WO1999059566A1 - Use of nitric oxide synthase inhibitors in the manufacture of a medicament for the prophylaxis or treatment of bacterial infection - Google Patents

Use of nitric oxide synthase inhibitors in the manufacture of a medicament for the prophylaxis or treatment of bacterial infection Download PDF

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Publication number
WO1999059566A1
WO1999059566A1 PCT/EP1999/003265 EP9903265W WO9959566A1 WO 1999059566 A1 WO1999059566 A1 WO 1999059566A1 EP 9903265 W EP9903265 W EP 9903265W WO 9959566 A1 WO9959566 A1 WO 9959566A1
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formula
group
compound
pharmaceutically acceptable
nitric oxide
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PCT/EP1999/003265
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French (fr)
Inventor
Wendy Karen Alderton
Richard Graham Knowles
Christoph Hubertus Ladel
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Glaxo Group Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of nitric oxide synthase inhibitors for the prophylaxis or treatment of bacterial infections.
  • nitric oxide has been implicated in the pathophysiology of hypotension induced by endotoxin and inflammatory sequelae of viral or bacterial infections.
  • the biosynthesis of nitric oxide from L-arginine is catalysed by the enzyme NO synthase.
  • WO 91/04024 and other references teach that inhibitors of nitric oxide may be useful for the treatment of septic shock.
  • nitric oxide synthase namely those which selectively inhibit the inducible isoform of the enzyme, can decrease the number of bacteria in infected animals and can reduce mortality in infected animals and so may have utility in the prophylaxis or treatment of a bacterial infection.
  • the present invention provides a method for prophylaxis or treatment of a bacterial infection in a mammal, such as a human, which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase.
  • a method for reducing the number of bacteria in an infected mammal, such as a human which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase.
  • an inhibitor of inducible nitric oxide synthase for the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection.
  • an inhibitor of inducible nitric oxide synthase for the manufacture of a medicament for reducing the number of bacteria in an infected mammal, such as a human.
  • treatment is meant the improvement of clinical outcome, for example, a reduction in mortality. Such improvement of clinical outcome may be as a result of a reduction of the number of bacteria in the infected mammal.
  • bacterial infection is meant an infection due to intracellular or extracellular bacteria, in particular due to extracellular bacteria, more particularly, any gram- positive or gram-negative bacteria.
  • the infection may be bacterial pneumonia, for example, caused by Streptococcus pneumoniae or Klebsiella pneumoniae, or bacterial septicemia induced by Streptococcus pneumoniae or Haemophilus influenzae.
  • the infection may be community acquired pneumonia, such as may be caused by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Chlamydia pneumoniae, Mycoplasma pneumoniae, or Legionella pneumophila, in particular by Streptococcus pneumoniae or Haemophilus influenzae.
  • community acquired pneumonia such as may be caused by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Chlamydia pneumoniae, Mycoplasma pneumoniae, or Legionella pneumophila, in particular by Streptococcus pneumoniae or Haemophilus influenzae.
  • the infection may be hospital acquired pneumonia, such as may be caused by Streptococcus pneumoniae, Haemophilus influenzae, a Klebsiella species such as Klebsiella pneumoniae, Eschenchia coli, an Enterobacter species, Pseudomonas aeruginosa, or an Actinetobacter species.
  • the infection is one caused by bacteria not primarily resident in red blood cells.
  • Other conditions caused by bacterial infection include urinary tract infections e.g. pyelonephritis, otitis (media or externa), ocular infections, bronchitis, and other clinical situations where bacterial infection may arise as a complication e.g. during surgery.
  • inhibitor of inducible nitric oxide synthase is meant a compound which inhibits the inducible isoform of nitric oxide synthase (iNOS) to a significantly greater extent than the constitutive (neuronal and endothelial) isoforms (nNOS and eNOS).
  • the inhibitor would exhibit at least 5-fold selectivity, preferably at least 10-fold selectivity, and more preferably at least 50-fold selectivity for iNOS over eNOS and nNOS as demonstrated in an enzyme inhibition assay, such as the assays described below.
  • suitable compounds include some of those described in WO93/13055, WO 96/19440, and WO 98/30537.
  • inhibitors of iNOS may be found, for example, in the scope of the following patent applications: WO96/15120, WO 97/10204, WO 97/32844, WO 95/24382, WO 95/25717, WO 99/05131 , WO 96/33175, WO 96/35677, WO 98/30220, WO 95/11231 , WO 95/11014, EP 0897912, EP0724570, WO 96/14842, WO 96/14844, WO 97/38977, WO 98/45294, WO 98/46611, WO 99/01455, WO 97/14686, WO 96/30350, WO 96/30350, WO 97/02254, JP 10045751 -A, FR 2730733, FR 2727111 , WO 98/37079, and JP 08310945.
  • the present invention provides a method for prophylaxis or treatment of a bacterial infection in a mammal which comprises administering an effective amount of a compound of formula (I):
  • R 1 is a C ⁇ straight or branched chain alkyl group
  • R 2 is a group of formula wherein
  • R 3 is -(CH 2 ) n NH 2 wherein n is 0, 1 , or 2;
  • Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more (suitably, 1 to 4) C 1-3 alkyl groups; or
  • Q is a group of formula -(CH 2 )pX(CH 2 )q- where p is 2 or 3, q is 1 or 2 and X is S(0) x where x is 0, 1 or 2, O or NR 4 where R 4 is H or C M alkyl; or
  • Q is a group of formula -(CHy-A ⁇ H-)-- where r is 0, 1 or 2, s is 0, 1 or 2 and A is a 3 to 6 membered carbocyclic or heterocyclic ring which may optionally be substituted by one or more (suitably, 1 to 4) suitable substituents such as C ⁇ alkyl, C ⁇ alkoxy, hydroxy, halo, nitro, cyano, trifluoro C 1-6 alkyl, amino, C ⁇ alkylamino or di C ⁇ alkylamino.
