Sapropterin Dihydrochloride Synthesis Essay

Clinical data
License data
  • US:C (Risk not ruled out)
Routes of
By mouth
ATC code
Legal status
Legal status
Pharmacokinetic data
Biological half-life4 hours (healthy adults)
6–7 hours (PKU patients)

IUPAC name

  • (6R)-2-Amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydropteridin-4(1H)-one
CAS Number
Chemical and physical data
Molar mass241.25 g/mol
3D model (JSmol)


  • O=C2\N=C(/NC=1NC[C@@H](NC=12)[C@@H](O)[C@@H](O)C)N


  • InChI=1S/C9H15N5O3/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)/t3-,4+,6-/m0/s1 Y
 NY (what is this?)  (verify)

Tetrahydrobiopterin (BH4, THB), also known as sapropterin, is a naturally occurring essential cofactor of the three aromatic amino acid hydroxylase enzymes, used in the degradation of amino acid phenylalanine and in the biosynthesis of the neurotransmittersserotonin (5-hydroxytryptamine, 5-HT), melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), and is a cofactor for the production of nitric oxide (NO) by the nitric oxide synthases.[1] Chemically, its structure is that of a reduced pteridine derivative.

Medical use[edit]

Tetrahydrobiopterin is available as a tablet for oral administration in the form of tetrahydrobiopterin dihydrochloride (BH4*2HCL).[2] BH4*2HCL is FDA approved under the trade name Kuvan. The typical cost of treating a patient with Kuvan is $100,000 per year.[3]BioMarin holds the patent for Kuvan until at least 2024, but Par Pharmaceutical has a right to produce a generic version by 2020.[4]

BH4*2HCL is indicated in tetrahydrobiopterin deficiency caused by GTPCH deficiency or PTPS deficiency.[5] Also, BH4*2HCL is FDA approved for use in phenylketonuria, along with dietary measures.[6] Most people with phenylketonuria, however, have little or no benefit of BH4*2HCL.[7]

Adverse effects[edit]

The most common adverse effects, observed in more than 10% of patients, include headache and a running or obstructed nose. Diarrhea and vomiting are also relatively common, seen in at least 1% of patients.[8]


No interaction studies have been conducted. Because of its mechanism, tetrahydrobiopterin might interact with dihydrofolate reductase inhibitors like methotrexate and trimethoprim, and NO-enhancing drugs like nitroglycerin, molsidomine, minoxidil, and PDE5 inhibitors. Combination of tetrahydrobiopterin with levodopa can lead to increased excitability.[8]


Tetrahydrobiopterin has the following responsibilities as a cofactor:

Tetrahydrobiopterin has multiple roles in human biochemistry. One is to convert amino acids such as phenylalanine, tyrosine, and tryptophan to precursors of dopamine and serotonin, major monoamine neurotransmitters. Due to its role in the conversion of L-tyrosine into L-dopa, which is the precursor for dopamine, a deficiency in tetrahydrobiopterin can cause severe neurological issues unrelated to a toxic buildup of L-phenylalanine; dopamine is a vital neurotransmitter, and is the precursor of norepinephrine and epinephrine. Thus, a deficiency of BH4 can lead to systemic deficiencies of dopamine, norepinephrine, and epinephrine. In fact, one of the primary conditions that can result from GTPCH-related BH4 deficiency is dopamine-responsive dystonia;[9] currently, this condition is typically treated with carbidopa/levodopa, which directly restores dopamine levels within the brain.

BH4 also serves as a catalyst for the production of nitric oxide. Among other things, nitric oxide is involved in vasodilation, which improves systematic blood flow. The role of BH4 in this enzymatic process is so critical that some research points to a deficiency of BH4 – and thus, of nitric oxide – as being a core cause of the neurovascular dysfunction that is the hallmark of circulation-related diseases such as diabetes.[10]


Tetrahydrobiopterin was discovered to play a role as an enzymatic cofactor. The first enzyme found to use tetrahydrobiopterin is phenylalanine hydroxylase (PAH).[11]

Biosynthesis and recycling[edit]

Tetrahydrobiopterin is biosynthesized from guanosine triphosphate (GTP) by three chemical reactions mediated by the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR).[12]

BH4 is catabolized to BH3 and BH2, which are pro-oxidants (free radicals). Research shows that ascorbic acid (also known as ascorbate or vitamin C) is effective at recycling BH3 into BH4,[13] preventing the BH3 radical from reacting with other free radicals (superoxide and peroxynitrite specifically). Without this recycling process, uncoupling of the endothelial nitric oxide synthase (eNOS) enzyme and reduced bioavailability of the vasodilatornitric oxide occur, creating a form of endothelial dysfunction.[14] Ascorbic acid is oxidized to dehydroascorbic acid during this process, although it can be recycled back to ascorbic acid.


