HK1246165B - Agent for enhancing atp in cells - Google Patents

Description

Intracellular ATP enhancer

Technical Field

The present invention relates to an intracellular ATP enhancer for humans or animals. More specifically, the present invention relates to an intracellular ATP enhancer for humans or animals comprising a combination of: A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof, and B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof.

Background Art

ATP (adenosine triphosphate, hereinafter sometimes referred to as ATP) is the most important compound for storing energy in an organism and supplying it when needed. A decrease in ATP is thought to be associated with the pathological conditions of various diseases. For example, a decrease in ATP in red blood cells is believed to be the cause of many types of hereditary hemolytic anemias. Examples include sickle cell disease (Non-Patent Document 1), pyruvate kinase deficiency (Non-Patent Document 2), spherocytosis (Non-Patent Document 3), elliptocytosis (Non-Patent Document 3), stomatocytosis (Non-Patent Document 4), and thalassemia (Non-Patent Document 5).

Furthermore, it is proposed that a decrease in intracellular ATP is a mechanism of myocardial damage in ischemic heart disease (Non-Patent Document 6), and it is reported that the symptoms of chronic stable angina pectoris are suppressed by high-dose administration of allopurinol, a xanthine oxidase/xanthine dehydrogenase inhibitor (Non-Patent Document 7). The authors suggest that the increase in ATP due to allopurinol has a beneficial effect on ischemic heart disease (Non-Patent Document 7).

In addition, ATP enhancement therapy may be effective for heart failure. Heart failure patients in the United States usually undergo heart transplantation, and the period from the occurrence of heart failure to death, but the period from the occurrence of heart failure to heart transplantation is used as a measurement of the early progression speed of heart failure. The shorter period until heart transplantation illustrates that the progress of heart failure is faster. In Europe and the United States, the frequency of hereditary muscle AMP deaminase (AMPD) deficiency is extremely high, and about 20% of the general population has heterozygous deficiency (heterozygous deficiency). It is known from research that people with muscle hereditary AMPD deficiency have a longer period from heart failure to heart transplantation (non-patent literature 8). It is also proposed that AMPD inhibitors improve the heart failure of mice (non-patent literature 9). Usually, the ATP of muscle is reduced due to exercise. However, it is reported that people with hereditary muscle AMPD deficiency have intramuscular ATP that does not decrease after exercise or suppresses reduction (non-patent literature 10). In particular, because AMP is not converted into IMP (inosine monophosphate, hereinafter sometimes referred to as IMP), AMP reduction can be prevented and ATP reduction does not occur (Fig. 1). From the above, it is considered that hereditary muscle AMPD deficiency is less likely to cause a decrease in ATP in cardiomyocytes and suppress the progression of heart failure.

Enhancing ATP in this manner would be expected to ameliorate the pathology of diseases in which decreased ATP is associated with the pathology.

Although it has been reported that inosine enhances muscle movement under the expectation of the occurrence of an enhancement effect of muscle movement due to the increase in ATP by inosine administration, a report has recently been made denying the effect of inosine (Non-Patent Document 11). However, the reason why the muscle enhancement effect of inosine cannot be confirmed is probably because inosine alone is insufficient to achieve an ATP enhancement effect.

Nishino et al. have found that xanthine oxidase/xanthine dehydrogenase inhibitors such as febuxostat, administered to model mice of amyotrophic lateral sclerosis (hereinafter sometimes referred to as ALS), inhibit disease progression (non-patent literature 12). Allopurinol does not inhibit disease progression. Nishino et al. speculate that the increase in ATP in nerve cells administered with febuxostat inhibits disease progression (non-patent literature 12). Nishino et al. speculate that the reason why allopurinol has no effect is because allopurinol consumes PRPP and, on the contrary, inhibits ATP synthesis (non-patent literature 12). In fact, it is reported that knockout of Na/K-ATPase in ALS model mice inhibits the degeneration of nerve cells (non-patent literature 12). It is also reported that Na/K-ATPase activity increases in ALS patients (non-patent literature 13). Therefore, it is believed that the activation of Na/K-ATPase that reduces ATP promotes the onset or progression of ALS, and the inhibition of Na/K-ATPase that inhibits ATP reduction inhibits the progression of ALS.

In addition, report inosine administration alleviates the symptom of Parkinson's disease (non-patent literature 14) and multiple sclerosis (non-patent literature 15). The author of two documents believes that the minimizing of serum uric acid value can be relevant to disease. In order to improve serum uric acid value and produce the purpose of therapeutic effect by using inosine, clinical trial is carried out. However, in previous report, effect is not enough.

It has been reported that due to the single administration of inosine, intracellular ATP increases slightly. In fact, the people such as Ogasawara report that when the erythrocyte that is allowed to be placed at low temperature for 20 to 30 days and ATP reduces is placed 1 hour after the addition of inosine, ATP increases (non-patent literature 16), but inosine is rapidly metabolized to uric acid (Fig. 1) by hypoxanthine and xanthine in human body. Therefore, independent inosine is not enough to produce enough ATP enhancing effects. In addition, although it is expected that the single administration of febuxostat enhances intracellular ATP slightly, independent febuxostat may be insufficient.

