CN101365452A - Methods and compositions for the treatment of vascular disease - Google Patents

Abstract

The present invention is directed to a novel methods and compositions for the therapeutic intervention of vascular complications associated with diabetes, hyperlipidemias, and various cardiovascular disorders including but not limited to recalcitrant hypertension, coronary artery disease, pulmonary arterial hypertension, congestive heart failure, and hemolytic anemias. More specifically, the specification describes methods and compositions for treating such vascular disorders using compositions comprising BH4 and derivative thereof. Combination therapies of BH4 and other therapeutic regimens are contemplated.

Description

The method and composition of treatment disease

The application requires the U.S. the 60/742nd of December in 2005 submission on the 5th, the U.S. the 60/764th that No. 578 provisional application, on February 3rd, 2006 submit to, the U.S. the 60/817th that No. 979 provisional application and on June 30th, 2006 submit to, the priority of No. 847 provisional application is incorporated herein by the full content of quoting as proof above-mentioned each application at this.

Technical field

The present invention relates generally to the treatment intervention of angiopathy.More specifically, the present invention relates to be used for the treatment of the method and composition of the endothelial function disturbance relevant with dysfunction of blood vessel.

Background technology

Diabetes and cardiovascular complication thereof are the main causes that the U.S. and the Western countries are dead and fall ill.These advancings of disease are relevant with some paathogenic factors, comprise the genetic predisposition, sex of disease, such as life style factor, age, insulin resistant, hypertension and hyperlipidemia (comprising hypercholesterolemia) such as smoking and diet.

The treatment possibility of diabetes and relevant cardiovascular disease comprises various medicines, for example anticholesteremic agent (as, Statins), vasoactive agent (as, PPAR-γ part, beta blocker), ACE inhibitor, angiotensin-ii receptor blockers, calcium channel blocker, vitamin and antioxidant (as, nicotinic acid, ascorbic acid or vitamin C).The ultimate principle that reduces plasma cholesterol with his spit of fland medicine can't be interpreted as what coronary heart attack appear at usually block and not serious individuality in, and why block and do not occur in capillary tube or the vein.During use, the risk that his spit of fland medicine only can coronary event take place once more reduces by 30%～40%.Thereby the ultimate principle of vasoactive agent is to bring high blood pressure down by the resistance of blood flow that direct or indirect smooth muscle vasoactive and/or heart reduces blood vessel.Yet such medicine is not treated the cause of hypertension and undesired blood flow, but attempts to reduce the influence that this disorder causes.Such medicine stimulates sympathetic nervous system by baroreceptor reflex, thereby heart rate and myocardial contraction are increased, and this is not useful or desirable effect.Vitamin E, vitamin C, probucol (probucol) and beta-carotene have constituted the most of antioxidants that are used for the treatment of diabetes at present.Yet, unfortunately, use separately or with other medicaments when co-administered, neither one can solve the disease of cell (that is, skin or endothelium) dysfunction and the mediation of other oxidative stresss fully in these medicaments.Because their effect " mode ", tissue absorb and other correlated characteristics, all present available antioxidants all can only the relaxing factor metabolism of remote-effects endothelium source, and only acts on some active oxygen (ROS).In addition, if dosage is incorrect, they have adverse effect to disease process.

And the treatment to these disorders is a short-term at present, the long-term efficacy wretched insufficiency.Up to now, do not produce favourable long-term effect effectively when medicine is used for the acute and chronic occlusion angiopathy of diabetes and relevant heart central vessel system and peripheral vascular system, and can't treat potential disease or the 26S Proteasome Structure and Function of blood vessel is returned to normal condition.

Also there is serious adverse in described each medicine when the patient is had some advantageous effects, and usually need be with higher non-physiological dose administration.These side effect are relevant with dosage usually.The untoward reaction of the medicine of above-mentioned those kinds comprises the teratogenesis after hypoglycemia, renal dysfunction, myopathy (comprising rhabdomyolysis), liver poisoning, airway resistance and maternity dress are used.Other side effect of such medicine comprise headache, cardiopalmus, anxiety, slight depression, myocardial infarction, congestive heart failure, fatigue and weak.In addition, pharmacology dosage may not have specificity to the influence of the initial molecule cause of disease activity, and the curative effect scope of disease---is depended on a number of factors---limitation is also arranged.Under some situation, described untoward reaction may be the same with dyspepsia common as blushing, but can make the patient to therapeutic scheme famine compliance.In order to offset the untoward reaction of medicine, various conjoint therapys have been proposed as the treatment possibility.

Therefore, still need continuous and effective and have specific medicament and under the situation that does not cause serious adverse effects, treat endothelial function disturbance as the cause of disease of angiopathy.The objective of the invention is to satisfy such demand.

Summary of the invention

Generally speaking, the invention describes treatment intervention to the endothelial function disturbance that causes angiopathy.The present invention aims to provide and is used for the treatment of compositions and the method for suffering from the disease that is characterised in that endothelial function disturbance or disorderly object, described method comprises that the compositions that will comprise tetrahydrobiopterin (BH4) or its precursor or derivant is applied to described object or co-administered in described object with therapeutic agent separately, wherein with the situation of the compositions of not using the described BH4 of containing under described endothelial function disturbance compare the described endothelial function disturbance that can effectively alleviate described object of using.The present invention also will provide a kind of treatment to suffer from the method for the object of endothelial function disturbance, described method comprises a kind of factor of using the generation that can strengthen vasodilation thing nitric oxide (NO) separately or the combination of the multiple factor, or the combination of the described a kind of factor or the multiple factor is co-administered with therapeutic agent.

In a scheme, the invention provides the method that a kind of treatment is diagnosed as the object of suffering from the vascular complication relevant with diabetes, described method comprises uses BH4 or its precursor or derivatives thereof separately, or BH4 or its precursor or derivatives thereof and other medicaments is co-administered, wherein said medicament is that therapeutic agent maybe can strengthen the factor that vasodilation nitric oxide (NO) produces.

In one embodiment, the vascular complication relevant with diabetes be including, but not limited to general vasofunctional disturbances, for example unusual vascular compliance, endothelial function disturbance and hypertension; The disorder of intractable hypertension and insulin sensitivity and glycemic control.In another embodiment, the vascular complication relevant with diabetes is including, but not limited to unusual periphery perfusion, and for example intermittent claudication, periphery perfusion reduce, skin blood flows minimizing and wound healing is incomplete.In another embodiment, the vascular complication relevant with diabetes be including, but not limited to heart disease, for example congestive heart failure, is with or without the hypertensive pulmonary vascular disease of congestive heart failure, angina pectoris, coronary heart disease and the relevant atherosclerosis relevant with motion; Ophthalmic, for example optic atrophy and the retinopathy that causes by diabetes; And nephropathy, for example microalbuminuria, renal failure and the glomerular filtration rate in the nephropathy that is caused by diabetes reduces.

In a relevant embodiment, described therapeutic agent is the medicament that is used for the treatment of diabetes, including, but not limited to improving the medicament of insulin sensitivity, PPAR-γ part (Thiazolidine ketone, glitazone, troglitazone class, rosiglitazone (Avandia) and pioglitazone) for example; Insulin secretagogue agent, for example sulphanylureas (gliquidone, tolbutamide, glimepiride, chlorpropamide, glipizide, glibenclamide and acetohexamide) and meglitinide (meglitinide, repaglinide, Nateglinide); And the medicament that reduces the hepatic glucose generation, for example metformin.

In alternative plan, the invention provides the method that treatment is diagnosed as the object of suffering from the angiopathy that has nothing to do with diabetes, described method comprises uses BH4 or its precursor or derivatives thereof separately, or BH4 or its precursor or derivatives thereof and other medicaments is co-administered, wherein said medicament is that therapeutic agent maybe can strengthen the factor that vasodilation thing nitric oxide (NO) produces.Like this with the irrelevant angiopathy of diabetes be selected from by pulmonary vascular disease, hemolytic anemia, apoplexy and relevant ischemic angiopathy (for example apoplexy, heart disease or coronary heart disease, arteriosclerosis or peripheral blood vessel), thrombosis, with transplant the group that relevant endothelial function disturbance and heart disease or coronary heart disease are formed.In one embodiment, hypertensive pulmonary vascular disease, idiopathic pulmonary hypertension disease and the neonate persistent pulmonary hypertensis (PPHN) of pulmonary vascular disease in sicklemia and other hemoglobinopathies.In another embodiment, hemolytic anemia comprises hereditary hemolytic anemia and acquired hemolytic anemia.Hereditary hemolytic anemia is including, but not limited to sicklemia, thalassemia, by G6PD lacks, deficiency of pyruvate kinase causes hemolytic anemia, hereditary elliptocytosis, hereditary spherocytosis, hereditary stomatocytosis, heritability ovalocyte increase disease, paroxysmal sleep type hemoglobinuria and hemoglobin meniscocyte (SC) disease.Non-immunity hemolytic anemia and Secondary cases immune hemolytic anemia that acquired hemolytic anemia causes including, but not limited to microangiopathic hemolytic anemia, idiopathic autoimmune hemolytic anemia, by chemicals or physical factor or device (left ventricular assist device, mechanical heart valve and shunting device).

In another embodiment, apoplexy and relevant ischemic angiopathy be including, but not limited to vasospasm, for example the cerebral vasospasm after the apoplexy.Thrombosis is including, but not limited to thrombosis, thrombosis, hemopexis (clotting) and blood coagulation (coagulation).In another embodiment, with transplant dysfunction of blood vessel and the Ciclosporin A inductive endothelial function disturbance of relevant endothelial function disturbance behind the solid organ transplantation.In another embodiment, heart disease and coronary heart disease are including, but not limited to congestive heart failure, dysfunction of blood vessel and the angina pectoris relevant with hypercholesterolemia, and dysfunction of blood vessel relevant with smoking and angina pectoris.

In a relevant embodiment, described therapeutic agent is the medicament that is used for the treatment of angiopathy, described medicament is including, but not limited to being generally used for treating the endothelin-receptor antagonists of hypertension and other disorders relevant with endothelial function disturbance, for example bosentan, darusentan, enrasentan, tezosentan, atrasentan, An Beishengtan and sitaxentan; Smooth muscle relaxant, for example PDE5 (phosphodiesterase 5) inhibitor (indirect action) and minoxidil (directly effect); Angiotensin-Converting (ACE) inhibitor, for example captopril, enalapril, lisinopril, fosinopril, perindopril, quinapril, trandolapril, benazepril and ramipril; Angiotensin-ii receptor blockers, for example irbesartan, losartan, valsartan, Eprosartan, Olmesartan, Candesartan and telmisartan; Beta blocker, for example atenolol, metoprolol, nadolol, bisoprolol, pindolol, acebutolol, betaxolol and Propranolol; Diuretic, for example hydrochlorothiazide, furosemide, torasemide and metolazone; Calcium channel blocker, for example amlodipine, felodipine, nisoldipine, nifedipine, verapamil and diltiazem (diltiazem); Alpha-receptor blocker doxazosin, terazosin, alfuzosin and Tamsulosin; And maincenter alfa agonists, for example clonidine.

In one embodiment, the combination of the precursor of BH4 or BH4 or derivant and PDE5 inhibitor provides the unexpected advantageous effects to one or more vascular pressure parameters.Therefore, the untoward reaction that BH4 or its precursor or derivant are expected to alleviate these medicines, the rising that for example alleviates blood pressure.

In a relevant programme, the invention provides by with 200mg/ days at least (for example, each administration 100mg, twice of every day), or 150mg/ days at least, or 100mg/ days at least dosage is used purified 6R BH4 and is treated the hypertensive method of people to suffering from hypertensive people.In such illustrative embodiments, the dosage range of BH4 also can be less than 500mg/ days, or less than 400mg/ days, or less than 300mg/ days.In one embodiment, the day application dosage of BH4 is 5mg/kg/ days at least, or 10mg/kg/ days at least, 20mg/kg/ days or 30mg/kg/ days at the most.BH4 can use separately, also can be co-administered with the other treatment agent that is used for the treatment of angiopathy or hyperlipemia (for example or any therapeutic agent in those therapeutic agents of listing below).

In third party's case, the invention provides the method that treatment is diagnosed as the object of suffering from hyperlipemia, described method comprises uses BH4 or its precursor or derivatives thereof separately, or BH4 or its precursor or derivatives thereof and other medicaments is co-administered, wherein said medicament is the factor that therapeutic agent maybe can strengthen the generation of vasodilation thing nitric oxide (NO).In a relevant embodiment, described therapeutic agent is the medicament that is used for the treatment of hyperlipemia, including, but not limited to reducing the medicament of LDL (low density lipoprotein, LDL), Statins (atorvastatin for example, fluvastatin, lovastatin, pravastatin, rosuvastatin calcium and simvastatin) and nicotinic acid, cetp inhibitors (for example torcetrapib), PPAR-α stimulant (the special class of shellfish for example, gemfibrozil, fenofibrate, bezafibrate, ciprofibrate), conjugated bile acid also prevents the heavily absorption of bile acid and the medicament of cholesterol reducing level (bile acid chelating agent for example, colestyramine and colestipol, and cholesterol absorption inhibitor).BH4 or its precursor or derivatives thereof can be used with the combination (for example combination of Ta Ting and cetp inhibitors) of such medicament, and are expected to alleviate the ill effect relevant with hypertension.

In cubic case; the present invention also will provide a kind of treatment to suffer from the method for the object of endothelial function disturbance; described method comprises uses generation enhancing or a kind of factor of normalization or the combination of the multiple factor that can make vasodilation thing nitric oxide (NO) separately, or the combination and the therapeutic agent of the described a kind of factor or the multiple factor is co-administered.In one embodiment, such factor has strengthened from the beginning biosynthetic activity or the expression of BH4, and is selected from the group of being made up of GTP (guanosine triphosphate) cyclohydrolase I (GTPCH1), 6-pyruvoyl tetrahydro pterin synzyme (PTPS) and sepiapterin reductase.In a preferred embodiment of the present invention, by using any or multiple cyclic adenosine monophosphate (cAMP) analog or agonist to increase the expression that GTPCH1 expresses, thereby it is synthetic to increase BH4, described cyclic adenosine monophosphate (cAMP) analog or agonist comprise forskolin (forskolin), 8-bromine cAMP or play a part increases other medicaments of the cell signalling of cAMP mediation, for example cytokine and somatomedin, comprise interleukin-1, interferon-(IFN-γ), tumor necrosis factor-alpha (THF-α), the c-reactive protein, HMG-CoA-reductase (such as Statins such as atorvastatins), nerve growth factor (NGF), epidermal growth factor (EGF), hormones (comprising adrenomedullin and ovostab), and other chemical compounds, for example NADPH (NADPH) and NADPH analog, caffeine, Ciclosporin A, methyl-xanthine (comprising 3-isobutyl-1-methylxanthine), theophylline, reserpine and hydrogen peroxide.

Therefore, an embodiment of the invention aim to provide by suppress the degraded of 3 ', 5 '-nucleolus glycosides with 11 di-phosphate ester enzyme families (PDE1-11) (comprising PDE1, PDE3 and PDE5) inhibitor, to increase the GTPCH1 level.PDE inhibitor of the present invention comprises viagra/sldenafil, Tadalafil/tadanafil, Vardenafil/Ai Lida (levitra), You Dina non-(udenafil), 8-methoxy-IBMX, UK-90234, dexamethasone, hesperetin, Hesperidin, irsogladine, vinpocetine, cilostamide, rolipram (rolipram), B-carboline-3-carboxylic acid, ethyl ester (β-CCE), tetrahydrochysene-beta-carboline derivatives, 3-O-methyl Quercetin etc.

Another embodiment of the invention aims to provide the level that increases the BH4-synzyme by the endothelium targeted delivery of the polynucleotide of gene therapy or BH4 synthetic method, thereby increases the level of BH4.Another embodiment of the invention aims to provide by replenishing BH4-synzyme GTPCH1, PTPS, SR, PCD, DHPR and DHFR increases the BH4 level.Described BH4-synzyme is intended to contain all native forms and the non-natural form of these enzymes, comprises proteinic mutant.

Another embodiment of the invention relates to by suppressing alkali phosphatase (AP) activity so that substrate 7, and 8-triphosphoric acid dihydroneopterin turns to BH4 synzyme PTPS rather than alkali phosphatase, thereby increases the BH4 level.Suppress active reagent of AP or chemical compound and comprise phosphate ester analog, levamisole and L-phenylalanine.Another embodiment of the invention aims to provide medicament or the chemical compound that suppresses alkali phosphatase, comprises suppressing the synthetic siRNA of alkali phosphatase (siRNA), antisense RNA, dsDNA, micromolecule, neutralizing antibody (strand, chimeric and/or humanized) and antibody fragment.

Another embodiment of the invention comprises the catalyst that the enzymatic synthesis that strengthens the synthetic de novo synthesis route of BH4 is required or the active reagent or the chemical compound of cofactor.

Another embodiment of the invention comprises the reagent or the chemical compound of the enzyme generation degraded that prevents that BH4 is synthetic required.Another embodiment of the present invention comprises that the reagent or the chemical compound of degraded take place the synzyme (comprising GTPCH1, PTPS and SR) that prevents synthetic required catalyst of BH4 and BH4.

Thereby another embodiment of the invention relates to the reduction increase BH4 level that increases BH2 via salvage pathway.In vivo, BH4 can be oxidized to BH2.With quinoid (qBH2) and 7, the BH2 that 8-dihydro pterin form exists is reduced to BH4 by DHPR (dihydro pterin reductase) and DHFR (dihydrofolate reductase) respectively.A preferred embodiment of the present invention relates to by with the active of regulatory enzyme PCD, DHPR such as the reagent on this approach or compound N ADPH, thio-alcohol, mercuric p-chlorbenzoate (perchloromercuribenzoate), hydrogen peroxide and DHFR with synthesize and increase the regeneration from BH2 to BH4 or remedy.

Another embodiment of the invention relates to by using the oxidation that reduces BH4 such as reagent such as antioxidant or chemical compound to stablize the reagent of BH4, and described antioxidant comprises ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), tocopherols (for example vitamin A), selenium, beta-carotene, carotenoid, flavone, flavonoid, folic acid, flavone, flavanone, isoflavone, catechol, anthocyanidin and chalcone derivative.

In further embodiment, described one or more factors can increase the active of nitric oxide synthetase or express, thereby strengthen the generation of NO.

In another embodiment, the present invention aims to provide the factor that suppresses GTPCH feedback regulation albumen (GFRP).Thereby a preferred embodiment of the present invention relates to and suppresses reagent or the chemical compound that BH4 and combining of GTPCH1/GFRP complex prevent the feedback suppression that caused by BH4.Described reagent of the present invention or chemical compound comprise competitive inhibitor, for example have change to the alternative form of the BH4 of the affinity of complex and analog etc.Another embodiment of the present invention comprises bonded reagent or the chemical compound that strengthens the L-phenylalanine and can induce the synthetic CTPCH1/GFRP of BH4.Another embodiment of the invention comprises reagent or the chemical compound such as the level of increase L-phenylalanine (playing inhibition GFRP and the BH4 feedback suppression to GTPCH1) such as the precursor of L-phenylalanine.

Another embodiment of the present invention aims to provide active or synthetic reagent or the chemical compound of regulating GFRP.A preferred embodiment of the present invention comprises inhibition active reagent of GFRP or chemical compound.Another embodiment of the invention comprises synthesizing with inhibition GFRP such as siRNA, micromolecule, antibody and antibody fragments.

The present invention further provides reagent or chemical compound, comprise GTP (guanosine triphosphate), 7,8-dihydro-triphosphoric acid 1-(2-amino-4-hydroxy-6-pteridinyl)-1,2,3-propanetriol and 6-pyruvoyl-tetrahydrobiopterin as the BH4 precursor.

The day amount of application of BH4 is about 0.1mg/kg～about 30mg/kg, for example about 5mg/kg～10mg/kg.BH4 single dose every day can be used, perhaps every day, multiple dose was used.In some embodiments, the BH4 therapy is not successive, but is basic administration with the sky, is kept until improve (blood pressure of for example, suffering from the patient of intractable hypertension becomes normal) of clinical endpoint.

In another scheme of the present invention, carry out using of BH4 derivant with unexpected low dosage, still can reach therapeutic efficiency.Estimate that such BH4 derivant has the biological nature of improvement with respect to natural BH4.In one embodiment, estimate that the BH4 derivant will be lower than the common dosage of the BH4 that is used for the treatment of other BH4 reactive disorderly (for example hyperphenylalaninemia) to the effective dose of hypertension, angiopathy or any disease as herein described.In particular, the present invention estimates that any 1 ', 2 '-diacyl described herein-(6R, S)-5,6,7,8-tetrahydrochysene-L-biopterin or lipidal tetrahydrobiopterin all can show the biological nature of improving under low dosage.

The present invention also is to provide a kind of compositions, and described compositions comprises can be longer than BH4 and pharmaceutically useful carrier, diluent or the excipient of crystalline state of 8 hours stabilisation in the time of ambient-temp-stable.Preferably, the BH4 that is used has the more BH4 of the stabilized crystalline attitude of high stability than amorphous stabilisation BH4.More preferably, the BH4 of described stabilized crystalline attitude comprises the 6R BH4 of at least 99.5% or 99.9% purity.Also can use such as precursors such as dihydrobiopterin (BH2) and L-Sepiapterins.BH4 can be oral.

Can by intramuscular administration, subcutaneous administration or intravenously administrable, through feeding drug into pulmones use separately BH4 or with its with the other treatment agent or the intervening measure that is generally used for treating the endothelial function disorder use, wherein said intervening measure is including, but not limited to being used to keep homeostatic medicament and intervening measure, complementary therapy and specific therapy.Specific therapy can comprise the medicament that is used for the treatment of diabetes, described medicament is including, but not limited to improving the medicament of insulin sensitivity, for example PPAR-γ part (Thiazolidine ketone, glitazone, troglitazone class, rosiglitazone (Avandia) and pioglitazone); Insulin secretagogue agent, for example sulphanylureas (gliquidone, tolbutamide, glimepiride, chlorpropamide, glipizide, glibenclamide and acetohexamide) and meglitinide (meglitinide, repaglinide, Nateglinide); And the medicament that reduces the generation of hepatic glucose, for example metformin.Specific therapy can comprise the medicament that is used for the treatment of angiopathy, described medicament is including, but not limited to being generally used for treating hypertension and other disorderly endothelin-receptor antagonists relevant with endothelial function disturbance, for example bosentan, darusentan, enrasentan, tezosentan, atrasentan, An Beishengtan and sitaxentan; Smooth muscle relaxant, for example PDE5 inhibitor (indirect action) and minoxidil (directly effect); Angiotensin-Converting (ACE) inhibitor, for example captopril, enalapril, lisinopril, fosinopril, perindopril, quinapril, trandolapril, benazepril and ramipril; Angiotensin-ii receptor blockers, for example irbesartan, losartan, valsartan, Eprosartan, Olmesartan, Candesartan and telmisartan; Beta blocker, for example atenolol, metoprolol, nadolol, bisoprolol, pindolol, acebutolol, betaxolol and Propranolol; Diuretic, for example hydrochlorothiazide, furosemide, torasemide and metolazone; Calcium channel blocker, for example amlodipine, felodipine, nisoldipine, nifedipine, verapamil and diltiazem (diltiazem); Alpha-receptor blocker, for example doxazosin, terazosin, alfuzosin and Tamsulosin; And maincenter alfa agonists, for example clonidine.Specific therapy can comprise the medicament that is used for the treatment of hyperlipemia, described medicament is including, but not limited to reducing the medicament of LDL, for example Statins (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin calcium and simvastatin) and nicotinic acid, PPAR-α stimulant (for example special class of shellfish, gemfibrozil, fenofibrate, bezafibrate, ciprofibrate), conjugated bile acid and prevent the heavily absorption of bile acid and the medicament of cholesterol reducing level (for example bile acid chelating agent, colestyramine and colestipol, and cholesterol absorption inhibitor).The medicament that is used to keep the homeostasis level that BH4 and/or NO produce can comprise from the beginning biosynthetic activity or the factor of expression, for example GTP (guanosine triphosphate) cyclohydrolase I (GTPCH1), 6-pyruvoyl tetrahydro pterin synzyme (PTPS) and the sepiapterin reductase that strengthens BH4; Can be used for stablizing the factor of BH4, for example vitamin C, ascorbic acid, alpha-tocopherol; Increase the activity of nitric oxide synthetase and express and also therefore strengthen the factor that NO produces; And the factor that suppresses GTPCH feedback regulation Protein G FRP.

The present invention aims to provide and uses the BH4 that is selected from by one or more crystal forms in crystal polymorphic A, crystal polymorph b, crystal polymorphic F, crystal polymorphic J, crystal polymorphic K, crystal water mould assembly C, crystal water mould assembly D, crystal water mould assembly E, crystal water mould assembly H, crystal water mould assembly O, solvation crystal type G, solvation crystal type I, solvation crystal type L, solvation crystal type M, solvation crystal type N and the group formed thereof.

