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WO2024214791A1 - Promoteur de phosphorylation, inhibiteur de liaison et composition pharmaceutique utilisant lesdits promoteur et inhibiteur - Google Patents

Promoteur de phosphorylation, inhibiteur de liaison et composition pharmaceutique utilisant lesdits promoteur et inhibiteur Download PDF

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Publication number
WO2024214791A1
WO2024214791A1 PCT/JP2024/014720 JP2024014720W WO2024214791A1 WO 2024214791 A1 WO2024214791 A1 WO 2024214791A1 JP 2024014720 W JP2024014720 W JP 2024014720W WO 2024214791 A1 WO2024214791 A1 WO 2024214791A1
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rabeprazole
phosphorylation
erk
wdr5
pharmaceutical composition
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Japanese (ja)
Inventor
茂弘 大戸
彰夫 王子田
直哉 進藤
直哉 松永
優哉 吉田
賢吾 ▲濱▼村
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Kyushu University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a phosphorylation promoter and a binding inhibitor used mainly for improving symptoms of cognitive impairment, a pharmaceutical composition for treating hippocampal-dependent memory learning disorder containing the same, and a pharmaceutical composition for treating chronic kidney disease (CKD)-induced cognitive impairment.
  • CKD chronic kidney disease
  • CKD Chronic kidney disease
  • cardiovascular disorders and liver dysfunction are observed in CKD, and among them, central nervous system disorders represented by cognitive impairment, depression-like symptoms, and sleep disorders are frequently observed in CKD patients.
  • cognitive impairment has a prevalence of 9.8% in dialysis patients, making it difficult to manage the patient's condition and is considered a risk factor that affects the prognosis of life.
  • Patent Document 1 discloses a method for inhibiting SARM1 NADase activity and/or treating a neurodegenerative or neurological disease or disorder in a patient in need of such treatment, comprising administering a specific composition to the patient.
  • the neurodegenerative disease Alzheimer's disease, which is a type of cognitive dysfunction, is disclosed.
  • the specific composition the use of a proton pump inhibitor having a therapeutic effect on axonal damage is disclosed.
  • Patent Document 1 does not disclose any treatment methods or compositions for other cognitive functions in general besides Alzheimer's disease, and does not clarify any specific mechanisms other than those for the treatment of axonal damage.
  • the inventors have focused on the hippocampus in cognitive dysfunction, and on p-ERK, a rhythm control factor in the memory and learning brain that is expressed in the hippocampus. Since phosphorylation of ERK (extracellular signal-regulated kinase) shows a circadian rhythm that is involved in the memory and learning brain, the inventors focused on the possibility that ERK phosphorylation may be involved in the mechanism that improves the symptoms of cognitive dysfunction.
  • ERK extracellular signal-regulated kinase
  • the present invention has been made in consideration of the above circumstances, and aims to provide a phosphorylation promoter, a binding inhibitor, and a pharmaceutical composition using the same, which can be effectively used for the treatment of cognitive dysfunction, by inhibiting the binding of WD repeat-containing protein 5 (WDR5) to IQ motif-containing GTPase-activating protein 1 (IQGAP1) and promoting the phosphorylation of ERK (extracellular signal-regulated kinase).
  • WDR5 WD repeat-containing protein 5
  • IQGAP1 IQ motif-containing GTPase-activating protein 1
  • a first aspect of the present invention is an agent for promoting phosphorylation of extracellular signal-regulated kinase (ERK), which comprises rabeprazole, a rabeprazole derivative, or a salt thereof as an active ingredient.
  • ERK extracellular signal-regulated kinase
  • Aspect 2 of the present invention is an inhibitor of the binding of WD repeat-containing protein 5 (WDR5) to IQ motif-containing GTPase-activating protein 1 (IQGAP1), which contains rabeprazole, a rabeprazole derivative, or a salt thereof as an active ingredient.
  • WDR5 WD repeat-containing protein 5
  • IQGAP1 IQ motif-containing GTPase-activating protein 1
  • Aspect 3 of the present invention is a pharmaceutical composition for treating hippocampal-dependent memory and learning disorders, comprising the phosphorylation promoter described in aspect 1 or the binding inhibitor described in aspect 2, and a carrier.
