Inflammatory Bowel Diseases®
`7(3):221 -225 0 2001 Crohn's & Colitis Foundation of America, Inc.
`
`Measurement of Colonic Mucosal Concentrations of
`5-Aminosalicylic Acid Is Useful for Estimating Its Therapeutic
`Efficacy in Distal Ulcerative Colitis: Comparison of Orally
`Administered Mesalamine and Sulfasalazine
`
`*Makoto Naganuma, -Yasushi Iwao, *Haruhiko Ogata, *Nagamu Inoue, *Shinsuke Funakoshi,
`*Shojirou Yamamoto, *Yuji Nakamura, *Hiromasa Ishii, and *fToshifumi Hibi
`
`*Department of Internal Medicine, School of Medicine, Keio University, Tokyo; and tKeio Cancer Center, Tokyo, Japan
`
`Summary: Objectives: Oral 5-aminosalicylic acid (5-ASA)
`preparations have been used frequently in the treatment of ul-
`cerative colitis. However, there have been few reports investi-
`gating the relationship between colonic mucosal concentrations
`of 5-ASA and its clinical efficacy when oral sulfasalazine or
`5-ASA compounds were administered. The aim of this study is
`to compare the mucosal concentrations of 5-ASA ensured by
`sulfasalazine or mesalamine, and to define the clinical signifi-
`cance of the measurement of 5-ASA concentrations in the treat-
`ment of distal ulcerative colitis. Materials and Methods: Biop-
`sies were taken from the rectum and sigmoid colon of the oral
`sulfasalazine group (n = 13) and the slow-release 5-ASA (me-
`salamine) group with (n = 5) or without (n = 11) rectal
`administration of 5-ASA. High-pressure liquid chromatogra-
`phy was used to measure the tissue concentrations of 5-ASA
`and its metabolites. We compared the 5-ASA concentrations of
`the sulfasalazine group with the mesalamine group. Further-
`
`more, we analyzed the relationship between tissue 5-ASA con-
`centrations and the Disease Activity Index (DAI). Results: The
`concentrations of 5-ASA and acetyl-5-ASA in the sulfasalazine
`group were higher than those in the group taking oral mesala-
`mine alone (p < 0.01). The concentration of 5-ASA was much
`higher in the patients who received oral and rectal mesalamine
`in an enema than in the patients who had oral mesalamine
`alone. There was a significant inverse correlation between the
`DAI and concentrations of 5-ASA in the rectum (r = 0.712, p
`< 0.001). Conclusions: We demonstrated that the colonic mu-
`cosal concentration of 5-ASA was significantly higher in the
`sulfasalazine group than in the mesalamine group. Further-
`more, the concentrations of mucosal 5-ASA may be a good
`marker for the estimation of its efficacy in the treatment of
`ulcerative colitis. Key Words: 5-Aminosalicylates, mucosal
`concentration—Ulcerative colitis, distal—Aminosalicylates.
`
`INTRODUCTION
`
`In ulcerative colitis (UC), no fundamental therapy has
`been established because the etiology of this disease re-
`mains unknown. Corticosteroids and sulfasalazine have
`been used as conventional treatment. In addition, mesala-
`mine (a formulation of 5-aminosalicylic acid [ASA]),
`which is marketed in a variety of controlled-release
`forms to release the active ingredient 5-ASA near the
`affected area in the large intestine, is widely used. Oral
`
`Received November 16, 2000; accepted May 18, 2001.
`Address correspondence and reprint requests to Dr. T. Hibi, Depart-
`ment of Internal Medicine, School of Medicine, Keio University, 35
`Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: thibi@
`mc. med.keio.ac.jp
`
`mesalamine is effective for not only UC but also Crohn's
`disease (CD) with ileal and/or colonic involvement. Fur-
`thermore, due to minimal side effects, it is also used to
`maintain remission. In comparative studies of mesala-
`mine and sulfasalazine, oral delayed-release mesalamine
`was reported to be as effective as sulfasalazine in the
`maintenance of remission (1).
`However, recent studies have reported that the com-
`bination of oral mesalamine tablets plus mesalamine ad-
`ministered by suppositories or in enemas is more effec-
`tive than oral mesalamine tablets alone (2,3). Currently,
`numerous clinicians prefer this combination therapy to
`oral mesalamine alone for distal UC. It is supposed that
`the lower mucosal concentration of the effective ingre-
`dient in the distal colon could be the reason for the lower
`
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`M. NAGANUMA ET AL
`
`observed efficacy of oral mesalamine therapy. However,
`there have been few studies (4,5) investigating the co-
`lonic mucosal concentration of the 5-ASA ingredient and
`its participation in the therapeutic effect of 5-ASA com-
`pounds.
