TETRAHYDROBIOPTERIN FORMULATIONS

INTRODUCTION

Aspects of the present application relate to oral pharmaceutical formulations of tetrahydrobiopterin or analogs or derivatives, and to uses thereof for the treatment of metabolic disorders such as hyperphenylalaninemia in humans. In embodiments, the present application relates to stable solid formulations of tetrahydrobiopterin that are easily solubilized.

Tetrahydrobiopterin (BH4, or THB, or sapropterin) is a naturally occurring biogenic amine that is an essential cofactor for a number of different enzymes including the aromatic amino acid hydroxylase enzymes used in the degradation of amino acid phenylalanine and biosynthesis of various neurotransmitters. Of the naturally occurring pterins, only the 6R enantiomer of 5, 6, 7, 8-tetrahydrobiopterin is biologically active. Phenylalanine hydroxylase (PAH) is an enzyme which converts the amino acid phenylalanine to other essential compounds in the body. PAH deficiency due to absent or mutated PAH enzyme or a deficiency in its cofactor BH4, causes a spectrum of disorders including classic phenylketonuria (PKU) and hyperphenylalaninemia (a less severe accumulation of phenylalanine). When there is a defect in the biosynthesis or recycling of the cofactor tetrahydrobiopterin (BH4) by the patient, the coenzyme (also called bio-pterin) can be supplemented as treatment.

Although the role of BH4 deficiency in hyperphenylalaninemia is known, treatment with BH4 has not been widely suggested because such treatment is very expensive (Hanley, New England Journal of Medicine 348(17): 1723, 2003), the compound is unstable and readily undergoes aerobic oxidation at room temperature (Davis et al., European Journal of Biochemistry, Vol 173, 345-351, 1988; U.S. Patent No. 4,701,455) and has a shelf-life of less 8 hours at room temperature (Berneggar and Blau, Molecular Genetetics and Metabolism 77:304-313, 2002). Tetrahydrobiopterin products available on the market need to be specially packaged or kept frozen. For example the labeling on the tablets sold by Schirck's Laboratory specifies that the tablets should be kept frozen and states that the product has a shelf life at room temperature of only 2 months. Biopten® (tetrahydrobiopterin granules) requires expensive, hermetically-sealed foil packaging to maintain room temperature stability. The instability of such BH4 compositions is commercially undesirable and significant degradation due to improper storage could hinder therapy of patients. Kuvan™ (sapropterin dihydrochloride) tablets contain ascorbic acid as antioxidant and are highly sensitive to moisture. The tablets are supplied in high-density polyethylene bottles with a silica gel desiccant cartridge, and a pharmaceutical-grade polyester coil.

Thus there is a need for solid formulations of tetrahydrobiopterin that are both affordable and stable and for processes for the preparation of the same.

SUMMARY

Aspects of the present application relate to solid formulations of tetrahydrobiopterin, such as tablets, processes for producing such formulations, and treatment methods using such formulations.

Embodiments of the present application provide stable solid formulations of tetrahydrobiopterin, or precursors or derivatives or analogs thereof that comprise a stable, crystalline form of BH4 and a carrier, diluent, or other excipient.

The present application also provides processes for the preparation of stable solid formulations of tetrahydrobiopterin such as tablets, capsules, granules, or powders.

In an aspect, the application includes granulation of tetrahydrobiopterin with suitable excipients and the processing of such granules into tablets, or filling of the granules into capsules.

In embodiments, the tetrahydrobiopterin compound is dry blended with suitable pharmaceutically acceptable excipients and compressed into tablets.

In an aspect, formulations of the present application contain the stable crystalline polymorphic Form B of (6R)-5, 6, 7, 8-tetrahydrobiopterin, in amounts ranging from about 20 mg to about 1000 mg.

In an aspect, formulations of the present application are designed to disintegrate rapidly after administration, to enable ease of administration.

In an aspect, stable pharmaceutical compositions of the present application contain one or more of additional ingredients such as a binder, disintegrant, antioxidant, lubricant, or any combinations thereof.

The stable solid formulations may optionally include other therapeutic agents suitable for the condition to be treated, e.g., folates, including folate precursors, folic acids, or folate derivatives, and/or vitamins such as vitamin C and/or vitamin B12, and/or neurotransmitter precursors such as L-dopa or carbidopa, and/or 5-hydroxytryptophan, and/or arginine.

