JP6709476B1 - Citrine deficiency treatment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel therapeutic agent for citrin deficiency. SOLUTION: The active ingredient contains (1) ornithine or a salt thereof, and (2) one or more of aspartic acid, alanine, or glutamic acid, or a salt thereof. [Selection diagram] Figure 1

Description

The present invention relates to a therapeutic agent for citrin deficiency.

Citrine deficiency is a genetic disease that causes deficiency of citrin, which is a transporter existing in the inner mitochondrial membrane. Symptoms caused by citrin deficiency include hypercitrullineemia, hyperammonemia, accumulation of NADH metabolism-related substances in the cytoplasm or blood, and the like. Due to these symptoms, patients with citrin deficiency suffer from disorientation, abnormal behavior, consciousness disorder, jaundice, cholestasis, fatty liver, and energy deficiency (caused by carbohydrate repellency associated with excessive cytoplasmic NADH). It is believed that) occurs. It is also known that due to these symptoms, citrin deficiency patients are more likely to suffer from diseases such as liver damage and hepatic encephalopathy. On the other hand, it is known that when liver transplantation is performed as a treatment for citrin deficiency, blood biochemical findings in patients are normalized and these symptoms disappear.

It is known that a general method for coping with hyperammonemia such as a low protein diet exacerbates the symptoms of citrin deficiency (Non-patent Document 1). As a therapeutic agent for citrin deficiency, it is considered to use arginine preparation (Argi U (registered trademark)), medium-chain fatty acid triglyceride (MCT) preparation (non-patent document 2), pyruvic acid preparation (patent document 1), etc. Has been done.

Japanese Patent No. 4912574

Fukushima, K. et al. Intern. Med. 49, 243-247 (2010). Saheki, T. et al. Mol. Genet. Metab. 107, 322-329 (2012).

Known therapeutic agents for citrin deficiency are not effective for all patients. An object of the present invention is to provide a new therapeutic agent for citrin deficiency and to increase the options in the treatment of citrin deficiency.

The present inventors have surprisingly found that ornithine, which is changed into citrulline by metabolism in the body, is administered in combination with a specific amino acid, whereby the symptoms of citrin deficiency can be suppressed, The present invention has been completed based on this discovery.

In order to achieve the object of the present invention, for example, the therapeutic agent for citrin deficiency of the present invention has the following constitution. That is,
It is characterized by containing L-ornithine L-aspartate as an active ingredient.

A new therapeutic agent for citrin deficiency can be provided.

FIG. 3 is a graph showing changes in blood ammonia concentration and blood citrulline concentration when various amino acids are administered enterally. The figure which shows the change of a urea synthesis rate and L/P ratio at the time of perfusing alanine or aspartic acid in addition to ornithine in a liver perfusion experiment. The figure which shows the alanine concentration difference in a portal vein and an artery when various amino acids are enterally administered.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. The following embodiments do not limit the invention according to the claims, and not all combinations of the features described in the embodiments are essential to the invention. Two or more features of the plurality of features described in the embodiments may be arbitrarily combined. Further, the same or similar configurations are designated by the same reference numerals, and duplicated description will be omitted.

The therapeutic agent for citrin deficiency according to one embodiment of the present invention contains (1) ornithine or a salt thereof, and (2) one or more of aspartic acid, alanine, or glutamic acid, or a salt thereof, as active ingredients. ..

The therapeutic agent for citrin deficiency refers to an agent for alleviating or suppressing the symptoms caused by citrin deficiency in patients with citrin deficiency. For example, the citrin deficiency therapeutic agent may be a therapeutic agent for hypercitrullineemia or hyperammonemia caused by citrin deficiency, that is, a blood citrulline concentration reducing agent or a blood ammonia concentration reducing agent. .. Further, the therapeutic agent for citrin deficiency may be an agent for lowering intracytoplasmic NADH caused by citrin deficiency.

