4 Chlorobutanol Synthesis Essay

IUPAC Name

1,1,1-trichloro-2-methylpropan-2-ol

InChI

InChI=1S/C4H7Cl3O/c1-3(2,8)4(5,6)7/h8H,1-2H3

InChI Key

OSASVXMJTNOKOY-UHFFFAOYSA-N

Canonical SMILES

CC(C)(C(Cl)(Cl)Cl)O

MeSH Synonyms

Acetonchloroform, Anhydrous Chlorobutanol, Chlorbutol, Chloretone, Chlorobutanol, Chlorobutanol, Anhydrous, Trichlorbutanol

Depositor-Supplied Synonyms

Chlorobutanol, Chlorbutol, CHLORETONE, Acetonchloroform, 1,1,1-Trichloro-2-methyl-2-propanol, Acetochlorone, Chlorbutanol, Chloreton, Chlortran, Clortran, Coliquifilm, Dentalone, Khloreton, Methaform, Sedaform, Anhydrous chlorobutanol, Acetonechloroform, Trichloro-tert-butanol, Chlorobutanol, anhydrous, 2-Propanol, 1,1,1-trichloro-2-methyl-, Trichlorisobutylalcohol, 57-15-8, Chlorbutanolum, Chlorbutolum, 1,1,1-trichloro-2-methylpropan-2-ol, 2-(Trichloromethyl)-2-propanol, Trichloro-t-butyl alcohol, tert-Trichlorobutyl alcohol, Trichloro-tert-butyl alcohol, 1,1,1-Trichloro-tert-butyl alcohol, 2-(Trichloromethyl)propan-2-ol, Caswell No. 185, Clorobutanolo [DCIT], beta,beta,beta-Trichloro-tert-butyl alcohol, UNII-HM4YQM8WRC, NSC 44794, Clorobutanol [INN-Spanish], 1,1,1-trichloro-2-methyl-propan-2-ol, Chlorobutanolum [INN-Latin], 2-Propanol, 2-methyl-1,1,1-trichloro-, Trichloro-2-methylpropan-2-ol, HSDB 2761, 2-Propanol, trichloro-2-methyl-, OSASVXMJTNOKOY-UHFFFAOYSA-N, EINECS 200-317-6, SBB058738, EPA Pesticide Chemical Code 017501, 2,2,2-Trichloro-1,1-dimethylethanol, 28471-22-9, BRN 0878167, NCGC00159392-02, NCGC00159392-05, AI3-00048, DSSTox_CID_21217, DSSTox_RID_79651, WLN: QX1&1&XGGG, DSSTox_GSID_41217, .beta.,.beta.,.beta.-Trichloro-tert-butyl alcohol, Chlorobutanol hemihydrate, Trichlorbutanol, 1,1-Trichloro-tert-butyl alcohol, 6001-64-5, 1,1-Trichloro-2-methyl-2-propanol, 2-Propanol,1,1-trichloro-2-methyl-, CAS-57-15-8, .beta.,.beta.-Trichloro-tert-butyl alcohol, beta,beta,beta-Trichloro-tert-butyl alcohol hemihydrate, chloretol, chloretonl, chlortral, Chlorobutanolum, Clorobutanol, Clorobutanolo, Trichlorobutanol, Acetonechloroform, Chloretone (TN), t-Trichlorobutyl alcohol, HM4YQM8WRC, AC1Q1NNR, AC1Q3GOR, C4H7Cl3O, AC1L1LJ9, SCHEMBL1040, 20801_RIEDEL, 36681_RIEDEL, 2-Propanol,trichloro-2-methyl-, CHEMBL1439973, Chlorobutanol (JP16/NF/INN), Chlorobutanol [USAN:INN:JAN], 36681_FLUKA, 91210_FLUKA, CTK4G1557, 2-(trichloromethyl)-propan-2-ol, NSC4596, NSC5208, MolPort-001-784-250, 20801_SIAL, HMS3264C17, Pharmakon1600-01506102, NSC-4596, NSC-5208, NSC44794, EINECS 249-042-3, Tox21_111629, AR-1I2031, NSC-44794, NSC760101, ZINC01482005, AKOS003619059, Tox21_111629_1, 2-methyl-1,1,1-trichloro-2-propanol, CCG-213842, MCULE-7565024869, NE21463, NSC-760101, NCGC00159392-03, NCGC00159392-04, AN-23577, CJ-23632, N743, LS-122688, FT-0605936, ST51037685, EN300-19331, D01942, 47768-EP2269977A2, 47768-EP2272817A1, 47768-EP2311811A1, 4-01-00-01629 (Beilstein Handbook Reference), I14-7607, I14-19299, 3B3-033157, InChI=1/C4H7Cl3O/c1-3(2,8)4(5,6)7/h8H,1-2H

Removed Synonyms

‘Chlorobutanol’, ‘Acetonechloroform’, ‘Chlorobutanol’, CID5977, T0386, 1,1,1-Trichloro-2-methyl-2-propanol hemihydrate, D002724, HCP, 5887-83-2

This application is a continuation of U.S. patent application Ser. No. 12/508,209, now U.S. Pat. No. 8,067,416, filed Jul. 23, 2009, which in turn is a continuation of U.S. patent application Ser. No. 11/143,887, now U.S. Pat. No. 7,566,714, filed Jun. 1, 2005 which in turn is a continuation of U.S. patent application Ser. No. 10/991,573, which was filed on Nov. 17, 2004 and which claimed the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/520,767, which was filed Nov. 17, 2003. The entire disclosure of each of these applications is hereby incorporated herein by reference.

1. Field

The present invention is generally directed to the therapeutic intervention of metabolic disorders, particularly those involving amino acid metabolism. More particularly, the present invention is directed to methods and compositions for the treatment of phenylketonuria, vascular diseases, ischemic or inflammatory diseases, or insulin resistance, or conditions and patients that would benefit from enhancement of nitric oxide synthase activity.

