EP1575593A2

EP1575593A2 - Use of von tetrahydrobiopterine derivatives in the treatment and nutrition of patients with amino acid metabolic disorders

Info

Publication number: EP1575593A2
Application number: EP03785822A
Authority: EP
Inventors: Ania Muntau-Heger; Adelbert A. Roscher
Original assignee: Orphanetics Pharma Entwicklungs GmbH
Current assignee: Biomarin Pharmaceutical Inc
Priority date: 2002-12-20
Filing date: 2003-12-15
Publication date: 2005-09-21
Also published as: US20060211701A1; DE10260263A1; WO2004058268A3; US20170042899A1; WO2004058268A2; AU2003294856A1; AU2003294856A8

Abstract

The invention relates to the use of tetrahydrobiopterine and the derivatives thereof in the production of a medicament to improve protein tolerance for the treatment of diseases arising for an amino acid metabolic disorder, e.g. hyperphenylalaninemia. The invention also relates to a composition which contains tetrahydrobiopterine or derivatives thereof in addition to a special mixture of amino acids. The invention can, for instance, be used as a food which is low in phenylalanine in the complete nutrition of hyperphenylalaninemic patients. Tests carried out within the context of said invention revealed that by treating patients who had phenylalanine concentrations of more than 200 ñmol/l in their blood with tetrahydrobiopterine, it was possible to reduce the concentrations of phenylalanine by 37 % to 92 %.

Description

description

Use of tetrahydrobiopterin derivatives for the treatment and nutrition of patients with amino acid metabolism disorders

The present invention relates to the use of tetrahydrobiopterin derivatives according to claim 1, a composition according to claim 13, a use of tetrahydrobiopterin derivatives as dietary supplements according to claim 26, a special food according to claim 28, a phenylalanine-poor special food according to claim 40, and a diagnostic agent for the diagnosis of tetrahydrobiopterin sensitive Diseases associated with disturbed amino acid metabolism according to claim 43.

Diseases as a result of amino acid metabolism disorders are relatively widespread in their entirety, diseases that are mostly genetic. As a pathophysiological correlate, there are reduced activities of certain enzymes with the result of increased or decreased concentrations of amino acids and of neurotransmitters and messenger substances synthesized from them, as well as impaired tolerance (protein tolerance) of certain protein components in food.

For the purposes of the present invention, the term

"Diseases as a result of a disturbed amino acid metabolism can be understood as the following pathophysiological conditions:

Conditions with increased phenylalanine or decreased tyrosine,

Serotonin or dopamine in body fluids, tissues or cells, especially in conditions with reduced phenylalanine hydroxylase, tyrosine hydroxylase tryptophan hydroxylase and NO synthase activity. These conditions can include, but are not limited to, the following clinical pictures: phenylketonuria, especially mild Phenylketonuria, classic phenylketonuria; Skin pigment disorders, especially vitiiigo; as well as conditions due to reduced cellular availability of catecholamines, in particular orthostatic hypotension (Shy-Drager syndrome), muscular dystonia; and neurotransmitter disorders, especially schizophrenia; Conditions due to reduced cellular availability of dopamine or serotonin as a result of tyrosine hydroxylase or tryptophan hydroxylase deficiency, in particular Parkinsonism, depressive disorders and dystonic movement disorders, conditions with reduced NO synthase activity, in particular endothelial dysfunction, inadequate defense against infection.

A known amino acid metabolism disorder, which is due to the lack or reduced metabolizability of phenylalanine, is hyperphenylalanemia, which is caused by a lack of phenylalanine hydroxylase. At least half of the affected patients manifest mild clinical phenotypes. The only possible treatment in the prior art for most amino acid metabolic diseases, such as, for example, hyperphenylalaninemia, is to feed the patients on a diet that uses products that do not contain the amino acid affected by the specific metabolic disorder or contain it only in very small amounts ,

Hyperphenylalaninemia was one of the first genetic disorders that could be treated. In most cases, hyperphenylalaninemia is caused by a phenylalanine hydroxylase deficiency caused by mutations on the phenylalanine hydroxylase gene. The severity of the phenotypes associated with this range from classic phenylketonuria (Online Mendelian inheritance theory in humans No. 261600) (Online Mendelian Inheritance in Man number 261600) to mild phenylketonuria and mild hyperphenylalaninemia. At least half of the affected patients have one of the milder clinical phenotypes. Both patients with classic phenylketonuria sufferers, as well as patients suffering from mild phenylketonuria, must have a low-protein diet throughout their lives to prevent neurological sequelae and ensure normal cognitive development, whereas patients with mild hyperphenylalaninemia may not need treatment. In connection with the very strict diet, there is a risk of nutritional deficiency symptoms and it places a heavy burden on the patients and their families.

A causal therapy has not yet existed in the prior art, so that there is no other option for the affected patients than to follow a strict diet if they do not want to risk experiencing significant sequelae of the amino acid metabolism disorder and the associated hyperphenylalaninemia, for example. The neurological sequelae include, for example, irreversible damage to the nervous system and the brain, mental retardation and even complete idiocy. In addition, kidney damage, liver damage and damage to the sensory organs are described.

For the affected patients, this means - using the example of

Hyperphenylalaninemia - that you have to provide them with a low-phenylalanine diet. Since phenylalanine is an important protein building block, especially in the animal world, it is naturally difficult to feed patients with amino acid metabolism disorders - without provoking unwanted and toxic increases in phenylalanine. In addition, nutritional deficiency symptoms can occur.

In the prior art, protein hydrolyzates were previously used for this purpose, which were prepared from proteins low in phenylalanine by acidic or alkaline hydrolysis.

Such products had a more than bad taste and were often unsustainable for the patient in the long run. Besides these

Hydrolyzates could only be used as food for the affected patients according to the corresponding dietary concept, strictly selected dishes, mostly of a vegetarian nature. In contrast, the synthetic amino acid mixtures that do not contain the amino acid that is affected by the metabolic disorder are already a major improvement over the traditional hydrolysates.

Phenylalanine-free products on this basis are known, for example, from US Pat. No. 5,393,532 and have since been used as a special food for hyperphenylalaninemia and phenylketonuria patients.

Furthermore, it is known from WO 98/08402 A1 to produce special foods based on casein-glyco-macropeptides in combination with amino acid mixtures in order to provide patients with e.g. to be able to feed phenylalanine-free.

In terms of taste, however, such amino acid mixtures are far below the level of ordinary foods.

In summary, it should be noted that a strict, lifelong diet plan based on a special

Amino acid metabolism disorder is tailored to be a strong psychosocial

Represents stress and other treatment methods have so far not been successful.

Starting from this prior art, it is therefore an object of the present invention to provide substances which, on the one hand, are used in the context of a therapeutic treatment of

Amino acid metabolism disorders can be used and on the other hand can be used for the production of foodstuffs, in particular special dietetic foods for patients affected by amino acid metabolism disorders.

The above task is accomplished by using

Tetrahydrobiopterin derivatives according to claim 1, a composition according to claim 13, a use of tetrahydrobiopterin derivatives as

Food supplement according to claim 26, a special food according to claim 28 and a low phenylalanine special food according to claim 40.

It is a further object of the present invention to provide a diagnostic agent for those amino acid metabolism disorders which can be influenced favorably by tetrahydrobiopterin derivatives.

This object is achieved by a diagnostic agent according to claim 43.

In particular, the present invention relates to the use of at least one compound having the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH3) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H7) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms;

wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ ,

C ₂ Hs! wherein R4 and R6 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are independently selected from the

Group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; such as

their pharmaceutically acceptable salts;

for the manufacture of a medicament for improving protein tolerance for the treatment of diseases as a result of a disturbed amino acid metabolism.