  • R 1 is a C ⁇ e straight or branched chain alkyl group
  • Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more (suitably, 1 to 4) C ⁇ alkyl groups; or
  • Q is a group of formula -(CH 2 )pX(CH 2 )q- where p is 2 or 3, q is 1 or 2 and X is S(O) x where x is 0, 1 or 2, O or NR 4 where R 4 is H or C ⁇ alkyl; or
  • Q is a group of formula -(CH 2 )-A(CH 2 ) S - where r is 0, 1 or 2, S is 0, 1 or 2 and A is a 3 to 6 membered carbocyclic or heterocyclic ring which may optionally be substituted by one or more (suitably 1 to 4) suitable substituents such as C,. 6 alkyl, C ⁇ alkoxy, hydroxy, halo, nitro, cyano, trifluoro C ⁇ alkyl, amino, C, .6 alkylamino or di C L galkylamino.
  • R 1 is methyl
  • R 2 is a group of formula -(CH 2 ) p X(CH 2 ) q -CH(NH 2 )CO 2 H where p, X, and q are as defined above, most preferably R 2 is a group of formula -(CH 2 ) p S(O) x (CH 2 ) q -CH(NH 2 )CO 2 H where p, x, and q are as defined above.
  • R 3 is -(CH 2 ) n NH 2 wherein n is 0, 1 , or 2.
  • Some compounds of formula (I) include an asymmetric centre in the amino acid functionality and although the natural L configuration is preferred, it is intended that the formula includes both isomers as well as mixtures thereof in any proportion.
  • Preferred compounds of formula (I) for the methods and uses of the present invention include: N-(3-aminomethylbenzyl)acetamide;
  • Suitable pharmaceutically acceptable salts of the compounds of formula (I) include those formed with both organic and inorganic acids or bases.
  • Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic, and isethionic acids.
  • Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine.
  • esters and amides of the compounds of formula (I) may have the acid group converted to a C ⁇ alkyl, aryl, aryl C ⁇ alkyl, or amino acid ester or amide.
  • Pharmaceutically acceptable amides of the compounds of formula (I) may have an amino group converted to a C,. 6 alkyl, aryl, aryl C 1- ⁇ alkyl, or amino acid amide.
  • the amount of inhibitor of inducible nitric oxide synthase, such as a compound of formula (I), or a pharmaceutically acceptable salt, ester or amide thereof which is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, and the particular disorder or disease being treated.
  • the compounds of the invention may be administered orally or via injection (either as a bolus, for example twice a day or as a continuous infusion) at a dose of from 0.02 to 1500mg/kg per day, suitably 0.1 to 500mg/kg per day, more suitably 1.0 to 25mg/kg per day.
  • the dose range for adult humans is generally from 1mg to 35g/day, suitably 5mg to 2g/day, more suitably 70mg to 1.8g/day.
  • Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5mg to 500mg, usually around 10mg to 200mg.
  • the inhibitor of inducible nitric oxide synthase such as the compound of formula (I), or pharmaceutically acceptable salt, ester, or amide thereof to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • active ingredient means an inhibitor of inducible nitric oxide synthase, such as a compound of formula (I), or a pharmaceutically acceptable salt, ester, or amide thereof.
  • the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.
  • Formulations for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose an acacia.
  • Preferred unit dosage formulations are those containing an effective dose, as hereinbefore recited, or an appropriate fraction thereof, of the active ingredient.
  • formulations used according to the invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the inhibitors of iNOS used according to the present invention may be used in combination with a further active ingredient, for example an antibiotic.
  • compositions comprising an inhibitor of inducible nitric oxide synthase (for example , a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof) and an antibiotic (as defined below) together with a pharmaceutically acceptable diluent or carrier comprise a further aspect of the invention.
  • an inhibitor of inducible nitric oxide synthase for example , a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof
  • an antibiotic as defined below
  • the present invention comprises a method for prophylaxis or treatment of a bacterial infection in a mammal which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase (for example, a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof) and an antibiotic (as defined below).
  • an inhibitor of inducible nitric oxide synthase for example, a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof
  • an antibiotic as defined below.
  • Suitable antibiotics for use in combination with an iNOS inhibitor in accordance with the present invention may be selected from a sulphonamide (e.g. sulphamethoxazole), a diaminopyrimidine (e.g. trimethoprim), a combination of a sulphonamide and a diaminopyrimidine (e.g. sulphamethoxazole-trimethoprim), a quinolone, particularly a fluoroquinolone (e.g. ciprofloxacin, levofloxacin and trovafloxacin), a nitroimidazole, a penicillin (e.g. amoxycillin), a cephalosporin (e.g.
  • a sulphonamide e.g. sulphamethoxazole
  • a diaminopyrimidine e.g. trimethoprim
  • the antibiotic for use in combination with an iNOS inhibitor in accordance with the present invention may be selected from a quinolone, a penicillin, or a cephalosporin (such as cefuroxime, or a prodrug of cefuroxime).