  • Aspect 4 of the present invention is a pharmaceutical composition for treating chronic kidney disease (CKD)-induced cognitive impairment, comprising the phosphorylation promoter described in aspect 1 or the binding inhibitor described in aspect 2, and a carrier.
  • CKD chronic kidney disease
  • Aspect 5 of the present invention is a pharmaceutical composition for treating chronic kidney disease (CKD)-induced cognitive impairment, comprising a proton pump inhibitor and a carrier.
  • CKD chronic kidney disease
  • the present invention provides a phosphorylation promoter, a binding inhibitor, and a pharmaceutical composition using the same, which can be effectively used for the treatment of cognitive impairment by inhibiting the binding of WD repeat-containing protein 5 (WDR5) to IQ motif-containing GTPase-activating protein 1 (IQGAP1) and promoting the phosphorylation of ERK (extracellular signal-regulated kinase).
  • WDR5 WD repeat-containing protein 5
  • IQGAP1 IQ motif-containing GTPase-activating protein 1
  • FIG. 1 is a graph showing the results of measuring BUN in 5/6Nx mice in this example.
  • FIG. 1 is a graph showing the memory learning ability and ERK phosphorylation of 5/6Nx mice of this example.
  • FIG. 1 is a schematic and graphical representation showing the initial process of the primary screening in this example.
  • FIG. 2 is a graph showing the results of investigating the activity of each compound of this example.
  • FIG. 2 is a graph showing the results of measuring SRE:Luc activity for various proton pump inhibitors of this example.
  • FIG. 2 is a graph showing the effect of rabeprazole on ERK phosphorylation in the hippocampus in this example.
  • FIG. 1 is a graph showing the effect of rabeprazole on memory and learning impairment in 5/6Nx mice of this example.
  • FIG. 1 is a photographic and graphical representation showing the effect of overexpression or knockdown of WDR5 on ERK phosphorylation in NIH3T3 cells in this example.
  • FIG. 1 is a graph showing the effect of OICR-9429 in this example.
  • FIG. 1 is a photograph showing the preparation of cells in which human WDR5 and IQGAP1 proteins are overexpressed in this example, and the expression levels of each protein in the cells.
  • the phosphorylation promoter of the present embodiment is an extracellular signal-regulated kinase (ERK) phosphorylation promoter containing rabeprazole, a rabeprazole derivative, or a salt thereof as an active ingredient.
  • ERK extracellular signal-regulated kinase
  • any of rabeprazole, rabeprazole derivatives, or salts thereof can be used as the active ingredient.
  • rabeprazole derivatives that can be used in this embodiment include: Formula (1): NS-18-008, Deoxyrabeprazole, molecular weight 343.45 Formula (2): NS-18-009, Oxyrabeprazole, molecular weight 375.44 Formula (3): NS-18-001, molecular weight 373.47 Formula (4): NS-17-100, molecular weight 357.47 Formula (5): NS-15-094, molecular weight 413.49 Formula (6): NS-15-092, molecular weight 397.49 The following formula (7): 2-( ⁇ [4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl ⁇ sulfinyl)-1H-benzo[d]imidazole (rabeprazole) The following formula (8): N-(2- ⁇ 2-[2-(2- ⁇ [2-( ⁇ [2-(
  • the active ingredient in this embodiment is particularly preferably rabeprazole.
  • the phosphorylation promoter of this embodiment is an agent for promoting phosphorylation of extracellular signal-regulated kinase (ERK). It is known that phosphorylated p-ERK is expressed in the hippocampus as a rhythmic regulator of memory learning ability. It is also known that cAMP is expressed in the same way. ERK has a circadian rhythm in its phosphorylation stage, and is thought to contribute to rhythm formation as a factor that improves memory learning ability.
  • ERK extracellular signal-regulated kinase
  • the above-mentioned active ingredient inhibits the binding of WD repeat-containing protein 5 (WDR5) to IQ motif-containing GTPase activating protein 1 (IQGAP1). As a result, it promotes the binding of MEK protein, which promotes the phosphorylation of ERK, to IQGAP1, and promotes the phosphorylation of ERK. As a result, it has the effect of improving hippocampus-dependent memory learning disorder, such as CKD-induced cognitive dysfunction, by improving memory learning ability. In addition, the active ingredient of this embodiment promotes phosphorylation of ERK, thereby widely improving disorders involving ERK phosphorylation.