Prior art literature

Non-patent literature

Non-patent document 1: Palek J, Liu SC, Liu PA. Crosslinking of the nearest membrane protein neighbors in ATP depleted, calcium enriched and irreversiblysickled red cells. Prog Clin Biol Res. 1978; 20:75-91.

Non-patent document 2: Glader BE. Salicylate-induced injury of pyruvate-kinase-deficient erythrocytes. N Engl J Med. 1976 Apr 22; 294(17): 916-8.

Non-patent literature 3: de Jong K, Larkin SK, Eber S, Franck PF, Roelofsen B, Kuypers FA. Hereditary spherocytosis and elliptocytosis erythrocytes show a normal transbilayer phospholipid distribution. Blood. 1999Jul 1;94(1):319-25.

Non-patent document 4: Mentzer WC Jr, Smith WB, Goldstone J, Shohet SB. Hereditary stomatocytosis: membrane and metabolism studies. Blood. 1975 Nov; 46(5): 659-69.

Non-patent document 5: Wiley JS.Increased erythrocyte cation permeability inthalassemia and conditions of marrow stress.J Clin Invest.1981Apr;67(4):917-22.

Non-patent literature 6: Vogt AM, Ackermann C, Yildiz M, Schoels W, Kubler W. Lactateaccumulation rather than ATP depletion predicts ischemic myocardial necrosis:implications for the development of lethal myocardial injury. Biochim BiophysActa. 2002Mar 16; 1586(2):219-26

Non-patent literature 7: Noman A, Ang DS, Ogston S, Lang CC, Struthers AD. Effect of high-dose allopurinol on exercise in patients with chronic stable angina: arandomised, placebo controlled crossover trial. Lancet. 2010 Jun 19; 375 (9732): 2161-7.

Non-patent literature 8: Loh E, Rebbeck TR, Mahoney PD, DeNofrio D, Swain JL, Holmes EW. Common variant in AMPD1gene predicts improved clinical outcome in patients with heart failure. Circulation. 1999Mar 23;99(11):1422-5.

Non-patent literature 9: Borkowski T, Slominska EM, Orlewska C, Chlopicki S, Siondalski P, Yacoub MH, Smolenski RT. Protection of mouse heart against hypoxicdamage by AMP deaminase inhibition. Nucleosides Nucleotides NucleicAcids. 2010Jun; 29(4-6): 449-52.

Non-patent literature 10: Norman B, Sabina RL, Jansson E. Regulation of skeletalmuscle ATP catabolism by AMPD1genotype during sprint exercise in asymptomatic subjects. J Appl Physiol (1985). 2001Jul; 91 (1): 258-64.

Non-patent literature 11: McNaughton L, Dalton B, Tarr J. Inosine supplementation has no effect on aerobic or anaerobic cycling performance. Int J Sport Nutr. 1999 Dec; 9(4): 333-44.

Non-Patent Document 12: Yasuko Abe, Shinsuke Kato, Takeshi Nishino. Therapeutic agent for amyotrophic lateral sclerosis. US Patent US 8318792B2, European Patent EP2050467A1

Non-patent literature 13: Gallardo G, Barowski J, Ravits J, Siddique T, Lingrel JB, Robertson J, Steen H, Bonni A. Anα2-Na/K ATPase/α-adducin complex in astrocytestriggers non-cell autonomous neurodegeneration. Nat Neurosci. 2014Dec; 17(12):1710-9.

Non-patent literature 14: Parkinson Study Group SURE-PD Investigators, Schwarzschild MA, Ascherio A, Beal MF, Cudkowicz ME, Curhan GC, Hare JM, Hooper DC, Kieburtz KD, Macklin EA, Oakes D, Rudolph A, Shoulson I, Tennis MK, Espay AJ, Gartner M, Hung A, Bwala G, Lenehan R, Encarnacion E, Ainslie M, Castillo R, Togasaki D, Barles G, Friedman JH, Niles L, Carter JH, Murray M, Goetz CG, Jaglin J, Ahmed A, Russell DS, Cotto C, Goudreau JL, Russell D, Parashos SA, Ede P, Saint-Hilaire MH, Thomas CA, James R, Stacy MA, Johnson J. Gauger L,Antonelle deMarcaida J,Thurlow S,Isaacson SH,Carvajal L,Rao J,Cook M,Hope-Porche C,McClurg L,Grasso DL,Logan R,Orme C,Ross T,Brocht AF,Constantinescu R,SharmaS,Venuto C,Weber J,Eaton K.Inosine to increase serum and cerebrospinal fluidurate in Parkinson disease: a randomized clinical trial.JAMA Neurol.2014Feb;71(2):141-50.

Non-patent literature 15: Markowitz CE, Spitsin S, Zimmerman V, Jacobs D, Udupa JK, Hooper DC, Koprowski H. The treatment of multiple sclerosis with inosine. JAltern Complement Med. 2009Jun; 15(6): 619-25.

Non-patent literature 16: Ogasawara N, Goto H, Yamada Y, Nishigaki I, Itoh T, HasegawaI, Park KS. Deficiency of AMP deaminase in erythrocytes. Hum Genet. 1987Jan; 75(1):15-8.

Summary of the Invention

Problems to be solved by the invention

The problem to be solved by the present invention is to provide a composition having an effect of enhancing intracellular ATP, in particular an ATP enhancer that exceeds the enhancing effect of inosine or febuxostat alone.

