EP4096409A1

EP4096409A1 - Method, comprising bacteriophages, for reducing the population of at least one adipogenic bacterial species, and bacteriophages and the use thereof

Info

Publication number: EP4096409A1
Application number: EP21703367.9A
Authority: EP
Inventors: Oxana Karpf
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-01-27
Filing date: 2021-01-20
Publication date: 2022-12-07
Also published as: CN115334886A; CA3165924A1; DE102020101859A1; WO2021151759A1; AU2021213377A1; JP2023518653A; US20230145361A1

Abstract

The invention relates to a method for reducing the population of at least one adipogenic bacterial species, which method comprises the following steps: a) preparing a biological sample, which contains bacteria of at least one adipogenic bacterial species; and b) preparing bacteriophages of at least one bacteriophage species, which are specific to at least one adipogenic bacterial species and which comprise at least one nucleic acid that is functionally linked to a promotor and/or a regulatory element; and c) bringing the biological sample in contact with and incubating the sample with the bacteriophages, wherein the incubation is carried out until the population of the adipogenic bacterial species is reduced by at least 70%. The invention also relates to bacteriophages and the use thereof.

Description

Method for reducing the population of at least one adipogenic bacterial species comprising bacteriophages and bacteriophages and their use

The invention relates to a method for reducing the population of at least one adipogenic bacterial species, as well as bacteriophages and their use according to the preamble of the independent claims.

It is widely known that the interaction between bacteria and their host, i.e. humans and / or animals, can have both positive and negative effects. On the one hand, bacteria can be symbionts or commensals for humans or animals and thereby use them or live with them without causing them any damage. On the other hand, a large number of types of bacteria are known which, as pathological pathogens or parasites, attack and damage the human or animal organism, and are suspected of triggering and / or promoting a number of diseases and related secondary diseases, such as, for example the development of obesity and excessive weight gain. Such damage can lead to death of the organism.

To combat bacterial colonization or bacterial infection, a number of chemical substances have been established as antibiotics, which are currently considered conventional therapy for humans and animals. Such substances are characterized by a broad spectrum of activity, as they target molecular structures or molecules of the bacteria, which occur in a large number of types of bacteria. As a result, a wide range of bacteria can be reduced or eliminated by means of antibiotics. The disadvantage here is that the initially mentioned commensal or symbiotic bacteria are also killed by the use of antibiotics, which can lead to consequential damage to humans, animals and / or the environment. For example, damage to commensal and / or symbiotic bacteria can result in a shift in the balance of the microbiome in favor of pathogenic microorganisms (e.g. bacteria and / or fungi), which, for example, can lead to a condition lasting several months with often serious consequences .

It is also known that chemical substances that are used as antibiotics can interact with physiological metabolic processes. Here is z. B. known that certain antibiotics have an anabolic effect, which is used, for example, in agriculture. However, it is disadvantageous that the antibiotics used in agriculture get into the human and / or animal organism via the food chain and can also lead to fattening effects such as acceleration of growth and / or hormonal development. Other forms of intolerance to chemical substances used as antibiotics are also described. These include the development of allergies to precisely these chemical substances and the consequences of a resulting allergic reaction through to allergic shock syndrome and / or the development of various (food) intolerances. In addition, if the antibiotic therapy fails, repeated therapy with the same medicinal active ingredient is not possible, so that the therapy is usually switched to a different antibiotic active ingredient group, which has an even broader spectrum of activity and thus increases an even more significant imbalance of the microbiota, including the emergence of multi-resistant bacteria. Rather, a certain waiting time must be observed between two antibiotic therapies, which, however, can in turn lead to the spread of the pathological bacteria. In this case, and also if antibiotics are discontinued too early in the event of a supposedly successful therapy, the bacteria that have come into contact with the chemical substance can develop resistance mechanisms and develop so-called resistance to antibiotics without dying off. If resistance develops, such a bacterium is insensitive to antibiotic therapy with the corresponding chemical substance. In this case, treatment or therapy is no longer successful. This can also arise because residual amounts of an antibiotic or its degradation products get into the drinking water and / or the Elmwelt and the bacteria in the Elmwelt come into contact with this substance. Last but not least, the penetration of chemical substances that are used as antibiotics into bacterial biofilms is made more difficult, since the biofilms are characterized by a particularly sugar-containing and therefore polar surface, through which a number of known antibiotics cannot penetrate.

Obesity, obesity and / or excessive weight gain can now be described as a global epidemic and are considered problematic, especially in terms of physical and mental health. These mental or physical ailments can arise as health consequences from overweight, obesity and / or excessive weight gain, as well as being favored due to an increased risk, such as diseases of the metabolism, the cardiovascular system, the hormonal balance, the musculoskeletal system or other organ systems. These include diseases or conditions such as arteriosclerosis, diabetes, joint problems, including those affecting the spine, hips and knees, digestive disorders and cancer, such as breast, colon and pancreatic cancer. Loss of weight in the case of overweight and / or obesity or the stabilization of the ideal weight is difficult for many sufferers, however, since the modern way of life and / or a family disposition make it difficult to reduce weight in the case of overweight, obesity and / or excessive weight gain. However, it is precisely this weight reduction in overweight people or people who have gained a lot of weight, since in particular overweight, obesity and / or excessive weight gain can cause complications in the therapy of a number of diseases or delay or prevent the success of the therapy . Furthermore, excessive fatty tissue and / or organs in the case of obesity can disrupt the functionality of the organs and even cause organ failure. Other diseases in which obesity regularly contributes at least to their development or which causes complications in their therapy relate to the medical fields of oncology, immunology, gastroenterology, endocrinology, psychiatry, psychosomatics, orthopedics, paediatrics, surgery, urology and others.