  • amyloid ⁇ expression is known to be a cause of cognitive dysfunction
  • p-ERK expression is known to be reduced in amyloid ⁇ -expressing mice.
  • the active ingredient of this embodiment may be widely applicable to improving disorders in which reduced p-ERK expression is observed by promoting phosphorylation of ERK.
  • the amount of the active ingredient contained in the phosphorylation promoter of this embodiment is not particularly limited, but as a guideline, in the case of surgical administration such as intraperitoneal administration, it is preferable that the amount is 10 to 250 mg/kg, and more preferably 25 to 100 mg/kg, of the body weight (kg) of the organism to be administered. In addition, in the case of oral administration, it is preferable that the amount is 10 times the amount mentioned above, or about 10 to 200 mg once, 1 to 3 times the clinical amount.
  • the binding inhibitor of this embodiment is an inhibitor of the binding of WD repeat-containing protein 5 (WDR5) to IQ motif-containing GTPase-activating protein 1 (IQGAP1).
  • WDR5 WD repeat-containing protein 5
  • IQGAP1 IQ motif-containing GTPase-activating protein 1
  • the binding inhibitor contains rabeprazole, a rabeprazole derivative or a salt thereof as an active ingredient.
  • the rabeprazole, rabeprazole derivatives, or salts thereof may be the same as those of the phosphorylation promoter described above.
  • the content of the rabeprazole, rabeprazole derivatives, or salts thereof may be selected from the same as those of the phosphorylation promoter described above.
  • these active ingredients have the effect of improving hippocampal-dependent memory and learning disorders, such as CKD-induced cognitive dysfunction, by inhibiting the binding of WDR5 to IQGAP1.
  • the pharmaceutical composition of this embodiment contains the phosphorylation promoter or the binding inhibitor, and a carrier.
  • the pharmaceutical composition of this embodiment is for treating hippocampal-dependent memory and learning disorders.
  • Hippocampal-dependent memory and learning disorders broadly refer to disorders of memory and learning functions associated with the function of the hippocampus, which is the brain region that controls memory and learning, which are considered to be higher cognitive functions. For example, it is known that lesions in hippocampal tissue occur when cognitive function is impaired.
  • the hippocampus-dependent memory and learning disorder refers to a memory and learning disorder caused by a decrease in the expression of p-ERK (phosphorylated ERK) in the hippocampus. This decrease occurs, for example, by a decrease in the amount of ERK phosphorylation in the hippocampus.
  • p-ERK phosphorylated ERK
  • the pharmaceutical composition of this embodiment promotes ERK phosphorylation by the phosphorylation promoter or the binding inhibitor, and has the effect of treating hippocampus-dependent memory and learning disorders.
  • the pharmaceutical composition of the present embodiment improves the cognitive function of a subject.
  • the cognitive function broadly refers to the functions of an organism to collect and perceive external information, memorize and make decisions, and functions related thereto.
  • cognitive functions include attention, judgment, and memory.
  • Cognitive function refers to the cognitive functions possessed by all living organisms, and in particular those possessed by humans. Improving cognitive function refers broadly to improving these functions, but in particular refers to a subject whose cognitive function has declined recovering to the state it was before the decline. Factors that can cause cognitive function to decline include various diseases, as well as aging.
  • Specific indicators of cognitive function can include those that are conventionally known, but in this embodiment, memory learning ability is particularly preferable. Memory learning ability can be measured, in particular, by a passive avoidance test.
  • the carrier contained in the pharmaceutical composition of the present embodiment may generally contain a pharma- ceutical acceptable carrier.
  • pharma- ceutical acceptable carrier those generally used in the preparation of pharmaceutical compositions can be used without any particular limitation. More specifically, for example, binders such as gelatin, cornstarch, tragacanth gum, gum arabic, etc.; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; and solvents for injections such as water, ethanol, and glycerin.
  • the pharmaceutical composition of the present embodiment may contain the above-mentioned components and may also contain optional components described below.
