Abstract:
Drug delivery systems for delivering agents capable of reducing the quantity of residual antibiotics reaching the colon following oral or parenteral antibiotic therapy, and for delivering metallo-dependent enzymes, and methods of using the drug delivery systems, are disclosed. The drug delivery systems include pectin beads that encapsulate the active agent (which can be a metallo-dependent enzyme), where the pectin is crosslinked with zinc or any divalent cation of interest and the pectin beads are coated with Eudragit®-type polymers. The drug delivery systems are orally administrable, but can deliver the active agents to the colon. In some embodiments, they can administer the agents to various positions in the gastro-intestinal tract, including the colon. One metallo-dependent enzyme is the β-lactamase L1 from  Stenotrophomonas maltophilia , and agents that inactivate macrolide, quinolone, fluoroquinolone or glycopeptide antibiotics can also be used. The delivery of the active agent can be modulated to occur at various pre-selected sites of delivery within the intestinal tract by gelling/crosslinking a mixture of the active agent, such as a metallo-dependent enzyme, and pectin, with divalent metallic cations such as Ca +2  or Zn +2 . A stable metallo-dependent enzyme formulation can be delivered to the lower intestine or colon. The use of zinc cations to crosslink the pectin is particularly preferred when specific metallo-dependent enzymes, which are Zn 2+  dependent, could interact with other cationic species if they were used to gel the pectin beads and thus adversely affect the activity of such metallo-dependent enzymes.

Description:
REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to U.S.S.N. 60/859,599, filed on Nov. 17, 2006, the contents of which are hereby incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention is in the area of oral drug delivery systems to administer active agents to the colon. More specifically, the present invention relates to the delivery of metallo-dependent enzymes. 
       BACKGROUND OF THE INVENTION 
       [0003]    Following their oral administration, antibiotics pass through the stomach and are then absorbed in the small intestine to diffuse in the whole organism and treat the infectious outbreak site(s) for which they have been administered. All the same, a fraction of antibiotics ingested (the importance of this fraction varies with the characteristics of each antibiotic) is not absorbed and continues its progress to the colon before being eliminated in the stool. 
         [0004]    These residual antibiotics are combined, in the large intestine, with a fraction of the antibiotics absorbed, but which are re-excreted in the digestive tract by means of biliary elimination. This fraction is of variable importance as a function of metabolism and elimination pathways for each antibiotic. Finally, for certain antibiotics, a fraction of the dose absorbed is directly eliminated from the blood through the intestinal mucosa back into the lumen of the digestive tract, a good example is known with ciprofloxacin. Thus, whether administered orally or parenterally, a residual fraction of active antibiotics is generally found in the colon. This is the case, to varying degrees, for the great majority antibiotics from the various families used in therapeutics, with the sole notable exception of antibiotics from the amino-glycoside family for which intestinal excretion is negligible. For other antibiotics, intestinal excretion of a residual antibiotic activity will have a variety of consequences, all harmful. Indeed, the colon harbors a complex and very dense bacterial ecosystem (several hundreds of different bacterial species; more than 10 11  bacteria per gram of colonic content) which will be affected by the arrival of active antibiotic residues. The following can be observed: 
         [0005]    1. Flora imbalance which is the main cause of banal diarrhea occurring following antibiotic treatments (Bartlett J. G. (2002) Clinical practice. Antibiotic associated diarrhea, New England Journal of Medicine, 346, 334). Even though this diarrhea is generally not serious and ceases rapidly, either spontaneously, or upon completion of the antibiotic treatment, it is adversely perceived by patients and adds to the discomfort of the original illness for which the antibiotic was prescribed; 
         [0006]    2. interference with the resistance to colonization by exogenic bacteria (or “barrier effect”) with possible risk of infection, such as alimentary salmonella intoxication (Holmberg S. D. et al. (1984) Drug resistant Salmonella from animals fed antimicrobials, New England Journal of Medicine, 311, 617); 
         [0007]    3. selection of microorganisms resistant to the antibiotic. These microorganisms can be of various types: 
         [0008]    a) first they can be pathogenic bacteria such as for example,  Clostridium difficile , a species capable of secreting toxins causing a form of colitis known as pseudomembranous colitis (Bartlett J. G. (1997)  Clostridium difficile  infection: pathophysiology and diagnosis, Seminar in Gastrointestinal Disease, 8, 12); 
         [0009]    b) they can also be microorganisms that are relatively weakly pathogenic, but whose multiplication can lead to an associated infection (vaginal Candidosis or  Escherichia coli  resistant cystitis). 
         [0010]    c) they can finally be non-pathogenic commensal drug-resistant bacteria whose multiplication and fecal elimination will increase dissemination of antibiotic resistance in the environment. It is well documented that antibiotic resistance genes are carried by mobile or transposable genetic elements that may contain up to 5 or 6 antibiotic resistance genes, and are readily transmitted to other bacteria, even across species. Consequently, these resistant commensal bacteria may constitute an important source leading to drug resistance for pathogenic species. This risk is currently considered seminal in terms of the disquieting character of the evolution towards drug multiresistance by numerous species pathogenic for humans. 
         [0011]    It would be advantageous to provide drug delivery systems and methods for reducing the quantity of residual antibiotics reaching the colon following oral or parenteral antibiotic therapy. The present invention provides such drug delivery systems and methods. 
       SUMMARY OF THE INVENTION 
       [0012]    Drug delivery systems for delivering metallo-dependent enzymes, and methods of treatment using the drug delivery systems, are disclosed. Also disclosed are drug delivery systems for delivering agents capable of reducing the quantity of residual antibiotics reaching the colon following oral or parenteral antibiotic therapy, and methods for using the drug delivery systems. In some embodiments, metallo-dependent enzymes are used to reduce the quantity of residual antibiotics reaching the colon. β-lactamase L1 from  Stenotrophomonas maltophilia  is one example of a metallo-dependent enzyme useful for reducing the quantity of residual antibiotics reaching the colon. 
         [0013]    In other embodiments, one can use β-lactamases which are not metallo-enzymes (i.e., classes A, C or D). Moreover, one can use enzymes, metallo-dependent or otherwise, to inactivate other classes of antibiotics such as macrolides, quinolones and fluoriquinolones, glycopeptides, lipopeptides, cyclins, oxazolidinones, and other classes of antibiotics. The enzymes can have the full sequence of the native enzyme, or can be truncated or otherwise modified so long as they maintain acceptable activity. 
         [0014]    The drug delivery systems include pectin beads crosslinked with zinc or any divalent cation of interest, which beads are then coated with Eudragit®-type polymers. When a metallo-dependent enzyme is encapsulated in the pectin bead, the cation used to crosslink the pectin comprises the same cation to which the enzyme is dependent. 
         [0015]    The drug delivery systems are orally administrable, but can deliver the active agents to the colon. In some embodiments, they can administer the agents to various positions in the gastro-intestinal tract, including the colon. 
         [0016]    Colon-specific delivery is obtained by formulating a metallo-dependent enzyme or an agent capable of reducing the quantity of residual antibiotics reaching the colon following oral or parenteral antibiotic therapy (which can be a metallo-dependent enzyme) with specific polymers that degrade in the colon, such as pectin. The pectin is gelled/crosslinked with a cation such as a zinc cation. The formulation, typically in the form of ionically crosslinked pectin beads, is subsequently coated with a specific polymer, such as a Eudragit® polymer. 
         [0017]    The delivery can be modulated to occur at various pre-selected sites of delivery within the intestinal tract by gelling/crosslinking a mixture of the encapsulated agent, such as the metallo-dependent enzyme, and pectin, with divalent metallic cations such as Ca 2 + or Zn 2 +. 
         [0018]    Previous efforts have focused on coating pectin beads with cationic polymers such as polyethylene imine (PEI), chitosan or other cationic polymers, to prevent the pectin beads from degrading in the upper gastrointestinal tract. Such efforts are described, for example, in U.S. patent application Ser. No. 10/524,318, and U.S. Patent Application No. 60/651,352, the contents of which are hereby incorporated by reference. 
         [0019]    The present invention relates to coating the pectin beads with Eudragit® polymers such as FS30D, L30D (also known as L30D-55), NE30D, mixtures thereof or other desirable types of Eudragit® to achieve the desired release of the encapsulated agent, such as β-lactamase L1, at predefined levels of the gastro-intestinal tract (GIT). 
         [0020]    When the Eudragit® coating is dissolved, according to certain parameters such as pH or time, the beads are preferentially degraded by pectinolytic enzymes found in the lower part of the intestinal tract. Degradation of pectin then releases the agent encapsulated within the bead. 
         [0021]    One aspect of the invention is to provide a stable metallo-enzyme formulation for the lower intestinal or colonic delivery of such an enzyme. The use of zinc cations to crosslink the pectin is particularly preferred when specific metallo-dependent enzymes, which are Zn 2+  dependent, could interact with other cationic species if they were used to gel the pectin beads. Such interactions could drastically affect the activity of such metallo-dependent enzymes. Accordingly, one embodiment of the drug delivery system involves using Zn 2 + ions as a crosslinking agent for the pectin beads and in association with Zn 2+  dependent enzymes which are very sensitive to the presence of other competitive cations. Of course, if the enzymes are dependent on other metal cations, such other metal cations (if they have a valence exceeding +1) can be used to crosslink the pectin. 
         [0022]    The processes to obtain such beads can involve specific process conditions, such as time for gelification, washing, and drying that can be optimized to provide the highest quality beads, with optimized efficacy in vitro and in vivo. Therefore, another embodiment of the invention relates to processes for preparing zinc-crosslinked and Eudragit®-coated pectin beads. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES  
         [0023]      FIG. 1  is a graph showing the efficiency of water rinsing to remove excess metallic cations from a formulation of β-Lactamase L1 in pectin beads crosslinked with zinc acetate, measured in terms of conductivity (mS/cm) per sample following various washes. 
           [0024]      FIG. 2  is a graph showing the effect of gelification time, rinsing process, and drying time on recovery of β-Lactamase L1 activity. 
           [0025]      FIG. 3  is a graph showing the enzymatic activity of β-lactamase L1 using CENTA as a substrate, measured in terms of response (OD/min) versus L1 concentration (μg/ml). 
           [0026]      FIG. 4  is a series of scanning electron micrographs showing Eudragit-coated beads prepared using the methods described herein, and a cross-section of the beads showing the approximate thickness of Eudragit layer. 
           [0027]      FIG. 5  is a chart showing the release kinetics of β-lactamase L1 from uncoated beads, and Eudragit-coated beads with or without hydroxypropyl methyl cellulose (HPMC) pre-coating, measured in terms of activity (μg/mg beads) versus time (minutes). Blue triangles represent uncoated beads; red circles represent beads coated with 40% Eudragit L30D-55 without pre-coating; green squares represent beads pre-coated with 5% HPMC and coated with 40% Eudragit L30D-55. 
           [0028]      FIG. 6  is a chart showing the hydrolysis of amoxicillin by uncoated, and. Eudragit-coated beads with or without a hydroxypropyl methylcellulose (HPMC) pre-coating, measured in terms of residual amoxicillin (%) versus time (minutes). Blue. triangles represent uncoated beads; red circles represent beads coated with 40% Eudragit L30D-55 without pre-coating; green squares represent beads pre-coated with HPMC and coated with Eudragit L30D-55. 
           [0029]      FIG. 7  is a chart showing the effect of Eudragit-coated pectin beads containing β-lactamase L1 on the emergence of antibiotic-resistant bacteria in piglets treated with amoxicillin, measured in terms of amoxicillin resistant bacteriacae (%) versus treatment duration (days). Blue triangles represent untreated animals (n=12); red diamonds represent animals treated with amoxicillin and placebo pectin beads (n=12); green squares represent animals treated with amoxicillin together with Eudragit-coated pectin beads containing β-lactamase L1 (n=4). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0030]    The drug delivery systems described herein will be better understood with reference to the following detailed description. 
       I. Pectin Beads 
       [0031]    The pectin beads are formed from pectin, zinc ions, and further coating with Eudragit® polymers and encapsulate one or more active agents. 
         [0032]    Stability and protection of the pectin beads in gastric medium and intestinal medium is ensured by the Eudragit® polymer coating. In contrast, uncoated beads of pectin are not stable in such an environment and do not adequately protect their contents against degradation and/or inactivation. The Eudragit® coating ensures that they resist long enough so that their contents are able to reach the colon intact. 
         [0033]    Pectin 
         [0034]    Pectin is a polysaccharide isolated from the cellular walls of superior plants, used widely in the agricultural food industry (as a coagulant or thickener for jams, ice creams and the like) and pharmaceutics. It is polymolecular and polydisperse. Its drug delivery system varies depending on the source, extraction conditions and environmental factors. 
         [0035]    Pectins are principally composed of linear chains of beta-1,4-D)-galacturonic acid, at times interspersed by units of rhamnose. The carboxylic groups of galacturonic acid can be partially esterified to yield methylated pectins. Two types of pectins are distinguished according to their degree of methylation (DM: number of methoxy groups per 100 units of galacturonic acid):
       highly methylated pectin (HM: high methoxy) where the degree of methylation varies between 50 and 80%. It is slightly soluble in water and forms gels in acidic medium (pH&lt;3.6) or in the presence of sugars;   weakly methylated pectin (LM: low methoxy), with a degree of methylation varying from 25 to 50%. More soluble in water than HM pectin, it gives gels in the presence of divalent cations such as Ca 2+  ions. Indeed, Ca 2+  ions form “bridges” between the free carboxylated groups of galacturonic acid moities. The network that is formed has been described by Grant et al. under the name of &lt;&lt;egg-box model&gt;&gt; (Grant G. T. et al. (1973) Biological interactions between polysaccharides and divalent cations: the egg-box model, FEBS Letters, 32, 195).       
 