Being overweight can lead to illnesses and / or ailments, such as metabolic syndrome, diabetes mellitus type 2, gallbladder diseases, chronic intestinal inflammation, over-acidification of the stomach, high blood pressure, lipid metabolism disorders, breathing difficulties, sleep apnea, and coronary heart disease , Osteoarthritis, gout, cancer, such as uterine, breast, cervical, colon, prostate and gallbladder cancer, sex hormone disorders, decreased libido, joint and back pain, increased risk of thrombosis and embolism, increased risk during operations and Anesthesia, psychosocial problems and the associated restrictions in quality of life, for example due to depression, a reduced self-esteem, for example due to a lack of recognition from the environment. Bacteria in the intestinal flora can also contribute to the development of obesity. It is conceivable that a reduction in the population of the corresponding bacteria within the intestinal flora prevents or at least limits the development of obesity or excessive weight gain. The disadvantage here is that conventional antibiotic therapy not only eliminates the desired, obesity-promoting bacteria, but also commensal bacteria, which have positive effects on the host organism, ie the human and / or animal organism and, for example, promote digestion. This would also mean putting antibiotics on the market in excess, which can promote the development of resistance. Antibiotic use is generally not appropriate for weight reduction, as this would have to take place over a very long period (e.g. months), which would mean excessive damage to the commensal flora and prolonged occurrence of the side effects associated with the antibiotics. A pure antibiotic therapy is therefore not advantageous in the treatment and / or therapy of obesity or excessive weight gain. Due to the mentioned disadvantages of conventional therapies with chemical substances that are used as antibiotics, as well as in the general conventional control of bacterial populations, in particular in therapy and / or. Treatment of overweight, obesity and / or excessive weight gain, there is a great and steadily growing need to modulate at least one adipogenic bacterial species in a simple, effective, tolerable, harmless, low-side-effect, inexpensive, targeted and specific manner fight and / or eliminate. At the same time, it is necessary to achieve long-term therapeutic success. The invention has therefore set itself the task of providing bacteriophages and a method for reducing the population of at least one adipogenic bacterial species to the abovementioned Avoid disadvantages of the prior art and which is characterized by high effectiveness, specificity, tolerability and / or reliability, as well as simple, low-error, time- and cost-efficient application. This object is achieved in a surprisingly simple but effective way by a method and bacteriophages according to the teaching of the independent claims.

According to the invention, a method for reducing the population of at least one adipogenic bacterial species is proposed, which comprises the following steps: a) providing a biological sample which comprises bacteria at least one adipogenic bacterial species; and b) providing bacteriophages of at least one bacteriophage species which are specific for at least one adipogenic bacterial species and which comprise at least one nucleic acid functionally linked to a promoter and / or a regulatory element, the nucleic acid being selected from the group comprising: i . a nucleic acid sequence which codes for at least one antibacterial nucleic acid molecule; and ii. a nucleic acid sequence which encodes a nucleic acid molecule that is at least 50% identical to the nucleic acid molecule which is encoded by the nucleic acid sequences from i); and iii. a nucleic acid sequence which codes for at least one antibacterial polypeptide; and iv. a nucleic acid sequence which encodes a polypeptide that is at least 50% identical to a polypeptide which is encoded by the nucleic acid sequences from iii); and V. a nucleic acid sequence for a fragment of a nucleic acid from i), ii), iii) or iv), the fragment encoding a nucleic acid molecule or a polypeptide; and c) bringing the biological sample into contact and incubating it with the bacteriophages, the incubation taking place until the population of the adipogenic bacterial species is reduced by at least 70%.

The method according to the invention is based on the basic idea that by using bacteriophages specific for the at least one adipogenic bacterial species it is possible to reliably, quickly, simply, specifically and permanently reduce the population of this at least one adipogenic bacterial species. It has been found that due to the simultaneous host dependency, other organisms such as fungi, bacteria, viruses or the like, and / or cells of multicellular tissue and / or organisms advantageously remain unaffected, so that no damage, impairment and / or disruption occurs. Due to the host dependency of the bacteriophage, the method according to the invention can be repeated at any time without any resistance occurring. As is known to a person skilled in the art, there is simultaneous host dependency if, in the absence of the respective adipogenic bacterial species within the biological sample, there is no reduction of another bacterial species within the biological sample for which the bacteriophage is not specific. This also applies to organs and / or tissues of the host organism, i.e. the human and / or animal organism.

In the context of the invention it has been recognized that it is sufficient to reduce the population of at least one bacterial species to provide a biological sample which comprises bacteria of at least one adipogenic bacterial species and to administer this with the To incubate bacteriophages of at least one bacteriophage species which are specific for the at least one adipogenic bacterial species.

The term “population” is known to a person skilled in the art and generally relates to the totality of all individuals of the same species that occur in a certain area or in a certain area of the environment. In the context of the invention, the population relates to the entirety of the bacteria of at least one adipogenic bacterial species. The terms “bacterium” and “bacteria” can be understood by a person skilled in the art as interchangeable synonyms and relate to representatives of one or more types of bacteria.

The term “adipogenic bacterial species” is known to a person skilled in the art and relates to at least one species of bacteria, preferably 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more types of bacteria, which due to their way of life, for example through their molecular metabolism and / or their interaction with other types of bacteria, fungi and / or cells influence a human and / or animal organism directly or indirectly in such a way that obesity, adiposity, overweight and / or weight gain are initiated by the build-up, storage and / or reduced breakdown of fatty tissue, preferably white fat and / or increased. A person skilled in the art is aware of direct or indirect influencing of the organism. For example, they can influence the organism directly by destroying tissue of the organism, or they can indirectly influence the metabolism or other processes of the organism, which among other things initiates and / or promotes weight gain and / or non-reduction of obesity can. Furthermore, it is understandable to the person skilled in the art that “adipogenic” in this context can also mean that the corresponding bacterial species does not itself have to produce a direct adipogenic effect in the organism, but that this is conducive to the growth, lifestyle and / or survival of an anti-adipogenic bacterial species can oppose and thus indirectly has an adipodic effect on the organism. In the context of the invention, it is conceivable that the adipogenic bacterial species can occur within the gastrointestinal tract of the human and / or animal organism, such as in the stomach and / or in the intestine, for example.

