Patent Application
20210213109
The present invention relates to a method and a pharmaceutical composition for treating Hunter syndrome.
Hunter syndrome or mucosaccharidosis type II is one of lysosomal storage diseases (LSDs) in which mucopolysaccharides such as glycosaminoglycans (GAGs) are not degraded in the body due to deficiency of iduronate-2-sulfatase (IDS or 12S) and thus accumulated in lysosomes. GAGs are accumulated in cells throughout the body and cause various symptoms. The symptoms include prominent facial features, large head, abdominal distension caused by enlarged liver or spleen, and the like, which occur with hearing loss, heart valve disease, obstructive respiratory disease, sleep apnea, and the like. In addition, accumulated GAGs may also limit joint movement, and may cause neurological symptoms and delayed development when the central nervous system is affected. Hunter syndrome is known to occur in approximately 1 in 162,000 live births and is inherited in an X-linked recessive pattern.
Enzyme replacement therapy (ERT), which is one of the treatment methods for Hunter syndrome, is a method in which externally produced IDS is administered into the body. Such a method has an advantage of being simple in terms of administration while having a disadvantage of high treatment cost in that the enzyme needs to be continuously administrated. Elaprase® (Shire Pharmaceuticals Group), which is a recombinantly produced enzyme replacement therapy and has been approved by the US FDA, is on the market, and has disadvantages in that unit price thereof is very high and it exhibits low efficacy and safety.
The enzyme replacement therapy is achieved by systemic administration of naturally-occurring or recombinant-derived proteins and/or enzymes to a subject. Approved enzyme replacement therapies are typically administered intravenously and are generally effective in treating physical symptoms resulting from an enzyme deficiency that is responsible for a disease or condition. However, these enzyme replacement therapies are problematic in that the intravenously administered proteins and/or enzymes are limitedly distributed in cells and tissues of the central nervous system (CNS). In particular, for treatment of diseases with CNS etiology, such enzyme replacement therapies have been problematic because the intravenously administered proteins and/or enzymes do not pass the blood-brain barrier (BBB).
In order to overcome these problems with the blood-brain barrier, attempts have been made to develop a treatment method in which proteins and/or enzymes are directly administered into the subject's cerebrospinal fluid by intrathecal (IT) injection; however, such attempts have not yet been successful in terms of stability and effectiveness.
Accordingly, there is a need for the development of a treatment method that can effectively treat Hunter syndrome patients with a central nervous disorder and ultimately bring about improved brain function in these patients.
The present inventors have found that in a case where iduronate-2-sulfatase beta is repeatedly administered into the cerebral lateral ventricle of a subject for a long time, such administration has a surprising effect of suppressing and improving brain function decline observed in patients with Hunter syndrome; and thus have completed the present invention.
An object of the present invention is to provide a therapeutic method and a pharmaceutical composition for substantially treating or improving central nervous system degeneration, in particular, brain function decline, caused by Hunter syndrome.
In order to achieve the above object, the present invention provides a pharmaceutical composition for treating Hunter syndrome in a subject, which comprises iduronate-2-sulfatase beta and is administered once every 4 weeks into the cerebral lateral ventricle, and a method for treating Hunter syndrome using the same.
The pharmaceutical composition according to an aspect of the present invention exhibits effects such as actually improving brain function in a subject suffering from Hunter syndrome. In the present invention, it has been identified that administration of such a composition can sufficiently suppress brain function loss even in clinical practice.
In addition, the pharmaceutical composition according to the present invention, which is repeatedly administered over a long period of time, has a better therapeutic effect on Hunter syndrome than a case where the same dose of the active ingredient is given by single administration, and further has effects, which cannot be anticipated from the results obtained by the single administration, such as treating or restoring damaged brain structures, and substantially treating or improving brain function, in particular, improving memory and learning function.
In order to better understand the present invention, certain terms are defined as follows. Further explanations for the terms as indicated below and other terms are set forth throughout the present specification.
