WO2023063909A1 - Nanoformulations that include antimicrobial agent(s), targeted by peptide 6.2 - Google Patents

Nanoformulations that include antimicrobial agent(s), targeted by peptide 6.2 Download PDF

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Publication number
WO2023063909A1
WO2023063909A1 PCT/TR2022/051103 TR2022051103W WO2023063909A1 WO 2023063909 A1 WO2023063909 A1 WO 2023063909A1 TR 2022051103 W TR2022051103 W TR 2022051103W WO 2023063909 A1 WO2023063909 A1 WO 2023063909A1
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Prior art keywords
peptide
nanostructure
nanocarriers
drug
antimicrobial
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PCT/TR2022/051103
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French (fr)
Inventor
Serap DERMAN
Irem COKSU
Yigit Can BAYCILI
Tulin OZBEK
Pelin PELIT ARAYICI
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Yildiz Teknik Universitesi
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Priority claimed from TR2021/015840 external-priority patent/TR2021015840A2/en
Application filed by Yildiz Teknik Universitesi filed Critical Yildiz Teknik Universitesi
Publication of WO2023063909A1 publication Critical patent/WO2023063909A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid

Definitions

  • AMPs antimicrobial peptides
  • AMPs are low molecular weight proteins with broad spectrum antimicrobial and immuno modulator activities against Gram positive and Gram negative infectious bacteria, viruses and funghi (Bardan et al., Antimicrobial peptides and the skin. Expert opinion on biological therapy, (2004), 4(4), 543-549).
  • AMPs that are classified in accordance with their net load, secondary structural content and physicochemical properties such as solubility, include both hydrophobic and hydrophylic side chain which allows solvation in aqueous media (Bahar, Controlling biofiim and persister cells by targeting cell membranes. (2015). Dissertations - ALL. 377.; Elias & Choi, Interactions among stratum corneum defensive functions. Experimental dermatology, (2005), 14(10), 719-726).
  • Primary object of the present invention is provision of a nanoformulation that is targeted and loaded with one or more antimicrobial agents.
  • the term "active substance" corresponds to one or more antimicrobial agents.
  • a nanoformulation is proposed that corresponds to a nanostructure that includes one or more antimicrobial agents with a high extent of activity on antibiotic resistant bacteria. The present invention enables controlled release of the antimicrobial agent as a result of integration of antimicrobial agents with nanoformulations.
  • the present invention proposes one or more nanostructures (100) comprising one or more nanocarriers (10) loaded with one or more active substances (11), an outer surface of said nanocarriers (10) being provided with one or more one or more targeting means (12).
  • the nanostructure (100) comprises Peptide 6.2 (abbreviated as P6.2) as targeting means (12), and the nanostructure (100) can further comprise one or more different targeting means (12) other than Peptide 6.2. Thanks to the fact that the surface thereof is provided with Peptide 6.2, the nanostructure (100) according to the present invention enables a high- selectivity targeting of bacteria that are suitable for being targeted with Peptide 6.2.
  • a building material of the nanocarrier (10) can comprise one or more materials selected from the list consisting of one or more polymers, one or more lipids, one or more proteins, and one or more carbohydrates.
  • the active substance (11) can comprise one or more substances selected from the list consisting of peptide Hf-18, and/or one or more antibiotics and/or one or more antimicrobial substances.
  • the active substance (11) can include a content in accordance with any of the following alternatives (hence, throughout the present specification, the alternative "more” in the wording "one or more” shall be considered in this manner):
  • the active substance (11) includes peptide Hf-18.
  • Peptide Hf-18 can be also referred to as HF-18.
  • said one or more targeting means (12) is Peptide 6.2.
  • the active substance (11) includes Hf-18 or more preferably where the active substance (11) is Hf-18, said synergistic effect becomes more significant; thus, at relatively low antimicrobial substance concentrations, a relatively high extent of biological activity can be achieved more effectively.
  • the present invention further proposes a method for obtaining a nanostructure (100) that is loaded with one or more active substances (11), and that includes a nanocarrier (10) which has an outer surface that is provided with one or more targeting means (12) that includes Peptide 6.2.
  • the method according to the present invention comprises the following steps of: a) loading of a building material that includes one or more materials selected from the list consisting of one or more polymers, one or more lipids, one or more proteins, and one or more carbohydrates, with one or more active substances (11) selected from the list consisting of Peptide Hf-18, one or more antibiotics and one or more antimicrobial substances; thereby obtaining one or more nanocarriers (10) that is loaded with one or more active substances (11); b) binding of one or more targeting means (12) that include Peptide 6.2, onto a surface of the one or more nanocarriers (10) that are obtained in the step a.
  • the loading operation in the step a of the method according to the present invention enables the protection of the active substance (11) against enzymatic degradation; thus prolongs the half life and enables controlled release.
  • the step b of the method according to the present invention enables the provision of a nanostructure (100) that has high selectivity against bacteria that are suitable for being targeted by Peptide 6.2.
  • the loading operation in the step a of the method according to the present invention can be performed with any of suitable methods such as single-emulsion, double-emulsion, emulsification, desolvation or ionic gelation.
  • the loading operation in the step a is implemented with a process selected from single-emulsion, double-emulsion, emulsification, desolvation or ionic gelation.
  • the binding operation in the step b can is performed with chemical binding via bioconjugation, or with physical binding via electrostatic interaction.
  • one or more active substances (11) that include Peptide Hf-18 is loaded onto the building material.
  • the one or more active substance (11) in such implementation includes Peptide Hf-18.
  • a nanostructure (100) is obtained in which the active substance (11) includes Hf-18 and the targeting means (12) includes Peptide 6.2.
  • step b Peptide 6.2 is attached onto a surface of the one or more nanocarriers (10) that are obtained in the step a.