The adipogenic bacterial species is not finally selected from: Acinetobacter, Actinobacteria, Actinomyces, Bacilli, Bacteriodes, Bartonella, Bordetella, Borrelii, Brucella, Citrobacter, Campylobacter, Chlamydia, Clostridia, Corynebacteria, Desulfovibrio, Enterocacteria, Ehrlichia, obociacobcus , Ery sipelotrichia, Escheri chia, Faecalibacterium, Firmicutes, Francisella, Helicobacter, Hemophili, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria, Methano- brevibacter, Moraxella, Mycobacterium, Mycoplasmi, Neisseria, Pa- serotcilin- dacosa, Pa- serotcilin- dia , Propionibacterium, Proteus, Pseudomonas, Rickettsia, Ruminococci, Salmonella, Shigella, Spirrillum, Spirochetes, Staphylococci, Stenotrophomonas, Streptobacil li, Streptococci, Treponema, Vibrio, Yersenia, Bifidobacterium, Dialacnia, Orocospacterium, Copacnacea, Orocospacterium, Bulacospacterium, Copacnacterium, Bulacospacterium, Bulacospacterium, Roseburia, Christensenellaceae, Erwinia, Flavonifractor, Oscillospira, Phasco larctobacterium, Prevotella, Succinivibrio, Ruminococcus and / or Veillonella.

Furthermore, the adipogenic bacterial species is not conclusively selected from: Acinetobacter baumannii, Bacillus cereus, Bacillus anthracis, Bacillus subtilis, Bacteriodes thetaiotaomicron, Bacteriodes vulgatus, Bartonella henselae, Bordetella pertussis, Borrelia recurrenticis, Borrelydlobacteria buni, Borrelatuedlobacteria buni, Borrelatuedlobacteri, Borrelatuedlobferjuni, Borrelatuedlobacteri jejuni, Borreliarelia hermsii fruendii, Chlamydia psittaci, Chlamydia trachomatis, Chla mydia pneumoniae, Clostridium botulinum, Clostridium difficle, Clostri dium tetani, Clostridium perfringens, Clostridium ramosum, Clostridium novyi, Clostridia digeria septicum, Clostridia septicum, Clostridium fawnlichum, Clostridium fawnlichum, Clostridium façade, Clostridium septicum , Escherichia coli (e.g. EHEC, EIEC, ETEC), Faecalihacterium prausnit- zii, Francisella tularensis, Helicobacter pylori, Hemophilus influenzae, Hemophilus parainfluenzae, Hemophilus aegyptila, Klebsiella pneumo niae, Lactobacospillus pneumo niae, Lactobacospillus pneumo peptiae, Lactobacospillus reuterorrex, Lactobacospillus pneumo niae, Lactobacospillus pneumo peptiae, Lactobacospillus reptirae, Lactobacospillus reptirae monocytogenes, Methaobrevibacter smithii, Moraxella catarrhalis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacterium intracellulare, Mycobacterium avium-intracellulars, Myobacterium menoniae, Mycobacterium meniseria, Mycobacterium, Mycobacterium avium johnei, mycobacterium, Mycobacterium avium, neonorrhoissium, Mycobacterium. Rickettsia prowozekii, Rickettsia rickettsii, Rickettsia akari, Propionibacterium acnes, Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella typhimurium, Salmonella typhi, Salmonella paraty- phi, Salmonella schottmulleri, Salmonella hirshieldyus, Spir- lumellae dysaphrum- cus, Shilligellae au- shieldii reus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Strep tococcus pneumoniae, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguis, Streptococcus pyogenes, Streptococcus pyogenes, Streptocococcus pyogenes, Streptocococcus pyrogenes, Streptocococcus bertain, Treponovema, Streptocococcus, Treponovema, Streptocococcus, Streptococalcus, Streptocococcus, Streptocococcus, Streptocococcus, Streptocococcus, Treponovema, Streptocococcus, Streptocococcus, Streptocococcus, Trepono carateum, Vibrio cholera, Vibrio parahaemolyticus, Yersenia pestis, Yersinia enterocolitica, Bacteroides fragilis, Blautia hydrogenotorophica, Coprococcus catus, Eubacterium ventriosum, Bacteroides faecichin- chillae, Buminidobor- bacterium boltis, Bacteroides bruteocacti, Buminidobacterium boltis, Clostromii gasseri, Clostromii gasseri, Clostridobacterium boltis, Clostromii ravillasserium, Clostromii and / or Ruminococcus obeum. It is assumed here that the corresponding bacterial species has a known and / or suspected adipogenic effect on a human and / or animal organism. In the first step of the method according to the invention, it is necessary to provide a biological sample comprising bacteria at least one adipogenic bacterial species. The term "biological sample" relates to a material of a human and / or animal organism and / or a material which has at least briefly come into contact with a human and / or animal organism. It has been recognized as essential that the biological sample comprises bacteria of at least one adipogenic bacterial species, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more types of bacteria. Such samples are known to a person skilled in the art, such as, for example, samples containing cells and nuclei (containing DNA and / or RNA) from, among other things, saliva, urine, blood, stool, sweat, cellular tissue, organ puncture, organs, parts of organs , whole organisms (including dead ones), soil samples, water samples or similar samples.

In the next step of the method according to the invention, it is necessary to provide bacteriophages of at least one bacteriophage species which are specific for at least one adipogenic bacterial species. The term “bacteriophage” or “bacteriophage species” is understandable to a person skilled in the art and relates to infectious organic structures that can spread outside of cells and multiply within a cell. The cells serve as a host and, in the case of bacteriophages, are essentially bacteria, with bacteriophages having a pronounced host specificity, ie, in other words, specific for at least one type of bacteria and thus only these bacteria for which they are specific for distribution and Use multiplication.