In another embodiment, can select BH4 is used with folate (comprising folate precursors), folic acid and folate derivatives.Such folate is including, but not limited to tetrahydrofolate; 5-formoxyl-(6S)-tetrahydrofolic acid and salt thereof, 5-methyl-(6S)-tetrahydrofolic acid and salt, 5 thereof for example; the 10-methylene-(6R)-tetrahydrofolic acid and salt, 5 thereof; 10-methine-(6R)-tetrahydrofolic acid and salt thereof, 10-formoxyl-(6R)-tetrahydrofolic acid and salt thereof, 5-formimino group-(6S)-tetrahydrofolic acid and salt, (6S)-tetrahydrofolic acid and salt thereof, and the combination of above-claimed cpd.In another embodiment, can select BH4 is used with arginine.

From following detailed, other features of the present invention and advantage will become apparent.Yet, be to be understood that, detailed description and specific embodiment, when pointing out preferred implementation of the present invention, only being that the mode of explanation provides, because from the detailed description of this paper, the variations and modifications in main idea of the present invention and the scope all will become apparent to those skilled in the art.

Description of drawings

Following accompanying drawing has formed the part of this description, and is used for further specifying the solution of the present invention.By with reference to the accompanying drawings and in conjunction with the detailed description of the specific embodiment described herein, the present invention may be better understood.

The generation of Fig. 1: NO.

Fig. 2: endothelial function disturbance and BH4 lack.

The pathophysiology that Fig. 3: Secondary cases BH4 lacks.

Fig. 4: Secondary cases BH4 lacks and uncoupling eNOS.

Fig. 5: diabetes and BH4 lack.

Fig. 6: the characteristic X-ray diffraction pattern that Type B (6R)-BH4 shows.

Fig. 7: the characteristic X-ray diffraction pattern that A type (6R)-BH4 shows.

Fig. 8: the characteristic X-ray diffraction pattern that F type (6R)-BH4 shows.

Fig. 9: the characteristic X-ray diffraction pattern that J type (6R)-BH4 shows.

Figure 10: the characteristic X-ray diffraction pattern that K type (6R)-BH4 shows.

Figure 11: the characteristic X-ray diffraction pattern that C type (6R)-BH4 shows.

Figure 12: the characteristic X-ray diffraction pattern that D type (6R)-BH4 shows.

Figure 13: the characteristic X-ray diffraction pattern that E type (6R)-BH4 shows.

Figure 14: the characteristic X-ray diffraction pattern that H type (6R)-BH4 shows.

Figure 15: the characteristic X-ray diffraction pattern that O type (6R)-BH4 shows.

Figure 16: the characteristic X-ray diffraction pattern that G type (6R)-BH4 shows.

Figure 17: the characteristic X-ray diffraction pattern that I type (6R)-BH4 shows.

Figure 18: the characteristic X-ray diffraction pattern that L type (6R)-BH4 shows.

Figure 19: the characteristic X-ray diffraction pattern that M type (6R)-BH4 shows.

Figure 20: the characteristic X-ray diffraction pattern that N type (6R)-BH4 shows.

Figure 21: use blood pressure response with the people of BH4 with 5mg/kg/ days dosage.

Figure 22: with every day 2 times altogether the dosage of 200mg be applied blood pressure response with the people of BH4.

Figure 23: with every day 2 times altogether the dosage of 100mg be applied blood pressure response with the people of BH4.

Figure 24: after BH4 and independent treatment of sldenafil or therapeutic alliance, the average systolic during behind the dosed administration 24 hours.

Figure 25: after BH4 and independent treatment of sldenafil or therapeutic alliance, the AvDP during behind the dosed administration 24 hours.

Figure 26: after BH4 and independent treatment of sldenafil or therapeutic alliance, the mean arterial pressure during behind the dosed administration 24 hours.

Figure 27: after BH4 and independent treatment of sldenafil or therapeutic alliance, the average arterial pulse pressure during behind the dosed administration 24 hours.

Figure 28: after BH4 and independent treatment of sldenafil or therapeutic alliance, the average (+dP/dt during behind the dosed administration 24 hours _Max).

Figure 29: after BH4 and independent treatment of sldenafil or therapeutic alliance, the average heart rate during behind the dosed administration 24 hours.

The specific embodiment

The effect of I.BH4 in endothelial function disturbance

As shown in Figure 1, BH4 is the essential cofactor during from arginine biosynthesis NO by endothelial type nitric oxide synthase (eNOS).Owing to can't be by due to the endothelium zone, therefore be called endothelial function disturbance from arginine and BH4 generation NO.

As shown in Figure 2, endothelial function disturbance is characterised in that endothelium (forming the monolayer lining of the barrier between blood vessel wall and blood) can't normally utilize the eNOS of endothelium to produce vasodilation thing NO (Alp etc., J Clin Invest 2003; 112 (5): 725-735; Katusic, Am J Physiol HeartCirc Physiol 2001; 281 (3): H981-H986; Meininger etc., Biochem J 2000; 349 (Pt 1): 353-356; Pieper, J Cardiovasc Pharmacol 1997; 29 (1): 8-15; Fukuda etc., Heart 2002; 87 (3): 264-269; Ueda etc., J Am Coll Cardiol 2000; 35 (1): 71-75; Maier etc., J Cardiovasc Pharmacol 2000; 35 (2): 173-178; Kakoki etc., J Am SocNephrol 2000; 11 (2): 301-309; De Vriese etc., Br J Pharmacol 2000; 130 (5): 963-974; Pannirselvam etc., Br J Pharmacol 2002; 136 (2): 255-263; Shinozaki etc., Circ Res 2000; 87 (7): 566-573; Cosentino etc., Eur Heart J 1998; 19 Suppl G:G3-G8).

The direct effect that NO lacks is to cause vasoconstriction excessive, and secondary effect is that the free radical that produces increases (Channon, Trends Cardiovasc Med 2004; 14 (8): 323-327; Meininger etc., Biochem J 2000; 349 (Pt 1): 353-356; Berka etc., Biochemistry 2004; 43 (41): 13137-13148; Vasquez-Vivar etc., Biochem J 2002; 362 (Pt 3): 733-739; Cosentino etc., Cardiovasc Res 1999; 43 (2): 274-278; Guzik etc., Circulation2002; 105 (14): 1656-1662), blood vessel injury and atherosclerosis quicken (Cosentino etc., Eur Heart J 1998; 19 Suppl G:G3-G8; Vasquez-Vivar etc., Biochem J 2002; 362 (Pt 3): 733-739) and blood coagulation (thrombogenicity) and coagulability (coagulation) increase.Total data shows that NO supports blood flow, and under the situation that lacks NO, blood flow reduces, and the probability of atherosclerosis and blood coagulation increases.Therefore, insufficient, atherosclerosis acceleration, blood coagulation increase of endothelial function disturbance and vasoconstriction/hypertension, diastole, select plain increase higher relevant with the cardiac event incidence rate.

The consistent display organization NOS level of research and the basic NOS activity of carrying out with the transgene mouse model of suffering from drepanocytosis increase, and vasodilation is to the response weakening (Reiter etc., Current Opinions in Hematology 10:99-107 (2003)) of endothelium dependent form agonist (for example acetylcholine).Therefore, show from the discovery of zooscopy, as response to the chronic blood vessel injury relevant with drepanocytosis, but the NO redeeming.Yet, in people's research, as if the bioavailability of other factor affecting NO is arranged.In suffering from the women of drepanocytosis, the estrogen that ovary produces is protected endothelial function by the expression of enhancing NOS and the generation of basic endothelial NO.Therefore, the women who suffers from drepanocytosis has normal basic NO and produces and be excited NO and produce.Yet the basic NO that suffers from the male of drepanocytosis and suffer from vasoocclusive crisis and acute syndromic all patients in thoracic cavity produces and is excited NO and produces and reduce to some extent.The circulation comprehensive effect of blood plasma hematochrome and superoxides causes the destruction of NO.In drepanocytosis, the increase that NO produces has reduced the endothelium of adhesion molecule to be expressed and erythrocyte subsequently and leukocytic adhesion, thereby prevents the development (Space etc., Am.J.Hematology63:200-204 (2000)) of vasoocclusive crisis.NO also can reduce the generation of hematoblastic activation and thrombin, thereby prevents blood coagulation.NO comprises at other advantageous effects aspect the treatment of drepanocytosis makes the intravital active oxygen of the object of suffering from asthma and drepanocytosis (ROS) inactivation, makes smooth muscle loosening.

Recent years, the principle that BH4 influences endothelial function disturbance is clear, as shown in table 1, and it becomes crucial Pathophysiology step (Meininger etc., Biochem J 2000 by the various mechanism that are attributable to Secondary cases BH4 shortage; 349 (Pt 1): 353-356; Meininger etc., FASEB J2004).Can prepare from arginine the eNOS enzyme reaction of NO, BH4 is the essential cofactor (Fig. 1) that 1 molar ratio is participated in 1 mole, shows, and in many cases, the BH4 shortage that is caused by various factors is the basic reason that can't produce NO.Fig. 3 provides the diagram of the pathophysiology of expression Secondary cases BH4 shortage.

As second function, recent data show that BH4 also is essential for the eNOS (Fig. 4) of the coupling of stablizing operate as normal, dimerization form.When BH4 lacks, eNOS is dissociated into the dysfunction monomer and uncoupling takes place, and causes the generation such as free radicals such as peroxynitrite salt, these free radicals and then cause the further destruction in BH4 storehouse, hidden hunger (Channon, the Trends Caxdiovasc Med 2004 of BH4 have been aggravated; 14 (8): 323-327; Werner etc., Exp BiolMed (Maywood) 2003; 228 (11): 1291-1302; Vasquez-Vivar etc., Biochem J2002; 362 (Pt 3): 733-739; Wei, Chem Rev 2003; 103 (6): 2365-2383).

In diabetics, the hyperglycemia level can directly suppress biosynthesis (Meininger etc., the Biochem J 2000 of BH4 by the expression that reduces first kind of enzyme GTP cyclohydrolase (GTPCH); 349 (Pt 1): 353-356; Meininger etc., FASEB J 2004; Cai etc., Cardiovasc Res2005; 65 (4): S23-831), and cause (Alp etc., J Clin Invest 2003 in the animal body; 112 (5): 725-735; Yu etc., J Ocul Pharmacol Ther 2001; 17 (2): 123-129; Meininger etc., Biochem J 2000; 349 (Pt 1): 353-356; Pieper, J CardiovascPharmacol 1997; 29 (1): 8-15; Pannirselvam etc., Br J Pharmacol 2002; 136 (2): 255-263; Hamon etc., Clin Chim Acta 1989; 181 (3): 249-253) with human body interior (Nystrom etc., Am J Physiol Endocrinol Metab 2004; Channon, TrendsCardiovasc Med 2004; 14 (8): 323-327; Werner etc., Exp Biol Med (Maywood) 2003; 228 (11): 1291-1302; Heitzer etc., Diabetologia 2000; 43 (11): 1435-1438; Guzik etc., Circulation 2002; 105 (14): dysfunction of blood vessel 1656-1662), this dysfunction of blood vessel can respond to the BH4 substitute.As if excessive cholesterol also can be suppressed GTPCH in the hypercholesterolemiapatients patients body, this compacting can use by BH4 and be reversed (Fukuda etc., Heart 2002; 87 (3): 264-269).As if the patient who suffers from heart failure and coronary heart disease also have endothelial function disturbance (Setoguchi etc., the J Cardiovasc Pharmacol 2002 that is caused by the BH4 shortage; 39 (3): 363-368; Maier etc., J Cardiovasc Pharmacol 2000; 35 (2): 173-178).During smoking, destroyed the BH4 storehouse from the excessive oxidative compound of cigarette, but can pass through BH4 substitute restore funcitons (Katusic, Am J Physiol Heart Circ Physiol 2001; 281 (3): H981-H986; Ueda etc., J Am Coll Cardiol 2000; 35 (1): 71-75).In hemolytic anemia, but free haemachrome produces the also free radical in loss BH4 storehouse (Rother etc., JAMA2005; 293 (13): 1653-1662).The BH4 shortage that takes place thus causes producing NO.In suffering from the object of drepanocytosis, the BH4 substitute can effectively be treated vascular occlusion crisis and the blood coagulation that causes because of pulmonary hypertension, ischemic damage and reperfusion damage and the organ injury that the blood vessel insufficient flow causes, by erythrocyte and leukocytic obstruction and/or adhesion.

On the basis of recent document, be suitable for using the possible indication inventory of 6R-BH4 to comprise diabetes (Nystrom etc., Am J Physiol Endocrinol Metab 2004; Channon, Trends Cardiovasc Med 2004; 14 (8): 323-327; Werner etc., Exp Biol Med (Maywood) 2003; 228 (11): 1291-1302; Alp etc., J Clin Invest 2003; 112 (5): 725-735; Katusic, Am J Physiol Heart Circ Physiol 2001; 281 (3): H981-H986; Yu etc., J Ocul Pharmacol Ther 2001; 17 (2): 123-129; Heitzer etc., Diabetologia 2000,43 (11): 1435-1438; .Meininger etc., Biochem J 2000; 349 (Pt 1): 353-356; Pieper etc., J Cardiovasc Pharmacol 1997; 29 (1): 8-15), hypercholesterolemia (Fukuda etc., Heart 2002; 87 (3): 264-269), smoking (Lowe etc., Drug Metab Dispos 2004; Ueda etc., J Am Coll Cardiol 2000; 35 (1): 71-75), congestive heart failure (Setoguchi etc., J Cardiovasc Pharmacol 2002; 39 (3): 363-368), atherosclerosis (Channon, Trends Cardiovasc Med 2004; 14 (8): 323-327; Katusic, Am J Physiol Heart Circ Physiol 2001; 281 (3): H981-H986; Maier etc., J Cardiovasc Pharmacol 2000; 35 (2): 173-178), hypertensive pulmonary vascular disease (Pritchard etc., Circulation 2005; 111 (16): 2022-2024; Khoo etc., Circulation 2005; 111 (16): 2126-2133), coronary heart disease (Nystrom etc., Am J PhysiolEndocrinol Metab 2004; Maier etc., J Cardiovasc Pharmacol 2000; 35 (2): 173-178; Setoguchi etc., J Am Coll Cardiol 2001; 38 (2): 493-498), reperfusion injury/dysfunction of blood vessel (Yamashiro etc., J Cardiovasc Surg (Torino) 2003 after the organ transplantation; 44 (1): 37-49; Kakoki etc., J Am Soc Nephrol 2000; 11 (2): 301-309; Duranski etc., J Clin Invest 2005), the vasospasm after the apoplexy (Pluta etc., JAMA 2005; 293 (12): 1477-1484) and hemolytic anemia, comprise drepanocytosis (No. the 2003/0078231st, U.S. Patent application; Wood etc., Free Radical Biology ﹠amp; Medicine 40:1443-1453 (2006)).In all these indications, BH4 all occurs and lack, and the deficiency and the vascular system that cause nitric oxide (NO) to produce can't be reacted to normal relaxation signals.

Generally speaking, the invention describes treatment intervention to the endothelial function disturbance that causes angiopathy.The present invention aims to provide the method and composition that treatment suffers from the disease that is characterised in that endothelial function disturbance and disorderly object, described method comprises that the compositions that will comprise tetrahydrobiopterin (BH4) or its precursor or derivatives thereof is applied to described object or described compositions is co-administered in described object with therapeutic agent separately, wherein with the situation of the compositions of not using the described BH4 of comprising under described endothelial function disturbance compare described using separately or the co-administered endothelial function disturbance that can effectively alleviate described object.The present invention also will provide treatment to suffer from the method for the object of endothelial function disturbance, described method comprises a kind of factor of using the generation that can strengthen vasodilation thing nitric oxide (NO) separately or the combination of the multiple factor, or the described a kind of factor or the multiple factor is co-administered with therapeutic agent.

1. the vascular complication relevant with diabetes

Diabetes and other cardiovascular disease states are characterised in that the bioactive forfeiture of nitric oxide (NO), and this causes vasodilation thing and the change of the balance between the vasoconstriction thing in the endothelium and causes endothelial function disturbance.Since such as active oxygen (ROS) free-radical generating such as (for example superoxides molecules) excessive due to, endothelial function disturbance has constituted the fundamental cause of following each symptom: the vasoconstriction amplitude increase (causing hypertension), stretching reaction deficiency, thrombosis and platelet aggregation to blood flow or other signals increase, such as cell surface adhesion molecules such as selecting element increase, thrombin increases and atherosclerosis is quickened.Because NO plays an important role aspect the blood vessel homeostasis keeping,, and become the sign of the badness come-off of described disease so the bioactive forfeiture of NO has promoted the pathogeny of angiopathy.

Recent discovery shows that the accelerated decomposition metabolism of BH4 in being exposed to the tremulous pulse of oxidative stress promoted the known pathogeny that is present in the endothelial function disturbance in the diabetics tremulous pulse.In addition, blood sugar increasing has hindered the increase of the cellular level of BH4.Observed ascorbic acid deficiency also causes the BH4 level that the availability of eNOS is reduced in diabetics.Fortunately, in animal body and human body, confirmed that experimental the replenishing of BH4 has advantageous effects to endothelial function.Utilization suffers from the such viewpoint of research support that high concentration BH4 that the mammary gland arterial ring of the vascular ring of animal of diabetes or atherosclerosis and diabetics carries out replenishes: BH4 can potentially improve endothelial function disturbance and recover vascular function.About BH4 some examples of the positive role of cardiovascular diseases and diabetics are comprised: use the effect that as if BH4 can increase NO mediation the patient's that suffers from diabetes or hypercholesterolemia forearm blood flow, and to normal subjects to no effect (Heitzer etc., Diabetologia.43 (11): 1435-8 (2000)).Using BH4 in a large number can recover to stand the vein transplantation thing of diabetics of coronary artery bypass graft (CAB) and the vascular function in the tremulous pulse (Guzik etc., Circulation 105 (14): 1656-1662 (2002)).The experimenter compares with matched group, and BH4 can increase the patient's who suffers from type ii diabetes and coronary heart disease insulin sensitivity (Nystrom etc., AmJ Physiol Endocrinol Metab.2004 Nov; 287 (5): E919-25.Epub (2004)).In biosynthesis pathway, replenish before the BH4 private savings show help increase intracellular BH4 level, to improve in the body NO synthetic and improve endothelial function.Fig. 5 represents that BH4 lacks the influence in diabetes.

2. unsteered hypertension

Effect to BH4 in the hyperpietic is studied.Give and to suffer from the relatively poor hypertension of control (tremulous pulse shrinks BP〉135mmHg) and be in 8 oral BH4 of patient (5mg/kg/ days, 10mg/kg/ days) of traditional resisting hypertension therapy for treating phase, continued for 8 weeks.The evaluation patient behind buccal nitroglycerine at the baseline place, the vasodilation (FMD) and the expansion of treatment 8 week backs, the brachial artery blood flow mediation in treatment 1 week of back, and the Zhou Bianhua of the blood pressure in this 9 week and treatment 6 weeks of back.The BH4 treatment back FMD in 8 weeks significantly increases, and stops BH4 treatment 1 all FMD afterwards and returns to baseline.Oral BH4 significantly reduces systolic pressure and diastolic pressure, and stops the treatment back during 6 weeks, and blood pressure returns to baseline value.After treating for 3 weeks, blood pressure significantly reduces, and the BH4 of two kinds of dosage produces similar effect.This discovery shows that BH4 has improved the endothelium dependent form vasodilation (Lefeve etc. that control relatively poor hyperpietic, American Heart Association Scientific Sessions 2003, Session No.AOP.43.3, Presentation Number 2378).

3. coronary heart disease

The feature of coronary heart disease may be the atherosclerotic combination of endothelial function disturbance and acceleration.Show all in the multinomial research that BH4 can recover vascular function.BH4 has increased the blood flow (Fukuda etc., 2002) of coronary vasodilator.In the patient who suffers from based on the endothelial function disorder of coronary flow velocity variations, perfusion BH4 has prevented from the abnormal vascular of acetylcholine is shunk (Maier etc. (2000) J.Cardiovasc Pharmacol 35:173-178) in angiographic procedure.The BH4 treatment both provided the coronary flow that improves, and the atherosclerosis/blood coagulation that reduces is provided again.The mortality rate that the MI repeatedly (myocardial infarction) of diabetics causes is very high.

4. pulmonary vascular disease---hypertensive pulmonary vascular disease

Evidence suggests and have the NO deficiency in hypertensive pulmonary vascular disease (PAH): (1) PAH patient demonstrates the exhalation NO level with reduction; (2) having knock out mice development that BH4 biosynthesis defective and BH4 lack defective hypertensive pulmonary vascular disease and mainly shows (Nandi etc., 2005, Circulation 111:2086) as it; (3) recent data about sldenafil show that the conduction of NO signal is not enough in PAH.Yet, still unmanned so far BH4 substitute data.

5. hemolytic anemia comprises drepanocytosis

More existing data show in the hemolytic anemia patient and endothelial function disturbance occurs, and the shortage of NO is the basic reason that this problem occurs.The shortage of BH4 is likely to be destroyed by the oxidation in BH4 storehouse and causes.Zooscopy shows, as response to the chronic blood vessel injury relevant with drepanocytosis, and the NO redeeming.The joint effect of circulation blood plasma hematochrome and superoxides can cause the destruction (Reiter etc., Current Opinions in Hematology 10:99-107 (2003)) of NO.Considered that new treatment increases bioavailability or counteracting oxidative stress and the unsteered free radical propagation relevant with drepanocytosis of NO.The co-administered generation that can increase NO of arginine and hydroxyurea, and (Morris etc., J.Pediatric Hematology 25 (8): 629-634 (2003)) to improve utilization among SCD (cardiac sudden death) patient of arginine under being in stable state.Except hydroxyurea and arginine, considered other therapies, for example suck NO to increase the NO level, using allopurinol destroys to reduce NO, and use Statins and sldenafil to strengthen NO response (Mack etc., Intl.J.Biochem.Cell Biol. is in sending to press (2006)).U.S. Patent Application Publication No. 2003/0078231 has been described with sulfo group-S-adenosylmethionine derivant and has been treated several diseases (comprising sicklemia disease) that caused by oxidative stress and unsteered free radical propagation as nutrient substance with antioxidant properties or food supplement.U.S. Patent Application Publication No. 2005/0239807A1 has described with comprising the active oxygen generation enzyme inhibitor (for example allopurinol) that the bioactive group of NO donor is provided and has treated and oxidative stress diseases associated (for example sicklemia disease).

In drepanocytosis, NO reduces the endothelium of adhesion molecule to express and subsequently erythrocyte and leukocytic adhesion, thereby prevents the development (Space etc., Am.J.Hematology63:200-204 (2000)) of vasoocclusive crisis.Confirmed that the nadph oxidase relevant with cell is that (Wood etc., FASEB are (8) J.19: 989-991 (2005)) for the source of superoxides.The quick generation of the superoxides group relevant with drepanocytosis can cause the generation such as secondary active oxygen such as OH and ONOO and nitrogen metabolism thing of known oxidable BH4, thereby causes BH4 to lack.In a research, use L-Sepiapterin (precursor of a kind of BH4) and hemocyte adhesion weakening relevant (Wood etc., J.Free Radical Biology ﹠amp for meniscocyte's transgenic (β S) mice; Medicine 40:1443-1453 (2006)).Though consistent with the present invention, this author specifically notes that L-Sepiapterin lacks anti-oxidation characteristics and the autoxidation characteristic of external source BH4, and the present invention aims to provide the use of external source BH4.In addition, as mentioned above, transgenic meniscocyte disease mouse model possibly can't accurately be reflected in the observed homeostasis mechanism (Reiter etc., CurrentOpinion in Hematology 10:99-107 (2003)) of controlling the complexity of NOS, NO and BH4 level among the mankind.

