IngredientsBiopterins are pterin derivatives that function as endogenous enzyme cofactors in many species of animals and in some bacteria and fungi. The prototypical compound of the class is biopterin, chemically designated as 6-(1,2-dihydroxypropyl)-pterin. More formally, biopterin is defined as the pteridine analogue in which the heterocyclic ring is substituted with amino, carbonyl oxygen, and 1,2-dihydroxypropyl at the 2, 4, and 6 positions, respectively. The compound belongs to the broader class of pteridines โ bicyclic molecules characterized by a pyrimidine ring fused to a pyrazine ring.
Compounds containing the pterin ring are called pteridines and include folates. Like folates, pterins exist in different states of reduction according to the number of hydrogen ions attached to the ring. The redox activity of biopterins is conferred by their heteroatomic rings, which can exist in several alternative oxidation and protonation states. Notably, the biopterin rings can be oxidized (biopterin), partially reduced (dihydrobiopterin, BH2), and fully reduced (tetrahydrobiopterin, BH4).
In humans, tetrahydrobiopterin (BH4) is the endogenous cofactor for aromatic amino acid hydroxylase (AAAH) enzymes. The fully oxidized form, simply called "biopterin," is the most stable form and the one most commonly detected in urine and biological fluids; the pharmacologically and biochemically active form is the fully reduced tetrahydrobiopterin (BH4), also known by its International Nonproprietary Name (INN) sapropterin. Chemically, BH4 is (6R)-5,6,7,8-tetrahydrobiopterin, a pteridine defined by its unique heterocyclic ring structure.
Pterins are compounds derived from guanosine triphosphate via a magnesium-dependent cyclohydrolase. They are found in all living organisms, from bacteria, blue-green algae, and trypanosomes to mammals; they have also been detected in the chloroplasts of spinach leaves and the cotyledons of pea sprouts.
The biological synthesis of pteridines was first discovered in 1889, when Sir Frederick Gowland Hopkins isolated the yellow pigments from the wings of English butterflies. The identity of these compounds was not established until the 1940s, when three compounds were isolated and shown to share a novel pyrimidine ring system. The name "pterin" was given to these compounds after the Greek word ptera, meaning wing, for the source from which these molecules were isolated.
A wide variety of pterins exist in bacteria, such as the various pterins with three-carbon side chains of Escherichia coli, the glucuronidated pterins of Bacillus subtilis, the three ribityl pteridines of the marine bioluminescent bacterium Photobacterium phosphoreum, the molybdopterins of photosynthetic bacteria, and the tetrahydromethanopterin of methanogenic bacteria.
The body can produce adequate amounts of biopterin in the absence of dietary intake; synthesis requires GTP, niacin, and magnesium. While many animal foods contain various bioactive forms of biopterin, the amounts have not been well investigated. Forms present in food include tetrahydrobiopterin, dihydrobiopterin, and neopterin. The quantities available from specific foods or typical intake levels are not yet well characterized.
Evidence has been presented that the rodent and human intestinal microbiota are capable of generating biopterin. Over the last two decades, both green sulfur bacteria and cyanobacteria have been postulated as BH4 producers based on the discovery of PTPS-2 orthologs in their genomes. Pteridines are involved in pigmentation, and thus the biopterin-generating ability of these bacteria may relate to photosynthetic requirements.
Data suggest that major contributors to the BH4 endogenous pool are breast milk early in life, followed by the intestinal microbiota as it becomes established into adulthood.
Tetrahydrobiopterin is available as a tablet for oral administration in the form of sapropterin dihydrochloride (BH4ยท2HCl). It is a cofactor of the three aromatic amino acid hydroxylase enzymes, used in the metabolism of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotonin, melatonin, dopamine, norepinephrine, epinephrine, and adrenaline, and is a cofactor for the production of nitric oxide (NO) by the nitric oxide synthases.
Sapropterin dihydrochloride, the active pharmaceutical ingredient, is a synthetic preparation of the dihydrochloride salt of naturally occurring tetrahydrobiopterin (BH4). Sapropterin dihydrochloride is a white to pale yellow powder. The pharmaceutical product is available both as tablets and as a powder for oral solution. Bacteria produce several unique glycosides of biopterin (and of other pterins as well), using a specific biopterin glucosyltransferase.
