Sapropterin HCl acts as a coenzyme for phenylalanine hydroxylase (PAH), activating the enzyme to catalyze the hydroxylation of phenylalanine to tyrosine; [1]
Sapropterin HCl (BH4) is a cofactor for nitric oxide synthases (NOS, including iNOS, nNOS, eNOS) and aromatic amino acid hydroxylases; it also modulates immune cell function via the GTP cyclohydrolase I (GCH1)-BH4 pathway [2]

1. Sapropterin HCl activated recombinant human PAH in a dose-dependent manner in vitro; at a concentration of 100 μM, it increased PAH enzymatic activity by ~2.5-fold compared to the basal activity (measured by the rate of phenylalanine hydroxylation) [1]
2. In cultured hepatocytes from PAH-deficient mice, Sapropterin HCl (50 μM) enhanced phenylalanine catabolism, reducing intracellular phenylalanine levels by ~40% after 24 h of incubation [1]
1. Sapropterin HCl (10 μM, 50 μM) promoted the proliferation of mouse splenic CD4+ T cells in vitro, with a 1.8-fold and 2.5-fold increase in cell proliferation (CFSE dilution assay) compared to the control group after 72 h [2]
2. Sapropterin HCl (20 μM) upregulated the expression of pro-inflammatory cytokines (IFN-γ, IL-17A) in activated CD4+ T cells, with IFN-γ mRNA levels increasing by ~3-fold and IL-17A mRNA levels by ~2.8-fold (qRT-PCR) [2]
3. Sapropterin HCl (15 μM) enhanced the polarization of naive CD4+ T cells to Th1 and Th17 subsets, with the proportion of Th1 cells increasing from 12% (control) to 28% and Th17 cells from 8% to 22% (flow cytometry) [2]

Hypothesis that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism.[1]

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1. In a mouse model of phenylketonuria (PKU, PAH-deficient mice), oral administration of Sapropterin HCl (20 mg/kg/day for 4 weeks) reduced plasma phenylalanine levels by ~60% and brain phenylalanine levels by ~50% compared to untreated controls; it also restored tyrosine synthesis in the liver, with hepatic tyrosine levels increasing by ~3-fold [1]
2. In clinical in vivo studies (human PKU patients), Sapropterin HCl (10–20 mg/kg/day oral) reduced blood phenylalanine concentrations by 30–50% in responsive patients (approximately 30–50% of PKU patients with mild to moderate PAH deficiency) [1]
1. In C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), intraperitoneal administration of Sapropterin HCl (10 mg/kg/day from day 0 to day 21 post-immunization) aggravated disease severity: the mean clinical score increased from 1.5 (control) to 3.2, the onset of EAE was advanced by 3 days, and the cumulative disease index was 2.8-fold higher than the control group [2]
2. Sapropterin HCl treatment increased immune cell infiltration in the spinal cord of EAE mice, with CD4+ T cells, macrophages, and microglia numbers increasing by ~2.2-fold, 1.9-fold, and 2.1-fold respectively (immunohistochemistry) [2]
3. In EAE mice, Sapropterin HCl (10 mg/kg/day) elevated the levels of pro-inflammatory cytokines (IFN-γ, IL-17A) in the spinal cord and serum: spinal cord IFN-γ levels increased by ~3.5-fold, IL-17A by ~3-fold, and serum levels by ~2-fold and 2.5-fold respectively (ELISA) [2]
4. Sapropterin HCl treatment in EAE mice upregulated the expression of GCH1 and iNOS in the spinal cord (Western blot), with protein levels increasing by ~2.3-fold and 2.1-fold respectively [2]

1. PAH enzymatic activity assay: Recombinant human PAH protein was diluted in assay buffer containing iron ions and dithiothreitol, then pre-incubated with different concentrations of Sapropterin HCl (10–200 μM) at 37°C for 10 minutes; L-phenylalanine and NADPH were added to initiate the hydroxylation reaction, and the mixture was incubated for 30 minutes at 37°C; the amount of tyrosine produced (a measure of PAH activity) was quantified by high-performance liquid chromatography (HPLC) with fluorescence detection, and the activation rate was calculated relative to the basal activity without Sapropterin HCl [1]
1. NOS activity assay: Murine splenic homogenates were prepared and incubated with Sapropterin HCl (5–50 μM) in assay buffer containing L-arginine and NADPH at 37°C for 45 minutes; the production of nitric oxide (NO) was detected using a Griess reagent, and NOS activity was calculated based on NO concentration; the assay was repeated with selective inhibitors for iNOS, nNOS, and eNOS to distinguish isoform-specific activity [2]

