Use of vitamin composition in preparing drug for preventing, treating, or delaying Alzheimer's disease

Abstract

A use of a vitamin composition in preparing a drug for preventing, treating, or delaying Alzheimer's disease. The composition comprises a B vitamins composition or an analog or derivative thereof, vitamin C or an analog or derivative thereof, or a combination of a B vitamins composition or an analog or derivative thereof with vitamin C or an analog or derivative thereof.

Claims

1. A method for treating or Alzheimer's disease, wherein the method comprises administering a composition to a subject in need thereof, wherein the composition consists of a combination of vitamins, and wherein the combination of vitamins comprises the following components based on weight ratio: 100 parts of vitamin B1, 100 parts of vitamin B2, 100 parts of vitamin B3, 100 parts of vitamin B5, 100 parts of vitamin B6, 0.1 parts of vitamin B7, 0.4 parts of vitamin B9, 0.1 parts of vitamin B12, and 150 parts of vitamin C.

2. The method of claim 1, wherein the composition further comprises an effective amount of other vitamins.

3. The method of claim 2, wherein the other vitamins comprise (i) one or more fat-soluble vitamins, (ii) vitamin A, or (iii) both one or more fat-soluble vitamins and vitamin A.

4. The method of claim 3, wherein the one or more fat-soluble vitamins are selected from the group consisting of vitamin D, vitamin E, and vitamin K.

5. The method of claim 1, wherein the vitamin B1 is thiamine, the vitamin B2 is riboflavin, the vitamin B3 is niacin, the vitamin B5 is pantothenic acid, B7 is biotin, B9 is folic acid, B12 is cyanocobalamine, and the vitamin C is ascorbic acid.

6. The method of claim 1, wherein the composition administered to the subject in need thereof is in an oral dosage form.

7. The method of claim 6, wherein the oral dosage form is chewable tablets, dispersible tablets, granules, capsules, or oral liquids.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: The latency to platform in acquisition test (D1-D5) in each group of animals.

(2) FIG. 2: The retention time of each group animals in the target quadrant (1 is the target quadrant).

(3) FIG. 3: Swimming distance of each group animals in the target quadrant (1 is the target quadrant).

(4) FIG. 4: The activity of energy metabolism related enzymes of cerebral cortex.

(5) FIG. 5: The gene expression of energy metabolism related enzyme of cerebral cortex.

DETAILED DESCRIPTION

Example 1: Therapeutic Effect of Multivitamin on ICV-STZ Indeuced AD Rats

1. Purpose of the Study

(6) The purpose of this study was to evaluate the relationship between Alzheimer's disease and energy metabolism in the brain, and further to evaluate whether vitamin compound can play a role in the prevention and treatment of Alzheimer's disease by regulating energy metabolism in the brain.

2. Experimental Materials

(7) 2.1 Reagents

(8) Streptozotocin, vitamin BC tablets (ZENSUN) containing components by weight ratio: 100 parts of vitamin B1, 100 parts of vitamin B2, 100 parts of vitamin B3, 100 parts of vitamin B5, 100 parts of vitamin B6, 0.1 parts of vitamin B7, 0.4 parts of vitamin B9, 0.1 parts of vitamin B12, 150 parts of vitamin C, liquid magnesium, vitamins (D, E, K). Chloral hydrate.

(9) Mitochondrial enzyme activity kit of Solarbio (item number of PDH:MS2103, item number of α-KGDH: MS2100, ICDHm: MS2104, SDH: MS2102), TAKARA SYBR II, total RNA Isolation Kit (TIANGEN), RT-PCR Kit (TOYOBO), Primers (Synthesized by Sangon Biotech).

(10) 2.2 Experimental Instruments and Equipment

(11) 1. Animal behavior testing equipment: Morris Water Maze of rats

(12) 2. Animal behavior video recording and analysis software

(13) 3. Rat/Mouse digital brain stereo locator

(14) 4. Rat/Mouse Brain stereotactic injection pump

(15) 5. Rat/Mouse cranial drill

(16) 6. Automated Tissue Homogenization device: Shanghai Jingxin Industrial Development Co., Ltd. JXFSTPRP-24

(17) 7. Ultrasonic cell crusher: Ningbo Xinzhi Biological Technology Co., Ltd. JY92-IIDN

(18) 8. Thermostatic Water Bath: Shanghai Pingxuan Scientific Instrument Co., Ltd. DK-8D

