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Efficacy of Polaprezinc for Acute Radiation Proctitis in a Rat Model
Int. J. Radiation Oncology Biol. Phys., Vol. 80, No. 3, pp. 877–884, 2011 Copyright Ó 2011 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/$ - see front matter
doi:10.1016/j.ijrobp.2011.01.009
BIOLOGY CONTRIBUTION
EFFICACY OF POLAPREZINC FOR ACUTE RADIATION PROCTITIS IN A RAT MODEL HIROSHI DOI, M.D.,* NORIHIKO KAMIKONYA, M.D., PH.D.,* YASUHIRO TAKADA, M.D., PH.D.,* MASAYUKI FUJIWARA, M.D., PH.D.,* KEITA TSUBOI, M.D., PH.D.,* HIROYUKI INOUE, M.S.,* MASAO TANOOKA, B.S.,* TAKESHI NAKAMURA, M.S.,y TOSHIYUKI SHIKATA, B.S.,y TOHRU TSUJIMURA, M.D., PH.D.,z AND SHOZO HIROTA, M.D., PH.D.* Departments of *Radiology and zPathology, Hyogo College of Medicine, Hyogo, Japan; yDepartment of Pharmacy, The Hospital of Hyogo College of Medicine, Hyogo, Japan Purpose: The purpose of the present study was to standardize the experimental rat model of radiation proctitis and to examine the efficacy of polaprezinc on radiation proctitis. Methods and Materials: A total of 54 female Wistar rats (5 weeks old) were used. The rats were divided into three groups: those treated with polaprezinc (PZ+), those treated with base alone, exclusive of polaprezinc (PZ), and those treated without any medication (control). All the rats were irradiated to the rectum. Polaprezinc was prepared as an ointment. The ointment was administered rectally each day after irradiation. All rats were killed on the 10th day after irradiation. The mucosal changes were evaluated endoscopically and pathologically. The results were graded from 0 to 4 and compared according to milder or more severe status, as applicable. Results: According to the endoscopic findings, the proportion of mild changes in the PZ+, PZ, and control group was 71.4%, 25.0%, and 14.3% respectively. On pathologic examination, the proportion of low-grade findings in the PZ+, PZ, and control group was 80.0%, 58.3%, and 42.9% for mucosal damage, 85.0%, 41.7%, and 42.9% for a mild degree of inflammation, and 50.0%, 33.3%, and 4.8% for a shallow depth of inflammation, respectively. The PZ+ group tended to have milder mucosal damage than the other groups, according to all criteria used. In addition, significant differences were observed between the PZ+ and control groups regarding the endoscopic findings, degree of inflammation, and depth of inflammation. Conclusions: This model was confirmed to be a useful experimental rat model for radiation proctitis. The results of the present study have demonstrated the efficacy of polaprezinc against acute radiation-induced rectal disorders using the rat model. Ó 2011 Elsevier Inc. Radiation proctitis, Rats, Polaprezinc, Irradiation, Colitis.
These effects decrease the quality of life of some patients and often require either an interruption of therapy or other modifications that could forestall the optimal completion of the original treatment plan. Several therapeutic modalities have been used for the treatment of acute and chronic radiation proctitis, including medical therapy, such as corticosteroids (2, 3), sucralfate (4, 5), metronidazole (6), and formalin (7, 8), endoscopic treatment, such as various lasers, including argon plasma coagulation (9), yttrium-aluminum-garnet laser (10), and treatment with hyperbaric oxygen (11).
INTRODUCTION Radiation proctitis is a major clinical complication of irradiation for cancer in the pelvic region, including cancer of the uterus, bladder, and prostate. The rectum is exposed to high radiation doses during radiotherapy for pelvic malignancies. The rate of complications has been reported to be 5–20% (1). The incidence of intestinal radiation toxicity depends on the radiation dose, treatment term, and fractionation schedule. Acute radiation proctitis entails significant acute morbidity, including diarrhea, abdominal cramps, and hematochezia.
Conflict of interest: none. Acknowledgments—The authors thank all medical radiation technologists from the Department of Clinical Radiology, The Hospital of Hyogo College of Medicine (Nishinomiya, Hyogo, Japan) and Ms. Michiko Kakihana from Department of Pathology, Hyogo College of Medicine (Nishinomiya, Hyogo, Japan) for valuable her technical assistance. We also acknowledge Mr. Brian Quinn for assistance in the language revision of our report. Received May 5, 2010, and in revised form Dec 24, 2010. Accepted for publication Jan 10, 2011.
Reprint requests to: Hiroshi Doi, M.D., Department of Radiology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya City, Hyogo 663-8501 Japan. Tel: (81) 798-45-6362; Fax: (81) 798-45-6361 E-mail: [email protected] Presented in part at the American Society for Radiation Oncology 52nd Annual Meeting, October 31 to November 4, 2010, San Diego, CA, and the Joint 15th Congress of the European Cancer Organisation and 34th Congress of the European Society for Medical Oncology, September 20–24, 2009, Berlin, Germany. Supported in part by funding from Grants-in-Aid for Scientific Research (Grant 21591622) and unrestricted funding from Zeria Pharmaceutical Co., Ltd. 877
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However, these medical approaches have not been sufficiently effective and have had only a limited benefit. Therefore, the search for a more effective therapy continues. Polaprezinc (Zeria Pharmaceutical, Tokyo, Japan), an antiulcer drug, is a chelate compound consisting of a zinc ion, L-carnosine, a b-alanine dipeptide, and L-histidine. Its antioxidant effects have been reported in previous studies (12–14). The purpose of the present study was to standardize the experimental animal model of radiation-induced rectal disorders and to examine the efficacy of polaprezinc to prevent radiation-induced rectal disorders. METHODS AND MATERIALS Preliminary study A total of 14 Wistar rats (5 weeks old) were used in the preliminary study. To confirm the validity of the technique to irradiate the rectum in a vertical position, Gastrografin was injected into a rat anus under pentobarbital anesthesia, and X-rays were performed in the vertical position. In addition, to confirm the adhesiveness of the ointment to the rectum in the rat, ointment containing barium sulfate was rectally administered to another rat. X-rays were then taken twice after application of the ointment, immediately after administration and 10 minutes after administration. To verify that the damage to the mucosa from the rectal administrations was minimal, the catheter to administer the ointment was inserted into the rectum daily for 5 days using 6 rats, and the mucosal changes were compared macroscopically and pathologically with those from the other 6 rats that had not undergone any procedures. The rats enrolled in the preliminary study were excluded from the present study.
Rats A total of 54 female Wistar rats, weighing 90–120 g at 5 weeks of age were obtained from CLEA Japan (Tokyo, Japan). All rats were acclimated for 7 days. They were housed 3/cage and fed with a laboratory rodent pellet formula and tap water ad libitum. The Hyogo College of Medicine Institutional Animal Care and Use Committee approved all animal procedures before the initiation of the project. The rats were assigned to one of three groups as follows. Group A contained 21 rats treated with polaprezinc. Group B included 12 rats treated with base alone (without polaprezinc). Group C contained 21 rats treated without any medication. The homogeneity of the groups was confirmed by analyzing the body weights using Bartlett’s test (p = .210).