An aspect of the application provides treatment methods using solid formulations. The formulations of the present application are useful for intervention in metabolic disorders, in patients exhibiting elevated phenylalanine levels or decreased tyrosine levels, for example, subjects suffering from hyperphenylalanemia, mild phenylketonuria, or classic severe phenylketonuria, and for the treatment of subjects suffering from conditions that would benefit from enhancement of nitric oxide synthase activity, such as vascular diseases, and ischemic and inflammatory diseases. The total dose required for each treatment may be administered in multiple doses or in a single dose. The stable formulations may be administered daily or at some other interval, e.g., every other day or even weekly.

DETAILED DESCRIPTION

Aspects of the present application provide stable formulations containing an anhydrous polymorphic form of (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride that is stable at room temperature to atmospheric oxygen and normal humidity, designated "polymorph B."

Tetrahydrobiopterin (sometimes referred to as BH4) is a biogenic amine of the naturally-occurring pterin family that is a cofactor for a number of different enzymes, including phenylalanine hydroxylase (PAH), tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase. Pterins are present in physiological fluids and tissues in reduced and oxidized forms, however, only the 5,6,7,8-tetrahydrobiopterin is biologically active. Sapropterin dihydrochloride is a synthetic preparation of the dihydrochloride salt of naturally occurring tetrahydrobiopterin (BH4) having a chemical name (6R)-2-amino-6-[(1 R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro-4(1H)-pteridone dihydrochloride and the following structural formula.

For the purposes of this application, "tetrahydrobiopterin" is meant to include tetrahydrobiopterin, sapropterin, and any salts, precursors, derivatives or analogs thereof.

As described in US Pat. Nos. 7,566,462 and 7,727,987, polymorph B is a slightly hygroscopic anhydrate having thermodynamic stability above about 20°C. Polymorph B exhibits an X-ray powder diffraction pattern, expressed in d-spacing values (A), with peaks at: 8.7 (vs), 6.9 (w), 5.90 (vw), 5.63 (m), 5.07 (m), 4.76 (m), 4.40 (m), 4.15 (w), 4.00 (s), 3.95 (m), 3.52 (m), 3.44 (w), 3.32 (m), 3.23 (s), 3.17 (w), 3.11 (vs), 3.06 (w), 2.99 (w), 2.96 (w), 2.94 (m), 2.87 (w), 2.84 (s), 2.82 (m), 2.69 (w), 2.59 (w), and 2.44 (w). As used herein, the abbreviations in parentheses have the meanings: vs=very strong intensity; s=strong intensity; m=medium intensity; w=weak intensity; and vw=very weak intensity.

In embodiments of the compositions and methods of the present application, a composition including polymorph B of may be prepared by dissolving a solid form of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in water at ambient temperature, adding a non-solvent in an amount sufficient to form a suspension, optionally stirring the suspension for a certain time, and thereafter isolating the formed crystals. The composition may be further processed into a suitable pharmaceutical formulation. Suitable non-solvents are for example methanol, ethanol, isopropanol, acetic acid, acetonitrile, and acetone.
Pharmaceutical dosage forms of the present application may be in the form of tablets, capsules, and particulates (e.g., granules, beads, powders, and pellets).

The pharmaceutical formulations of the present application may, in addition to tetrahydrobiopterin and other desired active ingredients, also contain one or more additional formulation ingredients chosen from a wide variety of excipients known in the art. According to the desired properties of the formulation, any number of ingredients may be used, alone or in combination, based upon their functions. Such ingredients include, but are not limited to, diluents, binders, disintegrants, compression aids, lubricants, flavors, sweeteners, colorants, and preservatives.

The term "tablet" as used herein is intended to encompass compressed pharmaceutical dosage forms of any shapes and sizes, whether coated or uncoated.

For soluble powder formulations, the composition of a powder is similar to that of tablets, except that a lubricant is generally present in lesser amounts and a binder is a dry binder. In embodiments, a powder is granulated.
The term "capsule" as used herein refers to a hard gelatin capsule filled with one or more tablets, mini-tablets, granules, and/or powders of the present application.

In embodiments, the present application is directed to oral pharmaceutical formulations comprising sapropterin dihydrochloride, an acidic antioxidant, a binder, a disintegrant, an acidifying agent, and a lubricant, and optionally one or more additional agents such as flavoring agents, colorants, and sweeteners.

