Journal of Pharmacy & Pharmaceutical Sciences
Canadian Society for Pharmaceutical Sciences

Sample Publication

"The following is a modified, previously published article which is included as an example only. Xprofen is a fictitious name."

Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgment
References

Chiral Inversion of Xprofen by the Rat Gut

S. Sattari1 and F. Jamali, Faculty of Pharmacy and Pharmaceutical Sciences
University of Alberta, Edmonton, Alberta, Canada, T6G 2N8

Received for publication: June 14, 1996

Correspondence author: Dr. F. Jamali
Faculty of Pharmacy and Pharmaceutical Sciences
University of Alberta, Edmonton, Alberta, Canada, T6G 2N8
E-mail: fjamali@pharmacy.ualberta.ca
Present address, College of Pharmacy, Medical University of Tehran, Tehran, Iran

Abstract

Purpose. In the presence of excised human and rat gut, the pharmacologically inactive R enantiomers of both ibuprofen and fenoprofen are bioinverted to their antiinflammatory antipodes. We attempted to further localize the site of inversion. Methods. We incubated Xprofen, in oxygenated (O2, CO2, 95:5) Krebs-Henseleit solution (37o, pH, 7.4) for 3 h in the presence of the intestinal contents, epithelium and muscular layer of upper jejunum and everted jejunum sack of antibiotic treated (500 mg/kg neomycin and erythromycin b.i.d. for 3 days) and control adult female Sprague-Dawley rats. The formation of S-FN and acylglucuronidated FN was examined in the incubation medium using a stereospecific HPLC assay.  The metabolic activities are reported per g of wet tissue. Results. The extent of inversion by the everted rat gut was substantial (30.7±5.1%) but no significant differences between the control and germ-eradicated rats was observed. The epithelial cells were found to be the major site of inversion in the intestinal wall (37.5±4.7%) with the muscular layer (7.8±2.1%) and intestinal contents (5.7±2.2%) contributing only to a small extent to the process. Both enantiomers were substantially acyl-glucuconjugated in  the epithelial and muscular layers, and the intestinal content. Conclusion. The gut epithelium is efficient in bioinversion of xprofen.

Introduction

The nonsteroidal anti-inflammatory drugs (NSAIDs) are important in the treatment of rheumatoid arthritis and osteoarthritis. 2-arylpropionic acids (e.g. fenoprofen, FN) are a main sub-group of NSAIDs which have an asymmetric carbon. Therefore, they are usually available and used as racemates. However, the antiinflammatory activities of 2-arylpropionate NSAIDs are attributed mainly to the S enantiomer (Reviewed in 1, 2). A unique metabolic pathway of the R enantiomers of some 2-arylpropionate is an unidirectional bioinversion to their active antipode (3). Various sites have been proposed for chiral inversion. The formation of S-ibuprofen after iv administration to humans of R-ibuprofen is a strong evidence of systemic inversion (4) perhaps in the liver as has been shown for xprofen (5) and ibuprofen (6, 7) in the rat. On the other hand, a higher S:R plasma concentration observed after oral administration of ibuprofen oral formulations with slower absorption as compared with rapidly absorbed dosage forms is suggestive of presystemic inversion in the gastrointestinal (GI) tract (8-11). Direct evidence for inversion in the intestine has been provided for ibuprofen in humans (10), and benoxoprofen (12), ibuprofen (11) and xprofen (5) in the rat. In addition, a greater extent of inversion has been reported for xprofen in the serosal as compared with the mucosal side of the rat everted gut (5). This has been suggested to be an indicative of the involvement of the enterocyte of basolateral membrane in the process (5). The objective of this study was to investigate the extent of involvement in the chiral inversion of each of the epithelial and muscular layers of the intestine and the intestinal contents using rat as the animal model and Xprofen as the drug model.

