Field of the invention

The present invention relates to the application of the enzyme fructosyl-amino acid oxidase (FAOD) -without the addition of proteases- cosmetical conditions which are characterized by the presence of advanced glycation end products (AGEs). More specifically, the present invention relates to the treatment of aged skin and hypertrophic scars due to bum injuries. At present no effective medical treatment is available for hypertrophic scars. Hypertrophic scarring is characterized by raised and thickened scars with colored pigmentation and skin retraction, which contain accumulations of AGEs.

Hence, the invention relates to the in vivo application of FAOD in order to deglycate and inactivate said AGEs.

Background of the invention

Protein glycation is a process of ageing, where metabolically important sugars react with primary amine groups, forming adducts that can rearrange and react further, eventually leading to cross-links between proteins (Maillard reaction) (1). This ageing process of protein glycation specifically occurs in organs with long-lived proteins such as the eye and the skin.

The chemical modification of collagen has a major effect on the loss of skin elasticity and skin discoloration, two typical sequelae of bum injuries (7). In every deep second degree or third degree (full thickness) burn wound, the thermal damage extends into the deep dermal layers or even into the subcutaneous tissue, which in humans inevitably results in substantial scar tissue formation, even after surgical therapy with skin grafts. For this reason, in all deep bum wounds or other skin defects, the initial period of healing and closure of the wound is obligatory followed by a prolonged period (12-24 months) of additional scar treatment consisting of the application of moisturizers, customized pressure garments and silicone application. Despite this cumbersome, intensive treatment regimen, burn sequelae never disappear and residual unaesthetic scarring with functional and psychological impairment often requires additional (painful) corticoid injections or secondary surgical corrections (7). However, no solution has been disclosed or suggested to effectively treat hypertrophic and excessive scar formation in heavily burned skin which is based on breaking down AGEs in the collagenous matrix by applying extracellularly one or a combination of deglycating enzymes such as FAOD and/or FN3K. In aging skin, UV rays induce accumulation of AGEs in the extracellular matrix of the dermis and epidermis, resulting in a dryer, thinner and less elastic skin (8). US2010159045 discloses the topical usage of botanical extracts on the skin which induce the expression of intracellular enzyme F3K in the skin in order to treat cutaneous signs resulting from non-pathological impairment of the barrier-function of the skin by -for example- UV radiation. However, F3K or FAOD as a deglycating enzyme has never been used for extracellular, topical and cosmetical usage on the skin.

Fmctosyl- amino acid oxidase (FAOD; fmctosyl-a-L-amino acid: oxygen oxidoreductase (defructosylating), is an enzyme found in many bacteria and yeasts (9,10). FAOD catalyzes the oxidation of the C-N bond linking the Cl of the fmctosyl moiety and the nitrogen of the amino group of fmctosyl amino acids. Flavin adenine dinucleotide (FAD) acts as its cofactor. It is active on both of fmctosyl lysine and fmctosyl valine. FAOD-based detection methods for glycated proteins -as for example described by Shen et al. (Abstract B44 of 2013 AACC Annual Meeting) and in US2005/0014935 have been commercially available since 1999. However FAOD is unable to react with most intact glycated proteins, and samples thus require an initial proteolytic digestion step to liberate glycated amino acids or glycated dipeptides (. Capuano et al. (J. Agric. Food Chem 2007:4189) show a deglycating effect of FAOD on some low molecular weight proteins such as insulin and conclude that FAOD could be used as a tool to inhibit protein glycation in food systems. However an in vivo therapeutic/cosmetic use of FAOD in AGEs-induced conditions in human or animal has never been considered

Figure 1. Hotelling plot of principal component (PCA) analysis of amide peaks (fig 1A) and carbohydrate region (fig IB) after treatment of human hypertrophic skin with FAOD alone or in combination with FN3K. NIR was performed on sections treated with PBS + ATP + Mg (dots), with FN3K + ATP + MgCh (squares), with FAOD + FAD (pentagons) or with the combination of FN3K + ATP + MgCh and FAOD + FAD (triangles). First t(l) and second t(2) principal components.