In embodiments, the present application is directed to soluble formulations comprising tetrahydrobiopterin
as an active ingredient, a superdisintegrant such as croscarmellose, crospovidone, or sodium starch glycolate, an antioxidant such as ascorbic acid, an acidifying agent, and other desired excipients.

As used herein, an antioxidant is an agent that acts to inhibit the oxidation of other substances.

Antioxidants help to stabilize tetrahydrobiopterin products, especially after dissolution. Non-limiting examples of antioxidants include ascorbic acid, fatty acid esters of ascorbic acid such as ascorbyl palmitate and ascorbyl stearate, amino acid salts such as L-cysteine hydrochloride, and salts of ascorbic acid such as sodium, calcium, or potassium ascorbate, all of which are acidic. Alternatively, non-acidic antioxidants such as beta-carotene or alpha-tocopherol may also be used in stable tablet formulations.
Exemplary concentrations of an antioxidant in a stable tablet formulation of the present application are between about 1 and about 3 wt %.

Suitable weight ratios of antioxidant to tetrahydrobiopterin in a stable tablet formulation of the present application are, for example, in the range of about 1:1 to about 1:40. For example, weight ratios of antioxidant to tetrahydrobiopterin may be about 1:1.3, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:36, about 1:40 etc.

An acidic additive or acidifying agent such as citric acid or malic acid may be added to enhance stability of the tablet formulation.

Typically, pharmaceutical formulations of the application have total weights of about 200 mg or more. In some embodiments, the formulations have total weights up to about 1000 mg.

Embodiments of formulations of the application can have disintegration times of about 180 seconds or less, upon contact with about 120-240 mL of water.

Formulations of the present application can be prepared using conventional methods that are well known in the art. Granules can be formed by any processes, using operations such as one or more of dry granulation, wet granulation, extrusion-spheronization, and the like. In embodiments, a granulating fluid used is nonaqueous.

In embodiments, the granulation of the active ingredient, optionally with one or more pharmaceutically acceptable excipients such as diluents or fillers, may be carried out in equipment such as planetary mixers, double cone blenders, rapid mixer granulators (RMG), fluid bed processors, and the like.

Alternatively, powder blends may be compacted using a roller compactor and then milled to produce granules that are suitable for compression. The granules obtained may further be compressed into tablets or filled into capsules, using techniques known in the art. Alternatively, tablets may be prepared by a direct compression technique, using powder blends. Alternatively, other technologies known in the art, such as freeze-drying, spray drying, mass extrusion and moulding, may also be used to prepare formulations in accordance with the present application.

In the context of the present application, during the processing of the pharmaceutical formulations into finished dosage forms, one or more pharmaceutically acceptable excipients may optionally be included, such as but not limited to one or more diluents, binders, disintegrants, lubricants, glidants, coloring agents, film-forming agents, and others.

Various useful fillers or diluents include, but are not limited to, starches, lactose, cellulose derivatives, confectioner's sugar and the like. Different grades of lactose include, but are not limited to, lactose monohydrate, lactose DT (direct tableting), lactose anhydrous, Flowlac™ (available from Meggle Products), Pharmatose1""tavailable from DMV) and others. Different starches include, but are not limited to, maize starch, potato starch, rice starch, wheat starch, pregelatinized starch (commercially available as PCS PC10 from Signet Chemical Corporation) and starch 1500, starch 1500 LM grade (low moisture content grade) from Colorcon, fully pregelatinized starch (commercially available as National 78-1551 from Essex Grain Products) and others. Different cellulose compounds that can be used include crystalline cellulose and powdered cellulose. Examples of crystalline cellulose products include, but are not limited to, Ceolus™ KG801, Avicel™ PH101, PH102, PH301, PH302 and PH-F20, PH-112 microcrystalline cellulose 114, microcrystalline cellulose 112, and silicified microcrystalline cellulose (e.g., Prosolv™ supplied by JRS Pharma). Other useful diluents include, but are not limited to, carmellose, sugar alcohols such as mannitol (Pearlitol™ SD200), sorbitol, and xylitol, calcium carbonate, magnesium carbonate, dibasic calcium phosphate, and tribasic calcium phosphate.