BH4 represents 65โ80% of total biopterins in plasma, and large variations in biopterin concentration from 6.9 nmol/L to 23.6 nmol/L have been observed, which have been attributed to BH4 lability. Despite the importance of BH4 in pathological conditions, studies of natural fluctuation of endogenous BH4 in humans have been rare, possibly because BH4 is subject to autoxidation under physiologic conditions.
Biopterin, as a chemically defined compound, is not associated with a traditional herbal or ethnobotanical history of use. Its identity as a distinct pteridine molecule was established only through 20th-century biochemistry. The biological synthesis of pteridines was first discovered in 1889, when Hopkins isolated the yellow pigments from butterfly wings, but the identity of these compounds was not established until the 1940s.
Tetrahydrobiopterin was discovered to play a role as an enzymatic cofactor. The first enzyme found to use tetrahydrobiopterin is phenylalanine hydroxylase (PAH). The clinical relevance of biopterin deficiency in human disease โ specifically in atypical phenylketonuria โ was recognized in the 1970s and 1980s, when it became clear that some forms of hyperphenylalaninemia were caused not by mutations in PAH itself but by defects in BH4 biosynthesis or recycling enzymes.
The first therapeutic applications of exogenous biopterin compounds emerged from inborn-error-of-metabolism clinics, where some ingested tetrahydrobiopterin was shown to be absorbed, as demonstrated by the rapid lowering of phenylalanine levels in a patient with defective biopterin synthesis. Clinical development of biopterin-based therapy for PKU accelerated through the 1990s and 2000s, culminating in regulatory approval. There is no documented ethnobotanical or traditional-medicine history of deliberate biopterin supplementation as a distinct practice.
The most significant pteridines include folic acid, biopterin, and their derivatives. Within the biopterin family, the principal members relevant to biochemistry and therapeutics are:
BH4 biosynthesis proceeds from guanosine triphosphate (GTP) via 7,8-dihydroneopterin triphosphate and 6-pyruvoyl-5,6,7,8-tetrahydropterin. The first and rate-limiting step in the pathway is GTP cyclohydrolase I (GTPCH). The following steps are catalyzed by the enzymes 6-pyruvoyl tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR).
BH4 biosynthesis involves up to eight different proteins supporting six alternate de novo and two alternate salvage pathways. Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin: (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (DHFR) activity.
Exogenous sepiapterin can be reduced in all cells by sepiapterin reductase to BH2, and further by dihydrofolate reductase (DHFR) to form BH4, the so-called "salvage pathway." The principal oxidant species leading to BH4 oxidation to BH2 is peroxynitrite.
Concentrations of BH4 are maintained by de novo synthesis and by a salvage mechanism that reduces quinoid dihydrobiopterin back to BH4. Two enzymes required in the regeneration of BH4 include pterin-4ฮฑ-carbinolamine dehydratase (PCBD1) and dihydropteridine reductase also known as quinoid dihydropteridine reductase (QDPR).
GTP cyclohydrolase I (GTPCH) is the first and rate-determining enzyme in the pathway for de novo synthesis of tetrahydrobiopterin, an essential cofactor that governs the activity of all three nitric-oxide synthases and the aromatic amino acid hydroxylases.
BH4 is a required cofactor for the aromatic amino acid hydroxylases (phenylalanine, tyrosine, and tryptophan hydroxylases), although for these enzymes it functions as a redox-active cofactor in a manner that is fundamentally different from its mechanism of use by nitric oxide synthases.
BH4 is the essential cofactor of enzymatic hydroxylation reactions mediated by phenylalanine hydroxylase, tyrosine hydroxylase (TH), and tryptophan hydroxylase. These convert phenylalanine to tyrosine, tyrosine to L-dopa, and tryptophan to 5-hydroxytryptophan, respectively. Downstream effects encompass the entire monoamine neurotransmitter system: within the brain, BH4 is absolutely required for the synthesis of dopamine, norepinephrine, epinephrine, and serotonin, as well as the gaseous neurotransmitter nitric oxide.