Figure 2. Fluorometry of human hypertrophic skin treated with FAOD alone or in combination with FN3K. Autofluorescence was measured before treatment (baseline, black bars) and after treatment with different deglycating enzymes (grey bars). Mean autofluorescence values and standard deviation of 3 measurements of emission spectra (400 - 600 nm) of human hypertrophic skin treated with FN3K + ATP + MgC12 (fig 2A), FAOD + FAD (fig 2B), combination FN3K + ATP + MgCF and FAOD + FAD (fig 2C). As a negative control, hypertrophic skin was treated with PBS with ATP and MgCF (fig 2D), ns = not significant (P > 0.05), *P < 0.05, ** P < 0.01, **** P < 0.0001.

Figure 3. Fluorometry of healthy human breast skin treated with FAOD alone or in combination with FN3K. Healthy human breast skin was first glycated with glycolaldehyde and then treated with different deglycating enzymes. Autofluorescence was measured before treatment (baseline, white bars), after treatment with glycolaldehyde (GA, black bars) and after treatment with different deglycation enzymes (grey bars). Mean autofluorescence values and standard deviation of 3 measurements of emission spectra (400 - 600 nm) of human breast skin treated with FN3K + ATP + MgC12 (fig 3A), FAOD + FAD (fig 3B), combination FN3K + ATP + MgCF and FAOD + FAD (fig 3C). As a negative control, AGE-modified human breast tissue was treated with PBS with ATP and MgCF (fig 3D), ns = not significant (P > 0.05), *P < 0.05, ** p < 0.01, *** P < 0.001.

Figure 4. Proposed structure for the compound with m/z 133,09695 at RT 0.82 (Compound # Al), detected in arginine-containing samples.

Figure 5. Proposed structure for the compound with m/z 337,17098 at RT 0.88 (Compound # A3), detected in arginine-containing samples.

Figure 6. Proposed structure for the compound with m/z 319,16064 at RT 0.88 (Compound # A4), detected in arginine-containing sampleFigure 7. Proposed structure for the compound with m/z 309,16554 at RT 0.85 (Compound # LI), detected in lysine-containing samples.

Figure 8. Proposed structure for the compound with m/z 219,13387 at RT 0.89 (Compound # L2), detected in lysine-containing samples.

Figure 9. Proposed structure for the compound with m/z 205,11825 at RT 0.91 (Compound # L3), detected in lysine-containing samples.

Figure 10. Proposed structure for the compound with m/z 351,15047 at RT 0.95 (Compound # A9), detected in arginine-containing samples.

Figure 11. Proposed structure for the compound with m/z 131,12904 at RT 0.83 (Compound # A2), detected in arginine-containing samples.

Summary of invention The present invention relates to the usage of compositions comprising a fmctosyl amino oxidase to treat aging skin or hypertrophic and excessive scar formation in the skin.

The present invention further relates to the usage of compositions as described above further comprising flavin adenine dinucleotide (FAD).

The present invention further relates to the usage of compositions as described above further comprising a fmctoseamine-3-kinase and adenosine triphosphate (ATP).

The present invention further relates to the usage of compositions as described above further comprising magnesium ions.

The present invention also relates to the usage of compositions as described above wherein said compositions are administered topically, by injection or by microneedling.

The present invention further relates to the usage of compositions as described above wherein said topical administration, by injection or by microneedling is further enhanced using ultrasound.

The present invention also relates to the usage of a composition comprising a fmctosamine-3- kinase and adenosine tri phosphate to treat an aging skin or hypertrophic and excessive scar formation in the skin, and further relates to the latter composition which further comprises magnesium ions.

The present invention relates to the surprising finding that FAOD -which is shown not to be able to revert protein glycation without the preceding usage of proteinases- is capable to deglycate proteins within the skin in vivo and has -as such- a cosmetic effect. Furthermore, the present invention further discloses that FAOD deglycates different proteins than F3K so that using both enzymes simultaneously on the same tissues has an improved (additive) cosmetic effect.

The present invention relates in first instance to a composition comprising a fmctosyl amino oxidase for use to treatAGEs-related appearances of the skin.

The present invention thus relates to a composition comprising a fructosyl-amino acid oxidase. The term ‘a fructosyl- amino acid oxidase (FAOD; fructosyl-a-L-amino acid: oxygen oxidoreductase (defmctosylating)’ relates to any enzyme classified as catalyzing the oxidation of the C-N bond linking the Cl of the fructosyl moiety and the nitrogen of the amino group of fructosyl amino acids. The reaction proceeds to an unstable Schiff base intermediate, which hydrolyzes to produce glucosone and an amino acid. The enzyme's reduced flavin adenine dinucleotide (FAD) cofactor is then reoxidized by molecular oxygen with the release of hydrogen peroxide.