  • a nanostructure (100) is obtained in which the targeting means (12) is Peptide 6.2.
  • the building material is loaded with one or more active substances (11) that include Peptide Hf-18; and in the step b, Peptide 6.2 is attached onto a surface of the one or more nanocarriers (10) obtained in the step a.
  • said one or more active substances (11) include Peptide Hf-18 and the targeting means (12) is Peptide 6.2.
  • a nanostructure (100) is obtained in which the active substance (11) includes Hf-18 and the targeting means (12) is Peptide 6.2; thereby enabling the synergistic effect of Hf- 18 peptide and Peptide 6.2 in combination with each other.
  • nanostructure can also be referred to as “nanoformulation” or “nanoparticular carrier system”
  • nanoformulation or “nanoparticular carrier system”
  • the building material of the nanocarrier (10) can include one or more components selected from polymers, lipids, proteins and carbohydrates.
  • One or more active substances (11) selected from Peptide Hf-18, antibiotics or antimicrobial molecules can be loaded onto said building material.
  • One or more nanocarriers (10) that are loaded with one or more active substances (11) can be thus obtained.
  • the targeting means (12) can include Peptide 6.2 or can be Peptide 6.2.
  • a nanoformulation or in other words, a nanostructure (100) can be obtained, which is targeted with Peptide 6.2 and which includes nanocarrier (10) loaded with active substance (11).
  • Building material of the nanocarrier (10) enhances biobiodistribution and pharmacocinetics of the active substance (11) by enhancing the protection thereof against biological degradation; further enhances the solubility, stability and transportation to the target area by modification of physicochemical properties such as surface and shape; decreases its toxicity and enables a prolonged circulation thanks to the controlled release.
  • Said building material can include, but not limited to, one or more materials that can be selected from the following:
  • one or more homopolymers that can be selected from PCL and PLA;
  • one or more lipids that can be selected from stearic acid, palmitic acid and phosphatidylcholine;
  • Peptide 6.2 can be used as the targeting means (12) in view of that the Peptide 6.2 has a higher affinity and rapid binding kinetics against bacterial membranes that include phosphatidylglycerol (PG), thereby having a high antibacterial activity and low hemolytic activity.
  • PG phosphatidylglycerol
  • Hf-18 is a peptide that is disclosed in the following exemplary source.
  • the sequence of Hf- 18 can be synthesized based on the content that is available in the reference publication provided below:
  • Peptide 6.2 is a peptide that is disclosed in the following exemplary sources.
  • the sequence of Peptide 6.2 can be synthesized based on contents available in any of the reference publications provided below:
  • Hf-18 and Peptide 6.2 can be produced by a person that is skilled in peptid synthesis within the professional specialized knowledge, starting from the respective sequence listings; or commercially obtained from the market, prepared in accordance with the latter principle. Production methods of Hf-18 and Peptide 6.2 mentioned in the present application are exemplified below.
  • Example 1 production of Hf-18 and Peptide 6.2
  • Peptide 6.2 that shows a higher affinity against procariotic membranes rather than eucariotic membranes (Mar nez et al., 2020, Antibacterial, anti-biofilm and in vivo activities of the antimicrobial peptides P5 and P6. 2.
  • the synthesis can be performed in a peptide synthesis device, for instance, based on Fmoc chemistry.
  • a peptide synthesis device a commercially available microwave assisted solid phase peptide synthesis device can be used; for instance, one supplied by the company CEM Liberty.
  • resins that bear Leu and Lys amino acids are to be used, respectively.
  • Such resin can be, e.g., swollen for 3 hours in DMF (dimethyl formamide), to enable the exposure of active ends on the resin that is a polymeric particle, is enabled.
  • a 20% solution of piperidine in DMF can be prepared as a deprotector (an agent for breaking side group protector); a mixture of HBTU/HOBt (0- (Benzotriazole-l-yl) - N,N,N',N'- tetramethyluronium hexafluorophosphate) I N- Hydroksybenzotriazole) can be prepared as an activator (activating agent); and a mixture of DIEA/NMP (Diisopropyl ethyl amine I N-Methyl-2-pyrrolidone) can be prepared as an activator base (activating base).
  • the synthesis can be initiated by starting a respective program over the device.
  • the microwave assisted solid phase peptide synthesis process is complete, the synthesis is to be brought to an end by collecting the peptide chain that is attached to resin, said peptide that is attached to resin is washed with one or more suitable chemicals such as dichloro methane (DCM), then filtered and dried.
  • DCM dichloro methane
  • the synthesized peptide chain is separated from the resin by a cleavage cocktail such as TFA (trifluoro acetic acid) I EDT (1,2 - ethane dithiol) / Thioanisol I H2O (090/2.5/2.5/5) v/v.
  • a cleavage cocktail such as TFA (trifluoro acetic acid) I EDT (1,2 - ethane dithiol) / Thioanisol I H2O (090/2.5/2.5/5) v/v.
  • the gelled solution can be transferred into a centrifuge tube, cold diethyl ether can be added thereonto, then subjected to centrifuge, such that the peptide is precipitated in the form of a white powder.
  • the precipitated peptide can be dried for storing; said drying operation can be performed at a pressure under 1 atmosphere (e.g., under vacuum).
  • the storing operation can be performed at a low temperature, for instance at -40 °C.
  • the dried peptide can be named as a "crude peptide".
  • molecular weight determination can be performed by using an LC-MS system (such as Shimadzu LC-MS 2010 EV) that includes an electrospray ionization (ESI) probe and that performs separation by reverse phase chromatography (RP-HPLC) (for instance, using a suitable gradient elution).
