Composition for treatment of glutaric aciduria and administration method therefor

Abstract

To provide a composition for treatment of glutaric aciduria.SOLUTION: The present invention provides a pharmaceutical composition for treatment of glutaric aciduria comprising recombinant human glutaryl-CoA dehydrogenase (rhGCDH).SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present disclosure relates to compositions and methods of administration for treating glutaric aciduria, specifically, recombinant human glutaryl-CoA dehydrogenase (recombinant ... The present invention relates to a pharmaceutical composition for treating glutaric aciduria, comprising glutaryl-CoA dehydrogenase (rhGCDH), and a method for treating glutaric aciduria, comprising administering the pharmaceutical composition. [Background technology]

[0002] Glutaric aciduria, or glutaric acidemia, is a condition in which the body This is a disease in which the body is unable to break down the amino acids lysine, hydroxylysine, and tryptophan, and intermediate metabolic products such as glutaric acid and 3-hydroxyglutaric acid accumulate in the body. It is divided into glutaric aciduria type 1 (GA-1) and type 2 (GA-2), and is a disease caused by an autosomal recessive inborn error.

[0003] Most children with glutaric aciduria develop macrocephaly, and may have a range of other neurological symptoms, including irritability, opisthotonus, hypotonia, easy tonicity, chorea, seizures, and developmental delay. Many children have macrocephaly at birth or soon after birth, and the increase in head circumference usually occurs within the first few weeks to months of life. However, some children develop normally until age 2 years, while others have late-onset symptoms after age 6 years.

[0004] Glutaric aciduria type 1 (GA-1) is a metabolic disorder characterized by the inhibition of mitochondrial glutaryl-CoA dehydrogenase ( It is known that glutaric aciduria type 2 (GA-2) is caused by a defect or deficiency of glutary-CoA dehydrogenase (GCDH), and glutaric aciduria type 2 (GA-2) is caused by mutations in the ETFA, ETFB, and ETFDH genes.

[0005] Glutaric aciduria is a fatal disease that can lead to death within the age of 10 if left untreated, but patients with mild symptoms who are diagnosed, treated and managed early can achieve normal development through protein restriction and carnitine and riboflavin supplementation. However, treatment is very ineffective on brains that have already been damaged, so early diagnosis, treatment and management are essential. Treatment includes first aid, dietary therapy, arginine, carnitine, clofibrate and / or riboflavin supplementation, neurological drugs and multifaceted support. Therapy is used.

[0006] Children with glutaric aciduria must be placed on a low protein, lysine-free diet, with reduced tryptophan, but a total tryptophan-free diet can cause serious side effects, so a low tryptophan diet is required. Dietary therapy and carnitine supplementation are not known to reverse the major clinical symptoms in children with neurological symptoms, but they are known to modulate the progression of the disease.

[0007] For diseases caused by defects or deficiencies in a single enzyme, enzyme replacement treatment can be considered, which involves administering the deficient enzyme into the body. However, in many diseases that cause brain damage, unless the therapeutic agent is administered directly into the ventricles, it is often not possible to prevent brain damage. This is because the blood-brain barrier (BLB) restricts the exchange of substances between the brain parenchyma and the blood. Most of the proteins and components with large molecular weights are related to the BBB. Can't get through the gate.

[0008] For example, among the mucopolysaccharidoses, Hunter's syndrome and Hurler's syndrome drome, Sanfilippo syndrome, etc., are specific This is a disease in which a lack of enzymes prevents the precursors from being broken down, resulting in brain damage. In this case, even if the deficient enzyme is injected into the bloodstream, it cannot pass through the blood-brain barrier, and only the soft tissue improves, but the brain damage and decline in brain function cannot be prevented (Lamp, C. et al., J. Inherit. Metab. Dis., 2014; Bradley, LA et al., G Orphanet. Med., 2017;Sohn, YB et al., Orphanet J. Rare Dis., 2013;Muenzer, J. et al., Genet. Metab., 2007;Muenzer, J. et al., Genet. Med., 2006;Muenzer, J. et al. In this case, the only way to prevent the decline in brain function is to administer the missing enzyme directly into the ventricles of the brain, using a special device inserted through brain surgery (Ommay et al., Genet. Med., 2011). A device was then used to directly administer the missing enzymes, which allowed the precursors stored in the brain to be broken down (Sohn, YB, et al., J. Inherit. Metab. D, 2003). is., 2018;Giugliani, R. et al., Expert Opin. Orpha In addition to the diseases listed above, other diseases such as GM1 gangliosidosis and Krabbe disease are also included. There are various diseases such as pulmonary embolism (PE) and chronic kidney disease (CKD).

