Human milk oligosaccharides (HMOs) for the prevention of infections on implants

Abstract

The present invention relates to human milk oligosaccharides (HMOs) for use in the prevention and / or treatment of infections on implants. These oligosaccharides derived from breast milk are particularly suitable and effective as an allergen-free antibacterial agent for the intended use.

Description

[0001] The invention relates to human milk oligosaccharides (HMOs) intended for use in preventing infections on implants. These oligosaccharides derived from breast milk are particularly suitable and effective as an allergen-free antibacterial agent for the intended use.

[0002] Breast milk contains fats, proteins, and carbohydrates as its main components, with mature breast milk typically having a low protein and high carbohydrate content. In addition to all essential nutrients, breast milk also contains growth factors, hormones, micronutrients, vitamins, enzymes, and trace elements necessary for the infant's growth. The nutrients in breast milk are characterized by low allergenicity. This is also described, for example, in patent application WO 2022 / 200526 A1 of the current applicant.

[0003] Implants are used for reconstruction in various medical fields. They are generally made of artificial materials such as metals, ceramics, or carbon. Bacteria can easily adhere to these materials and form a bacterial biofilm, which can lead to inflammatory reactions and, subsequently, to implant dysfunction and significant impairment for the patient. Periprosthetic (PPI) and implant-associated infections remain a dreaded complication despite highly standardized countermeasures. With multiple revisions, the risk of an unmanageable situation increases dramatically.

[0004] The problem of implant-associated infections can be combated, for example, by coating the implant with silver. Furthermore, it is known to subject metallic implants to anodizing, acid treatment, and iodine treatment to obtain an antibacterial implant, as described, for example, in EP 3 586 883 B1. In addition, certain antibacterial polymers are known that can be applied to implants, as described in EP 3 153 186 A1.

[0005] The current inventor has now surprisingly discovered that oligosaccharides derived from breast milk are particularly suitable for preventing and / or treating infections of implants and prostheses, as well as for obtaining an antibacterial coating for implants.

[0006] The present invention therefore relates to oligosaccharides from human breast milk, which are intended to be used for the treatment and / or prevention of infections of implants and prostheses.

[0007] These human milk oligosaccharides are obtained primarily by collecting and pooling breast milk, i.e., mixing milk from different women, and then separating it into fat, protein, and sugar / carbohydrate fractions, from which the oligosaccharides can then be concentrated. This fraction can then be dried, for example, by freeze-drying, spray-drying, or roller-drying to make it more shelf-stable.

[0008] A possible method for separating the different components of breast milk is described in WO 2022 / 200526 A1 of the current applicant.

[0009] The sugar and carbohydrate content of human breast milk consists primarily of lactose and oligosaccharides made up of monosaccharides such as glucose, galactose, N-acetylglucosamine, fucose, and sialic acid. Currently, approximately 200 different oligosaccharides are known to exist in human milk. The ratio of lactose to oligosaccharides is approximately 5:1, meaning about 80% by weight of lactose to about 20% by weight of oligosaccharides.

[0010] From this sugar or carbohydrate content, the HMOs can then be further enriched by removing the lactose.

[0011] For the present invention, preferably 5 to 15 wt.%, in particular 7 to 13 wt.%, and most preferably 9 to 11 wt.% purified HMOs from human breast milk are used in a suitable liquid medium. It is further preferred that the HMOs are used in the form of a powder or a paste.

[0012] The implants to be treated or coated with HMOs are primarily selected from artificial joints, especially hip and knee joints, and dental implants. Other orthopedic implants suitable for this treatment include ankle joint endoprostheses, arthrodesis nails, spinal surgical implants such as internal fixators (spondylodesis), trauma surgery implants, intramedullary nails, angle-stable and non-angle-stable plate osteosyntheses, wires, cerclage wires, and screws, as well as cardiological implants such as pacemakers, defibrillators, and ports.

[0013] These implants preferably feature surfaces made of metal, ceramic, or carbon materials. Plastic materials such as polyethylene can also be used for implants.

[0014] A key aspect of the invention therefore relates to the use of human milk oligosaccharides to prevent bacterial infections on implants.

[0015] Finally, an implant coated or treated with antibacterial human milk oligosaccharides as described above represents a further aspect of the present invention. For this purpose, a paste or powder containing HMOs is applied to the implant.

[0016] The invention will now be described with reference to particularly preferred embodiments, which are intended to illustrate the invention but not to limit it.