Irradiation Each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (46.8 2.1 mg/kg) and weighed. Next, 3 rats at a time were restrained and taped by the tail to an acryl plate in the vertical position. Lead shielding was used to cover the rat, except for a 2.5-cm-long area of the lower pelvis containing the rectum in the middle of the field. All rats were irradiated with a single X-ray fraction of 25 Gy, at 4 MV, with a dose rate of 200 monitor units/min. No filtration was used in the present study. Several rats were selected as samples to confirm the consistency between the actual dose and the delivered monitor units. The absorbed X-ray dose in the rectum
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was measured using a semiconductor detector inserted into the rectum, and the delivered monitor units were then revised, as appropriate.
Treatment with ointment Polaprezinc was prepared as an ointment with a compound of white petrolatum and a purified lanolin base. Group A (polaprezinc) was irradiated and treated with ointment that included polaprezinc at 100 mg/kg/body weight. Group B (no polaprezinc) was irradiated and treated with ointment consisting of base alone, containing white petrolatum and purified lanolin without polaprezinc. Finally, Group C (control) was irradiated and was not treated with any medication. The ointment was administrated to the rat anus without any anesthesia daily after irradiation. A syringe (1 mL) with a catheter that had been especially designed for the present study was used to administer the ointment.
Evaluation of rectal damage Each rat was observed daily for signs of proctitis, such as diarrhea and rectal bleeding, after irradiation. The clinical findings were scored as follows: 0, no symptoms; 2, diarrhea; and 4, gross bleeding. All rats were killed on the 10th day after irradiation and weighed. Colonoscopy was performed on the rats using a 4-mm-diameter laryngendoscope immediately after death. The rectal lumen images of the rats were obtained using a digital camera. The endoscopic findings were evaluated and graded using a method previously described by Northway et al. (15) as follows: 0, normal mucosa; 1, edema, mild hyperemia, or decrease vascularity; 2, diffuse hyperemia, multiple punctuate areas of hemorrhage, or confluent areas of hemorrhage; 3, the presence of erosion or frank hemorrhage; and 4, ulcers. Mild findings were defined as Grade 0 and 1 and severe findings as Grade 2–4. The distal rectum was excised for pathologic evaluation after colonoscopy and immediately fixed in 10% neutral buffered formalin solution. The colon was sectioned, divided into two or three equal segments (from the proximal rectum to the anus), and submitted for histologic analysis. All slides were stained with hematoxylin-eosin and examined using light microscopy by a pathologist who was unaware of the treatment groups. The severity of proctitis in each specimen was evaluated by the pathologic examination findings. The morphologic mucosal damage, degree of inflammation, and depth of inflammation are all listed in Table 1. The areas of morphologic mucosal damage in the rectum were evaluated mainly by the crypt changes found and graded. Mild findings were defined as Grades 0, 1, and 2 and severe findings as Grades 3 and 4. The degree of inflammation was evaluated by investigating the density of inflammatory cell infiltration, according to the submucosal edema present and the destruction of the muscularis propria. The degree of inflammation was graded as listed in Table 1. In addition, mild findings were defined as Grades 0–3 and severe findings as Grade 4. The depth of inflammation was evaluated and graded according to the depth of inflammatory cells infiltration into the rectal mucosa. Mild findings included Grades 0–2 and severe findings Grades 3 and 4. In addition, the apparent loss of the columnar shape on microscopic examination was measured, and the length of the apparent
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Table 1. Grading of pathologic changes on microscopic examination Score
Description
Morphologic mucosal damage 0 Normal or minor alterations that could not be ascribed with certainty to radiation 1 Slight crypt change without loss of epithelium 2 Crypt change with loss of epithelium equaling less than one-half 3 Crypt change with loss of epithelium greater than one-half 4 Loss of epithelium through muscularis mucosa Degree of inflammation 0 Normal mucosa 1 Minor alterations 2 Apparent inflammation (mild inflammation) 3 More significant inflammation (moderate inflammation) 4 Severe inflammation Depth of inflammation 0 No apparent inflammation 1 Inflammatory cells extend up to edge of mucosa (epithelium, lamina propria) but not beyond 2 Inflammatory cells extend through mucosa and into submucosa 3 Inflammatory cells infiltrate through submucosa and extends into muscularis propria 4 Inflammatory cells extend through muscularis propria into subserosa mucosal change was recorded to estimate the craniocaudal length of inflammation.
Statistical analysis The results are presented as the mean standard deviation. The results of the mucosal damage evaluation were compared according to milder or more severe status, as applicable. The relationship among the groups was assessed using the chi-square test. p Values of < .05 were considered statistically significant. Tukey’s allpairwise-comparison test was used to identify differences in the length of inflammation among the three groups.
RESULTS The findings of the enema study showed that the distal colon and sigmoid colon moved outside of the lower pelvis, and the length of the rectum was approximately $3 cm in our rat model (Fig. 1). The spreading of the barium ointment was limited to the rectal region 10 minutes after administration, as well as immediately after administration. No apparent differences were observed between the rats with and without insertion of the catheter or between the rectum and more proximal colon. No irradiated rats died during the follow-up period. The body weight on the 10th day after irradiation was 151.7 9.9 g in Group A, 153.6 7.4 g in Group B, and 158.1 8.5 g in Group C. No significant difference was found in the weight among the three groups. The clinical findings of proctitis were diarrhea, including mucus and pollution around the anus, and gross bleeding
Fig. 1. Findings from enema study. Distal colon and sigmoid colon moved outside low pelvis and rectal length was approximately 3 cm in vertical position.
(Fig. 2). The results of the clinical findings are listed in Table 2. The proportion of Grade 0 (no symptoms) was 19.0% in Group A, 16.7% in Group B, and 4.8% in Group C. Group A had a greater frequency of Grade 0 than did the other groups; however, the difference was not significant. The results of the endoscopic findings are shown in Fig. 3 and Table 3. Only in Group A, Grade 0 results were observed. The proportion of mild changes in the endoscopic findings was 71.4% in Group A, 25.0% in Group B, and 14.3% in Group C. Group A showed significantly milder endoscopic findings than did the other two groups (p < .05). One specimen from Group A was lost in the fixation process. Apparent mucosal changes were observed in the pathologic examinations of all the specimens (Fig. 4). The epithelial changes included crypt architectural distortion with cryptitis, characterized by an infiltration of inflammatory cells. Also, the columnar shape was lost to planarization. Varying levels of loss of the epithelium were seen. Severe changes were characterized by the loss of epithelium through the muscularis mucosa and mucosal ulceration.
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Fig. 2. Clinical findings. (a) Grade 0, no symptoms; (b) Grade 2, diarrhea (included effusion around anus); (c) Grade 4, gross bleeding.