As used herein, the term “about,” as applied to one or more numerical values, refers to a numerical value that is similar to a stated reference numerical value. In an aspect of the present invention, the term “about” refers to a range of numerical values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9% 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or lower than) of the stated numerical value unless otherwise stated or the context clearly indicates otherwise (except a case where such number exceeds 100% of a possible numerical value).
As used herein, the term “treatment” refers to any administration of a therapeutic protein (for example, lysosomal enzyme) that partially or completely alleviates, ameliorates, relieves, suppresses, delays onset of, decreases severity of and/or decreases incidence of one or more symptoms or features of a particular disease, disorder, and/or condition (for examples, Hunters syndrome). Such treatment may be applied to a subject who does not exhibit signs of the relevant disease, disorder, and/or condition, and/or to a subject who exhibits only early signs of the disease, disorder, and/or condition. Optionally or additionally, such treatment may be applied to a subject who exhibits one or more established signs of the relevant disease, disorder, and/or condition.
Hereinafter, the present invention will be described in detail.
In an aspect, the present invention may be directed to a pharmaceutical composition comprising iduronate-2-sulfatase beta (“IDS-β”).
In an aspect of the present invention, the pharmaceutical composition may be for treating Hunter syndrome in a subject.
In an aspect of the present invention, the pharmaceutical composition may be for treating or inhibiting ventriculomegaly in a subject suffering from Hunter syndrome.
In an aspect of the present invention, the pharmaceutical composition may be for improving brain function in a subject suffering from Hunter syndrome.
In an aspect of the present invention, the IDS-β may include a protein having the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 1 is a recombinant human iduronate-2-sulfatase protein. In an aspect of the present invention, the IDS-β may further include a protein having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 is a recombinant human iduronate-2-sulfatase protein in which cysteine at amino acid position 59 is substituted with formylglycine (Fgly).
In an aspect of the present invention, the IDS-β may include equal to or lower than about 20 to 35 mol % of a protein having the amino acid sequence of SEQ ID NO: 1 and equal to or lower than about 65 to 80 mol % of a protein having the amino acid sequence of SEQ ID NO: 2. Specifically, In an aspect of the present invention, the IDS-β may include equal to or lower than about 35 mol % of a protein having the amino acid sequence of SEQ ID NO: 1 and equal to or lower than about 65 mol % of a protein having the amino acid sequence of SEQ ID NO: 2.
In an aspect of the present invention, the IDS-β may include a protein having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 1. In an aspect of the present invention, the IDS-β may include a protein having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 2.
In an aspect of the present invention, the IDS-β may be at a concentration of about 0.1 mg/ml to about 60 mg/ml, about 1 mg/ml to about 10 mg/ml, or about 6 mg/ml.
In an aspect of the present invention, a dose of the IDS-β at the time of single administration may be about 1 μg to 60 μg, about 15 μg to about 60 μg, about 20 g to about 40 μg, about 25 μg to about 35 μg, about 29 μg to about 31 μg, or about 30 μg; and such a dose may be a dose in a case of being administered to a mammal such as a mouse. However, the present invention is not limited thereto. In addition, in an aspect of the present invention, a dose of the IDS-β at the time of single administration may be about 1 mg to 60 mg, about 15 mg to about 60 mg, about 20 mg to about 40 mg, about 25 mg to about 35 mg, about 29 mg to about 31 mg, or about 30 mg; and such a dose may be a dose in a case of being administered to a human. However, the present invention is not limited thereto.
In an aspect of the present invention, the pharmaceutical composition may be administered once every 4 weeks into the cerebral lateral ventricle of the subject.
In an aspect of the present invention, the pharmaceutical composition may be administered directly to either or both of the cerebral lateral ventricles of the subject.
In an aspect of the present invention, administration of the pharmaceutical composition may decrease or inhibit ventriculomegaly in a subject with Hunter syndrome; improve the subject's brain function; improve the subject's learning and memory ability; suppress the subject's hyperactivity characteristics; decrease cellular vacuolization in brain cells; or inhibit expression of lysosome-associated membrane protein 2 (LAMP2) in brain cells.
