Trans-epithelial intestinal elimination of moxifloxacin in rabbits

Albert Musafijaa, Jacob Ramonb, Yuri Shtelmana, Gila Yosepha, Bina Rubinovitza, Shlomo Segeva and Ethan Rubinsteina,*

a The Infectious Diseases Unit, and b Department of Urology, Chaim Sheba Medical Center, Tel Hashomer 52621, Tel Aviv University School of Medicine, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The intestinal trans-epithelial elimination of moxifloxacin was measured in the jejunum, ileum, caecum and in the bile in a rabbit model. Over a 120 min period following a single parenteral administration of moxifloxacin 15 mg/kg, peak serum concentration was 3.1 (± 1.1) mg/L. The elimination constants were: 0.019 (± 0.017) µg/cm2/min, 0.011 (± 0.014) µg/cm2/min and 0.002 (± 0.002) µg/cm2/min in the jejunum, ileum and caecum, respectively. Per loop, over 120 min, the respective eliminated quantities were: 9.59 (± 9.37) µg, 8.26 (± 6.74) µg and 1.92 (± 1.86) µg. Biliary moxifloxacin concentrations varied between 1.30 and 5.16 mg/L and exceeded serum levels from 45 min onwards. Intestinal concentrations paralleled serum moxifloxacin levels. Altogether, approximately 4.5% of the moxifloxacin dose was eliminated trans-epithelially in the digestive tract over the 120 min experimental period.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The intestinal tract has recently gained attention as an important pathway in eliminating fluoroquinolones.16 Biliary elimination and trans-epithelial intestinal secretion are the most important secretory routes in the intestine. In previous experiments it was demonstrated that a certain fraction of sparfloxacin, lomefloxacin and ciprofloxacin administered intravenously was secreted in the intestine by direct trans-epithelial elimination through the jejunal, ileal and caecal walls in rabbits, rats and humans.36

The aim of this study was to investigate whether moxifloxacin, a new fluoroquinolone with anti-Gram-positive and anti-anaerobe activity7,8 might be eliminated through the same sections of the intestine. We used a technique identical to that used in earlier studies, i.e. the measurement of eliminated fluoroquinolone every 15 min during the first 2 h after parenteral administration of the antibiotic. In addition, in the present study we measured the concentration of moxifloxacin in the bile at the same time intervals, because recent reports document substantial secretion via the hepatic route, when the agent was administered intravenously.7,8


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Twenty New Zealand White albino male rabbits, weighing c. 3.0 kg, were used. Following a 24 h fast, but with free access to water, the animals were anaesthetized with a combination of diazepam (Teva, Petah Tikva, Israel), 3 mg/kg and ketamine (Parke Davis, Pontypool, UK), 25 mg/kg. A midline abdominal incision was performed and the jejunum, ileum and caecum were identified. In each section a segment of c. 5 cm length was isolated with its intact blood supply and the ligated segments were copiously lavaged.

A catheter was inserted into the femoral artery and jugular vein. The first blood sample was withdrawn (time zero) and a freshly prepared solution of moxifloxacin (Bayer AG, Leverkusen, Germany) (15 mg/kg bodyweight) dissolved in phosphate-buffered saline pH 7.3 (Unipath, Denver, CO, USA) was administered via the jugular vein. Blood and bile samples, as well as the fluid samples from each intestinal loop, were withdrawn at 15 min intervals during 120 min. After centrifugation at 2000 rpm for 10 min (Heraeus, Germany, 23 cm rotor) in a cooled centrifuge (+4°C), all samples were frozen until assay.

At the end of the experiment, the animals were killed with sodium barbiturate (pentothal, Abbot SPA, Milan, Italy), and the isolated segments were excised. Each segment was then spread wet and the surface area was measured.

