SAME Technical Field of the Invention

The present invention relates to a method and device for separating unwanted chemicals and their metabolites from breast milk. More particularly, the invention pertains to a novel method and device for the elimination of unsafe compounds found in breast milk by using of nanosurfaces comprising antibodies targeting the chemical compounds whereby removal of these compounds from the breast milk is visualized by the user.

Background of the Invention

Milk is the primary source of nutrition for newborns before they are able to eat and digest other foods. An infant is generally needed to be fed with breast milk at least for 6 months but supplemented breastfeeding is recommended until at least age two and then for as long as the mother and child wish. The benefits of the breast milk for an infant is well established. Breastfeeding offers health benefits to mother and child even after toddlerhood. These benefits of breastmilk include, for instance decreased risk of sudden infant death syndrome, increased intelligence, decreased likelihood of contracting middle ear infections, cold and flu resistance, a tiny decrease in the risk of childhood leukaemia, lower risk of childhood onset diabetes, decreased risk of asthma and eczema, decreased dental problems, decreased risk of obesity later in life, and a decreased risk of developing psychological disorders, including in adopted children.

However, during the term of the breastfeeding, the mother can be taking consciously or unconsciously various chemicals that can be harmful for the health of the infant. Especially, various factors such as food additives, cosmetics, hair dyes, agrochemicals, sweeteners, tobacco, alcohol and drugs are known to be transmitted to breast milk and threatening infant's health (Ito S, Lee A. Drug excretion into breast milk— Overview Advanced Drug Delivery Reviews 55(5); 617-627, 2003). If the mother is medicated continuously because of a chronic disease such as diabetes, nephropathy or cardiac diseases, breastfeeding may not be recommended and therefore the infant is fed with baby foods being deprived of the benefits obtainable with breast milk. The concentration of most drugs in the breast milk is close to their concentration in maternal plasma (Berlin CM, Briggs GG. Drugs and chemicals in human milk. Semin Fetal Neonatal Med 10:149-159, 2005). Mothers receiving beta-blockers, salicylates, lithium and antineoplastic agents are generally not allowed for breastfeeding of infants younger than 6 months. Particularly, there are various problems reported so far in nerve transmission of the infants fed with breast milk of mothers receiving antidepressant agents (Sagraves R. Drugs in Breast Milk: a Scientific Explanation. J Pediatr Health Care. 11(5): 230-235,1997). It is also shown that nicotine and its metabolite cotinine transmitted to the baby through the breast milk and has adverse effects as well (Bogen DL, Davies ED, Barnhart WC, Lucero CA, Moss DR. What do mothers think about concurrent breast-feeding and smoking? Ambul Pediatr. 8(3):200-204, 2008; Mennella JA, Yourshaw LM, Morgan LK. Breastfeeding and smoking: short-term effects on infant feeding and sleep. Pediatrics. 120(3):497-502, 2007). Additionally, cosmetics, hair dyes, sweeteners, food additives, preservatives and agrochemicals with continuous uptake in daily life are reported to be excreting to the breast milk and causing various health problems like autism, hyperactivity and cancer in the infant (Hill HJ. Food additives and contaminants and their contribution to the human cancer load. Eur J Cancer Prev 2:291-295, 1993, Pollock I. Hyperactivity and food additives. Bibl Nutr Dieta 48:81-89, 1991).

It is therefore, desirable to separate these harmful chemical compounds from the breast milk before breastfeeding the infant. There have been various attempts for filtering and removing of undesired components of the breast milk for preventing harmful effects thereof on the newborn infants. WO 2004/082558 A2, for instance discloses devices and methods for removal of leukocytes from breast milk whereby a filter is attached to the nipple shield or baby bottle nipple at a location permitting removal of leukocytes. The filters mentioned therein are noted to be leukocyte depletion filters and leukocyte reduction filters which require liquid to pass through the said filters. In other words, the liquid (i.e. breast milk) is forced to pass through very small openings which require excessive pressure. Another device for removing organic/inorganic toxins such as halogenated endocrine disruptors, heavy metals and radionuclides is disclosed in US 2007/0005006 Al. Accordingly, a filter device such as activated carbon and/or a resin that absorbs/adsorbs cations and anions, organic and inorganic toxins, and endocrine disruptors is provided in a nipple shield or within a baby bottle. These arrangements have the very strict requirement of screening the milk through the pores having very small openings, and filtering therefore is carried out according to the particle size of the unwanted moiety. Very small pore openings would readily be noted as disadvantageous as they can fail in screening of toxic moieties or they can be screening the beneficial ingredients of the breast milk besides the toxic moieties. Therefore, it is quite hard with the conventional devices targeting solely of the undesired toxic moieties with high precision and efficiency, and also without drawbacks such as high pressure gradients and complicated system requirements.