More specifically, the terms ‘a fructosyl-amino acid oxidase (FAOD; fructosyl-a-L-amino acid: oxygen oxidoreductase (defmctosylating)’ relates to the enzyme encoded by the gene encoding the fructosyl- amino acid oxidase (fructosyl-a-L-amino acid: oxygen oxidoreductase(defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 which was cloned and expressed in Escherichia coli as described by Sakaue et al. (11). The latter enzyme -as a non-limiting example of an enzyme which can be used in the present invention- can be purchased from-for example- Creative Enzymes, Shirley, NY or can be made using well-known recombinant methods as is for example described by Sakaue et al. (11)

The term 'a fmctosamine-3-kinase' relates to enzymes classified as enzymes 2.7.1.171 in -for example-the Brenda enzyme database (www.brenda-enzvmes.org). The latter enzymes are part of an ATP-dependent system for removing carbohydrates from non-enzymatically glycated proteins and catalyze the following reaction: ATP + [protein]-N6-D-fmctosyl-L-lysine = ADP + [protein]-N6-(3-0-phospho-D-fructosyl)-L-lysine. More specifically, the term 'a fructosamine-3 -kinase' relates to -as a non-limiting example- to the human fmctosamine-3- kinase having accession number or the National Center for Biotechnology Information (NCBI) Reference sequence number :NP_071441.1 (see https://www.ncbi.nlm.nih.gov/protein/NP BothWO2019149648 and W02020053188 describe -for example- the recombinant production of F3K in Pichia pastoris.

It should be further clear that the term ‘a fmctosamine-3-kinase’ and ‘a fructosyl amino acid oxidase’ relates to the enzymes as described above, but also to functional fragments and variants thereof. The term “functional fragments and variants” relates to fragments and variants of the naturally occurring enzymes. Indeed, for many applications of enzymes, part of the protein may be sufficient to achieve an enzymatic effect. The same applies for variants (i.e. proteins in which one or more amino acids have been substituted with other amino acids, but which retain functionality or even show improved functionality), in particular for variants of the enzymes optimized for enzymatic activity (as is also described further with regard to recombinant enzymes). The term ‘fragment’ thus refers to an enzyme containing fewer amino acids than the 309 amino acid sequence of the human fructosamine-3-kinase having NCBI Reference sequence number :NP_071441.1 or the 372 amino acid sequence of the fructosyl amino acid oxidase as disclosed by Sakaue et al. (11) and that retains said enzyme activity. Such fragment can -for example- be a protein with a deletion of 10% or less of the total number of amino acids at the C- and/or N-terminus. The term “variant” thus refers to a protein having at least 50 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with the 309 amino acid sequence of the human fructosamine-3- kinase having NCBI Reference sequence number :NP_071441.1 or with or the 372 amino acid sequence of the fructosyl amino acid oxidase as disclosed by Sakaue et al. (11) and that retains said enzyme activity.

Hence, orthologues, or genes in other genera and species (than the human fmctosamine-3- kinase having NCBI Reference sequence number :NP_071441.1 or than the fructosyl amino acid oxidase as disclosed by Sakaue et al. (11) with at least 50 % identity at amino acid level, and having said enzyme activity are part of the present invention. The percentage of amino acid sequence identity is determined by alignment of the two sequences and identification of the number of positions with identical amino acids divided by the number of amino acids in the shorter of the sequences x 100. The latter ‘variant’ may also differ from the protein having NCBI Reference sequence number :NP_071441.1 or the protein as disclosed by Sakaue et al. (11) only in conservative substitutions and/or modifications, such that the ability of the protein to have enzymatic activity is retained. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of protein chemistry would expect the nature of the protein to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be proteins as described herein modified by, for example, the deletion or addition of amino acids that have minimal influence on the enzymes activity as defined above, secondary structure and hydropathic nature of the enzyme.

The terms ‘adenosine tri phosphate’ (ATP), flavin adenine dinucleotide (FAD) and ‘magnesium ions’ relate to well-known cofactors of the latter enzymes. The present invention further relates to a composition for use as described above which further comprises a peroxidase. The term ‘peroxidase’ relates to any well-known enzyme having EC number 1.11.1.x and are capable to break up peroxides, more specifically which are capable to break up hydrogen peroxides which are released during the oxidation of the reduced FAD which is the cofactor of FAOD.The term 'animal' may relate to any animal such as mammals (dogs, cats, horses, ...), birds and reptiles.

