WO2009141264A2 - Gallidermine pour traiter les infections de l’appareil respiratoire - Google Patents

Gallidermine pour traiter les infections de l’appareil respiratoire Download PDF

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Publication number
WO2009141264A2
WO2009141264A2 PCT/EP2009/055852 EP2009055852W WO2009141264A2 WO 2009141264 A2 WO2009141264 A2 WO 2009141264A2 EP 2009055852 W EP2009055852 W EP 2009055852W WO 2009141264 A2 WO2009141264 A2 WO 2009141264A2
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Prior art keywords
gallidermin
pharmaceutical composition
composition according
active substance
pharmaceutically active
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PCT/EP2009/055852
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English (en)
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WO2009141264A3 (fr
Inventor
Hubert Muellner
Thierry Bouyssou
Juergen Daemmgen
Bernd Disse
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Boehringer Ingelheim Vetmedica Gmbh
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Publication of WO2009141264A2 publication Critical patent/WO2009141264A2/fr
Publication of WO2009141264A3 publication Critical patent/WO2009141264A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • the present invention lies in the field of the treatment and prophylaxis of infectious diseases. It relates to the prophylaxis and treatment of bacterial infections of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient, comprising the lantibiotic Gallidermin or a pharmaceutically active variant thereof as active substance, and related subject matters.
  • Infectious diseases especially those which are caused by gram-positive bacteria are still a severe danger for humans and animals. In the livestock industry they lead to severe losses.
  • Important genera of gram-positive pathogenic bacteria are Staphylococcus and Streptococcus.
  • Staphylococcus aureus and Streptococcus pneumoniae can cause life-threatening diseases of the lung, in humans as well as in mammals like cattle and pigs.
  • Other examples for critical pathogenes of the human lung are Haemophilus influenza and Moraxella catarrhalis.
  • Especially the growing rates of immunity against classical antibiotica treatments make it necessary to develop alternative concepts for therapies against such infections.
  • Lantibiotics are a class of small peptidic, i.e. peptide or peptide-derived molecules that exert an antibiotic effect, especially on bacteria which effect can be bacteriostatic or bacteriocidal. They are naturally produced by gram positve bacteria as gene encoded precursor peptides (e.g. gdmA as the gene for the Gallidermin precursor peptide) and undergo posttranslational modification of the primary transcript (prolantibiotic) to generate the mature, active peptide. Therefore many of them are characterized by significant posttranslational modifications, including the modification of amino acids into unusual, sometimes even bridged thioether amino acids, like lanthionine and 3- methyllanthionine.
  • Lantibiotics are generally divided into two classes in respect to their mode of action: lantibiotics of class A (Nisin- type) usually integrate into the plasma membrane and form pores which leads to a loss of low-molecula intracellular components and of the electric membrane potential. Lantibiotics of class B (Mersacidin-type or Cinnamycin-type) usually block the synthesis of peptidoglycan by binding tightly to the LIPID Il and thus to a direct inhibition of the synthesis of the bacterial cell wall.
  • the 4-ring lantibiotic Gallidermin like other 4-ring lantibiotics (Epidermin and Mutacin) is an example for a class A-type lantibiotic.
  • Other examples are the 5-ring lantibiotics Nisin and Subtilin and the 3-ring lantibiotic Pep5.
  • Gallidermin comprises the structure that is represented by figures 1 and 2 of this application.
  • Gallidermin The natural variant of Gallidermin is described by Furmanek et al. (1999), J. Appl. Microbiol., Vol. 87, 856-866. It exerts antimicrobial activity especially against the
  • Staphylococcus species which could - as assumed by the authors - be used for the treatment of staphylococcal infections.
  • Gallidermin for the treatment of bacterial infections of the skin is disclosed by EP 342486 A1. There it is described as being particularly efficient against Propionibacterium acnes.
  • EP 427912 A1 discloses a method of inhibiting procaryotic microbial growth especially useful in the food industry which method involves adding a synergistically effective combination of a lantibiotic and lysozyme to an environment in which such microbial growth is to be inhibited.
  • Preferred lantibiotics are selected from the group consisting of Nisin, Subtilin, Pep 5, Epidermin, Gallidermin, Cinnamycin, Ro09-0198, Duramycin or Ancovenin.
  • the procaryotic microorganism is a gram-positive bacteria. Respective medical uses are not disclosed.
  • compositions containing bacteriocin are disclosed in VVO 93/13793 A1.
  • Such bacteriocins are selected from Nisin, Subtilin, Epidermin, Pep5, Ancovenin, Gallidermin, Duromycin and Cinnamycin.
  • These compositions are described and/or claimed to be useful as antibacterial agents, especially with respect to infections of bacteria in the gastrointestinal tract of humans, e.g. infections caused by the gram-positive as well as gram-negative bacteria Helicobacter, Escherichia, Salmonella, Bacillus, Clostridia, Bacteroides, Campylobacter or Yersinia.
  • WO 94/28726 A2 describes pharmaceutical uses of the lantibiotic Duramycin, which facilitates the clearance of retained pulmonary secretions in a patient for example with cystic fibrosis.
  • the lantibiotic is preferably administered topically to the respiratory epithelia, such as by generating an aerosol thereof which is then inhaled by the subject.
  • a method of combatting tuberculosis comprising administering a lantibiotic to a subject in need of such treatment. This is exemplified again by Duramycin.
  • Gallidermin and/or Epidermin in the field of veterinary medicine is disclosed by WO 95/05844 A1. These lantibiotics are described to be useful for the prevention or treatment of bovine mastitis and for the sterilization of milk products without affecting microorganisms involved in the production of cheese and yoghurt. Relevant pathogens of mastitis are gram-positive Streptococcus spp. However, in the meantime Epidermin has found to be less active than Gallidermin in this technical field.
  • lantibiotics for killing antibiotic-resistant pathogenic bacteria, especially gram-positives
  • Claimed are especially Nisin, Subtilin, Epidermin, Gallidermin, Pep 5, Cinnamycin, Duramycin and Ancovenin, preferred Nisin, which is also supported by the examples.
  • Nisin is also described to be useful for preventing or treating infections by antibiotic- resistant or multidrug-resistant pathogenic bacterial strains of Streptococcus pneumoniae, by US patent US 5910479.
  • the use of other lantibiotics like Gallidermin is also claimed but not exemplified.
  • Nisin has found to be less active than Gallidermin in this technical field.
  • Goldstein et al. (1998), J. Antimicrob. Chemother., 42(2), 277-278 describe the activity of a certain antibacterial peptide, i.e. of Nisin against S. pneumoniae in vitro and in a mouse infection model.
  • This model consists of a certain mouse strain that has been infected intraperitoneal ⁇ .
  • This publication is also silent about infections of other regions of the patient's body, especially about the respiratory tract and about the efficacy of other antibacterial peptides.
  • Tobramycin For sake of completeness the use of Tobramycin should be mentioned here because it is state of the art to use this agent for the treatment of infections of the respiratory tract in the form of an aerosol. Chemically, Tobramycin belongs to the group of aminoglycosids. However, this molecule exerts nephrotoxic and ototoxic side effects which cause risks for the treatment of patients. It is therefore desirable to identify antibiotic molecules for the treatment of infections of the respiratory tract which do not exert these negative side effects.
  • the present invention provides a pharmaceutical composition for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient, comprising Gallidermin or a pharmaceutically active variant thereof as active substance.
  • a further aspect of the present invention concerns a combined product, comprising a pharmaceutical composition according to the invention and a technical apparatus which allows the application of metered doses of the pharmaceutical composition.
  • a further aspect of the present invention concerns the respective uses of Gallidermin or a pharmaceutically active variant thereof as active substance for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient.
  • a further aspect of the present invention concerns the respective methods for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient, wherein the method comprises administration of Gallidermin or a pharmaceutically active variant thereof as active substance to said patient.
  • a further aspect of the present invention concerns the respective uses of Gallidermin or a pharmaceutically active variant thereof as active substance for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient.
  • Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission (Biochemistry, 1972, vol. VV, pages 1726-1732).
  • One aspect of the present invention provides a pharmaceutical composition for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient, comprising Gallidermin or a pharmaceutically active variant thereof as active substance.
  • Gallidermin is illustrated by figures 1 and 2 of this application, both showing the wildtype molecule of Gallidermin as produced naturally by Staphylococcus gallinarum (DSM 4616) and disclosed in EP 342486 A2.
  • DSM 4616 Staphylococcus gallinarum
  • Gallidermin according to the invention also comprises variants of Gallidermin that are additionally characterized by point mutations in one, two or three positions of the molecule shown in figures 1 and 2 which counts 22 amino acids in its wildtype form, some of them modified as explained in the above mentioned literature.
  • Point mutations leading to Gallidermin variants according to the invention comprise the deletion, the insertion or the substitution of - in total - up to three amino acids of the wildtype molecule.
  • the molecule Epidermin is also a variant of Gallidermin and is thus comprised by the definition Gallidermin or a pharmaceutically active variant according to the invention because it differs from the wildtype Gallidermin in only one position, i.e. I instead of L in position 6.
  • variants according to the invention are exemplified by the scientific publication from B. Ottenwalder et al. (1995) in Appl. Environ. Microbiol., Nov. 1995, pages 3894-3903.
  • Preferred variants according to the invention are variants L6V, Dhb14Dha, A12L and Dhb14P, as described by Ottenwalder et al. They can be derived from the wildtype Gallidermin by the substitution (exchange) of leucin in position 6 against valine, of Dhb in position 14 against Dha (2,3-didehydroalanine), of alanine in position 12 against leucine and Dhb in position 14 against proline, and combinations of up to three of these exchanges. According to the invention these substitutions can be combined with other sequence variations as long as the total number of variations in comparison to the wildtype Gallidermin is not larger than 3.
  • Gallidermin and pharmaceutically active variants thereof comprised by the invention can be produced accordingly. Further ameliorations of that process might be developed in the future and can consequently be exploited for the production of Gallidermin and pharmaceutically active variants thereof according to the pending application. Sequence variations can be introduced as described by Ottenwalder et al. or analogously.
  • Gallidermin and other active variants according to the invention like Epidermin can be ascribed to type-A lantibiotics which act on the bacterial membrane by pore formation. It is believed that such a pore leads to an efflux of low-molecular intracellular components and the depolarization of the cytoplasmic membrane resulting in an instant termination of essential biosynthetic processes.
  • Gram-negative and mammalian cells are usually less affected by this mechanism which makes lantibiotics according to the invention especially suitable for the treatment of infections caused by gram-positive bacteria. The proof for the efficacy of this treatment in given in he examples of this application.
  • compositions for the prophylaxis or treatment of a bacterial infection by such susceptible bacteria are therefore also comprised by the invention.
  • composition refers to all compositions of chemical substances that are suitable for the application to a human or non-human patient and thus for the treatment or prophylaxis of a disease.
  • Ingredients for pharmaceutical compositions are in principal known from the state of the art or can be derived from the ongoing progress in this technical field. They all belong to the claimed area as far as they are designed for for the prophylaxis or treatment of a bacterial infection of the respiratory tract and comprise Gallidermin or a pharmaceutically active variant thereof as active substance.
  • Suitable preparations for administering the active substance(s) according to the invention include tablets, capsules, suppositories, solutions, etc. Of particular importance according to the invention is the administration of the compounds according to the invention by inhalation (see below).
  • the proportion of pharmaceutically active compound or compounds should be in the range from 0.05 to 90% by weight, preferably 0.1 to 50% by weight of the total composition.
  • Suitable tablets may be obtained, for example, by mixing the active substance(s) with known excipients, for example inert diluents such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatine, lubricants such as magnesium stearate or talc and/or agents for delaying release, such as carboxymethyl cellulose, cellulose acetate phthalate, or polyvinyl acetate.
  • excipients for example inert diluents such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatine, lubricants such as magnesium stearate or talc and/or agents for delaying release, such as carboxymethyl cellulose, cellulose acetate phthalate, or polyvinyl acetate.
  • excipients for example inert dilu
  • Coated tablets may be prepared accordingly by coating cores produced analogously to the tablets with substances normally used for tablet coatings, for example collidone or shellac, gum arabic, talc, titanium dioxide or sugar.
  • the core may also consist of a number of layers.
  • the tablet coating may consist of a number or layers to achieve delayed release, possibly using the excipients mentioned above for the tablets.
  • Syrups or elixirs containing the active substance(s) or combinations thereof according to the invention may additionally contain a sweetener such as saccharine, cyclamate, glycerol or sugar and a flavour enhancer, e.g. a flavouring such as vanillin or orange extract. They may also contain suspension adjuvants or thickeners such as sodium carboxymethyl cellulose, wetting agents such as, for example, condensation products of fatty alcohols with ethylene oxide, or preservatives such as p-hydroxybenzoates.
  • a sweetener such as saccharine, cyclamate, glycerol or sugar
  • a flavour enhancer e.g. a flavouring such as vanillin or orange extract.
  • They may also contain suspension adjuvants or thickeners such as sodium carboxymethyl cellulose, wetting agents such as, for example, condensation products of fatty alcohols with ethylene oxide, or preservatives such as p-hydroxybenzoates.
  • Solutions are prepared in the usual way, e.g. with the addition of isotonic agents, preservatives such as p-hydroxybenzoates, or stabilisers such as alkali metal salts of ethylenediamine tetraacetic acid, optionally using emulsifiers and/or dispersants, whilst if water is used as the diluent, for example, optionally organic solvents may optionally be used as solvating agents or dissolving aids, and transferred into injection vials or ampoules or infusion bottles.
  • isotonic agents e.g. with the addition of isotonic agents, preservatives such as p-hydroxybenzoates, or stabilisers such as alkali metal salts of ethylenediamine tetraacetic acid, optionally using emulsifiers and/or dispersants, whilst if water is used as the diluent, for example, optionally organic solvents may optionally be used as solvating agents or dissolving aids
  • Capsules containing one or more active substances or combinations of active substances may for example be prepared by mixing the active substances with inert carriers such as lactose or sorbitol and packing them into gelatine capsules.
  • Suitable suppositories may be made for example by mixing with carriers provided for this purpose, such as neutral fats or polyethyleneglycol or the derivatives thereof.
  • Excipients which may be used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins (e.g. petroleum fractions), vegetable oils (e.g. groundnut or sesame oil), mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carriers such as e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk), synthetic mineral powders (e.g. highly dispersed silicic acid and silicates), sugars (e.g. cane sugar, lactose and glucose), emulsifiers (e.g.
  • pharmaceutically acceptable organic solvents such as paraffins (e.g. petroleum fractions), vegetable oils (e.g. groundnut or sesame oil), mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carriers such as e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk), synthetic mineral powders (e.g. highly disper
  • the tablets may contain, apart from or additionally to the abovementioned carriers, additives such as sodium citrate, calcium carbonate and dicalcium phosphate together with various additives such as starch, preferably potato starch, gelatine and the like.
  • additives such as sodium citrate, calcium carbonate and dicalcium phosphate together with various additives such as starch, preferably potato starch, gelatine and the like.
  • lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used at the same time for the tabletting process.
  • the active substances may be combined with various flavour enhancers or colourings in addition to the excipients mentioned above.
  • the dosage of the compound(s) according to the invention is naturally greatly dependent on the route of administration and the disease to be treated.
  • the compound(s) according to the invention can be used effectively in the ⁇ g range or above, for example in the gram range.
  • the compounds according to the invention may be given in higher doses (in the range from 1 to 1000 mg, for example, although this does not imply any limitation).
  • a solution of a Gallidermin variant according to the invention can be prepared that comprises this active variant in a concentration of between 0.1 and 10 mg/ml, and increasingly preferred between 0.25 and 7.5 mg/ml, between 0.5 and 5 mg/ml, between 0.75 and 2.5 mg/ml, mostly preferred between 0.9 and 1.1 mg/ml (see below).