  • the antibiotic may be cefuroxime, or a prodrug of cefuroxime , such as cefuroxime axetil.
  • the inhibition of purified human NO synthases may be determined using a preparation of human nNOS (Furfine et al. (1993) Biochem. 32, 8512-8517), iNOS (Sherman et al. (1993) Biochem. 32, 11600-11605) and eNOS (Garvey et al. (1994) Arch. Bioch. Biophys., 235-241). Their activity may be monitored by the conversion of [ ⁇ C]-L-arginine to citrulline as described by Schmidt et al. ((1991) Proc. Natl. Acad. Sci.
  • reaction mixtures 100 ⁇ l containing 50mM HEPES pH 7.0, 8 ⁇ M tetrahydrobiopterin, 1mM NADPH and 0.5 ⁇ M [ 1 4C]-L-arginine (30,000 cpm) at 30°C.
  • CFU colony forming units
  • mice infected with S. pneumoniae were infected intranasally (i.n.) with S. pneumoniae 3496 and treated twice daily, orally, with an aqueous solution of (S)-[2-(1-iminoethylamino)ethyl]- L-homocysteine (Compound 1) (30 mg/kg). Control mice were treated with water only. Treatment started 1 hour before infection with live bacteria. Survival of the mice was monitored. Viable bacterial counts per lung were performed at different time points after infection.
  • Plasma levels of nitrite/nitrate were determined using Griess reagent ( 1g/100ml sulfanilamide and 0.1 g/100ml N- naphthyl)ethylendiamine dihydrochloride in approximately 5% phosphoric acid; Green e a/ (1982), Anal. Biochem. 126, 131-138).
  • the pathological consequences of infection with S. pneumoniae were monitored by histo- pathology. Lungs were fixed in formalin and sectioned using a rotary microtome. Staining was performed according to standard methods with hematoxilin-eosin stain.
  • the specific immune response was monitored by measuring serum antibodies against S. pneumoniae by Enzyme Linked Immunosorbent Assay (ELISA) technique, using a lysate of S. pneumoniae for coating, in order to detect the overall content of anti-pneumococcal antibodies. After incubation of the respective sera, specific binding was detected using a polyclonal rabbit anti- mouse serum, conjugated with alkaline-phosphatase (DAKO, Milan, Italy). The read-out was performed using PNPP (4-Nitrophenyl Phosphate; Fluka Chemie, Buchs, Switzerland) as a substrate for the alkaline phosphatase. Measurement of absorbance at 405nm was performed in a plate reader. Additionally, survivors of an infection with S. pneumoniae with and without treatment with compound 1 were challenged with a high dose of bacteria (>20 LD 50 ) and the survival was monitored. Results
  • EXPERIMENT 1 mouse strain: CBA/J (highly susceptible to S. pneumoniae; Charles River Italia, Milan) (a) Median Survival Time (MST)
  • mice showed antibody titers in serum between 1/800 and 1/1600. This represents normal anti-Streptococci titers compared with non-treated mice in our hands, infected i.n.
  • mice of the experiment were challenged with lethal dose of S. pneumoniae 3496 after resolution of the infection (4 weeks after the initial infection).
  • non-treated but infected mice as well as non-infected (therefore non-immune) mice were included. All of the immunized mice survived whereas the naive mice all died within 4 days.
  • EXPERIMENT 2 mouse strain: Balb/c (intermediate susceptibility to S. pneumoniae, Charles River Italia, Milan) (a) Median Survival Time (MST) Experimental observation time: 6 days
  • Formalin fixed lungs were processed, included in paraffin, cut at the rotatory microtome, stained with Haemotoxilin and Eosin (Sigma) and microscopically examined.
  • mice Microscopic examination of the lungs revealed areas of pneumonia in all examined mice from control or compound 1 treated groups. Changes were consistent with those observed in preliminary background study in mice infected with the same bacterial strain; in addition, pleuritis was observed starting from day 2 after infection.
  • mice from control group showed marked changes, while mice treated with the test compound showed mild pictures of pneumonia, particularly on day 2 after infection. Only mice receiving the test compound survived on Day 7 after infection and in these animals clear signs of recovery from pneumonia were observed.
  • Compound 2 administered subcutaneously via osmotic mini-pump.
  • mice In vivo treatment of mice infected with Klebsiella pneumoniae. Mice were infected intranasally (i.n.) with 1.3 X10 7 CFU/mouse K. pneumoniae 1976E and treated twice daily, orally, with a saline solution of Compound 1 (5 mg/kg). Control mice were treated with saline only. Treatment started Ihour before infection with live bacteria. Survival of the mice was monitored. Viable bacterial counts per lung were performed at different time points after infection.
  • EXPERIMENT 4 mouse strain: Balb/c female 20-22 g
  • CFU Viable bacterial counts ex vivo Bacterial counts
  • EXAMPLE 2 Septicemia (A) In vivo treatment of mice infected with S. pneumoniae. Mice were infected intraperitoneally (i.p.) with S. pneumoniae 3496 and treated twice daily, orally, with an aqueous solution of (S)-[2-(1-iminoethylamino)ethyl]- L-homocysteine (Compound 1) (30 mg/kg). Control mice were treated with water only. Treatment started 1 hour before infection with live bacteria. Survival of the mice was monitored.
