Methods for treating hyperphenylalaninemia

Abstract

To provide a method for treating phenylketonuria in a subject having a blood phenylalanine concentration greater than 120 micromole per liter.SOLUTION: Provided is a method including a step of administering to a subject an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

Background Art

[0001] Phenylketonuria (PKU) is a congenital metabolic disorder mainly caused by mutations in the phenylalanine hydroxylase (PAH) gene. Mutations in the PAH gene result in a decrease in catalytic activity leading to hyperphenylalaninemia (HPA). There are many mutations in the PAH gene (>400), resulting in phenotypic differences in the amount of enzyme produced and / or enzyme activity. In severe PKU cases, PAH enzyme activity is completely absent or highly deficient, typically with very high blood phenylalanine (Phe) concentrations (>1200 micromol / liter). Partial deficiency of PAH activity reduces the degree of elevated blood phenylalanine (e.g., about 360 - 1200 micromol / liter). High concentrations of phenylalanine are toxic to the brain. If left untreated, severe and irreversible intellectual disability may occur. Due to the almost worldwide introduction of neonatal screening, PKU is now diagnosed at birth. It has been reported in all ethnic groups and is estimated to occur at a rate of about 1 in 10,000 people.

[0002] Currently, there is no cure for PKU. Initial treatment consists of the rapid implementation of a phenylalanine-restricted diet supplemented with specially designed medical foods. Adherence to the diet can be difficult for older children, adolescents, and adults. Tetrahydrobiopterin (BH4) supplementation has demonstrated a clinically significant reduction in plasma phenylalanine concentrations in approximately 30% of PKU patients. BH4 is an essential cofactor for PAH in the conversion of Phe to tyrosine (Tyr). As a result of BH4 supplementation, increased intracellular BH4, improved PAH function, and decreased plasma phenylalanine have been shown. Kuvan® (sapropterin dihydrochloride), a synthetic formulation of BH4, is approved as a pharmaceutical for use in the treatment of hyperphenylalaninemia (HPA) in tetrahydrobiopterin-responsive adult and pediatric PKU patients in several countries. However, control by dietary phenylalanine restriction and / or BH4 supplementation is not optimal, and the majority of patients maintain elevated plasma phenylalanine concentrations. Another treatment for PKU, pegvaliase-pqpz, is approved only for adult patients and is a method of administering a phenylalanine-metabolizing enzyme by daily injection. However, this method has the potential for complications due to side effects such as anaphylaxis, and there is a problem that many patients maintain elevated phenylalanine levels even with treatment. Therefore, a further method for treating PKU is needed.

Summary of the Invention

[0003] The present invention features a method of reducing the blood phenylalanine concentration in a subject in need thereof by administering sepiapterin or a pharmaceutically acceptable salt thereof. The inventors have found that sepiapterin is effective in reducing blood phenylalanine concentration. In particular, the inventors have found that sepiapterin is effective in reducing the blood phenylalanine concentration to levels similar to those of healthy individuals and / or in reducing the blood phenylalanine concentration in subjects who have failed prior treatment with sapropterin dihydrochloride and / or pegvaliase pqpz.

[0004] In one aspect, the present invention features a method of reducing the level of phenylalanine (e.g., blood phenylalanine concentration) in a subject (e.g., a subject with hyperphenylalaninemia, a subject with hyperphenylalaninemia due to phenylketonuria, a subject with phenylketonuria, a subject with tetrahydrobiopterin-responsive phenylketonuria, or a subject with sepiapterin-responsive phenylketonuria). This method includes the step of administering an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.

[0005] In one aspect, the present invention features a method of treating hyperphenylalaninemia (e.g., hyperphenylalaninemia due to phenylketonuria such as tetrahydrobiopterin-responsive phenylketonuria, or a subject with sepiapterin-responsive phenylketonuria) in a subject in need thereof. This method includes the step of administering an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.

[0006] In one aspect, the present invention features a method of treating phenylketonuria (e.g., a subject with tetrahydrobiopterin-responsive phenylketonuria, or a subject with sepiapterin-responsive phenylketonuria, classical phenylketonuria, and / or non-classical phenylketonuria) in a subject in need thereof. This method includes the step of administering an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments of any of the methods described herein, the subject has a blood phenylalanine concentration (e.g., an uncontrolled phenylalanine blood concentration) greater than 120 micromoles / liter (e.g., greater than 200 micromoles / liter, greater than 300 micromoles / liter, greater than 360 micromoles / liter, greater than 400 micromoles / liter, greater than 450 micromoles / liter, greater than 500 micromoles / liter, greater than 550 micromoles / liter, greater than 600 micromoles / liter, greater than 650 micromoles / liter, greater than 700 micromoles / liter, greater than 800 micromoles / liter, greater than 900 micromoles / liter, greater than 1000 micromoles / liter, greater than 1100 micromoles / liter, or greater than 1200 micromoles / liter). In some embodiments of any of the methods described herein, the subject has a blood phenylalanine concentration (e.g., an uncontrolled phenylalanine blood concentration) of 120 - 360 micromoles / liter, 360 - 600 micromoles / liter, 600 - 1200 micromoles / liter, or greater than 1200 micromoles / liter.

[0008] In some embodiments of any of the methods described herein, the subject has a blood phenylalanine concentration (e.g., an uncontrolled phenylalanine blood concentration) greater than 400 micromoles / liter (e.g., greater than 450 micromoles / liter, greater than 500 micromoles / liter, greater than 550 micromoles / liter, greater than 600 micromoles / liter, greater than 650 micromoles / liter, greater than 700 micromoles / liter, greater than 800 micromoles / liter, greater than 900 micromoles / liter, greater than 1000 micromoles / liter, greater than 1100 micromoles / liter, or greater than 1200 micromoles / liter) in treatment with existing management such as a phenylalanine-restricted diet and / or sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride). In some embodiments, treatment with sapropterin comprises administration of 5 - 20 mg / kg of sapropterin or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments of any of the methods described herein, the subject did not respond to treatment with sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride). In some embodiments, administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) resulted in a plasma concentration decrease of less than 15-30% in the subject. In some embodiments, administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) resulted in a plasma concentration decrease of less than 30% in the subject. In some embodiments, administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) resulted in a plasma concentration decrease of less than 20% in the subject. In some embodiments, administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) resulted in a plasma concentration decrease of less than 15% in the subject. For example, administration of at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg) of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) for at least 8 days (e.g., at least 14 days, at least 21 days, at least 28 days, at least 30 days) resulted in a plasma concentration decrease of less than 30% in the subject. In some embodiments, after administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride), the plasma concentration of the subject was greater than 120 micromoles per liter (e.g., greater than 200 micromol / liter, greater than 300 micromol / liter, greater than 360 micromol / liter, greater than 400 micromol / liter, greater than 450 micromol / liter, greater than 500 micromol / liter, greater than 550 micromol / liter, greater than 600 micromol / liter, greater than 650 micromol / liter, greater than 700 micromol / liter, greater than 800 micromol / liter, greater than 900 micromol / liter, greater than 1000 micromol / liter, greater than 1100 micromol / liter, or greater than 1200 micromol / liter).For example, after administration of at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg) of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) for at least 8 days (e.g., at least 14 days, at least 21 days, at least 28 days, at least 30 days), the plasma concentration of the subject was greater than 120 micromoles / liter (e.g., greater than 200 micromoles / liter, greater than 300 micromoles / liter, greater than 360 micromoles / liter, greater than 400 micromoles / liter, greater than 450 micromoles / liter, greater than 500 micromoles / liter, greater than 550 micromoles / liter, greater than 600 micromoles / liter, greater than 650 micromoles / liter, greater than 700 micromoles / liter, greater than 800 micromoles / liter, greater than 900 micromoles / liter, greater than 1000 micromoles / liter, greater than 1100 micromoles / liter, or greater than 1200 micromoles / liter).

[0010] In some embodiments of any of the methods described herein, the subject did not respond to treatment with pegvaliase-pqpz. In some embodiments, administration of pegvaliase-pqpz resulted in less than a 20% decrease in the plasma concentration of the subject. For example, the plasma concentration of the subject decreased by less than 30% or less than 20% when administered at least about 20 mg once daily (e.g., at least about 30 mg once daily, at least 40 mg once daily) of pegvaliase-pqpz for at least 16 weeks (e.g., at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24 weeks). In some embodiments, after administration of pegvaliase-pqpz, the plasma concentration of the subject is greater than 120 micromoles per liter (e.g., greater than 200 micromoles per liter, greater than 300 micromoles per liter, greater than 360 micromoles per liter, greater than 400 micromoles per liter, greater than 450 micromoles per liter, greater than 500 micromoles per liter, greater than 550 micromoles per liter, greater than 600 micromoles per liter, greater than 650 micromoles per liter, greater than 700 micromoles per liter, greater than 800 micromoles per liter, greater than 900 micromoles per liter, greater than 1000 micromoles per liter, greater than 1100 micromoles per liter, or greater than 1200 micromoles per liter).For example, the plasma concentration of the subject after administering at least about 20 mg (e.g., at least about 30 mg, at least about 40 mg) of pegloticase-pqpz for at least 16 weeks (e.g., at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24 weeks) was greater than 120 micromoles / liter (e.g., greater than 200 micromoles / liter, greater than 300 micromoles / liter, greater than 360 micromoles / liter, greater than 400 micromoles / liter, greater than 450 micromoles / liter, greater than 500 micromoles / liter, greater than 550 micromoles / liter, greater than 600 micromoles / liter, greater than 650 micromoles / liter, greater than 700 micromoles / liter, greater than 800 micromoles / liter, greater than 900 micromoles / liter, greater than 1000 micromoles / liter, greater than 1100 micromoles / liter, or greater than 1200 micromoles / liter).

[0011] In some embodiments of any of the methods described herein, the subject discontinued treatment with pegloticase pqpz due to an adverse reaction (e.g., anaphylaxis, injection site reaction, arthralgia, hypersensitivity reaction, headache, systemic skin reaction lasting at least 14 days, pruritus, nausea, abdominal pain, oral pharyngeal pain, vomiting, cough, diarrhea, and / or fatigue) and / or tolerance.

[0012] In some embodiments of any of the methods described herein, administering sepiapterin or a pharmaceutically acceptable salt thereof reduces the blood phenylalanine concentration of a subject to less than 600 micromoles per liter. In some embodiments of any of the methods described herein, administering sepiapterin or a pharmaceutically acceptable salt thereof reduces the blood phenylalanine concentration of a subject to less than 360 micromoles per liter. In some embodiments of any of the methods described herein, administering sepiapterin or a pharmaceutically acceptable salt thereof reduces the blood phenylalanine concentration of a subject to less than 120 micromoles per liter. In some embodiments of any of the methods described herein, administering sepiapterin or a pharmaceutically acceptable salt thereof reduces the blood phenylalanine concentration of a subject to 120 to 360 micromoles per liter. In some embodiments of any of the methods described herein, administering sepiapterin or a pharmaceutically acceptable salt thereof reduces the blood phenylalanine concentration of a subject to 360 to 600 micromoles per liter.

[0013] In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to less than 600 micromoles per liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to less than 360 micromoles per liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to less than 120 micromoles per liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to 120 - 360 micromoles per liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to 360 - 600 micromoles per liter.

[0014] In some embodiments of any of the methods described herein, by the step of administering sepiapterin or a pharmaceutically acceptable salt thereof, the blood phenylalanine concentration of the subject is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 10% - about 30%, about 20% - about 40%, about 30% - about 50%, about 40% - about 60%, about 50% - about 70%, about 60% - about 80%, or about 70% - about 90%). The amount of reduction may be measured after administration over at least one week (e.g., two weeks, three weeks, four weeks, five weeks, six weeks).

[0015] In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration in a subject by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 10% to about 30%, about 20% to about 40%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, about 60% to about 80%, or about 70% to about 90%).

[0016] In some embodiments of any of the methods described herein, by administering sepiapterin or a pharmaceutically acceptable salt thereof, the blood phenylalanine concentration of a subject is reduced to less than 600 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day). In some embodiments of any of the methods described herein, by administering sepiapterin or a pharmaceutically acceptable salt thereof, the blood phenylalanine concentration of a subject is reduced to less than 360 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day).In some embodiments of any of the methods described herein, by administering sepiapterin or a pharmaceutically acceptable salt thereof, the blood phenylalanine concentration of a subject is reduced to less than 120 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day). In some embodiments of any of the methods described herein, by administering sepiapterin or a pharmaceutically acceptable salt thereof, the blood phenylalanine concentration of a subject is reduced to 120 - 360 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day).In some embodiments of any of the methods described herein, by administering sepiapterin or a pharmaceutically acceptable salt thereof, the blood phenylalanine concentration of a subject is reduced to 360 - 600 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day - 1400 mg / day, 1200 mg / day - 1600 mg / day, 1300 mg / day - 1700 mg / day, 1600 mg / day - 2000 mg / day, 1800 mg / day - 2400 mg / day, 2000 mg / day - 3000 mg / day, 3000 mg / day - 4000 mg / day, 4000 - 5000 mg / day).