The present invention relates to the findings that the application of a F3K or a FAOD alone, or a combination of both enzymes, and their co-factor(s), results in less AGEs in aging skin or hypertrophic scars. In other words, the latter treatments improve skin properties in patients with aging skin or hypertrophic scars.

With the term ‘hypertrophic scar’ is meant “a cutaneous condition characterized by deposits of excessive amounts of collagen which gives rise to a raised scar, but not to the degree observed with keloids.”

With the term ‘fluorescence in hypertrophic scars’ is meant the UV fluorescence signal observed after illuminating the scarred skin with UV light (360 nm).

With the term ‘elasticity in hypertrophic scars is meant the ability of the skin (mainly the dermis) to stretch and snap back to its original shape.

With the term ‘skin pigmentation in hypertrophic scars’ is meant the colour of the skin due to the presence of pigments in the skin.

In other words, in another embodiment, the present invention relates to the surprising finding that the administration of fructosyl-amino acid oxidase alone or in combination with fmctosamine 3 kinase, and their cofactor(s), or in combination with peroxidase, can be used to treat hypertrophic and excessive scar formation in the skin or to treat aging skin. The present invention thus relates to a composition for use as described above wherein said treatment of aging skin or hypertrophic and excessive scar formation involves a deglycation of skin advanced glycation end products.

Furthermore the present invention relates to a composition for use as described above wherein said composition is administered topically, by injection or by microneedling.

The terms ‘topical administration’ relates to application to the skin including creams, foams, gels, lotions, and ointments. The word topical derives from Greek topikόV topikos, "of a place". The application technique is used under controlled aseptic conditions.

The term ‘injection’, is a shallow or superficial injection of a substance into the dermis, which is located between the epidermis and the hypodermis.

The term ‘microneedling’ refers to the application of microneedles which are microscopic applicators used to deliver drugs across the skin barrier.

The present invention further relates to a composition for use as described above wherein said topical administration, by injection or by microneedling is further enhanced using ultrasound. With the terms ‘is further enhanced using ultrasound’ is meant any device producing ultrasound waves between 20 kHz- 100 MHz for penetrating epidermis, dermis and subcutis.

The present invention also relates to composition comprising a fructosamine-3-kinase and adenosine tri phosphate for use to treat aging skin and/or hypertrophic and excessive scar formation in the skin, and further relates to a composition comprising a fructosamine-3-kinase and adenosine tri phosphate which further comprises magnesium ions.

The present invention thus relates -in other words- to a method to treat aging skin or hypertrophic and excessive scars in a subject in need thereof wherein said method comprises administering a therapeutically effective amount of a compound comprising a F3K and ATP, or a F3Kand ATP and magnesium ions, or, a F3K and ATP and a FAOD and FAD, or a F3K and ATP and a FAOD and FAD and peroxidase, or, a F3K and ATP and magnesium ions and a FAOD and FAD, or, a F3K and ATP and magnesium ions and FAOD and FAD and peroxidase, or, FAOD and FAD , or FAOD and FAD and peroxidase, or FAOD and peroxidase, or FAOD alone on the skin of said subject.

For the preparation of a pharmaceutical formulation for topical F3K and FAOD treatment, one can produce a methylcellulose base gel. Hypromellose, short for hydroxypropyl methylcellulose (HPMC), is an inert, viscoelastic polymer used as eye drops, as well as an excipient and controlled-delivery component in oral medicaments. HPMC is an excellent vehicle for bringing F3K and/or FAOD onto the damaged skin.

The term ‘a therapeutically effective amount’ -with regard to F3K- relates to an amount ranging from lO pl to 100 pi taken from a therapeutic dose ranging between about 4,17 and 12.5 pg/ml fmctosamine-3 -kinase, 2.50 and 4,17 mM ATP and 1.00 and 1.67 mM MgCF. The latter therapeutic doses can be obtained by mixing 1:1, 1:2, 1:3 or 1:5 a solution of 25 pg/ml fmctosamine-3 -kinase with a fresh solution of 5mM ATP/2mM MgCF. The term ‘a therapeutically effective amount’ -with regard to FAOD- relates to an amount ranging from 10 mΐ to 100 mΐ taken from a therapeutic dose ranging between 1 U/mL and 100 U/mL fructosyl-amino acid oxidase. FAD concentrations of 2 mmol FAD/mol FAOD can be used for optimal functioning of the enzyme. The solution buffer should have a pH values between 6.8 and 7.7.