In addition, it is known that in patients with citrin deficiency, various symptoms can be prevented or eliminated by bringing the blood biochemical findings of the patients closer to normal. Therefore, the therapeutic agent for citrin deficiency may be a therapeutic agent or preventive agent for disorientation, abnormal behavior, consciousness disorder, jaundice, cholestasis, fatty liver, insufficient energy, liver disorder, or hepatic encephalopathy.

Ornithine, aspartic acid, alanine, and glutamic acid are each amino acids. Each amino acid may be a simple substance or a salt. The salt of the amino acid can be any pharmaceutically acceptable salt. For example, the amino acid salt may be a sodium salt, a potassium salt, a hydrochloride, a carbonate or the like. Moreover, the salt of an amino acid may be a salt of a basic amino acid and an acidic amino acid. In one embodiment, the therapeutic agent for citrin deficiency contains ornithine aspartate as a combination of ornithine or a salt thereof and aspartic acid or a salt thereof.

The therapeutic agent for citrin deficiency may contain the above amino acid in the form of peptide or protein. That is, the therapeutic agent for citrin deficiency according to one embodiment contains (1) ornithine, and (2) a peptide or protein containing at least one of aspartic acid, alanine, or glutamic acid as a constituent element.

In one embodiment, (1) the weight of ornithine or a salt thereof (calculated as the weight of free amino acid (ornithine), the same applies hereinafter) contained in the therapeutic agent for citrin deficiency is 10% or more, and preferably It is 20% or more, more preferably 30% or more, and further preferably 40% or more. Further, in one embodiment, (2) one or more weights of aspartic acid, alanine, or glutamic acid contained in the therapeutic agent for citrin deficiency (the total weight of aspartic acid, alanine, and glutamic acid, and free amino acid ( Aspartic acid, alanine, and glutamic acid), the same hereinafter) is 10% or more, preferably 20% or more, more preferably 30% or more, and further preferably 40% or more. is there. By increasing the amino acid content in this way, a more effective therapeutic effect can be obtained while reducing the burden upon administration.

From the viewpoint of improving the therapeutic effect, in one embodiment, (1) the weight of ornithine or a salt thereof and (2) the weight of one or more of aspartic acid, alanine, or glutamic acid contained in the therapeutic agent for citrin deficiency. The ratio ((1):(2)) is 2:8 or more, preferably 3:7 or more, more preferably 4:6 or more, while it is 8:2 or less, preferably 7 :3 or less, and more preferably 6:4 or less.

From the viewpoint of improving the therapeutic effect, in one embodiment, one or more of (1) ornithine or a salt thereof and (2) aspartic acid, alanine, or glutamic acid contained in the citrin deficiency therapeutic agent (aspartic acid, alanine, And (total amount of glutamic acid) ((1):(2)) is 2:8 or more, preferably 3:7 or more, more preferably 4:6 or more, while 8: It is 2 or less, preferably 7:3 or less, and more preferably 6:4 or less.

In one embodiment, the mole of (1) ornithine or a salt thereof and (2) one or more of aspartic acid, alanine, or glutamic acid (the total amount of aspartic acid, alanine, and glutamic acid) contained in the therapeutic agent for citrin deficiency. The ratio ((1):(2)) is 5:5. For example, the citrin deficiency therapeutic agent may contain ornithine aspartate, which is a salt of ornithine and aspartic acid in an equimolar ratio. It has been confirmed that such a therapeutic agent for citrin deficiency is particularly effective.

In one embodiment, the therapeutic agent for citrin deficiency contains a small amount of amino acids other than ornithine, aspartic acid, alanine, and glutamic acid. For example, in one embodiment, the total weight of ornithine, aspartic acid, alanine, and glutamic acid contained in the therapeutic agent for citrin deficiency is 50% or more of the total amount of amino acids contained in the therapeutic agent for citrin deficiency, and preferably 70%. % Or more, more preferably 90% or more, and particularly preferably 100%. The therapeutic agent for citrin deficiency according to one embodiment comprises (1) ornithine or a salt thereof, and (2) one or more of aspartic acid, alanine, or glutamic acid, or a salt thereof, and is, for example, ornithine aspartate. It may be.

