Novel olsalazine derivative compound and composition for preventing or treating thrombocytopenia, comprising same as active ingredient

Abstract

One aspect relates to: olsalkene; a preparation method therefor; a composition for preventing or treating thrombocytopenia, comprising same; and use thereof. According to one aspect, it has been identified that olsalkene is not cleaved by azoreductase (AzoR) and binds to biliverdin reductase (BLVRB) in the body, thereby inhibiting the reduction of biliverdin (BV) to bilirubin (BR). In addition, it has been identified that platelet production is promoted through the inhibition of biliverdin reductase B activity. Therefore, the olsalkene can be effectively used as a therapeutic agent for thrombocytopenia or as a health functional food through the role of promoting platelet production by inhibiting the activity of biliverdin reductase B.

Description

Novel olsalazine derivative compound and composition for preventing or treating thrombocytopenia containing the same as an active ingredient

[0001] An example of the present invention relates to Olsalkene (OSK), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, a method for preparing the same, a composition comprising the same as an active ingredient for preventing or treating thrombocytopenia, and a use of the composition.

[0002] Platelets are derived from megakaryocytes (MKs), which differentiate from pluripotent hematopoietic stem cells. Platelets play a central role in diverse biological processes, including hemostasis and thrombosis, as well as inflammation, angiogenesis, innate and adaptive immunity, and tumor metastasis. Therefore, regulating platelet counts and controlling specific platelet responses are key goals for new drugs treating platelet-related diseases. Platelet dysregulation can lead to various bleeding disorders, including thrombocytopenia. Common treatments for thrombocytopenia involve increasing platelet counts through platelet induction or platelet transfusions. Conversely, thrombocytosis, in contrast to thrombocytopenia, refers to an excess of platelets in the blood. Large-scale platelet transcriptome sequencing analysis of primary (essential) and secondary (reactive) thrombocythemia cohorts revealed that biliverdin reductase B (BLVRB) regulates platelet production through MK differentiation by modulating reactive oxygen species (ROS).

[0003] In the heme degradation pathway, BLVRB utilizes NAD(P)H downstream of heme oxygenase(s)-1 (inducible HMOX1) and -2 (constitutive HMOX2) to reduce biliverdin (BV)-IXβ to bilirubin (BR)-IXβ. The product of this process, BR, acts as a potent antioxidant and exhibits cytoprotective effects, although high concentrations can induce toxicity. Therefore, the BV / BR redox cycle under the control of BLVRB plays a crucial role in regulating reactive oxygen species (ROS). Studies using induced pluripotent stem cells (iPSCs) expressing a loss-of-function mutant of BLVRB (BLVRBS111L) revealed that transformed CD34+ / BLVRBS111L hematopoietic stem cells (HSCs) exhibited significantly increased ROS accumulation and proliferation, as measured by MK colony formation (CFU-MK). In contrast, wild-type BLVRB did not exhibit these effects. The loss-of-function mutant (BLVRBS111L) induces MK differentiation and, consequently, promotes platelet production through ROS accumulation. Therefore, effectively removing the antioxidant BR by inhibiting BLVRB activity may provide a novel strategy to enhance platelet production by leveraging BLVRB's unique redox regulation within the heme degradation pathway.

[0004] Xanthene dyes and acridine-containing compounds have been developed to inhibit BLVRB activity. These compounds are structurally similar to the natural BLVRB substrate, flavin mononucleotide (FMN). Among the xanthine dyes, erythrosin B and phloxine B have emerged as the most potent inhibitors. Molecular modeling and X-ray crystallography studies have revealed that erythrosin B and phloxine B bind to the active site of BLVRB. Furthermore, a hydrogen bonding network within the BLVRB active site has been elucidated, highlighting the important role of the S111 residue in catalytic activity. Interestingly, linear Weaver-Burk plot analysis revealed that these inhibitors bind similarly to the BLVRB binding pocket, but in a noncompetitive manner, although not as deeply as the natural substrate, FMN. Both erythrosin B and phloxine B have been utilized as food colorants, and phloxine B has also exhibited antibacterial activity against various Gram-positive bacteria. However, chronic administration of erythrosin B has been associated with the promotion of thyroid tumors in rats. Furthermore, NMR and dynamic light scattering (DLS) experiments confirmed that both erythrosin B and phloxin B induce multimerization of BLVRB, thereby reducing its therapeutic efficacy. Therefore, new drug candidates targeting BLVRB are needed to treat platelet disorders.

[0005] To pursue this goal, a drug repurposing approach was used to screen novel candidates, resulting in the identification of 20 potential inhibitors. Among the inhibitors derived from repurposing FDA-approved drugs, olsalazine (OSA) exhibited the strongest inhibitory affinity. Recent studies have reported that olsalazine is degraded in various human gastrointestinal environments. Olsalazine contains a diazenylated bond that is readily cleavable by abundant azoreductase (AzoR).

[0006] Therefore, the present inventors focused on developing a new compound that retains the inhibitory, biochemical, and biophysical properties of olsalazine while being cleavable by AzoR. To this end, the inventors devised a novel chemical scheme that replaced the diazenylic bond in olsalazine with an alkene bond, resulting in olsalkene (OSK, (E)-5,5'-(ethene-1,2-diyl)bis(2-hydroxybenzoic acid)).

[0007] One aspect is to provide Olsalkene, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, represented by the following chemical formula 1.