  • Such a solution preferably further comprises an appropriate buffer, preferably in the weak acid pH range between 4 and 7, preferably 5 and 6 pH units.
  • appropriate buffers are weak organic acids plus a salt of that same acid, e.g. lactic acid, maleic acid or acetic acid, plus the respective salt of sodium, pottassium, magnesium or calcium or a phosphate buffer.
  • Such a solution preferably further comprises stabilisers to protect it physically, chemically and/or biologically.
  • stabilizers are in principal known from the state of the art. Stabilisation against physical effects like freezing and thawing can be reached by e.g. polymeric substances like polyols. Stabilisation against chemical effects is reached by molecules which protect the chemical and structural identity of the Gallidermin molecule. These are for example protease inhibitors like benzamidin hydrochloride, Phenylmethylsulfonyl fluoride (PMSF) and derivatives of boric acids, and reducing agents like dithiothreitol (DTT), ⁇ -Mercaptoethanol, Cystein or Glutathion to stabilize the disulfide bridges.
  • PMSF Phenylmethylsulfonyl fluoride
  • DTT dithiothreitol
  • a pharmaceutical composition according to the invention can comprise one or more biocompatible surfactants to optimize the spreading of the active substance.
  • the pharmaceutical compositions according to the invention are preferably substantially free from surfactants, especially when they are designed for the administration by inhalation.
  • a patient is to be understood in its broadest sense and thus includes all animals that comprise a respiratory tract that can be infected by bacteria.
  • a patient according to the invention is a bird or a human or other mammal, more preferably human or a mammal that selected from the species of industrial importance like chicken or pig.
  • respiratory tract is to be understood in its broadest sense, comprising all organs that are involved in the oxygen supply and carbon dioxide detoxication of the body of a human or animal, esp. of a bird, mammal or human.
  • the elements of the respiratory tract comprise nose, oral cavity, pharynx, larynx, trachea, bronchial tubes and the lungs.
  • treatment comprises all sorts of application of a pharmaceutical composition in order to inhibit the desease-causing activity of the relevant bacterial agent against the infected patient.
  • prophylaxis comprises all sorts of treatment of a patient that are applied before an infection of the respiratory tract by the relevant bacterial agent has taken place in order to protect against such an infection.
  • a pharmaceutical composition according to the invention is characterized by the fact that Gallidermin or the pharmaceutically active variant thereof is the wildtype Gallidermin or a variant Gallidermin with one, two or three point mutations in comparison to the wildtype Gallidermin, preferably the wildtype Gallidermin, Gallidermin L6V, Gallidermin A12L, Gallidermin Dhb14P or Gallidermin Dhb14Dha.
  • Gallidermin L6V is described as a variant with an enhanced antimicrobial activity.
  • Gallidermin A12L, Gallidermin Dhb14P are disclosed as variants with high resistance against proteolytic degradation.
  • Gallidermin Dhb14Dha also exerts antimicrobial activity and is additionally characterized by a higher sensitivity against tryptic cleavage.
  • the most effective Gallidermin variants against certain subsets of bacterial populations are selected in different scenarios of infections with respect to the genus and/or species of the bacteria causing the infection, to the species of the patient and/or to the exact part of the respiratory tract to be treated.
  • a pharmaceutical composition according to the invention is characterized by the fact that the infected respiratory tract comprises at least partially the lung.
  • the elements of the respiratory tract comprise nose, oral cavity, pharynx, larynx, trachea, bronchial tubes and the lungs.
  • influenza epedimies examples for such infections are influenza epedimies.
  • Haemophilus influenzae formerly called Pfeiffer's bacillus or Bacillus influenzae
  • Bacillus influenzae is a non-motile gram-negative coccobacillus first described in 1892 by Richard Pfeiffer during an influenza pandemic.
  • Another example of such life-threatening, human epidemic infections mostly of the lung is tuberculosis which is caused by gram-positive Mycobacterium tuberculosis.
  • Related species infect e.g. bovine (M. bovis) and poultry (M. avium-intracellulare).
  • Example 10 proves an antibiotic effect of Gallidermin against Mycobacterium tuberculosis.
  • multiple infections by more than one of these pathogens are treated at the same time, increasingly preferred the infectious by at least 2, 3, 4, 5, 6, 7, 8, 9 and 10 of these pathogenes.
  • Bacillus species' comprise pathogenic and non-pathogenic gram-positive species'.
  • Pathogenic bacillus species' are e.g. B. anthracis and B. cereus, the treatment of / protection against those species' therefore characterizes preferred embodiments of the pending application.
  • Staphylococcus Another genus of relevant, gram-positive pathogenic infectious bacteria is Staphylococcus.
  • Staphylococcus species esp. S. aurus can cause infections of the skin as well as of mucous membranes, esp. of the nose and of the pharynx.
  • Other pathogenic species are e.g. S. intermedius, S. epidermidis and S. saprophytics.
  • the efficiency of Gallidermin against Staphylococcus aureus as a representative from this group of pathogenes is demonstrated by the examples of this application.
  • Streptococcus Another genus of relevant, gram-positive pathogenic infectious bacteria is Streptococcus.
  • S. pneumoniae is a causative of human pneumonia
  • S. pyogenes is a causative of tonsillitis, pharyngitis, scarlet fever and rheumatoid fever
  • S. agalactiae can cause e.g. meningitis
  • S. dysgalactiae and S. uberis can cause bovine mastitis
  • S. milleri, S. mutans and S. salivarius are causatives of infections in the oral cavity and of the teeth.
  • Corynebacterium Another genus of relevant, gram-positive pathogenic infectious bacteria is Corynebacterium. Esp. the species Corynebacterium diphteriae is known as a pathogenic bacteria causing diphteria, an infectious disease esp. of the tonsils, and the mucous membranes of the nose and the pharynx. C. bovis is known as another agent to cause bovine mastitis.
  • Further gram-positive pathogenic infectious bacteria relevant for the pending application are: Propionibacterium, Listeria (e.g. L monocytogenes) and Micrococcus.
  • Further gram-negative pathogenic infectious bacteria relevant for the pending application are Neisseriaceae, esp. N. catarrhalis which can cause infections esp. of the mouth and pharynx.
  • Helicobacter Another genus of relevant susceptible, gram-negative pathogenic infectious bacteria is Helicobacter (formerly Campylobacter). Esp. the species Helicobacter pylori is a known causative as infections in humans and animals, esp. infections of the gastric mucous membrane. Examples 13 and 14 of this application prove the antimicrobial activity of Gallidermin against this pathogen.
  • the minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) of Gallidermin against H. pylori have been determined as being 8 ⁇ g/ml Gallidermin and 16 ⁇ g/ml Gallidermin, respectively. However more refined results might be possible with more sensitive methods.
  • the shown assays are also applicable to the other pathogenes discussed above, if accomodated to the respective growing conditions of these diverse species.
  • the genus Mycoplasma is assigned to the bacteria, though it might also be classified separately, close to virus.
  • This genus comprises pleomorphous microorganisms, with species sizes ranging between 125 nm and 150 ⁇ m.
  • Mycoplasmas lack a cell wall but are characterized by a thin cell membrane (of usually about 10 nm) which is also susceptible to Gallidermin and Gallidermin variants according to the invention.
  • Mycoplasma pneumoniae is a causative of an atypical pneumoniae of humans.
  • Mycoplasma mycoides is the causative of bovine pleuropneumonia which infection can lead to severe damages of cattle herds. Both, M. pneumoniae and M. mycoides are thus called pleuropneumonia-like organisms (PPLO).
  • Mycoplasma hyopneumoniae is a swine pathogen, also infecting esp. the lungs.
  • Mycoplasma gallisepticum causes subacute to chronic infections of the respiratory tract (sinusitis) of birds, esp. of chicken.
  • Mycoplasma hominis is a related human pathogen, mainly infecting the throat or the urogenital tract.