[0017] In some embodiments of any of the methods described herein, administering sepiapterin or a pharmaceutically acceptable salt thereof reduces the subject's blood phenylalanine concentration to less than 600 micromoles / liter at a natural protein intake of greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or between about 10 g / day and about 30 g / day, between about 20 g / day and about 40 g / day, between about 30 g / day and about 50 g / day, between about 40 g / day and about 60 g / day, between about 50 g / day and about 70 g / day, between about 60 g / day and about 80 g / day). In some embodiments of any of the above-described methods, administering sepiapterin or any pharmaceutically acceptable salt thereof reduces the subject's blood phenylalanine concentration to less than 360 micromoles / liter at a natural protein intake of greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or between about 10 g / day and about 30 g / day, between about 20 g / day and about 40 g / day, between about 30 g / day and about 50 g / day, between about 40 g / day and about 60 g / day, between about 50 g / day and about 70 g / day, between about 60 g / day and about 80 g / day). In some embodiments of any of the methods described herein, administering sepiapterin or any pharmaceutically acceptable salt thereof reduces the subject's blood phenylalanine concentration to less than 120 micromoles / liter at a natural protein intake of greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or between about 10 g / day and about 30 g / day, between about 20 g / day and about 40 g / day, between about 30 g / day and about 50 g / day, between about 40 g / day and about 60 g / day, between about 50 g / day and about 70 g / day, between about 60 g / day and about 80 g / day).In some embodiments of any of the methods described herein, administration of sepiapterin or any of its pharmaceutically acceptable salts results in a decrease in the blood phenylalanine concentration of the subject to 120 - 360 micromoles per liter at a natural protein intake greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or about 10 g / day to about 30 g / day, about 20 g / day to about 40 g / day, about 30 g / day to about 50 g / day, about 40 g / day to about 60 g / day, about 50 g / day to about 70 g / day, about 60 g / day to about 80 g / day). In some embodiments of any of the methods described herein, administration of sepiapterin or any of its pharmaceutically acceptable salts results in a decrease in the blood phenylalanine concentration of the subject to 360 - 600 micromoles per liter at a natural protein intake greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or about 10 g / day to about 30 g / day, about 20 g / day to about 40 g / day, about 30 g / day to about 50 g / day, about 40 g / day to about 60 g / day, about 50 g / day to about 70 g / day, about 60 g / day to about 80 g / day).

[0018] In some embodiments of any of the methods described herein, administration of sepiapterin or a pharmaceutically acceptable salt thereof results in a decrease in the blood phenylalanine concentration of the subject of at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) from the blood phenylalanine concentration prior to administration of sepiapterin or a pharmaceutically acceptable salt thereof.

[0019] In some embodiments of any of the methods described herein, by administering sepiapterin or a pharmaceutically acceptable salt thereof, within 10 hours after administration, a BH4 concentration of at least 50 ng / ml (e.g., at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, and 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) is produced in the plasma of the subject.

[0020] In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to lower the blood phenylalanine concentration of a subject to less than 600 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to lower the blood phenylalanine concentration of a subject to less than 360 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day).In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to less than 120 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to reduce the blood phenylalanine concentration of a subject to 120 - 360 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day).In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is an amount sufficient to lower the blood phenylalanine concentration of a subject to 360 - 600 micromoles per liter with a phenylalanine intake of at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day - 1400 mg / day, 1200 mg / day - 1600 mg / day, 1300 mg / day - 1700 mg / day, 1600 mg / day - 2000 mg / day, 1800 mg / day - 2400 mg / day, 2000 mg / day - 3000 mg / day, 3000 mg / day - 4000 mg / day, 4000 - 5000 mg / day).

[0021] In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or from about 10 g / day to about 30 g / day, from about 20 g / day to about 40 g / day, from about 30 g / day to about 50 g / day, from about 40 g / day to about 60 g / day, from about 50 g / day to about 70 g / day, from about 60 g / day to about 80 g / day) of natural protein intake and is an amount sufficient to reduce the subject's blood phenylalanine concentration to less than 600 micromoles per liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or from about 10 g / day to about 30 g / day, from about 20 g / day to about 40 g / day, from about 30 g / day to about 50 g / day, from about 40 g / day to about 60 g / day, from about 50 g / day to about 70 g / day, from about 60 g / day to about 80 g / day) of natural protein intake and is an amount sufficient to reduce the subject's blood phenylalanine concentration to less than 360 micromoles per liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or from about 10 g / day to about 30 g / day, from about 20 g / day to about 40 g / day, from about 30 g / day to about 50 g / day, from about 40 g / day to about 60 g / day, from about 50 g / day to about 70 g / day, from about 60 g / day to about 80 g / day) of natural protein intake and is an amount sufficient to reduce the subject's blood phenylalanine concentration to less than 120 micromoles per liter.In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or about 10 g / day to about 30 g / day, about 20 g / day to about 40 g / day, about 30 g / day to about 50 g / day, about 40 g / day to about 60 g / day, about 50 g / day to about 70 g / day, about 60 g / day to about 80 g / day) of natural protein intake and is an amount sufficient to reduce the subject's blood phenylalanine concentration to 120 - 360 micromoles / liter. In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is greater than 10 g / day (e.g., greater than 20 g / day, greater than 30 g / day, greater than 40 g / day, greater than 50 g / day, greater than 60 g / day, greater than 70 g / day, greater than 80 g / day, or about 10 g / day to about 30 g / day, about 20 g / day to about 40 g / day, about 30 g / day to about 50 g / day, about 40 g / day to about 60 g / day, about 50 g / day to about 70 g / day, about 60 g / day to about 80 g / day) of natural protein intake and is an amount sufficient to reduce the subject's blood phenylalanine concentration to 360 - 600 micromoles / liter.

[0022] In some embodiments of any of the methods described herein, the effective amount is an amount sufficient to reduce the subject's blood phenylalanine concentration by at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) from the blood phenylalanine concentration prior to administration of sepiapterin or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments of any of the methods described herein, the effective amount is an amount sufficient (e.g., 2.5 mg / kg to 100 mg / kg per administration) to produce a BH4 concentration of at least 50 ng / ml (e.g., at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) in the plasma of the subject within 10 hours of administration of sepiapterin or a pharmaceutically acceptable salt thereof.

[0024] In some embodiments of any of the methods described herein, the effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is from about 2.5 mg / kg to about 100 mg / kg per administration (e.g., from about 20 mg / kg to about 60 mg / kg, or about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg).

[0025] In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered with food. In some embodiments of any of the methods described herein, the effective amount is sufficient to produce a BH4 concentration of at least 50 ng / ml (e.g., at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) in the plasma of the subject within 10 hours after administration with food (e.g., 2.5 mg / kg to 100 mg / kg per administration). In some embodiments, the effective amount is at least 5% lower (e.g., at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) than the dose sufficient to result in a maximum BH4 plasma concentration (Cmax) of at least 50 ng / ml in the plasma of the subject within 10 hours after administering sepiapterin or a pharmaceutically acceptable salt thereof without food.

[0026] In some embodiments of any of the methods described herein, administration to a subject is performed less than 30 minutes before ingesting food, or after ingesting food, for example, immediately before or up to 1 hour after ingesting food. In some embodiments, administration to a subject is substantially simultaneous with food. In some embodiments of any of the methods described herein, the food is a high-protein food. In some embodiments of any of the methods described herein, the food is a high-fat food (e.g., at least 25, 30, 40, or 50% of the calories are from fat). In some embodiments of any of the methods described herein, the food is a high-protein and high-fat food. In some embodiments, the food is a high-calorie food (e.g., the food contains at least 100 calories, for example, at least 200 calories, at least 300 calories, at least 400 calories, at least 500 calories, for example, 500 - 1500 or 800 - 1000 calories). In some embodiments of any of the methods described herein, the food is a meal, for example, breakfast, lunch, or dinner.

[0027] In some embodiments of any of the methods described herein, administration with food (e.g., performed less than 30 minutes before ingesting food, or after ingesting food, for example, between immediately before and 1 hour after ingesting food) results in an increase in Cmax of BH4 (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%) compared to administration without food (e.g., performed more than 2 hours after ingesting food and up to 30 minutes before ingesting food again).

[0028] In some embodiments of any of the methods described herein, administration with food (e.g., performed less than 30 minutes before ingesting the food, or after ingesting the food, e.g., performed from immediately before to 1 hour after ingesting the food) results in an increase in the degree of BH4 production and the resulting plasma exposure (AUC 0-last ) compared to administration without food (e.g., performed less than 30 minutes before ingesting the food, or after ingesting the food, e.g., performed from immediately before to 1 hour after ingesting the food) (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%).

[0029] In some embodiments of any of the methods described herein, sepiapterin or a pharmaceutically acceptable salt thereof is provided in a composition separate from the ingested food (e.g., sepiapterin is not incorporated into a food product). In some embodiments of any of the methods described herein, ingestion of the food is performed before administration of sepiapterin or a pharmaceutically acceptable salt thereof (e.g., ingestion of the food is performed from 1 hour before to immediately before administration of sepiapterin). In some embodiments of any of the methods described herein, ingestion of the food is performed after administration of sepiapterin or a pharmaceutically acceptable salt thereof (e.g., ingestion of the food is performed from immediately after to 30 minutes after administration).

[0030] In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered in two equal doses (e.g., two doses at different times of the day). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered as two 60 mg / kg doses (e.g., one 60 mg / kg dose in the morning and one 60 mg / kg dose in the evening). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered as two 40 mg / kg doses (e.g., one 40 mg / kg dose in the morning and one 40 mg / kg dose in the evening). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered as two 30 mg / kg doses (e.g., one 30 mg / kg dose in the morning and one 30 mg / kg dose in the evening). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered as two 20 mg / kg doses (e.g., one 20 mg / kg dose in the morning and one 20 mg / kg dose in the evening). In some embodiments of any of the methods described herein, an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is administered as two 10 mg / kg doses (e.g., one 10 mg / kg dose in the morning and one 10 mg / kg dose in the evening).

[0031] In some embodiments of any of the methods described herein, the risk of adverse events (e.g., headache, rhinorrhea, pharyngolaryngeal pain, diarrhea, vomiting, cough, and / or nasal congestion) is reduced compared to subjects administered at least 10 mg / kg (e.g., at least 15 mg / kg, at least 20 mg / kg) of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride). In some embodiments of any of the methods described herein, the risk of adverse events (e.g., headache, rhinorrhea, pharyngolaryngeal pain, diarrhea, vomiting, cough, and / or nasal congestion) is reduced compared to subjects administered at least 20 mg / kg of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride).

[0032] In some embodiments of any of the methods described herein, the subject is a child (e.g., the subject is less than 18 years old, less than 17 years old, less than 16 years old, less than 15 years old, less than 14 years old, less than 13 years old, less than 12 years old, less than 11 years old, less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old). In some embodiments of any of the methods described herein, the subject is an adult (e.g., the subject is over 18 years old).

[0033] In some embodiments of any of the methods described herein, the subject is diagnosed with tetrahydrobiopterin-responsive phenylketonuria. In some embodiments of any of the methods described herein, the subject is diagnosed with hyperphenylalaninemia due to sepiapterin-responsive PKU.

[0034] In some embodiments of any of the methods described herein, the subject is on a phenylalanine-restricted diet (e.g., the subject is on a diet that includes milk substitutes or formulations such as Phenyl-Free 2, and measured amounts of fruits, vegetables, bread, pasta, and cereal). In some embodiments of any of the methods described herein, the subject has a median phenylalanine intake of less than 3000 mg / day (e.g., less than 2500 mg / day, less than 2000 mg / day, less than 1500 mg / day, less than 1000 mg / day, less than 500 mg / day).

[0035] In some embodiments of any of the methods described herein, the subject is not on a phenylalanine-restricted diet (e.g., the subject has a median phenylalanine intake greater than 1000 mg / day (e.g., greater than 1500 mg / day, greater than 2000 mg / day, greater than 2500 mg / day, greater than 3000 mg / day)).

[0036] In some embodiments of any of the methods described herein, less than 25% (e.g., less than 20%, less than 15%, less than 10%, or less than 5%) of the protein in the subject's diet is natural protein. In some embodiments of any of the methods described herein, more than 25% (e.g., more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%) of the protein in the subject's diet is natural protein. In some embodiments of any of the methods described herein, the subject has a natural protein intake of less than 10 g / day (e.g., less than 5 g / day, or from about 5 g / day to about 10 g / day). In some embodiments of any of the methods described herein, the subject has a natural protein intake of more than 10 g / day (e.g., more than 20 g / day, more than 30 g / day, more than 40 g / day, more than 50 g / day, more than 60 g / day, more than 70 g / day, more than 80 g / day, or from about 10 g / day to about 30 g / day, from about 20 g / day to about 40 g / day, from about 30 g / day to about 50 g / day, from about 40 g / day to about 60 g / day, from about 50 g / day to about 70 g / day, from about 60 g / day to about 80 g / day).

[0037] In some embodiments of any of the methods described herein, sepiapterin or a pharmaceutically acceptable salt thereof is formulated as an oral powder for suspension. In some embodiments of any of the methods described herein, sepiapterin or a pharmaceutically acceptable salt thereof is administered as a suspension in a flavored vehicle for suspension (e.g., MEDISCA® Oral Mix).

[0038] In some embodiments of any of the methods described herein, the administering step results in an increase in the subject's neurocognitive function (e.g., an increase in executive function, a decrease in anxiety, a decrease in attention deficit / hyperactivity disorder symptoms, and / or a decrease in instances of brain fog). In some embodiments, the administering step results in an increase in executive function as measured by an evaluation such as (e.g., Behavioral Assessment of the Dysexecutive Syndrome (BADS), Behavior Rating Inventory of Executive Function (BRIEF or Mini-BRIEF), Berkeley Deficits in Executive Function Scale (BDEFS), Behavioral Disorder Scale (BDS), ASEBA Child Behavior Checklist (CBLC), Comprehensive Executive Function Inventory (CEFI), CogScreen, Continuous Performance Task (CPT), Controlled Oral Word Association Test (COWAT), d2 Test of Attention, Delis-Kaplan Executive Function System (D-KEFS), Digit Vigilance Test, Figural Fluency Test, Halstead Category Test, Hayling Test and Brixton Test, Iowa Gambling Task, Kaplan Baycrest Neurocognitive Assessment (KBNA), Kaufman Short Neuropsychological Assessment, Mental Clutter Scale, Paced Auditory Serial Addition Test (PASAT), Phenylketonuria - Quality of Life (PKU-QOL), Rey-Osterrieth Complex Figure, Rough Figural Fluency Test, Stroop Task, Executive Control Task, Test of Variables of Attention (T.O.V.A.), Tower of London Test, Trail Making Test (TMT) or Trail A & B, Wisconsin Card Sorting Test (WCST), or Symbol Digit Modalities Test). In some embodiments of any of the methods described herein, the administering step results in an increase in executive function as measured by a Cambridge Neuropsychological Test Automated Battery (CANTAB) assessment such as (e.g., as measured by reaction speed, spatial span, spatial working memory, rapid digit information processing, sustained attention, and / or stop signal task).In some embodiments of any of the methods described herein, the administering step results in an improvement in attention and / or mood (such as as measured by, for example, the ADHD-RS 5 scale (or its inattention assessment) and / or the Profile of Mood States (POMS) scale).