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, ADHD, hypertension, endothelial dysfunction, and chronic kidney disease.[15][16] Experimental studies suggest that tetrahydrobiopterin regulates deficient production of nitric oxide in cardiovascular disease states, and contributes to the response to inflammation and injury, for example in pain due to nerve injury. A 2015 BioMarin-funded study of PKU patients found that those who responded to tetrahydrobiopterin also showed a reduction of ADHD symptoms.[17]


In 1997, a small pilot study was published on the efficacy of tetrahydrobiopterin (BH4) on relieving the symptoms of autism, which concluded that it "might be useful for a subgroup of children with autism" and that double-blind trials are needed, as are trials which measure outcomes over a longer period of time.[18] In 2010, Frye et al. published a paper which concluded that it was safe, and also noted that "several clinical trials have suggested that treatment with BH4 improves ASD symptomatology in some individuals."[19]

Cardiovascular disease[edit]

Since nitric oxide production is important in regulation of blood pressure and blood flow, thereby playing a significant role in cardiovascular diseases, tetrahydrobiopterin is a potential therapeutic target. In the endothelial cell lining of blood vessels, endothelial nitric oxide synthase is dependent on tetrahydrobiopterin availability.[20] Increasing tetrahydrobiopterin in endothelial cells by augmenting the levels of the biosynthetic enzyme GTPCH can maintain endothelial nitric oxide synthase function in experimental models of disease states such as diabetes,[21] atherosclerosis, and hypoxic pulmonary hypertension.[22] However, treatment of patients with existing coronary artery disease with oral tetrahydrobiopterin is limited by oxidation of tetrahydrobiopterin to the inactive form, dihydrobiopterin, with little benefit on vascular function.[23]

See also[edit]


External links[edit]