The present invention further relates to a composition as indicated above wherein said fructosamine-3 -kinase and said fructosyl amino acid oxidase is a recombinant enzyme. The term ‘recombinant’ refers to fructosamine-3-kinase or fructosyl amino acid oxidase obtained as an outcome of the expression of recombinant DNA encoding for a fructosamine-3-kinase or fructosyl amino acid oxidase inside living cells such as bacteria or yeast cells. Practitioners are further directed to Sambrook et al. Molecular Cloning: A laboratory Manual, 4 ^th ed. , Cold Spring Harbor press, Plainsview, New York (2012) and Ausubel et al. Current Protocols in Molecular Biology (supplement 114), John Wiley & Sons, New York (2016).

More specifically the present invention relates to a recombinant fructosamine-3-kinase which is obtainable by recombinant production in Pichia pastoris and, even more specifically, wherein said recombinant fructosamine-3-kinase obtainable by recombinant production in Pichia pastoris has the amino acid sequence as given by SEQ ID N° 1 or SEQ ID N°2. SEQ ID N°1 is a construct with an N-terminal cleavable HIS-tag and a caspase 3-cleavable Asp-Glu-Val- Asp (DEVD) linker between the His6 tag and the protein coding sequence which allows for clean removal of the tag. SEQ ID N° 2 is the cleaved version of SEQ ID N°l.

The amino acid sequences of SEQ ID N°1 and SEQ ID N°2 (and their encoding nucleic acid sequences SEQ ID N3 and SEQ ID N° 4, respective) are as follows:

SEQ ID N° 1:

Type: amino acid 1-letter (underlined: His6-tag, italics: linker , bold underlined: caspase cleavage site)

MHHHHHHVZVGPGSDEVDEQLLRAELRTATLRAFGGPGAGCISEGRAYDTDAGPVFV KVNRRT QARQMFEGEVASLEALRSTGLVRVPRPMKVIDLPGGGAAFVMEHLKMKSLSSQASKLGEQ MA DLHLYNQKLREKLKEEENTVGRRGEGAEPQYVDKFGFHTVTCCGFIPQVNEWQDDWPTFF AR HRLQAQLDLIEKDYADREARELWSRLQVKIPDLFCGLEIVPALLHGDLWSGNVAEDDVGP I I YDPASFYGHSEFELAIALMFGGFPRSFFTAYHRKIPKAPGFDQRLLLYQLFNYLNHWNHF GR EYRSPSLGTMRRLLK* 5 10 The present invention indeed relates –in addition- to the finding that the recombinant fructosamine-3-kinase obtainable by recombinant production in Pichia pastoris and having the amino acid sequence as given by SEQ ID N° 1 and 2 are preferred enzymes for treating said AGEs-related conditions . Indeed, the latter enzymes are preferred as 1) their production in Pichia resulted in higher yields of the enzyme compared with the production in –for example- 15 E. coli, 2) the enzymes had a higher purity when analysed on SDS page, and 3) the presence of endotoxin, which is known to provoke an inflammation following administration, can be avoided. The following examples are provided to better illustrate the present invention and should not 20 be considered as limiting the scope of the invention. Examples Example 1. Hotelling plot of principal component (PCA) analysis of amide peaks ( fig 1A) and carbohydrate region (fig 1B) after treatment of human hypertrophic skin by FAOD 25 alone and in combination with FN3K. Hypertrophic scar tissue was obtained as waste material after surgical procedure. Subcutaneous fat tissue and fascia was carefully removed surgically by a skilled plastic surgeon. Skin fragments were then placed in black 96-well-plate (FluoroNunc PolySorp, Thermo Fisher Scientific, Massachusetts, USA) and near infrared (NIR) spectra were recorded off-line using a NIR spectrometer equipped with an immobilized 30 reflection probe of seven 400 µm fibers, an InGaAs detector and a halogen lamp (AvaSpecNIR256-2.5-HSC with an FCR-7UVIR400-2-BX reflection probe, Avantes). As glycation results in a spectral shift in the near-infrared spectrum of proteins, it is possible to observe specific peak sharpening and spectral variations in NIR spectra due to deglycation of proteins. This allows us to distinguish glycated skin from control treated skin. The use of non- 11 invasive NIR monitoring enables us to assess the treatment in a non-destructive way. Next, skin fragments were incubated for 6 hr at 37°C with 20 mΐ of a solution of FN3K (250 mg/mL) and its cofactors (5 mM ATP-2 mM MgCF), 20 mΐ of a solution of FAOD 10 U/mL + FAD 1 mmol/L, or combination of 20 mΐ FN3K 250mg/mL + cofactors (ATP 5 mmol/L + MgCF 2 mmol/L) and 20 mΐ FAOD 10 U/mL + FAD 1 mmol/L. FAOD was purchased from Creative Enzymes, Shirley, NY. FAD was purchased from Sigma- Aldrich, Tokyo, Japan. As a control, PBS with only cofactors 5 mM ATP-2 mM MgCF was used. After 6 hr incubation, skin fragments were washed 4 times with PBS and stored for 24 hr at 4°C and NIR spectra were recorded again.