[0039] In some embodiments of any of the methods described herein, the effective amount is an amount sufficient to produce an increase in the subject's neurocognitive function (e.g., an increase in executive function, a decrease in anxiety, a decrease in attention deficit / hyperactivity disorder symptoms, and / or a decrease in instances of brain fog). In some embodiments, the effective amount is an amount sufficient to result in an increase in executive function as measured by an evaluation such as (e.g., Behavioral Assessment of the Dysexecutive Syndrome (BADS), Behavior Rating Inventory of Executive Function (BRIEF or Mini-BRIEF), Berkeley Deficits in Executive Function Scale (BDEFS), Behavioral Disorder Scale (BDS), ASEBA Child Behavior Checklist (CBLC), Comprehensive Executive Function Inventory (CEFI), CogScreen, Continuous Performance Task (CPT), Controlled Oral Word Association Test (COWAT), d2 Test of Attention, Delis-Kaplan Executive Function System (D-KEFS), Digit Vigilance Test, Figural Fluency Test, Halstead Category Test, Hayling Test and Brixton Test, Iowa Gambling Task, Kaplan Baycrest Neurocognitive Assessment (KBNA), Kaufman Short Neuropsychological Assessment, Mental Clutter Scale, Paced Auditory Serial Addition Test (PASAT), Phenylketonuria - Quality of Life (PKU-QOL), Rey-Osterrieth Complex Figure, Rough Figural Fluency Test, Stroop Task, Task of Executive Control, Test of Variables of Attention (T.O.V.A.), Tower of London Test, Trail Making Test (TMT) or Trail A & B, Wisconsin Card Sorting Test (WCST), or Symbol Digit Modalities Test). In some embodiments, the effective amount is an amount sufficient to result in an increase in executive function as measured by a Cambridge Neuropsychological Test Automated Battery (CANTAB) assessment such as (e.g., as measured by reaction speed, spatial span, spatial working memory, rapid digital information processing, sustained attention, and / or stop signal task).In some embodiments of any of the methods described herein, the effective amount is an amount sufficient to produce an improvement in attention and / or mood (such as, for example, as measured by the ADHD-RS 5 scale (or its inattention assessment) and / or the Profile of Mood States (POMS) scale).

[0040] In some embodiments of any of the methods described herein, the administering step results in an increase in the quality of sleep and / or a decrease in symptoms associated with sleep deprivation. In some embodiments of any of the methods described herein, the effective amount is an amount sufficient to result in an increase in the quality of sleep and / or a decrease in symptoms associated with sleep deprivation.

[0041] Definitions In this application, unless the context clearly dictates otherwise: (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "comprises" may be understood to include the listed elements or steps, whether presented by themselves or in conjunction with one or more additional elements or steps; and (iv) the term "about" may be understood to allow for standard variations as understood by those of ordinary skill in the art; and (v) when ranges are provided, the endpoints are included.

[0042] The descriptions of the compounds, compositions, formulations, and treatment methods described herein should be understood to include the embodiments "comprising," "consisting of," and "consisting essentially of." In some embodiments, for all of the compositions described herein and all of the methods of using the compositions described herein, the composition can include the recited ingredients or steps, or can "consist essentially of" the recited ingredients or steps. When a composition is described as "consisting essentially of" the recited ingredients, the composition includes the recited ingredients and may include other ingredients that do not substantially affect the condition being treated, but does not include any other ingredients that substantially affect the condition being treated other than those specifically recited; or, if the composition actually includes additional ingredients that substantially affect the condition being treated other than the recited ingredients, the composition does not include a sufficient concentration or amount of the additional ingredients to substantially affect the condition being treated. When a method is described as "consisting essentially of" the recited steps, the method includes the recited steps and may include other steps that do not substantially affect the condition being treated, but the method does not include any other steps that substantially affect the condition being treated other than those specifically recited. By way of non-limiting specific example, when a composition is described as "consisting essentially of" a certain ingredient, the composition may further include any amount of a pharmaceutically acceptable carrier, vehicle, or diluent and other such ingredients that do not substantially affect the condition being treated.

[0043] Unless otherwise apparent from the context, all references to sepiapterin contained herein refer to sepiapterin or a pharmaceutically acceptable salt of sepiapterin.

[0044] As used herein, the term "about" refers to a value within the range of ±10% of the value that follows the term "about". In this specification, referring to a value or parameter as "about" includes (and describes) variations directed to the value or parameter itself. For example, a description referring to "about X" includes the description of "X". As used herein, the term "administering" refers to the administration of a composition (e.g., a compound described herein or a preparation containing it) to a subject or system. Administration to an animal subject (e.g., administration to a human) may be by any suitable route. For example, in some embodiments, administration is by bronchus (including bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including tracheal instillation), transdermal, intravaginal, and intravitreal.

[0045] "Measuring the level of phenylalanine" means detecting phenylalanine by a method known in the art, either directly or indirectly. "Measuring directly" means performing a process for obtaining a physical entity or value (e.g., assaying or testing a sample, or "analyzing a sample" as the term is defined herein). "Measuring indirectly" means receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Methods for measuring phenylalanine levels typically include, but are not limited to, fluorescence assays in which by-products of the reaction of phenylalanine react with a fluorescent probe.

[0046] The "effective amount" of a compound may vary depending on factors such as the individual medical condition, age, sex, and weight, as well as the ability of the compound to induce the desired response. A therapeutically effective amount includes an amount where the therapeutically beneficial effect exceeds any toxic or detrimental effect of the compound. An effective amount also includes an amount sufficient to provide a benefit, e.g., a clinical benefit.

[0047] As used herein, the term "non-responsive to prior therapy" means a subject whose condition has remained stable, worsened, or improved less than the standard tolerated by that therapy despite treatment with a specific therapy (e.g., a therapy as described in the FDA-approved label for that therapy).

[0048] As used herein, the term "food" refers to a solid food having sufficient bulk and fat content such that it is not rapidly dissolved and absorbed in the stomach. For example, a meal such as breakfast, lunch, or dinner. The term "with food" as used herein refers to administering the composition between about 30 minutes before eating (e.g., eating food) and about 2 hours after eating. The terms "without food", "fasting", or "fasted" refer to a state in which no solid food has been consumed for at least about 2 hours, up to about 30 minutes before further solid food is ingested.

[0049] As used herein, the term "hyperphenylalaninemia" refers to a medical condition in which a subject has elevated levels of phenylalanine in the blood compared to a healthy subject. For example, in some embodiments, a subject is considered to have hyperphenylalaninemia if the concentration of phenylalanine in their blood is greater than 60 micromoles / liter. In some embodiments, a subject is considered to have hyperphenylalaninemia if the concentration of phenylalanine in their blood is greater than 120 micromoles / liter (e.g., from about 120 micromoles / liter to about 2400 micromoles / liter).

[0050] "Level" means the level of phenylalanine when compared to a reference. The reference can be any useful reference as defined herein, such as the level in the subject prior to administration, i.e., the baseline level. A "decreased level" or "increased level" of phenylalanine means a decrease or increase in the phenylalanine level when compared to the reference (e.g., a decrease or increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or a decrease or increase greater than those; a decrease or increase greater than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200% when compared to the reference; a decrease or increase of less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or those; or an increase of about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). The level of phenylalanine may be expressed as mass / vol (e.g., g / dL, mg / mL, μg / mL, ng / mL), concentration / vol (e.g., nanomoles / liter, micromoles / liter, micromoles / milliliter), or as a percentage of the total amino acids in the sample.

[0051] "Native protein" means a protein derived from a natural source (e.g., animal, plant, or fungus) containing Phe.

[0052] As used herein, the term "pharmaceutical composition" refers to a composition comprising a compound described herein formulated with a pharmaceutically acceptable excipient. The pharmaceutical composition can be formulated, for example, for oral administration in unit dosage form (e.g., tablets, capsules, caplets, gelcaps, suspensions, solutions, or syrups), for topical administration (e.g., as creams, gels, lotions, or ointments), for intravenous administration (e.g., as a sterile solution in a solvent system suitable for intravenous use, free of particulate matter), or in any other pharmaceutically acceptable formulation.

[0053] As used herein, the term "pharmaceutically acceptable salt" means any salt that is suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977, and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base groups with a suitable organic acid.

[0054] In many cases, the compounds are prepared or used as pharmaceutically acceptable salts, which are prepared as addition products of pharmaceutically acceptable acids. Suitable pharmaceutically acceptable acids, as well as methods for preparing suitable salts, are well known in the art. The salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.

[0055] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, besylate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gentisate, glucoheptonate, glycerophosphate, glycolate, hemisulfate, heptonate, hexanate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, maleate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts.

[0056] "The blood phenylalanine concentration decreases" means that the level of phenylalanine in the blood and / or plasma of the subject decreases. The level of phenylalanine can be measured using any method known in the art. For example, the concentration of phenylalanine in the blood of the subject can be measured by dried blood spot or plasma analysis.

[0057] "Sepiapterin-responsive phenylketonuria" means phenylketonuria in which the subject shows a decrease in blood phenylalanine from baseline after administration of sepiapterin or a pharmaceutically acceptable salt thereof for up to one month.

[0058] As used herein, the term "substantially free" means a qualitative state indicating the absence of all or almost all of the compound or class of compounds of interest. A person of ordinary skill in the biological arts will understand that biological and chemical phenomena are rarely, if ever, determined to be exactly zero due to inherent errors in any measurement. Thus, the term "substantially free" is used herein to capture the potential lack of completeness inherent in many biological and chemical measurements.

[0059] As used herein, the term "subject" or "patient" refers to any organism to which a compound or composition according to the present invention can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may be in need of treatment, may be undergoing treatment, may be receiving treatment in the future, or may be a human or animal being cared for by a professional trained in a particular disease or condition.

[0060] As used herein, the terms "treating," "treated," or "treatment" mean both therapeutic treatment and prophylactic or suppressive measures, the purpose of which is to inhibit or slow down (reduce) an undesirable physiological state, disorder, or disease, or to obtain a beneficial or desirable clinical outcome. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of a state, disorder, or disease, stabilization (i.e., not worsening) of a state, disorder, or disease, onset or delay of the progression of a state, disorder, or disease, improvement (partial or total) of a state, disorder, or medical condition or remission, whether detectable or not, improvement of at least one measurable physical parameter, which need not be distinguishable by the subject, or improvement or amelioration of a state, disorder, or disease. Treatment includes eliciting a clinically significant response without undue levels of side effects. Treatment also includes prolonging survival as compared to the expected survival in the absence of treatment.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present disclosure, and other, suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0062] Details of one or more embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the specification and claims.

Brief Description of the Drawings

[0063]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

[0064] The inventors have surprisingly found that sapropterin is effective in reducing blood phenylalanine concentration. In particular, the inventors have found that sapropterin is effective in reducing blood phenylalanine concentration to a level similar to that of healthy subjects and / or is effective in subjects who did not respond to prior treatment with sapropterin dihydrochloride and / or pegvaliase-pqpz. Accordingly, the present invention features a method for reducing the blood phenylalanine concentration of a subject by administering sapropterin or a pharmaceutically acceptable salt thereof.

[0065] Sapropterin Sapropterin passes into the cell and is converted to 7,8-dihydrobiopterin by sapropterin reductase. Subsequently, 7,8-dihydrobiopterin is converted to BH4 via reduction by dihydrofolate reductase.

[0066] Sapropterin has the following structure. JPEG2025111476000001.jpg3765Sapropterin

[0067] Sepiapterin or a pharmaceutically acceptable salt thereof can be formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition of the present invention contains 20% to 30%, for example, about 20%, 22%, 25%, 27%, or 30% of sepiapterin or a salt thereof based on the total weight. In some embodiments, the pharmaceutical composition contains more than 20%, for example, more than 25%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of sepiapterin based on the total weight. In some embodiments, the pharmaceutical composition contains less than 20%, for example, less than 20%, less than 15%, less than 10%, or less than 5% of sepiapterin based on the total weight.

[0068] In some embodiments, the present invention features a pharmaceutical composition comprising sepiapterin or a salt thereof, and an antioxidant in an amount less than 10% by total weight (e.g., about 9%, 7%, 5%, 3%, 1%, 0.5%, 0.25%, 0.1%, or no antioxidant). The antioxidant may be ascorbic acid. In some embodiments, the ratio of sepiapterin or a pharmaceutically acceptable salt thereof to the antioxidant is 1:1 or greater than 1:1 by weight, e.g., 2:1, 5:1, 7:1, or 10:1. The pharmaceutical composition may comprise sepiapterin or a pharmaceutically acceptable salt thereof in an amount of 20-30% by total weight, e.g., about 20%, 22%, 25%, 27%, or 30%. The pharmaceutical composition may further comprise a dispersant, e.g., croscarmellose sodium. The pharmaceutical composition may comprise the dispersant in an amount of 0.1-1.5% by total weight, e.g., 0.1%, 0.5%, 1%, or 1.5%. In some embodiments, the pharmaceutical composition comprises at least one anti-caking agent, e.g., colloidal silicon dioxide or microcrystalline cellulose. The pharmaceutical composition may comprise the anti-caking agent in an amount of 65-75% by total weight, e.g., about 65%, 67%, 70%, 73%, or 75%. In some embodiments, the pharmaceutical composition comprises both colloidal silicon dioxide and microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprises microcrystalline cellulose in an amount of 60-65% by total weight and colloidal silicon dioxide in an amount of 5-7% by total weight. In some embodiments, the sepiapterin crystal form is formulated as particles less than 140 μm (e.g., about 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, or 5 μm). In some embodiments, the pharmaceutical composition comprises impurities such as lactoyl pterin in an amount less than 1% by total weight, e.g., less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, or less than 0.2%.