         [0038]    There are also amidated pectins. Treatment of pectin by ammonia transforms some methyl carboxylate groups (—COOCH 3 ) into carboxamide groups (—CONH 2 ). This amidation confers novel properties to the pectins, in particular better resistance to variations in pH. Amidated pectins tend to be more tolerant to the variations in pH, and have also been studied for the manufacture of matricial tablets for colonic delivery (Wakerly Z. et al. (1997) Studies on amidated pectins as potential carriers in colonic drug delivery, Journal of Pharmacy and Pharmacology. 49, 622). 
         [0039]    Pectin is degraded by enzymes originating from higher plants and various microorganisms (fungi, bacteria, and the like) among which bacteria from the human colonic flora. The enzymes produced by the microflora encompass a mixture of polysaccharidases, glycosidases and esterases. 
         [0040]    Metal Cations 
         [0041]    Some metallo-dependent enzymes rely on zinc cations to function. Divalent zinc cations from various zinc salts can be used to crosslink pectin, as well as interact with the enzyme. Examples include zinc sulfate, zinc chloride, and zinc acetate. 
         [0042]    Eudragit® Polymers 
         [0043]    The coating of drug-loaded cores such as tablets, capsules, granules, pellets or crystals offers many advantages, such as higher physicochemical stability, better compliance and increased therapeutic efficiency of the active ingredients. Indeed, the. effectiveness of a medication depends not only on the actives it contains, but also on formulation and processing. 
         [0044]    Poly(meth)acrylates have proven particularly suitable as coating materials. These polymers, of which only a few milligrams are employed, are pharmacologically inactive, i.e., are excreted unchanged. 
         [0045]    EUDRAGIT® is the trade name for copolymers derived from esters of acrylic and methacrylic acid, whose properties are determined by functional groups. The individual EUDRAGIT® grades differ in their proportion of neutral, alkaline or acid groups and thus in terms of physicochemical properties. The skillful use and combination of different EUDRAGIT® polymers offers ideal solutions for controlled drug release in various pharmaceutical and technical applications. EUDRAGIT® provides functional films for sustained-release tablet and pellet coatings. The polymers are described in international pharmacopeias such as Ph. Eur., USP/NF, DMF and JPE. 
         [0046]    EUDRAGIT® polymers can provide the following possibilities for controlled drug release:
       Gastrointestinal tract targeting (gastroresistance, release in the colon)   Protective coatings (taste and odor masking, protection against moisture)   Delayed drug release (sustained-release formulations).       
 