ATR-IR spectra of human hypertrophic skin were recorded and normalized in the region of interest from 4000-400 cm ^"1 . PCA scoreplots in both the region of amides (figure 1A) and carbohydrate region (figure IB) show clustering in spectra of control treated (dots) and spectra after deglycation (triangles, F3K treatment; squares, FAOD treatment; pentagons, combination F3K and FAOD treatment).

Example 2. Fluorometry of human hypertrophic skin treated by FAOD alone or in combination with FN3K.

Hypertrophic scar tissue was obtained as waste material after surgical procedure. Subcutaneous fat tissue and fascia was carefully removed surgically by a skilled plastic surgeon. Skin fragments were then placed in black 96-well-plate (FluoroNunc PolySorp, Thermo Fisher Scientific, Massachusetts, USA). AGEs were quantified based on Maillard-type autofluorescence (AF) measurements (excitation 365 nm, emission 390-700 nm) using a Flame miniature spectrometer (FLAME-S-VIS-NIR-ES, 350-1000 nm, Ocean Optics, Dunedin, FL, USA) equipped with a high-power LED light source (365 nm, Ocean Optics) and a reflection probe (QR400-7-VIS-BX, Ocean Optics). Next, skin fragments were incubated for 6 hr at 37°C with 20 mΐ of a solution of FN3K (250 pg/mL) and its cofactors (5 mM ATP-2 mM MgCF), 20 mΐ of a solution of FAOD 10 U/mL + FAD 1 mmol/L, or combination of 20 mΐ FN3K 250pg/mL + cofactors (ATP 5 mmol/L + MgCF 2 mmol/L) and 20 mΐ FAOD 10 U/mL + FAD 1 mmol/L; FAOD was purchased from Creative Enzymes, Shirley, NY. FAD was purchased from Sigma- Aldrich, Tokyo, Japan. As a control, PBS with only cofactors 5 mM ATP-2 mM MgCF was used. After 6 hr incubation, skin fragments were washed 4 times with PBS and stored for 24 hr at 4°C and autofluorescence was recorded again. Following a 6 h incubation with FN3K, a 40% decrease of the AGE concentration in the skin fragment was obtained: baseline 0,08242 ± 0,004372 and after 6 hr FN3K treatment 0,04985 ± 0,001353 (fig 2A). After FAOD treatment, 40% decrease of autofluorescent values was noticed: baseline 0,04285 ± 0,004217 and after 6 hr treatment 0,02577 ± 0,002101 (fig 2B). After treatment of combination of FN3K and FAOD, 83% decrease was measured, baseline 0,09161 ± 0,001510 and after treatment 0,01571 ± 0,0008990 (fig 2C). No significant differences were noticed in autofluorescence in control experiment, baseline 0,06371 ± 0,001830 and after 6 hr PBS treatment (AF-waarde 0,06569 ± 0,002286).