[0017] The attached Figs. Figures 1 to 4 show growth curves of S. aureus at different concentrations of HMOs. Example 1: Production of a milk powder using human milk oligosaccharides

[0018] As already described in the above-mentioned WO 2022 / 200526 A1, a carbohydrate powder is obtained by centrifuging pooled breast milk, removing the fats, and producing cream and whey with proteins, which are filtered so that a carbohydrate powder remains as the permeate.

[0019] The saturation of human milk oligosaccharides was then achieved by solid-phase extraction (FPE or SPE). First, the carbohydrates were absorbed onto a graphite structure. The interaction of lactose, the only disaccharide present in human breast milk, is not strong enough for proper absorption. The unabsorbed lactose was washed off with water. The absorbed oligosaccharides, which mostly contain the more complex structures, were released using organic solvents (ethanol or acetonitrile). Instead of the expensive standard graphite (PGC – porous graphitic carbon), simple graphite powder was used, which also yielded good results. This allowed for the enrichment of HMOs on a gram scale.

[0020] The exceptional effectiveness of this composition was demonstrated in the experiments described below. Example 2: Growth experiments with purified HMOs from breast milk in different concentrations

[0021] In the experiments described below, the antibacterial efficacy of HMOs against a bacterium commonly found in implant infections, Staphylococcus aureus (S. aureus, strain EDCC 5055).

[0022] Previous biofilm assays using titanium fragments had already shown a reduction in bacterial growth. Furthermore, a preliminary sterility test revealed that the HMO samples were initially sterile: Plating 200 µl of HMO solution onto a blood agar plate without sterile filtration showed only slight growth of approximately 4-8 clear, aqueous colonies after 5 days. The liquid medium only gradually became cloudy after 1 week at room temperature.

[0023] This provided the opportunity to test the HMO solution with a growth curve without sterile filtration to determine whether sterile filtration leads to a loss of HMOs (the cloudy original solution at all concentrations gradually clogs the sterile filter during filtration and is completely clear afterward) and whether this has an effect on growth. Such an experiment was not possible with the carbohydrate solution, as the powder was contaminated with a few microbes and led to strong bacterial growth during enrichment.

[0024] HMO concentrations: - Powder in 4 different concentrations of 5%, 7%, 9% and 11% of HMO milk powder, produced as described in Example 1 - Positive control: S. aureus EDCC 5055 - Measurement against TSB (tryptone soy broth medium) with (positive control) and without S. aureus (negative control)

[0025] 1. The HMO solutions with 5%, 7%, 9% and 11% in TSB were prepared as follows: - Amount in g of HMO milk powder for 6 ml: 5% 0.3g / 6ml 7% 0.42 g / 6 ml 9% 0.54g / 6ml 11% 0.66 g / 6 ml Weigh the powder into a sterile 50 ml Greiner tube using a cleaned (alcohol and water) spatula on a precision balance. (The powder is statically charged.) - Add 6 ml of TSB under the sterile workbench and vortex briefly. Then place the powder in the incubator for 30 minutes at 37°C (otherwise the powder was very difficult to dissolve) and subsequently vortex until all components were visibly dissolved. - 6 ml were prepared because a biofilm assay was also being performed in parallel.

[0026] 2. Setting up the TECAN solutions (see www.Tecan.com): - The day before, prepare an overnight culture from a blood agar plate with 20 ml TSB and S. aureus EDCC 5055, 37°C, 150 rpm - Preparation of biofilm solutions (HMO powder solutions with and without S. aureus, control): 6 ml of HMO solution / concentration (5, 7, 9, 11%) from the 50 ml Greiner tube is divided as follows: Pipette 2.5 ml into a fresh 5 ml Eppi-Cap (non-sterile filtered). Draw up 3.5 ml using a syringe and needles and press through one sterile filter (0.22 µm per concentration) and filter the HMO solution directly into a 5 ml cap (volume loss due to filtration). Pipette 3 ml of the resulting sterile HMO solution into a fresh 5 ml cap. - To prepare the 1:100 dilutions of the test solutions with S. aureus (for biofilm assay and Tecan), the following is pipetted in each case: Add 25 µl of S. aureus ÜNK (non-sterile filtered; pur=p) to 2.5 ml of HMOs. Add 30 µl of S. aureus ÜNK (sterilized filtered) to 3 ml of HMOs. Add 50 µl S. aureus ÜNK (positive control without HMOs) to 5 ml TSB. - all vortices Dividing the prepared samples for Biofilm assay in the 96-well plate as further described Growth curve in Tecan with the remaining volume (separate protocol)