2. Background of the Related Technology

Phenylketonuria (PKU) is an inherited metabolic disorder that was first identified in the 1930s. In most cases, and until the mid-1990s, it was thought that this is a disorder of amino acid metabolism resulting from a deficiency in the liver enzyme phenylalanine hydroxylase (PAH). Deficiencies in PAH in turn result in an excess of phenylalanine (Phe) in the brain and plasma. The deficiency in PAH ultimately manifests in a lack of tyrosine, which is a precursor for the neurotransmitters.

Left undetected and untreated early in the life of an infant, PKU leads to irreversible damage of the nervous system, severe mental retardation and poor brain development. Features other than mental retardation in untreated patients include brain calcification, light pigmentation, peculiarities of gait, stance, and sitting posture, eczema, and epilepsy. It has been reported that an infant suffers a loss of 50IQ points within the first year of infancy and PKU is invariably accompanied by at least some loss of IQ. Once detected, the condition is treated by providing the infant, and later the child, with a low Phe diet. In adults, the protein supplements routinely taken by classic PKU patients may be Phe-free with the assumption that such adults will receive sufficient quantities of Phe through the remaining diet, controlled under a strict regimen, so that the overall diet is a low Phe diet. Also, pregnant women who suffer from the condition are recommended a diet that is low in Phe to avoid the risk of impairment of the development of the fetus and congenital malformation (maternal PKU syndrome).

In more recent years it has been shown that pathological symptoms which manifest from the condition of excess of Phe, collectively termed hyperphenylalaninemia (HPA), may be divided into multiple discrete disorders, which are diagnosed according to plasma Phe concentrations and responsiveness to a cofactor for PAH. At an initial level, HPAs may be divided into HPA caused as a result of a deficiency in the cofactor 6R-L-erythro-5,6,7,8, tetrahydrobiopterin (BH4; malignant PKU) and HPA resulting from a deficiency in PAH. The latter category is further subdivided into at least three categories depending on the plasma concentration of Phe in the absence of dietary or other therapeutic intervention (referred to herein as “unrestricted plasma Phe concentration”).

Normal plasma Phe homeostasis is tightly controlled resulting in a plasma Phe concentration of 60 μmol/L±15 won. Classical PKU (OMIM No. 261600) is the most severe form of PKU and it results from null or severe mutations in PAH, which lead to unrestricted plasma Phe concentrations greater than 1200 won when left untreated. Individuals with classical (or severe) PKU must be treated with a strict dietary regimen that is based on a very low Phe diet in order to reduce their Phe concentrations to a safe range. Milder forms of HPA also have been characterized. A less severe form of PKU is one which manifests in plasma Phe concentrations of 10-20 mg/dL (600-1200 μmol/L), and is generally termed “mild PKU”. This moderate form of PKU is managed through the use of moderate dietary restrictions, e.g., a low total protein diet, but otherwise not necessarily Phe-free. Finally, mild HPA, also referred to as benign or non-PKU HPA is characterized by plasma Phe concentrations of between 180-600 μmol/L. The individuals with non-PKU HPA are not routinely treated as they are considered to have plasma Phe levels that are within the “safe” range. Nevertheless, as mentioned above, these Phe levels are still significantly elevated in these individuals as compared to normal, non-PKU subjects and may present detrimental sequelae in at least pregnant women and very young patients. For a more detailed review of HPA resulting from PAH deficiency, those of skill in the art are referred to Scriver et al., 2001 (Hyperphenylalaninemia: Phenylalanine Hydroxylase Deficiency, In: Scriver C R, Beaudet A L, Sly W S, Valle D, Childs B, Vogelstein B, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill, 2001: 1667-1724). NIH Guidelines indicate that for children with PKU, it is preferable reduce the plasma Phe to be 360-420 μmol/L.

HPA also results from defects in BH4 metabolism. BH4 is an essential cofactor of both tyrosine and tryptophan hydroxylase, the rate limiting enzymes in the biosynthesis of the neurotransmitters dopamine and serotonin. The effects of deficiencies in dopamine and serotonin are collectively known as “atypical” or “malignant” HPA. Thus, traditional diagnoses of HPA have involved a determination of whether the HPA is a result of BH4 deficiency or PAH deficiency. Typically, diagnosis of PKU is established on the basis of a persistently elevated blood Phe concentration. Following a positive screen for elevated blood Phe (plasma Phe >120 μmol; Weglage et al., J. Inherit. Metab. Dis., 25:321-322, 2002), a differential screen is performed in which it is determined whether the elevated Phe is a result of BH4 deficiency or PAH deficiency. The differential diagnosis involves determining whether the elevated Phe concentration is decreased as a result of BH4 administration (BH4 loading test). The BH4 loading test typically involves a one-time load of BH4 e.g., 5-20 mg/kg being administered to the subject who is on a normal (i.e., unrestricted) diet and determining whether the subject experiences a decrease in Phe levels (see e.g., Ponzone et al., Eur. J. Pediatr. 152:655-661, 1993; Weglage et al., J. Inherit. Metab. Dis., 25:321-322, 2002.)

Typically, individuals that respond to a BH4 loading test by a decrease in plasma Phe levels are diagnosed as having a defect in BH4 homeostasis. However, there have been various reports of patients with a BH4 responsive type of PAH deficiency (Kure et al., J. Pediatr. 135:375-378, 1999; Lassker et al., J. Inherit. Metabol. Dis. 25:65-70, 2002; Linder et al., Mol. Genet. Metab. 73:104-106, 2001; Spaapen et al., Mol. Genet. and Metabolism, 78:93-99, 2003; Trefz et al., 2001). These subjects have plasma Phe levels that are typical of moderate PKU, i.e., less than 1000 μmol/L and typically less than 600 μmol/L. Patients that have severe classical PKU are not responsive to typical 24 hour BH4 loading tests (Ponzone et al., N. Engl. J. Med 348(17):1722-1723, 2003).