[0008] [Chemical Formula 1]

[0009]

[0010] Another aspect is to provide a method for producing the above Olsalkene.

[0011] Another aspect is to provide a pharmaceutical composition for preventing or treating thrombocytopenia, comprising the above Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Another aspect is to provide a pharmaceutical preparation for preventing or treating thrombocytopenia, comprising the pharmaceutical composition described above.

[0013] Another aspect is to provide a health functional food composition for preventing or improving thrombocytopenia, comprising the above-mentioned Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another aspect provides a method for preventing or treating thrombocytopenia comprising administering Olsalkene or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0015] Another aspect provides the use of Olsalkene and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the prevention or treatment of thrombocytopenia.

[0016] To achieve the above purpose, Olsalkene represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[0017] [Chemical Formula 1]

[0018]

[0019] In addition, the present invention provides a method for producing Olsalkene, comprising the following steps:

[0020] A step of preparing an intermediate by reacting 5-formyl-2-hydroxybenzoic acid with methyl iodide;

[0021] A step of reacting the above intermediate with TiCl4 to produce a compound represented by the following chemical formula 2; and

[0022] A step of reacting a compound represented by chemical formula 2 with BBr3.

[0023] [Chemical Formula 2]

[0024]

[0025] In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia, comprising Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0026] In one embodiment of the present invention, the thrombocytopenia may be at least one selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

[0027] In another embodiment of the present invention, the composition may inhibit the activity of biliverdin (BV)-Ixβ reductase B (BLVRB).

[0028] In another embodiment of the present invention, the composition may inhibit the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

[0029] In addition, the present invention provides a pharmaceutical preparation for preventing or treating thrombocytopenia, comprising the pharmaceutical composition.

[0030] In one embodiment of the present invention, the formulation may be in the form of an injection, an infusion, a spray, a liquid or a patch.

[0031] In addition, the present invention provides a health functional food composition for preventing or improving thrombocytopenia, comprising Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0032] In one embodiment of the present invention, the thrombocytopenia may be at least one selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

[0033] In another embodiment of the present invention, the composition may inhibit the activity of biliverdin (BV)-Ixβ reductase B (BLVRB).

[0034] In another embodiment of the present invention, the composition may inhibit the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

[0035] In addition, the present invention provides a method for preventing or treating thrombocytopenia, comprising administering Olsalkene or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0036] The present invention also provides the use of Olsalkene and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the prevention or treatment of thrombocytopenia.

[0037] The present inventors developed olsakene, a derivative that retains the biochemical and biophysical properties of olsalazine. Unlike the existing olsalazine, olsakene is not cleaved by azoreductase (AzoR) in the body, but binds to biliverdin reductase B (BLVRB), thereby inhibiting the reduction of biliverdin (BV) to bilirubin (BR). Furthermore, they confirmed that it promotes platelet production by inhibiting the activity of biliverdin reductase B.

[0038] Through this, Olsalken can be usefully utilized as a treatment for thrombocytopenia and as a health functional food, as it acts to promote platelet production by inhibiting the activity of biliverdin reductase B.

[0039] Figures 1A and 1B illustrate the partial purification and enzyme kinetics of the AzoR enzyme. Figure 1A shows SDS-PAGE results showing the protein profile after purification via anion exchange chromatography. Figure 1B shows the enzyme activity assay of AzoR, which is indicated by the decrease in absorbance at 340 nm over time, measured by measuring the reduction of menadione in the eluted fraction.

[0040] Figures 2A to 2D illustrate the cleavage of the diazenylic bond of olsalazine (OSA) to generate 5-aminosalicylic acid (5-ASA) by azoreductase (AzoR) containing cell extracts. Figure 2A shows olsalazine in Figure 2B, and NAD in Figure 2C. + and 1D 1H NMR spectra of OSA treated with AzoR activity and NADH fractions along with the reference spectrum of 5-ASA in Figure 2D. Figure 2B shows all chemical structures of OSA, and Figure 2C shows NAD +, and Figure 2D shows the products of the AzoR catalyst of 5-ASA, with all chemical structures using NADH and OSA indicated above the corresponding spectrum.

[0041] Figures 3A to 3D show the binding of half-holo BLVRB to 5-ASA. Figure 3A shows the binding of 0.1 mM half-holo BLVRB in the absence (black) and presence (red) of 1 mM 5-ASA. 1 H- 15 N HSQC spectra are shown. Figure 3B shows the chemical shift differences between half-holo BLVRB and BLVRB in complex with OSK (black) and 5-ASA (gray). Figure 3C shows NMR titration data for binding to half-holo BLVRB. Figure 3D shows a surface representation of half-holo BLVRB bound to 5-ASA, generated using the crystal symmetry of the previously reported PDB structure (7ERA).

[0042] Figures 4A to 4C show 1D 1H NMR spectra, where Figure 4A shows the fraction of AzoR activity and OSK treated with NADH, Figure 4B shows OSK, and Figure 4C shows NADH.

[0043] Figures 5A and 5B are isothermal titration calorimetry thermograms of BLVRB through titration of OSK (Figure 5A) and OSA (Figure 5B).