Furthermore, it is desirable to provide a novel ATP enhancer and ATP enhancing method that suppresses an increase in serum uric acid value caused by inosine when inosine is administered to enhance ATP.

Solutions for solving problems

In order to solve the above-mentioned problems, the present invention has the following configurations.

<1> A human or animal intracellular ATP enhancer containing a combination of A) and B):

A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof; and

B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof.

<2> The intracellular ATP enhancer according to <1>, wherein the xanthine oxidase/xanthine dehydrogenase inhibitor is any one or more selected from febuxostat, topiroxostat (FUJIYAKUHIN), allopurinol, hydroxyalkane, carprofen, and Y-700 (Mitsubishi Tanabe Pharma).

<3> The ATP enhancer according to <1> or <2>, wherein the ATP enhancer is in the form of a combination drug or a kit formulation.

<4> Febuxostat used in combination with inosine as an intracellular ATP enhancer.

<5> Pharmaceuticals, which are compound preparations or kit preparations containing a combination of A) and B):

A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof; and

B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof.

The present invention also provides the following administration method.

<6> A method for enhancing intracellular ATP in humans or animals, comprising administering to humans or animals the steps of A) and B):

A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof; and

B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof.

<7> The method for enhancing intracellular ATP according to <6>, wherein the xanthine oxidase/xanthine dehydrogenase inhibitor is any one or more selected from febuxostat, topiroxetine (FUJIYAKUHIN), allopurinol, hydroxyalkane, carprofen, and Y-700 (Mitsubishi Tanabe Pharma).

<8> The method for enhancing intracellular ATP according to <6> or <7>, wherein A) and B) are a compound pharmaceutical comprising A) and B).

<9> A method for enhancing intracellular ATP, comprising administering to a patient with hemolytic anemia, ischemic heart disease, heart failure, amyotrophic lateral sclerosis, Parkinson's disease, or ADSL deficiency the steps of A) and B):

A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof; and

B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof.

Effects of the Invention

The ATP-enhancing effect of the combined administration of A) and B) of the present invention makes it possible to provide therapeutic agents for various diseases in which a decrease in ATP forms part of the pathological condition, and further various diseases in which the progression of the pathological condition is suppressed by an excess supply of ATP:

A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof; and

B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[Figure 1] Figure 1 is a diagram of the pathway of ATP synthesis.

[Figure 2] Figure 2 shows the regimen of combined administration of febuxostat and inosine and the transition of serum uric acid levels. The horizontal axis represents the serum uric acid level measurement day, and the vertical axis represents the serum uric acid level (unit: mg/dL).

[Figure 3] Figure 3 is a graph showing the results of a regression analysis of the relationship between inosine dosage and serum uric acid level. The horizontal axis represents the daily dose of inosine (unit: g/day), the vertical axis represents the serum uric acid level (unit: mg/dL), and the equation in the figure is an equation representing a regression line. In this equation, x is the daily dose of inosine, and y is the serum uric acid level.

[Figure 4] Figure 4 is a diagram showing the transition of serum uric acid levels for the single administration of febuxostat or inosine and the combination of the two drugs. The horizontal axis represents the serum uric acid value measurement day (e.g., 20150501 represents May 1, 2015), and the vertical axis represents the serum uric acid value (unit: mg/dL).

Figure 5 compares the levels of various purines in peripheral blood when Febuxostat and inosine are used in combination and when the drugs are not taken. The purine levels are expressed as moles per liter of whole blood. Febuxostat (40 mg) + inosine (2 g): Values when Febuxostat and inosine are used in combination. None: Values when the drugs are not taken.

[Fig. 6] Fig. 6 is a diagram of the conversion of serum uric acid values in the case of administration to groups A to E. The horizontal axis represents the measurement period (week), and the vertical axis represents the serum uric acid value (unit mg/dL). Group A: Febuxostat 20 mg, twice a day, 14 days, Group B: Inosine 500 mg, twice a day, 14 days, Group C: Febuxostat 20 mg+Inosine 500 mg, twice a day, 14 days, Group D: Febuxostat 20 mg+Inosine 1000 mg, twice a day, 14 days, Group E: Febuxostat 30 mg, twice a day, 14 days (same as the following figure).

[ Fig. 7] Fig. 7 is a graph showing the transition of urine uric acid concentration/creatinine concentration in the case of administration to Groups A to E. The horizontal axis represents the measurement period (week), and the vertical axis represents urine uric acid concentration/creatinine concentration (ratio).

Figure 8 is a graph comparing ATP and ADP in the blood when the drug was administered to Groups A to E. The horizontal axis represents the measurement period (weeks), and the vertical axis represents the ATP or ADP concentration (unit: μM). ATP: solid line, ADP: dotted line.

Figure 9 is a graph comparing Hx (hypoxanthine) and X (xanthine) in blood when administered to Groups A to E. The horizontal axis represents the measurement period (weeks), and the vertical axis represents the Hx or X concentration (unit: μM). Hx: solid line, X: dotted line.

[ Fig. 10] Fig. 10 is a graph comparing the concentrations (µM) of various purines in urine when administered to Groups A to E.

DETAILED DESCRIPTION

An active ingredient of the present invention is A) a xanthine oxidase/xanthine dehydrogenase inhibitor, or a pharmaceutically acceptable salt thereof. Examples of xanthine oxidase/xanthine dehydrogenase inhibitors include febuxostat, topiramate (FUJIYAKUHIN), allopurinol, hydroxyalkanes, carprofen, and Y-700 (Mitsubishi Tanabe Pharma), of which febuxostat is desirable.