It has been suggested that individuals that are responsive to BH4 do not require dietary intervention, but rather should be treated with BH4. Likewise, the converse has been suggested for subjects that have been diagnosed as non-responsive to the BH4 loading test, i.e., these subjects should be treated with dietary restriction and not BH4 therapy. Ponzone et al. particularly noted that individuals that have severe phenylketonuria will not respond to BH4 therapy and such therapy should not be used on these patients (Ponzone et al., N. Engl. J. Med 348(17):1722-1723, 2003). Thus, presently there are divergent therapeutic regimens for treatment of HPA depending on whether or not the individual is responsive to BH4. Moreover, it has been suggested that very few patients will benefit from BH4 therapy. In fact, it is thought that the only individuals with a PAH-deficient form of HPA that will benefit from BH4 therapy are those with mild PKU. As these individuals will typically have Phe levels in the safe range (i.e., less than 600 μM), the disease state can be controlled using moderate dietary restriction (see Hanley, N. Engl. J. Med 348(17):1723, 2003). Thus, BH4 therapy either alone, or in combination with any other therapeutic intervention, has not being considered as a viable therapeutic intervention for the vast majority of individuals with HPA.

BH4 is a biogenic amine of the naturally-occurring pterin family. Pterins are present in physiological fluids and tissues in reduced and oxidized forms, however, only the 5,6,7,8, tetrahydrobiopterin is biologically active. This is a chiral molecule and the 6R enantiomer of the cofactor is known to be the biologically active enantiomer. For a detailed review of the synthesis and disorders of BH4 see Blau et al., 2001 (Disorders of tetrahydrobiopterin and related biogenic amines. In: Scriver C R, Beaudet A L, Sly W S, Valle D, Childs B, Vogelstein B, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill, 2001: 1275-1776). Despite the elucidation of the role of BH4 deficiency in HPA, treatment with BH4 has not been suggested because such treatment is very expensive, as high as $30,000 per year for an adolescent or adult, as compared with $6,000 for phenylalanine-restricted dietary therapy (Hanley, N. Engl. J. Med 348(17):1723, 2003). Another significant problem with BH4 is that this compound is unstable and readily undergoes aerobic oxidation at room temperature (Davis et al., Eur. J. Biochem., Vol 173, 345-351, 1988; U.S. Pat. No. 4,701,455) and has a shelf-life of less 8 hours at room temperature (Berneggar and Blau, Mol. Genet. Metabol. 77:304-313, 2002).

Thus, to date, dietary intervention is the typical therapeutic intervention used for all patients with severe classical PKU and in many patients with moderate PKU. Such dietary intervention typically entails restricting the patient to foodstuff that is composed of natural foods which are free from, or low in, Phe. However, in addition to eliminating Phe, such a dietary regimen eliminates many sources of other essential amino acids, vitamins and minerals. Consequently, without supplementation, such a diet provides inadequate protein, energy, vitamins and minerals to support normal growth and development. As PKU is a manifestation of a lack of tyrosine, which arises due to the lack of hydroxylation of phenylalanine, tyrosine becomes an essential amino acid and dietary supplements for PKU must contain a tyrosine supplement. Therefore, it is common to use nutritional formulas to supplement the diets of PKU patients. Also, for babies, it is common to use infant formulas which have a low Phe content as the sole or primary food source.

However, dietary protein restriction is at best an ineffective way of controlling PKU in many classes of patients. For example, treatment is of paramount importance during pregnancy because high Phe levels may result in intrauterine retardation of brain development. However, a low protein diet during pregnancy may result in retarded renal development and is thought to produce a subsequent reduction in the number of nephrons and potentially leads to hypertension in adulthood. (D'Agostino, N. Engl. J. Med. 348(17)1723-1724, 2003).

Poor patient compliance with a protein-restricted diet also is a problem. The Phe-free protein formulae available are bitter tasting making it difficult to ensure that the patient consumes sufficient quantities of the protein to maintain the required daily intakes of protein, amino acids, vitamins, minerals, and the like. This is particularly a problem with older children who may require up to 70 g, dry weight, of the formulas per day. For example, Schuett, V. E.; 1990; DHHS Publication No HRS-MCH-89-5, reports that more than 40% of PKU patients in the US of eight years or older no longer adhere to the dietary treatment. (U.S. Pat. No. 6,506,422). Many adolescent patients fail to rigorously follow the protein-restricted diet due to fears of peer attitude.

Thus, there remains a need for a therapeutic medicament to replace or supplement and alleviate the dietary restrictions under which a PKU patient is placed. The present invention is directed to addressing such a need.

The invention describes intervention in metabolic disorders, particularly those involving amino acid metabolism. More particularly, the present invention is directed to methods and compositions for the treatment of subjects exhibiting elevated phenylalanine levels, for example, subjects suffering from hyperphenylalanemia, mild phenylketonuria or classic severe phenylketonuria; and methods and compositions for the treatment of subjects suffering from conditions that would benefit from enhancement of nitric oxide synthase activity; and methods and compositions for treatment of subjects suffering from vascular diseases, ischemic or inflammatory diseases, diabetes, or insulin resistance.

In one aspect, the invention describes methods of treating classic severe phenylketonuria (PKU) in a subject comprising administering to the subject a protein-restricted diet in combination with a composition comprising tetrahydrobiopterin (BH4) or a precursor or derivative thereof, wherein the combined administration of the protein-restricted diet and BH4 is effective to lower the phenylalanine concentration in the plasma of the subject as compared to the concentration in the absence of the combined administration. In specific embodiments, the subject is one who does not manifest a deficiency in BH4 homeostasis. The subject may be an individual that does not manifest symptoms of L-dopa neurotransmitter deficiency.

A subject selected from treatment according to the methods of the invention will have an elevated plasma Phe concentration, such a concentration may be greater than 1800 μM/L in the absence of the therapeutic. Other embodiments contemplate that has a plasma phenylalanine concentration of greater than 1000 μM in the absence of a therapeutic regimen. In preferred embodiments, the combined administration methods of the invention decrease the plasma phenylalanine concentration of the subject to less than 600 μM. More preferably, it is decreased to less than 500 μM. Even more preferably, the combined administration decreases the plasma phenylalanine concentration of the subject to 36 μM±15 μM.