`The present study was undertaken to clarify the rela-
`tionship between the mucosal concentration of 5-ASA
`and the disease activity index (DAI) and to compare the
`amounts of intramucosal 5-ASA ensured by oral sul-
`fasalazine or mesalamine. In addition, a rationale for
`choosing rectal administration of 5-ASA was evaluated
`by the measurement of mucosal 5-ASA concentration.
`
`PATIENTS AND METHOD
`
`Twenty-four patients with distal UC taking 3 g per day
`of oral sulfasalazine (Salazopyrin; Yoshitomi Pharma.
`Co., Ltd., Tokyo, Japan) (n = 13) or 2.25 g per day of
`oral slow-release mesalamine (Pentasa; Nisshin-Kyorin
`Pharma. Co., Ltd., Tokyo, Japan) (n = 11) (11 male and
`13 female, age range, 22-58 years) were enrolled in this
`study. There were five additional patients who received
`both oral mesalamine and 5-ASA in an enema containing
`1 g of mesalamine (1 g/100 m1). The current therapy had
`been continued for more than 2 weeks before colonos-
`copy. Histories of salicylate allergy, previous bowel re-
`section, or hepatic or renal disease were grounds for
`exclusion. The patients with inflammation beyond
`splenic flexure at the time of this study procedure were
`also excluded. None were receiving steroids or immuno-
`suppressants. Informed consent was obtained from each
`patient. The study was held from January 1998 to Feb-
`ruary 1999.
`Three biopsied specimens from the sigmoid colon and
`rectum were collected by the same colonoscopist. To
`minimize any possible effects on the assay, colonoscopy
`was performed without pretreatment (an intestinal
`cleansing). On the day of the examination, oral admin-
`istrations of mesalamine and sulfasalazine were given as
`usual. The last dosage of oral mesalamine or sulfasala-
`zine was taken between 7:00-9:00 a.m. The last 5-ASA
`enema was given at night (8:00-11:00 p.m.), before the
`day of colonoscopy.
`High-pressure liquid chromatography (HPLC) was
`performed to measure the concentrations of 5-ASA and
`its metabolite, acetyl-5-ASA. We modified the method
`as previously described by Hogezand et al. (6). Briefly,
`the wet weight of the biopsied specimens was measured
`and the entire amount was placed in a Teflon potter-type
`homogenizer. Then 1 mL of normal saline solution was
`added, and the mixture was homogenized for 20 minutes
`in an ice bath. Furthermore, 0.4 mL of the samples con-
`
`lnJlammcaory Bowel Diseases®, Vol. 7, No. 3, August 2001
`
`taining methanol were mixed and centrifuged. Propionic
`anhydride (20 µ1) were added to the supemate (37°C, 60
`min). After 0.2 mL of NaOH (2N) and HC1 (2N) were
`added, centrifugation was performed. The supemate was
`filtered through a 0.45 p.,m disposable filter, and 40 µ1 of
`the fluid were injected into the HPLC apparatus. HPLC
`analysis was performed on a Cosmosil 5C18-AR chro-
`matograph (Nakaraitesuku Co., Kyoto, Japan) equipped
`with a 6 x 250 mm column. Detection was performed
`using a fluorescence monitor, with excitation set at 310
`nm and emission at 410 nm. The mobile phase consisted
`of deionized water adjusted to pH 2.5 with 0.01M
`Na2HPO4 and methanol. A flow rate of 0.8 ml/min was
`established. The detection limit for 5-ASA concentration
`was 0.1 µg/g.
`Disease activity index values were also calculated.
`Four variables having a range of values for severity, with
`0 representing no abnormality and 3 representing the
`most severe (7), were used. SAS computer software
`(SAS, Inc., Cary, NC, U.S.A.) was used for the statistical
`analysis of the data. The data was analyzed using the
`Mann-Whitney U test, the X2 test, and Pearson's corre-
`lation coefficient with a statistical level of significance of
`5% or less.