         [0050]    EUDRAGIT® polymers are available in a wide range of different concentrations and physical forms (aqueous solution, aqueous dispersion, organic solution, solid substances). 
         [0051]    The pharmaceutical properties of EUDRAGIT® polymers are determined by the chemical properties of their functional groups. A distinction is made between
       poly(meth)acrylates, soluble in digestive fluids (by salt formation) p EUDRAGIT® L, S, FS and E polymers with acidic or alkaline groups enable pH-dependent release of the active ingredient.       
 
         [0053]    Applications: from simple taste masking via resistance solely to gastric fluid, to controlled drug release in all sections of the intestine
       poly(meth)acrylates, insoluble in digestive fluids       
 
         [0055]    EUDRAGIT® L and RS polymers with alkaline and EUDRAGIT® NE polymers with neutral groups enable controlled time release of the active by pH-independent swelling. 
         [0056]    Enteric Coatings: Gastoresistance and Release in the Colon 
         [0057]    Enteric EUDRAGIT® 0  coatings provide protection against drug release in the stomach and enable controlled release in the intestine. Targeted drug release in the gastrointestinal tract is recommended for particular applications or therapeutic strategies, for example when the drug is sparingly soluble in the upper digestive tract, or when the drug may be degraded by gastric fluid. Secondly, this dosage form is very patient-friendly as it does not stress the stomach and the number of doses of the therapeutic drug can be considerably reduced, thanks to prolonged delivery. The dominant criterion for release is the pH-dependent dissolution of the coating, which takes place in a certain section of the intestine (pH 5 to over 7) rather than in the stomach (pH 1-5). For these applications, anionic EUDRAGIT® grades containing carboxyl groups, can be mixed with each other. This makes it possible to finely adjust the dissolution pH, and thus to define the drug release site in the intestine. EUDRAGIT® L and S grades are suitable for enteric coatings. EUDRAGIT® FS 30 D is specifically used for controlled release in the colon. 
         [0058]    Application benefits of enteric EUDRAGIT® coatings include:
       pH-dependent drug release   protection of actives sensitive to gastric fluid   protection of the gastric mucosa from aggressive actives   increase in drug effectiveness   good storage stability   controlled release in the colon/GI targeting       
 
         [0065]    Active Agents 
         [0066]    The active agent can be introduced into the drug delivery system as a powder, a solution, a suspension, or complexed with a solubilizing agent, such as a cyclodextrin or any other suitable compound. 
         [0067]    Some of the active agents described herein can be administered in the form of prodrugs. Prodrugs have been widely studied for the colonic targeting of various active ingredients (such as steroid and non-steroid anti-inflammatory drugs, and spasmolytics). These systems are based on the capacity of the enzymes produced by the colonic flora to act on the prodrugs to release the active form of the active ingredient. 
         [0068]    The prodrugs can be based on the action of bacterial azoreductases, so that the active agents are targeted to the colon with the drug delivery systems described herein, and the active agents are formed by reaction of the prodrug with a bacterial azoreductase, which provides a dual mechanism for ensuring that the drugs are administered to the colon. Representative chemistry for forming such prodrugs is described, for example, in Peppercorn M. A. et al. ( 1972 ) The role of intestinal bacteria in the metabolism of salicylazosulfapyridin,  The Journal of Pharmacology and Experimental Therapeutics,  181, 555 and 64, 240. 
         [0069]    Another approach consists in using bacterial hydrolases such as glycosidases and polysaccharidases (Friend D. R. (1995) Glycoside prodrugs: novel pharmacotherapy for colonic diseases,  S.T.P. Pharma Sciences,  5, 70; Friend D. R. et al. (1984) A colon-specific drug-delivery system based on drug glycosides and the glycosidases of colonic bacteria,  Journal of Medicinal Chemistry,  27, 261; Friend D. R et al. (1985) Drug glycosides: potential prodrugs for colon-specific drug delivery,  Journal of Medicinal Chemistry,  28, 51; and Friend D. R. et al. (1992) Drug glycosides in oral colon-specific drug delivery,  Journal of Controlled Release,  19, 109). Prodrugs have thus been developed by coupling, for example, sugar with steroids (glucose, galactose, cellobiose, dextrane (international application WO 90/09168)), cyclodextrins Hirayama F. et al. (1996) In vitro evaluation of Biphenylyl Acetic Acid-beta-Cyclodextrin conjugates as colon-targeting prodrugs: drug release behavior in rat biological media,  Journal of Pharmacy and Pharmacology,  48, 27). 
         [0070]    a) Agents that Inactivate Antibiotics 
         [0071]    Any agent that inactivates an antibiotic can be administered. When the antibiotic is a beta-lactam antibiotic, β-lactamases can be used, and when the antibiotic is from another class of antibiotics, enzymes or other molecules that inactivate such antibiotics can be used. One such example would be to use an erythromycin esterase to inactivate macrolide antibiotics. 
         [0072]    One representative enzyme is β-lactamase L1, a Zn 2+ -dependent β-lactamase from  Stenotrophomonas maltophilia , which was chosen from a series of β-lactamases because its characteristics showed the best profile for the targeted application. Also, it has demonstrated to have an excellent stability profile. The characteristics of various β-lactamases evaluated are described hereafter. 
         [0073]    One representative erythromycin esterase is that disclosed by Andremont A. et al. ((1985) Plasmid mediated susceptibility to intestinal microbial antagonisms in  Escherichia coli Infect. Immun.  49(3), 751), the contents of which are hereby incorporated by reference. 
         [0074]    When the antibiotic is a quinolone, the active agent can be one capable of inactivating quinolones. Representative agents include those disclosed by Chen Y et al. ((1997) Microbicidal models of soil metabolisms biotransformations of danofloxacin  Journal of Industrial Microbiology and Biotechnology  19, 378). 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
             
             
               
                   
                 FEZ-1 * 
                   
                 K cat /K m   
                 L-1(wt) ** 
                   
                 K cat /K m   
               
               
                 Antibiotics 
                 K cat  (s −1 ) 
                 K m  (μM) 
                 (μM/s −1 ) 
                 K cat  (s −1 ) 
                 K m  (μM) 
                 (μM/s −1 ) 
               
               
                   
               
               
                 Penicillin 
                   
                   
                   
                   
                   
                   
               
               
                 Benzylpenicillin 
                 70 
                 590 
                 0.11 
                 600 
                 38 
                 16 
               
               
                 Ampicillin 
                 &gt;5.5 
                 &gt;5000 
                 0.011 
                 520 
                 55 
                 9.5 
               
               
                 Cabenicillin 
                 35 
                 1600 
                 0.023 
               
               
                 Pipericillin 
                 50 
                 4200 
                 0.012 
               
               
                 Azlocillin 
               
               
                 Mezlocillin 
               
               
                 Ticarcillin 
                 &gt;65 
                 &gt;5000 
                 0.013 
               
               
                 Timocillin 
               
               
                 Cephalosporin 
               
               
                 Cephaloridin 
                 16 
                 1000 
                 0.016 
               
               
                 Cephalothin 
                 300 
                 120 
                 2.5 
                 82 
                 8.9 
                 9.2 
               
               
                 Cefoxitin 
                   
                   
                   
                 1 
                 3 
                 0.33 
               
               
                 Cefuroxin 
               
               
                 Cefotaxim 
                 165 
                 70 
                 2.4 
                 270 
                 10 
                 27 
               
               
                 Ceftazidin 
               
               
                 Cefepim 
                 &gt;6 
                 &gt;1000 
                 0.006 
               
               
                 Cefpirom 
               
               
                 Nitrocefin 
                 90 
                 100 
                 0.9 
                 41 
                 4 
                 10 
               
               
                 Moxalactam 
                 3 
                 18 
                 0.17 
               
               
                 Carbapenem 
               
               
                 Imipenem 
                 &gt;200 
                 &gt;1000 
                 0.2 
                 370 
                 57 
                 6.5 
               
               
                 Meropenem 
                 45 
                 85 
                 0.5 
                 157 
                 15 
                 10 
               