  • LC-MS system such as Shimadzu LC-MS 2010 EV
  • ESI electrospray ionization
  • RP-HPLC reverse phase chromatography
  • Teknokroma Tracer Exel 120 ODS-A 5pm / 20 x 0.21cm
  • a [in accordance with peptidine solubility, (water, 0.1% (v/v) TFA) or (water, 0.1% (v/v) formic acid)] and B [in accordance with peptidine solubility, (Acetonitrile, 0.1% (v/v) TFA)) or (Acetonitrile, 0.1% (v/v) formic acid)] can be used as gradient elution; 0.2 mU/min can be selected as flow rate.
  • ESI can be utilized in positive ion mode within a range between 200 - 2000 m/z.
  • Capillary temperature can be set at 250 °C.
  • Nebulisator gas (N 2 ) flow rate can be e.g., 1.5 L / min.
  • a step of drying can be applied, e.g., using a lyophilization device; thereby pure peptide is obtained in the form of powder.
  • a gradient elution can be employed [such as, in accordance with peptidine solubility (water, 0.1% (v/v) TFA) or (water, 0.1% (v/v) formic acid)] and B [in accordance with peptidine solubility (Acetonitrile, 0.1% (v/v) TFA)) or (Acetonitrile, 0.1% (v/v) formic acid)].
  • Purified peptide is obtained as a result of the purification step.
  • the purified peptide can be lyophilized and stored for instance at -40 °C (T Acar, PP Arayici, B Ucar, M Karahan, Z Mustafaeva, Synthesis, characterization and lipophilicity study of Brucella abortus' immunogenic peptide sequence that can be used in the future vaccination studies, International Journal of Peptide Research and Therapeutics 25 (3), 911-918, 2019, DOI: 10.1007/S10989-018-9739-0); (Derman S., Kizilbey K., Mansuroglu B., Mustafaeva Z., Synthesis and characterization of Canine parvovirus (CPV) VP2 W-7L-20 synthetic peptide for synthetic vaccine, Fresenius Environmental Bulletin 23(2A): 558-566, 2014).
  • CPV Canine parvovirus
  • the development that is the subject of the present application is industrially applicable.
  • the nanostructure (100) obtained according to the present application sets an innovative alternative to antibiotics group drugs that are already used in treatment of bacterial infections.
  • the development of the present application enables a nanoformulation or nanostructure (100) system that targets the surfaces of bacteria that can be targeted by Peptide 6.2; the system includes an antimicrobial compound and provides controlled release of said compound as an active substance (11).
  • the system that can be obtained in accordance with the present invention has the potential of use in modern exemplary commercial usage forms; as a drug, in any of the forms of injection vial, tablet or capsule; or as a final product, in any of the forms of pomade, cream or granule.
  • the present invention enables the following drugs:
  • a drug in the form of capsules that includes the nanostructure (100) according to the present invention
  • a drug in the form of pomade that includes the nanostructure (100) according to the present invention

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Abstract

The present invention proposes one or more nanostructures (100) that are loaded with one or more active substances (11), said nanostructures (100) comprising one or more nanocarriers (10), an outer surface of said nanocarriers (10) being provided with one or more targeting means (12). Said one or more targeting means (12) comprise Peptide 6.2. The present invention further proposes a method for obtaining such nanostructures (100).

Description

NANOFORMULATIONS THAT INCLUDE ANTIMICROBIAL AGENT(S), TARGETED BY PEPTIDE 6.2
Technical Field of the Invention
The present invention relates to a therapeutic agent that is in the form of a nanoformulation, or that includes a nanoformulation, for being supplied to the pharmaceutical industry.
Background of the Invention
Antibiotics were effectively used between the years 1930-1960 as their golden age, yet nowadays antibiotics resistance caused the emergence and spread of antimicrobial resistance (AMR) and multiple drug resistant bacteria negatively affected the public health and damaged economies of countries (Aslam et al., Antibiotic resistance: a rundown of a global crisis. (2018), Infection and drug resistance, 11, 1645). In particular, due to the sepses and pneumonia based on hospital-acquired infections (HAIs) related to antimicrobial resistant pathogens annually cause decease of 99 thousands of patients along with an annual loss of 8 billion dollars (America, Combating antimicrobial resistance: policy recommendations to save lives. Clinical Infectious Diseases, (2011), 52 (suppl_5), S397-S428). Reportedly, the costs related to treatment of HAI patients reach to a level of up to 29,000 US Dollars per patient. In the USA, a total economic loss of about 20 billion dollars are recorded, whereas a further ca. 35 billion dollars of annual loss is recorded in relation with efficiency drop based on antimicrobial resistance in healthcare systems (Ventola, The antibiotic resistance crisis: part 1: causes and threats. (2015), Pharmacy and therapeutics, 40(4), 277). Meanwhile in Europe it is estimated that 25,000 patients die each year due to multiple drug resistant bacterial infections, costing 1.5 billion Euros to the economy of the European Union (Kunkalekar et al., Siiver-doped manganese dioxide and trioxide nanopartides inhibit both gram positive and gram negative pathogenic bacteria. (2014), Colloids and Surfaces B: Biointerfaces, 113, 429-434.; Li 8i Webster, Bacteria antibiotic resistance: New challenges and opportunities for implant-associated orthopedic infections. (2018) Journal of Orthopaedic Research, 36(1), 22-32).