[0044] Figures 6A to 6D show the binding topology of OSK within the substrate pocket as revealed by NMR spectroscopy. Figure 6A shows the binding topology of half-holo BLVRB titrated with OSK. 1 H- 15N HSQC spectra are shown. Figure 6B shows the chemical shift differences between half-holo BLVRB and BLVRB complexed with OSK. Figure 6C shows the surface of half-holo BLVRB containing FMN generated using the crystal symmetry of the previously reported PDB structure (1HE4). Figure 6D shows the surface of half-holo BLVRB using OSK derived from the crystal structure.

[0045] Figures 7A to 7C illustrate structural comparisons of BLVRB and olasalazine (OSA) and olsalkene (OSK) within the complex. Figure 7A depicts the backbone of BLVRB as a ribbon. Figure 7B superimposes the interaction sites of BLVRB with OSA or OSK. Figure 7C is a schematic diagram of the interaction network of the BLVRB:OSK complex.

[0046] Figures 8A to 8C show linear Weaver-Burk plots of BLVRB using the inhibition mechanism (Figure 8A) and OSK (Figure 8B) and OSA (Figure 8C).

[0047] Figures 9A to 9E are apo-BLVRB 1 H- 15 N HSQC spectra, with data overlaid for OSK (Fig. 9A), OSA (Fig. 9B), and PhlB (Fig. 9C) in the absence (black) and presence (gray) of PhlB. Figure 9D shows NMR titration data for apo-BLVRB binding of OSK and OSA, respectively.

[0048] Hereinafter, the present invention will be described in detail.

[0049] To achieve the above purpose, Olsalkene represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[0050] [Chemical Formula 1]

[0051]

[0052] The term "olsalkene" above refers to (E)-5,5'-(ethene-1,2-diyl)bis(2-hydroxybenzoic acid), an olsalazine derivative in which the diazenyl bond of olsalazine is replaced with an alkene bond. The present inventors confirmed that it inhibits BLVRB activity without being cleaved by azoreductase (AzoR) in the body.

[0053] The term "olsalazine" refers to a 5-aminosalicylic acid (5-ASA) derivative, an anti-inflammatory drug primarily used to treat ulcerative colitis. Olsalazine is known to inhibit BLVRB activity. However, studies have shown that olsalazine is degraded in the human gastrointestinal environment because it contains a diazenylated bond that is readily cleaved by abundant azoreductases.

[0054] The above term "stereoisomer" refers to compounds having the same molecular formula and bonding structure but different atomic arrangements in three-dimensional space.

[0055] “Pharmaceutically acceptable salt” may mean a pharmaceutically acceptable salt, hydrate, solvate or prodrug of a compound that retains the desired biological activity of the compound and exhibits minimal or no undesirable toxicological effects.

[0056] The term "salt" as used herein refers to an acidic and / or basic salt formed from an inorganic and / or organic acid and base. In one embodiment, a salt of a compound may be formed by reacting the compound of the present invention with an equivalent amount of an acid or base in a medium such as a salt precipitate or in an aqueous medium.

[0057] In addition, the present invention provides a method for producing Olsalkene of claim 1, comprising the following steps:

[0058] A step of preparing an intermediate by reacting 5-formyl-2-hydroxybenzoic acid with methyl iodide;

[0059] A step of reacting the above intermediate with TiCl4 to produce a compound represented by the following chemical formula 2; and

[0060] A step of reacting a compound represented by chemical formula 2 with BBr3.

[0061] [Chemical Formula 2]

[0062]

[0063] Meanwhile, the method for manufacturing Olsalken is not limited to the specific synthetic conditions of the examples, and a person with ordinary knowledge in the field of organic chemistry can appropriately modify and perform the method using known organic chemistry knowledge.

[0064] Unless otherwise specified, all numbers, values, and / or expressions expressing quantities of ingredients, reaction conditions, compositions, and combinations used herein are approximations that inherently reflect, among other things, the various uncertainties of measurement that arise in obtaining such values, and therefore should be understood as being modified in all instances by the term "about." Furthermore, whenever a numerical range is disclosed herein, such range is continuous and includes every value from the minimum value to the maximum value inclusive, unless otherwise indicated. Furthermore, whenever such a range refers to integers, every integer from the minimum value to the maximum value inclusive, unless otherwise indicated, is included.

[0065] In this specification, when a range is described for a variable, the variable will be understood to include all values ​​within the described range including the described endpoints of the range. For example, the range "5 to 10" will be understood to include the values ​​5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any value between integers that fall within the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, a range of "10% to 30%" will be understood to include all integers up to and including 30%, as well as any subranges such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between reasonable integers within the stated range, such as 10.5%, 15.5%, 25.5%, etc.

[0066] In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia, comprising Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0067] The term "thrombocytopenia" refers to a condition in which the number of platelets in the blood is lower than normal. Platelets play a crucial role in promoting hemostasis through clotting during bleeding. Thrombocytopenia can cause symptoms such as easy bruising and persistent bleeding. The causes of thrombocytopenia are diverse, including autoimmune diseases, infections, drug side effects, and bone marrow abnormalities.

[0068] In one embodiment of the present invention, the thrombocytopenia may be at least one selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

[0069] In another embodiment of the present invention, the composition may inhibit the activity of biliverdin (BV)-Ixβ reductase B (BLVRB).