  • Legionella spp. L. pneumophila, L bozemanii, L micdadei, L. dumoffii
  • Coxiella spp. C. burneti).
  • a pharmaceutical composition according to the invention is characterized by the fact that the patient to be treated is a bird or a mammal, preferably chicken (Gallus gallus), human ⁇ Homo sapiens), bovine (Bos bovis), pig (Sus domesticus), sheep (Ovis, esp. Ovis aries), goat (Capra, esp. Capra aegagrus), horse (Equus), cat (Felidae), dog (Canidae), more preferably human (Homo sapiens).
  • the non-human species selected here are of high economic importance which made it necessary to define alternative ways for treatment and prophylaxis against infections of strains that might no longer be susceptible to a treatment with other antibiotics. Untreated, such infections of lifestock could lead to severe economical damage.
  • a pharmaceutical composition according to the invention is characterized by the fact that is prepared to be administered topically to the lungs, preferably by inhalative administration, more preferred by the use of a technical apparatus which allows the application of metered doses of the pharmaceutical composition.
  • Topical application of Gallidermin or the selected Galiidermin variant according to the invention has got the advantage that this anti-microbial agent gets in direct contact to the infectious bacteria or that there is just a short distance of diffusion left for getting in contact with the bacteria.
  • This allows that the total amount of this antimicrobial agent in the pharmaceutical composition to be applied can be relatively low in comparision to a composition administered orally because it is not diluted by the circulation the blood in the whole body. Additional side effects of the agent in the body of the patient are then kept relatively low.
  • the inhalative administration method has been developed especially for the topical application of agents to the lungs and can thus be appropriate for the administration of Gallidermin or a pharmaceutically active variant thereof as active substance to the organ to be treated.
  • the active substance has to be made available in forms suitable for inhalation, lnhalable preparations according to the invention include inhalable powders, propellant-containing metered dose aerosols or propellant-free inhalable solutions.
  • Gallidermin or a pharmaceutically active variant thereof as active substance may consist of the active substances on their own or of a mixture of the active substances with physiologically acceptable excipients.
  • the term carrier may optionally be used instead of the term excipient.
  • propellant-free inhalable solutions also includes concentrates or sterile inhalable solutions ready for use.
  • the preparations according to the invention may contain the active substances and optional further active substances either together in one formulation or in two separate formulations. These formulations which may be used within the scope of the present invention are described in more detail below.
  • Inhalable powders according to the invention may contain Gallidermin or a pharmaceutically active variant thereof as active substance on its own or in admixture with suitable physiologically acceptable excipients.
  • physiologically acceptable excipients may be used to prepare these inhalable powders according to the invention: monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextran), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these excipients with one another.
  • monosaccharides e.g. glucose or arabinose
  • disaccharides e.g. lactose, saccharose, maltose
  • oligo- and polysaccharides e.g. dextran
  • polyalcohols e.g. sorbitol, mannitol, xylitol
  • salts e.g. sodium chloride, calcium carbonate
  • the excipients have a maximum average particle size of up to 250 ⁇ m, preferably between 10 and 150 ⁇ m, most preferably between 15 and 80 ⁇ m. It may sometimes seem appropriate to add finer excipient fractions with an average particle size of 1 to 9 ⁇ m to the excipient mentioned above. These finer excipients are also selected from the group of possible excipients listed hereinbefore.
  • micronised active substance preferably with an average particle size of 0.5 to 10 ⁇ m, more preferably from 1 to 6 ⁇ m, is added to the excipient mixture.
  • Processes for producing the inhalable powders according to the invention by grinding and micronising and by finally mixing the ingredients together are known from the prior art.
  • the inhalable powders according to the invention may be prepared and administered either in the form of a single powder mixture which contain Gallidermin or a pharmaceutically active variant thereof as active substance and an optional further active substance together or in the form of separate inhalable powders which contain only one of them separately.
  • the inhalable powders according to the invention may be administered using inhalers known from the prior art.
  • Inhalable powders according to the invention which contain one or more physiologically acceptable excipients in addition to the active substance according to the invention may be administered, for example, by means of inhalers which deliver a single dose from a supply using a measuring chamber as described in US 4570630A, or by other means as described in DE 3625685 A.
  • the inhalable powders according to the invention which contain the active substance(s) optionally in conjunction with a physiologically acceptable excipient may be administered, for example, using the inhaler known by the name Turbuhaler ® or using inhalers as disclosed for example in EP 237507 A.
  • the inhalable powders according to the invention which contain physiologically acceptable excipient in addition to the active substance(s) are packed into capsules (to produce so-called inhalettes) which are used in inhalers as described, for example, in WO 94/28958 A.
  • FIG. 1 A particularly preferred inhaler for using the pharmaceutical combination according to the invention in inhalettes is shown in Figure 1 of WO 03/087097 A1 to which reference is hereby made.
  • This inhaler (Handyhaler ® ) for inhaling powdered pharmaceutical compositions from capsules is characterised by a housing 1 containing two windows 2, a deck 3 in which there are air inlet ports and which is provided with a screen 5 secured via a screen housing 4, an inhalation chamber 6 connected to the deck 3 on which there is a push button 9 provided with two sharpened pins 7 and movable counter to a spring 8, and a mouthpiece 12 which is connected to the housing 1 , the deck 3 and a cover 11 via a spindle 10 to enable it to be flipped open or shut, as well as airholes 13 for adjusting the flow resistance.
  • the quantities packed into each capsule should be 1 to 30 mg per capsule. These capsules containthe possible separate doses mentioned hereinbefore for each single dose.
  • propellant gas-driven inhalation aerosols containing the active substances according to the invention can be applied.
  • Inhalation aerosols containing propellant gas according to the invention may contain the active substances dissolved in the propellant gas or in dispersed form.
  • Gallidermin or a pharmaceutically active variant thereof as active substance and the optional further active substance may be present in separate formulations or in a single preparation, in which they are either both dissolved, both dispersed or only one component is dissolved and the other is dispersed.
  • the propellant gases which may be used to prepare the inhalation aerosols according to the invention are known from the prior art.
  • Suitable propellant gases are selected from among hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane.
  • hydrocarbons such as n-propane, n-butane or isobutane
  • halohydrocarbons such as fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane.
  • the propellant gases mentioned above may be used on their own or in mixtures thereof.
  • propellant gases are halogenated alkane derivatives selected from TG 11 , TG12, TG134a (1 ,1 ,1 ,2-tetrafluoroethane) and TG227 (1 ,1 ,1 ,2,3,3,3- heptafluoropropane) and mixtures thereof, of which the propellant gases TG 134a, TG227 and mixtures thereof are preferred.
  • the propel lant-d riven inhalation aerosols according to the invention may also contain other ingredients such as co-solvents, stabilisers, surfactants, antioxidants, lubricants and pH adjusters. All these ingredients are known in the art.
  • the inhalation aerosols containing propellant gas according to the invention may contain up to 5 wt.-% of each active substance. Aerosols according to the invention contain, for example, 0.002 to 5 wt.-%, 0.01 to 3 wt.-%, 0.015 to 2 wt.-%, 0.1 to 2 wt.-%, 0.5 to 2 wt.-% or 0.5 to 1 wt.-% of each active substance.
  • the particles of active substance preferably have an average particle size of up to 10 ⁇ m, preferably from 0.1 to 6 ⁇ m, more preferably from 1 to 5 ⁇ m.
  • the present invention relates to pharmaceutical compositions in the form of propellant-driven aerosols as hereinbefore described combined with one or more inhalers suitable for administering these aerosols.
  • the present invention relates to inhalers which are characterised in that they contain the propellant gas-containing aerosols described above according to the invention.
  • the present invention also relates to cartridges fitted with a suitable valve which can be used in a suitable inhaler and which contain one of the above-mentioned propellant gas-containing inhalation aerosols according to the invention. Suitable cartridges and methods of filling these cartridges with the inhalable aerosols containing propellant gas according to the invention are known from the prior art.
  • propellant-free inhalable solutions or suspensions containing the active substances according to the invention can be applied.