[0069] In some embodiments, sepiapterin is a salt of sepiapterin, for example, a salt with sulfuric acid, p-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, malonic acid, tartaric acid (e.g., L-tartaric acid), phosphoric acid, gentisic acid, fumaric acid, glycolic acid, acetic acid, or nicotinic acid.

[0070] In some embodiments, sepiapterin or a pharmaceutically acceptable salt thereof is in crystalline form. The crystalline sepiapterin free base or the crystalline form of a salt of sepiapterin can occur as an anhydrate (e.g., having no bound water or solvent or hydration or solvation), or as a hydrate, a partial hydrate (e.g., hemihydrate, sesquihydrate, etc.), a dihydrate, a trihydrate, etc. Here, the crystalline form binds the water of hydration or solvent molecules associated with the sepiapterin crystalline form or its salt. In one embodiment, crystalline sepiapterin occurs as a monohydrate or a hemihydrate.

[0071] In some embodiments, sepiapterin is present in crystalline form, for example, as described in WO 2018 / 102314 and WO 2018 / 102315, the entire disclosures of which are incorporated herein by reference.

[0072] In some embodiments, the sepiapterin crystal form is characterized by a powder X-ray diffraction pattern obtained by irradiation with CuKα X-rays having peaks represented by 2θ of at least 9.7° ± 0.5, for example, 9.7° ± 0.2, 10.2° ± 0.5, for example, 10.2° ± 0.2, and 11.3° ± 0.5, for example, 11.3° ± 0.2. In other embodiments, the sepiapterin crystal form is characterized by a powder X-ray diffraction pattern obtained by irradiation with CuKα X-rays having peaks represented by 2θ of at least 9.7° ± 0.5, for example, 9.7° ± 0.2, 10.2° ± 0.5, for example, 10.2° ± 0.2, 11.3° ± 0.5, for example, 11.3° ± 0.2, 14.0° ± 0.5, for example, 14.0° ± 0.2, 14.6° ± 0.5, for example, 14.6° ± 0.2, 19.9° ± 0.5, for example, 19.9° ± 0.2, 22.2° ± 0.5, for example, 22.2° ± 0.2, 25.3° ± 0.5, for example, 25.3° ± 0.2, and 32.4° ± 0.5, for example, 32.4° ± 0.2. In an essentially pure form of this crystal form, peaks can be observed at the refractive angles 2θ as shown in Table 1. Alternatively or additionally, this crystal form is characterized by a DSC curve showing two endotherms at 71.6 °C and 233.4 °C.

[0073]

Table 1

[0074] In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, or 3 peaks) at a diffraction angle 2θ (°) of 8.4° ± 0.5, e.g., 8.4° ± 0.2, 16.9° ± 0.5, e.g., 16.9° ± 0.2, or 25.4° ± 0.5, e.g., 25.4° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, or 5 peaks) at a diffraction angle 2θ (°) of 8.4° ± 0.5, e.g., 8.4° ± 0.2, 14.9° ± 0.5, e.g., 14.9° ± 0.2, 16.9° ± 0.5, e.g., 16.9° ± 0.2, 25.4° ± 0.5, e.g., 25.4° ± 0.2, and 34.1° ± 0.5, e.g., 34.1° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In an essentially pure material of this crystalline form, peaks may be observed at the refractive angle 2θ as shown in Table 2. Alternatively or additionally, this crystalline form is characterized by a DSC curve showing a melting event at 195.2 °C.

[0075]

Table 2

[0076] In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, or 3 peaks) at a diffraction angle 2θ (°) of 5.7° ± 0.5, e.g., 5.7° ± 0.2, 7.8° ± 0.5, e.g., 7.8° ± 0.2, or 25.4° ± 0.5, e.g., 25.4° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 peaks) at a diffraction angle 2θ (°) of 5.7° ± 0.5, e.g., 5.7° ± 0.2, 7.8° ± 0.5, e.g., 7.8° ± 0.2, 9.1° ± 0.5, e.g., 9.1° ± 0.2, 11.5° ± 0.5, e.g., 11.5° ± 0.2, 15.3° ± 0.5, e.g., 15.3° ± 0.2, 16.0° ± 0.5, e.g., 16.0° ± 0.2, 20.1° ± 0.5, e.g., 20.1° ± 0.2, 25.4° ± 0.5, e.g., 25.4° ± 0.2, and 26.6° ± 0.5, e.g., 26.6° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In an essentially pure material of this crystalline form, peaks may be observed at the refractive angles 2θ as shown in Table 3. Alternatively or additionally, this crystalline form is characterized by a DSC curve showing five endothermic peaks at 58.3°C, 101.8°C, 129.8°C, 156.5°C, and 168.3°C.

[0077]

Table 3

[0078] In some embodiments, the crystal form of sepiapterin has at least one peak (e.g., 1, 2, or 3 peaks) at diffraction angles 2θ (°) of 8.9° ± 0.5, e.g., 8.9° ± 0.2, 10.3° ± 0.5, e.g., 10.3° ± 0.2, or 26.0° ± 0.5, e.g., 26.0° ± 0.2, as measured by X-ray diffraction with CuKα X-ray irradiation or calculated from X-ray diffraction. In some embodiments, the crystal form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 peaks) at diffraction angles 2θ (°) of 8.9° ± 0.5, e.g., 8.9° ± 0.2, 10.3° ± 0.5, e.g., 10.3° ± 0.2, 10.9° ± 0.5, e.g., 10.9° ± 0.2, 17.8° ± 0.5, e.g., 17.8° ± 0.2, 24.9° ± 0.5, e.g., 24.9° ± 0.2, 26.0° ± 0.5, e.g., 26.0° ± 0.2, 26.7° ± 0.5, e.g., 26.7° ± 0.2, 26.8° ± 0.5, e.g., 26.8° ± 0.2, and 28.3° ± 0.5, e.g., 28.3° ± 0.2, as measured by X-ray diffraction with CuKα X-ray irradiation or calculated from X-ray diffraction. In an essentially pure material of this crystal form, peaks may be observed at the refractive angles 2θ as shown in Table 4. Alternatively or additionally, this crystal form is characterized by a DSC curve showing three endotherms at 42.7 °C, 66.3 °C, and 232.9 °C.

[0079]

Table 4

[0080] In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, or 3 peaks) at a diffraction angle 2θ (°) of 4.7° ± 0.5, e.g., 4.7° ± 0.2, 7.4° ± 0.5, e.g., 7.4° ± 0.2, or 26.2° ± 0.5, e.g., 26.2° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 peaks) at a diffraction angle 2θ (°) of 4.7° ± 0.5, e.g., 4.7° ± 0.2, 7.4° ± 0.5, e.g., 7.4° ± 0.2, 9.5° ± 0.5, e.g., 9.5° ± 0.2, 11.3° ± 0.5, e.g., 11.3° ± 0.2, 15.6° ± 0.5, e.g., 15.6° ± 0.2, 16.4° ± 0.5, e.g., 16.4° ± 0.2, 26.2° ± 0.5, e.g., 26.2° ± 0.2, or 27.2° ± 0.5, e.g., 27.2° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In an essentially pure material of this crystalline form, peaks may be observed at the refractive angle 2θ as shown in Table 5. Alternatively or additionally, this crystalline form is characterized by a DSC curve showing endothermic peaks at 82.8 °C and 179.8 °C.

[0081]

Table 5

[0082] In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, 5, or 6 peaks) at diffraction angles 2θ (°) of 6.0° ± 0.5, 6.0° ± 0.2, 10.6° ± 0.5, 10.6° ± 0.2, 12.1° ± 0.5, for example, 12.1° ± 0.2, 15.9° ± 0.5, for example, 15.9° ± 0.2, 20.9° ± 0.5, for example, 20.9° ± 0.2, or 24.6° ± 0.5, for example, 24.6° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In some embodiments, the crystalline form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 peaks) at diffraction angles 2θ (°) of 6.0° ± 0.5, for example, 6.0° ± 0.2, 10.6° ± 0.5, for example, 10.6° ± 0.2, 12.1° ± 0.5, for example, 12.1° ± 0.2, 15.9° ± 0.5, for example, 15.9° ± 0.2, 18.1° ± 0.5, for example, 18.1° ± 0.2, 20.9° ± 0.5, for example, 20.9° ± 0.2, 22.1° ± 0.5, for example, 22.1° ± 0.2, 24.6° ± 0.5, for example, 24.6° ± 0.2, 26.1° ± 0.5, for example, 26.1° ± 0.2, 28.1° ± 0.5, for example, 28.1° ± 0.2, 28.9° ± 0.5, for example, 28.9° ± 0.2, 32.1° ± 0.5, for example, 32.1° ± 0.2, or 37.0° ± 0.5, for example, 37.0° ± 0.2, as measured by X-ray diffraction upon irradiation with CuKα X-rays or calculated from X-ray diffraction. In an essentially pure form of this crystalline form, peaks may be observed at refractive angles 2θ as shown in Table 6. Alternatively or additionally, this crystalline form is characterized by a DSC curve showing two endothermic peaks at 112.9 °C and 195.8 °C.

[0083] [Table 6]

[0084] In some embodiments, the crystal form of sepiapterin has at least one peak (e.g., 1, 2, or 3 peaks) at a diffraction angle 2θ (°) of 10.0° ± 0.5, e.g., 10.0° ± 0.2, 10.6° ± 0.5, e.g., 10.6° ± 0.2, or 25.7° ± 0.5, e.g., 25.7° ± 0.2, as measured by X-ray diffraction with CuKα X-ray irradiation or calculated from X-ray diffraction. In some embodiments, the crystal form of sepiapterin has at least one peak (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 peaks) at a diffraction angle 2θ (°) of 10.0° ± 0.5, e.g., 10.0° ± 0.2, 10.6° ± 0.5, e.g., 10.6° ± 0.2, 11.2° ± 0.5, e.g., 11.2° ± 0.2, 15.3° ± 0.5, e.g., 15.3° ± 0.2, 15.9° ± 0.5, e.g., 15.9° ± 0.2, 22.8° ± 0.5, e.g., 22.8° ± 0.2, 24.4° ± 0.5, e.g., 24.4° ± 0.2, 25.0° ± 0.5, e.g., 25.0° ± 0.2, 25.7° ± 0.5, e.g., 25.7° ± 0.2, or 26.6° ± 0.5, e.g., 26.6° ± 0.2, as measured by X-ray diffraction with CuKα X-ray irradiation or calculated from X-ray diffraction. In an essentially pure material of this crystal form, peaks may be observed at the refractive angles 2θ as shown in Table 7.

[0085]

Table 7

[0086] In some embodiments, the crystal form of sepiapterin hydrochloride has at least one peak (e.g., 1, 2, or 3 peaks) at diffraction angles 2θ (°) of 7.8° ± 0.5, e.g., 7.8° ± 0.2, 12.9° ± 0.5, e.g., 12.9° ± 0.2, or 26.2° ± 0.5, e.g., 26.2° ± 0.2, as measured by X-ray diffraction with CuKα X-ray irradiation or calculated from X-ray diffraction. In some embodiments, the strongest peak in the X-ray diffraction pattern of the crystal form of sepiapterin hydrochloride is observed at a refractive angle 2θ of 7.8° ± 0.5, e.g., 7.8° ± 0.2. This substantially pure material of the crystal of sepiapterin hydrochloride may have peaks at refractive angles 2θ as shown in Table 8. Alternatively or additionally, the crystalline hydrochloride of sepiapterin is characterized by a DSC curve showing an endotherm at 225.9°C.

[0087] [Table 8]

[0088] In some embodiments, the crystal form 1 methanesulfonate of sepiapterin is characterized by peaks in the X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.8° ± 0.5, e.g., 7.8° ± 0.5, e.g., 7.8° ± 0.2, 23.5° ± 0.5, e.g., 23.5° ± 0.2, and 29.0° ± 0.5, e.g., 29.0° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a refractive angle 2θ of 23.5° ± 0.5, e.g., 23.5° ± 0.2. In a substantially pure material of the crystal form 1 methanesulfonate of sepiapterin, peaks may be observed at refractive angles 2θ as shown in Table 9. Alternatively or additionally, the crystal form 1 methanesulfonate of sepiapterin is characterized by a DSC curve showing two endotherms at 186.0°C and 229.1°C.

[0089] [Table 9]

[0090] In some embodiments, the crystalline form 2 methanesulfonate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.8° ± 0.5, such as 7.9° ± 0.5, such as 7.9° ± 0.2, 23.4° ± 0.5, such as 23.4° ± 0.2, and 28.9° ± 0.5, such as 28.9° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 23.5° ± 0.5, such as 23.5° ± 0.2. In an essentially pure material of the crystalline form 2 methanesulfonate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 10. Alternatively or additionally, the crystalline form 2 methanesulfonate of sepiapterin is characterized by a DSC curve showing three endotherms at 75.5 °C, 182.6 °C and 234.9 °C.

[0091]

Table 10

[0092] In some embodiments, the crystalline form 3 methanesulfonate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.8° ± 0.5, such as 21.7° ± 0.5, such as 21.7° ± 0.2, 26.0° ± 0.5, such as 26.0° ± 0.2, and 28.9° ± 0.5, such as 28.9° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 26.0° ± 0.5, such as 26.0° ± 0.2. In an essentially pure material of the crystalline form 3 methanesulfonate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 11. Alternatively or additionally, the crystalline form 3 methanesulfonate of sepiapterin is characterized by a DSC curve showing two endotherms at 195.1 °C and 240.1 °C.

[0093]

Table 11

[0094] In some embodiments, the crystalline nicotinate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 9.5° ± 0.5, such as 9.5° ± 0.2, 9.9° ± 0.5, such as 9.9° ± 0.2, and 24.5° ± 0.5, such as 24.5° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 24.5° ± 0.5, such as 24.5° ± 0.2. In an essentially pure material of the crystalline nicotinate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 12. Alternatively or additionally, the crystalline nicotinate of sepiapterin is characterized by a DSC curve showing an endotherm at 221.9 °C.