[0070] In another embodiment of the present invention, the composition may inhibit the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

[0071] The above term "biliverdin (BV)-Ixβ reductase B (BLVRB)" refers to an enzyme that plays an important role in the heme metabolism process. Specifically, biliverdin reductase refers to an enzyme that catalyzes the reaction that reduces biliverdin to bilirubin. Here, "biliverdin (BV)" is a green pigment produced during the process of heme breakdown, and "bilirubin (BR)" is a yellow pigment that is ultimately excreted from the body through bile. Biliverdin reductase uses NADPH as a coenzyme to carry out the above reaction. This enzyme is also found in liver cells, spleen, and other tissues, and is known to have antioxidant and cytoprotective functions.

[0072] The term "prevention" above means any action that inhibits or delays the onset of thrombocytopenia, and the term "treatment" above means any action that improves or beneficially changes thrombocytopenia by administering a pharmaceutical composition according to one aspect.

[0073] In one aspect, the "pharmaceutical composition" may be provided as a pharmaceutical composition comprising the active ingredient alone or including one or more pharmaceutically acceptable carriers, excipients or diluents.

[0074] Specifically, the carrier may be, for example, a colloidal suspension, a powder, a saline solution, a lipid, a liposome, microspheres, or nano-spheres. These may be complexed or associated with a carrier vehicle and may be transported in vivo using carrier systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.

[0075] When the above pharmaceutical composition is formulated, it can be prepared using diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above pharmaceutical composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. When manufacturing in the form of eye drops, known diluents or excipients may be used.

[0076] The above pharmaceutical composition may be administered parenterally or orally, such as by external application to the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, or intrathoracic injection.

[0077] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field.

[0078] The above administration may be administered once daily or in several divided doses. For example, it may be administered every other day or once a week.

[0079] The above pharmaceutical composition may be provided mixed with a conventionally known pharmaceutical composition for the prevention or treatment of thrombocytopenia or a newly developed pharmaceutical composition for the prevention or treatment of thrombocytopenia. When the above pharmaceutical composition further includes a pharmaceutical composition for the prevention or treatment of thrombocytopenia, it is important to mix the pharmaceutical composition in an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0080] Furthermore, in one aspect, the pharmaceutical composition may be administered alone or in combination with another thrombocytopenia treatment. Specifically, the pharmaceutical composition may be administered in combination with a known composition having a preventive or therapeutic effect on thrombocytopenia or another thrombocytopenia treatment, and may be administered simultaneously, separately, or sequentially, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves maximum effect with the minimum amount possible without causing side effects, and this can be readily determined by those skilled in the art.

[0081] In addition, the present invention provides a pharmaceutical preparation for preventing or treating thrombocytopenia, comprising the pharmaceutical composition.

[0082] The above term "injection" usually refers to a solution, suspension, emulsion, or solid sterile preparation that is administered directly into body tissues or organs such as subcutaneously, intramuscularly, or into blood vessels, or that is dissolved or suspended in a solvent when used.

[0083] The above term "injectable" refers to a drug that is inserted into the body through the urethra, anus, vagina, etc. and is dissolved by body temperature or secretions before its effect is revealed.

[0084] The above term "spray" refers to a medicine that is sprayed like a mist using a device.

[0085] The above term "liquid formulation" refers to a pharmaceutical form that provides a drug in liquid form, and refers to a formulation that contains an active ingredient in liquid form.

[0086] The term "patch" refers to a preparation that adheres to the skin and provides sustained therapeutic effects. Patches have a lower risk of adverse effects, such as gastrointestinal or liver damage, due to oral medications. They can be administered to patients who have difficulty taking oral medications. Furthermore, they are absorbed directly into the bloodstream through the skin, bypassing liver metabolism. Therefore, compared to oral medications, they can be administered in lower doses while maintaining the same efficacy without adverse effects.

[0087] In one embodiment of the present invention, the formulation may be in the form of an injection, an infusion, a spray, a liquid or a patch.

[0088] In addition, the present invention provides a health functional food composition for preventing or improving thrombocytopenia, comprising Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0089] In one embodiment of the present invention, the thrombocytopenia may be at least one selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

[0090] In another embodiment of the present invention, the composition may inhibit the activity of biliverdin (BV)-Ixβ reductase B (BLVRB).

[0091] In another embodiment of the present invention, the composition may inhibit the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

[0092] The term "improvement" above may mean any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0093] In one aspect, the health functional food may be used simultaneously with or separately from a drug for treatment before or after the onset of the disease in order to prevent or improve thrombocytopenia.

[0094] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0095] The above health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further comprising one or more of a carrier, diluent, excipient, and additive. Foods to which compounds according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0096] Specific examples of the carrier, excipient, diluent and additive may include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

[0097] In addition to containing the above-mentioned effective ingredient, the above-mentioned health functional food may contain other ingredients as essential ingredients without special limitations. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, erythritol, etc. In addition to the above-mentioned flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the above-mentioned natural carbohydrates can be appropriately determined by a person skilled in the art.

[0098] In addition to the above, health functional foods according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0099] The above health functional food may be provided in combination with a conventionally known health functional food for preventing or improving thrombocytopenia, or a newly developed health functional food for preventing or improving thrombocytopenia. If the above health functional food further includes a health functional food for preventing or improving thrombocytopenia, it is important to mix the two in an amount that achieves maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0100] In addition, the health functional food may be consumed alone or in combination with a health functional food for preventing or improving thrombocytopenia. The health functional food may be consumed in combination with a known composition having a preventive or improving effect on thrombocytopenia or another health functional food for preventing or improving thrombocytopenia, and may be consumed simultaneously, separately, or sequentially, and may be consumed singly or in multiple doses. It is important to take all of the above factors into consideration and consume an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0101] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0102] Example

[0103] Experimental materials and methods

[0104] 1. Chemical substances

[0105] Olsalazine, Phloxine-B, FMN, NADH, NAD + , NADPH, NADP + , All chemicals including methylhydroquinone (2-MHQ) were purchased from Sigma Aldrich (USA) unless otherwise stated. 15 N-ammonium chloride, 13 C-glucose and 2 H-Dimethyl sulfoxide (d6-DMSO) was purchased from Cambridge Isotope Laboratories.