For each molecule of phenylalanine converted to tyrosine, one molecule of BH4 is converted to quinonoid-dihydropterin (q-BH2). Dihydropteridine reductase (DHPR) transfers hydrogen ions from NADPH to q-BH2 for the resynthesis of BH4, in which form most of the biopterins in the tissues exist.
BH4 is an obligate cofactor of nitric oxide (NO) synthases. It plays a redox role in the catalysis of NO formation from L-arginine, O2, and NADPH. In addition to its established effects on the NOS heme spin state, substrate affinity, and enzyme dimerization, BH4 is required as a one-electron donor to oxyferrous [Fe(II)ยทO2] heme that is formed as an intermediate in the catalytic cycle.
Nitric oxide synthase (NOS) exhibits two alternative activities depending on the relative availability of its cofactor BH4. In the presence of sufficient BH4, NOS catalyzes the production of NO and citrulline from the amino acid arginine. When the availability of BH4 is low, as it is in chronic inflammatory conditions and tumors, electron transfer in the active site of NOS becomes uncoupled from the oxidation of arginine. This results in the production of radical species that are capable of a direct attack on tetrahydrobiopterin, further depleting its local availability.
The pteridine cofactor BH4 has emerged as a critical determinant of eNOS activity: when BH4 availability is limiting, eNOS no longer produces NO but instead generates superoxide. BH4 is highly reductive and therefore easily oxidized to its non-productive metabolite 7,8-dihydrobiopterin (BH2) by free radicals such as superoxide or peroxynitrite.
The fully reduced biopterin BH4 is capable of both scavenging and generating superoxide radical. Thus, although BH4 is generally considered to be antioxidant, in some settings it can be pro-oxidant. It also has a protective role in the cell as an antioxidant and scavenger of reactive nitrogen and oxygen species.
The influence of BH4 on tyrosine hydroxylase (TH) protein levels is explained by an additional role for BH4 as a molecular chaperone that stabilizes both TH and phenylalanine hydroxylase (PAH) proteins. Free BH4 in solution is capable of reducing the catalytic iron in PAH and TH; however, structural studies show that BH4 bound at the active site does not directly interact with iron.
Background and mechanism: PKU is caused by an inherited deficiency of the enzyme phenylalanine hydroxylase (PAH), and the pathophysiology of the disorder is related to chronic accumulation of the free amino acid phenylalanine in tissues. Contemporary therapy has been based upon restriction of dietary protein intake, which leads to reduction of blood phenylalanine levels.
Regulatory status: In December 2007, the United States Food and Drug Administration approved sapropterin (Kuvanยฎ, BioMarin Pharmaceutical), a pharmaceutical form of tetrahydrobiopterin (BH4), for the treatment of BH4-responsive PKU. With the FDA and European Commission approvals of sapropterin dihydrochloride (Kuvanยฎ), a new era in PKU treatment began.
Clinical evidence: Sapropterin has been compared with placebo in one Phase 2 and one Phase 3 clinical trial, demonstrating significantly better response rates. Among individuals with hyperphenylalaninemia and some residual phenylalanine hydroxylase function, sapropterin can enhance activity of this enzyme to decrease serum phenylalanine concentrations.
Approximately 20% to 55% of patients โ primarily those with milder forms of PKU โ respond to treatment. Treatment can reduce blood phenylalanine levels by approximately 29% and increase dietary protein tolerance. All the people with PKU who respond positively to BH4 have at least some residual PAH enzyme activity. Sapropterin, by enhancing the activity of residual PAH enzyme, may increase tolerance to phenylalanine and allow a less restrictive diet.
Among 245 different genotypes, 40 were responsive to sapropterin, 26 were ambiguously responsive, and 179 were unresponsive.
Sepiapterin (next-generation BH4 precursor): Sepiapterin, a precursor of tetrahydrobiopterin (BH4), enhances enzyme activity and stability, offering therapeutic benefit even in patients unresponsive to BH4 alone. Results from the pivotal APHENITY Phase III trial demonstrated a substantial 63% mean reduction in blood phenylalanine and significant dietary liberalization, with 97% of participants able to increase natural protein intake safely. Importantly, 43% of prior non-responders to sapropterin showed clinical improvement.