`
`RESULTS
`Concentration of 5-ASA and Its Metabolites in the
`Sigmoid Colon and Rectum
`
`Table 1 shows the clinical characteristics of the 29
`patients receiving oral sulfasalazine or mesalamine in
`whom the mucosal concentrations were measured. The
`average DAI in the mesalamine group tends to be higher
`than those in the sulfasalazine group; however, there was
`no significant difference.
`Rectal concentrations of 5-ASA, acetyl-5-ASA, and
`total 5-ASA (5-ASA plus acetyl-5-ASA) were signifi-
`cantly higher in the sulfasalazine group (19.3 ± 5.5 µg/g,
`
`TABLE 1. Clinical characteristics of the sulfasalazine and
`mesalamine groups in ulcerative colitis
`
`Sutfasalazine
`(n = 13)
`
`Mesalamine
`oral alone
`(n = 11)
`
`Mesalamine
`oral + enema
`(n = 5)
`
`Age (yr)
`Sex (M:F)
`Type
`One attack
`Relapse-remission
`Chronic continuous
`Location of disease
`Proctitis
`Left-sided colitis
`Disease Activity Index
`
`41.0
`4:9
`
`2(18%)
`9 (69%)
`2 (18%)
`7 (54%)
`
`40.7
`8:3
`
`1 (9%)
`9 (82%)
`1 (9%)
`6 (55%)
`
`35.2
`2:3
`
`0 (0%)
`4 (80%)
`1 (20%)
`2 (40%)
`
`6 (46%)
`4.1 t 2.5
`
`5 (45%)
`5.8* 2.9
`
`3 (60%)
`4.6 *3.2
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`MEASUREMENT OF MUCOSAL 5-ASA CONCENTRATION
`
`223
`
`30.1 ± 9.0 µg/g, and 49.4 ± 12.3 µg/g, respectively) in
`comparison with those in the group taking oral mesala-
`mine alone (0.7 ± 0.6 µg/g, 5.8 ± 3.1 µg/g, and 6.6 ± 3.6
`µg/g, respectively) (p < 0.01) (Fig!). Similarly, those
`concentrations in the sigmoid colon were higher in the
`sulfasalazine group than in the group taking mesalamine
`alone (p < 0.05) (Fig. 1B). Furthermore, the concentra-
`tion of total 5-ASA was much higher in the patients who
`received oral and rectal mesalamine (110.4 ± 77.0 µg/g
`[rectum], 7.3 ± 26.7 µg/g [sigmoid colon]) than in the
`patients who had oral mesalamine alone (6.6 ± 3.6 µg/g
`[rectum], 22.2 ± 10.3 µg/g [sigmoid colon]) (Fig. 2A and
`B) (p < 0.05, respectively).
`
`Relationship Between Mucosal 5-ASA
`Concentrations and Clinical Activity
`
`Rectal concentration of 5-ASA in the patients without
`blood in stool (56.3 ± 17.8 µg/g) was significantly higher
`than those with blood in stool (9.8 ± 5.4 µg/g) (p < 0.01)
`as shown in Figure 3. There was a significant inverse
`correlation between the DAI and concentrations of
`5-ASA in the rectum (r = 0.712, p < 0.001) (Fig. 4).
`
`DISCUSSION
`
`In the present study, we demonstrated that the colonic
`mucosal concentration of 5-ASA was significantly
`higher in patients with UC receiving sulfasalazine as
`compared with those receiving mesalamine. Sulfasala-
`zine is composed of 5-ASA joined by an azo bond to
`sulfapyridine. Colonic bacteria split the azo bond to re-
`lease 5-ASA, the active ingredient, into the colonic lu-
`men. 5-ASA is not absorbed from the colon; it appears to
`have a local therapeutic effect by acting intraluminally
`
`(8). On the other hand, oral 5-ASA exhibits maximum
`efficacy in the small bowel or right colon (5,9). Conse-
`quently, as we indicated in the current study, the mucosal
`concentration of 5-ASA and its metabolite in the rectal
`mucosa of the sulfasalazine group was higher than that of
`the mesalamine group.