[0095] [Table 12]

[0096] In some embodiments, the crystalline p-toluenesulfonate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 6.5° ± 0.5, such as 6.5° ± 0.2, 15.1° ± 0.5, such as 15.1° ± 0.2, and 23.4° ± 0.5, such as 23.4° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 6.5° ± 0.5, such as 6.5° ± 0.2. In an essentially pure material of the p-toluenesulfonate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 13. Alternatively or additionally, the crystalline p-toluenesulfonate of sepiapterin is characterized by a DSC curve showing three endotherms at 77.2 °C, 202.4 °C, and 260.2 °C.

[0097] [Table 13]

[0098] In some embodiments, the crystalline benzenesulfonate of sepiapterin is characterized by peaks in the X-ray diffraction pattern observed at diffraction angles 2θ of at least 6.5° ± 0.5, such as 6.5° ± 0.2, 14.8° ± 0.5, such as 14.8° ± 0.2, and 19.6° ± 0.5, such as 19.6° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 6.5° ± 0.5, such as 6.5° ± 0.2. In an essentially pure material of the benzenesulfonate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 14. Alternatively or additionally, the crystalline benzenesulfonate of sepiapterin is characterized by a DSC curve showing two endotherms at 202.3 °C and 265.5 °C.

[0099]

Table 14

[0100] In some embodiments, the crystalline phosphate of sepiapterin is characterized by peaks in the X-ray diffraction pattern observed at diffraction angles 2θ of at least 16.6° ± 0.5, such as 16.6° ± 0.2, 22.2° ± 0.5, such as 22.2° ± 0.2, and 25.6° ± 0.5, such as 25.6° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 25.6° ± 0.5, such as 25.6° ± 0.2. In an essentially pure material of the crystalline phosphate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 15. Alternatively or additionally, the crystalline phosphate of sepiapterin is characterized by a DSC curve showing three endotherms at 125.9 °C, 152.1 °C, and 157.6 °C.

[0101]

Table 15

[0102] In some embodiments, the crystalline malonate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 6.9° ± 0.5, such as 6.9° ± 0.2, 22.7° ± 0.5, such as 22.7° ± 0.2, and 23.8° ± 0.5, such as 23.8° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 6.9° ± 0.5, such as 6.9° ± 0.2. In an essentially pure material of the crystalline malonate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 16. Alternatively or additionally, the crystalline malonate of sepiapterin is characterized by a DSC curve showing a melting event at 115.8°C.

[0103]

Table 16

[0104] In some embodiments, the crystalline L-tartrate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.3° ± 0.5, such as 7.3° ± 0.2, 14.2° ± 0.5, such as 14.2° ± 0.2, and 21.8° ± 0.5, such as 21.8° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 6.9° ± 0.5, such as 6.9° ± 0.2. In an essentially pure material of the crystalline L-tartrate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 17. Alternatively or additionally, the crystalline L-tartrate of sepiapterin is characterized by a DSC curve showing two endotherms at 97.2°C and 160.6°C.

[0105]

Table 17

[0106] In some embodiments, the crystalline gentisate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.1° ± 0.5, such as 7.1° ± 0.2, 8.7° ± 0.5, such as 8.7° ± 0.2, and 26.7° ± 0.5, such as 26.7° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 7.1° ± 0.5, such as 7.1° ± 0.2. In an essentially pure material of the crystalline gentisate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 18. Alternatively or additionally, the crystalline gentisate of sepiapterin is characterized by a DSC curve showing three endotherms at 70.5 °C, 128.2 °C, and 184.7 °C.

[0107]

Table 18

[0108] In some embodiments, the crystalline fumarate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 11.3° ± 0.5, such as 11.3° ± 0.2, 24.0° ± 0.5, such as 24.0° ± 0.2, and 28.2° ± 0.5, such as 28.2° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of at least 24.0° ± 0.5, such as 24.0° ± 0.2. In an essentially pure material of the crystalline fumarate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 19. Alternatively or additionally, the crystalline fumarate of sepiapterin can be characterized by a DSC curve showing two endotherms at 114.3 °C and 229.7 °C.

[0109]

Table 19

[0110] In some embodiments, the crystalline glycolate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.6° ± 0.5, such as 7.6° ± 0.2, 10.7° ± 0.5, such as 10.7° ± 0.2, and 24.0° ± 0.5, such as 24.0° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 7.6° ± 0.5, such as 7.6° ± 0.2. In an essentially pure material of the crystalline glycolate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 20. Alternatively or additionally, the crystalline glycolate of sepiapterin is characterized by a DSC curve showing two endotherms at 133.9 °C and 147.7 °C.

[0111]

Table 20

[0112] In some embodiments, the crystalline acetate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 6.2° ± 0.5, such as 6.2° ± 0.2, 12.0° ± 0.5, such as 12.0° ± 0.2, and 18.1° ± 0.5, such as 18.1° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of at least 6.2° ± 0.5, such as 6.2° ± 0.2. In an essentially pure material of the crystalline acetate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 21. Alternatively or additionally, the crystalline acetate of sepiapterin is characterized by a DSC curve showing two endotherms at 146.1 °C and 175.4 °C.

[0113]

Table 21

[0114] In some embodiments, the crystalline 1 sulfate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 5.1° ± 0.5, such as 5.1° ± 0.2, 7.8° ± 0.5, such as 7.8° ± 0.2, and 23.0° ± 0.5, such as 23.0° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 5.1° ± 0.5, such as 5.1° ± 0.2. In an essentially pure material of the crystalline form 1 sulfate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 22. Alternatively or additionally, the crystalline form 1 sulfate of sepiapterin is characterized by a DSC curve showing three endotherms at 94.5 °C, 158.3 °C, and 209.9 °C.

[0115]

Table 22

[0116] In some embodiments, the crystalline 2 sulfate of sepiapterin is characterized by peaks in an X-ray diffraction pattern observed at diffraction angles 2θ of at least 7.8° ± 0.5, such as 7.8° ± 0.2, 8.8° ± 0.5, such as 8.8° ± 0.2, and 24.1° ± 0.5, such as 24.1° ± 0.2. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at a diffraction angle 2θ of 8.8° ± 0.5, such as 8.8° ± 0.2. In an essentially pure material of the crystalline form 2 sulfate of sepiapterin, peaks may be observed at diffraction angles 2θ as shown in Table 23.

[0117]

Table 23

[0118] The present invention can employ a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof. Examples of pharmaceutical compositions of sepiapterin and its salts can be found in WO 2019 / 046849 and WO 2019 / 232120, the compositions of which are incorporated herein by reference in their entirety.

[0119] The pharmaceutically acceptable excipient can be any of those conventionally used and is limited only by physicochemical considerations such as solubility and route of administration. Those skilled in the art will understand that, in addition to the pharmaceutical compositions described below, sepiapterin can be formulated as an inclusion complex such as a cyclodextrin inclusion complex, or as liposomes.

[0120] The pharmaceutically acceptable excipients described herein, such as vehicles, adjuvants, excipients, or diluents, are well known to those skilled in the art and are readily available. The pharmaceutically acceptable excipient is preferably chemically inert to sepiapterin and has no harmful side effects or toxicity under the conditions of use.

[0121] Formulations for increasing gastric and / or foregut residence time Gastric retention drug delivery is an approach in which a drug formulation is designed to remain in the stomach longer until drug release is complete.

[0122] Bioadhesive formulations utilize polymers that can adhere to surfaces and provide controlled release of drugs. Bioadhesive polymers can be anionic (e.g., carboxymethylcellulose, alginic acid, polyacrylic acid, pectin, carrageenan, polycarbophil, or carbomer), cationic (e.g., chitosan, polylysine, or polybrene), or nonionic (e.g., polyethylene glycol, polyvinylpyrrolidone, dextran, or hydroxypropylmethylcellulose).

[0123] The high-density formulation is designed to be present in the stomach at a level lower than the pyloric sphincter, and thus can avoid becoming empty. Excipients suitable for the high-density formulation include iron powder, barium sulfate, zinc oxide, titanium oxide, and the like.

[0124] The swellable formulation is designed to swell in the stomach to be larger than the pyloric sphincter, and thus can avoid fasting. For example, a formulation containing a drug core, a swellable hydrocolloid, and an outer semi-permeable polymer is suitable for the swellable formulation.

[0125] The superporous hydrogel formulation is designed to swell in the stomach to be larger than the pyloric sphincter, similar to the swellable formulation. The superporous hydrogel formulation may contain a polymer such as croscarmellose sodium.

[0126] The floating formulation is designed to have a density lower than that of gastric juice. The floating formulation may include a composition containing an ion exchange resin, a raft system, an inflatable chamber, a foaming mixture, a swellable hydrocolloid, or a multi-particle system.

[0127] Antioxidant Sepiapterin tends to oxidize rapidly when exposed to air. Accordingly, the pharmaceutical composition of the present invention may contain an antioxidant. The antioxidant may minimize the oxidative degradation of sepiapterin. Examples of antioxidants include, but are not limited to, ascorbic acid, tocopherol, retinol, ascorbyl palmitate, N-acetylcysteine, glutathione, ethylenediaminetetraacetic acid, sodium bisulfite, sodium metabisulfite, thiourea, butylated hydroxytoluene, butylated hydroxyanisole, and vitamin E. In some embodiments, the pharmaceutical composition of the present invention contains, as an antioxidant, ascorbic acid, tocopherol, retinol, ascorbyl palmitate, N-acetylcysteine, glutathione, butylated hydroxytoluene, and / or butylated hydroxyanisole.

[0128] In some embodiments, the pharmaceutical composition contains less than 10% by weight of an antioxidant, for example, less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, or substantially no antioxidant. In some embodiments, the pharmaceutical composition contains 2 - 9% of an antioxidant based on the total weight, for example, 2 - 4%, 3 - 5%, 4 - 6%, 5 - 7%, 6 - 8%, or 7 - 9%. In some embodiments, the pharmaceutical composition contains 5 - 100% of the USP maximum daily dose of an antioxidant, for example, in some embodiments, the pharmaceutical composition contains about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the USP maximum daily dose of an antioxidant. In some embodiments, the ratio of sepiapterin to the antioxidant is at least 1:1 by weight, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0129] Dispersant In some embodiments, the pharmaceutical composition of the present invention contains at least one dispersant. The dispersant may separate the particles in the formulation, for example, and may release the drug substance upon contact with moisture. Examples of dispersants include, but are not limited to, crosslinked polyvinylpyrrolidone, carboxymethylcellulose (e.g., croscarmellose salts, e.g., croscarmellose sodium), starch (e.g., sodium starch glycolate), or alginic acid. In some embodiments, the dispersant in the pharmaceutical composition is carboxymethylcellulose such as a pharmaceutically acceptable salt of croscarmellose. In some embodiments, the pharmaceutical composition may contain 0.1 - 1.5% (e.g., about 0.1%, 0.5%, 1%, or 1.5%) of a dispersant based on the total weight. In some embodiments, the pharmaceutical composition contains less than 1.5% (e.g., less than 1%, less than 0.5%, or less than 0.1%) of a dispersant based on the total weight.

[0130] Anticaking agent In some embodiments, the pharmaceutical composition of the present invention comprises at least one anti-caking agent. In some embodiments, the pharmaceutical composition of the present invention comprises at least two anti-caking agents. Exemplary anti-caking agents include colloidal silicon dioxide, microcrystalline cellulose, tricalcium phosphate, microcrystalline cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, calcium phosphate, sodium silicate, colloidal silicon dioxide, calcium silicate, magnesium trisilicate, talcum powder, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, and polydimethylsiloxane. In some embodiments, at least one anti-caking agent is colloidal silicon dioxide or microcrystalline cellulose. In some embodiments, the pharmaceutical composition may comprise 65 - 75% (e.g., about 65%, 67%, 70%, 73%, or 75%) anti-caking agent based on the total weight. In some embodiments, the pharmaceutical composition comprises both colloidal silicon dioxide and microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprises 60 - 65% microcrystalline cellulose and 5 - 7% colloidal silicon dioxide based on the total weight.

[0131] Administration vehicle In some embodiments, the pharmaceutical composition of the present invention is combined with an administration vehicle prior to administration (e.g., an administration vehicle having a viscosity of about 50 - 1750 centipoise (cP)). One type of suspension that can be used is a combination of glycerin and sucrose in water (e.g., MEDISCA oral mix containing 2.5% glycerin and 27% sucrose in water). An appropriate amount of the composition can be added to the administration vehicle mixture and stirred to suspend the composition immediately prior to administration. [[ID=⑦]] (R) [[ID=⑧]]oral mix).

[0132] Alternatively, other suspending agents may be used as the administration vehicle. Exemplary suspending agents include agar, alginic acid, sodium carboxymethyl cellulose, carrageenan, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, methyl cellulose, polyethylene glycol, povidone, tragacanth, xanthan gum, or other suspending agents known in the art.

[0133] Dosage Sepiapterin or a pharmaceutically acceptable salt thereof can be used at any appropriate dosage. Appropriate dosages and dosage regimens can be determined within the scope of the prior art. Generally, treatment is initiated at a dosage lower than the optimal dosage. Thereafter, the dosage is increased incrementally until the optimal effect is obtained under the circumstances. For convenience, the total daily dosage can be divided and administered throughout the day if desired. For appropriate dosages and appropriate administration of a particular compound, the present invention provides a wide range of responses. Typically, the dosage ranges from about 2.5 to about 150 mg / kg body weight / day (e.g., 60 mg / kg / day) of the subject being treated. For example, in embodiments, sepiapterin, or a pharmaceutically acceptable salt thereof, is administered one or more times a day at about 20 mg / kg to about 150 mg / kg, about 20 mg / kg to about 60 mg / kg, about 40 mg / kg to about 100 mg / kg, about 100 mg / kg to about 150 mg / kg, about 60 mg / kg to about 120 mg / kg, about 80 mg / kg to about 100 mg / kg, about 40 mg / kg to about 60 mg / kg, about 2.5 mg / kg to about 20 mg / kg, about 2.5 mg / kg to about 10 mg / kg, or about 2.5 mg / kg to about 5 mg / kg of subject body weight / day to obtain the desired therapeutic effect.