6. intermittent claudication

Intermittent claudication is the most remarkable symptom of peripheral arterial disease.It is narrowed down by the atherosclerotic of iliac artery and femoral artery usually and causes, often with the damage of lower limb tip tremulous pulse.In intermittent claudication, the requirement when blood flow is enough to satisfy people's rest.Yet when such people moved, the blood vessel obstruction that becomes was not enough to satisfy the level of the demand of motion muscle thereby limit blood flows and oxygen supply is reduced to.Correspondingly, health reduces such as the release of the vasodilatory factors such as NO and increases the release of the pressor factor (for example thromboxane, 5-hydroxy tryptamine, Angiotensin II, Endothelin and norepinephrine).Evidence suggests that also hemocyte can become unusual and be easy to form grumeleuse.The symptom of intermittent claudication comprises glittering tight, the toe pain ulcer of chaeta disappearance, toenail thickening, the skin on Calf muscle atrophy, toe and the foot, blackout but does not bleed that in some cases, lower limb tremulous pulse place can form blood clot.Intermittent claudication meeting influence is 5% the male more than 50 years old nearly.Patient's severity of symptoms of 10%～20%, and in 20 people, have 1 people and cause amputation because of the development of gangrene limbs.The treatment of intermittent claudication comprises repairs (Hiatt in change lifestyles (motion, smoking cessation, alleviating alcohol addiction), exercise therapy, Drug therapy (pentoxifylline, naftidrofuryl, antithrombotic agents, phosphodiesterase inhibitor cilostazol) and enriching substance (vitamin E, B and C), low-cholesterol diet and the blood vessel, Atherosclerosis Supplements (2005) is in the printing; Wolosker etc., Clinics 60 (3): 193-200 (2005)).The BH4 that studies show that in the diabetics uses with to improve the blood flow and the FMD that are caused by ischemia relevant.

7. neonate persistent pulmonary hypertensis

The term infant of suffering from neonate persistent pulmonary hypertensis (PPHN) has higher mortality rate, and quantity may be annual 40,000 babies.Mortality rate is very high, probably is 20%～50%.Many these babies are LGA (large for gestational age infants) and diabetes mother's children, meet the relation that BH4 that hyperglycemia causes lacks.

8. apoplexy and relevant ischemic angiopathy

Show that from the data of dog apoplexy model the vasospasm around the blood clot position causes damage to spread after the apoplexy, and more serious than initial incident, this vasospasm can be prevented by the perfusion nitrite solution.

The treatment of II.BH4 and blood vessel disorder

The present invention describes with BH4 and uses pharmaceutical intervention into the blood vessel disorder on basis.The BH4 (stable form or other forms) that the present invention also will provide any kind comprises purposes among the patient crowd of the object of suffering from various forms of angiopathys in treatment, and described angiopathy is including, but not limited to the intractable hypertension when having and do not have diabetes to exist or do not control hypertension, intermittent claudication, coronary heart disease, hypertensive pulmonary vascular disease and hemolytic anemia (comprising drepanocytosis).The compositions based on BH4 like this can be used separately, also can be with any other therapeutic agent and/or to be generally used for treating the intervention of blood vessel disorder co-administered.Such therapeutic agent is including, but not limited to being used for the treatment of the medicament of diabetes, the described medicament that is used for the treatment of diabetes is including, but not limited to improving the medicament of insulin sensitivity, for example PPAR-γ part (Thiazolidine ketone, glitazone, troglitazone class, rosiglitazone (Avandia) and pioglitazone); Insulin secretagogue agent, for example sulphanylureas (gliquidone, tolbutamide, glimepiride, chlorpropamide, glipizide, glibenclamide and acetohexamide) and meglitinide (meglitinide, repaglinide, Nateglinide); And the medicament that reduces the hepatic glucose generation, for example metformin.Such medicament is including, but not limited to being used for the treatment of the medicament of angiopathy, the described medicament of angiopathy that is used for the treatment of is including, but not limited to being generally used for treating the endothelin-receptor antagonists of hypertension and other disorders relevant with endothelial function disturbance, for example bosentan, darusentan, enrasentan, tezosentan, atrasentan, An Beishengtan and sitaxentan; Smooth muscle relaxant, for example PDE5 inhibitor (indirect action) and minoxidil (directly effect); Angiotensin-Converting (ACE) inhibitor, for example captopril, enalapril, lisinopril, fosinopril, perindopril, quinapril, trandolapril, benazepril and ramipril; Angiotensin-ii receptor blockers, for example irbesartan, losartan, valsartan, Eprosartan, Olmesartan, Candesartan and telmisartan; Beta blocker, for example atenolol, metoprolol, nadolol, bisoprolol, pindolol, acebutolol, betaxolol and Propranolol; Diuretic, for example hydrochlorothiazide, furosemide, torasemide and metolazone; Calcium channel blocker, for example amlodipine, felodipine, nisoldipine, nifedipine, verapamil and diltiazem (diltiazem); Alpha-receptor blocker doxazosin, terazosin, alfuzosin and Tamsulosin; And maincenter alfa agonists, for example clonidine.Such medicament is including, but not limited to being used for the treatment of the medicament of hyperlipemia, the described medicament of hyperlipemia that is used for the treatment of is including, but not limited to reducing the medicament of LDL, Statins (atorvastatin for example, fluvastatin, lovastatin, pravastatin, rosuvastatin calcium and simvastatin) and nicotinic acid, PPAR-α stimulant (the special class of shellfish for example, gemfibrozil, fenofibrate, bezafibrate, ciprofibrate), conjugated bile acid also prevents the heavily absorption of bile acid and the medicament of cholesterol reducing level (bile acid chelating agent for example, colestyramine and colestipol, and cholesterol absorption inhibitor).

Some embodiments of the present invention aim to provide by the compositions that will comprise BH4 or its precursor or derivatives thereof and are applied to object separately or said composition is treated dysfunction of blood vessel with conventional vascular treatment is co-administered in object, wherein with do not carry out using separately or comparing of BH4 with the vascular therapeutic described concentration when co-administered of routine, use BH4 separately or all can effectively improve the clinical relevant terminal point (clinically relevantendpoint) of described object with the co-administered BH4 of conventional angiogenic therapy.

An embodiment of the invention need be applied to any individuality with unusual terminal point with the BH4 compositions with the amount that can effectively make numerical value normalization.In a preferred implementation, such individuality suffers from specific angiopathy after diagnosing.The present invention aims to provide stable BH4 compositions described herein is applied to the amount that terminal point effectively can be improved to normal level and is diagnosed as the patient who suffers from the particular blood vessel disease, and described particular blood vessel disease is characterised in that specific symptoms and/or is used to diagnose the conventionally test of particular blood vessel disease.

Skilled person in the art will appreciate that thereby the present invention aims to provide the normal value that the patient who has a classical symptom with BH4 treatment produces clinical relevant terminal point.In addition, for clinical relevant terminal point, any variation of the end point values in minimum normal range all will be considered to the treatment final result of therapeutic scheme that the patient is adopted.

1. therapeutic alliance

The present invention also to provide by use separately BH4 or be generally used for treating the medicament of dysfunction of blood vessel or intervene the treatment intervention that co-administered BH4 carries out to various types of dysfunctions of blood vessel.Should be appreciated that and BH4 can be treated and be used for the treatment of the conventional dose or the intervention associating of dysfunction of blood vessel, thereby reach the therapeutic effect of the clinical relevant terminal point raising of the such disease that makes among such patient.As mentioned above, the purpose that dysfunction of blood vessel is treated is to keep homeostasis, auxiliary treatment is provided and provides specific treatment to improve clinical relevant terminal point.Be subject to the factor (comprising that the level of BH4 and NO produce) of dysfunction of blood vessel influence and homeostasis is kept in the generation that do not increase destructive free radical by correction.Auxiliary treatment is made up of the medicament of using the effectiveness that can increase first-selected treatment or intervention.The purpose of specific treatment is to keep normal clinical relevant terminal point.Above the conventional dose and the intervention that are generally used for treating dysfunction of blood vessel are discussed.Some routine interventions that are used to control or treat dysfunction of blood vessel comprise the anti-diabetic medicament, are used for the treatment of the medicament and the treatment of hyperlipidemia disease of various types of angiopathys.The medicament that is used for the treatment of diabetes is including, but not limited to improving the medicament of insulin sensitivity, for example PPAR-γ part (Thiazolidine ketone, glitazone, troglitazone class, rosiglitazone (Avandia) and pioglitazone); Insulin secretagogue agent, for example sulphanylureas (gliquidone, tolbutamide, glimepiride, chlorpropamide, glipizide, glibenclamide and acetohexamide) and meglitinide (meglitinide, repaglinide, Nateglinide); And the medicament that reduces the hepatic glucose generation, for example metformin.The medicament that is used for the treatment of angiopathy is including, but not limited to being generally used for treating the endothelin-receptor antagonists of hypertension and other disorders relevant with endothelial function disturbance, for example bosentan, darusentan, enrasentan, tezosentan, atrasentan, An Beishengtan and sitaxentan; Smooth muscle relaxant, for example PDE5 inhibitor (indirect action) and minoxidil (directly effect); Angiotensin-Converting (ACE) inhibitor, for example captopril, enalapril, lisinopril, fosinopril, perindopril, quinapril, trandolapril, benazepril and ramipril; Angiotensin-ii receptor blockers, for example irbesartan, losartan, valsartan, Eprosartan, Olmesartan, Candesartan and telmisartan; Beta blocker, for example atenolol, metoprolol, nadolol, bisoprolol, pindolol, acebutolol, betaxolol and Propranolol; Diuretic, for example hydrochlorothiazide, furosemide, torasemide and metolazone; Calcium channel blocker, for example amlodipine, felodipine, nisoldipine, nifedipine, verapamil and diltiazem; Alpha-receptor blocker doxazosin, terazosin, alfuzosin and Tamsulosin; And maincenter alfa agonists, for example clonidine.The medicament that is used for the treatment of hyperlipemia is including, but not limited to reducing the medicament of LDL, for example Statins (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin calcium and simvastatin) and nicotinic acid, PPAR-α stimulant (for example special class of shellfish, gemfibrozil, fenofibrate, bezafibrate, ciprofibrate), conjugated bile acid or prevent the heavily absorption of bile acid and the medicament of cholesterol reducing level (for example bile acid chelating agent, colestyramine and colestipol, and cholesterol absorption inhibitor).

Use separately or with therapeutic agent with intervene the BH4 compositions that co-administered BH4 with control and/or treatment dysfunction of blood vessel might not be a stabilisation described herein.Those skilled in the art will know that unsettled BH4 preparation of compositions method under room temperature and illumination condition.Though the unstability of this BH4 compositions has limited the treatment of adopting such compositions to carry out, in some therapeutic alliance of the patient who suffers from dysfunction of blood vessel with BH4 treatment and the routine treatment treatment that is used for the treatment of angiopathy, still can adopt unsettled BH4 compositions.

The present invention has described in further detail BH4 method for compositions and the composition for preparing such stabilisation.The BH4 compositions of stabilisation of the present invention is included in room temperature can be longer than 8 hours the stable time, or is at least 24 hours, 3 months, 6 months, 9 months, 12 months or longer BH4 crystal.Method and composition of the present invention aims to provide the Pharmaceutical composition of the independent BH4 of stabilisation, and described Pharmaceutical composition can be sent by any conventional route of administration, including, but not limited to oral, intramuscular injection, subcutaneous injection and intravenous injection etc.Compositions of the present invention can further be that the BH4 compositions of antioxidant, described antioxidant are arranged is the antioxidant that helps to prolong the stability of BH4 compositions in combination.

Certain methods of the present invention comprises BH4 and conventional dose and intervention is used in combination, thereby reach the treatment final result in suffering from the patient of angiopathy.In order to reach suitable treatment final result in the therapeutic alliance that relates to herein, can BH4 compositions and described medicament/intervention be applied to object with the combined amount that can effectively produce required treatment final result usually.This method can comprise uses BH4 compositions and described medicament/intervention simultaneously.This can realize by using the single compositions or the pharmacological preparation that exist with combination dosage forms that not only comprise therapeutic agent but also comprise BH4, or realize by use the BH4 preparation when intervening.As selection, can almost take the described intervention of described medicament/carry out simultaneously what use the pharmacological preparation of BH4 (tablet, injection, potus).In other selection schemes, the BH4 treatment can be carried out before or after medicament/intervention, and interval can be a few minutes to arrive several hours.Medicament/treatment is intervened and the embodiment of BH4 compositions separate administration in, should guarantee usually that two kinds of medicaments can be worked in coordination with to play a role, so that BH4 still can bring into play the advantageous effect to the patient.Under these circumstances, imagination can be in about 2～6 hours of medicament/intervention (before or after) use BH4, most preferably only be about 1 hour time delay.Yet, should be appreciated that 2～6 hours time range between two kinds of pharmacy applications only is exemplary, the interval between the using of BH4 and second kind of medicament/intervention can be longer, for example, 24 hours, 36 hours, 48 hours, 72 hours, a week or longer.In some embodiments, the BH4 treatment will be continuous treatment, wherein daily dose BH4 will be applied to the patient indefinitely.

2. the BH4 compositions that is used for the treatment of

The compositions that can be used for related treatment is herein discussed in this part.

United States Patent (USP) the 5th, 698, No. 408, the 2nd, 601, No. 215, No. 3505329, the 4th, 540, No. 783, the 4th, 550, No. 109, the 4th, 587, No. 340, the 4th, 595, No. 752, the 4th, 649, No. 197, the 4th, 665, No. 182, the 4th, 701, No. 455, the 4th, 713, No. 454, the 4th, 937, No. 342, the 5th, 037, No. 981, the 5th, 198, No. 547, the 5th, 350, No. 851, the 5th, 401, No. 844, the 5th, 698,408 and each self-described of Canada application CA 2420374 (by quoting as proof content separately being introduced) at this can be used as the dihydrobiopterin of the present composition, the preparation method of BH4 and derivant thereof.Any such method all can be used for preparing BH4 compositions used in the Therapeutic Method of the present invention.

United States Patent (USP) the 4th, 752, No. 573, the 4th, 758, No. 571, the 4th, 774, No. 244, the 4th, 920, No. 122, the 5th, 753, No. 656, the 5th, 922, No. 713, the 5th, 874; No. 433, the 5th, 945, No. 452, the 6th, 274, No. 581, the 6th, 410, No. 535, the 6th, 441, No. 038, the 6th, each self-described of 544, No. 994 and U.S. Patent Publication US 20020187958, US 20020106645, US 2002/0076782, US 20030032616 (by quoting as proof content separately being introduced) at this use the BH4 compositions to carry out the method for various treatments.These patents all are incorporated herein by quoting as proof at this, as general instruction of using the BH4 method for compositions well known by persons skilled in the art, described method can be suitable for treating angiopathy described herein.

In particular, United States Patent (USP) the 4th, 540, the BH4 derivant of having described to can be used for Therapeutic Method of the present invention for No. 783 promptly 1 ', 2 '-diacyl-(6R, S)-5,6,7,8-tetrahydrochysene-L-biopterin and inorganic salt or organic salt.Preferably pharmaceutically useful salt is used for Therapeutic Method of the present invention.United States Patent (USP) the 4th, 540, the chemical compound of describing in No. 783 has general formula (I):

R wherein ¹And R ²Identical or different, and each acyl group naturally.

Described acyl group preferably has 1～10 carbon atom, particularly 3～10 carbon atoms.Preferred acyl group is by general formula R ⁵CO representative---R wherein ⁵Be hydrogen or have 1 above carbon atom, the particularly alkyl of 2～9 carbon atoms.By R ⁵The preferred embodiment of described alkyl of representative is, for example, and the having 1 above carbon atom, be preferably the saturated or unsaturated aliphatic group of 2～9 carbon atoms of straight chain or branching; Having substituent or not having substituent phenyl by the representative of following general formula:

R wherein ⁶, R ⁷, R ⁸, R ⁹And R ¹⁰Be the alkyl (R wherein of hydrogen or straight chain or branching ⁶, R ⁷, R ⁸, R ⁹And R ¹⁰The sum of carbon atom be preferably be not more than 3); Having substituent or not having substituent phenyl by the representative of following general formula:

R wherein ¹¹And R ¹²Be hydrogen, methyl or ethyl (R wherein ¹¹And R ¹²The total number of carbon atoms be preferably be not more than 2); With the having substituent or do not have substituent aryl alkyl of following general formula representative:

R wherein ¹³Be hydrogen or methyl.In the above-mentioned acyl group, most preferably formoxyl, acetyl group, propiono, bytyry, isobutyryl, valeryl, isovaleryl and benzoyl.R preferably ¹With R ²Identical.

The chemical compound of general formula (I) has two kinds of diastereomers, promptly at 6 diastereoisomeric 1 ', 2 '-diacyls-(6R)-5,6,7, and 8-tetrahydrochysene-L-biopterin and 1 ', 2 '-diacyl-(6S)-5,6,7,8-tetrahydrochysene-L-biopterin.Chemical compound of the present invention comprises these two kinds of diastereomers and composition thereof.

Chemical compound of the present invention (I) can be the form such as inorganic salts such as hydrochlorate, sulfate or phosphate, perhaps such as the form of organic salts such as acetate, oxalates, and the perhaps form of double salt.

United States Patent (USP) the 4th, 550, lipoid biopterin and the tetrahydrobiopterin described as the BH4 derivant for No. 109.These lipoids BH4 derivant can be used as pharmaceutical salts by Therapeutic Method according to the present invention.At United States Patent (USP) the 4th, 550, the chemical compound of describing in No. 109 is represented by following structure:

Wherein

---when among the ring B two pairs of keys being arranged, there is not R;

---when not having two pairs of keys among the ring B, R is a hydrogen; And

---R ' and R " be that saturated or unsaturated, molecular weight balance is so that it has the aliphatic alkyl of the lipoid characteristic of lipids independently.

Usually R ' and R " be selected from alkyl with 1～31 carbon atom, restrictive condition is R '+2R " the summation of carbon unit greater than 10 and less than 33.

In these derivants, the 2-N-acyl group preferably has 9～32 carbon units, is preferably 9～20, so that end product has the lipoid feature.The example of 2-N-acyl group is capryl, palmityl, stearyl and inferior oleoyl.The 2-N-acyl group can be saturated (for example stearyl), also can be undersaturated (for example inferior oleoyl).In addition, also can make end product have required lipoid feature as this nontoxic aromatic series 2-N-acyl group of phenylacetyl group.Ideal 1 ', 2 '-two-O-acyl group is low molecular weight alkyl and the thiazolinyl with 2～8 (being preferably 2～4) carbon units, and the example is an acetyl group.

United States Patent (USP) the 4th, 550, No. 109 the biopterin chemical compound of following formula has been described also:

Wherein R is naturally occurring saturated or undersaturated fatty acid, and Ac=COCH ₃Can be with these biopterin hydrogenation of compounds to form corresponding tetrahydrobiopterin, tetrahydrobiopterin can be used for Therapeutic Method of the present invention.The exemplary chain length of fatty acid group R is C ₁₀～C ₁₈The unit.Exemplary compounds comprises 2-N-acetyl group-1 '; 2 '-two-O-acetyl group-L-biopterin, 2-N-capryl-1 '; 2 '-two-O-acetyl group-L-biopterin, 2-N-palmityl-1 '; 2 '-two-O-acetyl group-L-biopterin, 2N-stearyl-1 '; 2 '-two-O-acetyl group-L-biopterin, the inferior oleoyl-1 ' of 2-N-; 2 '-two-O-acetyl group-L-biopterin and 2-N-phenylacetyl group-1 '; 2 '-two-O-acetyl group-L-biopterin, and corresponding tetrahydrobiopterin.

Except the conventional method of above-mentioned preparation BH4, the present invention also specifically comprises preparation and uses the BH4 compositions, and the BH4 compositions that described BH4 compositions is a stabilisation is preferably the 6R BH4 of 99.5% or 99.9% purity.If use BH4 self, then can use U.S. Patent application the 11/143rd with the form of purification, No. 887 (with the corresponding International Application PCT/US04/38296 that submitted on November 17th, 2004, publication number is WO 2005/049000) and U.S. Patent application the 10/990th, any salt or polymorphic or crystal form described in No. 316 (with corresponding International Application PCT/IB04/04447 that on November 17th, 2004 submitted to, publication number is WO 2005/065018).Various crystal forms can be made easily and be used for oral tablet, powder or other solid dosage formss.Described crystal form also can be used as the additive of general food.Of International Application PCT/US05/41252 (publication number is WO 2006/055511, its full content is introduced by quoting as proof at this) of submitting on November 16th, 2005, BH4 can be used as stable solid preparation.Hereinafter the form that will use BH4 and approach are done further and are gone through.

In a preferred embodiment, imagining method of the present invention, will to provide daily dose to the patient who needs BH4 be the BH4 of about 10mg/kg～about 20mg/kg.Certainly, those skilled in the art can adjust this dosage up and down according to the effect that reaches by using.Described daily dose can single dose administration, also can be with interval multiple dose administration easily.In the exemplary embodiment, daily dose can be 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 22mg/kg, 24mg/kg, 26mg/kg, 28mg/kg, 30mg/kg, 32mg/kg, 34mg/kg, 36mg/kg, 38mg/kg, 40mg/kg, 42mg/kg, 44mg/kg, 46mg/kg, 48mg/kg, 50mg/kg or more.

In low dose therapy method of the present invention, included low dosage is, for example every day 0.1mg/kg～5mg/kg, comprise 0.1mg/kg～2mg/kg, 0.1mg/kg～3mg/kg or 1mg/kg～5mg/kg.Preferably less than the dosage of 5mg/kg every day.According to the present invention, such dosage is expected to improve relevant research terminal point, estimates that the BH4 derivant has the biological nature of improvement with respect to the natural B H4 of such dosage.In particular, the present invention includes, any 1 ', 2 '-diacyl described herein-(6R, S)-5,6,7,8-tetrahydrochysene-L-biopterin or lipoid tetrahydrobiopterin all can show the biological nature of improving at low dosage.

Particularly, the present invention aims to provide any morbid state that BH4 or its precursor or derivatives thereof mention in treatment the application or the U. S. application of submitting on June 1st, 2005 the 11/143rd, No. 887 (and the corresponding International Application PCT/US04/38296 that submitted on November 17th, 2004, publication number is WO 2005/049000) purposes in (by quoting as proof its full content being introduced) related any angiopathy state at this, wherein dosage is 0.1mg/ kg body weight/sky～5mg/ kg body weight/sky, can pass through any administration (including, but not limited to oral), but be administered once every day, also but every day repeatedly (for example, 2 times or 3 times or 4 times) administration in batches, the administration persistent period was at least 1 week, 2 weeks, 3 weeks, 4 weeks or longer, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer.Exemplary dosage comprise be less than 5mg/kg/ days, 4.5mg/kg/ they is following, 4mg/kg/ is days following, 3.5mg/kg/ is days following, 3mg/kg/ is days following, 2.5mg/kg/ is days following, 2mg/kg/ is days following, 1.5mg/kg/ is days following, 1mg/kg/ is days following or below 0.5mg/kg/ days.The dosage of the unit bodies surface area of equivalence also within the scope of the invention.

For the people of the body weight/body surface area with average level (for example 70kg), the present invention also comprises the total daily dose that is less than 400mg.The example of total daily dose like this comprises 360mg/ days, 350mg/ days, 300mg/ days, 280mg/ days, 210mg/ days, 180mg/ days, 175mg/ days, 150mg/ days or 140mg/ days.For example, can easily realize the dosage of 350mg/ days or 175mg/ days by the peroral dosage form of oral 175mg once or twice every day.Other examples of total daily dose comprise 320mg/ days following, 160mg/ days following or below 80mg/ days.Such dosage can obtain by the peroral dosage form of 80mg or 160mg being easy to use.Other examples of total daily dose comprise 45mg/ days, 90mg/ days, 135mg/ days, 180mg/ days, 225mg/ days, 270mg/ days, 315mg/ days or below 360mg/ days, can obtain by the peroral dosage form of 45mg or 90mg being easy to use.Other examples of total daily dose comprise 60mg/ days, 120mg/ days, 180mg/ days, 240mg/ days, 300mg/ days or 360mg/ days, can obtain by the peroral dosage form of 60mg or 120mg being easy to use.Other examples of total daily dose comprise 70mg/ days, 140mg/ days, 210mg/ days, 280mg/ days or 350mg/ days, can obtain by the peroral dosage form of 70mg or 140mg being easy to use.The example of total daily dose also comprises 55mg/ days, 110mg/ days, 165mg/ days, 220mg/ days, 275mg/ days or 330mg/ days, can obtain by the peroral dosage form of 55mg being easy to use.Other examples of total daily dose comprise 65mg/ days, 130mg/ days, 195mg/ days, 260mg/ days or 325mg/ days, or 75mg/ days, 150mg/ days, 225mg/ days, 300mg/ days or 375mg/ days, can use by the dosage form of for example 65mg or 75mg.

3. pharmaceutical composition

The pharmaceutical composition that is used to use of the present invention can comprise first compositions, and described first compositions comprises the BH4 of medicinal forms, can make up with pharmaceutical carrier in case of necessity.The any-mode of intended purposes that can be by reaching them is used these compositionss.Compositions of the present invention is used for the treatment of the amount of application and the application program of angiopathy and can be easily determined by those skilled in the art.As mentioned above, those skilled in the art can adopt amount and the scheme of the current BH4 that advises at first under medical background, for example those compositionss that propose in order to regulate the NOS activity.The described any application program that is used to use BH4 in load experiment, administered formulation, route of administration etc. easily can be improved, for use in the present invention.