               
                 Biapenem 
                 70 
                 &gt;1000 
                 0.07 
                 134 
                 32 
                 4.2 
               
               
                 Monobactams 
               
               
                 Aztreonam 
               
               
                 Carumonam 
               
               
                 Mechanism- 
               
               
                 Based 
               
               
                 Inactivators 
               
               
                 Sulbactam 
               
               
                 Tazobactam 
                 40 
                 700 
                 1.06 
               
               
                 Clavulanic Acid 
                 &lt;0.01 
                 &gt;1000 
                 &lt;0.00001 
                 11 
                 22 
                 0.5 
               
               
                   
               
             
          
           
               
                   
                 IMP-1 *** 
                   
                 K cat /K m   
                 VIM-2 **** 
                   
                 K cat /K m   
               
               
                 Antibiotics 
                 K cat  (s −1 ) 
                 K m  (μM) 
                 (μM/s −1 ) 
                 K cat  (s −1 ) 
                 K m  (μM) 
                 (μM/s −1 ) 
               
               
                   
               
               
                 Penicillin 
                   
                   
                   
                   
                   
                   
               
               
                 Benzylpenicillin 
                 320 
                 520 
                 0.62 
                 56 
                 49 
                 1.114 
               
               
                 Ampicillin 
                 950 
                 200 
                 4.8 
                 125 
                 90 
                 1.4 
               
               
                 Cabenicillin 
                   
                   
                 0.02 
                 185 
                 205 
                 0.9 
               
               
                 Pipericillin 
                   
                   
                 0.72 
                 300 
                 125 
                 2.4 
               
               
                 Azlocillin 
                   
                   
                   
                 200 
                 200 
                 1 
               
               
                 Mezlocillin 
                   
                   
                   
                 200 
                 125 
                 1.4 
               
               
                 Ticarcillin 
                 1.1 
                 740 
                 0.0015 
                 180 
                 125 
                 1.6 
               
               
                 Timocillin 
                   
                 &gt;2000 
                 &lt;0.0001 
                 7.7 
                 390 
                 0.002 
               
               
                 Cephalosporin 
               
               
                 Cephaloridin 
                 53 
                 22 
                 2.4 
                 140 
                 50 
                 2.8 
               
               
                 Cephalothin 
                 48 
                 21 
                 2.4 
                 130 
                 11 
                 12 
               
               
                 Cefoxitin 
                 16 
                 8 
                 2 
                 15 
                 13 
                 1.2 
               
               
                 Cefuroxin 
                 8 
                 37 
                 0.22 
                 8 
                 20 
                 0.4 
               
               
                 Cefotaxim 
                 1.3 
                 4 
                 0.45 
                 70 
                 12 
                 5.8 
               
               
                 Ceftazidin 
                 8 
                 44 
                 0.18 
                 3.6 
                 72 
                 0.05 
               
               
                 Cefepim 
                 7 
                 11 
                 0.66 
                 &gt;40 
                 &gt;400 
                 0.1 
               
               
                 Cefpirom 
                 9 
                 14 
                 0.64 
                 180 
                 180 
                 1 
               
               
                 Nitrocefin 
                 63 
                 27 
                 2.3 
                 770 
                 18 
                 43 
               
               
                 Moxalactam 
                   
                   
                   
                 90 
                 55 
                 1.6 
               
               
                 Carbapenem 
               
               
                 Imipenem 
                 46 
                 39 
                 1.2 
                 34 
                 9 
                 3.8 
               
               
                 Meropenem 
                 50 
                 10 
                   
                 5 
                 2 
                 2.5 
               
               
                 Biapenem 
                 160 
                 28 
                 6 
                 8.5 
                 15 
                 0.55 
               
               
                 Monobactams 
               
               
                 Aztreonam 
                 &gt;0.01 
                 &gt;1000 
                 &lt;0.0001 
                 &lt;0.01 
                 &gt;1000 
                 &lt;0.0001 
               
               
                 Carumonam 
                 &gt;0.01 
                 &gt;1000 
                 &lt;0.0001 
               
               
                 Mechanism- 
               
               
                 Based 
               
               
                 Inactivators 
               
               
                 Sulbactam 
                   
                   
                   
                 23 
                 320 
                 0.072 
               
               
                 Tazobactam 
                   
                   
                   
                 28 
                 875 
                 0.032 
               
               
                 Clavulanic Acid 
               
               
                   
               
               
                 * (Mercuri et al., Antimicrob. Agents. Chemother. 2001 April; 45(4): 1254-1262) 
               
               
                 ** (Carenbauer et al., BMC Biochem. 2002; 3: 4. Epub 2002 February 13; Frere, 2005, unpublished data) 
               
               
                 *** (Murphy et al., 2003, Antimicrob. Ag. Chemother. 2003 February, 47(2): 582-7; Laraki et al., Antimicrob. Ag. Chemother. 1999 April, 43(4): 902-6) 
               
               
                 **** (Docquier et al. J. Antimicrob. Chemother. 2003 February, 51(2): 257-266) 
               
             
          
         
       