  • mice infected with H. influenzae were infected intraperitoneally (i.p.) with H. influenzae 10211 (ATCC, USA) and treated twice daily, orally, with an aqueous solution of (S)-[2-(1- iminoethylamino)ethyl]-L-homocysteine (Compound 1) (5 mg/kg). Control mice were treated with water only. Treatment started 1 hour before infection with live bacteria. Survival of the mice was monitored. Results
  • EXAMPLE 3 Pneumonia treated with a combination of an iNOS inhibitor and an antibiotic.
  • EXPERIMENT 5 Combination with Ceftazidime in subcutaneous (s.c.) administration twice daily and (R)-2-amino-6-(1-iminoethylamino)-4,4-dioxo-4- thiahexanoic acid (Compound 2) in continuous s.c. administration via osmotic mini-pumps
  • mice ⁇ steria monocytogenes was applied intravenously (i.v.) in 0.2 ml PBS pH 7.4 to C57BL/6 mice (Charles River) (1.8x10 4 CFU per mouse).

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Abstract

The invention relates to use of an inhibitor of inducible nitric oxide synthase for the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection where the inhibitor of inducible nitric oxide synthase is, for example, a compound of formula (I), or a pharmaceutically acceptable salt, ester, or amide thereof, in which R1 is a C¿1-6? straight or branched chain alkyl group; and R?2¿ is a group of formula (II) or (III).

Description

USE OF NITRIC OXIDE SYNTHASE INHIBITORS IN THE MANUFACTURE OF A MEDICAMENT FOR THE PROPHYLAXIS OR TREATMENT OF BACTERIAL INFECTION
The present invention relates to the use of nitric oxide synthase inhibitors for the prophylaxis or treatment of bacterial infections.
Excess production of nitric oxide has been implicated in the pathophysiology of hypotension induced by endotoxin and inflammatory sequelae of viral or bacterial infections. The biosynthesis of nitric oxide from L-arginine is catalysed by the enzyme NO synthase. Hence, WO 91/04024 and other references teach that inhibitors of nitric oxide may be useful for the treatment of septic shock.
Previous literature evidence indicates that bacterial infection would be exacerbated in the presence of a nitric oxide synthase inhibitor (for example, the review by Macmicking et al, Annu. Rev. Immunol. 1997, 15, 323-50). Further it has been shown that exogenous NO has an antimicrobial effect in infected cells, which in itself contraindicates treatment of bacterial infection with an inhibitor of nitric oxide synthase (for example, the review by Bogdan: Behring Inst. Mitt., No. 99, 58-72 (1997). Further discussion of the role of nitric oxide in infection may be found in articles by F.C. Fang, J Clin. Invest, 99 (12), 2818-2825, 1997 and ASM News, 63 (12), 668-673.
We have now surprisingly found that certain inhibitors of nitric oxide synthase, namely those which selectively inhibit the inducible isoform of the enzyme, can decrease the number of bacteria in infected animals and can reduce mortality in infected animals and so may have utility in the prophylaxis or treatment of a bacterial infection.
Accordingly, the present invention provides a method for prophylaxis or treatment of a bacterial infection in a mammal, such as a human, which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase. In particular, there is provided a method for reducing the number of bacteria in an infected mammal, such as a human, which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase.
In the alternative, there is provided the use of an inhibitor of inducible nitric oxide synthase for the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection. In particular, there is provided the use of an inhibitor of inducible nitric oxide synthase for the manufacture of a medicament for reducing the number of bacteria in an infected mammal, such as a human.
By "treatment" is meant the improvement of clinical outcome, for example, a reduction in mortality. Such improvement of clinical outcome may be as a result of a reduction of the number of bacteria in the infected mammal.
By "bacterial infection" is meant an infection due to intracellular or extracellular bacteria, in particular due to extracellular bacteria, more particularly, any gram- positive or gram-negative bacteria. In particular, the infection may be bacterial pneumonia, for example, caused by Streptococcus pneumoniae or Klebsiella pneumoniae, or bacterial septicemia induced by Streptococcus pneumoniae or Haemophilus influenzae. More particularly, the infection may be community acquired pneumonia, such as may be caused by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Chlamydia pneumoniae, Mycoplasma pneumoniae, or Legionella pneumophila, in particular by Streptococcus pneumoniae or Haemophilus influenzae. Alternatively, the infection may be hospital acquired pneumonia, such as may be caused by Streptococcus pneumoniae, Haemophilus influenzae, a Klebsiella species such as Klebsiella pneumoniae, Eschenchia coli, an Enterobacter species, Pseudomonas aeruginosa, or an Actinetobacter species. In a particular aspect of the invention the infection is one caused by bacteria not primarily resident in red blood cells. Other conditions caused by bacterial infection include urinary tract infections e.g. pyelonephritis, otitis (media or externa), ocular infections, bronchitis, and other clinical situations where bacterial infection may arise as a complication e.g. during surgery.
By "inhibitor of inducible nitric oxide synthase" is meant a compound which inhibits the inducible isoform of nitric oxide synthase (iNOS) to a significantly greater extent than the constitutive (neuronal and endothelial) isoforms (nNOS and eNOS). Suitably, the inhibitor would exhibit at least 5-fold selectivity, preferably at least 10-fold selectivity, and more preferably at least 50-fold selectivity for iNOS over eNOS and nNOS as demonstrated in an enzyme inhibition assay, such as the assays described below. Examples of suitable compounds include some of those described in WO93/13055, WO 96/19440, and WO 98/30537.
