1 Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, PO Box 57, Helsinki; 2 Institute of Biomedicine/Pharmacology, PO Box 63, University of Helsinki, FIN-00014 Helsinki; 3 Ipsat Therapies Ltd, Sinimäentie 10B, FIN-02630 Espoo, Finland
Received 8 August 2002; returned 29 October 2002; revised 12 November 2002; accepted 20 November 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: ß-lactamase, ß-lactams, jejunum, microflora
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although ß-lactam antibiotics are by far the most effective, safe and widely used antibiotics, they may alter the normal intestinal microflora.5 The disturbance in bacterial composition reflects the antibacterial spectrum and the activity of the drug used.6 After parenteral injection, ampicillin is distributed rapidly and widely, resulting in a high concentration of the drug in bile.7 From bile it is excreted into the gut and is known to cause disruption of the normal intestinal microflora,8 by diminishing the main flora and increasing the number of yeasts9 as well as inducing a high risk of Clostridium difficile colitis.10
The purpose of our research was to evaluate a novel enzymic therapy, namely targeted recombinant ß-lactamase (TRBL), and establish whether this could reduce the concentrations of parenterally administered ß-lactam antimicrobials in the gut lumen. The hypothesis is if the intestinal concentrations of these agents can be reduced, then the adverse effects on the normal flora might also be reduced. The study was conducted in a jejunum-fistulated beagle model,11,12 which permits ready access to the intestinal contents, with the animals receiving the ß-lactamase therapy by the oral route and ampicillin by the parenteral route.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant ß-lactamase containing amino acid residues 41268 of the PenP protein of Bacillus licheniformis 749/C13 was manufactured by Ipsat Therapies Oy (Espoo, Finland). The purified ß-lactamase was freeze-dried and used as the biologically active substance in manufacturing enteric-coated Eudragit L 30D-55 pellets (methacrylic acid/ethyl acrylate copolymer, total diameter 0.9 mm), which dissolve above pH 5.5.
Determination of active ß-lactamase content in enteric-coated pellets
ß-Lactamase was extracted from the pellets with 20 mM sodium citrate buffer (pH 6.5), shaking the mixture occasionally at room temperature for 60 min. Insoluble material was removed by centrifugation (8000g, 10 min at room temperature). For quality control the ß-lactamase activity of the supernatant was determined spectrophotometrically by using nitrocefin (Oxoid) as the substrate.14
Experimental protocol
The laboratory beagles used in the study were obtained from the National Laboratory Animal Centre (University of Kuopio, Finland) and the Faculty of Veterinary Medicine (University of Helsinki, Finland). The dogs ranged from 1 to 6 years of age and their weight from 9 to 16.5 kg. The dogs were given 1.5 cans of commercial, canned dog food (Pedigree, Fortivil 400 g; Waltham, Masterfoods Oy, Helsinki, Finland) twice a day.
The experimental protocol had been approved by the local ethics committee for animal experiment action in Helsinki, Finland, and was conducted in accordance with valid guidelines.15
An intussuscepted nipple valve fistula was operated into the jejunum of each dog.11,12 To study the doseresponse effect of TRBL on the jejunum, we used intravenous (iv) Na-ampicillin 40 mg/kg (A-PEN inject 1 g; Orion, Espoo, Finland) and peroral TRBL. The TRBL was administered at the following doses: 0 mg/kg (placebo, n = 6), 0.003 mg/kg (n = 5), 0.03 mg/kg (n = 6) and 0.3 mg/kg (n = 5). Each dog was given a standard dose of ampicillin (40 mg/kg) and the dogs received a single TRBL/ampicillin combination.
The ß-lactamase pellets were packed into gelatin capsules (Capsugel, size 0; Warner-Lambert, Zaventem, Belgium) according to the treatment dose for each dog. Empty capsules were used as the peroral placebo treatment. The TRBL/placebo capsules were administered perorally 3 min before the iv ampicillin, which was injected 30 min after feeding.
Serum samples were collected at 15, 30, 45, 60 and 120 min after iv ampicillin administration, and jejunal samples every 15 min during the first 2 h, then every 30 min during the next 3 h. After collection, the blood samples were centrifuged (room temperature, 1000g, 15 min). The samples were then kept at 80°C to await analysis.
Jejunal sample pH was measured immediately after collection (Sentron pH-meter; Sentron Europe B.V., Roden, The Netherlands). Thereafter, the samples were stored at 80°C until analysed. For analysis, the jejunal samples were centrifuged (4°C, 1800g, 15 min) and filtered through 0.22 µm filters (Millex-GP; Millipore, Bedford, MA, USA). Faecal samples were prepared for analysis by mincing and mixing 0.51.5 g of stool with a five-fold amount of NaCl (Braun 9 mg/mL, 0.9%, 100 mL; Braun Medical Oy, Espoo, Finland), and then centrifuged (4°C, 1800g, 20 min). After centrifugation, the faecal samples were handled in the same manner as the intestinal samples.
