AU604117B2 - Pharmaceutical composition, for oral administration, designed to attenuate the effects of beta-lactamines - Google Patents

Description

AV,-AI-15786/88 p~~3 ORGANISC N AVDj.

4 D!DE L POP.I'ETE INTELLECTUELLE 0 PYBf u7 DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU TR ITE D CERATION EN MATIERE DE BREVETS (PCT) (51) Classification Internationale des brevets 4 (11) Numiro de publication internationale: WO 88/ 07865 A61K 35/74 Al (43) Date de publication internationale: octobre 1988 (20.10.88) (21) Numeiro de la demnande internationale: PCT/FR88/00172 (74) Mandataire: LACHASSAGNE, Jacques; B.P. 07, F- 78430 Louveciennes (FR).

(22) Date de dip6t international: 8 avril 1988 (08.04.88) (81) Etats disignks: AT (brevet europ~en), AU, BE (brevet (31) Numnio de la demande prioritaire: 87/05110 europ~en), BJ (brevet QAPI), CF (brevet QAPI), CG (brevet QAPI), CH (brevet europ~en), CM (brevet (32) Date de priorite: 10 avril 1987 (10.04.87) QAPI), DE (brevet europ~en), DK, FR (brevet europ~en), GA (brevet.GAPI), GB (brevet europ~en), IT (33) Pays de prioriti: FR (brevet europ~en), JP, KP, LU (brevet europ~en), ML (brevet OAPI), MR (brevet OAPI), NL (brevet europ~en), SE (brevet europ~en), SN (brevet QAPI), TD) (71) Deposant (pour tous les Etats d~sign~s sauf US): INSTI- (brevet OAPI), TG (brevet OAPI), US.

TUT GUSTAVE ROUSSY [FR/FR]; Rue Camille- Desmoulins, F-94805 Villejuif C~dex t")CD (72) Inventeurs; et Pubiec rapport de recherche internationale. n Inventeurs/Diposants (US seulement) TANCREDE, Cyrille [FR/FR]; 15, avenue Joffre, F-92420 Vaucresson ANDREMONT, Antoine [FR/FR]; 252, rue A. 0. J. P. c de Vaugirard, F-75015 Paris LABIA, Roger [FR/ 0 DI E C 1988 FR]; 1, rue Falret, F-92170 Vanves LEONARD, Florence [FR/CH]; Chemnin des Ormeaux, CH-1066 Epalinges F AUSTRA LIAN0 4 N0VW988 PATENT OFFICE 0 (54) Title: PHAMACEUTICAL COMPOSITION, FOR ORAL ADMINISTRATION, DESIGNED TO ATTENUATE THE EFFECTS OF g-LACTAMINES (54)Titre: COMPOSITION PHARMACEUTIQUE, ADMINISTRABLE PAR VOlE ORALE, DESTINEE A RE- DUIRE LES EFFETS DES S-LACTAMINES (57) Abstract A pharmaceutical composition for oral administration is designed to attenuate the effects of 9-lactamines on the intestinal flora in humans and is characterized in that it contains strict anaerobic bacteria which are non pathogenic in humans and produce g-lactamases.

(57) Abrigi La pr~sente invention a pour objet une composition pharmaceutique, administrable par voie orale, destin~e A 6 duire les effets des R-lactamines sur la flore intestinale chez l'homme, et caract~ris~e en ce qu'elle comprend des bacteries ana~robies strictes non pathog~nes pour l'homme et productrices de E-lactamases.

The present invention relates to an orally administrable pharmaceutical composition intended to reduce the effects of (-lactamines on the intestinal flora. The invention relates more particularly to a composition containing bacteria which are non-pathogenic to man.

It is well known that treatment with broad spectrum ,'-lactamines causes important changes in the normal microbial flora, and more particularly in the intestinal flora in treatment with jV -lactamines substantially eliminated by biliary route.

These changes may concern in particular the strictly anaerobic bacteria which are the dominant populations of the intestinal flora and which normally prevent the cblonization of potentially pathogenic microorganisms such as enterobacteria, pseudomonas, staphylococci, yeasts, etc. Thus, the decrease in the dominant flora may lead to the development of infectious germs, which is I particularly dangerous for some patients.