[0009] On the other hand, glutaric aciduria has a poor prognosis even if it is controlled by extreme dietary restriction, and a therapeutic agent that can effectively control the disease has not yet been developed. There is a demand for the development of a therapeutic agent and a therapeutic method for effectively treating this disease, and there is a need to develop a therapeutic agent and a therapeutic method that can be administered intravenously or subcutaneously without the trouble of having to administer directly into the ventricles as in diseases that cause brain damage.

[0010] [Prior art document] (Patent Document 1) International Patent Publication WO 2013133691 A1 (Patent Document 2) International Patent Publication WO 2013133711 A1 (Non-patent document 1) Kolker et al., “Diagnosis and manag ment of glutaric aciduria type I - revised recommendations”;Journal of Inherited Met abolic disease, Published online Mar 23, 2011;34(3):pp 677-694. Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present disclosure is to provide a therapeutic agent capable of effectively treating glutaric aciduria and an administration method thereof.

[0012] For this reason, the inventors of the present disclosure have made extensive efforts to develop a therapeutic agent capable of effectively treating glutaric aciduria, and as a result have developed a pharmaceutical composition for treating glutaric aciduria, which contains recombinant human glutaryl-CoA dehydrogenase (rhGCDH), and a method for administering the pharmaceutical composition in vivo (intravenously and subcutaneously). iv and sc) administration methods have been developed, and when used, The present invention was accomplished by confirming a significant decrease in glutaric acid (GA) in mice with GA. [Means for solving the problem]

[0013] To achieve the above object, one aspect of the present disclosure provides a pharmaceutical composition for treating glutaric aciduria, comprising recombinant human glutaryl-CoA dehydrogenase (rhGCDH).

[0014] The composition of the present disclosure may be a pharmaceutical composition for treating glutaric aciduria for internal administration, in particular, a pharmaceutical composition for treating glutaric aciduria for intravenous or subcutaneous administration.

[0015] Another aspect of the present disclosure is to provide a method for treating glutaric aciduria comprising the step of intravenously or subcutaneously administering said pharmaceutical composition. Effect of the Invention