Various useful binders include, but are not limited to, hydroxypropyl celluloses, also called HPC (e.g., Klucel™ LF, Klucel EXF) and useful in various grades, hydroxypropyl methylcelluloses, also called hypromelloses or HPMC (e.g., Methocel™ products) and useful in various grades, polyvinylpyrrolidones, also called PVP or povidones (such as grades K25, K29, K30, and K90), copovidone (e.g., Plasdone™ S 630), powdered acacia, gelatin, guar gum, carbomers (e.g., Carbopol™ products), microcrystalline cellulose or MCC, methylcelluloses, polymethacrylates, and starches.

Various useful disintegrants include, but are not limited to, carmellose calcium (Gotoku Yakuhin Co., Ltd.), carboxymethylstarch sodium (Matsutani Kagaku Co., Ltd., Kimura Sangyo Co., Ltd., etc.), croscarmellose sodium (Ac-di-sol™ from FMC-Asahi Chemical Industry Co., Ltd.), crospovidones, examples of commercially available crospovidone products including but not limited to crosslinked povidone, Kollidon™ CL from BASF (Germany), Polyplasdone™ XL, XI-10, and INF-10 from ISP Inc. (USA), and low-substituted hydroxypropylcelluloses. Examples of low-substituted hydroxypropylcelluloses include, but are not limited to, low-substituted hydroxypropylcellulose LH11, LH21, LH31, LH22, LH32, LH20, LH30, LH32 and LH33 (all manufactured by Shin-Etsu Chemical Co., Ltd.). Other useful disintegrants include sodium starch glycolate, colloidal silicon dioxide, and starches.

In certain embodiments, superdisintegrants may be used to provide rapid disintegration of the formulation on contact with aqueous fluids such as water and fruit juices. Super disintegrants provide quick disintegration due to combined effect of swelling and water absorption by the formulation. Non-limiting examples of superdisintegrants are croscarmellose (e.g., Ac-Di-Sol by FMC BioPolymer), crospovidone (e.g., Kollidon CL, Kollidon CL-F, Kollidon CL-SF, and Kollidon CL-M from BASF), and sodium starch glycolate.

An effective amount of any pharmaceutically acceptable tableting lubricant can be added to assist with compressing tablets and improving particle flow characteristics. Useful tablet lubricants include magnesium stearate, glyceryl monostearates, palmitic acid, talc, carnauba wax, calcium stearate sodium, sodium or magnesium lauryl sulfate, calcium soaps, zinc stearate, polyoxyethylene monostearates, calcium silicate, silicon dioxide, hydrogenated vegetable oils and fats, stearic acid and combinations thereof.

One or more glidant materials, which improve the flow of powder blends and minimize dosage form weight variation, can be used. Useful glidants include, but are not limited to, silicon dioxide, talc, and combinations thereof.

The compositions of the present application may optionally contain a coloring agent. Suitable coloring agents include, without limitation, natural and/or artificial materials such as FD&C coloring agents, natural juice concentrates, pigments such as titanium oxide, iron oxides, silicon dioxide, and zinc oxide, combinations thereof, and the like.

Equipment suitable for processing pharmaceutical compositions of the present application include rapid mixer granulators, planetary mixers, double cone blenders, mass mixers, ribbon mixers, fluid bed processors, mechanical sifters, homogenizers, blenders, roller compacters, extrusion-spheronizers, compression machines, capsule filling machines, rotating bowls or coating pans, tray dryers, fluid bed dryers, rotary cone vacuum dryers, and the like, multimills, fluid energy mills, ball mills, colloid mills, roller mills, hammer mills, and the like, equipped with a suitable screen.

The pharmaceutical dosage forms of the present application are intended for oral administration to a patient in need thereof.

The pharmaceutical formulations of the present application are useful in the therapeutic or prophylactic treatment of metabolic disorders, in patients exhibiting elevated phenylalanine levels or decreased tyrosine levels, for example, subjects suffering from hyperphenylalanemia, mild phenylketonuria or classic severe phenylketonuria; and for the treatment of subjects suffering from conditions that would benefit from enhancement of nitric oxide synthase activity, such as vascular diseases, ischemic or inflammatory diseases. The amounts of active ingredient required for a therapy may be administered in multiple doses or in a single dose. The stable formulations may be administered daily or at some other interval, e.g., every other day or even weekly.