It has already been proposed in the past to orally administer various bacteria or yeasts. However, this was done only to replace the bacteria constituting the original intestinal flora.

The present invention is aimed at providing a new composition containing non-pathogenic bacteria, which is intended not to replace the initial flora, but to prevent the disappearance of the naturally occurring intestinal flora.

To this end, the object of the present invention is an orally administrable pharmaceutical composition which is intended to reduce the effects of -lactamines on the human intestinal flora and which is characterized in that it comprises strictly anaerobic bacteria which produce (-lactamases: h Bacteroides fragilis Bacteroides melaninoger Bacteroides intermediuE Bacteroides nonfragiliE Bacteroides asaccharol Bacteroides bivius Bacteroides disiens Bacteroides oralis Bacteroides ruminicola Bacteroides capillosus Bacteroides uniformis Clostridium butyricum Fusobacterium nucleatur 1 (Nord C.E. 1986 Rev. InfE These bacteria form part subjects, and there is nc the intestine of patients The composition accordin orally a few hours beforE treatment with P-lactamir doses during the (-lacta I I1 2 nicus iticus ct Dis. 8-5 543, 548).

ect Dis. 8-5 543, 548).

of the intestinal flora of normal objection to introducing them into Swho are deprived of them.

I to the invention is usually administered e or practically at the same time as the ne. It is possible to envisage repeated nine treatment.

The composition according to the invention is used during treatment with (-lactamines such as: penicillins especially penicillin G and phenoxymethylpenicillins, methicillin and isoxazolylpenicillins (for example oxacillin and cloxacillin), aminopenicillins (for example ampicillin and amoxicillin), amidinopenicillins (for example pivmecillinam), carboxypenicillins (for example carbenicillin, ticarcillin), methoxycarboxypenicillins (for example temocillin), acylureidopenicillins (for example Mezlocillin, Azlocillin, Piperacillin), acylpenicillins (for example Apalcillin) cephalosporins especially cefalotin, cefazolin, cefamandole, cefuroxime, cefotaxime, ceftizoxime, ceftazidime, ceftriaxone.

monobactams (with an azetidin ring), for example aztreonam.

L

xI I 3 The strictly anaerobic bacterial strains used according to the present invention are, in particular, strains producing f.-lactamases belonging to the Bacteroides genus.

The strains capable of producing P-lactamases can be determined by an in vitro test. To this end a method can be used which consists in dosing the quantity of 3 -lactamine remaining after contact with the solution presumed to contain a 3-lactamase activity (Rolfe RD and coll. J. Infect Dis. 147, 227, 1983).

The strains showing an enzymatic activity of at least 0.02 /mole i of cephaloridin/minute/mg of protein are selected.

j Strains producing -lactamases may be isolated from human faeces.

Strictly anaerobic and non-pathogenic bacteria producing r-lactamases may be presented under lyophilized form and added to a drinkable excipient just before administration. They may also be in the form of gelatin capsules, tablets or similar solid forms mixed with excipients. In order to prevent any destruction of the bacteria during passage through the stomach.

it is possible to use forms comprising an antacid excipient or an enteric coating.

In man, the quantity of bacteria which produce P-lactamases 8 1011 administered orally is from about 10 to 10 live bacterial cells.

The activity of the compositions according to the invention has been shown on the model of the heteroxenic mouse treated with ceftriaxone. Strains of anaerobic bacteria producing 3-lactamases were obtained beforehand from human faecal samples, and the cultures issued from these bacteria were used for the administration.

1. Isolation of strains of strictly anaerobic bacteria which produce r-lactamase.

Strictly anaerobic bacteria were isolated from the faeces of KL5 4 healthy subjects under anaerobiosic conditions. In each subject the dominant clones were identified and the /-lactamase activity of the isolated strains was-measured in vitro.

The (-lactamases are detected by the semi-quantitative microbiological method described by Rolfe RD and coll. Infect Dis.