[0016] A pharmaceutical composition for treating glutaric aciduria comprising recombinant human glutaryl-CoA dehydrogenase (rhGCDH) according to one aspect of the present disclosure and administration of the pharmaceutical composition in vivo (intravenously and subcutaneously) IV and sc administration of ribavirin has been proposed as a new treatment for glutaric aciduria. The compound has excellent effects even when administered internally, which is significantly easier than intracerebroventricular administration, and is therefore expected to be able to regulate and treat glutaric aciduria more effectively. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the purity of the purified recombinant human Glutaryl-CoA dehydrogenase (rhGCDH) confirmed by Coomassie Blue staining (FIG. 1A) and Western blot (FIG. 1B). [Diagram 2]Figure 2 shows the results of measuring the concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in the urine of normal mice and GCDH KO mice using GC-MSD (gas chromatography mass spectrometry), confirming that the GCDH KO mice are an animal model for glutaric aciduria. In the figure, the peak on the left side of the red box is the glutaric acid peak, and the peak on the right side is the hydroxyglutaric acid peak. [Diagram 3] FIG. 3 is a schematic diagram of the primary animal experiment on GCDH KO mice, an animal model of glutaric aciduria. Recombinant human GCDH was administered once at 50 and 100 μg through the tail vein, and urine was collected one week before and two days after administration, and glutaric acid levels were measured by LC-MS. [Figure 4] FIG. 4 shows the results of a primary animal experiment in which recombinant human GCDH was administered to GCDH KO mice, an animal model of glutaric aciduria, and is a graph showing the glutaric acid levels measured by LC-MS in urine samples taken one week before and two days after administration. [Diagram 5] FIG. 5 shows the results of a primary animal experiment in which recombinant human GCDH was administered to GCDH KO mice, an animal model of glutaric aciduria. Urine samples were collected one week before and two days after administration, and the glutaric acid levels were measured by LC-MS. The graph shows the percentage calculated based on the level before administration (100%). [Figure 6] FIG. 6 is a schematic diagram of the second animal experiment on GCDH KO mice, an animal model of glutaric aciduria. Recombinant human GCDH was administered in a single dose of 50 and 100 μg via the tail vein and 50, 150, and 250 μg subcutaneously. Urine samples were collected 3 days before, 2 days after, and 1 week after administration, and glutaric acid levels were measured by LC-MS. [Figure 7]FIG. 7 shows the results of a second animal experiment in which recombinant human GCDH was intravenously administered to GCDH KO mice, an animal model of glutaric aciduria. Urine samples were collected 3 days before administration, 2 days after administration, and 1 week after administration, and the glutaric acid values ​​were calculated as a percentage based on the values ​​before administration (100%). [Figure 8] FIG. 8 shows the results of a second animal experiment in which recombinant human GCDH was subcutaneously administered to GCDH KO mice, an animal model of glutaric aciduria. Urine samples were collected 3 days before administration, 2 days after administration, and 1 week after administration, and the glutaric acid values ​​were calculated as a percentage based on the values ​​before administration (100%). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The embodiments or aspects of the present disclosure are illustrated for the purpose of explaining the technical idea of ​​the present disclosure, and the scope of the present disclosure is not limited to the embodiments or aspects presented below or the specific description thereof.

[0019] Additionally, those skilled in the art to which this disclosure pertains will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein, which equivalents are intended to be encompassed by this disclosure.

[0020] All technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs, unless otherwise defined. All terms used in this disclosure are selected for the purpose of more clearly describing this disclosure, and are not selected to limit the scope of the rights of this disclosure.

[0021] As used in this disclosure, terms such as "including," "comprising," "having," and the like, should be understood as open-ended terms that include the possibility of including other embodiments, unless otherwise stated in the phrase or sentence in which the term appears.

[0022] In the present disclosure, expressions such as "consisting only of" the configuration are closed-ended terms that exclude the possibility of including other configurations other than the configuration in question. should be understood as such.

[0023] Unless otherwise specified, singular expressions described in this disclosure may include plural meanings, and this also applies to singular expressions described in the claims.

[0024] In one aspect of the present disclosure, the term "recombinant human glutaryl-CoA dehydrogenase (recombinant human glutaryl-CoA dehydrogenase)" refers to mbinant human glutaryl-CoA dehydrogenase; The composition containing human glutaryl-CoA dehydrogenase (rhGCDH) is a recombinant human glutaryl-CoA dehydrogenase gene. The term "pharmaceutical composition" refers to a composition containing a protein expression product obtained by recombinantly expressing the rhGCDH. The pharmaceutical composition of the present disclosure includes, without limitation, a composition containing the rhGCDH of the present disclosure. In one embodiment of the present disclosure, the pharmaceutical composition of the present disclosure may contain a pharmaceutical effective amount of the rhGCDH of the present disclosure and may further contain a pharmaceutical acceptable carrier.