Inflammatory cells included neutrophils, lymphocytes, and, characteristically, eosinophils. The density of the inflammatory cells became greater and they had migrated deeper, with an increasing severity of inflammation. Other prominent changes included varying degrees of edema in the submucosa, degeneration of the muscle fibers, and thickening of the vessel walls. These pathologic findings were limited to the distal rectum, and the columnar shape remained outside the irradiated field (Fig. 5). The findings regarding the presence of morphologic mucosal damage are listed in Table 4. The morphologic mucosal damage ranged from Grade 2 to 4 in all groups. The proportion of low grades was 80.0% in Group A, 58.3% in Group B, and 42.9% in Group C. No significant differences were observed; however, Group A tended to have milder damage than the other two groups. The results regarding the degree of inflammation are listed in Table 5. The degree of inflammation in Groups A and B ranged from Grades 2 to 4. However, in Group C, no Grade 0–2 results were seen regarding the degree of inflammation. The proportion with a mild degree of inflammation was 85.0% in Group A, 41.7% in Group B, and 42.9% in Group C. Group A showed significantly milder damage than did the two other groups in the degree of inflammation (p < .05). The results of the depth of inflammation are listed in Table 6. Group A only showed Grade 1 in the depth of inTable 2. Clinical findings
Characteristic Grade 0 Grade 2 Grade 4 Mild (Grade 0) Severe (Grade 2, 4)
Group A (PZ+; n = 21)
Group B (PZ; n = 12)
Group C (control; n = 21)
4 (19.0) 16 (76.2) 1 (4.8) 4 (19.0) 17 (81.0)
0 (0.0) 10 (83.3) 2 (16.7) 0 (0.0) 12 (100.0)
1 (4.8) 19 (90.5) 1 (4.8) 1 (4.8) 20 (95.2)
Abbreviation: PZ = polaprezinc. Data presented as numbers, with percentages in parentheses. No significant difference found among 3 groups (p < .05).
flammation. The proportion with a shallow depth of inflammation was 50.0% in Group A, 33.3% in Group B, and 4.8% in Group C. Group A had significantly milder damage than Group C (p < .05). The average length of crypt damage was 15.6 3.8 mm in Group A, 15.6 3.0 mm in Group B, and 18.1 3.5 mm in Group C. No apparent differences were seen among the three groups. DISCUSSION Radiotherapy is a well-established treatment of pelvic malignancies such as prostate cancer, and proctitis is a common adverse event. Experimental animal models of radiation-induced gastrointestinal toxicities have been described in previous studies (15–28). However, irradiation plus differing surgical techniques could have produced different results. Hubmann (16) originally described a method to irradiate the rectum of the rat without surgical procedures and with minimal exposure to other organs. This technique has been used in several reports (15, 17–20). Kiszel et al. (17) reported that the kidney and proximal ureters move well outside the irradiated field on vertical positioning with retrograde pyelography. The present enema study showed that the distal colon and sigmoid colon moved outside the lower pelvis, and the length of the rectum was approximately 3 cm. According to the imaging studies, this method seems to make it possible to perform selective irradiation of the rectum in the rat while inducing minimal damage to other organs. In addition, the length of the apparent morphologic changes was measured, and the mean value for all groups was 16.6 3.6 mm (no apparent differences were observed among the three groups regarding the length of crypt damage). Therefore, this irradiation technique allows for selective irradiation to the distal colon without damage to other organs. Kan et al. (18) reported that the erosion of the surface epithelium, which started on the sixth day after irradiation, progressed to multifocal ulceration with severe inflammation until the 10th day after irradiation, with healing of the
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Fig. 3. Endoscopic findings. (a) Grade 1, edema and d mild hyperemia found on the rectal mucosa; (b) Grade 3, apparent erosion present.
ulceration with extensive regenerative changes on the 14th day after irradiation. Northway et al. (15) and Korkut et al. (19) reported that apparent mucosal damage occurred on the 10th day after irradiation. The present study evaluated the mucosal changes on the 10th day after irradiation. As a result, apparent mucosal changes (i.e., loss of epithelium, apparent extension of inflammation) were observed in all three groups. However, Group A showed milder mucosal changes and inflammation than the other two groups. These findings have demonstrated that the suitable day to evaluate irradiation-induced rectal damage is the 10th day after irradiation. The pathologic findings were consistent with those from previous reports (15–23) and were also consistent with the pathologic findings of radiation proctitis in the human rectum (29). The mucosal damage in the rectum was evaluated pathologically to investigate both the morphologic changes and inflammation. The morphologic changes included a loss of
Table 3. Endoscopic findings
Characteristic Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0–1) Severe (Grade 2–4) Chi-square test p value (p < .05)
Group A (PZ+ n = 21)
Group B (PZ; n = 12)
Group C (control; n = 21)
6 (28.6) 9 (42.9) 4 (19.0) 1 (4.8) 1 (4.8) 15 (71.4)
0 (0.0) 3 (25.0) 3 (25.0) 6 (50.0) 0 (0.0) 3 (25.0)
0 (0.0) 3 (14.3) 12 (57.1) 3 (14.3) 3 (14.3) 3 (14.3)
6 (28.6)
9 (75.0)
18 (85.7)
PZ+ vs. PZ
PZ+ vs. control .0006
PZ vs. control NS
.03
Abbreviations: PZ = polaprezinc, NS = not significant. Data presented as numbers, with percentages in parentheses.
columnar shape and epithelial organization. Other changes were also observed, including submucosal edema, degeneration of the muscle fibers, and thickening of the vessel walls. However, it was difficult to perform a quantitative analysis to evaluate the mucosal damage using these changes. Therefore, these findings were not used for the analysis in the present study. Despite the identical administration techniques in Groups A and B, Group B still exhibited milder rectal damage than Group C. The base treatment consisted of white petrolatum and purified lanolin without any additional agents, and this alone seemed to have a certain level of effectiveness in healing the mucosa. Therefore, our study has shown that it is possible to safely administer the ointment into the rectum. The present method has demonstrated that it is possible to administer agents into the rat anus both safely and appropriately. The rats that received polaprezinc (Group A) tended to show milder mucosal damage than the other groups for all criteria of the present study. In addition, significant differences were observed between Groups A and C regarding the degree (p < .05) and the depth (p < .01) of inflammation. In addition, Group A showed greater efficacy than Group B in the present study. Thus, polaprezinc was confirmed to have additive benefits compared with the administration of the base alone. Polarezinc [N-(3-aminopropionyl)-L-histidinatozinc], an antiulcer agent, is a chelated compound consisting of zinc ion and L-carnosine. This agent has not only prevented gastric mucosal lesions in various experimental models but has shown a healing promoting effect on gastric ulcers. The latter action can be accounted for owing to antioxidant properties, stimulation of mucus production, and maintenance of the gastric mucosal barrier (12–14, 30). Yoshikawa et al. (12) reported the antioxidant activities of polaprezinc, such as the superoxide scavenging activity, inhibition of superoxide protection, inhibition of the Fenton reaction, hydroxyl radical scavenging activity, inhibition of lipid peroxidation, and singlet oxygen quenching in vitro. In addition, this compound’s
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Fig. 4. Pathologic findings. (a) Surface epithelium showed crypt architectural distortion, characterized by prominent flattening of columnar cells and invasion of inflammatory cells. In addition, significant mucosal edema observed in lamina propria. (b) Severe mucosal damage (Grade 4), with loss of epithelium reaching submucosa through muscularis mucosa and leading to ulceration. (c) Grade 4 severity in degree of inflammation. Density of inflammatory cell infiltration very high, with destruction of muscularis propria also observed. In addition, inflammatory cells extended into muscularis propria for Grade 3 severity in depth of inflammation.