     
         [0075]    Patients can be treated with combinations of these agents. 
         [0076]    b) Metallo-Dependent Enzymes 
         [0077]    There are a variety of enzymes that are known to be metallo-dependent, in addition to the β-lactamase L1 enzyme discussed above. When it is desired to administer such enzymes to a patient via oral administration, care must be taken to avoid having the enzyme digested in the stomach or upper intestine. Accordingly, the drug delivery system described herein can advantageously be used to deliver such metallo-dependent enzymes. The cation used to crosslink the pectin comprises the cation on which the enzyme depends. 
       II. Methods for Preparing the Pectin Beads 
       [0078]    Pectin beads can be prepared using methods known to those of skill in the art, including by mixing the active agent in a pectin solution, and gelification of the pectin anionic moieties by a divalent cation such as divalent zinc in the form of acetate solution for example. 
         [0079]    This is typically done by stirring a solution, suspension or dispersion of the active agent, for example, β-lactamase L1, and pectin, adjusting the pH of the solution if necessary and adding this solution dropwise to a zinc acetate solution, or other solution comprising zinc ions, under agitation. 
         [0080]    The technologies for adding the pectin solution dropwise to the zinc acetate solution are known to those of skill in the art; it includes the multi nozzle system from Nisco Engineering AG or any other relevant technology to produce drops from a pectin solution. 
         [0081]    The pectin drops undergo a gelification process, ideally during a predetermined time to obtain the best encapsulation yield and subsequent release efficiency. 
         [0082]    The concentration of the pectin solution is advantageously from 4 to 10% (w/v), preferably 4 to 7%, the zinc acetate solution is advantageously from 2 to 20% (w/v), preferably from 5 to 15%. More preferably, the pectin solution is about 5% (w/v), the zinc acetate solution is about 12% (w/v). 
         [0083]    The pectin beads are advantageously stirred in the zinc acetate solution at a pH of about 6, at room temperature under slow agitation for at least 12 minutes up to 20 hours, preferably from 20 minutes to 2 hours. 
         [0084]    The beads can then be recollected and rinsed in distilled water until conductivity of the rinsing solution reaches a plateau. Rinsing is preferably done at least twice or under a continuous process to minimize the amount of residual zinc acetate recovered in the rinsing solution. 
         [0085]    The rinsed beads can then be collected and subjected to a drying process using methods known to those of skill in the art, including heated incubator or fluid bed technologies. 
         [0086]    The beads are preferably dried at a temperature of between 20 and 40° C. for 30 min to 24 hours, preferably at 35° C. overnight. Drying is performed preferentially until the weight of the beads reaches a plateau. 
         [0087]    The diameter of the particles can be finely tuned using needles of appropriate internal diameter to form the pectin drops added to the zinc acetate solution. The beads are preferably between about 600 and 1500 μm in diameter. 
         [0088]    When the active agent is β-lactamase L1, the encapsulation yields are between 50 and 100%, measured in terms of enzymatic activity. 
       III. Formation of Drug Delivery Systems Including Pectin Beads 
       [0089]    The pectin beads can be collected, and combined with appropriate excipients and formulated into a variety of oral drug delivery systems. For example, the beads can be combined with a solid excipient, and tableted, or included in a capsule. 
         [0090]    The pectin beads can also be combined with liquid/gel excipients which do not degrade the pectin beads, and the mixture/dispersion can be incorporated into a capsule, such as a gel-cap. 
         [0091]    The tablets or capsules can be coated, if desired, with a suitable enteric coating so as to assist in passing through the stomach without degradation. The pH in the stomach is of the order of 1 to 3 but it increases in the small intestine and the colon to attain values close to 7 (Hovgaard L. et al. (1996) Current Applications of Polysaccharides in Colon Targeting,  Critical Reviews in Therapeutic Drug Carrier Systems,  13, 185). The drug delivery systems, in the form of tablets, gelatin capsules, spheroids and the like, can reach the colon, without being exposed to these variations in pH, by coating them with a pH-dependent polymer, insoluble in acidic pH but soluble in neutral or alkaline pH (Kinget et al., op. cit.). The polymers most currently used for this purpose are derivatives of methacrylic acid, Eudragit® L and S (Ashford M. et al. (1993), An in vivo investigation of the suitability of pH-dependent polymers for colonic targeting,  International Journal of Pharmaceutics,  95, 193 and 95, 241; and David A. et al. (1997) Acrylic polymers for colon-specific drug delivery,  S.T.P. Pharma Sciences,  7, 546), and, more recently, Eudragit® FS. 
         [0092]    The drug delivery systems are administered in an effective amount suitable to provide the adequate degree of treatment or prevention of the disorders for which the compounds are administered. The efficient amounts of these compounds are typically below the threshold concentration required to elicit any appreciable side effects. The compounds can be administered in a therapeutic window in which some the disorders are treated and certain side effects are avoided. Ideally, the effective dose of the compounds described herein is sufficient to provide the desired effects in the colon but is insufficient (i.e., is not at a high enough level) to provide undesirable side effects elsewhere in the body. 
         [0093]    Most preferably, effective doses are at very low concentrations, where maximal effects are observed to occur, with minimal side effects, and this is optimized by targeted colonic delivery of the active agents. The foregoing effective doses typically represent that amount administered as a single dose, or as one or more doses administered over a 24-hour period. 
       IV. Methods of Treatment Using the Drug Delivery Systems Described Herein 
       [0094]    The drug delivery systems described herein can be used to treat disorders which result from exposure of the colon to antibiotics, such as diarrhea, modification of the commensal flora, and the development of bacterial resistance to antibiotics, when the drug delivery systems contain agents which inactivate antibiotics. The active agents can be administered in a therapeutically effective dosage to a patient who has been, is being, or will be treated with one or several antibiotics. 
         [0095]    When the metallo-dependent enzyme is an enzyme other than one which inactivates antibiotics, such enzyme can be administered to treat the specific disorders treated by such enzymes. 
         [0096]    The present invention will be better understood with reference to the following non-limiting examples. 
       EXAMPLE 1 
     Development of a Sensitive, Quantitative and Specific Assay for β-lactamase L1  
       [0097]    Hydrolysis of nitrocefin is a well known technique used to quantify penicillinase activity. However, the usual format is in single tubes and is not adapted for analysis of a large number of samples. This example describes the development and fit for purpose qualification of this assay in 96 wells microplate format 
         [0098]    Stock solution of Nitrocefin was obtained by solubilization of the Nitrocefin dried powder at a concentration of 10 mM in dimethylsulfoxide (DMSO). The stock solution was stored at −20° C. and diluted 100-fold immediately prior to use in 50 mM sodium phosphate buffer (Hepes buffer) pH 7.0 containing 0.1 mg/ml bovine serum albumin (BSA). Buffer selection is described in table I. 
         [0099]    20 μl containing the solution to be analyzed were added to 180 μl of diluted Nitrocefin. Kinetics of Nitrocefin hydrolysis are followed at 37° C. with a measure of absorbance at 492 nm each 30 seconds using a Multiskan Ascent (Thermo Labsystems) plate reader. 
         [0100]    The slope (difference in absorbance/second) was calculated using Excel Adds In Cellular (Prism Technologies, Cambridge UK). 
         [0101]    β-lactamase L1 (Eurocent, Belgium, approx. 10 mg/mol as determined by μBCA assay) was diluted 500×, 1000×, 2000× and 4000× in each solubilization buffer and reaction was initiated by addition of 20 μl of solution containing enzyme to 180 μl of buffers containing nitrocefin at 100 μM. 
         [0102]    Activity of β-lactamase L1 was tested in 10 mM Hepes, 145 mM NaCl buffer pH 7.4. The interference of EDTA with the activity of the metallo-dependent enzyme and need for carrier protein (Bovine Serum Albumin abbreviated as BSA) were tested. As illustrated in Table 1, EDTA (that can be used to solubilize beads in vitro to assay their contents) should be avoided. The inclusion of BSA or another carrier protein is beneficial. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Selection of buffer drug delivery system for 
               
               
                 β-lactamase L1 activity quantification 
               
             
          
           
               
                 Buffer 
                 Slope 
                 Yield 
               
               
                   
               
             
          
           
               
                 10 mM Hepes, 145 mM NaCl pH 7.4 
                 0.142 
                 100% 
                   
               
               
                 10 mM Hepes, 145 mM NaCl, 1% EDTA pH 7.4 
                 0.026 
                 18.8% 
               
               
                 10 mM Hepes, 145 mM NaCl, 0.1 mg/ml BSA pH 
                 0.167 
                 118.2% 
               
               
                 7.4 
               
               
                 10 mM Hepes, 145 mM NaCl, 0.1 mg/ml BSA, 1% 
                 0.084 
                 59.0% 
               
               
                 EDTA pH 7.4 
               
               
                   
               
             
          
         
       
     
         [0103]    As illustrated in Table 1, EDTA interferes in enzymatic activity assay, whereas BSA enhances the recovery of enzymatic activity. 
       EXAMPLE 2 
     Instability of β-lactamase L1 In Original Pectin Mix and Effect of Metallic Counter-Ion 
       [0104]    0.3 ml of β-lactamase L1 (Eurogentec, Belgium, approx. 10 mg/mL as determined by μBCA assay) was mixed to 10 g of a 6% pectin solution (Low methoxylated amidated pectin (Unipectine), Texturant Systems, cat# OG175C) made in water; the pH of the pectin solution was not adjusted. 
         [0105]    The pectin/β-lactamase L1 mixture was added drop-wise over a period of 2 minutes using a peristaltic pump and a needle of 0.8 mm inner diameter to a beaker containing 40 ml of calcium chloride (6%) under agitation (200 rpm) at room temperature. 
         [0106]    After further incubation to allow equilibration between free and bound calcium ions, beads were recovered by filtration and washed 3 times in 200 ml of purified water to eliminate excess of free calcium. At this stage, beads are referred to as “gelled beads”. 
         [0107]    Beads were dried 2 hours at 37° C. in an oven, yielding dried beads. 
         [0108]    2×5 droplets and 2×15 droplets were sampled at the exit of the needle to measure the initial β-lactamase L1 activity. Protein-free beads were also prepared as negative controls. 
         [0109]    The β-lactamase L1 enzymatic activity (nitrocefin hydrolysis) was quantified with and without Zn ions (0.1 mM ZnCl 2 ) as described in Example 1. 
         [0110]    As illustrated in Table 2, no enzymatic activity was found in the β-lactamase L1/pectin mix while significant activity was recovered in the beads assayed in buffer containing Zn. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Inactivation of β-lactamase L1 in non pH-adjusted pectin solution 
               
             
          
           
               
                 Slope/min 
                 Without ZnAc 
                 With ZnAc 
               
               
                   
               
             
          
           
               
                 Before mix with pectin 
                 0.105 
                 (100.0%) 
                 0.103 
                 (100.0%) 
               
               
                 β-Lactamase/pectin mix 
                 0.000 
                 (0.0%) 
                 0.000 
                 (0.0%) 
               