[0134] In some embodiments, sepiapterin or a pharmaceutically acceptable salt thereof can be formulated as a unit solid oral dosage formulation such as a particle. In these embodiments, each unit solid oral dosage formulation, e.g., sachet, can contain any suitable amount of sepiapterin or a pharmaceutically acceptable salt thereof. For example, each solid oral dosage formulation can contain about 2.5 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 750 mg, about 1 g, about 1.25 g, or about 1.5 g.

[0135] Sepiapterin or a pharmaceutically acceptable salt thereof can be used in the preparation of liquid formulations such as in the form of a solution, suspension or emulsion. Formulations suitable for oral administration include, but are not limited to, (a) solids or granules such as capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, (b) powders, (c) solutions of an effective amount of the compound dissolved in a diluent such as water, physiological saline, or orange juice, (d) suspensions in a suitable liquid, and (e) suitable emulsions. Preferably, they are solid oral formulations such as in capsule form, tablet form, and powder form. The capsule form can be of the usual hard or soft shell gelatin type, containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch. The tablet form can contain one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, coloring agents, diluents, buffering agents, disintegrants, wetting agents, preservatives, flavors, and pharmacologically compatible excipients. The lozenge form can contain the active ingredient within a flavor (usually sucrose and acacia or tragacanth), and similarly the pastille can contain the active ingredient within an inert base such as gelatin and glycerin, or sucrose and acacia, emulsion, gel, etc., and such excipients are known in the art.

[0136] Formulations suitable for oral and / or parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The compounds of the present invention can be administered in a physiologically acceptable diluent in a pharmaceutical excipient such as a sterile liquid or a mixture of liquids, including water, saline, aqueous glucose and related sugar solutions, ethanol, alcohols such as benzyl alcohol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol and polyethylene alcohols, glycerol ketals such as 2,2-dimethyl-l,3-dioxolan-4-methanol, ethers such as poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents, and other pharmaceutical adjuvants.

[0137] The present invention features a pharmaceutically acceptable formulation of a pharmaceutical composition comprising a therapeutically effective amount of sepiapterin and less than 10% antioxidant. In some embodiments, the pharmaceutical composition is a granule formulation dispersed in a pharmaceutically acceptable excipient, for example, the composition can be mixed with water and ingested by the subject (e.g., within 5 to 10 minutes). Suitable formulations for use in the present invention are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA 22nd ed., 2010. The use thereof in the pharmaceutical compositions of the present invention is contemplated, except when any conventional excipient is incompatible with the active ingredient. Further, for animal (e.g., human) administration, it will be understood that the formulation should meet the standards of sterility, pyrogenicity, general safety and purity as required by the biological standards of the FDA office.

[0138] Solid preparation for oral administration Formulations for oral use contain particles comprising the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients, and such formulations are known to those skilled in the art (e.g., U.S. Patent Nos.: 5,817,307, 5,824,300, 5,830,456, 5,846,526, 5,882,640, 5,910,304, 6,036,949, 6,036,949, 6,372,218. These are incorporated herein by reference.). Excipients are, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates or alginic acid), binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinyl pyrrolidone, or polyethylene glycol), and lubricants, glidants, antiadhesion agents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc), and anticaking agents (e.g., colloidal silicon dioxide, microcrystalline cellulose, tricalcium phosphate, microcrystalline cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, calcium phosphate, sodium silicate, colloidal silicon dioxide, calcium silicate, magnesium trisilicate, talcum powder, sodium aluminosilicate, potassium aluminosilicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, polydimethylsiloxane). Other pharmaceutically acceptable excipients can be coloring agents, flavors, plasticizers, humectants, and buffering agents. In some embodiments, the excipient (e.g., flavor) is packaged with the composition.In some embodiments, the excipient (e.g., flavoring) is packaged separately from the composition (e.g., combined with the composition prior to administration).

[0139] The solid composition of the present invention may include a coating adapted to protect the composition from unwanted chemical changes (e.g., chemical decomposition prior to release of the active substance). The coating may be applied to the solid dosage form in a manner similar to that described in the Encyclopedia of Pharmaceutical Technology noted above.

[0140] Using the above components, powders and granules may be prepared by conventional methods, for example, using a mixer, a fluidized bed apparatus, a melt solidification apparatus, a rotor granulator, an extrusion / spheronization apparatus, or a spray drying apparatus, etc.

[0141] Methods of Treatment Sepiapterin can function as a useful therapeutic agent for diseases associated with elevated blood phenylalanine concentrations, such as hyperphenylalaninemia or phenylketonuria. As described herein, various forms of these diseases, for example, phenylketonuria, tetrahydrobiopterin-responsive phenylketonuria, sepiapterin-responsive phenylketonuria, classical phenylketonuria, or hyperphenylalaninemia resulting from non-classical phenylketonuria can be treated. Accordingly, various forms of sepiapterin or its salts according to the present invention can be administered to a subject in an effective amount to obtain treatment or amelioration of a disease, disorder or condition.

[0142] Subjects treated by the methods of the present specification typically have a blood phenylalanine concentration (e.g., an uncontrolled blood phenylalanine concentration) greater than 120 micromoles per liter (e.g., greater than 200 micromoles per liter, greater than 300 micromoles per liter, greater than 360 micromoles per liter, greater than 400 micromoles per liter, greater than 450 micromoles per liter, greater than 500 micromoles per liter, greater than 550 micromoles per liter, greater than 600 micromoles per liter, greater than 650 micromoles per liter, greater than 700 micromoles per liter, greater than 800 micromoles per liter, greater than 900 micromoles per liter, greater than 1000 micromoles per liter, greater than 1100 micromoles per liter, or greater than 1200 micromoles per liter). Also, the subjects may be stratified by having a blood phenylalanine concentration (e.g., an uncontrolled blood phenylalanine concentration) of 120 - 360 micromoles per liter, 360 - 600 micromoles per liter, 600 - 1200 micromoles per liter, or greater than 1200 micromoles per liter.

[0143] Subjects with insufficient reduction of blood phenylalanine under existing therapies (e.g., phenylalanine-restricted diet, sapropterin, or pegvaliase-pqpz) can also be treated by the methods described herein. For example, the blood phenylalanine concentration of subjects being treated with a therapy other than sepiapterin is greater than 120 micromoles per liter under existing management (e.g., greater than 200 micromoles per liter, greater than 300 micromoles per liter, greater than 360 micromoles per liter, greater than 400 micromoles per liter, greater than 450 micromoles per liter, greater than 500 micromoles per liter, greater than 550 micromoles per liter, greater than 600 micromoles per liter, greater than 650 micromoles per liter, greater than 700 micromoles per liter, greater than 800 micromoles per liter, greater than 900 micromoles per liter, greater than 1000 micromoles per liter, greater than 1100 micromoles per liter, or greater than 1200 micromoles per liter).

[0144] By administering sepiapterin or a pharmaceutically acceptable salt thereof, the phenylalanine concentration in the blood of a subject can be reduced to less than 600 micromoles per liter, for example, less than 360 micromoles per liter or less than 120 micromoles per liter, for example, 120 to 360 micromoles per liter or 360 to 600 micromoles per liter. Alternatively or additionally, by administering sepiapterin, for example, the phenylalanine concentration in the blood of a subject before administration of sepiapterin can be reduced by at least 10% (for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 10% to about 30%, about 20% to about 40%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, about 60% to about 80%, or about 70% to about 90%). The reduction can be measured after administration over at least one week (for example, two weeks, three weeks, four weeks, five weeks, six weeks, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks). Also, administration of sepiapterin can result in a BH4 concentration of at least 50 ng / ml (for example, at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) in the plasma of the subject within 10 hours after administration.

[0145] The subject may be a subject who did not respond to treatment with sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) or pegvaliase-pqpz. For example, upon administration of pegvaliase-pqpz, the plasma concentration of the subject may have decreased by less than 20%. For example, upon administration of at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg) of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride), the plasma concentration of the subject decreased by less than 30%, or upon administration of at least about 20 mg of pegvaliase pqpz once daily for at least 8 days (e.g., at least 14 days, at least 21 days, at least 28 days, at least 30 days), it decreased by less than 20%. In some embodiments, upon administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride), e.g., at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg), the plasma concentration of the subject decreased by less than 15% to less than 30%. In some embodiments, upon administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride), e.g., at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg), the plasma concentration of the subject decreased by less than 30%. In some embodiments, upon administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride), e.g., at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg), the plasma concentration of the subject decreased by less than 20%. In some embodiments, upon administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride), e.g., at least about 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg), the plasma concentration of the subject decreased by less than 15%.In some embodiments, the plasma concentration of the subject after administering sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) for at least 8 days (e.g., at least 14 days, at least 21 days, at least 28 days, at least 30 days), or pegloticase-pqpz at least about 20 mg once a day for at least 16 weeks (e.g., at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24 weeks) was greater than 120 micromoles / liter (e.g., greater than 200 micromoles / liter, greater than 300 micromoles / liter, greater than 360 micromoles / liter, greater than 400 micromoles / liter, greater than 450 micromoles / liter, greater than 500 micromoles / liter, greater than 550 micromoles / liter, greater than 600 micromoles / liter, greater than 650 micromoles / liter, greater than 700 micromoles / liter, greater than 800 micromoles / liter, greater than 900 micromoles / liter, greater than 1000 micromoles / liter, greater than 1100 micromoles / liter, or greater than 1200 micromoles / liter).For example, the plasma concentration in a subject after administration of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride) at at least 10 mg / kg (e.g., at least about 15 mg / kg, at least about 20 mg / kg) for at least 8 days (e.g., at least 14 days, at least 21 days, at least 28 days, at least 30 days), or pegvaliase-pqpz at at least about 20 mg once daily for at least 16 weeks (e.g., at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24 weeks) was greater than 120 micromoles / liter (e.g., greater than 200 micromoles / liter, greater than 300 micromoles / liter, greater than 360 micromoles / liter, greater than 400 micromoles / liter, greater than 450 micromoles / liter, greater than 500 micromoles / liter, greater than 550 micromoles / liter, greater than 600 micromoles / liter, greater than 650 micromoles / liter, greater than 700 micromoles / liter, greater than 800 micromoles / liter, greater than 900 micromoles / liter, greater than 1000 micromoles / liter, greater than 1100 micromoles / liter, or greater than 1200 micromoles / liter).

[0146] The subject can be administered sepapterin even after treatment with pegvaliase pqpz has been discontinued due to an adverse reaction (e.g., anaphylaxis, injection site reaction, joint pain, hypersensitivity reaction, headache, a systemic skin reaction lasting at least 14 days, pruritus, nausea, abdominal pain, oropharyngeal pain, vomiting, cough, diarrhea, and / or fatigue) and / or tolerability. The risk of adverse events (e.g., headache, rhinorrhea, pharyngolaryngeal pain, diarrhea, vomiting, cough, and / or nasal congestion) can be reduced compared to subjects administered at least 10 mg / kg (e.g., at least 15 mg / kg, at least 20 mg / kg) of sapropterin or a pharmaceutically acceptable salt thereof (e.g., sapropterin dihydrochloride).

[0147] In some embodiments, the subject is on a phenylalanine-restricted diet (e.g., the subject is on a diet that includes milk alternatives or formulations such as Phenyl-Free 2, and measured amounts of fruits, vegetables, bread, pasta, and cereal). In some embodiments, the subject is not on a phenylalanine-restricted diet (e.g., the subject is not on a diet that includes milk alternatives or formulations such as Phenyl-Free 2, and measured amounts of fruits, vegetables, bread, pasta, and cereal). In some embodiments, the subject has a median phenylalanine intake of less than 3000 mg / day (e.g., less than 2500 mg / day, less than 2000 mg / day, less than 1500 mg / day, less than 1000 mg / day, less than 500 mg / day). Treatment with sepiapterin may enable a decrease in blood phenylalanine concentration (e.g., less than 600, 360, or 120 micromoles / liter) in combination with phenylalanine intake or natural protein intake. For example, the subject may consume at least about 1000 mg / day (e.g., at least about 1100 mg / day, at least about 1200 mg / day, at least about 1300 mg / day, at least about 1400 mg / day, at least about 1500 mg / day, at least about 1600 mg / day, at least about 1700 mg / day, at least about 1800 mg / day, at least about 1900 mg / day, or at least about 2000 mg / day, or 1000 mg / day to 1400 mg / day, 1200 mg / day to 1600 mg / day, 1300 mg / day to 1700 mg / day, 1600 mg / day to 2000 mg / day, 1800 mg / day to 2400 mg / day, 2000 mg / day to 3000 mg / day, 3000 mg / day to 4000 mg / day, 4000 to 5000 mg / day) of phenylalanine. In some embodiments, less than 25% (e.g., less than 20%, less than 15%, less than 10%, or less than 5%) of the protein in the subject's diet is natural protein, or more than 25% (e.g., more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%) of the protein in the subject's diet is natural protein.In some embodiments, the subject has a natural protein intake of more than 10 g / day (e.g., more than 20 g / day, more than 30 g / day, more than 40 g / day, more than 50 g / day, more than 60 g / day, more than 70 g / day, more than 80 g / day, or about 10 g / day to about 30 g / day, about 20 g / day to about 40 g / day, about 30 g / day to about 50 g / day, about 40 g / day to about 60 g / day, about 50 g / day to about 70 g / day, about 60 g / day to about 80 g / day). In some embodiments of any of the methods described herein, the subject has a natural protein intake of less than 10 g / day (e.g., less than 5 g / day or about 5 g / day to about 10 g / day).

[0148] In some embodiments, the subject has PKU defined as at least one measurement >450 μmol / L in addition to any previous blood Phe measurement >360 μmol / L, or an average of the most recent three blood Phe measurements >450 μmol / L. In some embodiments, the subject does not have PKU defined as at least one measurement >450 μmol / L in addition to any previous blood Phe measurement >360 μmol / L, or an average of the most recent three blood Phe measurements >450 μmol / L. In some embodiments, the subject has classical PKU defined as any documented past measurement of blood Phe of at least 1,200 μmol / L. In some embodiments, the subject does not have classical PKU defined as any documented past measurement of blood Phe of at least 1,200 μmol / L.