Another active ingredient of the present invention is B) any one or more compounds selected from inosine, inosinic acid, hypoxanthine, and pharmaceutically acceptable salts thereof, among which inosine is desirable.

The intracellular ATP enhancer of the present invention refers to the effect of the active ingredient of the present invention on increasing ATP production in cells. Increase is used to mean increasing from a steady state, inhibiting a decrease, or bringing a decreased state closer to a steady state. The effects of the present invention can be confirmed by directly measuring intracellular ATP concentrations and indirectly by measuring products of other metabolic pathways induced by the increase in ATP.

The term "combination comprising A) and B)" as used herein refers to any combination of components A) and B) that is administered to exert an ATP-enhancing effect in the body of a subject. This includes, for example, the mixing of a compound pharmaceutical of components A) and B) to form a composition, or the administration of co-existing drugs, although physically separate without mixing, within the same time period of administration. Examples of compound pharmaceuticals of components A) and B) that are mixed to form a composition include those mixed as pharmaceutical preparations. Examples of pharmaceutical preparations include oral preparations such as granules, powders, solid preparations, and liquids. Examples of co-existing drugs that are administered within the same time period of administration, although physically separate, include so-called kit preparations and forms packaged in a single bag. The same time period does not necessarily mean the same time in the strict sense, and the same time period of the present invention includes situations where there is a gap within the range of exerting the ATP-enhancing effect of the present invention. For example, when one drug is taken before a meal and the other is taken after a meal, this corresponds to the situation of administration within the same time period of the present invention.

The dosage of the present invention is desirably 10 to 80 mg/day for A) febuxostat. The dosage is desirably 0.5 to 4.0 g/day for B) inosine.

In terms of administration, each dose can be administered once a day or twice a day. Wherein, Febuxostat is desirably administered twice a day rather than once a day in the conventional use of Febuxostat. It is also desirable that inosine is administered twice a day rather than once a day. Therefore, inosine and Febuxostat are desirably administered twice a day separately.

In the case of a combination drug, adjustment can be made in consideration of the daily dose and administration method, and febuxostat and inosine can be desirably adjusted by adding 0.5 g, 1 g, 1.5 g, or 2 g of inosine to 20 mg or 40 mg of febuxostat.

The subject of administration of the present invention is a human or an animal, and the human or animal is under a condition requiring ATP enhancement.

Target diseases include the following diseases, for which ATP reduction is strongly associated with pathological conditions, namely (1) hemolytic anemia, (2) ischemic heart disease, (3) heart failure, (4) amyotrophic lateral sclerosis, (5) Parkinson's disease, and (6) ADSL deficiency. Among them, the present invention is particularly effective for (2) ischemic heart disease, (3) heart failure, and (4) amyotrophic lateral sclerosis.

The ATP enhancer of the present invention may be further combined with other drugs within the range not impairing the effects of the present invention.

The present invention will now be specifically described based on Examples; however, the present invention is not limited thereto.

Example

[Test Example 1] First Dosage Test (Combination Administration)

1. Measurement of serum uric acid

As a clinical chemistry automatic analysis apparatus, a dry clinical chemistry analysis measuring unit manufactured by ARKRAY, Inc. was used, and the serum uric acid value was measured by using a uricase-peroxidase method.

2. Drug administration test

(1) Target group

Human, age 67, male, height 180cm, weight 77kg

(2) Dosage regimen

Dosage regimen is shown in Figure 2. The first clinical trial is by first using febuxostat (single administration of febuxostat) with 40mg/ days, after a period of time, inosine (combination administration of febuxostat and inosine) is additionally used, and the dosage of inosine is gradually increased from 0.5 to 3g/ days. Meanwhile, blood is collected at the opportunity (timing) shown on the horizontal axis of Figure 2 to measure serum uric acid value.

3. Serum uric acid measurement results

The serum uric acid value of each administration period is shown in Fig. 2.According to the result, due to the administration of febuxostat with 40mg/ days, uric acid value is as expected reduced to 4.7mg/dL (2014.07.05) from 8.4mg/dL (2014.07.12).Subsequently, as the result of using inosine in a gradually increased mode from 0.5g/ days to 3g/ days except febuxostat, the serum uric acid value during the administration of inosine 0g, 0.5g, 1g, 2g and 3g is respectively 4.9 (2014.08.23), 5.1 (2014.9.01), 5.5 (2014.09.06), 6.0 (2014.09.11) and 6.4 (2014.09.26) mg/dL, and regression analysis discloses that serum uric acid value increases (Fig. 3) with the speed of the inosine 0.51mg/dL of every 1g. This can be seen from the regression line with a slope of 0.51 in Figure 3. Therefore, it is believed that when x (inosine) increases by 1, y (serum uric acid level) increases by 0.51. Even 40 mg/day of febuxostat and 3 g/day of inosine administration did not cause any adverse events.

[Test Example 2] Second Dosage Test

1. Measurement of serum uric acid

Same as Test Example 1.

2. Drug administration test

(1) The subjects of administration were the same as those in Test Example 1.