According to most recent data published by the World Health Organization, the ratio of nosocomial infections in Turkiye is 13.4%. In the 2020 National Nosocomial Infections Surveillance Network (UHIESA) report published by Turkish Public Health Institute, rank number one and two in distribution of pathogens in bacteria that cause nosocomial infections are shared by carbapenem resistant Acinetobacter boumannii by 73.18 % and methicillin resistant coagulase negative staphylococcus (MRKNS) by 66.51 %, respectively (Gbzel, M. G. (2019), Ulusal Hastane Enfeksiyonlan Surveyans Agi (UHESA), Turkiye Halk Sagligi Kurumu, https://www.tmc-online.org/images/37_kongre/gokhan_guzel.pdf (available as of 23.09.2021). Turkiye Halk Sagligi Kurumu, https://hsgm.saglik.gov.tr/depo/birimler/Bulasici-hastaliklar- db/hastaliklar/SHIE/Raporlar/USHIESA_OZET_RAPORU_2020.pdf (available as of 23.09.2021)).
Facing this situation, the most important scenario recently developed by WHO is that no powerful antimicrobial agent will remain available for therapeutic use, and that the whole humanity may die because of infections. It is indicated that as of the year 2050, about 444 millions of people will be defeated by infections and the birth ratios will rapidly drop in this scenario (Bartlett, et al., Seven ways to preserve the miracle of antibiotics. Clinical Infectious Diseases, (2013), 56(10), 1445-1450; Gould 8i Bal, New antibiotic agents in the pipeline and how they can help overcome microbial resistance. Virulence, (2013), 4(2), 185-191). In the light of these information, it is nowadays necessary to develop an alternative antimicrobial strategy.
In the recent 20 years, antimicrobial peptides (AMPs) have become attractive in designing novel antimicrobial agents, due to their natural antimicrobial propereties and low tendency to develop bacterial resistance. AMPs are considered promising candidates for effectively treating multiple drug resistance bacteria and overcoming present antibiotic resistance thereof. AMPs are low molecular weight proteins with broad spectrum antimicrobial and immuno modulator activities against Gram positive and Gram negative infectious bacteria, viruses and funghi (Bardan et al., Antimicrobial peptides and the skin. Expert opinion on biological therapy, (2004), 4(4), 543-549). AMPs that are classified in accordance with their net load, secondary structural content and physicochemical properties such as solubility, include both hydrophobic and hydrophylic side chain which allows solvation in aqueous media (Bahar, Controlling biofiim and persister cells by targeting cell membranes. (2015). Dissertations - ALL. 377.; Elias & Choi, Interactions among stratum corneum defensive functions. Experimental dermatology, (2005), 14(10), 719-726). Alpha-helical cationic AMPs which are abundant and widely available in nature, can disintegrate the bacterial cytoplasmic membrane that causes cellular death, via osmotic shock (Hancock & Rozek, Role of membranes in the activities of antimicrobial cationic peptides. FEMS microbiology letters, (2002), 206(2), 143-149). Along with cationic AMPs, also anionic AMPs are defined that have a low tendency to develop resistance, as the most important property thereof (Harris et al., Anionic antimicrobial peptides from eukaryotic organisms. Current Protein and Peptide Science, (2009), 10(6), 585-606). These peptides initially interact with bacterial cell wall and membrane and transit into the cytosol (Santos et al., Nanomateriais and molecular transporters to overcome the bacterial envelope barrier: Towards advanced delivery of antibiotics. Advanced drug delivery reviews, (2018), 136, 28-48; Sun et al., Host defense (antimicrobial) peptides, Peptide applications in biomedicine, biotechnology and bioengineering (2018), pp. 253-285, Elsevier); and contrary to the antibiotics that bind to the proteins to inhibit peptydoglucane synthesis, these peptides bind to the proteins; to cause lysis by forming pores in the complex that forms the membrane, over constituents that are present in the membrane (Kumar et al., Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and biocompatibiiity in vivo. Biomolecules, (2018), 8(1), 4; Le et al., Intracellular targeting mechanisms by antimicrobial peptides. Antimicrobial agents and chemotherapy, (2017), 61(4)). AMPs participate the innate immune response that constitutes the primary response against infections, thereby increasing its efficiency. Furthermore, AMPs emerged as one of the alternative antimicrobial compounds by having a great potential for clinical use thanks to their multiple action mechanisms and broad activity spectra.
Objects of the Invention Primary object of the present invention is provision of a nanoformulation that is targeted and loaded with one or more antimicrobial agents.
Another object of the present invention is the provision of a controlled release and targeted nanoformulation that has a high extent of antimicrobial effect on G+ S. aureus, G" E. co/ian G" P. aeruginosa.
Brief Description of the Invention
With the development that is the subject of the present application, it is aimed to increase the biological activity of a nanoformulation with one or more trapped active substances, as well as to achieve selectivity in treatment by Peptide 6.2 that targets bacterial cells. Within the context of the present application, the term "active substance" corresponds to one or more antimicrobial agents. With the present development, a nanoformulation is proposed that corresponds to a nanostructure that includes one or more antimicrobial agents with a high extent of activity on antibiotic resistant bacteria. The present invention enables controlled release of the antimicrobial agent as a result of integration of antimicrobial agents with nanoformulations. Said controlled release enables the plasma concentration to be maintained within a desired range; the surface modification achieved by that the surfaces of the nanostructure is equipped with Peptide 6.2 enables targeting of the active substance along with the nanostructure onto a target microorganism; systemic toxicity of the active substance is decreased; cellular uptake ratio of the active substance is increased thanks to nanometric dimensions of the nanostructure; and bacterial lysis can be achieved via a plurality of mechanisms depending to the respective active substances. In particular, in the case where the active substance includes Hf 18, the nanostructure of the present application can be successfully used in treatment of infections related to Gram positive and Gram-negative AMR and MDR bacteria.
Brief description of the Figure
The present invention is exemplified with the appended drawing briefly described below; said example merely an exemplary implementation of the present invention, without delimiting the other possible ways of implementation and general functions that provide solution to the related technical problem.
Fig.l is a schematic section view of a nanostructure according to the present invention.