It is also important to visualize removal of the unwanted chemical moieties by the user. The user needs to be assured of whether the moieties have been properly removed from the breast milk for the sake of providing user's confidence and proper usage of a removal system.

Accordingly, an object of the present invention is providing a novel device and method for the elimination of chemical moieties that are found in the breast milk and are harmful for the baby's health in a selective manner targeting only the harmful chemical compounds.

Another, object of the present invention is providing a novel device and method which does not necessitate filtering of breast milk through a porous structure such as a filter.

Another object of the present invention is developing a device and method for removing of harmful chemicals found in the breast milk even in very low amounts (i.e. ng/mL). A further object of the present invention is developing a device and method for removing of unwanted chemicals from breast milk specifically targeting chemicals such as pharmaceutical drugs, agrochemicals, food additives and cosmetics. Still a further object of the present invention is eliminating shortcomings of prior art with a reliable and consistent device visualizing removal of unwanted chemicals from breast milk.

Further objects and advantages of the present invention will be apparent from the description below. These and other objectives of the present invention are attained with a novel device and method according to the appended claims. Brief Description of the Invention

In an aspect of the present invention, a device for removing an unwanted moiety from milk of a mammalian is provided. The device comprises a nanosurface at least a portion of which is combined with at least one antibody targeting said unwanted moiety wherein the antibody is immobilized on the nanosurface and said portion of the nanosurface comprises means for visualizing binding of the chemical or chemical moiety to the antibody. The nanosurface may comprise, for instance, a matrix of polymers, nanofibers, silica beads, aerogels or hydrogels suitable for immobilization of an antibody used in the context of the present invention. The antibody, as a specific capturing molecule, recognize and binds its antigen or ligand. Capturing molecules should be covalently attached to the nanosurface by reacting -COOH with -NH ₂ groups on said nanosurface. The milk as mentioned herein is preferably human breast milk.

The means for visualization as mentioned above comprises preferably a labelling material configured to change colour once the unwanted chemical or chemical moiety is bound to the antibody. More particularly the visualization means may include a coloured material selected from the group of a latex, and nanosized particles of gold, carbon, silver, selenium or platinum to be bound to the antibody. Most preferably the material comprises colloidal gold particles. In another aspect the nanosurface may further comprise lactase and/or an antibacterial agent. The antibacterial agent is preferably selected from the group of chitosan, and an oil or extract of a plant selected from anise, thyme, sage, rosemary and cinnamon. In this aspect, lactase and/or antibacterial agent may be incorporated into a membrane provided with the visualization means as mentioned above.

The device according to the present invention can be a receptacle (1) comprising said nanosurface integrally in an inner wall (11) and/or bottom portion (13) thereof. Alternatively the device can be contemplated as an infuser (2) immersible into milk stored in a receptacle (1) whereby the nanosurface as such constitutes the infuser or the nanosurface is provided in an inner volume of the infuser (2). In a variation, the infuser (2) comprises an open end portion (21) and a closed bottom portion (22) such that the milk can be poured from the open end portion (21) so that the milk can be contacting with infuser's inner surface and gradually released into a receptacle (1). In another variation, the infuser (2) is in the form of a bag comprising the nanosurface on its structure or in an inner volume thereof. Still in a further variation, the infuser (2) is of the type a prism or tablet immersible into the milk stored in a receptacle (1). Additionally, the device can be a baby bottle, nipple or nipple shield incorporated with the nanosurface mentioned above. Further, the device can be a filter which can be affixed to a device for feeding an infant.