(2) Dosage regimen

Dosage regimen is shown in Fig. 4.After having passed enough periods from test example 1, once again febuxostat is applied to patient with 40mg/ days, and serum uric acid level is measured after 3 days.Subsequently, febuxostat is stopped, after a period of time, the inosine administration (after breakfast and dinner, respectively with 10g is applied) of 2g/ days is started, and then the inosine of 2g/ days and the febuxostat of 40mg (after breakfast and dinner, respectively with 20mg is applied) are used in combination.The time point shown in the horizontal axis of Fig. 4 is measured serum uric acid level.

3. Serum uric acid measurement results

The serum uric acid values at each dosing period are shown in Figure 4. The serum uric acid value measured after 3 days of dosing with 40 mg/day of febuxostat decreased by 3.4 mg/dL from 8.2 mg/dL before dosing (May 1, 2015) to 4.8 mg/dL after dosing (May 15, 2015). This is almost the same as the decrease of 3.7 mg/dL in the previous trial.

Subsequently, stop febuxostat, and confirm that serum uric acid value returns to 8.1mg/dL (20150605) and 8.3mg/dL (20150703) (Fig. 4) that are almost identical with previous value. From 20150704, start the inosine administration of 2g/ days (administration of each 1g after breakfast and dinner), and the serum uric acid value measured after 6 days extremely rises to 13.4mg/dL. Especially, because due to the inosine administration of 2g/ days, described value rises 5.1mg/dL, so the described value of every 1g/ days inosine administration rises 2.55mg/dL. Subsequently, as the result of the combination use of 40mg febuxostat (each with 20mg administration after breakfast and dinner), serum uric acid value is 4.4mg/dL (20150717) (Fig. 4). No adverse event occurs.

[Test Example 3] Third Dosage Test

1. Analytical methods

(1) Purine measurement method.

Various purines in peripheral blood were measured according to the literature (1). Briefly, peripheral blood was collected with EDTA, mixed with 500 μL + 500 μL ice-cold 8% PCA, immediately vortexed for 5 seconds, and centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was stored at -80°C. The sample was dissolved in the collected state, and 40 μL of 2M K ₂ CO ₃ in 6M KOH was added to 650 μL of the solution to simultaneously precipitate PCA and neutralize the solution. After the solution was centrifuged at 12,000 × g for 10 minutes at 4°C, 160 μL of mobile phase was added to 40 μL of supernatant and HPLC was performed. The conditions of HPLC were also set according to the following literature (1). The amount of purine is expressed as the molar amount contained in 1 L of whole blood.

Literature (1): Coolen EJ, Arts IC, Swennen EL, Bast A, Stuart MA, Dagnelie PC. Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP-HPLC and UV-detection. J Chromatogr B AnalytTechnol Biomed Life Sci. 2008Mar 15; 864(1-2):43-51.

(2) Uric acid measurement method

Same as Test Example 1.

2. Drug administration test

(1) The subjects of administration were the same as those in Test Example 1.

(2) Dosage regimen

The combination of 2g/day inosine (each administered with 1g after breakfast and dinner) and 40mg of febuxostat (each administered with 20mg after breakfast and dinner) was used, and blood was collected after 7 days for the measurement of multiple purines in peripheral blood (febuxostat (40mg)+inosine 2g in Figure 5). Subsequently, all drugs were stopped, and blood was collected after 7 days for the measurement of multiple purines in peripheral blood (none in Figure 5).

3. Test results

The test results are shown in Figure 5. Although the uric acid value during the combination of inosine and febuxostat is 0.54 times higher than when not taking the drug, this is a reasonable value, because the result of the second administration test is that the serum uric acid value without taking the drug is 8.2, 8.1, and 8.3 mg/dL, and the serum uric acid value during the inosine of 2g/ days (each with 1g administration after breakfast and dinner) and the febuxostat of 40mg (each with 20g administration after breakfast and dinner) is 4.4 mg/dL, therefore, 4.4/8.1=0.54 is obtained. Therefore, the serum uric acid value during the combination of inosine of 2g/ days and the febuxostat of 40mg/ days is about 1/2 when not applying the drug.

Although ATP increases 1.05 times due to drug administration, this is reasonable because the drug administration test is carried out on normal people with no identified ATP reduction. Normal people have enough ATP and do not need to be further enhanced. Compared to ATP, the amount of IMP is extremely small (IMP amount is 1/204 of ATP amount without drug), and increases 1.16 times (Fig. 5) due to drug administration. Therefore, the combined use of inosine and febuxostat causes a slight increase in IMP. As expected, the purine that increases most significantly is hypoxanthine. Hypoxanthine increases 27.3 times (Fig. 5). Inosine and xanthine increase 1.65 times and 5.76 times respectively. Although inosine is administered as 2g per day, inosine only increases slightly, which shows that consumption of inosine is used to increase hypoxanthine. Fortunately, the increase of xanthine related to the report of stones is limited. Obviously, due to febuxostat, the increase of uric acid is significantly suppressed.

This experiment is carried out for normal people, and normal people have enough ATP and do not need further enhancement.Due to the combined use of inosine and febuxostat, no significant increase is produced.Yet, due to the significant increase of hypoxanthine, it is believed that when ATP is insufficient, the circuit of replenishing ATP works immediately (Fig. 1).

