Use of Thiamphenicol for Thetreatment of Chlamydia

Europäisches Patentamt *EP001212049B1* (19) European Patent Office

Office européen des brevets (11) EP 1 212 049 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: A61K 31/165, A61P 31/04 of the grant of the patent: 06.07.2005 Bulletin 2005/27 (86) International application number: PCT/EP2001/003709 (21) Application number: 01940275.9 (87) International publication number: (22) Date of filing: 02.04.2001 WO 2001/076585 (18.10.2001 Gazette 2001/42)

(54) USE OF THIAMPHENICOL FOR THETREATMENT OF CHLAMYDIA PNEUMONIAE INFECTIONS VERWENDUNG VON THIAMPHENICOL IN DER BEHANDLUNG VON INFEKTIONEN VERURSACHT DURCH CHLAMYDIA PNEUMONIAE UTILISATION DE THIAMPHENICOL ET DE SES DERIVES DANS LA PREPARATION DE COMPOSITIONS PHARMACEUTIQUES UTILISEES POUR TRAITER DES INFECTIONS A CHLAMYDIA PNEUMONIAE

(84) Designated Contracting States: (74) Representative: Longoni, Alessandra AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU Zambon Group S.p.A MC NL PT SE TR Intellectual Property Department Via Lillo del Duca, 10 (30) Priority: 11.04.2000 IT MI000776 20091 Bresso (Milano) (IT)

(43) Date of publication of application: (56) References cited: 12.06.2002 Bulletin 2002/24 WO-A-00/01378

(73) Proprietor: ZAMBON GROUP S.p.A. • LOMBARDI ALESSANDRA ET AL: 36100 Vicenza (IT) "Antimicrobial activity of thiamphenicol-glycinate-acetylcysteinate and (72) Inventors: other drugs against Chlamydia pneumoniae." • COLOMBO, Giovanni,Battista ARZNEIMITTEL-FORSCHUNG, vol. 51, no. 3, I-20060 Cassina de Pecchi (IT) 2001, pages 264-267, XP001019117 ISSN: • UNGHERI, Domenico 0004-4172 I-20015 Parabiago (IT) • F BLASI: "Clinical featrus of chlamydia • LICCIARDELLO, Luciano pneumoniae acute respiratory infection" I-20052 Monza (IT) CLINICAL MICROBIOLOGY AND INFECTION, • GISMONDO, Maria,Rita vol. 1, no. 1, 1 March 1996 (1996-03-01), pages I-20157 Milano (IT) s14-s18, XP001037674 lONDON, GREAT • DRAGO, Lorenzo BRITAIN I-20157 Milano (IT)