Evidence strength: This is the strongest area of evidence for biopterin-family compounds. There are multiple Phase 2 and Phase 3 randomized controlled trials and regulatory approval in both the US and EU. The limitation is that the benefit is confined to the subset (~20โ55%) of PKU patients with BH4-responsive genotypes.
Mechanistic basis: Recent evidence supports potential cardiovascular benefits from BH4 replacement for the treatment of hypertension, ischemia-reperfusion injury, and cardiac hypertrophy with chamber remodeling. Such disorders exhibit BH4 depletion because of its oxidation and/or reduced synthesis, which can result in functional uncoupling of nitric oxide synthase (NOS).
Reduced vascular BH4 levels and eNOS uncoupling have been observed in patients with endothelial dysfunction resulting from hypertension and diabetes mellitus.
Systematic review evidence: A 2014 systematic review published in Evidence-Based Complementary and Alternative Medicine searched MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials. Fourteen articles were selected with a total of 370 patients. Ten of the fourteen studies showed a significant improvement in endothelial dysfunction of various cardiovascular disease groups with BH4 supplementation compared with control groups or placebos. Three studies showed no positive outcome, and one study showed that low-dose BH4 had no effect but that high-dose BH4 did have a significantly different result.
BH4 improves endothelial function in those who smoke, diabetic subjects, hypertensive subjects, patients with hypercholesterolemia, and those with coronary artery disease.
Limitations of cardiovascular evidence: Genetic mouse models of augmented endothelial cell BH4 synthesis have shown proof of concept that endothelial cell BH4 can alter cardiovascular disease pathogenesis, but clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation and limited endothelial cell uptake of BH4. Most studies have been short-term and small in sample size, and no large phase 3 cardiovascular outcome trials have been completed. The tendency of BH4 to be oxidized to BH2 in vivo limits its efficacy as a therapeutic agent in conditions of oxidative stress. Evidence in this area is preliminary to moderate in strength.
Mechanistic rationale: BH4 is an essential cofactor for several critical metabolic pathways that have been reported to be abnormal in autism spectrum disorder (ASD). In addition, the cerebrospinal fluid concentration of BH4 is reported to be depressed in children with ASD.
Clinical evidence: Over the past 25 years, several clinical trials have suggested that treatment with BH4 improves ASD symptomatology in some individuals. In an open-label study, children with ASD who had low concentrations of cerebrospinal fluid or urine pterins were treated with 20 mg/kg per day of BH4. The majority of children (63%) responded positively to treatment, with minimal adverse events.
In a randomized double-blind placebo-controlled trial, 46 children aged 3โ7 years diagnosed with an ASD were randomly assigned to double-blind treatment with 20 mg/kg/day BH4 or placebo for 16 weeks. These results indicated that BH4 offers promise in reducing symptoms of ASD.
Evidence strength: Evidence is preliminary. While several clinical trials suggest benefit in some children, sample sizes have been small, the mechanism of action in ASD is not fully elucidated, and the open-label design of many studies limits conclusions. Larger, adequately powered, double-blind trials are needed.
Other than PKU studies, tetrahydrobiopterin has participated in clinical trials studying other approaches to solving conditions resultant from a deficiency of tetrahydrobiopterin. These include autism, depression, ADHD, hypertension, endothelial dysfunction, and chronic kidney disease.
In psychiatry, tetrahydrobiopterin has been hypothesized to be involved in the pathophysiology of depression, although evidence is inconclusive to date. Regarding depression, the evidence in existing smaller studies is mixed and limited with contrasting BH4 treatment results.
A 2015 BioMarin-funded study of PKU patients found that those who responded to tetrahydrobiopterin also showed a reduction of ADHD symptoms.
Evidence strength: Evidence for neuropsychiatric conditions other than PKU-related neurotoxicity is very preliminary. Studies are small, often uncontrolled, and their results are mixed. No definitive clinical trials have established a therapeutic indication.
Multiple inborn errors of metabolism are closely related to the biosynthesis and regeneration of BH4; therefore, it serves as an important drug target to treat metabolic disorders such as phenylketonuria (PKU), primary tetrahydrobiopterin deficiencies (PBD), and tyrosine hydroxylase deficiency.