Preferred embodiments of the use according to the invention are described below:

One is particularly suitable for the use according to the invention

Compound selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride or sulfate, and a compound with the following structure:

(-) - (1 ^, R, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride; and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

In particular, hydrochlorides or sulfates are used as salts.

The above-mentioned compounds are particularly suitable as medicaments for the treatment of the following diseases or amino acid metabolism disorders:

Conditions with increased phenylalanine or reduced tyrosine in body fluids, tissues or cells, in particular conditions with reduced phenylalanine hydroxylase activity; Phenylketonuria, especially mild phenylketonuria, classic phenylketonuria; Skin pigmentation disorders, especially vitiligo; Conditions due to reduced cellular availability of catecholamines, especially orthostatic hypotension (Shy-Drager syndrome), muscular dystonia; and neurotransmitter disorders, especially schizophrenia.

A hydrochloride, in particular a dihydrochloride, is preferably used as the pharmaceutically acceptable salt.

In addition, the present invention is important by at least one compound having the following general formula as chaperone, in particular chemical chaperone, or so-called protein folding aid:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) _2. N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ H _δ ; wherein R4 and R6 are independently selected from the

Group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; such as their pharmaceutically acceptable salts.

Even when used as a chaperone, it is preferred that the compound is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-) - (1'R, 2'S, 6R) -2-amino-6- (r, 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

The compounds mentioned have proven to be outstandingly suitable for reducing protein misfolding and thereby for improving enzyme activity, in particular in the case of structural anomalies in enzymes which require tetrahydrobiopterin as a cofactor, for example in the case of defects in phenylalanine hydroxylase. As a result of this mechanism of action, they are preferably suitable for the production of medicaments which are suitable for the treatment of clinical pictures which Structural anomalies of the following enzymes are attributable: phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, or NO synthase.

The chaperones according to the invention are therefore suitable for the therapy of conditions

with increased phenylalanine or reduced tyrosine, serotonin or dopamine in body fluids, tissues or cells, especially in conditions with reduced phenylalanine hydroxylase, tyrosine hydroxylase tryptophan hydroxylase and NO synthase activity.

This aspect of the present invention relates to the use of at least one compound according to the following general formula as a neurotransmitter or messenger enhancer, in particular for

Catecholamines and / or serotonin and / or dopamine and / or nitrogen oxide (NO):

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical 1 contains up to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ ,

C ₂ H ₅ ; wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; and their pharmaceutically acceptable salts.

As a neurotransmitter or messenger enhancer, a compound is preferably selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound with the following structure:

(-H1Η _l 2'S _I 6R) -2-amino-6- (1 ^, _I 2'-di ydrox propyl) -5.6 _I 7 _I 8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and or

2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-r, 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

The present invention further relates to a composition which contains at least one compound having the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; where R2 is selected from the group consisting of: H, OH, SH,

NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ ,

wherein R4 and 'R6 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; such as

their pharmaceutically acceptable salts; such as

containing at least one amino acid which is selected from the group consisting of the essential amino acids: isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, histidine; and from the non-essential amino acids, in particular alanine, arginine, aspartic acid, asparagine, cysteine, in particular acetylcysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine.

A preferred composition is characterized in that it contains the essential amino acids selected from the group consisting of: isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, histidine and additionally at least one of the amino acids alanine, arginine, aspartic acid, asparagine, cysteine , in particular acetylcysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine.

It is further preferred that the composition according to the invention additionally contains carbohydrates, in particular glucose, and / or vitamins. The composition according to the invention can preferably be formulated as a preparation to be administered orally or intravenously.

The preparation can be in the form of a powder, tablet, capsule, dragee, in drop form or for topical applications, in particular ointments; as well as a solution for intravenous use.

Of course, such preparations can be in the form of a pharmaceutical composition, optionally with pharmaceutical-pharmaceutical auxiliary substances.

However, the composition according to the invention can also be in the form of a dietary composition, optionally with auxiliary substances customary in food technology, in particular emulsifiers, preferably lecithin or choline.

In addition, it is preferred that the composition according to the invention additionally contains minerals and / or electrolytes which are selected from: mineral salts; Saline salts; Sea salts; Trace elements, in particular selenium, manganese, copper, zinc, molybdenum, iodine, chromium; Alkali ions, especially lithium, sodium, potassium; Alkaline earth ions, especially magnesium, calcium; Iron.

In the context of a dietetic food for patients with hyperphenylalaninemia, the composition according to the invention can even additionally contain phenylalanine without the risk of a toxic accumulation of phenylalanine in the serum, cerebrospinal fluid and / or the brain. It is further preferred that the composition additionally contains L-carnitine and / or myoinosit and / or choline.

Furthermore, it can be useful that the composition according to the invention also contains the antioxidants customary in food technology, in particular vitamin C, whereby the oxidative decomposition of the tetrahydrobiopterin derivatives can be at least largely avoided and the storage stability of the composition is improved.

A composition with a compound is preferably used, the compound being selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-) - (1 ^, R, 2 ^, S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin. The present invention is of particular importance in the production of dietary supplements which are suitable for enabling patients affected by amino acid metabolism disorders to have a largely normal diet despite their disease.

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH _2l F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ H _δ ; wherein R4 and R6 are independently selected from the

Group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; and their suitable salts; as a dietary supplement.

Such a compound is particularly suitable as a food supplement for the addressed patient group, which is selected from the group consisting of: 5,6,7,8-

Tetrahydrobiopterin, sapropterin, especially its hydrochloride, and a compound with the following structure:

(-) - (1'R, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin. The present invention is of outstanding importance in the production of a special food based on essentially phenylalanine-free amino acid mixtures with which, in particular, patients with hyperphenylalaninemia can be optimally fed.

Such special food contains at least one compound with the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ H _δ ; wherein R4 and R6 are independently selected from the

Group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; such as

their food-technically acceptable salts.

A special food for hyperphenylalaninemia patients is particularly suitable which contains at least one compound selected from the group consisting of: 5,6,7,8-

(-) - (1'R, 2 ^, S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin. To ensure the complete food supply, it is preferred that the special food according to the invention additionally contains carbohydrates, in particular glucose, maltodextrin, starch and / or fats, such as fish oil, in particular salmon oil, herring oil, mackerel oil or tuna oil; contains.

It is particularly preferred that the special food is hypoallergenic and / or essentially gluten-free.

Since most amino acid metabolism disorders are genetic hereditary diseases, it is necessary to provide the patient with the right food from birth. It is therefore a particular advantage that the special food of the present invention can be formulated as a baby food, in particular as a milk substitute for both infants and older children and adults.

Such a milk substitute for infants additionally has a fat content, in particular approximately 90% as triglycerides, 10% as mono- and diglycerides.

The special food is made available as a powder, in particular as a lyophilisate, for easier packaging and to increase storage stability.

Furthermore, it is preferred to additionally provide the special food according to the invention with fatty acid supplements, in particular unsaturated fatty acids, preferably omega-3 fatty acids, in particular alphalinolenic acid, docosahexaenoic acid, eicosapentaenoic acid, or omega-6 fatty acids, in particular arachidonic acid, linoleic acid, linolenic acid; or oleic acid. It is further preferred that the special food contains fish oil additives, in particular from salmon, herring, mackerel or tuna oil.

In addition, the special food can have a fat content that includes vegetable oils, in particular safflower oil and / or soybean oil and / or coconut oil.