It is essential that the bacteriophages which are used for the method according to the invention comprise at least one nucleic acid functionally linked to a promoter and / or a regulatory element or have a promoter and / or a regulatory element in combination with a nucleic acid, the Nucleic acid encodes either an antibacterial nucleic acid molecule, a bacterial polypeptide and / or a fragment thereof. Before- the bacteriophages additionally comprise a nucleic acid with at least 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more promoters and / or regulatory elements. The nucleic acid sequences which code for promoters or regulatory sequences are sufficiently known to the person skilled in the art.

It is understandable to a person skilled in the art that the terms “nucleic acids”, “genes”, “DNA”, “RNA”, “mRNA”, “cRNA”, “miRNA” and the compositions “nucleic acid sequence” and “nucleic acid molecule “Used as interchangeable synonyms for each other to denote deoxyribonucleotides or ribonucleotides and polymers thereof, either single or double stranded.

The terms “promoter” and “regulatory sequence” or “regulatory element” are also understandable to the person skilled in the art and relate to nucleic acid sequences of the DNA which enable and / or increase the expression, i.e. the writing of a gene in mRNA. In this case, regulatory elements can be, for example, enhancer elements, i.e. binding sites for growth factors, hormones, oncogenes and / or the like.

A person skilled in the art can also understand that the terms “polypeptide”, “peptide”, “amino acids” and “protein” are used as interchangeable synonyms for one another to denote a polymer of amino acid residues. Naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that can later be modified. Polypeptides can be present as reserve substances, as transport and / or signaling molecules, as structural molecules, as protective and / or defense molecules and / or as metabolically active molecules and can perform various functions inside and / or outside a cell or in the organism. For example, polypeptides can have an antibacterial effect and thus counteract the way of life of the bacteria due to their properties or functions. In the context of the method according to the invention, at least one antibacterial nucleic acid molecule, an antibacterial polypeptide and / or a fragment thereof is provided; the bacteriophages can preferably also be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more antibacterial nucleic acid molecules, antibacterial polypeptides and / or fragments thereof. Antibacterial nucleic acid molecules can come from the area of the RNAs species and can be, for example, functional mRNA or miRNA. Polypeptides can be present as simple linear polypeptides or also as folded structural proteins. The proteins that come into question are molecules that act as toxins, growth-inhibiting substances, as well as enzymes or molecules to inhibit cell division. Possible nucleic acid molecules, for example RNA molecules, are those which, for example, inhibit metabolic processes, hinder cell division or promote malfunctions in biological processes within the bacteria.

In the context of the method according to the invention, a nucleic acid sequence is conceivable which encodes a nucleic acid molecule, a polypeptide and / or a fragment thereof which is at least 50% identical to the aforementioned antibacterial nucleic acid sequence or the antibacterial polypeptide. A nucleic acid sequence is also preferably conceivable which contains at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99% or 100% identical to the aforementioned antibacterial nucleic acid sequence or the antibacterial len polypeptide. The nucleic acid sequence is a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence which is already present in bacteriophages or which has been introduced into the respective bacteriophage, for example by molecular biological methods. For example, these molecular biological methods can be carried out in vivo and / or in vitro Recombination, gene transfer, CRISPR / Cas, Talen or the like be.

The bacteriophages can belong to the order Caudovirales, Ligamenvirales or the like. the bacteriophage preferably includes the Ackermanviridae family Ampullaviridae, Bicaudaviridae, Clavaviridae, corticovirus, Fuselloviridae, Globuloviridae, guttaviridae, Lipoth- rixviridae, myoviridae, Plasmaviridae, podoviridae, Portogloboviridae, rudivirus, Salterprovirus, Sphaerolipoviridae, Siphoviridae, Tectiviri- dae, Tristomaviridae, Turriviridae, Inoviridae, Microviridae, Spiraviridae, Pleolipoviridae, Cystoviridae or Leviviridae. In the context of the invention it is conceivable that bacteriophages are provided at least one type of bacteriophage, preferably from 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bacteriophage species. Furthermore, it is conceivable that the bacteriophages of a bacteriophage species specifically for at least one, and preferably 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more adipogenic bacterial species. A targeted selection of a suitable bacteriophage for the corresponding bacterial species is known to the person skilled in the art and can be easily selected from databases, for example. In the next step of the method according to the invention, it is necessary to bring the biological sample into contact with the bacteriophage and to incubate it. The “bringing into contact” can take place, for example, by dripping on, injecting, laying on and / or another method known to the person skilled in the art for bringing bacteriophage into contact with the biological sample.

The term “incubation” in connection with the method according to the invention relates to a method step in which the biological sample is incubated together with the bacteriophages over a certain period of time, with a multiplication cycle of the bacteriophages, preferably under the appropriate conditions known to a person skilled in the art within the biological sample comprising the for the Bacteriophage-specific bacteria of at least one type of bacteria comes from. In addition to incubating a biological sample with bacteriophages, this method step also includes monitoring this until the population of the at least one bacterial species has been reduced by at least 70%. The implementation of the monitoring is sufficiently known to the person skilled in the art and can, for example, take place on the basis of a defined period of time or also be based on the experience of the person skilled in the art, for example by observing the sample. Alternatively, a colored or otherwise optically visible indication can be provided, which indicates that the population of the bacterial species has been reduced by at least 70%. This can be done, for example, by achieving the desired bacterial density or by means of a chemical and / or biochemical indicator, for example by means of a coloration, a color change in the coloration or a loss of coloration. The steps of incubation and monitoring can take place alternately until the bacterial species has been reduced by at least 70%.