[0025] In this disclosure, glutaric aciduria refers to glutaric aciduria type 1, which is caused by a deficiency or lack of GCDH, resulting in the loss of the amino acids lysine, hydroxylysine, and It cannot break down lysine and tryptophan, and cannot produce the intermediate metabolic products It is a disease that shows various symptoms due to accumulation of toxins in the body. Most affected children have various degrees of neurological symptoms, including macrocephaly (irritability, opisthotonus, hypotonia, irritability, hypertonia ... Symptoms include catatonia, chorea, convulsions, and developmental delay.

[0026] In the present disclosure, glutaryl-CoA dehydrogenase (GCDH) is generated in the degradation pathway of lysine or tryptophan. An enzyme that catalyzes the oxidative decarboxylation of glutaryl-CoA to produce crotonoyl-CoA, and may be GCDH, which has the enzyme number EC 1.3.99.7 of the International Union of Biochemistry and Molecular Biology. In the present disclosure, GCDH may comprise the amino acid sequence of SEQ ID NO: 1, which is based on the Homo sapiens GCDH sequence of NCBI accession No. NP_000150.1 ( https: / / www.ncbi.nlm.nih.gov / protein / NP_000150.1 ).

[0027] The amino acid may include not only the amino acid sequence of SEQ ID NO: 1, but also an amino acid sequence having about 80% or more, specifically about 90% or more, more specifically about 95% or more, even more specifically about 97% or more, and most specifically about 99% or more homology with the amino acid sequence of SEQ ID NO: 1. Such a homologous sequence includes, without limitation, an amino acid sequence that shows a protein having substantially the same or corresponding activity as a protein containing the amino acid sequence of SEQ ID NO: 1. It is also obvious that such an amino acid sequence having a homology includes an amino acid sequence in which a part of the sequence is modified, changed, substituted, or added. For example, a part of the amino acid sequence may be modified, changed, substituted, or added in order to optimize the protein expression and yield process, and specifically, the signal amino acid sequence of SEQ ID NO: 2 may be further included, but is not limited thereto. In the present disclosure, GCDH was expressed by adding a signal amino acid sequence of SEQ ID NO: 2 near the N-terminus of the amino acid sequence of SEQ ID NO: 1 in order to improve expression and yield in CHO cells.

[0028] In the present disclosure, "homology" refers to the degree of identity of bases or amino acid residues between sequences in the amino acid or nucleic acid sequences of a gene encoding a protein after aligning the sequences to maximize identity in a specific comparison region. If the homology is sufficiently high, the expression product of the gene may have the same or similar activity. The percentage (%) of sequence identity may be determined using a known sequence comparison program (e.g., Blast (NCBI), etc.).

[0029] In one aspect of the present disclosure, the term "about" is used with the intention of including the manufacturing process error included in the specific numerical value and some numerical adjustments included in the scope of the technical idea of ​​the present disclosure. For example, the term "about" means a range of ±10%, in one aspect ±5%, and in another aspect ±2% of the value it refers to. In the field of the present disclosure, approximations of this level are appropriate unless a value is specifically stated to require a narrower range.

[0030] In the present disclosure, proteins may be expressed and produced by methods known in the art to which the present disclosure pertains. In the present disclosure, host cells suitable for expressing and producing proteins include higher eukaryotic cells as described herein, including vertebrate host cells. Growth of vertebrate cells in culture has become commonplace. Examples of useful mammalian host cell lines include monkey kidney cells (CV1), monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line (293 cells), baby hamster kidney cells (BHK), Chinese hamster ovary cells (CHO), mouse Sertoli cells (TM4), African green monkey kidney cells (VERO), and the like. -76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), and Buffaloa Examples of such cells include, but are not limited to, rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells.

[0031] The host cells used to produce the proteins of the present disclosure may be cultured in a variety of media. Commercially available media, such as Ham's F10 medium, minimal essential medium, RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), and any other media used in the technical field to which the present disclosure pertains, may be used for host cell culture. Any of these media may contain, if necessary, hormones and / or growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, etc.), and / or other nutrients. and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin TM Drugs) , trace elements, and glucose or an equivalent energy source. Any other essential / auxiliary supplements may also be included at appropriate concentrations known to one of ordinary skill in the relevant art. Other culture conditions such as temperature, pH, CO2 concentration, etc. may be set to appropriate conditions known in association with the host cell selected for protein expression, which may be set to appropriate conditions known in the relevant art. It should be apparent to one of ordinary skill in the art.