A particularly preferred embodiment of the special food of the present invention, because of its character as a milk substitute, is also to form it as a milk mix drink, in particular fruit milk mix drink or cocoa, which is particularly suitable for patients with an amino acid metabolism disorder, in particular hyperphenylalaninemia.

The present invention is of outstanding importance in the nutrition of patients with hyperphenylalaninemia: the merit of the inventors of the present invention makes it possible for the first time to provide such patients with a special food low in phenylalanine, which is suitable by the addition of tetrahydrobiopterin derivatives Increase protein tolerance and the breakdown of phenylalanine.

According to the present invention, such a special phenylalanine food contains a low-protein staple food and at least one compound with the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ ,

wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; and their food-technically acceptable salts. For the low-phenylalanine special food according to the invention, it is also preferred to use a compound which is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-) - (1'R, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

It is possible and preferred to train the low-phenylalanine special food as: ready meals; Pasta, especially pasta; Baked goods, in particular bread, cakes, cookies; Confectionery, in particular chocolate, candy, ice cream; Beverages, in particular milk substitutes, in the form of a milk mix drink, in particular a fruit milk mix drink or cocoa; as well as beer. This allows hyperphenylalaninaemia patients to eat significantly higher amounts of normal food for the first time - without putting themselves at risk due to their amino acid metabolism disorder - and without relying exclusively on the malodorous products of the prior art.

As a result of the rapid onset of the action of tetrahydrobiopterin derivatives, it is finally possible within the scope of the present invention to provide a diagnostic agent for the detection of tetrahydrobiopterin-sensitive diseases of the amino acid metabolism, which contains at least one compound with the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ Hs; wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ ,

C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Ci, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is GH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ ,

C ₂ H ₅ , and - - represents an optional double bond; especially 5,6,7,8-tetrahydrobiopterin; and their pharmaceutically acceptable salts.

In summary, it can be stated that it is possible for the first time with the compounds described in the context of the present invention to treat certain genetically caused amino acid metabolism disorders with medication, so that the patients improve the protein tolerance and largely normalize their disturbed enzyme activity and the concentrations of the amino acids concerned and / or have their metabolites in body fluids and body cells.

Furthermore, the present invention proposes compositions of nutritional supplements and special foods which simultaneously contain the compounds described in the invention for improving the protein tolerance and the degradation of phenylalanine. This makes it possible for the first time to feed patients with amino acid metabolism disorders practically normally, ie with almost all taste and compositional nuances. In addition to the connections already mentioned several times above, however, the following connections can also be used as preferred embodiments for all claim categories:

All individual connections and their different

Enantiomers which, with the substituents R1 to R10 and X disclosed in each case, result from the general formulas shown and all subcombinations thereof.

In particular, the following sub-combinations of connections are intended

Be part of the revelation:

wherein R1 is selected from the group consisting of: H, OH, SH; and or

wherein R1 is selected from the group consisting of: F, Cl, Br, I; and or

wherein R1 is selected from the group consisting of: NH ₂ , N (CH3) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H7) ₂ ; and or

wherein R1 is NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; and or wherein R2 is selected from the group consisting of: H, OH, SH; and or

wherein R2 is selected from the group consisting of: NH ₂ , F, Cl,

Br, I, O, S; and or

wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ ; and or

wherein R4 and R6 are independently selected from the group consisting of: H, OH, SH, NH ₂ ; and or

wherein R4 and R6 are independently selected from the group consisting of: F, Cl, Br, I; and or

wherein R4 and R6 are independently acetyl; and or

wherein R4 and R6 are selected independently of one another from the group consisting of: OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; and or

wherein R5 is selected from the group consisting of: CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; and or

wherein R5 is phenyl; and or

wherein R7 and R8 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO; and or wherein R7 and R8 are independently selected from the group consisting of: COOR9, wherein R9 is CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl; and or

wherein R10 is selected from the group consisting of: H, CH ₃ ,

C ₂ H, and - - represents an optional double bond.

It has also been found that lipophilic tetrahydrobiopterin derivatives, such as are described, for example, in EP 0 164 964 A1, are particularly suitable for increasing the serum half-life compared to tetrahydrobiopterin from approximately 8 hours to over 18 hours. On the other hand, such are lipophilic

Tetrahydrobiopterin derivatives are particularly suitable for producing special foods and food supplements, since they also dissolve well in fat-containing mixtures, for example in milk substitutes.

Another advantage of the lipophilic compounds is their reduced sensitivity to oxidation.

Such lipophilic compounds are in particular those in which

R1 in the above general formula is an NH acyl, the acyl radical containing in particular 9 to 32, preferably 9 to 20 carbon atoms; and or

R4 and R6 are selected independently of one another from the group consisting of: OX, where X is in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; where the substituents R2, R3, R5, R7, R8, R9, R10 can be selected as disclosed in the context of the present invention.

The following lipophilic tetrahydrobiopterin derivatives can preferably be used by way of example for the purposes of the present invention:

2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopteriπ; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

Tetrahydrobiopterin is currently commercially available, for example as sapropterin hydrochloride which is available under the name BIOPTEN ^® from Suntory and which is used for the therapy of genetically caused tetrahydrobiopterin synthesis disorders.

In addition, tetrahydrobiopterin and its derivatives can be produced synthetically. An example of this is EP 0 164 964 A1, which i.a. describes the preparation of a series of acylated tetrahydrobiopterin derivatives. The US also discloses

4,665,182 the organic chemical synthesis of biopterin derivatives.

Thus, the preparation of the compounds used is not difficult for the person skilled in the art.

Further advantages and features result from the description of exemplary embodiments and from the drawing.

It shows:

1 shows the phenylalanine concentrations in the blood before provocation with phenylalanine and before and after administration of tetrahydrobiopterin in mild hyperphenylalaninaemia, mild phenylketonuria, mild, phenylketonuria not responding to tetrahydrobiopterin and classic phenylketonuria;

2 shows the effects of short-term treatments with tetrahydrobiopterin on phenylalanine oxidation;

3 shows a relation between the cumulative recovery rate of ¹³ C-labeled CO ₂ during after administration of ¹³ C-labeled phenylalanine and the phenylalanine blood concentrations before and after administration of tetrahydrobiopterin;

4 shows the effect of tetrahydrobiopterin on peripheral phenylalanine clearance and oxidation rates in patients with hyperphenylalaninemia; and

5 shows the structural localization of phenylalanine hydroxylase missense mutations.

Table 1 shows the correlation between genotypes and clinical phenotypes.

Examples

procedure

In order to investigate the therapeutic effectiveness of tetrahydrobiopterin, we carried out a combined phenylalanine-tetrahydrobiopterin stress test for diagnostics and analyzed the effect in vivo by determining the Rates in 38 people with a deficiency of phenylalanine hydroxylase before and after administration of tetrahydrobiopterin derivatives The response to tetrahydrobiopterin was related to certain genotypes and we localized mutations based on the structural model of the phenylalanine hydroxylase monomer and the resulting protein misfolding. Results

In 27 of the 31 patients (87 percent) with mild hyperphenylalaninemia (n = 10) or mild phenylketonuria (n = 21), tetrahydrobiopterin significantly reduced the phenylalanine content in the blood and increased / improved phenylalanine oxidation. Conversely, none of the seven patients with classic phenylketonuria (n = 7) met the criterion of strong response to tetrahydrobiopterin, as defined in the study. However, minor effects were demonstrated in individual patients with classic phenylketonuria. Long-term therapy with tetrahydrobiopterin, which was carried out in five children, significantly increased the daily phenylalanine tolerance from 8.7 + 8.6 mg / kg body weight (range 8.8-30) to 61.4 ± 27.9 mg / kg body weight ( Range 17.9-90) with medication (P = 0.0043), allowing them to stop their special diet. Seven mutations of the phenylalanine hydroxylase gene (P314S, Y417H, V177M, V245A, A300S, E290G and IVS4-5C-> G) and the resulting structural anomaly and misfolding of the enzyme were most likely to be causally related to the response to tetrahydro mutations , F39L, D415N, S310Y, R158Q and I65T) have been classified as possibly related. Four mutations (Y414C, L48S, R261Q and 165V) were inconsistently related to this phenotype. Mutations associated with response to tetrahydrobiopterin were localized primarily in the catalytic region of the protein and were not directly involved in cofactor binding.