In the context of the invention it has proven to be advantageous if the reduction in the population of the bacterial species is at least 70%. The reduction in the population can preferably also be more than 70%, for example at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99 , 5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100%, with a reduction of at least 70% of the population of the bacterial species being significant and a reduction of at least 95% of the population of the bacterial species is considered significant. The person skilled in the art knows that a reduction is only 100% in the rarest cases, but this can certainly be the case when using suitable bacteriophage in a suitable biological sample he is sufficient.

By reducing the population of the bacterial species from 70% to 100%, the person skilled in the art also recognizes the success of the method, which establishes an adequate antibacterial effect and thus an adequate reduction in the adipogenic bacterial species within the biological sample. The reduction in the population of the bacterial species can then be used as a measure of the effectiveness or the success of a weight-reducing therapy based on the use of the corresponding bacteriophage. Alternatively, a reduction of a bacterial species can be aimed at, which is contrary to the life or a way of life of an anti-adipogenic bacterial species. In this way, the existence or growth of an anti-adipogenic bacterial species can be achieved by reducing the bacterial species that counteracts it, but is specific for the bacteriophage. It is assumed here that a growth of a population of an anti-adipogenic bacterial species can ultimately have the same effect on an organism as the reduction of a population of an adipogenic bacterial species.

Using the method according to the invention, it is thus possible to reduce the population of at least one adipogenic bacterial species specifically, effectively, with few side effects, tolerable, harmless to health, permanently and for a long time within a biological sample, without bacteria or other cells, tissues and / or organisms within to damage and / or influence the same biological sample for which the bacteriophages have no specificity. In this way, in contrast to the use of antibiotics, damage to other types of bacteria, as well as fungi, tissues and / or organs in the human and / or animal organism, is completely avoided. The method according to the invention can thus not only be carried out reliably and cost-effectively, but also quickly and easily, especially since the few procedural steps can be carried out in a standardized manner and a high success rate is achieved through a significant (more than 70% reduction) or significant (more than 95% reduction) reduction in the type of bacteria. The method can also be carried out with simple means and only requires devices known to the person skilled in the art for providing the biological sample and the bacteriophage, for bringing them into contact and for incubating them and for evaluating the reduction of the corresponding bacterial species within the population.

Advantageous further developments of the invention, which can be implemented individually or in combination, are presented in the subclaims.

In a further development of the invention, it is conceivable that an evaluation of the reduction in the population of the adipogenic bacterial species takes place after step c). The term “evaluate” is understandable to a person skilled in the art and relates to the analysis, evaluation and / or evaluation of step c). Since a reduction in the population of the bacterial species by at least 70% was previously monitored, this process step is used in particular to check the reduction achieved and optionally to record the result by making written records, photographs and film recordings, and to collect and document data on counts , Density measurements, growth determinations or similar methods and can be used for statistics with regard to the repeated implementation of the method according to the invention. In this step, it is also possible to determine the vitality or viability of the bacteria remaining in the biological sample which were not or not sufficiently killed by the bacteriophages. It is also possible to evaluate the vitality of the bacteria for which the corresponding bacteriophage has no specificity. Common methods for this are also known to the person skilled in the art. It is also conceivable that in step b) the nucleic acid has two or more promoters and / or regulatory elements, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more promoters and / or regulatory elements can be provided. It is essential that a particularly strong activation of the expressions of the nucleic acid and thus a particularly increased and efficient production of the antibacterial nucleic acid, the antibacterial nucleic acid molecule, the antibacterial polypeptide and / or the fragment thereof can be achieved. A bacteriophage with such a nucleic acid can contribute particularly efficiently to the reduction of the population of the bacterial species and, for example, significantly accelerate or significantly shorten step c) of the method according to the invention (incubation).

In yet another development, it is conceivable that the nucleic acid and nucleic acid sequences encode two or more nucleic acid molecules, polypeptides and / or fragments thereof, the nucleic acid preferably 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18,

19 or 20 nucleic acid molecules, polypeptides and / or fragments thereof. These nucleic acid molecules, polypeptides and / or fragments thereof can have an antibacterial effect for one or more types of bacteria; it is preferred here that bacteriophages can infect several types of bacteria with such a nucleic acid. In addition, it can be provided that the nucleic acid sequences are specific for killing a type of bacteria. The method can in turn be accelerated or a clear or significant reduction in the population of the bacterial species within the biological sample can be achieved. This leads to an increase in the efficiency of the method and to a reduction in the duration and / or costs when carrying out the method.

In a further development of the method, it is conceivable that the adipogenic bacterial species is anaerobic and / or aerobic and the bacterial species dem Strain Actinobacteria, Bacteroidetes, Firmicutes or Proteobacteria can be assigned.

The terms “aerobic” and “anaerobic” are well known to the person skilled in the art and describe a preferred way of life of the organism in question. An “aerobic” way of life means that the organism needs atmospheric oxygen to survive or is dependent on oxygen from the air. In contrast, “anae rob” describes a way of life that gets by without oxygen, which means that the organism in question can maintain its molecular metabolism without oxygen. A distinction is made between “obligatory”, “tolerant” and “facultative” aerobic or anaerobic organisms, whereby the person skilled in the art can understand that “obligatory” means an exclusively aerobic or anaerobic way of life excluding the other oxygen situation. “Tolerant”, on the other hand, means that the organisms live aerobically or anaerobically, but tolerate the opposite oxygen conditions, at least for a certain period of time. Finally, the term “facultative” means that, for example, anaerobic organisms basically have a corresponding metabolism, but can switch to an aerobic metabolism in the presence of oxygen, and vice versa. Thus, bacterial species can have an exclusively aerobic or anaerobic way of life or a combination thereof.

It is assumed that the definition and / or the explanations of the terms mentioned above apply to all aspects described below in this description, unless otherwise stated.