  • Propellant-free inhalable solutions and suspensions according to the invention contain, for example, aqueous or alcoholic, preferably ethanolic solvents, optionally ethanolic solvents mixed with aqueous solvents. If aqueous/ethanolic solvent mixtures are used the relative proportion of ethanol compared with water is not limited but preferably the maximum is up to 70 percent by volume, more particularly up to 60 percent by volume of ethanol. The remainder of the volume is made up of water.
  • the solutions or suspensions containing the active substance(s) according to the invention separately or together, are adjusted to a pH of 2 to 7, preferably 2 to 5, using suitable acids and/or the respective acids which have already formed an acid addition salt with one of the active substances.
  • organic acids ascorbic acid, fumaric acid and citric acid are preferred. If desired, mixtures of the above acids may be used, particularly in the case of acids which have other properties in addition to their acidifying qualities, e.g. as flavourings, antioxidants or complexing agents, such as citric acid or ascorbic acid, for example.
  • editic acid or one of the known salts thereof, sodium editate, can be added as a stabiliser or complexing agent.
  • Co-solvents and/or other excipients may be added to the propellant-free inhaiable solutions according to the invention.
  • excipients and additives in this context denote any pharmacologically acceptable substance which is not an active substance but which can be formulated with the active substance or substances in the pharmacologically suitable solvent in order to improve the qualitative properties of the active substance formulation.
  • these substances Preferably, these substances have no pharmacological effect or, in connection with the desired therapy, no appreciable or at least no undesirable pharmacological effect.
  • excipients and additives include, for example, surfactants, stabilisers, complexing agents, antioxidants and/or preservatives which guarantee or prolong the shelf life of the finished pharmaceutical formulation, flavourings, vitamins and/or other additives known in the art.
  • the additives also include pharmacologically acceptable salts such as sodium chloride as isotonic agents.
  • the preferred excipients include antioxidants such as ascorbic acid, for example, provided that it has not already been used to adjust the pH, vitamin A, vitamin E, tocopherols and similar vitamins and provitamins occurring in the human body.
  • Preservatives may be used to protect the formulation from contamination with pathogens. Suitable preservatives are those which are known in the art, particularly cetyl pyridinium chloride, benzalkonium chloride or benzoic acid or benzoates such as sodium benzoate in the concentration known from the prior art.
  • the preservatives mentioned above are preferably present in concentrations of up to 50mg/100ml, more preferably between 5 and 20mg/100ml.
  • Preferred formulations contain, in addition to the solvent water and the active substance(s), only benzalkonium chloride and sodium editate. In another preferred embodiment, no sodium editate is present.
  • the propellant-free inhalable solutions according to the invention are administered in particular using inhalers of the kind which are capable of nebulising a small amount of a liquid formulation in the therapeutic dose within a few seconds to produce an aerosol suitable for therapeutic inhalation.
  • preferred inhalers are those in which a quantity of less than 100 ⁇ l, preferably less than 50 ⁇ l, more preferably between 20 and 30 ⁇ l of active substance solution can be nebulised in preferably one spray action to form an aerosol with an average particle size of less than 20 ⁇ m, preferably less than 10 ⁇ m, in such a way that the inhalable part of the aerosol corresponds to the therapeutically effective quantity.
  • This nebuliser can advantageously be used to produce the inhalable aerosols according to the invention containing the combination of active substance(s). Because of its cylindrical shape and handy size of less than 9 to 15 cm long and 2 to 4 cm wide, this device can be carried at all times by the patient.
  • the nebuliser sprays a defined volume of pharmaceutical formulation using high pressures through small nozzles so as to produce inhalable aerosols.
  • the preferred atomiser essentially consists of an upper housing part, a pump housing, a nozzle, a locking mechanism, a spring housing, a spring and a storage container, characterised by a pump housing which is secured in the upper housing part and which comprises at one end a nozzle body with the nozzle or nozzle arrangement, - a hollow plunger with valve body, a power takeoff flange in which the hollow plunger is secured and which is located in the upper housing part, a locking mechanism situated in the upper housing part, a spring housing with the spring contained therein, which is rotatably mounted on the upper housing part by means of a rotary bearing, a lower housing part which is fitted onto the spring housing in the axial direction.
  • the hollow plunger with valve body corresponds to a device disclosed in VVO 97/12687 A. It projects partially into the cylinder of the pump housing and is axially movable within the cylinder. Reference is made in particular to Figures 1 to 4, especially Figure 3, and the relevant parts of the description.
  • the hollow plunger with valve body exerts a pressure of 5 to 60 Mpa (about 50 to 600 bar), preferably 10 to 60 Mpa (about 100 to 600 bar) on the fluid, the measured amount of active substance solution, at its high pressure end at the moment when the spring is actuated. Volumes of 10 to 50 ⁇ l are preferred, while volumes of 10 to 20 ⁇ l are particularly preferred and a volume of 15 ⁇ l per spray is most particularly preferred.
  • the valve body is preferably mounted at the end of the hollow plunger facing the valve body.
  • the nozzle in the nozzle body is preferably microstructured, i.e. produced by microtechnology.
  • Microstructured nozzle bodies are disclosed for example in WO 94/07607 A2; reference is hereby made to the contents of this specification, particularly Figure 1 therein and the associated description.
  • the nozzle body consists for example of two sheets of glass and/or silicon firmly joined together, at least one of which has one or more microstructured channels which connect the nozzle inlet end to the nozzle outlet end.
  • the directions of spraying of the nozzles in the nozzle body may extend parallel to one another or may be inclined relative to one another in the direction of the nozzle opening.
  • the directions of spraying may be at an angle of 20 to 160° to one another, preferably 60 to 150°, most preferably 80 to 100°.
  • the nozzle openings are preferably arranged at a spacing of 10 to 200 ⁇ m, more preferably at a spacing of 10 to 100 ⁇ m, most preferably 30 to 70 ⁇ m. Spacings of 50 ⁇ m are most preferred.
  • the directions of spraying will therefore meet in the vicinity of the nozzle openings.
  • the liquid pharmaceutical preparation strikes the nozzle body with an entry pressure of up to 600 bar, preferably 200 to 300 bar, and is atomised into an inhalable aerosol through the nozzle openings.
  • the preferred particle or droplet sizes of the aerosol are up to 20 ⁇ m, preferably 3 to 10 ⁇ m.
  • the locking mechanism contains a spring, preferably a cylindrical helical compression spring, as a store for the mechanical energy.
  • the spring acts on the power takeoff flange as an actuating member the movement of which is determined by the position of a locking member.
  • the travel of the power takeoff flange is precisely limited by an upper and lower stop.
  • the spring is preferably biased, via a power step-up gear, e.g. a helical thrust gear, by an external torque which is produced when the upper housing part is rotated counter to the spring housing in the lower housing part.
  • the upper housing part and the power takeoff flange have a single or multiple V-shaped gear.
  • the locking member with engaging locking surfaces is arranged in a ring around the power takeoff flange. It consists, for example, of a ring of plastic or metal which is inherently radially elastically deformable.
  • the ring is arranged in a plane at right angles to the atomiser axis. After the biasing of the spring, the locking surfaces of the locking member move into the path of the power takeoff flange and prevent the spring from relaxing.
  • the locking member is actuated by means of a button.
  • the actuating button is connected or coupled to the locking member. In order to actuate the locking mechanism, the actuating button is moved parallel to the annular plane, preferably into the atomiser; this causes the deformable ring to deform in the annular plane. Details of the construction of the locking mechanism are given in WO 97/20590 A.
  • the lower housing part is pushed axially over the spring housing and covers the mounting, the drive of the spindle and the storage container for the fluid.
  • the upper housing part When the atomiser is actuated the upper housing part is rotated relative to the lower housing part, the lower housing part taking the spring housing with it.
  • the spring is thereby compressed and biased by means of the helical thrust gear and the locking mechanism engages automatically.
  • the angle of rotation is preferably a whole-number fraction of 360°, e.g. 180°.