[0027] For the growth curve in Tecan further: - 200 µl each of the test solutions and control solutions are pipetted 3 times into a 96 well Tecan microtiter plate according to a pipetting scheme. Column A1-H1: Negative control (blank) TSB only Columns A2-H2: empty Column A3-C3: Positive control S. aureus Column A4-C3: Positive control S. aureus Column A5-C5+D3: 5% HMO sterile filtered in TSB with S. aureus Column A5-C5: 5% HMO non-sterile filtered in TSB with S. aureus Column A5-C5+E3: 7% HMO sterile filtered in TSB with S. aureus Column A5-C5: 7% HMO non-sterile filtered in TSB with S. aureus Column A5-C5+F3: 9% HMO sterile filtered in TSB with S. aureus Column A5-C5: 9% HMO non-sterile filtered in TSB with S. aureus Columns A5-C5: 11% HMO sterile filtered in TSB with S. aureus Columns A5-C5: 11% HMO non-sterile filtered in TSB with S. aureus -Place the lid on and insert the microtiter plate directly into the Tecan device (Infinite 200Pro) and begin the measurement over 23:59 (highest value) hours.

[0028] Measurement conditions: Wavelength: 600nm Temperature: 37°C Kinetic interval: 20 min Duration 360 sec Evaluate the data after the run using an Excel file.