The BH4 is preferably administered in an amount of between about 1 mg/kg to about 30 mg/kg, more preferably between about 5 mg/kg to about 30 mg/kg. The BH4 may be administered in a single daily dose or in multiple doses on a daily basis. In some embodiments, the BH4 therapy is not continuous, but rather BH4 is administered on a daily basis until the plasma phenylalanine concentration of the subject is decreased to less than 360 μM. Preferably, wherein the plasma phenylalanine concentration of the subject is monitored on a daily basis and the BH4 is administered when a 10% increase in plasma phenylalanine concentration is observed. Preferably, the BH4 being administered is a stabilized crystallized form of BH4 that has greater stability than non-crystallized stabilized BH4. More preferably, the stabilized crystallized form of BH4 comprises at least 99.5% pure 6R BH4. Precursors such as dihydrobiopterin (BH2), and sepiapterin also may be administered. BH4 may be administered orally.

The protein-restricted diet administered in the methods herein is one that is a phenylalanine-restricted diet wherein the total phenylalanine intake of the subject is restricted to less than 600 mg per day. In other embodiments, the protein-restricted diet is a phenylalanine-restricted diet wherein the total phenylalanine is restricted to less than 300 mg per day. In still other embodiments, the protein-restricted diet is one which is supplemented with amino acids, such as tyrosine, valine, isoleucine and leucine. In certain embodiments, protein-restricted diet comprises a protein supplement and the BH4 is provided in the same composition as the protein supplement.

In specific embodiments, the subject is one which has been diagnosed as having a mutant phenylalanine hydroxylase (PAH). The mutant PAH may comprise a mutation in the catalytic domain of PAH. Exemplary such mutations include one or more mutations selected from the group consisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C.

Also contemplated herein is a method for the treating a pregnant female having hyperphenylalaninemia (HPA) comprising administering to the subject a protein-restricted diet in combination with a composition comprising tetrahydrobiopterin (BH4) or a precursor or derivative thereof, wherein the combined administration of the protein-restricted diet and BH4 is effective to lower the phenylalanine concentration in the plasma of the subject as compared to the concentration in the absence of the combined administration. In certain embodiments, the subject has an unrestricted plasma phenylalanine concentration of greater than 180 μM but less than 60 μM. In other embodiments, the subject has an unrestricted plasma phenylalanine concentration of greater than 50 μM but less than 1200 μM. In still other embodiments, the subject has an unrestricted plasma phenylalanine concentration of greater than 1000 μM.

Also contemplated is a method of treating a patient having above normal concentration of plasma phenylalanine (e.g., greater than 180 μM/L and more preferably, greater than 36 μM/L) comprising administering to the patient a stabilized BH4 composition in an amount effective to produce a decrease in the plasma phenylalanine concentration of the patient. Preferably, the stabilized BH4 composition is stable at room temperature for more than 8 hours. The patient will likely have a plasma phenylalanine concentration greater than 18 μM prior to administration of the BH4. More particularly, the patient has a plasma phenylalanine concentration of between 120 μM and 200 μM. In other embodiments, the patient has a plasma phenylalanine concentration of between 200 μM and 600 μM. In still other embodiments, the patient has a plasma phenylalanine concentration of between 600 μM and 1200 μM. Yet another class of patients to be treated are those that have an unrestricted plasma phenylalanine concentration greater than 1200 μM. In specific embodiments, the patient is an infant, more particularly, an infant having a plasma phenylalanine concentration greater than 1200 μM. In other embodiments, the patient is pregnant and pregnant patient has a plasma phenylalanine concentration of between about 200 μM to about 600 μM. Pregnant patients with a plasma phenylalanine concentration greater than 1200 μM are particularly attractive candidates for this type of therapy, as are patient who are females of child-bearing age that are contemplating pregnancy. In those embodiments, in which the patient has a plasma phenylalanine concentration greater than 1200 μM, and the method further comprises administering a protein-restricted diet to the patient.

The invention also contemplates a method of treating a patient having phenylketonuria, comprising administering to the patient a stabilized BH4 composition in an amount effective to produce a decrease in the plasma phenylalanine concentration of the patient wherein the patient has been diagnosed as unresponsive to a single-dose BH4 loading test. Preferably, the patient is unresponsive within 24 hours of the BH4 load.

Another related aspect of the invention provides a multiple dose loading test that involves administration of more than one dose of BH4. The data described herein demonstrates that subjects who are considered “unresponsive” to a single dose BH4 loading test can respond to multiple doses of BH4 with a significant reduction in phenylalanine levels. In one embodiment, at least two doses of BH4 which may be between about 5 mg to 40 mg are administered to a subject over a time period of more than one day, preferably 7 days.

The treatment methods according to the invention may comprise administering between about 10 mg BH4/kg body weight to about 200 mg BH4/kg body weight. The BH4 may be administered through any route commonly used in practice, e.g., orally, subcutaneously, sublingually, parenterally, per rectum, per and nares. The BH4 may be administered daily or at some other interval, e.g., every alternative day or even weekly. The BH4 is preferably administered in combination with a protein-restricted diet, and optionally concurrently with folates, including folate precursors, folic acids, and folate derivatives.

It is contemplated that that BH4 will be administered as part of a component of a therapeutic protein formulation. The protein-restricted diet may comprise a normal diet of low-protein containing foodstuff. Alternatively, the protein-restricted diet comprises an intake of protein formula that is phenylalanine-free protein diet, and the subject obtains his essential amount of Phe from remaining components of a very low protein diet. In certain embodiments, the protein-restricted diet is supplemented with non-phenylalanine containing protein supplements. More particularly, the non-phenylalanine containing protein supplements comprise tyrosine or other essential amino acids. In other embodiments, the protein supplements may also comprise folates, including folate precursors, folic acids, and folate derivatives.