Traces of ampicillin were measured from all of the samples by HPLC. The detection limit for serum samples was 1 mg/L and the detection limit for both jejunal and faecal samples was 0.5 mg/L (United Laboratories, Helsinki, Finland). The method used for ampicillin determination is a modification of a method described previously.16 ß-Lactamase activity was determined in duplicate by a microtitre plate assay (Microtitre plate, 96-well/flat bottom; Bibby Sterilin, Staffordshire, UK)14,17 using nitrocefin (Oxoid) as the substrate (detection limit 10 ng/mL; quantification limit 30 ng/mL). The spectrophotometer measured the linear activity every 10 s at 10 time points, 20 s after the nitrocefin substrate had been added to the microtitre plate by a pipetting machine (Labsystems Multiskan RC reader, Finland).
Statistical analysis
The mean concentrations ± S.E.M. of ampicillin and ß-lactamase were computed for the samples in each treatment group at each time point, and the areas under the concentrationtime curves (AUCs) were calculated. ANOVA for the results was carried out using the program S-plus 2000 Mixed Effects Linear Models.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibiotic treatment frequently alters the normal intestinal flora and may result in diarrhoea and overgrowth of potentially pathogenic microorganisms.18 This effect largely occurs in the large intestine and colon, and if antibiotics can be prevented from reaching the luminal contents of these organs, many of the adverse effects may be prevented.
In this study, we used a gastroresistant formula of TRBL, which has been shown to release in vitro >80% of its total ß-lactamase activity within 15 min at pH values >5.5 (data not shown). For an orally administered agent such as this, certain characteristics, such as a high capacity to degrade the desired ß-lactam antibiotic and resistance to the action of proteolytic enzymes (so that it can remain active in the gut for a long time), are required. Our results show that recombinant ß-lactamase of B. licheniformis 749/C resists the harsh intestinal conditions and is able to break down ampicillin in vivo, and therefore it would appear to be an excellent candidate for use in designing a drug substance to be applied in the inactivation of unabsorbed ampicillin in the gut.
There was some fluctuation in the jejunal pH values during the sampling period, even within an individual dog (data not shown). However, the pH value was within the required limits (pH 5.56.5) in all dogs at most of the time points for the dissolution of the TRBL-containing pellets used in the study. Furthermore, the diameter of the pellets was optimal for expeditious passage through the canine pylorus, as it is known that <1.2 mm granules can pass rapidly from the stomach into the gut.1921 Because ampicillin is rapidly distributed after iv medication and its half-life is quite short (t1/2 in human serum is 1.01.2 h), we timed the ß-lactamase dosing 3 min before iv ampicillin injection to ensure that the enzyme would be available in the gut when ampicillin entered it. The highest concentrations of both ampicillin and ß-lactamase were detected in the jejunal samples 15 min to 1 h after dosing, suggesting that we had been successful in this respect, and that these two different medication forms, parenteral and oral, can be combined.
The results also very clearly indicate that ampicillin can be detected in jejunal samples by HPLC and that the ampicillin concentration in the jejunal samples depends on the ß-lactamase dose used. With a ß-lactamase dose of 0.03 mg/kg, we were able to lower the jejunal ampicillin concentration to an almost undetectable level, and thus prevent it from reaching the colon. To our knowledge, there are no published articles in which ampicillin has been quantified in faecal samples by HPLC, and in this study we were unable to measure any ampicillin in the faecal samples, even in the group treated with iv ampicillin and oral placebo.
It is likely that bacteria with ß-lactamase activity, present in the gut, may have degraded the ampicillin, and had an agent with greater ß-lactamase stability been used it is possible that we may have been able to detect it in the faecal samples. The absence of any ß-lactamase activity in the faecal samples would suggest that the ß-lactamase is broken down in the gut, in line with unpublished data we have obtained from studies with rats (data not shown).
Enzymic and non-enzymic inactivation of ß-lactam antibiotics occurs naturally inside the intestinal tract,2224 and it is not to be expected that any biological enzyme in its active form would penetrate the gastrointestinal tract and reach the parenteral system. Unfortunately, the nitrocefin assay as such does not enable the determination of ß-lactamase activity from serum samples,14 so we were unable to look directly for ß-lactamase activity. Nevertheless, our results give indirect evidence of the non-absorption of TRBL from the gastrointestinal tract into the systemic circulation, as even the highest ß-lactamase dose used did not change the AUC of serum ampicillin. This is an important result considering the aim of parenteral antibiotic therapy, since it means that our treatment does not affect the antibiotic concentration in the systemic circulation and that the antibiotic is effective systemically.
In conclusion, the findings of this doseresponse study suggest that this novel treatment modality could be developed into a method for degrading parenterally injected ß-lactam antibiotics in the gut. This, for its part, offers the opportunity to prevent antibiotic-induced adverse effects on the gut. However, as we have only been able to demonstrate a reduction in the concentration of ampicillin in the gut lumen, further studies will be required to establish whether this actually prevents changes in the gut microflora.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Nord, C. E. & Edlund, C. (1990). Impact of antimicrobial agents on human intestinal microflora. Journal of Chemotherapy 4, 21837.
