Enzymic degradation of a ß-lactam antibiotic, ampicillin, in the gut: a novel treatment modality

Jaana Harmoinen1,*, Kirsi Vaali2, Pertti Koski3, Kaisa Syrjänen3, Outi Laitinen1, Kai Lindevall3 and Elias Westermarck1

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics can cause severe alterations in the gut microflora and promote diarrhoea and overgrowth of pathogenic bacteria. The present study investigated the potency of targeted recombinant ß-lactamase (TRBL) to degrade a ß-lactam antibiotic in the jejunum of fistula-operated beagles. We used different peroral doses of purified ß-lactamase (PenP) of Bacillus licheniformis in enteric-coated pellets together with intravenous ampicillin. Serum and jejunal samples were collected for ampicillin and ß-lactamase analysis. A dose–response effect of TRBL on ampicillin concentrations in the jejunal samples could be observed. The highest doses applied decreased the jejunal ampicillin concentrations to undetectable levels. In the serum samples, the ampicillin concentrations were not affected by the ß-lactamase dose used. Our results indicate that it may be possible to evolve a targeted treatment to degrade ß-lactam antibiotics intestinally and, thus, decrease antibiotic-induced adverse effects on the gut microflora.

Keywords: ß-lactamase, ß-lactams, jejunum, microflora


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Normal gastrointestinal microflora form a relatively stable ecosystem.1 However, certain factors, like the administration of antibiotics, can profoundly disrupt this microbial balance.2 The most common antibiotic-associated adverse effects include overgrowth of pre-existing microorganisms as a consequence of a systemic infection or severe diarrhoea.35

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Form of TRBL dosage

Recombinant ß-lactamase containing amino acid residues 41–268 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 dose–response 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 0.5–1.5 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 concentration–time curves (AUCs) were calculated. ANOVA for the results was carried out using the program S-plus 2000 Mixed Effects Linear Models.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serum ampicillin was detectable in all the dogs during the experiment period, and maximal concentrations were reached 15 min after iv dosing. There was no significant difference in serum ampicillin concentrations between the different treatment groups (P = 0.32; Figure 1). The mean AUCs of serum ampicillin from the different treatment groups are presented in Table 1.



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Figure 1. Effect of orally administered ß-lactamase pellets on the serum ampicillin level in beagle dogs. Different doses of encapsulated enteric-coated ß-lactamase pellets were given orally 3 min before iv-administered ampicillin (40 mg/kg). The values for each test group [white stars, ampicillin 40 mg/kg iv + placebo per os (n = 6); white squares, ampicillin 40 mg/kg iv + TRBL 0.003 mg/kg per os (n = 5); black squares, ampicillin 40 mg/kg iv + TRBL 0.03 mg/kg per os (n = 6); black stars, ampicillin 40 mg/kg iv + TRBL 0.3 mg/kg per os (n = 5)] are presented as mean serum ampicillin concentrations ± S.E.M. at different time points.

 

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Table 1.  Mean area under the concentration–time curve (AUC) for serum and jejunal ampicillin and ß-lactamase (TRBL) concentrations in each test group
 
A dose–response effect of TRBL on ampicillin concentrations in the jejunal samples was observed (Figure 2). The highest concentration of secreted ampicillin was detected in the placebo group 15 min after antibiotic administration. The lowest dose of TRBL (0.003 mg/kg) used reduced the jejunal ampicillin concentration below that of placebo. However, concentrations remained detectable for the first 2 h of sampling. With a 0.03 mg/kg dose of TRBL, the ampicillin concentrations in the jejunal samples were close to the detection limit of the assay (0.5 mg/L) and several concentrations slightly above this were observed during the study period (Figure 2). With a TRBL dose of 0.3 mg/kg, the jejunal ampicillin concentration was above the detection limit at the first sampling point (15 min), after which the concentrations dropped and remained below the detection level throughout the 5 h study period (Figure 2 and Table 1). There was a significant relationship between the concentration of ampicillin in the jejunal fluid and the ß-lactamase dose (P = 0.001).



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Figure 2. Effect of orally administered ß-lactamase pellets on the concentrations of ampicillin in the jejunum of beagle dogs after iv administration of ampicillin (40 mg/kg). The values for each test group [white stars, ampicillin 40 mg/kg iv + placebo per os (n = 6); white squares, ampicillin 40 mg/kg iv + TRBL 0.003 mg/kg per os (n = 5); black squares, ampicillin 40 mg/kg iv + TRBL 0.03 mg/kg per os (n = 6); black stars, ampicillin 40 mg/kg iv + TRBL 0.3 mg/kg per os (n = 5)] are presented as mean jejunal ampicillin concentrations ± S.E.M. at different time points.

 
The ß-lactamase activities determined in the jejunal samples of the different groups were directly related to dose given (Figure 3 and Table 1), with most of the activity seen within the first hour after administration.



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Figure 3. ß-Lactamase activity in the jejunum of beagle dogs after different oral doses. The values for each test group [white stars, ampicillin 40 mg/kg iv + placebo per os (n = 6); white squares, ampicillin 40 mg/kg iv + TRBL 0.003 mg/kg per os (n = 5); black squares, ampicillin 40 mg/kg iv + TRBL 0.03 mg/kg per os (n = 6); black stars, ampicillin 40 mg/kg iv + TRBL 0.3 mg/kg per os (n = 5)] are presented as the mean jejunal ß-lactamase activity ± S.E.M. at different time points.

 
No ampicillin or ß-lactamase was detectable in the faecal samples, either in the time zero samples or in the samples taken the following morning.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We introduce a novel peroral treatment modality, TRBL, for inactivating a parenterally administered and secreted but unabsorbed ß-lactam antibiotic, ampicillin, in the canine jejunum.

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 5.5–6.5) 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 1.0–1.2 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 dose–response 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
 
We would like to thank Rafael Frias, DVM, for his assistance in preparing the samples for analysis, as well as laboratory technicians Seppo Lasanen, Pirkko Nokkala-Wahrman and Martti Siimekselä for their professional help in handling and taking care of the animals.


    Footnotes
 
* Corresponding author. Tel: +358-9-191-49551; Fax: +358-9-191-49670; E-mail: jaana.harmoinen{at}helsinki.fi Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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