[0032] In the present disclosure, the protein may be purified by any protein purification method known in the art to which the present disclosure pertains. For example, various known purification methods may be used, including fractionation on hydrophobic interaction, immunoaffinity or ion-exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica or on anion or cation-exchange resins, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration, or genetic engineering methods (e.g., tagging), and multiple purification methods may be used in any combination to increase the purity of the protein.

[0033] In the present disclosure, proteins purified through the methods described above can have a high level of purity such that they can be administered to animals, particularly humans, for experimental, clinical, and therapeutic purposes.

[0034] In the present disclosure, recombinant human GCDH may be contained in the composition at a concentration of about 0.001 to about 500 mg / ml, but is not limited thereto.

[0035] In the present disclosure, the pharmaceutical composition comprising recombinant human GCDH may be formulated and administered in a manner consistent with medical practice. Factors to be considered in this regard include the disease being treated, the particular animal being treated, the clinical condition of the individual subject, the cause of the disease, the site of delivery of the substance, the method of administration, the administration schedule, and other factors known to the clinician. In the present disclosure, the dosage form may include a liquid dosage form or a lyophilized powder dosage form, etc. The pharmaceutical composition of the present disclosure may be prepared in the form of an ampoule, a vial, a bottle, a cartridge, a reservoir, a lyoject, or a pre-filled syringe, etc., and may be prepared in a single dose or a multi-dose form, etc.

[0036] The "pharmaceutical effective amount", "administration dose", "therapeutically effective amount" or "effective dose" of recombinant human GCDH to be administered is determined by the above-mentioned considerations, and is the minimum amount necessary for the prevention, amelioration or treatment of a particular disease. In the present disclosure, the "pharmaceutical effective amount", "administration dose", "therapeutically effective amount" or "effective dose" of recombinant human GCDH may be, for example, about 0.0001 mg / kg to about 100 mg / kg per administration. This allows the composition of the present disclosure to be The composition contains about 0.001 to about 1000 mg of recombinant human GCDH per dose. The pharmaceutical compositions of the present disclosure may be administered periodically, such as once daily, three times weekly, twice weekly, once weekly, three times monthly, twice monthly, or once monthly, at the discretion of an experienced clinician, or may be administered aperiodically in situations such as, but not limited to, acute progression of disease.

[0037] Meanwhile, in the present disclosure, the pharmaceutical composition may be administered to a patient in parallel with an existing glutaric aciduria treatment therapy. Specifically, the pharmaceutical composition of the present disclosure reduces lysine oxidation and increases the physiologic detoxification of glutaryl-CoA, which are commonly performed as existing glutaric aciduria treatments. Therapy for arginine, carnitine, clofibrate and / or or riboflavin supplementation, dietary modifications such as low-protein diets and low-lysine / lysine-free diets. etc. More specifically, the pharmaceutical compositions of the present disclosure may be for administration to a patient in conjunction with routine dietary adjustments.

[0038] In the present disclosure, pharmaceutical compositions can be prepared by mixing recombinant human GCDH having a desired degree of purity with pharma- ceutical or physiologically acceptable carriers, excipients or stabilizers using standard methods known in the art. Acceptable carriers include saline or buffers, such as phosphates, citric acid and other organic acids; antioxidants, such as ascorbic acid; low molecular weight polypeptides (containing less than about 10 amino acid residues); proteins, such as Serum albumin, gelatin or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or monosaccharides, disaccharides and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic Surfactants, such as TWEEN TM , PLURONICS TM , or PEG.