  1. ^Całka, Jarosław (2006). "The role of nitric oxide in the hypothalamic control of LHRH and oxytocin release, sexual behavior and aging of the LHRH and oxytocin neurons". Folia Histochemica et Cytobiologica. 44 (1): 3–12. PMID 16584085. 
  2. ^Schaub J, Däumling S, Curtius HC, Niederwieser A, Bartholomé K, Viscontini M, Schircks B, Bieri JH (1978). "Tetrahydrobiopterin therapy of atypical phenylketonuria due to defective dihydrobiopterin biosynthesis". Arch. Dis. Child. 53 (8): 674–6. doi:10.1136/adc.53.8.674. PMC 1545051. PMID 708106. 
  3. ^Matthew Herper (2016-07-28). "How Focusing On Obscure Diseases Made BioMarin A $15 Billion Company". Forbes. Retrieved 2017-10-09. 
  4. ^"BioMarin Announces Kuvan (sapropterin dihydrochloride) Patent Challenge Settlement". PR Newswire. 2017-04-13. Retrieved 2017-10-09. 
  5. ^"Tetrahydrobiopterin Deficiency". National Organization for Rare Disorders (NORD). Retrieved 2017-10-09. 
  6. ^"What are common treatments for phenylketonuria (PKU)?". NICHD. 2013-08-23. Retrieved 12 September 2016. 
  7. ^Camp, Kathryn M.; Parisi, Melissa A.; Acosta, Phyllis B.; Berry, Gerard T.; Bilder, Deborah A.; Blau, Nenad; Bodamer, Olaf A.; Brosco, Jeffrey P.; Brown, Christine S.; Burlina, Alberto B.; Burton, Barbara K.; Chang, Christine S.; Coates, Paul M.; Cunningham, Amy C.; Dobrowolski, Steven F.; Ferguson, John H.; Franklin, Thomas D.; Frazier, Dianne M.; Grange, Dorothy K.; Greene, Carol L.; Groft, Stephen C.; Harding, Cary O.; Howell, R. Rodney; Huntington, Kathleen L.; Hyatt-Knorr, Henrietta D.; Jevaji, Indira P.; Levy, Harvey L.; Lichter-Konecki, Uta; Lindegren, Mary Lou; et al. (2014). "Phenylketonuria Scientific Review Conference: State of the science and future research needs". Molecular Genetics and Metabolism. 112 (2): 87–122. doi:10.1016/j.ymgme.2014.02.013. PMID 24667081. 
  8. ^ abHaberfeld, H, ed. (1 March 2017). Austria-Codex (in German). Vienna: Österreichischer Apothekerverlag. Kuvan 100 mg-Tabletten. 
  9. ^"Genetics Home Reference: GCH1". National Institutes of Health. 
  10. ^Wu, Guoyao; Meininger, Cynthia J. (2009). "Nitric oxide and vascular insulin resistance". BioFactors. 35 (1): 21–7. doi:10.1002/biof.3. PMID 19319842. 
  11. ^Kaufman, S (1958). "A new cofactor required for the enzymatic conversion of phenylalanine to tyrosine". The Journal of Biological Chemistry. 230 (2): 931–9. PMID 13525410. 
  12. ^Thöny, Beat; Auerbach, Günter; Blau, Nenad (2000). "Tetrahydrobiopterin biosynthesis, regeneration and functions". Biochemical Journal. 347: 1–16. doi:10.1042/0264-6021:3470001. PMC 1220924. PMID 10727395. 
  13. ^Kuzkaya, N.; Weissmann, N.; Harrison, D. G.; Dikalov, S. (2003). "Interactions of Peroxynitrite, Tetrahydrobiopterin, Ascorbic Acid, and Thiols: Implications For Uncoupling Endothelial Nitric-Oxide Synthase". Journal of Biological Chemistry. 278 (25): 22546–54. doi:10.1074/jbc.M302227200. PMID 12692136. 
  14. ^Muller-Delp, J. M. (2009). "Ascorbic acid and tetrahydrobiopterin: looking beyond nitric oxide bioavailability". Cardiovascular Research. 84 (2): 178–9. doi:10.1093/cvr/cvp307. PMID 19744948. 
  15. ^ Search results for Kuvan
  16. ^"BioMarin Initiates Phase 3b Study to Evaluate the Effects of Kuvan on Neurophychiatric Symptoms in Subjects with PKU". BioMarin Pharmaceutical Inc. 17 August 2010. 
  17. ^Burton, B.; Grant, M.; Feigenbaum, A.; Singh, R.; Hendren, R.; Siriwardena, K.; Phillips, J.; Sanchez-Valle, A.; Waisbren, S.; Gillis, J.; Prasad, S.; Merilainen, M.; Lang, W.; Zhang, C.; Yu, S.; Stahl, S. (2015). "A randomized, placebo-controlled, double-blind study of sapropterin to treat ADHD symptoms and executive function impairment in children and adults with sapropterin-responsive phenylketonuria". Molecular Genetics and Metabolism. 114 (3): 415–24. doi:10.1016/j.ymgme.2014.11.011. PMID 25533024. 
  18. ^Fernell, Elisabeth; Watanabe, Yasuyoshi; Adolfsson, Ingrid; Tani, Yoshihiro; Bergström, Mats; Phd, Per Hartvig; Md, Anders Lilja; Phd., Anne-Liis von Knorring MD.; Phd., Christopher Gillberg MD.; Phd., Bengt Lángström (2008). "Possible effects of tetrahydrobiopterin treatment in six children with autism - clinical and positron emission tomography data: A pilot study". Developmental Medicine & Child Neurology. 39 (5): 313–8. doi:10.1111/j.1469-8749.1997.tb07437.x. PMID 9236697. 
  19. ^Frye, Richard E.; Huffman, Lynne C.; Elliott, Glen R. (2010). "Tetrahydrobiopterin as a novel therapeutic intervention for autism". Neurotherapeutics. 7 (3): 241–9. doi:10.1016/j.nurt.2010.05.004. PMC 2908599. PMID 20643376. 
  20. ^Channon, Keithm. (2004). "Tetrahydrobiopterin". Trends in Cardiovascular Medicine. 14 (8): 323–7. doi:10.1016/j.tcm.2004.10.003. PMID 15596110. 
  21. ^Alp, Nicholas J.; Mussa, Shafi; Khoo, Jeffrey; Cai, Shijie; Guzik, Tomasz; Jefferson, Andrew; Goh, Nicky; Rockett, Kirk A.; Channon, Keith M. (2003). "Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression". Journal of Clinical Investigation. 112 (5): 725–35. doi:10.1172/JCI17786. PMC 182196. PMID 12952921. 
  22. ^Khoo, J. P.; Zhao, L; Alp, N. J.; Bendall, J. K.; Nicoli, T; Rockett, K; Wilkins, M. R.; Channon, K. M. (2005). "Pivotal Role for Endothelial Tetrahydrobiopterin in Pulmonary Hypertension". Circulation. 111 (16): 2126–33. doi:10.1161/01.CIR.0000162470.26840.89. PMID 15824200. 
  23. ^Cunnington, C.; Van Assche, T.; Shirodaria, C.; Kylintireas, I.; Lindsay, A. C.; Lee, J. M.; Antoniades, C.; Margaritis, M.; Lee, R.; Cerrato, R.; Crabtree, M. J.; Francis, J. M.; Sayeed, R.; Ratnatunga, C.; Pillai, R.; Choudhury, R. P.; Neubauer, S.; Channon, K. M. (2012). "Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients with Coronary Artery Disease". Circulation. 125 (11): 1356–66. doi:10.1161/CIRCULATIONAHA.111.038919. PMC 5238935. PMID 22315282. 