pharmacologic potency has been demonstrated by examining its adherence to the mucosa (31). Matsuu-Matsuyama et al. (22) treated rats that had received whole body X-ray irradiation with oral polaprezinc. Ohkawara et al. (32) reported that polaprezinc can be administered intrarectally to combat dextran sulfate-induced colitis in mice. However, no studies, to date, have reported the rectal administration of polaprezinc to reduce radiation-induced intestinal injury. Oxygen radicals in radiation-induced gastrointestinal disorders have been previously reported (33). The rectal administration of polaprezinc could therefore have greater efficacy against mucosal damage compared with peroral administration. No significant differences were seen regarding the morphologic mucosal damage; however, Group A did show a milder degree of damage and also, apparently, lower grades of inflammation than the other two groups. These results might be attributable to the anti-inflammatory effects of polaprezinc. Therefore, polaprezinc was found to exhibit an apparent efficacy against radiation-induced rectal disor-
ders. These data raise the possibility that the administration of polaprezinc could reduce the incidence of severe radiation-induced proctitis. In the present study, the administration of polaprezinc was performed after irradiation to irradiate all rats equally. However, the administration of polaprezinc as a premedication before irradiation might make both the anti-inflammatory effect and the efficacy stronger than when used alone after irradiation. Therefore, the optimal timing to administer polaprezinc should be investigated further to enhance the efficacy of this treatment modality. Chronic radiation injury is thought to result from tissue ischemia secondary to thrombogenesis induced by thrombin formation in the microvessels. Ulceration, rectal obstruction, and carcinogenesis in the rat intestine have been reported as late adverse effects 3–14 months after irradiation, according to long-term observations (16, 17, 23–28). A long-term observation study after irradiation is presently underway to establish an experimental rat model of radiation-induced rectal disorders and late adverse events and also to assess the efficacy of polaprezinc against late rectal toxicity.
Table 4. Morphologic mucosal damages on microscopic examination
Characteristic Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0–2) Severe (Grade 3-4) Fig. 5. Measurement of apparent loss of columnar shape. Crypt architectural distortion limited to distal rectum, with columnar shape remaining outside irradiated field.
Group A (PZ+; n = 20)
Group B (PZ; n = 12)
Group C (control; n = 21)
0 (0.0) 0 (0.0) 16 (80.0) 2 (10.0) 2 (10.0) 16 (80.0)
0 (0.0) 0 (0.0) 7 (58.3) 2 (16.7) 3 (25.0) 7 (58.3)
0 (0.0) 0 (0.0) 9 (42.9) 3 (14.3) 9 (42.9) 9 (42.9)
4 (20.0)
5 (41.7)
12 (57.1)
Abbreviation: PZ = polaprezinc. Data presented as numbers, with percentages in parentheses. No significant differences found among 3 groups (p < .05).
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Table 5. Degrees of inflammation on microscopic examination
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Table 6. Depth of inflammation on microscopic examination
Characteristic
Group A (PZ+; n = 20)
Group B (PZ; n = 12)
Group C (control; n = 21)
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0–3) Severe (Grade 4) Chi-square test
0 (0.0) 0 (0.0) 6 (30.0) 11 (55.0) 3 (15.0) 17 (85.0) 3 (15.0) PZ+ vs. PZ
p value (p < .05)
.03
0 (0.0) 0 (0.0) 1 (8.3) 4 (33.3) 7 (58.3) 5 (41.7) 7 (58.3) PZ+ vs. control .01
0 (0.0) 0 (0.0) 0 (0.0) 9 (42.9) 12 (57.1) 9 (42.9) 12 (57.1) PZ vs. control NS
Abbreviations as in Table 3. Data presented as numbers, with percentages in parentheses.
CONCLUSIONS Polaprezinc has therefore been proved to be effective against radiation-induced rectal disorders in a rat model.
Characteristic
Group A (PZ+; n = 20)
Group B (PZ; n = 12)
Group C (control; n = 21)
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0, 1, 2) Severe (Grade 3, 4) Chi-square test
0 (0.0) 2 (10.0) 8 (40.0) 0 (0.0) 10 (50.0) 10 (50.0) 10 (50.0) PZ+ vs. PZ
0 (0.0) 0 (0.0) 4 (33.3) 0 (0.0) 8 (66.7) 4 (33.3) 8 (66.7) PZ+ vs. control .004
0 (0.0) 0 (0.0) 1 (4.8) 4 (19.0) 16 (76.2) 1 (4.8) 20 (95.2) PZ vs. control NS
p value (p < .05)
NS
Abbreviations as in Table 3. Data presented as numbers, with percentages in parentheses.
The present study demonstrated an anti-inflammatory effect of polaprezinc against acute radiation induced proctitis using an experimental rat model.
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13. Yoshikawa T, Yamaguchi T, Yoshida N, et al. Effect of Z-103 on TNB-induced colitis in rats. Digestion 1997;58:464–468. 14. Ohkawara T, Nishihira J, Nagashima R, et al. Polaprezinc protects human colon cells from oxidative injury induced by hydrogen peroxide: Relevant to cytoprotective heat shock proteins. World J Gastroenterol 2006;12:6178–6181. 15. Northway MG, Scobey MW, Geisinger KR. Radiation proctitis in the rat: Sequential changes and effects of anti-inflammatory agents. Cancer 1988;62:1962–1969. 16. Hubmann FH. Effect of X irradiation on the rectum of the rat. Br J Radiol 1981;54:250–254. 17. Kiszel Z, Spiethoff A, Trott KR. Large bowel stenosis in rats after fractionated local irradiation. Radiother Oncol 1984;2: 247–254. 18. Kan S, Chun M, Jin YM, et al. A rat model for radiationinduced proctitis. J Korean Med Sci 2000;15:682–689. 19. Korkut C, Asoglu O, Aksoy M, et al. Histopathological comparison of topical therapy modalities for acute radiation proctitis in an experimental rat model. World J Gastroenterol 2006; 12:4879–4883. 20. Geisinger KR, Scobey MW, Northway MG, et al. Radiation-induced proctitis cystica profunda in the rat. Dig Dis Sci 1990;35: 833–839. 21. Ikeda Y, Ito M, Matsuu M, et al. Expression of ICAM-1 and acute inflammatory cell infiltration in the early phase of radiation colitis in rats. J Radiat Res 2000;41:279–291. 22. Matsuu-Matsuyama M, Shichijo K, Okaichi K, et al. Protection by polaprezinc against radiation-induced apoptosis in rat jejunal crypt cells. J Radiat Res 2008;49:341–347. 23. Hayashida M, Shichijo K, Matsuu M, et al. Prolonged radiation damage in rat colon and urokinase expression in epithelium. Acta Med Nagasaki 2001;46:39–45. 24. Hauer-Jensen M, Sauer T, Davik F, et al. Late change following single dose roentgen irradiation of rat small intestine. Acta Radiol Oncol 1983;22:299–303. 25. Poulakos L, Elwell JH, Osborne JW, et al. The prevalence and severity of late effects in normal rat duodenum following intraoperative irradiation. Int J Radiat Oncol Biol Phys 1990;18: 841–848.