Compositions in the scope of the invention comprises that all are can realize effectively that the amount of its intended purposes comprises the compositions of BH4 of the present invention, BH4 analog and derivant.Similarly, because some Therapeutic Method of the present invention aims to provide a kind of like this conjoint therapy: except being generally used for treating the medicament of angiopathy and intervening, also use compositions based on BH4, so pharmaceutical composition of the present invention also aims to provide all such compositionss: when with said composition and the conventional dose that is used for the treatment of angiopathy with intervene when co-administered, said composition comprises the therapeutic agent based on BH4, its analog or homologue of the amount that can effectively alleviate one or more angiopathy symptoms at least.Certainly, the most significant symptom that can alleviate is that therapeutic alliance makes clinical relevant terminal point be improved, but also other symptoms etc. is monitored.These indications utilize technology monitoring well known by persons skilled in the art.

(6R)-the crystal polymorphic of L-tetrahydrobiopterin dihydrochloride

Find the dihydrochloride of BH4, particularly BH4, shown crystalline polytropism.The structure of BH4 is as follows:

(6R) type of BH4 is known biologically active form, yet also known BH4 is at room temperature unstable.A kind of crystalline state polymorphic of having found BH4 is more stable, at room temperature is difficult for decomposing.

BH4 is owing to have hygroscopicity and oxidation sensitive, thereby be difficult to handle, therefore be made into the form of dihydrochloride and sell (Schircks Laboratories, Jona with this form, Switzerland), under nitrogen, it is encapsulated to prevent the degraded of material with ampoule bottle.United States Patent (USP) the 4th, 649,197 disclose because 6 (R, S)-crystallinity of L-erythro form-tetrahydrobiopterin dihydrochloride is relatively poor, so be difficult to (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride and (6S)-L-erythro form-tetrahydrobiopterin dihydrochloride is separated into its diastereomer.European patent has been described the preparation of tetrahydrobiopterin for No. 0079574, has wherein obtained the solid tetrahydrobiopterin dihydrochloride as intermediate.S.Matsuura etc. have described 6 (R)-tetrahydrobiopterins of colourless acicular crystalline solid in " Chemistry Letter; 1984; 735-738 " and " Heterocycles; Vol.23; No.12,1985,3115-3120 ", the x-ray analysis that discloses among the J.Biochem.98,1341-1348 (1985) characterizes this crystalline solid.The optical rotation of finding described crystalline product is 6.81 °, and 6.51 ° optical rotation of the crystalline solid of the white crystal form of report is very approaching among the embodiment 6 of this and European patent EP-A2-0191335.

The result of gained shows that may there be different crystal formations in described chemical compound, comprises a plurality of polymorphics and solvate in the development process of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.The interest that continues in this field need effectively and reliably the method for the independent crystal formation of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride and controlled crystallization condition so that crystal formation to be provided, described crystal formation is preferably stable and be easy to handle and processing in the manufacturing of preparation and preparation, and described crystal formation all can provide higher storage stability in material form or as formulation products, more unsettled form perhaps is provided, and it is suitable as controlled crystalline intermediate to be used to prepare stable form.

Polymorph b

Be called " Type B " at this most stable crystallization polymorphic that will find, perhaps be called " polymorph b ".The result of gained demonstration has prepared several known crystalline solids in the research and development process of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, but nobody recognizes polytropism and the influence of this polytropism to the crystalline stability of BH4.

Polymorph b is slightly hygroscopic anhydride, has the highest thermodynamic stability about more than 20 ℃.In addition, because the heat stability of Type B, can be by the probability of goal condition preparation, suitable form and particle diameter, so Type B is easy to processing and handles.The fusing point of Type B is about 260 ℃ (Δ Hf〉140J/g), but since its before fusion He in the melting process, will decompose, so can't measure fusing point clearly.These outstanding characteristics make the polymorph b of high temperature preparation be specially adapted to medicinal usage.Can obtain the polymorph b of fine-powdered, particle diameter can be 0.2 μ m～500 μ m.

The X-ray powder diffraction pattern that Type B shows with the d value (

) be expressed as: 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), 2.44 (w).Fig. 6 is the characteristic X-ray diffraction pattern that Type B (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

The meaning of the following abbreviation in the bracket used herein is: (vs)=and very high strength; (s)=high strength; (m)=moderate strength; (w)=weak intensity; (vw)=utmost point weak intensity.Characteristic X-ray powder diffraction pattern is shown in Fig. 6.

Other polymorphics of having found BH4 have gratifying chemistry and physical stability, can handle safely in preparation and process for preparation, and all provide higher storage stability in pure substance form or dosage form.In addition, found Type B and other polymorphics of (for example, the 100kg scale) preparation BH4 in enormous quantities, and it has been stored the long time.

All crystal formations (polymorphic, hydrate and solvate) comprise crystal form B, all can be used for preparing the most stable polymorph b.Can the balancing each other of float in suitable polar nonaqueous solvent obtain polymorph b by amorphous form or other forms except polymorph b (for example polymorphic A).Therefore, medication preparation described herein is meant the preparation of the polymorph b of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.

Can in room temperature suspension be stirred to the time that is enough to produce polymorph b by being dispersed in the solvent, then isolate crystalline Type B, and remove the solvent in the isolated Type B, thereby other forms of BH4 are converted into Type B in room temperature other forms with BH4.Room temperature used herein refers to the temperature in 0 ℃～60 ℃ scopes, is preferably 15 ℃～40 ℃.In processing and whipping process, can change the temperature that is applied by progressively reducing temperature or reducing temperature continuously.Be applicable to the suitable solvent that other forms are converted into Type B including, but not limited to methanol, ethanol, isopropyl alcohol, other C3～C4 alcohol, acetic acid, acetonitrile, oxolane, methyl tertiary butyl ether(MTBE), 1,4-diox, ethyl acetate, isopropyl acetate, other C3～C6 alcohol acetic ester, methyl ethyl ketone and other methyl-C3～C5 alkyl ketone.Finishing the time of balancing each other can be up to 30 hours, is preferably maximum 20 hours or less than 20 hours.

Also can obtain polymorph b by crystallization from the mixed solvent that contains the mixed solvent of about at the most 5% water, particularly ethanol, acetic acid and water.Having found can be by being dissolved in the preferred of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride in the mixed solvent that comprises ethanol, acetic acid and water for energy solid form or the Type B lower than Type B, this dissolving in case of necessity can be carried out at higher temperature, crystal seed is added in the described solution then, suspension cooling with gained, and separate formed crystal, thereby the polymorph b of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.Dissolving can be carried out in room temperature, or carries out at the highest 70 ℃, is preferably the highest 50 ℃.Can dissolve with final solvent mixture, also parent material can be dissolved in earlier in the water, add other solvents then together, or add other solvents one by one.The composition of solvent mixture can be that volume ratio is the water of 1:3:2～1:9:4 (being preferably 1:5:4): acetic acid: oxolane.Preferably stir described solution.Cooling can refer to that temperature reduces to-40 ℃～0 ℃, is preferably and reduces to 10 ℃～30 ℃.Suitable crystal seed is polymorph b of other batches or crystal with similar or same modality.After the separation, available Type B such as non-solvent wash crystallizations such as acetone or oxolanes, and dry in due form.

Also can from aqueous solution, obtain polymorph b such as non-solvents such as methanol, ethanol or acetic acid by adding.Advantageously, can carry out crystallization and separation process in room temperature, and needn't cooling solution.Therefore this method is fit to carry out with commercial scale very much.

In the embodiment of described herein compositions and method, be dissolved in the water in room temperature by other solid forms except Type B or Type B (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, to be enough to form the amount interpolation non-solvent of suspension, in case of necessity described float is stirred certain hour, subsequently with formed crystal separation, thereby preparation comprises the compositions of the polymorph b of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.Described compositions further can be modified into pharmaceutical composition as described below.

With respect to solution, (6R)-the L-erythro form-concentration of tetrahydrobiopterin dihydrochloride in aqueous solution can be 10 weight %～80 weight %, 20 weight %～60 weight % more preferably.Preferred non-solvent (that is used solvent in the suspension of preparation BH4) is methanol, ethanol and acetic acid.Described non-solvent can be added in the aqueous solution.More preferably, described aqueous solution is added in the non-solvent.The mixing time that forms behind the suspension can be 30 hours at the most, is preferably at the most 20 hours or is less than 20 hours.Carry out isolated by filtration and drying with aforesaid known way.

Polymorph b is very stable crystalline state, is easy to filter, dry and be ground to the required particle diameter of pharmaceutical preparation.These outstanding characteristics make polymorph b be specially adapted to medicinal usage.

Polymorphic A

The another kind of crystal polymorphic of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " A type " or " polymorphic A " at this.Polymorphic A is the tool hygroscopicity slightly, and water absorption is about 3 weight %, and when with 10 ℃/minute speed heating, the water that is absorbed discharges between 50 ℃～200 ℃ continuously.Polymorphic A is the hygroscopicity anhydride, is a kind of metastable state for Type B; Yet if be kept in the container of tight seal, it can stablize the several months under environmental condition.The A type is particularly suitable as preparation and stablizes polymorphous intermediate and initiation material.Polymorphic A can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that polymorphic A shows with the d-value (

) be expressed as: 15.5 (vs.), 12.0 (m), 6.7 (m), 6.5 (m), 6.3 (w), 6.1 (w), 5.96 (w), 5.49 (m), 4.89 (m), 3.79 (m), 3.70 (s), 3.48 (m), 3.45 (m), 3.33 (s), 3.26 (s), 3.22 (m), 3.18 (m), 3.08 (m), 3.02 (w), 2.95 (w), 2.87 (m), 2.79 (w), 2.70 (w).Fig. 7 is the characteristic X-ray diffraction pattern that A type (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

The feature raman bands that polymorphic A shows wave number (cm ^-1) be expressed as: 2934 (w), 2880 (w), 1692 (s), 1683 (m), 1577 (w), 1462 (m), 1360 (w), 1237 (w), 1108 (w), 1005 (vw), 881 (vw), 813 (vw), 717 (m), 687 (m), 673 (m), 659 (m), 550 (w), 530 (w), 492 (m), 371 (m), 258 (w), 207 (w), 101 (s), 87 (s) cm ^-1

Can by with (6R) thereby-the aqueous solution lyophilization of L-erythro form-tetrahydrobiopterin dihydrochloride or remove water and obtain polymorphic A.Can be by (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride being dissolved in the water in room temperature, (1) described solution is cooled to low temperature so that solution solidifies, and under reduced pressure remove water, or (2) remove water from described aqueous solution, thus the polymorphic A of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.

Can pass through the crystalline A type of isolated by filtration, the dry then so that water that is absorbed volatilizees from product.Drying condition and method are known, and dewatering in the dry or described herein variation scheme (2) of separator can be carried out applying under the pyritous condition, for example the highest 80 ℃, is preferably 30 ℃～80 ℃, or carries out under vacuum, or carry out under high-temperature vacuum.Before the precipitate and separate that in will changing scheme (2), will obtain, suspension can be stirred certain hour and balance each other with realization.With respect to solution, (6R)-the L-erythro form-concentration of tetrahydrobiopterin dihydrochloride in aqueous solution can be 5 weight %～40 weight %.

Preferably cool off fast to obtain solid solution as raw material.Reduce pressure up to removing solvent fully.Lyophilization is a technology well-known in the art.The time of finishing removal of solvents is depended on the vacuum (can be 0.01 millibar～1 millibar) that is applied, employed solvent and cryogenic temperature.

In room temperature or be stable when being lower than room temperature, this can be confirmed by in room temperature the suspension in oxolane or the t-butyl methyl ether being stirred the test of carrying out in 5 days and 18 hours that balances each other respectively under nitrogen under the substantially anhydrous condition of polymorphic A.Filter during room temperature with air-dry and obtain unchanged polymorphic A.

Polymorphic F

The another kind of crystal polymorphic of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " F type " or " polymorphic F " at this.Polymorphic F is the tool hygroscopicity slightly, and water absorption is about 3 weight %, and when with 10 ℃/minute speed heating, the water that is absorbed discharges between 50 ℃～200 ℃ continuously.Polymorphic F is a metastable state, and is the hygroscopicity anhydride, and is more stable than A type when room temperature, stable not as Type B when higher temperature, and the F type is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic F can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that polymorphic F shows with the d-value (

) be expressed as: 17.1 (vs.), 12.1 (w), 8.6 (w), 7.0 (w), 6.5 (w), 6.4 (w), 5.92 (W), 5.72 (w), 5.11 (w), 4.92 (m), 4.86 (w), 4.68 (m), 4.41 (w), 4.12 (w), 3.88 (w), 3.83 (w), 3.70 (m), 3.64 (w), 3.55 (m), 3.49 (s), 3.46 (vs), 3.39 (s), 3.33 (m), 3.31 (m), 3.27 (m), 3.21 (m), 3.19 (m), 3.09 (m), 3.02 (m) and 2.96 (m).Fig. 8 is the characteristic X-ray diffraction pattern that F type (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can obtain polymorphic F by balancing each other of the suspension of polymorphic A in suitable polar nonaqueous solvent, described solvent dissolves described low-yield form hardly, particularly such as alcohols such as methanol, ethanol, propanol and isopropyl alcohols.Also can be by when being lower than room temperature, the solid particle of described A type (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride being scattered in the nonaqueous solvent that dissolves described (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride hardly, this float is stirred to the time that is enough to produce polymorphic F in described temperature, the F type of fractional crystallization and from the isolating F type of institute, remove and desolvate then, thus the polymorphic F of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride obtained.The removal of solvent and drying can or be lower than room temperature (for example being low to moderate 0 ℃) in room temperature and carry out at air, dry air or under such as dry-run protection gases such as nitrogen or rare gas.Temperature in the process that balances each other is preferably 5 ℃～15 ℃, most preferably is about 10 ℃.

Polymorphic J

The another kind of crystal polymorphic of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " J type " or " polymorphic J " at this.Polymorphic J is the tool hygroscopicity slightly, and when handling in humid air, it can absorb water.Polymorphic J is a metastable state, and is the hygroscopicity anhydride, it can be transformed back E type as described below, and the E type is to obtain by the J type being exposed under the high relative humidity condition of (for example being higher than 75% relative humidity).The J type is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic J can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that polymorphic J shows with the d-value (

) be expressed as: 14.6 (m), 6.6 (w), 6.4 (w), 5.47 (w), 4.84 (w), 3.29 (vs) and 3.21 (vs).Fig. 9 is the characteristic X-ray diffraction pattern that J type (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can be by under vacuum, the dehydration of E type being obtained the J type when the moderate temperature.In particular, can obtain the J type with the water of removing in the E type by obtaining the E type and in vacuum desiccator, handling the E type in moderate temperature, thereby the polymorphic J of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, described moderate temperature can be 25 ℃～70 ℃, most preferably is 30 ℃～50 ℃.

Polymorphic K

The another kind of crystal polymorphic of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " K type " or " polymorphic K " at this.Polymorphic K is the tool hygroscopicity slightly, and water absorption is about 2.0 weight %, and when with 10 ℃/minute speed heating, the water that is absorbed discharges between 50 ℃～100 ℃ continuously.Polymorphic K is a metastable state, and is the hygroscopicity anhydride, and is stable not as Type B when higher temperature, and the K type is particularly suitable as intermediate and raw material that polymorphic (particularly Type B) stablized in preparation.Polymorphic K can be prepared into pressed powder with required moderate particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that polymorphic J shows with the d-value ( ) be expressed as: 14.0 (s), 9.4 (w), 6.6 (w), 6.4 (w), 6.3 (w), 6.1 (w), 6.0 (w), 5.66 (w), 5.33 (w), 5.13 (vw), 4.73 (m), 4.64 (m), 4.48 (w), 4.32 (vw), 4.22 (w), 4.08 (w), 3.88 (w), 3.79 (w), 3.54 (m), 3.49 (vs), 3.39 (m), 3.33 (vs), 3.13 (s), 3.10 (m), 3.05 (m), 3.01 (m), 2.99 (m) and 2.90 (m).Figure 10 is the characteristic X-ray diffraction pattern that K type (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can by under having the situation of a small amount of ascorbic acid from the polar solvent mixture that contains low amounts of water crystallization obtain polymorphic K.The used solvent of solvent mixture can be selected from acetic acid and such as alcohol such as methanol, ethanol, normal propyl alcohol or isopropyl alcohols.In particular, can be by (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride being dissolved in the acetic acid and alcohol or tetrahydrofuran compound that comprises low amounts of water and a small amount of ascorbic acid when the higher temperature, temperature is reduced to is lower than room temperature so that described dihydrochloride crystallization, precipitation separation, and in the dry isolated precipitation of higher temperature, can under vacuum, carry out described drying in case of necessity, thus the polymorphic K of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.Suitable alcohol is for example methanol, ethanol, propanol and isopropyl alcohol, preferably ethanol.The ratio of acetic acid and alcohol or oxolane can be 2:1～1:2, is preferably about 1:1.Can under the situation that has more high-load water, carry out the dissolving of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, and can add more anti-solvent mixture (anti-solvent mixture), thereby be precipitated completely.With respect to described solvent mixture, the amount of the water in the final composition can be 0.5 weight %～5 weight %, and the amount of ascorbic acid can be 0.01 weight %～0.5 weight %.Solution temperature can be 30 ℃～100 ℃, is preferably 35 ℃～70 ℃, and baking temperature can be 30 ℃～50 ℃.It is available such as pure washing precipitations such as ethanol to separate back (for example filtering the back).Can be when being higher than room temperature (for example 30 ℃～40 ℃) balancing each other in isopropyl alcohol for example polymorphic K easily is converted into the most stable Type B, in case of necessity with the Type B crystal as crystal seed.

(6R)-hydrate forms of L-erythro form-tetrahydrobiopterin dihydrochloride

Further describe as following, find that there are many crystalline hydrates in (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, described and it is defined as C type, D type, E type, H type and O type at this.These hydrate forms can be used as pharmaceutical preparation described herein stable form with BH4, and can be used in the polymorphous preparation of compositions of the stable crystal that comprises BH4.

C type hydrate

A kind of hydrate crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " C type " or " hydrate C " at this.C type hydrate is the tool hygroscopicity slightly, and water content is about 5.5 weight %, shows that the C type is a monohydrate.The fusing point of hydrate C is about 94 ℃ of (Δ H _fBe about 31J/g), and C type hydrate is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic C can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the C type shows with the d-value (

) be expressed as: 18.2 (m), 15.4 (w), 13.9 (vs), 10.4 (W), 9.6 (w), 9.1 (w), 8.8 (m), 8.2 (w), 8.0 (w), 6.8 (m), 6.5 (w), 6.05 (m), 5.77 (w), 5.64 (w), 5.44 (w), 5.19 (w), 4.89 (w), 4.76 (w), 4.70 (w), 4.41 (w), 4.25 (m), 4.00 (m), 3.88 (m), 3.80 (m), 3.59 (s), 3.50 (m), 3.44 (m), 3.37 (m), 3.26 (s), 3.19 (vs), 3.17 (s) 3 3.11 (m), 3.06 (m), 3.02 (m), 2.97 (vs), 2.93 (m), 2.89 (m), 2.83 (m) and 2.43 (m).Figure 11 is the characteristic X-ray diffraction pattern that the C type hydrate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Obtain C type hydrate such as balancing each other of polymorphous suspensions such as polymorph b in the time of can be by room temperature in the non-solvent,, comprise the water that is preferably about 5 weight % in the described suspension wherein with respect to described solvent.Can be by (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride be suspended in such as heptane, C1～C4 alcohol (methanol for example, ethanol, normal propyl alcohol or 2-propanol), acetas (for example ethyl acetate), acetonitrile, acetic acid or ether (oxolane diox for example, t-butyl methyl ether) etc. in the binary mixture or ternary mixture of non-solvent or such non-solvent, to wherein adding enough water to form monohydrate, and in room temperature or (for example be lower than room temperature, 0 ℃～30 ℃) this suspension is stirred to the time that is enough to form monohydrate, thus the C type hydrate of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.With respect to the amount of solvent, enough water can be 1 weight %～10 weight %, be preferably the water of 3 weight %～8 weight %.Solid can be leached, and when being about room temperature at air drying.Described solid can absorb some water, thereby has the water content higher than the theoretical value of 5.5 weight %.With respect to D type and Type B, C type hydrate is also unstable, is converted into polymorph b in the time of about 40 ℃ in air and under lower relative humidity easily.Suspension balance in the time of can be by room temperature is converted into more stable hydrate D with the C type.

D type hydrate

The another kind of hydrate crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " D type " or " hydrate D " at this.D type hydrate is the tool hygroscopicity slightly, can have the water content of about 5.0 weight %～7.0 weight %, shows that the D type is a monohydrate.The fusing point of hydrate D is about 153 ℃ of (Δ H _fBe about 111J/g), have the stability more much higher than C type, even when all being stable when room temperature is exposed to atmospheric humidity.Therefore D type hydrate can be used to prepare preparation or stablize polymorphous intermediate and raw material as preparation.Polymorphic D can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the D type shows with the d-value (

) be expressed as: 8.6 (s), 6.8 (w), 5.56 (m), 4.99 (m), 4.67 (s), 4.32 (m), 3.93 (vs), 3.88 (w), 3.64 (w), 3.41 (w), 3.25 (w), 3.17 (m), 3.05 (s), 2.94 (w), 2.92 (w), 2.88 (m), 2.85 (w), 2.80 (w), 2.79 (m), 2.68 (w), 2.65 (w), 2.52 (vw), 2.35 (w), 2.34 (w), 2.30 (w) and 2.29 (w).Figure 12 is the characteristic X-ray diffraction pattern that the D type hydrate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can be by the concentrated aqueous solution of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride being added when about room temperature to excessive non-solvent (for example hexane, heptane, dichloromethane, normal propyl alcohol or 2-propanol, acetone, ethyl acetate, acetonitrile, acetic acid or such as ethers such as oxolane, diox, t-butyl methyl ether, or the binary mixture of such non-solvent or ternary mixture) in, and at this suspension of stirring at room, thereby obtain D type hydrate.This crystalline solid can be leached, dry under drying nitrogen during room temperature then.Preferred non-solvent is an isopropyl alcohol.The interpolation of aqueous solution can dropwise be carried out, to avoid rapid precipitation.Can be by adding in the excessive non-solvent at the concentrated aqueous solution of about room temperature with (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, and at this suspension of stirring at room, thereby the D type hydrate of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.Excessive non-solvent can be meant that the ratio of aqueous solution and nonaqueous solvent is 1:10～1:1000.The D type comprises the water excessive slightly with respect to monohydrate, it is believed that this water because the slight hygroscopicity of this crystal water compound but adsorbed water.D type hydrate is considered to be in room temperature and less than in the known hydrate under 70% the relative humidity the most stable one.D type hydrate is used in prepared preparation under the stable condition of this hydrate.Room temperature can be meant 20 ℃～30 ℃.

E type hydrate

The another kind of hydrate crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " E type " or " hydrate E " at this.The water content of E type hydrate is about 10 weight %～14 weight %, shows that the E type is a dihydrate.Hydrate E forms under subambient temperature.E type hydrate is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.The E type is particularly suitable for preparing anhydrous J type by (in case of necessity under vacuum) drying under nitrogen.The E type is not had a hygroscopicity, is still stable under quite high relative humidity (that is, be higher than about 60% and the highest about 85% relative humidity).Polymorphic E can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the E type shows with the d-value (

) be expressed as: 15.4 (s), 6.6 (w), 6.5 (w), 5.95 (vw), 5.61 (vw), 5.48 (w), 5.24 (w), 4.87 (w), 4.50 (vw), 427 (w), 3.94 (w), 3.78 (w), 3.69 (m), 3.60 (w), 3.33 (s), 3.26 (vs), 3.16 (w), 3.08 (m), 2.98 (w), 2.95 (m), 2.91 (w), 2.87 (m), 2.79 (w), 2.74 (w), 2.69 (w) and 2.62 (w).Figure 13 is the characteristic X-ray diffraction pattern that the E type hydrate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can add to excessive by concentrated aqueous solution and be cooled in the non-solvent of about 10 ℃～-10 ℃ (being preferably 0 ℃～-10 ℃) (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, and stir this suspension, thereby obtain E type hydrate in described temperature.This crystalline solid can be leached, dry under drying nitrogen during room temperature then.Non-solvent is, for example hexane, heptane, dichloromethane, normal propyl alcohol or 2-propanol, acetone, ethyl acetate, acetonitrile, acetic acid or such as ethers such as oxolane, diox, t-butyl methyl ether, or the mixture of such non-solvent.Preferred non-solvent is an isopropyl alcohol.The interpolation of aqueous solution can dropwise be carried out, to avoid rapid precipitation.Can add to excessive by concentrated aqueous solution and be cooled in about 10 ℃～-10 ℃ non-solvent (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, and in this float of stirring at room, thereby the E type hydrate of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.Excessive non-solvent can be meant that the ratio of aqueous solution and nonaqueous solvent is 1:10～1:1000.Preferred non-solvent is an oxolane.Another kind of preparation method comprises that it is 70%～90% that polymorph b is exposed to relative humidity, is preferably in about 80% the atmosphere.E type hydrate is considered to dihydrate, thus more adsorbable extra water.Can be by dry and polymorphic E is converted into polymorphic J in moderate temperature (can be that temperature is 20 ℃～50 ℃, pressure be 0 millibar～100 millibars) under vacuum.Because the stability of E type in higher relative humidity, it is particularly suitable for the preparation of semi-solid form.