Detailed Description of the Invention
Referring to the drawing briefly described above, the present invention is hereinbelow described in detail.
The present invention proposes one or more nanostructures (100) comprising one or more nanocarriers (10) loaded with one or more active substances (11), an outer surface of said nanocarriers (10) being provided with one or more one or more targeting means (12). The nanostructure (100) comprises Peptide 6.2 (abbreviated as P6.2) as targeting means (12), and the nanostructure (100) can further comprise one or more different targeting means (12) other than Peptide 6.2. Thanks to the fact that the surface thereof is provided with Peptide 6.2, the nanostructure (100) according to the present invention enables a high- selectivity targeting of bacteria that are suitable for being targeted with Peptide 6.2.
In a preferred embodiment according to the present invention, a building material of the nanocarrier (10) can comprise one or more materials selected from the list consisting of one or more polymers, one or more lipids, one or more proteins, and one or more carbohydrates.
In a preferred embodiment of the invention, the active substance (11) can comprise one or more substances selected from the list consisting of peptide Hf-18, and/or one or more antibiotics and/or one or more antimicrobial substances. The case where the active substance (11) includes one or more antibiotics and/or one or more antimicrobial substances shall be considered as that said one or more substances are different substances with regard to each other. As an example to such case, the active substance (11) can include a content in accordance with any of the following alternatives (hence, throughout the present specification, the alternative "more" in the wording "one or more" shall be considered in this manner):
- a plurality of antibiotics that are different from each other, or
- a plurality of antimicrobial substances that are different from each other, or
- an antimicrobial substance along with an antibiotic, or
- an antimicrobial substance along with a plurality of antibiotics that are different from each other, or
- an antibiotic along with one or more antimicrobial substances that are different from each other, or
- a plurality of antibiotics that are different from each other, along with one or more antimicrobial substances that are different from each other.
In a more preferred embodiment according to the present invention, the active substance (11) includes peptide Hf-18. The synergistic effect of the targeting means (12) that includes Peptide 6.2, and peptide Hf-18 as the active substance (11), a relatively high extent of biological activity can be achieved at relatively low concentrations of antimicrobial substance. Peptide Hf-18 can be also referred to as HF-18.
In a preferred embodiment of the invention, said one or more targeting means (12) is Peptide 6.2. Particularly in the case where the active substance (11) includes Hf-18 or more preferably where the active substance (11) is Hf-18, said synergistic effect becomes more significant; thus, at relatively low antimicrobial substance concentrations, a relatively high extent of biological activity can be achieved more effectively.
The present invention further proposes a method for obtaining a nanostructure (100) that is loaded with one or more active substances (11), and that includes a nanocarrier (10) which has an outer surface that is provided with one or more targeting means (12) that includes Peptide 6.2. The method according to the present invention comprises the following steps of: a) loading of a building material that includes one or more materials selected from the list consisting of one or more polymers, one or more lipids, one or more proteins, and one or more carbohydrates, with one or more active substances (11) selected from the list consisting of Peptide Hf-18, one or more antibiotics and one or more antimicrobial substances; thereby obtaining one or more nanocarriers (10) that is loaded with one or more active substances (11); b) binding of one or more targeting means (12) that include Peptide 6.2, onto a surface of the one or more nanocarriers (10) that are obtained in the step a.
The loading operation in the step a of the method according to the present invention enables the protection of the active substance (11) against enzymatic degradation; thus prolongs the half life and enables controlled release. On the other hand, the step b of the method according to the present invention enables the provision of a nanostructure (100) that has high selectivity against bacteria that are suitable for being targeted by Peptide 6.2.
The loading operation in the step a of the method according to the present invention can be performed with any of suitable methods such as single-emulsion, double-emulsion, emulsification, desolvation or ionic gelation. In a preferred version of the method according to the present invention, the loading operation in the step a is implemented with a process selected from single-emulsion, double-emulsion, emulsification, desolvation or ionic gelation.
In the binding operation in the step b can is performed with chemical binding via bioconjugation, or with physical binding via electrostatic interaction.
In a preferred version of the method according to the present invention, in the step a, one or more active substances (11) that include Peptide Hf-18 is loaded onto the building material. Hence, the one or more active substance (11) in such implementation includes Peptide Hf-18. Thus, a nanostructure (100) is obtained in which the active substance (11) includes Hf-18 and the targeting means (12) includes Peptide 6.2.
In a preferred version of the method according to the present invention, in the step b, Peptide 6.2 is attached onto a surface of the one or more nanocarriers (10) that are obtained in the step a. Hence, in such version, a nanostructure (100) is obtained in which the targeting means (12) is Peptide 6.2. In a more preferred version of the method according to the present invention, in the step а, the building material is loaded with one or more active substances (11) that include Peptide Hf-18; and in the step b, Peptide 6.2 is attached onto a surface of the one or more nanocarriers (10) obtained in the step a. Accordingly, said one or more active substances (11) include Peptide Hf-18 and the targeting means (12) is Peptide 6.2. As a result, a nanostructure (100) is obtained in which the active substance (11) includes Hf-18 and the targeting means (12) is Peptide 6.2; thereby enabling the synergistic effect of Hf- 18 peptide and Peptide 6.2 in combination with each other.
Within the context of the present application, the term "nanostructure" can also be referred to as "nanoformulation" or "nanoparticular carrier system" To the best of our knowledge, in the prior art, no nanostructure is encountered that is targeted with Peptide б.2 onto bacterial surfaces, and no nanoformulation is encountered in which Peptide Hf-18 is loaded into a nanocarrier for controlled release.