  • the pharmaceutical composition may be prepared in various forms, for example, in the form of a solid such as a powder or tablet, or in the form of a liquid.
  • the total weight of the pharmaceutical composition of this embodiment can be appropriately selected so long as the content of the active ingredient per one intake of the composition is within the above-mentioned range.
  • the pharmaceutical composition can be formulated by appropriately combining the active ingredient with the above-mentioned pharma- ceutically acceptable carriers and additives, and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.
  • the pharmaceutical composition may contain additives.
  • additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, etc.
  • lubricants such as calcium stearate and magnesium stearate
  • sweeteners such as sucrose, lactose, saccharin and maltitol
  • flavorings such as peppermint and saffron oil
  • stabilizers such as benzyl alcohol and phenol
  • buffers such as phosphates and sodium acetate
  • solubilizers such as benzyl benzoate and benzyl alcohol
  • antioxidants preservatives, etc.
  • the pharmaceutical composition of this embodiment may be in a dosage form for oral use or a dosage form for parenteral use, but a dosage form for parenteral use is preferred.
  • Dosage forms for oral use include, for example, tablets, capsules, elixirs, microcapsules, etc.
  • Dosage forms for parenteral use include, for example, injections, etc.
  • the pharmaceutical composition may be used in combination with at least one selected from the group consisting of therapeutic agents for other diseases. These therapeutic agents may be mixed and administered as a single pharmaceutical composition, or may be administered as separate formulations. Furthermore, each formulation may be administered by the same administration route, or by separate administration routes. Furthermore, each formulation may be administered simultaneously, sequentially, or separately with a certain time or period between them. In one embodiment, the pharmaceutical composition for treating osteoarthritis and the other drug may be prepared as a kit that includes them.
  • the subjects to which the pharmaceutical composition of this embodiment is administered are not particularly limited, and examples include humans, monkeys, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, and cells thereof. Among these, mammals or mammalian cells are preferred, and humans or human cells are particularly preferred.
  • the administration route is preferably a parenteral administration route such as subcutaneous administration, transdermal administration, intramuscular administration, or intra-articular administration, and intra-articular administration is particularly preferred.
  • the pharmaceutical composition of this embodiment is preferably for intra-articular injection.
  • CKD chronic kidney disease
  • the pharmaceutical composition of this embodiment is for treating CKD-induced cognitive dysfunction.
  • CKD refers to a condition in which kidney damage or a decrease in glomerular filtration rate, which is an indicator of kidney function, continues for more than three months, and may be accompanied by central nervous system disorders such as cognitive dysfunction, depression-like symptoms, and sleep disorders. In this embodiment, it broadly refers to cognitive dysfunction induced by CKD.
  • CKD-induced cognitive dysfunction may also be the aforementioned hippocampus-dependent memory and learning impairment.
  • the component composition of the pharmaceutical composition for treating CKD-induced cognitive impairment of this embodiment can be selected from the same components as those of the pharmaceutical composition for treating hippocampal-dependent memory and learning disorders described above.
  • the action and effect (mechanism) is also similar to that of the pharmaceutical composition for treating hippocampal-dependent memory and learning disorders described above.
  • the inventors focused on the relationship between p-ERK signals expressed in the mouse hippocampus and the diurnal rhythm of memory and learning ability, and targeted CKD-induced cognitive impairment, using pathological model mice to search for and identify novel compounds from approved drugs that activate hippocampal p-ERK and improve cognitive impairment.
  • CKD Chronic kidney disease
  • WDR5 is involved in rabeprazole's ERK phosphorylation activation.
  • CKD model mice created by 5/6Nx showed a significant decrease in hippocampal-dependent memory and learning ability compared to sham mice in the passive avoidance test, and tended to develop CKD-induced memory and learning impairment.
  • this memory and learning impairment corresponded to a decrease in hippocampal ERK phosphorylation.
  • the increased function of ERK phosphorylation expressed in the hippocampus is completely important for memory and learning ability in CKD-induced memory and learning impairment.
  • Rabeprazole is originally a drug that acts as a proton pump inhibitor, but it is also known as a drug that exhibits antitumor effects. However, its effect on memory and learning ability and its effect of activating ERK phosphorylation were not yet known.