Example 3. Fluorometry of healthy human breast skin treated with FAOD alone or in combination with FN3K. Human healthy breast tissue was recovered after surgery. Subcutaneous fat tissue and fascia were carefully removed by a skilled plastic surgeon. Healthy human breast skin is then incubated for 3 hr with 25 mM glycolaldehyde (Sigma-Aldrich, Tokyo Japan) in PBS at 37°C [46, 47], rinsed with PBS for 60 min in an ultrasonic bath (Branson 3510MT, Danbury, USA) and stored overnight in fresh PBS at 4°C. Breast skin fragments were then placed in a black 96-well-plaat (FluoroNunc PolySorp, Thermo Fisher Scientific, Massachusetts, USA) and were incubated for 6 hr at 37°C with 20 mΐ of a solution of FN3K (250 pg/mL) and its cofactors (5 mM ATP-2 mM MgCF), 20 mΐ of a solution of FAOD 10 U/mL, or combination of 20 mΐ FN3K 250pg/mL + cofactors (ATP 5 mmol/L + MgCF 2 mmol/L) and 20 mΐ FAOD 10 U/mL. As a control, PBS with only cofactors 5 mM ATP-2 mM MgCF were used. FAOD was purchased from Creative Enzymes, Shirley, NY. AGEs were quantified based on Maillard-type autofluorescence (AF) measurements (excitation 365 nm, emission 390-700 nm) using a Flame miniature spectrometer (FLAME-S-VIS-NIR- ES, 350-1000 nm, Ocean Optics, Dunedin, FL, USA) equipped with a high-power LED light source (365 nm, Ocean Optics) and a reflection probe (QR400-7-VIS-BX, Ocean Optics). Autofluorescence was measured after 6 hr treatment. As a control, PBS with 5 mM ATP-2 mM MgCF was used. Autofluorescence raised significantly after incubation with 25 mM glycolaldehyde. FN3K treatment resulted in a decrease of autofluorescence with 45 % (fig 3A), FAOD treated resulted in a decrease of autofluorescence with 41 % (fig 3B) and combination treatment resulted in a decrease with 60 % (fig3C). No significant changes occurred in control treated skin (fig 3D).

Example 4. Fluorometry of human skin treated with FAOD alone or in combination with FN3K and ultrasound or microneedling. Human skin tissue is recovered after surgery. Subcutaneous fat tissue and fascia is carefully removed by a skilled plastic surgeon. Human skin is then incubated for 3 hr with 25 mM glycolaldehyde (Sigma-Aldrich, Tokyo Japan) in PBS at 37°C [46, 47], rinsed with PBS for 60 min in an ultrasonic bath (Branson 3510MT, Danbury, USA) and stored overnight in fresh PBS at 4°C. Skin fragments are then placed in a black 96-well-plaat (FluoroNunc PolySorp, Thermo Fisher Scientific, Massachusetts, USA) and are treated with ultrasound waves between 5-50 MHz to penetrate epidermis, dermis and subcutis or with microneedling (13) and incubated for 6 hr at 37°C with 20 mΐ of a solution of FN3K (250 mg/mL) and its cofactors (5 mM ATP-2 mM MgCF), 20 mΐ of a solution of FAOD 10 U/mL + FAD 1 mmol/L, or combination of 20 mΐ FN3K 250mg/mL + cofactors (ATP 5 mmol/L + MgCF 2 mmol/L) and 20 mΐ FAOD 10 U/mL + FAD 1 mmol/L. As a control, PBS with only cofactors 5 mM ATP-2 mM MgCF are used. FAOD is purchased from Creative Enzymes, Shirley, NY. FAD is purchased from Sigma-Aldrich, Tokyo, Japan. AGEs are quantified based on Maillard-type autofluorescence (AF) measurements (excitation 365 nm, emission 390-700 nm) using a Flame miniature spectrometer (FLAME-S-VIS-NIR-ES, 350- 1000 nm, Ocean Optics, Dunedin, FL, USA) equipped with a high-power LED light source (365 nm, Ocean Optics) and a reflection probe (QR400-7-VIS-BX, Ocean Optics). Autofluorescence is measured after 6 hr treatment. As a control, PBS with 5 mM ATP-2 mM MgCF is used.

Example 5

Mixtures of fructose and lysine as well as mixture of fructose and arginine, as well as glucose and arginine and glucose-lysine were incubated. After an incubation at 37°C for one week, a portion of the mixture was digested by respectively fructosyl amino oxidase (FAOD, 38.3 U/mL) and fructosamine 3 kinase (F3K, 250 pg/mL; ATP, 5 mmol/L; MgCF, 2 mmol/L).

An untargeted metabolite profiling approach based on ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS) was applied.

Material and methods

Samples

Mixtures prior to enzyme treatment (group 1): • Mixture of glucose (100 mg/mL) and arginine (100 mg/mL) in water, incubated at 37°C for one week.