In another embodiment, the therapeutic agent for citrin deficiency contains a low amount of a specific amino acid. For example, in one embodiment, the weight of glycine contained in the therapeutic agent for citrin deficiency is 10% or less, preferably 1% or less, and more preferably 1% or less of the total weight of ornithine, aspartic acid, alanine, and glutamic acid. It is 0.1% or less. Since it has been confirmed that glycine tends to increase blood ammonia concentration as described later, it is expected that such a therapeutic agent for citrin deficiency will be highly effective.

The therapeutic agent for citrin deficiency may further contain components such as an auxiliary agent or a carrier in addition to the above amino acids. Examples of auxiliaries include excipients, additives, coatings, sweeteners and the like. The dosage form of the therapeutic agent for citrin deficiency is not particularly limited. For example, the therapeutic agent for citrin deficiency may be a powder, a tablet, a capsule, a suspension, or the like.

As described above, the therapeutic agent for citrin deficiency according to one embodiment of the present invention is used for treating citrin deficiency. A treatment method according to an embodiment of the present invention is a method for treating a patient with citrin deficiency, which comprises the step of administering the above-mentioned therapeutic agent for citrin deficiency to the patient. Such a treatment method can alleviate or suppress the symptoms caused by citrin deficiency in a patient.

The patient is not specified. The patient may be, for example, a human or other mammal. The patient may be an adult patient or a pediatric patient.

The dose of the therapeutic agent for citrin deficiency can be appropriately selected according to the patient's age, symptoms, dosage form and the like. In one embodiment, the dose of the therapeutic agent for citrin deficiency is 2 g or more per day, preferably 4 g or more, and more preferably 6 g or more. In addition, in one embodiment, the dose is 40 g or more per day, preferably 30 g or less.

In addition, in one embodiment, the therapeutic agent for citrin deficiency is administered to a patient so that 1 g or more, preferably 2 g or more, and more preferably 3 g or more of ornithine is administered per day. On the other hand, in one embodiment, the therapeutic agent for citrin deficiency is administered to a patient so that 20 g or less, preferably 15 g or less ornithine is administered per day.

Furthermore, in one embodiment, the therapeutic agent for citrin deficiency has a weight of at least one of aspartic acid, alanine, and glutamic acid (total weight of aspartic acid, alanine, and glutamic acid) of 1 g or more per day, preferably Is administered to the patient in an amount of 2 g or more, more preferably 3 g or more. On the other hand, in one embodiment, the therapeutic agent for citrin deficiency is administered to a patient such that the weight of one or more of aspartic acid, alanine, and glutamic acid to be administered is 20 g or less, preferably 15 g or less per day. Is administered.

According to the above dose, a more effective therapeutic effect can be obtained while reducing the burden at the time of administration.

In addition, the therapeutic agent for citrin deficiency can be administered by an appropriate administration route, for example, oral administration, enteral administration, or injection (for example, perihepatic administration such as intrahepatic artery administration or portal vein administration). Injection) or the like can be used. The number of administrations and the timing of administration can also be appropriately selected according to the age, symptoms, dosage form of the patient. For example, the therapeutic agent for citrin deficiency may be administered daily or every other day. In one embodiment, the therapeutic agent for citrin deficiency is administered 2-4 times daily.

(Laboratory animals and reagents)
Animal experiments were performed under the approval of the University Ethics Committee and in accordance with the ARRIVE guidelines. Wild type (Wt) mice and citrin/glycerol-3-phosphate dehydrogenase double knockout (Double-KO) mice were congenic with C57BL/6. Mice were bred by the method described in Saheki, T. et al. J. Biol. Chem. 282, 25041-25052 (2007). Briefly, Wt mice and citrin knockout (Ctrn-KO) mice were obtained by mating heterozygous Ctrn-KO mice (Ctrn+/-/mGPD+/+), and glycerol-3-phosphate dehydrogenase knockout ( (mGPD-KO) mice and Double-KO mice were obtained by mating heterozygous Ctrn-KO/homozygous mGPD-KO mice (Ctrn+/-/mGPD-/-). Genotyping of DNA obtained from ear punches was performed in Ctrn-KO mice (Sinasac, DS, as described in Saheki, T. et al. Mol. Genet. Metab. 120, 306-316 (2017). et al. Mol. Cell. Biol. 24, 527-536 (2004).) and mGPD-KO mouse (Eto, K. et al. Science 283, 981-985 (1999).) for each target mutation. Was done using. Note that due to the racial difference in liver glycerophosphate shuttle activity between humans and mice, Double-KO mice are more phenotypically suitable as mouse models of citrin deficiency than Citrin-KO mice. .