147, 227, 1983). The amount of activity is measured on a scale ranging from 0+ to Maximum f-lactamase activity is represented by Intermediate activities marked as 1+ correspond to a partial hydrolysis of the antibiotic. 0+ is considered as the absence of 3 -lactamase activity. Gelose Medium 5 (Difco) and B. subtilis ATCC 6633 control strain were used.

The fecal samples are incubated for 30 minutes at 37 0 C with a known antibiotic concentration: Amoxicillin, Amoxicillin clavulanic acid, Ticarcillin, Cefotaxime, Ceftriaxone or Cefoperazone; the residual antibiotic activity is dosed.

The following Table I gives the activity profile of the isolated strains. The values provided are the percentages of hydrolysis of the various P-lactamines.

vi 41 vi,

L

TABLE I PROFILE OF SUBSTRATES OF (2-LACTAMASES EXTRACTED FROM 10 STRAINS OF STRICT ANAEROBES ISOLATED FROM THE FAECAL FLORA OF A NORMAL SUBJECT Hydrolysis. of %-actamines strains Aodxicillin Amoxicillin Ticarcillin 0efataxinha- Ceftriaxo- CPfC'PeraCrn clavulanic acid B.thetaictamicran Cl 100 4 15 7 100 100 C.claotridiformis C6 100 4 100 100 100 100 B. uniformis E9 100 0 100 26 100 100 B.uniformns V4E3 100 11 100 100 100 100 B.uniformis ES 100 0 100 100 100 100 B.uniformis E4 100 0 100 100 100 37 B.uniformls E7 100 0 100 100 100 100 B. uniformis E8 100 0 100 100 56 0 B.thetaiotacnlcron C10 100 0 100 100 100 100 B.thetaitaomicron C2 100 0 26 100 100 100 Results are expressed as percentage of antibiotic hydrolyzed in 30 minutes at 370.

L'

-6 The -lactamase activity was also tested according 'to a spectrophometric method (O'Callaghan and coll. Antimicrobial Agents Chemotheraby 1, 283, 1972) The results are reported in -the following Table II.

TABLE II -LACTAKASE ACTIVITY OF BACTERIAL EXTRACTS (,lmole of cephaloridine/mn/mar oil protein) STRAIN A C TIYTY B.uniformis E4 0.793 B.uniformis ES 5 1.065 B.uniformis E 7 0.712 B.uniforinis E 8 2.165 B.uniformis E9 0.578 B.thetaiotaomicron C1 0.337 B.thetaiotaomicron C2 0.109 C.c Io st r id if or m is C6 0.302 B.thetaiotaomicror CIO 0.162 B.thetaiotaomicron V4E3 0.039 It is believed that the strains used must have a activity 0.02 Pmole of cephaloridine/minute/mg bacterial proteins.

(3 -lactamase of total Vt~,~ 7 2. Evidencing in the mouse the properties of the strains isolated in the mouse.

a) Association of four anaerobic strains producing f-lactamase and treatment with ceftriaxone: Germ-free mice (axenic) C3H (Centre de Sel6ction des Animaux de Laboratoires, Orleans, France) are kept in a flexible plastic isolator of the Texler type. They receive drinking water of pH 3, sterilized by heat. Food is a commercially available product (R03, Villemoisson/Orge, France) sterilized by irradiation at 4 megarads.

Such animals are associated to four strains of anerobic bacteria: Cl: B. thetaiotaomicron C6: C. clostridiformis E9: B. uniformis V4E3: B. thetaiotaomicron Before the treatment with ceftriaxone, -lactamase activity cannot be detected whilst. the bacterial strains are implanted 10,02 0.31) C o\maing Y'\'t at 10 1002 3 e/g of faeces. When these same mice receive ceftriaxone per os (added to the drinking water at a rate of 2 mg/ml), the total counts are not modified and the enzymatic activity remains non-detectable, but the antibiotic is not found back in the faeces.

b) Influence of the introduction of strains of anaerobes producing p-lactamases in mice having a human intestinal flora and treated with ceftriaxone: Axenic mice were also associated to a complex human flora. The preparation of the feeding solution is carried out in an anaerobic chamber from freshly collected human faeces. The sample is crushed by means of an Ultraturrax device and diluted 100 times in-LCY medium; This diluted product is placed in the isolator containing the axenic mice. In order to protect this bacterial Ssolution from contact with oxygen, hermetically closed tubes S are used for placing the same in the anaerobic chamber. As in 8 the previous case, the mice previously deprived of liquid were force-fed by gastric and rectal route. Feeding is repeated after 24 hours. Ten to fifteen days are required to obtain the balance with barrier effects comparable with those observed in the donor.