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 212 049 B1

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Description

[0001] The present invention relates to the use of thiamphenicol and derivatives thereof for the preparation of pharmaceutical compositions useful in the treatment of Chlamydia pneumoniae infections. 5 [0002] Chlamydia pneumoniae is an intracellular bacterium recently considered responsible of respiratory infections both of the upper tract and the lower tract. [0003] This bacterium is one of the most widespread human pathogens and primary infections in children from 5 to 14 years have been supported by documentary evidence. In children the infection is generally mild and asymptomatic, but can be more serious in adult and elderly. 10 [0004] Chlamydia pneumoniae is responsible for about 10% of cases of atypical pneumonia and of 5% of cases of bronchitis. It has also been associated with respiratory airways diseases and with new onset asthma and asthmatic bronchitis in the adults. Sinusitis caused by Chlamydia pneumoniae also associated with infections of the lower respiratory tract has been described and, moreover, Chlamydia pneumoniae has been isolated from middle ear fluids of patients with otitis media. 15 [0005] For a survey of the pathologies associated to Chlamydia pneumoniae infections see F. Blasi, [0006] Clinical Microbiology and Infections , vol. 1. Suppl. 1, March 1996, S14-S18. [0007] Among the antibiotics more commonly used in therapy, azithromycin and, in particular, clarithromycin resulted active in vitro against Chlamydia pneumoniae and others agents involved in these infections and therefore they are potential therapeutical agents in the treatment of Chlamydia pneumoniae infections. 20 [0008] Some quinolonic antibiotics too, offer a potential therapy for Chlamydia pneumoniae infections. [0009] In the cases of Chlamydia pneumoniae infection the antibiotic treatment can require a long period and cases of Chlamydia pneumoniae chronic persistent infections in which the antibiotic therapy has failed have already been reported. [0010] The restricted number of antibiotics useful in the treatment of Chlamydia pneumoniae infections and the in- 25 creasing importance that these infections are assuming from a clinical point of view, make necessary the identifying of antibiotics active against Chlamydia pneumomae. Thiamphenicol (The Merck Index, XII ed., No 9436, page 1587) is a known antibiotic used for the treatment of Gram-positive and Gram-negative bactenal infection In the treatment of respiratory pathologies thiamphenicol is often used as glycinate hydrochloride or acetylcysteinate, i.e. an ester of thiamphenicol salified with hydrochloric acid or with acetylcysteine respectively. 30 [0011] To our knowledge no data concerning the activity of thiamphenicol or derivatives thereof versus Chlamydia pneumomae is reported in the literature. It is known instead that thiamphemcol is active against Chlamydia trachomatis, a pathogen responsible of urogenital apparatus infections, but its activity is markedly lower than that of other antibiotics, such as for example erythromycin [G Ridgeway et al., J Antimicrob. Chemother. (1979), 5(4), 483-4]. [0012] We have now found that thiamphenicol is particularly effective in the treatment of infections caused by Chlamy- 35 dia pneumoniae. [0013] It is therefore an object of the present invention the use of thiamphenicol and derivatives thereof for the preparation of a pharmaceutical compositions useful in the treatment of Chlamydia pneumoniae infections. [0014] The pharmaceutical compositions useful in the present invention are compositions for enteral or parenteral use containing thiamphenicol or derivatives thereof such as, for example, thiamphenicol glycinate and salts thereof. 40 [0015] Particularly preferred is the use of thiamphenicol glycinate acetylcystemate. [0016] Also preferred is the use of thiamphenicol glycinate hydrochloride. [0017] The amount of active ingredient, expressed as thiamphenicol, contained in the pharmaceutical composition may change depending on the administration way and on the senousness of the infection but is generally comprised between 250 mg and 5000 mg per dose, more preferably between 500 mg and 2000 mg. 45 [0018] The pharmaceutical compositions can be in a solid or liquid form, suitable for administering by injectable, oral or aerosol route. [0019] Preferred are the pharmaceutical compositions suitable for administering by aerosol or injectable route. [0020] More preferred are the pharmaceutical compositions suitable for administering by aerosol or injectable route containing thiamphenicol glycinate hydrochloride or acetylcystemate. 50 [0021] Particularly suitable are the pharmaceutical compositions already on the market with the trademark FLUIMU- CIL ANTIBIOTICO® and GLITISOL®. [0022] The thiamphenicol efficacy against strains of Chlamydia pneumomae of recent clinical isolation has been demonstrated in vitro by calculating the MIC (Minimum Inhibitory Concentration) in comparison with other antibiotics. It is important to underline as thiamphenicol showed a MIC completely comparable, or better, with respect to that of 55 reference antibiotics already used in therapy for the treatment of Chlamydia pneumoniae infections. [0023] With the aim to better illustrate the present invention the following example is now given.

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Materials and methods

Chlamydia pneumoniae strains

5 [0024] Chlamydia pneumoniae TW183 and Chlamydia pneumoniae 2023 were obtained from American Type Culture Collection. The other strains (No 9 isolated) were clinically isolated in the period 1997-1999.

Antibiotics

10 [0025] The following antibiotics were used: thiamphenicol glycinate acetylcysteinate (TGA), azithromycin, ciprofloxacin, ceftriaxone, amoxicillin, clarithromycin, doxycycline and tetracycline hydrochloride.

Cell cultures

15 [0026] Monolayers of Hep-2 cells were prepared by seeding 2 x 105 cell/ml in EMEM with 10% fetal calf serum supplemented with L-glutammine, on 12 mm cover slips and left at 35°C, 5% CO2 for 24 to 48 hours for confluent growth. The growth medium was removed and Hep-2 monolayers were inoculated with the different strains at prede- terminated concentration calculated to give 3-5 x 102 inclusions for well. [0027] The monolayers, inoculated with Chlamydia pneumoniae were centrifuged at 1700 g for 60 minutes at 30°C. 20 [0028] Supernatant was removed and was replaced with 2.0 ml of EMEM containing 2% fetal calf serum, L-glutammine, cycloheximide 1 µg/ml and different dilutions of the tested antibiotics. [0029] in positive control no antibiotic was added and negative control were considered wells not inoculated with Chlamydia pneumoniae. [0030] Cells were incubated at 35°C in atmosphere additionated with 5% CO2, for 3 days, then the monolayers were 25 fixed with acetone for 10 minutes at -20°C and stained with a fluorescein-contugated antibody specific for Chlamydia pneumoniae (Argene Biosoft) and observed under a fluorescence microscope. [0031] All tests were run in duplicate. The number of inclusions were counted and the MIC (the lowest concentration at which complete inhibition of inclusion formation was observed) was determined.

30 Results

[0032] The activities of the tested antibiotics are shown in table 1.

Table 1 35 Antimicrobic activity in vitro against stains of Chlamydia pneumoniae ANTIBIOTIC MIC (µg/ml) Clarithromycin 0.03-0.25 Azithromycin 0.06-0.5 40 Amoxicillin > 16 Doxycycline 0.06-0.25 Ciprofloxacin 0.5-2 45 Ceftnaxone > 16 Tetracycline 0.06-0.5 TGA 0.03-0.25

50 [0033] Clarithromycin and thiamphemcol glycinate acetylcystemate (TGA) are the most active antibiotics (MIC range 0.03 and 0.25 µg/ml for both). [0034] In table 2 the MIC values per strain of each test are shown.