`There are some investigations on concentrations of
`5-ASA and its metabolites in serum, urine, and feces
`(6,10,11). Relay described that oral dose loading results
`in an increase in serum, urine, and feces (12). However,
`increasing the oral dose does not produce a therapeutic
`effect in UC (12,13). Mucosal tissue concentration of
`5-ASA may be an important determinant of therapeutic
`response, as antiinflammatory effect of 5-ASA is thought
`to be dose related (14). Hussain et al. (15) described that
`rectal mucosal concentrations of aminosalicylates were
`lower at relapse. More recently, Frieri et al. demonstrated
`the relationship between colonic mucosal concentrations
`of 5-ASA and activity of UC (4,5). Our results also in-
`dicated that there was an inverse correlation between the
`DAI and mucosal concentrations of 5-ASA, and that the
`mucosal 5-ASA concentration was low in patients with
`active disease. Taken together, mucosal inflammation
`may flare up because of low 5-ASA concentration. The
`duration of active inflammation may arise from low con-
`centrations of 5-ASA. Alternatively, low 5-ASA concen-
`tration may reflect the influence of active lesions such as
`ulcers, or the secondary loss of absorption ability due to
`the presence of active lesions.
`Low rectal concentrations of 5-ASA in the group tak-
`ing oral mesalamine alone support recent data of the
`efficacy of 5-ASA when administered in an enema (16).
`Actually, our results indicate that the mucosal concen-
`trations of 5-ASA following enema administration were
`
`Rectum
`
`Sigmoid colon
`
`(p g/g)
`
`(pg/g)
`
`<1! (cid:9)
`
`1 40
`
`1 ' -
`
`1110
`
`201
`
`60
`
`40
`
`20
`
`11
`
`Sulfasalazine Nlesalamine
`
`11
`
`Sulfasalazine Mesalamine
`
`FIG. 1. Mucosal concentrations of
`5-ASA and acetyl-5-ASA (total
`5-ASA) in the rectum (A) and sigmoid
`colon (B). The concentrations in the
`sulfasalazine group (n = 13) was sig-
`nificantly higher (**p <0.01, "p < 0.05,
`respectively) than in the group taking
`mesalamine alone (n = 11).
`
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`
`M. NAGANUMA ET AL
`
`Rectum
`
`'igmoid colon
`
`p/g i
`
`(p g/g)
`
`FIG. 2. Mucosal concentrations of
`5-ASA and acetyl-5-ASA (total
`5-ASA) in the rectum (A) and sigmoid
`colon (B). The concentrations in the
`oral and rectal administration (en-
`ema) group (n = 5) was significantly
`higher ("p < 0.05) than in the group
`taking mesalamine alone (n = 11).
`
`Mesalamine Mesalamine
`p.o. alone (cid:9)
`p.o.+enema B
`
`Mesalamine Mesalamine
`p.o. alone (cid:9)
`p.o.+ enema
`
`higher than those of oral mesalamine alone and equiva-
`lent to those of oral sulfasalazine. One study reported
`that mucosal concentrations of 5-ASA in the rectum
`were significantly higher in patients receiving oral plus
`topical mesalamine than in those receiving oral mesala-
`mine alone (5). Our results confirm these data and sup-
`port that mesalamine administered in an enema has the
`same efficacy as sulfasalazine with minimal side effects
`during the active stage of distal UC (16). Another report
`has revealed that the combination of mesalamine admin-
`
`istered in an enema and orally is more effective than oral
`administration of mesalamine alone (2). Our results,
`showing that the rectal concentration of 5-ASA in pa-
`tients without blood in stool was higher than those in
`patients with blood in stool, supported the hypothesis
`that increasing the mucosal 5-ASA concentration in the
`distal colon is important in achieving clinical effect. The
`current study suggests that lower mucosal concentrations
`of 5-ASA may be associated with inefficacy of oral sul-
`fasalazine or mesalamine and a risk factor for relapse.
`
`(p g/ g)
`
`**
`
`CONCLUSION
`
`In conclusion, we demonstrated higher concentrations
`of 5-ASA provided by oral sulfasalazine and rectal ad-
`ministration of 5-ASA as compared with oral mesala-
`
`(+) (cid:9)
`(-)
`n=13) (cid:9)
`(11.11)
`Blood in stool
`FIG. 3. Rectal concentrations of 5-ASA in patients without blood
`in stool (n = 11) was significantly higher (**p < 0.01) than in those
`with blood in stool (n = 13).
`
`lnJlammatory Bowel Diseases®, Vol. 7, No. 3, August 2001
`
`so (cid:9)
`100 (cid:9)
`150
`Concentration of 5-ASA 0.1 gig)
`FIG. 4. Correlation between rectal mucosal concentrations of
`5-ASA and the Disease Activity Index (DA!).