[0149] In some embodiments, the subject is a child (e.g., the subject is less than 18 years old, less than 17 years old, less than 16 years old, less than 15 years old, less than 14 years old, less than 13 years old, less than 12 years old, less than 11 years old, less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old). In some embodiments, the subject is an adult (e.g., the subject is over 18 years old).

[0150] In some embodiments, the treatment increases an increase in the subject's neurocognitive function (e.g., an increase in executive function, a decrease in anxiety, a decrease in attention deficit / hyperactivity disorder symptoms, and / or a decrease in instances of brain fog). Changes in executive function can be measured by assessment such as the Behavioral Assessment of the Dysexecutive Syndrome (BADS), the Behavior Rating Inventory of Executive Function (BRIEF or Mini-BRIEF), the Berkeley Deficits in Executive Function Scale (BDEFS), the Behavioral Disorder Scale (BDS), the ASEBA Child Behavior Checklist (CBLC), the Comprehensive Executive Function Inventory (CEFI), CogScreen, the Continuous Performance Task (CPT), the Controlled Oral Word Association Test (COWAT), the d2 test of attention, the Delis-Kaplan Executive Function System (D-KEFS), the Digit Vigilance Test, the Figural Fluency Test, the Halstead Category Test, the Heiling Test and the Brixton Test, the Iowa Gambling Task, the Kaplan Baycrest Neurocognitive Assessment (KBNA), the Kaufman Short Neuropsychological Assessment, the Mental Clutter Scale, the Paced Auditory Serial Addition Test (PASAT), the Phenylketonuria - Quality of Life (PKU-QOL), the Rey-Osterrieth Complex Figure, the Rough Figural Fluency Test, the Stroop Task, the Executive Control Task, the Test of Variables of Attention (T.O.V.A.), the London Tower Test, the Trail Making Test (TMT) or Trail A & B, the Wisconsin Card Sorting Test (WCST), the Symbol Digit Modalities Test, or the Cambridge Neuropsychological Test Automated Battery (CANTAB) assessment (e.g., by measurement of reaction speed, spatial span, spatial working memory, rapid digital information processing, sustained attention, and / or stop signal task). The treatment can result in an improvement in attention and / or mood (e.g., as measured by the ADHD-RS 5 scale (or its inattention assessment) and / or the Profile of Mood States (POMS) scale). The treatment can result in an increase in the quality of sleep and / or a decrease in symptoms associated with sleep deprivation).

[0151] Sepiapterin may or may not be administered with food. Without being bound by theory, when sepiapterin is administered with food, plasma exposure of BH4 increases (for example, by reducing the absorption rate of sepiapterin). If the administered sepiapterin is rapidly absorbed, for example, when administered on an empty stomach, the intracellular sepiapterin reductase and / or dihydrofolate reductase will be saturated beyond V max and as a result, at least a portion of the administered sepiapterin may exit the cell without being reduced to 7,8-dihydrobiopterin and subsequent BH4. This excess sepiapterin is then excreted without being converted to BH4, resulting in a lower plasma BH4 level compared to when sepiapterin is administered with food that results in a reaction rate below, at, or slightly above the Vmax of substrate saturation of sepiapterin reductase and / or dihydrofolate reductase, which reduces or prolongs the absorption rate of sepiapterin. Administration of sepiapterin with food unexpectedly results in a lower maximum BH4 plasma concentration (Cmax) and area under the concentration-time curve (AUC of BH4 from time zero to the final concentration) compared to administration without food 0-last) results in an increase in the degree of exposure measured thereby. For example, the effective amount of sepiapterin is an amount sufficient (e.g., 2.5 mg / kg to 100 mg / kg per administration) to produce a BH4 concentration of at least 50 ng / ml (e.g., at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) in the plasma of the subject within 10 hours of administration with food. The effective amount is at least 5% less (e.g., at least 60 ng / ml, at least 100 ng / ml, at least 200 ng / ml, at least 400 ng / ml, at least 600 ng / ml, at least 1000 ng / ml, or at least 2000 ng / ml, or 50 ng / ml to 100 ng / ml, 60 ng / ml to 400 ng / ml, 200 ng / ml to 600 ng / ml, 400 ng / ml to 1000 ng / ml, or 600 ng / ml to 1500 ng / ml) than the dose sufficient to result in a maximum BH4 plasma concentration (Cmax) of at least 50 ng / ml in the plasma of the subject within 10 hours of administering sepiapterin without food.

[0152] In some embodiments of any of the methods described herein, the food is a high-protein food. In some embodiments of any of the methods described herein, the food is a high-fat food (e.g., at least 25, 30, 40, or 50% of the calories are from fat). In some embodiments of any of the methods described herein, the food is a high-protein and high-fat food. In some embodiments, the food is a high-calorie food (e.g., the food contains at least 100 calories, e.g., at least 200 calories, at least 300 calories, at least 400 calories, at least 500 calories, e.g., 500 - 1500 or 800 - 1000 calories). In some embodiments of any of the methods described herein, the food is a meal, e.g., breakfast, lunch, or dinner. Sepiapterin may be provided in a composition separate from the ingested food (e.g., sepiapterin is not incorporated into the food product).

[0153] Administration to the subject may be performed less than 30 minutes before ingesting the food, or after ingesting the food, e.g., from immediately before to 1 hour after ingesting the food, e.g., substantially simultaneously with the food. By administering with the food (e.g., performed less than 30 minutes before ingesting the food, or after ingesting the food, e.g., from immediately before to 1 hour after ingesting the food), compared to administration without the food (e.g., performed more than 2 hours after ingesting the food and up to 30 minutes before ingesting the food again), an increase in the Cmax of BH4 or the degree of BH4 production and the resulting plasma exposure (AUC 0-last ) (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%) can be brought about.

[0154] The actual dosage of the composition of the present invention to be administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, characteristics of the subject, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or number of administrations of an effective amount may vary according to the response of the subject. The physician responsible for administration shall determine, in any event, the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

[0155] In some embodiments, the subject is administered about 2.5 mg / kg to 120 mg / kg per administration (e.g., about 20 mg / kg to about 60 mg / kg, or about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg). The subject may be administered the pharmaceutical composition containing sepiapterin once, twice, or three times a day during treatment. In some embodiments, the subject continues other current treatments for high phenylalanine blood concentration (e.g., phenylalanine-restricted diet) except for BH4 supplementation (if taking BH4). In some embodiments, the subject may not be permitted to take drugs known to inhibit folic acid synthesis (e.g., methotrexate, pemetrexed, or trimethoprim). Sepiapterin or a pharmaceutically acceptable salt thereof may be administered in two equal doses (e.g., two doses at different times of the day), for example, two 60 mg / kg doses (e.g., one 60 mg / kg dose in the morning and one 60 mg / kg dose at night), two 40 mg / kg doses (e.g., one 40 mg / kg dose in the morning and one 40 mg / kg dose at night), two 30 mg / kg doses (e.g., one 30 mg / kg dose in the morning and one 30 mg / kg dose at night), two 20 mg / kg doses (e.g., one 20 mg / kg dose in the morning and one 20 mg / kg dose at night), or two 10 mg / kg doses (e.g., one 10 mg / kg dose in the morning and one 10 mg / kg dose at night).

[0156] In some embodiments, subjects taking BH4 discontinue BH4 administration (i.e., BH4 washout, e.g., before or simultaneously with the start of sepiapterin treatment). Blood samples for Phe concentration may be obtained during the BH4 washout period 7, 5, 3, and 1 days prior to treatment with the pharmaceutical composition of the invention, or at any time during the BH4 washout period when blood Phe concentration is > 360 μmol / L. In some embodiments, pre-dose blood samples are tested for sepiapterin, Phe, BH4, and tyrosine (Tyr).

[0157] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0158] Furthermore, it is understood that any particular embodiments of the invention that fall within the prior art may be explicitly excluded from any one or more of the claims. Such embodiments are considered to be known to those skilled in the art in the relevant art, and thus can be excluded even if the exclusion is not explicitly recited herein. Any particular embodiments of the compositions of the invention (e.g., any compound, any production method, any method of use, etc.) can be excluded from one or more claims for any reason, whether or not related to the existence of the prior art.

Examples

[0159] Example 1. Evaluation of the Food Effect during Sepiapterin Administration Methods Subjects were orally administered sepiapterin (10 mg / kg) twice, one week apart, in a fasting and fed state. Subjects were fed a standard high-fat (about 50% of the total calorie content of the diet) and high-calorie (about 800 - 1000 calories) diet starting 30 minutes before the second oral administration of sepiapterin on day 8.

[0160] Sampling for PK analysis was performed before dosing on days 1 and 8 (within 30 minutes before dosing) and at 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after dosing on days 1 and 8.

[0161] The blood concentrations of sepiapterin and BH4 were analyzed at MNG Labs.

[0162] The cerebrospinal fluid (CSF) of the selected subjects was collected by lumbar puncture approximately 30 minutes after the time of the maximum observed plasma BH4 concentration (Tmax) measured by blood analysis on day 1 (before dosing) and day 7 (i.e., after daily dosing for 7 days).

[0163] The cerebrospinal fluid (CSF) was analyzed at MNG Labs. Descriptive statistics are provided to characterize any changes in neurotransmitter metabolism between the sample results on days 1 and 7.

[0164] Results As shown in Tables 24 and 25 and Figure 1 below, surprisingly, the Cmax of BH4 in plasma was much higher in subjects who had eaten before dosing compared to subjects who had fasted before dosing. Furthermore, when sepiapterin was administered in the fed state versus the fasted state, a decrease in plasma sepiapterin concentration was seen, but an increase in BH4 concentration was seen (Figure 1). The geometric mean ratio (fasted / fed, 90% CI) of plasma sepiapterin concentration was 1.29 (0.84 - 2.00) for AUC last and 1.57 (1.21 - 2.0) for C max . The corresponding ratio (90% CI) of BH4 in plasma was 0.58 (0.47 - 0.71) for AUC 0-inf and 0.55 (0.45 - 0.68) for C max . The overall exposure of BH4 measured by AUC0- inf and AUC last increased 1.7-fold when sepiapterin was administered in the fed state compared to the fasted state.

[0165]

Table 24

[0166]

Table 25

[0167] Furthermore, as shown in Tables 26 and 27 below, surprisingly, the Cmax of sepiapterin in plasma was much lower in subjects who had eaten before administration compared to subjects who had fasted before administration.

[0168]

Table 26

[0169]

Table 27

[0170] Example 2. Comparison of Adverse Events between Fed and Fasting Subjects Method Single-dose administration of sepiapterin (10 mg / kg) was performed on 12 subjects under fasting conditions, and then administration was carried out under fed conditions 7 days later. The standard definition of adverse events (AE) was used. All-cause AEs occurred at any time, and treatment-emergent adverse events (TEAEs) occurred at the time of or after administration of the test drug. TEAEs related to the test drug were based on the investigator's opinion. Serious AEs were defined as life-threatening, death, hospitalization or prolongation of an existing hospitalization, or persistent or significant disability / incapacity, or substantial disruption of the ability to perform normal life functions, or congenital abnormality / birth defect.

[0171] Results As shown in Table 28, surprisingly, the incidence of adverse events decreased when sepiapterin was administered to fed subjects compared to fasting subjects.

[0172]

Table 28

[0173] Example 3. Pharmacokinetic analysis by multiple administrations of sepiapterin Method Three cohorts including eight fed subjects were randomly assigned at a 6:2 ratio to receive once-daily sepiapterin or placebo for 7 days. The sentinel dosing strategy was also used for the highest dose of sepiapterin administration.

[0174] Results The plasma time-concentrations of sepiapterin and BH4 were similar after 1 and 7 days of treatment with sepiapterin, and there was no drug accumulation. Pharmacokinetic data are shown in Table 29 below.

[0175] [Table 29]

[0176] Example 4. Decrease in blood phenylalanine concentration in PKU patients by sepiapterin administration After eligibility screening, a 7-day run-in period was initiated. Thereafter, patients were orally administered once-daily for 7 days either PTC923 at 20 mg / kg / day (“low dose”), or either PTC923 at 60 mg / kg / day or sapropterin dihydrochloride in random order. PTC923 (formerly CNSA-001) is oral sepiapterin. Each patient was randomly assigned to one of six treatment sequences (Figure 2). Randomization across six different treatment sequences was intended to minimize carry-over effects, treatment period effects, patient fatigue, and natural variation in blood Phe levels. A 7-day washout period was provided between each active treatment period. Patients were asked to maintain their pre-test normal diet, including intake of amino acid mixtures if prescribed, and were monitored by a dietitian during the study period.

[0177] Blood samples for Phe measurement were collected 7, 5, 3, and 1 days before randomization. During the treatment period, blood samples were taken before administration on day 1, and after administration on days 3, 5, and 7. During the washout period, blood samples for Phe measurement were taken on days 1, 3, 5, and 7. All samples were collected at the same time of day, either fasting or 3 hours after a meal. Phe in blood was measured at the Central Bioanalytical Laboratory (Agilex Biolabs Pty Ltd) using an effective LC-MS-MS technique for dried blood sampling by Volumetric Absorptive Microsampling (VAMS®) with Mitra® 4 sampler clam shell.

[0178] Twenty-four patients were randomized and all completed the study and were included in both the efficacy and safety populations. The mean age of the patients was 26 years and the mean weight was 69 kg (Table 30). Two-thirds of the patients were female (67%). All patients had no prior treatment history with sapropterin. Eleven of the 24 patients (46%) had classical PKU defined as any documented past measurement of blood Phe of at least 1,200 μmol / L. Supplements containing amino acids for PKU management were reported to have been received by 13 patients (54%) before the study and 14 patients (58%) during the study.

[0179]

Table 30

[0180] All patients entered the study on a low-protein, Phe-restricted diet and were instructed to keep their daily protein intake constant during the study. The mean [SD] daily dietary Phe intake was similar for the three groups at baseline (PTC923 60 mg / kg 2,550 [1,371] mg; PTC923 20 mg / kg 2,265

[946] mg; sapropterin dihydrochloride 20 mg / kg 2,510 [1,395] mg). The mean change in daily dietary Phe intake up to day 7 increased in all groups and was 19 [1,260] mg, 650 [1,262] mg, and 523 [1,132] mg, respectively.