  • the present inventors investigated the relationship between proton pump inhibitors, which include rabeprazole, and cognitive function, and reviewed Patent Document 1 and other documents, but the mechanism by which proton pump inhibitors are involved other than in axonal damage, and the therapeutic effect on cognitive impairment other than Alzheimer's disease, were unknown.
  • rabeprazole improves memory and learning impairment in CKD model mice, as described in the Examples below. As a result, after five consecutive days of administration, memory and learning ability was significantly improved. Furthermore, it was assumed from the blood BUN and creatinine values that this effect was not mediated by improvement of renal function. Furthermore, the mechanism by which rabeprazole increases ERK phosphorylation was analyzed. In leukemia, fusion proteins are found due to gene translocation. Among these, there is the MLL1-WDR5 protein, and it is thought that the action of rabeprazole may exhibit antitumor activity. Therefore, hippocampal proteins were used to examine whether rabeprazole interacts with WDR5. As a result, it was revealed that rabeprazole interacts with WDR5 expressed in the hippocampus.
  • IQGAP1 was identified by mass spectrometry as a protein with which WDR5 interacts.
  • rabeprazole increases the phosphorylation activity of ERK via MEK by inhibiting the binding of WDR5 to IQGAP was clarified.
  • rabeprazole may reduce the risk of developing dementia or may prevent it, and therefore found that in humans, rabeprazole may also have an effect on preventing the onset of dementia by affecting the hippocampus in the central nervous system and inducing phosphorylation activation of ERK.
  • This technology may be applicable to elucidating the pathology of CKD-induced central nervous system disorders by targeting WDR5, elucidating their relationship with the biological clock, and developing new diagnostic and therapeutic methods using these findings.
  • rabeprazole is not thought to have the ability to regulate central nervous transmission itself, the memory and learning ability improving effect discovered in this study is thought to be applicable as a seed compound for future drug discovery.
  • the present invention provides a method for treating hippocampal-dependent memory and learning impairment, comprising administering to a patient in need of treatment an effective amount of a pharmaceutical composition containing the phosphorylation promoter or the binding inhibitor as an active ingredient.
  • the present invention provides a method for treating chronic kidney disease (CKD)-induced cognitive impairment, comprising administering to a patient in need of treatment an effective amount of a pharmaceutical composition containing the phosphorylation promoter or the binding inhibitor as an active ingredient.
  • CKD chronic kidney disease
  • the present invention provides a use of rabeprazole, a rabeprazole derivative, or a salt thereof for producing the phosphorylation promoter or the binding inhibitor.
  • the present invention provides use of the phosphorylation promoter or the binding inhibitor for producing the pharmaceutical composition.
  • CKD model mice were prepared using 5/6 nephrectomy mice (5/6Nx) and used in the experiment.
  • 5/6Nx 5/6 nephrectomy mice
  • 7-week-old ICR male mice were first shaved backward from the last rib under anesthesia and disinfected with alcohol. The posterior part of the thorax was palpated to confirm the position of the kidney, and the skin was incised parallel to the midline using straight surgical scissors.
  • the abdominal external oblique muscle was incised in the same way as the skin in a place without blood vessels.
  • the kidney was confirmed, and the adrenal gland and kidney were separated, and the adrenal gland were returned to the abdominal cavity.
  • the renal artery and vein were simultaneously clamped with an arterial clamp to stop bleeding. Thereafter, the renal artery and vein were cut with straight surgical scissors at the top and bottom, and the cut surface was subjected to hemostasis with a biological instant adhesive (Aron Alpha (registered trademark)).
  • Mouse fetal fibroblast-derived NIH3T3 cells were obtained from the Medical Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University, and mouse neuroblastoma C-1300N cells were obtained from the RIKEN BioResource Center Cell Bank (Ibaraki, Japan). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Moregate Biotech, Bulimba, Australia) and 0.5% penicillin-streptomycin (Invitrogen, Grand Island, NY, USA) at 37°C under 5% CO2 /95% conditions and used in each experiment.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Moregate Biotech, Bulimba, Australia
  • penicillin-streptomycin Invitrogen, Grand Island, NY, USA
  • mice The hippocampus-dependent memory learning ability of mice was evaluated by a passive avoidance test.