The mice with human flora are inoculated intragastrically with 1 ml of TGY broth (Trypticase 30 g/l, yeast extract 20 g/l, glucose 5 g/l, sodium thioglycolate 1 g/l, pH 7.4) containing 5.108 ufc/ml of 4 P-lactamase producing strains of anaerobes (Cl, C6, E9, V4E3) before oral administration of Ceftriaxone (2 mg/ml in drinking water). The results are reported in Table III.

During the treatment period, no antibiotic concentration is found in the faeces. A (3-lactamase activity against ceftriaxone is detected after 14 days of treatment, it ranges between 1+ and 4+.

Ceftriaxone-susceptible enterobacteria are eliminated. Enterococci counts are equivalent to those found for E. flora in an untreated control. The resistance to a colonization by exogenic microorganisms resistant to ceftriaxone (Ent. cloacae IGR67, C. albicans IGR66) is maintained in mice in which the active Bacteroides strains were implanted previously.

Anaerobic bacteria are still present in the group having received Bacteroides and the total counts made in the anaerobic chamber are not significantly changed by the presence of these bacteria, compared to the control. MIC 50 and 90 of these bacteria are 512 and ~1024 P/g of ceftriaxone per ml respectively. Gramnegative bacilli represent 84% of this flora and there are also 13% of sporulated gram-positive bacilli and 3% of cocci.

The identification using the Api 20A system allows the detection among these strains of C. clostridiformis and B. uniformis having the same characteristics as those administered immediately before starting the treatment with Ceftriaxone.

9 TABLE III EFFECT OF ANAEROBES PRODUCING [-LACTAMASE ON THE ECOLOGICAL IMPACT OF CEFTRIAXONE IN MICE WITH HUMAN FLORA

I

Treated flora (s-4) Treated flora Bacteroides (s-5) Non-treated flora Ceftriaxonesusceptible enterobacteria Ent. Cloacae IGR67 Enterococci C. albicans IGR66 Total anaerobes Ceftriaxone concentration pg/ml f-lactamase activity <2.00 7.23 0.86 <2.00 6.79 0.53 <5.00 890-1400* (n=40) 0+ (n=50) <2.00 <2.00 5.90 0.24 ,A 5.50 0.19 10.48 0.11 <0.1 (n=50) 1+to 4+ (n=50) 5.10 1.01 <2.00 6.13 0.31 4.88 0.25 10.22 0.10 <0.1 0+ Average SEM ufc/g of faeces (10 g) on 6 takings Underlined results: significant differences p Extreme values s number of mice tested n number of faecal samples studied In human therapy, 108 to 1011 bacteria are administered per day of antibiotic treatment in the form of gastroresistant gelatin capsules with enteric effect (gelatin capsules containing 5.10 9 of bacteroides).

i

Claims (4)

1. Orally administrable pharmaceutical composition intended to reduce the effects of P-lactamines on the human intestinal flora, characterized in that it comprises strictly live anaerobic bacteria having a p-lactamase activity of at least 0.02 pmole of cephaloridin/minute/mg of total bacterial proteins and which are non-pathogenic to man, which are in a concentration from 108 to 10 1 cells and which produce P-lactamases; Bacteroides fragilis Bacteroides melaninogenicus Bacteroides intermedius Bacteroides nonfragilis Bacteroides asaccharolyticus S* Bacteroides bivius Bacteroides disiens Bacteroides oralis Bacteroides ruminicola Bacteroides capillosus Bacteroides uniformis L Clostridium butyricum Fusobacterium nucleatum in adjunct with a drinkable excipient.