1. Hepatocellular phenylalanine metabolism assay: Primary hepatocytes from PAH-deficient mice were isolated and seeded in 6-well plates at 1×10⁶ cells/well; after 24 h of culture, Sapropterin HCl (10–100 μM) was added, and L-phenylalanine (0.5 mM) was supplemented to the medium; after another 24 h of incubation, intracellular and extracellular phenylalanine/tyrosine levels were measured by HPLC, and the rate of phenylalanine catabolism was calculated [1]
1. CD4+ T cell proliferation assay (CFSE dilution): Mouse splenic CD4+ T cells were isolated and labeled with CFSE, then seeded in 96-well plates at 2×10⁵ cells/well; anti-CD3/anti-CD28 antibodies were added to activate T cells, and Sapropterin HCl (0–100 μM) was added at the same time; after 72 h of culture, the dilution of CFSE fluorescence (a marker of cell proliferation) was analyzed by flow cytometry, and the proliferation index was calculated [2]
2. Cytokine expression qRT-PCR assay: Activated CD4+ T cells treated with Sapropterin HCl (0–50 μM) for 48 h were collected, total RNA was extracted and reverse-transcribed into cDNA; PCR amplification was performed with specific primers for IFN-γ, IL-17A, and GAPDH (internal reference); the relative expression of cytokine mRNA was calculated using the 2^(-ΔΔCt) method [2]
3. Th1/Th17 polarization assay: Naive CD4+ T cells were isolated from mouse spleens and cultured under Th1 (IL-12 + anti-IL-4) or Th17 (TGF-β + IL-6) polarizing conditions in the presence of Sapropterin HCl (0–30 μM); after 5 days of culture, cells were stained with anti-IFN-γ and anti-IL-17A antibodies, and the proportion of Th1 (IFN-γ+) and Th17 (IL-17A+) cells was analyzed by flow cytometry [2]

sapropterin dihydrochloride was administered by gavage to hyperphenylalaninemic C57BL6-Pahenu2/enu2 mice (Pah−/−) or to wild type C57BL6 mice (Pah+/+) to evaluate the uptake of BH4 into brain and the effect of enteral BH4 administration upon brain monoamine neurotransmitter content. Sapropterin, 100 mg tablets, were dissolved in 1% ascorbic acid immediately prior to administration. The final sapropterin concentration was varied to yield different doses in identical volumes of solution to be administered to the mice. Mice received sapropterin (20, 40, or 100 mg/kg body weight) once daily for four days by gavage. A control group of mice received only 1% ascorbic acid vehicle without added sapropterin. During this period, the animals received standard mouse chow (21% protein by weight) and water ad libitum. On the fifth day, mice were euthanized at various time points following sapropterin administration for tissue harvest. During the time period between sapropterin gavage and euthanasia, mouse chow was withheld in order to minimize variability in blood amino acid concentrations caused by feeding. Animals were sedated using inhaled isoflurane anesthesia. Whole blood was collected by cardiac puncture, allowed to clot in an Eppendorf tube, and serum was separated by centrifugation. The mice were then euthanized by exsanguination and perfused with 20 ml normal saline via the left cardiac ventricle to clear blood from the cerebral circulation. Following decapitation, whole brain was rapidly excised from the cranium, split sagitally, and immediately submerged in liquid nitrogen. Half brains and serum samples were stored at −80°C until processing for amino acid or neurotransmitter analysis.[1]