  • the experimental equipment used was a MPB-M010 sensor-equipped step-through cage (for mice), an ST-10 step-through timer, and an SG-1000E shock generator (MELQUEST, Toyama, Japan).
  • the test was conducted on a two-day schedule consisting of Training, which conditions memory formation, and Test, which evaluates memory formation and retention (Hippocampus 18:11-19, 2008, Proc Natl Acad Sci USA 107: 4436-4441, 2010., J Radiat Res 49: 517-526, 2008.).
  • the mouse was placed in the light room of the apparatus and allowed to acclimate for 2 minutes, after which the partition door was opened and time measurement was started.
  • the door was quickly closed and a foot shock (0.5 mA, 3 seconds) was presented (time measurement stopped automatically).
  • a foot shock 0.5 mA, 3 seconds
  • the mouse was kept in the dark room for 1 minute, and the mouse was given electrical stimulation and spatial stimulation in the dark room more firmly to promote hippocampus-dependent memory formation.
  • the mouse was then removed from the light room and placed in a normal breeding environment. The test was performed 24 hours after training.
  • the mouse was again placed in the device from the light room and allowed to acclimate for 20 seconds, and the door was opened and the time it took to move from the light room to the dark room (latency time; LT) was measured.
  • the test during the dark period was performed under red light.
  • the memory learning ability of each individual was evaluated using the LT extension time at the test based on the training time as an index, and a score set according to the degree of extension.
  • the measurement conditions of the test device were set as follows. END TIME: 5min FOOT SHOCK DELAY: 2.0min SENSOR ACCEPT TIME: 1.0s SHOCK STOP TIME: 3.0s
  • Rabeprazole administration experiment in mice In this study, rabeprazole hydrochloride was used as a p-ERK activator. Rabeprazole was prepared as a suspension in physiological saline just before use, and administered to mice by intraperitoneal injection at a dose of 50 mg/kg once a day for five days. Training was performed on the final day of administration, and behavioral tests were performed at a later date.
  • Nuclear and cytoplasmic proteins were extracted from cells and mouse hippocampal tissue using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc., IL, USA) according to the attached protocol.
  • the prepared proteins were separated on SDS-polyacrylamide gel, transferred to a PVDF membrane, and reacted with anti-p-ERK antibody (#9101; Cell Signaling, Japan), anti-ERK antibody (#9102; Cell Signaling, Japan), anti-WDR5 antibody (#13105; Cell Signaling), or anti- ⁇ -actin antibody (sc-539599; Santa Cruz), followed by peroxidase-labeled secondary antibody.
  • Bands were detected using Chemi-Lumi One (Nacalai Tesque Inc., Kyoto, Japan), and images were captured using LAS-3000 (Fuji Film, Tokyo, Japan).
  • NIH3T3 cells, astrocytes, and C1300 cells cultured to semi-confluence were transfected with SRE::Luc reporter vector (100 ng) using Lipofectamine LTX and PLUS reagent (Life Technologies Cor.). Correction of transfection efficiency was performed using phRL-TK vector (Promega). After 24 hours from transfection, cells were harvested using Passive lysis buffer (Promega), and luciferase activity was measured according to the protocol of Dual-Luciferase reporter assay system (Promega).
  • the SRE::Luc reporter vector plasmid was introduced into NIH3T3 cells by lipofection using Lipofectamin-LTX reagent (Invitrogen) according to the recommended protocol. After 24 hours of culture from transfection, the cells were exposed to G418 sulfate at a concentration of 8000 ⁇ g/mL, and the medium was repeatedly exchanged to select cells into which the gene had been introduced. Single cell cloning was then performed by limiting dilution.
  • the prepared SRE::Luc NIH3T3 cells were seeded at 4000 cells/well on a 384-well LIA white plate (Cat No. 781080, Greiner).
  • a micro plate dispenser Multidrop combi (Thermo Fisher Scientific, Massachusetts, MA) was used for cell seeding.
  • 78 ⁇ L of DMEM Phenol Red Free, FBS 10%
  • epMotion 96 Eppendorf was used, and the compound solution was added to the cell plate at 5 ⁇ L/well (final volume 50 ⁇ L, final compound concentration 2.5 ⁇ M, final DMSO concentration 0.25%), and luciferase activity was measured 24 hours later.