The invention contemplates methods of treating an infant having phenylketonuria, comprising administering a stabilized BH4 composition to the patient in an amount effective to produce a decrease in the plasma phenylalanine concentration of the infant wherein the infant is between 0 and 3 years of age and the infant has a plasma phenylalanine concentration of between about 360 μM to about 4800 μM. Prior to the administering of BH4, the infant has a phenylalanine concentration of about 1200 μM and the administering of BH4 decreases the plasma phenylalanine concentration to about 1000 μM. In other embodiments, prior to the administering of BH4 the infant has a phenylalanine concentration of about 800 μM and the administering of BH4 decreases the plasma phenylalanine concentration to about 600 μM. In still further embodiments, prior to the administering of BH4 the infant has a phenylalanine concentration of about 400 μM and the administering of BH4 decreases the plasma phenylalanine concentration to about 300 μM. The therapeutic methods contemplated herein should preferably reduce the plasma phenylalanine concentration of the infant to 360±15 μM.

Also contemplated is a composition comprising a stabilized, crystallize form of BH4 that is stable at room temperature for more than 8 hours and a pharmaceutically acceptable carrier, diluent or excipient. The composition may further comprise a medical protein supplement. In other embodiments, the BH4 composition is part of an infant formula. In still other embodiments, the protein supplement is phenylalanine free. The protein supplement preferably is fortified with L-tyrosine, L-glutamine, L-carnitine at a concentration of 20 mg/100 g supplement, L-taurine at a concentration of 40 mg/100 g supplement and selenium. It may further comprise the recommended daily doses of minerals, e.g., calcium, phosphorus and magnesium. The supplement further may comprise the recommended daily dose of one or more amino acids selected from the group consisting of L-leucine, L-proline, L-lysine acetate, L-valine, L-isoleucine, L-arginine, L-alanine, glycine, L-asparagine monohydrate, L-tryptophan, L-serine, L-threonine, L-histidine, L-methionine, L-glutamic acid, and L-aspartic acid. In addition, the supplement may be fortified with the recommended daily dosage of vitamins A, D and E. The supplement preferably comprises a fat content that provides at least 40% of the energy of the supplement. Such a supplement may be provided in the form of a powder supplement or in the form of a protein bar.

Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Dietary intervention is the therapeutic intervention used for all patients with severe classical PKU and in many patients with moderate PKU. However, such dietary protein restriction leads to an inadequate supply of protein, energy, vitamins and minerals to support normal growth and development. Thus, dietary protein restriction is at best an ineffective way of controlling the PKU in many classes of patients, especially in pregnant women and in young children, both categories of subjects that require elevated amounts of protein as compared to normal adult individuals. Use of dietary restriction also is hampered by poor patient compliance with a protein-restricted diet. In October 2000, the National Institutes of Health issued a consensus statement on PKU screening and management in which “research on nondietary alternatives to treatment of PKU [was] strongly encouraged.” Thus, there is an art-recognized need for a therapeutic medicament to replace and/or supplement and alleviate the dietary restrictions under which a PKU patient is placed.

The present application for the first time describes a pharmaceutical intervention of PKU based on the administration of a stabilized form of BH4. The methods and compositions for producing such a stabilized BH4 compositions are described in further detail in Example 2. The stabilized BH4 compositions of the present invention comprise BH4 crystals that are stable at room temperature for longer than 8 hours. The methods and compositions of the present invention contemplate pharmaceutical compositions of the stabilized BH4 alone that may be delivered through any conventional route of administration, including but not limited to oral, intramuscular injection, subcutaneous injection, intravenous injection and the like. The compositions of the present invention may further comprise BH4 compositions in combination with an antioxidant that aids in prolonging the stability of the BH4 composition. In addition, discussed in greater below, the present invention further comprises foodstuffs that comprise BH4. For example, the invention contemplates conventional protein powder compositions such as PHENEX, LOFENALAC, PHENYL-FREE and the like that have been modified by the addition of BH4.

The present invention further contemplates the therapeutic intervention of various PKU phenotypes by administration of BH4 in combination with a protein-restricted diet. The BH4 to be administered in combination with the diet may, but need not necessarily, be a stabilized BH4 composition described herein. Those of skill in the art are aware of methods of producing a BH4 composition that is unstable at room temperature and in light. While therapies using such a composition are hindered by the instability of the BH4 composition, its use is still contemplated in certain combination therapies where BH4 non-responsive patients suffering from severe classical PKU are treated with a course of BH4 treatment and dietary protein restriction.

Methods and compositions for effecting the treatment of metabolic disorders, including PKU, are described in further detail herein below.

The present invention is directed to the treatment of a variety of HPA patient populations with methods that comprise the use of stabilized BH4 compositions, or unstabilized BH4 compositions, either alone or in combination with other therapeutic regimens, for managing HPA and/or PKU. In particular, it is contemplated that any type of BH4, in a stabilized or other form may be used to treat that patient population that has phenylalanine concentrations that are low enough that dietary intervention is not normally used (i.e., patients with mild HPA). Such patients that are amenable to all forms treatment with BH4 compositions to ameliorate the effects of mild HPA, include pregnant women and infants with serum concentrations of less than 200 μM. The various patient populations, and their different therapeutic needs, are discussed in further detail in the present section.

Certain embodiments of the present invention are directed to treating classic severe PKU by administering to the subject a protein-restricted diet in combination with a composition comprising BH4 or a precursor or derivative thereof, wherein the combined administration of the protein-restricted diet and BH4 is effective to lower the phenylalanine concentration in the plasma of said subject as compared to said concentration in the absence of said combined administration. In addition, the invention also contemplates treating a pregnant female that has HPA by administering to the female a protein-restricted diet in combination with BH4 or a precursor or derivative thereof, such that the combined administration of the protein-restricted diet and BH4 is effective to lower the phenylalanine concentration in the plasma of the pregnant woman as compared to such a concentration in the absence of said combined administration. In specific embodiments, therapy is contemplated for patient who manifest Phe levels greater than 420 μM.

Other embodiments of the invention entail administering a stabilized BH4 composition to any individual that has HPA, characterized by a plasma Phe concentration greater than 180 μM prior to the administration of the BH4, in an amount effective to produce a decrease in such a plasma Phe concentration of the patient. The methods of the invention also may be used in the treatment of PKU patients that that have been diagnosed as unresponsive to a BH4 loading test. The methods of the invention will be useful in treating an infant having PKU characterized by an elevated Phe concentrations of between greater than 300 μM/L with the stabilized BH4 compositions described herein. By “infant” the present application refers to a patient that is between the ages of 0 to about 36 months.