Conclusions:

A response to tetrahydrobiopterin derivatives - characterized by an improvement in • protein tolerance, extensive normalization of disturbed phenyalanine hydroxylase activity and a decrease in increased Phenylalanine concentration - often occurs in patients with a mild phenotype of hyperphenylalaninemia. The response cannot be reliably predicted on the basis of the genotype, which is particularly the case with composite double heterozygous genotypes. Drug treatment with tetrahydrobiopterin derivatives and / or the addition of compounds to food could free many patients from their very stressful low-phenylalanine diet and thereby make their diet easier.

After filing the present patent application, the

Data of the invention published and documented in scientifically reviewed form: New England Journal of Medicine, 2002, 347 (26), 2122-2132 (26.12.02).

introduction

Hyperphenylalaninemia, a widespread inheritable

Metabolic disease was one of the first genetic disorders that could be treated. In most cases, hyperphenylalaninemia results from a lack of phenylalanine hydroxylase (EC1.14.16.1) caused by mutations in the phenylalanine hydroxylase gene. The severity of the phenotypes associated with this range from classic phenylketonuria (MIM261600) to mild phenylketonuria and mild hyperphenylalaninemia. At least half of the affected patients have one of the milder clinical phenotypes. Both patients who have classic phenylketonuria and patients who have mild phenylketonuria must have a low-protein diet throughout their lives to prevent neurological sequelae and ensure normal cognitive development. In connection with the very A strict special diet poses the risk of nutritional deficiency symptoms, and it is a burden for patients and their families. Only patients who have mild hyperphenylaninemia may not need treatment. The search for alternative treatment methods without changing the diet was stimulated.

In about 50 genetically related diseases in humans, treatment with a high dose of a cofactor can stimulate enzyme activity. Tetrahydrobiopterin is a natural cofactor of aromatic amino acid hydroxylases and nitrogen oxide synthase. Substitution of this cofactor component is an established treatment for rare cases of hyperphenylalanine anemia resulting from congenital errors in tetrahydrobiopterin biosynthesis. However, more than 98 percent of patients with hyperphenylalaninemia have mutations in the phenylalanine hydroxylase gene and they have an increased rather than a decreased plasma concentration of biopterin, which is due to the activity of the guanosine triphosphate cyclohydroxylase I feedback regulation protein. For this reason, a possible therapeutic effect of tetrahydrobiopterin in patients with a lack of phenylalanine hydroxylase has not been considered.

Recently, it has been shown that individual patients with mutations in the phenylalanine hydroxylase gene have lower levels of phenylalanine in the blood after being given tetrahydrobiopterin for diagnostic purposes. However, peripheral phenylalanine levels are known to be regulated by various genetic sites and modifying factors and there is no evidence that the positive effects of tetrahydrobiopterin occur at the phenylalanine hydroxylation level. In this study, which was conducted on randomly selected patients, the following questions were addressed: (1) How widespread is the response to tetrahydrobiopterin? (2) Does tetrahydrobiopterin restore phenylalanine oxidation ability? (3) Is the response to tetrahydrobiopterin related to certain genotypes and are there related mutations at certain locations in the protein structure? (4) Does it improve protein tolerance in long-term treatment?

method

patients

We received a written consent form from the families of 38 children with various sub-forms of hyperphenylalaninemia. The classification was made depending on the plasma phenylalanine concentration before treatment: <600 μmol / l, mild hyperphenylalaninemia, n = 10, age 15 days to 10 years; 600-1200 μmol / l, mild phenylketonuria, n = 21, ages 8 days to 17 years; > 1200μmol / l, classic phenylketonuria, n = 7, age 1 day to 9 years. A defect in the tetrahydrobiopterin biosynthesis or in the recycling of tetrahydrobiopterin was excluded by an analysis of the pterin values in the urine and the dihydropteridine reductase activity in erythrocytes. We examined 7 patients during the newborn period and 31 when they were older. Sick siblings (n = 5) were also included in the study because it is known that non-genetic factors influence phenylalanine homeostasis.

Combined phenylalanine and tetrahydrobiopterin

stress test Phenylalanine intake was achieved by letting the patient eat a meal containing 100 mg phenylalanine per kilogram of body weight. One hour after the end of the meal, the patients ingested 20 mg tetrahydrobiopterin per kilogram (Schircks Laboratories, Jona, Switzerland). The phenylalanine concentration in the blood was determined by electrospray ionization tandem mass spectroscopy - before the absorption of phenylalanine and before and after (at 4, 8 and 15 hours) provocation with tetrahydrobiopterin. During the test phase, the newborns were fed breast milk, while the older children received a standardized protein intake (10 mg phenylalanine per kilogram) between six and eight hours after exposure to tetrahydrobiopterin.

In Vivo Analysis of L-Phenylalanine Oxidation The tests were performed after four hours of fasting in young children and one overnight fast in older children. A total of 6 mg (Euriostop, Paris, France) administered orally per kilogram of body weight. The tracer was dissolved in a 25% dextrose solution (2 mg per milliliter). Breath samples were then taken over a period of 180 minutes and stored in air-empty glass tubes until analysis by means of isotope mass spectroscopy (deltaS, Thermoquest, Bremen). The recovery of carbon-13 in the breath samples was calculated as described by Treacy et al. described, assuming a total ^{carbon dioxide production} of 300 mmol per hour x square meter of the body surface. ¹³ CO ₂ production was presented as a cumulative percentage of the dose administered versus time. The validity of the results in newborns could be affected by diet or the fact that breath sampling is more difficult for them than for older children. The Baseline percentage of ¹³ C, measured at time 0, however, did not differ significantly in the newborn and older children. The values were considered to be below detectability if the signal intensity of the atomic excess at time t, obtained by subtracting the average base value, did not allow a sufficient differentiation from atmospheric ¹³ CO ₂ . On average, fewer than 1 (older children) and less than 2 (newborns) out of 27 consecutive ¹³ CO ₂ measurements obtained during the 180 minutes of a single test were not interpretable. This has a negligible impact on the final evaluation.

Analysis of the mutations

DNA was extracted from the leukocytes by the standard method. 13 genome fragments, which contain the entire coding sequence, as well as the exon-flanking, intronic sequence of the phenylalanine hydroxylase gene were identified by a

Polymerase chain reaction amplified, followed by direct sequencing.