(19) 9. Desk centrifuge: Thermo Scientific FRESCO 17

(20) 10. Microplate reader for elisa: Molecular Devices SpectraMax M2

(21) 11. PCR instrument: BIORAD CFX-Connect TM Real-Time System Thermal Cycler Block 5020

(22) 2.2 Experimental Animals

(23) Male Wistar rat (250-320 g)

3. Experimental Design and Methods

(24) 3.1 Intraventricular Injection of Streptozocin in Rats-Preparing the Model of AD (Induced Glucose Metabolism Abnormal in the Brain)

(25) This model was an AD animal model that simulates sporadic AD (SAD) (Icv-STZ model), intracerebral glucose/energy metabolism disorder caused by streptozocin (STZ) injection through the lateral ventricle, successfully mimicked various pathological manifestations of SAD, such as oxidative stress, activation of inflammatory responses, abnormalities in the cholinergic pathway, hyperphosphorylation of tau and aggregation of AB, learning and memory dysfunction. The model needs a short time and there were some cognitive dysfunction and pathological features of AD within a few weeks. It was a relatively quick and easy method to construct AD model, which overcome the time cost of the APPIPS1/tauP301L triple transgenic mouse with AD model. Therefore, this project intends to breed the triple transgenic animal models while building the Icv-STZ model, and verify the effects of vitamins on energy metabolism, prevention and treatment of AD by the same treatment and detection methods. It is expected to repeatedly demonstrate the efficacy of vitamins through different models.

(26) The Icv-STZ model was built as below:

(27) Wistar rats (250-320 g) were anesthetized, fixed on the digital brain stereo locator, depilate the top of the head and disinfect the skin. The median incision at the top of the head was made, exposed the anterior bony, injection was made by microinjector at 0.9 mm behind the bregma, 1.5 mm to the left and right sides of the midline, 3.6 mm vertically from the surface. 5 μL of STZ (3 mg/kg) was slowly inject into the left and right ventricles, the injection time was 8 min, and the needle was left for 2 min, then was slowly withdrawn. All operations are performed under sterile conditions. The skin incision was sterilized with penicillin. Sew up the wound. The sham group received equal volume injection of normal saline.

(28) 3.2 Grouping and Administration of Icv-STZ Model Rats

(29) After the Icv-STZ model was established, rats were cultured for one week, and then drug was administered intragastrically for a period of 2 months. Experimental animals were divided into 4 groups: normal control group, model group, treatment group and positive drug group, each group has 20 animals. The specific groups are shown in the table below:

(30) TABLE-US-00001 TABLE 1 Icv-STZ model rats groups (n = 20/group) Groups Drug therapy Normal control (lcv-saline injection) vehicle Model (lcv-STZ injection) vehicle Vitamin (lcv-STZ injection) multivitamins Positive drug (lcv-STZ injection) donepezil
3.3 Verification of Icv-STZ Animal Model

(31) In order to confirm the stability and reliability of the Icv-STZ rat model, at the same time as the formal administration experiment, two other groups as the normal control group (Icy-saline) and the model group (Icv-STZ injection) were set, each group has 20 animals. Behavioral tests were performed 1 month and 2 months after the completion of modeling.

(32) I. Conversion of multivitamin BC tablets to mouse dose=(÷60 kg×12.3 mouse coefficient×5 times dose)

(33) II. Conversion of multivitamin BC tablets to rat dose=(÷60 kg×6.2 rat coefficient×5 times dose)

(34) III. Rats converted to mouse dose=1.98×

(35) Mouse converted to rat dose=0.5×

(36) IV. Animal subcutaneous dose conversion to intragastric administration dose=3×