[0039] The pharmaceutical compositions of the present disclosure may contain pharma- ceutical acceptable salts at approximately physiological concentrations. Optionally, the dosage forms of the present invention may contain a pharma- ceutical acceptable preservative. Specifically, the concentration of the preservative may be 0.1-2.0% (typically v / v). In the present disclosure, the preservative is a pharma- ceutical acceptable salt. The surfactant may be any of those known in the art, specifically, benzyl alcohol, phenol, m-cresol, methylparaben, propylparaben, or a combination thereof. The pharmaceutical composition of the present disclosure may contain a pharma- ceutical acceptable surfactant at a concentration of 0.005-0.02%.

[0040] In the present disclosure, the pharmaceutical composition may further contain one or more active compounds necessary for the treatment of glutaric aciduria, specifically, active compounds having complementary activities to recombinant human GCDH that do not adversely affect each other. The compounds may be contained in the composition in an amount effective to achieve the intended purpose.

[0041] In one aspect of the present disclosure, there is provided a method for treating glutaric aciduria, comprising administering a pharmaceutical composition comprising recombinant human GCDH. The terms "recombinant human GCDH", "pharmaceutical composition comprising recombinant human GCDH" and "glutaric aciduria" in the present disclosure are as defined above.

[0042] In the present disclosure, a pharmaceutical composition comprising recombinant human GCDH can be administered to a human or animal subject by known methods, such as intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, subcutaneous, or inhalation routes.

[0043] As will be seen in the examples below, the pharmaceutical composition comprising the recombinant human GCDH of the present disclosure exhibits excellent effects when administered intravenously or subcutaneously, and therefore intravenous or subcutaneous administration is particularly preferable. EXAMPLES

[0044] The configuration and effects of the present disclosure will be described in more detail below with reference to examples and test examples. However, these examples and test examples are provided only for illustrative purposes to facilitate understanding of the present disclosure, and the scope and scope of the present disclosure are not limited to the following examples.

[0045] [Example] Example 1. Production and purification of recombinant human glutaryl-CoA dehydrogenase (GCDH) In order to produce recombinant human Glutaryl-CoA Dehydrogenase (GCDH), an optimized plasmid was constructed from the pcDNA3.4-TOPO backbone plasmid so that it could be suitable for GCDH expression in CHO cells (Cricetulus griseus; Chinese Hamster cells). As a result, a plasmid expressing the GCDH protein containing the amino acid sequence of SEQ ID NO:1 was constructed (p18AFUOQC_GCDH_pcDNA3.4-TOPO). was obtained by expressing a GCDH protein to which the amino acid sequence of SEQ ID NO: 2, which is a signal sequence, was added near the N-terminus for the optimization of expression and yield.

[0046] The p18AFUOQC plasmid prepared above was cultured in CHO cells (Expi TM CHO cells) After transforming the vector into the vector and culturing and expressing the vector at 37°C and 8% CO2 for 5 days, the cells were Collected.

[0047] This was purified using an immunoaffinity column (HisTrap column), and the purification was The purity was confirmed by Coomassie Blue staining and Western blot (Figure 1). The protein concentration was 7.06 mg / ml.

[0048] Example 2. Glutaric aciduria animal model GCDH KO mice, which are used as a glutaric aciduria disease model mouse, have already been widely This is a commercially available mouse strain (e.g., Cyagen, strain name: KO-C57BL / 6J-Gcdh em1cyagen ;Conditional K O-C57BL / 6J-Gcdh em1.1cyagen ). In this disclosure, however, each inventor Separately, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene scissors technology We created GCDH KO mice using the gene. The KO mice were created by targeting the Exon 2 region of mouse GCDH, and the specific production was outsourced to Macrogen. Genotyping was performed using T7 endonuclease I reaction to confirm whether the glutaric aciduria disease model mice (GCDH KO mice) created had been knocked out (KO). The mice that were knocked out through genotyping were primarily were identified (Table 1).

[0049] [Table 1]

[0050] Specifically, 21 F0 mutant mice were examined for the presence or absence of mutations using the T7 endonuclease I reaction. As a result, it was confirmed that mutations occurred in all 21 mice, and in particular, F0 #4, #9, #13, and #20 mice had KO mutations in both chromosomes. Among these, F0 #4 individuals were selected and propagated for further experiments.