Other inhibitors of iNOS may be found, for example, in the scope of the following patent applications: WO96/15120, WO 97/10204, WO 97/32844, WO 95/24382, WO 95/25717, WO 99/05131 , WO 96/33175, WO 96/35677, WO 98/30220, WO 95/11231 , WO 95/11014, EP 0897912, EP0724570, WO 96/14842, WO 96/14844, WO 97/38977, WO 98/45294, WO 98/46611, WO 99/01455, WO 97/14686, WO 96/30350, WO 96/30350, WO 97/02254, JP 10045751 -A, FR 2730733, FR 2727111 , WO 98/37079, and JP 08310945.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.
Thus in a further aspect, the present invention provides a method for prophylaxis or treatment of a bacterial infection in a mammal which comprises administering an effective amount of a compound of formula (I):
Ri
^
(I)
HN NH
or a pharmaceutically acceptable salt, ester, or amide thereof , in which R1 is a C^ straight or branched chain alkyl group; and
R2 is a group of formula
Figure imgf000006_0001
wherein
R3 is -(CH2)nNH2 wherein n is 0, 1 , or 2; Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more (suitably, 1 to 4) C1-3 alkyl groups; or
Q is a group of formula -(CH2)pX(CH2)q- where p is 2 or 3, q is 1 or 2 and X is S(0)x where x is 0, 1 or 2, O or NR4 where R4 is H or CM alkyl; or
Q is a group of formula -(CHy-A^H-)-- where r is 0, 1 or 2, s is 0, 1 or 2 and A is a 3 to 6 membered carbocyclic or heterocyclic ring which may optionally be substituted by one or more (suitably, 1 to 4) suitable substituents such as C^ alkyl, C^ alkoxy, hydroxy, halo, nitro, cyano, trifluoro C1-6 alkyl, amino, C^alkylamino or di C^alkylamino.
In the alternative, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof as defined above for the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection.
Preferably for the methods and uses described above, the compound of formula (I) is
Figure imgf000007_0001
or a pharmaceutically acceptable salt, ester, or amide thereof, in which:
R1 is a C^e straight or branched chain alkyl group;
Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more (suitably, 1 to 4) C^ alkyl groups; or
Q is a group of formula -(CH2)pX(CH2)q- where p is 2 or 3, q is 1 or 2 and X is S(O)x where x is 0, 1 or 2, O or NR4 where R4 is H or C^ alkyl; or
Q is a group of formula -(CH2)-A(CH2)S- where r is 0, 1 or 2, S is 0, 1 or 2 and A is a 3 to 6 membered carbocyclic or heterocyclic ring which may optionally be substituted by one or more (suitably 1 to 4) suitable substituents such as C,. 6alkyl, C^ alkoxy, hydroxy, halo, nitro, cyano, trifluoro C^ alkyl, amino, C,.6 alkylamino or di CLgalkylamino.
Preferably, in the compound of formula (I), R1 is methyl.
Preferably, in the compound of formula (I), R2 is a group of formula -(CH2)pX(CH2)q-CH(NH2)CO2H where p, X, and q are as defined above, most preferably R2 is a group of formula -(CH2)pS(O)x(CH2)q-CH(NH2)CO2H where p, x, and q are as defined above.
In a further aspect, in the methods and uses described above, the compound of formula (I) is
Figure imgf000008_0001
or a pharmaceutically acceptable salt, ester, or amide thereof, in which: R3 is -(CH2)nNH2 wherein n is 0, 1 , or 2.
Some compounds of formula (I) include an asymmetric centre in the amino acid functionality and although the natural L configuration is preferred, it is intended that the formula includes both isomers as well as mixtures thereof in any proportion.
Preferred compounds of formula (I) for the methods and uses of the present invention include: N-(3-aminomethylbenzyl)acetamide;
(R)-2-amino-6-(1 -iminoethylamino)-4,4-dioxo-4-thiahexanoic acid; and (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine; and pharmaceutically acceptable salts, esters, or amides thereof.
Compounds of formula (I) which are particularly preferred for the methods and uses of the present invention, include:
(R)-2-amino-6-(1-iminoethylamino)-4,4-dioxo-4-thiahexanoic acid; and
(S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine; and pharmaceutically acceptable salts, esters, or amides thereof.
Suitable pharmaceutically acceptable salts of the compounds of formula (I) include those formed with both organic and inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic, and isethionic acids. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine.
Pharmaceutically acceptable esters and amides of the compounds of formula (I) may have the acid group converted to a C^alkyl, aryl, aryl C^ alkyl, or amino acid ester or amide. Pharmaceutically acceptable amides of the compounds of formula (I) may have an amino group converted to a C,.6alkyl, aryl, aryl C1-β alkyl, or amino acid amide.
The amount of inhibitor of inducible nitric oxide synthase, such as a compound of formula (I), or a pharmaceutically acceptable salt, ester or amide thereof which is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, and the particular disorder or disease being treated. The compounds of the invention may be administered orally or via injection (either as a bolus, for example twice a day or as a continuous infusion) at a dose of from 0.02 to 1500mg/kg per day, suitably 0.1 to 500mg/kg per day, more suitably 1.0 to 25mg/kg per day. The dose range for adult humans is generally from 1mg to 35g/day, suitably 5mg to 2g/day, more suitably 70mg to 1.8g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5mg to 500mg, usually around 10mg to 200mg.
