akol, E. Bloemena, A.S. Pena, S.G. Meuwissen, et al., Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines, Clin. Exp. Immunol. 114 (3) (1998) 385–391. L.D. Quan, P.E. Purdue, X.M. Liu, M.D. Boska, S.M. Lele, G.M. Thiele, et al., Development of a macromolecular prodrug for the treatment of inflammatory arthritis: mechanisms involved in arthrotropism and sustained therapeutic efficacy, Arthritis Res. Ther. 12 (5) (2010) R170. K. Ren, A. Dusad, F. Yuan, H. Yuan, P.E. Purdue, E.V. Fehringer, et al., Macromolecular prodrug of dexamethasone prevents particle-induced peri-implant osteolysis with reduced systemic side effects, J. Control. Release 175 (2014) 1–9. R.I. Scheinman, P.C. Cogswell, A.K. Lofquist, A.S. Baldwin Jr., Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids, Science 270 (5234) (1995) 283–286. Y. Tao, C. Williams-Skipp, R.I. Scheinman, Mapping of glucocorticoid receptor DNA binding domain surfaces contributing to transrepression of NF-kappa B and induction of apoptosis, J. Biol. Chem. 276 (4) (2001) 2329–2332. P.J. Barnes, Glucocorticosteroids: current and future directions, Br. J. Pharmacol. 163 (1) (2011) 29–43. T. Rhen, J.A. Cidlowski, Antiinflammatory action of glucocorticoids—new mechanisms for old drugs, N. Engl. J. Med. 353 (16) (2005) 1711–1723. M.J.R. Nikhil Singh, M. Jane Tucker, Mechanisms of glucocorticoid-mediated antiinflammatory and immunosuppressive action, Paediatr. Perinat. Drug Ther. 6 (2) (2004) 107–115. H.S. Cooper, S.N. Murthy, R.S. Shah, D.J. Sedergran, Clinicopathologic study of dextran sulfate sodium experimental murine colitis, Lab. Invest. 69 (2) (1993) 238–249. O.H. Kang, D.K. Kim, Y.A. Choi, H.J. Park, J. Tae, C.S. Kang, et al., Suppressive effect of non-anaphylactogenic anti-IgE antibody on the development of dextran sulfate sodiuminduced colitis, Int. J. Mol. Med. 18 (5) (2006) 893–899. M. Silbermann, U. Kleinhaus, E. Livne, T. Kedar, Retardation of bone growth in triamcinolone-treated mice, J. Anat. 121 (Pt 3) (1976) 515–535. G. Ortoft, H. Gronbaek, H. Oxlund, Growth hormone administration can improve growth in glucocorticoid-injected rats without affecting the lymphocytopenic effect of the glucocorticoid, Growth Horm. IGF Res. 8 (3) (1998) 251–264. D.B. Allen, M. Mullen, B. Mullen, A meta-analysis of the effect of oral and inhaled corticosteroids on growth, J. Allergy Clin. Immunol. 93 (6) (1994) 967–976. S. Wirtz, C. Neufert, B. Weigmann, M.F. Neurath, Chemically induced mouse models of intestinal inflammation, Nat. Protoc. 2 (3) (2007) 541–546. M. Kawada, A. Arihiro, E. Mizoguchi, Insights from advances in research of chemically induced experimental models of human inflammatory bowel disease, World J. Gastroenterol. 13 (42) (2007) 5581–5593.
R O
Acknowledgments
P
544
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
Potential of P-Dex to improve the clinical management of IBD
chain-length fatty acids in the colon, PLoS One 7 (3) (2012) e32084. [42] L.A. Dieleman, B.U. Ridwan, G.S. Tennyson, K.W. Beagley, R.P. Bucy, C.O. Elson, Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice, Gastroenterology 107 (6) (1994) 1643–1652. [43] S.Y. Oh, K.A. Cho, J.L. Kang, K.H. Kim, S.Y. Woo, Comparison of experimental mouse models of inflammatory bowel disease, Int. J. Mol. Med. 33 (2) (2014) 333–340. [44] F.R. Byrne, J.L. Viney, Mouse models of inflammatory bowel disease, Curr. Opin. Drug Discov. Dev. 9 (2) (2006) 207–217.
N C O
R
R
E
C
T
E
D
P
R O
O
F
[38] S. Wirtz, M.F. Neurath, Mouse models of inflammatory bowel disease, Adv. Drug Deliv. Rev. 59 (11) (2007) 1073–1083. [39] I. Okayasu, S. Hatakeyama, M. Yamada, T. Ohkusa, Y. Inagaki, R. Nakaya, A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice, Gastroenterology 98 (3) (1990) 694–702. [40] M. Mahler, I.J. Bristol, E.H. Leiter, A.E. Workman, E.H. Birkenmeier, C.O. Elson, et al., Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis, Am. J. Physiol. 274 (3 Pt 1) (1998) G544–G551. [41] H. Laroui, S.A. Ingersoll, H.C. Liu, M.T. Baker, S. Ayyadurai, M.A. Charania, et al., Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-
U
675 676 677 678 679 680 681 682 683 684 685 686 687 700
11
Please cite this article as: K. Ren, et al., Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.03.027
688 689 690 691 692 693 694 695 696 697 698 699