[0106] 2. Chemical synthesis of olsalken (OSK)

[0107] All reagents, starting materials, and anhydrous organic solvents with a purity of 99.9% or higher were procured from Sigma-Aldrich, TCI. Thin layer chromatography (TLC) was performed on silica gel 60 F. 254 The results were performed on Merck aluminum sheets using UV and visualized by staining with phosphomolybdic acid and KMnO4. Column chromatography was performed on Merck silica gel 60 (230-400 mesh) for purification of the compounds. 1 H NMR spectra were recorded on a Bruker DRX-400 spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to an internal standard, and coupling constants (J) are expressed in hertz (Hz). Product masses were determined using a Shimadzu (MALDI-TOF) mass spectrometer.

[0108] [Olsalken's Synthesis]

[0109]

[0110] 5-Formyl-2-hydroxybenzoic acid (1 g, 0.0060 mol), methyl iodide (1.49 ml, 0.0240 mol), and dried K2CO3 (2.91 g, 0.0210 mol) were added to dry DMF solvent in a 100 ml double-necked round-bottom flask stored under a nitrogen atmosphere with a magnetic stirrer overnight, and the reaction progress was monitored by TLC analysis. After completion, the crude product was quenched with water (3 × 25 ml), extracted with dichloromethane, and purified by column chromatography to obtain methyl 5-formyl-2-methoxybenzoate (950 mg). Separately, Zn powder (1.01 g, 0.0224 mol) was taken in THF at 0°C, TiCl4 (2.12 g, 0.0112 mol) was added over 30 min, the solution was continuously heated from room temperature to 60°C for 2 h, and again the reaction mixture, methyl 5-formyl-2-methoxybenzoate (500 mg, 0.0028 mol) was added dropwise in THF, refluxed for 4 h, further quenched with K2CO3 and extracted with (E)-dimethyl 5,5'-(ethene-1,2-diyl) bis(2-methoxybenzoate) (150 mg) provided in DCM.

[0111] - BBr3 (240 μL, 0.0014) was added to a solution of (E)-dimethyl 5,5'-(ethene-1,2-diyl)bis(2-methoxybenzoate) (100 mg, 0.0003 mol) in DCM at -78°C in a 10-mL round-bottom flask and stirred at room temperature for 5 h. Then, 1 mL of 1 M HCl was added for quenching, stirred well for 15 min, the solvent was removed under reduced pressure, and the crude product was dissolved in ethyl acetate and washed twice with water and brine. The resulting mixture was dissolved in 95% ethanol, followed by the addition of potassium hydroxide solution and stirred under reflux for 3 h. After precipitation occurred, the reaction mixture was acidified again with 1 M HCl and brine, extracted, and recrystallized from ethyl acetate. Finally, a colorless crystalline solid of (E)-5,5'-(ethene-1,2-diyl)bis(2-hydroxybenzoic acid) (60 mg, 72%) was obtained. 1 Further confirmation was achieved through H NMR and mass spectral analyses. 1 H NMR (CDCl3, 400MHz) δ6.96 (d, 2H,J= 8.64 Hz), 7.12 (s, 2H), 7.79 (d, 2H,J= 8.56 Hz), 7.96 (s, 2H); C 16 H 12 MALDI-TOF m / z calcd for O6: 300.27, 322.5044 (M+Na) respectively.

[0112] 3. Expression and partial purification of AzoR

[0113] Escherichia coli DH5α cells were cultured in LB medium at 37°C to obtain AzoR protein. Various hydrophilic quinones enhance the mRNA level of the acpD gene encoding AzoR protein, so that the AzoR protein was detected at 600 nm (OD 600) when the optical density of the cells reached 0.8, 0.5 mM methylhydroquinone (2-MHQ) was added to the culture medium. The cells were harvested by centrifugation after 3–4 h, and anion exchange column chromatography was performed to concentrate the AzoR protein using a Hitrap-Q HP column (Cytiva). The column was pre-calibrated with buffer (pH 8.0, 25 mM Tris-HCl) and the protein was eluted using a 1.0 M NaCl gradient. The AzoR activity of the eluted fraction was measured by an enzyme activity assay using 0.1 mM menadione and 0.1 mM NADH, and the decrease in absorption at 340 nm was measured as previously reported. A 0.1 mM stock solution of menadione was prepared in 100% DMSO. The eluted fraction was diluted 100-fold before analysis. Finally, the highest active eluted fraction was diluted 1000-fold in phosphate-based saline (PBS) buffer and used for the cleavage assay of olsalazine, to which 1% DMSO was additionally added to dissolve 0.1 mM menadione.