The data described herein demonstrates that subjects who are considered “unresponsive” to the single dose BH4 loading test may in fact respond to multiple doses of BH4 with a significant reduction in phenylalanine levels. Thus, another aspect of the invention provides a multiple dose loading test that involves administration of more than one dose of BH4. Exemplary multiple dose loading tests include administration of between 5 and 40 mg/kg tetrahydrobiopterin, or more preferably 10 to 20 mg/kg, over a time period of at least 1 day, or at least 2 days, or at least 3, 4, 5, 6, 7, 10 or 14 days, preferably 2-14, 3-14, or 5-10 days, and most preferably 7 days.

The invention provides methods of using any of the tetrahydrobiopterin polymorphs described herein, or stable pharmaceutical preparations comprising any of such polymorphs, for treatment of conditions associated with elevated phenylalanine levels or decreased tyrosine levels, which may be caused, for example, by reduced phenylalanine hydroxylase, tyrosine hydroxylase, or tryptophan hydroxylase activity. Conditions associated with elevated phenylalanine levels specifically include phenylketonuria, both mild and classic, and hyperphenylalaninemia as described elsewhere herein, and exemplary patient populations include the patient subgroups described herein as well as any other patient exhibiting phenylalanine levels above normal.

The invention further provides methods of using any of the polymorphs described herein, or stable pharmaceutical preparations comprising any of such polymorphs, for treatment of subjects suffering from conditions that would benefit from enhancement of nitric oxide synthase activity and patients suffering from vascular diseases, ischemic or inflammatory diseases, or insulin resistance. The treatment may, for example alleviate a deficiency in nitric oxide synthase activity or may, for example provide an increase in nitric oxide synthase activity over normal levels. It has been suggested that a patient suffering from a deficiency in nitric oxide synthase activity would benefit from treatment with folates, including folate precursors, folic acids, or folate derivatives. Thus, it is also contemplated, that compositions and methods disclosed herein include the concurrent treatment with folates, including folate precursors, folic acids, or folate derivatives. Exemplary folates are disclosed in U.S. Pat. Nos. 6,011,040 and 6,544,994, both of which are incorporated herein by reference, and include folic acid (pteroylmonoglutamate), dihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid, 5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid (leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolic acid, one or more of the folylpolyglutamates, compounds in which the pyrazine ring of the pterin moiety of folic acid or of the folylpolyglutamates is reduced to give dihydrofolates or tetrahydrofolates, or derivatives of all the preceding compounds in which the N-5 or N-10 positions carry one carbon units at various levels of oxidation, or pharmaceutically compatible salts thereof, or a combination of two or more thereof. Exemplary tetrahydrofolates include 5-formyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolic acid or (6S)-tetrahydrofolic acid, and salts thereof. as is treatment with a pharmaceutical composition or foodstuff that comprises both a tetrahydrobiopterin polymorph and a folate.

Nitric oxide is constitutively produced by vascular endothelial cells where it plays a key physiological role in the regulation of blood pressure and vascular tone. It has been suggested that a deficiency in nitric oxide bioactivity is involved in the pathogenesis of vascular dysfunctions, including coronary artery disease, atherosclerosis of any arteries, including coronary, carotid, cerebral, or peripheral vascular arteries, ischemia-reperfusion injury, hypertension, diabetes, diabetic vasculopathy, cardiovascular disease, peripheral vascular disease, or neurodegenerative conditions stemming from ischemia and/or inflammation, such as stroke, and that such pathogenesis includes damaged endothelium, insufficient oxygen flow to organs and tissues, elevated systemic vascular resistance (high blood pressure), vascular smooth muscle proliferation, progression of vascular stenosis (narrowing) and inflammation. Thus, treatment of any of these conditions is contemplated according to methods of the invention.

It has also been suggested that the enhancement of nitric oxide synthase activity also results in reduction of elevated superoxide levels, increased insulin sensitivity, and reduction in vascular dysfunction associated with insulin resistance, as described in U.S. Pat. No. 6,410,535, incorporated herein by reference. Thus, treatment of diabetes (type I or type II), hyperinsulinemia, or insulin resistance is contemplated according to the invention. Diseases having vascular dysfunction associated with insulin resistance include those caused by insulin resistance or aggravated by insulin resistance, or those for which cure is retarded by insulin resistance, such as hypertension, hyperlipidemia, arteriosclerosis, coronary vasoconstrictive angina, effort angina, cerebrovascular constrictive lesion, cerebrovascular insufficiency, cerebral vasospasm, peripheral circulation disorder, coronary arteriorestenosis following percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass grafting (CABG), obesity, insulin-independent diabetes, hyperinsulinemia, lipid metabolism abnormality, coronary arteriosclerotic heart diseases or the like so far as they are associated with insulin resistance. It is contemplated that when administered to patients with these diseases, BH4 can prevent or treat these diseases by activating the functions of NOS, increasing NO production and suppressing the production of active oxygen species to improve disorders of vascular endothelial cells.

A. Characteristics of Severe Classical PKU and Methods of Treatment Thereof According to the Present Invention.

As indicated herein above in the background section, severe PKU manifests in a plasma Phe concentration greater than 1200 μM/L and may be found to be as high as 4800 μM/L. Patients that have this disorder must be treated with a Phe-free diet in order to bring their plasma Phe concentrations down to a level that is clinically acceptable (typically, less than 600 μM/L, and preferably less than 300 μM/L). These patients are only able to tolerate a maximum of between 250-350 mg dietary Phe per day (Spaapen et al., Mol. Genet. and Metab. 78:93-99, 2003). As such, these patients are started on a Phe-restricted formula diet between 7-10 days after birth and are burdened with this dietary restriction for the remainder their lifespan. Any alleviation of the strict dietary restrictions that these individuals are encumbered with would be beneficial.