Structure-based localization of phenylalanine hydroxylase gene mutations

An overall length model of the tetrahydrobiopterin-bound phenylalanine hydroxylase was created from the crystal structures of various truncated forms by overlaying the catalytic areas using the tools provided by SWISS-MODEL / Swiss-Pdb Viewer. Results

Effects of tetrahydrobiopterin on blood phenylalanine concentration and phenylalanine oxidation rates

Patients were classified as responding to tetrahydrobiopterin when the phenylalanine concentration in the blood decreased by at least 30% 15 hours after exposure to tetrahydrobiopterin compared to the value before taking tetrahydrobiopterin. Response to tetrahydrobiopterin was observed in all 10 patients with mild phenylalaninemia and in 17 of the 21 patients with mild phenylketonuria. Only 4 patients with mild phenylketonuria and all 7 patients with classic phenylketonuria did not meet the criterion of response to tetrahydrobiopterin (Fig. 1). In some patients, the phenylalanine concentration dropped rapidly, similar to that seen in patients with a tetrahydrobiopterin synthetic defect, while others reacted slowly and reached the lowest phenylalanine concentration only 15 hours after cofactor administration (data not shown). Patients with varying degrees of clinical severity

Disease reached basal cumulative ¹³ CO ₂ recovery rates, each with their individual residual phenylalainin oxidation capacity (classic phenylketonuria, mean 1, 4%; mild phenylketonuria, 3.1%; mild hyperphenylalaninemia, 5.6%; healthy comparison group 9.0% ) reflected. After treatment with tetrahydrobiopterin (10 mg / kg body weight, 24 hours), the total ¹³ CO ₂ recovery increased significantly in the same patients who had responded to the exercise test. The increase was more pronounced in patients with mild phenylketonuria than in patients with mild Hyperphenylalanine anemia (Fig. 2A). It is noteworthy that 8 out of 11 patients who did not respond had a mild increase in phenylalanine oxidation. after short-term therapy with tetrahydrobiopterin, whereby the phenylalanine content in the blood was also influenced in 3 of these patients. This suggests that with prolonged therapy, even with severe forms of hyperphenylalaninemia, little improvement could be achieved with tetrahydrobiopterin derivatives. The time kuπ / s of the fractional ¹³ CO ₂ formation showed clear deviations from the normal oxidation phenotype (Fig. 2B, C, D and E). After cofactor administration, the curve dropped to normal in patients who responded to tetrahydrobiopterin (Figures 2B and C), but remained unchanged in patients who did not respond to tetrahydrobiopterin.

Before treatment with tetrahydrobiopterin, all patients had phenylalanine concentrations in the blood above 200 μmol / l, and the cumulative ¹³ CO ₂ recovery was below 7% with a considerable overlap between the values of the patients who responded and those who did not , After the administration of tetrahydrobiopterin, the two patient groups formed two non-overlapping clusters. Among the patients who were sensitive to tetrahydrobiopterin, 4 children were found who showed a moderate response to tetrahydrobiopterin (FIG. 3).

Considerable inter-individual variability was observed: Exposure to tetrahydrobiopterin reduced phenylalanine concentrations by 37 to 92% when the blood values were compared before and 15 hours after tetrahydrobiopterin administration. In 23 of the 27 patients reacting to tetrahydrobiopterin, the phenylalanine concentrations in the blood decreased to values below 200 μmol / l, with 4 patients reaching values between 200 and 400 μmol / l. In patients who did not respond, the concentration of phenylalanine exceeded after Exposure to tetrahydrobiopterin always 400 μmol / l. Tetrahydrobiopterin increased the ¹³ C-phenylalanine oxidation rates by 10 to 91% and 22 of the 27 people who responded to tetrahydrobiopterin reached normal levels. The remaining 5 patients showed improvement, but a normal level was not reached. Although generally consistent, some patients showed remarkable inconsistencies in the tetrahydrobiopterine effect on the two endpoints analyzed. (Examples given in Fig. 4). A patient with classic phenylketonuria experienced a slight increase in the phenylalanine concentration in the blood and an improvement in the phenylalanine oxidation rate, but the patient did not meet the criterion of strong response to tetrahydrobiopterin (Fig. 4).

Long-term treatment with tetrahydrobiopterin The families with five children aged 4 to 14 years with milder

Phenylketonuria agreed to a therapy attempt in which the diet low in phenylalanine was replaced by oral administration of tetrahydrobiopterin in daily doses between 7.1 and 10.7 mg / kg body weight. Treatment lasted 207 + 51.3 days (mean ± SD; range 166-263). The cofactor treatment resulted in an increase in the daily phenylalanine tolerance of 8.7 + 8.6 mg / kg body weight (range 8.8-30 previously at 61.4 + 27.9 mg / kg body weight (range 17.9-90)) Treatment (P = 0.0043) with little effect on the phenylalanine concentration in the blood (during dietary treatment, 366 + 120 μmol / l; during pure cofactor treatment, 378 + 173 μmol / l). Identification and structure-based localization of phenylalanine hydroxylase gene mutations

Two mutant alleles were identified in 37 of 38 patients (Table 1). We classified 7 mutations (P314S, Y417H, V177M, V245A, A300S, E390G, IVS4-5C> G) as most likely responsible for the response to tetrahydrobiopterin, since they were detected in either homozygous or functional hemizygotic form. Six other mutations may be related to response to tetrahydrobiopterin due to significant in vitro residual enzyme activity (A403V, F39L, D415N, R158Q, I65T) or a known serious mutation on the second allele (S310Y) , Four mutations (Y414C, L48S, R261 Q, I65V) showed an inconsistent association with the response to tetrahydrobiopterin. Eight out of 12 missense mutations related to tetrahydrobiopterin response are on the catalytic, whereas two are on the regulatory and two on the tetramerization domain. None of them had an effect on residues of the active site or on amino acids that interact directly with the cofactor (FIG. 5).

discussion

We show several lines of evidence to make it clear that the metabolic phenotype of the lack of phenylalanine hydroxylase can be significantly modified by pharmacological doses of tetrahydrobiopterin or its derivatives. First, ingestion of tetrahydrobiopterin resulted in normal or approximately normal phenylalanine concentrations in the blood in most patients with residual phenylalanine hydroxylase enzyme activity, suggesting that the response to tetrahydrobiopterin in patients who were phenotypically mild Symptoms are common. Second, tetrahydrobiopterin increased the remaining phenylalanine oxidizing ability in these patient groups. Third, long-term treatment with tetrahydrobiopterin led to a significant improvement in protein tolerance and the elimination of the need for restrictive diet therapy.

We show that the in vitro phenylalanine oxidation test allows patients with hyperphenylalaninemia to be divided into different classes of different severity. These results are consistent with the data on the ability of the method to measure the phenylalanine hydroxylase gene dose. Because of the multifactorial nature of hyperphenylalaninemia, the phenyialanine oxidation rate throughout the body is not a simple equivalent of phenylalanine hydroxylase activity. The decrease in blood phenylalanine was accompanied by an improvement in in vivo phenylalanine oxidization ability in all patients who responded to tetrahydrobiopterin. All in all, these observations are consistent with the hypothesis that the misfolding of the enzyme and the disrupted phenylalanine hydroxylase activity can be improved by tetrahydrobiopterin. The extent of improvement in phenylalanine degradation has not always been the improvement in phenylalanine oxidation, a not unexpected result for a genetically determined enzyme deficiency in general and for a lack of phenylalanine hydroxylase in particular. We observed slow and fast reactions, as well as differences in the time course and in the relative extent of the ¹³ CO ₂ formation, which indicates that tetrahydrobiopterin exerts its effects through different modes of action and - depending on the extent of protein misfolding - with different efficiency. In addition to the suggestion that high-dose tetrahydrobiopterin treatment could compensate for a reduced affinity of the defective phenylalanine hydroxylase for tetrahydrobiopterin, additional modes of action must be considered. Treatment with tetrahydrobiopterin could additionally upregulate phenylalanine hydroxylase gene expression, stabilize phenylalanine hydroxylase mRNA, facilitate the functional phenylalanine hydroxylase tetramer formation or protect a wrongly folded enzyme protein from proteolytic digestion.