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26. Denman DL, Kirchner FR, Osborne JW. Induction of colonic adenocarcinoma in the rat by X-Irradiation. Cancer Res 1978;38:1899–1905. 27. Wang J, Zheng H, Ou X, et al. Hirudin ameliorates intestinal radiation toxicity in the rat: Support for thrombin inhibition as strategy to minimize side-effects after radiation therapy and as countermeasure against radiation exposure. J Thromb Haemost 2004;2:2027–2035. 28. Black WC, Gomez LS, Yuhas JM, et al. Quantitation of the late effects of X-radiation on the large intestine. Cancer 1980;45: 444–451. 29. Hovdenak N, Fajardo LF, Hauer-Jensen M. Acute radiation proctitis: A sequential clinicopathologic study during pelvic radiotherapy. Int J Radiat Oncol Biol Phys 2000;48:1111–1117.
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30. Arakawa T, Saitoh H, Nakamura A, et al. Effect of zinc L-carnosine on gastric mucosal and cell damage caused by ethanol in rats: Correlation with endogenous prostaglandin E2. Dig Dis Sci 1990;35:559–566. 31. Katayama S, Nishizawa K, Hirano M, et al. Effect of polaprezinc on healing of acetic acid-induced stomatitis in hamsters. J Pharma Pharma Sci 2000;3:114–117. 32. Ohkawara T, Takeda H, Kato K, et al. Polaprezinc ([N-(3-aminopropionyl)-L-histidinato zinc] ameliorates dextran sulfate sodium-induced colitis in mice. Scand J Gastroenterol 2005; 40:1321–1327. 33. Otamiri T, Sj€ odahl R. Oxygen radicals: Their role in selected gastrointestinal disorders. Dig Dis 1991;9:133– 141.
doi:10.1016/j.ijrobp.2011.01.009
BIOLOGY CONTRIBUTION
EFFICACY OF POLAPREZINC FOR ACUTE RADIATION PROCTITIS IN A RAT MODEL HIROSHI DOI, M.D.,* NORIHIKO KAMIKONYA, M.D., PH.D.,* YASUHIRO TAKADA, M.D., PH.D.,* MASAYUKI FUJIWARA, M.D., PH.D.,* KEITA TSUBOI, M.D., PH.D.,* HIROYUKI INOUE, M.S.,* MASAO TANOOKA, B.S.,* TAKESHI NAKAMURA, M.S.,y TOSHIYUKI SHIKATA, B.S.,y TOHRU TSUJIMURA, M.D., PH.D.,z AND SHOZO HIROTA, M.D., PH.D.* Departments of *Radiology and zPathology, Hyogo College of Medicine, Hyogo, Japan; yDepartment of Pharmacy, The Hospital of Hyogo College of Medicine, Hyogo, Japan Purpose: The purpose of the present study was to standardize the experimental rat model of radiation proctitis and to examine the efficacy of polaprezinc on radiation proctitis. Methods and Materials: A total of 54 female Wistar rats (5 weeks old) were used. The rats were divided into three groups: those treated with polaprezinc (PZ+), those treated with base alone, exclusive of polaprezinc (PZ), and those treated without any medication (control). All the rats were irradiated to the rectum. Polaprezinc was prepared as an ointment. The ointment was administered rectally each day after irradiation. All rats were killed on the 10th day after irradiation. The mucosal changes were evaluated endoscopically and pathologically. The results were graded from 0 to 4 and compared according to milder or more severe status, as applicable. Results: According to the endoscopic findings, the proportion of mild changes in the PZ+, PZ, and control group was 71.4%, 25.0%, and 14.3% respectively. On pathologic examination, the proportion of low-grade findings in the PZ+, PZ, and control group was 80.0%, 58.3%, and 42.9% for mucosal damage, 85.0%, 41.7%, and 42.9% for a mild degree of inflammation, and 50.0%, 33.3%, and 4.8% for a shallow depth of inflammation, respectively. The PZ+ group tended to have milder mucosal damage than the other groups, according to all criteria used. In addition, significant differences were observed between the PZ+ and control groups regarding the endoscopic findings, degree of inflammation, and depth of inflammation. Conclusions: This model was confirmed to be a useful experimental rat model for radiation proctitis. The results of the present study have demonstrated the efficacy of polaprezinc against acute radiation-induced rectal disorders using the rat model. Ó 2011 Elsevier Inc. Radiation proctitis, Rats, Polaprezinc, Irradiation, Colitis.
These effects decrease the quality of life of some patients and often require either an interruption of therapy or other modifications that could forestall the optimal completion of the original treatment plan. Several therapeutic modalities have been used for the treatment of acute and chronic radiation proctitis, including medical therapy, such as corticosteroids (2, 3), sucralfate (4, 5), metronidazole (6), and formalin (7, 8), endoscopic treatment, such as various lasers, including argon plasma coagulation (9), yttrium-aluminum-garnet laser (10), and treatment with hyperbaric oxygen (11).
INTRODUCTION Radiation proctitis is a major clinical complication of irradiation for cancer in the pelvic region, including cancer of the uterus, bladder, and prostate. The rectum is exposed to high radiation doses during radiotherapy for pelvic malignancies. The rate of complications has been reported to be 5–20% (1). The incidence of intestinal radiation toxicity depends on the radiation dose, treatment term, and fractionation schedule. Acute radiation proctitis entails significant acute morbidity, including diarrhea, abdominal cramps, and hematochezia.
Conflict of interest: none. Acknowledgments—The authors thank all medical radiation technologists from the Department of Clinical Radiology, The Hospital of Hyogo College of Medicine (Nishinomiya, Hyogo, Japan) and Ms. Michiko Kakihana from Department of Pathology, Hyogo College of Medicine (Nishinomiya, Hyogo, Japan) for valuable her technical assistance. We also acknowledge Mr. Brian Quinn for assistance in the language revision of our report. Received May 5, 2010, and in revised form Dec 24, 2010. Accepted for publication Jan 10, 2011.