While it is possible for the inhibitor of inducible nitric oxide synthase, such as the compound of formula (I), or pharmaceutically acceptable salt, ester, or amide thereof to be administered alone, it is preferable to present it as a pharmaceutical formulation.
Hereinafter, the term "active ingredient" means an inhibitor of inducible nitric oxide synthase, such as a compound of formula (I), or a pharmaceutically acceptable salt, ester, or amide thereof.
The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.
Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose an acacia.
Preferred unit dosage formulations are those containing an effective dose, as hereinbefore recited, or an appropriate fraction thereof, of the active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations used according to the invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents. Furthermore, the inhibitors of iNOS used according to the present invention may be used in combination with a further active ingredient, for example an antibiotic.
The combination referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical compositions comprising an inhibitor of inducible nitric oxide synthase (for example , a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof) and an antibiotic (as defined below) together with a pharmaceutically acceptable diluent or carrier comprise a further aspect of the invention.
The individual components of such a combination may also be administered either sequentially or simultaneously in separate pharmaceutical formulations.
Therefore, in a further aspect, the present invention comprises a method for prophylaxis or treatment of a bacterial infection in a mammal which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase (for example, a compound of formula (I) or a pharmaceutically acceptable salt, ester, or amide thereof) and an antibiotic (as defined below).
Appropriate doses of known therapeutic agents, such as antibiotics, for use in combination with the iNOS inhibitor, for example, a compound of formula (I), will be readily appreciated by those skilled in the art.
Suitable antibiotics for use in combination with an iNOS inhibitor in accordance with the present invention may be selected from a sulphonamide (e.g. sulphamethoxazole), a diaminopyrimidine (e.g. trimethoprim), a combination of a sulphonamide and a diaminopyrimidine (e.g. sulphamethoxazole-trimethoprim), a quinolone, particularly a fluoroquinolone (e.g. ciprofloxacin, levofloxacin and trovafloxacin), a nitroimidazole, a penicillin (e.g. amoxycillin), a cephalosporin (e.g. cefuroxime or ceftazidime), a glycopeptide (e.g. vancomycin), a tetracycline, an aminoglycoside, chloramphenicol, or a macrolide. In one aspect of the invention, the antibiotic for use in combination with an iNOS inhibitor in accordance with the present invention may be selected from a quinolone, a penicillin, or a cephalosporin (such as cefuroxime, or a prodrug of cefuroxime). In a particular aspect, the antibiotic may be cefuroxime, or a prodrug of cefuroxime , such as cefuroxime axetil.
Enzyme inhibition assays
The inhibition of purified human NO synthases may be determined using a preparation of human nNOS (Furfine et al. (1993) Biochem. 32, 8512-8517), iNOS (Sherman et al. (1993) Biochem. 32, 11600-11605) and eNOS (Garvey et al. (1994) Arch. Bioch. Biophys., 235-241). Their activity may be monitored by the conversion of [^C]-L-arginine to citrulline as described by Schmidt et al. ((1991) Proc. Natl. Acad. Sci. USA 88, 365-369) in reaction mixtures (100μl) containing 50mM HEPES pH 7.0, 8μM tetrahydrobiopterin, 1mM NADPH and 0.5μM [14C]-L-arginine (30,000 cpm) at 30°C.
The present invention will now be illustrated by way of example only using data gathered in initial experiments. Throughout the examples, the abbreviation "CFU" means colony forming units.
Example 1 : Pneumonia
(A) In vivo treatment of mice infected with S. pneumoniae. Mice were infected intranasally (i.n.) with S. pneumoniae 3496 and treated twice daily, orally, with an aqueous solution of (S)-[2-(1-iminoethylamino)ethyl]- L-homocysteine (Compound 1) (30 mg/kg). Control mice were treated with water only. Treatment started 1 hour before infection with live bacteria. Survival of the mice was monitored. Viable bacterial counts per lung were performed at different time points after infection. Plasma levels of nitrite/nitrate were determined using Griess reagent ( 1g/100ml sulfanilamide and 0.1 g/100ml N- naphthyl)ethylendiamine dihydrochloride in approximately 5% phosphoric acid; Green e a/ (1982), Anal. Biochem. 126, 131-138). The pathological consequences of infection with S. pneumoniae were monitored by histo- pathology. Lungs were fixed in formalin and sectioned using a rotary microtome. Staining was performed according to standard methods with hematoxilin-eosin stain.
The specific immune response was monitored by measuring serum antibodies against S. pneumoniae by Enzyme Linked Immunosorbent Assay (ELISA) technique, using a lysate of S. pneumoniae for coating, in order to detect the overall content of anti-pneumococcal antibodies. After incubation of the respective sera, specific binding was detected using a polyclonal rabbit anti- mouse serum, conjugated with alkaline-phosphatase (DAKO, Milan, Italy). The read-out was performed using PNPP (4-Nitrophenyl Phosphate; Fluka Chemie, Buchs, Switzerland) as a substrate for the alkaline phosphatase. Measurement of absorbance at 405nm was performed in a plate reader. Additionally, survivors of an infection with S. pneumoniae with and without treatment with compound 1 were challenged with a high dose of bacteria (>20 LD50) and the survival was monitored. Results
EXPERIMENT 1 : mouse strain: CBA/J (highly susceptible to S. pneumoniae; Charles River Italia, Milan) (a) Median Survival Time (MST)
Experimental observation time : 20 days
Figure imgf000015_0001
(b) Bacterial load in the lungs of infected animals at different time points after infection
Figure imgf000016_0001
mean of n = 5 mice per group and time point; note that on day 6 in the control group only 4 of 8 mice were alive, therefore mean is of n = 4 mice at this point for this group
(c) Determination of nitrite in plasma of the mice: data from pooled sera of n = 6 mice; determination in triplicates
(n.d. = not determined). The fact that no reduction in plasma nitrite was observed is to be expected from the teaching of Bergeron, Y. et al. (1998),
Infect. Immun. 66, 912-922.