[0051] The GCDH KO mouse was identified as an animal model for glutaric aciduria as described below. Specifically, the levels of glutaric acid (GA) and 3-hydroxyglutaric acid (3-Hydroxyglutaric acid; 3 -OHGA concentrations and blood glutarylcarnitine (C5DC) concentrations were measured.

[0052] The GA and 3-OHGA values ​​were determined by organic acid analysis of mouse urine using GC-MS, and the C5DC values ​​were determined by dried blood spot analysis. The results are shown in Table 2 below and in FIG.

[0053] [Table 2]

[0054] As can be seen from the above results, the GCDH KO mice produced were able to express C5DC and glutamate. The levels of glutaric acid and other substances were significantly high, and the symptoms were very similar to those of patients with glutaric aciduria type 1, so it was confirmed that this mouse can be used as an animal model for GA1.

[0055] Example 3. Experiments with recombinant human glutaryl-CoA dehydrogenase administration in an animal model of glutaric aciduria type 1 The recombinant human glutaryl-CoA dehydrogenase (GCDH) produced in Example 1 was administered to the glutaric aciduria type 1 animal model produced / confirmed in Example 2, and the therapeutic effect was confirmed twice. I checked it thoroughly.

[0056] Example 3-1.1 In vivo experiment A single injection of recombinant human GCDH was administered to 5-week-old GCDH KO mice via the tail vein. Specifically, rhGCDH was administered in single doses of 50 and 100 μg, and the glutaric acid (GA) levels in urine were measured using LC-MS / MS. Urine samples were collected from each experimental group a week before and two days after administration and measurements were performed. The outline of the first animal experiment is shown in Figure 4, and the experimental groups and numbers of animals are shown in Table 3 below.

[0057] [Table 3]

[0058] The glutaric acid level in urine collected using the method described above was measured using LC-MS / MS analysis. The results are shown in Table 4 below. On the other hand, the amount of urine required for LC-MS / MS analysis is Because there were many samples, urine samples from all individuals in each experimental group were combined and analyzed as one sample.

[0059] [Table 4]

[0060] As can be seen from Table 4 and Figures 4 and 5, the glutaric acid level in wild-type mice was maintained at about 100 mmol / mol or less, whereas the glutaric acid level in GCDH KO mice was about 6400-7500 mmol / mol without administration of rhGCDH. In the group that received hGCDH, it was confirmed that glutaric acid levels decreased by approximately 30-45% two days later, regardless of the dose.

[0061] Example 3-2. Second in vivo experiment In an experiment, recombinant human GCDH was administered once to 5-6 week-old GCDH KO mice via the tail vein (iv) or subcutaneous (sc). A single dose of 100 μg was administered, and rhGCDH was administered subcutaneously in single doses of 50, 150, and 250 μg. In general, the subcutaneous dose had a higher bioavailability than the intravenous dose. Since the BA (ba) was 20% (see Korean Patent Publication No. 2017-0004814), the dose was set at 1, 3, and 5 times the dose of 50 μg administered intravenously.

[0062] The glutaric acid (GA) level in urine was measured by LC-MS / MS. Urine samples were collected from each individual in each experimental group 3 days before, 2 days after, and 1 week after CDH administration. The outline of the second animal experiment is shown in Figure 6, and the experimental groups and numbers of individuals are shown in Tables 5 and 6 below.

[0063] [Table 5]

[0064] [Table 6]

[0065] The glutaric acid level in urine collected using the method described above was measured using LC-MS / MS analysis. The results are shown in Table 7 below. In the second experiment, the values ​​were measured for each individual and the average values ​​were calculated.