According to the invention, a kit for carrying out the method according to the invention is also proposed, comprising a) a container comprising bacteriophages of at least one bacteriophage species which is specific for at least one adipogenic Species of bacteria are and which are at least one functional with a

Promoter and / or nucleic acid linked to a regulatory element; and b) instructions for carrying out the procedure.

The term “kit” as used here refers to a collection of the above-mentioned components (kit-of-parts), which are preferably provided separately or within a single container.

The term “instruction for carrying out the method” relates to a numerical, written and / or graphic representation of the description, by means of which a simplification of the carrying out of the method objective according to the invention is achieved. These can preferably be in the form of a manual or can be provided by a computer program such as an application. Furthermore, it is conceivable that the computer program has an implemented algorithm which is able to carry out the determination, the comparison and / or the result thereof, to which reference is made in the method of the present invention. The computer program can be provided on a data storage medium or device such as an optical storage medium (e.g. compact disc) or directly on a computer or a data processing device. In addition, the instructions may preferably include standards for use levels as known to a person skilled in the art. In addition, the instructions include requirements for the storage and disposal of the container and the biological sample. In addition, the instructions for carrying out the method can contain tables, registers, databases or excerpts from the same about adipogenic, anti-adipogenic and / or other types of bacteria and types of bacteriophage known to the person skilled in the art, with information about the specificity and / or efficiency of the types of bacteriophage can be listed. According to the invention, a bacteriophage is also proposed which comprises at least one nucleic acid functionally linked to a promoter and / or to a regulatory element, the bacteriophage being specific for the at least one adipogenic bacterial species and the nucleic acid being selected from the group comprising: a ) a nucleic acid sequence which codes for at least one antibacterial nucleic acid molecule; and b) a nucleic acid sequence which encodes a nucleic acid molecule that is at least 50% identical to the nucleic acid molecules that is encoded by the nucleic acid sequences from a); and c) a nucleic acid sequence which codes for at least one antibacterial polypeptide; and d) a nucleic acid sequence which encodes a polypeptide that is at least 50% identical to a polypeptide that is encoded by the nucleic acid sequences from c); and e) a nucleic acid sequence for a fragment of a nucleic acid from a), b), c) or d) encodes a nucleic acid molecule or a polypeptide via the fragment.

The bacteriophage according to the invention is described in detail elsewhere, the nucleic acid sequence being a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence which is already present in the bacteriophage or which, for example, was introduced into the bacteriophage by molecular biological methods . For example, these molecular biological methods can relate to recombination, gene transfer, CRISPR / Cas, Talen or the like in vivo and / or in vitro, as is known to a person skilled in the art. In a further development, it is conceivable that the nucleic acid has two or more promoters and / or regulatory elements. Significant is that a particularly strong activation of the expressions of the nucleic acid and thus a particularly increased and efficient production of the antibacterial nucleic acid, the antibacterial nucleic acid molecule, the antibacterial polypeptide and / or the fragment thereof are achieved. The bacteriophage with such a nucleic acid can contribute particularly efficiently to the reduction of the population of the adipogenic bacterial species or contribute to the reduction of a bacterial species which is contrary to the way of life of an anti-adipogenic bacterial species. It is also conceivable that the nucleic acid encodes nucleic acid sequences for two or more nucleic acid molecules, polypeptides and / or fragments thereof. It is preferred here that the bacteriophage can infect several types of bacteria with such a nucleic acid.

In yet another development, it is conceivable that the adipogenic bacterial species is anaerobic and / or aerobic and the bacterial species can be assigned to the strain Actinobacteria, Bacteroidetes, Firmicutes or Proteobacteria.

According to the invention are also bacteriophages and / or a pharmaceutical composition comprising bacteriophages and at least one other component as a drug to change the composition of the intestinal flora, as a drug to achieve and / or maintain weight reduction, as a drug to prevent weight gain and / or as a drug proposed in the treatment of overweight, obesity, metabolic disease, cardiovascular disease, bacterial infection, metabolic disease, metabolic syndrome and / or cancer.

The term “pharmaceutical composition” used here refers to a mixture of bacteriophages, which are defined in more detail elsewhere in this document, and at least one another component. Such further constituents can preferably be stabilizers, wetting agents, pharmaceutical carriers, pharmaceutically acceptable carriers, diluents, pharmaceutically acceptable diluents, additional active pharmaceutical ingredients, release agents and the like. Preferred diluents include water, alcohols, physiological salt solutions, buffers such as phosphate-buffered salt solutions, syrup, oil, water, emulsions, various types of wetting agents, and the like. The carrier must be acceptable in the sense that it is compatible with the other ingredients of the composition and that it is human and / or animal

Organism is not harmful. The pharmaceutical carrier used can contain a solid, a gel or a liquid. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Similarly, the carrier or diluent may contain a time release material well known in the art, such as glycerol monostearate or glycerol distearate alone or with a wax. These suitable carriers include those mentioned above and others known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, European Pharmacopoeia, Homeopathic Pharmacopoeia of the USA or HAB. The pharmaceutically acceptable diluent is chosen so that the biological activity of the combination is not impaired. Examples of such diluents are distilled water, physiological saline solution, Ringer's solutions, Dextroselö solution and Hank's solution. In addition, the pharmaceutical composition can also contain other carriers, auxiliary substances or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. The bacteriophages and / or the pharmaceutical composition are to be adapted for the corresponding use. Accordingly, it is assumed that the bacteriophages and / or the pharmaceutical Chemical composition can be formulated for systemic or topical application depending on the desired mode of administration. The bacteriophages and / or the pharmaceutical composition are preferably to be formulated for systemic or local application. Oral application, for example in the form of tablets, solutions or drinking ampoules, or topical application in gel form or application by injection is preferably provided. However, depending on the nature and mode of action, the bacteriophages and / or the pharmaceutical composition can also be administered by other routes, including dermal, intramuscular, subcutaneous, oral, rectal, retrograde or intravenous administration. The exact, individually recommended dosage can in principle depend on further parameters which are well known to those skilled in the art. For example, children may receive a different dose than adults. A person skilled in the art can easily determine whether the dosage needs to be adjusted with the aid of various known calculation tools.