In the experiment, 60- to 160-day-old mice were used. The mice were bred with free access to water and CE2 feed (manufactured by CLEA Japan, Inc.).

In each of the following experiments, L-arginine hydrochloride was used as L-arginine, L-ornithine hydrochloride was used as L-ornithine, L-aspartate sodium was used as L-aspartic acid, and L-glutamic acid was used as L-glutamic acid. Sodium hydrogen glutamate was used, L-lactic acid was sodium L-lactate, and pyruvic acid was sodium pyruvate, including ammonium chloride, L-alanine, glycine, and sucrose. It was manufactured by Kojunkaku Co., Ltd. or Nacalai Tesque, Inc. Furthermore, as the L-ornithine L-aspartate, one manufactured by Tokyo Chemical Industry Co., Ltd. was used. As the medium-chain fatty acid triglyceride, one manufactured by Kissei Pharmaceutical Co., Ltd. was used.

[Example 1: Effect of amino acids on ammonia concentration and citrulline concentration]
Blood ammonia concentration and plasma citrulline concentration were investigated by administering various amino acids to mice, which are models of citrin deficiency.

According to the preliminary study by the present inventors, administration of sucrose or glycine to Double-KO mice increases blood ammonia concentration, and administration of both sucrose and glycine further increases blood ammonia concentration. It was confirmed. Therefore, changes in blood ammonia concentration and plasma citrulline concentration were examined by further administration of various amino acids and the like to Double-KO mice to which both sucrose (Suc) and glycine (Gly) were administered. ..

Specifically, a drug solution of 20 ml/kg body weight was administered to a Wt mouse or a Double-KO mouse using a stomach probe. As the drug solution, physiological saline (Saline), 1M glycine aqueous solution (Gly), 20% sucrose aqueous solution (Suc), 20% sucrose+1M glycine aqueous solution (Suc+Gly), and 20% sucrose+1M glycine+0. A 5M aqueous solution of various amino acids (10 mmol/kg body weight) was used. As the amino acid, L-alanine (Ala), L-glutamic acid (Glu), L-aspartic acid (Asp), L-arginine (Arg), or L-ornithine (Orn) was used. Further, instead of the amino acid, 0.5 M pyruvic acid (Pyr) or 5% medium chain fatty acid triglyceride (MCT) (1 g/kg body weight) was also used. Furthermore, as an amino acid, a combination of L-ornithine and L-aspartic acid (Orn+Asp) (each 10 mmol/kg body weight), or a combination of L-ornithine and L-alanine (Orn+Ala) (each 10 mmol/kg) Body weight) was also used.

One hour after administration, the mice were sacrificed between 10 am and 11 am. Blood samples were taken from the heart under anesthesia with Somnopentyl. The blood ammonia concentration was determined using Ammonia-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

The experimental results are shown in FIGS. 1(A) and 1(B). As shown in FIG. 1(A), pyruvic acid (Pyr), MCT, L-alanine (Ala), L-glutamic acid (Glu), L-aspartic acid (Asp), L-arginine (Arg), and L-. When each of ornithine (Orn) was administered in addition to sucrose + glycine, it was confirmed that the blood ammonia concentration decreased, but the blood ammonia concentration was still higher than that of the Wt mouse (Saline administration), It was confirmed that the effect of reducing blood ammonia concentration was not sufficient. On the other hand, as shown in FIG. 1(B), particularly when arginine or ornithine was additionally administered, the plasma citrulline concentration was significantly higher than that of Double-KO mice (Suc+Gly) not additionally administered with various amino acids. It was confirmed that it would rise.