               
                 Gelled milli particles 
                 0.0078 
                 (7.4%) 
                 0.042 
                 (40.7%) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 3 
     Optimization of Metallic Ion Used to Gel the Pectin, and the Effect of pH of the Pectin Solution. 
       [0111]    In order to decipher the effects of the pectin solution parameters and Zinc ions, an experiment comparing four formulations was performed (design was build according to factorial design, Design Expert 6.0.10, Stat-Ease, Mineapollis). Two parameters were tested: 
         [0112]    (a) pH of the pectin solution: 4.0 and 7.0 
         [0113]    (b) metallic cation in the gelification bath: Ca 2+  (CaCl 2 ) or Zn 2+  (Zinc acetate abbreviated ZnAc) 
         [0114]    Beads were prepared as described in Example 2. However, the concentration of the pectin solution was decreased from 6% to 4% due to the decrease in solubility of pectin with increased pH. The encapsulation yield was measured by assaying the enzymatic activity of β-Lactamase L1 as described in Example 1. 
         [0115]    5 beads were solubilized in 20 ml of 10 mM Hepes, 145 mM NaCl, 0.1 mg/ml BSA pH 7.4 in the presence or absence 1% pectinase (Pectinases from Aspergillus Aculeatus, Pectinex SP-L Ultra, SIGMA, France) overnight at 4° C. 
         [0116]    The positive control consisted in diluting the same amount of β-lactamase L1 as should be contained in 5 beads in 20 ml of 10 mM Hepes, 145 mM NaCl, 0.1 mg/ml BSA pH 7.4. 
         [0117]    As illustrated in Table 3, β-lactamase L1 is inactivated irrespective of the cation used for pectin gelification when the pectin solution is at pH 4.0 (4.3% residual activity in Calcium and 3.8% in Zinc), whereas nearly full activity is retained after buffering the pectin solution to pH 7.0 (86.7% in Calcium and 64.0% in Zinc). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effect of cation used for gelification and pH of pectin 
               
               
                 on stability (recovery of β-Lactamase activity) 
               
             
          
           
               
                 Sample 
                 CaCl 2  pH 4 
                 CaCl 2  pH 7 
                 ZnAc pH 4 
                 ZnAc pH 7 
               
               
                   
               
             
          
           
               
                 Before mix 
                 0.102 
                 (100%) 
                 0.090 
                 (100%) 
                 0.108 
                 (100%) 
                 0.090 
                 (100%) 
               
               
                 Gelled beads 
                 0.004 
                 (4.3%) 
                 0.072 
                 (80.0%) 
                 0.004 
                 (3.8%) 
                 0.072 
                 (80.0%) 
               
               
                 Dried beads 
                 0.003 
                 (31.7%) 
                 0.078 
                 (86.7%) 
                 0.037 
                 (35.0%) 
                 0.058 
                 (64.0%) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 4 
     Determination of Critical Parameters to Formulate β-Lactamase L1 for Colon-Specific Delivery and Optimization of These Parameters 
       [0118]    Five parameters were tested: 
         [0119]    (a) Concentration of the pectin solution (Low methoxylated amidated pectin (Unipectine), Texturant Systems, cat# OG175C): 4% and 5% (w/v) 
         [0120]    (b) Cation for gelification: Ca 2+  or Zn 2+   
         [0121]    (c) Secondary coating of the gelled beads with polyethyleneimine (PEI) solution (PEI, High molecular weight, water-free, SIGMA-ALDRICH, France) 
         [0122]    (d) pH of the PEI solution: 7 and 11 (original non ph-adjusted solution). 
         [0123]    (e) Solubilization of the beads to assay the encapsulated enzymatic activity with and without 1% pectinase 
         [0124]    Table 4 summarizes the experimental design 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Experimental design for the optimization of critical 
               
               
                 parameters involved in β-Lactamase L1 formulation 
               
             
          
           
               
                   
                 A: Pectin 
                   
                 C: PEI 
                 D: pH of 
                   
               
               
                 Run 
                 (%) 
                 B: Ion 
                 Coating 
                 PEI 
                 E: Pectinase 
               
               
                   
               
             
          
           
               
                 1 
                 5 
                 Zn 2+   
                 Yes 
                 11 
                 Yes 
               
               
                 2 
                 4 
                 Zn 2+   
                 Yes 
                 7 
                 Yes 
               
               
                 3 
                 5 
                 Ca 2+   
                 No 
                   
                 Yes 
               
               
                 4 
                 4 
                 Ca 2+   
                 Yes 
                 7 
                 No 
               
               
                 5 
                 5 
                 Ca 2+   
                 Yes 
                 7 
                 Yes 
               
               
                 6 
                 4 
                 Ca 2+   
                 No 
                   
                 Yes 
               
               
                 7 
                 4 
                 Zn 2+   
                 Yes 
                 11 
                 No 
               
               
                 8 
                 5 
                 Ca 2+   
                 Yes 
                 11 
                 No 
               
               
                 9 
                 4 
                 Ca 2+   
                 Yes 
                 11 
                 Yes 
               
               
                 10 
                 4 
                 Zn 2+   
                 No 
                   
                 No 
               
               
                 11 
                 5 
                 Zn 2+   
                 No 
                   
                 Yes 
               
               
                 12 
                 4 
                 Zn 2+   
                 No 
                   
                 Yes 
               
               
                 13 
                 5 
                 Zn 2+   
                 Yes 
                 7 
                 No 
               
               
                 14 
                 5 
                 Ca 2+   
                 No 
                   
                 No 
               
               
                 15 
                 4 
                 Ca 2+   
                 No 
                   
                 No 
               
               
                 16 
                 5 
                 Zn 2+   
                 No 
                   
                 No 
               
               
                   
               
             
          
         
       
     
         [0125]    These 16 experiments were performed in duplicate (32 results), and run 13 was replicated (34 results). The pH of the 4% and 5% pectin solutions was adjusted to 7.0. However, it was discovered that the pH of the 5% pectin solution was unstable and decreased to pH 5.4 by the end of the experiments. A 5% pectin solution was therefore also adjusted to pH 8.5 for comparison. Finally, the 48 results were analyzed using Factorial Design. 
         [0126]    Beads were prepared as described in Example 2 except that the gelification time in the cation bath was reduced from 20 min to 10 min to allow a smart timing of the experiments. 
         [0127]    Samples (5 beads) were solubilized overnight at 4° C. in 20 ml of 10 mM Hepes, 145 mM NaCl, 0.1 mg/ml BSA pH 7.4 with and without 1% pectinase before measuring enzymatic activity (nitrocefin hydrolysis as described in Example 1). 
         [0128]    Tale 5 summarizes the experimental results obtained. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Full results of Experimental design for optimizing critical 
               
               
                 parameters involved in β-Lactamase L1 formulation 
               
             
          
           
               
                   
                 % 
                 pH 
                   
                   
                 pH of 
                 pec- 
                   
               
               
                 Run 
                 pectin 
                 pectin 
                 ion 
                 PEI 
                 PEI 
                 tinase 
                 yield 
               
               
                   
               
             
          
           
               
                  1 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 11 
                 yes 
                 1.201 
               
               
                 17 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 11 
                 yes 
                 1.13 
               
               
                  3b 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 11 
                 yes 
                 1.39 
               
               
                 11 
                 5 
                 5.4 
                 Zn 2+   
                 no 
                   
                 yes 
                 1.272 
               
               
                 27 
                 5 
                 5.4 
                 Zn 2+   
                 no 
                   
                 yes 
                 1.36 
               
               
                  2b 
                 5 
                 5.4 
                 Zn 2+   
                 no 
                   
                 yes 
                 1.044 
               
               
                  1b 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 7 
                 yes 
                 1.045 
               
               
                 13 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 7 
                 no 
                 0.687 
               
               
                 29 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 7 
                 no 
                 0.72 
               
               
                 33 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 7 
                 no 
                 0.661 
               
               
                 34 
                 5 
                 5.4 
                 Zn 2+   
                 yes 
                 7 
                 no 
                 0.691 
               