• Mixture of fructose (100 mg/mL) and arginine (100 mg/mL) in water, incubated at 37 °C for one week.

• Mixture of glucose (100 mg/mL) and lysine (100 mg/mL) in water, incubated at 37°C for one week.

• Mixture of fructose (100 mg/mL) and lysine (100 mg/mL) in water, incubated at 37°C for one week.

•

• Mixtures after enzyme treatment (group 2):

• Mixture Glu-Arg treated with FAOD,

• Mixture Glu-Arg treated with FN3K,

• Mixture Fru-Arg treated with FAOD,

• Mixture Fru-Arg treated with FN3K,

• Mixture Glu-Lys treated with FAOD,

• Mixture Glu-Lys treated with FN3K, .

• Mixture Fru-Lys treated with FN3K,

In addition, solutions of glucose, fructose, arginine and lysine were also provided for the purpose of optimization and the study of the MS fragmentation pattern of the these compounds.

UHPLC-HRMS conditions

The chromatographic separation was achieved on an Accela 1250 pump (Thermo Fisher Scientific), using a Zorbax RRHD Eclipse Plus reverse-phase C18 column (100A, 1.8 pm, 100 mm x 2.1 mm). The mobile phase consisted of 0.1 % (v/v) formic acid in water (eluent A), and 0.1 % (v/v) formic acid in methanol (eluent B). A gradient elution programme was applied as follows: 0 - 0.5 min: 5 % B, 0.5 - 20.0 min: 5 - 99 % B, 20.0 - 21.0 min: 99 % B, 21.0 - 24.0 min: 99 - 5 % B, 24.0 - 28.0 min: 5 % B. The mobile phase flow rate was 0.3 mL/min. The column temperature was set at 40°C and temperature of the autosampler was 10°C. The injection volume was 5 pL.

High-resolution accurate mass and tandem mass spectrometry (MS/MS) fragmentation data were obtained using a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a with heated-electrospray ionization (HESI-II) interface. The instrument was operated in the positive ionization mode. Data acquisition included full MS and data dependent MS/MS scans. The ionization source parameters were as follows: a spray voltage of 3.0 kV, a capillary temperature of 350°C, a heater temperature of 375°C, a sheath gas flow rate of 45 arbitrary units (a.u.), an auxiliary gas flow rate of 10 a.u. Daily external calibration of the HRMS was performed using the Calmix solution from Thermo Scientific over a mass range of 138-1721 Da. Online mass calibration using diisooctyl phthalate (C24H3804) as a lock mass was enabled.

Instrument control was carried out by Xcalibur 4.2 software (Thermo Fisher Scientific). For data processing, both Xcalibur and Compound Discoverer 3.3 software (Thermo Fisher Scientific) were used.

Results

Detection of relevant compounds in the investigated samples

Processing of data generated by the UHPLC-HRMS/MS analysis revealed the presence of a large number of compounds in the investigated samples (± 800 distinct compounds were detected in each sample). The majority of compounds that were detected in a given sample from group 2 (i.e. mixture of a specific amino acid and sugar incubated at 37°C, and subsequently treated by an enzyme) were also present in the corresponding sample from group 1 (i.e. mixture of that specific amino acid and sugar incubated at 37°C, but not treated enzymatically). Therefore a detected MS peak was considered to be relevant (i.e. a potential advanced glycation end-product (AGE)) when the following criteria were met:

• The compound is not present in the blank (water).

• For a given sample from group 1, the detected compound is not present in the sample obtained using the same sugar and a different amino acid.

• For a given sample from group 2, the detected compound is not present in the sample obtained using the same sugar and enzyme but a different amino acid.

Forty and 19 relevant compounds (that could possibly be AGEs) were selected for arginine- containing and lysine-containing samples, respectively.

COMPARATIVE ANALYSIS MS FAOD AND F3K digestion

Disappearing AGEs (A = arginine based, L = lysine -based)

STRONG =: more than 80% reduction (vs. untreated sample)

WEAK: between 20 and 40% reduction NOT ACTIVE: less than 20% difference

Formed products

CONCLUSION: Both F3K and FAOD can break down a variety of Advanced glycation end products. FAOD recognize substrates that are not recognized by F3K (compounds #A3 and #A4). The effect of FAOD on AGE degradation is generally more pronounced than that of F3K

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