In addition, the combined use of inosine and febuxostat only resulted in a slight increase in inosine and xanthine, and even led to a decrease in uric acid. Only hypoxanthine increased significantly. The increase in xanthine was also relatively small and was an amount that did not cause problems compared to the amount of uric acid. This suggests that the combined use of inosine and febuxostat has a lower likelihood of crystalluria and stones.

4. Discussion of the results of the first to third tests

Comparing the results of the second test with those of the first test, since in the first test, the serum uric acid value increased by 0.51 mg/dL per 1 g/day of inosine in the presence of 40 mg/day of febuxostat, febuxostat inhibited the increase in serum uric acid value caused by inosine to 0.51/2.55=1/4.47.

Therefore, although it is found that respectively Febuxostat has the effect of reducing serum uric acid value and inosine has the effect of increasing serum uric acid value, it is thought that the serum uric acid increasing effect of inosine is suppressed by Febuxostat (to 1/4.47 or 22.4%).In other words, Febuxostat not only reduces serum uric acid value, but also significantly suppresses the serum urea increasing effect of inosine.

Febuxostat significantly suppresses the data explanation of the serum urea increasing effect of inosine, because the effect of febuxostat, due to the inhibition of xanthine oxidase/xanthine dehydrogenase, the hypoxanthine obtained from inosine significantly increases. This is confirmed by the result of the third test (Fig. 5). It is believed that this increase is much larger than the single administration of inosine. This is because it is believed that as seen in the increase of serum uric acid value, due to being metabolized to uric acid immediately, the single inosine administration has insufficient hypoxanthine increasing effect, and is also insufficient with respect to ATP enhancing. In fact, in the third test, the combination of inosine and febuxostat causes a slight increase of inosine. Consume most of thereby increase hypoxanthine. It is believed that this hypoxanthine helps ATP to enhance.

Possibly, this is because purine nucleoside phosphorylase (purine nucleoside phosphorylase, PNP) acts on inosine, causes being immediately converted into hypoxanthine and 1-phosphate ribose (Fig. 1).It is thought that hypoxanthine and 1-phosphate ribose produced in this way both play the effect (Fig. 1) of strengthening ATP.In addition, it is thought that because Febuxostat strongly suppresses xanthine oxidase/xanthine dehydrogenase, suppresses the decomposition of hypoxanthine to xanthine, and the concentration of hypoxanthine increases, and it further serves as the supply source of purine compound.This is supported by the fact (Fig. 5) that only the increase of hypoxanthine is very large when Febuxostat and inosine are used in combination.Hypoxanthine reacts with PRPP due to HPRT enzyme, and synthesizes IMP (Fig. 1).By two kinds of reactions, IMP is converted into AMP, and described AMP is converted into ATP.On the other hand, it is thought that 1-phosphate ribose is as the supply source of PRPP by 5-phosphate ribose, and due to adenine phosphoribosyltransferase (APRT), PRPP is attached to adenine and produces AMP, thus further increases ATP concentration. It is also believed that PRPP plays a significant role by increasing de novo purine synthesis and serving as a substrate for HPRT, thereby increasing the action of IMP.

It is important to note that the administration method of febuxostat when inosine and febuxostat are used in combination. When using the febuxostat of 40mg/ days and the inosine of 2g/ days, in the first and second tests, inosine was applied with 1g after breakfast and dinner. However, in the first test, febuxostat was applied once with 40mg after breakfast, and in the second test, it was applied twice with 20mg after breakfast and dinner. In the former, serum uric acid value was 6.0mg/dL, and in the latter, it was 4.4mg/dL, resulting in a larger difference of 1.5mg/dL. This is considered to be due to the stronger effect of reducing serum uric acid value by applying 20mg twice a day than applying 40mg febuxostat once a day. Therefore, when febuxostat is used in combination with inosine, it is desirable that febuxostat and inosine are applied equally twice a day, rather than once a day as in the conventional use of febuxostat.

[Test Example 4] Clinical trial (combination administration)

Although the administration subjects were the same in the first to third administration tests, in this experiment, the test subjects were expanded to confirm that the results of the first to third tests were correct.

1. Multiple measurement methods

(1) Clinical trials

Except for the following items, the measurement was performed by a conventional method.

(2) Serum uric acid level

Same as Test Example 1

(3) Urine uric acid concentration/creatinine concentration

Since the urine uric acid concentration varies with the urine volume, the urine uric acid amount was evaluated by using the urine uric acid/creatinine value obtained by dividing by the urine creatinine concentration. The uric acid value was measured in the same manner as in Test Example 1.

(4) Blood purine concentration

Same as Test Example 3.

(5) Urine purine concentration

Same as Test Example 3.

2. Drug administration test

(1) Target group

The following drug administration test was conducted on 16 healthy Japanese adult males, 1 subject in Phase I and 15 subjects divided into Groups A to E with 3 subjects in each group in Phase II.

(2) Dosage regimen

(2-1)Phase I

Safety was confirmed by concurrent administration of 20 mg of febuxostat and 500 mg of inosine twice daily for 14 days to one subject.

(2) Phase II

After the completion of Phase I, the three subjects in each group were dosed as follows:

Group A: febuxostat 20 mg, twice a day, 14 days;

Group B: inosine 500 mg, twice a day, 14 days;

Group C: Febuxostat 20 mg + inosine 500 mg, twice a day, 14 days;

Group D: Febuxostat 20 mg + inosine 1000 mg, twice a day, 14 days; and

Group E: Febuxostat 30 mg, twice a day, for 14 days.