[0114] 4. Expression and purification of BLVRB

[0115] N-terminal His6-tagged BLVRB was expressed in Escherichia coli strain BL21 DE3 using pET-21b protein expression vector. Transformed E. coli cells were cultured in LB and M9 media at 37°C, and unlabeled and isotope-labeled ( 15 N or 13 C / 15 N) BLVRB protein was expressed. To induce protein expression, the culture medium was adjusted to OD 600When reaching 0.8, 0.5 mM IPTG was added, and cells were harvested after 3–4 h and lysed by sonication in buffer (pH 8.0, 25 mM Tris-HCl, 500 mM NaCl, 10 mM β). BLVRB protein was first purified by His-tag affinity column chromatography using a HisTrap HP column (Cytiva). To remove the His-tag, the protein eluate was dialyzed against buffer (pH 8.0, 50 mM Tris-HCl, 1 mM dithiothreitol) overnight at 4°C after adding thrombin (~5 units / mg protein). Since thrombin bound to the HiTrap-Q HP column at pH 8.0, ion exchange column chromatography was performed with buffer (pH 8.0, 25 mM Tris-HCl, and 1 mM dithiothreitol). Elution was performed with a 1.0 M NaCl gradient, eluting BLVRB and thrombin at ~150 and ~300 mM NaCl, respectively. To separate His6-tagged BLVRB from fully cleaved BLVRB, a HisTrap HP column was attached parallel to the HiTrap-Q column. The BLVRB protein was further purified by size exclusion chromatography (SEC) using a HiLoad 16 / 600 Superdex 75 (Cytiva) with unbuffered solution (50 mM NaCl and 1 mM dithiothreitol). The protein fractions were concentrated using a 10 kDa MWCO (Millipore) ultracentrifugal filter and stored at -70°C until use.

[0116] Purified BLVRB was denatured in buffer (pH 7.5, 25 mM Tris-HCl, 10 mM β-mercaptoethanol, and 6.0 M urea) and then applied to a HisTrap HP column. After washing with buffer (pH 5.0, 25 mM Na-acetate, 10 mM β-mercaptoethanol, and 6.0 M urea), the solution was eluted with a gradient of 1.0 M NaCl. After diluting the solution twofold with buffer (pH 5.0, 20 mM Na-acetate, 10 mM β-mercaptoethanol, and 6.0 M urea), apo-BLVRB was further purified by cation-exchange column chromatography using a HiTrap-SP column (Cytiva). Protein elution was performed with a gradient of 1.0 M NaCl in the presence of 6.0 M urea. The protein eluate was reconjugated by dialysis against buffer (pH 6.5, 50 mM Bis-tris, 150 mM NaCl, and 1 mM DTT). After removing aggregates (~20%) by centrifugation, the protein was purified by SEC using HiLoad 16 / 600 Superdex 75 with buffer (pH 6.5, 10 mM Bis-tris, 50 mM NaCl, 1 mM DTT). The apo-BLVRB solution was concentrated and stored at -70°C. When the frozen sample was thawed, approximately 10% of apo-BLVRB was precipitated, but the structure was not maintained. 1 H- 15 Confirmed through N HSQC spectrum.

[0117] 5. Crystallization and structural determination of the BLVRB:OSK complex

[0118] 1mM NADPH +was added to the BLVRB protein solution and further purified by size exclusion chromatography in buffer (pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, and 1 mM DTT) using HiLoad 16 / 600 Superdex 75. The protein fraction was concentrated using a 10 kDa MWCO ultracentrifugal filter. The protein supernatant (14.8 mg / ml) was integrated and stored at -70°C before use.

[0119] BLVRB crystals were grown in buffer (pH 6.5, 0.1 M Bis-Tris, 1.9–2.0 M ammonium sulfate) using the hanging drop method vapor diffusion at 18°C. To soak OSK molecules, crystals were grown in a solution of 1 mM OSK and 0.2 mM NADP. + The crystals were incubated at 18°C ​​for 1 day in the same crystallization buffer containing 1% DMSO. Since the OSK stock solution (100 mM) was prepared in 100% DMSO, 1% DMSO was additionally present in the immersion solution. Crystals of the BLVRB:OSK complex were looped and flash frozen in liquid nitrogen using 20–25% glycerol as a cryoprotectant.

[0120] X-ray diffraction data were collected at beamline 5C of the Pohang Accelerator Laboratory. The diffraction data were processed using the HKL-2000 program, and the structure was solved by molecular replacement (MR) using the previously reported BLVRB coordinates (PDB code, 7ERA). The structural model was refined by alternating model building with the Coot program and refinement with the PHENIX software package. Structural visualization was performed using the Chimera program. A schematic diagram of the interaction network for OSK and OSA complexed with BLVRB was generated using LIGPLOT.

[0121] 6. Enzyme activity analysis.

[0122] Enzyme kinetics were monitored using the absorbance of NADPH at 340 nm using a SpectraMax Gemini EM Microplate Reader (Molecular Devices). Enzyme reactions were performed with 0.5 μM BLVRB, 100 μM FMN, and 100 μM NADPH in PBS buffer containing 0.5% DMSO in the presence of various concentrations of inhibitors (OSK and OSA). The initial reaction rate was estimated from data collected over 5 min and fitted to the Michaelis–Menten equation. The inhibition model and inhibition constant were determined using an in-house Python script for nonlinear least-squares regression fitting.