  • the power takeoff part in the upper housing part is moved along by a given distance, the hollow plunger is withdrawn inside the cylinder in the pump housing, as a result of which some of the fluid is sucked out of the storage container and into the high pressure chamber in front of the nozzle.
  • a number of exchangeable storage containers which contain the fluid to be atomised may be pushed into the atomiser one after another and used in succession.
  • the storage container contains the aqueous aerosol preparation according to the invention.
  • the atomising process is initiated by pressing gently on the actuating button.
  • the locking mechanism opens up the path for the power takeoff member.
  • the biased spring pushes the plunger into the cylinder of the pump housing.
  • the fluid leaves the nozzle of the atomiser in atomised form.
  • the components of the atomiser are made of a material which is suitable for its purpose.
  • the housing of the atomiser and, if its operation permits, other parts as well, are preferably made of plastics, e.g. by injection moulding. For medical purposes, physiologically safe materials are used.
  • Figures 2a/b of WO 03/087097 A1 to which reference is hereby made show the nebuliser (Respimat ® ) which can advantageously be used for inhaling the aqueous aerosol preparations according to the invention.
  • Figure 2a shows a longitudinal section through the atomiser with the spring biased while Figure 2b shows a longitudinal section through the atomiser with the spring relaxed.
  • the upper housing part (51) contains the pump housing (52) on the end of which is mounted the holder (53) for the atomiser nozzle.
  • the hollow plunger (57) fixed in the power takeoff flange (56) of the locking mechanism projects partially into the cylinder of the pump housing.
  • the hollow plunger carries the valve body (58).
  • the hollow plunger is sealed off by means of the seal (59).
  • Inside the upper housing part is the stop (60) on which the power takeoff flange abuts when the spring is relaxed.
  • On the power takeoff flange is the stop (61) on which the power takeoff flange abuts when the spring is biased.
  • the locking member (62) moves between the stop (61) and a support (63) in the upper housing part.
  • the actuating button (64) is connected to the locking member.
  • the upper housing part ends in the mouthpiece (65) and is sealed off by means of the protective cover (66) which can be placed thereon.
  • the spring housing (67) with compression spring (68) is rotatably mounted on the upper housing part by means of the snap-in lugs (69) and rotary bearing.
  • the lower housing part (70) is pushed over the spring housing.
  • Inside the spring housing is the exchangeable storage container (71) for the fluid (72) which is to be atomised.
  • the storage container is sealed off by the stopper (73) through which the hollow plunger projects into the storage container and is immersed at its end in the fluid (supply of active substance solution).
  • the spindle (74) for the mechanical counter is mounted in the covering of the spring housing.
  • the drive pinion (75) At the end of the spindle facing the upper housing part.
  • the slider (76) sits on the spindle.
  • the nebuliser described above is suitable for nebulising the aerosol preparations according to the invention to produce an aerosol suitable for inhalation.
  • the quantity delivered should correspond to a defined quantity with a tolerance of not more than 25%, preferably 20% of this amount in at least 97%, preferably at least 98% of all operations of the inhaler (spray actuations).
  • a tolerance of not more than 25% preferably 20% of this amount in at least 97%, preferably at least 98% of all operations of the inhaler (spray actuations).
  • the formulation according to the invention may also be nebulised by means of inhalers other than those described above, e.g. jet stream inhalers or other stationary nebulisers.
  • the invention relates to pharmaceutical formulations in the form of propellant-free inhalable solutions or suspensions as described above combined with a device suitable for administering these formulations, preferably in conjunction with the Respimat ® .
  • the physicochemical parameter of the pharmaceutical compositions according to the invention especially the viscosity of the respective solution or dispersion, and the technical design of the apparatus are co-ordinated to each other in order to allow the desired distribution of particle sizes.
  • Such variations can be performed by a person skilled in the art of designing pharmaceuticals and/or pharmaceutical devices.
  • a pharmaceutical composition according to the invention is characterized by the fact that the technical apparatus for the inhalative administration is selected from a dry powder inhaler or a nebulizer, preferably a nebulizer.
  • Example 12 of the pending application illustrates the successful use of the apparatus Respimat ® for the topical application of a composition according to the invention to guinea gigs as representatives of mammals, esp. of non-human patients.
  • a pharmaceutical composition according to the invention is characterized by the fact that it is prepared to be administered in the form of an inhalable powder with a maximum average particle size of up to 250 ⁇ m, preferably between 10 and 150 ⁇ m, most preferably between 15 and 80 ⁇ m or in the form of a micronised inhalable powder with an average particle size of 0.5 to 10 ⁇ m, more preferably from 1 to 6 ⁇ m.
  • a pharmaceutical composition according to the invention is characterized by the fact that it is prepared to be administered in the form of propellant gas-driven inhalation solution or aerosol or in the form of a propellant-free inhalable solution or aerosol containing each active substance in a concentration of 0.002 to 5 wt.-%, and increasingly preferred 0.01 to 3 wt.-%, 0.015 to 2 wt.-%, 0.1 to 2 wt.-%, 0.5 to 2 wt.-%, and mostly preferred 0.5 to 1 wt.-% of each active substance.
  • a pharmaceutical composition according to the invention is characterized by the fact that it is prepared to be administered in the form of a propellant gas-driven inhalation suspension or in the form of a propellant-free inhalable suspension containing the active substances in dispersed form with the particles of active substance in an average particle size of up to 10 ⁇ m, preferably from 0.1 to 6 ⁇ m, more preferably from 1 to 5 ⁇ m.
  • a pharmaceutical composition according to the invention is characterized by the fact that the inhalative administration of Gallidermin or the pharmaceutically active variant thereof is to be administered to reach dissolved concentrations on the airway surface of the subject of from 0.125 to 256 ⁇ g/ml.
  • This concentration range has been found to be effective for treating the respiratory tract (examples 11 and 12) and applies especially to the organ to be treated, mostly preferred to the lung.
  • a pharmaceutical composition according to the invention is characterized by the fact that the therapeutically effective amount of Gallidermin or the pharmaceutically active variant thereof is to be administered between 0.01 and 1.2 mg per kg body weight of the patient.
  • a pharmaceutical composition according to the invention is characterized by the fact that it comprises a second or further pharmaceutically active substance, preferably a further pharmaceutically active substance selected from the group of antibacterial or antiviral substances, more preferred another pharmaceutically active variant of Gallidermin according to the invention.
  • the state of the art comprises a large quatity of pharmaceutical compositions for the treatment of the respiratory tract. Accordingly it is within the ambit of the invention to combine the pharmaceutically active substance according to the invention with a second or further pharmaceutically active substances to one pharmaceutical composition in order to apply them at once, whenever advantageous.
  • Such a combined pharmaceutical composition can be prepared by the manufacturer of the fully formulated pharmaceutical or by a pharmacist shortly before the treatment or prophylaxis of the patient.
  • such a further pharmaceutically active substance is selected from the group of antibacterial or antiviral substances in order to broaden the scope of antimicrobial activity of the pharmaceutical composition according to the invention.
  • a further pharmaceutically active substance is selected from the group of antibacterial or antiviral substances in order to broaden the scope of antimicrobial activity of the pharmaceutical composition according to the invention.
  • antibiotics for combating infections of gram-positive, of gram-negative or of viruses can be used for such a combination.
  • a combination therapy with different antibiotics, including Gallidermin or variants thereof is thus possible. This allows the treatment of multiple infection of the same organ, caused by more than one pathogen. Also the combination of three or more antimicrobial activities can be advantageous.
  • such other agent is an peptide or enzyme with antibiotic or bacteriostatic activity, especially Lysozyme.
  • such a further pharmaceutically active substance is selected from the group of the other pharmaceutically active variants of Gallidermin according to the invention.
  • the specific advantages of the diverse variants of Gallidermin, esp. those defined above, can be combined with each other.
  • a further aspect of the present invention concerns a combined product, comprising a pharmaceutical composition according to the invention and a technical apparatus which allows the application of metered doses of the pharmaceutical composition.