3 . Nord, C. E. (1993). The effect of antimicrobial agents on the ecology of the human intestinal microflora. Veterinary Microbiology 3, 1937.
4 . van den Bogaard, A. E. & Stobberingh, E. E. (1999). Antibiotic usage in animals: impact on bacterial resistance and public health. Drugs 58, 589607.[ISI][Medline]
5 . Edlund, C., Stark, C. & Nord, C. E. (1994). The relationship between an increase in ß-lactam activity after oral administration of three new cephalosporins and protection against intestinal ecological disturbances. Journal of Antimicrobial Chemotherapy 34, 12738.[Abstract]
6 . Nakaya, R., Chida, T. & Sibaoka, H. (1981). Antimicrobial agents and intestinal microflora. Bifidobacteria Microflora 1, 2537.
7 . Acred, P., Brown, D. M., Turner, D. H. & Wilson, M. J. (1962). Pharmacology and chemotherapy of ampicillina new broad-spectrum penicillin. British Journal of Pharmacology 18, 35669.
8 . Sullivan, Å., Edlund, C. & Nord, C. E. (2001). Effect of antimicrobial agents on the ecological balance of human microflora. Lancet Infectious Diseases 1, 10114.[CrossRef][Medline]
9 . Amtsberg, G., Stock, V., Treschnak, E. & Ringel, U. (1989). Composition of intestinal microorganisms in the dog in relation to diet and decontamination of the intestinal tract with various antibacterial substances. Advances in Animal Physiology and Animal Nutrition 19, 12030.
10 . Gorbach, S. L. (1993). Perturbation of intestinal microflora. Veterinary and Human Toxicology 35, 1523.
11 . Harmoinen, J., Mättö, J., Rinkinen, M., Wilson-Rahmberg, M. & Westermarck, E. (2001). Permanent jejunum fistula: promising method for obtaining small intestinal chyme without disturbing intestinal function. Comparative Medicine 51, 2526.[ISI]
12 . Wilson-Rahmberg, M. & Jonsson, O. (1997). Method for long-term intestinal access in the dog. Laboratory Animals 31, 23140.[ISI][Medline]
13 . Neugebauer, K., Sprengel, R. & Schaller, H. (1981). Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium. Nucleic Acids Research 9, 257788.[Abstract]
14 . OCallaghan, C. H., Morris, A., Kirby, S. & Shingler, A. H. (1972). Novel method for detection of ß-lactamase by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 2828.
15 . Legislation for the use of animals in scientific procedures: Animals (Scientific Procedures) Act 1986. http://www.homeoffice. gov.uk/new_indexs/index_anima.htm (25 June 2002, date last accessed).
16 . Vree, T. B., Hekster, Y. A., Baars, A. M. & van der Kleijn, E. (1978). Rapid determination of amoxycillin and ampicillin in body fluids of many by means of high-performance liquid chromatography. Journal of Chromatography 145, 496501.[CrossRef][Medline]
17 . Simons, K., Sarvas, M., Garoff, H. & Helenius, A. (1975). Membrane-bound and secreted forms of penicillinase from Bacillus licheniformis. Journal of Molecular Biology 126, 67390.[CrossRef]
18 . Nord, C. E., Kager, L. & Heimdahl, A. (1984). Impact of antimicrobial agents on the gastrointestinal microflora and the risk of infections. American Journal of Medicine 76, 99106.[CrossRef][ISI][Medline]
19 . Davis, S. S., Hardy, J. G. & Hara, J. W. (1986). Transit of pharmaceutical dosage through the small intestine. Gut 27, 88692.[Abstract]
20 . Guilford, W. G. & Strombeck, D. R. (1990). Gastric structure and function. In Small Animal Gastroenterology, 2nd edn (Strombeck, D. R. & Guilford, W. G., Eds), pp. 16786. W. B. Saunders Company, Philadelphia, PA, USA.
21 . Aoyagi, N., Ogata, H., Uchiyama, M., Yasuda, Y. & Tanioka, Y. (1992). Gastric emptying of tablets and granules in humans, dogs, pigs, and stomach-emptying-controlled rabbits. Journal of Pharmacological Science 81, 11704.
22 . van der Waaij, D. & Nord, C. E. (2000). Development and persistence of multi-resistance to antibiotics in bacteria; an analysis and a new approach to this urgent problem. International Journal of Antimicrobial Agents 16, 1917.[CrossRef][ISI][Medline]
23 . Hazenberg, M. P., Pennock-Schröder, A. M. & van der Merwe, J. P. (1984). Binding to and antibacterial effect of ampicillin, neomycin and polymyxin B on human faeces. Journal of Hygiene Cambridge 93, 2734.
24 . Welling, G. W., Holtrop, A., Slootmaker-van der Meulen, C., Meijer-Severs, C. J., van Santen, E., Tonk, R. H. et al. (1992). Inactivation of ceftriaxone by fecal enzyme preparations during ceftriaxone treatment. Journal of Antimicrobial Chemotherapy 30, 2345.[ISI][Medline]