The polymers that can be used in the context of the present invention as the nanosurface may be selected from the group consisting of polyvinyl alcohol (PVA), polyacrylic acid (PAA), methacrylic acids, N-butyl acrylate, N-butyl methacrylate, hexyl acrylate, 2- ethylbutyl acrylate, isooctyl acrylate, 2-ethylhexyl acrylate, decyl acrylate, decyl methacrylate, dodecyl acrylate, dodecyl methacrylate, tridecyl acrylate, tridecyl methacrylatepolyethylene (PE), polyurethane (PU), polycaprolactone, polycarbonate, polytetrafluoroethylene (PTFE), polyacetal (PA), polymethylmethacrylate (PMMA), polyethylene glycols, polyethylene terephthalate (PET), silicone rubber (SR), polysulfone (PS), polylactic acid (PLA), polyglycolic acid (PGA), poly(DTE carbonate), poly(propylene carbonate), polytetrafluoroethylene, polyacetal, polymethyl methacrylate, polyethylene terephthalate, polycaprolactone, polyvinylpyrrolidone, acrylic polymers, alpha hydroxy acid polyester, beta-l,3-glucan, natural rubber, cellulose, Type-I collagen, Type-II collagen, alginate, gelatine, chitosan; heteropolymer or copolymers including poly(lactic-co-glycolic acid), poly(ethylene glycol-co-lactide), poly(L-lactide-co-e-caprolactone), poly(ethylene-co-vinyl alcohol), styrene-divinyl benzene copolymer, polyfvinyl chlorite-co-vinyl acetate-co-vinyl alcohol), polyfethylene -alt-maleic anhydride, polypropylene-graft-maleic anhydride), Polyethylene-graft- maleic anhydride, polyfmaleic anhydride-alt- 1-octadecene), polyfisobutylene-alt-maleic anhydride), polystyrene-block-poly(ethylene-ran-butylene)-block-polystyr ene-graft- maleic anhydride and polyvinyl alcohol-polyacrylic acid.

The antibody as mentioned in the foregoing explanations is specific to a chemical or chemical moiety that is excreted in to the milk. These chemicals can be selected from antidepressants and antipsychotic drugs, alkaloids, analgesics, immunosuppressant agents, cephalosporins, drugs used for the treatment of respiratory diseases, and metabolites thereof.

In another aspect, the present invention relates to a method for removing of a chemical from milk of a mammalian comprising bringing into contact the device as mentioned above with the milk before or during feeding of an infant. The method may further comprise placing of the device against flow of the milk such as the milk from mother or a milk pump. Alternatively, the method may comprise immersion of the device into the milk stored in a receptacle. In a further aspect, the invention relates to a method for producing the device as mentioned above, comprising the following steps: providing a nanosurface in the form of a matrix,

immobilizing antibodies on the matrix targeting predetermined chemicals or chemical moieties in the milk,

incorporating means for visualization into the matrix, and

placing the polymer matrix comprising antibodies and means for visualization into a device for feeding an infant, or shaping the matrix as a strainer or infuser for treatment of the milk. Furthermore, the antibodies used in the present invention can be monoclonal or polyclonal, or can be obtained from cell lines as conventionally known in the art. Brief Description of the Figures

Figure 1 is a perspective view of an embodiment according to the present invention wherein the nanosurface is provided in inner wall and/or bottom surface of a receptacle. Figure 2 is a perspective view of another embodiment according to the present invention wherein the nanosurface is provided as an infuser device fillable with milk from an open end portion and being immersible into the milk inside of a receptacle.

Figure 3 is a perspective view of another embodiment according to the present invention wherein the nanosurface is provided as an infuser device in the form of a bag immersible into the milk inside of a receptacle.

Figure 4 is a perspective view of another embodiment according to the present invention wherein the nanosurface is provided as an infuser device in the form of a prism or tablet immersible into the milk inside of a receptacle.