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5 II 0.06 0.03 0.06 0.03 0.03 0.03 0.03 0.03 10 0.125 0.125 0.125 g/ml TGA µ I 0.03 0.03 0.25 0.03 0.03 0.03 0.06 0.125 0.125 0.125 0.125

15 II 0.5 0.5 0.5 0.06 0.06 0.25 0.06 0.06 0.125 0.125 0.125 g/ml µ I Tetracycline 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.125 0.125 0.125 0.125 20 1 2 1 1 2 1 2 1 II 0.5 0.5 0.5 g/ml µ I 25 1 1 2 2 ciprofloxacin 0.5 0.5 0.5 0.5 0.5 0.5 0.5 II 0.06 0.06 0.06 0.06 0.06 0.25 0.06 0.25 0.125 0.125 0.125 30 g/ml µ Table 2 Table I Doxycycline 0.06 0.06 0.06 0.06 0.25 0.25 0.06 0.125 0.125 0.125 0.125

35 II 0.5 0.25 0.06 0.25 0.25 0.25 0.25 0.25 0.125 0.125 0.125 g/ml µ I azithromycin 0.5 0.5 0.5 0.06 0.06 0.25 0.25 0.25 0.06 0.125 0.125

40 II 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.125 0.125 0.125 0.125 g/ml µ 45 I Clarithromycin 0.03 0.25 0.03 0.03 0.03 0.03 0.03 0.125 0.125 0.125 0.125

50 strain 1 2 3 4 5 6 7 8 9 2023 ATCC TW 183

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[0035] All the values are under the break point (thiamphenicol ≤16 µg/ml, clarithromycin ≤2 µg/ml, amoxicillin ≤2 µg/ ml, doxycycline ≤4 µg/ml, ciprofloxacin ≤1 µg/ml, tetracycline ≤2 µg/ml) except for azithromycin (≤1 µg/ml) and ceftriaxone (≤2 µg/ml). [0036] Moreover thiamphenicol has shown the best value of the ratio between MIC (0.03-0.25 µg/ml) and break point 5 (≤16 µg/ml) in comparison with the others antimicrobial compounds the break point values of which are lower than thiamphenicol.

Claims 10 1. Use of thiamphenicol and derivatives thereof for the preparation of a pharmaceutical composition useful for the treatment of Chlamydia pneumomae infections.

2. Use according to claim 1 wherein thiamphenicol is used as thiamphenicol glycinate or salts thereof. 15 3. Use according to claim 2 wherein thiamphenicol is used as thiamphemcol glycinate acetylcysteinate.

4. Use according to claim 2 wherein thiamphenicol is used as thiamphenicol glycinate hydrochloride.

20 5. Use according to claim 1 wherein the pharmaceutical composition is suitable for aerosol and injectable administration.

6. Use according to claim 1 wherein the amount of thiamphenicol is comprised between 250 mg and 5000 mg.

25 Patentansprüche

1. Verwendung von Thiamphenicol und Derivaten davon zur Herstellung eines Arzneimittels, das zur Behandlung von Infektionen mit Chlamydia pneumoniae nützlich ist. 30 2. Verwendung nach Anspruch 1, wobei Thiamphenicol als Thiamphenicolglycinat oder Salzen davon verwendet wird.

3. Verwendung nach Anspruch 2, wobei Thiamphenicol als Thiamphenicolglycinatacetylcysteinat verwendet wird. 35 4. Verwendung nach Anspruch 2, wobei Thiamphenicol als Thiamphenicolglycinathydrochlorid verwendet wird.

5. Verwendung nach Anspruch 1, wobei das Arzneimittel zur Aerosol- oder Injektionsverabreichung geeignet ist.

40 6. Verwendung nach Anspruch 1, wobei die Menge an Thiamphenicol zwischen 250 mg und 5000 mg liegt.

Revendications

45 1. Utilisation du thiamphénicol et dérivés correspondants pour la préparation d'une composition pharmaceutique servant au traitement des infections Chlamydia pneumoniae.

2. Utilisation selon la revendication 1, caractérisée en ce que le thiamphénicol est utilisé sous forme de glycinate de thiamphénicol ou de sels correspondants. 50 3. Utilisation selon la revendication 2, caractérisée en ce que le thiamphénicol est utilisé sous forme d'acétylcys- téinate de thiamphénicol glycinate.

4. Utilisation selon la revendication 2, caractérisée en ce que le thiamphénicol est utilisé sous forme de glycinate 55 de thiamphénicol chlorhydrate.

5. Utilisation selon la revendication 1, caractérisée en ce que la composition pharmaceutique convient à une administration aérosol ou par injection.

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6. Utilisation selon la revendication 1, caractérisée en ce que la quantité de thiamphénicol se trouve comprise entre 250 mg et 5000 mg.