H type hydrate

The another kind of hydrate crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " H type " or " hydrate H " at this.The water content of H type hydrate is about 5.0 weight %～7.0 weight %, shows that the H type is hygroscopic monohydrate.H type hydrate forms under subambient temperature.H type hydrate is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic H can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the H type shows with the d-value (

) be expressed as: 8.615.8 (vs), 10.3 (w), 8.0 (w), 6.6 (w), 6.07 (w), 4.81 (w), 4.30 (w), 3.87 (m), 3.60 (m), 3.27 (m), 3.21 (m), 3.13 (w), 3.05 (w), 296 (m), 2.89 (m), 2.82 (w) and 2.67 (m).Figure 14 is the characteristic X-ray diffraction pattern that the H type hydrate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can be by (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride being dissolved in the mixture of acetic acid and water when the room temperature, add non-solvent then to be settled out crystalline solid, stir certain hour with the suspension cooling of gained and with refrigerative suspension, thereby obtain H type hydrate.Described crystalline solid is leached, then drying at room temperature under vacuum.Non-solvent is, for example hexane, heptane, dichloromethane, normal propyl alcohol or 2-propanol, acetone, ethyl acetate, acetonitrile, acetic acid or such as ethers such as oxolane, diox, t-butyl methyl ether, or the mixture of such non-solvent.Preferred non-solvent is an oxolane.In can mixture by the water that in room temperature (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is dissolved in acetic acid and lacks than the amount of acetic acid, add non-solvent and the suspension of gained is cooled to-10 ℃～10 ℃, be preferably-5 ℃～5 ℃, and this suspension is stirred certain hour, thereby the H type hydrate of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride in described temperature.Certain hour can refer to 1 hour～20 hours.The weight ratio of acetic acid and water can be 2:1～25:1, is preferably 5:1～15:1.The weight ratio of acetic acid/water and non-solvent can be 1:2～1:5.H type hydrate seemingly contains the monohydrate of excessive slightly adsorbed water because of hygroscopicity.

O type hydrate

The another kind of hydrate crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " O type " or " hydrate O " at this.O type hydrate forms in the temperature near room temperature.O type hydrate is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic O can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.The characteristic peak of the characteristic X-ray powder diffraction pattern that the O type shows with the d-value (

) be expressed as: 15.9 (w), 14.0 (w), 12.0 (w), 8.8 (m), 7.0 (w), 6.5 (w), 6.3 (m), 6.00 (w), 5.75 (w), 5.65 (m), 5.06 (m), 4.98 (m), 4.92 (m), 4.84 (w), 4.77 (w), 4.42 (w), 4.33 (w), 4.00 (m), 3.88 (m), 3.78 (w), 3.69 (s), 3.64 (s), 3.52 (vs), 3.49 (s), 3.46 (s), 3.42 (s), 3.32 (m), 3.27 (m), 3.23 (s), 3.18 (s), 3.15 (vs), 3.12 (m), 3.04 (vs), 2.95 (m), 2.81 (s), 2.72 (m), 2.67 (m) and 2.61 (m).Figure 15 is the characteristic X-ray diffraction pattern that the O type hydrate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can be by polymorphic F be exposed about 24 hours in containing the nitrogen atmosphere of steam (final relative humidity be about 52%), thus prepare O type hydrate.The F type is the hygroscopic anhydride of summary tool, is used in 52% the following preparation of relative humidity O type, and this fact shows that the O type is a hydrate, and is more stable than F type under room temperature and wet condition.

(6R)-solvate forms of L-erythro form-tetrahydrobiopterin dihydrochloride

Further describe as following, find that there are many crystalline solvate forms in (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride, described at this, and it is defined as G type, I type, L type, M type and N type.These solvate forms can be used as pharmaceutical preparation described herein stable form with BH4, and can be used in the preparation of the polymorphous compositions of stable crystal that comprises BH4.

G type solvate

A kind of alcohol solvent compound crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " G type " or " hydrate G " at this.The ethanol content of G type alcohol solvent compound is about 8.0 weight %～12.5 weight %, shows that the G type is a hygroscopicity monoethanol solvate.G type solvate forms in subambient temperature.The G type is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic G can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the G type shows with the d-value (

) be expressed as: 14.5 (vs), 10.9 (w), 9.8 (w), 7.0 (w), 6.3 (w), 5.74 (w), 5.24 (vw), 5.04 (vw), 4.79 (w), 4.41 (w), 4.02 (w), 3.86 (w), 3.77 (w), 3.69 (w), 3.63 (m), 3.57 (m), 3.49 (m), 3.41 (m), 3.26 (m), 3.17 (m), 3.07 (m), 2.97 (m), 2.95 (m), 2.87 (w) and 2.61 (w).Figure 16 is the characteristic X-ray diffraction pattern that the G type solvate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can be by L-erythro form-tetrahydrobiopterin dihydrochloride be dissolved in the water, and add excess ethanol greatly, the suspension of gained is stirred in room temperature or subambient temperature, and when room temperature roughly that isolated solid is dry under air or nitrogen, thereby obtain G type alcohol solvent compound.At this, excess ethanol is meant that the water in the mixture of second alcohol and water of gained is less than 10%, is preferably about 3%～6% greatly.Can (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride be dissolved in water or water and the alcoholic acid mixture by in about room temperature～75 ℃ the time; with the hot solution cool to room temperature and reduce to 5 ℃～10 ℃; can add ethanol in case of necessity to finish precipitation; at 20 ℃～5 ℃ suspensions that stir gained; leach white crystalline solid; and when room temperature roughly with described solid under the air or dry under such as protective gas such as nitrogen, thereby the G type alcoholate of preparation (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride.In the first variation scheme, this method can be carried out like this: when about room temperature (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is dissolved in a spot of water, adds excess ethanol then, the suspension with gained is stirred to the time that is enough to balance each other then.In the second variation scheme, (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride can be suspended in the ethanol, add a spot of water in case of necessity, and with this suspension heating, make (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride dissolving, this solution is cooled to about 5 ℃～15 ℃, adds other ethanol in this float, the suspension with gained is stirred to the time that is enough to balance each other then.

I type solvate

A kind of acetic acid solvent thing crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " I type " or " hydrate I " at this.The acetic acid content of I type acetic acid solvent thing is about 12.7 weight %, shows that the I type is a hygroscopicity acetic acid list solvate.I type solvate forms in subambient temperature.I type acetic acid solvent thing is particularly suitable as preparation and stablizes polymorphous intermediate and raw material.Polymorphic I can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the I type shows with the d-value ( ) be expressed as: 14.5 (m), 14.0 (w), 11.0 (w), 7.0 (vw), 6.9 (vw), 6.2 (vw), 5.30 (w), 4.79 (w), 4.44 (w), 4.29 (w), 4.20 (vw), 4.02 (w), 3.84 (w), 3.80 (w), 3.67 (vs), 3.61 (m), 3.56 (w), 3.44 (m), 3.27 (w), 3.19 (w), 3.1l (s), 3.00 (m), 2.94 (w), 2.87 (w) and 2.80 (w).Figure 17 is the characteristic X-ray diffraction pattern that the I type solvate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can by with L-erythro form-tetrahydrobiopterin dihydrochloride in high-temperature digestion in the mixture of acetic acid and water, in described solution, further add acetic acid, be cooled to about 10 ℃, formed suspension is heated to about 15 ℃ then, then resulting suspension is stirred to the time that is enough to balance each other, mixing time can continue up to 3 days, thereby obtained I type acetic acid solvent thing.Leach this crystalline solid then, and at air or under such as protective gas such as nitrogen at the temperature drying of about room temperature.

L type solvate

The mixture of finding a kind of alcohol solvent compound/hydrate crystal form of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " L type " or " hydrate L " at this.The L type can contain 4% to high water to 13% ethanol and 0%～about 6%.When handling in ethanol for about 0 ℃～20 ℃, the L type can be converted into the G type.In addition, when when handling in organic solvent, the L type can be converted into Type B in room temperature (10 ℃～60 ℃).Polymorphic L can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the L type shows with the d-value (

) be expressed as: 14.1 (vs), 10.4 (w), 9.5 (w), 9.0 (vw), 6.9 (w), 6.5 (w), 6.1 (w), 5.75 (w), 5.61 (w), 5.08 (w) 54.71 (w), 3.86 (w), 3.78 (w), 3.46 (m), 3.36 (m), 3.06 (w), 2.90 (w) and 2.82 (w).Figure 18 is the characteristic X-ray diffraction pattern that the L type solvate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can by with E type hydrate in the room temperature low suspension in ethanol, and, be preferably about 5 ℃ at 0 ℃～10 ℃, this suspension is stirred to the time that is enough to balance each other, mixing time can be 10 hours～20 hours, thereby obtains the L type.Then crystalline solid is leached and drying, drying condition preferably reduces pressure in the time of 30 ℃, or under nitrogen.The L type that the analysis showed that TG-FTIR may contain the second alcohol and water of non-quantitative, that is, it can be used as polymorphic (anhydride) existence, or exists as the mixture of alcohol solvent compound/hydrate, even exists as hydrate.

M type solvate

A kind of alcohol solvent compound crystal form of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " M type " or " hydrate M " at this.The M type can contain 4% to high water to 13% ethanol and 0%～about 6%, shows that the M type is the hygroscopic alcohol solvent compound of tool slightly.M type solvate forms in room temperature.Because when when in ethanol, handling for about-10 ℃～15 ℃, the M type can be converted into the G type, when in such as ethanol, the pure and mild C4 alcohol of C3 or cyclic ethers organic solvents such as (for example THF are with diox), handling, the M type can be converted into Type B, so the M type is especially suitable for use as preparation and stablizes polymorphous intermediate and raw material.Polymorphic M can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the M type shows with the d-value (

) be expressed as: 18.9 (s), 6.4 (m), 6.06 (w), 5.66 (w), 5.28 (w), 4.50 (w), 4.23 (w) and 3.22 (vs).Figure 19 is the characteristic X-ray diffraction pattern that the M type solvate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can by room temperature (that is, 10 ℃～40 ℃) with L-erythro form-tetrahydrobiopterin dihydrochloride dissolve with ethanol in ethanol, and under nitrogen, make solution evaporation, thereby obtain M type alcohol solvent compound.Thereby also can be by being in speed under the dry nitrogen feeble QI air-flow of about 20ml/ minute～100ml/ minute with the dry M type that obtains of G type.Depend on the degree of drying under the nitrogen, alcoholic acid residual volume can change, and promptly about 3%～13%.

N type solvate

The another kind of solvate crystal forms of finding (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride is the preferred stable form that is used for the BH4 of pharmaceutical preparation described herein, is referred to as " N type " or " hydrate N " at this.The N type can contain the highest 10% isopropyl alcohol and water altogether, shows that the N type is to omit the hygroscopic isopropanol solvate of tool.Can obtain the N type at about 30 ℃ of vacuum dryings then by with washed with isopropyl alcohol D type.The N type is especially suitable for use as preparation and stablizes polymorphous intermediate and raw material.Polymorphic N can be prepared into pressed powder with required meta particle size range that is generally 1 μ m～about 500 μ m.

The characteristic peak of the characteristic X-ray powder diffraction pattern that the N type shows with the d-value (

) be expressed as: 19.5 (m), 9.9 (w), 6.7 (w), 5.15 (w), 4.83 (w), 3.91 (w), 3.56 (m), 3.33 (vs), 3.15 (w), 2.89 (w), 2.81 (w), 2.56 (w) and 2.36 (w).Figure 20 is the characteristic X-ray diffraction pattern that the N type solvate of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride shows.

Can be by L-erythro form-tetrahydrobiopterin dihydrochloride being dissolved in the isopropyl alcohol of 4.0ml and the mixture of water (mixed volume is than being for example 4:1), thus isopropyl alcohol type N obtained.Slowly add isopropyl alcohol (IPA, for example about 4.0ml) to this solution, the gained suspension is cooled to 0 ℃, and in this temperature stirred for several hour (for example, about 10 hours～18 hours).In room temperature this float is filtered, and use the washed with isopropyl alcohol solid residue.Crystalline material with gained descends dry a few hours (for example, about 5 hours～20 hours) in room temperature (for example, about 20 ℃～30 ℃) and decompression (about 2 millibars～10 millibars) then.TG-FTIR is presented between 25 ℃～200 ℃ 9.0% the loss in weight, and this is that isopropyl alcohol and water cause jointly.This result shows that the N type can exist with the form of isopropanol solvate, also can exist with the mixed form of isopropanol solvate/hydrate, perhaps exists with the non-solvent form that comprises low amounts of water.

For polymorphous preparation, can adopt crystallization technique well known in the art, for example the decomposition of stirred suspension (balancing each other), precipitation, recrystallization, evaporation, solvent (as water) absorption method or solvate.Available dilution, saturated or oversaturated solution carries out crystallization, can also can not add crystal seed with suitable nucleator as crystal seed.Can adopt the highest 100 ℃ temperature to form solution.Can reduce to-100 ℃, be preferably the cooling of reducing to-30 ℃ to cause crystallization and precipitation.Metastable polymorphic or pseudo-polymorphic can be used to prepare solution or suspension (this solution or suspension are used to prepare more stable form), and be used to the solution concentration that reaches higher.

Surprisingly, find that D type hydrate is a form the most stable in the hydrate, Type B and D type are particularly suitable for being used in the pharmaceutical preparation.Type B and D type possess some advantages, for example targeted preparation, the good processing that has because of the crystal size that suits and form, the extraordinary stability under the working condition of types of formulation, storage stability, higher water solublity and higher biocompatibility.Therefore, an embodiment in this disclosed compositions and method is to comprise the polymorph b of (6R)-L-erythro form-tetrahydrobiopterin dihydrochloride and/or the pharmaceutical composition of D type hydrate and pharmaceutical carrier or diluent.

(6R)-and the crystal form of L-erythro form-tetrahydrobiopterin dihydrochloride can be with a kind of use in folic acid or tetrahydrofolic acid or their pharmaceutical salts (for example sodium salt, potassium salt, calcium salt or ammonium salt), perhaps uses with wherein a kind of arginine of adding.Crystal form: folic acid or its salt: arginic weight ratio can be about 1:10:10～about 10:1:1.

The invention provides any tetrahydrobiopterin polymorphic described herein or comprise the method that any so polymorphous stabilised pharmaceutical is used for the treatment of the disease relevant with endothelial function disturbance.Also relate to a kind of like this method: carry out Synergistic treatment with folic acid class (comprising folic acid precursors, folic acid or folic acid derivatives), with not only comprise tetrahydrobiopterin or BH4 precursor or BH4 derivant but also comprise the pharmaceutical composition of folic acid or food to treat also be the same.United States Patent (USP) the 6th; 011; 040 and 6; 544; disclosed exemplary folic acid in 994 (these two parts of patents all being incorporated herein by quoting as proof) at this; described exemplary folic acid comprises folic acid (pteroylmonoglutamic acid); dihydrofoilic acid; tetrahydrofolic acid; the 5-methyl tetrahydrofolate; 5; the 10-methylene tetrahydrofolate; 5; the 10-anhydroleucovorin; 5, the 10-formiminotetrahydrofolic acid; 5-formoxyl tetrahydrofolic acid (folinic acid); 10-formoxyl tetrahydrofolic acid; the 10-methyl tetrahydrofolate; more than one leaf acyl polyglutamic acid; wherein the pyrazine ring of the pterin of folic acid or leaf acyl polyglutamic acid part is reduced to provide such chemical compound of dihydrofoilic acid or tetrahydrofolic acid; or on N-5 or N-10 position, have a derivant of all aforesaid compounds of a carbon unit of various oxidation level; or their pharmaceutical salts; or the combination of two or more above-claimed cpds.Exemplary tetrahydrofolic acid comprises 5-formoxyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5; the 10-methylene-(6S)-tetrahydrofolic acid, 5; 10-methine-(6S)-tetrahydrofolic acid, 10-formoxyl-(6S)-tetrahydrofolic acid, 5-formimino group-(6S)-tetrahydrofolic acid or (6S)-tetrahydrofolic acid, and their salt.

4. pharmaceutical preparation

Preferably preparation described herein is used as oral formulations.Oral formulations is preferably such as solid preparations such as capsule, tablet, pill and lozenge, or such as liquid preparations such as water slurry, elixir and syrup.The various forms of BH4 described herein directly can be used with powder (micronized particle), granule, suspension or solution mode, perhaps itself and other medicinal ingredient can be grouped together, comprise described component is mixed, in case of necessity with its fine pulverizing, the capsule of packing into then and forming by for example glutoid or soft gelatin, perhaps be pressed into tablet, pill or lozenge, perhaps with its suspension or be dissolved in and be used for suspension, elixir and syrupy carrier.Can be coated with coating after being compressed into pill.

The medicinal ingredient that is used for various forms of preparations is well-known, for example, can be various preparation types used such as binding agent, excipient, lubricant, surfactant, sweeting agent, aromatic, coating material, antiseptic, dyestuff, thickening agent, adjuvant, antibacterial, antioxidant and carriers such as natural or synthetic polymers.The limiting examples of used binding agent comprises Tragacanth, Radix Acaciae senegalis, starch, gelatin and biodegradable polymer in the compositions described herein, for example the equal polyester or the copolyesters of dicarboxylic acids, alkylene glycol, ployalkylene glycol and/or aliphatic hydroxyl carboxylic acid; The homopolyamide or the copolyamide of dicarboxylic acids, alkylene diamine and/or aliphatic amino carboxylic acid; Corresponding polyester-polyamide copolymer, poly-anhydride, poe, poly phosphazene and Merlon.Described biodegradable polymer can be straight chain, branching or crosslinked.Instantiation is polyglycolic acid, polylactic acid and poly--d, the l-lactide/glycolides.Other examples of polymer are such as polyoxyalkylene (water-soluble polymers such as the polyacrylamide of polyoxyethylene, polyoxypropylene and mixed polymer thereof, polyacrylamide and hydroxyalkylation, poly and ester thereof or amide, polyacrylic acid and ester thereof or amide, polyvinyl alcohol and ester thereof or ether, polyvinyl imidazole, polyvinylpyrrolidone, and such as natural polymers such as chitosans.

The limiting examples of used excipient comprises such as phosphate such as dicalcium phosphates in the compositions described herein.The limiting examples of used lubricant comprises natural or artificial oil, fat, wax or such as soaps such as magnesium stearate in the compositions described herein.

Used surfactant can be anionic, anionic, zwitterionic or neutral in the compositions described herein.The limiting examples of used surfactant comprises lecithin in the compositions described herein; phospholipid; the sulphuric acid monooctyl ester; sulphuric acid ester in the last of the ten Heavenly stems; the sulphuric acid dodecyl ester; sulphuric acid myristyl ester; sulphuric acid cetyl ester and sulphuric acid stearyl; enuatrol or Capric acid sodium salt; 1-acylamino-ethane-2-sulfonic acid (1-decoyl aminoethane-2-sulfonic acid for example; 1-caprinoyl aminoethane-2-sulfonic acid; 1-dodecanoyl aminoethane-2-sulfonic acid; 1-myristoyl aminoethane-2-sulfonic acid; 1-hexadecanoyl aminoethane-2-sulfonic acid; 1-stearyl aminoethane-2-sulfonic acid); and taurocholic acid and tauroursodeoxycholic acid; bile acid and their salt; cholic acid for example; deoxycholic acid and sodium glycocholate; Capric acid sodium salt or sodium laurate; enuatrol; sodium lauryl sulphate; sodium hexadecyl sulfate; sulfated castor oil and dioctyl sodium sulphosuccinate; cocamido propyl betaine and lauryl betaine; aliphatic alcohol; cholesterol; glyceryl monostearate or distearin; glyceryl monooleate or glyceryl dioleate; with monopalmitin or glycerol-1,3-dipalmitate, and stearic acid polyoxyethylene base ester.

The limiting examples of used sweeting agent comprises sucrose, fructose, lactose or aspartame in the compositions described herein.The limiting examples of used aromatic comprises Herba Menthae, wintergreen oil or such as fruit flavors such as Fructus Pruni pseudocerasi or Fructus Citri tangerinaes in the compositions described herein.The limiting examples of used coating material comprises gelatin, wax, Lac, sugar or other biodegradable polymer in the compositions described herein.The limiting examples of used antiseptic comprises methyl parahydroxybenzoate or propyl p-hydroxybenzoate, sorbic acid, methaform, phenol and thiomersalate in the compositions described herein.

Also D type hydrate described herein can be mixed with effervescent tablet or effervescent powder, they decompose in aqueous environment so that drinkable solution to be provided.Syrup or elixir can comprise polymorphic described herein, as the sucrose of sweeting agent or fructose, such as antiseptic such as methyl parahydroxybenzoate, dyestuff and aromatic.

For the controllable release of activating agent in gastrointestinal tract that realizes contacting, and in blood plasma, provide the activating agent of constant substantially and effective level, also polymorphic described herein can be mixed with slow releasing preparation with body fluid.For this purpose, this crystal form can be embedded in the polymeric matrix of biodegradable polymer, water-soluble type polymer or both mixture composition, also can comprise suitable surfactant in case of necessity.Embedding can refer to microgranule is mixed in the polymeric matrix in this article.Controlled release preparation also can make by by known dispersion or emulsifying packaging technique dispersed microparticles or emulsive microdroplet being sealed.

Though individual need is different, the optimum range of the effective dose of every kind of composition determine to belong to techniques well known.The typical doses of BH4 comprises about 1mg/ kg body weight/sky～about 20mg/ kg body weight/sky, is generally equal to about 5mg/ days (1mg/kg * 5 kg body weight)～3000mg/ days (30mg/kg * double centner body weight).Such dosage can single dose administration, also can be divided into multiple dose.Though can every day successive administration, it is desirable to bring up to and just stop the BH4 treatment when being higher than certain threshold level when concrete clinical indices.Certainly, under the situation that the clinical improvements index worsens, can restart this treatment.

The suitable dose that should be appreciated that compositions of the present invention will depend on kind, the therapeutic frequency of receiver's age, health status and body weight, Synergistic treatment (if any), the character of the desired effect that reaches (that is, desired the low amount of lung pressure drop).Administration frequency also depends on the drug effect that arterial oxygen is pressed.Yet, understand and confirmable as those skilled in the art, need not can customize most preferred dose by individual subject by undue experimentation.This generally includes the adjustment of standard dose, for example, if patient's body weight is lower, then reduces dosage.

As mentioned above, can be divided into multiple dosing with treat required accumulated dose at every turn, but also single dose administration.The BH4 compositions can be used separately, also can it is co-administered with the treatment at other symptoms of this disease or this disease.

From then on the disclosure that presents of place can find out obviously that in the broadest sense, the present invention aims to provide the clinical practice of the compositions that comprises crystalline BH4 preparation.Described compositions should be mixed with suitable pharmaceutical compositions, that is, and the form that in such therapeutic alliance, is suitable for using in the body.Usually, this will need to prepare and not contain pyrogen and can be to the compositions of deleterious other impurity of humans and animals substantially.Preferably, the preparation that comprises crystalline BH4 compositions can be such: can be directly used in the treatment angiopathy.

Usually need to adopt suitable salt and buffer agent so that BH4 is suitable for absorbing.Waterborne compositions of the present invention comprise the dissolving of effective dose be dispersed in pharmaceutical carrier or aqueous medium in BH4.Such composition palatable clothes administration or drug administration by injection.