               
                 16 
                 5 
                 5.4 
                 Zn 2+   
                 no 
                   
                 no 
                 0.762 
               
               
                 32 
                 5 
                 5.4 
                 Zn 2+   
                 no 
                   
                 no 
                 0.788 
               
               
                 45 
                 5 
                 8.5 
                 Zn 2+   
                 no 
                   
                 yes 
                 0.951 
               
               
                 38 
                 5 
                 8.5 
                 Zn 2+   
                 no 
                   
                 yes 
                 0.818 
               
               
                 41 
                 5 
                 8.5 
                 Zn 2+   
                 no 
                   
                 no 
                 0.245 
               
               
                 48 
                 5 
                 8.5 
                 Zn 2+   
                 no 
                   
                 no 
                 0.363 
               
               
                 46 
                 5 
                 8.5 
                 Zn 2+   
                 yes 
                 7 
                 no 
                 0.815 
               
               
                 39 
                 5 
                 8.5 
                 Zn 2+   
                 yes 
                 7 
                 no 
                 0.826 
               
               
                  2 
                 4 
                 7 
                 Zn 2+   
                 yes 
                 7 
                 yes 
                 1.01 
               
               
                 18 
                 4 
                 7 
                 Zn 2+   
                 yes 
                 7 
                 yes 
                 1.162 
               
               
                 12 
                 4 
                 7 
                 Zn 2+   
                 no 
                   
                 yes 
                 1.165 
               
               
                 28 
                 4 
                 7 
                 Zn 2+   
                 no 
                   
                 yes 
                 1.148 
               
               
                  7 
                 4 
                 7 
                 Zn 2+   
                 yes 
                 11 
                 no 
                 0.727 
               
               
                 23 
                 4 
                 7 
                 Zn 2+   
                 yes 
                 11 
                 no 
                 0.679 
               
               
                 10 
                 4 
                 7 
                 Zn 2+   
                 no 
                   
                 no 
                 0.674 
               
               
                 26 
                 4 
                 7 
                 Zn 2+   
                 no 
                   
                 no 
                 0.659 
               
               
                  3 
                 5 
                 5.4 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.094 
               
               
                  5 
                 5 
                 5.4 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.031 
               
               
                 19 
                 5 
                 5.4 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.108 
               
               
                 21 
                 5 
                 5.4 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.039 
               
               
                  8 
                 5 
                 5.4 
                 Ca 2+   
                 yes 
                 11 
                 no 
                 0.047 
               
               
                 24 
                 5 
                 5.4 
                 Ca 2+   
                 yes 
                 11 
                 no 
                 0.066 
               
               
                 14 
                 5 
                 5.4 
                 Ca 2+   
                 no 
                   
                 no 
                 0.488 
               
               
                 30 
                 5 
                 5.4 
                 Ca 2+   
                 no 
                   
                 no 
                 0.512 
               
               
                 35 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.35 
               
               
                 36 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.379 
               
               
                 42 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.363 
               
               
                 43 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.394 
               
               
                  4b 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 7 
                 yes 
                 0.53 
               
               
                  7b 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 7 
                 no 
                 0.704 
               
               
                 37 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 11 
                 no 
                 0.029 
               
               
                 44 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 11 
                 no 
                 0.029 
               
               
                  9b 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 11 
                 no 
                 0.737 
               
               
                 40 
                 5 
                 8.5 
                 Ca 2+   
                   
                   
                 no 
                 0.322 
               
               
                 47 
                 5 
                 8.5 
                 Ca 2+   
                   
                   
                 no 
                 0.656 
               
               
                  6b 
                 5 
                 8.5 
                 Ca 2+   
                 yes 
                 11 
                 yes 
                 0.517 
               
               
                  5b 
                 5 
                 8.5 
                 Ca 2+   
                 no 
                   
                 yes 
                 0.656 
               
               
                  8b 
                 5 
                 8.5 
                 Ca 2+   
                 no 
                   
                 no 
                 0.967 
               
               
                   
               
             
          
         
       
     
         [0129]    Simple mono-variate statistical analysis (decreasing yield of encapsulation sorting) highlighted that optimal formulation of β-lactamase L1 was obtained using the following parameters 
         [0130]    (a) A pectin concentration of 5% (maximum solubility at pH 5.4) 
         [0131]    (b) The pectin solution should be neutralized to a pH of at least 5.4 
         [0132]    (c) Zinc ion should be used 
         [0133]    (e) Pectinase should be used to quantify the enzymatic activity of encapsulated β-lactamase L1. 
       EXAMPLE 5 
     Improvement of Stability of the Beads Comprising β-Lactamase L1 in Simulated Intestinal Media (SIM) by Increased Zinc Ion Concentration and Duration of Drying 
       [0134]    Beads containing β-lactamase L1 were prepared as described in Example 4. Increasing Zinc acetate concentrations (6, 8, 10 and 12%) were tested. Further coating with or without PEI were compared. The time for drying the beads was also increased from 2 hours to overnight. The efficiency of washing the beads to remove excess metallic ion used for gelification was also monitored by measuring the conductivity of the water rinsing solution. As illustrated in  FIG. 1 , efficient washing was obtained after 3 water washes of the beads. 
         [0135]    As illustrated in Table 6, the higher concentration of zinc ions increased stability in SIM (Simulated Intestinal Medium, US Pharmacopeia 26) of the beads containing β-lactamase L1, while a PEI secondary coating decreased their stability. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Effect of Zinc acetate concentration and PEI secondary coating 
               
               
                 on stability of beads containing β-Lactamase L1 in SIM 
               
             
          
           
               
                   
                 SIM 
               
             
          
           
               
                   
                 Run# 
                 % Zn 
                 PEI 
                 1 h 
                 2 h 
                 3 h 
                 4 h 
                 5 h 
               
               
                   
                   
               
               
                   
                 8 
                 10% 
                 N 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 3 
                 12% 
                 Y 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 4 
                 12% 
                 N 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 2 
                  8% 
                 N 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 5 
                  6% 
                 Y 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 1 
                  8% 
                 Y 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                   
                 7 
                 10% 
                 Y 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                   
               
               
                   
                 +: stable beads 
               
               
                   
                 −: dissolved beads 
               
               
                   
                 Y: with PEI secondary coating 
               
               
                   
                 N: without PEI secondary coating 
               
             
          
         
       
     
       EXAMPLE 6 
     Effect of Zinc Concentration and Drying Time on the Stability of Beads in Simulated Intestinal Media (SIM) 
       [0136]    Beads containing β-lactamase L1 were prepared as previously described, and gelled with 6 or 12% zinc acetate solutions (see Example 5). The effect of drying time was also tested by drying beads for 2, 4 and 16 h at 35° C. (temperature preferred to 37° C. for industrialization purposes). Only beads gelled in the 12% zinc solution and dried for more than 4 h were stable in SIM after 5 h incubation at 37° C. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Stability of beads in Simulated Intestinal 
               
               
                 Medium for 5 h at 37° C. 
               
             
          
           
               
                   
                 Incubation at 
                   
                   
                   
               
               
                   
                 37° C. (h) 
                 2 h drying 
                 4 h drying 
                 Overnight 
               
               
                   
                   
               
             
          
           
               
                 milli-particles 
                 1 
                 5 
                 0 
                 0 
               
               
                 in 6% Zn 
                 2 
                 1 
                 0 
                 0 
               
               
                   
                 3 
                 1 
                 0 
                 0 
               
               
                   
                 4 
                 1 
                 0 
                 0 
               
               
                   
                 5 
                 1 
                 0 
                 0 
               
               
                 milli-particles 
                 1 
                 5 
                 5 
                 5 
               
               
                 in 12% Zn 
                 2 
                 5 
                 5 
                 5 
               
               
                   
                 3 
                 5 
                 5 
                 5 
               
               
                   
                 4 
                 4 
                 5 
                 5 
               
               
                   
                 5 
                 3 
                 4 
                 5 
               
               
                   
               
             
          
         
       
     