[0066] [Table 7]

[0067] [Table 8]

[0068] Wild-type mice maintained glutaric acid levels at or below an average of approximately 100 mmol / mol. The GCDH KO mice showed no significant decrease in glutaric acid (indicated by "-"). Without rhGCDH administration, the average glutaric acid level was about 3600 to 10,000. In the rhGCDH-administered group, the 2-day It was confirmed that the glutaric acid level had subsequently decreased by approximately 30-40%.

[0069] On the other hand, the effects of intravenous administration and subcutaneous administration are compared as shown in Table 9 below.

[0070] [Table 9]

[0071] Considering that the bioavailability of subcutaneous administration is generally 20% of that of intravenous administration (Korea (Reference to Patent No. 2017-0004814), (1) GCDH KO + rhGCDH 5 It can be confirmed that the (1) 0 μg iv experimental group and the (2) GCDH KO + rhGCDH 250 μg sc experimental group had similar GA reducing effects 2 and 7 days after administration.

[0072] As can be seen from the above data, the administration of GCDH to an animal model of glutaric aciduria was able to effectively regulate the glutaric acid level, and the effect was maintained even one week after administration.

[0073] Through this, the recombinant human glutaryl-CoA dehydrogenase (recombinant nt human Glutaryl-CoA dehydrogenase;rhGCD The pharmaceutical composition containing H) may be administered via a commonly used route of administration to the brain, even if it is not administered directly to the cerebral ventricles. It can be seen that the glutaric acid level can be significantly reduced and maintained during treatment, and thus the compound can be effectively used to treat glutaric aciduria.

[0074] From the above description, a person skilled in the art to which the present disclosure belongs can understand that the present disclosure can be embodied in other specific forms without changing the technical idea or essential features of the present disclosure. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present disclosure should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below rather than the above detailed description, and the equivalent concepts thereof.

Claims

1. Recombinant human glutaryl-CoA dehydrogenase A pharmaceutical composition for treating glutaric aciduria, comprising rhGCDH (rhGDH-glutaryl-CoA dehydrogenase).

2. The pharmaceutical composition for treating glutaric aciduria according to claim 1, wherein the rhGCDH comprises the amino acid sequence of SEQ ID NO:

1.

3. 2. The pharmaceutical composition for treating glutaric aciduria according to claim 1, which is administered intravenously or subcutaneously.

4. 2. The pharmaceutical composition for treating glutaric aciduria according to claim 1, wherein the glutaric aciduria is type 1 glutaric aciduria.

5. 2. The pharmaceutical composition for treating glutaric aciduria according to claim 1, which is administered once a week, twice a month, or once a month.

6. 2. The pharmaceutical composition for treating glutaric aciduria according to claim 1, which is administered in conjunction with dietary adjustments for glutaric aciduria.

7. The dietary modifications for glutaric aciduria include a low protein diet, a low-lysine diet, a lysine-free diet, arginine, carnitine, clofibrate and / or lipoprotein.

7. The pharmaceutical composition for treating glutaric aciduria according to claim 6, which is a flavin replacement therapy or a combination thereof.

8. Recombinant human glutaryl-CoA dehydrogenase The present invention relates to a method for treating glutaric aciduria, the method comprising the step of administering a pharmaceutical composition for treating glutaric aciduria, the pharmaceutical composition comprising rhGCDH.

9. The method for treating glutaric aciduria according to claim 8, wherein the rhGCDH comprises the amino acid sequence of SEQ ID NO:

1.

10. The method for treating glutaric aciduria according to claim 8, wherein the administration is intravenous or subcutaneous.

11. The method for treating glutaric aciduria according to claim 8, wherein the glutaric aciduria is type 1 glutaric aciduria.

12. 9. The method of claim 8, which is administered once a week, twice a month, or once a month.

13. 9. The method of claim 8, administered to a patient in conjunction with dietary adjustments for glutaric aciduria.

14. The dietary modifications for glutaric aciduria include a low protein diet, a low-lysine diet, a lysine-free diet, arginine, carnitine, clofibrate and / or lipoprotein. The method for treating glutaric aciduria according to claim 13, which is flavin replacement therapy or a combination thereof.

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