Predictions of the phenotype based on the genotype can be difficult in complex, genetically multifactorial diseases such as hyperphenylalanemia. In the group of patients - who responded to tetrahydrobiopterin, we identified predominantly "mild" genotypes, whereas the genotypes of patients who did not respond were predominantly "severe". The experimental indications of the relationship between different mutations and the response to tetrahydrobiopterin differ in their consistency, and it is therefore difficult to make predictions based on the genotype, especially in the presence of double heterozygosity. The Y414C mutation is known to occur in more than one clinical phenotype. We identified this mutation in a functional hemizygotic stage in two patients with identical genotypes but different reactions to tetrahydrobiopterin. This observation could be explained by the fact that the effects of multiple modifying gene locations have different effects on hyperphenylalanine anemia. In the homozygous state, which suggests homopolymeric tetramer formation, it was found that the Y414C and the L48S mutations cause a response to tetrahydrobiopterin. However, in the functional hemizygotic state, we discovered these mutations in individuals with classic phenylketonuria who did not respond to tetrahydrobiopterin. Under these circumstances, heteropolymerization could hinder the formation of functional tetramers.

Our data confirm the assumption that most missense mutations are related to the response to tetrahydrobiopterin lie in the catalytic domain of the protein, but do not relate to residues of the active center and are not directly involved in the cofactor binding. In the case of a monomer, these mutations can affect the interactions between the domains or influence residues on the contact surfaces of the dimers or tetramers and thus lead to misfolding of the proteins and reduced enzyme activity. Tetrahydrobiopterin thus acts as a chemical chaperone and prevents this.

Previously, in vitro expression analyzes were used to predict the functional influence of the phenylalanine hydroxylase gene mutations in vivo. An overestimation of the phenylalanine hydroxylase activities in vitro compared to those in vivo was observed. This could be explained by the fact that the in vitro expression analysis has hitherto been carried out almost exclusively in the presence of high concentrations of natural or synthetic cofactors, which complicates genotype-phenotype correlation. Revised experimental protocols,, should include a range of different tetrahydrobiopterin concentrations to assess the intrinsic severity of the mutations. Since one from the pretherapeutic

Plasma phenylaline concentrations cannot conclude whether and how tetrahydrobiopterin is responded to, a new clinical classification is advisable: (1) hyperphenylalaninaemia, which does not respond to tetrahydrobiopterine, (2) hyperphenylalaninaemia, where responses are made to tetrahydrobiopterine (include) a lack of phenylalanine hydroxylase responsive to tetrahydrobiopterin; and (b) disorders in the tetrahydrobiopterin biosynthetic pathway. A phenylalanine tetrahydrobiopterin exercise test with an extended observation phase (> 15 hours) can reliably differentiate between patients who respond and patients who do not respond should be carried out in all persons suffering from hyperphenylalaninemia to ensure reliable identification of the patients who could benefit from tetrahydrobiopterin treatment. Our short-term study does not rule out the possibility that slight effects may only become apparent after a long treatment, even in some patients with classic phenylketonuria.

Our results show that long-term therapy with tetrahydrobiopterin leads to increased phenylalanine tolerance. A cofactor treatment instead of the stressful special diet is suitable for many patients and one can expect that the treatment with tetrahydrobiopterin derivatives leads to a considerable improvement in the quality of life. In particular, the addition of these compounds to foods should make the design of the otherwise very difficult diet considerably easier. Tetrahydrobiopterin treatment could also be helpful for maternal phenylketonuria, since strict metabolic control during pregnancy is very difficult but very important to avoid serious negative effects in the newborn. However, it has not yet been determined how safe or side effect it is to take tetrahydrobiopterin during pregnancy. In total, more than 350 patients with a tetrahydrobiopterine deficiency were treated with the cofactor. When assessing safety, some dose-related undesirable side effects such as sleep disorders, polyuria and thin stools were observed (BIOPTEN ^® approval certificate, Suntory, Japan).

Some obstacles need to be removed before treatment with tetrahydrobiopterin can become routine. First, tetrahydrobiopterin is not yet an approved drug in most countries. Second, it is still expensive. Third, studies of doses to be administered, as well as clinical Studies with regard to the bioavailability and the as yet unknown long-term side effects of tetrahydrobiopterin in phenylalanine hydroxylase deficiency are required.

In summary, we can show that pharmacological doses of tetrahydrobiopterin can improve or completely normalize disturbed phenylalanine oxidation in most patients with hyperphenylalaninemia of a less severe phenotype by correcting protein misfolding. In addition, improved protein tolerance and relaxation of dietary measures can be achieved. These findings are important for the diagnostic procedure, the clinical classification and the therapeutic procedure. In the near future, cofactor treatment will free many patients from their very stressful dietary restrictions.

TABLE 1. GENOTYPES OF PATIENTS WITH TETRAHYDROBIOPTERIN-SENSITIVE AND NON-SENSITIVE HYPERPHENYLAI ANINEMIA

A3-CEL 1 ALI-EI-E 2 PE - MOTΩ? TEΩÄ-TΪ --- ROBIOP ERIN-S --- NSITIVITÄT

A403V IVS4 + 5G> T mild yes

A403V n.i. Mild yes

P314S * R408 ^f mild yes

F39L D415N Mild Yes

Y414C D415N Mild Yes

Y417H * Y417H * Mild Phenvl etonurie Yes

F55 S310Y * mild yes

R261Q Y414C Mild phenylketonuria Yes

V177M R408 ⁺ mild yes

P275L * Y414C Mild phenylketonuria Yes

V245A R408 ⁺ mild yes

L48S R158Q Mild phenylketonuria Yes

Y417H * Y417H * Mild phenylketonuria Yes

V245Ä R408 ^f mild yes

R2SlX ^t A300S Mild phenylketonuria Yes

R158Q E390G Mild phenylketonuria Yes

R261X * A300S Mild phenylketonuria Yes

Y414C IVS12 + lG> A ^t Mild phenylketonuria Yes

I65S *. A300S Mild Phenvlketonuria Yes

R261Q Y414C Mild phenylketonuria Yes

K274f5delllb E390G Mild phenylketonuria Yes

IVS4-50G R408W ^f Mild phenylketonuria Yes

R261X ⁺ A300S M-MHP Ph <= nv1 Vrpfnnπri <=>

I65T Y414C Mild Phenvlketonuria Moderate E390G IVS12 + lG> A ^f Mild Phenvlketonurie Moderate I65V R261Q Mild Moderat R158Q Y414C Mi l dt. PhsnvI RtoTiiiri fi Moderat Table 1 - continued

ATiI-EL 1 A --- I-EI-E 2 PHENOTYPE TETRAHYDROBIOPTERIN- SENSI TIVI TAT

Y414C IVS12 + 10A ^{1 "} Classic No.

P281 ^t Y414C Mild phenylketonuria No.

I 65V IVS12 + 1G> A mild phenolketonuria Ne n

I65V IVS12 + lG> A ^t Mild phenylketonuria No.

N61D * R261Q Mild phenylketonuria No.

R408 ^f . ] R413P Y414C Classic No.

P281L ^t P281L ⁺ Classic Ne n

R243X ⁺ Y414C Classic No.

L48S P2811 ^t Classic No.

R261 Q R408W ^t Classic No.

R243X * TV?; 7-.IΓ,> Ά Kl asai srhe WPIΠ

Mutations that are highly likely to be associated with tetrahydrobioptene sensitivity are grayed out

Mutations that are potentially associated with tetrahydrαbiopteπn sensitivity are printed in bold

Mutations that are associated with tetrahydrobiopterin sensitivity are always in italics

* Mutation not previously described ^for putative null mutation on nl not identified

Figure Legend

Figure 1

Effect of tetrahydrobiopterin on the phenylalanine concentration in the blood.