The term “change in the composition of the intestinal flora” describes a process that is understandable to a person skilled in the art, in which at least one population of an adipogenic bacterial species that is part of the intestinal flora of the human and / or animal organism increases, reduces and / or is otherwise impaired. Viewed in isolation, this can be measurable by determining the respective number of bacteria or can be determined relative to other types of bacteria within the intestinal flora. Suitable methods for this are known to the person skilled in the art.

The term “achieving and / or maintaining a weight reduction” is understandable to a person skilled in the art and relates to the short-term reduction of the body weight of the human and / or animal organism and / or, preferably, the permanent maintenance of the reduction in the body weight of the human and / or animal Organism. The term "preventing weight gain" is one of skill in the art also understandable and relates to the permanent and / or long-term maintenance of the reduced body weight of the human and / or animal organism. It is preferably conceivable that the achievement and / or maintenance of the weight reduction and the prevention of the weight gain of the human and / or animal organism are achieved by reducing at least one adipogenic bacterial species.

The use of the bacteriophages and / or the pharmaceutical composition as a medicament can be used, inter alia, in the treatment of diseases in the medical fields of oncology, immunology, infectology, dentistry, oral and maxillofacial medicine, otorhinolaryngology, ophthalmology, neurology, Gynecology, gastroenterology, endocrinology, psychiatry, psychosomatics, orthopedics, pediatrics, surgery, urology and / or the like. Possible diagnoses that may indicate the use of the bacteriophage and / or the pharmaceutical composition include the metabolic syndrome, type 2 diabetes mellitus, gall bladder diseases, chronic diseases of the digestive tract, chronic intestinal inflammation, hyperacidity of the stomach, high blood pressure, disorders of lipid metabolism, Breathing difficulties, sleep apnea, coronary heart disease, osteoarthritis, gout, cancers, such as uterine, breast, cervical, pancreatic, liver, stomach, colon, prostate and / or gallbladder cancer, sex hormone disorders, decreased libido, joint - and back pain, increased risk of thrombosis and embolism, increased risk during operations and anesthesia, psychosocial problems and the associated restrictions on quality of life, for example due to depression, reduced self-esteem and / or a perceived low level of recognition by the environment. The aim is preferably in each case to reduce at least one adipogenic bacterial species and thus to achieve and / or maintain the weight reduction and / or to prevent the increase in weight bringing about a human and / or animal organism as the main or secondary result in order to obtain a good, preferably improved, prognosis for the treatment of the respective disease and / or respective condition. A use of the bacteriophages and / or the pharmaceutical composition is conceivable regardless of the actual disease. Success is seen in the reduction of at least one adipogenic bacterial species, a weight reduction and / or a delayed or stopped weight gain. The term “treatment” relates to any improvement in the human and / or animal organism that occurs in comparison to an untreated human and / or animal organism, this improvement preferably being the reduction of at least one adipogenic bacterial species, an achievement and / or Preservation of weight reduction and / or prevention of weight gain in the human and / or animal organism is based. It is assumed that treatment may not be successful in 100% of the human and / or animal organisms to be treated. However, the term assumes that the treatment is successful for a statistically significant proportion of the test subjects (e.g. a cohort in a cohort study). A person skilled in the art can easily determine whether a part is statistically significant with the aid of various known statistical evaluation instruments, for example determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-values are preferably 0.05, 0.01, 0.005 or 0.0001.

The term “drug” refers to bacteriophages and / or a pharmaceutical composition in a therapeutically effective dose, as mentioned elsewhere in this document. The pharmaceutical composition preferably has bacteriophages, at least one pharmaceutically acceptable carrier and / or a diluent. The drug can be formulated for various routes of administration which are fully described elsewhere in this document. A therapeutically effective dose refers to the amounts required to change the composition of the intestinal flora, to reduce at least one adipogenic bacterial species, to achieve and / or maintain weight reduction, to prevent weight gain and / or to treat those mentioned elsewhere Illness is necessary. The therapeutic effectiveness and toxicity can be determined by standard pharmaceutical methods in cell cultures or test animals, for example ED50 (the dose that is therapeutically effective at 50%) and LD50 (the dose that is fatal at 50%). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. The dosage regimen is determined by the attending physician and other clinical factors. As is known in medicine, the appropriate dosage will depend on many factors known to those skilled in the art, including size, body surface area, age, substance to be administered, sex, time and route of administration, general health, and others concurrent medication. Progress can be monitored through a periodic assessment.

Furthermore, according to the invention bacteriophages and / or a pharmaceutical composition comprising bacteriophages and at least one further component for use in the treatment of obesity, obesity, a metabolic disease, a cardiovascular disease, a bacterial infection, a metabolic disease, the metabolic Syndrome and / or cancer. According to the invention are also bacteriophages and / or a pharmaceutical composition comprising bacteriophages and at least one further component for use in a therapeutic method or in a non-therapeutic method for changing the composition of the intestinal flora, in a therapeutic method or in a non-therapeutic method to achieve and / or maintain a weight reduction, in a therapeutic method or in a non-therapeutic method to prevent weight gain and / or in a therapeutic method or in a non-therapeutic method for the treatment of overweight, obesity, a Metabolic disease, cardiovascular disease, bacterial infection, metabolic disease, metabolic syndrome and / or cancer.

In a further development of the invention, it is conceivable that the bacterial infection is a bacterial infection of the respiratory tract, the teeth, the mouth, the jaw, the eye, the musculoskeletal system, the blood, the gastrointestinal tract, preferably a bacterial infection of the large intestine, the small intestine, of the duodenum, the stomach, the liver, the gall bladder and / or the pancreas, the skin, the cardiovascular system, the hormonal balance, the mind, the immune system, the nervous system, the metabolism, wounds and / or the urogenital tract .