In an aspect of the present invention, administration of the pharmaceutical composition may decrease accumulation of heparan sulfate (HS) in the brain (for example, brain cells) or cerebrospinal fluid of the subject. In an aspect of the present invention, the HS accumulation may be decreased by 20%, 40%, 50%, 60%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, or 3-fold as compared with a control.
In an aspect of the present invention, administration of the pharmaceutical composition may be performed three or more times to the subject. In an aspect of the present invention, administration of the pharmaceutical composition may be performed 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 or more times; and may specifically be performed 6 times to the subject.
In an aspect of the present invention, administration of the pharmaceutical composition may be performed for 12 weeks or longer and 48 weeks or shorter. In an aspect of the present invention, administration of the pharmaceutical composition may be performed for 12 weeks or longer, 16 weeks or longer, 20 weeks or longer, 24 weeks or longer, 28 weeks or longer, 32 weeks or longer, 36 weeks or longer, 40 weeks or longer, 44 weeks or longer, or 48 weeks or longer; and may specifically be performed for 24 weeks.
In an aspect of the present invention, the subject may be a human or mammal. In an aspect of the present invention, the mammal may be a mouse, rat, rodent, cat, rabbit, dog, pig, horse, or cow.
In an aspect of the present invention, administration of the pharmaceutical composition may be performed through an intraventricular catheter system including a reservoir and a catheter connected to the reservoir. In the test examples as described later, since the administration was made to mice, separate three-dimensional positioning device and three-dimensional positioning coordinate system were required. However, in clinical application, the pharmaceutical composition of the present invention can be administered through a simple intraventricular catheter system.
In an aspect of the present invention, administration of the pharmaceutical composition may be performed with a method comprising steps of: 1) surgically inserting the catheter system, in which the reservoir is located under the scalp of the subject and an end of the catheter is located in the cerebral lateral ventricle of the subject, so that a cerebral lateral ventricular inner space is connected, via a catheter inner space, to a reservoir inner space, thereby allowing cerebrospinal fluid to flow from the cerebral lateral ventricle to the reservoir and causing the reservoir to be filled with the cerebrospinal fluid; 2) withdrawing about 0.1 to 5 ml of the cerebrospinal fluid from the reservoir at a rate of 0.1 to 60 ml/min; 3) injecting 0.1 to 5 ml of the pharmaceutical composition into the reservoir at a rate of 0.1 to 60 ml/min; and 4) allowing the pharmaceutical composition to flow from the reservoir, via the catheter, to the cerebral lateral ventricle.
In an aspect of the present invention, administration of the pharmaceutical composition may be performed in combination with one or more additional enzyme replacement therapies for treating Hunter syndrome. Specifically, in an aspect of the present invention, the additional enzyme replacement therapy may be at least one of intravenous administration and subcutaneous administration of IDS-β. Such intravenous administration and subcutaneous administration may refer to conventional administration methods; and in clinical application, cerebral lateral ventricular administration of the present invention does not require setting a limit for the administration schedule.
In an aspect of the present invention, the pharmaceutical composition may further include sodium chloride and polysorbate 20, and may have a pH of about 5 to about 7. In an aspect of the present invention, the pharmaceutical composition may have a pH of about 6.
In an aspect of the present invention, sodium chloride contained in the pharmaceutical composition may have a concentration of about 100 mM to about 200 mM. Here, the concentration of sodium chloride may correspond to a range of all integer values that exist within the above-mentioned range. Specifically, sodium chloride may have a concentration of about 150 mM.
In an aspect of the present invention, polysorbate 20 contained in the pharmaceutical composition may have a concentration of about 0.01 mg/ml to 0.5 mg/ml. Here, the concentration of polysorbate 20 may correspond to a range of all decimal values that exist within the above-mentioned range. Specifically, polysorbate 20 may have a concentration of about 0.05 mg/ml. In an aspect of the present invention, administration of the pharmaceutical composition may not cause any severe adverse effects. In an aspect of the present invention, the severe adverse effect may refer to, but is not limited to, any effect that causes substantial immune response, toxicity, or death.