  • a further aspect of the present invention resides in the use of Gallidermin or a pharmaceutically active variant thereof as active substance for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient.
  • a further aspect of the present invention resides in a method for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient, wherein the method comprises administration of Gallidermin or a pharmaceutically active variant thereof as active substance to said patient.
  • a combined product can be applied for this method, which combined product comprises a pharmaceutical composition according to the invention and a technical apparatus which allows the application of metered doses of the pharmaceutical composition.
  • a further aspect of the present invention resides in the use of Gallidermin or a pharmaceutically active variant thereof as active substance for the prophylaxis or treatment of a bacterial infection of the respiratory tract caused by gram-positive bacteria or other susceptible bacterial pathogens in a patient.
  • a combined product can be applied for this use, which combined product comprises a pharmaceutical composition according to the invention and a technical apparatus which allows the application of metered doses of the pharmaceutical composition.
  • the effect of a lantibiotic like Gallidermin can be tested on epithelial cells like human epithelial cells, e.g. 293T cells (originally isolated from human kidney; available from Invitrogen GmbH, Düsseldorf, Germany) or e.g. bovine epithelial cells. They can be cultivated in the medium Dulbecco's modified Eagle minimal essential Medium (DMEM; available from Invitrogen GmbH), plus 10% calf serum (CS); bovine epithelial cells can be cultivated in DMEM, plus 10% CS plus 5 ⁇ g/ml Insulin and 1 ⁇ g/ml hydrocortisone.
  • DMEM Dulbecco's modified Eagle minimal essential Medium
  • CS calf serum
  • these cells can be infected with infectious bacteria like Staphylococcus aureus that due to their life cycle invade the cells.
  • Appropriate concentrations of these bacteria can be chosen as e.g. a MOI (multiplicity of infection; relation of cells vs. infectious bacteria) of 2 or of 20 or any other value in between. This is followed by an invasion phase of about 2h.
  • still extracellular bacteria in the supernantant are removed and fresh medium containing 20 ⁇ g/ml Lysostaphin and 50 ⁇ g/ml Gentamicin added in order to kill still remaining extracellular bacteria.
  • medium containing the lantibiotic to be tested e.g. Gallidermin at diverse concentration rates (e.g. 50 ⁇ g/ml) in 1 ml of fresh medium is to be added to the test wells and the controls.
  • the cells can be harvested, e.g. for taking a time kinetics after 2, 4, 6 and 24 h (or any other time value that seems to appropriate).
  • the medium is taken off and the cell lawn washed gently off with a solution of 1 % saponin (in destilled water). They are resuspended in 1 ml 1 % saponin and incubated at 37°C for at least 2 h. Dilutions of these suspensions can be plated on Tryptic Soy Broth (TSB), agar plates;(Difco, BBL, Detroit, Michigan, USA) and then incubated for example over night at 37°C.
  • TTB Tryptic Soy Broth
  • the value of CFU derived from the intracellular ⁇ surving bacterial cells is determined. This can be interpreted as a value for the rate of effectiveness of the protection against the respective bacteria, exerted by the applied lantibiotic.
  • human epithelial cells 293T in several parallel arrays have been infected with a strain of S. aureus as an example of pathogenic gram-positive bacteria at concentration values of 2 and of 20 MOI.
  • the analyzed lantibiotic Gallidermin has been applied at a concentration value of 50 ⁇ g/ml of the infection medium. Probes have been taken after 2, 4, 6 and 24h of incubation.
  • the result of the time kinetics with the MOI of 20 is given in table 1 ; the result of the time kinetics with the MOI of 2 is given in table 2.
  • Table 1 Time kinetics of an infection of 293T cells by S. aureus with an MOI of
  • Table 2 Time kinetics of an infection of 293T cells by S. aureus with an MOI of
  • Gallidermin values are given in 1 ,000 CFU of intracellular ⁇ surviving bacteria.
  • the cells After 24 h incubation with (probe) and without (control) Gallidermin, the cells have been plated on agar plates containing 1 , 2 or 4 ⁇ g/ml Gallidermin.
  • Example 2 has been repeated with bovine epithelial MAC-T cells and with the same S. aureus strain as in example 2, which were applied at a concentration value of 20 and of 2 MOI. Again, Gallidermin has been applied at a concentration value of 50 ⁇ g/ml of the infection medium. Probes have been taken after 2, 4, 6 and 24h of incubation. The result of the time kinetics with the MOI of 20 is given in table 3, the result with the MOI of 2 is given in table 4.
  • Table 3 Time kinetics of an infection of MAC-T cells by the tested S. aureus strain with an MOI of 20, treated with 50 ⁇ g/ml Gallidermin (probe) and control without Gallidermin; values are given in 1 ,000,000 CFU of intracellular ⁇ surviving bacteria.
  • Table 4 Time kinetics of an infection of MAC-T cells by the tested S. aureus strain with an MOI of 2, treated with 50 ⁇ g/ml Gallidermin (probe) and control without Gallidermin; values are given in 1 ,000 CFU of intracellular ⁇ surviving bacteria.
  • the respective values for an MOI of 2 can be estimated as 97%, 80%, 69% and 79%, respectively. Which means that the efficiency of the Gallidermin treatment is higher at smaller infection rates.
  • Gallidermin is also effective in the protection of bovine epithelial cells.
  • mutants of S. aureus which are phenotypically characterized by the growth in small colonies and an up to sixfold extended generation time. They are characterized by mutations in the electron transport chain, e.g. in the gene hemB. However, they can cause severe infections because they are able to resist in host cells without destroying them (Proctor, R.A., and Peters, G.; 1998: "Small colony variants in staphylococcal infections: diagnostic and therapeutic implications"; CHn. Infect. Dis., vol. 27, 419-422).
  • Table 5 Time kinetics of an infection of 293T cells by S. aureus 110 with an MOI of 20, treated with 50 ⁇ g/ml Gallidermin (probe) and control without Gallidermin; values are given in 1 ,000 CFU of intracellular ⁇ surviving bacteria.
  • probes of 200 ⁇ l each are transferred into the wells of 96 well microtiterplates (working volume 200 ⁇ l). For the sake of a solid statistical result is is recommended to run several identical probes in parallel, e.g. 8 wells in one row of the microtiterplate. Then these plates are incubated again, e.g. for 20 - 24 h at 37 0 C.
  • the medium is taken off and the settled lawn washed about 3 times with 1 x PBS (pH 7,0) (Phosphate buffered saline; 0.02 mol/l sodium phosphate buffer, 0.15 mol/l NaCI, pH 7.O.).
  • the remaining bacteria are heat fixed (incubation for 30 min at 80 -100°C) and stained with 150 ⁇ l saturated crystal violet (Serva, Heidelberg, Germany; Cat. no. 27335) in water for 5 min.
  • the excess crystal violet is washed off through rinsing with water and dried through gentle pushing of the microtiterplate.
  • the stained biofilm can then be solved on 150 ⁇ l EtOH (90 - 100%); the parallel probes can then be pooled and the optical density be measured by use of a photometer.
  • Table 6 Effect of Gallidermin on the development of a biofilm by S. aureus 325, treated with increasing Gallidermin concentrations; the control has been run without Gallidermin; values are given in % of the control.
  • MIC minimum inhibiting concentration
  • the test starts with an overnight culture of the respective bacteria (like Streptococci or Staphylococci) in Mueller-Hinton-Medium medium (Merck, Darmstadt, Germany; art. no. 1.05396.0500).
  • this culture is used to inoculate fresh medium at an initial bacterial concentration of 1% which then is let grown until the logarithmic phase (in most cases an optical density of around OD (60 o nm ) ⁇ 0,5) which equals about 0.5 - 1 x 10 8 CFU/ml.
  • This suspension is now diluted by the factor of 1 :500 and 100 ⁇ l of this dilution are mixed with decreasing concentrations of Gallidermin dissolved in Mueller-Hinton-Medium or medium to fill the wells of 96 well microtiterplates. In doing so each well is inoculated with about 1-2 x 10 5 CFU/ml per well.