`
`200
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`MEASUREMENT OF MUCOSAL 5-ASA CONCENTRATION
`
`225
`
`mine, and indicated that the measurement of mucosal
`concentrations may be important in predicting the effi-
`cacy of oral 5-ASA formulations. Our results may pro-
`vide a rationale for choosing 5-ASA agents in the treat-
`ment of distal UC. The development of proper and ef-
`fective measures to maintain sufficient mucosal
`concentrations of 5-ASA may yield better therapeutic
`effects from sulfasalazine and 5-ASA preparations.
`
`REFERENCES
`
`1. Mulder CJ, Tytget GN, Weterman IT, et al. Double-blind com-
`parison of slow-release 5-aminosalicylate and sulfasalazine in re-
`mission maintenance in ulcerative colitis. Gastroenterology
`1988,95:1449-53.
`2. Safdi M, DeMicco M, Sninsky C, et al. A double-blind comparison
`of oral versus rectal mesalamine versus combination therapy in the
`treatment of distal ulcerative colitis. Am J Gastroenterol
`1997,92:1867-71.
`3. D'Albasio G, Pacini F, Camarri E, et al. Combined therapy with
`5-aminosalicylic acid tablets and enemas for maintaining remis-
`sion in ulcerative colitis: a randomized double-blind study. Am J
`Gastroenterol 1997,92:1143-7.
`4. Frieri G, Giacomelli R, Pimpo M, et al. Mucosal 5-aminosalicylic
`acid concentration inversely correlates with severity of colonic
`inflammation in patients with ulcerative colitis. Gut 2000;47:
`410-14.
`5. Frieri G. Pimpo MT, Palumbo GC, et al. Rectal and colonic me-
`salazine concentration in ulcerative colitis: oral versus oral plus
`topical treatment. Aliment Pharmacol Ther 1999;13:1413-17.
`6. Hogezand RA, Balen HCJP, Schaik A, et al. Determination of
`
`sodium azodisalicylate, salazosulphapyridine and their metabolites
`in serum, urine and faeces by high-performance liquid chromatog-
`raphy. J Chromatogr 1984;305:470-6.
`7. Sutherland LR, Martin F, Greer S, et al. 5-aminosalicylic acid
`enema in the treatment of distal ulcerative colitis, proctosigmoid-
`itis, and proctitis. Gastroenterology 1987;92:1894-8.
`8. Stenson WF. Inflammatory bowel disease. In: Goldman L, Bennett
`JC, eds. Cecil Textbook of Medicine. 21st Ed. Philadelphia: W. B.
`Saunders, 2000:722-8.
`9. Bondesen S. Intestinal fate of 5-aminosalicylic acid: regional and
`systemic kinetic studies in relation to inflammatory bowel disease.
`Pharmacol Toxicol 1997;81:1-28.
`10. De Vos M, Verdievel H, Schoonjans R, et al. Concentrations of
`5-ASA and Ac-5-ASA in human ileocolonic biopsy homogenates
`after oral 5-ASA preparations. Gut 1992;33:1338-42.
`11. Stretch GL, Campbell BJ, Dwarakanath AD, et al. 5-aminosalicylic
`acid absorption and metabolism in ulcerative colitis patients re-
`ceiving maintenance sulphasalazine, olsalazine or mesalazine. Ali-
`ment Pharmacol Ther 1996;10:941-7.
`12. Relay SA. What dose of 5-aminosalicylic acid (mesalamine) in
`ulcerative colitis? Gut 1998;42:761-3.
`13. Sutherland LR, Roth DE, Beck PL. Alternatives to sulfasalazine:
`meta-analysis of 5-ASA in the treatment of ulcerative colitis. In-
`flammatory Bowel Diseases 1997;3:65-78.
`14. Greenfield SM. Punchard NA, Teare JP, et al. The mode of action
`of aminosalicylates in inflammatory bowel disease. Aliment Phar-
`macol Ther 1993;7:369-83.
`15. Hussain F, Ajjan R, Grumdman M, et al. Mucosal aminosalicylate
`levels correlate with clinical activity in patients with ulcerative
`colitis [Abstract]. Gut 1997;40:A23.
`16. Kam L. Cohen H, Dooley C. et al. A comparison of mesalamine
`suspension enema and oral sulfasalazine for treatment of active
`distal ulcerative colitis in adults. Am J Gastroenterol
`1996,91:1338-42.
`
`Inflammatory Bowel Diseases®, Vol. 7, No. 3, August 2001
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