Reprint requests to: Hiroshi Doi, M.D., Department of Radiology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya City, Hyogo 663-8501 Japan. Tel: (81) 798-45-6362; Fax: (81) 798-45-6361 E-mail: [email protected] Presented in part at the American Society for Radiation Oncology 52nd Annual Meeting, October 31 to November 4, 2010, San Diego, CA, and the Joint 15th Congress of the European Cancer Organisation and 34th Congress of the European Society for Medical Oncology, September 20–24, 2009, Berlin, Germany. Supported in part by funding from Grants-in-Aid for Scientific Research (Grant 21591622) and unrestricted funding from Zeria Pharmaceutical Co., Ltd. 877
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However, these medical approaches have not been sufficiently effective and have had only a limited benefit. Therefore, the search for a more effective therapy continues. Polaprezinc (Zeria Pharmaceutical, Tokyo, Japan), an antiulcer drug, is a chelate compound consisting of a zinc ion, L-carnosine, a b-alanine dipeptide, and L-histidine. Its antioxidant effects have been reported in previous studies (12–14). The purpose of the present study was to standardize the experimental animal model of radiation-induced rectal disorders and to examine the efficacy of polaprezinc to prevent radiation-induced rectal disorders. METHODS AND MATERIALS Preliminary study A total of 14 Wistar rats (5 weeks old) were used in the preliminary study. To confirm the validity of the technique to irradiate the rectum in a vertical position, Gastrografin was injected into a rat anus under pentobarbital anesthesia, and X-rays were performed in the vertical position. In addition, to confirm the adhesiveness of the ointment to the rectum in the rat, ointment containing barium sulfate was rectally administered to another rat. X-rays were then taken twice after application of the ointment, immediately after administration and 10 minutes after administration. To verify that the damage to the mucosa from the rectal administrations was minimal, the catheter to administer the ointment was inserted into the rectum daily for 5 days using 6 rats, and the mucosal changes were compared macroscopically and pathologically with those from the other 6 rats that had not undergone any procedures. The rats enrolled in the preliminary study were excluded from the present study.
Rats A total of 54 female Wistar rats, weighing 90–120 g at 5 weeks of age were obtained from CLEA Japan (Tokyo, Japan). All rats were acclimated for 7 days. They were housed 3/cage and fed with a laboratory rodent pellet formula and tap water ad libitum. The Hyogo College of Medicine Institutional Animal Care and Use Committee approved all animal procedures before the initiation of the project. The rats were assigned to one of three groups as follows. Group A contained 21 rats treated with polaprezinc. Group B included 12 rats treated with base alone (without polaprezinc). Group C contained 21 rats treated without any medication. The homogeneity of the groups was confirmed by analyzing the body weights using Bartlett’s test (p = .210).
Irradiation Each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (46.8 2.1 mg/kg) and weighed. Next, 3 rats at a time were restrained and taped by the tail to an acryl plate in the vertical position. Lead shielding was used to cover the rat, except for a 2.5-cm-long area of the lower pelvis containing the rectum in the middle of the field. All rats were irradiated with a single X-ray fraction of 25 Gy, at 4 MV, with a dose rate of 200 monitor units/min. No filtration was used in the present study. Several rats were selected as samples to confirm the consistency between the actual dose and the delivered monitor units. The absorbed X-ray dose in the rectum
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was measured using a semiconductor detector inserted into the rectum, and the delivered monitor units were then revised, as appropriate.
Treatment with ointment Polaprezinc was prepared as an ointment with a compound of white petrolatum and a purified lanolin base. Group A (polaprezinc) was irradiated and treated with ointment that included polaprezinc at 100 mg/kg/body weight. Group B (no polaprezinc) was irradiated and treated with ointment consisting of base alone, containing white petrolatum and purified lanolin without polaprezinc. Finally, Group C (control) was irradiated and was not treated with any medication. The ointment was administrated to the rat anus without any anesthesia daily after irradiation. A syringe (1 mL) with a catheter that had been especially designed for the present study was used to administer the ointment.
Evaluation of rectal damage Each rat was observed daily for signs of proctitis, such as diarrhea and rectal bleeding, after irradiation. The clinical findings were scored as follows: 0, no symptoms; 2, diarrhea; and 4, gross bleeding. All rats were killed on the 10th day after irradiation and weighed. Colonoscopy was performed on the rats using a 4-mm-diameter laryngendoscope immediately after death. The rectal lumen images of the rats were obtained using a digital camera. The endoscopic findings were evaluated and graded using a method previously described by Northway et al. (15) as follows: 0, normal mucosa; 1, edema, mild hyperemia, or decrease vascularity; 2, diffuse hyperemia, multiple punctuate areas of hemorrhage, or confluent areas of hemorrhage; 3, the presence of erosion or frank hemorrhage; and 4, ulcers. Mild findings were defined as Grade 0 and 1 and severe findings as Grade 2–4. The distal rectum was excised for pathologic evaluation after colonoscopy and immediately fixed in 10% neutral buffered formalin solution. The colon was sectioned, divided into two or three equal segments (from the proximal rectum to the anus), and submitted for histologic analysis. All slides were stained with hematoxylin-eosin and examined using light microscopy by a pathologist who was unaware of the treatment groups. The severity of proctitis in each specimen was evaluated by the pathologic examination findings. The morphologic mucosal damage, degree of inflammation, and depth of inflammation are all listed in Table 1. The areas of morphologic mucosal damage in the rectum were evaluated mainly by the crypt changes found and graded. Mild findings were defined as Grades 0, 1, and 2 and severe findings as Grades 3 and 4. The degree of inflammation was evaluated by investigating the density of inflammatory cell infiltration, according to the submucosal edema present and the destruction of the muscularis propria. The degree of inflammation was graded as listed in Table 1. In addition, mild findings were defined as Grades 0–3 and severe findings as Grade 4. The depth of inflammation was evaluated and graded according to the depth of inflammatory cells infiltration into the rectal mucosa. Mild findings included Grades 0–2 and severe findings Grades 3 and 4. In addition, the apparent loss of the columnar shape on microscopic examination was measured, and the length of the apparent
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Table 1. Grading of pathologic changes on microscopic examination Score
Description
Morphologic mucosal damage 0 Normal or minor alterations that could not be ascribed with certainty to radiation 1 Slight crypt change without loss of epithelium 2 Crypt change with loss of epithelium equaling less than one-half 3 Crypt change with loss of epithelium greater than one-half 4 Loss of epithelium through muscularis mucosa Degree of inflammation 0 Normal mucosa 1 Minor alterations 2 Apparent inflammation (mild inflammation) 3 More significant inflammation (moderate inflammation) 4 Severe inflammation Depth of inflammation 0 No apparent inflammation 1 Inflammatory cells extend up to edge of mucosa (epithelium, lamina propria) but not beyond 2 Inflammatory cells extend through mucosa and into submucosa 3 Inflammatory cells infiltrate through submucosa and extends into muscularis propria 4 Inflammatory cells extend through muscularis propria into subserosa mucosal change was recorded to estimate the craniocaudal length of inflammation.
Statistical analysis The results are presented as the mean standard deviation. The results of the mucosal damage evaluation were compared according to milder or more severe status, as applicable. The relationship among the groups was assessed using the chi-square test. p Values of < .05 were considered statistically significant. Tukey’s allpairwise-comparison test was used to identify differences in the length of inflammation among the three groups.