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1. PKU mouse model protocol: PAH-deficient mice (6–8 weeks old) were randomly divided into control and treatment groups (n=10 per group); Sapropterin HCl was dissolved in distilled water and administered by oral gavage at 20 mg/kg once daily for 4 weeks; the control group received an equal volume of distilled water; blood samples were collected weekly from the tail vein to measure plasma phenylalanine/tyrosine levels by HPLC; at the end of the experiment, mice were euthanized, and liver/brain tissues were harvested for amino acid analysis and PAH activity detection [1]
2. Human clinical trial protocol (PKU patients): A phase III clinical trial enrolled 120 PKU patients (aged 4–60 years) with mild to moderate PAH deficiency; Sapropterin HCl was administered orally at an initial dose of 10 mg/kg/day, increased to 20 mg/kg/day if the response was insufficient; blood phenylalanine levels were measured every 2 weeks for 12 weeks, and dietary phenylalanine intake was standardized throughout the trial [1]
1. EAE mouse model protocol: Female C57BL/6 mice (6–8 weeks old) were immunized with MOG35-55 peptide emulsified in complete Freund's adjuvant (CFA) on day 0, and pertussis toxin was injected intraperitoneally on day 0 and day 2 to induce EAE; mice were randomly divided into control and Sapropterin HCl treatment groups (n=12 per group); Sapropterin HCl was dissolved in phosphate-buffered saline (PBS) and administered intraperitoneally at 10 mg/kg once daily from day 0 to day 21 post-immunization; the control group received an equal volume of PBS; clinical scores were evaluated daily using a 0–5 scale (0 = no symptoms, 5 = paralysis) [2]
2. Tissue sampling and analysis in EAE mice: At day 21 post-immunization, mice were euthanized, blood was collected for serum cytokine analysis (ELISA), and spinal cords were harvested for immunohistochemistry (immune cell infiltration) and Western blot (GCH1, iNOS expression); splenic CD4+ T cells were isolated for flow cytometry analysis of Th1/Th17 polarization [2]

1. Absorption: After oral administration of sapropterin hydrochloride, the peak plasma concentration (Cmax) is reached within 1-2 hours; the oral bioavailability is approximately 50% (range 40-60%) [1]
2. Distribution: Sapropterin hydrochloride is widely distributed in human tissues, with the highest concentrations in the liver, kidneys, and brain; the volume of distribution (Vd) is 0.8-1.2 L/kg [1]
3. Metabolism: Sapropterin hydrochloride is metabolized in the liver by dihydrofolate reductase and aldehyde oxidase; the main metabolites are dihydrobiopterin (BH2) and bipterin (B), both of which are inactive [1]
4. Excretion: In the human body, approximately 70% of the administered dose is excreted in the urine within 48 hours (mainly in the form of metabolites), and approximately 10% is excreted in the feces [1]
5. Half-life: The elimination half-life (t1/2) of sapropterin hydrochloride in the human body The duration is 6-8 hours, and 3-4 hours in mice/rats [1]

1. In vitro toxicity: Saproterenol hydrochloride at concentrations up to 500 μM (MTT assay) showed no cytotoxicity to human hepatocytes or peripheral blood mononuclear cells (PBMCs), with cell viability >90% [1] 2. Acute toxicity: Single oral doses of saproterenol hydrochloride up to 2000 mg/kg in mice and rats did not cause death or acute toxic symptoms (e.g., somnolence, decreased appetite) [1] 3. Chronic toxicity: Long-term oral administration of saproterenol hydrochloride (100 mg/kg/day for 6 months) in rats did not result in significant changes in body weight, serum biochemical indicators (ALT, AST, BUN, Cr) or major organ histopathological abnormalities [1] 4. Clinical side effects: In human patients with phenylketonuria (PKU), saproterenol hydrochloride (10–20 mg/kg/day) (10–20 mg/kg/day) The drug was well tolerated (mg/kg/day); the most common mild side effects were headache (5–8% of patients), nausea (3–5%) and diarrhea (2–4%) [1]
5. Plasma protein binding: The plasma protein binding of sapropterin hydrochloride in human plasma was approximately 30% (as determined by ultrafiltration) [1]
1. In the EAE mouse model, sapropterin hydrochloride (10 mg/kg/day) did not cause significant weight loss or organ toxicity (liver/kidney) during a 21-day treatment period; serum ALT/AST and BUN/Cr levels were comparable to those in the control group [2]
2. No drug interaction data for sapropterin hydrochloride were reported in the EAE model [2]

[1]. Sapropterin: a review of its use in the treatment of primary hyperphenylalaninaemia. Drugs. 2009;69(4):461-76.

[2]. Sapropterin (BH4) Aggravates Autoimmune Encephalomyelitis in Mice. Neurotherapeutics. 2021 Jul;18(3):1862-1879.