As used herein, the term “substantial immune responses” may refer to severe or serious immune responses, such as substantial adaptive T-cell mediated immune response.
In an aspect of the present invention, the pharmaceutical composition may be provided in a form filled into a container, such as a vial, a syringe, or a pre-filled syringe, and may be provided in a form filled into a disposable plastic syringe in a case of being administered in a single dose. In an aspect of the present invention, the pharmaceutical composition may be administered in a volume of about 1 μL to about 10 μL, a volume of about 3 μL to about 7 μL, a volume of about 4 μL to about 6 μL, or a volume of about 5 μL; however, the present invention is not limited thereto. In addition, in an aspect of the present invention, the pharmaceutical composition may be administered in a volume of about 1 mL to about 10 mL, a volume of about 3 mL to about 7 mL, a volume of about 4 mL to about 6 mL, or a volume of about 5 mL; however, the present invention is not limited thereto.
In an aspect, the present invention may be directed to a pharmaceutical composition for treating or inhibiting ventriculomegaly in a subject suffering from Hunter syndrome, comprising about 150 mM sodium chloride, about 0.05 mg/ml of polysorbate 20, and about 5 mg/ml of iduronate-2-sulfatase beta (IDS-β), in which the pharmaceutical composition has a pH of about 6 and is administered once every 4 weeks into the cerebral lateral ventricle.
In an aspect, the present invention may be directed to a pharmaceutical composition for improving brain function in a subject suffering from Hunter syndrome, comprising about 150 mM sodium chloride, about 0.05 mg/ml of polysorbate 20, and about 5 mg/ml of iduronate-2-sulfatase beta (IDS-β), in which the pharmaceutical composition has a pH of about 6 and is administered once every 4 weeks into the cerebral lateral ventricle.
In an aspect, the present invention provides a method for treating Hunter syndrome, comprising a step of applying to a subject the pharmaceutical composition of the present invention.
In an aspect of the present invention, there is provided a use of the pharmaceutical composition of the present invention for treating Hunter syndrome.
In an aspect of the present invention, there is provided a use of the pharmaceutical composition of the present invention for manufacture of a medicament for treating Hunter syndrome.
Hereinafter, the configuration and effects of the present specification will be described in more detail by way of examples and test examples. However, these examples and test examples are provided for illustrative purposes only to help understand the present specification, and the scope of the present specification is not limited by the following examples.
As a carrier solution, 150 mM sodium chloride and 0.05 mg/ml of Tween 20 solution (Merck Millipore, Darmstadt, Germany) were used. An IDS-β solution (50 mg/ml or 15 mg/ml) obtained from Green Cross Co., Ltd. was diluted in the above carrier solution, to prepare an IDS-β formulation at a concentration of 6 mg/ml. The thus prepared IDS-β formulation was used in the test examples below.
The mice used in the test examples as described in the present specification were in-house-produced mice, and were produced in such a manner that a part of iduronate 2-sulfatase (IDS) gene was deleted. Briefly, deletion in the IDS gene was made from exons 2 to 3. IDS KO mice (IDS knock-out mice) were bred from a C57BL/6.1295 background strain and had a null mutation in the IDS gene. Wild-type (WT) control mice were bred from a C57BL/B6.1295 strain. The genotypes of all mice were checked by polymerase chain reaction (PCR) using DNA obtained from a tail snip. The test examples in the present specification were approved by the Institutional Animal Care and Use Committee (Approval No. 20140925005) and conducted in accordance with the animal welfare policy of the Samsung Biomedical Research Institute (Seoul, Korea).
The mice obtained by breeding were classified and assigned as shown in Table 1 below.