Genetic defects of biopterin synthesis or activation cause severe phenylketonuria (PKU) with neurological damage and weak muscle tonus. Primary BH4 deficiency may arise from genetic mutations in any of the biosynthesis enzymes (GTPCH, PTPS, SR) or recycling enzymes (DHPR, PCBD1), each presenting with distinct clinical profiles affecting neurotransmitter synthesis and phenylalanine metabolism.
Urinary pterins have been found as potential biomarkers in many pathophysiological conditions including inflammation, viral infections, and cancer. Activation of the human immune system during some pathological processes (e.g., cancer, viral infection, renal dysfunction) leads to significantly elevated levels of pterins excreted to urine.
Among the eight pteridines studied in one analysis, 6-biopterin, 6-hydroxymethylpterin, pterin, xanthopterin, and isoxanthopterin levels were significantly higher in samples from cancer patients than in those from healthy subjects. The authors concluded that some pteridine levels can be used as biomarkers for noninvasive diagnosis of cancer; however, more data is needed to support this hypothesis.
Recently, tetrahydrobiopterin metabolism has received increasing attention in the field of cancer immunology and immunotherapy due to its involvement in the cytotoxic T cell response. A feedforward loop may act like a molecular switch, reinforcing low tetrahydrobiopterin levels leading to altered NO signaling, restrained immune effector activity, and perpetual vascular inflammation.
Evidence strength: The use of biopterin and related pterins as cancer biomarkers is investigational. Findings are based on relatively small observational studies. There is no established clinical application for biopterin measurement in cancer diagnosis at this time.
Sapropterin dihydrochloride tablets are indicated to reduce blood phenylalanine (Phe) levels in adult and pediatric patients one month of age and older with hyperphenylalaninemia (HPA) due to tetrahydrobiopterin-responsive phenylketonuria (PKU). Sapropterin dihydrochloride tablets are to be used in conjunction with a Phe-restricted diet.
In PKU patients who are responsive to BH4 treatment, blood Phe levels decrease within 24 hours after a single administration of sapropterin dihydrochloride, although maximal effect on Phe level may take up to a month, depending on the patient. A single daily dose of sapropterin dihydrochloride is adequate to maintain stable blood Phe levels over a 24-hour period.
Based on available studies, sapropterin appears to be safe and well tolerated, with adverse event rates similar to those of placebo. No deaths were reported in any of the BioMarin-sponsored studies. Adverse events reported in all studies were generally graded mild; severe adverse events were infrequent. Incidence and frequency of adverse events had little or no correlation with study drug dose.
The most common adverse effects observed in more than 10% of people include headache and a running or obstructed nose. Diarrhea and vomiting are also relatively common, seen in at least 1% of people.
Children aged less than 7 years treated with 20 mg/kg/day are at increased risk for low levels of blood Phe compared with patients aged 7 years or older. The official prescribing information warns that prolonged levels of blood Phe that are too low are associated with catabolism and protein breakdown, requiring active management of dietary Phe intake while receiving therapy.
As a cofactor for nitric oxide synthase, tetrahydrobiopterin supplementation has shown beneficial results for the treatment of endothelial dysfunction in animal experiments and clinical trials, although the tendency of BH4 to become oxidized to BH2 remains a problem. This oxidation is relevant both to the stability of pharmaceutical preparations and to the in vivo efficacy of supplementation under conditions of high oxidative stress.
Inhibitors of folate synthesis (e.g., methotrexate, valproic acid, phenobarbital, trimethoprim) can decrease endogenous BH4 levels; blood Phe levels should be monitored more frequently and sapropterin dihydrochloride dosage adjusted as needed.
Specifically documented interactions include:
Although pregnancy outcome data are limited, sapropterin may be used in pregnant females with BH4-responsive phenylketonuria (PKU) in conjunction with a phenylalanine-restricted diet when appropriate. Data collection to monitor pregnancy and infant outcomes following exposure to sapropterin is ongoing.
Additional studies are warranted to assess the long-term safety and efficacy of sapropterin therapy. The risk from using supplemental biopterin at any level in non-PKU contexts is not well documented. Trimethoprim inhibits reactivation of oxidized biopterin; whether this causes deficiency symptoms in the general population is not known. The risk from using supplemental biopterin at any level is not well documented.
Health conditions that Biopterin may help support.
Body systems that Biopterin may help support.
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