Materials and Methods

Chemicals
Racemate and the enantiomers (>98% stereochemically pure) of XN were gifts from Bio Rule Inc. (Edmonton, Canada). Neomycin sulfate and erythromycin were purchased from Sigma Chemical Company (St. Louis, Mo, U.S.A.). Nutrient Agar and Plate Count Agar were obtained from Difco Laboratories (Detroit, Michigan, U.S.A.) and Becton, Dikinson & Company (Cockeysville, MD, U.S.A.), respectively. Except for the acetonitrile, which was HPLC grade, all other chemicals were analytical grade.

Animals and Treatments
Female Sprague-Dawley rats with body weight of 250-300 g were used.  Animals had free access to water and food ad libitum. They were anesthetized with diethyl ether and through a vertical midline incision, 15 cm of the upper jejunum was excised. To examine the effect of intestinal microflora on the extent of inversion, four female Sprague-Dawley rats received 500 mg/kg b.i.d. of both erythromycin and neomycin sulfate (treated group) or 1 mL of distilled water (control group) orally for 3 days. The sterility of intestinal preparations was tested using two standard microbiological media (nutrient and plate count agar). Under aseptic conditions, samples of the intestinal contents were taken from the end of the excised jejunum using a sterile swab, transferred to the petri dishes containing the media and incubated at 37o for 3 days.

Preparation of Tissues and Intestinal contents
After removal of jejunum (n=4), the intestinal contents were removed using a glass rod. The tissue was flushed with 20 mL of ice-cold isotonic sodium chloride and everted. The clean everted jejunum segments and their contents were incubated separately in the presence of R-XN. For the antibiotic treated rats and their controls, the jejunum was everted and one end was tied off with surgical thread. The formed sack was filled with drug free warm oxygenated (O2, CO2, 95:5) Krebs-Henseleit solution and incubated in an organ bath containing R-XN. The procedure was similar to the method used by Esposito et al (13). The epithelial layer was isolated utilizing both the scraping (14) and shaking methods (15). However, as the results obtained for the two methods were almost identical only those of the shaking method are reported. In a cold room (4o) Jejunum  was cut into 7.5 cm segments and directly everted onto the plastic pegs which were securely placed in a specially designed container filled with 600 mL of continually oxygenated EDTA-Mannitol buffer (pH 7.4, containing 2.5 mM EDTA, 300 mM D-Mannitol and 10 mM Tris-HCl). The container was vibrated at 1400 HZ for 15 min. The formed suspension was centrifuged in 20 mL test tubes at 1500 g for 3 min. The pellets were collected and pooled. The cells and the remaining muscular layer (washed with 20 mL of oxygenated cold Krebs-Henseleit solution) were incubated separately.

In-vitro Incubation and Sampling
Jacketed warm (37o) glass organ baths were filled with 50 mL of continually oxygenated (O2,CO2, 95:5) Krebs-Henseleit solution containing 5mg/L of R-XN. Intestinal contents, isolated epithelial cells (0.85±0.06 g, n=3), gut muscular layer (1.0±0.15 g, n=3) or everted rat jejunum sack (15 cm, 1.2±0.17, n=4), were placed in the organ bath and incubated for 3 h. For everted rat gut sacks, samples were taken from both serosal and mucosal sides. At the end of each experiment, samples were taken, centrifuged at 1500 g for 3 min and the supernatants were placed in acid washed tubes (to prevent spontaneous hydrolysis of conjugated XN) and kept at -20o until analyzed.

Assay and Sample Analysis
Concentrations of R-and S-FN were measured using a stereospecific assay (16). The extent of conjugation was determined by comparing the concentration of enantiomers before and after alkaline hydrolysis (5).

Data Analysis
The observed metabolic activities were corrected for the weight and are reported per g wet weight. Two tailed paired (related samples) and unpaired (unrelated samples) Student's t test were used to test the significance of differences. When comparing more than two means one-way ANOVA followed by Duncan's Multiple Range test were used. The level of significance was set at a=0.05. The results are expressed as mean±standard deviation.