  • Bright-GloTM Luciferase Assay System Promega was used, and after addition at 10 ⁇ L/well, the cells were incubated at 37° C. for 10 minutes and then measured.
  • EnSpire Multimode Plate Reader Perkin Elmer, Massachusetts, MA
  • the SER-Luc vector was purchased from Promega, and the Wdr5 (Myc-DDK-tagged) expression vector was purchased from ORIGEN.
  • WDR5 shRNA WDR5 (NM_080848_518) shRNA was purchased from Invitrogen.
  • Renal function assessment in 5/6Nx mice First, renal function in CDK was evaluated to examine whether 5/6Nx mice could be used as a test system for evaluating renal function. As a CKD model mouse, a 5/6 nephrectomy mouse (5/6Nx), which is a model mouse of progressive renal failure, was used. First, to examine the effect of 5/6Nx on renal function, blood urea nitrogen (BUN) was measured weekly for 8 weeks after surgery.
  • BUN blood urea nitrogen
  • FIG. 1 is a graph showing the results of measuring BUN in 5/6Nx mice.
  • the BUN concentration in 5/6Nx mice showed a transient increase in the first week after surgery, but converged between the second and fourth weeks after surgery, and then tended to increase gradually until the eighth week after surgery.
  • no change in the BUN concentration was observed in the Sham mice throughout the eight weeks after surgery.
  • mice exhibit hippocampal-dependent memory and learning impairment, which is thought to be one symptom of CKD-induced cognitive impairment, and that the pathology may involve inhibition of ERK phosphorylation in the hippocampus.
  • FIG. 4 is a graph showing the results of investigating the activity of each compound.
  • the graph in (a) shows a comparison of the ERK phosphorylation activity of the compounds, designated as compound numbers 1 to 13, in terms of luciferase activity.
  • FIG. 5 is a graph showing the results of measuring the SRE:Luc activity of various proton pump inhibitors.
  • (b) shows the names of the compounds A to H in (a).
  • FIG. 6 is a graph showing the effect of rabeprazole on ERK phosphorylation in the hippocampus. (a) shows the concentration of rabeprazole in the hippocampus. After administration of rabeprazole (50 mg/kg, i.p.
  • FIG. 7 is a graph showing the effect of rabeprazole on the impairment of memory and learning ability in 5/6Nx mice.
  • (a) shows the results of the passive avoidance test of WAY-100635 in each mouse. Rabeprazole (50 mg/kg) or saline was administered i.p. (intraperitoneal administration) for 5 days.
  • the latency time extension score which is an index of memory learning ability
  • 5/6Nx mice were administered rabeprazole, an ERK phosphorylation activator, and the effect on hippocampal memory learning disorders was examined.
  • rabeprazole an ERK phosphorylation activator
  • Rabeprazole is a proton pump inhibitor, and its interaction with the fusion mutant protein MLL1-WD repeat-containing protein 5 (WDR5) observed in leukemia has been suggested (Eur J Med Chem 188,112027, 2020.). Therefore, we investigated whether biotinylated rabeprazole (biotinylated rabeprazole) binds to WDR5 protein using mouse hippocampal proteins.
  • FIG. 8 is a photographic and graphical representation showing the effect of overexpression or knockdown of WDR5 on ERK phosphorylation in NIH3T3 cells.
  • (a) shows the interaction of WDR5 protein with 2.5 ⁇ M biotin-rabeprazole.
  • (b) is a gel photograph showing the effect of overexpression or knockdown of WDR5 on ERK phosphorylation.
  • WDR5 is associated with the pathology of leukemia by promoting histone H3K4 trimethylation as an MLL fusion protein, and a WDR5 inhibitor (OICR-9429) has been developed as a target protein for anticancer drugs. Therefore, we used the existing WDR5 inhibitor OICR-9429 to examine its effect on the phosphorylation activity of ERK using SER::Luc as an indicator. 9 is a graph showing the effect of OICR-9429. The effect of OICR-9429 on SRE::Luc activity is the value examined 24 hours after the addition of each concentration in the figure to NIH3T3 cells.