The building material of the nanocarrier (10) can include one or more components selected from polymers, lipids, proteins and carbohydrates. One or more active substances (11) selected from Peptide Hf-18, antibiotics or antimicrobial molecules can be loaded onto said building material. One or more nanocarriers (10) that are loaded with one or more active substances (11) can be thus obtained.
The targeting means (12) can include Peptide 6.2 or can be Peptide 6.2. By konjugation of the targeting means (12) onto the surface of the nanocarrier (10); a nanoformulation or in other words, a nanostructure (100) can be obtained, which is targeted with Peptide 6.2 and which includes nanocarrier (10) loaded with active substance (11).
The active substance (11) can include one or more substances with antibiotic and/or antibacterial properties suitable for causing lysis of bacteria by showing antimicrobial behavior towards targeted bacteria; preferably including Peptide Hf-18. Targeting means (12) chemically attached onto nanocarrier (10) surface provides targeting towards bacteria, and thus enabling the one or more active substances (11) to directly access to the targeted bacteria.
EXAMPLE-1: nanocarrier building material
Building material of the nanocarrier (10) enhances biobiodistribution and pharmacocinetics of the active substance (11) by enhancing the protection thereof against biological degradation; further enhances the solubility, stability and transportation to the target area by modification of physicochemical properties such as surface and shape; decreases its toxicity and enables a prolonged circulation thanks to the controlled release. Said building material can include, but not limited to, one or more materials that can be selected from the following:
- for instance, one or more homopolymers that can be selected from PCL and PLA;
- for instance, one or more block copolymers that can be selected from PLGA and PEG-PCL;
- for instance, one or more lipids that can be selected from stearic acid, palmitic acid and phosphatidylcholine;
- for instance, one or more proteins that can be selected from HSA, BSA and ovalbumin;
- for instance, one or more carbohydrates that can be selected from chitosan, dextran, hyaluronic acid, cellulose and alginate.
EXAMPLE-2: active substance
The active substance (11) can be selected from antibiotics or antimicrobial substances that are used in treatment of infectious diseases and that shows a lytic effect on microorganisms or growth inhibition on microorganisms. Hf-18 can be selected as a nonlimiting example to active substance (11). Hf-18 does not show a significant haemolytic effect on mammalian cells, is not cytotoxic, yet has a strong antimicrobial activity. In pg/mL, MIC values of Peptide Hf-18 against G+ S. aureus, G' E. coliaxx G' P. aeruginosa are 8, 8 and 16, respectively. EXAMPLE-3: targeting means
For selectively targeting bacterial cells, Peptide 6.2 can be used as the targeting means (12) in view of that the Peptide 6.2 has a higher affinity and rapid binding kinetics against bacterial membranes that include phosphatidylglycerol (PG), thereby having a high antibacterial activity and low hemolytic activity. As antibacterial activity, in pg/mL, MIC value of Peptid 6.2 against each of G+ S. aureus, G' E. coiiax G' P. aeruginosa is 32.
Hf-18 is a peptide that is disclosed in the following exemplary source. The sequence of Hf- 18 can be synthesized based on the content that is available in the reference publication provided below:
- Jiang, M. et al. (2020), "An active domain HF-18 derived from hagfish intestinal peptide effectively inhibited drug-resistant bacteria in vitro/vivo" Biochemical Pharmacology, 172, 113746 (DOI: 10.1016/j.bcp.2019.113746).
Sequence listing of HF- 18 is as follows:
Gly-Phe-Phe-Lys-Lys-Ala-Trp-Arg-Lys- Val-Lys-Lys-Ala-Phe-Arg-Arg-Val-Leu GFFKKAWRKVKKAFRRVL
Peptide 6.2 is a peptide that is disclosed in the following exemplary sources. The sequence of Peptide 6.2 can be synthesized based on contents available in any of the reference publications provided below:
- Mar nez, M. et al., (2020), Antibacterial, anti-biofilm and in vivo activities of the antimicrobial peptides P5 and P6. 2. Microbial Pathogenesis, 139, 103886 (DOI: 10.1016/j.micpath.2019.103886);
- Maturana, P., et al. (2017), Lipid selectivity in novel antimicrobial peptides: implication on antimicrobial and hemolytic activity. Colloids and Surfaces B: Biointerfaces, 153, 152-159 (DOI: 10.1016/j.colsurfb.2017.02.003);
- Del Cogliano, M. E. et al., (2017). Cationic antimicrobial peptides inactivate shiga toxin-encoding bacteriophages. Frontiers in chemistry, 5, 122 (DOI: 10.3389/fchem.2017.00122).
Sequence listing of Peptid 6.2 is as follows: Gly-Leu-Leu-Arg-Lys-T rp-Gly-Lys-Lys-T rp-Lys-Glu-Phe-Leu-Arg-Arg-Val-T rp-Lys GLLRKWGKKWKEFLRRVWK
Hf-18 and Peptide 6.2 can be produced by a person that is skilled in peptid synthesis within the professional specialized knowledge, starting from the respective sequence listings; or commercially obtained from the market, prepared in accordance with the latter principle. Production methods of Hf-18 and Peptide 6.2 mentioned in the present application are exemplified below.