Cerebral Lateral Ventricular Administration
The mouse brain coordinates are required for accurate cerebral lateral ventricular administration. This information is described in [Paxinos, George and Franklin, Keith B. J., Paxinos and Franklin's The Mouse Brain in Stereotaxic Coordinates, Academic Press, 4th Edition, 2012] and is as illustrated in
Intracerebral lateral ventricular administration was performed for 6-week-old mice obtained by the above breeding. Each group was administered the carrier solution and the IDS-β solution of Preparation Example 1 in a volume of 5 μL at the time of single administration. On the day of administration, the mice were anesthetized with isoflurane (HANA PHARM CO., LTD., Korea) by inhalation and placed in a stereotactic instrument (Stoelting, USA). Then, the scalp of the mouse was minimally peeled off, and the exposed skull was cleaned. Each of the carrier solution and IDS-β solution prepared in Preparation Example 1 was placed in a disposable plastic syringe, and injected into the cerebral lateral ventricle of the mouse with a 31-gauge needle at a constant rate of 10 μL/min using a syringe pump (KD Scientific, Switzerland). During administration, the position of the syringe needle in the brain was monitored using the stereotaxic coordinates (Stoelting, USA). The coordinates of the needle at the start of administration were as follows: −0.58 mm caudal to bregma, 1.25 mm lateral to sagittal suture, and −1.77 mm in depth. After completing administration of the volume of 5 μL, an act of lifting the syringe needle by 0.15 mm per 30 seconds for removal thereof from the injection site was repeated 4 to 5 times. Then, an act of lifting the needle at a height of 0.3 mm per 30 seconds from the −1.00 mm in depth position was repeated several times to completely remove the syringe needle. These acts were introduced to prevent backflow of the administered solution, and the time required for the entire administration process was about 30 minutes per subject. After the administration, the exposed skin area including the injection site was simply sutured with a medical stapler.
After this administration, the mice did not show any serious adverse effects. No subject dropout, which is due to substantial immune response at the administration site, abnormal body function, or the like, was observed. Autopsy was performed 28 days after the last administration date so that the following analysis was conducted.
Collection of Brain and Cerebrospinal Fluid of Mouse
At the time of autopsy, which is 28 days after the last administration date, for each group according to Table 1, the cerebrospinal fluid was first collected using a borosilicate glass micropipette (outer diameter: 10 mm, inner diameter: 0.75 mm) at the cistema magna area of the mouse of interest (see
HS Analysis Method
HS levels in the cerebrospinal fluid and brain tissue samples collected from mouse were measured using liquid chromatography tandem-mass spectrometry (LC-MS/MS). A2 μL sample of the mouse cerebrospinal fluid or brain tissue, which had been homogenized in PBS, was aliquoted for pretreatment. Each of the samples was heated in hydrochloric acid-methanol to be mathanolyzed. The resulting methanolysate was evaporated to dryness under a stream of nitrogen gas. As internal standards, deuterated dermatan sulfate and heparan sulfate (DS-d6 and HS-d6) were used. The thus obtained solution was transferred to a centrifugal filter and centrifugation was performed. The resulting filtrate was analyzed. LC-MS/MS analysis was performed using an ultra performance liquid chromatography system (UPLC, Waters) and atriple quadrupole mass spectrometer (API5000, AB/MDS Sciex). Data were processed using the Analyst 1.5.1. software. The analysis results are illustrated in
Results
Amounts of HS accumulated in the brain and the cerebrospinal fluid in the respective groups were compared. Referring to
Referring to
Brain MRI analysis of the mice was performed to identify whether the cerebral lateral ventricular administration according to the present invention not only improves HS, which is a biochemical indicator of brain and cerebrospinal fluid, but also has an effect of inhibiting structural destruction or damage of the brain.
Specifically, after completion of 6 administrations, all mouse images were obtained using a 7.0-Tesla MRI system (Bruker-Biospin, Fallanden, Switzerland), and this system was equipped with a 20-cm gradient set capable of supplying up to 400 mT/m with a rise time of 100 ms. A birdcage coil (inside diameter, 72 mm; Bruker Biospin) was used for excitation, and an actively decoupled phased array coil was used for signal reception.