Detailed Description of the Invention

The present invention provides a novel method and device which involve use of a nanosurface incorporated with antibodies targeting antigens, namely the unsafe organic and inorganic chemical moieties found in the breast milk. The device for separating unwanted compounds from breast milk can be a receptacle such as a baby bottle with or without a nipple, or a breast milk pump comprising said receptacle. Alternatively, the device can be a nipple or nipple shield as such, and in this case, the nanosurface incorporated with antibodies can be conveniently placed into these devices so that the breast milk can be treated in situ within the course of feeding an infant. Further, the device according the present invention can be formed as an infuser as elaborated in preferred embodiments hereinbelow comprising the nanosurfaces according to the instant invention. The nanosurface may comprise, for instance, a matrix of polymers, nanofibers, silica beads, aerogels or hydrogels suitable for immobilization of an antibody used in the context of the present invention. These structures provide a large surface and contact area so that the unwanted chemicals can be easily brought into contact with antibodies and separated from the milk. The present invention advantageously incorporates means for visualizing the removal state of the unwanted chemical substances to the corresponding antibody so that the user may be confident with removal of these substances from the breast milk. This also advantageously ensure that breast milk can be brought into contact with the device for a sufficient time so that the user can recognize removal of the chemical substances properly and then feed the infant with the milk sufficiently treated. This is particularly important because the device of the present invention works with the principle of bringing breast milk into contact with the nanosurface loaded with antibodies, and removing the breast milk before being captured in required concentrations may render the milk insufficiently treated.

Means for visualizing as mentioned herein may involve any method and arrangement conventionally known in the art such as labelling of antibodies so that a colour change can be observed once the antibodies bind to specific antigens. In fact, there are many types of ligand-receptor assays available in the art that have been used to detect the presence of various substances in body fluids. These assays involve antigen antibody reactions, synthetic conjugates comprising radioactive, enzymatic, fluorescent, or visually observable metal sol tags, and specially designed reactor chambers. In all these assays, there is a receptor, e.g., an antibody, which is specific for the selected ligand or antigen, and a means for detecting the presence, and often the amount, of the ligand- receptor reaction product. In the context of the present invention, however, it is preferred to employ a rapid detection means including a labelling material such as a latex or nanosized particles of gold, carbon, silver, selenium and platinum. In this context, latex or nanosized particles are conjugated with antibodies and they cause a change in colour once the antibodies are bound with corresponding antigens. In order to improve sensitivity of the means for visualization according to the current invention, colloidal gold is particularly preferred.

Breast milk, just like the other milks contains lactose, and it is known that a person lacking lactase may experience symptoms of lactose intolerance. This may cause serious health problems in infants fed with breast milk. Therefore, the nanosurface used in the device according to the present invention may further comprise lactase for use specifically to treat breast milk for feeding infants lacking lactase. This enzyme can be impregnated to or immobilized on the nanosurface.

In another aspect, the device according to the present invention may further comprise an antibacterial agent. The nanosurface of the current invention can therefore become a bactericidal surface which prevents bacterial proliferation. This is particularly advantageous because the device having a bactericidal surface become reusable repeatedly. Natural polymers such as chitosan are known to have antibacterial properties, and therefore a nanosurface made of chitosan for use in the context of the present invention would inherently provide antibacterial properties without the need for including further agents. Alternatively, an antibacterial agent preferably of natural origin such as the oils or extracts of anise, thyme, sage, rosemary, cinnamon or combinations thereof can be used in the nanosurface of the present invention.

Since the antibodies contained in the nanosurface mentioned above readily react with the antigens, structural limitations of the conventional filtering devices, such as the necessity of screening through a filter, are completely eliminated. Therefore, the nanosurface according to the present invention can be provided within a baby bottle, nipple, nipple shield or any receptacle for the breast milk in any form and location as long as the milky liquid can be brought into contact with the nanosurface containing specific antibodies. It is thus up to the designer to provide suitable embodiments of the invention. However, it would be preferable to provide the nanosurface in an arrangement such that the best possible contact area during the flow or storage of the breast milk can be obtained. For instance, in a nipple shield or baby bottle used for immediate feeding of the infant, providing nanosurfaces perpendicular to the direction of milk flow would be preferred because this design would increase retention time in contact area of the milk on the surface, and this would increase also efficiency of removing unwanted compounds from the liquid composition of the breast milk. In a receptacle used for the storage of the breast milk, however a different design can be contemplated where a simple contact of the nanosurface and breast milk would be sufficient. In a preferred embodiment as shown in Fig. 1, for instance, the nanosurface can be placed over the inner walls (11) and/or bottom surface (12) of a receptacle (1) (e.g. baby bottle) such that nanosurface and antibodies can be brought into contact with breast milk in a simple manner.