[0123] 7. Isothermal titration calorimetry (ITC)

[0124] Isothermal Titration Calorimetry (ITC) experiments were performed at 25°C in buffer (pH 6.5, 50 mM Bis-tris, 50 mM NaCl, 0.1 mM TCEP) using a Microcal Auto-iTC200 (Malvern Instruments). 0.09 mM BLVRB and 1.1 mM drug (OSK or OSA) were loaded into the calorimetric cell (200 μl) and syringe (40 μl), respectively. ITC data were analyzed after subtracting the heat of a blank injection measured under identical conditions but in the absence of BLVRB. Data processing was performed using Origin software provided by the manufacturer, and fitting was performed with a single-site binding model.

[0125] 8. NMR experiment

[0126] All NMR experiments were performed using a Bruker 800 MHz spectrometer at 25°C. 0.2 mM 15 N-labeled BLVRB or apo-BLVRB were prepared in buffer (pH 6.5, 50 mM Bis-tris, 50 mM NaCl, 5% D2O) with or without drug molecules. 1 H- 15N heteronuclear single quantum coherence (HSQC) spectra were recorded. One-dimensional (1D) for monitoring enzymatic reactions. 1 H spectra were performed in PBS buffer. Chemical shift perturbation (CSP) data were (Δ 2 + [(Δ 2 Using the square root formula 1 H and 15 The relative effect from CS of N was normalized and presented. Diffusion order spectroscopy (DOSY) spectra were measured using ledbpgppr2s with an 80-millisecond diffusion delay, a 2-second relaxation delay, and 32 gradient intensity increments.

[0127] Experimental results

[0128] 1. To improve stability, diazenyls were replaced with alkene bonds to prevent azoreductase cleavage.

[0129] We tested the cleavage of OSA into 5-aminosalicylic acid (5-ASA) using a partially purified Escherichia coli (E. coli) AzoR protein (Fig. 1). Addition of methylhydroquinone (2-MHQ) to cell cultures enhanced AzoR expression.

[0130] As a result, incubation of OSA with AzoR and NADPH results in cleavage of the diazenylic bond of OSA, forming 5-ASA, the active compound responsible for the therapeutic effects of OSA in intestinal diseases, its original purpose (Fig. 2). Furthermore, the binding of 5-ASA, a product of AzoR catalysis, to BLVRB is significantly reduced (Fig. 3).

[0131] To this end, the inventors developed a new synthetic method that replaces the diazenyl bond with an alkene bond while maintaining the trans configuration.

[0132] As a result, olsalken (OSK) was generated, and it was confirmed that this olsalken was maintained intact and not cleaved by AzoR (Fig. 4).

[0133] 2. Thermodynamic analysis of the BLVRB and OSK bond

[0134] Thermodynamic quantities such as ΔH and ΔS were characterized using isothermal titration calorimetry (ITC), which directly measures the heat generated during the formation of the BLVRB-OSK complex at a constant temperature.

[0135] As a result, the dissociation constant of OSK (K D ) was found to be 140 nm (Table 1, Figure 5), indicating that OSK acts as a potent and efficient binder for BLVRB. Comparative analysis with the previously identified xanthene-based inhibitor, phloxine B, revealed that OSK exhibited a closer binding affinity. Furthermore, as shown by the enthalpic contributions observed in the ITC data, OSK exhibited a more specific interaction with BLVRB than the xanthine-based inhibitors (Table 1). The binding of OSK is primarily driven by the enthalpy change, reflecting the specific interaction between BLVRB and the compound. The difference in thermodynamic values ​​between OSA and OSK can be explained by the replacement of the diazenylic bond with an alkene bond. In summary, the ITC findings support the existence of a specific interaction between BLVRB and OSK.

[0136] [Table 1] Representative ITC data for the combination of each drug and BLVRB.

[0137]

[0138] 3. Combined Analysis of BLVRB and OSK

[0139] Protein-based NMR spectroscopy is gaining recognition as a highly effective method for identifying intermolecular interactions, including those involving small molecules and proteins. Protein-based NMR methods also provide valuable insights into binding sites, which are crucial for optimizing the positioning of small molecules within complexes. Typically, these include: 1 H- 15This involves monitoring chemical shift changes in the N HSQC spectrum. Assigning amide resonances is essential for interpreting the HSQC spectrum, and this is accomplished by the HNCA and HN(CO)CA spectra, which provide 95% of the backbone resonance assignments.

[0140] Accordingly, the inventors of the present invention titrated OSK in gradually increasing amounts to monitor the chemical shift change upon binding. 15 Utilizing N-labeled BLVRB 1 H- 15 N HSQC experiments were performed. Previous studies have shown that the FMN binding site of BLVRB is utilized for OSA binding, and Ser111 is a key residue in the catalytic mechanism of BLVRB.

[0141] Here, by monitoring the amide chemical shift changes in the HSQC spectrum, we observed that binding of OSK to BLVRB induced a change in the amide resonance of the FMN binding site (Fig. 6A). Furthermore, by analyzing the chemical shift perturbation (CSP) (Fig. 6B) and mapping OSK onto the BLVRB surface structure (Fig. 6C), we successfully verified the binding effect of OSK to the BLVRB surface (Fig. 6D). This analysis clearly confirmed that the FMN binding site, particularly the xanthine ring of FMN, is the binding site of OSK.

[0142] 4. X-ray crystal structure analysis of the BLVRB-OSK complex

[0143] The present inventors analyzed the structure of the complex through crystallization of the BLVRB-OSK complex to analyze in detail the binding of Olsalken to BLVRB.