Phenylalanine concentration in the blood (Phe) before the phenylalanine exposure and before and after the challenge by tetrahydrobiopterin (BH ₄ ). The

Boxes represent the 50% confidence ink / all (25th - 75th percentile); the horizontal black bars represent the medians; the

Error bars indicate the range between minimum and maximum. The

P is the difference between the phenylalanine level in the blood before and 15 hours after the tetrahydrobiopterin administration.

Figure 2

Effect of short-term treatment with tetrahydrobiopterin on phenylalanine oxidation in vivo. A cumulative ¹³ CO ₂ (180 min.) Recovery before and after treatment with tetrahydrobiopterin (BH). The boxes represent the 50% confidence interval (25th - 75th percentile); the horizontal black bars represent the medians; the error bars show the range between minimum and maximum. BE Fractional analysis of ¹³ CO ₂ formation in representative patients with impaired phenylalanine hydroxylase before (D) and after (λ) short-term treatment with tetrahydrobiopterin.

Figure 3

Relationship between the cumulative ¹³ CO ₂ recovery (180 min.) And the phenylalanine concentration in the blood before and after treatment with Tetrahydrobiopterin (BH ₄ ). Patients who do not respond to tetrahydrobiopterin: O; Patients responding to tetrahydrobiopterin: λ; Patients who respond moderately to tetrahydrobiopterin: λ.

Figure 4

Effect of tetrahydrobiopterin on peripheral phenylalanine clearance and on oxidation rates in individual hyperphenylalaninemia patients. The phenylalanine concentration in the blood before (filled bar) and 15 hours after the administration of tetrahydrobiopterin (BH ₄ ) (dark gray bar). The positive effects obtained by tetrahydrobiopterin in individual patients are shown by a black arrow (upper field). Cumulative ¹³ CO ₂ recovery (180 min.) Before (light gray bar) and after the administration of tetrahydrobiopterin (filled bar). The improvement in individual patients caused by tetrahydrobiopterin is shown by a black arrow (lower field). The normal range (nr) for in vivo phenylalanine oxidation determined by a healthy control group aged 2 days to 13 years is given (8.3 + 2.8%; average + SD, n = 12). Irregularities in the effect of tetrahydrobiopterin: marked decrease in the phenylalanine concentration in the blood, but a slight increase in the phenylalanine oxidation in one patient (λ) and little effect on the phenylalanine concentration in the blood and a large increase in the phenylalanine oxidation in another patient (H). A mild response to tetrahydrobiopterin does not meet the criterion of response to tetrahydrobiopterin in a patient with classic phenylketonuria (v). Figure 5

Structural localization of phenylalanine hydroxylase missense mutations. The phenylalanine hydroxylase monomer, shown in the form of a band, is composed of three functional domains: the regulatory domain (residues 1-142), the catalytic domain (residues 143-410), and the tetramerization domain (residues 411-452) , The iron at the active center (brown area, partially covered) and the cofactor analog 7,8-dihydro-tetrahydrobiopterin stick model are on the catalytic domain. Mutations that are most likely related to response to tetrahydrobiopterin are shown in turquoise. Mutations that may be related to response to tetrahydrobiopterin are shown in green. Mutations that are inconsistently related to the response to tetrahydrobiopterin are shown in purple.

Claims

Expectations

1. Use of at least one compound with the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ Hs; wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; such as

their pharmaceutically acceptable salts;

for the manufacture of a medicament for improving the protein tolerance for the treatment of diseases as a result of a disturbed amino acid metabolism.

2. Use according to claim 1, characterized in that the compound is selected from the group consisting of: 5,6,7, 8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride or sulfate, and a compound having the following structure:

(-) - (RR, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride; and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiophene; and / or 2-N-linoleoyl-r, 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

3. Use according to claim 1 or 2, characterized in that hydrochlorides or sulfates are used as salts.

4. Use according to one of claims 1 to 3, characterized in that the amino acid metabolism disorders include: states with increased phenylalanine or reduced tyrosine in body fluids, tissues or cells, in particular states with reduced phenylalanine hydroxylase activity, in particular conditions due to reduced cellular availability of catecholamines, especially orthostatic hypotension (Shy-Drager syndrome), muscular dystonia; and neurotransmitter disorders, especially schizophrenia; Phenylketonuria, especially mild phenylketonuria, classic phenylketonuria; Skin pigmentation disorders, especially vitiligo.

5. Use according to one of claims 1 to 4, characterized in that one is pharmaceutically acceptable

Salt uses a hydrochloride.

6. Use of at least one compound with the following general formula as chaperone:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ Hs; wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ ', and - - represents an optional double bond; such as their pharmaceutically acceptable salts.

7. Use according to claim 6, characterized in that the compound is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-H1 ^, R _l 2 ^, S _l 6R) -2-amino-6- (1 ' _I 2 ^, -dihydroxypropyl) -5.6 _I 7 _l 8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2 ^, -di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-r, 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

8. Use according to claim 6 or 7 for improving protein misfolding, in particular in the case of structural anomalies in enzymes which require tetrahydrobiopterin as a cofactor.

9. Use according to one of claims 6 to 8, characterized in that the enzymes are selected from: phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, or NO synthase.

10. Use according to one of claims 6 to 9, characterized in that the chaperone is used as a neurotransmitter and / or messenger enhancer, especially in conditions with increased phenylalanine or reduced tyrosine, serotonin or dopamine in body fluids, tissues or cells, in particular is used in conditions with reduced phenylalanine hydroxylase, tyrosine hydroxylase tryptophan hydroxylase and NO synthase activity.

11. Use of at least one compound according to the following general formula as a neurotransmitter or as a messenger enhancer, in particular for catecholamines and / or serotonin and / or dopamine and or nitrogen oxide (NO):

where R1 is selected from the group consisting of: H, OH,

SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ Hs; wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; Residential R5 is selected from the group consisting of: phenyl,

CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H _{7 is} butyl; wherein R10 is selected from the group consisting of: H, CH ₃ ,

C ₂ H ₅ , and - - represents an optional double bond; and their pharmaceutically acceptable salts.

12. Use according to claim 11, characterized in that the compound is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-) - (RR, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

13. Composition comprising at least one compound with the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH,, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to

Contains 20 carbon atoms; where R2 is selected from the group consisting of: H, OH,

SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , wherein R4 and R6 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I,

Acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl,

CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ ,

COOH, CHO, COOR9, wherein R9 is CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl; wherein R10 is selected from the group consisting of: H, CH ₃ ,

C ₂ H ₅ , and - - represents an optional double bond; such as

their pharmaceutically acceptable salts; such as

14. The composition according to claim 13, characterized in that it contains the essential amino acids selected from the group consisting of: isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, histidine and additionally at least one of the amino acids alanine, arginine, aspartic acid, asparagine,

Contains cysteine, especially acetylcysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine.

15. The composition according to claim 13 or 14, characterized in that it additionally carbohydrates, in particular

Contains glucose, and / or vitamins.

16. The composition according to any one of claims 13 to 15, characterized in that it is formulated as a preparation to be administered orally or intravenously.

17. The composition according to claim 16, characterized in that the preparation as a powder, tablet, capsule, dragee in drop form or for topical applications, in particular ointments; as well as a solution for intravenous

Application that is formulated.

18. Composition according to one of claims 13 to 17, characterized in that it is in the form of a pharmaceutical composition, optionally with adjuvants customary in pharmaceutical-galenical form.

19. Composition according to one of claims 13 to 18, characterized in that it is used as a dietary composition, optionally with food technology customary auxiliaries, in particular emulsifiers, preferably lecithin, choline.