Example 1: production of Hf-18 and Peptide 6.2
Peptide 6.2 that shows a higher affinity against procariotic membranes rather than eucariotic membranes (Mar nez et al., 2020, Antibacterial, anti-biofilm and in vivo activities of the antimicrobial peptides P5 and P6. 2. Microbial Pathogenesis, 139, 103886 (DOI: 10.1016/j.micpath.2019.103886)) and Hf-18 that has antibacterial effect (Jiang et al., 2020, "An active domain HF-18 derived from hagfish intestinal peptide effectively inhibited drug-resistant bacteria in vitro/vivo" Biochemical Pharmacology, 172, 113746 (DOI: 10.1016/j.bcp.2019.113746)) are synthesized using microwave assisted solid phase peptide synthesis method (T Acar, PP Arayici, B dear, M Karahan, Z Mustafaeva, Synthesis, characterization and lipophilicity study of Brucella abortus' immunogenic peptide sequence that can be used in the future vaccination studies, International Journal of Peptide Research and Therapeutics 25 (3), 911-918, 2019, DOI: 10.1007/S10989-018- 9739-0) (Derman S., Kizilbey K., Mansuroglu B., Mustafaeva Z., Synthesis and characterization of Canine parvovirus (CPV) VP2 W-7L-20 synthetic peptide for synthetic vaccine, Fresenius Environmental Bulletin 23(2A): 558-566, 2014). The synthesis can be performed in a peptide synthesis device, for instance, based on Fmoc chemistry. As a peptide synthesis devices, a commercially available microwave assisted solid phase peptide synthesis device can be used; for instance, one supplied by the company CEM Liberty.
In this technique, a growing peptide or oligomer chain is attached onto a solid phase /resin, and remains attached on said resin throughout the synthesis (ZO Ozdemir, Z Mustafaeva Akdeste, Development of polyelectrolyte based bioconjugates using with synthetic viral peptides Sigma 29, 65-89, availed online on 10 October 2021 at the following address: https://eds.yildiz.edu.tr/AjaxTool/GetArticleByPublishedArticleId?
PublishedArtideId=1861). Cease of protection (Deprotection), activation of carboxyl group (Activation) and formation of peptide bond (Coupling) are three basic consecutive steps of the peptide synthesis, that is followed by the separation of the synthesized peptide from the solid phase (Cleavage). The steps that are implemented in this technique can be briefly described as follows:
- Necessary amounts of amino acids (and other chemicals that can be used in accordance with general knowledge in the related technical field) for the peptide sequences to be synthesized can be calculated using e.g., PepDriver 2.6.6 software in the peptide synthesis device.
- For the HF-18 peptide and Peptide 6.2 at the carboxy end of the peptide sequences that are to be synthesized, resins that bear Leu and Lys amino acids are to be used, respectively. Such resin can be, e.g., swollen for 3 hours in DMF (dimethyl formamide), to enable the exposure of active ends on the resin that is a polymeric particle, is enabled.
- a 20% solution of piperidine in DMF (dimethyl formamide) can be prepared as a deprotector (an agent for breaking side group protector); a mixture of HBTU/HOBt (0- (Benzotriazole-l-yl) - N,N,N',N'- tetramethyluronium hexafluorophosphate) I N- Hydroksybenzotriazole) can be prepared as an activator (activating agent); and a mixture of DIEA/NMP (Diisopropyl ethyl amine I N-Methyl-2-pyrrolidone) can be prepared as an activator base (activating base).
- The synthesis can be initiated by starting a respective program over the device. When the microwave assisted solid phase peptide synthesis process is complete, the synthesis is to be brought to an end by collecting the peptide chain that is attached to resin, said peptide that is attached to resin is washed with one or more suitable chemicals such as dichloro methane (DCM), then filtered and dried.
- The synthesized peptide chain is separated from the resin by a cleavage cocktail such as TFA (trifluoro acetic acid) I EDT (1,2 - ethane dithiol) / Thioanisol I H2O (090/2.5/2.5/5) v/v. For the cleaving operation, resin loaded peptide is brought into contact with cleavage cocktail; this contacting operation can be performed by gently shaking in the cocktail, for e.g. three hours. After said contacting, the cocktail is filtered and thereby a filtrate is obtained. The filtrate is evaporated in an evaporator, thus a gelled solution is obtained. The gelled solution can be transferred into a centrifuge tube, cold diethyl ether can be added thereonto, then subjected to centrifuge, such that the peptide is precipitated in the form of a white powder. The precipitated peptide can be dried for storing; said drying operation can be performed at a pressure under 1 atmosphere (e.g., under vacuum). The storing operation can be performed at a low temperature, for instance at -40 °C. The dried peptide can be named as a "crude peptide".
- In characterization of the raw peptide, molecular weight determination can be performed by using an LC-MS system (such as Shimadzu LC-MS 2010 EV) that includes an electrospray ionization (ESI) probe and that performs separation by reverse phase chromatography (RP-HPLC) (for instance, using a suitable gradient elution). At processing the samples, for instance, Teknokroma Tracer Exel 120 ODS-A (5pm / 20 x 0.21cm) can be used as column. For instance, A [in accordance with peptidine solubility, (water, 0.1% (v/v) TFA) or (water, 0.1% (v/v) formic acid)] and B [in accordance with peptidine solubility, (Acetonitrile, 0.1% (v/v) TFA)) or (Acetonitrile, 0.1% (v/v) formic acid)] can be used as gradient elution; 0.2 mU/min can be selected as flow rate. ESI can be utilized in positive ion mode within a range between 200 - 2000 m/z. Capillary temperature can be set at 250 °C. Nebulisator gas (N2) flow rate can be e.g., 1.5 L / min.