The following examples are presented to further illustrate various specific aspects and embodiments of the present application, but are not intended to limit the scope of the application in any manner.

EXAMPLES 1-8
Ingredient mg/Tablet
1 [2 [3 [4 [5 [6 p7 [8
Sapropterin 100 100 10() 100 100 100 100 100
dihydrochloride
Mannitol 173.68 171.18 171.18 173.68 171.18 98.68 71.18 123.68
Microcrystalline &54 6^54 6^54 - - : (T54 6.54
cellulose
L-Hydroxypropyl - - - 6^54 6^54 6^54 - -
cellulose
Croscarmellose T3T5 13^5 13^5 - - - 13^3 -
sodium
Sodium starch - - - 13!5 13^5 13^5 - 13^5
glycolate
Ascorbic acid Z5 Z5 275 - - 75 275 50
L-Cysteine - - - 275 275 - - -
hydrochloride
Citric acid or - Z5 Z25 - Z25 Z5 Z25 Z5
Malic acid
Riboflavin O03 O03 O03 O03 O03 O03 O03 O03
Sodium stearyl 375 375 375 375 375 375 375 375 fumarate

The compositions are processed by direct compression into tablets, by passing the ingredients through a sieve and blending them. The blend is then compressed to form tablets.

Alternatively, the above ingredients may be granulated by techniques known in the art and the granules may be compressed into tablets or filled into capsules.

While particular embodiments of the present specification have been illustrated and described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the application. It is therefore intended to cover all such changes and modifications that are within the scope of this application.

We claim:

1. A pharmaceutical formulation comprising (6R)-L-erythro-tetrahydrobiopterin dihydrochloride, an antioxidant, and a pharmaceutical^ acceptable excipient, diluent, or carrier in the form of a tablet, wherein the weight ratio of the antioxidant to the (6R)-L-erythro-tetrahydrobiopterin dihydrochloride is less than 1:5 or more than 1:30.

2. A pharmaceutical formulation comprising (6R)-L-erythro-tetrahydrobiopterin dihydrochloride, an antioxidant, and atleast one acidifying agent selected from citric acid, malic acid, formic acid and atleast one pharmaceutical^ acceptable excipient.

3. A pharmaceutical formulation comprising a crystalline (6R)-L-erythro-tetrahydrobiopterin dihydrochloride, and without polyvinylpyrrolidone and di-calcium phosphate as an excipients.

4. A pharmaceutical formulation of claim 1-3, where in (6R)-L-erythro-tetrahydrobiopterin dihydrochloride is crystalline or amorphous form, an antioxidant, an acidifying agent and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the weight ratio of the antioxidant to the (6R)-L-erythro-tetrahydrobiopterin dihydrochloride is less than 1:5 or more than 1:30.

5. A pharmaceutical formulation of claim 1-3, further comprising crospovidone, dibasic calcium phosphate, D-mannitol, riboflavin and sodium stearyl fumarate.

6. A pharmaceutical formulation comprising (6R)-L-erythro-tetrahydrobiopterin dihydrochloride, an antioxidant, and a pharmaceutically acceptable excipient, diluent, or carrier in the form of a tablet, wherein the weight ratio of the antioxidant to the (6R)-L-erythro-tetrahydrobiopterin dihydrochloride is about 1:4.8, about 1:4.6, about 1:4.5, about 1:3.5, about 1:3.9, about 1:31, about 1:31.5, about 1:32.5

7. A pharmaceutical formulation, comprising about 100 mg of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride and about 2.75 mg of ascorbic acid, about 2.5 mg of citric acid in the form of a tablet.

8. A pharmaceutical formulation of claim 7, further comprise crospovidone, dibasic calcium phosphate, D-mannitol, riboflavin and sodium stearyl fumarate.

9. A pharmaceutical formulation of claim 7, comprising a (6R)-L-erythrotetrahydro-biopterin dihydrochloride, and without a polyvinylpyrrolidone as a biologically degradable polymeric binder and dicalcium phosphate as an excipient.

10. A pharmaceutical formulation of claim 7, comprising a (6R)-L-erythrotetrahydro- biopterin dihydrochloride, an antioxidant, acidifying agent and atleast pharmaceutical excipient, wherein the formulation is processed with dry granulation or direct compression.