Figure imgf000016_0002
Further, the antibody response towards S. pneumoniae in the sera of the surviving mice was monitored with ELISA technique, using a crude lysate of the S. pneumoniae for coating (sera were taken at day 14 p.i.):
The mice showed antibody titers in serum between 1/800 and 1/1600. This represents normal anti-Streptococci titers compared with non-treated mice in our hands, infected i.n.
To get a final proof of the immune response, survivors of the experiment were challenged with lethal dose of S. pneumoniae 3496 after resolution of the infection (4 weeks after the initial infection). For comparison, non-treated but infected mice as well as non-infected (therefore non-immune) mice were included. All of the immunized mice survived whereas the naive mice all died within 4 days.
These data show, that the acquired immunity after treatment with the iNOS- inhibitor was not compromised.
EXPERIMENT 2: mouse strain: Balb/c (intermediate susceptibility to S. pneumoniae, Charles River Italia, Milan) (a) Median Survival Time (MST) Experimental observation time: 6 days
Figure imgf000017_0001
(b) Bacterial load in the lungs of infected animals at different time points after infection
Figure imgf000018_0001
mean of n = 4 mice per group and time point, except the value for the compound 1 treated on day 6: n=2 mice (2 died): note that on day 6 no mice in the control group were alive, therefore the count of CFU/lung is an arbitrary one with the value of >1 x 109 CFU per lung.
(c) Determination of nitrite in plasma of the mice data from pooled sera of n = 4 mice, except the value of day 6: n = 2 mice; determination in triplicates (n.d. = not determined)
Figure imgf000018_0002
(d) Histology results
Formalin fixed lungs were processed, included in paraffin, cut at the rotatory microtome, stained with Haemotoxilin and Eosin (Sigma) and microscopically examined.
Microscopic examination of the lungs revealed areas of pneumonia in all examined mice from control or compound 1 treated groups. Changes were consistent with those observed in preliminary background study in mice infected with the same bacterial strain; in addition, pleuritis was observed starting from day 2 after infection.
Mice from control group showed marked changes, while mice treated with the test compound showed mild pictures of pneumonia, particularly on day 2 after infection. Only mice receiving the test compound survived on Day 7 after infection and in these animals clear signs of recovery from pneumonia were observed.
EXPERIMENT 3
S. pneumoniae infected mice (i.n. infection with at least 10 times LD50), n=8 mice per group were treated with (R)-2-amino-6-(1-iminoethylamino)-4,4-dioxo- 4-thiahexanoic acid (Compound 2) administered subcutaneously via osmotic mini-pump.
Figure imgf000019_0001
(B) In vivo treatment of mice infected with Klebsiella pneumoniae. Mice were infected intranasally (i.n.) with 1.3 X107 CFU/mouse K. pneumoniae 1976E and treated twice daily, orally, with a saline solution of Compound 1 (5 mg/kg). Control mice were treated with saline only. Treatment started Ihour before infection with live bacteria. Survival of the mice was monitored. Viable bacterial counts per lung were performed at different time points after infection.
Results
EXPERIMENT 4: mouse strain: Balb/c female 20-22 g
(a) Survival of the infected mice
None of the infected mice died during the time course of infection
(b) Viable bacterial counts ex vivo Bacterial counts (CFU) were determined in lung/blood.
Viable counts from the lungs of infected Balb/c mice (n=4 per group and time point)
Figure imgf000020_0001
EXAMPLE 2 : Septicemia (A) In vivo treatment of mice infected with S. pneumoniae. Mice were infected intraperitoneally (i.p.) with S. pneumoniae 3496 and treated twice daily, orally, with an aqueous solution of (S)-[2-(1-iminoethylamino)ethyl]- L-homocysteine (Compound 1) (30 mg/kg). Control mice were treated with water only. Treatment started 1 hour before infection with live bacteria. Survival of the mice was monitored.
Results
(a) Median Survival Time (MST)
Experimental observation time: 5 days
Figure imgf000021_0001
(B) In vivo treatment of mice infected with H. influenzae. Mice were infected intraperitoneally (i.p.) with H. influenzae 10211 (ATCC, USA) and treated twice daily, orally, with an aqueous solution of (S)-[2-(1- iminoethylamino)ethyl]-L-homocysteine (Compound 1) (5 mg/kg). Control mice were treated with water only. Treatment started 1 hour before infection with live bacteria. Survival of the mice was monitored. Results
(a) Median Survival Time (MST)
Experimental observation time: 4 days
Figure imgf000022_0001
EXAMPLE 3: Pneumonia treated with a combination of an iNOS inhibitor and an antibiotic.
All data were generated in a Balb/c mouse model of pulmonary infection with S. pneumoniae 3496 (infected with at least 10 times the LD50) using a method analogous to that described in Example 1. Calculation of geometric mean was done using an arbitrary value of 250 CFU/lung for the counts below detection limit and an arbitrary value of 5x109 CFU/lung for dead mice.