3. Results

3-1.Phase I

(1) Adverse events

(1-1) Physical Finding, etc.

The subjects had no adverse events in terms of subjective and physical findings.

(1-2) Clinical trials

AST showed an abnormal value of 49 U/L on day 8 (reference value: 10 to 40), and recovered to the reference value of 29 U/L on day 15. Creatinine showed an abnormal value of 1.09 mg/dL on day 8 (reference value: 0.61 to 1.04), and recovered to the reference value of 0.98 mg/dL on day 15. Blood glucose levels showed abnormal values of 66 mg/dL on day 8 and 67 mg/dL on day 15 (reference value: 70 to 109).

(2) Changes in uric acid levels

Serum uric acid values were 4.9 mg/dL on day 1, 2.5 mg/dL on day 8, and 2.9 mg/dL on day 15. Administration of 40 mg of febuxostat and 1 g of inosine resulted in an average decrease of 2.2 mg/dL.

3-2. Stage II

(1) Adverse events

(1-1) Physical Discovery

There were no significant differences between the groups with respect to age, height, weight, BMI, systolic blood pressure, diastolic blood pressure, pulse rate, and body temperature. No significant changes were observed in systolic blood pressure, diastolic blood pressure, pulse rate, and body temperature, except for a significant increase in pulse rate observed in one subject.

(1-2) Adverse events of test values (excluding uric acid values)

Total protein, albumin, total bilirubin, AST, ALT, AL-P, LD, γ-GT, total cholesterol, neutral fat, HDL cholesterol, LDL cholesterol measurement, uric acid, urea nitrogen, creatinine, sodium, chloride, potassium, calcium, blood glucose test, HbA1c (NGSP), white blood cell count WBC, red blood cell count RBC, hemoglobin level Hb, hematocrit Ht, platelet count PLT, BASO, EOSINO, NEUTRO, LYMPH, and MONO were measured.

No specific differences were observed in the context of the groups. No specific significant changes were identified except for serum uric acid values.

(2) Changes in serum uric acid levels

Fig. 6 illustrates the figure of each A to E group.In the B group using only inosine, observe the significant increase (up to 8.1mg/dL) of serum uric acid value.In A, C to E group, observe the reduction of serum uric acid value.In any administration example of febuxostat of 40mg/ days, serum uric acid value is not reduced to less than 2mg/dL; However, in the example of the E group of febuxostat using 60mg/ days, observe the serum uric acid value less than 2mg/dL.

Serum uric acid values decreased by 2.53 mg/dL (Group A) due to administration of 40 mg/dL of febuxostat, and by 2.23 mg/dL (Group C) and 1.47 mg/dL (Group D), respectively, in the case where 1 g or 2 g of inosine was administered daily concurrently with febuxostat.

Due to the febuxostat administration of 60mg/dL, serum uric acid value reduces 3.93mg/dL (E group).Due to the inosine of 1g every day, serum uric acid value rises by an average of 2.57mg/dL (B group).About the serum uric acid value increase effect aspect of the inosine in the presence of febuxostat administration, check this result, under the presence of the febuxostat of 40mg/ days, due to the 1g/ days administration of inosine, serum uric acid value increases by 0.3mg/dL, due to the 2g/ days administration of inosine, serum uric acid value increases by 1.06mg/dL.As mentioned above, without febuxostat administration, due to the 1g/ days administration of inosine, serum uric acid value increases by 2.57mg/dL, therefore, under the presence of febuxostat administration, significantly suppress the serum urea rising effect of inosine.

(3) Urine uric acid concentration/creatinine concentration

The changes in urine uric acid concentration/creatinine concentration from week 0 to week 2 in each group are shown in FIG7 . Due to inosine administration, urine uric acid/creatinine significantly increased only in group B. Urine uric acid/creatinine decreased in all groups A and C to G. These changes in urine uric acid concentration/creatinine concentration were almost identical to the pattern of changes in serum uric acid values.

(4) Blood purine concentration

The changes in the blood purine concentration of each group from week 0 to week 2 are shown in Figures 8 and 9 .

Figure 8 shows the concentration of blood ATP/ADP in groups A to E. ATP concentration did not change in groups A and B, and groups C and D showed an increase in ATP. No specific trend was observed in group E. Therefore, although no increase in ATP was observed with the individual administration of febuxostat or inosine, an increase in ATP was observed in the combined use examples, particularly in the example of 40 mg/day of febuxostat and 1 to 2 g/day of inosine. No specific trend exceeding these amounts was observed with the combined use of febuxostat and inosine.

Fig. 9 shows the concentration of hypoxanthine (Hx) and xanthine (X) in the blood of each A to E group. In the group of independent 40mg/ day's febuxostat administration (A group), Hx concentration did not change, while X significantly increased. In the group of independent 1g/ day's inosine administration (B group), Hx or X did not change. Although the inosine concentration in blood was also measured, in any group of the group of independent administration including inosine, the rise of the concentration of inosine was not observed (A to E groups). From these results, it is believed that the concentration of the enzyme, i.e. purine nucleoside phosphorylase (PNP), which is believed to convert inosine into Hx, is extremely high in blood, resulting in the rapid decomposition of inosine to Hx and the further decomposition of Hx to X. In the example of febuxostat using 40mg/ day in combination with 1 to 2g/ day's inosine, significant rise was identified in both Hx and X. Therefore, due to combined use, the effect (Fig. 9) of "the rise of Hx in blood" not observed under independent febuxostat or independent inosine was observed.