[0144] As a result, NADP + The crystal structure of BLVRB bound to OSK was determined at a resolution of 1.7 Å. The crystals belong to the space group P21212, and each crystal asymmetric unit contains two BLVRB molecules.

[0145] The overall structure of BLVRB in complex with OSK is very similar to previously determined structures. For example, the backbone RMSD of the complex is BLVRB-NADP + -FMN (PDB ID: 1HE4) and BLVRB-NADP + -Compared to the OSA (PDB ID: 7ERA) complex, the structural similarity between BLVRB complexed with OSK and the basic substrate and BLVRB complexed with OSA was high, with 1.06 and 0.17 Å, respectively (Fig. 7A).

[0146] Specifically, the binding site of OSK is NADP + It is closely associated with the nicotinamide moiety of the cofactor, and the xanthine ring of FMN adopts an orientation similar to that of xanthine-based inhibitors such as phloxine B and erythrosine (Figure 7B). The main driving force of OSK binding is NADP. + Ring-stack interactions between the nicotinamide moiety and OSK and hydrophobic interactions involving residues S111, F113, W116, L125, V128, P152, and H153 (Figure 7C). Additionally, favorable interactions are promoted by positively charged residues R78, K120, R124, R170, and K178. In particular, OSK shifts the side chain of W116 (as observed in the FMN complex) by about 2.5 Å toward the binding pocket, and the side chain of R78 forms a hydrogen bond with OSK, potentially enhancing the binding of the drug compound. In addition, the orientation of R78 may strengthen the hydrophobic interactions between the drug compound and BLVRB.

[0147] 5. Confirmation of BLVRB activity inhibition using Olsalken

[0148] The enzyme kinetics of BLVRB in the presence of NADPH were investigated using the inhibitors OSK and OSA. The inhibition mechanism of BLVRB by OSK or OSA is shown in Figure 8A. (Here, E represents BLVRB, and I represents OSK or OSA.)

[0149] As a result, the linear Weaver-Burk plot is competitive (K ic ) and non-competitive (K iu ) showed a mixed inhibition pattern characterized by an inhibition constant (Figs. 8B to 8C). The linear Weaver-Burk plot of OSK showed a maximum rate (V m ) and Michaelis constant (K) of 61.73 ± 5.28 μM m ) showed a competitive inhibition constant (K) of 559.95 ± 100.98 nM. ic ) suggests that OSK competes with its substrate (FMN) for binding to the active site of BLVRB. Meanwhile, the non-competitive inhibition constant (K) of 606.14 ± 61.32 nM iu ) is OSK NADP + and NADPH complex BLVRB, altering its conformation and inhibiting its enzyme activity (Fig. 8B). For OSA, V of 4.71 ± 0.09 μM / min m and K of 61.57 ± 2.75 μM m Similar dynamics are observed in competitive (K ic = 551.83 ± 38.65 nM) and noncompetitive (K iu = 787.53 ± 55.17 nM) inhibition constant indicates that OSA also competes with the substrate for binding to the active site, NADP + and suggests that it interacts with the NADPH complex BLVRB (Fig. 8C).

[0150] Overall, the mixed inhibition pattern observed in OSK and OSA suggests that these inhibitors inhibit NADP +and NADPH complex BLVRB, suggesting that they can affect BLVRB activity. In addition, both OSK and OSA can bind to apo-BLVRB, which is distinct from the xanthine-based inhibitor phloxin-B, implying a better inhibitory activity of OSK (Fig. 9).

[0151] These experimental results indicate that the olsalken of the present invention can be usefully used for the prevention and treatment of thrombocytopenia by inhibiting the activity of BLVRB.

[0152] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Olsalkene represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] 2. A method for producing Olsalkene of claim 1, comprising the following steps: A step of preparing an intermediate by reacting 5-formyl-2-hydroxybenzoic acid with methyl iodide; A step of reacting the above intermediate with TiCl4 to produce a compound represented by the following chemical formula 2; and A step of reacting a compound represented by the following chemical formula 2 with BBr3. [Chemical Formula 2] 3. A pharmaceutical composition for preventing or treating thrombocytopenia, comprising Olsalkene represented by the following chemical formula 1 and a pharmaceutically acceptable salt thereof as an active ingredient. [Chemical Formula 1] 4. In claim 3, A pharmaceutical composition, characterized in that the above thrombocytopenia is at least one selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

5. In claim 3, A pharmaceutical composition characterized in that the composition inhibits the activity of biliverdin (BV)-Ixβ reductase B (BLVRB).

6. In claim 3, The composition is a pharmaceutical composition characterized in that it inhibits the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

7. A pharmaceutical preparation for preventing or treating thrombocytopenia, comprising the pharmaceutical composition of claim 3.

8. In claim 7, The above formulation is a pharmaceutical formulation in the form of an injection, infusion, spray, liquid or patch.

9. A health functional food composition for preventing or improving thrombocytopenia, comprising Olsalkene represented by the following chemical formula 1 and a pharmaceutically acceptable salt thereof as an active ingredient. [Chemical Formula 1] 10. In claim 9, A health functional food composition, characterized in that the above thrombocytopenia is at least one selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

11. In claim 9, The composition is a health functional food composition characterized in that it inhibits the activity of biliverdin (BV)-Ixβ reductase B (BLVRB).

12. In claim 9, The composition is a health functional food composition characterized in that it inhibits the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

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