The term "gastrointestinal tract" is understandable to a person skilled in the art and relates to the organs of the human and / or animal organism which are used to absorb, crush, transport and / or process nutrients with the aim of providing nutrients for the human and / or or animal organism, whereby the intestine, in particular through the intestinal flora, contributes significantly to the regulation of these. The features described here can each be implemented individually or in combination with one another. The invention is not restricted to the exemplary embodiments.

Claims

1. A method for reducing the population of at least one adipogenic bacterial species, which comprises the following steps: a) providing a biological sample which comprises bacteria of at least one adipogenic bacterial species; and b) providing bacteriophages of at least one bacteriophage species which are specific for at least one adipogenic bacterial species and which comprise at least one nucleic acid functionally linked to a promoter and / or a regulatory element, the nucleic acid being selected from the group comprising: i. a nucleic acid sequence which codes for at least one antibacterial nucleic acid molecule; and ii. a nucleic acid sequence which encodes a nucleic acid molecule which is at least 50% identical to the nucleic acid molecule which is encoded by the nucleic acid sequences from i); and iii. a nucleic acid sequence which codes for at least one antibacterial polypeptide; and iv. a nucleic acid sequence which encodes a polypeptide which is at least 50% identical to a polypeptide which is encoded by the nucleic acid sequences from iii); and V. a nucleic acid sequence for a fragment of a

Nucleic acid from i), ii), iii) or iv), the fragment encoding a nucleic acid molecule or a polypeptide; and c) bringing the biological sample into contact and incubating it with the bacteriophages, the incubation taking place until the population of the adipogenic bacterial species is reduced by at least 70%.

2. The method of claim 1, wherein following step c) a

Evaluating the reduction in the population of the adipogenic bacterial species is done.

3. The method according to claim 1 or 2, wherein the nucleic acid has two or more promoters and / or regulatory elements.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid nucleic acid sequences for two or more nucleic acid molecules,

Encodes polypeptides and / or fragments thereof.

5. The method according to any one of claims 1 to 4, wherein the adipogenic bacterial species is anaerobic and / or aerobic and wherein the bacterial species can be assigned to the strain Actinobacteria, Bacteroidetes, Firmicutes or Proteobacteria.

6 kit for performing the method according to one of the preceding claims comprising: a) a container comprising bacteriophages of at least one bacteriophage species which are specific for at least one adipogenic bacterial species and which comprise at least one nucleic acid functionally linked to a promoter and / or a regulatory element; and b) instructions for carrying out the procedure.

7. Bacteriophage comprising at least one functional with one

Nucleic acid linked to a promoter and / or a regulatory element, the bacteriophage being specific for at least one adipogenic bacterial species and the nucleic acid being selected from the group comprising: a. a nucleic acid sequence which codes for at least one anti-bacterial nucleic acid molecule; and b. a nucleic acid sequence which encodes a nucleic acid molecule which is at least 50% identical to the nucleic acid molecule which is encoded by the nucleic acid sequences from a); and c. a nucleic acid sequence which codes for at least one anti-bacterial polypeptide; and d. a nucleic acid sequence which encodes a polypeptide which is at least 50% identical to a polypeptide which is encoded by the nucleic acid sequences from c); and e. a nucleic acid sequence for a fragment of a nucleic acid from a), b), c) or d), the fragment encoding a nucleic acid molecule or a polypeptide.

8. Bacteriophage according to claim 7, wherein the nucleic acid has two or more promoters and / or regulatory elements.

9. Bacteriophage according to claim 7 or 8, wherein the nucleic acid encodes nucleic acid sequences for two or more nucleic acid molecules, polypeptides and / or fragments thereof.

10. Bacteriophage according to one of claims 7 to 9, wherein the adipogenic bacterial species is anaerobic and / or aerobic and wherein the bacterial species can be assigned to the strain Actinobacteria, Bacteroidetes, Firmicutes or Proteobacteria.

11. Bacteriophages according to one of claims 7 to 10 and / or a pharmaceutical composition comprising bacteriophages according to one of claims 7 to 10 and at least one further component for use as a drug to change the composition of the intestinal flora, as a drug to achieve and / or Maintaining weight loss, as a drug to prevent weight gain and / or as a drug in the treatment of overweight, obesity, a metabolic disease, a cardiovascular disease, a bacterial infection, a metabolic disease, the metabolic syndrome and / or of cancer.

12. Bacteriophages according to one of claims 7 to 10 and / or a pharmaceutical composition comprising bacteriophages according to one of claims 7 to 10 and at least one further component for use in the treatment of overweight, obesity, a metabolic disease, a cardiovascular disease Disease, bacterial infection, metabolic disease, metabolic syndrome and / or cancer.

13. Bacteriophages according to one of claims 7 to 10 and / or a pharmaceutical composition comprising bacteriophages according to one of claims 7 to 10 and at least one further loading component for use in a therapeutic method or in a non-therapeutic method for changing the composition of the intestinal flora, in a therapeutic method or in a non-therapeutic method for achieving and / or maintaining a weight reduction, in a therapeutic method or in a non-therapeutic method for preventing weight gain and / or in a therapeutic method or in a non-therapeutic method for the treatment of overweight, obesity, a metabolic disease, a cardiovascular disease, a bacterial infection, a metabolic disease, des metabolic syndrome and / or cancer.

14. bacteriophage and / or pharmaceutical composition according to any one of claims 11 to 13, wherein the bacterial infection is a bacterial infection of the respiratory tract, the teeth, the mouth, the

Of the jaw, the eyes, the musculoskeletal system, the blood, the gastrointestinal tract, the skin, the cardiovascular system, the hormone balance, the mind, the immune system, the nervous system, the metabolism, wounds and / or the urogenital tract.