1. Introduction

Phenylketonuria (PKU OMIM 261600) is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. It results in the accumulation of phenylalanine (Phe), an essential amino acid mainly metabolized in the liver by the phe hydroxylase (PAH) system. This enzyme hydroxylates Phe to tyrosine (Figure 1) requiring tetrahydrobiopterin (BH4) as a co-factor. Defects in either PAH or the production or recycling of BH4 may result in hyperphenilalaninemia which can cause intellectual disability if untreated [1].

Figure 1. Metabolic pathway of phenylalanine.

The natural history of the phenylketonuria consists in a progressive irreversible neurological impairment during infancy and childhood. The most common outcome is severe mental retardation often associated with a “mousy” odor, eczema and reduced hair skin and iris pigmentation; also reduced growth, microcephaly and neurological signs as tremor, epilepsy are present. All untreated patients have behavioral problems as hyperactivity, stereotypy and anxiety. The severity of the clinical phenotype directly correlates with blood phenylalanine levels that reflect the degree of enzymatic deficiency.

Neonatal screening by measuring Phe levels in blood spots on filter paper can be identify affected infants at birth. Early treated PKU patients have normal intellectual quotients (I.Q.), but can have an I.Q. gap when compared to their non-PKU siblings [2]. In addition, patients with PKU frequently have lower scores for certain neuropsychological functions, with executive function being the most affected area [3].

The prevalence of PKU varies by country ranging from between one in 10,000 and one in 20,000 births in U.S.A. and Europe [4]. Depending on the genotype and severity of the enzyme defect, various forms of PKU with different clinical outcomes have been described [5]. These can be classified on the basis of blood Phe levels at diagnosis and dietary Phe tolerance. Until recently, a strict low-Phe diet was the only therapy available. The PKU diet consists of are striction of natural proteins in the diet and supplementation with special medical formulas that supply vitamins, minerals, and all essential amino acids except Phe. Dietary treatment has been very effective in the prevention of impaired cognitive development, but still has its shortcomings. Growth delay and specific deficiencies of calcium, zinc, selenium, iron, and vitamin B12 were reported with the early formulas [6,7,8]. The PKU diet is also cumbersome for the patients and their families, often leads to a lack of compliance starting in childhood. For these reasons, new medical formulas with improved nutritional quality and palatability have been developed to lower the volume of food and to improve compliance.

This review discusses the evolution of dietary treatment leading to improved nutritional management using an increased variety of PKU formulas, some of which containing glycomacropeptide, the introduction of new formulations of Large Neutral Amino Acid (LNAA) supplementation and pharmacological therapy with tretahydrobiopterin cofactors and enzyme substitution therapy.