         [0137]    The numbers represent the number of beads still apparently intact in solution. After washing and further incubation in Simulated Colonic Medium (SCM: 10 mM Hepes, 145 mM NaCl (stock solution). 1% pectinase, 0.1 mg/ml BSA were added just before use; pH was adjusted to pH 6.0 with NaOH 1 M), 63% of the initial β-lactamase activity (nitrocefin hydrolysis) was recovered. 
       EXAMPLE 7 
     Effect of Gelification Time, Rinsing Process, and Drying Time on Recovery of β-Lactamase L1 Activity 
       [0138]    Different batches of beads were prepared using a multi-nozzle system from Nisco Engineering AG. The beads underwent various gelification times, rinsing process and time and drying process type and time. 
         [0139]    It appears clearly that the best encapsulation efficiency and enzyme activity are obtained when gelification time is less than 20 hours and when rinsing is performed such as to eliminate residual Zinc acetate from the beads. The results are presented in  FIG. 2 . 
       EXAMPLE 8 
     Development of a Sensitive, Quantitative and Specific Assay for β-lactamase  L1 
       [0140]    Hydrolysis of CENTA is a well known technique used to quantify β-lactamase activity. However, the usual format is in single tubes and is not adapted for analysis of a large number of samples. This example describes the development and fit for purpose qualification of this assay in 96 wells microplate format 
         [0141]    A stock solution of CENTA was obtained by solubilization of the CENTA dried powder at a concentration of 25 mM in water; it was stored in 25 μl aliquots at −20° C. The assay mix was done by diluting 22 μl of CENTA stock solution in the following assay buffer: 10 ml 30 mM Hepes buffer pH 7.5 containing 50 μm ZnCl 2 , hence yielding a CENTA concentration of 110 μM. For the assay, 20 μl containing the enzyme to be assayed were added to 180 μl of assay mix, hence using a final concentration of 100 μM CENTA In the assay. Kinetics of CENTA hydrolysis were followed at 37° C. with a measure of absorbance at 405 nm each 9 seconds using a Multiskan Ascent (Thermo Electron Corporation) plate reader. The slope (difference in absorbance/second) was calculated using Ascent Software for Multiskan Ascent version 2.6. 
         [0142]    β-lactamase L1 (Eurogentec, Belgium, approx. 10 mg/mL as determined by μBCA assay) was diluted to 0.2, 0.5, 1.0 and 2.0 μg/ml in assay buffer and the reaction was initiated by addition of 20 μl of enzyme-containing solution to 180 μl of assay mix. As shown in Figure below, the assay was linear in 3 independent assays with respect to enzyme concentration in that range. Standard deviation was less than 10%. 
       EXAMPLE 9 
     Release of β-lactamase L1 from Uncoated Beads, and Eudragit-coated Beads with or without HPMC Pre-coating 
       [0143]    A batch of pectin beads containing β-lactamase L1 was manufactured under the following conditions: beads were formed by adding dropwise through a 0.5 mm internal diameter needle a solution of 5% pectin containing 300 mg/l purified recombinant β-lactamase L1 (Eurogentec, Belgium) to a 12% bath of Zn acetate, 2H 2 O. Beads were gelified for 90 min in the Zn acetate bath, collected, washed with water untill the water conductivity had reached a stable plateau, signifying that rinsing is optimal and finally dried at 35° C. under vacuum. Dried beads obtained were 0.8-1.25 mm diameter, weighed on average 0.6 mg and contain approx 5 to 6 μg β-lactamase L1 per mg of beads. They were either left uncoated, or coated using a Glatt GPC 1.1 with Top spray according to the following formulas shown in Table 9. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                   
                   
                 Amount 
                   
                   
                   
                   
               
               
                   
                 Amount 
                 (g) 
                 Amount 
                 Amount 
                 Amount 
                 Amount 
               
               
                   
                 (g) 
                 Batch 
                 (g) 
                 (g) 
                 (g) 
                 (g) 
               
               
                 Raw materials 
                 Batch 83 
                 100 
                 Batch 82 
                 Batch 99 
                 Batch 81 
                 Batch 97 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Eudragit L30D-55 
                 1600.0 
                 149.5 
                 300.0 
                 31.9 
                   
                   
               
               
                 Eudragit NE 30 D 
                   
                   
                 700.0 
                 74.4 
               
               
                 Eudragit FS30D 
                   
                   
                   
                   
                 800.0 
                 85.0 
               
               
                 GMS (Glycerol 
                 24.0 
                 2.2 
                 15.0 
                 1.6 
                 12.0 
                 1.3 
               
               
                 monostearate) 
               
               
                 Sodium Hydroxide 
                 28.8 
                 2.7 
                 30.4 
                 1.9 
                   
                 1.5 
               
               
                 Tween 80 
                 48.0 
                 2.2 
                 18.0 
                 1.6 
                 14.4 
                 1.3 
               
               
                 (polysorbate) 
               
               
                 33% Aqueous 
               
               
                 solution 
               
               
                 Triethyl Citrate 
                 1107.2 
                 94.5 
                 4.50 
                 67.2 
                 10.0 
                 25.2 
               
               
                 Water 
                 1600.0 
                 149.5 
                 565.7 
                 1600.0 
                 505.6 
                 85.0 
               
               
                 Pre-coating with 5% 
                 NO 
                 YES 
                 NO 
                 YES 
                 NO 
                 YES 
               
               
                 HPMC 
               
               
                   
               
             
          
         
       
     
         [0144]    Pre-coating of beads was performed with HPMC using same material as for the coating with Eudragit. 
         [0145]    Scanning electron micrographs (SEMs) of Eudragit-coated beads are shown in  FIG. 4 . A cross-section shows the relative thickness of the Eudragit coating. 
         [0146]    In order to assess the release of β-lactamase L1, coated and uncoated beads were incubated under gentle mixing at 37° C. in 50 mM Hepes buffer pH 7.4 containing 0.1 M NaCl and 100 PG/ml pectinases from  Aspergillus aculeatus  (Sigma Aldrich). Medium was withdrawn at various times and assayed for β-lactamase activity using the nitrocephin assay described in Example 1. 
         [0147]    Release kinetics were measured using the coated and uncoated beads, and the results are shown in  FIG. 5 . 
       EXAMPLE 10  
     Efficiency of Released L1 to Hydrolyze Antibiotics In Vitro 
       [0148]    In order to assess whether coated beads would actually be able to hydrolyze antibiotics when they reach the colon, they were successively incubated for 1 h in simulated gastric medium (0.1N HCl), 3 h at 37° C. in simulated intestinal medium (50 mM Na/K phosphate buffer pH 6.8 containing 0.1 M NaCl) and finally for the indicated amounts of time in simulated colonic medium (50 mM Hepes buffer pH 7.4, 0.1 M NaCl) containing 100 PG/ml pectinases from  Aspergillus aculeatus  (Sigma Aldrich) and 2 mg/ml amoxicillin. Medium was withdrawn at various times and the amount of residual amoxicillin was measure by HPLC and UV absorption. The procedure was performed using a Bio-Diss III apparatus (Varian). Uncoated beads were only incubated in the simulated colonic medium with pectinases and amoxicillin. 
         [0149]    The results are shown in  FIG. 6 . 
       EXAMPLE 11 
     Effect of β-lactamase L1 Containing Beads on the Emergence of Bacterial Resistance in Piglets Treated with Amoxicillin 
       [0150]    6-7 week old piglets were either untreated, or orally treated with 20 mg/kg amoxicillin per day for 7 days. Half of the treated animals received, together with the daily dose of antibiotics, a gelatin capsule filled with 320 mg pectin beads containing β-lactamase L1, pre-coated with 5% HPMC and coated with 40% Eudragit L30D-55 (batch 100); the other half received similarly coated placebo pectin beads. Feces were collected 3 days before the onset of treatment, and each day during 7 days of treatment and analyzed for their content of total and amoxicillin-resistant enterobacteria on MacConkey agar plates containing 0 or 100 μg/ml amoxicillin. As shown in  FIG. 7 , the feces of untreated animals contained a minimal proportion of amoxicillin-resistant bacteria (&lt;5%), whereas this proportion rapidly increased in animals treated with amoxicillin, reaching a value between 50 and 80% after 7 days. In contrast, animals receiving β-lactamase containing beads together with amoxicillin only exhibited a transient and limited increase in antibiotic-resistant bacteria. This experiment shows that the co-administration of Eudragit-coated pectin beads containing β-lactamase L1 protected piglets against the emergence of antibiotic resistant bacteria induced by the treatment of animals with amoxicillin. 
         [0151]    All patents and publications disclosed herein are incorporated by reference in their entirety. Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description of the invention.