In the case of single administration of 60 mg/day of febuxostat, a slight increase in Hx was observed along with an increase in X ( FIG. 9E ).

(5) Urine purine concentration

The changes in the concentrations of urine inosine, Hx, X, and uric acid in each group from week 0 to week 2 are shown in Figure 10. Urine Hx showed a moderate increase in the example of single administration of febuxostat and showed a significant increase in the example of combined use of febuxostat and inosine. In the example of single administration of inosine, no increase in Hx or X was identified. Although the concentration of X increased significantly in the example of single administration of febuxostat, the concentration increased significantly further in the example of combined use of febuxostat and inosine.

The maximum urine X concentrations in each group were 556.0 μM for Group A, 61.9 μM for Group B, 2023.3 μM for Group C, 1474.8 μM for Group D, and 867.7 μM for Group E. Therefore, in the example of using 40 mg of febuxostat in combination with 1 and 2 g of inosine, the maximum urine X concentrations were 3.64 and 2.65 times greater than those in the group administered with 40 mg of febuxostat alone.

4. Discussion of Experimental Example 4

(1) In a two-week continuous administration trial, the combined use of febuxostat and inosine was safe at doses equal to or less than 40 mg/day of febuxostat and 2 g/day of inosine. Although an increase in blood ATP was observed in these combined use groups, no such changes were observed in the other single-dose groups.

(2) A significant decrease in serum uric acid values was observed in the single administration of febuxostat, a significant increase in serum uric acid values was observed in the single administration of inosine, and a moderate decrease was observed in the combination treatment.

(3) No increase in inosine was observed in any group, and it is believed that inosine was metabolized to Hx by PNP.

(4) In the inosine alone administration group, Hx or X did not increase in blood or urine, and it is considered that Hx and X changed to uric acid.

(5) In the group receiving febuxostat alone, X increased moderately and Hx increased slightly to moderately in both blood and urine.

(6) When febuxostat and inosine were used in combination, Hx and X increased significantly in both blood and urine. It is believed that the increase in Hx in the blood causes an increase in ATP.

(7) From the above clinical trials, pharmacological effects that were not possible with the single administration of inosine or febuxostat were identified from the combined use of inosine or febuxostat. The present invention enables the enhancement of hypoxanthine and ATP in the blood, which is a new pharmacological effect that did not exist previously.

[Preparation Examples] Examples of compound medicines

A compounded pharmaceutical preparation for oral administration (tablet type) having the following ingredients per tablet was manufactured.

Febuxostat: 20 mg

Inosine: 0.5g

Alpha starch (disintegrating binder): 70 mg

Silicified microcrystalline cellulose (filler): 32.656 mg

Cross-linked carboxymethylcellulose sodium (disintegrant): 10 mg

Magnesium stearate (lubricant): 0.8 mg

[Preparation Example] Example of Kit Preparation

Tablets containing the following component A of febuxostat and a drug containing the following component B of inosine were manufactured and placed in separate identical bags to prevent mixing, for adjustment to 1 dose. A kit preparation was manufactured by packaging 2 doses, i.e., daily doses, in the same box.

A. Febuxostat tablets

Febuxostat: 20 mg

α-starch (disintegrating binder): 70 mg

Silicified microcrystalline cellulose (filler): 32.656 mg

Cross-linked carboxymethylcellulose sodium (disintegrant): 10 mg

Magnesium stearate (lubricant): 0.8 mg

B. Inosine

Inosine: 0.5g

Industrial applicability

The combined administration of febuxostat and inosine can enhance intracellular ATP. This is a novel pharmacological action different from conventional drugs. Therefore, it is believed that the present invention is effective for various diseases with reduced ATP as a pathological condition.

In addition, the uric acid level-raising effect of inosine is suppressed by its combination with febuxostat. Therefore, for patients taking febuxostat for the treatment of hyperuricemia, ATP can be increased without reducing the therapeutic effect on hyperuricemia.

Claims (6)

1. An intracellular ATP enhancer for humans or animals, comprising a combination of A) and B): A) Febuxostat or a pharmaceutically acceptable salt thereof; and B) Inosine or a pharmaceutically acceptable salt thereof. 2. The ATP enhancer according to claim 1, wherein the ATP enhancer is in the form of a compound drug or a kit formulation. 3. Use of febuxostat in combination with inosine in the preparation of intracellular ATP enhancers. 4. A pharmaceutical product, which is a compound preparation or reagent kit containing a combination of A) and B): A) Febuxostat or a pharmaceutically acceptable salt thereof; B) Inosine or a pharmaceutically acceptable salt thereof. 5. The intracellular ATP enhancer according to claim 1 or 2, or the pharmaceutical product according to claim 4, characterized in that the intracellular ATP enhancer or pharmaceutical product is administered orally. 6. The intracellular ATP enhancer according to claim 1 or 2, or the pharmaceutical product according to claim 4, wherein the intracellular ATP enhancer or pharmaceutical product is administered to humans.