The review was based on literature search on PUBMED for articles between 2000 and 2014 with the term “phenylketonuria” and “PKU” in combination with the following terms “diet”, “enzyme therapy”, “gene therapy”, “glycomacropeptide”, “large neutral amino acids”, “sapropterin”, “tetrahydrobiopterin” and “chaperones”. Only publications in English were taken in consideration.

2. Dietary Treatment

Treatment of phenylketonuria is still not optimal, and so novel alternative formulas have been sought. New dietary therapies include more palatable formulas with improved caloric content allowing better compliance [9]. Generally the diet has inadequate amounts of taurine and other micronutrients derived from animal products. In addition, the diet is low in long-chain poly unsatured fatty acids (LC-PUFAs) such arachidonic acid (AA) and docosahexaenoic-acid (DHH). Many formulas are now supplemented with long-chain poly unsatured fatty acids after studies have shown that LC-PUFAs improve the maturation of the visual system and motor skills in patients with PKU [10,11]. Synthetic amino acids are the primary protein source in the nutritional management of PKU. However, di-tripeptides are more effectively absorbed from the intestine. Glycomacropeptide (GMP) is a naturally occurring protein derived from cheese whey, which is low in phenylalanine and an excellent source of protein for PKU patients. GMP improves the taste, variety, satiety [12], and convenience of the diet with consequent improved dietary compliance, metaboli ccontrol, and finally a better quality of life for patients [13]. GMP is an intact protein source and, compared to free amino acids in standard formulas, improves protein retention and phenylalanine utilization [13]. One reason for non-compliance is the financial burden that many families face with formula and low protein foods because in many countries, as in the USA, not all insurance companies are required to pay for formula or cover low protein foods. Recent efforts to improve adherence to dietary treatment by restricting dietary protein have shown to be an alternative way of maintaining metabolic control in PKU [14]. Overall, restriction of the intake of natural protein with supplementation of a Phe-free medical formula remains the cornerstone of PKU management and is being helped by the developments of a larger variety of medical foods, some of which made with GMP, a low-phe whey protein.

Dietary treatment is monitored by frequent measurement of plasma/blood phenylalanine levels. A portable Phe monitoring device could improve dietary adherence by providing Phe levels in real time and, thereby, provide immediate motivation to patients with PKU [15]. Unfortunately, nobody has developed such a device up to this point in time. Iontophoretic extraction of Phe from the skin correlated with plasma levels of Phe above 300 µmol/L. but was not sufficiently sensitive to detect low phenylalanine levels [16].

3. Large Neutral Amino Acid Supplementation

Other novel therapeutic approaches can be categorized by the site of action or target organ (Figure 2) [17]. These categories include enteral, systemic, liver-directed approaches. Dietary restriction of Phe intake is an example of enteral approach. Alternatively Large Neutral Amino Acid (LNAA) can be used. LNAA can compete with the same transporter of Phe across the gastrointestinal and blood brain barrier to reduce Phe absorption and entry into the brain [18]. A double blind, placebo-controlled study indicated a significant decline in blood Phe concentration in patients with PKU treated with LNAA for 2 weeks suggesting that LNAA compete with the transport of Phe in the gastrointestinal trac [19]. These studies suggest that adding LNAA to the diet of patients with PKU could reduce blood Phe concentrations.

Figure 2. Potential therapeutic approaches for phenylketonuria (PKU) (modified from Harding, C.O., et al.)

Oral LNAA supplementation can reduce brain Phe concentrations and improve neuropsychological functioning [20]. Differences in outcome may be related to composition, dosing route of administration, and duration of the supplementation period. Reduced brain LNAA concentrations have been reported in PKU mice [21], and restoring levels of large neutral amino acids in the brains may improve cognitive outcome in PKU. Hoeksma et al [22] demonstrated a significant negative correlation between plasma Phe concentration and cerebral protein synthesis in patients with PKU. This leads to development of new medical foods with higher concentrations of LNAA and fortification with vitamins and lutein, an antioxidant important for the development of the brain [23]. Studies in PAH enu2 mice provide support for the use of a variety of non-physiological amino acids to act as competitive inhibitors of brain transporters to reduce brain Phe concentrations with minimal impact on other down-stream intermediates [24].