Moxifloxacin concentrations in the fluids examined were measured in duplicate by bioassay with Bacillus subtilis (Bacto Subtilis Spore Suspension, Difco Laboratories, Detroit, MI, USA) on tryptone soya agar pH 9 (Beckton Dickinson Co., Cockeysville, MD, USA). The two major metabolites of moxifloxacin, M1 (N-sulphonate) and M2 (acyl-glucoronide), are pharmacologically inactive8 and thus did not interfere with the bioassay. Standards were prepared using rabbit serum for assay in serum and PBS pH 9.0 for assay in intestinal fluids and bile. Assay plates were incubated at 37°C overnight. The lower limit of sensitivity for the assay was 0.16 mg/L, both in serum and in saline. For statistical analysis the analysis of variance (ANOVA) was used.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following the systemic administration of a single dose of moxifloxacin (15 mg/kg), the serum sample at 15 min had a concentration of 3.11 (± 1.11) mg/L, susequent samples showing gradually decreasing concentrations, reaching a concentration of 1.21 (± 0.26) mg/L at 120 min. Simultaneous mean bile concentrations exceeded serum levels at 45 min, reaching 5.16 (± 3.05) mg/L at the collection period 105–120 min. Mean moxifloxacin concentrations in the intestinal loops are shown in Figure 1Go. The highest concentration, 3.85 (± 1.2) mg/L, was measured in the jejunum at 15 min. The lowest concentrations were measured in the caecum.



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Figure 1. Moxifloxacin concentrations (in mg/L) in the jejunum ({diamond}), ileum ({square}) and caecum ({triangleup}) (± S.E.) following a 15 mg/kg moxifloxacin dose (data from 20 animals).

 
Over 120 min the quantities of moxifloxacin eliminated were 9.59 (± 9.37) µg, 8.26 (± 6.74) µg and 1.92 (± 1.86) µg in the jejunum, ileum and caecum, respectively. The quantities of moxifloxacin excreted per cm2 of intestinal wall (over 120 min) were 2.30 (± 2.02), 1.29 (± 1.64) and 0.264 (± 0.255) µg/cm2 in the jejunum, ileum and caecum, respectively.

Figure 2Go demonstrates the relationship between serum AUC and intestinal segment AUCs. Moxifloxacin intestinal elimination rates were 0.019 (± 0.017), 0.011 (± 0.014) and 0.002 (± 0.002) µg/cm2/min, for the three intestinal segments, respectively. Comparison of means by ANOVA yielded the following differences between mean amounts of moxifloxacin eliminated per loop (P < 0.15), and between means of amounts per cm2 for 1 min (P < 0.01) and for 2 h (P < 0.1).



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Figure 2. Relationship between moxifloxacin serum AUC and intestinal AUC of secreted moxifloxacin quantities (AUC in mg/L/min). For symbols see legend to Figure 1Go.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present investigation, we demonstrated that in rabbits, moxifloxacin is eliminated as an active compound in the small intestine and bile, and only a small fraction is eliminated in the caecum. Initial data suggest that c. 20% of moxifloxacin is eliminated in the urine as the parent compound, c. 20% is eliminated in the urine as the glucorinated form and 40% is eliminated in the faeces, 25% of that as unchanged moxifloxacin and the remainder as M metabolites.7,8 Our investigation suggests that trans-epithelial secretion contributes c. 20% to the faecal excretion of the unchanged drug and the remaining 80% is excreted via the bile. The bioassay used did not measure the inactive M metabolites and thus their trans-epithelial and biliary excretion was not determined.

Elimination of fluoroquinolone has been demonstrated to occur in the small bowel in rats,3,5,6 in human Caco-2 intestinal cells (representing human small intestinal enterocytes) grown as monolayers4 and in isolated rabbit small intestinal mucosa mounted in Grass chambers.2 For sparfloxacin and ciprofloxacin, elimination through the jejunum, ileum and caecum walls has been demonstrated previously.14 Extra-biliary intestinal secretion of moxifloxacin after iv administration has also been demonstrated in rats.7

Assuming that the length of the jejunum in rabbits is 70–85 cm with an average diameter of 1 cm, and the length of the ileum is c. 240 cm with a diameter of 1.3 cm,9 the approximate surface of the jejunum is c. 220–270 cm2 and of the ileum is c. 980 cm2. The approximate length of the rabbit caecum is 40–45 cm length and the diameter is 3–4 cm,9 yielding a surface area of c. 380–565 cm2. We estimate that 0.6 mg moxifloxacin would be eliminated trans-epithelially through the jejunum, an additional 1.3 mg through the ileum and an additional 0.1 mg through the caecum during the first 2 h following a single iv dose of 15 mg/kg moxifloxacin. Thus, 2 mg or c. 4.5% of the administered dose would be eliminated trans-epithelially in the intestine over the first 2 h following a parenteral administration. It is not known whether this excretory pattern would change in a pharmacokinetic steady state, or if significantly more drug would have been recovered in the intestine if the experiment had lasted longer.