2. Composition according to claim 1, characterized in that the bacteria are selected from among strains of .Bacteroides uniformis which produce p-lactamases.

3. Composition according to claim 1, characterized in that the bacteria are selected from among strains of Bacteroides thetaiotaomicron which produce P-lactamases. DATED this 10th day of July, 1990. INSTITUT GUSTAVE ROUSSY WATERMARK, PATENT TRADEMARK ATTORNEYS, 290 BURWOOD ROAD, HAWTHORN, VIC. 3122. AUSTRALIA. SLCG:KJS:JZ (11.9) A/~i 4 L--C 11 ABSTRACT The object of the present invention is an orally administrable pharmaceutical composition intended to reduce the effects of $-lactamines on the human intestinal flora, characterized in that it comprises strictly anaerobic bacteria which are non- pathogenic to man and produce (-lactamases. RAN G NT Si- INTERNATIONAL SEARCH REPORT PCT/FR 88/00172 International Application No I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, Indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC 4 Int.Cl.4: A 61 K 35/74 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System I Classification Symbols Int.C. 4 A 61 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category I Citation of Document, 1 with indication, where appropriate, of the relevant passages 12 I Relevant to Claim No. Y Chemical Abstracts, vol. 104, no. 25, 1-7 23 June 1986 (Columbus,Ohio,US), K. Sawa et al.: "The effect of cefixime on bacterial flora in the intestinal tracts of healthy male volunteers", see page 22, abstract no. 218682m, Chemo- therapy (Tokyo) 1985, 33 (Suppl. 6) 169-