The mice were anesthetized with isoflurane (HANA PHARM CO., LTD., Korea) (5% for induction, 2% for the animal setup, and 1% to 1.5% during the MRI experiment) in a mixture of oxygen and nitrogen gases (3:7) delivered to a nose cone for spontaneous respiration throughout the experiment. The head of the mouse was very carefully fixed using a bite/ear bar. Coronal images containing the cerebellum were obtained with the following characteristics: T2-weighted, fast spin-echo sequence with 192×192 matrix size, 19.2×19.2 mm2 resolution, TR/TE=3,000/70 ms, NEX=6, 1.0 mm slice thickness with no interslice gap.
To assess the percent volume ratio of ventricles relative to whole brain, 9 coronal MRIs per mouse were obtained. The percent volume ratio was manually estimated by outlining serial images in a blinded manner using ParaVision 2.0.2 software (Bruker, BioSpin, Karlsruhe, Germany). The boundaries of whole brain and ventricle were outlined on each image, and the corresponding area was calculated using the above-mentioned software. The cerebral ventricle area was calculated as (lateral ventricle area/total brain area). These results are shown in
Results
Among Hunter syndrome patients, those with central nervous system degeneration were accompanied by ventriculomegaly. Similarly, even in Hunter syndrome mice (IDS KO mice), ventriculomegaly could be identified by brain MRI. In this test example, it was verified whether among the IDS KO mice, ventriculomegaly in the untreated group is alleviated by the administration of the present invention. As a result, it was found that the Example exhibited significantly decreased ventricles as compared with the untreated group, Comparative Example 1.
At the time of autopsy of the mice in each group, among mouse brain tissues, sections of the cerebellum, medulla, thalamus, and cortex were collected for tissue staining, fixed with a 4% paraformaldehyde solution (BIOSESANG, Korea) overnight, subjected to tissue processing, and then embedded in paraffin. 4 μm-thick sections were prepared for hematoxylin/eosin (H&E) staining.
Hematoxylin/Eosin Staining
Hematoxylin/eosin staining was performed on the brain tissue sections collected from the mice in each group.
Specifically, a slide, on which the brain tissue section was fixed, was immersed in a xylene solution to perform deparaffinization of the slide. Then, the slide was immersed sequentially in ethanol ranging from 100% ethanol to lower-concentration ethanol, to remove xylene. Subsequently, the slide was washed with running water, and then stained by being immersed in a hematoxylin solution for 5 to 8 minutes. After staining, the slide was washed with running water, immersed in 1% hydrochloric acid for a while, and then washed again with running water. The slide was immersed in a 1% ammonia solution, and then washed again with running water. Subsequently, the slide was stained by being immersed in an eosin solution for 2 minutes. Then, the slide was dehydrated with an ethanol solution, and sealed with xylene.
The stained slide was scanned with the ScanScope AT (Aperio, Leica, German) device, and then analyzed with the ImageScope (Aperio, Leica, German) device to evaluate histopathological morphology. The pictures for the scanned slides are illustrated in
Results
The hematoxylin/eosin staining showed that for Hunter syndrome mice, structural destruction was ongoing throughout the brain due to accumulation of glycosaminoglycans (GAGs). Specifically, for the mice with Hunter syndrome in Comparative Example 1, cellular vacuolization, which is a typical marker of lysosomal storage diseases, was found throughout the mouse brain, as compared with the mice in Reference Example (see
Immunohistochemical Staining
The brain tissue sections of the respective groups prepared in Test Example 4 were stained with anti-LAMP-2 IgG (Santa Cruz Biotechnology, USA), which is a primary antibody, and were developed by the DAB method to visualize the expression of LAMP2 protein. The LAMP2 protein is a lysosome-associated membrane protein and is used as an indicator of lysosome activity. After being developed by the DAB method, the brain tissue sections were counter-stained with hematoxylin for the LAMP2 protein-positive cells and nuclei.
Specifically, the 4-μm thick tissue sections were allowed to stand in 10% normal goat serum (Dako, Carpinteria, Calif., USA) for 20 minutes at room temperature, and then allowed to stand at room temperature for 30 minutes together with anti-LAMP-2 antibody in a ratio of 1:100. The tissue sections were washed several times with PBS. Then, the tissue sections were allowed to stand for 30 minutes at room temperature together with HRP-labeled polymer-conjugated secondary antibody. The tissue sections were allowed to stand in a 3,3-diaminobenzidine substrate-chromogen solution (Dako, Carpinteria, Calif., USA) for 5 minutes, and washed with water. Then, nuclear staining was performed with hematoxylin.