Term " medicinal () " be meant and when this molecular entity and compositions are applied to the animal or human, do not produce untoward reaction, anaphylaxis or other adverse effects." pharmaceutical carrier " used herein comprises solvent, disperse medium, coating, antibacterial and antifungal, isotonic agent and absorption delayed-action activator etc. arbitrarily.It is well-known in the art that such medium and medicament are used for pharmaceutically active substance.Unless incompatible with described therapeutic combination, any conventional media or the purposes of medicament in described therapeutic combination are also included within the scope of the present invention.Also the supplementary active component can be added in the described compositions.In the exemplary embodiment, the pharmaceutical protein preparation can comprise corn-syrup solids; high oleic safflower oil; Oleum Cocois; soybean oil; the L-leucine; tricalcium phosphate; L-tyrosine; the L-proline; the L-lysine acetate; DATEM (a kind of emulsifying agent); L-glutaminate; the L-valine; dikalium phosphate; the L-isoleucine; the L-arginine; the L-alanine; glycine; the altheine monohydrate; the L-serine; potassium citrate; the L-threonine; sodium citrate; magnesium chloride; the L-histidine; the L-methionine; ascorbic acid; calcium carbonate; L-glutamic acid; L-cystine dihydrochloride; the L-tryptophan; the L-aspartic acid; choline chloride; taurine; the m-inositol; ferrous sulfate; ascorbic palmitate; zinc sulfate; the L-carnitine; acetic acid α-fertility acid esters; sodium chloride; nicotiamide; mixed tocopherol; calcium pantothenate; copper sulfate; the thiamine chloride hydrochlorate; vitamin A palmitate; magnesium sulfate; riboflavin; pyridoxine hydrochloride; folic acid; beta-carotene; potassium iodide; phylloquinone; biotin; sodium selenate; Chlorizate chromium; sodium molybdate; vitamin D3 and cyanocobalamin.Aminoacid in the enlargement, the amount of providing of minerals and vitamins should provide the recommended of each component.

" pharmaceutical carrier " used herein comprises solvent, disperse medium, coating, antibacterial and antifungal, isotonic agent and absorption delay agent etc. arbitrarily.It is well-known in the art that such medium and medicament are used for pharmaceutically active substance.Unless incompatible with described active component, any conventional media or the purposes of medicament in described therapeutic combination are also included within the scope of the present invention.Also the supplementary active component can be added in the described compositions.

Active compound of the present invention comprises the standard drug preparation of BH4, these preparations and those preparations well known by persons skilled in the art is discussed at this.These compositionss of the present invention will be used by any conventional route that is used for meal supplement.The same with BH4, preferably that described protein is oral.

In some embodiments, the present invention includes and be mixed with suction preparation by inhalation with being used for the treatment of the BH4 of angiopathy or its precursor or derivatives thereof.Similarly, BH4 or its precursor or derivatives thereof can be prepared into aerosol formulations.Method with the combination treatment hypertensive pulmonary vascular disease that can suck is well known by persons skilled in the art, and at for example United States Patent (USP) the 6th, 756, in No. 033 (being introduced into by quoting as proof) description was arranged, thereby this patent provides the instruction of sending prostaglandin formulations treatment hypertensive pulmonary vascular disease by inhalation at this.The suction technology that is used for prostaglandin described in the patent of mentioning in front also can be used for preparing the suction preparation of BH4 and/or its precursor and derivant.In addition, also comprise can be by the co-administered endothelial function disturbance for the treatment of based on compositions and the prostaglandin formulations of BH4 in the present invention.

Described reactive compound can be mixed with the free alkali that suitably is mixed with surfactant (for example hydroxypropyl cellulose) or the aqueous solution of pharmaceutical salts, use being used to.Also can be mixed with the dispersion liquid in glycerol, liquid polyethylene glycol and composition thereof and oil.Under common storage and service condition, these preparations comprise antiseptic to prevent growth of microorganism.

The BH4 compositions can be mixed with and be fit to the medicament forms that injection is used.Such compositions can be aseptic aqueous solution or dispersion liquid, and the sterilized powder that is used to prepare sterile water for injection solution or this instant preparation of dispersion liquid (extemporaneous preparation).In all cases, described medicament forms all must be aseptic, and must be the fluid with easy injectivity.It must be stable under preparation and condition of storage, and must pollute to avoid microorganism (for example antibacterial and fungus) through preservative treatment.Carrier can be to comprise for example water, ethanol, polyhydric alcohol (for example, glycerol, propylene glycol and liquid macrogol etc.), their suitable mixture and the solvent or the disperse medium of vegetable oil.Can keep required particle diameter and use surfactant to keep suitable flowability by for example using such as coatings such as lecithin, under dispersive situation.Can pass through various antibacterial and antifungal (for example p-Hydroxybenzoate, methaform, phenol, sorbic acid and thiomersalate etc.), suppress action of microorganisms.Under many circumstances, preferably comprise isotonic agent, for example sugar or sodium chloride.Can be used in the absorption prolongation that makes this Injectable composition in the Injectable composition by postponing absorbent (for example aluminum monostearate and gelatin).

Add in the appropriate solvent by reactive compound, add above-named various other compositions in case of necessity, carry out filtration sterilization then aequum, thus the preparation aseptic injectable solution.Generally speaking, by being added to comprise in basic disperse medium and the required sterile media from other compositions in above-named those compositions, various active component through sterilization prepare dispersion liquid.With regard to the sterilized powder that is used to prepare aseptic injectable solution, preferred manufacturing procedure is the vacuum drying and the Freeze Drying Technique that can obtain comprising from the powder of the active component of previous aseptic filtration solution and any supplementary element of wanting.

The preferred formulation of BH4 compositions and the preferred formulation that uses with method described herein are tablets.Surprisingly find ascorbic acid added in the tablet and can improve stability of formulation.Do not wish to be confined to specific stabilizing mechanism, it is believed that when BH4 being sneaked in the pharmaceutical preparation that comprises multiple excipient, even very a spot of ascorbic acid (for example, being less than 2 weight %) also can produce complex with BH4, and suppress one or more approach that BH4 degrades.Therefore, as International Application PCT/US05/41252 (the publication number WO2006/055511 that submits on November 16th, 2005, its full content is introduced by quoting as proof at this) in institute's sets forth in detail, the exemplary tablet of BH4 used herein comprises ascorbic acid.

Exemplary stable oral formulations comprises more than one following supplementary elements that can improve described stability of formulation or other characteristics: binding agent, disintegrating agent, acid antioxidant or lubricant or their combination.Exemplary stable tablet comprises binding agent and disintegrating agent, comprises acid antioxidant in case of necessity, further comprises lubricant in case of necessity.The exemplary concentration of binding agent is about 1 weight %～about 5 weight %, or about 1.5 weight %～3 weight %; Binding agent is about 1:10～about 1:20 with the example weight ratio of BH4.The exemplary concentration of disintegrating agent is about 1 weight %～about 20 weight %; Disintegrating agent is about 1:5～about 1:10 with the example weight ratio of BH4.The exemplary concentration of antioxidant is about 1 weight %～about 3 weight %, and antioxidant is about 1:5～1:30 with the example weight ratio of BH4.In an example, ascorbic acid is exactly an antioxidant, and its consumption is less than 1:1 with the ratio of BH4, for example, and below the 1:2, or below the 1:10.The exemplary concentration of the lubricant in the stable tablet of the present invention is about 0.1 weight %～about 2 weight %; Lubricant is about 1:25～1:65 with the example weight ratio of BH4.

Described stable solid preparation can comprise the other treatment agent (for example, the folic acid class comprises folic acid precursors, folic acid or folic acid derivatives) that is suitable for the disease that will treat in case of necessity; And/or arginine; And/or vitamin (for example vitamin C and/or vitamin B2 (riboflavin) and/or vitamin B12); And/or such as precursors of neurotransmitters such as L-DOPA or carbidopas; And/or 5-hydroxyryptophan.

Preferably BH4 used in the compositions described herein is made dihydrochloride, yet, estimate that other the salt form of BH4 also possesses required biological activity, therefore, also can use other salt form of BH4.

Can form the medicinal basic addition salts with metal or amine (for example alkali metal and alkaline-earth metal or organic amine).Also can prepare chemical compound pharmaceutical salts with medicinal cation.Suitable medicinal cation is that those skilled in the art are well-known, comprises base cations, alkaline earth cation, ammonium and quaternary ammonium cation.Carbonate or bicarbonate also are fine.Example as cationic metal is sodium, potassium, magnesium, ammonium, calcium or ferrum (trivalent) etc.The example of suitable amine comprises 2-aminopropane., Trimethylamine, histidine, N, N '-dibenzyl-ethylenediamin, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucosamine and procaine.

Medicinal acid-addition salts comprises inorganic or acylate.The example of suitable hydrochlorate comprises hydrochlorate, acetate, citrate, Salicylate, nitrate, phosphate.Other suitable pharmaceutical salts are that those skilled in the art are well-known, for example comprise the salt that forms with following acid: acetic acid, citric acid, oxalic acid, tartaric acid or mandelic acid, hydrochloric acid, hydrobromic acid, sulphuric acid or phosphoric acid; Organic carboxyl acid, sulfonic acid, thio-acid (sulfo acid) or phosphonic acids or N-substituted-amino sulfonic acid, for example acetic acid, propanoic acid, glycolic, succinic acid, maleic acid, hydroxymaleic acid, citraconic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucosaccharic acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosallcylic acid, 2-phenoxy benzoic acid, 2-acetoxy-benzoic acid, pamoic acid, nicotinic acid or .gamma.-pyridinecarboxylic acid; And aminoacid, for example, such as 20 kinds of related a-amino acids in native protein synthetic such as glutamic acid or aspartic acid, and phenylacetic acid, methanesulfonic acid, ethyl sulfonic acid, 2-ethylenehydrinsulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-toluene sulfonic acide, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid ester or 3-phosphoglycerate, G-6-P ester, N-cyclohexyl sulfamic acid (having formed cyclohexyl-n-sulfonate) or other acidic organic compounds, for example ascorbic acid.

Particularly, preferably BH4 and mineral acid or organic acid salt.Optionally the limiting examples of the salt form of BH4 comprises BH4 acetate, citrate, oxalates, tartrate, fumarate and mandelate.

The administration frequency of BH4 will depend on the pharmacokinetic parameter and the route of administration of medicament.Best medicament will be determined according to route of administration and required dosage by those skilled in the art.Referring to, for example " Remington ' s Pharmaceutical Sciences " (the 18th edition, 1990, Mack Publ.Co, Easton (Pennsylvania) 18042), the 1435th～1712 page, be introduced into the application by quoting as proof at this.Such preparation can influence the interior rate of release of physical state, stability, body and the interior removing speed of body of the medicament of being used.Depend on route of administration, can calculate proper dosage according to body weight, body surface area or organ size.The further refinement of determining the suitable necessary calculating of therapeutic dose by those skilled in the art according to a conventional method, particularly according to dosage information disclosed herein and check, and the pharmacokinetic data that is observed in animal or human's clinical trial carries out, and need not to carry out undue experimentation.

Can be by using being used to of having established to measure the blood levels of phenylalanine and proper dosage is determined in the detection of relevant dose response data.The final dose scope will (for example consider to change pharmaceutically-active factor by the doctor in charge, the ratio of medicine is lived, the seriousness of the seriousness of injury and patient's response, patient's age, situation, body weight, sex and diet, any infection, administration time and other clinical factors) determine.Along with the carrying out of research, proper dosage level and the further information of treatment persistent period about disease specific and disease will appear.

Should be appreciated that pharmaceutical composition of the present invention and Therapeutic Method can be used for people's medicine field and field of veterinary.Therefore, the object that treat can be a mammal, preferably is people or other animals.For the veterinary drug purposes, object comprises that for example farm-animals comprises cattle, sheep, pig, horse and goat; House pet, for example Canis familiaris L. and cat; External animal and/or zoo animal; Laboratory animal comprises mice, rat, rabbit, Cavia porcellus and hamster; And poultry, for example chicken, turkey, duck and goose.

Of the present invention aspect some, can make all that be used for treating such disease must be component packaged in test kit with uniting by independent use BH4 or with BH4 and other medicaments that are generally used for treating disease described herein or intervention.Particularly, the invention provides and be used for the test kit that treatment of diseases described herein is intervened, described test kit comprises the medicine of packaging kit, but buffer agent and other compositions that described medicine comprises BH4 or derivatives thereof or its precursor and is used to prepare the delivery form of described medicine; And/or send the device of such medicine; And/or be used from any medicament of conjoint therapy with such medicine one based on BH4; And/or how to treat the description of disease described herein with the indication together of described drug packages.Described description can be fixed in any medium that can see, the disk of for example printing paper, or embodied on computer readable or CD, or quote the description of remote computer information source (for example web presence that can obtain by the Internet).

III. change the factor that BH4 is synthetic and/or NO produces

The present invention aims to provide disease or the disorderly method that treatment is characterized as endothelial function disturbance, described method comprises that the compositions that will comprise the medicament that can increase tetrahydrobiopterin (BH4) or its precursor or derivatives thereof is applied to described object or co-administered in described object with therapeutic agent separately, wherein with the situation of the compositions of not using the described BH4 of containing under described endothelial function disturbance compare the described endothelial function disturbance that can effectively alleviate described object of using.

An embodiment of the invention comprise more than one the medicament by the synthetic or active BH4 of the increasing level of expression that increases the enzyme (comprising first enzyme and rate controlled enzyme GTPCH1, PTPS and SR) in the BH4 route of synthesis.In a preferred embodiment of the present invention, by using any or multiple cyclic adenosine monophosphate (cAMP) analog or agonist to increase the expression of GTPCH1, thereby increase the synthetic of BH4, described cAMP analog or agonist comprise forskolin, 8-bromine cAMP or other play the medicament of the cell signalling of increase cAMP mediation, for example cytokine and somatomedin, comprise interleukin-1, interferon-(IFN-γ), tumor necrosis factor-alpha (TNF-α), the c reactive protein, HMB-CoA-reductase (Statins, atorvastatin for example), nerve growth factor (NFG), endothelial cell growth factor (ECGF) (EGF), hormone (comprising adrenomedullin and ovostab), and other chemical compound, for example NADPH and NADPH analog, caffeine, Ciclosporin A, methyl-xanthine (comprising 3-isopropyl-1-methylxanthine), theophylline, reserpine, hydrogen peroxide.

Di-phosphate ester enzymatic degradation 3 ', 5 '-nucleolus glycosides, for example cGMP and cAMP have been proved conclusively in this area.CAMP is the known activator of the rate controlled enzyme GTPCH1 of BH4 synthetic (the essential cofactor of eNOS).Therefore, the inhibitor of di-phosphate ester enzyme family has the secondary activation to BH4-synzyme GTPCH1.Thereby an embodiment of the invention relate to the degraded that suppresses 3 ', 5 '-nucleolus glycosides by the inhibitor with 11 di-phosphate ester enzyme families (PDE1～11) (comprising PDE1, PDE3 and PDE5), thereby increase the GTPCH1 level.PDE inhibitor of the present invention comprises viagra/tadanafil, Tadalafil/sldenafil, Vardenafil/Ai Lida (levitra), 8-methoxy-IBMX, UK-90234, dexamethasone, hesperetin, Hesperidin, irsogladine, vinpocetine, cilostamide, rolipram (rolipram), B-carboline-3-carboxylic acid, ethyl ester (β-CCE), tetrahydrochysene-beta-carboline derivatives, 3-O-methyl Quercetin etc.

Another embodiment of the invention relates to the level that increases the BH4-synzyme by the gene therapy of the polynucleotide of BH4 synthesis mechanism or endothelium targeted delivery, thereby increases the level of BH4.The claimed synthetic patent of submitting to gene of BH4 that is used for gene therapy comprises US20030198620.Another embodiment of the invention relates to by replenishing BH4 synzyme GTPCH1, PTPS, SR, PCD, DHPR and DHFR increases the level of BH4.In this statement, the application utilizes the BH4 synzyme to contain all natural and non-natural forms (comprising activity of proteins and nonactive mutant) of these enzymes.

Another embodiment of the invention relates to by suppressing alkali phosphatase (AP) activity so that substrate 7, and the new purine of 8-triphosphoric acid dihydro turns to BH4 synzyme PTPS rather than AP, thereby increases the BH4 level.Suppress active reagent of AP or chemical compound and comprise phosphate ester analog, levamisole and L-phenylalanine.Another embodiment of the invention relates to reagent or the chemical compound that suppresses alkali phosphatase, comprises suppressing the synthetic siRNA of alkali phosphatase (siRNA), antisense RNA, dsDNA, micromolecule, neutralizing antibody strand, chimeric, humanized and antibody fragment.

Another embodiment of the invention comprises the catalyst that the enzymatic synthesis that strengthens the synthetic de novo synthesis of BH4 is required or the active reagent or the chemical compound of cofactor.

Another embodiment of the invention comprises the reagent or the chemical compound of the enzymatic degradation that prevents that BH4 is synthetic required.Another embodiment of the present invention comprises and prevents the synthetic required catalyst of BH4 and the reagent or the chemical compound of BH4 synzyme (comprising GTPCH1, PTPS and SR) degraded.

Another embodiment of the invention relates to the feedback regulation to the GTPCH1/GFRP complex that is caused by BH4 by suppressing, thereby increases the BH4 level.A preferred embodiment of the present invention relates to and suppresses combining of BH4 and GTPCH1/GFRP complex, prevents the reagent or the chemical compound of the feedback suppression that caused by BH4 thus.Reagent of the present invention or chemical compound comprise competitive inhibitor, for example described complex are had the alternative form of BH4 of different affinitys and analog etc.Another embodiment of the invention comprises bonded reagent or the chemical compound that strengthens the L-phenylalanine and can induce the synthetic CTPCH1/GFRP of BH4.Another embodiment of the invention relates to for example reagent or the chemical compound of the level of the increase L-phenylalanine such as precursor of L-phenylalanine.

Another embodiment of the invention relates to active or synthetic reagent or the chemical compound of regulating GFRP.A preferred embodiment of the present invention comprises inhibition active reagent of GFRP or chemical compound.Another embodiment of the invention comprises synthesizing with inhibition GFRP such as siRNA, micromolecule, antibody, antibody fragments.

Another embodiment of the invention relates to the reduction that increases BH2 by salvage pathway, thereby increases the level of BH4.In vivo, BH4 can be oxidized to BH2.With quinoid form (qBH2) and 7, the BH2 that 8-dihydro pterin form exists is reduced to BH4 by DHPR (dihydro pterin reductase) and DHFR (dihydrofolate reductase) respectively.A preferred embodiment of the present invention relates to by with the active of regulatory enzyme PCD, DHPR such as the reagent on this approach or compound N ADPH, thio-alcohol, mercuric p-chlorbenzoate, hydrogen peroxide and DHFR with synthesize and increase the regeneration from BH2 to BH4 or remedy.

Another embodiment of the invention relates to by using the oxidation that reduces BH4 such as reagent such as antioxidant or chemical compound to increase the level of BH4, and described antioxidant comprises ascorbic acid (vitamin C), vitamin E, tocopherols (for example vitamin A), selenium, beta-carotene, carotenoid, flavone, flavonoid, folic acid, flavone, flavanone, isoflavone, catechol, anthocyanidin and chalcone derivative etc.United States Patent (USP) and patent application US6544994, US20050119270, US20030007961 and US20020052374 have described the use of BH4 and antioxidant.

Another embodiment of the invention comprises as the reagent of BH4 precursor and chemical compound, comprises GTP (guanosine triphosphate), 7,8-triphosphoric acid dihydroneopterin and 6-pyruvoyl-tetrahydrobiopterin.

VII. embodiment

Include following embodiment in to demonstrate preferred implementation of the present invention.It will be appreciated by those skilled in the art that disclosed technology is represented technology that the inventor finds, that can carry out well among the following embodiment in practice of the present invention, therefore can think that they have been formed preferred embodiment.Yet, according to present disclosure, it will be appreciated by those skilled in the art that under the situation that does not deviate from main idea of the present invention and scope, in the disclosed specific embodiment, can make many changes and still obtain identical or similar result.

Embodiment 1

The clinical evaluation of 6R-tetrahydrobiopterin

Following embodiment provides the guidance to the parameter of the clinical evaluation BH4 that is used for Therapeutic Method of the present invention.As literary composition herewith everywhere as described in, BH4 is with being used to treat relevant with diabetes and ND cardiovascular complication, including, but not limited to intractable hypertension, intermittent claudication, coronary artery high pressure, coronary artery disease, hypertensive pulmonary vascular disease and hemolytic anemia (comprising drepanocytosis).To carry out clinical trial to provide to BH4 every day oral safe dose, pharmacokinetics, the initial reaction of succedaneum and the evaluation of defined clinical endpoint.Each patient is minimum to carry out a week test (but not necessarily being confined to a week) at reverse correlational study terminal point (for example to estimate, the development of pain in the intermittent claudication patient gait processes) effectiveness the time, but and collect enough safety informations of 30 evaluate patient.

The initial dosage of test will be at about 2mg/kg～about 10mg/kg, changes in perhaps about 1mg/kg～about 20mg/kg scope.Do not improve patient's clinical endpoint at this dosage, perhaps do not have to produce under the situation of significant directly clinical effectiveness, dosage should be increased in case of necessity, and the additionally short-term (but it must be restricted to a week) of keeping a week determining safety, and further estimate effectiveness.Also can consider lower dosage, for example, the dosage of 0.1mg/kg～2mg/kg, and the dosage of 1mg/kg～5mg/kg.Expect that such dosage can improve the correlational study terminal point, including, but not limited to relating to those research terminal points of atherosclerosis complication or pulmonary's hyperbarism.

Particularly, the present invention aims to provide any angiopathy state that BH4 or its precursor or derivatives thereof mention in treatment the application or the U. S. application of submitting on June 1st, 2005 the 11/143rd, purposes in No. 887 (by quoting as proof its full content being introduced the application) mentioned any angiopathy state at this, wherein dosage is 1mg/ kg body weight/sky～5mg/ kg body weight/sky, can pass through any administration (including, but not limited to oral), but be administered once every day, also but every day repeatedly (for example, 2 times or 3 times or 4 times) administration in batches, the administration persistent period was at least 1 week, 2 weeks, 3 weeks, 4 weeks or longer, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer.Exemplary dosage comprise be less than 5mg/kg/ days, 4.5mg/kg/ they is following, 4mg/kg/ is days following, 3.5mg/kg/ is days following, 3mg/kg/ is days following, 2.5mg/kg/ is days following, 2mg/kg/ is days following, 1.5mg/kg/ is days following, 1mg/kg/ is days following or below 0.5mg/kg/ days.Also the dosage of Deng Xiao unit bodies surface area also within the scope of the invention.

For the people of average weight/body surface area (for example 70kg), the present invention also provides the total daily dose that is less than 400mg.The example of total daily dose like this comprises 360mg/ days, 350mg/ days, 300mg/ days, 280mg/ days, 210mg/ days, 180mg/ days, 175mg/ days, 150mg/ days or 140mg/ days.For example, can easily realize the dosage of 350mg/ days or 175mg/ days by the peroral dosage form of oral 175mg once or twice every day.Other examples of total daily dose comprise 320mg/ days following, 160mg/ days following or below 80mg/ days.Such dosage can obtain by the peroral dosage form of 80mg or 160mg being easy to use.Other examples of total daily dose comprise 45mg/ days, 90mg/ days, 135mg/ days, 180mg/ days, 225mg/ days, 270mg/ days, 315mg/ days or below 360mg/ days, can obtain by the peroral dosage form of 45mg or 90mg being easy to use.Other examples of total daily dose comprise 60mg/ days, 120mg/ days, 180mg/ days, 240mg/ days, 300mg/ days or 360mg/ days, can obtain by the peroral dosage form of 60mg or 120mg being easy to use.Other examples of total daily dose comprise 70mg/ days, 140mg/ days, 210mg/ days, 280mg/ days or 350mg/ days, can obtain by the peroral dosage form of 70mg or 140mg being easy to use.The example of total daily dose also comprises 55mg/ days, 110mg/ days, 165mg/ days, 220mg/ days, 275mg/ days or 330mg/ days, can obtain by the peroral dosage form of 55mg being easy to use.Other examples of total daily dose comprise 65mg/ days, 130mg/ days, 195mg/ days, 260mg/ days or 325mg/ days, or 75mg/ days, 150mg/ days, 225mg/ days, 300mg/ days or 375mg/ days, can pass through, and for example, the dosage form of 65mg or 75mg is used.

If use BH4 self, then can use the stable solid preparation described in any in salt described in No. the 11/143rd, 887, the U. S. application or the polymorphic forms and/or the International Application PCT/US05/41252 (by quoting as proof its full content being introduced the application) that on November 16th, 2005 submitted at this.Can keep the precursor of BH4 of its favorable activity and the example of derivant to describe to some extent for the 11/143rd, No. 887 at U. S. application.

Safety is measured and will be comprised untoward reaction, anaphylaxis, whole clinical chemistry test (renal function and liver function), urinalysis and complete blood cell differential counting.In addition, also can monitor other parameters.Pharmacokinetic parameter and population distribution and the half life determination of 6R-BH4 in blood during present embodiment also aims to provide described medicine circulated in vivo.Wish that these mensuration help dosage and clinical response are connected.

Method

To suffering from relevant with diabetes or carrying out the baseline test with the patient of the irrelevant blood vessel disorder of diabetes, comprise the recording medicine history, carry out physical examination, going forward side by side works be usually used in diagnosing in the clinical settings concrete indication (for example, the pain development of the patient who suffers from intermittent claudication in dull and stereotyped test process) various diagnostic tests are including, but not limited to blood pressure determination, walking test in 6 minutes and ultrasoundcardiogram research.The human dosage 2mg/kg～about 10mg/kg that is advised of BH4 is divided into 1～3 administration every day.With frequent time interval monitoring clinical endpoint.The treatment phase is comprehensively estimated in a week after finishing.Increase dosage if desired, then the patient follows above-mentioned same program.In whole test process, all will monitor safety.