20. Composition according to one of claims 13 to 19, characterized in that it additionally contains minerals and / or

Contains electrolytes which are selected from: mineral salts; Saline salts; Sea salts; Trace elements, in particular selenium, manganese, copper, zinc, molybdenum, iodine, chromium; Alkali ions, especially lithium, sodium, potassium; Alkaline earth ions, especially magnesium, calcium; Iron.

21. The composition according to any one of claims 13 to 20, characterized in that it additionally contains phenylalanine.

22. The composition according to any one of claims 13 to 21, characterized in that it additionally contains L-camitine.

23. The composition according to any one of claims 13 to 22, characterized in that it additionally myoinosit and

Contains choline.

24. The composition according to any one of claims 13 to 23, characterized in that it contains antioxidants, in particular vitamin C.

25. Composition according to one of claims 13 to 24, characterized in that the compound is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, Sapropterin, especially its hydrochloride, and a compound with the following structure:

(-) - (RR, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxyproρyl) -5,6,7,8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or ^{2-N-palmitoyl-1,} 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-r, 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

26. Use of at least one compound with the following general formula:

where R1 is selected from the group consisting of: H, OH,

SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ Hs; wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl,

C ₂ H ₅ , and - - represents an optional double bond; and their suitable salts; as a dietary supplement.

27. Use according to claim 26, characterized in that the compound is selected from the group consisting of: 5,6,7, 8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-H1 ^, R _l 2 ^, S, 6R) -2-amino-6- (1 ^, _I 2 ^, -dihydroxypropyl) -5,6,7 _I 8-tetrahydro-

4 (3H) -pteridinone, especially its dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-palmitoyl-1 ', 2 ^, -di-O-acetyl-5,6,7,8-tetrahydrobiophene; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

28. Special food based on essentially phenylalanine-free amino acid mixtures, characterized in that it contains at least one compound with the following general formula:

where R1 is selected from the group consisting of: H, OH,

SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , wherein R4 and R6 are selected independently of one another from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular a C9 to C32 acyl radical, is preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl,

C ₂ H ₅ , and - - represents an optional double bond; such as

their food-technically acceptable salts.

29. Special food according to claim 28, characterized in that it contains at least one compound which is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-) - (1'R, 2'S, 6R) -2-amino-6- (1 ', 2'-dihydroxypropyl) -5,6,7,8-tetrahydro-

30. Special food according to claim 28 or 29, characterized in that it additionally contains carbohydrates, in particular glucose, maltodextrin, starch and / or fats, such as fish oil, in particular salmon oil, herring oil, mackerel oil, or tuna oil; contain.

31. Special food according to one of claims 28 to 30, characterized in that it is hypoallergenic and / or essentially gluten-free.

32. Special food according to one of claims 28 to 31, characterized in that it is a baby food.

33. Special food according to one of claims 28 to 32, characterized in that it is in the form of a powder, in particular a lyophilisate.

34. Special food according to one of claims 28 to 33, characterized in that it additionally contains fatty acid supplements, in particular unsaturated fatty acids, preferably omega-3 fatty acids, in particular alphalinolenic acid,

Docosahexaenoic acid, eicosapentaenoic acid, or omega-6 fatty acids, especially arachidonic acid, linoleic acid,

linolenic acid; or contains oleic acid.

35. Special food according to one of claims 28 to 34, characterized in that it contains fish oil additives, in particular from salmon, herring, mackerel or tuna oil.

36. Special food according to one of claims 28 to 35, characterized in that it can be used as a milk substitute, in particular for infants.

37. special food according to claim 36, characterized in that the milk substitute has a fat content, in particular 90% as triglycerides, 10% as mono- and diglycerides.

38. Special food according to claim 37, characterized in that the fat portion comprises safflower oil and / or soybean oil and / or coconut oil.

39. Special food according to one of claims 28 to 38, characterized in that the milk substitute is designed as a milk mix drink, in particular fruit milk mix drink or cocoa.

40. Low-phenylalanine special food, containing a low-protein staple food and at least one compound with the following general formula:

wherein R1 is selected from the group consisting of: H, OH, SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; wherein R2 is selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ , C ₂ Hs; wherein R4 and R6 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, acetyl, OX, where X is a C1 to C32 acyl radical, in particular is a C9 to C32 acyl radical, preferably a C9 to C20 acyl radical; wherein R5 is selected from the group consisting of: phenyl, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are independently selected from the

Group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ , COOH, CHO, COOR9, where R9 is CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl; wherein R10 is selected from the group consisting of: H, CH ₃ , C ₂ H ₅ , and - - represents an optional double bond; and their food-technically acceptable salts.

41. Low-phenylalanine special food according to claim 40, characterized in that it contains at least one compound which is selected from the group consisting of: 5,6,7,8-tetrahydrobiopterin, sapropterin, in particular its hydrochloride, and a compound having the following structure:

(-) - (RR _l ^2, S _I 6R) -2-amino-6- ^(1, ^2, -dihydroxypropyl) _-5> 6 _I 7,8-tetrahydro-

4 (3H) -pteridinoπ, in particular their dihydrochloride or sulfate and / or 2-N-stearoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and or

2-N-decanoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and or 2-N-palmitoyl-1 ', 2 ^, -di-O-acetyl-5,6,7,8-tetrahydrobiopterin; and / or 2-N-linoleoyl-1 ', 2'-di-O-acetyl-5,6,7,8-tetrahydrobiopterin.

42. Low-phenylalanine special food according to claim 40 or 41, characterized in that it is selected from:

Prepared meals; Pasta, especially pasta; Baked goods, in particular bread, cakes, cookies; Confectionery, in particular chocolate, candy, ice cream; Beverages, in particular milk substitutes, in the form of a milk mix drink, in particular a fruit milk mix drink or cocoa; as well as beer.

43. Diagnostic agent for the diagnosis of tetrahydrobiopterin sensitivity in diseases of the amino acid metabolism, containing at least one compound with the following general formula:

where R1 is selected from the group consisting of: H, OH,

SH, F, Cl, Br, I, NH ₂ , N (CH ₃ ) ₂ , N (C ₂ H ₅ ) ₂ , N (C ₃ H ₇ ) ₂ ; NH-acyl, the acyl radical containing 1 to 32 carbon atoms, in particular CH ₃ O, preferably 9 to 32, preferably 9 to 20 carbon atoms; where R2 is selected from the group consisting of: H, OH,

SH, NH ₂ , F, Cl, Br, I, O, S; wherein R3 is selected from the group consisting of: H, CH ₃ ,

C ₂ Hs! wherein R4 and R6 are independently selected from the group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I,

CH ₃ , C ₂ H ₅ , C ₃ H ₇ , butyl, isobutyl, t-butyl; wherein R7 and R8 are independently selected from the

Group consisting of: H, OH, SH, NH ₂ , F, Cl, Br, I, CH ₃ ,

44. Use according to claim 10, characterized in that the conditions include: phenylketonuria, in particular mild phenylketonuria, classic phenylketonuria; Skin pigmentation disorders, especially vitiligo; as well as conditions due to reduced cellular availability of catecholamines, in particular orthostatic hypotension (Shy-Drager syndrome), muscular dystonia; and neurotransmitter disorders, especially schizophrenia; Conditions due to reduced cellular availability of dopamine or serotonin as a result of tyrosine hydroxylase or tryptophan hydroxylase deficiency, in particular Parkinsonism, • depressive disorders and dystone Movement disorders, conditions with reduced NO synthase activity, especially endothelial dysfunction, lack of defense against infections.