Results

Incubation with jejunum tubes amounted to 30.7±5.1% (per g of wet tissue) chiral inversion. As depicted in (Figure. 1), the epithelial cells showed 37.5±4.7% and the muscular layer exhibited only 7.8±2.1% inversion activities. Incubation of R-XN with intestinal contents, resulted in only 5.7 ± 2.2% inversion to the S enantiomer. Similarly, the everted rat jejunum was efficient in inverting R-IB to S-IB (Figure 2.). Treatment with antibiotics and consequent sterilization of jejunum from microflora did not affect inversion activity (Figure 2.). This was in spite of the fact that after 3 days of microbiological incubation of the jejunal contents, there was no microbial growth in the samples collected from antibiotic treated rats while all of the samples from the untreated rats showed considerable microbial growth. The extent of inversion was significantly greater in the serosal side (control, 39%; treated 38%) as compared with the mucosal side (control, 28%; treated 21%) of jejunum for both control and antibiotic treated rats (Figure 2).

Figure. 1 Percent of chiral inversion of R-(-)-XN (5 mg/mL) by the rat jejunum epithelial cells, muscle layer and intestinal content after 3 h incubation at 37o.  Error bars indicate standard deviation of the mean (n= 3). Mean of epithelium significantly different from other means.

All specimens demonstrated significant glucuronidation activity (Figure 2 and Figure 3). Despite its poor chiral inversion activity, the intestinal content was efficient in acyl-glucuronidation of the limited amount of the inverted S enantiomer (Figure 3).

Figure 2. Chiral inversion and acylconjugation in the serosal and mucosal side of everted jejunum excised from control and antibiotic treated rats following incubation of 5 mg/mL R-XN at 37 o. Key: S and ,R are S and R- XN; S-Glu and R-Glu are S- and R acyl- glucuconjugated XN, respectively. Error bars, standard deviation of the mean (n= 3); a, different from control.

Discussion

Previous reports suggest that xprofen undergoes extensive chiral inversion in both humans (17) and the rat (5). Indeed, chiral inversion is the main pathway of clearance for R-Xprofen. Both liver and GI tract have been shown to be involved in the process of chiral inversion of 2-arylpropionates in general (2), and xprofen, in particular (5). The rat jejunum has been shown to be very efficient in chiral inversion of xprofen (5). This is not unexpected as it has been well documented that the GI tract is capable of performing both Phase I (18) and Phase II (19) metabolic reactions. This, however, can be attributed to the metabolic activity of the gut wall and/or the intestinal microflora. The mucosa of the intestine consists of three distinct layers, i.e., epithelium, lamina propria and muscularis mucosa. The presence of numerous enzymes, including cytochrome-P450, in the brush border barrier of the epithelial cells makes this one-cell-thick layer the most metabolically active one.  Muscularis mucosa, which is a 3 to 10 cell thick layer of smooth muscle, separates the mucosa from submucosa. While, the abundance of enzymes in the areas adjacent to brush border barrier of the epithelial cells are well acknowledged the metabolic activity of muscularis mucosa has not been well studied (20). The high R to S inversion efficiency of the intestinal epithelium coupled with the minimal activity of the muscular layer observed by us suggest that, indeed, the former is the major site of intestinal metabolic inversion.

Figure 3. Chiral inversion and acyl-glucuiniation conjugation of R-XN and acyl-glucurinidation of inverted S-XN after 3 h incubation at 37o in the presence of the rat jejunum epithelial cells, muscle layer and intestinal content.  Error bars indicate standard deviation of the mean (n= 3). For S-XN, the mean of contents significant different from other means. Glu donated acyl-glucuronidate.