(37) TABLE-US-00002 TABLE 2 Composition of multivitamin BC tablets and dosage Rat administration dose mg/kg Component (Intragastric administration/day) Vitamin B1 10.28 Vitamin B2 10.28 Vitamin B3 10.28 Vitamin B5 10.28 Vitamin B6 10.28 Vitamin B7 0.0103 Vitamin B9 0.0411 Vitamin B12 0.0103 Vitamin C 15.425
3.4 Reagent Preparation
a) 0.5% CMC-Na solution: 2.0 g of CMC-Na powder was weighed and 300 ml ultra-pure water was slowly added thereto; the mixture was subjected to magnetic stirring until it was completely dissolved to reach a constant volume of 400 ml, thereby preparing a clear solution of 0.5%, which was stored at 4° C. for later use.
Administration dose of multivitamin BC tablets:
Mouse dose: 1 piece of multivitamin BC tablets÷60 kg×12.3 mouse coefficient×5 times dose=1.025 piece/kg
Rat dose: 1 piece of multivitamin BC tablets÷60 kg×6.2 rat coefficient×5 times dose=0.516 piece/kg
b) Preparation of multivitamin BC tablets suspension: after grinding of appropriate multivitamin BC tablets, 0.5% CMC-Na was added thereto, and the mixture was subjected to oscillation to become homogeneous, thereby forming a stable suspension. 1.025 piece/kg per mouse and 0.516 piece/kg per rat. Drugs were administrated once in the morning and once in the evening.
Dose of positive control drug—donepezil hydrochloride:
Mouse dose: clinical dose (5 mg/d)÷60 kg×12.3 mouse coefficient=1.025 mg/kg
Rat dose: clinical dose (5 mg/d)÷60 kg×6.2 rat coefficient=0.516 mg/kg
c) Donepezil hydrochloride solution: Weighted appropriate donepezil hydrochloride powder, 0.5% CMC-Na was added thereto, and the mixture was subjected to oscillation to become homogeneous, thereby forming a stable suspension. Donepezil hydrochloride was administrated as below: 1.025 mg/kg (mouse) and 0.516 mg/kg (rat). The animals were administrated two times one day, donepezil hydrochloride solution in the morning, equal volume 0.5% CMC-Na and multivitamin BC solution in the afternoon.
3.5 Administration Method
Intragastrical administration volume of mouse and rats were 20 mL/kg and 10 mL/kg, respectively. The normal control and the model group were administered with a corresponding volume of 0.5% CMC-Na vitamin BC tablet adjuvant solution by weight.
3.6 Behavioral Tests
Morris Water Maze

(38) The Morris water maze was used as a behavioral tests for studying spatial learning and memory. There were strong escape motivation for rodents in water, they tried to escape from the water environment. The process of learning to escape the water environment reflects the learning ability of animals. Spatially located the safe place (platform) in the water according to the surrounding environment, and swim to the platform purposefully, which could reflect the spatial reference memory ability of the animals. The Morris experimental system consists of a water maze device, an automatic image acquisition and a software analysis system. The Morris water maze unit consists mainly of a pool and a platform with an adjustable height and a movable position.

(39) There were two parts of the water maze experiment:

(40) 1) Acquisition Phase

(41) The pool was divided into four quadrants, and the platform was placed in one quadrant with the liquid level over 1 cm of the platform. The animals were placed toward the pool wall into the water, and the position was randomly taken in one of four starting positions in different directions. The time for the animals to find and climb on the platform (escape latency) was recorded to test the animals' learning ability. If an animal has not found the platform for more than 60 s, it needs be guided to the platform. The animals were allowed to stand on the platform for 10 s, then removed, dried under far infrared light and returned to the cage until the next round of experiments. Each animal was trained 4 times a day for 5 days (or longer, depending on the model's learning situation, and the model's average latency <20 s was considered successful).

(42) 2) Exploration Training

(43) Exploration training was performed 1.5 hours and 1 day after the acquisition phase. The platform was removed before the experiment, the mice were placed in the water from the opposite side of the original platform quadrant, and started recording 60 s. The spatial memory detection index included: 1) the time of the animal crossed the platform for the first time, 2) the time that the animal spent in the target quadrant (the quadrant where the platform was originally placed); 3) how many times the animal entered into the target quadrant.

(44) 3.7 Mitochondrial Enzyme Activity Assay

(45) 1) Sampling of Brain Tissue

(46) 10% chloral hydrate, deeply anesthetize AD rats, brain tissue was stripped quickly after decapitation the rats. The cerebral cortex was put into a clean and pre-cooled 2 mL centrifuge tube, weight quickly and put into liquid nitrogen for quick freezing, then were collect uniformly, and put in the refrigerator and stored at −80° C.

(47) 2) Mitochondrial Enzyme Extraction

(48) The mitochondrial enzyme activity detection kit of Solarbio was used in the experiment. Weighted about 0.1 g cerebral cortex, 1 mL of reagent 1 and 10 μL of reagent 3 were added to the cerebral cortex above, and grinded them with ice bath. The precipitate was removed by centrifugation at 4° C. The supernatant was transferred to another centrifugal tube, the supernatant was removed by centrifugation at 4° C. 200 μL of reagent 2 and 2 μL of reagent 3 were added to the precipitate, ultrasonic disruption, then be used in the enzyme activity detection assay.

