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EP-3937957-B1 - PROCESS FOR MAKING STABLE BACTERIAL EXTRACTS AND THEIR USE AS PHARMACEUTICALS

EP3937957B1EP 3937957 B1EP3937957 B1EP 3937957B1EP-3937957-B1

Inventors

  • BAUER, JACQUES
  • PASQUALI, Christian

Dates

Publication Date
20260506
Application Date
20200312

Claims (10)

  1. A bacterial extract from Gram positive or Gram negative bacterial species chosen among Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and / or Streptococcus sanguinis, wherein said bacterial extract is obtainable by alkaline lysis of said bacterial species and neutralization with one or more organic acid selected among acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoicacid, glutamic acid, aspartic acid, a combination thereof, followed by purification by filtration of the neutralized extract and adjusting to a final physiological pH by adding one or more organic acid selected among acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, and/or a combination thereof.
  2. The bacterial extract according to claim 1, wherein said alkaline lysis is conducted at pH greater than 10, with variations of ±0.1 of the pH.
  3. The bacterial extract according to any one of the preceding claims, wherein the final pH is adjusted between 5 and 8, between 6 and 8, between 6.3 and 7.8, or between 6.5 and 7.8.
  4. The bacterial extract according to any one of the preceding claims, wherein said stable purified bacterial extract comprises less than 100 micrograms/mL nucleic acids, at least 0.1mg/mLof saccharides.
  5. The bacterial extract according to any one of the preceding claims, said bacterial extract being stable in liquid form at room temperature, +4°C, -20 °C, or at -80 °C.
  6. Pharmaceutical composition comprising the bacterial extract according to any one of the preceding claims and a pharmaceutically acceptable excipient or vehicle, wherein said pharmaceutical composition may be in the form of a solid, semi-solid, liquid, or aerosol formulation, and wherein said composition may be formulated for intranasal, intratracheal, mucosal, transmucosal, external skin topical, buccal, sublingual, oral, pulmonary, intrabronchial, and/or intrapulmonary routes of administration.
  7. Pharmaceutical composition of claim 6, wherein said pharmaceutical composition is liquid or aerosol and is formulated in a solution in the final form of a spray, droplet, colloidal, mist, nebulae, and/or in atomized vapor, or wherein said composition is liquid or semi-solid and is formulated in form of emulsions, microemulsions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, creams, solutions, hydroalcoholic solutions, hydroglycolic solutions, hydrogels, serums, ointments, mousses, pastes, or transdermal patches, or wherein said composition is solid and is formulated in a powder, and/or crushable tablet.
  8. Pharmaceutical composition according to claim 6 or 7, for use in a therapy.
  9. A delivery device comprising the pharmaceutical composition according to claim 6, said delivery device being selected from the group comprising of nasal insufflator device, intranasal inhaler, intranasal spray device, atomizer, nasal spray bottle, unit dose container, pump, dropper, squeeze bottle, nebulizer, metered dose inhaler (MDI), pressurized dose inhalers, insufflators, bidirectional devices, dose ampoules, nasal pads, nasal sponges, and nasal capsules.
  10. A process for preparing a stable bacterial extract according to claim 1, comprising the following steps: a. culturing each bacterial strain species in a suitable culture medium, b. heat killing the bacteria, removing the culture medium and harvesting the concentrated biomass, c. lysing each strain at an initial pH of greater than 10, d. decreasing the pH of the extract(s) obtained in step (c) by 1 or 2 units by adding one or more organic acid selected among acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid,2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, a combination thereof, or pharmaceutically acceptable salts and esters thereof, e. passing the product of step (d) at least once through a microfilter and retaining the product on an ultrafilter so as to obtain a purified soluble extract.