- Preferably, upon specifying the gradient that provides a most favourable separation (for the elution program, e.g.: for a duration of up to 5 minutes, 20% of B; for a duration between 5 and 15 minutes, 20 - 40% of B; for a duration between 15 and 30 minutes, 40 - 80% of B; for a duration between 30 and 35 minutes, 80 - 20% of B), each peak fraction can be collected one by one using a preparative HPLC device. If the separation of peptide peaks is not in a sufficient extent in the processed gradient elution, peptide peaks can be obtained by applying a different gradient. An organic phase that is present in the solution can be partially removed, e.g., using a rotary evaporator. After said step of removal, a step of drying can be applied, e.g., using a lyophilization device; thereby pure peptide is obtained in the form of powder. For purification, for instance, in a preparative HPLC (RP-HPLC, SPD-M20A, FRC-10A, LC-8A, CBM-20A, Shimadzu, Tokyo, Japan) that is provided with a Shim-pack PRC-ODS column (20 mm x 25 cm) and an UV-PDA detector, a gradient elution can be employed [such as, in accordance with peptidine solubility (water, 0.1% (v/v) TFA) or (water, 0.1% (v/v) formic acid)] and B [in accordance with peptidine solubility (Acetonitrile, 0.1% (v/v) TFA)) or (Acetonitrile, 0.1% (v/v) formic acid)]. Purified peptide is obtained as a result of the purification step. The purified peptide can be lyophilized and stored for instance at -40 °C (T Acar, PP Arayici, B Ucar, M Karahan, Z Mustafaeva, Synthesis, characterization and lipophilicity study of Brucella abortus' immunogenic peptide sequence that can be used in the future vaccination studies, International Journal of Peptide Research and Therapeutics 25 (3), 911-918, 2019, DOI: 10.1007/S10989-018-9739-0); (Derman S., Kizilbey K., Mansuroglu B., Mustafaeva Z., Synthesis and characterization of Canine parvovirus (CPV) VP2 W-7L-20 synthetic peptide for synthetic vaccine, Fresenius Environmental Bulletin 23(2A): 558-566, 2014).
The development that is the subject of the present application is industrially applicable. The nanostructure (100) obtained according to the present application sets an innovative alternative to antibiotics group drugs that are already used in treatment of bacterial infections. The development of the present application enables a nanoformulation or nanostructure (100) system that targets the surfaces of bacteria that can be targeted by Peptide 6.2; the system includes an antimicrobial compound and provides controlled release of said compound as an active substance (11). As an alternative to the antibiotics, the system that can be obtained in accordance with the present invention has the potential of use in modern exemplary commercial usage forms; as a drug, in any of the forms of injection vial, tablet or capsule; or as a final product, in any of the forms of pomade, cream or granule. Hence, the present invention enables the following drugs:
- a drug in the form of injection vials, that includes the nanostructure (100) according to the present invention;
- a drug in the form of tablets, that includes the nanostructure (100) according to the present invention;
- a drug in the form of capsules, that includes the nanostructure (100) according to the present invention; - a drug in the form of pomade, that includes the nanostructure (100) according to the present invention;
- a drug in the form of cream, that includes the nanostructure (100) according to the present invention; - a drug in the form of granules, that includes the nanostructure (100) according to the present invention.
Reference signs:
10 nanocarrier 11 active substance
12 targeting means
100 nanostructure

Claims

CLAIMS One or more nanostructures (100) that are loaded with one or more active substances (11), said nanostructures (100) comprising one or more nanocarriers
(10), an outer surface of said nanocarriers (10) being provided with one or more targeting means (12); characterized in that said one or more targeting means (12) comprise Peptide 6.2. Nanostructure (100) according to the claim 1, wherein a building material of the nanocarrier (10) includes one or more materials selected from the list consisting of one or more polymers, one or more lipids, one or more proteins and one or more carbohydrates. Nanostructure (100) according to any of claims 1 or 2, wherein the active substance
(11) includes one or more substances selected from peptide Hf 18, one or more antibiotics and one or more antimicrobial molecules. Nanostructure (100) according to claim 3, wherein the active substance (11) includes peptide Hf 18. Nanostructure (100) according to any of claims 1 to 4, wherein said one or more targeting means (12) is Peptide 6.2. Nanostructure (100) according to claim 4, wherein said one or more targeting means
(12) is Peptide 6.2. A drug in the form of injection vial, comprising the nanostructure (100) according to any of claims 1 to 6. A drug in the form of tablets, comprising the nanostructure (100) according to any of claims 1 to 6. 9. A drug in the form of capsules, comprising the nanostructure (100) according to any of claims 1 to 6.
10. A drug in the form of pomade, comprising the nanostructure (100) according to any of claims 1 to 6.
11. A drug in the form of cream, comprising the nanostructure (100) according to any of claims 1 to 6.
12. A drug in the form of granules, comprising the nanostructure (100) according to any of claims 1 to 6.
13. A method for obtaining a nanostructure (100) that is loaded with one or more active substances (11), said nanostructure (100) comprising one or more nanocarriers (10), an outer surface of said nanocarriers (10) being provided with one or more targeting means (12) comprising Peptide 6.2; wherein the method includes the following steps of: a) loading one or more active substances (11) selected from peptide Hf 18, one or more antibiotics and one or more antimicrobial molecules, onto a building material that includes one or more materials selected from the list comprising one or more polymers, one or more lipids, one or more proteins and one or more carbohydrates, for obtaining one or more nanocarriers (10) that are loaded with one or more active substances (11); b) binding of one or more targeting means (12) that include Peptide 6.2, onto a surface of the one or more nanocarriers (10) that are obtained in the step a.
14. Method according to claim 13, wherein the loading in the step a is performed using a process selected from single-emulsion, double-emulsion, emulsification, desolvation or ionic gelation. -18-
15. Method according to any of claims 13 or 14, wherein the binding in the step b is performed with chemical binding via bioconjugation, or with physical binding via electrostatic interaction.
16. Method according to any of claims 13 to 15, wherein in the step a, the building material is loaded with one or more active substances (11) that include peptide Hf
18.
17. Method according to any of claims 13 to 16, wherein in the step b, Peptide 6.2 is attached onto a surface of the one or more nanocarriers (10) that are obtained in the step a. 18. Method according to claim 16, wherein in the step b, Peptide 6.2 is attached onto a surface of the one or more nanocarriers (10) that are obtained in the step a.
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