The tests used for the diagnosis of individuals with classical Phe are described in further detail below in Section III. These tests have revealed that patients with classical severe PKU are non-responsive to BH4 and require a low phenylalanine diet (Lucke et al., Pediatr. Neurol. 28:228-230, 2003). In the present invention however, it is contemplated that this class of PKU patients should be treated with BH4 in order that the need for a strict phenylalanine-free diet may be alleviated.

Thus, it is contemplated that the methods of the invention will entail determining that the patient is suffering from classical PKU by monitoring the plasma Phe concentration of the individual. The patient is then treated by administering a combined regimen of a low protein diet and BH4 such that there is produced at least a 25% decrease in the plasma Phe concentrations of the patient. Preferably, the method will produce a 30% decrease in the plasma Phe concentration. Even more preferably, the method will produce a 40%, 50%, 60%, 70%, 80%, 90% or greater decrease in the plasma Phe concentration of the individual (for example, where a patient with severe classical PKU has a Phe concentration of 4800 μM/L, a 90% decrease in the Phe concentration will produce a plasma Phe concentration of 480 μM/L, a concentration that is sufficiently low to require little dietary restriction). Of course, it should be understood that the treatment methods of the present invention (whether for treating severe classical PKU or any other HPA described herein), should attempt to lower the plasma Phe concentrations of the patient to levels as close to 360 μM/L±15 μM/L as possible.

In preferred embodiments the plasma Phe concentrations of the classical PKU patient being treated is reduced from any amount of unrestricted plasma Phe concentration that is greater than 1000 μM/L to any plasma Phe level that is less than 600 μM/L. Of course, even if the combined treatment with the BH4 and the protein-restricted diet produces a lesser decrease in plasma Phe concentration, e.g., to a level of between 800 μM/L to about 1200 μM/L, this will be viewed as a clinically useful outcome of the therapy because patients that have a plasma Phe concentration in this range can manage the disease by simply restricting the amount of protein in the diet as opposed to eating a Phe-restricted formula, thereby resulting in a marked improvement in the quality of life of the individual, as well as leading to greater patient compliance with the dietary restriction.

Any increase in the amount of dietary Phe levels that can be tolerated by the patient as a result of the treatment will be considered to be a therapeutically effective outcome. For example, it is contemplated that as a result of administering the BH4-based therapy, the patient will be able to increase his/her intake of dietary Phe from 250-350 mg/day to 350-400 mg/day (i.e., the Phe tolerance phenotype of the patient is altered from that of a classic PKU patient to a moderate PKU patient). Of course, it would be preferable that the therapeutic intervention taught herein would allow the patient to increase his/her intake of dietary Phe from 250-350 mg/day to 400-600 mg/day (i.e., the Phe tolerance phenotype of the patient is altered from that of a classic PKU patient to a mild PKU patient), or even more preferably, to allow the patient to have an intake of greater than 600 mg Phe/day (i.e., normal dietary intake).

B. Characteristics of BH4-Non Responsive PKU Patients and Methods of Treatment Thereof According to the Present Invention.

A second group of patients that can be treated with the methods of the present invention are those individuals that have a been determined to have an elevated plasma Phe concentrations i.e., any concentration that is greater than 200 μM/L, but have been diagnosed to be non-responsive to BH4 therapy (as determined by the BH4 loading test described below). Such patients may include those individuals that have mild PKU (i.e., plasma Phe concentrations of up to 600 μM/L), individuals that have moderate PKU (i.e., plasma Phe concentrations of between 600 μM/L to about 1200 μM/L), as well as patients that have classic severe PKU (i.e., plasma Phe concentrations that are greater than 1200 μM/L).

The patients that are non-responsive to BH4 therapy are given BH4 in combination with a reduced amount of protein in their diet in order to decrease the plasma Phe concentrations of the patient. The methods of the present invention are such that the administration of the BH4 therapy produces a greater decrease in the plasma Phe concentrations of the patient as compared to the decrease that is produced with the same dietary protocol administered in the absence of the BH4 therapy.

In preferred embodiments, the patients are administered a composition that comprises a stabilized, crystallized form of BH4 characterized in Example 2 described herein below. This BH4 composition differs from those previously available in the art in that it is more stable at room temperature than the preparations previously known to those of skill in the art, e.g., those available in the BH4 loading kits (Schircks Laboratories, Jona, Switzerland.) Thus, the BH4 formulation may be stored at either room temperature or refrigerated and retain greater potency than the previously available BH4 compositions. As such, it is contemplated that this form of BH4 will have a greater therapeutic efficacy than a similar concentration the previously available BH4 compositions. This greater efficacy may be used to produce a therapeutically effective outcome even in patients that were previously identified as being non-responsive to BH4.

As with the subset of patients described in Section IA above, the BH4 non-responsive patients described in the present section may be treated by the stabilized BH4 compositions either alone or in combination with dietary restrictions. The dietary restrictions may be as a diet that restricts the Phe intake by providing a synthetic medical protein formula that has a diminished amount of Phe or alternatively, the dietary restriction may be one which simply requires that the patient limit his/her overall protein intake but nevertheless allows the patient to eat normal foodstuffs in limited quantities.

The preferred therapeutic outcomes discussed for classical PKU patients in Section IA above are incorporated into the present section by reference. Preferred therapeutic outcomes for patients with moderate PKU (i.e., patients that has an unrestricted plasma Phe concentration of 600 μM/L to 1200 μM/L) include at least a 25% decrease in the plasma Phe concentrations of the patient. Preferably, the method will produce a 30% decrease in the plasma Phe concentration. Even more preferably, the method will produce a 40%, 50%, 60%, 70%, 80%, 90% or greater decrease in the plasma Phe concentration of the individual (for example, where a patient with moderate classical PKU has a Phe concentration of 1000 μM/L, a 90% decrease in the Phe concentration will produce a plasma Phe concentration of 100 μM/L, a concentration that is sufficiently low to require little dietary restriction).