Diagnosis and inclusion criteria/exclusion standard

The idagnostic logout of the concrete blood vessel disorder of determining according to sex, age with by common diagnostic test is selected the experimenter.

Dosage, approach and scheme

The patient will accept the BH4 of 5mg/kg dosage every day.If reasonably amount is not improved clinical endpoint, and does not observe clinical benefit, then can increase dosage in case of necessity, till total daily dose reaches 20mg/kg.The BH4 daily dose is by oral administration or as liquid, powder, tablet or the administration of capsule nasogastric tube.Can also can be divided into 2～3 daily dose administrations with total daily dose as single dose administration.The patient is carried out clinical monitoring, and monitor any untoward reaction.If observe any uncommon symptom, then stop to study medicament administration immediately, and make the decision that whether continues to study.

The BH4 safety

If significantly acute or chronic drug reaction in research process, do not occur, determine that then the BH4 treatment is safe.If in the clinical examination of clinical laboratory or other suitable research, do not observe significantly unusually, determine that then the chronic administration of medicine is safe.

Embodiment 2

The clinical evaluation of 6R-tetrahydrobiopterin in diabetics

1/2 or the diabetics of 2a phase in the determining of dose effect, spacing of doses phase and safety

Before dosage/effectiveness study of beginning any 2a phase, in various diabetic populations, carry out the short cumulative research of 1/2 phase dosage with definite dose effect, and should consider safety vascular compliance.Research for the first time will determine dosage range and scheme and can be monitored with the scope of the vascular function terminal point of supporting clinical endpoint.The experimenter of research suffers from tangible angiopathy (microvascular compliance reduction) and hypertensive diabetics for the first time, use BH4 in the mode that is administered once every day or be administered twice every day, a day oral dose is 0mg/kg, 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg and 20mg/kg.To during one-week treatment, monitor patient's vascular compliance, perfusion/again perfusion/FF and blood pressure, and in case just settle out at a dosage and in one day span, to monitor.Described research will estimate 2 interim suitable dosage ranges and every day dosage regimen, and set up and be used to support the quantitative effect of clinical development to measure, and the safety in this crowd.

To probing into of the indication in 2a and the diabetic population of 2b phase

Estimate suitable crowd, dosage/scheme and indication in 2a phase program, carry out twice above research, the time is 30 days～60 days relatively short life treatment phase (life treatment period).In any group of forming by the diabetics that has the special medical needs of being paid close attention to (any 2a phase all comprises such group in studying), to carry out parallel evaluation to various other measurements, with other clinical impacts of the variation in the evaluation dysfunction of blood vessel, and the evaluation of the biochemical biomarker of endothelial function and insulin resistance.

In suitable crowd/indication, carry out the 2b phase and study, thereby help 3 phases of design research in bigger crowd, and estimate concrete dosage with the longer treatment phase of abundant examination.

The 3 phases research of in the diabetics of selecting, first indication of choosing being carried out

The research of 3 phases is conceived to key index, but also comprises the supportive information as the vascular function of secondary terminal point or three grades of terminal points.To carry out the research of 3 phases of at least twice contrast, but consider to carry out 2 extra phases or the design of 3 phases, so that other high-risk group are included, for example use different concomitant drugs the patient, patient's (for example patients with renal failure) of special medical problem is arranged or the patient of the other end of spectrum of disease.

General vascular function

Vascular compliance/endothelial function disturbance/HTN: evaluation [vascular compliance C2 measurement in the various diabeticss of the variation sign that the unusual and HTN (hypertension) of vascular compliance, C2 (small artery elasticity index) is arranged, described evaluation is to carry out in the cumulative process of one-week dosage (from 0mg/kg, cumulative to 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg and 20mg/kg).To evaluate in various patient crowds increases the influence of BH4 to vascular compliance and HTN, and the dose effect of definite BH4.The drug effect of measuring in one day is set up dosage regimen with help.

Intractable hypertension: the beginning 2a phase is studied, and it is based on randomization, double blinding placebo research design.This research comprises that 80 blood pressures are higher than the patient of 140mmHg/90mmHg, and adopts three kinds of Drug therapys.Comprise 40 diabeticss in patient's sum.Every group of 20 patients comprise placebo group, four groups altogether.Exclusion standard comprises acute limb ischemia, heart failure and orthopaedics complication.Dosage with 0mg/kg～10mg/kg is used BH4 to the patient, around continuing.Systolic pressure and diastolic pressure to the patient are estimated, and the monitoring ambulatory blood pressure.

Insulin sensitivity/glycemic control: will be relatively poor based on the mensuration evaluation glycemic control of blood glucose/insulin level, HgbAlc (glycated hemoglobin), and to common antidiabetic oral medicine non-responsiveness or the relatively poor diabetics of response.Expectation BH4 uses the result that can improve in the glucose tolerance test, and improves glycemic control (Hgb Alc reduction).Can in different schemes, evaluate such patient.

The periphery perfusion

The research of the concrete indication relevant with diabetes

Intermittent claudication: will carry out the 2a phase based on the placebo-controlled study design of 1:1, double blinding and study.This research comprises 80 patients that suffer from intermittent claudication, comprising 40 diabeticss.Exclusion standard comprises acute limb ischemia, heart failure and orthopaedics complication.To use placebo or BH4 to the patient with the dosage of 10mg/kg, continue for 12 weeks.The ability of the patient who estimates shank pain and long walk limited ability walking a period of time and segment distance on flat board is till beginning pain.Particularly, be to estimate following clinical relevant terminal point: the walking distance of the longest walking time on the acclive flat board, no pain, blood pressure, blood flow, blood flow mediation property vasodilation (FMD) and quality of life (QoL).Using of expectation BH4 can improve the stretching reaction of blood vessel to motion, and improves the ability of long walk, and this is the dabbling most important clinical relevant terminal point of periphery.

The perfusion of relatively poor periphery: the relatively poor patient of extremity blood flow is estimated in the improvement of extremity blood flow and the minimizing of amputation frequency when using BH4 by cumulative mode.

Relatively poor SkBF and wound healing: the enhancing evaluation of the improvement of SkBF and wound healing suffers from diabetic ulcer or the relatively poor patient of SkBF when treating with BH4.

Heart disease

Congestive heart failure (CHF) or hypertensive pulmonary vascular disease: on the basis of the measurement result that provides by ultrasoundcardiogram, cardiac output/ejection fraction and walking in six minutes test, estimate diabetics because of increased blood pressure and the bad and concurrent CHF of vascular function.Thereby expectation BH4 treatment can bring high blood pressure down, improve the collaborative cardiac function enhancing of cardiac flow, and improves walking in six minutes test.

Angina pectoris in the motor process: measure the experimenter when accepting the BH4 treatment from beginning to occur the anginal time up the going to of flat board, and monitoring coronary flow suffers from the restricted angina pectoris of motion and needs the diabetics of itrated compound treatment with evaluation.Expectation BH4 treatment can improve coronary flow, thereby postpones or the angina pectoris when eliminating dull and stereotyped test.

Coronary heart disease (CAD) and relevant atherosclerosis: the minimizing evaluation of the prevention of atherosclerosis progress and/or MI (myocardial infarction)/apoplexy/death is suffered from early days or the atherosis disease of late arterial and coronary heart disease and the diabetics of myocardial infarction or ischemic event is arranged before.Carry out the 2a phase and study based on randomization, double blinding, placebo-comparative study design.This research comprises 80 patients, wherein have 40 suffer from CAD, the need coronary artery bypass graft (CAB) diabetics of (CABG).Every group of 20 patients comprise placebo group, totally four groups.Exclusion standard is the appearance of heart failure and unstable angina.Dosage with 0mg/kg～10mg/kg is used BH4 or placebo to the patient, around continuing.The gentle BH4 level of the tissue water of the coronary artery dysfunction of evaluate patient, trunk dysfunction, CAD, active oxygen.The frequency of tempo, minimizing MI and other vascular events of expectation BH4 treatment can reduction atherosclerosis.Importantly, expect that vasodilative improvement and thrombogenic attenuating can strengthen the coronary artery function, the atherosclerosis that expectation reduces subsequently can be improved the progress of angiopathy.

Ophthalmic diseases

The retinopathy that optic atrophy or diabetes cause: estimate because of the insufficient diabetics that causes vision to fail gradually of retinal vessel with vision and retinal blood flow measurement.Expectation BH4 can improve blood flow, thereby improves vision.

Nephropathy

Microalbuminuria in the glomerular filtration rate of the nephropathy that diabetes cause or renal failure/reduction (GFR): lose to estimate with the urine protein in 24 hours samples and occur becoming that consistent urine protein is lost or the diabetics of creatinine levels rising with renal vascular disease with the mensuration of GFR.Expectation BH4 treatment can reduce albuminuria and improve GFR.

Hypertensive pulmonary vascular disease in the diabetics

The diabetics that is with or without congestive heart failure of suffering from hypertensive pulmonary vascular disease with the mensuration evaluation of the development of the mensuration of the ability of carrying out walking in six minutes test, CHF and pulmonary artery pressure.Expectation BH4 treatment can improve pulmonary artery pressure and cardiovascular performance.It is relevant with hypertensive pulmonary vascular disease that some data that exist in the animal model show that BH4 lacks.

Embodiment 3

Research with irrelevant other cardiovascular indications of diabetes

The lung angiopathy

Primary pulmonary hypertension (PAH): it is clinical to carry out the 1b phase based on open-label formula dose titration research design.This research comprises 10～20 patients that suffer from constitutional PAH.Exclusion standard comprises the instability reaction to medicine.Begin to carry out the titration of BH4 dosage from 0mg/kg, continued for 4 week～12 weeks to 10mg/kg.Ultrasonic cardiography graph evaluation with walking test in six minutes, New York heart disease association (NYHA) score, Borg dyspnea score, pulmonary artery and cardiac function is estimated the patient.

Hypertensive pulmonary vascular disease in drepanocytosis or other hemoglobinopathies (Hb): the measurement result evaluation with the development of the ability of carrying out walking in six minutes test, CHF and pulmonary artery pressure suffers from hypertensive pulmonary vascular disease, is with or without the Hb-S patient of congestive heart failure.Expectation BH4 treatment can improve pulmonary artery pressure and cardiovascular performance.

Idiopathic pulmonary hypertension disease (IPH): with the measurement result evaluation of the development of walking in six minutes test, CHF and pulmonary artery pressure do not have known cardiovascular cause a disease because of IPH patient, particularly age less than 40 years old young patient.Expectation BH4 treatment can improve pulmonary artery pressure and cardiovascular (CV) performance.

Neonate persistent pulmonary hypertensis (PPHN): with the elimination of pressure, the recovery of Oxygenation and the full-term newborn infant that mortality rate evaluation suffers from PPHN.Expectation BH4 treatment can make hypertensive pulmonary vascular disease fast, fully reverse and turn to, thereby improve Oxygenation.Different with impracticable and deleterious nitric oxide suction treatment, BH4 is nontoxic and effective.

Apoplexy and relevant ischemic angiopathy

Cerebral vasospasm after the apoplexy: the experimenter comprises because patient after the apoplexy of acute apoplexy and hospitalization.With before infarct size and the treatment and the brain after the treatment pour into and estimate the experimenter.Expectation BH4 treatment can make spasm lax, and reduces infarct size.Show that from the data of dog apoplexy model the vasospasm around the blood clot position causes damage to enlarge after the apoplexy, make damage more serious than initial incident, this vasospasm can be prevented by the perfusion nitrite solution.

The endothelial function disturbance relevant with organ transplantation

Dysfunction of blood vessel behind the solid organ transplantation: this research comprises the patient who has experienced solid organ transplantation.Estimate the experimenter with the organ dysfunction that dysfunction of blood vessel was caused after transplanting.Expectation BH4 can improve the organ dysfunction after transplanting, and reduces the loss of transplant organ.

The inductive endothelial function disturbance of Ciclosporin A (CsA): estimate the patient who takes CsA after the organ transplantation with the organ dysfunction that dysfunction of blood vessel was caused after transplanting.Expectation BH4 treatment can improve the organ dysfunction after transplanting, and reduces the frequency of vascular complication.

Brandacher etc. are at Transplantation, and 81 (4): reported survival in 583 (2006) with tetrahydrobiopterin and Ciclosporin A significant prolongation allograft.Compare with untreated allogene matched group, the graft endoperoxides nitrite in all groups forms and all decreases.Briefly, two complete heterogenic inbred line male mice strains have been obtained.Carried out the cervical region heterotopic cardiac transplantation and by palpation and check the survival rate determine heart allotransplantation every day, heart beating stops to show serious rejection fully, this can confirm by the histology.Every group of 5～10 animals are transplanted the back with BH4 (per 8 hours 50mg/kg newly are dissolved in the phosphate buffered saline (PBS) intramuscular administration) treatment 7 days.Postoperative killed mice in 1 day or 6 days, and the graft that takes off is carried out Histological evaluation.Collect corresponding plasma sample to be used for measuring nitrate and nitrite by HPLC.Tissue sample is freezing and be stored in-75 ℃ in liquid nitrogen immediately, perhaps tissue sample is fixed, and imbedded in the paraffin, up to passing through H﹠amp with formalin; E dyeing or immunohistochemistry are analyzed.Untreated allograft is about 7 days and 8 days be ostracised (the graft mean survival time is 7.1 ± 0.7 days) after transplanting, and tetrahydrobiopterin extends to 12.3 ± 4.9 days with survival period (p＜0.05 is different from untreated allotransplantation).Aspect Histological evaluation, through heart demonstration mononuclear cell infiltrate and some inflammation infiltrate focuses of BH4 treatment.Untreated animal with in the animal of BH4 treatment, the 6th day nitrate and nitrite concentration raise.The survival of allograft that these data have shown the tetrahydrobiopterin significant prolongation.

Heart disease or coronary heart disease

Dysfunction of blood vessel/angina pectoris: estimate hypercholesterolemiapatients patients and smoker with prevention and/or MI, apoplexy or dead minimizing to the atherosclerosis progress.Expectation BH4 treatment can reduce atherosclerosis tempo, improve the coronary artery diastole and reduce blood coagulation.

Congestive heart failure (CHF): test with development, cardiac output/ejection fraction and the walking in six minutes of the CHF that measured by ultrasoundcardiogram and to estimate the ND who suffers from CHF.Thereby expectation BH4 treatment can bring high blood pressure down, improve the collaborative cardiac function enhancing of cardiac flow, and improves walking in six minutes test.

Hemolytic anemia---drepanocytosis

Hemolytic anemia is characterised in that the circulation erythrocyte number deficiency (anemia) that erythrocytic too early destruction causes.Some causes of disease of hemolytic anemia comprise infection, Drug therapy, autoimmune obstacle and genetic disorder.Various types of hemolytic anemias are arranged, including, but not limited to sicklemia, paroxysmal sleep type hemoglobinuria, hemoglobin sc disease, hereditary elliptocytosis, heritability ovalocyte increase disease, special non-immunologic pattern hemolytic anemia, Secondary cases immunologic pattern hemolytic anemia and the thalassemia that property autoimmune type hemolytic anemia, chemicals or physical means cause.Some conventional therapies of hemolytic anemia comprise folic acid, iron supplement and corticosteroid, and the blood transfusion when urgent.Treatment type, prognosis and complication can change with the type of hemolytic anemia.Complication comprises the deterioration of heart disease, pneumonopathy or the cerebrovascular of cardiovascular failure and preexist.

To estimate patient's symptom, comprise that shiver with cold, fatigue, skin color are pale, short of breath, heart rate acceleration, jaundice, urine color depth and splenomegaly.Raise by detecting the unconjugated bilirubin level, the serum hoptoglobin is lower, have in the urine have in hemoglobin, the urine that hemosiderin, urine and excremental urobilinogen increase, the skein cell absolute counting increases, red blood cell count(RBC) (RBC) and hemoglobin is low raises to determine hemolytic existence with Serum LDH.The direct mensuration to red blood cell life span of being undertaken by isotope labelling techniques will be used to measure the life-span.In case determined haemolysis, just determined the particular type of hemolytic anemia with test more specifically.Be subjected to other measurement of correlations of this sickness influence to comprise uric acid, TIBC, RBC index, protein electrophoresis-serum, potassium test, platelet count, peripheral blood film, LAP, serum levels of iron, packed cell volume, ferritin, Febrile Agglutinins or cold agglutinin, Donath-Lansteiner test, direct and indirect Coombs coomb's test Coomb, CBC, hemanalysis (blood differential), AST and twenty-four-hour urine albumen.

Drepanocytosis

Drepanocytosis is also referred to as sicklemia disease or hemoglobin SS disease (Hb SS), it is a kind of genetic disorder, be characterised in that unusual crescent shape erythrocyte, its unusual effect causes the formation of little blood clot, causes being called as the development of the recurrence type pain outbreak of " meniscocyte's pain crisis ".Drepanocytosis is that the hemoglobin by the Exception Type that is called as Hb-S causes, and Hb-S generation polymerization, and twist erythrocytic shape causes erythrocytic too early destruction and/or breaks.Frangible meniscocyte is seldom to the bodily tissue delivering oxygen, thereby and can be broken into fragment and hinder blood flow.Sicklemia disease is an autosomal recessive hereditary diseases, among per 500 non-descendants Americans a sicklemia disease patient is arranged probably, has also infected many people of other races.Though drepanocytosis just exists from birth, the symptom just development later at 4 months usually, but and life-threatening.Inaccessible blood vessel and impaired organ can cause acute pain outbreak or pain " crisis ", comprise that hemolytic crisis (impaired erythrocyte depletion), spleen are detained crisis (splenomegaly that is caused by gathering of hemocyte) and aplastic crisis (infection induced bone marrow stops to produce erythrocyte).These pain crisis can influence back skeleton, long bone and thoracic cavity, and can be seriously to needing in hospital to carry out the stage of pain relieving and venous transfusion.Crisis repeatedly can cause kidney, lung.Skeleton, eyes and central nervous system's damage.

Treatment is carried out for a long time, comprises Supplement of folic acid (fundamental when producing cell is because of erythrocytic quick replacement need replenish).Treatment is conceived to the processing and the control of symptom, and attempts the occurrence frequency of restriction crisis.Take in analgesics and enough liquid and to treat pain outbreak.Find that hydroxyurea can pass through to reduce the syndromic outbreak of occurrence frequency and acute thoracic cavity of pain crisis, and reduce blood transfusion and need help some patients.Worry that hydroxyurea may cause leukemia, but this be there is no final conclusion.Developed some other newer therapy, comprised and induce health to produce more fetal hemoglobin, perhaps increased oxygen and drepanocytic bonded medicine to reduce the medicine of drepanocytic amount.Bone marrow transplantation can be treated this disease, but owing to used medicine in the migration process has toxicity, is difficult to find suitable donor and costly, so only can be used for small number of patients.Available antimicrobial drug and vaccine prevent the normal bacterial infection of suffering from of the child patient of drepanocytosis.Additional treatment can comprise at the syndromic part exchange transfusion in acute thoracic cavity, blood transfusion or operation at nervous system incident (for example apoplexy), dialysis or renal transplantation at nephropathy, irrigation or operation at priapism, operation at the eyes problem, hip replacement at the hip joint ischemic necrosis, under the situation that serious cholelithiasis exists, remove gallbladder, wound care, zinc oxide, perhaps at the operation of leg ulcer, medicine at psychological complication restores and psychological counselling.

In the past, the meniscocyte patient usually dies from the organ failure, and most of meniscocyte patient was death in 20 years old～40 years old, but through improving treatment, survival rate is improved, and the patient can live 40 years old～50 years old.Dead reason comprises organ failure, infection and hypertensive pulmonary vascular disease.Complication comprises recurrent aplastic crisis and hemolytic crisis, its erection disturbance that causes anemia and cholelithiasis, multisystem disease (kidney, liver, lung), anesthetics abuse, spleen to be detained syndrome, acute thoracic cavity syndrome, priapism causing, blind/visual impairment, neurological symptom and apoplexy, destruction of joint, cholelithiasis, infection (comprise pneumonia, cholecystitis, osteomyelitis and urinary tract infection, cause the assays for parvovirus B 19 of aplastic crisis to infect), nephridial tissue necrosis, splenic function forfeiture and leg ulcer.

Sicklemia disease is produced by two carriers with sickle cell trait, and suggestion is carried out the heritability psychological counselling to the carrier (having 1 people that sickle cell trait is arranged approximately among 12 non-descendants Americans) of all sickle cell traits.The antenatal diagnosis of sicklemia disease is feasible.The quick treatment of infecting, enough Oxygenations and prevent that dehydration from can prevent erythrocytic sickling.But antibiotic and vaccination prevention infection.For pre-preventing tissue deoxygenation, the suggestion patient avoids violent sports (particularly when splenomegaly), avoids emotional stress, avoids being in such as lower environment of oxygen content such as high height above sea level and non-pressurized flights, avoid contacting the known source of infection, avoid dewatering.

With the common sympton of evaluate patient, comprise pale, yellow eyes/skin, fatigue, asthma, heart rate acceleration, growth and delayed puberty, easy infection, ulcer of the lower limb, jaundice, skeleton pain, stomachache outbreak and fever.Also monitor other symptoms, comprise hematuria (hematuria), frequent micturition, excessive thirst, erection pain (priapism, the sick male patient of this of 10%～40% has this symptom), thoracic cavity pain, vision relatively poor/blind.

Sicklemia disease patient is diagnosed and the test monitored will comprise full blood count (CBC), hemoglobin electrophoresis and meniscocyte's test.Other tests comprise that the drepanocytic peripheral blood film of demonstration, urinary cast or hematuria, serum hemoglobin reduce, bilirubin raises, numeration of leukocyte is higher, serum potassium raises, the serum creatinine raises and the blood oxygenation reduction.Under some situation, CT scan or MRI can show apoplexy.

Embodiment 4

In clinical research, BH4 is applied to the hyperpietic

Preliminary study

Parenteral perfusion or the single oral dose of BH4 adopted in research in advance; So whether the long-term oral BH4 that does not know a couple of days to several weeks can cause the lasting improvement of endothelial function disturbance.This research evaluation carries out the long-term oral medication of BH4 and whether can cause that arteriotony (BP) lowers because of the improvement of endothelial function disturbance in the hyperpietic.Carry out twice research with the acting duration of investigating oral BH4 and hypertension experimenter response to the BH4 of various dose.

Enlist 18 years old～75 years old, suffer from not controlled hypertension and treated (BP〉135/85) or newly be diagnosed as the experimenter of hypertension (BP〉140/90) with the stable resisting hypertension therapy of tradition.The patient continues their current antihypertensive therapy and remains unchanged.Exclusion standard is: the female subjects that may give a birth, recently have the medical history of coronary artery or peripheral blood vessel symptom, known hypertension secondary cause, serious not control hypertension (systolic pressure greater than 180mmHg and/or diastolic pressure greater than 110mmHg), make life expectancy less than 6 months serious complication, renal dysfunction or hepatic insufficiency, the change of any antihypertensive therapy of following in nearest 6 weeks, and any hemorrhagic disorder.The BH4 powder derives from Schircks Laboratories (Zuo Na, Switzerland).When using with vitamin C, with BH4 and vitamin C mixed with 1mg vitamin C/1mg BH4, and in the capsule of the suitable size of packing into.The suggestion experimenter is kept at this pill in fridge or the refrigerator.

Research 1: the onset time of oral BH4 and acting duration

Enlist 8 experimenters, 3 male wherein, 60.5 ± 10.9 years old mean age.The experimenter is dispensed in two groups one group.One winding is subjected to the BH4 of 10mg/kg/ days (n=4), and another winding is subjected to the BH4 of 5mg/kg/ days (n=4), and every day is oral administration at twice, continues for 8 weeks.These dosage are to determine according to BH4 dosed administration used in the phenylketonuria.In first week and the 6th week during the whole treatment and after stopping to treat, carry out BP weekly and measure.Mean blood pressure is calculated as: (SBP+2 * DBP)/3.