Then, as shown in FIG. 1(A), when a combination of L-ornithine and L-aspartic acid was administered, by adding L-aspartic acid or L-alanine to L-ornithine, It was confirmed that the ammonia concentration decreased. In particular, the administration of the combination of L-ornithine and L-aspartic acid significantly lowered the blood ammonia concentration to a level equivalent to that of Wt mice (Saline administration). Also, similar results were obtained when the combination of L-ornithine and L-alanine was administered.

Further, as shown in FIG. 1(B), when a combination of L-ornithine and L-aspartic acid was administered, plasma citrulline was obtained by adding L-aspartic acid or L-alanine to L-ornithine. It was confirmed that the increase in concentration could be significantly suppressed (*: P<0.05, ***: P<0.001). That is, it was confirmed that the administration of the combination of L-ornithine and L-aspartic acid reduced the plasma citrulline concentration as compared with Double-KO mice (Suc+Gly) to which amino acid was not additionally administered. Was done. Also, the combination of L-ornithine and L-alanine brought the plasma citrulline concentration to a level close to that of Double-KO mice (Suc+Gly).

Next, the relationship between the doses of L-ornithine and L-alanine and the blood ammonia concentration was examined. Specifically, in addition to 20% sucrose+1 M glycine, the drug solution contained 0.125 M (2.5 mmol) of L-ornithine L-aspartate (Orn+Asp# (not including NaCl) in the figure). /kg body weight), 0.25 M (5 mmol/kg body weight), or 0.5 M (10 mmol/kg body weight), except that the same experiment was performed.

The results are shown in Fig. 1(C). As shown in FIG. 1(C), it was confirmed that blood ammonia concentration decreased in a dose-dependent manner, and at each dose including 0.125M, Double-KO mice (Suc It was confirmed that the blood ammonia concentration was significantly lower than that of (+Gly) (*: P<0.05, ***: P<0.001). Here, surprisingly, as shown in FIG. 1(A), as compared with the case where L-ornithine hydrochloride (10 mmol/kg body weight) and L-aspartate sodium salt (10 mmol/kg body weight) were respectively administered. It was confirmed that when the same amount of L-ornithine L-aspartate (10 mmol/kg body weight) was administered, the range of decrease in blood ammonia concentration was larger and was lower than in Wt mice (Saline administration). Was done. It should be noted that, as described above, L-ornithine L-aspartate does not contain NaCl as compared to the combination of L-ornithine hydrochloride and sodium L-aspartate. Also, using 12.3 as the conversion factor for calculating the human equivalent dose from the mouse dose, the dose of L-ornithine in the mouse, 2.5 mmol/kg body weight (330 mg/kg body weight), is 0.27 mg/kg in human. This corresponds to a dose of kg body weight (1.6 g for a body weight of 60 kg).

These results indicate that the combination of L-ornithine and L-aspartic acid or L-alanine can significantly reduce the blood ammonia level in citrin deficiency, for example, suppressing the increase in blood ammonia level due to diet and the like. Is indicated. The results also show that such a combination can lower the plasma citrulline concentration, or at least suppress the increase in plasma citrulline concentration due to the action of L-ornithine. Furthermore, these results indicate that L-ornithine L-aspartate has a remarkable blood ammonia concentration lowering effect on patients with citrin deficiency.

[Example 2: Investigation of action mechanism of aspartic acid and alanine]
In order to investigate the mechanism of action of alanine and aspartic acid, the rate of urea production and the lactate/pyruvate ratio (L/P ratio) were measured using liver perfusion experiments. The L/P ratio is known as an index showing the cytoplasmic NADH/NAD ⁺ ratio, and a large L/P ratio indicates an increase in the cytoplasmic NADH.