In preferred embodiments, the plasma Phe concentrations of the moderate PKU patient being treated is reduced from any amount of unrestricted plasma Phe concentration that is between 600 μM/L to 1200 μM/L to any plasma Phe level that is less than 300 μM/L. A particularly preferred treatment with the BH4 (either alone or in combination with a dietary restriction) produces a decrease in plasma Phe concentration, e.g., to a level of between 200 μM/L to about 400 μM/L, which will be viewed as a clinically useful outcome of the therapy because patients that have a plasma Phe concentration in this range can manage the disease by simply restricting the amount of protein in the diet as opposed to eating a Phe-restricted formula. Indeed, in many studies, it is taught that such patients may even eat a normal diet.

Any increase in the amount of dietary Phe levels that can be tolerated by the patient as a result of the treatment will be considered to be a therapeutically effective outcome. For example, it is contemplated that as a result of administering the BH4-based therapy (either alone or in combination with other therapeutic intervention), the patient will be able to increase his/her intake of dietary Phe from 350-400 mg/day to 400-600 mg/day (i.e., the Phe tolerance phenotype of the patient is altered from that of a moderate PKU patient to a mild PKU patient). Of course, it would be preferable that the therapeutic intervention taught herein would allow the patient to increase his/her intake of dietary Phe from 350-400 mg/day to 400 to allow the patient to have an intake of greater than 600 mg Phe/day (i.e., normal dietary intake).

Even if the patient being treated is one who manifests only mild PKU, i.e., has a dietary allowance of 400-600 mg Phe intake/day) will benefit from the BH4-based therapies of the present invention because it is desirable to produce a normalized plasma Phe concentration that is as close to 360 μM/L±15 μM/L as possible. For such patients, a preferred therapeutic outcomes will include at least a 25% decrease in the plasma Phe concentrations of the patient. Preferably, the method will produce a 30% decrease in the plasma Phe concentration. Even more preferably, the method will produce a 40%, 50%, 60%, or greater decrease in the plasma Phe concentration of the individual (for example, where a patient with mild PKU has a Phe concentration of 600 μM/L, a 60% decrease in the Phe concentration will produce a plasma Phe concentration of 360 μM/L, i.e., an acceptable, normal concentration of plasma Phe).

In preferred embodiments, the plasma Phe concentrations of the mild PKU patient being treated is reduced from any amount of non-restricted plasma Phe concentration that is between 400 μM/L to 600 μM/L to any plasma Phe level that is less than 100 μM/L. Of course, even if the treatment with the BH4 (either alone or in combination with a dietary restriction) produces a lesser decrease in plasma Phe concentration, e.g., to a level of between 200 μM/L to about 400 μM/L, this will be viewed as a clinically useful outcome of the therapy.

Any increase the amount of dietary Phe levels that can be tolerated by the patient as a result of the treatment will be considered to be a therapeutically effective outcome. For example, it is contemplated that as a result of administering the BH4-based therapy (either alone or in combination with other therapeutic intervention), the patient will be able to increase his/her intake of dietary Phe from 400-600 mg/day (i.e., the Phe tolerance phenotype of the patient is altered from that of a mild PKU patient to a mild HPA patient) to allow the patient to have an intake of greater than 600 mg Phe/day (i.e., normal dietary intake).

Furthermore, even if the patient is one who only manifests the symptoms of non PKU HPA, i.e., has an elevated plasma Phe concentration of up to 600 μM/L, but is otherwise allowed to eat a normal protein diet will benefit from the BH4 therapies of the invention because it has been shown that elevated Phe concentrations have significant effects on the IQ of such individuals. Moreover, as discussed below, BH4-based therapeutic intervention of subjects with special needs, e.g., pregnant women and infants, is particularly important even if that patient's plasma Phe levels are within the perceived “safe” level of less than 200 μM/L.

C. Maternal PKU and Methods of Treatment Thereof According to the Present Invention.

Metabolic control of plasma Phe levels in PKU women planning conception and those who are pregnant is important because of the serious consequences to the fetus exposed to even moderately elevated Phe levels in utero, regardless of the PAH status of the fetus. Therapeutic control of plasma Phe concentration is especially important in the first trimester of pregnancy, as failure to achieve adequate control will result in disorders including microcephaly, mental deficiency and congenital heart disease.

For example, the NIH Consensus Statement (vol 17 #3, October 2000) on Phenylketonuria reported that exposure of a fetus to maternal Phe levels of 3-10 mg/dL produced a 24% incidence of microcephaly, whilst those exposed to greater than 20 mg/dL (i.e., greater than 1200 μM/L) had a 73% incidence of microcephaly. Likewise congenital heart disease was found in over 10% of children exposed to maternal Phe levels that were greater than 20 mg/dL. Importantly, it has been noted that levels of Phe greater than 6 mg/dL significantly decrease the IQ of the child. Thus, it is imperative to ensure that the plasma Phe concentration of women with all forms of phenylketonuria, even those manifesting the mildest HPA, must be tightly controlled in order to avoid the risk of maternal PKU syndrome. However, the acceptable target levels for the plasma Phe concentrations of PKU women that have been used in U.S. clinics have ranged between 10 mg/dL and 15 mg/dL, which are much higher than the 2-6 mg/dL levels recommended for pregnant women or the 1-4 mg/dL that are used in British and German clinics to diminish the risks of developing maternal PKU syndrome.

Another important consideration for pregnant women is their overall protein intake. During pregnancy, it is important that women eat sufficient protein because it has been suggested that a low protein diet during pregnancy will result in retarded renal development and subsequent reduction in the number of nephrons and potentially leads to hypertension in adulthood. (D'Agostino, N. Engl. J. Med. 348(17)1723-1724, 2003). The following table provides exemplary guidelines for the recommended total dietary protein intake for various individuals.

The actual amount of protein ingested depends on the Phe content of the protein. The amino acid profiles of plant proteins is different from animal proteins. For example, with a focus on starches and vegetables, a general rule of 45-50 mg/Phe per gram of protein may suffice. However, an accepted standard for evaluating the constituents amino acids of a protein is an egg white, which contains 3.5 grams of protein of which 204 mg is Phe.

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