The evidence to support the efficacy of LNAA supplementation to significantly reduce blood Phe levels in patients with PKU is still limited. The effects of LNAAs have been assessed only for short times and in a limited number of patients using variable dosages (250–1000 mg LNAA/Kg/day) and different formulations of LNAA [25].Patients with Phe levels above 1000 µmol/L had a~40% of decrease in plasma Phe levels [25]. One randomized controlled study reported a positive effect on executive functions [26].

In summary, LNAA supplementation either alone or in combination with a low-Phe diet has been shown to improve health outcome for individuals unable to follow the low Phe diet. However long term outcome studies assessing efficacy and safety of LNAA supplementation are needed.

4. Tetrahydropterin as Enzyme Enhancement Therapy for PKU

Some patients with PKU respond to pharmacological doses of tetrahydropterin (BH4) with reduced blood Phe levels as first shown in 1999 by Kure et al. [27]. At pharmacological doses, sapropterin hydrochloride acts as a molecular chaperone that promotes correct folding and stability of the PAH enzyme [28]. The recommendations on how to test patients with hyperphenylalaninemia for BH4 responsiveness are evolving [29,30,31]. All patients with Phe levels>360 µmol/L should be tested for responsiveness to sapropterin (20 mg/Kg/day). Multiple Phe levels need to be obtained at baseline and after starting BH4 to account for normal physiological variation sin Phe levels. The effect of BHA is evaluated after ashort-term (up to 48 h) [32] and long-term (up to several weeks) [33] to demonstrate consistent reduction of Phe levels as compared to baseline. A decrease in blood Phe of 30% or more from baseline indicates response to sapropterin therapy [34]. Patients with milder phenotype generally are responsive [35]. Long-term treatment with sapropterin of responsive patients with PKU improves Phe tolerance and, in some cases, allows them to discontinue restrictive diets [36,37,38].

In summary, treatment with sapropterin resulted in significant (at least 30%) and sustained reductions in blood Phe concentrations and increased dietary Phe tolerance in responsive PKU patients. This has been tested in adults and children with phenylketonuria (Long-term developmental progression in infants and young children taking sapropterin for phenylketonuria: a two-year analysis of safety and efficacy [39]. Successful treatment with sapropterin may lead to a relaxation of a Phe restricted diet, although continued monitoring of blood Phe is advisable [40].

The use of pharmacological chaperones to stabilize or promote correct folding of mutant proteins represents a promising new approach in the treatment of many genetic diseases causing protein misfolding. Proteins and small molecules in addition to tetrahydrobiopterin may act as chaperones to assist in the folding of PAH. Pey et al. [41] performed a high-throughput ligand screening of over 1000 pharmacological agents and identified four compounds that enhanced the stability of PAH activity. In particular, the administration of low doses of two of these compounds increased PAH activity in mouse liver. Further studies are necessary before these compounds can be used in clinical practice.

5. Enzyme Therapy

Enzyme therapy for PKU is another option whereby the harmful increased levels of Phe can be reduced by the introduction of Phe-metabolizing enzymes changing the metabolic phenotype of PKU, regardless of genotype.

Enzyme therapy can be done either by enzyme replacement with PAH or by enzyme substitution with phenylalanine ammonia-lyase [42]. The metabolism of Phe takes place for the most part in the liver, and orthotropic liver transplantation corrects the metabolic phenotype [43]. Liver transplantation is not a treatment option except for unusual PKU patients who need a liver transplant for another disease such as cirrhosis, because of the burden of daily therapy in transplanted patients. In mice, enzyme replacement with PAH-fusion proteins is a promising approach [44].

Enzyme substitution therapy with phenylalanine ammonia-lyase (PAL) appears more promising. It can act as a surrogate for the deficient PAH and converts the excess systemic Phe to trans-cinnamic acid and ammonia. Both pharmacological and physiological principles of therapy have been demonstrated following the use of PAL given orally or by injection in PKU mouse models [45]. The oral route is complicated by proteolytic degradation of the enzyme, which, however, remains active within the gut. Injected PAL is immunogenic and can cause reactions. The conjugation with polyethylene glycol of PAL (PEG-PAL) has been successful to decrease the immune response [46].

Clinical trials to assess the safety and efficacy of multiple repetitive PEG-PAL injections have been performed. Subcutaneous administration of PAL-PEG was safe, well tolerated, and seemed effective at reducing blood Phe in all participants who received the highest dose with a nadir about six days after injection and an inverse correlation between drug and Phe concentrations in plasma [47]. Modifications of oral PEG-PAL to prevent degradation by digestive enzymes have been initiated in the effort to develop an effective oral therapy [48].