In another embodiment of the present invention, the nanosurface as scrutinized in the foregoing explanations can be contemplated in the form of an infuser (2) as shown in Figs. 2, 3 and 4. The infuser (2) shown in Fig. 2 is configured to be placed inside a receptacle (1) from the mouth portion (13) thereof in a manner extending along the inner volume of the receptacle (1) and providing a large contact area with the milk stored therein. It has an open end portion (21) where breast milk can be poured into the inner volume thereof, and a bottom portion (22) which is actually closed. As can be appreciated by a person skilled in the art, this infuser device (2) can be formed in a porous structure (i.e. mesh) such that milk can be retained in its inner volume for a while until it passes through its porous structure to the inner volume of the receptacle (1). Of course, the infuser device (2) may directly be placed into a receptacle (1) which is already filled with breast milk, and in this case, breast milk is moved in the reverse direction, i.e. through the inner volume of the infuser device (2). In both of the usage modes, milk is brought into contact with the nanosurface of the infuser device (2) in an efficient way and unwanted chemicals are removed without exerting an extra effort. In another embodiment of the present invention, the infuser device (2) is contemplated as a bag (i.e. tea bag shaped) comprising a matrix of the nanosurface which is designed for immersion into the breast milk stored in a receptacle (1) as shown in Fig. 3. As can be appreciated by those skilled in the art, nanosurface may constitute the said infuser device (2) or it can be stored within an inner volume of the same. This arrangement also suggests removal of unwanted chemicals without extra effort because of the antibodies immobilized on the nanosurface catching and entrapping of the corresponding unwanted chemicals or moieties and it can be simply immersed into the breast milk in a receptacle (1). In a further embodiment as shown in Fig. 4, the infuser device (2) can be provided as a rigid prism or tablet which can be immersed into the receptacle (1) from a mouth portion (13) thereof so that it can be readily brought into contact with the milk stored in the receptacle (1). Similar to the infuser device (2) as shown in Fig. 3, this device also comprise a nanosurface and it can be stored within an inner volume of the same.

As noted in the foregoing embodiments, the nanosurface containing antibodies can be integrated into a device for feeding an infant, such as baby bottles, nipples, nipple shields or milking pumps, or it can be arranged in a disposable manner such as the infuser devices (2) exemplified in the above mentioned embodiments. Providing the nanosurface as a disposable device is advantageous because it suggests simplicity and eliminates the need for regeneration of the nanosurface after treatment of the breast milk. Whilst it is not necessary to screen the breast milk, nanosurfaces of the present invention can be designed in the form a conventional filter for screening and treating the breast milk in situ. However, the present invention is advantageous in that the need for very smaller openings (i.e. pores) in a filter is completely eliminated and no physical separation according to the molecule size is needed. Antibodies contained in the nanosurface allow the designer to use larger openings in a filter because the said antibodies attract and catch the antigens once they get into close proximity with the nanosurface.

The nanosurfaces according to the present invention may comprise, for instance, a matrix of polymers, nanofibers, silica beads, aerogels or hydrogels suitable for immobilization of an antibody. These nanosurfaces are well-known in the art, and are known to be advantageous for immobilization of organic molecules such as peptides, enzymes and antibodies because of their high aspect ratio, large surface area to volume ratio, thermal and chemical stability, high porosity, high directional strength, biocompatibility and biodegradability, good structural morphology and flexibility. In order to obtain the polymer in nano structure, mold copying, colloidal lithography, interfacial polymerization, nanoprecipitation, multiple solvent emulsion evaporation, nano-identification, electrospray, self-assembly, drawing, phase separation, and electrospinning methods may be used.

In the context of the present invention, nanofibers are particularly preferred because they suggest high surface area and are noted to be very suitable for immobilization of antibodies over the surface thereof. This method is also advantageous in obtaining increased porousness and providing antibody /polymer mixture in optimum homogeneity.