180. Y Chemical Abstracts, vol. 92, no. 7, 1-7 18,February 1980 (Columbus,Ohio,US), F.P. Tally et al.: "Inactivation of cephalosporins by Bacteroides", see pages 138-139, abstract no. 52714e, Antimicrob.1 Agents Chemother. 1979, 16(5), 565-71 Y Chemical Abstracts, vol. 99, no. 1, 1,2,4-7 4 July 1983 (Columbus,Ohio,US), M. Tajima et al.: "The beta-lactamases of genus Bacteroides", see page 276, abstract no. 2765w, J. Antibiot. 1983, 36(4), 423-8 SSpecial categories of cited documents: 10 later document published after the international filing date document defining the general state the art which is not or piority date and not in conflict with the application but considered to be of particular relevance cited to understand the principle or theory underlying the invention earlier document but published on or after the International document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance;' the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 14 June 1988 (14.06.88) 11 July 1988 (11.07.88) International Searching Authority Signature of Authorized Officer European Patent Office Form PCT/ISA/210 (second sheet) (January 1985) 7 kWnlems *nil Appicatton No. PCT/FR 88/00172 Ill. DOCUMENTS CONSIDERED TO0BE RELEVANT (CONTINUED FROM THE SECOND SHEE) Category Cftln ol Oaajnwnt, wltm ditiwtm lpop'06,t th w eiyl fa levnry to Claim No Y Chemical Abstracts, vol. 85, no. 13, 1-3,6,7 2September 1976 (Coluxnbus,Ohio,US) A.E. Weinrich et al.: "Beta-lactanase activity in anaerobic bacteria", see pagE 273, abstract no. 89887v, Antimicrob. Agents Chemother. 1976, 10(1), 106-11 Form PCT'ISAM2O (extra Shl) (Januar RAPPORT DE RECHERCHE INTERNATIONALE Doends lnteirritionaie t PCT/FR 88 /00172 1. CLASSIMENT Di WINVINi ION (s1 plusleurs Symboles do classification sont applicable@, lea Indiquer tous)I Solon is classification internationale des brevet& (CIS) ou Alas fols Woon Is cleasfication nationals at Is CIS CIB 4:A 61 K 35/74 II. DOMAINES SUR LKSQUELS LA RECHERCHE A PORTE Documentation minimalo consultie Syatlmo do classification Symbotats do clssitfication Documentation consutd outre quo Is documentation minimal* dons Is mature e0i do toe documents font partia des domainos our losquals Is recherchea a port& 9 Ill. DOCUMENTS CONSID11RIS COMME! PERTINENTS 1 Ca *oi Identification des documents cliA,," avoc Indication, ml nicessaire, N* des rovendicatlons Catglodes passages pertinenit 11 vli.& is Y Chemical Abstracts, vol. 104, no. 25, 1-7 23 juin 1986 (Columbus, Ohio, US), K. Sawa et al.: "The effect of cefixirne on bacterial flora in the intestinal tracts-of healthy male volunteers", voir page 22, r6sum6 no. 218682m, Chemotherapy (Tokyo) 1985, 33(Suppl. 6) 169-80 Y Chemical Abstracts, vol. 92, no. 7, 1-7 18 f~vrier 1980 (Columbus, Ohio, US), F.P. Tally et al.: "Inactivation of cephalosporins by Bacteroides", voir pages 138-139, r6suzn6 no. 52714e, Antimicrob. Agents Chemother. 1979, 16(5), 565-71 Y Chemical Abstracts, vol. 99, no. 1, 1,2,4-7 4 juillet 1983 (Columbus, Ohio, US), M. Tajima et-al.: "The beta-lactamases of genus Bacteroides", voir page 276, r6sum6 no. 2765w, J. Antibiot. 1983, 1 *Calitlories spicietam do documents citts: o a documnent utriour Pubti post6riourrment 6 slateodipt eA mdocmentd~fnisant ~ta ginratda s tehniueInternational ou A Is date do priorit at napportenant pas onsiderdno com totclirmntannnt6IMt dots technique pertinent, maim is pu comprendra C9 ocntaidr isr Co ma l p uflcuti I dante dlina t toers ax sPrincipe ou Is th6orie constituent Is base do Ilinvantion a Im ount n~tumnmpbi sdt odptItrs document parltcutllremnt pertinent: Ilinvenion ravendi- tinl @u sprbn Catte date qudo no pout to consldirda comma nouvelle ou comma eLm document pouvent setar un douta mur una rovandication do Imptiquant unsamctivitil inventive suiteritio ou pour duane ro Ispl date souiml 'dine sY a document particulibroment pertinent; l'invention raean. aute itaio oupor na aion p~tae (ate qinqu) diqu~a no pout Mrs considtr~m comma Imptiquent une acm document to rtfrant 6 une divulgatton orae, A un usage, A activltAl inventive lorsout ao document act sasocit A un ou una exposition @u loum autres moyans ptusieurs outres documants do m~ma nature, Catte combi. cps document publti *vant Is dale do dip~t international, mals naison Atant 4vidante pour une parsonne du m~tier. Post~rleuremoent A Is date do priort6 ravendiquto a Am document qul fait partis da o m~ma famtte do bravets IV. CERTIFICATION Data A laquelte Is recherche International* a did effactivement Date dexzpidition du prisant rapport do racherche internationals achevA. 14 juin 1988 1JLW Administration chargto do Is recherche Internationa S d nctton Ir outoris6 OFFICE EUROPEEN DES BREVET'S Formulitre PCT/ISAMI21 (douitlbme toutile) (Janvp 1985) -2- Demands Internationel. N- PCT/FR 88/00172 (SUITE ON$ RENSEIGNEMENTS INDIQUIS SUR LA Ill. DOCUMENTS CONSID11RIS COMME PERTWNIENTS DEUXI111ME1 FIEUILLE) gd.orl ketification des docmerit. aids, a~ee Inktiofl al Moeeft, W dee qVsniato 36(M, 423-8 Y Chemical Abstracts, vol. 85, no. 13, 1-3,6,7 27 septemnbre 1976 (Columbus, Ohio, US) A.E. Weinrich et al.: "Beta-lactamase activity in anaerobic bacteria" voir page 273, r6sum6 no. 89887v, Antimicrob. Agents Chemother. 1976, 10(1), 106-11 Petmutuire PCTflAAM0 (foullis addtionne.II.3W (Jviur I M)~