6. Cell Directed Therapy

Another treatment under investigation involves liver repopulation with PAH-expressing cells after hepatocyte or hematopoietic stem-cell transplantation. Hepatocyte transplantation is under investigation because donor cells, in order to be efficacious, need to have a selective growth advantage over native hepatocytes [49].

Hepatocyte transplantation has been performed in preclinical studies using various animal models as well in humans with metabolic disorders such us urea cycle defects or glycogen storage disorders. This cellular approach could be possible for the permanent treatment of PKU if a selective growth advantage could be achieved for donor hepatocytes [50]. This treatment has been reported to be successful in an animal model with a selective advantage for the donor cells [17]. However, cell-based therapies using stem cells or more differentiated progenitor cells may represent the future of cell transplantation for treatment of metabolic liver diseases such as PKU.

7. Gene Therapy

Gene therapy for the treatment of PKU has been the focus of multiple research group over the last two decades. In a mouse model of PKU, important progress has been made by the use of an adenovirus related gene directed into the liver [51].

However liver-directed gene therapy does not lead to a permanent correction of PAH activity. The vector’s genome is not integrated into the hepatocyte’s DNA, and hepatocyte regeneration leads to elimination of episomalr AAV vector genomes. Reinjection of the same serotype vector also leads to its destruction by antibody-mediated immune reactions. Studies of PKU mouse models have also shown that gene therapy can be successfully delivered to non-hepatic tissues such as the muscle [52]. In fact, the insertion into the muscle cells of vectors containing the necessary genes for PAH and tetrahydrobiopterin synthesis resulted in a system that could convert phenylalanine into tyrosine mimicking the role of hepatic phenylalanine metabolism. The improvement in viral vector design has led to human gene therapy trials for other inborn errors of metabolism including α1 antitrypsin deficiency and Canavan disease [53].

Ongoing research by optimization of the direct muscle approach and improved sustained ability in liver-directed gene therapy might lead to human trials in PKU in the next years.

Another approach is based on in vitro read-through of PAH nonsense mutations using aminoglycosides [54]. Approximately 10% of patients with PKU carry a nonsense mutation, which results in the premature insertion of a stop codon. In most cases, these mutations result in unstable mRNA that is rapidly cleared by nonsense-mediated RNA decay. In rare cases, an unstable truncated protein is generated. Aminoglycoside antibiotics such as gentamicin and G-418 can promote read-through of stop codons resulting in production of some protein. The efficacy of aminoglycosides was evaluated in an in vitro expression system in two mammalian cell lines (COS-7 and HEX 293). The read-through PAH products exhibited enzymatic activity to levels similar to that found in moderate PKU. Unfortunately, none of the mouse models for PKU carries a nonsense mutation to determine whether the level of read-through in the liver is sufficient to restore Phe tolerance. Further studies are necessary to determine the impact of this approach on the therapy for PKU.

8. Conclusions

Patients with PKU should still be treated with dietary therapy, but in the long term the introduction of a wide array of new treatment approaches such as more palatable foods based on the use of GMP products or the administration of LNAA or BH4 could decrease the need for phenylalanine restriction in the diet. Patients’ surveys show that GMP foods have improved taste and are preferred to standard formula. Sapropterin is a safe and effective alternative to conventional dietary treatment of responsive PKU patients by stabilizing their blood Phe concentrations and by lowering the burden of the strict low Phe diet. LNAA supplementation has been efficacious in the lessening the need of a strict Phe diet reduction. Human trials are also underway using an enzymatic approach (PEG-PAL) while preclinical work seems promising in the fields of gene and cellular therapy. Finally, in the future, it will be reasonable to think of individualized treatment depending on the genotype and other variables such as age or phenotype.

In conclusion, long-term outcome studies assessing efficacy and safety of GMP medical foods, BH4, LNAA supplementation and enzymatic therapy will be useful in providing the evidence allowing for standardization of management and will alternatively provide in a cost-effective way an individualized management plan for PKU patients.

Author Contributions

Pietro Strisciuglio and Daniela Concolino reviewed the literature and wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.


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