In line with a previous report (5), incubation of R-NX in the presence of everted intestinal sack resulted in substantial chiral inversion. Eradication of the intestinal microflora had no effect on the extent of inversion indicating that the microflora do not play an important role in the process. Accordingly, incubation of the R enantiomer with intestinal content also resulted in only a limited extent of inversion. More than 400 anaerobe and aerobe bacterial species with a variety of enzyme activities have been isolated from GI tract or feces of animals (18-20). Similar to our observation, for many xenobiotics, the metabolism by the gut wall is quantitatively more important than that of microflora (20). With respect to acyl-glucuronidation, on the other hand, the examined specimens exhibited similar metabolic activity for the R enantiomer (15-21%). For S-XN, however, a higher glucurinidation activity was observed for the intestinal content (Figure 2, Figure 3.). The concentration of the S enantiomer in the incubationvessel containing intestinal contents was, however, very small (approximately 0.3 mg/mL) due to the limited extent of chiral inversion (5.7%).  Interestingly, the only significant effect observed as a result of the eradication of the intestinal microflora was a reduction in acyl-glucuconjugation of the S enantiomer (Figure 2.) . This further indicates the significant involvement of the microflora in the process of acyl-glucuconjugation. The greater S:R concentration ratio found in serosal as opposed to the mucosal side of everted rat gut sack is in agreement with the finding of Berry and Jamali (5) and may suggest chiral inversion during the passage of R-XN across the gut wall. This is also in accordance with our observation that the epithelial cells rather than intestinal microflora are the main contributor to the process of inversion. Indeed, in a preliminary study, after 1 h of incubation of R-XN with jejunum that resulted in 10% inversion, we transferred the same jejunum to another organ bath which contained fresh medium and followed the process in both baths for another h. We noticed another 10% inversion in the incubator which contained the tissue but not in the bath with no jejunum. This indicates that inversion is an inter-cellular process and the responsible metabolic system is not released into the medium. In both humans (8-10) and rats (5, 11), evidence of presystemic intestinal chiral inversion has been seen only with formulations which are absorbed slowly. Hence a sufficiently long residence time in the GI tract is needed for higher S:R plasma concentrations.  A comparison of rapidly absorbed formulations with iv doses, as has been made by some investigators (e.g., 21) has not shown different S:R ratios for different formulations. In addition, administration of ibuprofen as a solution (peak concentration time, 20 min) to a rat in situ intestine/liver perfusion model did not provide sufficient time for inversion in the intestine (22).  The present data, however, provide further direct evidence of chiral inversion in the GI tract. In conclusion, similar to what has been observed in humans, rat gut is efficient in inverting R-XN to its antipode. The intestinal epithelial layer, rather than muscular layer or microflora, is mainly responsible for the inversion. Both enantiomers were acyl-glucuconjugated  in the epithelial and muscular layers, and the intestinal content.

Acknowledgment

This work was supported by a grant from the Medical Research Council of Canada and by a studentship from the Iranian Ministry of Health. The assistance of Dr. Derrick Freitag in the microbiological section of this work in greatly appreciated.