[0181] The safety of PTC923 was monitored and adverse events (AEs) were coded using MedDRA® (Medical Dictionary for Regulatory Activities) version 21.0. Treatment-emergent adverse events (TEAEs) were defined as AEs that occurred at or after the first dose of the study drug. TEAEs were considered related to the current study treatment or the last study treatment before onset. Treatment-related TEAEs were those that were considered possibly, probably, or definitely related to the treatment at the investigator's discretion. Serious AEs (SAEs) were defined as death, life-threatening AEs, hospitalization or prolongation of an existing hospitalization, persistent or significant disability / incapacity, substantial disruption of the ability to perform normal life functions, or those resulting in congenital anomalies / birth defects.

[0182] Results PTC923 significantly reduced plasma Phe from baseline at all doses (Table 31). The 60 mg / kg dose of PTC923 had a significantly greater effect on reducing plasma Phe than the 20 mg / kg dose of sapropterin dihydrochloride (p = 0.0098). The effect of the 20 mg / kg dose of PTC923 on plasma Phe was numerically greater than that of the 20 mg / kg dose of sapropterin dihydrochloride. As shown in Figure 1, the low dose of PTC923 resulted in an average absolute decrease in plasma phenylalanine of approximately 100 μmol / L, and PTC923 resulted in an average absolute decrease in plasma phenylalanine of greater than 200 μmol / L.

[0183]

Table 31

[0184] The least squares mean (LSM) change (SE) from baseline plasma Phe levels was -206.4 (41.8) μmol / L (p < 0.0001) for PTC923 60 mg / kg, -146.9 (41.8) μmol / L (p = 0.0010) for PTC923 20 mg / kg, and -91.5 (41.7) μmol / L (p = 0.0339) for sapropterin dihydrochloride (Figure 3). The effect of PTC923 appeared to be dose-related.

[0185] The percentage of patients achieving plasma Phe < 360 μmol / L was 12 / 24 (50%) for patients randomized to PTC923 60 mg / kg, 11 / 24 (46%) for patients randomized to PTC923 20 mg / kg, and 10 / 24 (42%) for patients randomized to sapropterin dihydrochloride. The proportion of patients achieving plasma Phe < 360 μmol / L at any dose of PTC923 was 13 / 24 (54%).

[0186] Blood Phe decreased rapidly during the trial treatment. The least-squares mean change (SE; p-value) from the baseline of blood Phe to the first Phe measurement after the start of treatment (day 3) was -206.6 (36.6; p<0.0001) μmol / L for PTC923 60 mg / kg, -167.5 (36.6; p<0.0001) μmol / L for PTC923 20 mg / kg, and -72.3 (36.6; p=0.0543) μmol / L for sapropterin dihydrochloride. The mean change in blood Phe decrease was significantly greater for both PTC923 doses compared to sapropterin dihydrochloride on day 3 (LSM differences of -131.3 [SE 33.8; p=0.0007] and -95.2 [SE 33.7; p=0.0135] for PTC923 60 mg / kg and PTC923 20 mg / kg, respectively) (Figure 4).

[0187] As shown in Figure 5, PTC923 resulted in a superior decrease in blood phenylalanine concentration compared to sapropterin dihydrochloride in this trial.

[0188] The least-squares mean change (SE; p-value) in blood Phe for 11 classical PKU patients was -150.8 (63.1; p=0.0287) μmol / L for PTC923 60 mg / kg, -71.5 (61.8; p=0.2629) μmol / L for PTC923 20 mg / kg, and -2.8 (62.0; p=0.9640) μmol / L for sapropterin dihydrochloride (Table 31). The comparison of PTC923 60 mg / kg versus sapropterin dihydrochloride approached statistical significance (LSM difference was -148.0 [SE 63.0; p=0.0566] μmol / L) (Figure 6).

[0189] A sensitivity analysis excluding the treatment periods of patients with baseline blood Phe < 300 μmol / L was performed. The least-squares mean change in blood Phe from baseline (SE; p-value) in the sensitivity analysis population was -226.9 (44.2; p<0.0001) μmol / L for PTC923 at 60 mg / kg, -167.8 (45.2; p = 0.0007) μmol / L for PTC923 at 20 mg / kg, and -105.5 (43.7; p = 0.0211) μmol / L for sapropterin dihydrochloride at 20 mg / kg. The mean reduction in blood Phe was significantly greater for PTC923 at 60 mg / kg compared to sapropterin dihydrochloride (LSM difference was -121.4 [SE 42.9] μmol / L, p = 0.0146) (Figure 7).

[0190] A cofactor responder analysis was also performed in 19 patients with baseline Phe ≥ 300 μmol / L over all treatment periods. Twelve of the 19 patients (in any treatment group) showed a ≥ 20% reduction in blood Phe. The least-squares mean change in blood Phe from baseline (SE; p-value) for this group of 12 responders was -322.2 (60.0; p<0.0001) μmol / L for PTC923 at 60 mg / kg, -234.8 (61.2; p = 0.0011) μmol / L for PTC923 at 20 mg / kg, and -139.70 (58.6; p = 0.0277) μmol / L for sapropterin dihydrochloride. The mean change in the reduction of blood Phe was significantly greater for PTC923 at 60 mg / kg compared to sapropterin dihydrochloride (LSM difference was -182.5 [SE 62.0] μmol / L, p = 0.0158) (Figure 8). The number of patients treated with PTC923 showing a ≥ 20% reduction in blood Phe was more than 50% greater than the number of patients treated with sapropterin. The absolute mean change rate and absolute Phe reduction from baseline for patients treated with PTC923 at 60 mg / kg and showing at least a 30% reduction in Phe were -72.5% and -485.3 μmol / L, respectively.

[0191] In patients with similar numbers (PTC923 60 mg / kg / PTC923 20 mg / kg / saproptenil dihydrochloride), certain adverse events (AE; 29% / 25% / 21%), severe AEs (0% / 4% / 0%), and treatment-related AEs (13% / 0% / 0%) were reported. Headache was the most common all-cause AE (17% / 4% / 4%), and it was transient in all cases.

[0192] No other individual AEs occurred in ≥1 patient. Most AEs were mild in severity (19 / 22, 86% of all AEs). Two patients reported moderate-severe AEs (conjunctivitis, dysmenorrhea), and one patient reported a severe AE (gastroesophageal reflux). None of these three moderate-to-severe AEs were related to treatment. There were no SAEs, and no patients were discontinued due to AEs. Changes in vital signs and ECG parameters were generally small and comparable across treatment groups.

[0193] The primary objective of this study was to evaluate the efficacy of PTC923 as measured by the mean change in blood Phe from baseline. The efficacy population consisted of all patients who were randomized and had blood Phe measurements obtained at baseline and on days 3, 5, and 7 of treatment from at least one treatment period. The safety population consisted of all patients who were randomized and received any dose of the investigational treatment. The sample size calculation was based on a previous placebo-controlled, regulated trial of saproptenil with an observed mean decrease in blood Phe of 99 μmol / L and a standard deviation of 220 μmol / L, and estimated a true mean decrease in blood Phe of 300 μmol / L with 80% power using a two-sided α of 0.05.

[0194] The responsiveness to PTC923 was evaluated by measuring the mean change in blood Phe from baseline (calculated as the mean of the measurements on days 3, 5, and 7 of each treatment period). The pre-dose blood Phe concentration on day 1 of each treatment period served as the individual patient-specific baseline for that period.

[0195] The change from baseline in blood Phe during each treatment period was modeled using a linear mixed model (MMRM) for repeated measures. The efficacy model included fixed effects for baseline in each treatment period, treatment group, sequence, period, and a random subject effect within each sequence, and had a first-order autoregressive AR(1) covariance structure. No unplanned covariates were included.

[0196] Pairwise comparisons in the change from baseline in Phe were performed between each PTC923 treatment and sapropterin treatment by calculating the least-squares means using Dunnett's adjustment method for multiple comparisons. The estimated change amount, its standard error (SE), and the associated p-value of blood Phe values were calculated for each pairwise comparison. Dunnett's adjustment method was used to account for multiple comparisons of the test groups against the control group. This adjustment confirmed that the overall type I error rate under the null hypothesis was maintained even though multiple test groups were being compared to a single control group.

[0197] The overall mean change in blood Phe for each treatment was calculated and the standard deviation (SD) was determined.

[0198] In a predefined sensitivity analysis, treatment periods with baseline Phe < 300 μmol / L were excluded, and in a responder analysis, patients with baseline ≥ 300 μmol / L and a response of ≥ 20% decrease with sapropterin and / or PTC923 treatment were examined.

[0199] The data were analyzed using SAS v.9.4.

[0200] Summary Surprisingly, in a study involving only 24 subjects, PTC923 demonstrated statistically significant superiority in reducing blood phenylalanine levels compared to sapropterin dihydrochloride. Additionally, approximately 45% of classical PKU subjects responded to PTC923, achieving a reduction in phenylalanine levels of 20% or more. This is remarkable considering that historically, only about 10% of classical PKU subjects responded to sapropterin dihydrochloride treatment. Moreover, PTC923 showed an overall surprisingly high level of responders compared to sapropterin dihydrochloride. Compared to treatment with sapropterin dihydrochloride, 60% of subjects responded to PTC923, and three to four times as many subjects achieved normalization (i.e., blood phenylalanine levels on day 7 were less than 120 micromol / liter). In fact, subjects administered PTC923 were 13.45 times more likely to reach normal blood phenylalanine levels (i.e., less than 120 micromol / liter) compared to those administered sapropterin dihydrochloride. Notably, 46% of non-classical subjects treated with PTC923 achieved normalization, compared to 15.4% with sapropterin dihydrochloride. Improvements in sleep quality and concentration were also reported in subjects administered PTC923.

Claims

**Claim 1** A method for treating phenylketonuria in a subject having a blood phenylalanine concentration greater than 120 micromoles per liter, the method comprising administering to the subject an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof. **Claim 2** The method according to claim 1, wherein the subject is on a phenylalanine-restricted diet. **Claim 3** The method according to claim 1 or 2, wherein the subject has a blood phenylalanine concentration greater than 600 micromoles per liter. **Claim 4** The method according to any one of claims 1 to 3, wherein the subject is one who has not responded to treatment with sapropterin or a pharmaceutically acceptable salt thereof. **Claim 5** The method according to any one of claims 1 to 4, wherein the subject is one who has not responded to treatment with pegvaliase-pqpz. **Claim 6** The method according to any one of claims 1 to 5, wherein the subject is one who has discontinued treatment with pegvaliase-pqpz due to adverse reactions and / or intolerance. **Claim 7** The method according to any one of claims 1 to 6, wherein the blood phenylalanine concentration of the subject is reduced to less than 360 micromoles per liter by the administering step. **Claim 8** The method according to claim 7, wherein the blood phenylalanine concentration of the subject is reduced to less than 120 micromoles per liter by the administering step. **Claim 9** The method according to any one of claims 1 to 8, wherein the blood phenylalanine concentration of the subject is reduced by at least 35% compared to the blood phenylalanine concentration before administration of sepiapterin or a pharmaceutically acceptable salt thereof by the administering step. **Claim 10** The method according to any one of claims 1 to 9, wherein the blood phenylalanine concentration of the subject is reduced to less than 360 micromoles per liter with a phenylalanine intake of at least about 1500 mg / day by the administering step. **Claim 11** The method according to any one of claims 1 to 10, wherein a BH4 concentration of at least 50 ng / ml is produced in the plasma of the subject within 10 hours after administration of sepiapterin or a pharmaceutically acceptable salt thereof by the administering step. **Claim 12** The method according to any one of claims 1 to 11, wherein the effective amount of sepiapterin or a pharmaceutically acceptable salt thereof is about 20 mg / kg to about 60 mg / kg per administration. **Claim 13** The method according to any one of claims 1 to 12, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is about 20 mg / kg per administration.

14. The method according to any one of claims 1 to 12, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is about 40 mg / kg per administration.

15. The method according to any one of claims 1 to 12, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is about 60 mg / kg per administration.

16. The method according to any one of claims 1 to 15, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is administered once a day.

17. The method according to any one of claims 1 to 15, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is administered twice a day.

18. The method according to claim 17, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is administered in two equal doses.

19. The method according to any one of claims 1 to 18, wherein the effective amount of the sepiapterin or a pharmaceutically acceptable salt thereof is administered together with food.

20. The method according to claim 19, wherein the administration to the subject is performed less than 30 minutes before ingesting food or after ingesting food.

21. The method according to claim 19, wherein the administration to the subject is substantially simultaneous with food.

22. The method according to any one of claims 19 to 21, wherein the food is a high-protein and / or high-fat food.

23. The method according to any one of claims 19 to 22, wherein the food is a high-calorie food.

24. The method according to any one of claims 1 to 23, wherein the risk of adverse events is reduced as compared to a subject administered with at least 10 mg / kg of sapropterin or a pharmaceutically acceptable salt thereof.

25. The method according to any one of claims 1 to 24, wherein the step of administration results in an increase in the neurocognitive function of the subject.

26. The method according to any one of claims 1 to 25, wherein the subject is a child.

27. The method according to claim 26, wherein the child is less than 7 years old.

28. The method according to any one of claims 1 to 26, wherein the subject is over 7 years old.

29. The method according to any one of claims 1 to 28, wherein the subject is diagnosed with tetrahydrobiopterin-responsive phenylketonuria.

30. The method according to any one of claims 1 to 29, wherein the subject is diagnosed with sepiapterin-responsive phenylketonuria. **Claim 31** The method according to any one of claims 1 to 30, wherein the sepiapterin or a pharmaceutically acceptable salt thereof is formulated as an oral powder for suspension. **Claim 32** The method according to any one of claims 1 to 31, wherein the sepiapterin or a pharmaceutically acceptable salt thereof is administered as a suspension in a flavored suspension vehicle. **Claim 33** The method according to any one of claims 1 to 32, wherein the subject has a blood phenylalanine concentration greater than 1200 micromoles per liter.

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