In previous investigations we demonstrated that 24–26% of ciprofloxacin and c. 5% of sparfloxacin were eliminated in the small intestine of the rabbit.2,3 Thus, in rabbits, the jejunum and ileum, and to a minor degree the caecum, serve as excretory organs for moxifloxacin with excretory constants close to that of sparfloxacin.5

In the present study a significant amount of moxifloxacin was also excreted via the bile, which may have therapeutic implications for the treatment of biliary infections. The significantly lower excretion of moxifloxacin compared with ciprofloxacin in the intestine may decrease the likelihood of development of resistance in intestinal pathogens such as Salmonella spp., Shigella spp. and enteropathogenic Escherichia coli strains.

Comparison of the patterns of elimination in bile, jejunum, ileum and caecum, with simultaneous blood levels, suggests that the elimination of moxifloxacin in the jejunum and ileum may be a passive diffusion process which is dependent on blood drug concentrations while caecal elimination of moxifloxacin may be an independent (and negligible) process. In contrast, hepatic elimination of moxifloxacin demonstrated a different pattern suggesting an active elimination process.

In summary, moxifloxacin is eliminated in the small bowel and the bile of the rabbit. The net fraction eliminated in the above-mentioned parts of intestine during a 120 min period is in the region of 4.5%.


    Notes
 
* Corresponding author. Tel: +972-3-5303500; Fax: +972-3-5347081; E-mail: unit{at}netvision.net.il Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Musafija, A., Barzilai, A., Ramon, J. & Rubinstein, E. (1998). Effect of cholera toxin on intestinal elimination of ciprofloxacin in rabbits. Antimicrobial Agents and Chemotherapy 42, 473–4.[Abstract/Free Full Text]

2 . Ramon, J., Dautrey, S., Farinotti, R., Carbon, C. & Rubinstein, E. (1994). Intestinal elimination of ciprofloxacin in rabbits. Antimicrobial Agents and Chemotherapy 38, 757–60.[Abstract]

3 . Rubinstein, E., Dautrey, S., Farinotti, R., St Julien, L., Ramonand, J. & Carbon, C. (1995). Intestinal elimination of sparfloxacin, fleroxacin and ciprofloxacin in rats. Antimicrobial Agents and Chemotherapy 39, 99–102.[Abstract]

4 . Rubinstein, E., Huneau, J. F., Dumontier, A. M. & Carbon, C. (1992). In vitro and ex vivo intestinal absorption and secretion of sparfloxacin. In Program and Abstracts of the Thirty-Second Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, CA. Abstract 1463, p. 354. American Society for Microbiology, Washington, DC.

5 . Rubinstein, E., St Julien, L., Ramon, J., Dautrey, S., Farinotti, R., Huneau, J. F. et al. (1994). The intestinal elimination of ciprofloxacin in the rat. Journal of Infectious Diseases 169, 218–21.[ISI][Medline]

6 . Vergin, H., Metz, R., Nau, R., Kinzig, M. & Soergel, F. (1993). Elimination of lomefloxacin in rat intestinal loop. Drugs 45, Suppl. 3, 252.

7 . Siefert, H. M., Buhner, K., Domdey-Bette, A., Goller, G., Kohlsdorfer, C. & Steinke, W. (1996). Bay 12-8039, a new 8-methoxyquinolone: Pharmacokinetics in rats and monkeys. In Program and Abstracts of the Thirty-Sixth International Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996. Abstract F19, p. 103. American Society for Microbiology, Washington, DC.

8 . Stass, H. & Dalhoff, A. (1997). Determination of Bay 12-9039, a new 8-methoxyquinolone, in human body fluids, by high performance liquid chromatography with fluorescence detection using on-column focusing. Journal of Chromatography. B, Biomedical Sciences and Applications 702, 163–74.

9 . Lebas, F., Coudert, P., Rouvier, R. & de Rochambeau, H. (1986). The Rabbit: Husbandry, Health and Production, Animal Production and Health Series, Publication No. 21, pp. 21–4. Food and Agriculture Organization, Rome.

Received 7 September 1999; returned 16 November 1999; revised 11 January 2000; accepted 21 January 2000





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