[0029] Tables 1 to 4 below show the values ​​obtained with the different HMO concentrations. Table 1 Time MW MW Blank S. aureus 1 S. aureus 2 5% HMO st 5% pure HMO 0,200 0,220 0,217 0,251 0,418 0,209 0,236 0,232 0,254 0,418 0,213 0,252 0,247 0,260 0,422 0,216 0,283 0,278 0,269 0,430 0,218 0,342 0,336 0,280 0,439 2 0,220 0,432 0,426 0,294 0,449 0,222 0,552 0,546 0,314 0,462 0,223 0,687 0,682 0,342 0,477 0,224 0,812 0,809 0,378 0,495 0,226 0,896 0,893 0,423 0,517 4 0,227 0,957 0,954 0,475 0,543 0,228 1,007 1,004 0,536 0,571 0,229 1,045 1,042 0,604 0,604 0,230 1,073 1,070 0,679 0,640 0,231 1,089 1,086 0,757 0,681 6 0,232 1,104 1,101 0,837 0,723 0,233 1,117 1,114 0,918 0,768 0,235 1,128 1,125 0,997 0,812 0,236 1,137 1,133 1,072 0,857 8 0,237 1,143 1,140 1,139 0,899 0,237 1,148 1,145 1,195 0,941 0,238 1,154 1,150 1,242 0,983 0,239 1,160 1,156 1,281 1,025 0,240 1,167 1,163 1,315 1,067 10 0,241 1,175 1,171 1,344 1,106 0,241 1,185 1, 181 1,370 1,142 0,242 1,195 1,191 1,388 1,174 0,242 1,206 1,202 1,400 1,203 0,243 1,218 1,214 1,410 1,228 12 0,243 1,231 1,227 1,419 1,251 0,244 1,246 1,242 1,430 1,271 0,245 1,260 1,256 1,440 1,290 0,245 1,275 1,270 1,452 1,309 0,245 1,289 1,285 1,463 1,324 14 0,245 1,305 1,301 1,474 1,340 0,245 1,321 1,316 1,485 1,353 0,246 1, 336 1,331 1,494 1,366 0,247 1,351 1,347 1,503 1,379 0,247 1,365 1,360 1,511 1,392 16 0,247 1,376 1,373 1,520 1,406 0,247 1,385 1,382 1,525 1,418 0,247 1,392 1,389 1,536 1,429 0,248 1,401 1,397 1,545 1,439 0,248 1,411 1,406 1,554 1,450 19 0,248 1,420 1,415 1,562 1,460 0,249 1,430 1,424 1,571 1,469 0,249 1,438 1,432 1,579 1,479 0,250 1,444 1,439 1,586 1,488 0,250 1,450 1,444 1,593 1,496 21 0,251 1,454 1,449 1,599 1,504 0,250 1,459 1,452 1,604 1,511 0,251 1,463 1,457 1,610 1,518 0,251 1,467 1,461 1,616 1,526 0,252 1,471 1,464 1,620 1,533 23 0,252 1,474 1,468 1,623 1,539 Table 2 Blank S. aureus 1 7% HMO st 7% pure HMO 0,200 0,220 0,252 0,541 0,209 0,236 0,254 0,547 0,213 0,252 0,259 0,560 0,216 0,283 0,266 0,572 0,218 0,342 0,275 0,584 2 0,220 0,432 0,284 0,595 0,222 0,552 0,296 0,605 0,223 0,687 0,312 0,615 0,224 0,812 0,334 0,627 0,226 0,896 0,359 0,641 4 0,227 0,9578 0,389 0,658 0,228 1,007 0,423 0,676 0,229 1,045 0,460 0,696 0,230 1,073 0,502 0,717 0,231 1,089 0,548 0,741 6 0,232 1,104 0,598 0,766 0,233 1,117 0,651 0,790 0,235 1,128 0,707 0,815 0,236 1,137 0,766 0,841 8 0,237 1,143 0,823 0,867 0,237 1,148 0,880 0,894 0,238 1,154 0,935 0,920 0,239 1,160 0,989 0,947 0,240 1,167 1,039 0,973 10 0,241 1,175 1,085 0,996 0,241 1,185 1,126 1,020 0,242 1,195 1,161 1,043 0,242 1,206 1,190 1,067 0,243 1,218 1,214 1,092 12 0,243 1,231 1,235 1,115 0,244 1,246 1,254 1,139 0,245 1,260 1,269 1,161 0,245 1,275 1,283 1,183 0,245 1,289 1,295 1,202 14 0,245 1,305 1,305 1,219 0,245 1,321 1,317 1,235 0,246 1,336 1,326 1,249 0,247 1,351 1,332 1,262 0,247 1,365 1,337 1,274 16 0,247 1,376 1,341 1,285 0,247 1,385 1,345 1,296 0,247 1,392 1,348 1,305 0,248 1,401 1,350 1,314 0,248 1,411 1,353 1,324 19 0,248 1,420 1,355 1,333 0,249 1,430 1,359 1,342 0,249 1,438 1,362 1,350 0,250 1,444 1,364 1,358 0,250 1,450 1,367 1,365 21 0,251 1,454 1,369 1,371 0,250 1,459 1,371 1,377 0,251 1,463 1,374 1,383 0,251 1,467 1,377 1,388 0,252 1,471 1,380 1,393 23 0,252 1,474 1,382 1,398 Table 3 Blank S. aureus 1 9% HMO st 9% pure HMO 0 0,200 0,220 0,255 0,609 0,209 0,236 0,257 0,620 0,213 0,252 0,261 0,637 0,216 0,283 0,268 0,651 0,218 0,342 0,275 0,664 2 0,220 0,432 0,282 0,676 0,222 0,552 0,291 0,687 0,223 0,687 0,303 0,697 0,224 0,812 0,317 0,707 0,226 0,896 0,333 0,718 4 0,227 0,957 0,353 0,730 0,228 1,007 0,376 0,744 0,229 1,045 0,402 0,759 0,230 1,073 0,429 0,775 0,231 1,089 0,458 0,792 6 0,232 1,104 0,490 0,810 0,233 1,117 0,525 0,829 0,235 1,128 0,562 0,847 0,236 1,137 0,601 0,866 8 0,237 1,143 0,642 0,884 0,237 1,148 0,685 0,903 0,238 1,154 0,730 0,922 0,239 1,160 0,775 0,941 0,240 1,167 0,819 0,960 10 0,241 1,175 0,861 0,978 0,241 1,185 0,901 0,997 0,242 1,195 0,941 1,016 0,242 1,206 0,979 1,035 0,243 1,218 1,014 1,053 12 0,243 1,231 1,046 1,069 0,244 1,246 1,076 1,086 0,245 1,260 1,100 1,102 0,245 1,275 1,121 1,118 0,245 1,289 1,137 1,134 14 0,245 1,305 1,151 1,150 0,245 1,321 1,162 1,167 0,246 1,336 1,171 1,182 0,247 1,351 1,179 1,198 0,247 1,365 1,186 1,212 16 0,247 1,376 1,193 1,227 0,247 1,385 1,198 1,240 0,247 1,392 1,203 1,252 0,248 1,401 1,208 1,262 0,248 1,411 1,212 1,272 19 0,248 1,420 1,216 1,280 0,249 1,430 1,220 1,288 0,249 1,438 1,225 1,296 0,250 1,444 1,228 1,303 0,250 1,450 1,232 1,310 21 0,251 1,454 1,236 1,317 0,250 1,459 1,239 1,322 0,251 1,463 1,243 1,329 0,251 1,467 1,246 1,335 0,252 1,471 1,250 1,342 23 0,252 1,474 1,253 1,348 Table 4 Blank S. aureus 1 11% HMO st 11% HMO pur 1 0,200 0,220 0,260 0,706 0,209 0,236 0,262 0,723 0,213 0,252 0,267 0,743 0,216 0,283 0,272 0,759 0,218 0,342 0,278 0,772 2 0,220 0,432 0,286 0,785 0,222 0,552 0,293 0,796 0,223 0,687 0,303 0,805 0,224 0,812 0,313 0,814 0,226 0,896 0,324 0,822 4 0,227 0,957 0,337 0,829 0,228 1,007 0,351 0,840 0,229 1,045 0,368 0,851 0,230 1,073 0,385 0,862 0,231 1,089 0,405 0,873 6 0,232 1,104 0,426 0,883 0,233 1,117 0,447 0,894 0,235 1,128 0,469 0,905 0,236 1,137 0,493 0,916 8 0,237 1,143 0,517 0,926 0,237 1,148 0,542 0,937 0,238 1,154 0,567 0,947 0,239 1,160 0,595 0,957 0,240 1,167 0,622 0,965 10 0,241 1,175 0,649 0,972 0,241 1,185 0,677 0,978 0,242 1,195 0,706 0,985 0,242 1,206 0,735 0,991 0,243 1,218 0,765 0,996 12 0,243 1,231 0,793 1,000 0,244 1,246 0,819 1,004 0,245 1,260 0,845 1,008 0,245 1,275 0,871 1,009 0,245 1,289 0,895 1,011 14 0,245 1,305 0,920 1,013 0,245 1,321 0,944 1,014 0,246 1,336 0,965 1,014 0,247 1,351 0,986 1,014 0,247 1,365 1,005 1,013 16 0,247 1,376 1,023 1,014 0,247 1,385 1,039 1,015 0,247 1,392 1,052 1,015 0,248 1,401 1,064 1,016 0,248 1411 1,074 1,017 19 0,248 1,420 1,082 1,018 0,249 1,430 1,090 1,020 0,249 1,438 1,098 1,023 0,250 1,444 1,104 1,025 0,250 1,450 1,110 1,028 21 0,251 1,454 1,115 1,030 0,250 1,459 1,120 1,033 0,251 1,463 1,125 1,035 0,251 1,467 1,131 1,038 0,252 1,471 1,136 1,039 23 0,252 1,474 1,141 1,041