The liver perfusion experiment was performed according to the method described in Moriyama, M. et al. Hepatol. 44, 930-938 (2006). In each experiment, the liver was perfused for 30 minutes in the absence of exogenous material before drug administration. The perfusate after passing through the liver was collected at a given time.

Specifically, in each of Wt mouse, Ctrn-KO mouse, mGPD-KO mouse, and Double-KO mouse, L-lactic acid (2 mM), pyruvic acid (0.2 mM), and ammonium chloride (2 mM) While perfusion, L-ornithine (2 mM) was further perfused, and then L-alanine (2 mM) or L-aspartic acid (2 mM) was further perfused. The results are shown in FIGS. 2(A) to (D).

As shown in FIGS. 2(A) and 2(C), perfusion of L-lactic acid (2 mM), pyruvic acid (0.2 mM), and ammonium chloride (2 mM) increased the urea production rate in Wt mice, while , L/P ratio did not increase so much, but urea production rate did not increase significantly in Double-KO mice, while L/P ratio increased significantly (both were significantly different from Wt mice). ..

As shown in FIGS. 2(A) and 2(C), in the Double-KO mouse, even when the perfusion of L-ornithine was started, the production rate of urea hardly increased, and the L/P ratio tended to gradually increase. Although seen, the addition of additional L-alanine significantly increased the urea production rate and significantly decreased the L/P ratio. On the other hand, as shown in FIGS. 2(B) and 2(D), even if L-aspartic acid was further added, the urea production rate hardly changed and the L/P ratio hardly changed.

Thus, in the liver perfusion experiment, the effectiveness of L-aspartic acid could not be confirmed, and the results contrary to Example 1 in which L-aspartic acid was enterally administered were obtained. In the first place, the results of Example 1 were surprising to the present inventors. This is because the absorbed aspartic acid is transported to the hepatic cells of the venous side (perivenous hepatocytes), but the urea cycle enzymes related to the blood ammonia concentration are present in the hepatocytes of the portal side (periportal hepatatocytes). Stoll, B. et al. Hepatology 13, 247-253 (1991).).

The present inventors argue that L-aspartic acid is converted to L-alanine in the small intestine and is transported to the liver through the portal vein, which is the reason why L-aspartic acid administered enterally was effective. (Neame, KD et al. J. Physiol. (Lond.) 135, 442-450 (1957). Parsons, DS et al. J. Physiol. (Lond.) 239, 677-694 (1974). Windmueller, HG et al. Arch. biochem. Biophys. 175, 670-676 (1976).).

For confirmation, L-aspartic acid, L-glutamic acid, L-ornithine, or glycine was enterally administered to a mouse (here, mGPD-KO mouse was used), 1 hour after the administration in the portal vein. The difference between the plasma concentration of alanine and the plasma concentration of alanine in the abdominal aorta was measured. This experiment was performed in the same manner as in Example 1. The blood samples were simultaneously collected from the portal vein and abdominal aorta under anesthesia with somnopentyl. Results are shown in FIG.

As shown in FIG. 3, it was confirmed that the enteral administration of L-aspartic acid or L-glutamic acid significantly increased the difference in alanine concentration as compared with the control (Saline). This shows that enteral administration of L-aspartic acid or L-glutamic acid increases the amount of alanine supplied to the liver via the portal vein. Even if glycine was administered enterally, no significant increase in alanine concentration could be confirmed. The results in FIG. 3 indicate that L-glutamic acid has a similar effect to L-aspartic acid, that is, the combination of L-ornithine and L-glutamic acid has a therapeutic effect on citrin deficiency.

The invention is not limited to the above embodiment, and various modifications and changes can be made within the scope of the invention.

Claims (3)

A therapeutic agent for citrin deficiency, which comprises L-ornithine L-aspartate as an active ingredient. The dosage of ornithine becomes more 1g per day, and in an amount that the dose is more than 1g per day of aspartic acid, characterized in that it is administered to Citrin deficiency patients, citrine claim 1 Deficiency therapeutic agent. The therapeutic agent for citrin deficiency according to claim 1 or 2 , which is used for reducing blood ammonia concentration in a patient with citrin deficiency.