  • the OD at 600 nm of the culture broth is determined. and compared with the controls without antibiotic.
  • the growth inhibition in % represents the sensitivity of bacterial strains against antibiotic substances, e.g. Gallidermin.
  • Tobramycin measured as MHC value.
  • cells of a strain of M. tuberculosis have been used to inoculate an appropriate volume of an appropriate medium, e.g. Lowenstein-Jensen Medium (comprising 2.5 g KH 2 PO 4 , 0.24 g MgSO 4 , 0.6 g Mg-Citrate, 3.6 g L-Asparagine, 30 g Potato flour, 0.4 g Malachite green in 600 ml distilled water; 12 ml Glycerol and 1000 ml fresh egg mixture (yolk and whites); commercially avialable under medium no. 354 at DSMZ).
  • an appropriate medium e.g. Lowenstein-Jensen Medium
  • an appropriate medium e.g. Lowenstein-Jensen Medium
  • Potato flour 0.4 g Malachite green in 600 ml distilled water; 12 ml Glycerol and 1000 ml fresh egg mixture (yolk and whites); commercially avialable under medium no. 354 at D
  • Table 8 Growth curves of M. tuberculosis in the presence of 200 ⁇ g/ml of
  • Gallidermin is a potent antibiotic which effectively inhibits the growth of the pathogen M. tuberculosis.
  • Gallidermin has been dissolved in a vehicle containing 10 mM acetate, 3% mannitol and 3% sucrose in distilled water, pH 5, at concentrations permitting intravenous administrations of 0.1- 1 mg/kg. After anaesthetization of the guinea pigs this solution of Gallidermin was injected intravenously in a cumulative fashion from 0.1 mg/kg to 1 mg/kg, in doses of 0.1 mg/kg each hour. The resulting bronchospasm as well as the corresponding heart beat rate have been measured as given in table 9.
  • Table 9 Effect of Gallidermin on overflow in anaesthetized guinea pigs; the measured bronchospasm is given in ml X 10 of the increase in overflow; the corresponding heart beat rate increase is given in additional beats per minute (bpm), on top of the basal rate of 189 ⁇ 16.
  • Gallidermin has been dissolved again in a vehicle containing 10 mM acetate, 3% mannitol and 3% sucrose in distilled water, pH 5, at concentrations permitting inhaled administrations of 10 - 300 ⁇ g/kg per actuation (10 ⁇ l) with a soft mist inhaler (Respimat ® ; described in Int. J. Pharmaceutics (2004), vol. 283, pages 1-9).
  • the inhaled administrations of 600 - 1200 ⁇ g/kg are performed by giving 2 - 4 actuations of 300 ⁇ g/kg (10 ⁇ l).
  • the control group receives the vehicle in the same conditions (1 - 4 actuations of 10 ⁇ l). After anaesthetization of the guinea pigs this solution of Gallidermin was injected inhaled in a cumulative fashion from 10 ⁇ g/kg to 1.2 mg/kg.
  • Table 10 Effect of gallidermin on overflow in anaesthetized guinea pigs; the measured bronchospasm is given in ml X 10 of the increase in overflow; the corresponding heart beat rate increase is given in additional beats per minute (bpm), on top of the basal rate of 189 ⁇ 16.
  • Gallidermin applied inhalatively to guinea pigs does not cause the (side-)effects of bronchospasm and tachycardia.
  • Gallidermin inhaled induced a slight decrease in heart rate of 14 - 15 beats per minutes at the doses between 0.6 and 1.2 mg/kg. This decrease in heart rate was not dose-dependent and lasted for less than 1 minute.
  • the vehicle tested in the same conditions did not produce any change in overflow and also induced a slight decrease in heart rate of 5 - 6 beats per minute lasting less than 1 minute.
  • Gallidermin inhaled up to 1.2 mg/kg (4 fold above the first intravenous dose which induced side-effects) did not produce neither bronchospasm nor tachycardia. Only a minor decrease in heart rate was recorded between 0.6 and 1.2 mg/kg i.h. This slight side effect proved at least that the compound was well absorbed in the lung.
  • Example 13 Short-time killing assay to test the principal antimicrobial activity of Gallidermin against Helicobacter pylori
  • H. pylori e.g. H. pylori DSM 4867 (commercially available at DSMZ, i.e. Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; Braunschweig, Germany) is to be precultivated for 24 h on a blood agar plate (e.g. medium 420; commercially available at DSMZ) at 37 0 C under microaerophilic conditions.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • DSMZ i.e. Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • Braunschweig, Germany i.e. Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • human epithelial cells infected by and supporting the growth of H. pylori can be grown as polarized monolayers on filter supports, inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO 2 at their basolateral surface (aerophilic conditions) and 5% CO 2 , 5% O 2 , 90% N 2 (microaerophilic conditions) in the apical compartment. Then the bacteria are washed with physiologic salt solution and diluted to ca. 1 x 10 8 bacteria per ml in physiologic salt solution. Then Gallidermin or the respective variant to be tested is to be added in diverse concentrations over a broad range, e.g. at 31.3; 62.5; 125 and 250 ⁇ g/ml, and incubated for 1 h at room temperature.
  • the bacteria from these different probes are plated on agar plates, preferably on blood agar plates again, and bread for 24 h at 37°C.
  • Table 11 Antimicrobial activity of Gallidermin against Helicobacter pylori after 1 h incubation at different concentrations; measured by subsequent plating.
  • MIC minimal inhibition concentration
  • MMC minimal bactericidal concentration
  • liquid media e.g. medium 420; commercially available at DSMZ
  • Gallidermin in concentrations of 2, 4, 8, 16, 32, and 64 ⁇ g/ml (each in duplicate).
  • equal amounts of bacteria in this case 1 x 10 8 per ml have been applied and the probes were cultivated over night at 37°C under microaerophilic conditions.
  • MBC minimal bactericidal concentration
  • a more detailed differentiation is possible by analogous testings of more cultures with more differentiated concentration steps of the applied lantibiotic.

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Abstract

La présente invention concerne le traitement et la prophylaxie des maladies infectieuses. Elle concerne la prophylaxie et le traitement des maladies infectieuses de l’appareil respiratoire provoquées par les bactéries à Gram positif ou par d’autres bactéries pathogènes sensibles chez un patient, comprenant un antibiotique, la gallidermine, ou une de ses variantes pharmaceutiquement actives en tant que principe actif et l’invention concerne aussi des sujets connexes.
PCT/EP2009/055852 2008-05-20 2009-05-14 Gallidermine pour traiter les infections de l’appareil respiratoire WO2009141264A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028726A2 (fr) * 1993-06-09 1994-12-22 The Wellcome Foundation Limited Procede de traitement de la retention de secretions pulmonaires
US5910479A (en) * 1996-10-18 1999-06-08 Ambi Inc. Method for the treatment of Streptococcus pneumoniae infection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028726A2 (fr) * 1993-06-09 1994-12-22 The Wellcome Foundation Limited Procede de traitement de la retention de secretions pulmonaires
US5910479A (en) * 1996-10-18 1999-06-08 Ambi Inc. Method for the treatment of Streptococcus pneumoniae infection

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOLDSTEIN B P ET AL: "Activity of nisin against Streptococcus pneumoniae, in vitro, and in a mouse infection model." THE JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY AUG 1998, vol. 42, no. 2, August 1998 (1998-08), pages 277-278, XP002511350 ISSN: 0305-7453 *
KOVACS MARTA ET AL: "A functional dlt operon, encoding proteins required for incorporation of D-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae" JOURNAL OF BACTERIOLOGY, vol. 188, no. 16, August 2006 (2006-08), pages 5797-5805, XP002511349 ISSN: 0021-9193 *
MAHER SAM ET AL: "Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro." BIOCHEMICAL PHARMACOLOGY 28 APR 2006, vol. 71, no. 9, 28 April 2006 (2006-04-28), pages 1289-1298, XP005351092 ISSN: 0006-2952 *

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