[0030] The corresponding growth curves of S. aureus are in the Figs. Shown 1 to 4.

[0031] The experiments carried out show a clear effectiveness of HMOs, especially in concentrations of 7% to 11%, against bacteria present on implants, which makes them particularly suitable as well-tolerated and highly effective antibacterial agents against implant infections. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] WO 2022 / 200526 A1 [0002, 0008, 0018] EP 3 586 883 B1

[0004] EP 3 153 186 A1

[0004]

Claims

[1] Human milk oligosaccharides (HMOs) for use in the prevention of bacterial infections on implants. [2] Human milk oligosaccharides according to claim 1, characterized by that these are obtained from the carbohydrate content of pooled breast milk. [3] Human milk oligosaccharides according to claim 1 or 2, characterized by that these are used in a concentration of 5 to 15 wt.%, in particular 7 to 13 wt.%, especially preferably 9 to 11 wt.% HMOs from human breast milk. [4] Human milk oligosaccharides according to any one of claims 1 to 3, characterized by that these are in the form of a powder or a paste. [5] Human milk oligosaccharides according to any one of claims 1 to 4, characterized by that the implants are selected from artificial joints, especially hip and knee joints, and dental implants. [6] Human milk oligosaccharides according to claim 5, characterized bythat the implants have surfaces made of metal, ceramic or carbon materials. [7] Use of human milk oligosaccharides for the prevention and / or treatment of bacterial infections on implants. [8] Implant treated or coated with antibacterial human milk oligosaccharides according to any one of claims 1 to 6.

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