RESULTS The findings of the enema study showed that the distal colon and sigmoid colon moved outside of the lower pelvis, and the length of the rectum was approximately $3 cm in our rat model (Fig. 1). The spreading of the barium ointment was limited to the rectal region 10 minutes after administration, as well as immediately after administration. No apparent differences were observed between the rats with and without insertion of the catheter or between the rectum and more proximal colon. No irradiated rats died during the follow-up period. The body weight on the 10th day after irradiation was 151.7 9.9 g in Group A, 153.6 7.4 g in Group B, and 158.1 8.5 g in Group C. No significant difference was found in the weight among the three groups. The clinical findings of proctitis were diarrhea, including mucus and pollution around the anus, and gross bleeding
Fig. 1. Findings from enema study. Distal colon and sigmoid colon moved outside low pelvis and rectal length was approximately 3 cm in vertical position.
(Fig. 2). The results of the clinical findings are listed in Table 2. The proportion of Grade 0 (no symptoms) was 19.0% in Group A, 16.7% in Group B, and 4.8% in Group C. Group A had a greater frequency of Grade 0 than did the other groups; however, the difference was not significant. The results of the endoscopic findings are shown in Fig. 3 and Table 3. Only in Group A, Grade 0 results were observed. The proportion of mild changes in the endoscopic findings was 71.4% in Group A, 25.0% in Group B, and 14.3% in Group C. Group A showed significantly milder endoscopic findings than did the other two groups (p < .05). One specimen from Group A was lost in the fixation process. Apparent mucosal changes were observed in the pathologic examinations of all the specimens (Fig. 4). The epithelial changes included crypt architectural distortion with cryptitis, characterized by an infiltration of inflammatory cells. Also, the columnar shape was lost to planarization. Varying levels of loss of the epithelium were seen. Severe changes were characterized by the loss of epithelium through the muscularis mucosa and mucosal ulceration.
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Fig. 2. Clinical findings. (a) Grade 0, no symptoms; (b) Grade 2, diarrhea (included effusion around anus); (c) Grade 4, gross bleeding.
Inflammatory cells included neutrophils, lymphocytes, and, characteristically, eosinophils. The density of the inflammatory cells became greater and they had migrated deeper, with an increasing severity of inflammation. Other prominent changes included varying degrees of edema in the submucosa, degeneration of the muscle fibers, and thickening of the vessel walls. These pathologic findings were limited to the distal rectum, and the columnar shape remained outside the irradiated field (Fig. 5). The findings regarding the presence of morphologic mucosal damage are listed in Table 4. The morphologic mucosal damage ranged from Grade 2 to 4 in all groups. The proportion of low grades was 80.0% in Group A, 58.3% in Group B, and 42.9% in Group C. No significant differences were observed; however, Group A tended to have milder damage than the other two groups. The results regarding the degree of inflammation are listed in Table 5. The degree of inflammation in Groups A and B ranged from Grades 2 to 4. However, in Group C, no Grade 0–2 results were seen regarding the degree of inflammation. The proportion with a mild degree of inflammation was 85.0% in Group A, 41.7% in Group B, and 42.9% in Group C. Group A showed significantly milder damage than did the two other groups in the degree of inflammation (p < .05). The results of the depth of inflammation are listed in Table 6. Group A only showed Grade 1 in the depth of inTable 2. Clinical findings
Characteristic Grade 0 Grade 2 Grade 4 Mild (Grade 0) Severe (Grade 2, 4)
Group A (PZ+; n = 21)
Group B (PZ; n = 12)
Group C (control; n = 21)
4 (19.0) 16 (76.2) 1 (4.8) 4 (19.0) 17 (81.0)
0 (0.0) 10 (83.3) 2 (16.7) 0 (0.0) 12 (100.0)
1 (4.8) 19 (90.5) 1 (4.8) 1 (4.8) 20 (95.2)
Abbreviation: PZ = polaprezinc. Data presented as numbers, with percentages in parentheses. No significant difference found among 3 groups (p < .05).
flammation. The proportion with a shallow depth of inflammation was 50.0% in Group A, 33.3% in Group B, and 4.8% in Group C. Group A had significantly milder damage than Group C (p < .05). The average length of crypt damage was 15.6 3.8 mm in Group A, 15.6 3.0 mm in Group B, and 18.1 3.5 mm in Group C. No apparent differences were seen among the three groups. DISCUSSION Radiotherapy is a well-established treatment of pelvic malignancies such as prostate cancer, and proctitis is a common adverse event. Experimental animal models of radiation-induced gastrointestinal toxicities have been described in previous studies (15–28). However, irradiation plus differing surgical techniques could have produced different results. Hubmann (16) originally described a method to irradiate the rectum of the rat without surgical procedures and with minimal exposure to other organs. This technique has been used in several reports (15, 17–20). Kiszel et al. (17) reported that the kidney and proximal ureters move well outside the irradiated field on vertical positioning with retrograde pyelography. The present enema study showed that the distal colon and sigmoid colon moved outside the lower pelvis, and the length of the rectum was approximately 3 cm. According to the imaging studies, this method seems to make it possible to perform selective irradiation of the rectum in the rat while inducing minimal damage to other organs. In addition, the length of the apparent morphologic changes was measured, and the mean value for all groups was 16.6 3.6 mm (no apparent differences were observed among the three groups regarding the length of crypt damage). Therefore, this irradiation technique allows for selective irradiation to the distal colon without damage to other organs. Kan et al. (18) reported that the erosion of the surface epithelium, which started on the sixth day after irradiation, progressed to multifocal ulceration with severe inflammation until the 10th day after irradiation, with healing of the
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Fig. 3. Endoscopic findings. (a) Grade 1, edema and d mild hyperemia found on the rectal mucosa; (b) Grade 3, apparent erosion present.
ulceration with extensive regenerative changes on the 14th day after irradiation. Northway et al. (15) and Korkut et al. (19) reported that apparent mucosal damage occurred on the 10th day after irradiation. The present study evaluated the mucosal changes on the 10th day after irradiation. As a result, apparent mucosal changes (i.e., loss of epithelium, apparent extension of inflammation) were observed in all three groups. However, Group A showed milder mucosal changes and inflammation than the other two groups. These findings have demonstrated that the suitable day to evaluate irradiation-induced rectal damage is the 10th day after irradiation. The pathologic findings were consistent with those from previous reports (15–23) and were also consistent with the pathologic findings of radiation proctitis in the human rectum (29). The mucosal damage in the rectum was evaluated pathologically to investigate both the morphologic changes and inflammation. The morphologic changes included a loss of
Table 3. Endoscopic findings
Characteristic Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0–1) Severe (Grade 2–4) Chi-square test p value (p < .05)
Group A (PZ+ n = 21)
Group B (PZ; n = 12)
Group C (control; n = 21)
6 (28.6) 9 (42.9) 4 (19.0) 1 (4.8) 1 (4.8) 15 (71.4)
0 (0.0) 3 (25.0) 3 (25.0) 6 (50.0) 0 (0.0) 3 (25.0)
0 (0.0) 3 (14.3) 12 (57.1) 3 (14.3) 3 (14.3) 3 (14.3)
6 (28.6)
9 (75.0)
18 (85.7)
PZ+ vs. PZ
PZ+ vs. control .0006
PZ vs. control NS
.03
Abbreviations: PZ = polaprezinc, NS = not significant. Data presented as numbers, with percentages in parentheses.