The stained slide was scanned with the ScanScope AT (Aperio, Leica, Germany) device, and then analyzed with the ImageScope (Aperio, Leica, Germany) device to evaluate histopathological morphology. The pictures for the scanned slides are illustrated in
Results
Referring to
In order to identify an actual effect of the administration of the present invention on brain function, a fear conditioning test and an open field test were performed which are tests for evaluating learning-memory ability among animal behavioral assessments. These two behavioral assessment items can be used to evaluate overall brain function by evaluating the exploration ability or movement of small animals such as rodents, and their degree of coping with sufficiently anticipated environmental changes. The methods for performing the respective tests and the test results are as follows.
At weeks 2 to 3 after the cerebral lateral ventricular administration of Test Example 1 (after the 1st, 3rd, and 6th administrations), fear memory of the mice belonging to each group was evaluated using the following fear conditioning system (Coulboum Instruments): On day 1 of evaluation, the mice were stimulated in a fear conditioning chamber with a 30-second auditory cue (80 dB, 3600 Hz tone) as a conditioned stimulus and a 2-second foot shock (0.6 mA direct current) as an unconditioned stimulus. On day 2 of evaluation which is 24 hours later, the mice were placed back in the same chamber, and video-recorded at a frequency of 40 Hz (30 frames per 0.75 seconds) for 300 seconds to evaluate associative memory. 1 hour after the associative memory evaluation, in order to evaluate the cue memory of the mice, the mice were placed in a new and different chamber and given stimuli with an auditory cue. Mouse movements were likewise video-recorded for 300 seconds at a frequency of 40 Hz (30 frames per 0.75 seconds).
The recorded videos were analyzed post hoc using Freeze Frame Software Ver 3.32, which automatically calculated motion index and relative absence of motion. Freezing duration was counted when the mice displayed a bout of relative absence of motion for longer than 0.75 seconds or 2 seconds. This freezing duration was divided by total observation duration (300 seconds). The resulting value was expressed as a percentage, and the graphs for contextual conditioning and cue-dependent conditioning items are illustrated in
Results
Referring to
After the cerebral lateral ventricular administration of Test Example 1 (after the 1st, 3rd, and 6th administration), the following open field test was used to evaluate exploratory behavior of the mice in each group: The mice were allowed to explore an open-top arena (44.5 cm×44.5 cm) sided with opaque walls (15.0 cm height) and their movement was video-recorded for 20 minutes. Recorded videos were analyzed for exploration distance, moving speed, resting time, time spent in the central and peripheral zones, and frequency of entry into each zone. The central zone means an area of 31.0 cm×31.0 cm marked in the center of the arena, and the peripheral zone means the rest of the arena excluding the central zone. The graphs for the respective analysis results are illustrated in
Results
It is known that Hunter syndrome mice usually exhibit hyperactivity characteristics. This was also identified in the open field test results of the present invention. 20-minute observation shows that the sum of distances traveled over the entire section (that is, horizontal path distance) steadily increased in the mice in Comparative Example 1. However, it was found that for the mice in the Example, the sum of distances traveled decreased, indicating suppression of hyperactivity characteristics. In the same sense, the mice in Comparative Example 1 also showed a decreasing trend of resting time, during which no movement is made, whereas the mice in the Example showed an increased resting time.
In certain space, rodents have an instinct to prefer sides which are easier to protect them from predators such as birds rather than the center. Therefore, in the open field test, it is a general behavioral characteristic of mice to explore the peripheral zone rather than the central zone in a prepared space. However, it was found that the mice in Comparative Example 1 showed increased frequency of center entries and time spent in the central zone with aging. On the other hand, the mice in the Example showed a similar tendency to the mice in the Reference Example which are normal mice.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0061741 | May 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/006515 | 5/30/2019 | WO | 00 |