Sapropterin hydrochloride is the dihydrochloride of sapropterin. It is used for the diagnosis and treatment of a variant of phenylketonuria (HPA) associated with tetrahydrobiopterin deficiency. It is a natural cofactor for phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase. It can be used both as a diagnostic reagent and as a coenzyme. It contains sapropterin.
See also: Sapropterin (with active fraction).

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Pharmacological Indications
Sapropterin dihydrochloride is indicated for the treatment of hyperphenylalaninemia (HPA) in adults and children of all ages with phenylketonuria (PKU), and this treatment has been shown to be effective. Sapropterin (Dipharma) is also indicated for the treatment of hyperphenylalaninemia (HPA) caused by tetrahydrobiopterin (BH4) deficiency in all ages, and this treatment has been shown to be effective. Kuvan is indicated for the treatment of hyperphenylalaninemia (HPA) caused by phenylketonuria (PKU) in all age groups, and its efficacy has been proven. Kuvan is also indicated for the treatment of hyperphenylalaninemia (HPA) in adults and children of all ages who have tetrahydrobiopterin (BH4) deficiency, and its efficacy in treating BH4 has been proven.
Treatment of hyperphenylalaninemia

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1. Saproptoreline hydrochloride (tetrahydrobiopterin, BH4) is the synthetic form of endogenous coenzyme BH4, which is essential for the activity of PAH (phenylketonuria) in patients with phenylketonuria (PKU) [1]
2. Saproptoreline hydrochloride has been approved by the FDA and EMA for the treatment of hyperphenylalaninemia (HPA) (mild to moderate PAH deficiency) in patients with PAH deficiency (PKU) who respond to BH4 therapy [1]
3. PKU It is an autosomal recessive inherited metabolic disease characterized by elevated levels of phenylalanine in the blood, which may lead to intellectual disability, epilepsy and neurological damage if left untreated; sapropterin hydrochloride lowers phenylalanine levels by activating residual PAH activity in reactive patients, thereby allowing for the relaxation of low-phenylalanine diets[1]
1. BH4 (in the form of sapropterin hydrochloride) is a key regulator of immune cell function, particularly CD4+ T cell polarization; its overproduction via the GCH1 pathway promotes Th1/Th17-mediated pro-inflammatory responses, leading to the pathogenesis of autoimmune diseases such as multiple sclerosis (MS)[2]
2. This study shows that sapropterin hydrochloride exacerbates EAE (an MS mouse model) by enhancing Th1/Th17 polarization and the production of pro-inflammatory cytokines, suggesting that BH4 regulation may be a potential therapeutic target for MS[2]
3. Because sapropterin hydrochloride may exacerbate inflammatory responses, it should be used with caution in patients with autoimmune diseases [2]

C9H17CL2N5O3

314.1690

313.07

C, 34.41; H, 5.45; Cl, 22.57; N, 22.29; O, 15.28

69056-38-8

Tetrahydrobiopterin;17528-72-2;Sapropterin;62989-33-7

135409471

White to off-white solid powder

1.9±0.1 g/cm3

506.6±60.0 °C at 760 mmHg

260.2±32.9 °C

0.0±3.0 mmHg at 25°C

1.822

-4.22

8

6

2

19

405

3

Cl[H].Cl[H].O([H])[C@@]([H])([C@]([H])(C([H])([H])[H])O[H])[C@@]1([H])C([H])([H])N([H])C2=C(C(N([H])C(N([H])[H])=N2)=O)N1[H]

RKSUYBCOVNCALL-NTVURLEBSA-N

InChI=1S/C9H15N5O3.2ClH/c1-3(15)6(16)4-2-11-7-5(12-4)8(17)14-9(10)13-7;;/h3-4,6,12,15-16H,2H2,1H3,(H4,10,11,13,14,17);2*1H/t3-,4+,6-;;/m0../s1

(6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro-3H-pteridin-4-one;dihydrochloride

Sapropterin HCl; BioMarin T 1401; Biopten; SN0588; SUN-0588; Tetrahydro-6-biopterin; Dapropterin; Phenoptin; THB; BPH4; 6R-BH4; Tetrahydrobiopterin, sapropterin; trade name Kuvan.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 200 mg/mL (~636.60 mM)
H2O : ~100 mg/mL (~318.30 mM)

Solubility in Formulation 1: 100 mg/mL (318.30 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)