  • FIG. 10 is a photographic diagram showing the preparation of cells overexpressing human WDR5 and IQGAP1 proteins and the expression levels of each protein in the cells.
  • (a) is a photograph showing the generation of cells overexpressing human WDR5 and IQGAP1 proteins, and the expression levels of GFP-IQGAP1 and Flag-WDR5 proteins, respectively.
  • (b) shows the expression levels of GFP-IQGAP1, WDR5, MEK and ERK proteins in cells treated with 1.25 ⁇ M or 2.5 ⁇ M rabeprazole.
  • rabeprazole inhibits the interaction of WDR5 with IQGAP1, promoting the binding of MEK, which promotes ERK phosphorylation, to IQGAP1, thereby activating ERK phosphorylation.
  • Rabeprazole is known as a proton pump inhibitor, which has traditionally been used as a drug to suppress the secretion of digestive fluids, such as gastric acid. In other words, it may be used for diseases unrelated to cognitive function, including the treatment and prevention of digestive diseases. However, even if the clinical data is for diseases unrelated to cognitive function, if it is recorded whether the patient was administered rabeprazole and whether they developed dementia, it is possible to verify the association between dementia and rabeprazole by examining the existing clinical data.
  • Table 1 examines whether not taking rabeprazole is a risk factor for cognitive impairment in all patients in the data set. In other words, it shows the correlation between taking rabeprazole and reducing the risk.
  • Table 2 shows the respective odds and odds ratios, and Table 3 shows the confidence intervals. Here, if the odds ratio is high, specifically if the odds are 1 or more, this indicates that the risk increases if rabeprazole is not taken.
  • Table 4 examines whether not taking proton pump inhibitors (PPIs) in general is a risk factor for cognitive impairment in the entire patient population. In other words, it shows the correlation between taking PPIs and reducing the risk.
  • Table 5 shows the respective odds and odds ratios, and Table 6 shows the confidence intervals. Here, if the odds ratio is high, specifically, if the odds are 1 or more, this indicates that the risk increases if PPI is not taken.
  • the present invention provides a phosphorylation promoter, a binding inhibitor, and a pharmaceutical composition using the same, which can be effectively used for the treatment of cognitive impairment by inhibiting the binding of WD repeat-containing protein 5 (WDR5) to IQ motif-containing GTPase-activating protein 1 (IQGAP1) and promoting the phosphorylation of ERK (extracellular signal-regulated kinase).
  • WDR5 WD repeat-containing protein 5
  • IQGAP1 IQ motif-containing GTPase-activating protein 1

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Abstract

L'invention concerne : un promoteur de phosphorylation pour des kinases régulées par un signal extracellulaire (ERK) et un inhibiteur de liaison permettant d'inhiber la liaison d'une protéine 5 contenant un motif de répétition WD (WDR5) à une protéine 1 d'activation de GTPase contenant un motif IQ (IQGAP1), qui peut être efficacement utilisé pour traiter une déficience cognitive ou similaire en inhibant la liaison de WDR5 à IQGAP1 et en favorisant la phosphorylation de l'ERK ; et une composition pharmaceutique comprenant le promoteur et l'inhibiteur, la composition étant destinée au traitement d'une déficience cognitive induite par une maladie rénale chronique (CKD). L'invention concerne : le promoteur de phosphorylation et l'inhibiteur de liaison qui contiennent, en tant que principe actif, du rabéprazole, un dérivé de rabéprazole ou un sel de celui-ci ; et la composition pharmaceutique comprenant le promoteur de phosphorylation et l'inhibiteur de liaison.
PCT/JP2024/014720 2023-04-11 2024-04-11 Promoteur de phosphorylation, inhibiteur de liaison et composition pharmaceutique utilisant lesdits promoteur et inhibiteur Pending WO2024214791A1 (fr)

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
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CHEEMA EJAZ: "Investigating the association of proton pump inhibitors with chronic kidney disease and its impact on clinical practice and future research: a review", JOURNAL OF PHARMACEUTICAL POLICY AND PRACTICE, BIOMED CENTRAL LTD, LONDON, UK, vol. 12, no. 1, 1 December 2019 (2019-12-01), London, UK , XP093221301, ISSN: 2052-3211, DOI: 10.1186/s40545-019-0167-0 *
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