columnar shape and epithelial organization. Other changes were also observed, including submucosal edema, degeneration of the muscle fibers, and thickening of the vessel walls. However, it was difficult to perform a quantitative analysis to evaluate the mucosal damage using these changes. Therefore, these findings were not used for the analysis in the present study. Despite the identical administration techniques in Groups A and B, Group B still exhibited milder rectal damage than Group C. The base treatment consisted of white petrolatum and purified lanolin without any additional agents, and this alone seemed to have a certain level of effectiveness in healing the mucosa. Therefore, our study has shown that it is possible to safely administer the ointment into the rectum. The present method has demonstrated that it is possible to administer agents into the rat anus both safely and appropriately. The rats that received polaprezinc (Group A) tended to show milder mucosal damage than the other groups for all criteria of the present study. In addition, significant differences were observed between Groups A and C regarding the degree (p < .05) and the depth (p < .01) of inflammation. In addition, Group A showed greater efficacy than Group B in the present study. Thus, polaprezinc was confirmed to have additive benefits compared with the administration of the base alone. Polarezinc [N-(3-aminopropionyl)-L-histidinatozinc], an antiulcer agent, is a chelated compound consisting of zinc ion and L-carnosine. This agent has not only prevented gastric mucosal lesions in various experimental models but has shown a healing promoting effect on gastric ulcers. The latter action can be accounted for owing to antioxidant properties, stimulation of mucus production, and maintenance of the gastric mucosal barrier (12–14, 30). Yoshikawa et al. (12) reported the antioxidant activities of polaprezinc, such as the superoxide scavenging activity, inhibition of superoxide protection, inhibition of the Fenton reaction, hydroxyl radical scavenging activity, inhibition of lipid peroxidation, and singlet oxygen quenching in vitro. In addition, this compound’s
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Fig. 4. Pathologic findings. (a) Surface epithelium showed crypt architectural distortion, characterized by prominent flattening of columnar cells and invasion of inflammatory cells. In addition, significant mucosal edema observed in lamina propria. (b) Severe mucosal damage (Grade 4), with loss of epithelium reaching submucosa through muscularis mucosa and leading to ulceration. (c) Grade 4 severity in degree of inflammation. Density of inflammatory cell infiltration very high, with destruction of muscularis propria also observed. In addition, inflammatory cells extended into muscularis propria for Grade 3 severity in depth of inflammation.
pharmacologic potency has been demonstrated by examining its adherence to the mucosa (31). Matsuu-Matsuyama et al. (22) treated rats that had received whole body X-ray irradiation with oral polaprezinc. Ohkawara et al. (32) reported that polaprezinc can be administered intrarectally to combat dextran sulfate-induced colitis in mice. However, no studies, to date, have reported the rectal administration of polaprezinc to reduce radiation-induced intestinal injury. Oxygen radicals in radiation-induced gastrointestinal disorders have been previously reported (33). The rectal administration of polaprezinc could therefore have greater efficacy against mucosal damage compared with peroral administration. No significant differences were seen regarding the morphologic mucosal damage; however, Group A did show a milder degree of damage and also, apparently, lower grades of inflammation than the other two groups. These results might be attributable to the anti-inflammatory effects of polaprezinc. Therefore, polaprezinc was found to exhibit an apparent efficacy against radiation-induced rectal disor-
ders. These data raise the possibility that the administration of polaprezinc could reduce the incidence of severe radiation-induced proctitis. In the present study, the administration of polaprezinc was performed after irradiation to irradiate all rats equally. However, the administration of polaprezinc as a premedication before irradiation might make both the anti-inflammatory effect and the efficacy stronger than when used alone after irradiation. Therefore, the optimal timing to administer polaprezinc should be investigated further to enhance the efficacy of this treatment modality. Chronic radiation injury is thought to result from tissue ischemia secondary to thrombogenesis induced by thrombin formation in the microvessels. Ulceration, rectal obstruction, and carcinogenesis in the rat intestine have been reported as late adverse effects 3–14 months after irradiation, according to long-term observations (16, 17, 23–28). A long-term observation study after irradiation is presently underway to establish an experimental rat model of radiation-induced rectal disorders and late adverse events and also to assess the efficacy of polaprezinc against late rectal toxicity.
Table 4. Morphologic mucosal damages on microscopic examination
Characteristic Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0–2) Severe (Grade 3-4) Fig. 5. Measurement of apparent loss of columnar shape. Crypt architectural distortion limited to distal rectum, with columnar shape remaining outside irradiated field.
Group A (PZ+; n = 20)
Group B (PZ; n = 12)
Group C (control; n = 21)
0 (0.0) 0 (0.0) 16 (80.0) 2 (10.0) 2 (10.0) 16 (80.0)
0 (0.0) 0 (0.0) 7 (58.3) 2 (16.7) 3 (25.0) 7 (58.3)
0 (0.0) 0 (0.0) 9 (42.9) 3 (14.3) 9 (42.9) 9 (42.9)
4 (20.0)
5 (41.7)
12 (57.1)
Abbreviation: PZ = polaprezinc. Data presented as numbers, with percentages in parentheses. No significant differences found among 3 groups (p < .05).
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Table 5. Degrees of inflammation on microscopic examination
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Table 6. Depth of inflammation on microscopic examination
Characteristic
Group A (PZ+; n = 20)
Group B (PZ; n = 12)
Group C (control; n = 21)
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0–3) Severe (Grade 4) Chi-square test
0 (0.0) 0 (0.0) 6 (30.0) 11 (55.0) 3 (15.0) 17 (85.0) 3 (15.0) PZ+ vs. PZ
p value (p < .05)
.03
0 (0.0) 0 (0.0) 1 (8.3) 4 (33.3) 7 (58.3) 5 (41.7) 7 (58.3) PZ+ vs. control .01
0 (0.0) 0 (0.0) 0 (0.0) 9 (42.9) 12 (57.1) 9 (42.9) 12 (57.1) PZ vs. control NS
Abbreviations as in Table 3. Data presented as numbers, with percentages in parentheses.
CONCLUSIONS Polaprezinc has therefore been proved to be effective against radiation-induced rectal disorders in a rat model.
Characteristic
Group A (PZ+; n = 20)
Group B (PZ; n = 12)
Group C (control; n = 21)
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Mild (Grade 0, 1, 2) Severe (Grade 3, 4) Chi-square test
0 (0.0) 2 (10.0) 8 (40.0) 0 (0.0) 10 (50.0) 10 (50.0) 10 (50.0) PZ+ vs. PZ
0 (0.0) 0 (0.0) 4 (33.3) 0 (0.0) 8 (66.7) 4 (33.3) 8 (66.7) PZ+ vs. control .004
0 (0.0) 0 (0.0) 1 (4.8) 4 (19.0) 16 (76.2) 1 (4.8) 20 (95.2) PZ vs. control NS
p value (p < .05)
NS
Abbreviations as in Table 3. Data presented as numbers, with percentages in parentheses.
The present study demonstrated an anti-inflammatory effect of polaprezinc against acute radiation induced proctitis using an experimental rat model.
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