Description

FIELD OF THE INVENTION The present invention relates to novel stable bacterial extract preparations having substantial increased stability overtime, novel methods of preparation thereof, pharmaceutical formulations based on these novel stabilized bacterial extracts as well as novel pharmaceutical compositions, routes of administration and delivery devices for use in a therapy. BACKGROUND OF THE INVENTION There is an ever increasing interest in the development of pharmaceutically formulations, either liquid or solid, stable overtime, which can be administered to human subjects for treating infections and inflammations which can be easily adaptable to broader routes of administration, such as oral, intranasal, intratracheal, intrapulmonary, transmucosal, topical, buccal, etc.. as well as to disease conditions and stages (acute stage, exacerbations, etc..) of the patients. Bacterial infections are often implicated in many respiratory conditions and antibiotic treatments are common. The efficacy of antibiotics in treating such disease conditions and exacerbations has been debated. Their overuse has been associated with increased cost and the potential for increased microbial antibiotic resistance. Bacterial extract lysate preparations, which contain antigens derived from several strains of bacteria, have been shown to increase resistance to infection by these organisms. The way in which these bacterial extract lysates may exert their effects could be multi-fold and is not yet fully understood. Numerous bacterial extracts have been used as immune stimulants and anti- tumor agents. By way of examples, we can cite Bacillus Calmette-Guerin (BCG), Polysaccharide, beta 1,3, glucan, the Maruyama vaccine, and extracts of Bifidobacterium, L. lactis, L. fermentum, L. acidophilus and, S. lactis. Such extracts are thought to stimulate the immune system in a number of ways. One important way is to stimulate lymphocytes, to grow and to produce cytokines. The ability to induce production of such cytokines has a very powerful effect on the immune system. In particular, several bacterial extract preparations have been already successfully developed by the Applicant for the treatment and/or the prevention of upper tract respiratory disorders. To this regard, we can cite the drug Broncho-Vaxom® which is a bacterial lysate extract from several pathogens frequently responsible for respiratory tract infections, as described inter alia in US Patent No. 9,463,209B2. Broncho-Vaxom® is an immunostimulant administered as a capsule, a sachet or drops via the enteral oral route for the prevention and prophylaxis of respiratory tract infections, recurrent respiratory tract infections, such as acute bronchitis, chronic bronchitis, asthma, chronic obstructive pulmonary disease, and emphysema. It has been shown to enhance the production of TNF-α and interferon-y from cultured human peripheral blood mononuclear cells, to lead to activation of alveolar macrophages, and to stimulate bacterial killing by polymorphonuclear leukocytes. Applicant also developed Lactobacillus bacteria extracts and showed that these bacterial lysate extracts were efficient when administered as a capsule via the enteral oral route for treating infections, allergies, autoimmune disorders and inflammations (See international publication WO2010/027344). The constant exposure of the mucosal surfaces like airways and lungs to inhaled viruses, bacteria and toxins presents a challenge to the immune system. The situation becomes even worse when the host is exposed to viral infection and subsequently is superinfected by microbes leading to an increased mortality rate. Accordingly, bacterial secondary infections following viral infections with influenza (H1N1 and the like), human rhinovirus (HRV), rhinosyncitial virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19 and the like) are a pressing problem facing respiratory medicine. Experimentally, numerous in vivo studies have demonstrated that the bacterial extract Broncho-Vaxom® administered by the enteral oral route provided a protection in respiratory tract infection models. Most studies used solid pharmaceutical form (lyophilizate) as well as liquid bacterial extract, however, with limited stability. An example of such in vivo mice model using the human dose (7 mg dry weight bacterial extract, 40 mg of lyophilizate) showed that it was able to enhance protection from secondary bacterial and/or viral infections following influenza infection (Pasquali et al, Frontiers in Medicine 2014, 1, 41). Additionally functional dysbiosis originating from "junk food" enriched in fat, sugars and proteins and leading to microbiota dysregulation (Clarence M. et al., Abstract, March 30 9, 2018 - St-John University, Queens) was normalized by supplementing Broncho-Vaxom® in mouse accompanied with decrease of associated sequelae and co-morbidities. For several years, Broncho-Vaxom® has thus been administered via the enteral routes in solid for