EXPERIMENT 1 : Combination with Cefuroxime-Axetil in oral administration twice daily for both compounds
Figure imgf000023_0001
EXPERIMENT 2. Combination with Amoxyclav in oral administration twice daily for both compounds data collected day 4 post infection; n=5 per group
Figure imgf000024_0001
EXPERIMENT 3: Combination with Ciprofloxacin in oral administration twice daily for both compounds
data collected day 4 post infection; n=4 per group
Figure imgf000025_0001
data collected day 5 post infection; n=5 per group
Figure imgf000025_0002
EXPERIMENT 4: Combination with Erythromycin in administration twice daily for both compounds data collected day 4 post infection; n=5 per group
Figure imgf000026_0001
data collected day 4 post infection; n=5 per group
Figure imgf000026_0002
EXPERIMENT 5: Combination with Ceftazidime in subcutaneous (s.c.) administration twice daily and (R)-2-amino-6-(1-iminoethylamino)-4,4-dioxo-4- thiahexanoic acid (Compound 2) in continuous s.c. administration via osmotic mini-pumps
data collected day 4 post infection; n=5 per group
Figure imgf000027_0001
EXAMPLE 4: Intracellular pathogen (Listeria monocytogenes)
ϋsteria monocytogenes was applied intravenously (i.v.) in 0.2 ml PBS pH 7.4 to C57BL/6 mice (Charles River) (1.8x104 CFU per mouse). On the respective day after infection, liver and spleen were prepared, homogenised, and the bacterial load determined by plating serial ten-fold dilutions on Trypticase-Soy-Agar plates. After 24 hours at 37°C, bacterial counts were taken. n=5 mice per group and day. Results
Mean bacterial counts
Figure imgf000028_0001

Claims

1. A method for prophylaxis or treatment of a bacterial infection in a mammal which comprises administering an effective amount of an inhibitor of inducible nitric oxide synthase.
2. A method for prophylaxis or treatment of a bacterial infection in a mammal which comprises administering an effective amount of a compound of formula (I):
R1
^
(I)
HN ' NH
or a pharmaceutically acceptable salt, ester, or amide thereof , in which R1 is a C1-6 straight or branched chain alkyl group; and
R2 is a group of formula
Figure imgf000029_0001
wherein
R3 is -(CH2)nNH2 wherein n is 0, 1 , or 2;
Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more C1-3 alkyl groups; or
Q is a group of formula -(CH2)pX(CH2)q- where p is 2 or 3, q is 1 or 2 and X is S(O)x where x is 0, 1 or 2, O or NR4 where R4 is H or a, alkyl; or Q is a group of formula -(CH2),A(CH2)S- where r is 0, 1 or 2, s is 0, 1 or 2 and A is a 3 to 6 membered carbocyclic or heterocyclic ring which may optionally be substituted by one or more suitable substituents such as C,.6 alkyl, C^ alkoxy, hydroxy, halo, nitro, cyano, trifluoro C^ alkyl, amino, C^alkylamino or di C^alkylamino.
A method according to claim 2 wherein the compound of formula (I) is:
Figure imgf000030_0001
or a pharmaceutically acceptable salt, ester, or amide thereof, in which:
R1 is a C,.6 straight or branched chain alkyl group;
Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbon atoms and which may optionally be substituted by one or more C^ alkyl groups; or
Q is a group of formula -(CH2)pX(CH2)q- where p is 2 or 3, q is 1 or 2 and X is S(0)x where x is 0, 1 or 2, O or NR4 where R4 is H or C^alkyl; or
Q is a group of formula -(CH2),A(CH2)S- where r is 0, 1 or 2, S is 0, 1 or 2 and A is a 3 to 6 membered carbocyclic or heterocyclic ring which may optionally be substituted by one or more suitable substituents such as C^alkyl, C^ alkoxy, hydroxy, halo, nitro, cyano, trifluoro C^ alkyl, amino, C^alkylamino or di C^alkylamino.
4. A method according to any of claims 1 to 3 wherein the compound of formula (I) is (R)-2-amino-6-(1-iminoethylamino)-4,4-dioxo-4- thiahexanoic acid or a pharmaceutically acceptable salt, ester, or amide thereof.
5. A method according to any of claims 1 to 3 wherein the compound of formula (I) is (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine or a pharmaceutically acceptable salt, ester, or amide thereof.
6. Use of an inhibitor of inducible nitric oxide synthase for the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection.
7. Use of a compound of formula (I) as defined in any of claims 2 to 5 or a pharmaceutically acceptable salt, ester, or amide thereof for the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection.
8. A method according to any one of claims 1 to 5 which further comprises administration of an antibiotic.
9. A pharmaceutical formulation comprising an inhibitor of inducible nitric oxide synthase and an antibiotic together with a pharmaceutically acceptable diluent or carrier.
10. A pharmaceutical formulation according to claim 9 wherein the inhibitor of inducible nitric oxide synthase is a compound of formula (I) as defined in any one of claims 2 to 5, or a pharmaceutically acceptable salt, ester, or amide thereof.
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US10231975B2 (en) 2009-09-04 2019-03-19 Horizon Orphan Llc Use of aerosolized levofloxacin for treating cystic fibrosis
US10792289B2 (en) 2009-09-04 2020-10-06 Horizon Orphan Llc Use of aerosolized levofloxacin for treating cystic fibrosis
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AR020587A1 (en) 2002-05-22
MA24854A1 (en) 1999-12-31

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