References

  1. Hutt AJ; Caldwell J. The metabolic chiral inversion of 2-arylpropionic acids- a novel route with pharmacological consequences. J. Pharm. Pharmacol. 35:693-704, 1983.
  2. Jamali F. Pharmacokinetics of enantiomers of chiral non-steroidal anti-inflammatory drugs.  Eur. J. Drug Metab. Pharmacokin. 13:1-9, 1988.
  3. Wechter W.J; Loughheed D G; Reischer R J; Van Giessen G J; Kaiser D G. Enzamatic inversion of saturated carbon: Nature and mechanism of the inversion of R(-)-p-isobutyl hydratropic acid. Biochem. Biophys. Res. Comm.61: 833-837, 1974.
  4. Cox S R. Effect of route of administration on chiral inversion of R-(-)-ibuprofen. Clin. Pharmacol. Ther. 43:146, 1988.
  5. Berry B W; Jamali F. Presystemic and systemic chiral inversion of R-(-)-fenoprofen in the rat. J. Pharmacol. Exp. Ther. 258:695-701, 1991.
  6. Knihinicki R D; Williams K M; Day R O. Chiral inversion of 2-arylpropionic acid non-steroidal anti-inflammatory drugs: In vitro studies of ibuprofen and flurbiprofen.  Biochem. Pharamcol. 38:4389-4395, 1989.
  7. Jamali F; Berry B W. Pharmacokinetics of ibuprofen enantiomers in the rat: Effect of route of administration. Eur. J. Drug Metab. Pharmafcokin. 15: Suppl.) S-402, 1990.
  8. Jamali F; Singh N N; Pasutto F M; Russell A S; Coutts R T. Pharmacokinetics of ibuprofen in humans following oral administration of tablets with different absorption rates. Pharm. Res. 5:40-43, 1988.
  9. Borin M T; Cox S R; Hageman J M; Szpunar G.J. Serum concentration of ibuprofen enantiomers following multiple dose administration of sustained release ibuprofen 800 mg tablets or Motrin 400 mg tablets. Pharm. Res. 10:159S, 1988.
  10. Jamali F Mehvar; R; Russell S A; Sattari S; Yakimets Koo J. Human pharmacokinetics of ibuprofen enantiomers following different doses and formulations: Intestinal chiral inversion. J. Pharm. Sci. 81:221-225, 1992.
  11. Sattari S; Jamali F. Evidence of Absorption Rate Dependency of Ibuprofen Inversion in the Rat. Chirality  6:435-439, 1994.
  12. Simmonds R G; Woodage T J; Duff S M; Green J N. Stereospecific inversion of R-(-)-benoxaprofen in rat and man. Eur.J. Drug Metab. Pharmacokin. 5:169-172, 1980.
  13. G Esposito; G Casky; T Z. Extracellular space in the epithelium of rat's small intestine.  Am. J. Physiol. 226:50-55, 1974.
  14. Konturex P K; Brzozowski T; Konturex S.T; Dembinski, A. Role of epidermal growth factor, prostaglandins, and sulfhydryls in stress-induced gastric lesions.  Gastroentrol. 9:1607-1615, 1990.
  15. Dawson J R; Bridges J W. Xenobiotic metabolism by isolated intestinal epithelial cells from guinea-pigs. Biochem. Pharmacol. 28:3299-3305, 1979.
  16. Mehvar R; Jamali F. Stereospecific high-performance liquid chromatographic (HPLC) assay of fenoprofen enantiomers in plasma and urine.  Pharm. Res. 5:53-56, 1988.
  17. Rubin A; Knadler M P; Ho P K; Bechtol D R; Wolen L R. Stereoselective inversion of (R)-fenoprofen to (S)-fenoprofen in humans.  J. Pharm. Sci. 74:82-84, 1985.
  18. Hartiala K. Metabolism of hormones, drugs and other substances by the gut. Pharmacol. Rev. 53:496-534, 1973.
  19. Koster A Sj; Frankhijzen-Sierevogels A C; Noordhoek J. Distribution of glucuronidation capacity (1-naphtol and morphine) along the rat intestine. Biochem. Pharmacol. 34:3527-3532, 1978.
  20. Ilett K F; Tee L B G; Reeves P T; Minchin R F. Metabolism of drugs and other xenobiotics in the gut lumen and wall. Pharmacol. Ter. 46:67-93, 1990.
  21. Tracy T S; Hall S D. Metabolic inversion of (R)-ibuprofen: Epimerization and Hydrolysis of ibuprofenyl-Coenzyme A. Drug Metab. Dispos. 20:322-327, 1992.
  22. Jeffrey P; Tucker G T; Bye A; Crewe H K; Wright P A. The site of inversion of R(-)-ibuprofen: Studies using rat in-situ isolated perfused intestine/liver preparation. J. Pharm. Pharmacol. 43: 715-720, 1991.

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