(49) 3.8 Energy Metabolism Related mRNA Detection

(50) 1) Brain Tissue Sampling

(51) 10% chloral hydrate, deeply anesthetize AD rats, brain tissue was stripped quickly after decapitation the rats. The cerebral cortex was put into a clean and pre-cooled 2 mL centrifuge tube, weight quickly and put into liquid nitrogen for quick freezing, then were collect uniformly, and put in the refrigerator and stored at −80° C.

(52) 2) mRNA Expression

(53) The TIANGEN Total RNA Extraction Kit was used in the experiment. Weighted about 0.1 g cerebral cortex, 1 mL of lysate RZ was added to the cerebral cortex above, then homogenate the sample. Set aside for 5 minutes, 200 μL of chloroform were added to the precipitate, oscillate violently for 15 s, set aside for 3 minutes at room temperature, centrifugation at 4° C. The aqueous phase was moved to a new tube, 0.5 times volume of absolute alcohol was added to the tube and mixed homogeneously. Transferred to CR3 absorption column, the liquid waste was removed by centrifugation at 4° C. Protein removal solution was added, the liquid waste was removed by centrifugation. Rinsed twice with rinsing buffer, the liquid waste was removed by centrifugation. Stored at room temperature for 2 minutes after adding 30-100 μL RNase-Free ddH.sub.2O, centrifugated at 4° C. The expression of mRNA was detected by real-time PCR after reverse transcription by TOYOBO RT-PCR Kit.

4 Results

(54) In the acquisition phase of Morris Water Maze (D1-D5), we made the time that the animals take to find the hidden platform as the detection index of spatial memory. The shorter the latency to platform, the better the memory. As showed in FIG. 1, the latency to platform of each group animal shorten significantly as the time of training increasing. It suggested that learning and memory skills improved with repeated training. The learning and memory ability of model animals from D1 to D5 was significantly worse than that of normal control animals. The learning and memory ability of model animals were significantly improved after the treatment of energy metabolize drug (multivitamin BC), indicating that multivitamin BC could significantly improve the learning and memory ability of dementia animals.

(55) In the exploration training, the animals were placed in the water from the opposite side of the original platform quadrant. The time that the animal spent in the target quadrant (the quadrant where the hidden platform was originally placed) and the swimming distances was recorded as the test index of spatial memory. The better the animal's memory ability, the longer time in the target quadrant, and the longer swimming distance. The results were shown in FIG. 2 and FIG. 3. The time and swimming distance of the normal control animals in target quadrant—quadrant 1 (the original hidden platform quadrant) were significantly higher than other group, indicating that their memory level was the best. The time and swimming distance of the Vitamin BC group animals in target quadrant were higher than the model group. It further suggested that VBC therapy for 2 months could effectively improve the memory ability of animals with dementia.

(56) The results of mitochondrial enzyme activity detection assay were shown in FIG. 4. The multivitamin BC significantly improved the activity of key enzymes (pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase, mitochondrial isocitrate dehydrogenase (ICDHm), and succinate dehydrogenase (SDH)) involved in the tricarboxylic acid cycle in the cerebral cortex, thus improving mitochondrial metabolism activity.

(57) The results of energy metabolism-related mRNA assay showed that the expressions of SDHA and ATP5B gene in the cerebral cortex of the model animals were lower than those in the normal group, as shown in FIG. 5. The multivitamin BC and combination therapy increased expression of SDHA and ATP5B, improved mitochondrial metabolism, and improve cognitive function.

5 Conclusion

(58) The learning and memory ability of ICV-STZ model rats decreased significantly in the Morris Water Maze experiment. The learning and memory ability of rats improved significantly after multivitamin BC therapy for 2 months. The activity of key enzymes involved in the tricarboxylic acid cycle increased and the expression level of energy metabolism-related mRNA improved in the cerebral cortex. It indicated that this drug has certain therapeutic effect on the treatment for cognitive dysfunction of AD, and it is expected to provide a new class of drugs for AD patients.

(59) In order to describe and understand the present invention more clearly, we describe the present invention by examples in detail. It is clear that modification and alterations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.