Treating 8 Zhou Houhe at baseline place, BH4 stops one week of treatment back using ultrasonic measuring brachial artery endothelium-dependent relaxation and non-endothelium-dependent relaxation function.The Circulation of determination techniques of being implemented such as Prasad etc., 2000,101 (20), described in the 2349-2354.In simple terms, in the temperature control room, on the fossa cubitalis of non-dominant arm, measure the brachial artery diameter with 11MHz high-resolution ultrasound pick off (Acuson Inc.).BP cuff on the forearm is inflated to is higher than 200mg Hg and continues 5 minutes, venting fast, and after hyperemia begins one minute measures the brachial artery diameter, thus measure the vasodilation (FMD) of blood flow mediation.In all measurements, measure three diameters three independent diastasiss on the picture, and average.Calculate the brachial artery diastole of the maximum of representing FMD with following formula: (average congested diameter-average baselining diameter) ÷ average baselining diameter * 100.In order to measure non-endothelium-dependent relaxation vasodilation reaction, experimenter's sublingual administration 0.4mg nitroglycerine is measured the brachial artery diameter as stated above after 5 minutes.Calculate non-endothelium-dependent relaxation vasodilation with following formula: (average diameter behind the buccal nitroglycerine-average baselining diameter) ÷ average baselining diameter * 100.

In research 1 and research 2, first terminal point behind the statistical analysis (primary end point) is the reduction of shrinking BP, average BP and diastole BP in the BH4 therapeutic process.Secondary terminal point (secondary end point) comprises the improvement of endothelial function (for example FMD).The BP that analyzes from baseline to the final point of treatment with the linear hybrid effect model of the data of repeated measure changes, and the difference of the variation between 100mg and the 200mg dosage group.The quadratic term that in the model that calculates the nonlinear trend in the treatment phase, comprises all numbers.Dosage, all numbers and all numbers square be main effects item in the described model; Weekly dose and weekly dose square be the interaction item of described model.The significant item that interacts shows that two changing patteries between the dosage group are distinct.With two tail student t-check analysis FMD data.All results are expressed as meansigma methods ± SD, p value＜0.05 can be considered as having significance,statistical.

The result of research 1 shows that in the therapeutic alliance group, the BH4 treatment significantly reduces systolic pressure (p=0.005, secondary trend) and average tremulous pulse BP (p=0.01).The reaction of figure A display of blood pressure.Numerical value is the standard error of meansigma methods ± meansigma methods.The SBP=systolic blood pressure, MBP=mean blood pressure, DBP=diastolic blood pressure; The p value is determined by ANOVA.

The result shows that shrinking BP descends, and 3 all after-contraction BP meansigma methodss are 13 ± 9mmHg (p=0.004), and 5 all after-contraction BP meansigma methodss are 15 ± 15mmHg (p=0.04), and continues in this 8 weeks that are reduced in treatment always.In the 6th week after treatment stops, BP is returned to the level (comparing p=ns (not having statistical significance) with baseline) before the treatment.Heart rate does not have significant change after using BH4, and the variation of diastole BP does not reach the significant degree of statistics.Between the BH4 of the BH4 of 5mg/kg dosage and 10mg/kg dosage, there is not statistical significant difference yet.

After the BH4 in the 8 weeks treatment, (p=0.05 n=6), and is returned to baseline values (3.7 ± 1.3%, compare p=ns with baseline) the 6th week to the vasodilative meansigma methods of brachial artery blood flow mediation after stopping to treat from 3.4 ± 1% being increased to 8.2 ± 3.4%.The vasodilation of nitroglycerine mediation remains unchanged, and (before the BH4 treatment is 11.9 ± 3%, is 16.1 ± 5% after the BH4 treatment, p=0.1).

Research 2: the dose response investigation of oral BH4

4 people are arranged in the experimenter of 20 satisfied participation conditions be excluded in the introduction period owing to can't return to accept to follow up a case by regular visits to.6 male subject are arranged, and the mean age is 59.5 ± 8.3 years old.The BH4 of 8 oral 200mg b.i.d of experimenter (one day twice), the BH4 of 8 oral 100mg b.i.d of experimenter (a day twice).Aspect baseline characteristic, there is not significant difference between these two groups.Because BH4 and vitamin C are with the mixed of 1:1, therefore all experimenters take vitamin C with specified BH4 dosage, twice of every day in first two week.After the introduction period in this two week, ensuing time experimenter all around only takes BH4 with prescribed dose.Interim in introduction period and treatment, measure heart rate and BP weekly.Mean blood pressure is calculated as: (SBP+2 * DBP)/3.Stop BH4 treatment then, and in the 1st week that stops to treat with the 4th week carried out follow-up investigation, measurement BP and heart rate.Before the beginning Drug therapy, BH4 treatment terminal point and stop BH4 and treat and carried out conventional chemical inspection and LFT (liver function test) in back month.

In research 2, as mentioned above, at the terminal point of the terminal point of vitamin C introduction period in 2 weeks, the oral BH4 treatment in 4 weeks and after stopping BH4 the 4th week measurement brachial artery endothelium-dependent relaxation and non-endothelium-dependent relaxation function.

In two groups the vitamin C introduction period process, heart rate or BP do not have significant change; Before introduction period, 16 experimenters' contraction BP, diastole BP and average tremulous pulse BP are respectively 152 ± 11mmHg, 84 ± 13 mmHg and 106 ± 10 mmHg, and vitamin C is respectively 148 ± 19mmHg, 86 ± 11mmHg and 106 ± 12mmHg (all p=ns) after treating for 2 weeks.

The experimenter's of the BH4 of oral 200mg b.i.d (one day twice) systolic pressure (p=0.03) and average BP significantly reduce (through linear trend analysis p=0.04).Figure B shows with the blood pressure response in the group of 200mg BH4 b.i.d treatment.Numerical value is the standard error of meansigma methods ± meansigma methods.The SBP=systolic blood pressure, MBP=mean blood pressure, DBP=diastolic blood pressure; The p value is determined by ANOVA.The reduction of diastole BP does not reach the significant degree of statistics (p=0.08).The one week average BP in back significantly reduces (p=0.02).In several weeks subsequently, BP further significantly reduces, and touches the bottom after 3 weeks, and the meansigma methods that shrink BP this moment has reduced 16mmHg; P=0.04 (figure B).After stopping to treat a week, BP keeps descending, but stops the 4th week behind the BH4, and BP raises and is returned to baseline values.

The BH4 treatment (n=7) of 200mg b.i.d significantly improves FMD.FMD is increased to 7.1 ± 4.9% (p=0.016) after 4 weeks of treatment from 3.7 ± 3% of BH4 when beginning treatment.Stop BH4 and treated back one month, FMD is returned to baseline values (3.2 ± 1.1%, compare p=0.6 with baseline).

Not observing the significant BP of statistics in the experimenter of the BH4 that takes 100mg b.i.d changes.Figure C shows the blood pressure response with the group of 100mg BH4 b.i.d treatment.Numerical value is the standard error of meansigma methods ± meansigma methods.The SBP=systolic blood pressure, MBP=mean blood pressure, DBP=diastolic blood pressure.All there is not significant changes in heart rate in the group with the BH4 of 100mg or 200mg b.i.d treatment.

In the experimenter of the BH4 that takes 100mg b.i.d, there is not significant FMD to change that (5.3 ± 2.5% is relative 6.2 ± 3.5%, p=0.55).In the research process, the vasodilation and the non-endothelium-dependent relaxation vasodilation of nitroglycerine mediation all do not have significant change in two groups.

In all research process, all there is not tangible adverse events.This result shows that the long-term treatment of oral BH4 can effectively reduce the tremulous pulse BP that hypertension is controlled relatively poor experimenter or newly is diagnosed as hypertensive experimenter.Dosage or higher dosage at 200mg b.i.d are observed this effect, and heart rate is without any variation.After the BH4 treatment of described dosage, experimenter's endothelial function disturbance significantly improves, and also makes BP reduce.BP is reduced in 1 week of treatment just clearly, and is maintained to the 8th week of continuous treatment, does not have quick drug resistance reaction.At least blood pressure keeps descending in the week after treatment stops, but is returned to baseline after 4 weeks.Oral BH4 seems to have good tolerability and without any serious ill effect.

Further clinical research

The parallel study that carries out 2 phases, multicenter, randomization, double blinding, placebo-contrast with estimate 10mg/kg/ days 6R BH4 (be administered twice every day (b.i.d) continued for 8 weeks) to the influence of the experimenter's that suffers from the relatively poor systemic hypertension of control blood pressure (BP).Assess quantity of parameters, comprised systolic arterial pressure (SBP), auterial diastole pressure (DBP), NOS3 (eNOS) activity and endothelial function disturbance.Control among the relatively poor patient type ii diabetes patient and hypertension, also assess effect insulin sensitivity.Antihypertensive therapy and treating diabetes (if any) remain unchanged in whole research process.

Inclusion criteria in the research comprise through the record the essential hypertension history (in 2 different tests, contraction BP is at least 140mm Hg and/or diastole BP is at least 90mm Hg), though and use at least two kinds of traditional antihypertensive drug that the mechanism of action is different simultaneously, and before selecting at random, adhered at least 3 months, but hypercalcinuria is controlled still relatively poor, and interim average SBP of the screening in initial two weeks and average DBP are in following ranges: average SBP is at least 135mm Hg but is not more than 160mm Hg; Average DBP is at least 85mm Hg but is not more than 110mm Hg.In addition, diabetic subjects must have the type ii diabetes history through record, and uses identical therapy for treating at least 3 months.

The standard of getting rid of from research comprises: plan is conceived or possibility is conceived; The pro-treatment of any BH4 preparation; Known anaphylaxis or allergy to the excipient of any 6R BH4; Known hypertension secondary cause; Can influence the concurrency disease or the disease of research property of participation or safety, for example hemorrhagic disorder, faintness history or dizzy history, serious gastro oesophageal reflux disease (GORD) (GERD), Symptomatic coronary artery or peripheral blood vessel, arrhythmia, organ transplantation, organ failure or type i diabetes; Make life expectancy less than 6 months any serious complication; The serum creatinine〉2.0mg/dL or liver enzyme level be higher than 2 times of the normal value upper limit; Follow treatment with following medicine: (a) any known medicine (for example methotrexate) that suppresses folic acid metabolism, (b) levodopa or (c) any phosphodiesterase (PDE)-5 inhibitor is (for example

Ai Li

Or Revatio ^TM) or any PDE-3 inhibitor (for example cilostazol, milrinone (milrinone) or vesnarinone (vesnarinone)).

Satisfy the experimenter that inclusion criteria is not excluded standard exclusion and accept 6R BH4 or placebo, the treatment phase continued for 8 weeks, and in the 9th week with followed up a case by regular visits in the 12nd week (that is the 1st of follow-up period all and the 4th week).During the treatment phase, measure BP and heart rate (HR) weekly, and when following up a case by regular visits to for twice, measure BP and HR.In all research centers, the method that obtains BP all is through standardized, and each experimenter is carried out the selection of time that BP measures also is through standardized.Collect blood sample and the urine sample that is used for the detection of routine clinical laboratory and is used as biological marker in the 0th week, the 4th week, the 8th week and the 12nd week.To diabetics, collect the blood that is used to test fasting insulin level and blood sugar level and Hgb AlC in the 0th week, the 8th week and the 12nd week.Estimate ECG in the 8th week and the 12nd week.

Vital sign: SBP and DBP are all write down in each visit, and unit is mm Hg; Heart rate (HR), unit are per minute heart beating number of times; And breathing rate (RR), unit is the per minute frequency of respiration.Physical examination comprises the evaluation to body weight, overall appearance, cervical region, thoracic cavity/lung, heart sounds, abdominal part and lower limb.

During the screening phase in two weeks, all measure BP three times (triplicate in 10 minutes) in three visits at every turn.Whether satisfy inclusion criteria with average SBP value of these 9 test result calculations and average DBP value with definite BP.After the randomization grouping, measure BP once more three times (visit of 0 week), the meansigma methods of these three values is exactly a baseline value.

During the treatment phase (since the 1st week), take the research medicine weekly in the morning and measure before trough BP.Should be in one day much at one time (in about 90 minutes) measure BP.

12 lead electrocardiogram of record standard, and collect following measured value: heart rate, the rhythm and pace of moving things, interval measured value (being PR, QRS, QT and QTc) and axle.In the 0th week and the 8th week of treatment, use full-automatic ambulatory blood pressure monitoring instrument (ABPM) to carry out monitoring in 24 hours.ABPM measures and record shrinks BP and diastole BP; A plurality of BP measured values can be shown in the chart with expression BP curve.Collect blood (blood plasma) sample and urine sample standard biological sign with evaluation endothelial function disturbance and oxidative stress.Some experimenters are also tested FMD, whole body blood vessel repellence and arterial compliance in addition with ultrasound cardiogram.

Clinical laboratory's evaluation to blood sample and urine sample comprises following content:

The hematology: differential blood count, red blood cell count(RBC), platelet count,

Hemoglobin and hematocrit

Serum chemistry: albumin, alkali phosphatase, ALT (SGPT), AST

(SGOT), bilirubin, BUN, calcium, chloride, total gallbladder

Sterin, sarcosine GGT, globulin, glucose, LDH,

Phosphorus, potassium, total protein, sodium and uric acid

On an empty stomach (4～6 hours) serum triglycerides, T-CHOL, low density lipoprotein, LDL (LDL),

Lipid high density lipoprotein (HDL)

That suffers from type ii diabetes is tried fasting glucose and insulin and Hgb A ₁C

Person's additional serum chemistry:

Urinalysis: routine urinalysis: outward appearance, color, pH, proportion, ketone, Portugal

Grape sugar, bilirubin, nitrite, urobilinogen and microscope

Check

Collection morning, primary urine sample was as the standard biological sign

(following): microalbuminuria

Nitrate/nitrite, cGMP and different prostaglandin are answered in endothelial function disturbance and oxidation

The biological marker that swashs: standard is given birth to

Thing sign (blood plasma and urine):

With body quality coefficient (BMI) with on an empty stomach serum insulin levels and blood sugar level result estimate the experimenter's who suffers from type ii diabetes insulin sensitivity.Also measure Hgb AlC.

After research was finished, the oral BH4 that evaluated 10mg/kg/ days treated the influence to blood pressure, endothelial function and insulin sensitivity.The objective of the invention is to observe the significance,statistical reduction of systolic blood pressure and/or diastolic blood pressure.After the BH4 treatment, endothelial function and/or insulin sensitivity also can improve.In further research, estimated the effect of the additional dose in every day 2mg/kg～every day 10mg/kg scope.

Embodiment 5

BH4 and PDE5 inhibitor be the effect of coupling in vivo

This research evaluation 6R-BH4 and PDE5 inhibitor citric acid sldenafil be the cardiovascular effect during the high dose coupling in vivo.According to two Latin square cross-over design of describing in the following table (double Latinsquare crossover design) with medicament administration in 8 male, tested and the Bi Erge sleuth of not test (N.T.).All animals all pass through gastric infusion.Use test article (cumulative volume is 10mL/kg during administering drug combinations) with the dose volume of 5mL/kg; Control animal is with the dose volume administration of 10mL/kg.The 6R-BH4 (Sapropterin dihydrochloride) that provides with the tablet of 300mg (the active 6R-BH4/ sheet of 100mg) is stored in room temperature with desiccant.

	The administration phase 1	The administration phase 2	The administration phase 3	The administration phase 4
					Animal	The 1st day	The 4th day	The 8th day	The 11st day
No. 1	6R-BH4 a	The 6R-BH4+ sldenafil b	Contrast c	Sldenafil d
					No. 2	The 6R-BH4+ sldenafil	Sldenafil		6R-BH4	Contrast
No. 3	Contrast	6R-BH4	Sldenafil	The							6R-BH4+ sldenafil
					No. 4	Sldenafil	Contrast	The 6R-BH4+ sldenafil	6R-BH4
No. 5	Contrast	The 6R-BH4+ sldenafil	Sldenafil							6R-BH4
					No. 6	The 6R-BH4+ sldenafil	6R-BH4	Contrast	Sldenafil
No. 7	Sldenafil	Contrast		6R-BH4						The	6R-BH4+ sldenafil
					No. 8	6R-BH4	Sldenafil	The	6R-BH4+ sldenafil			Contrast

A. use 6R-BH4 to animal, dosage is 100mg/kg, and dose volume is 5mL/kg.

B. 6R-BH4 (100mg/kg, dose volume are 5mL/kg) is accepted in animals received therapeutic alliance earlier, accepts citric acid sldenafil (30mg/kg, dose volume are 5mL/kg) then, and cumulative volume is 10mL/kg.

C. control animals received either reverse osmosis water, dose volume is 10mL/kg.

D. use the citric acid sldenafil to animal, dosage is 30mg/kg, and dose volume is 5mL/kg.

During the treatment beginning, at about 7 monthly age～10 monthly ages of animal, the body weight of animal is 7.8kg～12.0kg before the administration.

At least 2 weeks before the treatment beginning make the animal overnight fasting, with its anesthesia and with electrocardiogram (ECG) and blood pressure conveyer implantation abdominal part and be sewn on the stomach wall.The blood pressure conduit is placed tremulous pulse, and be advanced to abdominal aorta.Check mortality rate, abnormity and pain and the uncomfortable sign of twice (morning and afternoon) animal every day.According to the extra discovery of observed situation record.The research terminal point that all animals have all lived and have been scheduled to.

Carried out three detailed observation administration last stage (that day before comprising administration) and administration stage the 3rd day and the 10th day.Each administration day is all write down ECG and blood pressure measurement, carries out at least 90 minutes record before the administration, continues record at least 8 hours after the administration, per hour carries out at least 15 minutes record then, and lasts till after the administration at least 24 hours.Blood pressure measurement comprises systolic pressure, diastolic pressure and mean arterial pressure, and pulse pressure (contraction-diastole).Each administration day is all carried out the blood pressure evaluation in about 2 hours, 4 hours, 8 hours, 12 hours and 24 hours before administration and after the administration.

Carry out the quantitative assessment of ECG measurement result (comprise RR interval, QT and through the gauged QT of heart rate (QTc adopts the Fridericia method)).

Left indoor pressure calculated signals positive inotropic action (+dP/dt according to about 2 hours, 4 hours, 8 hours, 12 hours and 24 hours before the administration and after the administration _Max) and heart rate.

Based on mortality rate, clinical sign, body weight, abdomen temperature measurement, comprise cardio-vascular parameters, the muscle strength status (+dP/dt of ecg analysis and hemodynamic data (systolic pressure, diastolic pressure and mean arterial pressure) _Max) and heart rate carry out cardiovascular effect and toxic evaluation.Figure D shows the average systolic during 24 hours after the administration of various treatments.Figure E shows the AvDP during 24 hours after the administration of various treatments.Figure F shows the mean arterial pressure during 24 hours after the administration of various treatments.Figure G shows the average arterial pulse pressure during 24 hours after the administration of various treatments.Figure H shows average (+dP/dt _Max).Figure I shows average heart rate.

The citric acid sldenafil is individually dosed or accepted in the animal of citric acid sldenafil and 6R-BH4 administering drug combinations having accepted, after administration 2 hours and 4 hours, and the QT interval that records on the ECG, reduce.QT interval when the citric acid sldenafil is individually dosed, the QT interval during with the 6R-BH4 administering drug combinations, there is no different with the citric acid sldenafil.Do not have in having accepted the individually dosed animal of 6R-BH4 to find that the QT that compares with matched group changes.Therefore, QT is changed owing to using the citric acid sldenafil separately.Do not observe heart rate and proofread and correct the variation of QT interval (QTc).QTc makes the fact of QT interval normalization data show that it is to increase (RR interval reduce) and cause by using the heart rate of finding behind the citric acid sldenafil that the QT interval reduces.Before using test article and afterwards, two animals show paroxysmal ventricular tachycardiies, and other animals show isolated temporary transient arrhythmia; The conduit that arrhythmia is attributable to implant in left ventricle for blood pressure measurement, rather than cause by test article.

Do not observe with treat relevant systolic pressure, mean arterial pressure or+dP/dt _MaxVariation.Compare with matched group, the diastolic pressure of having accepted administering drug combinations or having accepted the individually dosed animal of citric acid sldenafil all significantly reduces at all time points.Do not observe this effect after using 6R-BH4 separately.Do not find significant difference in the effect of using the citric acid sldenafil separately with itself and 6R-BH4 between co-administered effect, show that this is mainly caused by the citric acid sldenafil.6R-BH4 used with the citric acid sldenafil do not increase or suppress this effect.

At all time points, having accepted citric acid sldenafil heart rate individually dosed or that accepted the animal of citric acid sldenafil and 6R-BH4 administering drug combinations all significantly increases.Using the citric acid sldenafil separately and itself and 6R-BH4 not being found significant difference between co-administered, show that this is mainly caused by the citric acid sldenafil; 6R-BH4 does not increase or weakens this effect.

After administration 2 hours, 4 hours and 8 hours, the arterial pulse pressure of having accepted the individually dosed animal of citric acid sldenafil significantly increases.After co-administered citric acid sldenafil and the 6R-BH4 2 hours and 4 hours, arterial pulse pressure significantly increases.Think that these variations are secondary to the diastolic pressure of being found these treatment backs and reduce.

With use the citric acid sldenafil separately and compare, time point 2 hours and 8 hours, the arterial pulse pressure of having accepted the animal of citric acid sldenafil and 6R-BH4 administering drug combinations significantly reduces, and shows that 6R-BH4 may have weakening effect to the pulse pressure increase that is caused by the citric acid sldenafil.

Result of study shows the citric acid sldenafil and makes the diastolic pressure of Canis familiaris L. reduce with the citric acid sldenafil of 6R-BH4 coupling.The reduction of this blood pressure is attended by the increase of heart rate and the increase of arterial pulse pressure.Blood pressure reduces and the heart rate increase is the effect of citric acid sldenafil in Canis familiaris L. that writes down in the document.Its conclusion is that heart rate increases and the pulse pressure increase is secondary to the blood pressure reduction.Do not mention and use the effect of 6R-BH4 separately measured any cardio-vascular parameters.

As pointed, this result shows so beyond thought effect: the coupling of PED5 inhibitor and 6R-BH4 has weakened in these Canis familiaris L.s the inductive pulse pressure of observed citric acid sldenafil and has increased, and increases and can observe this pulse pressure when using sldenafil separately.

The 6R-BH4 of 100mg/kg is added in the citric acid sldenafil of 30mg/kg the cardiovascular effect of citric acid sldenafil in these Canis familiaris L.s do not had adverse influence.

All disclosed herein and claimed compositionss and/or method all can be prepared and implement according to present disclosure, need not too much experiment.Though adopted preferred implementation that the compositions and methods of the invention are described, but those skilled in the art obviously can change the step of compositions described herein and/or method and described method or the order of step under the situation that does not deviate from theory of the present invention, purport and scope.More specifically, obviously some to be chemistry relevant be again that the medicament that the physiology is correlated with can replace medicament described herein and obtain same or analogous result.Similar substituting and revise that all these it will be apparent to those skilled in the art all is considered as within as purport of the present invention, scope and theory that appending claims limited.

Claims (15)

1. method for the treatment of endothelial function disturbance, described method comprises the step of the people of this treatment of needs being used (6R)-tetrahydrobiopterin with the effective dose that can treat described endothelial function disturbance.

2. the method for claim 1, wherein described endothelial function disturbance is a hypertension.

3. as claim 1 or the described method of claim 2, wherein, described people suffers from type ii diabetes.

4. as each described method in the claim 1～3, wherein, described using is to carry out in oral mode.

5. as each described method in the claim 1～4, wherein, the effective dose of described treatment is that every day is greater than about 15mg/kg 3mg/kg～every day.

6. as each described method in the claim 1～4, wherein, the effective dose of described treatment is that every day is greater than about 10mg/kg 3mg/kg～every day.

7. as each described method in the claim 1～3, wherein, with Orally administered described (the 6R)-tetrahydrobiopterin of accumulated dose of about 5mg/kg every day.

8. as each described method in the claim 1～3, wherein, with Orally administered described (the 6R)-tetrahydrobiopterin of accumulated dose of about 10mg/kg every day.

9. as each described method in the claim 1～3, wherein, with Orally administered described (the 6R)-tetrahydrobiopterin of accumulated dose of about 400mg every day.

10. as each described method in the claim 1～9, wherein, described (6R)-tetrahydrobiopterin is used twice every day.

11. as each described method in the claim 1～10, described method further comprises uses medicine except that (6R)-tetrahydrobiopterin to described people.

12. method as claimed in claim 11, wherein, described medicine comprises phosphodiesterase 5 inhibitor.

13. method as claimed in claim 12, wherein, described phosphodiesterase 5 inhibitor are selected from the group of being made up of non-solvate of the pharmaceutical salts of the solvate of the pharmaceutical salts of the solvate of the pharmaceutical salts of sldenafil, sldenafil, sldenafil, tadanafil, tadanafil, tadanafil, Vardenafil, Vardenafil, the solvate of Vardenafil, You Dina is non-, You Dina is non-pharmaceutical salts, You Dina and their mixture.

14. method as claimed in claim 12, wherein, described phosphodiesterase 5 inhibitor are the pharmaceutical salts of sldenafil or sldenafil or the solvate of sldenafil.

15. a method for the treatment of drepanocytosis, described method comprise the step of the people of this treatment of needs being used (6R)-tetrahydrobiopterin with the effective dose that can treat described drepanocytosis.

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