Nanosurfaces forming a matrix for immobilization of antibodies according to this invention can be made of the polymers which are preferably biocompatible and/or biodegradable polymers and they can be of natural and/or synthetic origin. Synthetic polymers may be selected from homopolymers, heteropolymers or copolymers and terpolymers. Nanostructures may be formed by use of one or a combination from the group of polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyethylene (PE), polyurethane (PU), polycaprolactone, polycarbonate, polytetrafluoroethylene (PTFE), polyacetal (PA), polymethylmethacrylate (PMMA), polyethylene glycols, polyethylene terephthalate (PET), silicone rubber (SR), polysulfone (PS), polylactic acid (PLA), polyglycolic acid (PGA), poly(DTE carbonate), poly(propylene carbonate), polytetrafluoroethylene, polyacetal, polymethylmethacrylate, polyethylene terephthalate, polycaprolactone, polyvinylpyrrolidone, acrylic polymers, alpha hydroxy acid polyester, beta-l,3-glucan, natural rubber, cellulose, Type-I collagen, Type-II collagen, alginate, gelatine, chitosan, fibrinogen, chitin, elastin, hydroxylapatite, dextran, laminin, hyaluronic acid, starch, silk and similar synthetic or natural biopolymers and/or as a combination of natural/synthetic biopolymers.

Heteropolymer or copolymers of the invention can be selected from alternative copolymer, periodic copolymer, statistical copolymer, block copolymer, terpolymer and graft copolymer. For instance; poly(lactic-co-glycolic acid), poly(ethylene glycol-co- lactide), poly(L-lactide-co-e-caprolactone), poly(ethylene-co-vinyl alcohol), styrene- divinylbenzene copolymer, poly(vinyl chlorite-co-vinyl acetate-co-vinyl alcohol), poly(ethylene-alt-maleic anhydride, polypropylene-graft-maleic anhydride), Polyethylene-graft-maleic anhydride, poly(maleic anhydride-alt- 1-octadecene), poly(isobutylene-alt-maleic anhydride), polystyrene-block-poly(ethylene-ran-butylene)- block-polystyrene-graft-maleic anhydride and polyvinyl alcohol-polyacrylic acid are preferred.

Acrylic polymers used in the invention are homopolymer, copolymer, terpolymer and acrylic acid-like polymers. Polyacrylates may be selected from the group containing acrylic acid, methacrylic acid, N-butyl acrylate, N-butyl methacrylate, hexyl acrylate, 2- ethylbutyl acrylate, isooctyl acrylate, 2-ethylhexyl acrylate, decyl acrylate, decyl methacrylate, dodecyl acrylate, dodecyl methacrylate, tridecyl acrylate, tridecyl methacrylate or mixtures thereof.

The antibodies used in the context of the present invention can be obtained biologically or by way of synthetic methods as conventionally known in the art. In a typical biological method, for instance, a specific antigen is bonded with a hapten and injected to a rabbit for the production of the corresponding antibody which can be isolated from rabbit's blood later on and used with the nanosurfaces of the present invention. Alternatively, antibodies can be produced by recombinant techniques. However, antibodies generally are commercially available and they can directly be used depending on the specific chemical moiety targeted in the breast milk. The novel device and method of the present invention is noted to be advantageous for elimination of chemicals such as food additives, cosmetics, hair dyes, agrochemicals, sweeteners, tobacco and drugs, or their metabolites from the breast milk even if they exist in trace amounts while providing the user's confidence by visual means. Various drugs have already been reported to be excreted to breast milk easily and being harmful for the infant. The same holds also various metabolites of tobacco and tobacco products. The present invention ensures elimination of these and other factors harmful for the health of the infant, and therefore the invention is useful for removing any kind of antigen with high efficiency even if the antigen itself exists in very low amounts in the breast milk, thanks to the antibodies immobilized on a nanosurface. This eliminates conventional techniques relying on screening based on molecule size of the antigen.

The present invention is particularly suitable for removing unwanted drugs and drug metabolites such as antidepressant and antipsychotic drugs, analgesics, alkaloids, immunosuppressant agents, antibiotics, as well as the drugs widely used in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma from breast milk. The means for visualizing removal of the unwanted chemicals advantageously ensures user's confidence and provides also an indication for the user about waiting time necessary for completing the reactions between the chemical substances and antibodies.